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EFFICIENT TRANSDUCTION AND TARGETED EXPRESSION OF LENTIVIRAL
VECTOR TRANSGENES IN THE DEVELOPING RETINA
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
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.
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
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
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
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
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
Jason Edward Coleman
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
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).
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
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
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
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
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).
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
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
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.
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.
A 4.0 KB FRAGMENT OF THE GUANYLATE CYCLASE ACTIVATING PROTEIN-
1 (GCAP1) PROMOTER TARGETS GENE EXPRESSION TO PHOTORECEPTOR
CELLS IN THE DEVELOPING RETINA
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.
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
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
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
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
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.
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
9.5- 0 -. m agw GC1
1.4- I lodopsin
10 12 16 20
I I I I
IS OS ERG
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
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
II I z ucferas I
[- -D CE I luciferase I
[ E- I-- D E luciferase
0 0 -
SE 30 60 -o
t 20 40 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
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
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
E12 [ -[ E16
I- low or no activity -1 1434
I in vitro I 3121
I activity in ONL -E16
activity in INL~-E12 and 292
I inONL E16 1436
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
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.
IN VIVO ANALYSES OF THE DEVELOPMENTAL AND CELL-SPECIFIC
ACTIVITY OF THE HUMAN RETINAL GUANYLATE CYCLASE-1 (GC1)
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.
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).
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.
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
AGGCCGGGGT CTCAGTCGCT CAGCCTGCTC CGTCTGTGTT CGCAG
+ 1 I nlacZ
Exon 2 tttaaacTT AAGCTTCCAC CATGCCTAAG AAGAAACGAAAG
P K KK RK
I Human GC1 Gene
GCE7 t nz |
GCE1 In.z |
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).
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.
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
El 8.5 [t
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.
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
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.
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
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.
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
A d.,Ra'i 24 pi
ag fllprmal) (li-IOOBbp)
Amp gl1gl ..) pTYF-linker
mp|Biar uxb) 7489 bp R l
SCPPT- FLAP (233n-0aa5bp
\ II, MCS
hulle lai --
din IM 2ll7MbDr
Wo1 (3q57 dl.R (2708-21S3bp]
\ pS ?1J06lbp)0
Amp tp)lE l2110bp)
uv-ta) 8922 bp rriAur mra3n3bp)
s -il MCS
I CMV-TATATR(9 -2T4bp)
,-. i -
r '\ -1
,t-"-i ,, MCS
polo IRES (40004D bp)
3' SIN LTR
- 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
#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
5' LTR I 3' LTR
pTYF T RRE
pTYF CMV-IE IR U5 R PLAP pA
(transducing vector) ASD pp AATAAA
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
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.
30 45 60 70
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)
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
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
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
) 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).
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
108 109 1010
Titer of injected vector (TU/ml)
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
TYF. E F1 a.PLAP
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
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
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
S0.4 GC1 +GCAP1; owCa2+
l GC1 + GCAP1; high Ca2+
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
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
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.
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.
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
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
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.
LIST OF REFERENCES
Acland G. M., Aguirre G. D., Ray J., Zhang Q., Aleman T. S., Cideciyan A. V., Pearce-
Kelling S. E., Anand V., Zeng Y., Maguire A. M., Jacobson S. G., Hauswirth W.
W., and Bennett J. (2001) Gene therapy restores vision in a canine model of
childhood blindness. Nat Genet 28, 92-95.
Adler R., Magistretti P. J., Hyndman A. G., and Shoemaker W. J. (1982) Purification and
cytochemical identification of neuronal and non-neuronal cells in chick embryo
retina cultures. Dev Neurosci 5, 27-39.
Adler R., Tamres A., Bradford R. L., and Belecky-Adams T. L. (2001)
Microenvironmental regulation of visual pigment expression in the chick retina.
Dev Biol 236, 454-464.
Aiuti A., Slavin S., Aker M., Ficara F., Deola S., Mortellaro A., Morecki S., Andolfi G.,
Tabucchi A., Carlucci F., Marinello E., Cattaneo F., Vai S., Servida P., Miniero R.,
Roncarolo M. G., and Bordignon C. (2002) Correction of ADA-SCID by stem cell
gene therapy combined with nonmyeloablative conditioning. Science 296, 2410-
Akimoto M., Miyatake S., Kogishi J., Hangai M., Okazaki K., Takahashi J. C., Saiki M.,
Iwaki M., and Honda Y. (1999) Adenovirally expressed basic fibroblast growth
factor rescues photoreceptor cells in RCS rats. Invest Ophthalmol Vis Sci 40, 273-
Ali R. R., Reichel M. B., Kanuga N., Munro P. M., Alexander R. A., Clarke A. R.,
Luthert P. J., Bhattacharya S. S., and Hunt D. M. (1998) Absence of p53 delays
apoptotic photoreceptor cell death in the rds mouse. Curr Eye Res 17, 917-923.
