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Enhancing Sleeping Beauty Mediated Gene Therapy Towards Endothelial Specificity

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Title:
Enhancing Sleeping Beauty Mediated Gene Therapy Towards Endothelial Specificity
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LAM, WEI JOON BRIAN ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Beauty ( jstor )
DNA ( jstor )
Endothelial cells ( jstor )
Gene therapy ( jstor )
Liver ( jstor )
Lungs ( jstor )
Plasmids ( jstor )
Transfection ( jstor )
Transgenes ( jstor )
Transposons ( jstor )

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University of Florida
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University of Florida
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Copyright Wei Joon Brian Lam. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/31/2008

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ENHANCING SLEEPING BEAUTY MEDIATED GENE THERAPY TOWARDS ENDOTHELIAL SPECIFICITY By WEI JOON BRIAN LAM A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Wei Joon Brian Lam

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iii ACKNOWLEDGMENTS First and foremost, I would like to tha nk my parents, Ban Siew Lam and Mary Leong, for their unconditional love and support. They have sacrificed so much in order for their three children to have it all. I am al so grateful to Kenneth Skuse for his constant encouragement and support through these year s. I must thank my committee members, Dr. Donna Duckworth and Dr. Sean Sulliv an, for their kind advice and guidance throughout my academic training. I am grateful for the assistance from Dr. Li Liu with the animal studies. Dr. Sonia Sanz and Mr. James Baus were kind and helpful during the initial stages of the project. I also thank Dr. Lucia Notterpek, Ms. Jocelyn Go, and other members of Dr. Notterpek’s lab for their w onderful help with the immunohistochemistry studies. I would also like to acknowledge Joy ce Conners for her excellent administration support. Last but not least, I owe a treme ndous amount of gratitude to my mentor and advisor, Dr. Bradley S. Fletch er, for his guidance, support, and advice for the past three years. He has taught me to respect and apprecia te science, to work hard, and to constantly strive for the best. It has been a privileg e to have studied under his mentorship.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 Gene Therapy Models: Viral and Non-Viral................................................................1 Transposons..................................................................................................................2 Sleeping Beauty Transposon System............................................................................3 Rationale for Using Sleeping Beauty in Gene Therapy................................................4 Minimal Host-specific Responses.........................................................................4 Controlled Delivery...............................................................................................4 Integration and Long-term Expression..................................................................5 Safety.....................................................................................................................5 Larger Gene Load..................................................................................................5 Ease of Production.................................................................................................5 Efficacy..................................................................................................................6 Structure of the Sleeping Beauty System......................................................................6 Delivery....................................................................................................................... .8 Transposition..............................................................................................................10 The Transposition Mechanism............................................................................10 Other Cellular Factors.........................................................................................10 Integration...................................................................................................................10 Expression...................................................................................................................11 Non-transient Expression....................................................................................11 Promoters.............................................................................................................13 Long-term Expression.........................................................................................13 Applications................................................................................................................13 Improvements to Sleeping Beauty ..............................................................................14 Moving Forward: Cell Specific Expression Using Sleeping Beauty ..........................16 Rationale for Endothe lia Cell Expression...................................................................17

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v 2 MATERIALS AND METHODS...............................................................................18 Plasmid Construction..................................................................................................18 Endothelial-specific Transposase........................................................................18 Endothelial-specific Transposon.........................................................................21 Cis Plasmid..........................................................................................................22 Cell Culture and Transient Transfection.....................................................................22 FACS Analysis...........................................................................................................23 In vitro Transposition Assays.....................................................................................23 Animals and In vivo Gene Delivery............................................................................25 SEAP Assay................................................................................................................27 Luciferase Assay.........................................................................................................27 Immunohistochemistry...............................................................................................28 3 RESULTS...................................................................................................................30 Endothelial-Specific Sleeping Beauty Transposition..................................................30 Transfection Efficiency.......................................................................................30 In vitro Transposition Assay Analysis................................................................31 Endothelial-Specific Sleeping Beauty Expression......................................................38 In vivo Long Term SEAP Secretion....................................................................38 Luciferase Assay and In Vivo Expression in Tissues..........................................40 Localization of Transgene in Organ Tissues..............................................................50 4 DISCUSSION.............................................................................................................52 LIST OF REFERENCES...................................................................................................57 BIOGRAPHICAL SKETCH.............................................................................................64

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vi LIST OF TABLES Table page 1-1. SB mediated gene therapy applications......................................................................14 1-2. Other documented research work using SB...............................................................14 2-1. Volumes of DNA for in vivo luciferase experiment...................................................26 3-1. Results from Groups 1 to 6 in HUVECs , conducted in duplicates, from three separately administ ered experiments........................................................................32 3-2. Results from Groups 7 to 12 in HeLa cells, conducted in duplicates, from three separately administ ered experiments........................................................................35 3-3. Results of SEAP detection at 6 different time points post-injection..........................38 3-4. Average relative light units and prot ein concentrations detected in lung...................42 3-5. Average relative light un its and protein concentrati ons detected in heart..................43 3-6. Average relative light un its and protein concentrati ons detected in liver..................43 3-7. Average relative light un its and protein concentrati ons detected in spleen...............44 3-8. Average relative light un its and protein concentrati ons detected in kidney...............44 3-9. Average relative light units per mg in lung................................................................45 3-10. Average relative light units per mg in liver..............................................................46 3-11. Average relative light units per mg in heart.............................................................47 3-12. Average relative light units per mg in spleen...........................................................48 3-13. Average relative light units per mg in kidney..........................................................49

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vii LIST OF FIGURES Figure page 1-1. Components of the Sleeping Beauty system.................................................................7 1-2. Delivery of SB into a cell.............................................................................................9 1-3. Overview of the transposition, integration, and repair processes...............................12 2-1. Generation of SB16 and poly(A) fragment................................................................18 2-2. Addition of SB16 to the plasmid c ontaining Tie2 promoter and enhancer................19 2-3. Tie2-driven Sleeping Beauty transposase with a third Sal I site removed...................19 2-4. Illustration of the endot helial-specific transposase backbone, a short sequence containing an origin of replication, a selective marker, and an SV40 Enhancer.....20 2-5. Final components of th e endothelial-specific tr ansposase, pTie-HSB16...................20 2-6. Endothelial-specific transposon..................................................................................21 2-7. Two-in-one plasmid containing both transposon and transposase components.........22 2-8. Example of stained Neomycin-resistant colonies after tr ansposition assay...............25 3-1. On the right is the evaluation of transfection using pMSZ-GFP in HUVECs...........30 3-2. On the right is the evaluation of tr ansfection using pMSZ-GFP in HeLa cells..........31 3-3. Results of HUVECs transposition..............................................................................34 3-4. Results of HeLa transposition.....................................................................................37 3-5. Long term SEAP expressi on in mice, up to day +84.................................................39 3-6. Luciferase expression in l ung illustrated on a log scale.............................................45 3-7. Luciferase expression in liv er illustrated on a log scale.............................................46 3-8. Luciferase expression in h eart illustrated on a log scale............................................47

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viii 3-9. Luciferase activity detected in spleen illustrated on a log scale.................................48 3-10. Luciferase expression in kidney illustrated on a log scale.......................................49 3-11. Summary of luciferase expression in organ tissues on day +28 post-injection illustrated on a log scale...........................................................................................50 3-12. Luciferase localization in lung tissue of Trans group...............................................51

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ix Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ENHANCING SLEEPING BEAUTY MEDIATED GENE THERAPY TOWARDS ENDOTHELIAL SPECIFICITY By Wei Joon Brian Lam May 2006 Chair: Bradley S. Fletcher Major Department: Pharmacology and Therapeutics Genetics based medicine offers exciting opportunities in permanently treating a condition or disease. Gene ther apy can be generally described as delivering a gene to a cell to enable production of a pa rticular protein. To facilitate gene therapy, viral or nonviral vectors are commonly used. This research investigated the possibility of using the non-viral Sleeping Beauty (SB) transposon system for deliv ering a gene systemically and enhancing that gene expressi on in endothelial cells. Endot helial cells were chosen because they are involved in regulati ng vascular homeostasis and controlling angiogenesis seen in tumor formation. Endothe lial cell-specific Ti e2 and endothelin-1 promoters were placed within the transpos ase and transposon system, respectively. Two SB configurations were evaluated. The first had the transposase delivered on a separate vector (Trans), while the s econd was configured with the transposase and transposon on the same vector (Cis). The Tie2-driven tran sposase was tested in a transposition assay, and achieved transposition activity similar to the CMV-driven transposase in an

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x endothelial cell line. Using the endothe lial-specific SB system complexed to polyethylenimine, secreted alkaline phosphata se (SEAP) and lucife rase expression were evaluated following administration into mice. Stable long term SEAP expression was detected at day +84 post-injection using both endothelial-specific SB configurations. In evaluating tissue expression, robus t luciferase activity was det ected in lungs. The highest activity was recorded in th e no specific cell-targeted (Nonspec) group, followed by the Trans and Cis groups. The heart and liver had lo wer levels of expression. However, there was a significantly higher level of activity in the liver for the Nonspec group compared to the Trans and Cis groups. The lower expressions in the Trans and Cis groups may be an indication of the endothelial-specific Tie2 promoter’s low activity in non-vascular endothelial cells in the liver. No activities we re detected in the kidne y or spleen. Overall, the Trans plasmid system had higher activities in organ tissues compared to Cis. These results suggest that SB can be adapted to target specific cells for expression. In this case, cells successfully targeted were of endothelial origin. In addition, the Trans configuration appears to work better in vivo . Adapting SB towards endothelial specificity may be useful for future gene therapy approaches in tr eating cardiovascular and other endothelialrelated diseases.

