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Efficient Transduction and Targeted Expression of Lentiviral Vector Transgenes in the Developing Retina


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EFFICIENT TRANSDUCTION AND TARG ETED EXPRESSION OF LENTIVIRAL VECTOR TRANSGENES IN THE DEVELOPING RETINA By JASON EDWARD COLEMAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Jason Edward Coleman

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I dedicate this work to my parents, brothers, sisters, and friends who have all been a great source of encouragement and support throughout this endeavor and to the inspiring and loving memory of my grandfather, Dr. Joseph Edward Coleman.

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iv ACKNOWLEDGMENTS First and foremost, I would like to tha nk my mentor, Susan Semple-Rowland, for all of her excellent support a nd encouragement over the past four-and-a-half years. She is a very gifted educator and committed scholar. I feel very fortunate to have had the opportunity to work in her laboratory under he r guidance over the y ears. Her enthusiasm for science has been inspirational and I th ank her for providing an environment where students are encouraged to be creative and to succeed at the highest level. Next, I would like to thank the members of my committ ee, William Hauswirth, Marieta Heaton and Adrian Timmers, for providing their expertise and input through my research endeavors. I thank all of the past and present memb ers of the Semple-Rowland laboratory for their great friendships and invalu able assistance over the years. I would especially like to thank Yan Zhang for stimulating discussions on circadian clocks, Miguel Tepedino and Gabby Fuchs for their excellent assistance in the lab, and Christina Appin for producing great virus and keeping several generations of cells alive. The completion of this dissertation woul d not have been possible without the tremendous and humbling encouragement from all of my teachers and friends throughout my life. I would like to express my apprec iation for my undergraduate mentor, Hazel Jones, who helped initiate my scientific career and introduced me to the neuroscience discipline (and the H-Tx rat). I would also like to thank Jake Streit for taking the time to share his knowledge and expert ise in histology and micros copy. I thank fellow graduate students Matt Huentelman and Josh Stopek fo r their friendship and collaboration time,

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v which has helped to make lentivirus engineer ing more fun. Very special thanks go to my girlfriend, Rachel, who has pa tiently stood by me and supported me through this final phase of my doctoral degree. I would like to extend a tremendous thank you to my family for providing all of the love and support over my doctoral years. Fi nally, I would like to extend the greatest acknowledgement to my mother, Denise Moor e, who has never stopped believing in my potential and has provided unwavering love and support over the past five years.

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vi TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 Retinal Disease as a Model for Gene Therapy Efficacy...............................................1 Leber Congenital Amaurosis........................................................................................2 Clinical Phenotype and Genetics...........................................................................2 Clinicopathology of LCA1....................................................................................4 Animal Models of LCA1.......................................................................................4 Gene Therapy Vectors..................................................................................................7 Parvoviridae and Adenoviridae -based Vectors...................................................7 Retroviridae -based Vectors...................................................................................9 Regulation of Vector-Med iated Gene Expression......................................................13 Gene Regulation in Retinal Photoreceptors........................................................13 2 A 4.0 KB FRAGMENT OF THE G UANYLATE CYCLASE ACTIVATING PROTEIN-1 (GCAP1) PROMOTER TARGETS GENE EXPRESSION TO PHOTORECEPTOR CELLS IN THE DEVELOPING RETINA.............................15 Note........................................................................................................................... ..15 Introduction.................................................................................................................15 Methods......................................................................................................................18 Northern Blot Analyses.......................................................................................18 Preparation of Constructs....................................................................................19 Cell Cultures and Transfections..........................................................................20 Luciferase and -galactosidase Assays...............................................................21 Lentivirus Production..........................................................................................22 Embryonic Injections...........................................................................................23 Histochemistry and In vivo Promoter Analyses..................................................24 Results........................................................................................................................ .25 Expression of GCAP1 in De veloping Chicken Retina........................................25 In vitro GCAP1 Promoter Activity......................................................................26

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vii Lentiviral Transduction of Avian Tissues...........................................................27 Analyses of GCAP1 Promoter Fragments In vivo ...............................................29 Discussion...................................................................................................................32 3 IN VIVO ANALYSES OF THE DEVELO PMENTAL AND CELL-SPECIFIC ACTIVITY OF THE HUMAN RETI NAL GUANYLATE CYCLASE-1 (GC1) PROMOTER...............................................................................................................39 Introduction.................................................................................................................39 Methods......................................................................................................................41 Preparation of Constructs....................................................................................41 Production of Lentiviral Vector and Titers.........................................................42 Embryonic Injections...........................................................................................42 Tissue Preparation, Histoche mistry and Microscopy..........................................42 Results........................................................................................................................ .43 Primary Sequence Analyses................................................................................43 Tissue Specificity of nlacZ Expression...............................................................46 Cell Specificity and Developm ental Expression of nlacZ...................................46 Discussion...................................................................................................................47 4 IMPROVEMENTS IN THE DESIGN A ND PRODUCTION OF HIV-1-BASED LENTIVIRAL VECTORS RESULTS IN HIGH TRANSDUCTION EFFICIENCY IN RETINA AND THE EFFICIEN T EXPRESSION OF A RETINAL GUANYLATE CYCLASE-1 (GC1) TRANSGENE.................................................51 Note........................................................................................................................... ..51 Introduction.................................................................................................................51 Materials and Methods...............................................................................................53 Lentiviral Vector Constructs...............................................................................53 Lentiviral Vector Producti on, Concentration and Titers.....................................55 Delivery of EF1 -PLAP Vector to Chicken Neural Tube..................................59 Analyses of GC1 Expression Vectors.................................................................59 Results........................................................................................................................ .62 Lentivirus Production and Concentration............................................................62 In Vivo Performance of the Lentiviral Vector.....................................................64 GC1 Immunocytochemistry................................................................................64 GC1 Enzyme Activity.........................................................................................67 Discussion...................................................................................................................68 5 CONCLUSIONS AND FUTURE DIRECTIONS.....................................................72 Targeted Gene Expression in Retina..........................................................................72 Lentiviral Vector Transduction in Retina...................................................................76 LIST OF REFERENCES...................................................................................................78 BIOGRAPHICAL SKETCH.............................................................................................95

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viii LIST OF FIGURES Figure page 2 1. Expression of GCAP1, GC1 and iodopsin genes in developing chicken retina.......26 2-2. GCAP1 promoter activity in transfect ed E12 primary chicken embryonic retinal cultures.....................................................................................................................28 2-3. Retinal whole mounts and cross-secti ons prepared from embryos that received injections of TY-EF1 -nlacZ...................................................................................30 2-4. Cell specificity and temporal onset of activity of the 292, 1436 and 4009 GCAP1 promoter fragments in embryonic chicken retina.....................................................33 3-1. Sequence and schematic of the re tinal GC1 5’ flanki ng region-nlacZ fusion constructs..................................................................................................................45 3-2. Cross-sections of pineal gland from E18.5 embryo that was injected with the TYFGCE7-nlacZ virus....................................................................................................46 3-3. Cross-sections of retin as containing human GC1 prom oter-nlacZ transgenes...........48 4-1. Maps of the modular cloning plasmid v ectors constructed for the SIN lentiviral vector system used in this study...............................................................................55 4-2. The HIV-1-based self-inactivat ing lentiviral vector system.......................................56 4-3. Production of lentivirus by transfected 293T cells as a function of time...................62 4-4. Outline and results of the vector production protocol................................................63 4-5. Lentiviral vector-media ted transduction in chicken neural progenitor cells..............65 4-6. PLAP expression in pos t-hatch chicken retinas.........................................................66 4-7. Expression of recombinant bovine GC1 in avian-derived retinal photoreceptor cells and DF-1 fibroblast cells..........................................................................................67 4-8. Expression of recombinant bovine GC1 from transiently transfected transgenes and transgenes packaged into the lentiviral vectors........................................................69

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ix 5-1. Immunolabeling of primary embryonic ch icken retinal cultures with cone (antiiodopsin) and rod cell ma rkers (anti-rhodopsin)......................................................74

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x Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFICIENT TRANSDUCTION AND TARGETED EXPRESSION OF LENTIVIRAL VECTOR TRANSGENES IN THE DEVELOPING RETINA By Jason Edward Coleman May 2003 Chair: Susan L. Semple-Rowland, PhD Major Department: Neuroscience Gene therapy holds great promise as an effective treatment for genetic diseases. Retinal diseases caused by genetic mutations are among the leading causes of blindness and are an excellent place to begin studying th e basic principles of gene transfer-based treatments. In addition to understanding the mo lecular basis of a target disease, perhaps the most difficult steps in the development of somatic gene therap ies are engineering a suitable method to deliver therapeutic transg enes to the diseased cells and achieving appropriate levels of expre ssion for extended periods. The primary goal of this study was to develop a lentivirus-based gene delivery vector that can be used to target the expression of a functional, therapeutic tran sgene to photoreceptor cells in the retina. Lentiviral vectors derived from the hu man immunodeficiency virus type 1 are emerging as the vectors of choice for long-term, stable in vitro and in vivo gene transfer. Several inherited retinal diseases are caused by mutations in genes that are expressed in photoreceptor cells and are required for nor mal function of thes e cells. Cell-specific

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xi promoters can be incorporated into viral vector gene expression constructs and are used to direct expression of transgenes to specifi c cell types, and the le vel of expression of these transgenes is controlled by selecting promoters that possess different intrinsic activity levels. A self-inactivating lentiviral vector system was used in a novel manner to study the intrinsic activity profiles of promoters that regulate the expression of photoreceptorspecific genes. Using these methods, we were ab le to identify regions of these promoters that are capable of targeting gene expressi on to retinal cells during development. Data from these studies provide clues regarding the cis -acting elements that are important for regulating photorecep tor-specific genes in vivo Furthermore, we have improved the design of and methods of producing lentiviral vectors that will f acilitate use of this system for delivering a normal, functional copy of a therapeutic transgen e to retinal cells. The results of these studies lay the foundati on for future experiments aimed at studying the potential use of lentiviral vector therapies for treating autosomal recessive retinal diseases such as Leber congenital amaurosis type 1 (LCA1).

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1 CHAPTER 1 INTRODUCTION Retinal Disease as a Model for Gene Therapy Efficacy The basic premise of gene therapy is to tran sfer (permanently or transiently) genetic material to genetically defective cells, enab ling these cells to func tion normally without further treatment. Researchers within the fi eld of gene therapy have recently made promising advances toward realizing this goal, but we are still in the early stages of gene therapy research. Thus, it is imperative that initial gene thera py studies be conducted using animal models of well-defined genetic di seases that will provide the framework for the development of treatments for more comple x diseases. For example, genetic diseases that affect tissues such as the retina could c ontribute greatly to th e basic principles and applications of gene therapy. One practical advantage of studying the efficacy of gene therapy for retinal diseases is that the eye is an easily accessible, imm une-privileged organ. Another advantage is that many of the genetic mutations affecting retin al function occur in genes that encode proteins critically involved in the phototransduction cascade and the visual cycle – two processes that are well underst ood in retina. Consequently, th e etiology of se veral retinal diseases has been defined at the mol ecular level (www.sph.uth.tmc.edu/RetNet). Therefore, this and the fact that there are se veral well-defined animal models available of these diseases (Lin et al. 2002;Peters en-Jones 1998;Semple-Rowland et al. 1998), strongly support a focus on retinal disease in e fforts to define princi ples of gene therapy. Within the past 5 years, results from several studies showed that th e expression of various

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2 transgenes in cells transduced with vi ral vectors can successfully slow retinal degeneration in animal models of primary re tinal disease (Acland et al. 2001;Ali et al. 2000;Bennett et al. 1998;LaVail et al. 2000;Lewin et al. 1998;McGee Sanftner et al. 2001;Takahashi et al. 1999). One notable a dvance has been the demonstration that functional vision can be rest ored in a canine model of a congenital retinal dystrophy, Leber congenital amaurosis (LCA), by transf erring a normal copy of the RPE65 gene to cells within the retina (Acland et al. 2001). In the remaining portion of this chapte r, I will focus on disc ussing LCA and recent progress toward development of gene therapies for retinal di sease. Specific topics will include descriptions of animal models of LCA1, the rationale for selection and use of specific viral vectors for the developmen t of therapies, and the importance and development of strategies to target gene expression to specific cell types affected by retinal disease. Leber Congenital Amaurosis Clinical Phenotype and Genetics Leber first described the condition know n as LCA in 1869 (Leber 1869). LCA is a family of clinically and genetically heteroge neous inherited retinal diseases that produce the earliest and most severe forms of conge nital blindness (Perrault et al. 1999). It is generally assumed that LCA accounts for 5% of all cases of retinal dystrophies, but may be even more frequent due to the high rate of consanguinity among LCA families (Cremers et al. 2002;Foxman et al. 1985;Perrault et al. 1999). Visual deficits are usually detected by the age of 6 months in infants a nd LCA patients rarely present with a visual acuity better then 20/400 through life (Cre mers et al. 2002). The electroretinographic responses of LCA patients are severely atte nuated or non-existent at birth and, based on

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3 some published criteria, the electroretinogram (ERG) should be extinguished before the age of 1 year (Foxman et al. 1985). Uncomplicated LCA is inherited in an autosomal recessive mode. By 1996, Perrault and her colleagues had identified the first gene linked to LCA, the gene encoding the enzyme retinal guanylate cyclase-1 (GC1 ; designated LCA1) (Perrault et al. 1996). Since this discovery, mutations in six addi tional genes expressed in retina have been linked to LCA. These genes, which encode prot eins that are involved in several aspects of rod and cone cell function, include RPE65 (M arlhens et al. 1997), CRX (Freund et al. 1998), AIPL1 (Sohocki et al. 2000a;Sohocki et al. 2000b), CRB1 (den Hollander et al. 2001;Lotery et al. 2001) and RP GRIP1 (Dryja et al. 2001). Several GC1 mutations have been identifie d and most of these are frameshift and missense mutations (Perrault et al. 2000). Th e frameshift mutations generate premature translation termination codons that are predic ted to lead to the absence of GC1 protein (Perrault et al. 1996;Perrault et al. 2000;Rozet et al. 2001). The missense mutations, many of which occur in the catalytic domain, have been shown to severely compromise or abolish GC1 activity (Rozet et al. 2001) Functional consequences of an F589S missense mutation in GC1 show that the muta tion reduces basal GC1 activity by 80% and disrupts the ability of GCAP 1 to stimulate GC1 under low-calcium conditions (Duda et al. 1999b). Most GC1 mutations identified so far are assumed to result in significant reductions in the intracellular levels of cG MP in photoreceptor cells, reductions that could lead to a situation equivalent to cons tant light exposure during retinal development (Perrault et al. 2000).

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4 Clinicopathology of LCA1 Ophthalmological examinations of LCA1 patients reveal that the fundus appears normal early in life, but abnormalities such as salt-and-pepper pigmentation and the attenuation of retinal vessels begin to appear after several years (Edwards et al. 1971). Most of the reported histopathol ogic studies of LCA1 retinas have revealed that the rods and cones degenerate late in life (Franc ois and Hanssens 1969; Mizuno et al. 1977;Noble and Carr 1978). Immunohistochemical analyses performed in a postmortem eye obtained from a young LCA1 patient (11.5 years old) re vealed that substantial numbers of rods and cones were retained in the macula and far periphery; however there was an overall reduction in the labeling of c one outer segment proteins (M ilam et al. 2003). From a therapeutic standpoint, the results of these an alyses are encouraging and suggest that the retinal circuitry was intact and functional. Further insight into th e pathophysiological and cellular consequences of the GC1 mutati ons linked to LCA1 have been obtained primarily from studies of two animal models of this disease, the GUCY1*B chicken and the GC1-knockout mouse. Animal Models of LCA1 There are currently two an imal models of LCA1, the GC1-knockout mouse and the GUCY1*B chicken. The phenotypes of these two animal models are strikingly different. Comparisons of the two models provide impor tant clues about the consequences of GC1 null mutations on the development, function a nd health of cone a nd rod photoreceptor cells. GC1-knockout mouse The retinas of GC1-knockout mice are mor phologically normal at birth, but exhibit reductions in the amplitudes of both the rod a nd cone cell responses to light stimulation

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5 (Yang et al. 1999). By one month of age, cone responses to light are barely detectable and the ERGs of both the rod aand b-waves are dramatically reduced (Yang et al. 1999). These mice do not display any detectable visu al deficits despite the changes in rod function that progress until 5 months of age. The first signs of photoreceptor degeneration occur between 4 and 5 weeks of age and is ma rked by a rapid and specific loss of cones, leaving a normal population of rod cells (Ya ng et al. 1999). This pattern of photoreceptor degeneration differs significantly from that observed in the GUCY1*B chicken, which is described below. GUCY1*B chicken The GUCY1*B chicken, formerly know n as the retinal degeneration or rd chicken (Semple-Rowland and Cheng 1999), is recogniz ed as a naturally occurring model of human LCA1 and is the only animal model of inherited retinal di sease that possesses a cone-dominant retina (Semple-Rowland et al. 1998;Semple-Rowland and Lee 2000). A deletion-rearrangement of the GC1 gene resu lts in loss of the transmembrane domain of GC1, destabilization of the transcript, and a total absence of the GC1 enzyme in the GUCY1*B retina. The retinas of GUCY1*B animals are mo rphologically indistinguishable from normal retinas at hatching. Earl y signs of retinal pathology ap pear 7 to 10 days post-hatch in the photoreceptor layer of the central reti na. Degeneration of the photoreceptor layer is progressive so that by 21 days of age, ma rked degeneration of the photoreceptor outer segments is apparent. By 60 days of age, the mid-peripheral retina shows signs of degeneration and at 115 days of age, loss of photoreceptors from the central retina is complete. By 6 to 8 months of age, cell loss from the inner retina is also apparent. The amplitudes of the ERGs recorded from GU CY1*B chickens under photopic and scotopic

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6 conditions are absent or less than 7% of t hose recorded from normal chickens (Ulshafer et al. 1984). The levels of cGMP in the photoreceptor layer of 1-2 day old GUCY1*B chicken retinas are only 10 to 20% of thos e measured in age-matched normal retinas (Semple-Rowland et al. 1998). These results have led to the hypothe sis that decreased levels of cGMP may result in a state of constitutive hyperpolarization of the photoreceptor cells, a condition that could mimi c the degenerative events associated with constant light exposure (Fain a nd Lisman 1999;Hao et al. 2002). Upon comparison of the mouse and chicken models, it is evident that the only common feature of the progressive retinal degenerations is that cone function and survival are severely compromised by the ab sence of GC1. Differences in the spatial organization and composition of photoreceptor cells in mouse and chicken retinas may provide a likely explanation as to why the phenotypes and pat hologies are so contrasting. The retina of the chicken is cone dominant (80% cones; 20% rods), whereas that of the mouse is rod dominant (3-5% cones; 95-97% rods). Theref ore, the effects of cone degeneration on rod survival and function in a rod-dominant retina appear to be different than in a cone-dominant retina. Furthermor e, the cone cells in mouse are evenly distributed among the rod cell population throughout the retina. The distribution of cone cells in the chicken is more analogous to th at found in the macula/fovea region of central retina in humans. The finding that visual f unction is severely compromised in both LCA1 patients and GUCY1*B chickens is consistent with the central role of cones in vision in humans and chickens. Therefore, the data obtained from animal models and LCA1 patients suggest that cone cells should be the primary targets for gene therapies aimed at treating this disease.

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7 Gene Therapy Vectors In general, vectors or gene delivery vehicles that facilitate the transfer of genetic material to cells can be grouped into two br oad categories: non-viral vectors and viralbased vectors. Several studies have shown that genetic material can gain entry into cells by forming complexes with liposomal or other cationic molecules. However, while the development of effective non-vi ral vectors is rapidly progr essing, the technology is still in its infancy (Brisson and Huang 1999;Johns on-Saliba and Jans 2001;Lechardeur and Lukacs 2002). Many of the recent advances in gene therapy have been facilitated by the use of viral-based vectors. These vector syst ems take advantage of the natural capabilities of viruses to deliver genetic material to cells In particular, gene tr ansfer vectors based on viruses from the Parvoviridae Adenoviridae and Retroviridae families have shown the most promise in this regard. The unique char acteristics of the different viruses and the vectors derived from them must be consid ered when choosing a vector for use in developing viral vector-based therapies. Fo r example, host immune response, longevity and/or kinetics of transgene expression, cargo capacity (size limit of a particular gene of interest), vector tropism and the performance of gene regu latory sequences within the context of vector-mediated gene expression can vary widely with each vector system. Parvoviridae and Adenoviridae -based Vectors Vectors based on adeno-associated vi rus (AAV), a non-pathogenic member of Parvoviridae have been widely used in retinal gene therapy research. Recombinant AAV (rAAV) vectors have been shown to efficiently transduce retinal cells of several species and are capable of long-term expression of tr ansduced genes (Acland et al. 2001;Ali et al. 1998;Bennett et al. 1997;Bennett et al. 1999;Fl annery et al. 1997; Grant et al. 1997).

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8 Some limitations are encountered with the use of rAAV vectors. Traditional versions of rAAV vectors have a cargo capacity of less than 5 kb, which can pose problems in applications where large cDNAs and regulatory sequences are required. To increase the rAAV payload, some groups are ex ploring the possibility of using a transsplicing strategy to assemble a complete tran sgene from two different vectors after dualtransduction of cells (Reich et al. 2003) Another feature of rAAV that could be problematic in the treatment of rapidly advancing retinal diseas es is that it may take up to 4 weeks to achieve maximal expression of tran sgenes after infection (Sarra et al. 2002). Despite these limitations, rAAV vectors carry ing normal copies of diseased genes (Acland et al. 2001), genes enc oding growth factors (Lau et al. 2000) and genes encoding ribozymes specifically targeted to cleave mu tated genes (Hauswirth et al. 2000;LaVail et al. 2000;Lewin et al. 1998) have been successfully used to restore visual function or slow retinal degeneration in animal mode ls of inherited retinal disease. Adenovirus (Ad) has also been developed as a gene transfer vector. As with rAAV, Ad vectors have been shown to transduce photoreceptors in vivo after subretinal injection (Akimoto et al. 1999;Bennett et al. 19 94;Bennett et al. 1996b;Bennett et al. 1998). Although Ad vectors have been routinely gene rated to high titer and although Ad vectors exhibit efficient transduction of retinal cells, some caveats and limitations exist. For example, Ad is only effective in situations where transient expression of a gene is required since the viral DNA does not integrat e into the host genome. The short-lived expression of Ad vectors is due, in part, to the induction of a host immune response triggered by expression of the adenoviral ge nes in the target cells (Bennett et al. 1996a;Hoffman et al. 1997; Reichel et al. 1998) Gutless Ad vectors have been developed

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9 to circumvent this problem, to increase cargo capacity and to allow for stable integration of the transgene (Kochanek et al. 200 1;Mitani and Kubo 2002; Yant et al. 2002). Subretinal injections of adenoviral vect ors carrying normal copi es of either the phosphodiesterase -subunit, neurotrophic factors, or anti-apoptotic factors have been shown to delay photorecept or degeneration in the rd mouse model of retinal degeneration (Bennett et al. 1996b;Bennett et al 1998;Cayouette and Gravel 1997). Retroviridae -based Vectors Retroviral vectors were among the first virus-based systems used to develop gene transfer therapies (Buchschacher, Jr. and Wong-Staal 2000). Retroviruses can integrate foreign genes into the host genome and su stain long-term expression. These viruses consist of a diploid RNA geno me surrounded by an enveloped capsid and can be divided into two major taxonomic groups: simple and complex. The simple and complex retroviral genomes consist of the conserved gag pol and env genes flanked by cis -acting long terminal repeat sequences (LTR s), and contain a packaging signal ( ) adjacent to the 5’ LTR. The 5’ LTR is comprises a vira l promoter-enhancer region and transcription start site; and the 3’ LTR contains sequences required for efficient polyadenylation of viral transcripts. Simple retroviruses, such as murine le ukemia virus (MLV), are well characterized. Vectors based on MLV were among the earliest developed and have been at the forefront of clinical gene transfer technology. Early versions contai ned a nearly complete viral genome. Over the years, these vectors have been streamlined to contain only the genes necessary to transduce cells and stably inte grate genetic material into the host genome. MLV vectors are now engineered to be repl ication defective by re moving viral genes and

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10 leaving only the cis -elements necessary for a single round of replication (Brenner and Malech 2003). MLV-based vectors have recently been used in human gene therapy trials designed to treat X-linked severe combined immune de ficiency (Aiuti et al. 2002;Cavazzana-Calvo et al. 2000;Hacein-Bey-Abina et al. 2002). Wh ile the therapeutic outcome of this experimental treatment has proven to be be neficial and promising, researchers have recently identified two patients in the trial th at have developed cases of a rare leukemia (Brenner and Malech 2003;Hacein-Bey-Abina et al. 2003). These results suggest that the MLV-based retroviral vector s have oncogenic potential in humans (Fox 2003). The adverse effects observed in these cases could be linked to an intrinsic property of the oncoretrovirus-based vector system or in sertional mutagenesis (Brenner and Malech 2003). Regardless of the cause, these results poi nt to a need for additional studies aimed at developing safer gene transfer vectors for future use in humans. Lentiviruses are complex retroviruses th at can transduce di viding and non-dividing cells. For this reason, these viruses have been the focus of intense research efforts aimed at developing more efficient a nd versatile gene transfer vector s. In recent years, several groups have described the generation of gene transfer vectors derived from the human immunodeficiency virus type 1 (HIV-1), a lent ivirus that holds great promise as a basis for gene transfer vectors (Iwakuma et al 1999;Quinonez and Sutton 2002;Zufferey et al. 1998). HIV-1 is the most well studied lentivirus. In addition to the essential retroviral gag pol and env genes that make up the HIV-1 genom e, several accessory proteins are encoded by the genome. The so-called non-essent ial accessory proteins include vif, vpu,

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11 vpr, and nef. The essential regulatory proteins, tat and rev, also unique to lentiviruses, interact with viral genomic cis -elements to promote transcription elongation and to facilitate nuclea r export of viral RNAs, respectively. Since HIV-1 is a lethal human pathogen, concerns about the biosafety of HIV-1derived vectors have been a primary focus of research efforts directed toward the development of these vectors for clinical use in humans. Several steps have been taken to diminish the possibility of generating w ild-type virus during p ackaging and following administration of these vectors. Firstly, the genome has been divided among three bacterial expression plasmids in firstand seco nd-generation lentiviral vector systems: (1) a transducing vector that carries cis -elements and the promoter /gene of interest, (2) a packaging plasmid that carries an attenuated viral genome and modified LTRs, and (3) an envelope glycoprotein expression plasmid. Co-t ransfecting these plasmids in a producer cell line generates a replication-incompetent lentiviral vector. S econdly, modifications have been made to the transducing and p ackaging vectors by mi nimizing the amount of homologous sequence between the two vectors, thereby greatly reduci ng the likelihood of recombination. Finally, the latest versions of the transducing vectors are self-inactivating (SIN) (Iwakuma et al. 1999;Zuffe rey et al. 1998). In SIN vect ors, nearly all sequence has been deleted in the 3’ LTR, which is used as a template to generate both LTRs in the integrated proviral form of the vector. Ther efore, both LTRs are inactivated (i.e. do not activate transcription) following integration and transcription of the inserted transgene is activated by a heterologo us internal promoter. In light of the problems encountered duri ng recent human gene therapy trials with oncoretroviral vectors (Hacein-Be y-Abina et al. 2003), it will be important to continue

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12 improving the biosafety of future lentiviral v ector systems for use in humans. Lentiviral vectors have the potential to in itiate oncogenic events because they integrate transgenes into host cell DNA. While the specific target sites for le ntiviral vector transgene integration have not been el ucidated, studies have shown that integrated HIV-1 DNA is primarily detected in non-coding regions of human DNA in blood lymphocytes (Lyn et al. 2001). Two possible ways to circumvent th e problem of insertional mutagenesis due to random integration events are (1) to deve lop vectors that integrate transgenes at specific, non-oncogenic sites a nd (2) to develop vectors th at do not interfere with endogenous gene expression. A mechanism fo r site-specific integration could be incorporated into the lentiviral vector syst em by manipulating integr ase activity or other “forces” that influence target site selection (Belteki et al. 2003;Bushman 2002). The incorporation of elements such as DNA insulator sequences (Chung et al. 1997;Pannell and Ellis 2001) into future vector system s is likely to significantly improve the performance of lentiviral vect ors and to improve their biosaf ety by isolating the effects of any enhancer/promoter elements cont ained in integrated transgenes. In regard to the performance of lentivir al vectors in retina, recent studies have shown that these vectors are capable of tran sducing photoreceptors following subretinal injection, resulting in the l ong-term expression of transgenes (Auricchio et al. 2001;Cheng et al. 2002;Lotery et al. 2002;Miyoshi et al. 1998). Lentiviral vectormediated expression of the -phosphodiesterase gene in photoreceptors in the rd mouse attenuates photoreceptor degene ration for up to 4 months after injection (Takahashi et al. 1999). We have chosen to utilize an HIV-1-de rived lentiviral vect or system for gene

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13 rescue studies in the GUCY1*B chicken m odel for LCA1 because of the large cargo capacity and the rapid kinetics of transduction/transgene expression. Regulation of Vector-Mediated Gene Expression The ability to regulate gene expression in a physiological manner and to target expression to specific cell types such as photor eceptors is crucial to the development of successful somatic gene rescue strategies. Several factors are known to influence gene expression (e.g. chromatin organization, gene copy number, and gene methylation). Regulation of transcription in itiation is the most straight forward mechanism of gene regulation. The interactions of cellular trans -acting transcription factors with cis -acting DNA elements found in the proximal promoter region play a central role in regulating transcription initiation. The proximal promot er region of some photoreceptor genes has been shown to be sufficient to confer tissu e-specific gene expre ssion (Flannery et al. 1997;Liou et al. 1991;Mani et al 1999), although additional cis -elements located in distal promoter regions or in the gene itself may act to enhance or repress levels of gene expression (DesJardin and Hauswirth 1996;Wang et al. 1992). Gene Regulation in Retinal Photoreceptors Examination of the transcription factors a nd promoters involved in the regulation of photoreceptor genes has begun to reveal the molecular mechanisms that control gene expression in these cells. NRL and CR X proteins are two photoreceptor-specific transcription factors that are crucial for the expression of several photoreceptor genes and for photoreceptor development (Chen et al 1997;Furukawa et al. 1997;Mears et al. 2001;Rehemtulla et al. 1996;Sw aroop et al. 1992). Significant progress has been made in identifying common cis -acting DNA elements that regulate the expression of a number of photoreceptor-specific genes. Re gulatory regions conserved in the proximal promoters of

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14 the photoreceptor genes encoding arrestin (Boatright et al. 1997b;Kikuchi et al. 1993;Mani et al. 1999), -phosphodiesterase (Di Polo et al. 1996;Mohamed et al. 1998), interphotoreceptor retinoid-binding protein (IRBP) (Boatright et al. 2001;Bobola et al. 1995;Liou et al. 1991), rod opsin (Chen and Zack 1996;Gouras et al. 1994;Nie et al. 1996), and cone opsins (Chen et al. 1994;Wa ng et al. 1992) have been identified and characterized using transgenic and in vitro analyses. Truncated murine opsin promoters have b een used successfully to target viral transgene expression to photoreceptor cells (Flannery et al. 1997). Both opsin and phosphodiesterase promoters have been used to drive expression of ribozymes and the phosphodiesterase gene, respectively, in photor eceptor cells (LaVail et al. 2000;Lewin et al. 1998;Takahashi et al. 1999). As mentioned previously, one of the main objectives of my dissertation research is to develop a lentiviral-based vector syst em that can be used to rescue the retinal degeneration phenotype in the GUCY1*B chicke n. In constructing this system, I have focused much of my effort on identifying and utilizing promoters th at limit expression of GC1 transgenes to photoreceptor cells. The 5' flanking regions from IRBP, guanylate cyclase activating protein-1 and GC1, all of which are known photoreceptor-specific genes, were characterized and tested in vivo to accomplish this objective. These studies are presented in Chapters 2 and 3.