Ali R. R., Sarra G. M., Stephens C., Alwis M. D., Bainbridge J. W., Munro P. M., Fauser
S., Reichel M. B., Kinnon C., Hunt D. M., Bhattacharya S. S., and Thrasher A. J.
(2000) Restoration of photoreceptor ultrastructure and function in retinal
degeneration slow mice by gene therapy. Nat Genet 25, 306-310.
Ameixa C. and Brickell P. M. (2000) Characterization of a chicken retinoid X receptor-
gamma gene promoter and identification of sequences that direct expression in
retinal cells. Biochem J347, 485-490.
Auricchio A., Kobinger G., Anand V., Hildinger M., O'Connor E., Maguire A. M.,
Wilson J. M., and Bennett J. (2001) Exchange of surface proteins impacts on viral
vector cellular specificity and transduction characteristics: the retina as a model.
Hum Mol Genet 10, 3075-3081.
Belteki G., Gertsenstein M., Ow D. W., and Nagy A. (2003) Site-specific cassette
exchange and germline transmission with mouse ES cells expressing phiC31
integrase. Nat Biotechnol 21, 321-324.
Bennett J., Duan D., Engelhardt J. F., and Maguire A. M. (1997) Real-time, noninvasive in
vivo assessment of adeno-associated virus-mediated retinal transduction. Invest
Ophthalmol Vis Sci 38, 2857-2863.
Bennett J., Maguire A. M., Cideciyan A. V., Schnell M., Glover E., Anand V., Aleman T.
S., Chirmule N., Gupta A. R., Huang Y., Gao G. P., Nyberg W. C., Tazelaar J.,
Hughes J., Wilson J. M., and Jacobson S. G. (1999) Stable transgene expression in
rod photoreceptors after recombinant adeno-associated virus-mediated gene
transfer to monkey retina. Proc NatlAcad Sci USA 96, 9920-9925.
Bennett J., Pakola S., Zeng Y., and Maguire A. (1996a) Humoral response after
administration of El-deleted adenoviruses: immune privilege of the subretinal
space. Hum Gene Ther 7, 1763-1769.
Bennett J., Tanabe T., Sun D., Zeng Y., Kjeldbye H., Gouras P., and Maguire A. M.
(1996b) Photoreceptor cell rescue in retinal degeneration (rd) mice by in vivo gene
therapy. Nat Med 2, 649-654.
Bennett J., Wilson J., Sun D., Forbes B., and Maguire A. (1994) Adenovirus vector-
mediated in vivo gene transfer into adult murine retina. Invest Ophthalmol Vis Sci
Bennett J., Zeng Y., Bajwa R., Klatt L., Li Y., and Maguire A. M. (1998) Adenovirus-
mediated delivery of rhodopsin-promoted bcl-2 results in a delay in photoreceptor
cell death in the rd/rd mouse. Gene Ther 5, 1156-1164.
Bessis A., Champtiaux N., Chatelin L., and Changeux J. P. (1997) The neuron-restrictive
silencer element: a dual enhancer/silencer crucial for patterned expression of a
nicotinic receptor gene in the brain. Proc Natl Acad Sci US A 94, 5906-5911.
Boatright J. H., Borst D. E., Peoples J. W., Bruno J., Edwards C. L., Si J. S., and
Nickerson J. M. (1997a) A major cis activator of the IRBP gene contains CRX-
binding and Ret-1/PCE-I elements. Mol Vis 3, 15.
Boatright J. H., Buono R., Bruno J., Lang R. K., Si J. S., Shinohara T., Peoples J. W., and
Nickerson J. M. (1997b) The 5' flanking regions of IRBP and arrestin have promoter
activity in primary embryonic chicken retina cell cultures. Exp Eye Res 64, 269-
Boatright J. H., Knox B. E., Jones K. M., Stodulkova E., Nguyen H. T., Padove S. A.,
Borst D. E., and Nickerson J. M. (2001) Evidence of a tissue-restricting DNA
regulatory element in the mouse IRBP promoter. FEBS Lett 504, 27-30.
Bobola N., Hirsch E., Albini A., Altruda F., Noonan D., and Ravazzolo R. (1995) A single
cis-acting element in a short promoter segment of the gene encoding the
interphotoreceptor retinoid-binding protein confers tissue-specific expression. J
Biol Chem 270, 1289-1294.
Brenner S. and Malech H. L. (2003) Current developments in the design of onco-
retrovirus and lentivirus vector systems for hematopoietic cell gene therapy.
Biochim Biophys Acta 1640, 1-24.
Brisson M. and Huang L. (1999) Liposomes: conquering the nuclear barrier. Curr Opin
Mol Ther 1, 140-146.
Bruhn S. L. and Cepko C. L. (1996) Development of the pattern of photoreceptors in the
chick retina. JNeurosci 16, 1430-1439.