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1 CHAPTER 1 INTRODUCTION Gene therapy holds the promise of re volutionizing health care in the 21st century for some diseases. The general concept of ge ne therapy can be described as the delivery of a functional gene into a patient who harbors a gene tic mutation, thereby enabling production of the missing protein. In other word s, gene therapy can replace defective or disease-causing genes. Alternatively, gene ther apy can also be used to add a new function to a cell. While the purpose of gene thera py varies with each me dical condition, the end result sought is similar. Such genetics-b ased medicine, rather than conventional chemistry-based approaches, offers exciti ng opportunities at pr oviding long-term and permanent treatment that addresses the root cause of some medi cal conditions. This approach alleviates the difficult and costly task of symptom management over prolonged periods of time. However, most current ge ne therapy techniques are still undergoing refinement and carry some elements of risk. At present, gene ther apy treatments are only offered after careful evaluation of the risk -return benefits. As more gene therapy improvements are made, we hope that the risk component will some day be negligible. Gene Therapy Models: Viral and Non-Viral By 2004, over 900 clinical trials on human gene therapy have been conducted, are on-going, or approved. Non-viral systems such as naked DNA and lipofection accounted for a quarter of those trials [1]. Gene ther apy approaches can be broadly categorized as either viral or non-viral vector based. Vira l systems include the use of retroviruses, adenoviruses, lentiviruses and adeno-asso ciated viruses (AAV), amongst others. Non-

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2 viral systems include the use of naked DNA, lipids, and transposons. Each of these systems has its own advantages and disadvantages [2]. Viral based approaches are traditionally fa vored for gene therapy due to viruses’ ability to infect cells. Many in tegrate their own viral nucleic acids into their hosts’ cell genetic material to produce progeny. These vi ruses offer the possibility of efficient chromosomal integration and thus, possible long-term expression of transgenes. Viralbased systems are traditionally more effi cient at gene delivery and chromosomal integration compared to non-viral technique s such as using naked DNA. Their natural viral capability of entering cells is their biggest advantage. However, while viral systems are popular platforms for gene therapy research, many issues remain unanswered. Depending on the viral vector of choice, there are concerns over host responses to viral components [3], nonsustainable gene expression [4], therapeutic gene size restriction [5], a nd the propensity of some viruses to prefer genes and integrate near transcri ption units [6]. These are all valid concerns pertaining to safety and efficacy. A recent study also suggested that viral vectors have negative effects on human endothelial cell phenotype, activation, and function [7]. Therefore, viral-based gene therapy may not be best suited for di seases affecting the endothelium such as cardiovascular diseases. Such concerns with viral vectors have fueled the development of non-viral methods. Because conventional non-viral methods also have limitations of their own [8], new non-viral systems such as C31 [9] and Sleeping Beauty (SB) were developed. Transposons Transposons are genetic elements that can move within a genome through a process called transposition. They have so me virus-like characteristics, but they do not cause cell

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3 lysis. Transposable elements are grouped into two classes. Class I Retrotransposons move via RNA intermediates while Class II tran sposase dependent elements move through DNA intermediaries with the aid of a transp osase protein [10]. Autonomous transposons encode their own transposase protein fo r transposition while non-autonomous ones depend on transposase from another transposon for its own mobility [11]. Transposons can be sub-classified under several families. The Tc1/ mariner superfamily of DNA transposons is one of th e most widespread in nature. They are found in nematodes, arthropods, fish, frogs, and humans. Tc1/ mariner elements are about 1300 – 2400 base pairs (bp) long and contain a single transposase gene that is flanked by inverted repeats [12]. Given their wide-rang e presence in hosts, this transposon family probably spread through horizonta l transmissions [13]. Evoluti onary pressure to survive may have led to this cross-species trans position capability [14]. Interestingly, such ancient evolutionary adaptation millions of years ago may be leading us into a whole new approach towards medical tr eatment in the future. Sleeping Beauty Transposon System The Sleeping Beauty system is a Tc1-like transposon (TCE) and is a member of the Tc1/ mariner superfamily. Ivics et. al . identified a nuclear loca lization signal in TCE and evidence of possible cross species transposition capabilities [15]. Mariner transposon’s cross-species capability was later demonstrated in 1996 [ 13]. Having these attractive features in a vertebrate transposon prompted the team to reconstruct a TCE transposon from fish in 1997 using phylogenetic data. W ith the data as a guide, the synthetic transposon was made to closely resemble an extinct 10-15 million year old TCE element that likely possessed high transposition cap ability. For the transposase, they reengineered the gene by using a majority-r uled consensus derived from the salmonid

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4 family. Mutations in the transposase’s presen t genetic sequence were removed to enable it to regain its function. A working transpos ase with 340 amino acids was recreated and named SB10. For the first time, an active vertebrate DNA transposon with transposition capability in a human cell line was available. The revived transposon system was aptly named Sleeping Beauty [16]. Rationale for Using Sleeping Beauty in Gene Therapy For the past few years, major improvements were made to SB as a gene therapy system. With a greater unders tanding of its tran sposition mechanism, SB vectors are being optimized to make it into a gene therapy vector. SB is a promising system for gene therapy because it possesses ma ny of the required attributes . This system offers many advantages over viral and existing non-viral technique s, such as: Minimal Host-specific Responses Because SB components are completely nonviral, it is unlikely that host antigenspecific responses will be mounted against repeated therapy attempts. Viral-based therapies, such as adenovirus models, elic it immune responses towa rds the viral vector which will neutralize any attempts to re-deliver the therapeutic gene to the host [3]. Such problems will severely limit the potential uses of intended multiple dosing treatments. Controlled Delivery Since SB transposition requires the transpos ase protein, delivery of the transposase can be easily controlled or stopped once the therapeutic gene is delivered. The newly introduced gene will not be mobile as long as no transposase protein is available. This requirement assures that transposition activity can be somewhat controlled once SB is introduced into the human body.

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5 Integration and Long-term Expression SB integrates into the chromosomal DNA. This capability overcomes the most significant disadvantage of previous non-viral m odels. Because of its integrating abilities, SB enables long-term expression of transgenes in hosts [17-19]. Safety There are no active DNA transposons found in humans. Humans harbor only 2-3% of transposon DNA in our genome and all have been inactivated [ 20]. More importantly, the TCE IR/DR element is not present in the human genome. As such, the introduction of SB transposase will have minimal risk of re-activating any reside nt DNA transposons in patients. Larger Gene Load Some viral-based methods, such as AAV, have a cargo load limit of about 4 kilobases (kb) long and cannot be used for delivery of bigger genes. SB is capable of delivering transgenes that ar e as large as 6 kb. Since most human genes are less than 7 kb, this attribute is attractive for gene th erapy [21]. When a sandwich SB transposon configuration is deployed, wh ere two pairs of transposon sequences flank a transgene, “cargo” as large as 10kb can be successfully transposed [22]. Ease of Production Components for gene therapy applications have to be easily produced on a large scale to meet clinical demands. SB compone nts can be manufactured easily. This feature is an advantage over viral vectors where produ ction quantity, cost, f easibility, and safety are pressing issues.

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6 Efficacy Remnants of Tc1/ mariner transposons are found in humans and across many species [12]. These inactive fragments suggest that Tc1/ mariner transposons may have transposed into human cells years ag o. As SB is a member of the Tc1/ mariner family, it is expected that it too can have the abili ty to transfect human cells. In addition, as mariner elements do not require species-specific enzymes for transposition [23], SB will also likely be able to transpose across a wide host range, especi ally in humans. Structure of the Sleeping Beauty System The revived SB structure is similar to other TCEs. However, SB consists of a separated transposon and transposase system that are supplied on plasmids. The removed transposase gene is replaced by a therapeutic gene of interest. The transposon is made up of inverted repeats that are about 230bp long that flank both e nds of the element. Within each of the inverted repeats ar e two direct repeats (DR) of about 30bp long that serve as binding domains. This direct repe at within inverted repeats is called the IR/DR sequence. The two DR in each inverted repeats ar e about 165bp apart, and studies have demonstrated they are not interchangeable. The outer DR has a stronger transposase binding affinity compared to the inner DR. Th e spacing between the DR elements is also important and affects transposition rates. Such length requirements and affinity characteristics suggest the likelihood of a synaptic complex formation prior to transposition [24]. The transposase contains conserved regi ons that are required for transposition. There is a Nuclear Localizing Si gnal (NLS) in the N-terminal half of the transposase. A typical recombination enzyme containing a DDE motif (two aspartic acids and one glutamic acid) is found on the catalytic C-terminal half. Th e center of the transposase

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7 contains a glycine-rich motif [16] . The transposase can be supplied in cis on the same plasmid or in trans on a separate plasmid. Figure 1-1. Components of the Sleeping Beauty system. IR/DR, inverted repeat-direct repeat; Lo, left outer repeat; Li, left inner repeat; Ro, right outer repeat; Ri, right inner repeat; NLS, nuclear loca lization signal. The upper figure depicts what the ancient element probably looked like. The revived SB system is split into two components (trans system), wh ere one plasmid carries the transposon and gene of interest while another plasmi d carries the transposase gene that is often driven by a promoter. Both plasmids can also be combined as one (cis system). Transposase IR/DR IR/DR Lo Ro Ri Li 230b p 230b p 340 amino acids Binding domain NLS DDE catalytic domain IR/DR IR/DR Therapeutic Gene Trans p oson vecto r Transposase Transposase vector Ancient elemen t Sleeping Beauty (trans system) Binding domain NLS DDE Domain

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8 Delivery Unlike viruses, transposons generally cannot pass through the cell membrane. Therefore, a mechanism must be employed in order to introduce the transposon into the cellular environment where th ey can then transpose into the chromosomal DNA. Polyethylenimine (PEI) is a frequently us ed non-viral delivery method. It tightly packages plasmid DNA together onto itsel f to form polyplexes. Their stronger DNA compaction capacity over other polycations makes them a popular choice for gene delivery. PEI works by essentially promoting ce llular uptake of the complex and taking the compacted plasmid DNA with it through r eceptor-mediated endocytosis on a cell’s membrane. Once inside the cell, the polyplexes are released into the cytoplasm and the cell contains many copies of the plasmid DNA [25]. PEI has successfully delivered SBcarrying plasmids into the cellular envi ronment in previous studies [26, 27]. Apart from systemic delivery into the bloodstream, SB can be delivered locally through directly injectin g an area [28] or even by using an adenovirus [29]. In addition to delivering the transposase ge ne on the same or on a separate plasmid, a transposonmRNA transposase combination was recently documented [30]. Delivering the transposase mRNA is viewed as a safer method compared to delivering the transposase gene. If a cell has continuously available tran sposase proteins, it may trigger constant and multiple transposition events causing ge nomic instability. Other proposed methods include delivering SB as a pr e-transposing complex [31, 32] . In short, there are many possibilities of delivering SB into cells. Cu stomized cell delivery and targeting for SB will be an interesting research area for the near future.