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15 CHAPTER 2 A 4.0 KB FRAGMENT OF THE GUANYL ATE CYCLASE ACTI VATING PROTEIN1 (GCAP1) PROMOTER TARGETS GENE EXPRESSION TO PHOTORECEPTOR CELLS IN THE DE VELOPING RETINA Note The work presented in this chapter was published in Investigative Ophthalmology and Visual Science 43 1335-1343 (2002). Patrick Larkin and Yan Zhang assisted with the northern blot analyses and Gabriela Fuchs assisted with the cryosectioning. Introduction Guanylate cyclase activating protein 1 (GCAP1) is an EF-hand calcium-binding protein that activates phot oreceptor guanylate cyclase 1 (GC1) under low intracellular calcium conditions, thereby hast ening the recovery phase of phototransduction (Dizhoor and Hurley 1999;Palczewski et al. 2000;Polans et al. 1996). The expression of GCAP1 and GC1 in vertebrate retina is limited to cone and rod photoreceptor cells, a distribution that is consistent with thei r roles in phototrans duction (Cooper et al. 1995;Dizhoor et al. 1994;Dizhoor et al. 1995;Frins et al. 1996;Gorczyca et al. 1995;Howes et al. 1998;Palczewski et al. 1994). Within photorecepto r cells, GCAP1 is localized to the inner and outer segments and synaptic regions and ap pears to be expressed at higher levels in the cone cells of human, monkey and bovine retinas (Cuenca et al. 1998;Kachi et al. 1999). The expression of GCAP1 has also been detected in the pine al glands of bovine and chicken (Semple-Rowla nd et al. 1999;Venkataraman et al. 2000). Studies of the interactions of GCAP1 with GC 1 suggest that these proteins exist in photoreceptors as a stable complex independent of intracellular ca lcium concentrations, a nd that activation of

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16 GC1 occurs as a result of a calcium-depende nt conformational change in the complex (Duda et al. 1996;Gorczyca et al. 1995;Otto -Bruc et al. 1997;Rudnicka-Nawrot et al. 1998;Tucker et al. 1997). While at least three variants of GCAP are expressed in retina (GCAP1-3) (Dizhoor et al. 1995;Gorczyca et al 1994;Haeseleer et al. 1999;Palczewski et al. 1994), recent studies of GCAP1/2 knockout mice suggest that only GCAP1 is capable of restoring normal light response kine tics to photoreceptor cells (personal communication with W. Baehr and cited refe rence) (Mendez et al 2001). The current view that GCAP1 is essential for no rmal phototransduction is supported by the observation that missense mutations in the GCAP1 gene (Y99C and E155G) have been linked to autosomal dominant cone dystrophy in humans (Downes et al. 2001;Payne et al. 1998;Wilkie et al. 2001). These mutations, which interfere with the binding of calcium to GCAP1, lead to persistent activation of GC1 even under high calcium conditions (Dizhoor et al. 1998;Sokal et al 1999;Wilkie et al. 2001). Thes e results clearly indicate that GCAP1 plays a pivotal role in phototransduction and retinal disease. Therefore, it is of interest to understand how the expression of GCAP1 is regulated in developing and mature retina. In retinal photoreceptors, the magnitude s, cellular specificities and temporal dynamics of expression of several photorec eptor-specific genes are regulated at the transcriptional level. The intrinsic activities and cellular specificities of these genes can be attributed to complex interactions between cis -acting regulatory elem ents within their promoters and the cell-specific transcription fact ors that interact with them. The onset of expression of these genes in developing retina has also been shown to be dependent upon the interactions between promoter cis -elements and transcripti on factors, and is often

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17 linked temporally to the diffe rentiation and maturation of th e photoreceptor cells (e.g. see cited references) (Hauswirth et al. 1992;He et al. 1998;Johns on et al. 2001;Kennedy et al. 2001;Livesey et al. 2000;Morro w et al. 1998;van Ginkel and Hauswirth 1994). Relatively few studies have been carried out to exam ine the activities of photoreceptor-specific promoters in developing retina in vivo (Chen et al. 1994;Kennedy et al. 2001;Lem et al. 1991;Mani et al. 2001;van Gi nkel and Hauswirth 1994). Recently, the importance of correct temporal regulation of gene expre ssion in developing retin a has been clearly demonstrated in studies of cone-rod homeobox (CRX) (Furuka wa et al. 1999;Livesey et al. 2000) and neural retina leucine zipper (M ears et al. 2001) knockout mice. The results of these studies show that th e absence of expression of thes e key trans-acting factors in retina results in the down-regulation of e xpression of several photoreceptor-specific genes and abnormal development and f unction of the photoreceptor cells. In this series of experiments, we have examined the expression characteristics of fragments of the chicke n GCAP1 promoter both in vitro and in vivo with the purpose of identifying regions of the promoter that pl ay a role in regulating the activity, cell specificity and developmental expression of GCAP1. Our previous analyses of the sequence of the 5’ flanking region of this gene (Semple-Rowland et al. 1999) served as a guide for the selection of the GCAP1 promoter fragments that were analyzed in these experiments. The intrinsic activities of th ese fragments were determined by measuring the expression levels of GCAP1-luciferase fu sion constructs in transiently transfected primary embryonic chicken retinal cultures, an in vitro system that has been used to characterize the activities of the promoters of photoreceptor-specific genes obtained from a variety of species (Boatrig ht et al. 1997b;Boatright et al. 1997a;Chen et al. 1997). We

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18 utilized lentiviral vectors as a novel tool to extend the in vitro analyses of promoter function to the in vivo environment of the developing chicken retina. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are ideal vectors for this type of analysis because they are capable of transducing several different cell types, exhibit rapid integration and expression of transgenes in transduced cells(Vigna and Naldini 2000), and have a large cargo capaci ty (>18 kb) (Kumar et al. 2001). The cell specificity and the onset of the activity of selected GC AP1 promoter fragments in developing retina was assessed in vivo by monitoring the activity of GCAP1 promoternlacZ transgenes in the retinas of animals th at had received injections of lentivirus carrying these transgenes prior to the devel opment of the neural retina. The onset of expression of each GCAP1 promoter-nlacZ transgene in developing retina was compared to the expression profiles of the GCAP1 and GC1 genes in normal, developing chicken retina. Methods Northern Blot Analyses Embryonic retina-pigmented epithelium-chor oid tissues were removed from both eyes of each embryo and total RNA from these tissues was isolated using an RNeasy total RNA kit (Qiagen). Samples containing 10 g of RNA were electrophoresed on a 1.1% formaldehyde gel and transferred to a nylon transfer membrane (Micron Separations Incorporated). Northern blots were hybrid ized consecutively with radiolabeled cDNA probes specific for GCAP1, GC1, and iodops in as previously described (SempleRowland and van der 1992). The GCAP1 a nd iodopsin results were confirmed by repeating the analyses on a second series of independent samples. Blots were exposed to Kodak BioMax film (Eastman Kodak) for 12-16 hours at -80 C and the resulting

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19 hybridization signals were imaged using a BioRad Gel Doc 1000 system. The 18S rRNA was visualized by staining the blot with methylene blue. Preparation of Constructs The GCAP1 promoter fragments were am plified from appropriate regions of GCAP1 cosmid clones, ccos16 and ccos24 (S emple-Rowland et al. 1999) using the polymerase chain reaction and Pfu DNA polymerase (Stratagene). For each of the GCAP1 promoter fragments, uniqu e upstream primers containing a Not I site were used in combination with three different sets of downstream primers containing a Pme I site to generate the following fragments (transcrip tion start point = +1): (1) –292/+302, (2) – 1436/+302, (3) -3121/+222, (4) –4009/+222, and (5) –1434/+29 (Fig. 2-2A). In this study, these fragments will be referr ed to as 292, 1436, 3121, 4009 and 1434, names based on the position of the 5’ nucleotide. The sequence-specif ic (GenBank AF172707) primers used to amplify the fragments were as follows: 292 (sense – 5’ ACC CGT GTG CTT TTC; antisense – 5’ GCT CCA GTC AC T CT), 1436 (sense – 5’ ACC CGA CTC CTT CAA; antisense – same as 292), 3121 (sense – 5’ AAT CCT GCC CAT CAC TGC CCT ATC; antisense – 5’ AGT TTT GAG GT C GGT GGG TGA GTC), 4009 (sense – 5’ GGG CGA TTG GCA GGG AGG AG; antisense – same as 3121), 1434 (sense – 5’ ACC CGA CTC CTT CAA; antisense – 5’ C GG GCA AAT GTA AAA GC). Products from the polymerase chain reaction were subcl oned into the pCR-TOPO-blunt II vector (Invitrogen) and the DNA sequences of pos itive clones were verified by sequence analyses. GCAP1 promoter fragments were ex cised from the pCR-TOPO-blunt II clones using Not I and Pme I and ligated into the appr opriate vectors. For the in vitro activity assay constructs, the multiple cloning site (MCS) of the pGL2 vector (Promega) was modified by ligating the Sac I /Xho I fragment of the MCS of th e pBluescript II SK vector

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20 into the MCS of the pGL2 vector. The m odified vector was then digested with Xho I, blunt-ended, digested with Not I and the Not IPme I GCAP1 promoter fragments were ligated into the vector. For the in vivo lentiviral constructs, Not IPme I fragments were ligated into pTY-nlacZ digested with Not I and Pme I. The murine IRBP promoter (mIRBP1783), which included nucleotides –1783 to +101, was amplified from the pIRBP1783-EGFP plasmid vector using the pol ymerase chain reaction. The same cloning strategy described for the GCAP1 promoter c onstructs was used to generate pGL2 and lentivirus pTY-nlacZ plasmid vector s containing the mIRBP1783 promoter. Transfection-grade DNA was prepared for each construct using an endotoxin-free DNA maxiprep kit (Qiagen). Cell Cultures and Transfections Dispersed embryonic day 12 (E12) chick retinal cultures were prepared and transiently transfected essentially as previous ly described (Adler et al. 1982;Ameixa and Brickell 2000;Boatright et al. 1997b;Kumar et al. 1996;Poli ti and Adler 1986). Isolated neural retina was incuba ted in 0.25% trypsin at 37 C for 20 minutes, dispersed by trituration using a flame-narro wed glass pipette, and plat ed at a density of 2 x 106 cells / well in 24-well culture plates that had been coated with poly-Lornithine (Sigma). Cultures were maintained in basal medium of eagle (Life Technologies) supplemented with 5 g/L glucose, 10% fetal bovi ne serum, and antibiotics at 37 C in 5% CO2. Cells were transfected the day af ter seeding using the calcium phosphate method. Briefly, 10 g of promoter vector DNA and 0.5 g of control vector DNA containing the nlacZ reporter gene driven by the CMV promoter were added to 125 l of 0.2 M CaCl2. Next, 125 l of 2x HEPES-buffered saline was added dropwise to the DNA/CaCl2 mixture. The

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21 transfection mixture was allowed to incubate for 20 minutes at room temperature and then 62.5 l of the transfection mixture was added to each well. Cells were incubated overnight at 37 C in 5% CO2 and rinsed 3 times with PBS the following day. The transfection experiments were replicated 4 times and a new preparation of cultured cells was used for each experiment ( n = 4). Within each experi ment, transfection of each promoter construct was carried out in duplicate using the sa me transfection mixture. The photoreceptor-specific mIRBP1783 promoter was used as a positive control in all experiments (Boatright et al. 1997a;Boa tright et al. 2001;Ch ang et al. 2000). Luciferase and -galactosidase Assays Cell lysates from the E12 primary retin al cultures were prepared 40-48 hours post-transfection by adding 200 l lysis buffer (p rovided in the assay kits, see below) to each well, scraping the cells using a rubber pol iceman and processing the lysates for the luciferase or -galactosidase chemiluminescent assays according to th e manufacturer’s protocols (Galacto-star or Luciferase Assay Kits, Tropix). Luciferase and -galactosidase activities were measured in 20 and 40 l a liquots of each lysate, respectively. Assays were run in duplicate and quantified using a TD20/20 luminometer (Turner Designs) with an integration time of 10 seconds. Activity values were corrected for transfection efficiency across experiments by no rmalizing luciferase values to -galactosidase values. Promoter activity was expressed as fold-activ ity over the promoterless pGL2 vector. Data were analyzed using one-way repeated measures ANOVA and post-hoc pairwise comparisons were performed using th e Student-Newman-Keuls post-hoc test (SigmaStat).

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22 Lentivirus Production Viruses pseudotyped with VSV-G were prepared using a self-inactivating lentiviral vector system(Iwakuma et al. 1999) Packaging cells (293T) were plated in 10 cm culture dishes at a density of 6 x 106 cells / dish in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum, and antibiotics (Life Technologies). The 293T cells were grown to 80-90% confluence a nd were then transiently transfected with 6 g pTY-nlacZ (transgene -carrying vector), 12 g pHP (packaging vector), 5.5 g pHEF (encodes VSV-G envelope) and 0.5 g pCEP4-tat (encodes tat protein) per dish using Superfect transfection reagent (Qiagen). All four of the plasmids were added to 300 l DMEM and the DNA mixture was vortexed and incubated at room temperature for 5 minutes. Superfect (50 l) was added to the DNA mixture, which was then vortexed and incubated at room temperature for an additi onal 5 minutes. During this time, the medium was removed from the cells and replaced with 4.5 ml of fresh me dium. The transfection mixture was then added dropwise to the cu ltures which were th en incubated at 37 C in 5% CO2 for 3-4 hours. Following the incubation pe riod, the cells were rinsed one time with medium. Fresh medium (6 ml) was adde d back to the cells and the cells were incubated overnight. The next day, the medi um was removed and 6 ml of fresh medium was added to the cells. The medium contai ning the virus was harvested 48 and 72 hours post-transfection and frozen at -80 C until concentration. To c oncentrate the virus, the medium was rapidly thawed and passed through a 0.45 m low-protein binding Durapore filter (Millipore, Bedford, MA) to remove cell debris. The filtered medium containing the virus was concentrated 140-fold by ultracentrifugation at 20,000 x g for 2.5 hours at 4C. The virus pellet was resuspe nded by gentle shaking at 4 C for 4 hours, aliquoted and

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23 stored at -80 C until use. Infectious titers of virus were determined by infecting 4 x 104 TE671 cells seeded in 24-well plates with limiting dilutions of TY-EF1 -nlacZ virus in the presence of 8 g/ml polybrene. After 3-4 hours of infection, fresh medium was added to the cells. After 48 hours, the cultures were stained with 5-bromo-4-chloro-3-indolyl-D-galactosidase (X-gal) substrate as previous ly described (Chang et al. 1999). Virus titer was determined by counting the number of bluenucleated cells and infe ctious titers were expressed as the number of transducing units per ml (TU/ml). Particle titers were determined using a p24 ELISA kit obtained from BD Biosciences following the protocols provided therein. Dilutions of 1 x 10-6 and 1 x 10-7 of lysed viral partic les were assayed to obtain the mean ng p24/ml of each sample. Th e average infectivity of virus using the methods described above was determined for TY-EF1 -nlacZ virus Infectious titers of virus carrying tissue-specific promoters were estimated by multiplying the particle titers of the GCAP1/IRBP promoter-containing viruse s by the infectious titer to particle titer ratio obtained for the virus. The titers of a ll virus preparations were approximately 1 x 107 TU/ml. Embryonic Injections All animals were handled according to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Chicken eggs were set on day 0 and incubated on their sides without rotation at 37.5 C and 60% humidity. Viral in jections were performed on Hamberger-Hamilton stage 10-12 embryos (~E2). A small opening was made in the eggshell overlying the embryo, the position of which was determined using an egg candling light. With the aid of a dissecting microscope, inj ection of the virus into the ventricular space of the neural tube was car ried out using a micromanipulator (Sutter

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24 Instrument Company) fitted with a pulled gla ss capillary needle that was connected to a Sutter manual microinjector. The virus was mixe d with fast green (1.0 l 0.3% fast green in PBS per 20 l virus) to assist in the visualization of the injected virus. Upon penetration of the embryo, 0.5-1.0 l of virus was slowly inject ed into the neural tube. The egg was then sealed with parafilm a nd incubation was conti nued until the embryo reached the desired age for analysis. Histochemistry and In vivo Promoter Analyses Neural retina was dissected from the eyes of injected embryos at selected ages and dispase was used as necessary to aid in th e removal of the pigmented epithelium. Retina whole mounts were prepared by placing the tissue photoreceptor side down on a Millipore-Millicell insert containing PBS and flattened using fine tipped glass rods. To detect expression of nlacZ, retinas were fixed in 4% paraformaldehyde for 15 minutes. The retinas were then rinsed three times in PBS and incubated in PBS (pH 7.9) containing 35 mM potassium ferrocyanide, 35 mM potassium ferricyanide, 2 mM magnesium chloride, 0.02% NP-40 a nd 40 mg/ml X-gal substrate at 37 C for 3-4 hours. Following this incubation, the retinas were ri nsed three times in PBS. Retinas were cryoprotected with 30% sucrose and mounted in OCT medium. Sixteen to twenty micron thick serial sections were cut through areas posit ive for X-gal staining using a cryostat, mounted on slides, and counterstained with DAP I. Thirty to eighty sections, taken from the retinas of at least two different animal s injected with TY-GCAP1 promoter-nlacZ virus were analyzed for each time point. In some cases the pineal glands and brains of E20 embryos that had been injected with virus were removed and fixed in 4% paraformaldehyde. Pineal glands were stained with X-gal in toto and processed for

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25 cryosectioning as described above. The brains of E20 embryos were cut into four regions using a microtome blade as follows: cerebe llum / brainstem, optic tectum, anterior forebrain and posterior forebrain. The brain regions were stained in toto with X-gal, rinsed in PBS and viewed under a Zeiss di ssecting microscope; in some cases 16-20 m thick sections were cut thr ough the various regions of the brain using a cryostat and the sections were stained with X-gal as de scribed above. Brightfield and fluorescence microscopy was performed using a Zeiss Axioplan 2 microscope (Carl Zeiss Incorporated) fitted with a SPOT 2 Enhan ced Digital Camera System (Diagnostics Imaging Incorporated) for imaging. The DAP I nuclear stain was visualized using a longpass DAPI filter. The images of the X-gal stained sections were produced by creating a negative image of the stained section that was then overlaid with the DAPI-stained image of that same section using the SPOT camera imaging software. Results Expression of GCAP1 in Developing Chicken Retina Northern blot analyses were carried out to determine the onset and relative level of expression of the gene encoding GCAP1 in developing embryonic chicken retina (Fig. 21). Since the functional relationship between GCAP1 and GC1 is clos ely linked, analyses of GC1 expression were included for compar ison. The expression of the gene encoding iodopsin was also included in our analyses as a control. GCAP1 and GC1 transcripts were first detected in developing chicken retina on E14-15 and E13-14, respectively. The relative levels of GCAP1 a nd GC1 transcripts, which were comparable at each developmental stage, increased gradually as a function of embryonic age, reaching maximum levels between E19 and E20. Iodopsin transcripts were firs t detected at E14, a result that agrees with previous studies of the expression of iodopsin in developing

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26 chicken retina (Adler et al. 2001;Bruhn and Cepko 1996). The onset of transcription of all of these genes in retina coincides with the onset of photoreceptor outer segment development and cGMP synthesis in deve loping chicken retina which occur around E15 (Meller and Tetzlaff 1976) and E18 (Sem ple-Rowland et al. 1998), respectively. GC1 GCAP1 Iodopsin10-10.5 11.5 13-13.5 13.5-14 14 14 15 16.5 17 17 19-19.5 20 18S 18S 9.5 2.4 1.4 Kb 10121620 ISOSERG Figure 2 1. Expression of GCAP1, GC1 and iodo psin genes in developing chicken retina. Northern blot hybridized consecutively with GCAP1, GC1 and iodopsin cDNA probes and then stained with methylene blue to show 18S rRNA (RNA loading control) at sel ected developmental stages. The 18S rRNA was used as a loading control. Th e numbers across the top of the figure correspond to embryonic age. The time line at the bottom of the figure indicates the developmental ages at wh ich the photoreceptor inner (IS) and outer segments (OS) first appear and the earliest age at which electroretinograms (ERG) can be reco rded. The GCAP1 and iodopsin results were confirmed by repeating the north ern analyses on a second series of independent samples. In vitro GCAP1 Promoter Activity The activity of each promoter fragment was measured in primary retinal cultures transiently transfected with the promoter-r eporter constructs. Cu ltures were prepared from the retinas of E12 embryos in our expe riments because preliminary studies showed

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27 that the promoters of the GCAP1 and IRBP ge nes are active in these cultures. The five GCAP1-luciferase fusion constructs tested in this series of experiments are shown in Fig. 2-2A. A comparison of the activities of the GCAP1 promoter fragments using ANOVA revealed that they were significantly di fferent from each other (F = 9.79, df = 4, p < 0.001) (Fig. 2-2B). The activities of th e 292, 1436 and 4009 fragments, which were comparable to each other, were significantly greater than the activit ies of either the 1434 or the 3121 fragments ( p < 0.05). The activity of the 1434 promoter fragment was significantly greater than that exhibited by the 3121 fragment ( p < 0.05). A comparison of the activities of the 292, 1436 and 4009 GCAP1 promoter fragments to that of the mIRBP1783 fragment revealed th at the activities of the GCAP 1 promoter fragments were approximately one half that of the IRBP promoter fragment assayed under identical conditions (Fig. 2-2B). Comp arable results were obtaine d in another series of experiments in which cultures were transien tly transfected with the pTY-based GCAP1 and IRBP promoter-nlacZ constructs that were used to generate th e lentiviral vectors (data not shown). Lentiviral Transducti on of Avian Tissues The goals of this experiment were to determine if lentivirus pseudotyped with VSV-G could transduce chicken re tinal progenitor cells and if le ntivirus could be used as a tool to examine the expressi on characteristics of promoters in vivo To address these questions, we examined the expressi on and cellular di stribution of EF1 -nlacZ and mIRBP1783-nlacZ lentiviral transgen es in the retinas of E6 (EF1 -nlacZ) and E20 (EF1 -nlacZ and mIRBP1783-nlacZ) embryos that ha d received injections of lentiviruses carrying either of these transgenes early in development (~E2). Examination of whole

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28 A B fold luciferase activity (GCAP1 promoter) 0 10 20 30 40 50 fold luciferase activity (mIRBP1783 promoter) 0 20 40 60 80 100 2921436312140091434 GCAP1 promoter fragment** *IRBP luciferase luciferase luciferase luciferase-4009 -3121 -1436 -292 +222 +222 +302 +302 luciferase-1434+29+1 luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase A B fold luciferase activity (GCAP1 promoter) 0 10 20 30 40 50 fold luciferase activity (mIRBP1783 promoter) 0 20 40 60 80 100 2921436312140091434 GCAP1 promoter fragment** *IRBP luciferase luciferase luciferase luciferase-4009 -3121 -1436 -292 +222 +222 +302 +302 luciferase-1434+29+1 luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase luciferase Figure 2-2. GCAP1 promoter activity in transfected E12 primary chicken embryonic retinal cultures. A. Diagram of the five GCAP1 promoter fragments cloned into the pGL2 vector Each promoter fragment, except for 1434, contains a 25 bp repeated sequence (h atched bars) located within the 5’ UTR and all constructs contain th ree proximal cone-rod homeobox (CRX)like binding elements (white bars) an d a putative TATA box region (black bars). B. Histogram showing levels of luci ferase activity in primary retinal cultures 48 hours post-transfection. Each bar represents the mean SEM activity obtained from 4 separate experiments for each promoter fragment. Open bars = GCAP1 promoter fragme nts; filled bar = mIRBP1783 promoter; = activity significantly less th an 292, 1436, 4009 and 1434, p < 0.05; ** = activity significantly less than 292, 1436 and 4009, p < 0.05. mounts of retinas taken from E6 and E20 em bryos that had been injected with TY-EF1 nlacZ virus and stained with Xgal revealed the presence of several discrete clusters of blue-nucleated cells that were distributed through the entire focal plane of the retina (Fig. 2-3A). Cross-sectional analyses of these retinas revealed th at the nlacZ reporter gene was

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29 being expressed in all cell laye rs of the retina. The staining in tensities of the cells in E6 and E20 retinas were comparable, sugge sting that the activity of the EF1 promoter was similar at both of these stages of devel opment (Fig. 2-3B, C and D). The column-like staining pattern that we obser ved in the retinas of the embryos injected with TY-EF1 nlacZ virus closely resembled the staining pattern that has been reported in retroviral studies of cell lineage in developing retina (F ekete et al. 1994). This result suggests that the TY-EF1 -nlacZ transgene carried by the lentiv irus was integrated into the DNA of retinal progenitor cells and pa ssed to subsequent clones. In contrast to the clustered, column-like nlacZ staining pattern observed in the retinas of embryos injected with TYEF1 -nlacZ lentivirus, nlacZ staining in th e retinas of embryos injected with TYmIRBP1783-nlacZ lentivirus was limited to cells on the surface of the retina (Fig. 2-3E). Cross-sectional analyses of these retinas revealed th at expression of the mIRBP1783 promoter was restricted to photoreceptor ce lls (Fig. 2-3F), a result that corroborates previous studies of the cell-specificity of this promoter fragment (Boatright et al. 1997a;Boatright et al. 2001;Chang et al. 2000). An analysis of selected pineal glands and brains taken from E20 embryos showed that transduction of these tissues was minimal or undetectable following neural t ube injection of lentivirus (data not shown). Together, these results indicated that lentivirus could be used to examine the expression characteristics of promoters in vivo Analyses of GCAP1 Promoter Fragments In vivo The three GCAP1 promoter fragments that showed comparable levels of activity in our in vitro assays (Fig. 2-2B) were analyzed in vivo Lentiviral vectors containing the 292, 1436 and 4009 GCAP1 promoter fragments driv ing the nlacZ report er gene were

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30 ONL INL GCL PR V ONL INL GCL ONL INL GCL PR V ONL INL GCL A BC D E F ONL INL GCL PR V ONL INL GCL ONL INL GCL PR V ONL INL GCL A BC D E F Figure 2-3. Retinal whole mounts and cross-s ections prepared from embryos that received injections of TY-EF1 -nlacZ (A-D) or TY-mIRBP1783-nlacZ lentivirus (E,F). A. Area from a whole mount of an E6 retina showing a cluster of infected clones in tensely stained with X-gal. B. Cross-section through retina shown in panel A. C. Cross-section through a retina taken from an E20 embryo showing presence of cells stained with X-gal in all retinal cell layers. D. Inverted brightfield image shown in panel C (nlacZ-positive cells in red) overlayed with the DAPI image (in blue) to clearly show the retinal cell layers. E. Area from a whole mount of an E20 retina taken from an embryo injected with TY-mIRBP1783-nlacZ lentiv irus and stained with X-gal. F. Cross-section through the retina from panel E showing that the mIRBP1783 promoter limits expression of the nlacZ reporter gene to photoreceptor cells within the outer nuclear layer (ONL). PR = photoreceptor side; V = vitreous side; INL = inner nuclear laye r; GCL = ganglion cell layer.

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31 generated and injected into the neural tubes of E2 embryos to obtain an estimate of the onset of expression of these fragments in de veloping retina and their cell-specificities. GCAP1 promoter-driven nlacZ expression was examined in the retinas of E12, E16 and E20 embryos, stages of development that were selected based on the results of our analyses of the onset of normal GCAP1 expr ession in developing re tina (Fig. 2-1) and on the milestones of photoreceptor development in chicken. These stages correspond to time points that precede the onset of GCAP1 expression in vivo (E12), that approximate the onset of GCAP1 expression and the devel opment of photoreceptor outer segments in vivo (E16), and that include the period when GC AP1 expression has reached maximal levels in vivo just prior to hatching (E20). Onset of expression in developing retina X-gal stained retinal cells could be de tected in whole mounts of retinas taken from embryos that had been injected with either the 292 or th e 1436 promoter-nlacZ lentiviral vector as early as E12. The overall number of cells expressing nlacZ driven by either of these promoter fragments was much lower in the retinas of E12 embryos (292, n = 2; 1436, n = 3) than in the retinas of E16 (292, n = 3; 1436, n = 2) and E20 (292, n = 5; 1436, n = 3) embryos (data not shown). No dete ctable X-gal staining was observed in retinas of E12 embryos that had received inje ctions of the 4009 promoter-nlacZ lentiviral vector ( n = 6). The first evidence of X-gal stai ning resulting from 4009 promoter-driven nlacZ expression was observed at E16 (1 positive retina out of n = 6). By E20, the X-gal staining in these embryos had increased sufficiently so that positively stained cells could be detected in all retinas examined ( n = 5).

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32 Cell-specificity of expression The cell-specificity of the activity of each promoter-nlacZ transgene was determined by examining cross-sections cut fr om whole mounts of the retinas that had been removed from embryos injected with the various lentiviral vect ors (Fig. 2-4A). Both the 292 and 1436 promoter-reporter transgenes we re expressed in cells located within the inner nuclear layer (INL) at E12. A few nlacZ -positive cells were also observed within the ganglion cell layer (GCL) in these retinas at this time. In E16 and E20 retinas, X-gal stained cells were also detected within the ou ter nuclear layer (ONL). In these retinas, the number of stained cells observed in the ONL was generally higher than that observed in the INL. In contrast to the ra ther non-specific cellular staining pattern observed in retinas transduced with either the 292 or the 1436 promoter-nlacZ transgenes, cross-sectional analyses of E16 and E20 retinas transduced with virus carrying the 4009 promoter-nlacZ transgene revealed that only photoreceptor cells within the ONL were stained in these retinas. Discussion The results of our in vitro and in vivo analyses of various fragments of the GCAP1 promoter suggest that cis -elements regulating the activity, developmental expression, and cell-specific expression of th e GCAP1 promoter are located in distinct regions of the promoter. The 292, 1436 and 4009 fragments all exhibited similar activity levels in vitro a result which suggests that the cis -elements essential for conferring activity to these fragments are located within the 292 fragment. In our analyses, we noted that removal of the 25 bp repeated sequence, which comprise s ~50% of the 5’ UTR, resulted in a significant reduction in the activity of the 1436 promoter fragment. Inclusion of the

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33 sequence between nucleotides -1437 and -3121 also produced a significant reduction in promoter activity that could be ameliora ted by addition of the sequence between 292 1436 4009A B low or no activity in vitro activity in INL ~E12 and in ONL ~E16 activity in ONL ~E16 1434 3121 292 1436 4009 ATG CRX CRX CRX OTX OTX PCE1 RET4 CRX OTX TATA ATG CRX CRX CRX OTX OTX PCE1 RET4 CRX OTX TATA Figure 2-4. Cell specificity and temporal onset of activity of the 292, 1436 and 4009 GCAP1 promoter fragments in embryonic chicken retina. A. Examples of cross-sections through X-gal stained retinal whole mounts (nlacZ-positive cells = red; DAPI = blue). The embryonic ages are indicated along the top axis of the figure and the GCAP1 promoter fragment is indicated along the side

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34 axis of the figure. At E12, X-gal stai ning was observed in the inner nuclear layer (INL) of retinas transduced with either the 292 or the 1436 promoternlacZ transgene. By E16 and E20, X-ga l staining was observed predominately in the outer nuclear layer (ONL) and to a lesser extent in the INL. For the 292 and 1436 promoter fragments, two panels containing images from different regions of the same E20 retinas are shown to illustrate the fact that in some areas, X-gal staining was restricted to the ONL and that in other areas, staining was present in both the ONL and the INL. No detectable X-gal staining was observed in retinas of E12 embryos injected with the 4009 promoter-nlacZ lentiviral vector and only light staining was observed in the ONL at E16. By E20, the level of st aining in these retinas had increased sufficiently to allow easy identification of the X-gal positive cells. Thirty to eighty cross-sections cut from two to six retinas were analyzed for each promoter fragment at the different de velopmental ages (see the Methods and Results sections for details). IPL = in ner plexiform layer; GCL = ganglion cell layer. B. Schematic of the chicken GCAP 1 promoter showing putative cisDNA binding elements for retinaand photoreceptor-specific transcription factors (not to scale). The bracket bars highlight the differe nt regions of the GCAP1 promoter containing included in the various fragments that were examined. Violet box = region betwee n nucleotides -1437 and -3121 that negatively affected promoter activity; red arrow = transcription start point; blue and white-striped box = 25 bp rep eated sequence within 5’ UTR; ATG = translation start codon. nucleotides -3122 and -4009 to the fragment. It is possible that the observed decrease in activity of the 1436 promoter with the truncat ed 5’ UTR is due to a reduction in the efficiency of translation of the transcri pts produced from this promoter/reporter transgene, and that interactions between cis -elements located with in the -1437 to -3121 and the -3122 to -4009 regions are required to co nfer significant levels of activity to the longer GCAP1 promoter fragment s. To test these possibilities, it will be necessary to assay the transcription levels and f unctional activities of additional GCAP1 promoter/reporter constructs. In vivo analyses of the expressi on characteristics of the GCAP1 promoter fragments were performed in order to identify regions within the GCAP1 promoter that control the cell specificity and developmental onset of expression of the native GCAP1 gene. Analyses of embryonic retinas transduced w ith lentiviral vector s carrying the nlacZ

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35 reporter gene driven by the 292, 1436 or 4009 GC AP1 promoter fragments revealed that the 292 and 1436 GCAP1 promoter fragments both of which exhibited activity in vitro and in vivo, did not possess the cis -elements required to restri ct their activities to photoreceptor cells. Furthermore, the 292 and 1436 promoter fragments exhibited activity in vivo prior to the normal onset of expression of the GCAP1 gene during developmental. By including additional upstream sequence in the 4009 GCAP1 promoter fragment, we obtained a fragment that exhibited the e xpression characterist ics of the endogenous GCAP1 gene. These results suggest that the general organization of the GCAP1 promoter differs from those of previously characteri zed photoreceptor gene promoters, such as IRBP and rhodopsin, in which many of the cis -elements in these promoters that are responsible for restricting prom oter activity to photoreceptor cells are located within 1 kb of the transcription start poi nt (Boatright et al. 2001;B obola et al. 1995;Fei et al. 1999;Kennedy et al. 2001;Mani et al. 2001;Yokoyama et al. 1992;Zack et al. 1991). Based on the in vivo expression characteristics of th e 1436 and 4009 promoter fragments, it appears that cis -elements located in the distal promot er region are required to delay the onset of expression, a result sim ilar to that reported in recent in vivo studies of the Xenopus rhodopsin promoter (Kennedy et al. 2001). The GCAP1 promoter contai ns a cluster of putative cis -elements between nucleotides -143 and -838 that include binding sites for transcription factors that have been shown to regulate the expression of re tinaand photoreceptor-specific genes (Fig. 24B). We have previously reported that at least two putative CRX-like binding sites (C/TTAATC/T) are present within the first 1kb upstream of the tran scription start point in the 5’ flanking region of the chicken GCAP1 gene (Semple-Rowland et al. 1999). In

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36 addition, one Ret-4-like element (-187 to -184) (Chen and Zack 1996), two OTX-like binding elements (-196 to –202 and –832 to 838) (Chen and Zack 1996;Kimura et al. 2000) and one PCE-1/Ret-1-like element (-818 to -825) (Kikuchi et al 1993;Morabito et al. 1991;Yu and Barnstable 1994) are also located within this region (see Fig. 2-4B). Our analyses show that the shorter GCAP1 promot er fragments that contain these elements (292 and 1436) do not exhibit the expressi on characteristics of the native GCAP1 promoter in developing re tina. Clearly, additional cis -acting elements located in the distal GCAP1 promoter region (-1437 to -4009) are required to produce the cell-specificity and developmental expression characteristics of the native GCAP1 gene. As mentioned above, our in vitro data indicates that sequence located in the region between nucleotides -1437 and -3121 suppresses GCAP1 promoter activity in retinal cells. Silencing mechanisms similar to those reported for the regulation of neuron-specific gene promoters (Bessis et al. 1997;Schoenherr et al. 1996;Weber and Skene 1997;Weber and Skene 1998) could play a role in supp ressing GCAP1 promoter activity in nonphotoreceptor cells and in producing the tem poral expression characteristics of this promoter in developing retina. Similar m echanisms have been postulated for other photoreceptor gene promoters such as the murine IRBP promoter where a -70/+101 fragment of this promoter containing ciselements that are highly conserved in retinaand photoreceptor-specific promoters exhibits signif icant activity in vitro but additional sequence located between nucleotides -70 and -156 is requir ed to restrict its activity to photoreceptor cells in vivo (Boatright et al. 2001). Recent studies of other photoreceptor gene promoters suggest that specific combinations of regulatory factors expresse d in photoreceptor cells that bind to and

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37 transactivate these promoters are required for photoreceptor-specific gene expression (Boatright et al. 1997a;Bobola et al. 1995; Fei et al. 1999;Kimura et al. 2000). Examination of the sequence located upstream of the 1436 promoter fragment revealed the presence of additional putative homeodomain proteinbinding elements (see Fig. 24B). The region between nucleotides –2413 and –2423 contains a head-to-tail arrangement of two CRX-like binding elemen ts (consensus CTAATNNGATT), which is similar to that recently identified in seve ral putative CRX-regulated photoreceptor genes (Livesey et al. 2000). Additional CRX-lik e (-3305 to –3310) and OTX-like (-3356 to 3362) DNA binding elements are located within the –3122 to –4009 region of the GCAP1 promoter, elements that could potentially influence the expression characteristics of the 4009 promoter fragment (see Fig. 2-4B). The results of these experiments provide a rough blueprint of the structural and f unctional organization of the chicken GCAP1 promoter. Additional studies will be required to confirm that the putative cis -elements identified within the GCAP1 promoter bind trans-acting factors and that these interactions serve to shape the activ ity characteristics of this promoter. In establishing the usefulness of lentiviral -mediated gene transfer as a tool for analyses of promoter functi on in the developing retina, we have demonstrated that lentivirus can transduce chicken retinal proge nitor cells. In additi on, we show that the expression of transgenes carried by lentiv irus, which transduces both progenitor and terminally differentiated retinal cells, can be targeted to specific cell types by selecting appropriate internal promoter s. The experimental paradigm presented here should be amenable for studies of photoreceptor gene promoters from other species that exhibit activity in primary cultures of chicken retinal cells and, t hus, should have broad appeal

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38 for in vivo analyses of promoter function. Furthermor e, we show that the lentivirus vector system used in this study is capable of ca rrying and expressing tr ansgenes up to 7.4 kb in size, a cargo well below the recently demonstrated capacity of this vector system of over 18 kb (Kumar et al. 2001). The la rge cargo capacity of this vect or is an important feature of this system that will make it useful for st udies of the expression characteristics of large promoter fragments in vivo Finally, it is important to note that the utility of this method is not compromised by the experimental variab ility due to differences in viral titer or injection procedure. In experiments in wh ich only small populations of progenitor cells were transduced by the virus, it was possibl e to obtain data concerning the expression characteristics of the internal promoter s carried by these viruses by examining the expression of the reporter gene in the clones derived from transduced cells.

