30. Ex Vivo Gene Transfer and Transplantation of Liver Stem Cell for Alpha-1 Antitrypsin Deficiency

NAKED DNA: METHODS 28. Hydrodynamics-Based Transfection of Mouse Liver: A Study with Isolated Hepatocytes Michèle Lecocq,1 Simone Wattiaux- De Coninck,1 Robert Wattiaux,1 Michel Jadot.1 1 Chimie Physiologique, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium. A very efficient method for mouse hepatocyte transfection “in vivo”, is obtained by quickly injecting into the tail vein a large volume of plasmid DNA solution (hydrodynamic -based transfection). The mechanism of gene transfer by this procedure is not clearly understood. It is probable that after such an injection, access of plasmid DNA to hepatocytes is facilitated owing to the enlargement of endothelial fenestrae caused by hydrostatic pressure generated by the injection. However, it is not sufficient to explain the high efficiency of hydrodynamic based transfection since a very significant amount of DNA is taken up by hepatocytes even after a conventional injection that does not lead to expression. It has been shown that after an hydrodynamic injection, a large proportion of intact DNA remains in the liver for a relatively long time (Liu et al., (1999) Gene Ther. 6, 1258-1266). Recently, we have found that such DNA is bound to outside face of hepatocyte plasma membrane (Lecocq et al., (2003) J. Gene Med.5, 142-156). The possible relationship between that DNA and the transfection process is not clear. In the work presented here, we have investigated the problem by using primary cultures of hepatocytes isolated after a hydrodynamic injection of plasmid DNA, with cDNA of luciferase as a reporter gene. The rationale of that approach is that it allows to specifically follow the fate of DNA taken up by hepatocytes and its relationship with expression without interference of other liver cells. Results show that a very short time after injection (less than 3 min) is required to obtain hepatocytes exhibiting a maximal luciferase expression and that the time dependent gene expression is similar in isolated hepatocytes and in the whole liver. The fate of pDNA taken up by hepatocytes was investigated by injecting 35S DNA and by establishing the intracellular distribution of radioactivity by centrifugation methods after increasing times of culture. Total cell radioactivity slowly decreases with time but remains mainly acidprecipitable. Its distribution after differential and isopycnic centrifugation strongly suggests that it is to a large extent, bound to plasma membrane. When hepatocytes are incubated with pancreatic DNAse, immediately after their isolation, luciferase expression is not affected although 70-80% of the 35S DNA located at the outside face of plasma membrane is hydrolysed. A plausible explanation of these results is that the pDNA required for expression is quickly internalised after injection, and that the molecules that remain associated to the plasma membrane do not contribute to expression.

TRANSDUCTION BIOLOGY AND APPLICATIONS 29. Functional Restoration of CFTR-Mediated Chloride Transport in CF Airway Epithelia Using a Full-Length CFTR rAAV Vector and Combined Proteasome Inhibitors Liang N. Zhang,1 Phil Karp,4 Ziying Yan,1,3 Simon Godwin,5 Richard Peluso,5 Joseph Zabner,2,3,4 John F. Engelhardt.1,2,3 1 Department of Anatomy and Cell Biology, The University of Iowa, Iowa City, IA; 2Department of Internal Medicine, The University of Iowa, Iowa City, IA; 3Center for Gene Therapy, The University of Iowa, Iowa City, IA; 4Howard Hughes Medical Institute, The University of Iowa, Iowa City, IA; 5Targeted Genetics Corporation, Seattle, WA. Our previous studies have shown that proteasome inhibitors can dramatically increase the transduction efficiency of recombinant S12