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9 Figure 1-2. Delivery of SB into a cell. SB, Sleeping Beauty ; PEI, polyethylenimine. SB vectors are packed into a polycomplex that facilitate s entry into a cell through a process called endocytosis. + SB-carrying plasmids PEI Polyplex Endocytosis nucleus Cell SB delivered into cell nucleus

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10 Transposition The Transposition Mechanism SB transposes through a cut and paste mechanism. Frequency is influenced by the amount of transposase protein available [33] . While some protein is needed, too much transposase can cause overproduction inhi bition, a condition where oversupply of transposase actually inhibits or interferes with transposition [34] . The transposase has helix-turn-helix sub domains that interact with the transposon’s DR. Cleavage of the donor transposon occurs in a st aggered manner, leaving GTC overhangs on the 3’ ends [35]. Before cleaving the transposon, the transposase mediates protein-protein interactions that result in stable tetramer s forming the synaptic complex. Transposition rates decrease when the transposon size become s larger, with a maximum size of 9 kb in order to achieve high transposition rates [36]. Other Cellular Factors A DNA bending protein named HMGB1 assists in the crucial synaptic formation process [37]. A scaffolding protein called DNA-dependent protein ki nase (DNA-PK), and the cell cycle signaling Ataxia-t elangiecstasia mutated (ATM) ki nase are also believed to be present prior to the trans position process [38]. Apart fr om proteins, CpG methylation of the transposon was demonstrated to e nhance the transposition process [39]. Integration TA dinucleotides are always the inserti on sites for SB transposition. Apart from helping form synaptic complexes, the tran sposase cleaves the TA base pair in the recipient DNA and creates staggered 5’ overhangs at the recipient site. The transposon carrying the transgene then integrates into th e donor site [40]. Res earchers believe that

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11 physical properties of the DNA such as bend-abil ity at the AT site, ra ther than base pair interactions, are the reasons for in tegration site selection [41, 42]. Once integrated into its ne w location, the DNA strands need to be repaired. Studies have indicated that the DNA-PK non-homologous end joining pathway is responsible for the majority of post-SB transposition repair [43]. At the donor site , a footprint sequence of TAC(A or T)GTA is usually left behind after each transposition occurrence. Excision assays have been used to semi-quant ify such transposition activities [44]. Compared to viral vectors, SB has a si gnificantly lower preference towards genes so it lessens the risk of gene disruption or unintended oncogene activat ion [45]. This trait is another advantage for using SB as a gene therapy vector. Expression Non-transient Expression Once integrated into a cell, the newly intr oduced gene can be expressed by cellular mechanisms. SB transposition enables chromosoma l integration so that a transgene is not merely transiently expressed. Transient expr ession occurs when a non-integrated plasmid is maintained only temporarily in the cellular environment. In such situations, transgene expression is only for the time being or “trans ient” and therefore, not indefinite. Studies show that a transgene can be successfully integrated into the chromosome for long-term expression using SB. Non-homologous reco mbination events where the transposon integrates into a cell without the aid of the transposase occu r at very low frequencies [46, 47]. Therefore, the transposase must be co-d elivered with the transposon to stimulate integrative transposition and to facilitate long-term expression.

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12 Figure 1-3. Overview of the transposition, in tegration, and repair processes. IR/DR, inverted repeat-direct repeat. Cellular repair co-factors Transposase Transposase binds to DR sites CTG TA GAC AT Trans g ene TA CAG AT GTC Trans p osase Trans p oson HMGB1 Transposase IR/DR Transgene GTC CTG T r ans g ene GTC CTG Transgene TAC(A)(T)G TA AT G(T)(A)CAT TA CAG AT GTC CTG TA GAC AT Transgene TAC(A)(T)G G ( T )( A ) CAT Synaptic complex formation Transposition after staggered cut on transposon Integration at recipient site Insertion site excision Cellular repair co-factors Repair at recipient site Permanently integrated transgene at recipient site Repair at donor site Footprints left on the donor site Cellular co-factors

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13 Promoters SB has demonstrated its transgene deliver y ability in mouse embryonic stem cells and human cells [21]. SB mediated transpos ition has maintained long-term transgene expression activity after 3 mont hs in mammalian cells [19]. A research study showed that the transposase may also play a ro le in transgene expression [48]. Weaker promoters driving the transposas e appear to lead to better long-term expression when compared to strong promoters like CM V. Weaker promoters are unlikely to cause overproduction inhibition, a condition where too much transposase inhibits or interferes w ith transposit ion [34]. Long-term Expression In theory, integrated transgenes will allo w the host to express the required protein. However, transgenes can undergo epigenetic modifications in hosts. Modifications such as methylation are meant to silence foreign genes as a safety pr ecaution. At present, studies are inconclusive on the nature of SB methylation. Some SB transpositions triggered the methylation process while others did not. A better understanding of methylation activation is required if longterm transgene expression is sought. Applications SB has successfully delivered transgenes into targeted tissues and organs. These studies demonstrate the potenti al uses of SB for gene th erapy treatment in diseases affecting the organs and tissues of inte rest. The following is a table summarizing successful experiments documenting SB uses fo r various treatments (Table 1-1). Apart from gene therapy, SB is also used in cancer-related studies and in other creative applications (Table 1-2).

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14 Table 1-1. SB mediated ge ne therapy applications Organ Application Lung Gene therapy targeting endothelial cells demonstrated [19, 26] Lung Gene therapy for Hemophilia A demonstrated [49] Liver Gene therapy for Tyrosinemia I demonstrated [50] Liver Gene therapy for Hemophilia B demonstrated [18] Skin Gene therapy for Epidermolysis bullosa demonstrated [51] Glia Gene therapy targeting hu man glial tumors demonstrated [27, 28] Liver Gene therapy for Hemophilia A demonstrated [52] Table 1-2. Other documented research work using SB Purpose Mutagenesis [53-57] Demonstrated chromosomal transpos ition in embryonic stem cell [58] Mutagenesis of somatic cells [59, 60] Cancer screening and discovery [61-64] Gene transfer into human primary T-cells [65] Knockdown cell-line generation [66] Improvements to Sleeping Beauty Since the awakening of SB, improvements we re made to make it into an efficient and safe gene therapy system. Most improvements were focused on increasing transposition rates by generating better trans poson and/or transposase sequences. Since SB was synthetically reconstructed, improveme nts were made to bring it closer to its perceived native form where transposition woul d be highly efficient. For example, SB10 was the initially rec onstructed transposase. By 2003, an improved transposase designated SB11 was made [21]. By 2005, SB16 with a 17fold activity increase compared to SB10 was successfully tested [67].

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15 Improvements to the transposon were also made to increase transposition rates. After studying the structurefunction relationship on the transposon sequences, improvements were made in 2002 resulting in a more efficient transposon named pT2. By 2005, variants of next generation and impr oved transposons have emerged [22, 67]. Apart from modifying the structure of the SB system, some studies also investigated the configurati on of the SB plasmids. Invest igators were curious to see whether transposition activity differed when the transposase was delivered in trans on a separate plasmid compared the delivery of the transposase on the same plasmid as the transposon (in cis ). The first cis -plasmid was tested in 2002, but transfection rate was low at an estimated 0.5%. A finding of lower transposition by a cis vector compared to a trans plasmid system was similarly report ed in 2003 [19]. However, Converse et al. reported that their cis -plasmid actually had bette r transfection rates in H uh7 cells, a traditionally difficult cell line to transfect [46]. These results suggest optim al SB transposase delivery may be dependent on and varies with cell type. Table 1-3. Milestones on Sleeping Beauty Improvements 1997 Reconstruction of Sleeping Beauty [16] Generation of an improved transposon, pT2 [24] 2002 Construction of a cis vector [68] 2003 Development of an improved transposas e, SB11 with 2.3 fold activity increase over SB10 [21] Development of endotheli al targeting SB [26] Development of hyperactive transposase 12 with 8-fold activity increase over SB10 and transposition usi ng the sandwich method [22] 2004 Development of hyperactive transposase mutants with 9-fold activity increase over SB10 [69] Development of SB16 with 17-fold in increased activity [67] 2005 Delivery of transposase in mRNA form [30]

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16 Moving Forward: Cell Specific Expression Using Sleeping Beauty Improvements have been made to optimize the SB transposition system. Studies are increasingly focused on optimizing delivery, directing site-speci fic integration, and targeting expression in specifi c cells or organs. Site sel ection using SB was attempted previously but has not been successful [32] . Once achieved, sustainability and long-term safety issues can be further investigated. As a natural progression, this study aims to achieve advances in the areas of delivery and expression. Cell specific expressi on will be attempted using the endothelin-1 and Tie2 endothelial cell-specific promot ers, with each driving the transposon and transposase respectively. The Tie2-driven transposase will be the fo cus of this study. The Tie2 promoter has demonstrated preferential activ ity in the arteriolar endothelium [70] and has uniform gene expression in endothelial cells from embryonic stage to adul thood [71]. Highly specific, it is also a weak promoter and therefore suitable for use to prevent overproduction inhibition [18]. Weaker promoters may also be better at driving long-term expression [34]. Endothelial targe ting using endothelin-1 for the transposon was previously successfully demonstrated [26, 49]. An endothelial-specific transposase has ne ver been tested before. This study hopes to demonstrate endothelial-specific expressi on by SB using both an endothelial-specific transposon and transposase. Using improved versions of the transposon and transposase, this study also seeks to investigate the optimum delivery configurati on of SB for endothelial cells by comparing cis and trans plasmids.

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17 Rationale for Endothelia Cell Expression Endothelial cells play impor tant roles in the human body. They are involved in vascular homeostasis regulation [70, 72, 73] and angiogenesis of tumor cells [74]. Therefore, any gene therapy applications that can be directed towards endothelial cells offer the promise of highly needed therapeu tic applications for car diovascular diseases. This study investigates the possibility of enhancing the SB system towards endothelial cell specificity where only such cells w ill express the delivered transgenes.

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18 CHAPTER 2 MATERIALS AND METHODS Plasmid Construction Endothelial-specific Transposase Construction of the endothelial-specific transposon and transposase were generated over several cloning steps using plasmids . The pTRUF-HSB16 plasmid contains a hyperactive SB16 transposase (1.0kb) and a poly(A) (0.2kb) signal. SB16 has a 17-fold activity over the original SB10 (67). The pT RUF-HSB16 plasmid was first restrictiondigested with Xba I. Using oligonucleotides and Po lymerase Chain Reaction (PCR), a Not I site and a Xho I site were introduced to flank th e SB16 and poly(A) sequence (Figure 2-1). Once the sites were created, the pl asmid was restriction-digested with Not I and Xho I, isolating the fragment containing the SB 16 and poly(A) signal cassettes. To remove an existing Sal I site between the SB16 and poly(A) sequences, the isolated fragment was cloned into pBluescriptIISK, generating pB luescript-SB. The pBluescript-SB plasmid was cut with Sal I, and that single Sal I site was replaced by a Pvu I. pTRUF-HSB167229 bp poly A CBA Promoter HSB16 XbaI (68) Figure 2-1. Generation of SB16 and poly(A) fragment. After Xho I and Not I sites were introduced into the plasmid via PCR, the Xho INot I fragment was isolated. X ho I No t I

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19 To obtain the Tie2 promoter (2.0kb) and enhancer (1.6kb) cassettes, the pSPTg.T2FXK plasmid (a kind gi ft from Dr. Tom Sato, Sout hwestern Medical Center, TX) was cut with Not I and Xho I. These two restriction sites are located between the Tie2 promoter and enhancer, there by allowing the insertion of a Not I / Xho I fragment in between the two cassettes (see Figure 2-2). pSPTg.T2FXK8004 bp Tie Enhance r Tie2 Promoter XhoI (1317) NotI (19) SalI (3016) SalI (5917) Figure 2-2. Addition of SB16 to the plasmid containing Tie2 prom oter and enhancer. XhoI and NotI fragment was removed and replaced with the Not I / Xho I fragment from pBlueScript-SB that contains SB16 and poly(A) sequences. The NotI / XhoI SB16 fragment from pBluescript-SB was then cloned into pSPTg.T2XK. The resulting plasmid called pBS-Tie-SB contained a Tie2 promoterdriven SB16 transposase (Figure 2-3) . pBS-Tie-SB7911 bp Tie2 Enhancer Poly A Tie2 Promoter HSB16 SalI site remove d SalI (3017) SalI (5824) Figure 2-3. Tie2-driven Sleeping Beauty transposase with a third Sal I site removed.