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39 CHAPTER 3 IN VIVO ANALYSES OF THE DEVELO PMENTAL AND CELL-SPECIFIC ACTIVITY OF THE HUMAN RETI NAL GUANYLATE CYCLASE-1 (GC1) PROMOTER Introduction Retinal guanylate cyclase (GC)-1 and GC 2 are two particulate GC enzymes that catalyze the conversion of guanosine tri phosphate (GTP) to cyclic guanosine monophosphate (cGMP), a key second messenge r molecule in the phototransduction cascade (Pugh, Jr. and Lamb 1990). The synthesis of cGMP is essential for recovery of the dark state following photoexcitation of photoreceptor cells (Pugh, Jr. and Lamb 1990). The activities of retinal GCs are modulated by guanylate cyclase activating proteins (GCAPs), a family of EF-hand calcium binding proteins that inhibit or stimulate enzyme activity under high or low intracellu lar calcium conditions, respectively (Mendez et al. 2001). The mature GC1 protein is localized to photoreceptor outer segment membranes and the results from some studies suggest that it is expressed at higher levels in cone cells than in rod cells (Cooper et al. 1995;Dizhoor et al. 1994;Liu et al. 1994). GC1 is also expressed in the pineal gland, indicating that GC1 may also play a role in pinealocyte phototransduction (Venkataraman et al. 2000). Mutations in the GC1 gene have been linke d to specific types of inherited retinal dystrophies including autosoma l recessive Leber congenital amaurosis type 1 (LCA1) and autosomal dominant cone-rod dystrophy (AD CRD) (Kelsell et al. 1998;Perrault et al. 1998). Most of the LCA1 mutations are frames hift and missense mutations that lead to

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40 the absence of GC1 or to the abolition of its activity in pho toreceptor cells (Rozet et al. 2001). All ADCRD mutations occu r in a three-codon sequence lo cated within the region of the GC1 gene encoding the dimerization domain of the enzyme. These mutations are predicted to alter the function of GC1 by enhanc ing or decreasing its ability to respond to GCAP1 stimulation (Duda et al. 1999;Tu cker et al. 1999;Wilkie et al. 2000). One of our research goals is to determ ine if photoreceptor function and vision can be restored in the avian model of LCA1, the GUCY1*B chicken (Semple-Rowland et al. 1998;Semple-Rowland and Lee 2000). The recent demonstration that viral vectormediated gene therapy can be used to rest ore functional vision in a canine model of LCA2 (a subtype of LCA caused by a mutation in the RPE65 gene) (Acland et al. 2001) support the use of viral vector s for the study and treatment of LCA. We are currently conducting studies to examine the feasibility of using a lentiviral vector to deliver a functional GC1 transgene to the retinal progenitor cells of these animals. The ability to target viral transgene expr ession to specific cell types and to control expression levels of the transgene are importa nt factors that must be addressed when developing gene therapy strate gies. Currently, cell-specific promoters are used in viral vectors to direct expression of transgenes to specific cell types, and the level of expression of these transgenes is controlled by selecting promoters that possess different intrinsic activity levels (Dejneka et al 2001;Harvey and Caskey 1998;Kafri et al. 2000;Reiser 2000;Takaha shi et al. 1999). In the previous chapter, we showed th at a 4.0 kb fragment of the chicken GCAP1 promoter fulfills many of the requirements that we deem important for appropriate expression of a GC1 transgene in chicken re tina. Shortly after the completion of these

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41 experiments, we initiated a collaboration with Dr. Hans-Jurgen Flle’s laboratory to examine the expression characterist ics of the human GC1 promoter in vivo using the experimental paradigm presented in Chapte r 2. The primary impetus for conducting these analyses was our goal to identify a promoter fragment that most closely mimics the expression characteristics of the native GC1 promoter and could be used to drive GC1 transgene expression in our vectors. In addi tion, previous efforts to clone the chicken GC1 gene and 5’ flanking region were unsucces sful and the human promoter was a viable alternative. Human GC1 promote r-nlacZ transgenes were packag ed into lentiviral vectors and their expression characteristics were examined in vivo using the experimental paradigm described in Chapter 2. Methods Preparation of Constructs Three fragments of the human GC1 pr omoter (named GCE1, GCE7 and GCE8) were selected for use in this study based on th e results of previous analyses showing that they exhibited significant levels of activity in human retinoblastoma cells (Fulle and Gallardo 2001). All cloning of the promoter fr agments into the pTYF transducing vector (modified pTY vector that is described in Chap ter 4) of the lentiviral vector system were carried out in the laboratory of Dr. Hans-J urgen Flle as described below. The three promoter fragments were amplified using the polymerase chain reaction (PCR) and Pfu DNA polymerase (Stratagene). The core sequenc es of the primers for the designated GC1 promoter fragments were as follows: GCE1 (sense = 5’ CAC TTG TTA CTT TCT GGC TGA; antisense = 5’ GGT CAT TGC CGG CCG GCT T); GCE7 (sense = 5’ TCT GCT CCT CAT CCA ACA TTT C; antisense = same as GCE1); GCE8 (sense = same as GCE7; antisense = 5’ CAC AGG TCT TCC TTG CCA G). Not1 and Pme1 restriction

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42 enzyme recognition sequences were added to the sense and antisense primers, respectively. The CMV promoter was excise d from the pTYF.CMV.nlacZ vector using Not1 and Pme1 and replaced with PCR products digested with Not1 and Pme1 from the aforementioned reactions to generate the GC1 promoter-nlacZ expression vectors depicted in Fig. 3-1. Transfection-grade plasmid DNA was prepared using Qiagen endotoxin-free MaxiPrep kits. Production of Lentiviral Vector and Titers The production, concentration and titering of viruses used for experiments in this study were performed as described in the Chapter 4 Methods section. Briefly, final, infectious titers were estimated by multiply ing the concentration of p24 antigen (ng/ml) in vector stocks by the average specific transducing activity (TU/ng p24) of vector standard that was produced using the me thods described in Chapter 4 (6.1 x 103 TU/ng p24). Each virus preparation yielded stocks w ith estimated infectious titers that were between 0.1-1.0 x 1010 TU/ml (0.5-1.0 x 104 ng p24/ml). Embryonic Injections Neural tube injections of stage 10-12 em bryos were performed as described in the Chapter 2 Methods section. Tissue Preparation, Histochemistry and Microscopy Neural retina was dissected from the eyes of injected embryos at selected ages and dispase was used as necessary to aid in the removal of the pigmented epithelium. Retinal whole mounts were prepared by placing the tissue photoreceptor side down on a Millipore-Millicell insert containing PBS and flattened using fine tipped glass rods. To detect expression of nlacZ, retinas were fixed in 4% paraformaldehyde for 15 minutes. The retinas were then rinsed three times in PBS and incubated in PBS (pH 7.9)

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43 containing 35 mM potassium ferrocyanide, 35 mM potassium ferricyanide, 2 mM magnesium chloride, 0.02% NP-40 and 40 mg/ml X-gal substrate at 37C for 16-18 hours. Following this incuba tion, the retinas were rinsed three times in PBS, cryoprotected with 30% sucrose and mounted in optimal cutting temperature (OCT) medium for cryosectioning. Serial sections (20 m) were cut through areas positive for X-gal staining using a cryostat, mounte d on slides, and counterstained with 4,6diamidino-2-phenylindole (DAPI; Molecula r Probes). The sections were then coverslipped using an aqueous-based mounti ng medium (Gel Mount, BioMedia). Thirty to eighty sections, taken from the retinas of at least three different animals injected with the human GC1 promoter-nlacZ viral vect ors were analyzed for each time point. In some cases the pineal glands and brains of E18.5 embryos that had been injected with virus were removed and fixed in 4% pa raformaldehyde. Pineal glands were stained with X-gal in toto and processed for cryosectioning as described above. The brains were cut into 100 to 200 m-thick sections using a vibratome and placed in the wells of a 12well tissue culture plate. The sections of br ain were stained with X-gal for 16-18 hours, rinsed in PBS and viewed under a Zeiss dissecting microscope Brightfield and fluorescence microscopy were perfor med as described in Chapter 2. Results Primary Sequence Analyses The general structure and or ganization of the human GC1 gene has been described previously (Yang et al. 1995;Yang et al. 1996) Recent analyses of the GC1 5’ flanking region revealed that the 5’ UTR is co mprised of a 110 bp non-coding exon and a 304 bp intron and that the signal peptid e and translation start codon of GC1 are located in exon 2

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44 as illustrated in (Fig. 3-1; (Fulle a nd Gallardo 2001)). In the present study, in silica analyses were performed using the webbased versions of TRANSFAC (v4.0, TESS; http://www.cbil.upenn.edu/tess/) and the Eukary otic Neural Network Promoter Prediction (http://www.fruitfly.org/seq_tool s/promoter.html) programs. Fig. 3-1A shows a summary of the results obtained from these analyses, which revealed that a putative transcription start point (tsp) of GC1 lies 1.338 kb upstream of the ATG and that a strong TATA-box consensus sequence (TATAa/tAa/t) lies ~20 bp ups tream of this site between nucleotides –1320 and –1327 (the first nucleotide of the GC 1 translation start site [ATG]= +1). However, in a previous study of the b ovine GC1 5’ flanki ng region, the tsp was experimentally shown to be located within exon 1 (equivale nt to nucleotide -425 in Fig. 3-1A) and was associated with an initiator (Inr) consensus site (Johnston et al. 1997). Since the overall homology of the bovine and human 5’ flanking sequences is high and the results of these analyses do not conc ur, additional studies will be required to determine the precise location of the ts p in the human GC1 gene. Two cone-rod homeobox protein (CRX)-binding elements (CBEs; consensus CTAATNAGCTY) organized in a head-to-tail arrangement we re identified at posi tions –459 to –469 and – 1539 to –1549 relative to the translation start site. A 12-bp AT-rich sequence (TATATAATTGCT) that is repeated five tim es was identified between nucleotides – 1195 and –1327; the significance of this repe at is unknown, but it harbors near-consensus sequences for binding of core promoter cons tituents such as TATA-binding protein and TFIID. The sequence and location of the –459/ –469 CBE is conserved in the bovine GC1 promoter (data not shown). Overall, these results suggest that the core promoter region of

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45 the human GC1 gene may be located near the non-coding exon 1 a nd contains putative CBEs that could contribute to the re stricted expressi on pattern of GC1 in vivo nlacZ Exon 1 Exon 2P K K K R KIntron 1 gcggccgc TCTGCTCCTCAT CCAACATTTC CCCCAGCTTT AGAATCCACT GATGATTCTT ACCTGATCCA ATCTTTGCCA CCAAGTCTGA AAATGATTCA -1655 TTTAAAAGTT TTTAAGTTTT ATTTTTCACA TATAGATCTT TGACCTGGAA TAGATTTTGT GTATGGTGTG AGATAGGGAT TGAATTCTAT TTTCCCCCCA -1555 GATGGCTAAT CAGCTCCATT TGTTGAAGTT TATTCTTTCC AAGTTGGTAA GAAATGTCCA CGTCTTCACA CATTTGCTGT GTTTGTTTCA GTACTGTATA -1455 TTCTATTTCA TTGGTCCATT TGTTTATCCT TGTGCCAAAA CTATGAAGTC TTTATTGCCA TAGCTTTATA ATATAT ATTT ATATAAA TAT ATAATTGCT G -1355 TCACTACATA ATGTGG A TTT ATATAA ATAT ATAA TATATA ATTGCT ATAG CTTTATAGTA TATATT TATA TAATTGCT GT TTTATATATC ATATAATTAT -1255 ATAAATA TAT AATTGCT ATG TTTTATATAT TATATATTTA TGTAGA TATA TAATTGCT AT GTTTTATATA TTATATATTT ACATAAATAC ATAATTGCTA -1155 TTGCTTTATA ATAAATTTGT ATATCTTATA GGGCATCTTC CATTATACTC TTCTCTAAAA TTGTTAGCTA TTCTTGCCAT CCGGGCGATT ATATTCATTT -1055 TCACAACAAC CCTCAGATTA AGCAATTGCC CAAGGTCCAA AAATCAGCAA GAGGGACTTG GAACCCAGGT CTGTCGGAGG CCAAAGCTCT TTTCATTACT -955 TCTTGAGGGT GGTTTTCTAG GCATGGAGAA GCAGAGGTCA GGGAATCAAG TGTGGCGAGA GAGAGAAGAG AAGTGAAAGA AGAARGGCAG GTGTCAGCTT -855 GGTGTGGGTT TGGTCTCTGG GATATAGACT TTGCCAGCCA AAGGATGGAG CTTGAACTTA GCCGGCAGAA CTGGAAACAG AAGATTGTAA GGAAAGGGAC -755 TGGGATCAGT GTTTCTTCTC CAGGACGGAT TACCCACAGC TGTCCACGGG CAGGCACTTG TTACTTTCTG GCTGAGCAGG GCAGTGTGGC CGACGGCTGA -655 AAGGGGAAGC TGCGGCTGCT TTTGCGCAGG GGTGGTGGTG ATGAGGGTGA TGTGGGGGGC TGGAAGGCAT GGAGGGGAAA GGATCTGGCT GACTACCTGG -555 AAGCCAGGAC AGATCCCACC CCAGAAAGGC GCAGTAGGGG CTCTCATCCT CCACTAGCCC GCCCCTCCCT ACCTAATTAA GGACCCTAAT CAGCTTTGGG -455 GAGATTAAGG GCTCTGGCCG GCTGTAC CC A CGCCCCCGCC CTGGCCTGGG CTGGCAAGGA AGACCTGTGG GCGGGCGTCA AAAGGGGGAC CGGCCCTGTG -355 ACCCCTCACC GGGGCCGTGG GCCCGAGCCC CGGACTTCCC T GTAAGTGTC AGAGGCCCCT CCGCTGGGAT AGGGTCGGTC TGAGGGCGCA GGCGAGTCCC -255 TGCTGACCCC TGACGCCTCC GACGGGGGGA GGGGCAGGCC GGGTGGGAGC GGGAAGCCGG GGCGGCAGAA GGGGGCTTCG GGGCGGTGTC CTTGGCCCCA -155 GTTAGTCTTC CCAGCCTCCG GAGGGGGCGG TAGCAGCAGA ATCATCCCAT GGGTTACTCG GGCTTGGAGA AACTCGGGGT TACGGGGAGA ACCCTAGGGG-55 AGGCCGGGGT CTCAGTCGCT CAGCCTGCTC CGTCTGTGTT CGCAG AAGCC GGCAATGACC gtttaaacTT AAGCTTCCAC C ATG CCTAAG AAGAAACGAAAG+1A B nlacZ nlacZ CBE Intron 1 1 2 CBE Human GC1 Gene(-408/+1344) (-408/+953) (+639/+1344) nlacZ GCE7 GCE8 GCE1 GCE7 GCE8 GCE1 Figure 3-1. Sequence and schematic of the retinal GC1 5’ flanking region-nlacZ fusion constructs. A. Partial sequence showing the intron-containing promoter-nlacZ fusion region of the pTYF-based constructs. CBE = CRXbinding element; red boxes = CBEs; orange box = putative TATA box; purple text = unique AT-rich repeated sequence; purple boxes = exons; light blue box = intron; red A = putative GC1 ts p identified using in silica analyses; yellow A = bovine GC1 tsp; red arrow = GC1 start codon (+1); blue text = nlacZ ORF with peptide sequence of the nuclear-l ocalizing sequence of the SV40 large Tantigen; italics text = vector sequence. B. Diagram of the human GC1 promoter and schemes of the nlacZ constructs. Orange diamond = TATA box; red arrow = GC1 ATG; blue arrow = nlacZ ATG; purple box = exon.

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46 Tissue Specificity of nlacZ Expression All of the GC1 promoters test ed drove expression of nl acZ in the retinas, but not the brains, of E18.5 embryos that had been in jected with lentivirus at developmental stage 12. No nlacZ-positive cells were detected in the pineal glands of embryos that had been injected with the GCE1or GCE8-nlacZ lentiviruses. One out of the two pineal glands examined from embryos injected w ith GCE7-nlacZ lentivirus contained nlacZpositive cells. These cells were positioned n ear the lumen suggesting that they were pinealocytes (Fig. 3-2). brightfield DAPI100x 400x Figure 3-2. Cross-sections of pineal gland from E18.5 embr yo that was injected with the TYF-GCE7-nlacZ virus. Arrows indicate lumen with pinealocytes positioned around the perimeter. X-gal stai ning is shown as blue (left panel) or red (right panel). Th e right panel shows the overlay of the negative brightfield image shown on the left and the DAPI image. Cell Specificity and Developmental Expression of nlacZ In Chapter 2, we showed that expressi on of the GC1 gene in developing chicken retina begins at approximately E14. Base d on this observation, we chose to examine

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47 retinas from injected embryos at times prio r to (E10), equivalent to (E13) and several days after (E18.5) the onset of GC1 expression in order to examine the developmental activities of the GC1 promoter fragments. Very few or no nlacZ-positive cells were detected in the retinas of E10 and E13 embr yos that were injected with GCE1-nlacZ lentivirus. By E18.5, a significant number of nlacZ-positive cells were detected in both the outer nuclear layer (ONL) and inner nuclear layer (INL) of these animals (Fig. 3-3, top panel). In contrast, several nlacZ-positiv e cells that were di stributed throughout all cell layers in the retinas of E10 and E13 embryos that had been injected with GCE7nlacZ lentivirus. By E18.5, the expression of nlacZ was restricted to photoreceptor cells in the ONL (Fig. 3-3, middle panel). Finally, nl acZ positive cells were restricted to cells located in the central band of the INL and to cells within the ONL in the retinas of E10 and E13 embryos that had been injected w ith GCE8-nlacZ lentivirus (Fig. 3-3, bottom panel). By E18.5, the expression of nlacZ was restricted to the ONL a pattern resembling that generated by the GCE7 promoter. Discussion In Chapter 2, we established the usefulness of lentiviral-mediated gene transfer for studying promoter function in vivo In the present study, we used a similar experimental paradigm to examine the expression charac teristics of human GC1 promoter-nlacZ transgenes in the developing retina. The results of this study demonstrate that a 1.0 kb region of the human GC1 promoter locat ed between nucleo tides –386 and –1745 is sufficient to direct gene expr ession specifically to photorecep tor cells and to the pineal gland in vivo Furthermore, our results show that th e cellular specificity of the activity of this region of the GC1 promoter increases as a function of retinal development. Between E10 and E13, promoter activity was observed in all retinal cell laye rs, but by E18, this

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48 E10E13 E18.5ONL INL GCL ONL INL GCL GCE1ONL INL GCL nlacZ GCE7 nlacZ GCE8 GCE7 GCE8 nlacZ E10E13 E18.5 E18.5E18.5E18.5 Figure 3-3. Cross-sections of retinas containing human GC1 promoter-nlacZ transgenes. A schematic of the lentiviral tr ansgenes used for injections is depicted above each panel (refer to Fig. 3-1B for details). X-gal staining is red (nlacZ-positive cells) and DAPI staining is blue (cell nuclei). ONL = outer nuclear layer; INL = inner nuclear la yer; GCL = ganglion cell layer; red ellipses = cone-rod homeobox protein binding element; orange triangle = putative TATA box; red arrow = GC1 transl ation start site; blue arrow = nlacZ translation start site.

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49 activity was restricted to the ONL. Overal l, the intensity of X-gal staining was significantly lower in GCE8 retinas than in GCE7 retinas. These results suggest that expression driven by the –386/–1745 region of the GC1 promoter is augmented by the presence of intron 1 (compare GCE7 and GCE8, Fig. 3-3). CRX is a photoreceptor-specific transcription factor that pl ays an important role in regulating the expressi on of several photoreceptor-speci fic and pineal-specific genes, including itself (Furukawa et al. 2002;Livesey et al. 2000). It also appears to have an important role in regulating photoreceptor de velopment (Chen et al. 1997;Furukawa et al. 2002;Furukawa et al. 1997;Fur ukawa et al. 1999). Comparis ons of the results obtained for all three GC1 promoter fragments suggest that the two consensus CBEs identified in the proximal and distal regions of the prom oter may be required for directing expression of GC1 to the photoreceptor cells in vivo These results are consistent with studies showing that the promoters of several photoreceptor-specific genes contain multiple copies of CBEs (Livesey et al. 2000). Further experiments will be required to confirm the importance of these and other cis -acting elements in regulating the activity of the GC1 promoter. The temporal expression profiles of th e GCE7 and GCE8 promoter fragments revealed that these fragments did not possess th e same temporal expression pattern as that exhibited by the intact GC1 gene in devel oping retina. One plausible explanation for our observations is that the endogenous GC1 transcri ption onset in developing retina occurs earlier than E14. Use of more sensitive detection methods, such as RT-PCR, may show that GC1 gene transcription does begin at an earlier stage of development. It is unclear why the activities of the GCE7 and GCE8 prom oter fragments are restricted to the ONL

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50 at E18.5, but not at earlier stages of deve lopment. The cellular specificity of GC1 expression has not been examined in the de veloping retina. One po ssible explanation for our observations is that the cis -elements required to silence GC1 expression in nonphotoreceptor cells early in development are not present in these fragments. Another possibility is that the expre ssion of the human GC1 promoter is not regulated in a normal manner in the avian retina. Finally, it is possi ble that the GC1 gene is expressed in all retinal cells early in development of the retin a and its expression becomes more restricted over the course of development so that its expression is limited to photoreceptor cells in the late stages of development. In summary, the results from these analyses s how that the GC1 promot er contains distinct elements that drive its activity, control its expression during retinal development, and limit its expression to specific retinal cell types in vivo By conducting these analyses in vivo we have identified a GC1 promoter fr agment, GCE7, which possesses expression characteristics that should be suitable for use in our future efforts to drive express lentiviral GC1 transgen es in chicken retina.

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51 CHAPTER 4 IMPROVEMENTS IN THE DESIGN A ND PRODUCTION OF HIV-1-BASED LENTIVIRAL VECTORS RESULTS IN HI GH TRANSDUCTION EFFICIENCY IN RETINA AND THE EFFICIENT EXPRES SION OF A RETINAL GUANYLATE CYCLASE-1 (GC1) TRANSGENE Note The work presented in this chapter was publis hed as part of a re search article that appeared in Physiological Genomics 12 221-228 (2002). The guanylate cyclase activity assays were performed by Izabela Sokal in the laboratory of Dr. Krzysztof Palczewski. Introduction Lentiviral vectors derived from the huma n immunodeficiency virus type 1 (HIV-1) are emerging as the vectors of choice for long-term, stable in vitro and in vivo gene transfer. These vectors are attr active because they can carry large transgenes (up to 18 kb in size) (Kumar et al. 2001) and they are capab le of stably transduc ing both dividing and quiescent cells (Iwakuma et al. 1999;Miyos hi et al. 1998;Zuffe rey et al. 1998). The increase in interest in these vectors has given rise to a need for efficient and reproducible methods to produce large quant ities of high-titer lentiviral vector. Traditionally, lentiviral vectors are produced by co-transfecting human cell lines with plasmid DNAs that encode the viral compone nts required for viral packaging. Transient transfection of these cell lines is often accomplished using the conventional calcium phosphate co-precipitation technique (Naldini et al. 1996). Disadvantages of this method include: (1) the large amount of plasmid DNA that is re quired for transfection; (2) the difficulties associated with scaling up the pr ecipitation reaction; and (3) the high degree

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52 of variability observed in tr ansfection efficiency and vira l production. Recently, several groups have developed packaging cell lines th at facilitate the pr oduction of lentiviral vectors by reducing the need for multi-plasmid transfections (Farson et al. 2001;Klages et al. 2000;Pacchia et al. 2001;Xu et al. 2001). Although the use of packaging cell lines has streamlined the packaging procedure, the result ing viral titers have not been significantly higher than those obtained using transient co-transfection methods. In addition, the advantages of these new cell lines are often offset by the need to develop new lines for each generation of improved lentiviral vector. To achieve large-scale production of high-tite r lentiviral vector it is critical that transfection of the virus-pr oducing cell cultures be both efficient and reproducible; however, little effort has been made to optim ize this step in vector production. The results from the experiments presented in Chapters 2 and 3 demonstrate that lentiviral vectors transduce cells in developi ng retina, but improvements to the vector system and production methods would make this system more suitable for our future studies in the GUCY1*B chicken model of LCA1. The goals of the experiments described here were (1) to design and produce lentiv iral vectors that exhibit hi gh transduction efficiency in developing chicken retina and (2) to construc t a vector that produ ces active guanylate cyclase-1 (GC1) and can be used as a base ve ctor to develop therapeutic vectors for gene therapy studies aimed at treating LCA1. We were able to accomplish our first goal by combining a transfection method that utilizes the activated dendrim er-based transfection reagent, Superfect, with a novel vector con centration protocol. By using our new method, we were able to reproducibly ge nerate lentiviral vector stocks with titers greater than 1 x 1010 transducing units per ml (TU/ml) using less than one-third of the total amount of

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53 plasmid DNA that is commonly required for pr oduction of this vect or To achieve our second goal, we constructed a modular lentiviral vector sy stem that encodes functional GC1 and carries a multiple cloning site that facilitates the interchange of transgene components into the vector. Materials and Methods Lentiviral Vector Constructs The transducing vector used in our experiments was de rived from a previously described self-inactivating vect or (Cui et al. 1999;Iwakuma et al. 1999). The pTY vector was modified by inserting a cPPT-DNA FLAP element upstream of the multiple cloning site, an element that has been shown to si gnificantly improve the transduction efficiency of recombinant lentiviral vectors in vitro and in vivo (Follenzi et al. 2000;Zennou et al. 2001). All polymerase chain reaction (PCR) produc ts used for cloning as described below were amplified using Pfu high-fidelity DNA polymerase and cloned into intermediate pTOPO-BluntII vectors (Invitrogen) for use in subsequent steps. pTYF.linker: A 186-bp fragment containing the c PPT-DNA FLAP sequence was amplified from the pNHP vector using core primers that have been previously described (Zennou et al. 2000). Eag1 and Not1 linkers were added to the sense and antisense primers, respectively. The resulting fragment was excised with Not1 and Eag1 and cloned into the Not1 site of the pTY vector in the sense orientation, creati ng the pTYF.linker vect or (Fig. 4-1 A). The integrity of this modificati on was verified by DNA sequencing. pTYF.EF1.linker: The human elongation factor-1 (EF1 ) promoter was amplified from pTY.EFGFP (Zaiss et al. 2002) using sense and antisense primers containing NotI and NheI linkers, respectively. The EF1a promoter was excised using Not1 and Nhe1 and then cloned into

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54 the Not1 and Nhe1 sites of pTYF-linker ther eby generating the pTYF.EF1 .linker vector (Fig. 4-1 B). pTYF.EF1.PLAP: The placental alkaline phos phatase (PLAP) reporter gene was amplified from pRISAP (Chen et al 1999) (gift from C. Cepko) with sense and antisense primers containing Pme1 and Kpn1 linkers, respectively. The PLAP Pme1 / Kpn1 fragment was then cloned into the Sma1 / Kpn1 sites of pTYF.EF1a.linker to make the pTYF.EF1 .PLAP vector (Fig. 4-2). pTYF.EF1_IRES.EGFP: The polio virus internal ribosome entry site (IRES ) was obtained from pTYAT.CBA_IRES.EGFP (gift from A. Timmers) using sens e and antisense primers containing Sma1-Cla1 and Mlu1 linkers, respectively. The cDNA encodi ng enhanced green fluorescent protein (EGFP) was amplified from pEGFP-N1 (Cl ontech) using sense and antisense primers with Kpn1 EcoRV and Mlu1 linkers, respectively. The IRES and EGFP fragments were excised using Sma1 / Mlu1 and Mlu1 / Kpn1 and ligated into the Sma1 and Kpn1 sites of pTYF.EF1 .linker, resulting in the pTYF.EF1 _IRES.EGFP cloning vector (Fig. 4-1 C). pTYF.mIRBP1783.bGC1: First, a pTYF-mIRBP1783-linke r vector was generated by excising the mIRBP1783 promoter from pTYF-mIRBP1783-nlacZ with Not1 and Pme1 The fragment was then cloned into the Not1 / Sma1 sites of the pTYF.linker vector. The cDNA encoding bovine GC1 was amplified from the pSVL GC1 clone using the following primers: sense – 5’-CCA TCG ATA GTT TAA ACG AGC CCC GGA CTT; antisense – 5’-CCA TCG ATG ACC CAG CCT CAC TTC C. The resulting fragment was cloned into pTYFmIRBP1783-linker using Cla1 The amplified bovine cDNA, which included the entire open reading frame (ORF), extends from nucleotide 26 to nucleotide 3393 (GenBank L37089). pTYF.EF1.bGC1-IRES-EGFP: The bGC1 ORF was excised with Cla1 and cloned into the Cla1 site of the pTYF.EF1 _IRES-EGFP vector. pTYF.GCE7.bGC1-IRES-