Buchschacher G. L., Jr. and Wong-Staal F. (2000) Development of lentiviral vectors for
gene therapy for human diseases. Blood 95, 2499-2504.
Bushman F. D. (2002) Integration site selection by lentiviruses: biology and possible
control. Curr Top Microbiol Immunol 261, 165-177.
Cavazzana-Calvo M., Hacein-Bey S., de Saint B. G., Gross F., Yvon E., Nusbaum P.,
Selz F., Hue C., Certain S., Casanova J. L., Bousso P., Deist F. L., and Fischer A.
(2000) Gene therapy of human severe combined immunodeficiency (SCID)-X1
disease. Science 288, 669-672.
Cayouette M. and Gravel C. (1997) Adenovirus-mediated gene transfer of ciliary
neurotrophic factor can prevent photoreceptor degeneration in the retinal
degeneration (rd) mouse. Hum Gene Ther 8, 423-430.
Chang L. J., Urlacher V., Iwakuma T., Cui Y., and Zucali J. (1999) Efficacy and safety
analyses of a recombinant human immunodeficiency virus type 1 derived vector
system. Gene Ther 6, 715-728.
Chang M. A., Horner J. W., Conklin B. R., DePinho R. A., Bok D., and Zack D. J. (2000)
Tetracycline-inducible system for photoreceptor-specific gene expression. Invest
Ophthalmol Vis Sci 41, 4281-4287.
Chen C. M., Smith D. M., Peters M. A., Samson M. E., Zitz J., Tabin C. J., and Cepko C.
L. (1999) Production and design of more effective avian replication-incompetent
retroviral vectors. Dev Biol 214, 370-384.
Chen J., Tucker C. L., Woodford B., Szel A., Lem J., Gianella-Borradori A., Simon M. I.,
and Bogenmann E. (1994) The human blue opsin promoter directs transgene
expression in short-wave cones and bipolar cells in the mouse retina. Proc Natl
AcadSci USA 91, 2611-2615.
Chen S., Wang Q. L., Nie Z., Sun H., Lennon G., Copeland N. G., Gilbert D. J., Jenkins
N. A., and Zack D. J. (1997) Crx, a novel Otx-like paired-homeodomain protein,
binds to and transactivates photoreceptor cell-specific genes. Neuron 19, 1017-1030.
Chen S. and Zack D. J. (1996) Ret 4, a positive acting rhodopsin regulatory element
identified using a bovine retina in vitro transcription system. JBiol Chem 271,
Cheng L., Chaidhawangul S., Wong-Staal F., Gilbert J., Poeschla E., Toyoguchi M., El
Bradey M., Bergeron-Lynn G., Soules K. A., and Freeman W. R. (2002) Human
immunodeficiency virus type 2 (HIV-2) vector-mediated in vivo gene transfer into
adult rabbit retina. Curr Eye Res 24, 196-201.
Chung J. H., Bell A. C., and Felsenfeld G. (1997) Characterization of the chicken beta-
globin insulator. Proc NatlAcad Sci USA 94, 575-580.
Coleman J. E., Fuchs G. E., and Semple-Rowland S. L. (2002) Analyses of the guanylate
cyclase activating protein-1 gene promoter in the developing retina. Invest
Ophthalmol Vis Sci 43, 1335-1343.
Coleman J. E., Huentelman M. J., Kasparov S., Metcalfe B. L., Paton J. F., Katovich M.
J., Semple-Rowland S. L., Raizada M. K. (2003) Efficient large-scale production
and concentration of HIV-1-based lentiviral vectors for use in vivo. Physiol
Genomics 12, 221-8.
Cooper N., Liu L., Yoshida A., Pozdnyakov N., Margulis A., and Sitaramayya A. (1995)
The bovine rod outer segment guanylate cyclase, ROS-GC, is present in both outer
segment and synaptic layers of the retina. JMolNeurosci 6, 211-222.
Cremers F. P., van den Hurk J. A., and den Hollander A. I. (2002) Molecular genetics of
Leber congenital amaurosis. Hum Mol Genet 11, 1169-1176.
Cuenca N., Lopez S., Howes K., and Kolb H. (1998) The localization of guanylyl cyclase-
activating proteins in the mammalian retina. Invest Ophthalmol Vis Sci 39, 1243-
Cui Y., Iwakuma T., and Chang L. J. (1999) Contributions of viral splice sites and cis-
regulatory elements to lentivirus vector function. J Virol 73, 6171-6176.
Dejneka N. S., Auricchio A., Maguire A. M., Ye X., Gao G. P., Wilson J. M., and Bennett
J. (2001) Pharmacologically regulated gene expression in the retina following
transduction with viral vectors. Gene Ther 8, 442-446.
den Hollander A. I., Heckenlively J. R., van den Born L. I., de Kok Y. J., van der Velde-
Visser SD, Kellner U., Jurklies B., van Schooneveld M. J., Blankenagel A.,
Rohrschneider K., Wissinger B., Cruysberg J. R., Deutman A. F., Brunner H. G.,
Apfelstedt-Sylla E., Hoyng C. B., and Cremers F. P. (2001) Leber congenital
amaurosis and retinitis pigmentosa with Coats-like exudative vasculopathy are
associated with mutations in the crumbs homologue 1 (CRB1) gene. Am JHum
Genet 69, 198-203.