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20 Since pBS-Tie-SB was about 7.9kb long, it may not be the best plasmid for transposition use as long plasmids may redu ce transposition efficiency. To remove excess sequences, the pBS-Tie-SB plasmid was cut with Sal I, isolating a fragment containing the Tie2 promoter, enhancer, SB16, and poly(A) ca ssettes (4.8 kb). This isolated fragment was then cloned into pRKZ, a small 0.9kb plasmid containing an SV40 enhancer, RKII replication origin and a Zeocin selective marker (Figure 2-4). The final endothelialspecific transposase plasmid was named pT ie-HSB16 and is only 5.8kb long (Figure 2-5). pRKZ908 bp SV40 Enhancer RKII Ori Zeocin SalI (537) Figure 2-4. Illustration of th e endothelial-specific transpos ase backbone, a short sequence containing an origin of replication, a selective marker, and an SV40 Enhancer. pTie-HSB165819 bp Tie2 Enhancer SV40 Enhancer RKII Ori Zeocin PolyA Tie2 Promoter HSB16 Figure 2-5. Final components of the endotheli al-specific transposase, pTie-HSB16. The transgene is driven by a Tie2 promoter.

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21 Endothelial-specific Transposon The transposon plasmid pMSZ contains the IR/DR elements (0.23kb each) that flank a unique Not I site, making it convenient for transg ene insertion. This transposon is driven by the endothe lial cell-specific promoter endot helin-1 (0.43kb, a kind gift from Dr. Sean Sullivan, University of Florida) with an enhancer (0.19kb), a poly(A) signal (0.08kb) and contains a Zeocin selective mark er as well (Figure 2-6). This modified transposon was previously constructed in our lab and has enhanced transposition activity over the originally revived pT transposon ( 26). Endothelial-specifi c transposons carrying a Neomycin resistance gene (0.88kb), a SEAP reporter gene (1.5kb), and a luciferase gene (1.67kb) were separately construc ted and named pMSZ-Neo (3.7kb), pMSZ-SEAP (4.2kb), and pMSZ-Luc (4.4kb) respectively. pMSZ-Neo3748 bp Neo (Transgene) Zeocin ET-1 Enhancer R IR/DR L IR/DR RKII Ori poly A ET-1 Promoter SalI (3745) NotI (2361) NotI (3257) Figure 2-6. Endothelial-specific transposon. Illu strated here is pMSZ-Neo. The transgene cassette is flanked by Not I sites that ease cloning of a transgene of choice into the plasmid.

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22 Cis Plasmid To generate the single transposon-transposase cis plasmid, the pMSZ-Neo, pMSZSEAP, and pMSZ-Luc transposons were cut with Sal I to obtain linear plasmids. After treating with 0.7L calf intestine phosphatase for 45 minutes at 37 C to prevent selfligation, the plasmids were ligated to the 4.9kb Sal I fragment of pTie-HSB16 containing the endothelial-specific tr ansposase. Correct orient ation was verified using Not I cuts that resulted in 5.0kb, 2.5kb and the transgene fragments. The resulting transposontransposase plasmids were named pM SZ-Neo-Tie-HSB16 (8.2kb), pMSZ-SEAP-TieHSB16 (9.0kb), and pMSZ-Luc-Tie -HSB16 (9.17kb) (Figure 2-7). pMSZ-SEAP-Tie-HSB169012 bp Tie2 Enhancer Zeocin poly A ET-1 Promoter RKII Ori IR/DR IR/DR SEAP (Transgene) Tie2 Promoter ET-1 Enhancer HSB16 Figure 2-7. Two-in-one plasmi d containing both transposon a nd transposase components. Illustrated above is the pMSZ-SEAP-Tie-HSB16. Cell Culture and Transient Transfection HeLa (cervical) and human um bilical vein endothelial cells (HUVECs) were grown in DMEM high glucose from Invitrogen (C arlsbad, CA, USA) in 10cm tissue culture dishes. Fetal Bovine Serum (10%) from JRH Bioscience (Lenexa, KS, USA) and

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23 penicillin-streptomycin-glutamine mixture (Invi trogen) were added as supplements to the DMEM prior to use. The day before transfection, approximately 400,000 HUVECs or 500,000 HeLa were plated on 6cm dishes in triplicates and incubated in a 37 C 5% CO2 environment. LipofectAMINE Plus (Invitrogen) reagent was used to transfect HeLa cells, while GeneJammer from Strategene (La Jolla, CA, USA) was used for HUVECs. FACS Analysis Two days following transfection, transfecti on control plates with pMSZ-GFP were trypsinized and the cells scraped off. Collect ed cells were subjected to FACS analysis using Becton-Dickinson FACScan (San Jose, CA, USA). The results were analyzed using WinMDI to plot forward scatter versus fluores cence to estimate transfection efficiency. In vitro Transposition Assays Both endothelial-specific SB configurations were first tested in HeLa and HUVECs for activities. Approximately 3g of total transposon and transposase plasmid DNA was used for each transfection experiments in spec ific ratios. Five tr ansfection groups were tested in duplicates and repeated three times independently. Transf ection control groups consisted of the reporter plasmid pGreen Lantern and were used to gauge overall transfection rates in ea ch of the experiments. Negative control groups (Neg) consisted of pMSZ-Neo and pUC19. For no specific celltargeted controls (Nonspec), groups comprised of pMSZ-Neo and the CMV promoter-driven HSB16 were used. Separatelydelivered transposase groups (Trans) were made up of pMSZ-Neo and pTie-HSB16. Lastly, the Cis group consis ted of the transposon-transposase 2-in-1 plasmids. Due to differences in plasmid length and we ight, transfection ratios were calculated based on molar amounts. Weight conversions were made using the formula: [g DNA x

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24 (660pg/pmol) x (106 pg/1 g) x (1/N)] where N = number of nucleotides and 660pg/pmol is the average molecular weight of a nucleotide pair. Once the respective molarity was determ ined, the amount of transposase was calculated based on a 2 to 1 transposon:trans posase ratio. Using 1.5g of pMSZ-Neo with 0.629 pmol, the amount of transposase volum e required was 0.6L pTRUF-HSB16 and 0.6L pTie-HSB16. As for pMSZ-Neo-Tie-H SB16, a total of 1.5L was used. When needed, pUC19 was used to bring the total DNA to 3.0g. For HeLa using LipofectAMINE, a prep aration containing 250L Serum Free Media (SFM), 3.0g of total DNA, and 10L PLUS reagent was made for each plate. After 15 minutes of incuba tion, a second tube of mixt ure containing 250L SFM and 12L LipofectAMINE was added into the earl ier mixture. When the second 15-minute incubation period was over, 500L of the tota l mixture was added as drops onto each plate. The plates were incubated for 3 hour s, and the media was replaced by 3mL fresh serum enriched DMEM. For HUVECs, 18L of GeneJammer was a dded to 250L of SFM. After 7 minutes of incubation time, 3g of DNA was added and mixed well. The mixture was further incubated for another 7 minutes. Fresh se rum-enriched DMEM (2mL) are added onto each plate. The transfection mixture was th en added drop-wise onto the plates. After incubating the plates for 3 hours, another 2.5mL of serum enriched DMEM was added. The day following transfection, the media in the plates was replaced with fresh serum enriched DMEM. Two days after LipofectAMINE or Gene Jammer transfection, the cells were trypsinized and counted. A total of 40,000 cells per 10cm plate were plated in duplicate

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25 in medium containing Neomycin (1mg/ml). Af ter 7 days of undisrupted incubation, fresh Neomycin-containing media replaced the old ones . At day 14, the cells were washed with PBS, fixed for 15 minutes using 3.7% formalde hyde in PBS, stained for 30 minutes with 0.35% methylene blue in PBS, and finally ri nsed with water to remove the staining solution. Individual colonies on each plate from the three in dependently run experiments were manually counted (Figure 2-8). The result s were then tabulated and are reported in the next chapter. Figure 2-8. Example of stained Neomycin-resi stant colonies after transposition assay. Left and center plates are controls. The plate on right has tissue cells that were successfully transfected with SB. Animals and In vivo Gene Delivery Once activity is tissue culture was establis hed, both endothelial-specific SB were tested in animals. Four-week old C57B1/6 SCID mice from Jackson Laboratories (Bar Harbor, ME, USA) were housed under speci fic pathogen-free conditions. The animals were treated according to NIH Guidelines for Animal Care with approval of the IACUC of the University of Florida. A total of 60g DNA was used for SEAP assays while a constant 50g of DNA was used for luciferase localization studies. As an example, a 3:1 transposon-transposase ratio for lucifera se studies was calculated as follows:

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26 Table 2-1. Volumes of DNA for in vivo luciferase experiment. Negative Control Non-specific Control Trans Cis Transposon None (pcDNA3.1 used) 18.2L (37.5g) 12.5L (37.5g) Transposase None 3.04L (12.5g) 5L (12.5g) 16.67L (50g) Total 50g 50g 50g 50g In SEAP experiments involving the use of mice, the negative control group (Neg) was injected through the tail-vein with a transposon and non-functional transposase combination. For the no specific cell-targete d control group (Nonspe c), non-endothelial specific transposase was co -delivered with the tran sposon. For Trans and Cis, endothelial-specific transposase were co-deliv ered with the transposon and delivered as part of the transposon respectively. In the luciferase expression study using mice, the Neg Group was injected through the tail-vein with pcDNA3.1 while the Nons pec Group was injected with CMV-driven transposons and transposase. Endothelial-specific transpos on and transposase were codelivered in the Trans Group and delivered in a single plasmid for the Cis Group. Endotoxin-free DNA was extracted using Qi agen’s Endofree Giga Kit (Hilden, Germany). For SEAP studies, 60g of DNA and 10.8L of linear PEI from MBI Fermentas (Hanover, MD, USA) were separate ly prepared in 100L 5% glucose solution. For luciferase studies, 50g of DNA and 10L of Qbiogene’s in vivo jetPEI (Irvine, CA, USA) were separately prepared in 100L 5% glucose solution. PEI solutions were immediately a dded into DNA aliquots, vortexed, and centrifuged briefly. The DNA-PEI complex wa s calculated at a PEI nitrogen to DNA phosphate ratio of 1:6 for SEAP and 1:10 for luciferase studies. After 15 minutes of