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55 EGFP: The GCE7 promoter was excised from pTYF-GCE7-nlacZ (see Chapter 3) using Not1 and Pme1 and cloned into the Not1 / Pme1 site of pTYF.EF1 .bGC1-IRES-EGFP. Transfection-grade DNA was prepared using endotoxin-free DNA megaor maxiprep kits (Qiagen). AB CD MCS MCS MCS EF1 EF1 pTYF-linker 7489 bp pTYF.EF1 8922 bp pTYF.EF1 IRES.EGFP 7489 bp EF1 bovine GC1 IRES EGFP mIRBP1783 bovine GC1 GCE7 bovine GC1 IRES EGFP 5’ LTR3’ SIN LTR Figure 4-1. A. – C. Maps of the modular cloning plasmid vectors constructed for the SIN lentiviral vector system used in this study. Note the extensive listing of unique cloning sites. D. Schematics of bovine GC1 expr ession cassettes cloned into pTYF-based vectors. The black arrow i ndicates the transcription start point. Lentiviral Vector Production Concentration and Titers VSV-G-pseudotyped lentiviruses carrying an EF1 -PLAP transgene were prepared using the lentiviral vector system illustrate d in Fig. 2. 293T cells (Invitrogen Corporation,

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56 #R70007) were seeded in 75 cm2 (T-75) culture flasks at a density of 1 x 107 cells per flask and grown in Dulbecco’s modified Ea gle medium (DMEM; Gibco) containing 10% fetal bovine serum and antibiotics (130 U/ml penicillin and 130 g/ml streptomycin; growth medium). The cultures were maintained at 37C in 5% CO2 throughout the virus production period. On the following day, when the cultures reached 90-95% confluency, the growth medium was replaced with 5.0 ml of fresh medium. gag pol tat RRE CMV-TATA/TAR RSV SD SV40 pApNHP (packaging vector) pHEF-VSVG (envelope vector) VSV-GSV40 pA pTYF (transducing vector) 5’ LTR3’ LTR ppt RRE SD SA R bGHpA cPPT-DNA FLAP EF1 rev CMV-IE R U5AATAAA AATAAA EF1 PLAP Figure 4-2. The HIV-1-based self-inact ivating lentiviral vector system. The helper construct, pNHP, contains deletions in the regions encoding the accessory proteins vif, vpr, vpu and ne f and has been previously described (Zaiss et al. 2002). The self-inactivating transduci ng construct, pTYF, has a central polypurine tract (cPPT)-DNA flap elemen t located just upstream of the multiple cloning site and carries an EF1 -PLAP transgene. The packaging construct, pHEF.VSVG, encodes the vesicular stomatitis virus G (VSV-G) glycoprotein for pseudotyping (Cha ng et al. 1999). The pTYF.EF1 .PLAP construct was used to produce vector fo r the in vitro and in vivo experiments unless stated otherwise. For one large-scale preparat ion of virus, 20 T-75 flasks of 293T cells were transfected as follows: Transfection mixture for all 20 flasks was prepared by gently mixing 142 g pNHP, 70 g pTYF and 56 g pHEF.VSVG plasmi d DNA and 8.0 ml DMEM in one 50 ml polystyrene tube. Afte r mixing, 560 l of Superfect was added to

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57 the DNA solution. The contents of the tube we re gently mixed and incubated at room temperature for 10 min. Next, 430 l of the Superfect-DNA mixture was added dropwise to the T-75 flask (transfection start poin t) and the flask was incubated for 4-5 h. Following the incubation period, the medium containing the transfection mixture was replaced with 7.0 ml of fresh growth medi um. The next day, the media containing the first batch of virus was harvested from each flask and 6.5 ml of fresh growth medium was added to the cells. Upon collection, all viru s-containing media wa s filtered through a 0.45 m low protein-binding Durapore filter (Millip ore) to remove cell debris. To prepare transfection mixture sufficient for one T75 flask, the amounts of DNA, DMEM and Superfect were each divided by 20 to scale the reaction down. We have also found that viral vector can be produced in larger or sm aller cell culture flasks or plates by simply scaling cell numbers and the amount of DNA, DM EM and Superfect linearly with respect to the cell growth area. For some experiments, virus-contai ning media was concentrated using ultrafiltration and centrifugation as outlined in Diagram 1. For ultrafiltration, the virus stock collected from 20 T-75 flasks at 30 h post-transfection (~120 ml ) was divided into two 60 ml aliquots and centrifuged thr ough Centricon-80 ultrafiltration columns (Millipore) for 1 h in 4C at 2,500 x g The retentate was retr ieved by centrifuging the inverted column for 1 min in 4C at 990 x g and was stored at 4C until further processing. On the following day, the virus-c ontaining retentate was added to the ~120 ml of virus-containing media collected at 45 h post-transfection. Four 30 ml conicalbottom tubes (polyallomer Konical tubes; B eckman), each containing a 220 l cushion of 60% iodixanol solution (used directly from the Optiprep stock solution obtained from

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58 Axis-Shield) were prepared. Iodixanol was used because of its demonstrated safety in human clinical trials (Jorgensen et al. 1992) Media containing virus (30 ml) was gently pipetted into each tube, taking care not to disturb the iodixanol, and the samples were centrifuged at 50,000 x g for 2.5 h at 4C using a Beckma n SW-28 swinging bucket rotor. The media just above the media/iodixanol interface was carefully removed from each tube and discarded, leaving ~750 l of the solution in each tube (220 l of iodixanol plus ~500 l of media). The residual media containi ng virus and the iodixanol were mixed gently by shaking at 200 r.p.m for 2-3 h at 4 C. The resulting mixtures were pooled into one 3 ml conical-bottom tube (polyallomer K onical Tubes; Beckman) and centrifuged at 6100 x g for 22-24 h at 4C using a Beckman SW-50.1 swinging bucket rotor. The resulting supernatant was removed and di scarded and the remaining pellet was resuspended in 50 l of PBS or artificial cereb rospinal fluid by incubating the virus at 4C for 10-14 h. The final viral vector was gently mixed by pipetting, aliquoted and stored at -80C until use. Infectious titers of the TYF.EF1 .PLAP virus were determined by incubating 1.75 x 105 TE671 cells seeded in 12-well plates with limiting dilutions of the viral stock (1/10, 1/100 and 1/1000) in the presence of 8 g/ml polybrene. After a 4-5 h incubation period, fresh medium was added directly to the cells and, after 48 h, cultures were fixed, rinsed in PBS, heated in PBS at 65C for 30 min a nd stained for PLAP activity using previously reported methods (Fekete and Cepko 1993). Th e number of transducing units (TU; defined as an infectious particle) was de termined by estimating the number of PLAPpositive cells per well and final infectious tite rs were expressed as TU/ml. Estimates of the infectious titers of vector s lacking a strong promoter or the PLAP marker gene were

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59 based on the titers of unconcentrated TYF.EF1 .PLAP virus that was produced in parallel each time. Delivery of EF1 -PLAP Vector to Chicken Neural Tube The neural tube injections and preparati on of retinal flat mounts were carried out using the methods described in Chapter 2 (Col eman et al. 2002). The brains of injected embryos were fixed overnight in 4% paraformaldehyde at 4 C. The next day, the tissues were rinsed thoroughl y in PBS and 100 m thick sections were cut using a vibratome. Floating brain sections and re tinal flat mounts were subsequently processed for routine PLAP histochemistry using the technique s described above and as described at http://genetics.med.harvard.edu/~cepko/protoc ol/xgalplap-stain.htm. All tissues were collected on embryonic day 7 (E7) or 2 days post-hatch, 5 or 23 days after injections, respectively. Digital images of retinal flat mounts were captured with a Nikon Coolpix 995 camera fitted to a Zeiss Stemi V6 micros cope. In some cases, the percent area of retina transduced by the vector was determined as follows: TIFF images at a resolution of 1024 x 768 pixels were reduced by 35%, convert ed to grayscale using Adobe Photoshop and imported into the Scion Image program (a vailable at http://www.scioncorp.com). The density slice setting was used to select all of the pixels within the area of the flat mount that represented PLAP-positive areas and these were expressed as a percent of the total retinal area. Three to seven retinas were analyzed for each dose of vector. Analyses of GC1 Expression Vectors GC1 immunocytochemistry was performed on dispersed primary chicken retinal cultures and DF-1 cells (immortalized chicke n fibroblast cells; obtained from American

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60 Tissue Culture Company) that were tr ansiently transfected with the pTYFIRBP1783.bGC1 or pTYF.EF1 .bGC1/EGFP plasmid vectors, respectively. Primary retinal cultures The preparation, maintenance and transien t transfection of th e primary retinal cultures were performed as described under the Methods section in Chapter 2 with the exception that the cells were grown on glass coverslips coated with poly-D-ornithine. DF-1 cell cultures On the day prior to transfection, DF-1 cells were seeded into wells of 12-well plates that contained tissue culture-treated glass coverslips (Fisherbrand) and maintained in culture media as described above. Briefly, 3 g DNA was added to 50 l plain DMEM and mixed with 10 l Superfect and incubated at RT for 10 min. While the DNASuperfect mixture was incubating, the culture media was removed from the DF-1 cells and replaced with 0.5 ml fresh media. The transfection mixture (25 l) was then added to each well and incubated at 37C and 5% CO2 for 4-5 hrs. Following the incubation period, fresh media was added to each we ll and replaced one time on the following day. Immunocytochemistry and fluorescence microscopy Forty-eight hours after transf ection, both the primary retinal cultures and DF-1 cells were fixed using 4% paraformaldehyde for 5 min at RT, rinsed three times in PBS and processed for immunocytochemistry as follo ws. The cells were first blocked in PBS containing 10% goat serum for 30 min at RT. The cells were then incubated overnight at 4C with a GC1 polyclonal antibody (1/333 dilution in PBS containing 1.0% BSA and 0.3% Triton X-100; GC2, gift fr om A. Yamazaki). On the following day, the cells were rinsed three times for 15 min each and then incubated for 1 h at RT with a goat anti-

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61 mouse secondary antibody (1/500 diluti on in PBS) tagged with the Alexa-594 fluorophore (Molecular Probes). Th e cells were subsequently rinsed three times for 15 min each and counterstained with DAPI. The c overslips were carefully removed from the wells and mounted in Gel Mount (Biomedia) on glass slides. The st ained cells and/or direct GFP fluorescence were viewed using th e appropriate fluores cent filter sets and digital images were acquired using a SPOT2 Enhanced Digital Camera System mounted in a Zeiss Axioplan 2 fluorescence microscope. Generation of stably transduced cell lines TE671 cells were seeded into the well s of a 24-well culture plate and grown overnight at 37C, 5% CO2. On the following day, 300 ml of fresh media was added to the wells and TYF.EF1 _IRES.EGFP, TYF.EF1 .bGC1/EGFP or TYF.GCE7.bGC1/EGFP virus was added to the media at an MOI of ~ 5. After 24 hours, the cells were seeded into T-25 flasks and maintained by passaging two times a week. GC1 activity assays GC activity was measured in washed me mbrane fractions obtained from TE671 cells (~50 passages) and purified bovi ne rod outer segments (ROS) (150 g total protein). The fractions were incubated for 15 min at 30C with 1.5 mM [ 32P]GTP (19,00022,000 dpm/nmol; DuPont NEN), 50 mM Hepes, pH 7.8, 60 mM KCl, 20 mM NaCl, 10 mM MgCl 2 0.4 mM EGTA, and either 1.0 M or 0.030 M free CaCl 2 in the presence or absence of GCAP1 protein (5 g). The assays were repeated twice, each with similar results.

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62 Results Lentivirus Production and Concentration The goals of our first series of experiments were to determine the optimum ratio of total plasmid DNA to Superfect reagent that produced the highest titer virus and the optimum time for viral harvest. This ratio wa s determined to be 1:2 (ratios of 1:1, 1:1.5, 1:2, 1:5, and 1:10 were tested; data not show n). The titers of virus-containing media harvested directly from transfected 293T cultures were determined 30, 45, 60, and 70 hours post-transfection to identify the timef rame during which viru s production by these cultures is at maximum levels (Fig. 4-3) The average titer values were 8.0 x 106, 6.8 x 106, 2.6 x 106 and 0.8 x106 TU/ml at 30, 45, 60 and 70 hours post-transfection, respectively. Therefore, we collected culture media 30 and 45 hours post-transfection for subsequent experiments. It should also be noted that 293T cells passaged between 2 and 60 times were used for transfections and that passage number did not significantly affect transfection efficiency or final vector titers. Hours post-transfection Virus titer (x 10 6 TU/ml) 0 2 4 6 8 10 30456070 Figure 4-3. Production of lentivirus by tran sfected 293T cells as a function of time. VSV-G-pseudotyped lentiviruses carrying an EF1 -PLAP transgene were prepared using the lentiviral vector system illustrated in Fig. 4-2. Each bar represents the mean titer SEM of unconcentrated virus-containing medium collected at each time point (n = 3).

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63 Harvest virus at 30 h post-transfection (~20 x 7.0 ml) Concentrate by ultrafiltration (2 x Centricon-80 units) Harvest virus at 45 h post-transfection (~20 x 6.5 ml) Combine virus from step 2 and step 3a Overlay 30 ml virus onto 220 l iodixanol (x 4 tubes) Centrifuge at 50,000 x g for 2.5 hDay 1 Remove supernatant down to DMEM-iodixanol interface Combine virus from 4 tubes (Step 4a) and add to 3 ml tube Centrifuge at 6100 x g for 22-24 h Remove supernatant and add buffer to resuspend virus pellet to achieve an approximate 3000-fold volume change.Day 2 Day 3 1. 2. 3.a. b. 4. 5. 6. a. b. a. b. c. *Mean SEM derived from 13 separate large-scale virus preparations*** Titer (TU/ml) Approx. volume change (fold) Titer increase (fold) %Virus recovered Step 3b 1.40 0.35 x 107 n/a n/a n/a Step 5b 3.59 0.70 x 108 40 33 4 84 9 Step 6 1.40 0.44 x 1010 3000 958 191 40 8 Concentration Titer Figure 4-4. Outline and results of the vector production protocol. The top panel shows a simplified flow diagram of th e concentration procedure that is described in detail under Methods. Th e bottom panel summarizes the viral titer results obtained following each step of the concentration procedure. The goal of our second series of experi ments was to develop a concentration protocol that would minimize virus loss and yi eld the highest titer vi rus in the smallest possible volume. The concentration procedure and results are summarized in Fig. 4-4. The average starting titer of the virus-cont aining media (Fig. 4-4, bottom panel, Steps 13) was 1.40 0.35 x 107 TU/ml. The next step in the c oncentration procedure (Fig. 4-4, bottom panel, Step 4) yielded an average titer of 3.59 0.70 x 108 TU/ml in a volume of

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64 ~3.0 ml, resulting in a 33-fold increase in tit er and an average recovery of 84%. Further concentration of the virus stock by low-speed centrifugation (Fig. 4-4, bottom panel, Steps 5c and 6) yielded 1.40 0.44 x 1010 TU/ml, a 958-fold increase over the average starting titer. The averag e overall percent recovery of the virus was 40%. In vivo Performance of the Lentiviral Vector Administration of ~0.5 l of TYF.EF1 .PLAP virus (1 x 1010 TU/ml) into the chicken neural tube resulted in efficient transduction of large numbers of neural progenitor cells (Fig. 4-5). Cross-sections of stained retinas revealed numerous PLAPpositive cell columns (Fig. 4-5 D, bottom pane l). Columns of PLAP-positive cells were also observed throughout the developing brai n (Fig. 4-5 E). We also examined the relationship between viral dose a nd the percent of the retina transduced by the virus and determined that the transduction efficiency of the virus in deve loping retina was dosedependent (Fig. 4-5 A-C). The percent of to tal retinal area exhibiting PLAP expression was estimated to be 5%, 63% and 85% in embryos receiving injections of 108, 109 and 1010 TU/ml vector, respectively (Fig. 4-5 D, t op panel). PLAP expression was maintained in retinas from injected embryos that were examined 2 days after hatching, 21 days postinjection (Fig. 4-6). The relati onship between the amount of vi rus injected and the extent of viral transduction was maintained in th ese retinas, the number of PLAP-expressing cells being significantly less in embryos injected with 107 TU/ml virus (Fig. 4-6, left panel) than embryos injected with 1010 TU/ml virus (Fig. 4-6, right panel). GC1 Immunocytochemistry A small percentage of cells in the pr imary GUCY1*B chicken retinal and DF-1 cultures transiently transfected with either the pTYF.IRBP1783.bGC1 or the pTYF.EF1 .bGC1/EGFP vector, respectively, staine d positively for the GC1 protein

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65 %PLAP-positive retina 0 20 40 60 80 100 10 8 10 9 10 10Titer of injected vector (TU/ml) n = 3 n = 5 n = 7D E Figure 4-5. Lentiviral vector -mediated transduction of PLAP in chicken neural progenitor cells. A. C. PLAP expression in represen tative flat mounts of E7 chicken retinas from embryos re ceiving injections of (A) 108, (B) 109 or (C) 1010 TU/ml virus. D. top panel: Histogram showing the quantification of the percent area of PLAP-positive retina following injections of different doses of vector. Bars represent the mean SEM for each group (n = 3-7). b ottom panel: Cross-section of the retina shown in C. E. Cross-section showing PLAP-positive cells in the lateral anteri or cortex of an E7 embryo that had received a neural tube injection of 1010 TU/ml virus. (Fig. 4-7). A limited number of cells stained po sitive for GC1 in the two culture systems, which was consistent with the respective tran sfection efficiencies usually achieved in these cells and suggested that the staining was specific. GC1 expression driven by the

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66 1010 TU/ml 107 TU/ml TYF.EF1 .PLAP Figure 4-6. PLAP expression in post-hatch chicken retinas. Embryos were injected with different doses of virus at stag e 10-12 (neural tube). The retinas were processed for PLAP staining 2 days after hatching, 21 days after the injections. The bottom panels show close-ups of areas from the retinas pictured in the corresponding t op panels. Scale bars = 2 mm. IRBP1783 promoter was limited to cells exhi biting photoreceptor cell morphology in the GUCY1*B retinal cultures (Fig. 4-7, top panel) and was limited to the membrane surrounding the nucleus and to the apical ends of the cells th at eventually differentiate into the outer segments. GC1 immunostaining of the DF-1 cells was present throughout the cell body and its processes and co-localized with GFP fluorescence, a result that indicates that both cistrons of the bicistronic EF1 -bGC1-IRES-EGFP transgene were expressed (Fig. 4-7, bottom pane l). We were unable to determine from these analyses if the GC1-labeled protein within the soma was associated with the cell membrane.

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67 EF1 bovine GC1 IRES EGFP mIRBP1783 bovine GC1 GC1GFPGC1 + GFP Figure 4-7. Expression of recombinant bovine GC1 in avian-derived retinal photoreceptor cells (top panel) and DF-1 fibroblast cells (bottom panel). Bovine GC1 protein is labeled red an d the green in the bottom panel is intrinsic GFP fluorescence. Cells were transfected with the plasmid vectors illustrated above each panel as describe d in the Methods section of this chapter. Scale bars = 5 m GC1 Enzyme Activity To assess the activity of the GC1 enzyme produced from the pTYF.EF1 .bGC1/EGFP and pTYF.GCE7.bGC1/EGFP expression vectors, TE671 cells were first transiently transfected with th e plasmid DNA and processed for GC1 activity analyses 48 h post-transfection. The results of these analyses are shown in Fig. 4-8 A. Three samples were analyzed in this expe riment: (1) bovine ROS membranes; (2) mocktransfected TE671 cells; TE671 cells transfected with the (3) pTYF.EF1 .bGC1/EGFP vector or the (4) pTYF.GCE7.bGC1/EGFP vector. The activity of the GC1 enzyme in

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68 each sample was assayed under low and high calcium conditions in the presence and absence of GCAP1 protein. The results of th ese analyses show that the GC1 enzyme produced by our vector exhib its activity characteristics closely resembling those of the native GC1 enzyme present in bovine ROS. The activity of the GC1 enzyme in the TE671 cells dramatically increased when calcium levels were reduced in the presence of GCAP1 protein. The function of the bicistronic transgene within the pTYF.EF1 .bGC1/EGFP vector was also assessed in TE 671 cells that were transduced with virus made from this construct. The results indicate that the viru s is capable of stably expressing the GC1 transgene and that the bicistronic transgene efficiently expressed functional GC1 (Fig. 48A) in conjunction with the GFP marker protei n (Fig. 4-8B). Together these results show that the pTYF-based constructs are suita ble for use as the backbone for the final therapeutic lentiviral vectors that will be used to express functional GC1 in the GUCY1*B retina. Discussion By optimizing both the DNA transfection and viral concentration steps for production of lentiviral vector, we have overcome many of the problems that we had previously encountered in our efforts to pr oduce large volumes of high-titer lentiviral vector in a consistent manner. We found th at Superfect-mediated transfection of viral packaging cells consistently yielded largescale vector stocks (~120 ml) with starting titers averaging >1.0 x 107 TU/ml, titers that were comparable to vector stocks prepared using other transfection reagents. Use of S uperfect greatly simplified the transfection protocol and significantly reduced the amount of plasmid DNA required for the

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69 b o v i n e R O S T E 6 7 1 c e l ls pTYF-EF-GC1/GFP p T YF-G C E7 GC1/GFP EF G C 1/GF P EF-GFPGC Activity (nmol cGMP/min) 0.0 0.1 0.2 0.3 0.4 0.5 GC1 GC1 + GCAP1; low Ca 2+ GC1 + GCAP1; high Ca 2+ transfection virusA B EF1 bovine GC1 IRES EGFP SIN LTRSIN LTR Figure 4-8. Expression of recombinant bovi ne GC1 from transiently transfected transgenes and transgenes packag ed into the lentiviral vectors. A. Histogram showing the GCAPand calci um-dependent enzymatic activity of GC1 in vitro TE671 cells were transfected with plasmid or transduced with virus as described in the Methods section. B. GFP fluorescence in TE671 cells transduced with TYF.EF1 .bGC1/EGFP virus and schematic of the integrated transgene. This image coupled with the data shown in A demonstrates that the bi-cistronic transgene is functional wh en packaged into the lentivirus.

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70 procedure. The viral concentra tion protocol that we developed consistently increased the titers of the viruses by appr oximately 1000-fold (~1 x 1010 TU/ml). Furthermore, all vectors that we produced usi ng these methods exhibited high transduction efficiencies in vivo One of the goals of this study was to pr oduce viral stocks that could be used to transduce a high percentage of cells in the retina following deli very of lentiviral vector into the neural tube of the developing ch icken embryo. The injected virus transduced several populations of neural pr ogenitor cells, including those fated to become the neural retina (Figs. 4-5 and 4-6). A majority of cells exposed to virus during this stage of development are mitotic and have not yet diffe rentiated (Prada et al. 1991). By varying the concentration of the virus injected, we found that the pe rcent of retina transduced could be controlled in a lin ear fashion using does between 108 and 109 TU/ml. Injections of virus at a concentration of 1010 TU/ml produced maximal leve ls of retinal transduction. In the previous chapters, we s howed that it is possible to specifically target lentiviral vector-mediated expression of transgenes to retinal photoreceptor cells by selecting appropriate promoter fragments. Together, these results illustrate the effectiveness of our vector to transduce cells within the devel oping nervous system and illustrate the potential use of this vector as a to ol for studies of mechanisms regulating gene expression in vivo We also demonstrate that e fficient expression of the EF1 -PLAP transgene persists in the fully developed retina and that the vector is well-suited for use in our future studies of GC1 expression in the GUCY 1*B chicken model LCA1. In summary, the transfection and concen tration protocols outlined here allow efficient, reproducible producti on of high-titer lentiviral vectors that exhibit robust

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71 transduction properties in vivo The transfection protocol itself is simple and can be easily implemented by investigators interested in producing lentiviral vector in their laboratories. Furthermore, the methods can be easily adapted to la rge-scale lentiviral production protocols that are curre ntly being developed for use in large animal studies or for possible use in clinical studies.

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72 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS The results presented in Chapters 2 and 3 demonstrate that lentiviral vectors are capable of efficiently transducing avian neural progenitor cells and that the promoters of photoreceptor cell-specific gene s can be incorporated into these vectors to achieve targeted transgene expression in vivo The improvements made to the production and concentration protocols used to generate lent iviral vectors as described in Chapter 4 will facilitate the use of these vectors in vivo In addition, we have successfully developed and tested a bicistronic lentiviral vector construct that is capab le of mediatin g the expression of functional retinal guanylate cyclase-1 (GC1). Together, these results lay the groundwork for future studies of somatic ge ne therapy in the GUCY1*B chicken model of Leber congenital amaurosis type 1 (LCA1). Targeted Gene Expression in Retina Overall, the results from the experiments presented in Chapters 2 and 3 show that the GCE7 or GCAP4009 promoter fragments are suitable for driving the cell-specific expression of a GC1 transgene in retina. These results also lead to more specific questions regarding the mechanisms that co ntrol the onset of e xpression and the cellspecific regulation of these promoters in the developing retina The experimental paradigm presented in Chapters 2 and 3 coul d be extended to help answer the following questions that are releva nt to future studies of gene re scue in the GUCY1*B chicken: (1) Do the GCAP1 and GC1 promoters exhib it cone-specific or cone/rod-specific expression? (2) What are the intrinsic activ ity levels exhibited by the GCAP1 and GC1

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73 promoters in vivo ? (3) What cell types express GC1 during retinal development and is there a switch in the cellular specificity of its expression? Understanding the photoreceptor subtypes in which the GCAP1 and GC1 promoters are expressed is relevant to our efforts to rescue retinal function in the GUCY1*B chicken. The results of immunohistoc hemical analyses show that GCAP1 and GC1 are present in higher concentrations in cone cells than in r od cells (Cooper et al. 1995;Liu et al. 1994). Furthermore, clinical studies provide ev idence that cone cells may be more dependent on GCAP1 and GC1 in term s of survival and function. For example, patients diagnosed with retinal diseases that are linked to mutations in GCAP1 and GC1 exhibit phenotypes (e.g. diminished cone cel l electroretinograms) and behaviors (e.g. photophobic behavior and decrease d visual acuity) that are indicative of compromised cone cell function (Milam et al. 2003;Perra ult et al. 1999;Wilkie et al. 2001). Thus, successful treatment of diseases like LCA1 ma y depend to some extent on our abilities to insure that cone cells are incl uded in the target cell population. The precise cellular specificities of the GCAP1 and GC1 promoters could be determined by performing co-localization studies using antibodies specific for nlacZ and for cone (iodopsin) and rod (rhodopsin) specif ic markers. A reasonable approach would be to perform the immunocytoc hemical analyses on disperse d primary retinal cultures that have been prepared from the retinas of embryos that received injections of the various promoter-nlacZ lentiviral vectors. We have found that accura te identification of rod and cone cells expressing re porter genes is faci litated by use of dispersed cultures. Preliminary data obtained from this type of analysis are shown in Fig. 5-1.

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74 iodopsinGFPiodopsin+GFP iodopsinGFPiodopsin+GFP rhodopsinGFPrhodopsin+GFP Fig. 5-1. Immunolabeling of primary embryon ic chicken retinal cultures with cone (anti-iodopsin) and rod cell markers (anti-rhodopsin). Cultures were transfected with a mIRBP1783-GFP plas mid vector and the preparation and transfection of the cultures was perf ormed as described in Chapter 2. In addition to cell-specificity, it is also important to examine the levels of transcriptional activity exhibi ted by promoters when developi ng gene therapy strategies.. Strong, ubiquitous promoters are generally used in experimental gene delivery systems because they are readily available and usually guarantee high levels of expression in many cell types; however, abnormally high levels of expression of therapeutic genes in targeted cells may have delete rious effects on the function of these cells. For example, it has been suggested that over expression of guanylate cyclase-1 (GC1) may result in protein aggregation and/or can interfere with proper trafficking of GC1 to its position in the membrane, both of which may be detrimen tal to photoreceptor cells (Rozet et al. 2001). The over expression of GC1 in photorecepto r cells could also in terfere with proper regulation of the catalytic activity of this enzyme by GCAP1. Therefore, we have put

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75 considerable effort into identification of different promoters that may provide optimal levels of expression of GC1 in photoreceptors. It should be noted that levels of GC1 as low as 50% of that present in wild-type re tina are sufficient to sustain photoreceptor survival and function in the chicken retina (Semple-Rowland et al. 1998). To assess the intrinsic activity levels of selected GCAP1 and GC1 promoter fragments, the activities of thes e fragments could be analyzed in vivo using the methods described in Chapter 2. Comparisons of the re sults obtained from these experiments with those obtained in vitro would provide a more detailed pict ure of the intrinsic activities of these promoters. Finally, since we plan to introduce the vi rus during the early stages of embryonic development, it is important to understand the expression characteristics of the selected promoters in developing retina. In our anal yses of the GCAP1 promoter, we found that inclusion of the distal region of the GCAP1 5’ flanking region in the promoter fragment resulted in delayed, but specific expression of nlacZ in photoreceptor cells (Fig. 2-4). In contrast, the cell-specificity of expression of GC1 promoter fragments changed over the course of development, expression being li mited to photoreceptor cells during the later stages of development. Studies are currently planned to determine if the absence of expression of the GC1 promoters in non-photorece ptor cells late in development is due to silencing of expression of the transgene in retinal cells within the inner nuclear and ganglion cell layers. Use of laser capture dissection techniques will allow us to excise groups of cells from the INL that are positioned in columns marked by nlacZ-positive cells positioned in the ONL. The cells will th en be genotyped to confirm the presence or absence of the integrated transgene. The pr esence of the transgene in the absence of

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76 reporter expression would be consistent with the hypothesis that the promoter is actively silenced in these cells. Lentiviral Vector Tran sduction in Retina One of the long-term research goals of this project is to determine if vision can be restored in hatchling GUCY1*B chickens by de livering a lentiviral GC1 transgene to the retinal progenitor cells of thes e animals. We chose to use the lentiviral vector system because it circumvents some of the limitations of other vector systems. For example, the size of the GC1 transgene that we plan to use in these studies exceeds the cargo capacity of traditional recombinant AAV (rAAV) vectors, a problem that is not one that arises when using lentiviral vectors. Another issu e of importance concerns the time required for transgenes to reach maximum levels of expression. Transgenes carried by lentiviral vectors begin to express and reach maximum expression levels more rapidly (within 3 days) than those carried by rAAV (within 2-4 weeks) (Sarra et al. 2002). To rescue the function of GC1-null photorec eptors in the retina and restore vision, it is desirable to be able to transduce a high percentage of th ese cells with the therapeutic vector. Our initial attempts to transduce chic ken retinal progenitor cells with lentiviral vector were disappointing in this regard, the number of re tinal cells being transduced representing less than 5% of the total population. In view of the potential impact that poor transduction efficiency could have on the out come of future gene rescue experiments, much of our research effort was devoted to improving the performance of the lentiviral vector in vivo As described in Chapter 4, two changes were made to the system that dramatically improved transduction efficiencies in vitro and in vivo Because of these efforts, we were able to produce vira l vectors with titers ranging from 109 – 1010 TU/ml (~100-fold increase over previous efforts) that were capable of transducing greater than

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77 80% of the retinal cell populati on when injected into the neural tube. These modifications should significantly improve th e outcome of our future efforts to rescue sight in the GUCY1*B chicken. In the future, DNA in sulator elements from the chicken -globin gene (Chung et al. 1997) could be added to th e transducing vector, flanking the transgene insert. Insulators have been shown to re duce variegation effects and to significantly decrease transcriptional silencing in retrovir al vector transgenes (Pannell and Ellis 2001). This addition would contribute to a further ga in in the biosafety and performance of our lentiviral vector system in vivo. Several recent advances in the biosafet y and performance of lentiviral vector systems are beginning to assuage concerns ove r use of these vectors in gene therapy applications. Since I began my research progr am, three generations of lentiviral vectors have been developed in efforts to improve the biosafety and performance of the virus (Vigna and Naldini 2000). The third-generati on vector system consists of a modified helper vector that does not contain the Tat encoding region and a fourth expression vector that encodes the Rev protein, a protein that enhances viral packaging. These vectors could be readily incorporated into the sec ond-generation system that we used in the current studies. It may be prudent to utili ze the third-generation p ackaging vector system in our future studies of gene rescue in the GUCY1*B chicken.

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95 BIOGRAPHICAL SKETCH Jason Edward Coleman was born in Evansville, Indiana. After moving with his family to Jacksonville, Florida, he received formal training in the visual arts in high school and during the first couple years of coll ege before pursuing a research career in biology. In the spring of 1998, he graduated from the University of Florida where he received his BS in Neurobiology. Following grad uation, he worked as a research assistant in the Neuroscience Department at the Univ ersity of Florida. In the fall of 1998, he entered the Interdisciplinary Program in Biomedi cal Sciences at the University of Florida, College of Medicine. He joined the Depart ment of Neuroscience in May of 1999 where he pursued his doctoral degree under the s upervision of Dr. Susan Semple-Rowland.