DesJardin L. E. and Hauswirth W. W. (1996) Developmentally important DNA elements
within the bovine opsin upstream region. Invest Ophthalmol Vis Sci 37, 154-165.
Di Polo A., Rickman C. B., and Farber D. B. (1996) Isolation and initial characterization
of the 5' flanking region of the human and murine cyclic guanosine
monophosphate-phosphodiesterase beta-subunit genes. Invest Ophthalmol Vis Sci
Dizhoor A. M., Boikov S. G., and Olshevskaya E. V. (1998) Constitutive activation of
photoreceptor guanylate cyclase by Y99C mutant of GCAP-1. Possible role in
causing human autosomal dominant cone degeneration. JBiol Chem 273, 17311-
Dizhoor A. M. and Hurley J. B. (1999) Regulation of photoreceptor membrane guanylyl
cyclases by guanylyl cyclase activator proteins. Methods 19, 521-531.
Dizhoor A. M., Lowe D. G., Olshevskaya E. V., Laura R. P., and Hurley J. B. (1994) The
human photoreceptor membrane guanylyl cyclase, RetGC, is present in outer
segments and is regulated by calcium and a soluble activator. Neuron 12, 1345-1352.
Dizhoor A. M., Olshevskaya E. V., Henzel W. J., Wong S. C., Stults J. T., Ankoudinova
I., and Hurley J. B. (1995) Cloning, sequencing, and expression of a 24-kDa Ca(2+)-
binding protein activating photoreceptor guanylyl cyclase. JBiol Chem 270, 25200-
Downes S. M., Holder G. E., Fitzke F. W., Payne A. M., Warren M. J., Bhattacharya S.
S., and Bird A. C. (2001) Autosomal dominant cone and cone-rod dystrophy with
mutations in the guanylate cyclase activator 1A gene-encoding guanylate cyclase
activating protein-1. Arch Ophthalmol 119, 96-105.
Dryja T. P., Adams S. M., Grimsby J. L., McGee T. L., Hong D. H., Li T., Andreasson S.,
and Berson E. L. (2001) Null RPGRIP1 alleles in patients with Leber congenital
amaurosis. Am JHum Genet 68, 1295-1298.
Duda T., Goraczniak R. M., and Sharma R. K. (1996) Molecular characterization of
S100A1-S100B protein in retina and its activation mechanism of bovine
photoreceptor guanylate cyclase. Biochemistry 35, 6263-6266.
Duda T., Krishnan A., Venkataraman V., Lange C., Koch K. W., and Sharma R. K.
(1999a) Mutations in the rod outer segment membrane guanylate cyclase in a cone-
rod dystrophy cause defects in calcium signaling. Biochemistry 38, 13912-13919.
Duda T., Venkataraman V., Goraczniak R., Lange C., Koch K. W., and Sharma R. K.
(1999b) Functional consequences of a rod outer segment membrane guanylate
cyclase (ROS-GC1) gene mutation linked with Leber's congenital amaurosis.
Biochemistry 38, 509-515.
Edwards W. C., Macdonald R., Jr., and Price W. D. (1971) Congenital amaurosis of retinal
origin (Leber). Am J Ophthalmol 72, 724-728.
Fain G. L. and Lisman J. E. (1999) Light, Ca2+, and photoreceptor death: new evidence
for the equivalent-light hypothesis from arrestin knockout mice [comment]. Invest
Ophthalmol Vis Sci 40, 2770-2772.
Farson D., Witt R., McGuinness R., Dull T., Kelly M. Song J., Radeke R., Bukovsky A.,
Consiglio A., and Naldini L. (2001) A new-generation stable inducible packaging
cell line for lentiviral vectors. Hum Gene Ther 12, 981-997.
Fei Y., Matragoon S., Smith S. B., Overbeek P. A., Chen S., Zack D. J., and Liou G. I.
(1999) Functional dissection of the promoter of the interphotoreceptor retinoid-
binding protein gene: the cone-rod-homeobox element is essential for
photoreceptor-specific expression in vivo. JBiochem (Tokyo) 125, 1189-1199.
Fekete D. M. and Cepko C. L. (1993) Replication-competent retroviral vectors encoding
alkaline phosphatase reveal spatial restriction of viral gene expression/transduction
in the chick embryo. Mol Cell Biol 13, 2604-2613.