adeno-associated virus (rAAV) infection from the apical surface of polarized human airway epithelia. Based on these findings, we assessed the efficacy of both the current clinically used ITR driven full-length CFTR rAAV vector (tgAAV2-CF), and a second generation vector harboring a short 83 bp synthetic promoter driving expression of a full-length CFTR cDNA (tgAAV2-CF83), for their ability to correct CFTR chloride transport in human CF airway epithelia with combined proteasome inhibitors administration. Additionally, since rAAV5 has been suggested to transduce the apical surface of human airway epithelia more efficiently than rAAV2, we also evaluated pseudotyped rAAV2/5 viruses with both types of vector genomes. rAAV2 or rAAV2/5 vectors utilizing the ITR or synthetic promoters were used to infect polarized CF airway epithelia from the apical surface in the presence or absence of a combined cocktail of proteasome inhibitors. Two weeks after infection, CFTR-mediated cyclic AMP (cAMP)-sensitive shortcircuit current (Isc) was assessed in the presence of IBMX (100uM) and forskolin (10uM) and compared to normal human airway epithelia. In the absence of proteasome inhibitors, only minimal restoration of cAMP-inducible chloride currents were seen with the minimal promoter vector (tgAAV2-CF83) of the type-2 serotype (0.76 +/-0.16 uA) and no significant function correction was seen with any of the other three viruses tested (tgAAV2-CF, tgAAV2/5CF, or tgAAV2/5-CF83). However, when proteasome inhibitors were provided only at the time of infection, tgAAV2-CF83 restored CFTR-mediated chloride current upon IBMX/forskolin stimulation in the CF epithelia at the highest level (2.9+/-0.3 uA, n=9) and was not significantly different from that seen in normal human airway epithelial (3.5+/-0.8 uA, n=5). Surprisingly, pseudotyped tgAAV2/ 5-CF83 with the same synthetic promoter gave significantly less correction of chloride currents in CF epithelia (1.0+/- 0.2 uA, n=6) and was even lower than that seen with the ITR promoter CFTR vector tgAAV2-CF (1.9+/-0.2 uA, n=4). For all rAAV vectors, the addition of proteasome inhibitors during infection significantly increased the level of restored chloride current. In conclusion, these studies demonstrate that the AAV2 based vector systems when used in conjunction with proteasome inhibitors provide higher levels of functional efficacy in correcting CFTR defects in CF human airway epithelial following apical infection than pseudotyped AAV2/5 viruses. Additionally, the use of a small synthetic promoter provides a tangible improvement in expression of full-length CFTR. Given the current findings in clinical trials for CF lung disease using tgAAV2-CF virus, this study support the therapeutic efficacy of this vector provided intracellular barriers involving the ubiquitin/ proteasome system are adequately modulated. This work was funded by NIH grant HL58340, DK54759, and a collaboration with Targeted Genetics Corporations

30. Ex Vivo Gene Transfer and Transplantation of Liver Stem Cell for Alpha-1 Antitrypsin Deficiency Sihong Song, Rafal Witek, Yuanqing Lu, Bryon E. Petersen, Terence R. Flotte. 1 Departments of Pharmaceutics, Pothology and Pediatrics, University of Florida, Gainesville, FL, United States. Alpha 1-antitrypsin (AAT), a 52kD serine proteinase inhibitor, is normally secreted from hepatocytes and circulates in the plasma, protecting lung elastin from degradation by neutrophil elastase and related proteases. Deficiency of AAT can lead to pan-acinar emphysema due to destruction of pulmonary interstitial elastin if serum levels fall below 11mM (approximately 800mg/ml). In a subset of patients homozygous for the PI*Z mutation, liver disease may also develop, apparently related to polymerization of the mutant protein within the endoplasmic reticulum of affected hepatocytes. Gene therapy for AAT deficiency-related liver disease will likely be Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts