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EFFICIENT TRANSDUCTION AND TARGETED EXPRESSION OF LENTIVIRAL
VECTOR TRANSGENES IN THE DEVELOPING RETINA















By

JASON EDWARD COLEMAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2003

































Copyright 2003

by

Jason Edward Coleman

































I dedicate this work to my parents, brothers, sisters, and friends who have all been a great
source of encouragement and support throughout this endeavor and to the inspiring and
loving memory of my grandfather, Dr. Joseph Edward Coleman.















ACKNOWLEDGMENTS

First and foremost, I would like to thank my mentor, Susan Semple-Rowland, for

all of her excellent support and encouragement over the past four-and-a-half years. She is

a very gifted educator and committed scholar. I feel very fortunate to have had the

opportunity to work in her laboratory under her guidance over the years. Her enthusiasm

for science has been inspirational and I thank her for providing an environment where

students are encouraged to be creative and to succeed at the highest level. Next, I would

like to thank the members of my committee, William Hauswirth, Marieta Heaton and

Adrian Timmers, for providing their expertise and input through my research endeavors.

I thank all of the past and present members of the Semple-Rowland laboratory for

their great friendships and invaluable assistance over the years. I would especially like to

thank Yan Zhang for stimulating discussions on circadian clocks, Miguel Tepedino and

Gabby Fuchs for their excellent assistance in the lab, and Christina Appin for producing

great virus and keeping several generations of cells alive.

The completion of this dissertation would not have been possible without the

tremendous and humbling encouragement from all of my teachers and friends throughout

my life. I would like to express my appreciation for my undergraduate mentor, Hazel

Jones, who helped initiate my scientific career and introduced me to the neuroscience

discipline (and the H-Tx rat). I would also like to thank Jake Streit for taking the time to

share his knowledge and expertise in histology and microscopy. I thank fellow graduate

students Matt Huentelman and Josh Stopek for their friendship and collaboration time,









which has helped to make lentivirus engineering more fun. Very special thanks go to my

girlfriend, Rachel, who has patiently stood by me and supported me through this final

phase of my doctoral degree.

I would like to extend a tremendous thank you to my family for providing all of the

love and support over my doctoral years. Finally, I would like to extend the greatest

acknowledgement to my mother, Denise Moore, who has never stopped believing in my

potential and has provided unwavering love and support over the past five years.
















TABLE OF CONTENTS
Page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF FIGURE S ......... ............................... ............. .......... ..... viii

A B STR A C T ................................................. ..................................... .. x

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Retinal Disease as a Model for Gene Therapy Efficacy..............................................1
Leber Congenital A m aurosis ......................................................... ...............
Clinical Phenotype and G enetics....................................... ......... ............... 2
Clinicopathology of LCA ............................................................................. 4
A nim al M odels of LCA 1 ............... .............. ........................................... 4
Gene Therapy Vectors ........................................ ...... ... ...... ........ .. 7
Parvoviridae- and Adenoviridae-based Vectors ................................................7
Retroviridae-based Vectors ................... .............. ..................................9..
Regulation of Vector-Mediated Gene Expression ............................. ............... 13
Gene Regulation in Retinal Photoreceptors ................................ ............... 13

2 A 4.0 KB FRAGMENT OF THE GUANYLATE CYCLASE ACTIVATING
PROTEIN-1 (GCAP1) PROMOTER TARGETS GENE EXPRESSION TO
PHOTORECEPTOR CELLS IN THE DEVELOPING RETINA .............................15

N ote ................... .......................................................... ................ 15
In tro d u ctio n ...................................... ................................................ 15
M eth o d s .............................................................................. 18
N northern Blot A nalyses ......................................................... .............. 18
Preparation of Constructs ................... ................................. 19
Cell Cultures and Transfections ........................................ ....... ............... 20
Luciferase and P-galactosidase A says .................................... ............... 21
L entivirus Production ................................................ .............................. 22
Em bryonic Injections ................. ............... ...................... .. .. ...... .... 23
Histochemistry and In vivo Promoter Analyses ...............................................24
Results .............................................................................................................................25
Expression of GCAP1 in Developing Chicken Retina.................... .......... 25
In vitro GCAP1 Promoter Activity............................................. ...............26









Lentiviral Transduction of Avian Tissues ........................................................27
Analyses of GCAP1 Promoter Fragments In vivo.............................................29
D iscu ssio n .........................................................................................3 2

3 IN VIVO ANALYSES OF THE DEVELOPMENTAL AND CELL-SPECIFIC
ACTIVITY OF THE HUMAN RETINAL GUANYLATE CYCLASE-1 (GC1)
PROMOTER ................ ......... ..... ................................... ... 39

Introduction ............... .......... .. .................... ........................ 39
M methods .........................................................................4 1
Preparation of C onstructs .............................................. ........... ............... 41
Production of Lentiviral Vector and Titers ...................................................42
Em bryonic Injections ............... .................... ........ ........ ... ..... .. .... .... 42
Tissue Preparation, Histochemistry and Microscopy ...............................42
Results ............... .... ...................................43
Prim ary Sequence A nalyses ........................................ .......................... 43
Tissue Specificity of nlacZ Expression.....................................46
Cell Specificity and Developmental Expression of nlacZ..............................46
D discussion ........... ........... .............................................................. ..... 47

4 IMPROVEMENTS IN THE DESIGN AND PRODUCTION OF HIV-1-BASED
LENTIVIRAL VECTORS RESULTS IN HIGH TRANSDUCTION EFFICIENCY
IN RETINA AND THE EFFICIENT EXPRESSION OF A RETINAL
GUANYLATE CYCLASE-1 (GC1) TRANSGENE ................................................51

N o te ................... .......................................................... ................ 5 1
Intro du action ...................................... ................................................ 5 1
M materials and M methods ....................................................................... ..................53
Lentiviral Vector Constructs ................... ........ ...............53
Lentiviral Vector Production, Concentration and Titers ....................................55
Delivery of EF 1c-PLAP Vector to Chicken Neural Tube.............................59
Analyses of GC 1 Expression V ectors .............................................................. 59
R e su lts ........................... .............. .............................. ................ 6 2
Lentivirus Production and Concentration.........................................................62
In Vivo Performance of the Lentiviral Vector ................. ............................64
G C 1 Im m unocytochem istry ........................................ .......................... 64
G C 1 E nzy m e A ctiv ity .............................................................. .....................67
D isc u ssio n ............................................................................................................. 6 8

5 CONCLUSIONS AND FUTURE DIRECTIONS ............................................. 72

Targeted Gene Expression in Retina ........................................ ....... ............... 72
Lentiviral Vector Transduction in Retina............... ............. ................................ 76

L IST O F R EFER EN CE S ........................................... ............................. ............... 78

B IO G R A PH IC A L SK E TCH ..................................................................... ..................95
















LIST OF FIGURES


Figure page

2-1. Expression of GCAP1, GC1 and iodopsin genes in developing chicken retina.......26

2-2. GCAP1 promoter activity in transfected E12 primary chicken embryonic retinal
cu ltu re s ........................................................................... 2 8

2-3. Retinal whole mounts and cross-sections prepared from embryos that received
injections of TY -EFl -nlacZ ....................... ......... ........................ .. ............. 30

2-4. Cell specificity and temporal onset of activity of the 292, 1436 and 4009 GCAP1
promoter fragments in embryonic chicken retina................................ ...............33

3-1. Sequence and schematic of the retinal GC1 5' flanking region-nlacZ fusion
constructs ............... ........... .......................... ...........................45

3-2. Cross-sections of pineal gland from E18.5 embryo that was injected with the TYF-
G CE7-nlacZ virus. ........................................... ... .... ........ ......... 46

3-3. Cross-sections of retinas containing human GC 1 promoter-nlacZ transgenes...........48

4-1. Maps of the modular cloning plasmid vectors constructed for the SIN lentiviral
vector system used in this study ................................................................... ......55

4-2. The HIV-1-based self-inactivating lentiviral vector system................. .......... 56

4-3. Production of lentivirus by transfected 293T cells as a function of time .................62

4-4. Outline and results of the vector production protocol. ..............................................63

4-5. Lentiviral vector-mediated transduction in chicken neural progenitor cells.. ............65

4-6. PLAP expression in post-hatch chicken retinas. .............................. ............... .66

4-7. Expression of recombinant bovine GC 1 in avian-derived retinal photoreceptor cells
and DF-1 fibroblast cells. ...... ........................... .......................................67

4-8. Expression of recombinant bovine GC1 from transiently transfected transgenes and
transgenes packaged into the lentiviral vectors......................................................69









5-1. Immunolabeling of primary embryonic chicken retinal cultures with cone (anti-
iodopsin) and rod cell markers (anti-rhodopsin). ............. ................ ....... ........ 74















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFICIENT TRANSDUCTION AND TARGETED EXPRESSION OF LENTIVIRAL
VECTOR TRANSGENES IN THE DEVELOPING RETINA


By

Jason Edward Coleman

May 2003

Chair: Susan L. Semple-Rowland, PhD
Major Department: Neuroscience

Gene therapy holds great promise as an effective treatment for genetic diseases.

Retinal diseases caused by genetic mutations are among the leading causes of blindness

and are an excellent place to begin studying the basic principles of gene transfer-based

treatments. In addition to understanding the molecular basis of a target disease, perhaps

the most difficult steps in the development of somatic gene therapies are engineering a

suitable method to deliver therapeutic transgenes to the diseased cells and achieving

appropriate levels of expression for extended periods. The primary goal of this study was

to develop a lentivirus-based gene delivery vector that can be used to target the

expression of a functional, therapeutic transgene to photoreceptor cells in the retina.

Lentiviral vectors derived from the human immunodeficiency virus type 1 are

emerging as the vectors of choice for long-term, stable in vitro and in vivo gene transfer.

Several inherited retinal diseases are caused by mutations in genes that are expressed in

photoreceptor cells and are required for normal function of these cells. Cell-specific









promoters can be incorporated into viral vector gene expression constructs and are used

to direct expression of transgenes to specific cell types, and the level of expression of

these transgenes is controlled by selecting promoters that possess different intrinsic

activity levels.

A self-inactivating lentiviral vector system was used in a novel manner to study the

intrinsic activity profiles of promoters that regulate the expression of photoreceptor-

specific genes. Using these methods, we were able to identify regions of these promoters

that are capable of targeting gene expression to retinal cells during development. Data

from these studies provide clues regarding the cis-acting elements that are important for

regulating photoreceptor-specific genes in vivo. Furthermore, we have improved the

design of and methods of producing lentiviral vectors that will facilitate use of this

system for delivering a normal, functional copy of a therapeutic transgene to retinal cells.

The results of these studies lay the foundation for future experiments aimed at studying

the potential use of lentiviral vector therapies for treating autosomal recessive retinal

diseases such as Leber congenital amaurosis type 1 (LCA1).














CHAPTER 1
INTRODUCTION

Retinal Disease as a Model for Gene Therapy Efficacy

The basic premise of gene therapy is to transfer (permanently or transiently) genetic

material to genetically defective cells, enabling these cells to function normally without

further treatment. Researchers within the field of gene therapy have recently made

promising advances toward realizing this goal, but we are still in the early stages of gene

therapy research. Thus, it is imperative that initial gene therapy studies be conducted

using animal models of well-defined genetic diseases that will provide the framework for

the development of treatments for more complex diseases. For example, genetic diseases

that affect tissues such as the retina could contribute greatly to the basic principles and

applications of gene therapy.

One practical advantage of studying the efficacy of gene therapy for retinal diseases

is that the eye is an easily accessible, immune-privileged organ. Another advantage is that

many of the genetic mutations affecting retinal function occur in genes that encode

proteins critically involved in the phototransduction cascade and the visual cycle two

processes that are well understood in retina. Consequently, the etiology of several retinal

diseases has been defined at the molecular level (www.sph.uth.tmc.edu/RetNet).

Therefore, this and the fact that there are several well-defined animal models available of

these diseases (Lin et al. 2002;Petersen-Jones 1998;Semple-Rowland et al. 1998),

strongly support a focus on retinal disease in efforts to define principles of gene therapy.

Within the past 5 years, results from several studies showed that the expression of various









transgenes in cells transduced with viral vectors can successfully slow retinal

degeneration in animal models of primary retinal disease (Acland et al. 2001;Ali et al.

2000;Bennett et al. 1998;LaVail et al. 2000;Lewin et al. 1998;McGee Sanftner et al.

2001;Takahashi et al. 1999). One notable advance has been the demonstration that

functional vision can be restored in a canine model of a congenital retinal dystrophy,

Leber congenital amaurosis (LCA), by transferring a normal copy of the RPE65 gene to

cells within the retina (Acland et al. 2001).

In the remaining portion of this chapter, I will focus on discussing LCA and recent

progress toward development of gene therapies for retinal disease. Specific topics will

include descriptions of animal models of LCA1, the rationale for selection and use of

specific viral vectors for the development of therapies, and the importance and

development of strategies to target gene expression to specific cell types affected by

retinal disease.

Leber Congenital Amaurosis

Clinical Phenotype and Genetics

Leber first described the condition known as LCA in 1869 (Leber 1869). LCA is a

family of clinically and genetically heterogeneous inherited retinal diseases that produce

the earliest and most severe forms of congenital blindness (Perrault et al. 1999). It is

generally assumed that LCA accounts for 5% of all cases of retinal dystrophies, but may

be even more frequent due to the high rate of consanguinity among LCA families

(Cremers et al. 2002;Foxman et al. 1985;Perrault et al. 1999). Visual deficits are usually

detected by the age of 6 months in infants and LCA patients rarely present with a visual

acuity better then 20/400 through life (Cremers et al. 2002). The electroretinographic

responses of LCA patients are severely attenuated or non-existent at birth and, based on









some published criteria, the electroretinogram (ERG) should be extinguished before the

age of 1 year (Foxman et al. 1985).

Uncomplicated LCA is inherited in an autosomal recessive mode. By 1996,

Perrault and her colleagues had identified the first gene linked to LCA, the gene encoding

the enzyme retinal guanylate cyclase-1 (GC1; designated LCA1) (Perrault et al. 1996).

Since this discovery, mutations in six additional genes expressed in retina have been

linked to LCA. These genes, which encode proteins that are involved in several aspects of

rod and cone cell function, include RPE65 (Marlhens et al. 1997), CRX (Freund et al.

1998), AIPL1 (Sohocki et al. 2000a;Sohocki et al. 2000b), CRB1 (den Hollander et al.

2001;Lotery et al. 2001) and RPGRIP1 (Dryja et al. 2001).

Several GC1 mutations have been identified and most of these are frameshift and

missense mutations (Perrault et al. 2000). The frameshift mutations generate premature

translation termination codons that are predicted to lead to the absence of GC 1 protein

(Perrault et al. 1996;Perrault et al. 2000;Rozet et al. 2001). The missense mutations,

many of which occur in the catalytic domain, have been shown to severely compromise

or abolish GC1 activity (Rozet et al. 2001). Functional consequences of an F589S

missense mutation in GC1 show that the mutation reduces basal GC1 activity by 80% and

disrupts the ability of GCAP1 to stimulate GC1 under low-calcium conditions (Duda et

al. 1999b). Most GC1 mutations identified so far are assumed to result in significant

reductions in the intracellular levels of cGMP in photoreceptor cells, reductions that

could lead to a situation equivalent to constant light exposure during retinal development

(Perrault et al. 2000).









Clinicopathology of LCA1

Ophthalmological examinations of LCA1 patients reveal that the funds appears

normal early in life, but abnormalities such as salt-and-pepper pigmentation and the

attenuation of retinal vessels begin to appear after several years (Edwards et al. 1971).

Most of the reported histopathologic studies of LCA1 retinas have revealed that the rods

and cones degenerate late in life (Francois and Hanssens 1969;Mizuno et al. 1977;Noble

and Carr 1978). Immunohistochemical analyses performed in a postmortem eye obtained

from a young LCA1 patient (11.5 years old) revealed that substantial numbers of rods

and cones were retained in the macula and far periphery; however there was an overall

reduction in the labeling of cone outer segment proteins (Milam et al. 2003). From a

therapeutic standpoint, the results of these analyses are encouraging and suggest that the

retinal circuitry was intact and functional. Further insight into the pathophysiological and

cellular consequences of the GC 1 mutations linked to LCA1 have been obtained

primarily from studies of two animal models of this disease, the GUCY1*B chicken and

the GC1-knockout mouse.

Animal Models of LCA1

There are currently two animal models of LCA1, the GC1-knockout mouse and the

GUCY1*B chicken. The phenotypes of these two animal models are strikingly different.

Comparisons of the two models provide important clues about the consequences of GC1

null mutations on the development, function and health of cone and rod photoreceptor

cells.

GC1-knockout mouse

The retinas of GC 1-knockout mice are morphologically normal at birth, but exhibit

reductions in the amplitudes of both the rod and cone cell responses to light stimulation









(Yang et al. 1999). By one month of age, cone responses to light are barely detectable

and the ERGs of both the rod a- and b-waves are dramatically reduced (Yang et al. 1999).

These mice do not display any detectable visual deficits despite the changes in rod

function that progress until 5 months of age. The first signs of photoreceptor degeneration

occur between 4 and 5 weeks of age and is marked by a rapid and specific loss of cones,

leaving a normal population of rod cells (Yang et al. 1999). This pattern of photoreceptor

degeneration differs significantly from that observed in the GUCY1*B chicken, which is

described below.

GUCY1*B chicken

The GUCY1*B chicken, formerly known as the retinal degeneration or rd chicken

(Semple-Rowland and Cheng 1999), is recognized as a naturally occurring model of

human LCA1 and is the only animal model of inherited retinal disease that possesses a

cone-dominant retina (Semple-Rowland et al. 1998;Semple-Rowland and Lee 2000). A

deletion-rearrangement of the GC 1 gene results in loss of the transmembrane domain of

GC1, destabilization of the transcript, and a total absence of the GC1 enzyme in the

GUCY1*B retina.

The retinas of GUCY *B animals are morphologically indistinguishable from

normal retinas at hatching. Early signs of retinal pathology appear 7 to 10 days post-hatch

in the photoreceptor layer of the central retina. Degeneration of the photoreceptor layer is

progressive so that by 21 days of age, marked degeneration of the photoreceptor outer

segments is apparent. By 60 days of age, the mid-peripheral retina shows signs of

degeneration and at 115 days of age, loss of photoreceptors from the central retina is

complete. By 6 to 8 months of age, cell loss from the inner retina is also apparent. The

amplitudes of the ERGs recorded from GUCY1*B chickens under photopic and scotopic









conditions are absent or less than 7% of those recorded from normal chickens (Ulshafer

et al. 1984). The levels of cGMP in the photoreceptor layer of 1-2 day old GUCY1*B

chicken retinas are only 10 to 20% of those measured in age-matched normal retinas

(Semple-Rowland et al. 1998). These results have led to the hypothesis that decreased

levels of cGMP may result in a state of constitutive hyperpolarization of the

photoreceptor cells, a condition that could mimic the degenerative events associated with

constant light exposure (Fain and Lisman 1999;Hao et al. 2002).

Upon comparison of the mouse and chicken models, it is evident that the only

common feature of the progressive retinal degenerations is that cone function and

survival are severely compromised by the absence of GC1. Differences in the spatial

organization and composition of photoreceptor cells in mouse and chicken retinas may

provide a likely explanation as to why the phenotypes and pathologies are so contrasting.

The retina of the chicken is cone dominant (80% cones; 20% rods), whereas that of the

mouse is rod dominant (3-5% cones; 95-97% rods). Therefore, the effects of cone

degeneration on rod survival and function in a rod-dominant retina appear to be different

than in a cone-dominant retina. Furthermore, the cone cells in mouse are evenly

distributed among the rod cell population throughout the retina. The distribution of cone

cells in the chicken is more analogous to that found in the macula/fovea region of central

retina in humans. The finding that visual function is severely compromised in both LCA1

patients and GUCY1*B chickens is consistent with the central role of cones in vision in

humans and chickens. Therefore, the data obtained from animal models and LCA1

patients suggest that cone cells should be the primary targets for gene therapies aimed at

treating this disease.









Gene Therapy Vectors

In general, vectors or gene delivery vehicles that facilitate the transfer of genetic

material to cells can be grouped into two broad categories: non-viral vectors and viral-

based vectors. Several studies have shown that genetic material can gain entry into cells

by forming complexes with liposomal or other cationic molecules. However, while the

development of effective non-viral vectors is rapidly progressing, the technology is still

in its infancy (Brisson and Huang 1999;Johnson-Saliba and Jans 2001;Lechardeur and

Lukacs 2002). Many of the recent advances in gene therapy have been facilitated by the

use of viral-based vectors. These vector systems take advantage of the natural capabilities

of viruses to deliver genetic material to cells. In particular, gene transfer vectors based on

viruses from the Parvoviridae, Adenoviridae and Retroviridae families have shown the

most promise in this regard. The unique characteristics of the different viruses and the

vectors derived from them must be considered when choosing a vector for use in

developing viral vector-based therapies. For example, host immune response, longevity

and/or kinetics of transgene expression, cargo capacity (size limit of a particular gene of

interest), vector tropism and the performance of gene regulatory sequences within the

context of vector-mediated gene expression can vary widely with each vector system.

Parvoviridae- and Adenoviridae-based Vectors

Vectors based on adeno-associated virus (AAV), a non-pathogenic member of

Parvoviridae, have been widely used in retinal gene therapy research. Recombinant AAV

(rAAV) vectors have been shown to efficiently transduce retinal cells of several species

and are capable of long-term expression of transduced genes (Acland et al. 2001;Ali et al.

1998;Bennett et al. 1997;Bennett et al. 1999;Flannery et al. 1997;Grant et al. 1997).









Some limitations are encountered with the use of rAAV vectors. Traditional

versions of rAAV vectors have a cargo capacity of less than 5 kb, which can pose

problems in applications where large cDNAs and regulatory sequences are required. To

increase the rAAV payload, some groups are exploring the possibility of using a trans-

splicing strategy to assemble a complete transgene from two different vectors after dual-

transduction of cells (Reich et al. 2003). Another feature of rAAV that could be

problematic in the treatment of rapidly advancing retinal diseases is that it may take up to

4 weeks to achieve maximal expression of transgenes after infection (Sarra et al. 2002).

Despite these limitations, rAAV vectors carrying normal copies of diseased genes

(Acland et al. 2001), genes encoding growth factors (Lau et al. 2000) and genes encoding

ribozymes specifically targeted to cleave mutated genes (Hauswirth et al. 2000;LaVail et

al. 2000;Lewin et al. 1998) have been successfully used to restore visual function or slow

retinal degeneration in animal models of inherited retinal disease.

Adenovirus (Ad) has also been developed as a gene transfer vector. As with rAAV,

Ad vectors have been shown to transduce photoreceptors in vivo after subretinal injection

(Akimoto et al. 1999;Bennett et al. 1994;Bennett et al. 1996b;Bennett et al. 1998).

Although Ad vectors have been routinely generated to high titer and although Ad vectors

exhibit efficient transduction of retinal cells, some caveats and limitations exist. For

example, Ad is only effective in situations where transient expression of a gene is

required since the viral DNA does not integrate into the host genome. The short-lived

expression of Ad vectors is due, in part, to the induction of a host immune response

triggered by expression of the adenoviral genes in the target cells (Bennett et al.

1996a;Hoffman et al. 1997;Reichel et al. 1998). Gutless Ad vectors have been developed









to circumvent this problem, to increase cargo capacity and to allow for stable integration

of the transgene (Kochanek et al. 2001;Mitani and Kubo 2002;Yant et al. 2002).

Subretinal injections of adenoviral vectors carrying normal copies of either the

phosphodiesterase P-subunit, neurotrophic factors, or anti-apoptotic factors have been

shown to delay photoreceptor degeneration in the rd mouse model of retinal degeneration

(Bennett et al. 1996b;Bennett et al. 1998;Cayouette and Gravel 1997).

Retroviridae-based Vectors

Retroviral vectors were among the first virus-based systems used to develop gene

transfer therapies (Buchschacher, Jr. and Wong-Staal 2000). Retroviruses can integrate

foreign genes into the host genome and sustain long-term expression. These viruses

consist of a diploid RNA genome surrounded by an enveloped capsid and can be divided

into two major taxonomic groups: simple and complex. The simple and complex

retroviral genomes consist of the conserved gag, pol and env genes flanked by cis-acting

long terminal repeat sequences (LTRs), and contain a packaging signal (y) adjacent to

the 5' LTR. The 5' LTR is comprises a viral promoter-enhancer region and transcription

start site; and the 3' LTR contains sequences required for efficient polyadenylation of

viral transcripts.

Simple retroviruses, such as murine leukemia virus (MLV), are well characterized.

Vectors based on MLV were among the earliest developed and have been at the forefront

of clinical gene transfer technology. Early versions contained a nearly complete viral

genome. Over the years, these vectors have been streamlined to contain only the genes

necessary to transduce cells and stably integrate genetic material into the host genome.

MLV vectors are now engineered to be replication defective by removing viral genes and









leaving only the cis-elements necessary for a single round of replication (Brenner and

Malech 2003).

MLV-based vectors have recently been used in human gene therapy trials designed

to treat X-linked severe combined immune deficiency (Aiuti et al. 2002;Cavazzana-Calvo

et al. 2000;Hacein-Bey-Abina et al. 2002). While the therapeutic outcome of this

experimental treatment has proven to be beneficial and promising, researchers have

recently identified two patients in the trial that have developed cases of a rare leukemia

(Brenner and Malech 2003;Hacein-Bey-Abina et al. 2003). These results suggest that the

MLV-based retroviral vectors have oncogenic potential in humans (Fox 2003). The

adverse effects observed in these cases could be linked to an intrinsic property of the

oncoretrovirus-based vector system or insertional mutagenesis (Brenner and Malech

2003). Regardless of the cause, these results point to a need for additional studies aimed

at developing safer gene transfer vectors for future use in humans.

Lentiviruses are complex retroviruses that can transduce dividing and non-dividing

cells. For this reason, these viruses have been the focus of intense research efforts aimed

at developing more efficient and versatile gene transfer vectors. In recent years, several

groups have described the generation of gene transfer vectors derived from the human

immunodeficiency virus type 1 (HIV-1), a lentivirus that holds great promise as a basis

for gene transfer vectors (Iwakuma et al. 1999;Quinonez and Sutton 2002;Zufferey et al.

1998).

HIV-1 is the most well studied lentivirus. In addition to the essential retroviral gag,

pol, and env genes that make up the HIV-1 genome, several accessory proteins are

encoded by the genome. The so-called non-essential accessory proteins include vif, vpu,









vpr, and nef The essential regulatory proteins, tat and rev, also unique to lentiviruses,

interact with viral genomic cis-elements to promote transcription elongation and to

facilitate nuclear export of viral RNAs, respectively.

Since HIV-1 is a lethal human pathogen, concerns about the biosafety of HIV-1-

derived vectors have been a primary focus of research efforts directed toward the

development of these vectors for clinical use in humans. Several steps have been taken to

diminish the possibility of generating wild-type virus during packaging and following

administration of these vectors. Firstly, the genome has been divided among three

bacterial expression plasmids in first- and second-generation lentiviral vector systems: (1)

a transducing vector that carries cis-elements and the promoter/gene of interest, (2) a

packaging plasmid that carries an attenuated viral genome and modified LTRs, and (3) an

envelope glycoprotein expression plasmid. Co-transfecting these plasmids in a producer

cell line generates a replication-incompetent lentiviral vector. Secondly, modifications

have been made to the transducing and packaging vectors by minimizing the amount of

homologous sequence between the two vectors, thereby greatly reducing the likelihood of

recombination. Finally, the latest versions of the transducing vectors are self-inactivating

(SIN) (Iwakuma et al. 1999;Zufferey et al. 1998). In SIN vectors, nearly all sequence has

been deleted in the 3' LTR, which is used as a template to generate both LTRs in the

integrated proviral form of the vector. Therefore, both LTRs are inactivated (i.e. do not

activate transcription) following integration and transcription of the inserted transgene is

activated by a heterologous internal promoter.

In light of the problems encountered during recent human gene therapy trials with

oncoretroviral vectors (Hacein-Bey-Abina et al. 2003), it will be important to continue









improving the biosafety of future lentiviral vector systems for use in humans. Lentiviral

vectors have the potential to initiate oncogenic events because they integrate transgenes

into host cell DNA. While the specific target sites for lentiviral vector transgene

integration have not been elucidated, studies have shown that integrated HIV-1 DNA is

primarily detected in non-coding regions of human DNA in blood lymphocytes (Lyn et

al. 2001). Two possible ways to circumvent the problem of insertional mutagenesis due

to random integration events are (1) to develop vectors that integrate transgenes at

specific, non-oncogenic sites and (2) to develop vectors that do not interfere with

endogenous gene expression. A mechanism for site-specific integration could be

incorporated into the lentiviral vector system by manipulating integrase activity or other

"forces" that influence target site selection (Belteki et al. 2003;Bushman 2002). The

incorporation of elements such as DNA insulator sequences (Chung et al. 1997;Pannell

and Ellis 2001) into future vector systems is likely to significantly improve the

performance of lentiviral vectors and to improve their biosafety by isolating the effects of

any enhancer/promoter elements contained in integrated transgenes.

In regard to the performance of lentiviral vectors in retina, recent studies have

shown that these vectors are capable of transducing photoreceptors following subretinal

injection, resulting in the long-term expression of transgenes (Auricchio et al.

2001;Cheng et al. 2002;Lotery et al. 2002;Miyoshi et al. 1998). Lentiviral vector-

mediated expression of the P-phosphodiesterase gene in photoreceptors in the rd mouse

attenuates photoreceptor degeneration for up to 4 months after injection (Takahashi et al.

1999). We have chosen to utilize an HIV-l-derived lentiviral vector system for gene









rescue studies in the GUCY1*B chicken model for LCA1 because of the large cargo

capacity and the rapid kinetics of transduction/transgene expression.

Regulation of Vector-Mediated Gene Expression

The ability to regulate gene expression in a physiological manner and to target

expression to specific cell types such as photoreceptors is crucial to the development of

successful somatic gene rescue strategies. Several factors are known to influence gene

expression (e.g. chromatin organization, gene copy number, and gene methylation).

Regulation of transcription initiation is the most straightforward mechanism of gene

regulation. The interactions of cellular trans-acting transcription factors with cis-acting

DNA elements found in the proximal promoter region play a central role in regulating

transcription initiation. The proximal promoter region of some photoreceptor genes has

been shown to be sufficient to confer tissue-specific gene expression (Flannery et al.

1997;Liou et al. 1991;Mani et al. 1999), although additional cis-elements located in distal

promoter regions or in the gene itself may act to enhance or repress levels of gene

expression (DesJardin and Hauswirth 1996;Wang et al. 1992).

Gene Regulation in Retinal Photoreceptors

Examination of the transcription factors and promoters involved in the regulation of

photoreceptor genes has begun to reveal the molecular mechanisms that control gene

expression in these cells. NRL and CRX proteins are two photoreceptor-specific

transcription factors that are crucial for the expression of several photoreceptor genes and

for photoreceptor development (Chen et al. 1997;Furukawa et al. 1997;Mears et al.

2001;Rehemtulla et al. 1996;Swaroop et al. 1992). Significant progress has been made in

identifying common cis-acting DNA elements that regulate the expression of a number of

photoreceptor-specific genes. Regulatory regions conserved in the proximal promoters of









the photoreceptor genes encoding arrestin (Boatright et al. 1997b;Kikuchi et al.

1993;Mani et al. 1999), P-phosphodiesterase (Di Polo et al. 1996;Mohamed et al. 1998),

interphotoreceptor retinoid-binding protein (IRBP) (Boatright et al. 2001;Bobola et al.

1995;Liou et al. 1991), rod opsin (Chen and Zack 1996;Gouras et al. 1994;Nie et al.

1996), and cone opsins (Chen et al. 1994;Wang et al. 1992) have been identified and

characterized using transgenic and in vitro analyses.

Truncated murine opsin promoters have been used successfully to target viral

transgene expression to photoreceptor cells (Flannery et al. 1997). Both opsin and P-

phosphodiesterase promoters have been used to drive expression of ribozymes and the P-

phosphodiesterase gene, respectively, in photoreceptor cells (LaVail et al. 2000;Lewin et

al. 1998;Takahashi et al. 1999).

As mentioned previously, one of the main objectives of my dissertation research

is to develop a lentiviral-based vector system that can be used to rescue the retinal

degeneration phenotype in the GUCY1*B chicken. In constructing this system, I have

focused much of my effort on identifying and utilizing promoters that limit expression of

GC1 transgenes to photoreceptor cells. The 5' flanking regions from IRBP, guanylate

cyclase activating protein-1 and GC 1, all of which are known photoreceptor-specific

genes, were characterized and tested in vivo to accomplish this objective. These studies

are presented in Chapters 2 and 3.