Fekete D. M., Perez-Miguelsanz J., Ryder E. F., and Cepko C. L. (1994) Clonal analysis in
the chicken retina reveals tangential dispersion of clonally related cells. Dev Biol
Flannery J. G., Zolotukhin S., Vaquero M. I., LaVail M. M., Muzyczka N., and Hauswirth
W. W. (1997) Efficient photoreceptor-targeted gene expression in vivo by
recombinant adeno-associated virus. Proceedings of the National Academy of
Sciences of the United States of America 94, 6916-6921.
Follenzi A., Ailles L. E., Bakovic S., Geuna M., and Naldini L. (2000) Gene transfer by
lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol
sequences. Nat Genet 25, 217-222.
Fox J. L. (2003) US authorities uphold suspension of SCID gene therapy. Nat Biotechnol
Foxman S. G., Heckenlively J. R., Bateman J. B., and Wirtschafter J. D. (1985)
Classification of congenital and early onset retinitis pigmentosa. Arch Ophthalmol
Francois J. and Hanssens M. (1969) Histopathological study of 2 cases of Leber's
congenital tapeto-retinal degeneration. Ann Ocul (Paris) 202, 127-155.
Freund C. L., Wang Q. L., Chen S., Muskat B. L., Wiles C. D., Sheffield V. C., Jacobson
S. G., Mclnnes R. R., Zack D. J., and Stone E. M. (1998) De novo mutations in the
CRX homeobox gene associated with Leber congenital amaurosis [letter]. Nat
Genet 18, 311-312.
Frins S., Bonigk W., Muller F., Kellner R., and Koch K. W. (1996) Functional
characterization of a guanylyl cyclase-activating protein from vertebrate rods.
Cloning, heterologous expression, and localization. JBiol Chem 271, 8022-8027.
Fuille, H.-J. and Gallardo, E. M. Organization of two human retinal guanylyl cyclase
genes: isolation, analysis and comparison of upstream regulatory elements. Invest
Ophthalmol Vis Sci ARVO E-abstract: 1908.
Furukawa A., Koike C., Lippincott P., Cepko C. L., and Furukawa T. (2002) The mouse
Crx 5'-upstream transgene sequence directs cell-specific and developmentally
regulated expression in retinal photoreceptor cells. JNeurosci 22, 1640-1647.
Furukawa T., Morrow E. M., and Cepko C. L. (1997) Crx, a novel otx-like homeobox
gene, shows photoreceptor-specific expression and regulates photoreceptor
differentiation. Cell 91, 531-541.
Furukawa T., Morrow E. M., Li T., Davis F. C., and Cepko C. L. (1999) Retinopathy and
attenuated circadian entrainment in Crx-deficient mice. Nat Genet 23, 466-470.
Gorczyca W. A., Gray-Keller M. P., Detwiler P. B., and Palczewski K. (1994) Purification
and physiological evaluation of a guanylate cyclase activating protein from retinal
rods. Proc Natl Acad Sci USA 91, 4014-4018.
Gorczyca W. A., Polans A. S., Surgucheva I. G., Subbaraya I., Baehr W., and Palczewski
K. (1995) Guanylyl cyclase activating protein. A calcium-sensitive regulator of
phototransduction. JBiol Chem 270, 22029-22036.
Gouras P., Kjeldbye H., and Zack D. J. (1994) Reporter gene expression in cones in
transgenic mice carrying bovine rhodopsin promoter/lacZ transgenes. Vis Neurosci
Grant C. A., Ponnazhagan S., Wang X. S., Srivastava A., and Li T. (1997) Evaluation of
recombinant adeno-associated virus as a gene transfer vector for the retina. Curr
Eye Res 16, 949-956.
Hacein-Bey-Abina S., Le Deist F., Carlier F., Bouneaud C., Hue C., De Villartay J. P.,
Thrasher A. J., Wulffraat N., Sorensen R., Dupuis-Girod S., Fischer A., Davies E.
G., Kuis W., Leiva L., and Cavazzana-Calvo M. (2002) Sustained correction of X-
linked severe combined immunodeficiency by ex vivo gene therapy. NEnglJMed
Hacein-Bey-Abina S., von Kalle C., Schmidt M., Le Deist F., Wulffraat N., McIntyre E.,
Radford I., Villeval J. L., Fraser C. C., Cavazzana-Calvo M., and Fischer A. (2003)
A serious adverse event after successful gene therapy for X-linked severe combined
immunodeficiency. NEngl JMed 348, 255-256.
Haeseleer F., Sokal I., Li N., Pettenati M., Rao N., Bronson D., Wechter R., Baehr W.,
and Palczewski K. (1999) Molecular characterization of a third member of the
guanylyl cyclase-activating protein subfamily. JBiol Chem 274, 6526-6535.
Hao W., Wenzel A., Obin M. S., Chen C. K., Brill E., Krasnoperova N. V., Eversole-Cire
P., Kleyner Y., Taylor A., Simon M. I., Grimm C., Reme C. E., and Lem J. (2002)
Evidence for two apoptotic pathways in light-induced retinal degeneration. Nat
Genet 32, 254-260.