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27 incubation at room temperatur e, the polyplexes were immedi ately administered into mice using tail-vein injection. SEAP Assay For the SEAP studies, serial blood sampling was obtained via tail bleeds on days +3, 7, 14, 28, 56, and 84. After tail bleeding, the samples were microcentrifuged (x8000 rpm) at 4C for 15 minutes. The serum was then stored at -20C. Using a chemiluminescence kit from Roche, Mol ecular Biochemicals (Mannheim, Germany), SEAP activity was detected and recorded. Brie fly, 10L of samples were diluted 1:5 in 1x reaction buffer and incubated for 30 minutes at 65C. After heat inactivation, the samples were centrifuged for 30 seconds at room temperature. It was then cooled on ice for 3 minutes. Next, the samples were transf erred to a 96-well plate prior to 50L of substrate reagent being added. The incubati on continued for another 10 minutes with gentle rocking. Once the incubation period is over, the plate was analyzed using a FLEX Station Benchtop Scanning Fluorometer from Molecular Devices Corp. (Sunnyvale, CA, USA). The amount of SEAP pr otein was quantified by compar ison to a standard curve, and plotted using the Prism 4 software from GraphPad (Aurora, CO, USA). Luciferase Assay Animals used for the luciferase assays we re sacrificed at +28 days post-injection. Following anesthesia, the mice were perfused with ice-cold PBS and the lungs, heart, liver, spleen and kidneys were harvested. The fresh organs were briefly washed in PBS, and immediately quick-frozen. After 5 minut es of immersion in liquid nitrogen, the tissues were stored at -80C until needed. The luciferase assay was performed by pr eparing a liquid-nitrogen chilled mortar and pestle. Frozen fresh tissues were indivi dually pulverized into a fine powder by hand

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28 grinding. After that, 300L of 1x Cell Culture Lysis Reagent from Promega (Madison, WI, USA) was added to the pulverized tissues for protein extraction. The lysis mixtures were vortexed regularly for 60 minutes. Next, the tubes contai ning the lysis mixture were quickly frozen in liquid nitrogen and thawed at 37C in a water bath. This process was repeated three times. The tubes were then cen trifuged at maximum speed for 5 minutes at 4C. The supernatants were collected in fresh tubes. Luciferase activity was analyzed by adding a specific volume of sample into 100L of Luciferase Assay Reagent. Lumines cence emitted over 10s by each sample was measured using Zylux’s Sirius Luminomete r (Oakridge, TN, USA). The readings were repeated three times for each sample. In order to determine the total protein concentration in the assayed tissue samples, Bio-Rad’s Brad ford DC Protein Kit (Hercules, CA, USA) was used. Luciferase activity is expressed as relative light units (RLU) per mg protein. Using results from the protein assays, each sample’s protein concentration were converted from g/ml to mg/l. Dividing the RLU units with the volume of sample used resulted in RLU/L. As such, RLU/mg prot ein can be calculated from the information obtained. Immunohistochemistry Animals were sacrificed at +28 days pos t-injection. Following anesthesia, the mice were perfused with ice-cold PBS and 4% paraformaldehyde (PFA) in PBS via cardiac puncture. The lungs were also inflated with this fixative followed by Tissue Tek Optimal Cutting Temperature compound in PBS in situ to prevent collapse. Lungs were harvested and post-fixed in 4% PFA overn ight at 4C. After that, the tissues were immersed overnight in 30% sucrose in PBS at 4C, qui ck-froze the next day and stored at -80C until needed.

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29 Cryosections (5m) were cut and the tissues were permeabilized by incubation with 100% methanol at -20C for 5 minutes. Next, the tissues we re blocked for 2 hours with 30% normal horse serum in PBS. The primary antibody for luciferase was a goat antiluciferase (Promega) and the primary antibody for endothelial cells wa s a rat anti-mouse CD31 (PECAM-1) (Becton Dickinson, Franklin Lakes, NJ). Samples were incubated with primary antibodies overnig ht at 4C at 1:50 dilutions. After 3 washes with PBS totaling an hour, Alexa Fluor red-conjugate d donkey anti-goat a nd Alexa Fluor greenconjugated rabbit anti-rat (Invitrogen) secondary antibodies were adde d at 1:300 at room temperature. After two hours, the slides were washed in PBS for 1 hour. Glass coverslips were then mounted using Vectashield Mounting Media (Vector Laboratories, Burlingame, CA) and samples were imaged us ing a Spot camera on a Nikon Eclipse 1000 Microscope. Images were prin ted using Adobe Photoshop 5.5.

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30 CHAPTER 3 RESULTS Endothelial-Specific Sleeping Beauty Transposition In order to further restrict expre ssion within endothelial cells, a Sleeping Beauty transposase driven by a Tie2 promoter was constructed. This transposase was meant to further augment the previously tested endot helin-1 driven transposon. That transposon has demonstrated robust endot helial expression [26]. Transfection Efficiency Before transposition assays commenced, transfection of HUVECs and HeLa cells was tested using pMSZ-GFP, a transposon th at contains a GFP reporter gene. This preliminary test was performed to gauge tr ansfection efficiency in the HUVECs and HeLa cell lines. FACS analysis showed tr ansfection rates of 13% 18% in HUVECs and 50% in HeLa cells. Figure 3-1. On the right is the evaluation of transfecti on using pMSZ-GFP in HUVECs. On the left is an analysis of cells transfected with pcDNA3.1. FACs analysis was performed at 48hours post-transfection.

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31 Figure 3-2. On the right is th e evaluation of transfection us ing pMSZ-GFP in HeLa cells. Graph on the left is the negative cont rol with pcDNA3.1-transfected cells. In vitro Transposition Assay Analysis Once transfection using the GFP expression ve ctor was verified, transposition assay in tissue culture commenced as desc ribed in Material and Methods. The in vitro assay was designed to evaluate tran sposition activity by compari ng Tie2-driven expression in HUVECs, an endothelial cell line, with the non-endothelial HeLa cell line. Experiments were carried out in dupli cates in three separately conducted experiments previously desc ribed. Colonies from Neg (negative control; pMSZ-Neo + pUC19), Nonspec (no specific cell-targe ted control; pMSZ-Neo + pTRUF-HSB16), Trans (separately delivered transposase; pMSZ-Neo + pTie-HSB16) and Cis (single plasmid containing transposon and transpos ase; pMSZ-Neo-Tie-HSB16) groups were manually counted. Each plate was divided in to 8 quadrants for ease of counting. The following results were representative of th e data obtained. Groups 1 to 6 are HUVECs while Groups 7 to 12 are HeLa cells.

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32 Table 3-1. Results from Groups 1 to 6 in HUVECs, conducted in duplicates, from three separately administered experime nts. Neg, negative group with only transposon; Nonspec, no specific cell -targeted group with transposon and CMV-driven transposase; Trans, tran s group with transposon co-delivered with endothelial-specific transposase; Cis, cis group with transposon and Tie2-driven transposase on a single plasmid. Group 1 Neg Nonspec TransCisGroup 2NegNonspec Trans Cis 0 13 5 21 3 16 6 10 3 9 9 19 7 17 7 12 4 6 9 17 2 17 10 17 4 13 10 14 3 11 10 19 4 12 5 28 1 15 15 18 1 14 4 25 1 9 14 18 1 16 7 15 6 9 6 15 1 13 14 14 4 17 6 12 5 12 18 16 0 7 2 10 1 8 17 12 0 4 3 11 1 6 16 14 1 5 2 16 2 8 14 14 0 11 4 9 3 13 14 11 0 8 7 11 4 8 13 15 0 6 6 10 5 8 11 16 2 9 3 11 Number of colonies 6 7 20 12 Number of colonies 2 13 7 13 Group 3 Neg Nonspec TransCis Group 4NegNonspec Trans Cis 3 16 20 22 2 17 19 30 3 19 14 13 1 20 16 30 2 20 9 17 4 24 13 22 5 25 6 22 7 21 12 29 2 20 6 25 6 16 12 30 2 20 9 26 6 9 14 24 2 23 14 23 6 19 13 25 4 24 17 28 4 31 16 26 0 14 15 34 6 18 10 22 7 20 17 36 1 15 10 19 1 22 10 31 2 23 13 27 0 23 10 28 3 17 11 25 2 24 7 18 5 20 10 27 1 13 17 24 2 20 18 33 2 15 24 21 1 15 19 29 Number of colonies 4 17 30 30 Number of colonies 3 15 15 25

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33 Group 5 Neg Nonspec TransCisGroup 6NegNonspec Trans Cis 4 4 0 2 1 7 2 6 2 4 2 5 4 9 3 7 1 11 6 4 3 8 1 1 0 7 4 8 1 1 5 3 2 12 2 6 1 11 3 3 1 11 6 2 0 8 1 5 0 7 3 3 0 14 3 4 0 5 6 3 3 9 6 2 2 6 8 3 2 11 7 4 4 5 4 5 3 9 5 5 0 5 5 5 3 7 3 7 1 14 3 2 2 7 7 4 3 4 2 1 0 7 1 4 0 17 4 5 3 14 4 7 5 6 5 2 0 8 4 11 Number of colonies 3 5 3 4 Number of colonies 2 12 4 7 Negative Nonspec Trans Cis Total 232 1220 862 1436 Mean 19.33 101.67 71.83 120.00 Standard Deviation 8.44 41.50 40.44 74.51 Standard Error 2.44 11.98 11.68 21.51 Comparable expression activity was achie ved in HUVECs between the endothelialdriven transposase and the CMV-driven transposase for the positive control group. When compared to the positive control, both th e Tie2-driven transposase groups achieved similar transposition ra tes. The mean trans position between the nonspecific control and the Trans and Cis groups were statistically indifferent (t-T est for differences in two means, p>0.05).

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34 Transposition Activity HUVECs120.00 71.83 19.33 101.67 0.00 50.00 100.00 150.00 200.00 250.00 NegNonspecTransCis Transfection Groups NumberofColonies Figure 3-3. Results of HUVECs transposition. Ne omycin resistant colonies were counted after 2 weeks post-transfection. Neg, ne gative group with only transposon; Nonspec, no specific cell-targeted gr oup with transposon and CMV-driven transposase; Trans, trans group with tr ansposon co-delivered with endothelialspecific transposase; Cis, cis gr oup with transposon and Tie2-driven transposase on a single plasmid. The numb er above each bar denotes the mean while the standard deviations ar e reported as Y-error bars.