CHAPTER 2
A 4.0 KB FRAGMENT OF THE GUANYLATE CYCLASE ACTIVATING PROTEIN-
1 (GCAP1) PROMOTER TARGETS GENE EXPRESSION TO PHOTORECEPTOR
CELLS IN THE DEVELOPING RETINA

Note

The work presented in this chapter was published in Investigative Ophthalmology

and Visual Science 43, 1335-1343 (2002). Patrick Larkin and Yan Zhang assisted with

the northern blot analyses and Gabriela Fuchs assisted with the cryosectioning.

Introduction

Guanylate cyclase activating protein 1 (GCAP1) is an EF-hand calcium-binding

protein that activates photoreceptor guanylate cyclase 1 (GC1) under low intracellular

calcium conditions, thereby hastening the recovery phase of phototransduction (Dizhoor

and Hurley 1999;Palczewski et al. 2000;Polans et al. 1996). The expression of GCAP1

and GC1 in vertebrate retina is limited to cone and rod photoreceptor cells, a distribution

that is consistent with their roles in phototransduction (Cooper et al. 1995;Dizhoor et al.

1994;Dizhoor et al. 1995;Frins et al. 1996;Gorczyca et al. 1995;Howes et al.

1998;Palczewski et al. 1994). Within photoreceptor cells, GCAP1 is localized to the inner

and outer segments and synaptic regions and appears to be expressed at higher levels in

the cone cells of human, monkey and bovine retinas (Cuenca et al. 1998;Kachi et al.

1999). The expression of GCAP1 has also been detected in the pineal glands of bovine

and chicken (Semple-Rowland et al. 1999;Venkataraman et al. 2000). Studies of the

interactions of GCAP1 with GC 1 suggest that these proteins exist in photoreceptors as a

stable complex independent of intracellular calcium concentrations, and that activation of









GC 1 occurs as a result of a calcium-dependent conformational change in the complex

(Duda et al. 1996;Gorczyca et al. 1995;Otto-Bruc et al. 1997;Rudnicka-Nawrot et al.

1998;Tucker et al. 1997). While at least three variants of GCAP are expressed in retina

(GCAP1-3) (Dizhoor et al. 1995;Gorczyca et al. 1994;Haeseleer et al. 1999;Palczewski et

al. 1994), recent studies of GCAP1/2 knockout mice suggest that only GCAP1 is capable

of restoring normal light response kinetics to photoreceptor cells (personal

communication with W. Baehr and cited reference) (Mendez et al. 2001). The current

view that GCAP1 is essential for normal phototransduction is supported by the

observation that missense mutations in the GCAP1 gene (Y99C and E155G) have been

linked to autosomal dominant cone dystrophy in humans (Downes et al. 2001;Payne et al.

1998;Wilkie et al. 2001). These mutations, which interfere with the binding of calcium to

GCAP1, lead to persistent activation of GC 1 even under high calcium conditions

(Dizhoor et al. 1998;Sokal et al. 1999;Wilkie et al. 2001). These results clearly indicate

that GCAP1 plays a pivotal role in phototransduction and retinal disease. Therefore, it is

of interest to understand how the expression of GCAP 1 is regulated in developing and

mature retina.

In retinal photoreceptors, the magnitudes, cellular specificities and temporal

dynamics of expression of several photoreceptor-specific genes are regulated at the

transcriptional level. The intrinsic activities and cellular specificities of these genes can

be attributed to complex interactions between cis-acting regulatory elements within their

promoters and the cell-specific transcription factors that interact with them. The onset of

expression of these genes in developing retina has also been shown to be dependent upon

the interactions between promoter cis-elements and transcription factors, and is often









linked temporally to the differentiation and maturation of the photoreceptor cells (e.g. see

cited references) (Hauswirth et al. 1992;He et al. 1998;Johnson et al. 2001;Kennedy et al.

2001;Livesey et al. 2000;Morrow et al. 1998;van Ginkel and Hauswirth 1994). Relatively

few studies have been carried out to examine the activities of photoreceptor-specific

promoters in developing retina in vivo (Chen et al. 1994;Kennedy et al. 2001;Lem et al.

1991;Mani et al. 2001;van Ginkel and Hauswirth 1994). Recently, the importance of

correct temporal regulation of gene expression in developing retina has been clearly

demonstrated in studies of cone-rod homeobox (CRX) (Furukawa et al. 1999;Livesey et

al. 2000) and neural retina leucine zipper (Mears et al. 2001) knockout mice. The results

of these studies show that the absence of expression of these key trans-acting factors in

retina results in the down-regulation of expression of several photoreceptor-specific

genes and abnormal development and function of the photoreceptor cells.

In this series of experiments, we have examined the expression characteristics of

fragments of the chicken GCAP1 promoter both in vitro and in vivo with the purpose of

identifying regions of the promoter that play a role in regulating the activity, cell

specificity and developmental expression of GCAP1. Our previous analyses of the

sequence of the 5' flanking region of this gene (Semple-Rowland et al. 1999) served as a

guide for the selection of the GCAP1 promoter fragments that were analyzed in these

experiments. The intrinsic activities of these fragments were determined by measuring

the expression levels of GCAPl-luciferase fusion constructs in transiently transfected

primary embryonic chicken retinal cultures, an in vitro system that has been used to

characterize the activities of the promoters of photoreceptor-specific genes obtained from

a variety of species (Boatright et al. 1997b;Boatright et al. 1997a;Chen et al. 1997). We









utilized lentiviral vectors as a novel tool to extend the in vitro analyses of promoter

function to the in vivo environment of the developing chicken retina. Lentiviruses

pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are ideal vectors for

this type of analysis because they are capable of transducing several different cell types,

exhibit rapid integration and expression of transgenes in transduced cells(Vigna and

Naldini 2000), and have a large cargo capacity (>18 kb) (Kumar et al. 2001). The cell

specificity and the onset of the activity of selected GCAP 1 promoter fragments in

developing retina was assessed in vivo by monitoring the activity of GCAP1 promoter-

nlacZ transgenes in the retinas of animals that had received injections of lentivirus

carrying these transgenes prior to the development of the neural retina. The onset of

expression of each GCAP 1 promoter-nlacZ transgene in developing retina was compared

to the expression profiles of the GCAP1 and GC1 genes in normal, developing chicken

retina.

Methods

Northern Blot Analyses

Embryonic retina-pigmented epithelium-choroid tissues were removed from both

eyes of each embryo and total RNA from these tissues was isolated using an RNeasy total

RNA kit (Qiagen). Samples containing 10 [tg of RNA were electrophoresed on a 1.1%

formaldehyde gel and transferred to a nylon transfer membrane (Micron Separations

Incorporated). Northern blots were hybridized consecutively with radiolabeled cDNA

probes specific for GCAP1, GC1, and iodopsin as previously described (Semple-

Rowland and van der 1992). The GCAP1 and iodopsin results were confirmed by

repeating the analyses on a second series of independent samples. Blots were exposed to

Kodak BioMax film (Eastman Kodak) for 12-16 hours at -800C and the resulting









hybridization signals were imaged using a BioRad Gel Doc 1000 system. The 18S rRNA

was visualized by staining the blot with methylene blue.

Preparation of Constructs

The GCAP1 promoter fragments were amplified from appropriate regions of

GCAP1 cosmid clones, ccosl6 and ccos24 (Semple-Rowland et al. 1999) using the

polymerase chain reaction and Pfu DNA polymerase (Stratagene). For each of the

GCAP1 promoter fragments, unique upstream primers containing a NotI site were used in

combination with three different sets of downstream primers containing a Pmel site to

generate the following fragments (transcription start point = +1): (1) -292/+302, (2) -

1436/+302, (3) -3121/+222, (4) -4009/+222, and (5) -1434/+29 (Fig. 2-2A). In this

study, these fragments will be referred to as 292, 1436, 3121, 4009 and 1434, names

based on the position of the 5' nucleotide. The sequence-specific (GenBank AF 172707)

primers used to amplify the fragments were as follows: 292 (sense 5' ACC CGT GTG

CTT TTC; antisense 5' GCT CCA GTC ACT CT), 1436 (sense 5' ACC CGA CTC

CTT CAA; antisense same as 292), 3121 (sense 5' AAT CCT GCC CAT CAC TGC

CCT ATC; antisense 5' AGT TTT GAG GTC GGT GGG TGA GTC), 4009 (sense 5'

GGG CGA TTG GCA GGG AGG AG; antisense same as 3121), 1434 (sense 5' ACC

CGA CTC CTT CAA; antisense 5' CGG GCA AAT GTA AAA GC). Products from

the polymerase chain reaction were subcloned into the pCR-TOPO-blunt II vector

(Invitrogen) and the DNA sequences of positive clones were verified by sequence

analyses. GCAP1 promoter fragments were excised from the pCR-TOPO-blunt II clones

using NotI and Pmel and ligated into the appropriate vectors. For the in vitro activity

assay constructs, the multiple cloning site (MCS) of the pGL2 vector (Promega) was

modified by lighting the Sacl/Xhol fragment of the MCS of the pBluescript II SK vector









into the MCS of the pGL2 vector. The modified vector was then digested with Xhol,

blunt-ended, digested with NotI and the Notl-Pmel GCAP 1 promoter fragments were

ligated into the vector. For the in vivo lentiviral constructs, Notl-Pmel fragments were

ligated into pTY-nlacZ digested with NotI and Pmel. The murine IRBP promoter

(mIRBP1783), which included nucleotides -1783 to +101, was amplified from the

pIRBP1783-EGFP plasmid vector using the polymerase chain reaction. The same cloning

strategy described for the GCAP1 promoter constructs was used to generate pGL2 and

lentivirus pTY-nlacZ plasmid vectors containing the mIRBP1783 promoter.

Transfection-grade DNA was prepared for each construct using an endotoxin-free DNA

maxiprep kit (Qiagen).

Cell Cultures and Transfections

Dispersed embryonic day 12 (E12) chick retinal cultures were prepared and

transiently transfected essentially as previously described (Adler et al. 1982;Ameixa and

Brickell 2000;Boatright et al. 1997b;Kumar et al. 1996;Politi and Adler 1986). Isolated

neural retina was incubated in 0.25% trypsin at 370C for 20 minutes, dispersed by

trituration using a flame-narrowed glass pipette, and plated at a density of 2 x 106 cells /

well in 24-well culture plates that had been coated with poly-L-omithine (Sigma).

Cultures were maintained in basal medium of eagle (Life Technologies) supplemented

with 5 g/L glucose, 10% fetal bovine serum, and antibiotics at 370C in 5% CO2. Cells

were transfected the day after seeding using the calcium phosphate method. Briefly, 10

[tg of promoter vector DNA and 0.5 [tg of control vector DNA containing the nlacZ

reporter gene driven by the CMV promoter were added to 125 [l of 0.2 M CaC12. Next,

125 [l of 2x HEPES-buffered saline was added dropwise to the DNA/CaC12 mixture. The









transfection mixture was allowed to incubate for 20 minutes at room temperature and

then 62.5 ul of the transfection mixture was added to each well. Cells were incubated

overnight at 370C in 5% CO2 and rinsed 3 times with PBS the following day. The

transfection experiments were replicated 4 times and a new preparation of cultured cells

was used for each experiment (n = 4). Within each experiment, transfection of each

promoter construct was carried out in duplicate using the same transfection mixture. The

photoreceptor-specific mIRBP1783 promoter was used as a positive control in all

experiments (Boatright et al. 1997a;Boatright et al. 2001;Chang et al. 2000).

Luciferase and P-galactosidase Assays

Cell lysates from the E12 primary retinal cultures were prepared 40-48 hours

post-transfection by adding 200 [l lysis buffer (provided in the assay kits, see below) to

each well, scraping the cells using a rubber policeman and processing the lysates for the

luciferase or P-galactosidase chemiluminescent assays according to the manufacturer's

protocols (Galacto-star or Luciferase Assay Kits, Tropix). Luciferase and P-galactosidase

activities were measured in 20 and 40 tl aliquots of each lysate, respectively. Assays

were run in duplicate and quantified using a TD20/20 luminometer (Turner Designs) with

an integration time of 10 seconds. Activity values were corrected for transfection

efficiency across experiments by normalizing luciferase values to P-galactosidase values.

Promoter activity was expressed as fold-activity over the promoterless pGL2 vector. Data

were analyzed using one-way repeated measures ANOVA and post-hoc pairwise

comparisons were performed using the Student-Newman-Keuls post-hoc test

(SigmaStat).









Lentivirus Production

Viruses pseudotyped with VSV-G were prepared using a self-inactivating

lentiviral vector system(Iwakuma et al. 1999). Packaging cells (293T) were plated in 10

cm culture dishes at a density of 6 x 106 cells / dish in Dulbecco's modified eagle medium

(DMEM) containing 10% fetal bovine serum, and antibiotics (Life Technologies). The

293T cells were grown to 80-90% confluence and were then transiently transfected with

6 tg pTY-nlacZ (transgene-carrying vector), 12 |tg pHP (packaging vector), 5.5 |tg

pHEF (encodes VSV-G envelope) and 0.5 |tg pCEP4-tat (encodes tat protein) per dish

using Superfect transfection reagent (Qiagen). All four of the plasmids were added to 300

pl DMEM and the DNA mixture was vortexed and incubated at room temperature for 5

minutes. Superfect (50 [il) was added to the DNA mixture, which was then vortexed and

incubated at room temperature for an additional 5 minutes. During this time, the medium

was removed from the cells and replaced with 4.5 ml of fresh medium. The transfection

mixture was then added dropwise to the cultures which were then incubated at 370C in

5% CO2 for 3-4 hours. Following the incubation period, the cells were rinsed one time

with medium. Fresh medium (6 ml) was added back to the cells and the cells were

incubated overnight. The next day, the medium was removed and 6 ml of fresh medium

was added to the cells. The medium containing the virus was harvested 48 and 72 hours

post-transfection and frozen at -800C until concentration. To concentrate the virus, the

medium was rapidly thawed and passed through a 0.45 |jm low-protein binding Durapore

filter (Millipore, Bedford, MA) to remove cell debris. The filtered medium containing the

virus was concentrated 140-fold by ultracentrifugation at 20,000 x g for 2.5 hours at 40C.

The virus pellet was resuspended by gentle shaking at 40C for 4 hours, aliquoted and









stored at -800C until use. Infectious titers of virus were determined by infecting 4 x 104

TE671 cells seeded in 24-well plates with limiting dilutions of TY-EFla-nlacZ virus in

the presence of 8 tlg/ml polybrene. After 3-4 hours of infection, fresh medium was added

to the cells. After 48 hours, the cultures were stained with 5-bromo-4-chloro-3-indolyl-3-

D-galactosidase (X-gal) substrate as previously described (Chang et al. 1999). Virus titer

was determined by counting the number of blue-nucleated cells and infectious titers were

expressed as the number of transducing units per ml (TU/ml). Particle titers were

determined using a p24 ELISA kit obtained from BD Biosciences following the protocols

provided therein. Dilutions of 1 x 10-6 and 1 x 10-7 oflysed viral particles were assayed to

obtain the mean ng p24/ml of each sample. The average infectivity of virus using the

methods described above was determined for TY-EFla-nlacZ virus Infectious titers of

virus carrying tissue-specific promoters were estimated by multiplying the particle titers

of the GCAP1/IRBP promoter-containing viruses by the infectious titer to particle titer

ratio obtained for the virus. The titers of all virus preparations were approximately 1 x

107 TU/ml.

Embryonic Injections

All animals were handled according to the ARVO statement for the Use of Animals

in Ophthalmic and Vision Research. Chicken eggs were set on day 0 and incubated on

their sides without rotation at 37.50C and 60% humidity. Viral injections were performed

on Hamberger-Hamilton stage 10-12 embryos (-E2). A small opening was made in the

eggshell overlying the embryo, the position of which was determined using an egg

candling light. With the aid of a dissecting microscope, injection of the virus into the

ventricular space of the neural tube was carried out using a micromanipulator (Sutter









Instrument Company) fitted with a pulled glass capillary needle that was connected to a

Sutter manual microinjector. The virus was mixed with fast green (1.0 tl 0.3% fast green

in PBS per 20 tl virus) to assist in the visualization of the injected virus. Upon

penetration of the embryo, 0.5-1.0 [tl of virus was slowly injected into the neural tube.

The egg was then sealed with parafilm and incubation was continued until the embryo

reached the desired age for analysis.

Histochemistry and In vivo Promoter Analyses

Neural retina was dissected from the eyes of injected embryos at selected ages and

dispase was used as necessary to aid in the removal of the pigmented epithelium. Retina

whole mounts were prepared by placing the tissue photoreceptor side down on a

Millipore-Millicell insert containing PBS and flattened using fine tipped glass rods. To

detect expression of nlacZ, retinas were fixed in 4% paraformaldehyde for 15 minutes.

The retinas were then rinsed three times in PBS and incubated in PBS (pH 7.9)

containing 35 mM potassium ferrocyanide, 35 mM potassium ferricyanide, 2 mM

magnesium chloride, 0.02% NP-40 and 40 mg/ml X-gal substrate at 370C for 3-4 hours.

Following this incubation, the retinas were rinsed three times in PBS. Retinas were

cryoprotected with 30% sucrose and mounted in OCT medium. Sixteen to twenty micron

thick serial sections were cut through areas positive for X-gal staining using a cryostat,

mounted on slides, and counterstained with DAPI. Thirty to eighty sections, taken from

the retinas of at least two different animals injected with TY-GCAP1 promoter-nlacZ

virus were analyzed for each time point. In some cases the pineal glands and brains of

E20 embryos that had been injected with virus were removed and fixed in 4%

paraformaldehyde. Pineal glands were stained with X-gal in toto and processed for









cryosectioning as described above. The brains of E20 embryos were cut into four regions

using a microtome blade as follows: cerebellum / brainstem, optic tectum, anterior

forebrain and posterior forebrain. The brain regions were stained in toto with X-gal,

rinsed in PBS and viewed under a Zeiss dissecting microscope; in some cases 16-20 |tm

thick sections were cut through the various regions of the brain using a cryostat and the

sections were stained with X-gal as described above. Brightfield and fluorescence

microscopy was performed using a Zeiss Axioplan 2 microscope (Carl Zeiss

Incorporated) fitted with a SPOT 2 Enhanced Digital Camera System (Diagnostics

Imaging Incorporated) for imaging. The DAPI nuclear stain was visualized using a

longpass DAPI filter. The images of the X-gal stained sections were produced by creating

a negative image of the stained section that was then overlaid with the DAPI-stained

image of that same section using the SPOT camera imaging software.

Results

Expression of GCAP1 in Developing Chicken Retina

Northern blot analyses were carried out to determine the onset and relative level of

expression of the gene encoding GCAP1 in developing embryonic chicken retina (Fig. 2-

1). Since the functional relationship between GCAP1 and GC1 is closely linked, analyses

of GC 1 expression were included for comparison. The expression of the gene encoding

iodopsin was also included in our analyses as a control. GCAP1 and GC1 transcripts

were first detected in developing chicken retina on E14-15 and E13-14, respectively. The

relative levels of GCAP and GC 1 transcripts, which were comparable at each

developmental stage, increased gradually as a function of embryonic age, reaching

maximum levels between E19 and E20. Iodopsin transcripts were first detected at E14, a

result that agrees with previous studies of the expression of iodopsin in developing









chicken retina (Adler et al. 2001;Bruhn and Cepko 1996). The onset of transcription of all

of these genes in retina coincides with the onset of photoreceptor outer segment

development and cGMP synthesis in developing chicken retina which occur around E15

(Meller and Tetzlaff 1976) and E18 (Semple-Rowland et al. 1998), respectively.


In LO I inO
d cto "7 to
"O C") '7 O
o T Co q o L cb r- r-- o o

Kb
9.5- 0 -. m agw GC1


2.4- GCAP1


1.4- I lodopsin

10 12 16 20
I I I I

IS OS ERG
18S



Figure 2-1. Expression of GCAP1, GC1 and iodopsin genes in developing chicken
retina. Northern blot hybridized consecutively with GCAP1, GC1 and
iodopsin cDNA probes and then stained with methylene blue to show 18S
rRNA (RNA loading control) at selected developmental stages. The 18S
rRNA was used as a loading control. The numbers across the top of the figure
correspond to embryonic age. The time line at the bottom of the figure
indicates the developmental ages at which the photoreceptor inner (IS) and
outer segments (OS) first appear and the earliest age at which
electroretinograms (ERG) can be recorded. The GCAP1 and iodopsin results
were confirmed by repeating the northern analyses on a second series of
independent samples.

In vitro GCAP1 Promoter Activity

The activity of each promoter fragment was measured in primary retinal cultures

transiently transfected with the promoter-reporter constructs. Cultures were prepared

from the retinas of El2 embryos in our experiments because preliminary studies showed









that the promoters of the GCAP1 and IRBP genes are active in these cultures. The five

GCAPl-luciferase fusion constructs tested in this series of experiments are shown in Fig.

2-2A. A comparison of the activities of the GCAP1 promoter fragments using ANOVA

revealed that they were significantly different from each other (F = 9.79, df = 4, p <

0.001) (Fig. 2-2B). The activities of the 292, 1436 and 4009 fragments, which were

comparable to each other, were significantly greater than the activities of either the 1434

or the 3121 fragments (p < 0.05). The activity of the 1434 promoter fragment was

significantly greater than that exhibited by the 3121 fragment (p < 0.05). A comparison of

the activities of the 292, 1436 and 4009 GCAP1 promoter fragments to that of the

mIRBP1783 fragment revealed that the activities of the GCAP1 promoter fragments were

approximately one half that of the IRBP promoter fragment assayed under identical

conditions (Fig. 2-2B). Comparable results were obtained in another series of

experiments in which cultures were transiently transfected with the pTY-based GCAP1

and IRBP promoter-nlacZ constructs that were used to generate the lentiviral vectors

(data not shown).

Lentiviral Transduction of Avian Tissues

The goals of this experiment were to determine if lentivirus pseudotyped with

VSV-G could transduce chicken retinal progenitor cells and if lentivirus could be used as

a tool to examine the expression characteristics of promoters in vivo. To address these

questions, we examined the expression and cellular distribution ofEFla-nlacZ and

mIRBP1783-nlacZ lentiviral transgenes in the retinas of E6 (EF la-nlacZ) and E20

(EFla-nlacZ and mIRBP1783-nlacZ) embryos that had received injections of lentiviruses

carrying either of these transgenes early in development (-E2). Examination of whole











A
+1
II I z ucferas I
-4009 +222
[- -D CE I luciferase I
-3121 +222
[ E- I-- D E luciferase
-1436 +302
[ ELI~luciferasel
-292 +302
Sluciferase
-1434 +29
B

50 100


40 80
3 -2-
0 0 -
SE 30 60 -o

o (n
t 20 40 0
__(3 0
10 20


0 0
292 1436 3121 4009 1434 IRBP
GCAP1 promoter fragment
Figure 2-2. GCAP1 promoter activity in transfected E12 primary chicken
embryonic retinal cultures. A. Diagram of the five GCAP1 promoter
fragments cloned into the pGL2 vector. Each promoter fragment, except for
1434, contains a 25 bp repeated sequence (hatched bars) located within the 5'
UTR and all constructs contain three proximal cone-rod homeobox (CRX)-
like binding elements (white bars) and a putative TATA box region (black
bars). B. Histogram showing levels of luciferase activity in primary retinal
cultures 48 hours post-transfection. Each bar represents the mean + SEM
activity obtained from 4 separate experiments for each promoter fragment.
Open bars = GCAP1 promoter fragments; filled bar = mIRBP1783 promoter;
= activity significantly less than 292, 1436, 4009 and 1434, p < 0.05; ** =
activity significantly less than 292, 1436 and 4009, p < 0.05.

mounts of retinas taken from E6 and E20 embryos that had been injected with TY-EFla-

nlacZ virus and stained with X-gal revealed the presence of several discrete clusters of

blue-nucleated cells that were distributed through the entire focal plane of the retina (Fig.

2-3A). Cross-sectional analyses of these retinas revealed that the nlacZ reporter gene was









being expressed in all cell layers of the retina. The staining intensities of the cells in E6

and E20 retinas were comparable, suggesting that the activity of the EF la promoter was

similar at both of these stages of development (Fig. 2-3B, C and D). The column-like

staining pattern that we observed in the retinas of the embryos injected with TY-EFla-

nlacZ virus closely resembled the staining pattern that has been reported in retroviral

studies of cell lineage in developing retina (Fekete et al. 1994). This result suggests that

the TY-EF la-nlacZ transgene carried by the lentivirus was integrated into the DNA of

retinal progenitor cells and passed to subsequent clones. In contrast to the clustered,

column-like nlacZ staining pattern observed in the retinas of embryos injected with TY-

EF la-nlacZ lentivirus, nlacZ staining in the retinas of embryos injected with TY-

mIRBP1783-nlacZ lentivirus was limited to cells on the surface of the retina (Fig. 2-3E).

Cross-sectional analyses of these retinas revealed that expression of the mIRBP1783

promoter was restricted to photoreceptor cells (Fig. 2-3F), a result that corroborates

previous studies of the cell-specificity of this promoter fragment (Boatright et al.

1997a;Boatright et al. 2001;Chang et al. 2000). An analysis of selected pineal glands and

brains taken from E20 embryos showed that transduction of these tissues was minimal or

undetectable following neural tube injection of lentivirus (data not shown). Together,

these results indicated that lentivirus could be used to examine the expression

characteristics of promoters in vivo.

Analyses of GCAP1 Promoter Fragments In vivo

The three GCAP 1 promoter fragments that showed comparable levels of activity

in our in vitro assays (Fig. 2-2B) were analyzed in vivo. Lentiviral vectors containing the

292, 1436 and 4009 GCAP1 promoter fragments driving the nlacZ reporter gene were
























i 0








I

'aC







Figure 2-3. Retinal whole mounts and cross-sections prepared from embryos that
received injections of TY-EFla-nlacZ (A-D) or TY-mIRBP1783-nlacZ
lentivirus (E,F). A. Area from a whole mount of an E6 retina showing a
cluster of infected clones intensely stained with X-gal. B. Cross-section
through retina shown in panel A. C. Cross-section through a retina taken from
an E20 embryo showing presence of cells stained with X-gal in all retinal cell
layers. D. Inverted brightfield image shown in panel C (nlacZ-positive cells in
red) overlayed with the DAPI image (in blue) to clearly show the retinal cell
layers. E. Area from a whole mount of an E20 retina taken from an embryo
injected with TY-mIRBP1783-nlacZ lentivirus and stained with X-gal. F.
Cross-section through the retina from panel E showing that the mIRBP1783
promoter limits expression of the nlacZ reporter gene to photoreceptor cells
within the outer nuclear layer (ONL). PR = photoreceptor side; V = vitreous
side; INL = inner nuclear layer; GCL = ganglion cell layer.









generated and injected into the neural tubes of E2 embryos to obtain an estimate of the

onset of expression of these fragments in developing retina and their cell-specificities.

GCAP1 promoter-driven nlacZ expression was examined in the retinas of E12, E16 and

E20 embryos, stages of development that were selected based on the results of our

analyses of the onset of normal GCAP1 expression in developing retina (Fig. 2-1) and on

the milestones of photoreceptor development in chicken. These stages correspond to time

points that precede the onset of GCAP1 expression in vivo (E12), that approximate the

onset of GCAP 1 expression and the development of photoreceptor outer segments in vivo

(E16), and that include the period when GCAP1 expression has reached maximal levels

in vivo just prior to hatching (E20).

Onset of expression in developing retina

X-gal stained retinal cells could be detected in whole mounts of retinas taken

from embryos that had been injected with either the 292 or the 1436 promoter-nlacZ

lentiviral vector as early as E12. The overall number of cells expressing nlacZ driven by

either of these promoter fragments was much lower in the retinas of E12 embryos (292, n

= 2; 1436, n = 3) than in the retinas of E16 (292, n = 3; 1436, n = 2) and E20 (292, n = 5;

1436, n = 3) embryos (data not shown). No detectable X-gal staining was observed in

retinas of E12 embryos that had received injections of the 4009 promoter-nlacZ lentiviral

vector (n = 6). The first evidence of X-gal staining resulting from 4009 promoter-driven

nlacZ expression was observed at E16 (1 positive retina out ofn = 6). By E20, the X-gal

staining in these embryos had increased sufficiently so that positively stained cells could

be detected in all retinas examined (n = 5).









Cell-specificity of expression

The cell-specificity of the activity of each promoter-nlacZ transgene was

determined by examining cross-sections cut from whole mounts of the retinas that had

been removed from embryos injected with the various lentiviral vectors (Fig. 2-4A). Both

the 292 and 1436 promoter-reporter transgenes were expressed in cells located within the

inner nuclear layer (INL) at E12. A few nlacZ-positive cells were also observed within

the ganglion cell layer (GCL) in these retinas at this time. In E16 and E20 retinas, X-gal

stained cells were also detected within the outer nuclear layer (ONL). In these retinas, the

number of stained cells observed in the ONL was generally higher than that observed in

the INL. In contrast to the rather non-specific cellular staining pattern observed in retinas

transduced with either the 292 or the 1436 promoter-nlacZ transgenes, cross-sectional

analyses of E16 and E20 retinas transduced with virus carrying the 4009 promoter-nlacZ

transgene revealed that only photoreceptor cells within the ONL were stained in these

retinas.

Discussion

The results of our in vitro and in vivo analyses of various fragments of the GCAP 1

promoter suggest that cis-elements regulating the activity, developmental expression, and

cell-specific expression of the GCAP 1 promoter are located in distinct regions of the

promoter. The 292, 1436 and 4009 fragments all exhibited similar activity levels in vitro,

a result which suggests that the cis-elements essential for conferring activity to these

fragments are located within the 292 fragment. In our analyses, we noted that removal of

the 25 bp repeated sequence, which comprises -50% of the 5' UTR, resulted in a

significant reduction in the activity of the 1436 promoter fragment. Inclusion of the









sequence between nucleotides -1437 and -3121 also produced a significant reduction in

promoter activity that could be ameliorated by addition of the sequence between


Developmental age


E12 [ -[ E16


U.Ii


I -E20I


I- low or no activity -1 1434
I in vitro I 3121


CRX


OTX


I activity in ONL -E16


activity in INL~-E12 and 292
I inONL E16 1436
I 4009


Figure 2-4. Cell specificity and temporal onset of activity of the 292, 1436 and 4009
GCAP1 promoter fragments in embryonic chicken retina. A. Examples of
cross-sections through X-gal stained retinal whole mounts (nlacZ-positive
cells = red; DAPI = blue). The embryonic ages are indicated along the top axis
of the figure and the GCAP1 promoter fragment is indicated along the side









axis of the figure. At E12, X-gal staining was observed in the inner nuclear
layer (INL) of retinas transduced with either the 292 or the 1436 promoter-
nlacZ transgene. By E16 and E20, X-gal staining was observed predominately
in the outer nuclear layer (ONL) and to a lesser extent in the INL. For the 292
and 1436 promoter fragments, two panels containing images from different
regions of the same E20 retinas are shown to illustrate the fact that in some
areas, X-gal staining was restricted to the ONL and that in other areas,
staining was present in both the ONL and the INL. No detectable X-gal
staining was observed in retinas of E12 embryos injected with the 4009
promoter-nlacZ lentiviral vector and only light staining was observed in the
ONL at E16. By E20, the level of staining in these retinas had increased
sufficiently to allow easy identification of the X-gal positive cells. Thirty to
eighty cross-sections cut from two to six retinas were analyzed for each
promoter fragment at the different developmental ages (see the Methods and
Results sections for details). IPL = inner plexiform layer; GCL = ganglion cell
layer. B. Schematic of the chicken GCAP1 promoter showing putative cis-
DNA binding elements for retina- and photoreceptor-specific transcription
factors (not to scale). The bracket bars highlight the different regions of the
GCAP1 promoter containing included in the various fragments that were
examined. Violet box = region between nucleotides -1437 and -3121 that
negatively affected promoter activity; red arrow = transcription start point;
blue and white-striped box = 25 bp repeated sequence within 5' UTR; ATG =
translation start codon.

nucleotides -3122 and -4009 to the fragment. It is possible that the observed decrease in

activity of the 1436 promoter with the truncated 5' UTR is due to a reduction in the

efficiency of translation of the transcripts produced from this promoter/reporter

transgene, and that interactions between cis-elements located within the -1437 to -3121

and the -3122 to -4009 regions are required to confer significant levels of activity to the

longer GCAP1 promoter fragments. To test these possibilities, it will be necessary to

assay the transcription levels and functional activities of additional GCAP1

promoter/reporter constructs.

In vivo analyses of the expression characteristics of the GCAP1 promoter fragments

were performed in order to identify regions within the GCAP1 promoter that control the

cell specificity and developmental onset of expression of the native GCAP1 gene.

Analyses of embryonic retinas transduced with lentiviral vectors carrying the nlacZ









reporter gene driven by the 292, 1436 or 4009 GCAP1 promoter fragments revealed that

the 292 and 1436 GCAP1 promoter fragments, both of which exhibited activity in vitro

and in vivo, did not possess the cis-elements required to restrict their activities to

photoreceptor cells. Furthermore, the 292 and 1436 promoter fragments exhibited activity

in vivo prior to the normal onset of expression of the GCAP 1 gene during developmental.