Harvey D. M. and Caskey C. T. (1998) Inducible control of gene expression: prospects for
gene therapy. Curr Opin Chem Biol 2, 512-518.
Hauswirth W. W., Langerijt A. V., Timmers A. M., Adamus G., and Ulshafer R. J. (1992)
Early expression and localization of rhodopsin and interphotoreceptor retinoid-
binding protein (IRBP) in the developing fetal bovine retina. Exp Eye Res 54, 661-
Hauswirth W. W., LaVail M. M., Flannery J. G., and Lewin A. S. (2000) Ribozyme gene
therapy for autosomal dominant retinal disease. Clin Chem Lab Med 38, 147-153.
He L., Campbell M. L., Srivastava D., Blocker Y. S., Harris J. R., Swaroop A., and Fox D.
A. (1998) Spatial and temporal expression of AP-1 responsive rod photoreceptor
genes and bZIP transcription factors during development of the rat retina. Mol Vis
Hoffman L. M., Maguire A. M., and Bennett J. (1997) Cell-mediated immune response
and stability of intraocular transgene expression after adenovirus-mediated
delivery. Investigative Ophthalmology & Visual Science 38, 2224-2233.
Howes K., Bronson J. D., Dang Y. L., Li N., Zhang K. Ruiz C., Helekar B., Lee M.,
Subbaraya I., Kolb H., Chen J., and Baehr W. (1998) Gene array and expression of
mouse retina guanylate cyclase activating proteins 1 and 2. Invest Ophthalmol Vis
Sci 39, 867-875.
Iwakuma T., Cui Y., and Chang L. J. (1999) Self-inactivating lentiviral vectors with U3
and U5 modifications. Virology 261, 120-132.
Johnson-Saliba M. and Jans D. A. (2001) Gene therapy: optimising DNA delivery to the
nucleus. Curr Drug Targets 2, 371-399.
Johnson P. T., Williams R. R., and Reese B. E. (2001) Developmental patterns of protein
expression in photoreceptors implicate distinct environmental versus cell-intrinsic
mechanisms. VisNeurosci 18, 157-168.
Johnston J. P., Farhangfar F., Aparicio J. G., Nam S. H., and Applebury M. L. (1997) The
bovine guanylate cyclase GC-E gene and 5' flanking region. Gene 193, 219-227.
Jorgensen N. P., Nossen J. O., Borch K. W., Kristiansen A. B., Kristoffersen D. T.,
Lundby B., and Theodorsen L. (1992) Safety and tolerability of iodixanol in healthy
volunteers with reference to two monomeric X-ray contrast media. Eur JRadiol 15,
Kachi S., Nishizawa Y., Olshevskaya E., Yamazaki A., Miyake Y., Wakabayashi T.,
Dizhoor A., and Usukura J. (1999) Detailed localization of photoreceptor guanylate
cyclase activating protein-1 and -2 in mammalian retinas using light and electron
microscopy. Exp Eye Res 68, 465-473.
Kafri T., van Praag H., Gage F. H., and Verma I. M. (2000) Lentiviral vectors: regulated
gene expression. Mol Ther 1, 516-521.
Kelsell R. E., Gregory-Evans K., Payne A. M., Perrault I., Kaplan J., Yang R. B., Garbers
D. L., Bird A. C., Moore A. T., and Hunt D. M. (1998) Mutations in the retinal
guanylate cyclase (RETGC-1) gene in dominant cone-rod dystrophy. Hum Mol
Genet 7, 1179-1184.
Kennedy B. N., Vihtelic T. S., Checkley L., Vaughan K. T., and Hyde D. R. (2001)
Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line
expressing enhanced green fluorescent protein in rod photoreceptors. JBiol Chem
Kikuchi T., Raju K., Breitman M. L., and Shinohara T. (1993) The proximal promoter of
the mouse arrestin gene directs gene expression in photoreceptor cells and contains
an evolutionarily conserved retinal factor-binding site. Mol Cell Biol 13, 4400-4408.
Kimura A., Singh D., Wawrousek E. F., Kikuchi M., Nakamura M., and Shinohara T.
(2000) Both PCE-1/RX and OTX/CRX interactions are necessary for
photoreceptor-specific gene expression. JBiol Chem 275, 1152-1160.
Klages N., Zufferey R., and Trono D. (2000) A stable system for the high-titer production
of multiply attenuated lentiviral vectors. Mol Ther 2, 170-176.
Kochanek S., Schiedner G., and Volpers C. (2001) High-capacity 'gutless' adenoviral
vectors. Curr Opin Mol Ther 3, 454-463.
Kumar M., Keller B., Makalou N., and Sutton R. E. (2001) Systematic determination of
the packaging limit of lentiviral vectors. Hum Gene Ther 12, 1893-1905.