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35 Table 3-2. Results from Groups 7 to 12 in HeLa cells, conducted in duplicates, from three separately administered experime nts. Neg, negative group with only transposon; Nonspec, no specific cell -targeted group with transposon and CMV-driven transposase; Trans, tran s group with transposon co-delivered with endothelial-specific transposase; Cis, cis group with transposon and Tie2-driven transposase on a single plasmid. Group 7 Neg Nonspec TransCisGroup 8NegNonspec Trans Cis 1 89 37 45 1 110 23 60 0 48 37 34 2 145 26 40 4 53 34 34 1 100 31 55 3 71 34 38 1 84 40 63 4 99 28 26 2 136 30 69 1 51 22 32 2 166 49 51 3 70 23 38 2 122 39 57 1 90 35 37 1 87 25 42 1 74 46 62 1 64 50 42 2 84 60 53 2 50 41 30 1 150 86 45 2 60 32 39 0 113 44 50 1 134 36 44 0 90 33 48 1 95 50 42 2 100 28 60 0 78 44 41 1 120 33 46 0 112 33 24 Number of colonies 1 101 34 65 Number of colonies 1 83 48 29 Group 9 Neg Nonspec TransCisGroup 10NegNonspec Trans Cis 2 90 29 34 1 155 35 37 0 153 23 33 1 136 26 40 0 148 19 29 0 94 15 41 2 153 31 28 2 89 20 34 1 98 29 38 1 102 16 25 3 94 32 40 1 115 33 36 1 100 17 32 0 132 24 30 0 78 19 25 1 140 29 24 2 122 35 34 0 150 20 46 0 145 34 36 0 112 40 39 0 89 22 23 0 85 40 32 2 83 19 44 1 102 16 39 1 84 21 30 0 90 17 30 0 89 24 32 0 73 19 27 0 140 27 23 3 86 30 35 Number of colonies 0 138 31 20 Number of colonies 0 120 30 47

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36 Group 11 Neg Nonspec TransCisGroup 12NegNonspec TransCis 0 134 36 32 3 125 24 20 0 93 36 22 1 95 20 20 3 81 9 19 1 70 21 32 1 111 23 27 3 103 27 40 2 73 25 25 1 93 26 34 1 94 20 29 0 57 19 30 1 102 29 26 0 81 20 31 1 94 19 20 0 110 21 26 0 95 21 28 0 125 32 18 0 73 18 23 2 95 20 34 0 62 14 23 0 70 20 37 0 50 19 28 0 103 21 23 2 80 23 32 0 93 21 31 3 80 18 27 2 57 30 26 1 100 22 25 0 81 28 20 Number of colonies 1 112 19 26 Number of colonies 0 110 18 19 Neg Nonspec Trans Cis Total 99 9516 2752 3357 Mean 8.25 793 229.33 279.75 Standard Deviation 3.60 124.20 63.26 78.44 Standard Error 1.04 35.85 18.26 22.64 In HeLa cells, the strong CMV promoter-dri ven transposase had almost three times the expression activity over the weaker endothe lial cell-specific Tie2 promoter. Using a tTest, the results were significantly differe nt between the non-specific control and the Trans and Cis groups (p<0.05). By comparing re sults from both transposition activities in HeLa and HUVECs, robust endot helial specific expression wa s observed using the Tie2 promoter in HUVECs as the activity was compar able to the CMV-driven transposase. As expected, the Tie2 promoter’s activity in th e non-endothelial HeLa cells appears to be more restricted when compared to the CMV promoter.

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37 Transposition Activity HeLa279.75 229.33 793 8.25 0 100 200 300 400 500 600 700 800 900 1000 NegNonspecTransCis Transfection Groups NumberofColonies Figure 3-4. Results of HeLa transposition. Ne omycin resistant colonies were counted after 2 weeks post-transfection. Neg, ne gative group with only transposon; Nonspec, no specific cell-targeted gr oup with transposon and CMV-driven transposase; Trans, trans group with tr ansposon co-delivered with endothelialspecific transposase; Cis, cis gr oup with transposon and Tie2-driven transposase on a single plasmid. The numb er above each bar denotes the mean while the standard deviations are reported as Y-error bars.

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38 Endothelial-Specific Sleeping Beauty Expression In vivo Long Term SEAP Secretion The previous experiment demonstrates that the designed constructs work in tissue culture, yet they still have to be tested in vivo to gauge their efficacy in a real biological environment. The SEAP studies serve to answ er not only whether the endothelial specific SB system works in an animal, but also wh ether such expression can be maintained over the long term. Mice were inject ed through the tail-vein with the four test groups as described under Materials and Methods. Blood was collected and SEAP expression in the bloodstream was analyzed over an 84-day period: Table 3-3. Results of SEAP detection at 6 different time points post-injection. Numbers reported represent ng/ml of SEAP prot ein present in the bloodstream. Neg, negative group with tran sposon and non-functional tr ansposase; Nonspec, no specific cell-targeted group with tran sposon and CMV-driven transposase; Trans, trans group with transposon co -delivered with endothelial-specific transposase; Cis, cis group with tran sposon and Tie2-driven transposase on a single plasmid. Day 3 Avg Day 7 Avg Day 14 Avg Day 28 Avg Day 56 Avg Day 84 Avg 897.9 101.5 45.9 62.4 1.6 16 1414.7 101.8 71.3 61.5 28 57.2 Neg 1168 1160 107.7 103.6 39.5 52.2 20 47.9 60.7 30.1 28.2 33.8 1142.3 305 337.4 353.4 244.2 284 1588.8 265.3 266 254.2 303 244.8 Nonspec 1229.3 1321 269.5 279.9 256.6 286.6 237.2 281.6 364.1 303.7 335.2 288 864.5 235 152.7 160.4 207.6 154.8 1425.7 196.3 150.6 207.5 233 181 Trans 1300.1 1197 150.2 193.8 152.2 151.8 158.6 175.5 211 217.1 292.1 209.3 1719.4 149.1 115.2 147.2 228.2 244.9 861.3 185.3 102.1 172.9 165.6 198.6 Cis 1392.9 1325 136.7 157 82.2 99.8 174.6 164.9 210.9 201.5 138.8 194.1

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39 Long Term SEAP Activity in Mice 0 10 20 30 40 50 60 70 80 90 0 100 200 300 400 500Pos Nonspec Trans Cis 900 1400Time (days)SEAP Activity (ng/ml) Figure 3-5. Long term SEAP expression in mice, up to day +84. Neg, negative group with transposon and non-functional tran sposase; Nonspec, no specific celltargeted group with transposon and CM V-driven transposase; Trans, trans group with transposon co-delivered with endothelial-specific transposase; Cis, cis group with transposon and Tie2-d riven transposase on a single plasmid. Long term SEAP activity was observed at 12 weeks in mice injected with SB transposon and transposase. The amount of SEAP protein for each sample was quantified by comparison to a standard curve and reported as ng/ml. The Nonspec group that was injected with the CMV-driven transposase had the most robust activity, while the Neg group that was injected with just a transpos on but with no functiona l transposase had only background levels of activity. The two groups injected with the endothelial-driven

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40 transposase, Trans and Cis, also showed stab le long term activity. Both the trans and cis plasmids have similar expression levels. Thes e data suggests that the endothelial-driven transposase works in an animal model, f acilitates integration, and enables long term expression in vivo . Luciferase Assay and In Vivo Expression in Tissues Once systemic long-term expression of the transgene in mice was demonstrated, non-specific and endothelial-specific SB ca rrying the luciferase reporter gene was injected into mice thr ough the tail-vein. This part of the experiment sought to determine the location and amount of luciferase prot ein being expressed in major organs. The organs investigated were lungs, heart, liver , spleen, and kidneys. Detection was through the measurement of relative light units (RLU ) emitted per mg protein. When luciferase protein is detected in an organ, it is an i ndication that the luciferase transgenes were successfully delivered and are being e xpressed by cells in the organs. The following tables are representative of luciferase expression detected in lungs, heart, liver, spleen, and kidne ys at day +28 post-injection. There were three mice in the Nonspec and Trans test groups, while two mi ce were used for the Neg and Cis groups. Supernatant was taken from each tissue type of each animal and read three times for RLU detection on a luminometer. The protein amount in each sample was also determined and expressed as mg/l. Tables 3-4 to 38 summarize the average RLU and protein concentrations detected in the various orga ns. In order to calculate RLU per mg of protein, the average RLU/mg of each mous e was determined. Next, averages were calculated for each group. Tables 3-9 to 3-13 summarize the RLU per mg of protein calculations.

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41 Robust luciferase expression was detected in lungs on day +28 post-injection in the Nonspec, Trans and Cis groups (see Figure 36). Negative control re adings of the lung registered at a low 2.5 X 102. Readings of 6.9 X 104 from the Nonspec tissue were highest. The Trans and Cis tissues had lo wer readings, but still in the robust 104 and 103 ranges respectively. Trans also had twice the activity of Cis. Interestingly, there was a high level of activity in the Nonspec liver tissue (see Figure 3-7). The results were ra ther unexpected, as one might expect only low levels of activity in liver tissues following tail-vein administration. The luciferase activities in Nonspec tissues turned out to be much stronger and were in the 1.4 X 104 range. However, much lower activity was detected in Trans and Cis tissues when compared to Nonspec. This lower reading could signal the effects of the Tie2 pr omoters. Activity in Trans tissues were again highe r than the Cis counterpart. RLU / mg readings of 1.4 X 103 were detected in heart tissues from the Trans group, while results from the Cis group were lower at 8.5 X 102 (see Figure 3-8). Both levels of expression in Trans and Cis were similar to the Nonspec group. In addition, the results suggest positive activity in the heart when compared to the negative control. However, activity is not as robust as those observed in the lung. There was no luciferase expression detect ed in spleen. Nonspec, Trans, and Cis tissues had no significant activ ity levels (see Figure 3-9). There were no luciferase activities detected in the kidneys in any of the groups. All RLU / mg readings were registered in the 102 range (see Figure 3-10). In summary, results from the lucifera se study indicate that the most robust expression obtained was in lung tissues. The N onspec, Trans, and Cis groups registered

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42 high levels of activity. A high level of activity was also detected in the liver for the nonendothelial specific Nonspec group. Trans and Cis on the other hand, had low levels of activity in the liver. The second highest level of activity for th e Trans and Cis groups was detected in the heart. However, the levels detected in the heart were much lower than the activities found in the lungs. This finding of the Tran s and Cis groups differs from the Nonspec group. In Nonspec, the second highest level of activity was found in the liver, not the heart. No activities were detected in either spleen or kidney for all groups. Figure 3-11 summarizes the activities detected in each of the organ tissues. Table 3-4. Average relative light units and pr otein concentrations detected in lung. Neg, negative group with only pcDNA3.1; Nons pec, no specific cell-targeted group with CMV-driven transposon and CMVdriven transposase; Trans, trans group with ET-1 driven transposon co-deliv ered with Tie2-driven transposase; Cis, cis group with ET-1 driven trans poson and Tie2-driven transposase on a single plasmid. Samples from the tissue from each mouse in each group were read three times. N.A., not applicable. Lung Mouse 1 (RLU/5l) Protein (mg/l) Mouse 2 (RLU/5l) Protein (mg/l) Mouse 3 (RLU/5l) Protein (mg/l) Neg 42 0.037 46 0.031 N.A. N.A. Nonspec 9069 0.025 17301 0.036 4306 0.020 Trans 557 0.033 4865 0.033 2741 0.015 Cis 1871 0.024 838 0.026 N.A. N.A.