By including additional upstream sequence in the 4009 GCAP1 promoter fragment, we

obtained a fragment that exhibited the expression characteristics of the endogenous

GCAP1 gene. These results suggest that the general organization of the GCAP1 promoter

differs from those of previously characterized photoreceptor gene promoters, such as

IRBP and rhodopsin, in which many of the cis-elements in these promoters that are

responsible for restricting promoter activity to photoreceptor cells are located within 1 kb

of the transcription start point (Boatright et al. 2001;Bobola et al. 1995;Fei et al.

1999;Kennedy et al. 2001;Mani et al. 2001;Yokoyama et al. 1992;Zack et al. 1991).

Based on the in vivo expression characteristics of the 1436 and 4009 promoter fragments,

it appears that cis-elements located in the distal promoter region are required to delay the

onset of expression, a result similar to that reported in recent in vivo studies of the

Xenopus rhodopsin promoter (Kennedy et al. 2001).

The GCAP 1 promoter contains a cluster of putative cis-elements between

nucleotides -143 and -838 that include binding sites for transcription factors that have

been shown to regulate the expression of retina- and photoreceptor-specific genes (Fig. 2-

4B). We have previously reported that at least two putative CRX-like binding sites

(C/TTAATC/T) are present within the first 1kb upstream of the transcription start point

in the 5' flanking region of the chicken GCAP1 gene (Semple-Rowland et al. 1999). In









addition, one Ret-4-like element (-187 to -184) (Chen and Zack 1996), two OTX-like

binding elements (-196 to -202 and -832 to -838) (Chen and Zack 1996;Kimura et al.

2000) and one PCE-1/Ret-1-like element (-818 to -825) (Kikuchi et al. 1993;Morabito et

al. 1991;Yu and Bamstable 1994) are also located within this region (see Fig. 2-4B). Our

analyses show that the shorter GCAP1 promoter fragments that contain these elements

(292 and 1436) do not exhibit the expression characteristics of the native GCAP1

promoter in developing retina. Clearly, additional cis-acting elements located in the distal

GCAP1 promoter region (-1437 to -4009) are required to produce the cell-specificity and

developmental expression characteristics of the native GCAP1 gene. As mentioned

above, our in vitro data indicates that sequence located in the region between nucleotides

-1437 and -3121 suppresses GCAP1 promoter activity in retinal cells. Silencing

mechanisms similar to those reported for the regulation of neuron-specific gene

promoters (Bessis et al. 1997;Schoenherr et al. 1996;Weber and Skene 1997;Weber and

Skene 1998) could play a role in suppressing GCAP1 promoter activity in non-

photoreceptor cells and in producing the temporal expression characteristics of this

promoter in developing retina. Similar mechanisms have been postulated for other

photoreceptor gene promoters such as the murine IRBP promoter where a -70/+101

fragment of this promoter containing cis-elements that are highly conserved in retina- and

photoreceptor-specific promoters exhibits significant activity in vitro, but additional

sequence located between nucleotides -70 and -156 is required to restrict its activity to

photoreceptor cells in vivo (Boatright et al. 2001).

Recent studies of other photoreceptor gene promoters suggest that specific

combinations of regulatory factors expressed in photoreceptor cells that bind to and









transactivate these promoters are required for photoreceptor-specific gene expression

(Boatright et al. 1997a;Bobola et al. 1995;Fei et al. 1999;Kimura et al. 2000).

Examination of the sequence located upstream of the 1436 promoter fragment revealed

the presence of additional putative homeodomain protein-binding elements (see Fig. 2-

4B). The region between nucleotides -2413 and -2423 contains a head-to-tail

arrangement of two CRX-like binding elements (consensus CTAATNNGATT), which is

similar to that recently identified in several putative CRX-regulated photoreceptor genes

(Livesey et al. 2000). Additional CRX-like (-3305 to -3310) and OTX-like (-3356 to -

3362) DNA binding elements are located within the -3122 to -4009 region of the

GCAP1 promoter, elements that could potentially influence the expression characteristics

of the 4009 promoter fragment (see Fig. 2-4B). The results of these experiments provide

a rough blueprint of the structural and functional organization of the chicken GCAP 1

promoter. Additional studies will be required to confirm that the putative cis-elements

identified within the GCAP1 promoter bind trans-acting factors and that these

interactions serve to shape the activity characteristics of this promoter.

In establishing the usefulness of lentiviral-mediated gene transfer as a tool for

analyses of promoter function in the developing retina, we have demonstrated that

lentivirus can transduce chicken retinal progenitor cells. In addition, we show that the

expression of transgenes carried by lentivirus, which transduces both progenitor and

terminally differentiated retinal cells, can be targeted to specific cell types by selecting

appropriate internal promoters. The experimental paradigm presented here should be

amenable for studies of photoreceptor gene promoters from other species that exhibit

activity in primary cultures of chicken retinal cells and, thus, should have broad appeal









for in vivo analyses of promoter function. Furthermore, we show that the lentivirus vector

system used in this study is capable of carrying and expressing transgenes up to 7.4 kb in

size, a cargo well below the recently demonstrated capacity of this vector system of over

18 kb (Kumar et al. 2001). The large cargo capacity of this vector is an important feature

of this system that will make it useful for studies of the expression characteristics of large

promoter fragments in vivo. Finally, it is important to note that the utility of this method

is not compromised by the experimental variability due to differences in viral titer or

injection procedure. In experiments in which only small populations of progenitor cells

were transduced by the virus, it was possible to obtain data concerning the expression

characteristics of the internal promoters carried by these viruses by examining the

expression of the reporter gene in the clones derived from transduced cells.














CHAPTER 3
IN VIVO ANALYSES OF THE DEVELOPMENTAL AND CELL-SPECIFIC
ACTIVITY OF THE HUMAN RETINAL GUANYLATE CYCLASE-1 (GC1)
PROMOTER

Introduction

Retinal guanylate cyclase (GC)-1 and GC2 are two particulate GC enzymes that

catalyze the conversion of guanosine triphosphate (GTP) to cyclic guanosine

monophosphate (cGMP), a key second messenger molecule in the phototransduction

cascade (Pugh, Jr. and Lamb 1990). The synthesis of cGMP is essential for recovery of

the dark state following photoexcitation of photoreceptor cells (Pugh, Jr. and Lamb

1990). The activities of retinal GCs are modulated by guanylate cyclase activating

proteins (GCAPs), a family of EF-hand calcium binding proteins that inhibit or stimulate

enzyme activity under high or low intracellular calcium conditions, respectively (Mendez

et al. 2001).

The mature GC 1 protein is localized to photoreceptor outer segment membranes

and the results from some studies suggest that it is expressed at higher levels in cone cells

than in rod cells (Cooper et al. 1995;Dizhoor et al. 1994;Liu et al. 1994). GC1 is also

expressed in the pineal gland, indicating that GC1 may also play a role in pinealocyte

phototransduction (Venkataraman et al. 2000).

Mutations in the GC1 gene have been linked to specific types of inherited retinal

dystrophies including autosomal recessive Leber congenital amaurosis type 1 (LCA1)

and autosomal dominant cone-rod dystrophy (ADCRD) (Kelsell et al. 1998;Perrault et al.

1998). Most of the LCA1 mutations are frameshift and missense mutations that lead to









the absence of GC1 or to the abolition of its activity in photoreceptor cells (Rozet et al.

2001). All ADCRD mutations occur in a three-codon sequence located within the region

of the GC1 gene encoding the dimerization domain of the enzyme. These mutations are

predicted to alter the function of GC 1 by enhancing or decreasing its ability to respond to

GCAP1 stimulation (Duda et al. 1999;Tucker et al. 1999;Wilkie et al. 2000).

One of our research goals is to determine if photoreceptor function and vision can

be restored in the avian model of LCA1, the GUCY1*B chicken (Semple-Rowland et al.

1998;Semple-Rowland and Lee 2000). The recent demonstration that viral vector-

mediated gene therapy can be used to restore functional vision in a canine model of

LCA2 (a subtype of LCA caused by a mutation in the RPE65 gene) (Acland et al. 2001)

support the use of viral vectors for the study and treatment of LCA. We are currently

conducting studies to examine the feasibility of using a lentiviral vector to deliver a

functional GC 1 transgene to the retinal progenitor cells of these animals.

The ability to target viral transgene expression to specific cell types and to control

expression levels of the transgene are important factors that must be addressed when

developing gene therapy strategies. Currently, cell-specific promoters are used in viral

vectors to direct expression oftransgenes to specific cell types, and the level of

expression of these transgenes is controlled by selecting promoters that possess different

intrinsic activity levels (Dejneka et al. 2001;Harvey and Caskey 1998;Kafri et al.

2000;Reiser 2000;Takahashi et al. 1999).

In the previous chapter, we showed that a 4.0 kb fragment of the chicken GCAP1

promoter fulfills many of the requirements that we deem important for appropriate

expression of a GC 1 transgene in chicken retina. Shortly after the completion of these









experiments, we initiated a collaboration with Dr. Hans-Jurgen Fulle's laboratory to

examine the expression characteristics of the human GC1 promoter in vivo using the

experimental paradigm presented in Chapter 2. The primary impetus for conducting these

analyses was our goal to identify a promoter fragment that most closely mimics the

expression characteristics of the native GC 1 promoter and could be used to drive GC 1

transgene expression in our vectors. In addition, previous efforts to clone the chicken

GC1 gene and 5' flanking region were unsuccessful and the human promoter was a viable

alternative. Human GC1 promoter-nlacZ transgenes were packaged into lentiviral vectors

and their expression characteristics were examined in vivo using the experimental

paradigm described in Chapter 2.

Methods

Preparation of Constructs

Three fragments of the human GC1 promoter (named GCE1, GCE7 and GCE8)

were selected for use in this study based on the results of previous analyses showing that

they exhibited significant levels of activity in human retinoblastoma cells (Fulle and

Gallardo 2001). All cloning of the promoter fragments into the pTYF transducing vector

(modified pTY vector that is described in Chapter 4) of the lentiviral vector system were

carried out in the laboratory of Dr. Hans-Jurgen Fille as described below. The three

promoter fragments were amplified using the polymerase chain reaction (PCR) and Pfu

DNA polymerase (Stratagene). The core sequences of the primers for the designated GC 1

promoter fragments were as follows: GCE1 (sense = 5' CAC TTG TTA CTT TCT GGC

TGA; antisense = 5' GGT CAT TGC CGG CCG GCT T); GCE7 (sense = 5' TCT GCT

CCT CAT CCA ACA TTT C; antisense = same as GCE1); GCE8 (sense= same as

GCE7; antisense = 5' CAC AGG TCT TCC TTG CCA G). Not] and Pmel restriction









enzyme recognition sequences were added to the sense and antisense primers,

respectively. The CMV promoter was excised from the pTYF.CMV.nlacZ vector using

Not1 and Pmel and replaced with PCR products digested with Not1 and Pmel from the

aforementioned reactions to generate the GC 1 promoter-nlacZ expression vectors

depicted in Fig. 3-1. Transfection-grade plasmid DNA was prepared using Qiagen

endotoxin-free MaxiPrep kits.

Production of Lentiviral Vector and Titers

The production, concentration and titering of viruses used for experiments in this

study were performed as described in the Chapter 4 Methods section. Briefly, final,

infectious titers were estimated by multiplying the concentration of p24 antigen (ng/ml)

in vector stocks by the average specific transducing activity (TU/ng p24) of vector

standard that was produced using the methods described in Chapter 4 (6.1 x 103 TU/ng

p24). Each virus preparation yielded stocks with estimated infectious titers that were

between 0.1-1.0 x 1010 TU/ml (0.5-1.0 x 104 ng p24/ml).

Embryonic Injections

Neural tube injections of stage 10-12 embryos were performed as described in the

Chapter 2 Methods section.

Tissue Preparation, Histochemistry and Microscopy

Neural retina was dissected from the eyes of injected embryos at selected ages and

dispase was used as necessary to aid in the removal of the pigmented epithelium. Retinal

whole mounts were prepared by placing the tissue photoreceptor side down on a

Millipore-Millicell insert containing PBS and flattened using fine tipped glass rods. To

detect expression of nlacZ, retinas were fixed in 4% paraformaldehyde for 15 minutes.

The retinas were then rinsed three times in PBS and incubated in PBS (pH 7.9)









containing 35 mM potassium ferrocyanide, 35 mM potassium ferricyanide, 2 mM

magnesium chloride, 0.02% NP-40 and 40 mg/ml X-gal substrate at 370C for 16-18

hours. Following this incubation, the retinas were rinsed three times in PBS,

cryoprotected with 30% sucrose and mounted in optimal cutting temperature (OCT)

medium for cryosectioning. Serial sections (20 [tm) were cut through areas positive for

X-gal staining using a cryostat, mounted on slides, and counterstained with 4,6-

diamidino-2-phenylindole (DAPI; Molecular Probes). The sections were then

coverslipped using an aqueous-based mounting medium (Gel Mount, BioMedia). Thirty

to eighty sections, taken from the retinas of at least three different animals injected with

the human GC1 promoter-nlacZ viral vectors were analyzed for each time point.

In some cases the pineal glands and brains of E18.5 embryos that had been injected

with virus were removed and fixed in 4% paraformaldehyde. Pineal glands were stained

with X-gal in toto and processed for cryosectioning as described above. The brains were

cut into 100 to 200 [tm-thick sections using a vibratome and placed in the wells of a 12-

well tissue culture plate. The sections of brain were stained with X-gal for 16-18 hours,

rinsed in PBS and viewed under a Zeiss dissecting microscope. Brightfield and

fluorescence microscopy were performed as described in Chapter 2.

Results

Primary Sequence Analyses

The general structure and organization of the human GC1 gene has been described

previously (Yang et al. 1995;Yang et al. 1996). Recent analyses of the GC1 5' flanking

region revealed that the 5' UTR is comprised of a 110 bp non-coding exon and a 304 bp

intron and that the signal peptide and translation start codon of GC1 are located in exon 2









as illustrated in (Fig. 3-1; (Fulle and Gallardo 2001)). In the present study, in silica

analyses were performed using the web-based versions of TRANSFAC (v4.0, TESS;

http://www.cbil.upenn.edu/tess/) and the Eukaryotic Neural Network Promoter Prediction

(http://www.fruitfly.org/seq tools/promoter.html) programs. Fig. 3-1A shows a summary

of the results obtained from these analyses, which revealed that a putative transcription

start point (tsp) of GC1 lies 1.338 kb upstream of the ATG and that a strong TATA-box

consensus sequence (TATAa/tAa/t) lies -20 bp upstream of this site between nucleotides

-1320 and -1327 (the first nucleotide of the GC1 translation start site [ATG]= +1).

However, in a previous study of the bovine GC 1 5' flanking region, the tsp was

experimentally shown to be located within exon 1 (equivalent to nucleotide -425 in Fig.

3-1A) and was associated with an initiator (Inr) consensus site (Johnston et al. 1997).

Since the overall homology of the bovine and human 5' flanking sequences is high and

the results of these analyses do not concur, additional studies will be required to

determine the precise location of the tsp in the human GC1 gene. Two cone-rod

homeobox protein (CRX)-binding elements (CBEs; consensus CTAATNAGCTY)

organized in a head-to-tail arrangement were identified at positions -459 to -469 and -

1539 to -1549 relative to the translation start site. A 12-bp AT-rich sequence

(TATATAATTGCT) that is repeated five times was identified between nucleotides -

1195 and -1327; the significance of this repeat is unknown, but it harbors near-consensus

sequences for binding of core promoter constituents such as TATA-binding protein and

TFIID. The sequence and location of the -459/-469 CBE is conserved in the bovine GC1

promoter (data not shown). Overall, these results suggest that the core promoter region of














the human GC1 gene may be located near the non-coding exon 1 and contains putative


CBEs that could contribute to the restricted expression pattern of GC1 in vivo.



A gcggccgcTC TGCTCCTCAT CCAACATTTC CCCCAGCTTT AGAATCCACT
GATGATTCTT ACCTGATCCA ATCTTTGCCA CCAAGTCTGA AAATGATTCA 1655
TTTAAAAGTT TTTAAGTTTT ATTTTTCACA TATAGATCTT TGACCTGGAA
TAGATTTTGT GTATGGTGTG AGATAGGGAT TGAATTCTAT TTTCCCCCCA 1555
GATGG T GC ATT TGTTGAAGTT TATTCTTTCC AAGTTGGTAA
GAAATGTCCA CGTCTTCACA CATTTGCTGT GTTTGTTTCA GTACTGTATA 1455
TTCTATTTCA TTGGTCCATT TGTTTATCCT TGTGCCAAAA CTATGAAGTC
TTTATTGCCA TAGCTTTATA ATATATATTT ATAT AT T GCTG -1355
TCACTACATA ATGTGGATTT ATATAAATAT ATAATATATA ATTGCTATAG
CTTTATAGTA TATATTTATA TAATTGCTGT TTTATATATC ATATAATTAT -1255
ATAAATATAT AATTGCTATG TTTTATATAT TATATATTTA TGTAGATATA
TAATTGCTAT GTTTTATATA TTATATATTT ACATAAATAC ATAATTGCTA -1155
TTGCTTTATA ATAAATTTGT ATATCTTATA GGGCATCTTC CATTATACTC
TTCTCTAAAA TTGTTAGCTA TTCTTGCCAT CCGGGCGATT ATATTCATTT -1055
TCACAACAAC CCTCAGATTA AGCAATTGCC CAAGGTCCAA AAATCAGCAA
GAGGGACTTG GAACCCAGGT CTGTCGGAGG CCAAAGCTCT TTTCATTACT -955
TCTTGAGGGT GGTTTTCTAG GCATGGAGAA GCAGAGGTCA GGGAATCAAG
TGTGGCGAGA GAGAGAAGAG AAGTGAAAGA AGAARGGCAG GTGTCAGCTT -855
GGTGTGGGTT TGGTCTCTGG GATATAGACT TTGCCAGCCA AAGGATGGAG
CTTGAACTTA GCCGGCAGAA CTGGAAACAG AAGATTGTAA GGAAAGGGAC -755
TGGGATCAGT GTTTCTTCTC CAGGACGGAT TACCCACAGC TGTCCACGGG
CAGGCACTTG TTACTTTCTG GCTGAGCAGG GCAGTGTGGC CGACGGCTGA -655
AAGGGGAAGC TGCGGCTGCT TTTGCGCAGG GGTGGTGGTG ATGAGGGTGA
TGTGGGGGGC TGGAAGGCAT GGAGGGGAAA GGATCTGGCT GACTACCTGG -555
AAGCCAGGAC AGATCCCACC CCAGAAAGGC GCAGTAGGGG CTCTCATCCT
CCACTAGCCC GCCCCTCCCT ACCTAATTAA GGACC[TAAT CAGCTTTGGG -455


Exon 1




Intron 1


AGGCCGGGGT CTCAGTCGCT CAGCCTGCTC CGTCTGTGTT CGCAG
+ 1 I nlacZ
Exon 2 tttaaacTT AAGCTTCCAC CATGCCTAAG AAGAAACGAAAG
P K KK RK


B
I Human GC1 Gene
CBE CBE





GCE7 t nz |
(-408/+1344)

GCE8 z
(-408/+953)

GCE1 In.z |
(+639/+1344)

Figure 3-1. Sequence and schematic of the retinal GC1 5' flanking region-nlacZ

fusion constructs. A. Partial sequence showing the intron-containing

promoter-nlacZ fusion region of the pTYF-based constructs. CBE = CRX-

binding element; red boxes = CBEs; orange box = putative TATA box; purple

text = unique AT-rich repeated sequence; purple boxes = exons; light blue box

= intron; redA = putative GC1 tsp identified using in silica analyses; yellow A

= bovine GC1 tsp; redarrow = GC1 start codon (+1); blue text = nlacZ ORF

with peptide sequence of the nuclear-localizing sequence of the SV40 large T-

antigen; italics text = vector sequence. B. Diagram of the human GC 1

promoter and schemes of the nlacZ constructs. Orange diamond= TATA box;

red arrow = GC 1 ATG; blue arrow = nlacZ ATG; purple box = exon.


GAGATTAAGG GCTCTGGCCG GCTGTA









Tissue Specificity of nlacZ Expression

All of the GC 1 promoters tested drove expression of nlacZ in the retinas, but not

the brains, of E18.5 embryos that had been injected with lentivirus at developmental

stage 12. No nlacZ-positive cells were detected in the pineal glands of embryos that had

been injected with the GCE1- or GCE8-nlacZ lentiviruses. One out of the two pineal

glands examined from embryos injected with GCE7-nlacZ lentivirus contained nlacZ-

positive cells. These cells were positioned near the lumen suggesting that they were

pinealocytes (Fig. 3-2).


brightfield







,-










100.
Figure 3-2. Cross-sections of pineal gland from E18.5 embryo that was injected with
the TYF-GCE7-nlacZ virus. Arrows indicate lumen with pinealocytes
positioned around the perimeter. X-gal staining is shown as blue (left panel)
or red (right panel). The right panel shows the overlay of the negative
brightfield image shown on the left and the DAPI image.

Cell Specificity and Developmental Expression of nlacZ

In Chapter 2, we showed that expression of the GC1 gene in developing chicken

retina begins at approximately E14. Based on this observation, we chose to examine









retinas from injected embryos at times prior to (E10), equivalent to (E13) and several

days after (El 8.5) the onset of GC 1 expression in order to examine the developmental

activities of the GC1 promoter fragments. Very few or no nlacZ-positive cells were

detected in the retinas of E10 and E13 embryos that were injected with GCEl-nlacZ

lentivirus. By E18.5, a significant number of nlacZ-positive cells were detected in both

the outer nuclear layer (ONL) and inner nuclear layer (INL) of these animals (Fig. 3-3,

top panel). In contrast, several nlacZ-positive cells that were distributed throughout all

cell layers in the retinas of E10 and E13 embryos that had been injected with GCE7-

nlacZ lentivirus. By E18.5, the expression of nlacZ was restricted to photoreceptor cells

in the ONL (Fig. 3-3, middle panel). Finally, nlacZ positive cells were restricted to cells

located in the central band of the INL and to cells within the ONL in the retinas of E10

and E13 embryos that had been injected with GCE8-nlacZ lentivirus (Fig. 3-3, bottom

panel). By E18.5, the expression of nlacZ was restricted to the ONL, a pattern resembling

that generated by the GCE7 promoter.

Discussion

In Chapter 2, we established the usefulness of lentiviral-mediated gene transfer for

studying promoter function in vivo. In the present study, we used a similar experimental

paradigm to examine the expression characteristics of human GC1 promoter-nlacZ

transgenes in the developing retina. The results of this study demonstrate that a 1.0 kb

region of the human GC1 promoter located between nucleotides -386 and -1745 is

sufficient to direct gene expression specifically to photoreceptor cells and to the pineal

gland in vivo. Furthermore, our results show that the cellular specificity of the activity of

this region of the GC 1 promoter increases as a function of retinal development. Between

E10 and E13, promoter activity was observed in all retinal cell layers, but by E18, this













GCE1 nacZ


0]l


INll




GCo) l
El 8.5 [t


GCE7


0N


S ZacZ


GCE8


0N


Figure 3-3. Cross-sections of retinas containing human GC1 promoter-nlacZ
transgenes. A schematic of the lentiviral transgenes used for injections is
depicted above each panel (refer to Fig. 3-1B for details). X-gal staining is red
(nlacZ-positive cells) and DAPI staining is blue (cell nuclei). ONL = outer
nuclear layer; INL = inner nuclear layer; GCL = ganglion cell layer; red
ellipses = cone-rod homeobox protein binding element; orange triangle =
putative TATA box; red arrow = GC1 translation start site; blue arrow = nlacZ
translation start site.


nlacZ


E18.5


E18.5


El 8.5


I E18.5 I









activity was restricted to the ONL. Overall, the intensity of X-gal staining was

significantly lower in GCE8 retinas than in GCE7 retinas. These results suggest that

expression driven by the -386/-1745 region of the GC 1 promoter is augmented by the

presence of intron 1 (compare GCE7 and GCE8, Fig. 3-3).

CRX is a photoreceptor-specific transcription factor that plays an important role in

regulating the expression of several photoreceptor-specific and pineal-specific genes,

including itself (Furukawa et al. 2002;Livesey et al. 2000). It also appears to have an

important role in regulating photoreceptor development (Chen et al. 1997;Furukawa et al.

2002;Furukawa et al. 1997;Furukawa et al. 1999). Comparisons of the results obtained

for all three GC1 promoter fragments suggest that the two consensus CBEs identified in

the proximal and distal regions of the promoter may be required for directing expression

of GC 1 to the photoreceptor cells in vivo. These results are consistent with studies

showing that the promoters of several photoreceptor-specific genes contain multiple

copies of CBEs (Livesey et al. 2000). Further experiments will be required to confirm the

importance of these and other cis-acting elements in regulating the activity of the GC 1

promoter.

The temporal expression profiles of the GCE7 and GCE8 promoter fragments

revealed that these fragments did not possess the same temporal expression pattern as that

exhibited by the intact GC1 gene in developing retina. One plausible explanation for our

observations is that the endogenous GC1 transcription onset in developing retina occurs

earlier than E14. Use of more sensitive detection methods, such as RT-PCR, may show

that GC1 gene transcription does begin at an earlier stage of development. It is unclear

why the activities of the GCE7 and GCE8 promoter fragments are restricted to the ONL









at E18.5, but not at earlier stages of development. The cellular specificity of GC1

expression has not been examined in the developing retina. One possible explanation for

our observations is that the cis-elements required to silence GC 1 expression in non-

photoreceptor cells early in development are not present in these fragments. Another

possibility is that the expression of the human GC1 promoter is not regulated in a normal

manner in the avian retina. Finally, it is possible that the GC1 gene is expressed in all

retinal cells early in development of the retina and its expression becomes more restricted

over the course of development so that its expression is limited to photoreceptor cells in

the late stages of development.

In summary, the results from these analyses show that the GC1 promoter contains distinct

elements that drive its activity, control its expression during retinal development, and

limit its expression to specific retinal cell types in vivo. By conducting these analyses in

vivo, we have identified a GC1 promoter fragment, GCE7, which possesses expression

characteristics that should be suitable for use in our future efforts to drive express

lentiviral GC1 transgenes in chicken retina.














CHAPTER 4
IMPROVEMENTS IN THE DESIGN AND PRODUCTION OF HIV-1-BASED
LENTIVIRAL VECTORS RESULTS IN HIGH TRANSDUCTION EFFICIENCY IN
RETINA AND THE EFFICIENT EXPRESSION OF A RETINAL GUANYLATE
CYCLASE-1 (GC1) TRANSGENE

Note

The work presented in this chapter was published as part of a research article that

appeared in Physiological Genomics 12, 221-228 (2002). The guanylate cyclase activity

assays were performed by Izabela Sokal in the laboratory of Dr. Krzysztof Palczewski.

Introduction

Lentiviral vectors derived from the human immunodeficiency virus type 1 (HIV-1)

are emerging as the vectors of choice for long-term, stable in vitro and in vivo gene

transfer. These vectors are attractive because they can carry large transgenes (up to 18 kb

in size) (Kumar et al. 2001) and they are capable of stably transducing both dividing and

quiescent cells (Iwakuma et al. 1999;Miyoshi et al. 1998;Zufferey et al. 1998).

The increase in interest in these vectors has given rise to a need for efficient and

reproducible methods to produce large quantities of high-titer lentiviral vector.

Traditionally, lentiviral vectors are produced by co-transfecting human cell lines with

plasmid DNAs that encode the viral components required for viral packaging. Transient

transfection of these cell lines is often accomplished using the conventional calcium

phosphate co-precipitation technique (Naldini et al. 1996). Disadvantages of this method

include: (1) the large amount of plasmid DNA that is required for transfection; (2) the

difficulties associated with scaling up the precipitation reaction; and (3) the high degree









of variability observed in transfection efficiency and viral production. Recently, several

groups have developed packaging cell lines that facilitate the production of lentiviral

vectors by reducing the need for multi-plasmid transfections (Farson et al. 2001;Klages et

al. 2000;Pacchia et al. 2001;Xu et al. 2001). Although the use of packaging cell lines has

streamlined the packaging procedure, the resulting viral titers have not been significantly

higher than those obtained using transient co-transfection methods. In addition, the

advantages of these new cell lines are often offset by the need to develop new lines for

each generation of improved lentiviral vector.

To achieve large-scale production of high-titer lentiviral vector it is critical that

transfection of the virus-producing cell cultures be both efficient and reproducible;

however, little effort has been made to optimize this step in vector production. The results

from the experiments presented in Chapters 2 and 3 demonstrate that lentiviral vectors

transduce cells in developing retina, but improvements to the vector system and

production methods would make this system more suitable for our future studies in the

GUCY1*B chicken model of LCA1. The goals of the experiments described here were

(1) to design and produce lentiviral vectors that exhibit high transduction efficiency in

developing chicken retina and (2) to construct a vector that produces active guanylate

cyclase-1 (GC1) and can be used as a base vector to develop therapeutic vectors for gene

therapy studies aimed at treating LCA1. We were able to accomplish our first goal by

combining a transfection method that utilizes the activated dendrimer-based transfection

reagent, Superfect, with a novel vector concentration protocol. By using our new method,

we were able to reproducibly generate lentiviral vector stocks with titers greater than 1 x

1010 transducing units per ml (TU/ml) using less than one-third of the total amount of









plasmid DNA that is commonly required for production of this vector To achieve our

second goal, we constructed a modular lentiviral vector system that encodes functional

GC 1 and carries a multiple cloning site that facilitates the interchange of transgene

components into the vector.

Materials and Methods

Lentiviral Vector Constructs

The transducing vector used in our experiments was derived from a previously

described self-inactivating vector (Cui et al. 1999;Iwakuma et al. 1999). The pTY vector

was modified by inserting a cPPT-DNA FLAP element upstream of the multiple cloning

site, an element that has been shown to significantly improve the transduction efficiency

of recombinant lentiviral vectors in vitro and in vivo (Follenzi et al. 2000;Zennou et al.

2001). All polymerase chain reaction (PCR) products used for cloning as described below

were amplified using Pfu high-fidelity DNA polymerase and cloned into intermediate

pTOPO-BluntII vectors (Invitrogen) for use in subsequent steps. pTYF.linker: A 186-bp

fragment containing the cPPT-DNA FLAP sequence was amplified from the pNHP

vector using core primers that have been previously described (Zennou et al. 2000). Eag]

and Not] linkers were added to the sense and antisense primers, respectively. The

resulting fragment was excised with Not] and Eag] and cloned into the Not] site of the

pTY vector in the sense orientation, creating the pTYF.linker vector (Fig. 4-1 A). The

integrity of this modification was verified by DNA sequencing. pTYF.EF lalinker: The

human elongation factor- la (EFla) promoter was amplified from pTY.EFGFP (Zaiss et

al. 2002) using sense and antisense primers containing NotI and Nhel linkers,

respectively. The EFla promoter was excised using Notl and Nhel and then cloned into









the Not] and Nhel sites of pTYF-linker thereby generating the pTYF.EF la.linker vector

(Fig. 4-1 B). pTYF.EFIaPLAP: The placental alkaline phosphatase (PLAP) reporter

gene was amplified from pRISAP (Chen et al. 1999) (gift from C. Cepko) with sense and

antisense primers containing Pmel and Kpnl linkers, respectively. The PLAP

Pmel/Kpnl fragment was then cloned into the Smal/Kpnl sites of pTYF.EFla.linker to

make the pTYF.EFla.PLAP vector (Fig. 4-2). pTYF.EFla_IRES.EGFP: The polio

virus internal ribosome entry site (IRES) was obtained from pTYAT.CBAIRES.EGFP

(gift from A. Timmers) using sense and antisense primers containing Smal-Clal and

Mlul linkers, respectively. The cDNA encoding enhanced green fluorescent protein

(EGFP) was amplified from pEGFP-N1 (Clontech) using sense and antisense primers

with Kpn]-EcoRV and Mlul linkers, respectively. The IRES and EGFP fragments were

excised using Smal/Mlul and Mlul/Kpnl and ligated into the Smal and Kpnl sites of

pTYF.EFla.linker, resulting in the pTYF.EFlaIRES.EGFP cloning vector (Fig. 4-1 C).

pTYF.mlRBP1783.bGCI: First, a pTYF-mIRBP 1783-linker vector was generated by

excising the mIRBP1783 promoter from pTYF-mIRBP1783-nlacZ with Not] and Pmel.

The fragment was then cloned into the Notl/Smal sites of the pTYF.linker vector. The

cDNA encoding bovine GC1 was amplified from the pSVL GC1 clone using the following

primers: sense 5'-CCA TCG ATA GTT TAA ACG AGC CCC GGA CTT; antisense 5'-CCA

TCG ATG ACC CAG CCT CAC TTC C. The resulting fragment was cloned into pTYF-

mIRBP 1783-linker using Clal. The amplified bovine cDNA, which included the entire open

reading frame (ORF), extends from nucleotide 26 to nucleotide 3393 (GenBank L37089).

pTYF.EFlaobGCI-IRES-EGFP: The bGC1 ORF was excised with Clal and cloned

into the Clal site of the pTYF.EFl IRES-EGFP vector. pTYF.GCE7.bGCI-IRES-













EGFP: The GCE7 promoter was excised from pTYF-GCE7-nlacZ (see Chapter 3) using


Not] and Pmel and cloned into the Notl/Pmel site of pTYF.EFla..bGC1-IRES-EGFP.