Kumar R., Chen S., Scheurer D., Wang Q. L., Duh E., Sung C. H., Rehemtulla A.,
Swaroop A., Adler R., and Zack D. J. (1996) The bZIP transcription factor Nrl
stimulates rhodopsin promoter activity in primary retinal cell cultures. JBiol Chem
Lau D., McGee L. H., Zhou S., Rendahl K. G., Manning W. C., Escobedo J. A., and
Flannery J. G. (2000) Retinal degeneration is slowed in transgenic rats by AAV-
mediated delivery of FGF-2. Invest Ophthalmol Vis Sci 41, 3622-3633.
LaVail M. M., Yasumura D., Matthes M. T., Drenser K. A., Flannery J. G., Lewin A. S.,
and Hauswirth W. W. (2000) Ribozyme rescue of photoreceptor cells in P23H
transgenic rats: long-term survival and late-stage therapy. Proc Natl Acad Sci US A
Leber T. (1869) Ueber Retinitis pigmentosa und angeborene Amaurose. Albrecht von
Graefes Arch Ophthal 15, 1-25.
Lechardeur D. and Lukacs G. L. (2002) Intracellular barriers to non-viral gene transfer.
Curr Gene Ther 2, 183-194.
Lem J., Applebury M. L., Falk J. D., Flannery J. G., and Simon M. I. (1991) Tissue-
specific and developmental regulation of rod opsin chimeric genes in transgenic
mice. Neuron 6, 201-210.
Lewin A. S., Drenser K. A., Hauswirth W. W., Nishikawa S., Yasumura D., Flannery J.
G., and LaVail M. M. (1998) Ribozyme rescue of photoreceptor cells in a transgenic
rat model of autosomal dominant retinitis pigmentosa. Nat Med 4, 967-971.
Lin C. T., Gould D. J., Petersen-Jonest S. M., and Sargan D. R. (2002) Canine inherited
retinal degenerations: update on molecular genetic research and its clinical
application. JSmall Anim Pract 43, 426-432.
Liou G. I., Matragoon S., Yang J., Geng L., Overbeek P. A., and Ma D. P. (1991) Retina-
specific expression from the IRBP promoter in transgenic mice is conferred by 212
bp of the 5'-flanking region. Biochem Biophys Res Commun 181, 159-165.
Liu X., Seno K., Nishizawa Y., Hayashi F., Yamazaki A., Matsumoto H., Wakabayashi
T., and Usukura J. (1994) Ultrastructural localization of retinal guanylate cyclase in
human and monkey retinas. Exp Eye Res 59, 761-768.
Livesey F. J., Furukawa T., Steffen M. A., Church G. M., and Cepko C. L. (2000)
Microarray analysis of the transcriptional network controlled by the photoreceptor
homeobox gene Crx. Curr Biol 10, 301-310.
Lotery A. J., Derksen T. A., Russell S. R., Mullins R. F., Sauter S., Affatigato L. M.,
Stone E. M., and Davidson B. L. (2002) Gene transfer to the nonhuman primate
retina with recombinant feline immunodeficiency virus vectors. Hum Gene Ther 13,
Lotery A. J., Jacobson S. G., Fishman G. A., Weleber R. G., Fulton A. B.,
Namperumalsamy P., Heon E., Levin A. V., Grover S., Rosenow J. R., Kopp K. K.,
Sheffield V. C., and Stone E. M. (2001) Mutations in the CRB1 gene cause Leber
congenital amaurosis. Arch Ophthalmol 119, 415-420.
Lyn D., Bennett N. A., Shiramizu B. T., Herndier B. G., and Igietseme J. U. (2001)
Sequence analysis of HIV-1 insertion sites in peripheral blood lymphocytes. Cell
Mol Biol (Noisy -le-grand) 47, 981-986.
Mani S. S., Batni S., Whitaker L., Chen S., Engbretson G., and Knox B. E. (2001)
Xenopus rhodopsin promoter: Identification of immediate upstream sequences
necessary for high level, rod-specific transcription. JBiol Chem.
Mani S. S., Besharse J. C., and Knox B. E. (1999) Immediate upstream sequence of
arrestin directs rod-specific expression in Xenopus. JBiol Chem 274, 15590-15597.
Marlhens F., Bareil C., Griffoin J. M., Zrenner E., Amalric P., Eliaou C., Liu S. Y., Harris
E., Redmond T. M., Arnaud B., Claustres M., and Hamel C. P. (1997) Mutations in
RPE65 cause Leber's congenital amaurosis [letter]. Nat Genet 17, 139-141.
McGee Sanftner L. H., Abel H., Hauswirth W. W., and Flannery J. G. (2001) Glial cell
line derived neurotrophic factor delays photoreceptor degeneration in a transgenic
rat model of retinitis pigmentosa. Mol Ther 4, 622-629.
Mears A. J., Kondo M., Swain P. K., Takada Y., Bush R. A., Saunders T. L., Sieving P.