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43 Table 3-5. Average relative light units and prot ein concentrations detected in heart. Neg, negative group with only pcDNA3.1; Nons pec, no specific cell-targeted group with CMV-driven transposon and CMVdriven transposase; Trans, trans group with ET-1 driven transposon co-deliv ered with Tie2-driven transposase; Cis, cis group with ET-1 driven trans poson and Tie2-driven transposase on a single plasmid. Samples from the tissue from each mouse in each group were read three times. N.A., not applicable. Heart Mouse 1 (RLU/5l) Protein (mg/l) Mouse 2 (RLU/5l) Protein (mg/l) Mouse 3 (RLU/5l) Protein (mg/l) Neg 60 0.028 47 0.035 N.A. N.A. Nonspec 120 0.042 242 0.043 164 0.042 Trans 124 0.029 147 0.046 317 0.022 Cis 154 0.033 103 0.025 N.A. N.A. Table 3-6. Average relative light units and pr otein concentrations detected in liver. Neg, negative group with only pcDNA3.1; Nons pec, no specific cell-targeted group with CMV-driven transposon and CMVdriven transposase; Trans, trans group with ET-1 driven transposon co-deliv ered with Tie2-driven transposase; Cis, cis group with ET-1 driven trans poson and Tie2-driven transposase on a single plasmid. Samples from the tissue from each mouse in each group were read three times. N.A., not applicable. Liver Mouse 1 (RLU/2l) Protein (mg/l) Mouse 2 (RLU/2l) Protein (mg/l) Mouse 3 (RLU/2l) Protein (mg/l) Neg 79 0.141 79 0.135 N.A. N.A. Nonspec 4840 0.164 3600 0.153 4237 0.128 Trans 105 0.144 673 0.153 210 0.136 Cis 195 0.172 89 0.144 N.A. N.A.

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44 Table 3-7. Average relative light units and prot ein concentrations detected in spleen. Neg, negative group with only pcDNA3.1; Nons pec, no specific cell-targeted group with CMV-driven transposon and CMVdriven transposase; Trans, trans group with ET-1 driven transposon co-deliv ered with Tie2-driven transposase; Cis, cis group with ET-1 driven trans poson and Tie2-driven transposase on a single plasmid. Samples from the tissue from each mouse were read three times. N.A., not applicable Spleen Mouse 1 (RLU/30l) Protein (mg/l) Mouse 2 (RLU/30l) Protein (mg/l) Mouse 3 (RLU/30l) Protein (mg/l) Neg 48 0.012 73 0.008 N.A. N.A. Nonspec 80 0.005 121 0.014 103 0.006 Trans 76 0.009 91 0.015 83 0.008 Cis 98 0.012 69 0.007 N.A. N.A. Table 3-8. Average relative light units and protein concentrations detected in kidney. Neg, negative group with only pcDNA3.1; N onspec, no specific cell-targeted group with CMV-driven transposon a nd CMV-driven transposase; Trans, trans group with ET-1 driven transpos on co-delivered with Tie2-driven transposase; Cis, cis group with ET1 driven transposon and Tie2-driven transposase on a single plasmid. Samples from the tissue from each mouse were read three times. N.A., not applicable. Kidney Mouse 1 (RLU/5l) Protein (mg/l) Mouse 2 (RLU/5l) Protein (mg/l) Mouse 3 (RLU/5l) Protein (mg/l) Neg 50 0.097 54 0.093 N.A. N.A. Nonspec 71 0.098 46 0.060 58 0.087 Trans 47 0.086 51 0.076 65 0.112 Cis 67 0.109 65 0.099 N.A. N.A.

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45 Table 3-9. Average relative light units pe r mg in lung. Neg, negative group with only pcDNA3.1; Nonspec, no specific cell -targeted group with CMV-driven transposon and CMV-driven transposase; Trans, trans group with ET-1 driven transposon co-delivered with Tie2-drive n transposase; Cis, cis group with ET1 driven transposon and Tie2-driven transposase on a single plasmid. N.A., not applicable. Lung Mouse 1 RLU/mg Mouse 2 RLU/mg Mouse 3 RLU/mg Group Average RLU/mg Neg 223 287 N.A. 255 Nonspec 72349 95139 42382 69957 Trans 3327 29307 35311 22648 Cis 15624 2895 N.A. 9259 Luciferase Expression in Lung1 10 100 1000 10000 100000 NegNonspecTransCisRLU / mg Figure 3-6. Luciferase expressi on in lung illustrated on a l og scale. Neg, negative group with only pcDNA3.1; Nonspec, no speci fic cell-targeted group with CMVdriven transposon and CMV-driven tran sposase; Trans, trans group with ET-1 driven transposon co-delivered with Ti e2-driven transposase; Cis, cis group with ET-1 driven transposon and Tie2-dri ven transposase on a single plasmid.

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46 Table 3-10. Average relative light units per mg in liver. Neg, negative group with only pcDNA3.1; Nonspec, no specific cell -targeted group with CMV-driven transposon and CMV-driven transposase; Trans, trans group with ET-1 driven transposon co-delivered with Tie2-drive n transposase; Cis, cis group with ET1 driven transposon and Tie2-driven transposase on a single plasmid. N.A., not applicable. Liver Mouse 1 RLU/mg Mouse 2 RLU/mg Mouse 3 RLU/mg Group Average RLU/mg Neg 279 291 N.A. 285 Nonspec 14707 11738 16555 14333 Trans 367 2191 770 1109 Cis 567 307 N.A. 437 Luciferase Expression in Liver1 10 100 1000 10000 100000 NegNonspecTransCisRLU / mg Figure 3-7. Luciferase expressi on in liver illustrated on a l og scale. Neg, negative group with only pcDNA3.1; Nonspec, no speci fic cell targeted group with CMVdriven transposon and CMV-driven tran sposase; Trans, trans group with ET-1 driven transposon co-delivered with Ti e2-driven transposase; Cis, cis group with ET-1 driven transposon and Tie2-dri ven transposase on a single plasmid.

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47 Table 3-11. Average relative light units per mg in heart. Neg, negative group with only pcDNA3.1; Nonspec, no specific cell -targeted group with CMV-driven transposon and CMV-driven transposase; Trans, trans group with ET-1 driven transposon co-delivered with Tie2-drive n transposase; Cis, cis group with ET1 driven transposon and Tie2-driven transposase on a single plasmid. N.A., not applicable. Heart Mouse 1 RLU/mg Mouse 2 RLU/mg Mouse 3 RLU/mg Group Average RLU/mg Neg 426 264 N.A. 345 Nonspec 563 1123 773 820 Trans 830 627 2863 1440 Cis 912 794 N.A. 853 Luciferase Expression in Heart1 10 100 1000 10000 NegNonspecTransCisRLU / mg Figure 3-8. Luciferase expression in heart il lustrated on a log scal e. Neg, negative group with only pcDNA3.1; Nonspec, no speci fic cell-targeted group with CMVdriven transposon and CMV-driven tran sposase; Trans, trans group with ET-1 driven transposon co-delivered with Ti e2-driven transposase; Cis, cis group with ET-1 driven transposon and Tie2-dri ven transposase on a single plasmid.

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48 Table 3-12. Average relative light units per mg in spleen. Neg, negative group with only pcDNA3.1; Nonspec, no specific cell -targeted group with CMV-driven transposon and CMV-driven transposase; Trans, trans group with ET-1 driven transposon co-delivered with Tie2-drive n transposase; Cis, cis group with ET1 driven transposon and Tie2-driven transposase on a single plasmid. N.A., not applicable. Spleen Mouse 1 RLU/mg Mouse 2 RLU/mg Mouse 3 RLU/mg Group Average RLU/mg Neg 127 288 N.A. 208 Nonspec 499 287 559 448 Trans 267 200 324 264 Cis 258 298 N.A. 278 Luciferase Expression in Spleen1 10 100 1000 10000 NegNonspecTransCisRLU / mg Figure 3-9. Luciferase activity detected in spleen illustrated on a log scale. Neg, negative group with only pcDNA3.1; Nonspec, no specific cell-targeted group with CMV-driven transposon and CMV-driven transposase; Trans, trans group with ET-1 driven transposon co-delivered with Tie2-driven transposase; Cis, cis group with ET-1 driven transposon and Tie2-driven transposase on a single plasmid.

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49 Table 3-13. Average relative light units per mg in kidney. Neg, negative group with only pcDNA3.1; Nonspec, no specific cell -targeted group with CMV-driven transposon and CMV-driven transposase; Trans, trans group with ET-1 driven transposon co-delivered with Tie2-drive n transposase; Cis, cis group with ET1 driven transposon and Tie2-driven transposase on a single plasmid. N.A., not applicable. Kidney Mouse 1 RLU/mg Mouse 2 RLU/mg Mouse 3 RLU/mg Group Average RLU/mg Neg 102 116 N.A. 109 Nonspec 144 154 134 144 Trans 109 135 117 120 Cis 123 131 N.A. 127 Luciferase Expression in Kidney1 10 100 1000 10000NegNonspecTransCisRLU / mg Figure 3-10. Luciferase expres sion in kidney illustrated on a log scale. Neg, negative group with only pcDNA3.1; Nonspec, no specific cell-targeted group with CMV-driven transposon and CMV-driven transposase; Trans, trans group with ET-1 driven transposon co-delivered with Tie2-driven transposase; Cis, cis group with ET-1 driven transposon and Tie2-driven transposase on a single plasmid.

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50 Luciferase Expression in Tissues1 10 100 1000 10000 100000 LungLiverHeartSpleenKidneyRLU / mg Neg Nonspec Trans Cis Figure 3-11. Summary of lucife rase expression in organ tissu es on day +28 post-injection illustrated on a log scale. Expression reported on a log scale. Neg, negative group with only pcDNA3.1; Nonspec, no specific cell-targeted group with CMV-driven transposon and CMV-driven transposase; Trans, trans group with ET-1 driven transposon co-delivered with Tie2-driven transposase; Cis, cis group with ET-1 driven transposon and Tie2-driven transposase on a single plasmid. Localization of Transgene in Organ Tissues Once expression in some tissues was established, immunohistochemistry was performed to determine cellula r localization of luciferase in lung through delivery of SB using PEI. Using anti-lucifer ase and anti-endothelia l primary antibodies, perfusion-fixed tissues were labeled with red-conjugated secondary antibody for Luciferase and greenconjugated secondary antibody for endothelial cells as described in Materials and Methods. Localization of luciferase in endot helial cells was evaluated by locating cells that exhibit both colors.