Transfection-grade DNA was prepared using endotoxin-free DNA mega- or maxiprep


kits (Qiagen).


/CMV/TATA)-TAR (B-274bp)

A d.,Ra'i 24 pi
ag fllprmal) (li-IOOBbp)




Amp gl1gl ..) pTYF-linker
mp|Biar uxb) 7489 bp R l
RRE (18734110bp)

SCPPT- FLAP (233n-0aa5bp


\ II, MCS

hulle lai --
din IM 2ll7MbDr
Wo1 (3q57 dl.R (2708-21S3bp]
bGHpA(2S37-1Sbp)


A//TATATATR 74hp
B 2


\ pS ?1J06lbp)0

Amp tp)lE l2110bp)
pTYF.EFlca -
uv-ta) 8922 bp rriAur mra3n3bp)



SEFla


dlJU3RbGHpA(41024465bp) MG.J

s -il MCS

Pl(
\ p]I(wq)


sl'~llg


Aii6&65l ,
dI.UR bCHpA(PSOGflbp

UKV

I CMV-TATATR(9 -2T4bp)

.AIN.4 I
,-. i -


5' LTR





E11LAN


EFla
r '\ -1

MCS
,t-"-i ,, MCS

polo IRES (40004D bp)
.GFP (4-6Fi74bp)


3' SIN LTR
111I


];I


- M -E


Figure 4-1. A. C. Maps of the modular cloning plasmid vectors constructed for the SIN
lentiviral vector system used in this study. Note the extensive listing of unique
cloning sites. D. Schematics of bovine GC1 expression cassettes cloned into
pTYF-based vectors. The black arrow indicates the transcription start point.


Lentiviral Vector Production, Concentration and Titers


VSV-G-pseudotyped lentiviruses carrying an EFla-PLAP transgene were prepared


using the lentiviral vector system illustrated in Fig. 2. 293T cells (Invitrogen Corporation,


--Ilbovnr ,I1e I


r.. cjd


_Ir- wwm









#R70007) were seeded in 75 cm2 (T-75) culture flasks at a density of 1 x 107 cells per

flask and grown in Dulbecco's modified Eagle medium (DMEM; Gibco) containing 10%

fetal bovine serum and antibiotics (130 U/ml penicillin and 130 Ltg/ml streptomycin;

growth medium). The cultures were maintained at 370C in 5% CO2 throughout the virus

production period. On the following day, when the cultures reached 90-95% confluency,

the growth medium was replaced with 5.0 ml of fresh medium.

CMV-TATA/TAR SVo40 pA
pNHP gag RREI
(packaging vector) R t -rev-__a
RSV SD

cPPT-DNA FLAP
5' LTR I 3' LTR
pTYF T RRE
pTYF CMV-IE IR U5 R PLAP pA
(transducing vector) ASD pp AATAAA


pHEF-VSVG
(envelope vector)


SV40 pA
I
VSV-G


Figure 4-2. The HIV-1-based self-inactivating lentiviral vector system. The helper
construct, pNHP, contains deletions in the regions encoding the accessory
proteins vif, vpr, vpu and nef and has been previously described (Zaiss et al.
2002). The self-inactivating transducing construct, pTYF, has a central
polypurine tract (cPPT)-DNA flap element located just upstream of the
multiple cloning site and carries an EFla-PLAP transgene. The packaging
construct, pHEF.VSVG, encodes the vesicular stomatitis virus G (VSV-G)
glycoprotein for pseudotyping (Chang et al. 1999). The pTYF.EFla.PLAP
construct was used to produce vector for the in vitro and in vivo experiments
unless stated otherwise.

For one large-scale preparation of virus, 20 T-75 flasks of 293T cells were

transfected as follows: Transfection mixture for all 20 flasks was prepared by gently

mixing 142 Ltg pNHP, 70 Ltg pTYF and 56 Ltg pHEF.VSVG plasmid DNA and 8.0 ml

DMEM in one 50 ml polystyrene tube. After mixing, 560 pl of Superfect was added to









the DNA solution. The contents of the tube were gently mixed and incubated at room

temperature for 10 min. Next, 430 [tl of the Superfect-DNA mixture was added dropwise

to the T-75 flask (transfection start point) and the flask was incubated for 4-5 h.

Following the incubation period, the medium containing the transfection mixture was

replaced with 7.0 ml of fresh growth medium. The next day, the media containing the

first batch of virus was harvested from each flask and 6.5 ml of fresh growth medium was

added to the cells. Upon collection, all virus-containing media was filtered through a 0.45

|tm low protein-binding Durapore filter (Millipore) to remove cell debris. To prepare

transfection mixture sufficient for one T-75 flask, the amounts of DNA, DMEM and

Superfect were each divided by 20 to scale the reaction down. We have also found that

viral vector can be produced in larger or smaller cell culture flasks or plates by simply

scaling cell numbers and the amount of DNA, DMEM and Superfect linearly with respect

to the cell growth area.

For some experiments, virus-containing media was concentrated using

ultrafiltration and centrifugation as outlined in Diagram 1. For ultrafiltration, the virus

stock collected from 20 T-75 flasks at 30 h post-transfection (-120 ml) was divided into

two 60 ml aliquots and centrifuged through Centricon-80 ultrafiltration columns

(Millipore) for 1 h in 40C at 2,500 x g. The retentate was retrieved by centrifuging the

inverted column for 1 min in 40C at 990 x g and was stored at 40C until further

processing. On the following day, the virus-containing retentate was added to the -120

ml of virus-containing media collected at 45 h post-transfection. Four 30 ml conical-

bottom tubes (polyallomer Konical tubes; Beckman), each containing a 220 [l cushion of

60% iodixanol solution (used directly from the Optiprep stock solution obtained from









Axis-Shield) were prepared. Iodixanol was used because of its demonstrated safety in

human clinical trials (Jorgensen et al. 1992). Media containing virus (30 ml) was gently

pipetted into each tube, taking care not to disturb the iodixanol, and the samples were

centrifuged at 50,000 x g for 2.5 h at 40C using a Beckman SW-28 swinging bucket rotor.

The media just above the media/iodixanol interface was carefully removed from each

tube and discarded, leaving -750 [tl of the solution in each tube (220 [tl of iodixanol plus

-500 [tl of media). The residual media containing virus and the iodixanol were mixed

gently by shaking at 200 r.p.m for 2-3 h at 40C. The resulting mixtures were pooled into

one 3 ml conical-bottom tube (polyallomer Konical Tubes; Beckman) and centrifuged at

6100 x g for 22-24 h at 40C using a Beckman SW-50.1 swinging bucket rotor. The

resulting supernatant was removed and discarded and the remaining pellet was

resuspended in 50 tl of PBS or artificial cerebrospinal fluid by incubating the virus at

4C for 10-14 h. The final viral vector was gently mixed by pipetting, aliquoted and

stored at -800C until use.

Infectious titers of the TYF.EF 1 .PLAP virus were determined by incubating 1.75

x 105 TE671 cells seeded in 12-well plates with limiting dilutions of the viral stock (1/10,

1/100 and 1/1000) in the presence of 8 [tg/ml polybrene. After a 4-5 h incubation period,

fresh medium was added directly to the cells and, after 48 h, cultures were fixed, rinsed

in PBS, heated in PBS at 650C for 30 min and stained for PLAP activity using previously

reported methods (Fekete and Cepko 1993). The number of transducing units (TU;

defined as an infectious particle) was determined by estimating the number of PLAP-

positive cells per well and final infectious titers were expressed as TU/ml. Estimates of

the infectious titers of vectors lacking a strong promoter or the PLAP marker gene were









based on the titers of unconcentrated TYF.EF 1 .PLAP virus that was produced in

parallel each time.

Delivery of EFloc-PLAP Vector to Chicken Neural Tube

The neural tube injections and preparation of retinal flat mounts were carried out

using the methods described in Chapter 2 (Coleman et al. 2002). The brains of injected

embryos were fixed overnight in 4% paraformaldehyde at 40C. The next day, the tissues

were rinsed thoroughly in PBS and 100 |tm thick sections were cut using a vibratome.

Floating brain sections and retinal flat mounts were subsequently processed for routine

PLAP histochemistry using the techniques described above and as described at

http://genetics.med.harvard.edu/-cepko/protocol/xgalplap-stain.htm. All tissues were

collected on embryonic day 7 (E7) or 2 days post-hatch, 5 or 23 days after injections,

respectively. Digital images of retinal flat mounts were captured with a Nikon Coolpix

995 camera fitted to a Zeiss Stemi V6 microscope. In some cases, the percent area of

retina transduced by the vector was determined as follows: TIFF images at a resolution of

1024 x 768 pixels were reduced by 35%, converted to grayscale using Adobe Photoshop

and imported into the Scion Image program (available at http://www.scioncorp.com). The

density slice setting was used to select all of the pixels within the area of the flat mount

that represented PLAP-positive areas and these were expressed as a percent of the total

retinal area. Three to seven retinas were analyzed for each dose of vector.

Analyses of GC1 Expression Vectors

GC1 immunocytochemistry was performed on dispersed primary chicken retinal

cultures and DF-1 cells (immortalized chicken fibroblast cells; obtained from American









Tissue Culture Company) that were transiently transfected with the pTYF-

IRBP 1783.bGC1 or pTYF.EF 1 a.bGC1/EGFP plasmid vectors, respectively.

Primary retinal cultures

The preparation, maintenance and transient transfection of the primary retinal

cultures were performed as described under the Methods section in Chapter 2 with the

exception that the cells were grown on glass coverslips coated with poly-D-ornithine.

DF-1 cell cultures

On the day prior to transfection, DF-1 cells were seeded into wells of 12-well plates

that contained tissue culture-treated glass coverslips (Fisherbrand) and maintained in

culture media as described above. Briefly, 3 |tg DNA was added to 50 [tl plain DMEM

and mixed with 10 [l Superfect and incubated at RT for 10 min. While the DNA-

Superfect mixture was incubating, the culture media was removed from the DF-1 cells

and replaced with 0.5 ml fresh media. The transfection mixture (25 tl) was then added to

each well and incubated at 370C and 5% CO2 for 4-5 hrs. Following the incubation

period, fresh media was added to each well and replaced one time on the following day.

Immunocytochemistry and fluorescence microscopy

Forty-eight hours after transfection, both the primary retinal cultures and DF-1 cells

were fixed using 4% paraformaldehyde for 5 min at RT, rinsed three times in PBS and

processed for immunocytochemistry as follows. The cells were first blocked in PBS

containing 10% goat serum for 30 min at RT. The cells were then incubated overnight at

4C with a GC1 polyclonal antibody (1/333 dilution in PBS containing 1.0% BSA and

0.3% Triton X-100; GC2, gift from A. Yamazaki). On the following day, the cells were

rinsed three times for 15 min each and then incubated for 1 h at RT with a goat anti-









mouse secondary antibody (1/500 dilution in PBS) tagged with the Alexa-594

fluorophore (Molecular Probes). The cells were subsequently rinsed three times for 15

min each and counterstained with DAPI. The coverslips were carefully removed from the

wells and mounted in Gel Mount (Biomedia) on glass slides. The stained cells and/or

direct GFP fluorescence were viewed using the appropriate fluorescent filter sets and

digital images were acquired using a SPOT2 Enhanced Digital Camera System mounted

in a Zeiss Axioplan 2 fluorescence microscope.

Generation of stably transduced cell lines

TE671 cells were seeded into the wells of a 24-well culture plate and grown

overnight at 37C, 5% CO2. On the following day, 300 ml of fresh media was added to the

wells and TYF.EFla IRES.EGFP, TYF.EFla.bGC1/EGFP or TYF.GCE7.bGC1/EGFP

virus was added to the media at an MOI of 5. After 24 hours, the cells were seeded into

T-25 flasks and maintained by passaging two times a week.

GC1 activity assays

GC activity was measured in washed membrane fractions obtained from TE671

cells (-50 passages) and purified bovine rod outer segments (ROS) (150 |tg total protein).

The fractions were incubated for 15 min at 300C with 1.5 mM [a 32P]GTP (19,000-

22,000 dpm/nmol; DuPont NEN), 50 mM Hepes, pH 7.8, 60 mM KC1, 20 mM NaC1, 10

mM MgCl2, 0.4 mM EGTA, and either 1.0 [tM or 0.030 [tM free CaCl2 in the presence

or absence of GCAP1 protein (5 Gjg). The assays were repeated twice, each with similar

results.









Results

Lentivirus Production and Concentration

The goals of our first series of experiments were to determine the optimum ratio of

total plasmid DNA to Superfect reagent that produced the highest titer virus and the

optimum time for viral harvest. This ratio was determined to be 1:2 (ratios of 1:1, 1:1.5,

1:2, 1:5, and 1:10 were tested; data not shown). The titers of virus-containing media

harvested directly from transfected 293T cultures were determined 30, 45, 60, and 70

hours post-transfection to identify the timeframe during which virus production by these

cultures is at maximum levels (Fig. 4-3). The average titer values were 8.0 x 106, 6.8 x

106, 2.6 x 106 and 0.8 x106 TU/ml at 30, 45, 60 and 70 hours post-transfection,

respectively. Therefore, we collected culture media 30 and 45 hours post-transfection for

subsequent experiments. It should also be noted that 293T cells passage between 2 and

60 times were used for transfections and that passage number did not significantly affect

transfection efficiency or final vector titers.


10

E 8
I--
Co 6

o4

2

0
30 45 60 70
Hours post-transfection
Figure 4-3. Production of lentivirus by transfected 293T cells as a function of time.
VSV-G-pseudotyped lentiviruses carrying an EFla-PLAP transgene were
prepared using the lentiviral vector system illustrated in Fig. 4-2. Each bar
represents the mean titer SEM of unconcentrated virus-containing medium
collected at each time point (n = 3).










1. Harvest virus at 30 h post-transfection (-20 x 7.0 ml)

Day 1 -
2. Concentrate by ultrafiltration (2 x Centricon-80 units)

I
3. a. Harvest virus at 45 h post-transfection (-20 x 6.5 ml)
O b. Combine virus from step 2 and step 3a


Q)4. a. Overlay 30 ml virus onto 220 pl iodixanol (x 4 tubes)
SDay 2 b. Centrifuge at 50,000 x g for 2.5 h
0 a
5. a. Remove supernatant down to DMEM-iodixanol interface
b. Combine virus from 4 tubes (Step 4a) and add to 3 ml tube
c. Centrifuge at 6100 x g for 22-24 h

I
Day 3 6. Remove supernatant and add buffer to resuspend virus pellet to
L achieve an approximate 3000-fold volume change.

Approx. volume *
Titer (TU/ml) change fol Titer increase (fold) %Virus recovered
change (fold)
) Step 3b 1.40 0.35 x 107 n/a n/a n/a
I- Step5b 3.59 0.70 x10 40 33 4 84 9
SStep 6 1.40 0.44 x 1010 3000 958 191 40 8
*Mean SEM derived from 13 separate large-scale virus preparations
Figure 4-4. Outline and results of the vector production protocol. The top panel
shows a simplified flow diagram of the concentration procedure that is
described in detail under Methods. The bottom panel summarizes the viral
titer results obtained following each step of the concentration procedure.

The goal of our second series of experiments was to develop a concentration

protocol that would minimize virus loss and yield the highest titer virus in the smallest

possible volume. The concentration procedure and results are summarized in Fig. 4-4.

The average starting titer of the virus-containing media (Fig. 4-4, bottom panel, Steps 1-

3) was 1.40 + 0.35 x 107 TU/ml. The next step in the concentration procedure (Fig. 4-4,

bottom panel, Step 4) yielded an average titer of 3.59 0.70 x 108 TU/ml in a volume of









-3.0 ml, resulting in a 33-fold increase in titer and an average recovery of 84%. Further

concentration of the virus stock by low-speed centrifugation (Fig. 4-4, bottom panel,

Steps 5c and 6) yielded 1.40 + 0.44 x 1010 TU/ml, a 958-fold increase over the average

starting titer. The average overall percent recovery of the virus was 40%.

In vivo Performance of the Lentiviral Vector

Administration of -0.5 [tl of TYF.EFla.PLAP virus (1 x 1010 TU/ml) into the

chicken neural tube resulted in efficient transduction of large numbers of neural

progenitor cells (Fig. 4-5). Cross-sections of stained retinas revealed numerous PLAP-

positive cell columns (Fig. 4-5 D, bottom panel). Columns of PLAP-positive cells were

also observed throughout the developing brain (Fig. 4-5 E). We also examined the

relationship between viral dose and the percent of the retina transduced by the virus and

determined that the transduction efficiency of the virus in developing retina was dose-

dependent (Fig. 4-5 A-C). The percent of total retinal area exhibiting PLAP expression

was estimated to be 5%, 63% and 85% in embryos receiving injections of 108, 109 and

1010 TU/ml vector, respectively (Fig. 4-5 D, top panel). PLAP expression was maintained

in retinas from injected embryos that were examined 2 days after hatching, 21 days post-

injection (Fig. 4-6). The relationship between the amount of virus injected and the extent

of viral transduction was maintained in these retinas, the number of PLAP-expressing

cells being significantly less in embryos injected with 107 TU/ml virus (Fig. 4-6, left

panel) than embryos injected with 1010 TU/ml virus (Fig. 4-6, right panel).

GC1 Immunocytochemistry

A small percentage of cells in the primary GUCY1*B chicken retinal and DF-1

cultures transiently transfected with either the pTYF.IRBP1783.bGC1 or the

pTYF.EFla.bGC1/EGFP vector, respectively, stained positively for the GC1 protein









"D
100


i..


80

60

40

20


n=7


n=5
-T


108 109 1010
Titer of injected vector (TU/ml)


[1u


Figure 4-5. Lentiviral vector-mediated transduction of PLAP in chicken neural
progenitor cells. A. C. PLAP expression in representative flat mounts of E7
chicken retinas from embryos receiving injections of (A) 108, (B) 109 or (C)
1010 TU/ml virus. D. top panel: Histogram showing the quantification of the
percent area of PLAP-positive retina following injections of different doses of
vector. Bars represent the mean + SEM for each group (n = 3-7). bottom
panel: Cross-section of the retina shown in C. E. Cross-section showing
PLAP-positive cells in the lateral anterior cortex of an E7 embryo that had
received a neural tube injection of 1010 TU/ml virus.
(Fig. 4-7). A limited number of cells stained positive for GC1 in the two culture systems,

which was consistent with the respective transfection efficiencies usually achieved in

these cells and suggested that the staining was specific. GC1 expression driven by the


L


.,,. BRT,,









TYF. E F1 a.PLAP


107 TU/ml


1010 TU/ml


Figure 4-6. PLAP expression in post-hatch chicken retinas. Embryos were injected
with different doses of virus at stage 10-12 (neural tube). The retinas were
processed for PLAP staining 2 days after hatching, 21 days after the
injections. The bottom panels show close-ups of areas from the retinas
pictured in the corresponding top panels. Scale bars = 2 mm.

IRBP1783 promoter was limited to cells exhibiting photoreceptor cell morphology in the

GUCY1*B retinal cultures (Fig. 4-7, top panel) and was limited to the membrane

surrounding the nucleus and to the apical ends of the cells that eventually differentiate

into the outer segments. GC 1 immunostaining of the DF-1 cells was present throughout

the cell body and its processes and co-localized with GFP fluorescence, a result that

indicates that both cistrons of the bicistronic EFl a-bGC1-IRES-EGFP transgene were

expressed (Fig. 4-7, bottom panel). We were unable to determine from these analyses if

the GC1-labeled protein within the soma was associated with the cell membrane.










___


Figure 4-7. Expression of recombinant bovine GC1 in avian-derived retinal
photoreceptor cells (top panel) and DF-1 fibroblast cells (bottom panel).
Bovine GC1 protein is labeled red and the green in the bottom panel is
intrinsic GFP fluorescence. Cells were transfected with the plasmid vectors
illustrated above each panel as described in the Methods section of this
chapter. Scale bars = 5 |tm

GC1 Enzyme Activity

To assess the activity of the GC 1 enzyme produced from the

pTYF.EFla.bGC1/EGFP and pTYF.GCE7.bGC1/EGFP expression vectors, TE671 cells

were first transiently transfected with the plasmid DNA and processed for GC 1 activity

analyses 48 h post-transfection. The results of these analyses are shown in Fig. 4-8 A.

Three samples were analyzed in this experiment: (1) bovine ROS membranes; (2) mock-

transfected TE671 cells; TE671 cells transfected with the (3) pTYF.EFlau.bGC1/EGFP

vector or the (4) pTYF.GCE7.bGC1/EGFP vector. The activity of the GC1 enzyme in


I M miBP73 ovneGC









each sample was assayed under low and high calcium conditions in the presence and

absence of GCAP1 protein. The results of these analyses show that the GC1 enzyme

produced by our vector exhibits activity characteristics closely resembling those of the

native GC1 enzyme present in bovine ROS. The activity of the GC1 enzyme in the

TE671 cells dramatically increased when calcium levels were reduced in the presence of

GCAP1 protein.

The function of the bicistronic transgene within the pTYF.EF cl.bGC1/EGFP

vector was also assessed in TE671 cells that were transduced with virus made from this

construct. The results indicate that the virus is capable of stably expressing the GC1

transgene and that the bicistronic transgene efficiently expressed functional GC1 (Fig. 4-

8A) in conjunction with the GFP marker protein (Fig. 4-8B). Together, these results show

that the pTYF-based constructs are suitable for use as the backbone for the final

therapeutic lentiviral vectors that will be used to express functional GC1 in the

GUCY1*B retina.

Discussion

By optimizing both the DNA transfection and viral concentration steps for

production of lentiviral vector, we have overcome many of the problems that we had

previously encountered in our efforts to produce large volumes of high-titer lentiviral

vector in a consistent manner. We found that Superfect-mediated transfection of viral

packaging cells consistently yielded large-scale vector stocks (-120 ml) with starting

titers averaging >1.0 x 107 TU/ml, titers that were comparable to vector stocks prepared

using other transfection reagents. Use of Superfect greatly simplified the transfection

protocol and significantly reduced the amount of plasmid DNA required for the






69



A
0.5 virus
I GC1
S0.4 GC1 +GCAP1; owCa2+
l GC1 + GCAP1; high Ca2+
(D
o 0.3
E
0.2
transfection

0.1

0.0







B














SIN LTR SIN LTR


Figure 4-8. Expression of recombinant bovine GC1 from transiently transfected
transgenes and transgenes packaged into the lentiviral vectors. A.
Histogram showing the GCAP- and calcium-dependent enzymatic activity of
GC1 in vitro. TE671 cells were transfected with plasmid or transduced with
virus as described in the Methods section. B. GFP fluorescence in TE671 cells
transduced with TYF.EF 1c.bGC1/EGFP virus and schematic of the integrated
transgene. This image coupled with the data shown in A demonstrates that the
bi-cistronic transgene is functional when packaged into the lentivirus.









procedure. The viral concentration protocol that we developed consistently increased the

titers of the viruses by approximately 1000-fold (-1 x 1010 TU/ml). Furthermore, all

vectors that we produced using these methods exhibited high transduction efficiencies in

vivo.

One of the goals of this study was to produce viral stocks that could be used to

transduce a high percentage of cells in the retina following delivery of lentiviral vector

into the neural tube of the developing chicken embryo. The injected virus transduced

several populations of neural progenitor cells, including those fated to become the neural

retina (Figs. 4-5 and 4-6). A majority of cells exposed to virus during this stage of

development are mitotic and have not yet differentiated (Prada et al. 1991). By varying

the concentration of the virus injected, we found that the percent of retina transduced

could be controlled in a linear fashion using does between 108 and 109 TU/ml. Injections

of virus at a concentration of 1010 TU/ml produced maximal levels of retinal transduction.

In the previous chapters, we showed that it is possible to specifically target lentiviral

vector-mediated expression of transgenes to retinal photoreceptor cells by selecting

appropriate promoter fragments. Together, these results illustrate the effectiveness of our

vector to transduce cells within the developing nervous system and illustrate the potential

use of this vector as a tool for studies of mechanisms regulating gene expression in vivo.

We also demonstrate that efficient expression of the EFl c-PLAP transgene persists in the

fully developed retina and that the vector is well-suited for use in our future studies of

GC1 expression in the GUCY1*B chicken model LCA1.

In summary, the transfection and concentration protocols outlined here allow

efficient, reproducible production of high-titer lentiviral vectors that exhibit robust






71


transduction properties in vivo. The transfection protocol itself is simple and can be easily

implemented by investigators interested in producing lentiviral vector in their

laboratories. Furthermore, the methods can be easily adapted to large-scale lentiviral

production protocols that are currently being developed for use in large animal studies or

for possible use in clinical studies.














CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS

The results presented in Chapters 2 and 3 demonstrate that lentiviral vectors are

capable of efficiently transducing avian neural progenitor cells and that the promoters of

photoreceptor cell-specific genes can be incorporated into these vectors to achieve

targeted transgene expression in vivo. The improvements made to the production and

concentration protocols used to generate lentiviral vectors as described in Chapter 4 will

facilitate the use of these vectors in vivo. In addition, we have successfully developed and

tested a bicistronic lentiviral vector construct that is capable of mediating the expression

of functional retinal guanylate cyclase-1 (GC 1). Together, these results lay the

groundwork for future studies of somatic gene therapy in the GUCY1 *B chicken model

of Leber congenital amaurosis type 1 (LCA1).

Targeted Gene Expression in Retina

Overall, the results from the experiments presented in Chapters 2 and 3 show that

the GCE7 or GCAP4009 promoter fragments are suitable for driving the cell-specific

expression of a GC1 transgene in retina. These results also lead to more specific

questions regarding the mechanisms that control the onset of expression and the cell-

specific regulation of these promoters in the developing retina. The experimental

paradigm presented in Chapters 2 and 3 could be extended to help answer the following

questions that are relevant to future studies of gene rescue in the GUCY1 *B chicken: (1)

Do the GCAP1 and GC1 promoters exhibit cone-specific or cone/rod-specific

expression? (2) What are the intrinsic activity levels exhibited by the GCAP1 and GC1









promoters in vivo? (3) What cell types express GC1 during retinal development and is

there a switch in the cellular specificity of its expression?

Understanding the photoreceptor subtypes in which the GCAP1 and GC1

promoters are expressed is relevant to our efforts to rescue retinal function in the

GUCY1*B chicken. The results of immunohistochemical analyses show that GCAP1 and

GC1 are present in higher concentrations in cone cells than in rod cells (Cooper et al.

1995;Liu et al. 1994). Furthermore, clinical studies provide evidence that cone cells may

be more dependent on GCAP 1 and GC 1 in terms of survival and function. For example,

patients diagnosed with retinal diseases that are linked to mutations in GCAP1 and GC1

exhibit phenotypes (e.g. diminished cone cell electroretinograms) and behaviors (e.g.

photophobic behavior and decreased visual acuity) that are indicative of compromised

cone cell function (Milam et al. 2003;Perrault et al. 1999;Wilkie et al. 2001). Thus,

successful treatment of diseases like LCA1 may depend to some extent on our abilities to

insure that cone cells are included in the target cell population.

The precise cellular specificities of the GCAP1 and GC 1 promoters could be

determined by performing co-localization studies using antibodies specific for nlacZ and

for cone (iodopsin) and rod (rhodopsin) specific markers. A reasonable approach would

be to perform the immunocytochemical analyses on dispersed primary retinal cultures

that have been prepared from the retinas of embryos that received injections of the

various promoter-nlacZ lentiviral vectors. We have found that accurate identification of

rod and cone cells expressing reporter genes is facilitated by use of dispersed cultures.

Preliminary data obtained from this type of analysis are shown in Fig. 5-1.











Uoopi




6666si




Uhdpi


Fig. 5-1. Immunolabeling of primary embryonic chicken retinal cultures with cone
(anti-iodopsin) and rod cell markers (anti-rhodopsin). Cultures were
transfected with a mIRBP1783-GFP plasmid vector and the preparation and
transfection of the cultures was performed as described in Chapter 2.
In addition to cell-specificity, it is also important to examine the levels of
transcriptional activity exhibited by promoters when developing gene therapy strategies..
Strong, ubiquitous promoters are generally used in experimental gene delivery systems
because they are readily available and usually guarantee high levels of expression in
many cell types; however, abnormally high levels of expression of therapeutic genes in
targeted cells may have deleterious effects on the function of these cells. For example, it
has been suggested that over expression of guanylate cyclase-1 (GC 1) may result in
protein aggregation and/or can interfere with proper trafficking of GC 1 to its position in
the membrane, both of which may be detrimental to photoreceptor cells (Rozet et al.
2001). The over expression of GC1 in photoreceptor cells could also interfere with proper
regulation of the catalytic activity of this enzyme by GCAP Therefore, we have put


Vk
iodopsin+GFP


AW,
iodopsin+GFP




rhodopsin+GFP









considerable effort into identification of different promoters that may provide optimal

levels of expression of GC 1 in photoreceptors. It should be noted that levels of GC 1 as

low as 50% of that present in wild-type retina are sufficient to sustain photoreceptor

survival and function in the chicken retina (Semple-Rowland et al. 1998).

To assess the intrinsic activity levels of selected GCAP1 and GC1 promoter

fragments, the activities of these fragments could be analyzed in vivo using the methods

described in Chapter 2. Comparisons of the results obtained from these experiments with

those obtained in vitro would provide a more detailed picture of the intrinsic activities of

these promoters.

Finally, since we plan to introduce the virus during the early stages of embryonic

development, it is important to understand the expression characteristics of the selected

promoters in developing retina. In our analyses of the GCAP 1 promoter, we found that

inclusion of the distal region of the GCAP1 5' flanking region in the promoter fragment

resulted in delayed, but specific expression of nlacZ in photoreceptor cells (Fig. 2-4). In

contrast, the cell-specificity of expression of GC 1 promoter fragments changed over the

course of development, expression being limited to photoreceptor cells during the later

stages of development. Studies are currently planned to determine if the absence of

expression of the GC 1 promoters in non-photoreceptor cells late in development is due to

silencing of expression of the transgene in retinal cells within the inner nuclear and

ganglion cell layers. Use of laser capture dissection techniques will allow us to excise

groups of cells from the INL that are positioned in columns marked by nlacZ-positive

cells positioned in the ONL. The cells will then be genotyped to confirm the presence or

absence of the integrated transgene. The presence of the transgene in the absence of









reporter expression would be consistent with the hypothesis that the promoter is actively

silenced in these cells.

Lentiviral Vector Transduction in Retina

One of the long-term research goals of this project is to determine if vision can be

restored in hatchling GUCY1*B chickens by delivering a lentiviral GC1 transgene to the

retinal progenitor cells of these animals. We chose to use the lentiviral vector system

because it circumvents some of the limitations of other vector systems. For example, the

size of the GC1 transgene that we plan to use in these studies exceeds the cargo capacity

of traditional recombinant AAV (rAAV) vectors, a problem that is not one that arises

when using lentiviral vectors. Another issue of importance concerns the time required for

transgenes to reach maximum levels of expression. Transgenes carried by lentiviral

vectors begin to express and reach maximum expression levels more rapidly (within 3

days) than those carried by rAAV (within 2-4 weeks) (Sarra et al. 2002).

To rescue the function of GC 1-null photoreceptors in the retina and restore vision,

it is desirable to be able to transduce a high percentage of these cells with the therapeutic

vector. Our initial attempts to transduce chicken retinal progenitor cells with lentiviral

vector were disappointing in this regard, the number of retinal cells being transduced

representing less than 5% of the total population. In view of the potential impact that poor

transduction efficiency could have on the outcome of future gene rescue experiments,

much of our research effort was devoted to improving the performance of the lentiviral

vector in vivo. As described in Chapter 4, two changes were made to the system that

dramatically improved transduction efficiencies in vitro and in vivo. Because of these

efforts, we were able to produce viral vectors with titers ranging from 109 1010 TU/ml

(-100-fold increase over previous efforts) that were capable of transducing greater than









80% of the retinal cell population when injected into the neural tube. These modifications

should significantly improve the outcome of our future efforts to rescue sight in the

GUCY1*B chicken. In the future, DNA insulator elements from the chicken 3-globin

gene (Chung et al. 1997) could be added to the transducing vector, flanking the transgene

insert. Insulators have been shown to reduce variegation effects and to significantly

decrease transcriptional silencing in retroviral vector transgenes (Pannell and Ellis 2001).

This addition would contribute to a further gain in the biosafety and performance of our

lentiviral vector system in vivo.

Several recent advances in the biosafety and performance of lentiviral vector

systems are beginning to assuage concerns over use of these vectors in gene therapy

applications. Since I began my research program, three generations of lentiviral vectors

have been developed in efforts to improve the biosafety and performance of the virus

(Vigna and Naldini 2000). The third-generation vector system consists of a modified

helper vector that does not contain the Tat encoding region and a fourth expression vector

that encodes the Rev protein, a protein that enhances viral packaging. These vectors

could be readily incorporated into the second-generation system that we used in the

current studies. It may be prudent to utilize the third-generation packaging vector system

in our future studies of gene rescue in the GUCY1*B chicken.















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