A., and Swaroop A. (2001) Nrl is required for rod photoreceptor development. Nat
Genet 29, 447-452.
Meller K. and TetzlaffW. (1976) Scanning electron microscopic studies on the
development of the chick retina. Cell Tissue Res 170, 145-159.
Mendez A., Burns M. E., Sokal I., Dizhoor A. M., Baehr W., Palczewski K., Baylor D.
A., and Chen J. (2001) Role of guanylate cyclase-activating proteins (GCAPs) in
setting the flash sensitivity of rod photoreceptors. Proc NatlAcadSci USA 98,
Milam A. H., Barakat M. R., Gupta N., Rose L., Aleman T. S., Pianta M. J., Cideciyan A.
V., Sheffield V. C., Stone E. M., and Jacobson S. G. (2003) Clinicopathologic
effects of mutant GUCY2D in Leber congenital amaurosis. Ophthalmology 110,
Mitani K. and Kubo S. (2002) Adenovirus as an integrating vector. Curr Gene Ther 2, 135-
Miyoshi H., Blomer U., Takahashi M., Gage F. H., and Verma I. M. (1998) Development
of a self-inactivating lentivirus vector. J Virol 72, 8150-8157.
Mizuno K., Takei Y., Sears M. L., Peterson W. S., Carr R. E., and Jampol L. M. (1977)
Leber's congenital amaurosis. Am J Ophthalmol 83, 32-42.
Mohamed M. K., Taylor R. E., Feinstein D. S., Huang X., and Pittler S. J. (1998)
Structure and upstream region characterization of the human gene encoding rod
photoreceptor cGMP phosphodiesterase alpha-subunit. JMolNeurosci 10, 235-250.
Morabito M. A., Yu X., and Bamstable C. J. (1991) Characterization of developmentally
regulated and retina-specific nuclear protein binding to a site in the upstream region
of the rat opsin gene. JBiol Chem 266, 9667-9672.
Morrow E. M., Furukawa T., and Cepko C. L. (1998) Vertebrate photoreceptor cell
development and disease. Trends CellBiol 8, 353-358.
Naldini L., Blomer U., Gallay P., Ory D., Mulligan R., Gage F. H., Verma I. M., and
Trono D. (1996) In vivo gene delivery and stable transduction of nondividing cells
by a lentiviral vector. Science 272, 263-267.
Nie Z., Chen S., Kumar R., and Zack D. J. (1996) RER, an evolutionarily conserved
sequence upstream of the rhodopsin gene, has enhancer activity. JBiol Chem 271,
Noble K. G. and Carr R. E. (1978) Leber's congenital amaurosis. A retrospective study of
33 cases and a histopathological study of one case. Arch Ophthalmol 96, 818-821.
Otto-Bruc A., Buczylko J., Surgucheva I., Subbaraya I., Rudnicka-Nawrot M., Crabb J.
W., Arendt A., Hargrave P. A., Baehr W., and Palczewski K. (1997) Functional
reconstitution of photoreceptor guanylate cyclase with native and mutant forms of
guanylate cyclase-activating protein 1. Biochemistry 36, 4295-4302.
Pacchia A. L., Adelson M. E., Kaul M., Ron Y., and Dougherty J. P. (2001) An inducible
packaging cell system for safe, efficient lentiviral vector production in the absence
of HIV-1 accessory proteins. Virology 282, 77-86.
Palczewski K., Polans A. S., Baehr W., and Ames J. B. (2000) Ca(2+)-binding proteins in
the retina: structure, function, and the etiology of human visual diseases. Bioessays
Palczewski K., Subbaraya I., Gorczyca W. A., Helekar B. S., Ruiz C. C., Ohguro H.,
Huang J., Zhao X., Crabb J. W., Johnson R. S., and. (1994) Molecular cloning and
characterization of retinal photoreceptor guanylyl cyclase-activating protein.
Neuron 13, 395-404.
Pannell D. and Ellis J. (2001) Silencing of gene expression: implications for design of
retrovirus vectors. Rev Med Virol 11,205-217.
Payne A. M., Downes S. M., Bessant D. A., Taylor R., Holder G. E., Warren M. J., Bird
A. C., and Bhattacharya S. S. (1998) A mutation in guanylate cyclase activator 1A
(GUCA1A) in an autosomal dominant cone dystrophy pedigree mapping to a new
locus on chromosome 6p21.1. Hum Mol Genet 7, 273-277.
Perrault I., Rozet J. M., Calvas P., Gerber S., Camuzat A., Dollfus H., Chatelin S., Souied
E., Ghazi I., Leowski C., Bonnemaison M., Le Paslier D., Frezal J., Dufier J. L. ,
Pittler S., Munnich A., and Kaplan J. (1996) Retinal-specific guanylate cyclase gene
mutations in Leber's congenital amaurosis. Nat Genet 14, 461-464.