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51 Figure 3-12. Luciferase locali zation in lung tissue of Trans group. Green denotes staining of the endothelial marker CD31. Red denot es luciferase protein while blue denotes nuclei stain. Luciferase marker in red was observed in what are possibly capillary tissues, which are immuno-labeled in green. This prelim inary observation sugge sts the likelihood of specific transgene expression w ithin endothelial cells. We are working on the antibody dilution factors to obtain even higher quality pi ctures of the transgen e localization in lung tissues.

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52 CHAPTER 4 DISCUSSION For any gene therapy technique to advance to the clinical stage, it must first be proven effective and safe in preclinical studies. In Sleeping Beauty’s (SB) case, most current research is focused on three major areas that will he lp advance its gene therapy candidacy: overall efficacy, best delivery method, and tissue-specific targeting. This research project was focused on two of the th ree mentioned areas. More specifically, this research sought to answer the following questions: 1) Can SB be adapted to induce specific expr ession in targeted cell types? Since SB relies on popular co-delivery platforms such as polyplexes to penetrate cell walls, transposition is usually random in regard s to cell type. The majority of such delivery mechanisms are also usually non-sp ecific. As a result, SB will integrate into many different cell types. To minimize expression of transgene in certain cell types, promoters can be used to driv e expression in selected cells. This countermeasure strategy is especially useful if targeted treatment of an area or cell type is sought. In this study, endothelial ce lls were chosen as target cells because of their important roles in vascular ho meostasis, blood pressure regulation and cardiovascular diseases. In a ddition, they play a role in vascular angiogenesis and thus are important for tumor growth. Usi ng the Tie2 and endothelin-1 promoters, an endothelial-specific Sleeping Beauty transposon system may therefore potentially be a useful tool for treating cardiovascular diseases or targeting tumor vasculature in the future. This study is the first demonstrated case where each

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53 component of the SB system is driv en in a cell-specific manner targeting endothelial cells. 2) What is the best delivery configuration for an endothelial-specific SB? Previous reports have conflicting fi ndings (19, 46). Whether a trans or cis configuration is more efficient for transgene delivery is probably dependent on cell and tissue types, as well as the site and method of delivery. This research hopes to provide us with a better understanding of SB’s best configuration for systemic delivery in a controlled setting. To answer both questions, a series of experi ments was planned to test the use of an endothelial-specific SB system. Transpositi on assays were first performed by comparing an endothelial-specific SB to a non-endothe lial specific SB. The endothelial-specific SB’s activity was lower compared to the non-en dothelial SB in the HeLa cell line (Figure 3-4). Its activity was almost three times lo wer when compared to the CMV-driven SB. However, endothelial-specific SB activity was robust in the endothelial HUVECs. In fact, it reached levels of activity similar to the CMV promoter-driven SB (Figure 3-3). Statistical analysis suggests there were no differences in tr ansposition activity for the CMV-driven and Tie2-driven SB within e ndothelial cells. This finding supports the notion that SB expression can be restricted in specific cell types by using an appropriate promoter. Results of the transposition assay also s uggest that both Trans and Cis vectors had similar in vitro activities in HUVECs . Using a t-Test, both results were found to be statistically similar (p<0.05) . Thus, preliminary findings suggested that the plasmid configuration may not influe nce transposition activity in vitro .

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54 Once evidence of possible e ndothelial specificity in tissue cultur e cells was documented, confirmatory tests were conducte d to gauge an endothe lial-specific SB’s overall activity in vivo . Because the cellular environment in a body is different and more complex than a tissue culture’s controlled setting, in vivo tests using mice were performed to evaluate SB expression and stability. Four-week old mice were injected through the tail-vein with the four plasmid groups containing either the controls or endothelial-specific SB carryi ng a SEAP gene. Blood was colle cted at various time points and long-term SEAP expression in the bl oodstream was detected up to day +84 postinjection (Figure 3-5). Similar long-term SE AP expression using an endothelial-specific transposon was previously reported (26). Fo r the first time, this study demonstrates prolonged SEAP expression using SB with an endothelial-restricted transposase. This finding is promising, as it suggests successful transposition within endothelial cells in vivo enabling long-term tr ansgene expression in an animal model. As indicated from the in vitro studies, the Trans and Cis groups had similar activities in vivo. Both SEAP secretions in the bloodstream were of similar levels. Again, preliminary findings suggest that trans position may not be affected by plasmid configuration in vivo , so long as delivery and chromosomal integr ation are presumably successful to allow transgene protein secretion. Once expression in an animal model was demonstrated, the next step was to determine the transposition sites. Hence, a lu ciferase assay was conducted to evaluate the amount of transgene protein expression in various organs of mice in each test group. Again, four-week old mice were injected with the controls and e ndothelial-specific SB carrying a luciferase reporter gene. This part of the experi ment looks at sites where SB

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55 delivery in mice was most successful wh en administered systemically through intravenous injection. According to the results, lungs were the most targeted tissue when SB is administered through the tail vein (Figure 3-11). The finding was expected as previous studies have demons trated successful delivery of SB to the lungs through veins (19, 26, 49). There was some activity detected in the heart for Trans and Cis, but Cis had lower activity compared to Trans (Figure 3-8) . There was a high level of activity in liver in the Nonspec group, but significantly less readin gs were detected in the livers of Trans and Cis groups (Figure 3-7). This muted activ ity may be the result of the Tie2 promoters that may not be as active in non-vascular e ndothelial cells. No ac tivity was found in the kidney or spleen (Figures 3-9 and 3-10). Because there were three animals in the Nonspec and Trans test groups, the finding is significant and suggests an interesting outcome that warrants further evaluation. We are in the process of analyzing more animals to reach statistical significance in ot her groups, but we expect similar results will be obtained. Based on data from the transposition assay and SEAP expression study, one might have expected that both Trans and Cis woul d have similar expression activity in organ tissues. Surprisingly, there is preliminary evidence to suggest that Trans may have better expression activity compared to Cis in lung, h eart, and liver tissues. Trans has twice the activity compared to Cis in lungs (Figure 3-6) . It will be interesting to see the outcome when an additional mouse is used for the Cis group in a future study, making it three animals for the group studied.

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56 Last, for physical verifica tion, immunohistochemistry wa s performed to identify expression sites within lung tissues in the Tr ans animal. Present staining results suggest the likelihood of luciferase e xpression in endothelial cells. As for the next steps, an expanded study involving more animals per group may be helpful to verify and substantiate lucifera se expression within organ tissues. We can further study the possibility of gene therapy targeting the lung and liver using vein administration. Immunohistochemistry of the other major organs can also be performed to look for additional physical evidence of endothelial targeting. For the future, studies can be done to inve stigate the possibility of targeted sitedelivery using SB and special polyplexes with specific conjugated peptides or ligands so that only targeted cells will engulf the transf ection complexes. Such strategies have been demonstrated previously (75). If the developm ent of a targeted SB delivery is successful, then expression within specific cell types w ill be further enhanced on both delivery and expression aspects. Along with expected continued developments in increasing transposition efficiency, these advancements will likely propel SB to the forefront of gene therapy. In conclusion, a newly cons tructed endothelial-specific Sleeping Beauty system was successfully developed that showed activity in both cell culture and in animal model systems. Tie2 promoters can be used to rest rict expression to endot helial cells and to minimize levels in other cell types. Trans a ppears to be more efficient as a delivery configuration in vivo . It is hoped that this re search has helped advance Sleeping Beauty towards becoming a gene therapy applicati on for cardiovascular and cancer-related diseases.

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57 LIST OF REFERENCES 1. Edelstein, M.L., Abedi, M.R., Wixon, J., Edelstein, R.M. (2004). Gene therapy clinical trials worldwide 1989-2004 – an overview. J. Gene Med . 6 : 597-602. 2. Gardlik, R., Palffy, R., Hodosy, J., Lukacs, J., Turna, J., Celec, P. (2005). Vectors and delivery systems in gene therapy. Med. Sci. Monit . 11 (4): RA110-121. 3. Wilson, J.M. (2004). Adeno-associated vi rus and lentivirus pseudotypes for lungdirected gene therapy. Proc. Am. Thorac. Soc . 1 : 309-314. 4. Moss, R.B., Rodman, D., Spencer, L.T., Aitken, M.L., Zeitlin, P.L., Waltz, D., Milla, C., Brody, A.S., Clancy, J.P., Rams ey, B., Hamblett, N., Heald. A.E. (2004). Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to th e lungs of patients with cystic fibrosis: A multicenter, double blind, pl acebo-controlled trial. Chest . 125 : 509-521. 5. Flotte, T.R. (2005). Adeno-associated vi rus-mediated gene transfer for lung diseases. Hum. Gene Ther . 16 : 643-648. 6. Mitchell, R.S., Beitzel, B.F., Schroder, A.R.W., Shin, P., Chen, H., Berry, C.C., Ecker, J.R., Bushman, F.D. (2004). Retrov iral DNA Integration: ASLV, HIV, and MLV show distinct targ et site preferences. PLoS Biol . 2 (8): e234. 7. Tan, P.H., Xue, S., Manunta, M., Beutelspacher, S.C., Fazekasova, H., Alam, A.K.M.S., McClure, M.O., George, A.J. T. (2006). Effects of vectors on human endothelial cell signal transduction. Impli cations for cardiovascular gene therapy. Arterioscler. Thromb. Vasc. Biol . 26 (3): 462-467. 8. Patil, S.D., Rhodes, D.G., Burgess, D. J. (2005). DNA-based therapeutics and DNA delivery systems: A comprehensive review. AAPS J. 7 (1): E61-77. 9. Ehrhardt. A., Xu, H., Huang, Z., Engl er, J.A., Kay, M.A. (2005). A direct comparison of two nonviral gene therapy v ectors for somatic integration: In vivo evaluation of the bacteriophage integrase c31 and the Sleeping Beauty transposase. Mol. Ther . 11 (5): 695-706. 10. Lam, W.L., Seo, P., Robison, K., Virk, S., Gilbert, W. (1996). Discovery of amphibian Tc1 -like transposon families. J. Mol. Biol . 257 : 359-366. 11. Osborne, B.I., Baker, B. (1995). Movers a nd shakers: maize transposons as tools for analyzing other plant genomes. Curr. Opin. Cell. Biol. 7 : 406-413.

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64 BIOGRAPHICAL SKETCH Wei Joon Brian Lam was born in Ipoh, Mala ysia, in 1973 and is a citizen of the Republic of Singapore. After completing his high school education at St. Michael’s Institution, he attended St. Lawrence University in upstate New York. Brian received his Bachelor of Science degree in May 1995, with a major in biology and a multifield minor in theatre and music. In Apr il 1999, he received his Graduate Diploma in marketing from the Marketing Institute of Singapore. In his professional career, Brian has held positions in marketing, sales, and operations in the av iation and healthcare industries. He moved to Gainesville, Florida, in 2003 to attend gr aduate school. In May 2006, he received his Master of Science degree in medical scie nces and Master of Business Administration degree with concentrations in competitive strategy and finance.