Copyright © The American Society of Gene Therapy

TRANSDUCTION BIOLOGY AND APPLICATIONS dependent upon transduction of a high percentage of hepatocytes. Ex vivo gene delivery to liver stem cells followed by transplantation has great potential for increasing the rate of long-term transduction. In order to optimize the transduction efficiency of rAAV vectors, rat liver stem cells were infected with five serotypes of rAAV-CBhAAT vector (type 1, 2, 3, 4 and 5). Results from this experiment showed that rAAV1 mediated the highest hAAT secretion into the culture medium. The transgene expression can be clearly detected at 4 days after infection with AAV1 (supernatant AAT concentration of 15 mcg/ml), while no detection obtained until 10 days after infection with AAV2, 3 and 5. AAV4 did not mediate any transgene expression in this study. Subsequently, liver stem cells from C57 bl/ 6 mice were transduced with the rAAV1-CB-AAT vector at 50,000 MOI for 2h. After transducion cells were washed and resuspended in saline solution at appropriate concentration to give approximately 1x106 cells per 100 μl. Transduced liver stem cells were transplanted into the liver of partial hepatectomized C57bl/6 recipients (1x106 cells/mouse, n=6). The transgene expression was monitored by measuring the serum level of hAAT. Two weeks after transplantation, hAAT were detected from recipient mice. The expression levels (1 to 4 mg/ml) were sustained at least 6 weeks after stem cell transplantation. These results indicate that AAV1 infect liver stem cells more efficient than other serotypes, and that ex vivo manipulation and transplantation of liver stem cells provide an alternative approach for liver gene therapy. Supported in part by grants from NIH (DK58327, HL59412), the Alpha One Foundation, and the Juvenile Diabetes Research Foundation. US 6,461,606 B1

31. An Androgen Dependent Pathway Is Responsible for Gender Specific Differences in Transduction of Murine Liver by Recombinant Adeno-Associated Viral Vectors Andrew M. Davidoff,1 Catherine Y. C. Ng,1 Junfang Zhou,1 Yunyu Spence,1 Amit C. Nathwani.2 1 Department of Surgery and Division of Experimental Hematology, St. Jude Children’s Research Hospital, Memphis, TN; 2Haematology, University College London and National Blood Service, London, United Kingdom. A systematic evaluation of the influence of gender on transduction by recombinant adeno-associated viral vectors (rAAV) indicated that transgene expression after liver targeted delivery of vector particles was between 5-13 fold higher in male mice compared to females, irrespective of the proviral promoter or cDNA, and mouse strain. Molecular analysis revealed that the rAAV genome was stably retained in male liver at levels that were 7-fold higher than those observed in females. Further, the gender difference in transduction was observed with AAV–2 and AAV-5 based vectors, which utilize distinct receptor complexes for infection, suggesting that differences in transduction efficiency were not due to differential vector binding. In concordance with the differences in AAV transduction, gel shift analysis with nuclear extracts derived from the liver of mice and humans, revealed substantially higher binding of host nuclear proteins to the rep-binding site (RBS) of AAV ITR in male mice compared to females. Transduction efficiency and binding of nuclear protein to RBS was dramatically reduced in male mice by castration. In contrast, although oophorectomy did not significantly influence rAAV transduction, administration of 5a dihydrotestosterone, prior to gene transfer, increased stable hepatocyte gene transfer in females to levels observed in male mice, implying that androgens significantly influence hepatocyte gene transfer. Interestingly, gender did not have a significant impact on AAV gene transfer into non-hepatic tissue indicating that there are distinct tissue and gender specific differences Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy

in the mechanisms responsible for efficient transduction with this vector. These results have significant implications for gene therapy of autosomal and acquired disorders affecting the liver.

32. Adeno-Associated Virus 2-Mediated Gene Transfer: Role of Cellular Heat-Shock Protein 90 in Transgene Expression Li Zhong,1 Keyun Qing,1 Yue Si,1 Linyuan Chen,1 Mengqun Tan,2 Arun Srivastava.1,3 1 Department of Microbiology and Immunology, Walther Oncology Center, Walther Cancer Institute, Indiana University School of Medicine, Indianapolis, IN, United States; 2Department of Hematology, Central South University Xiang-Ya School of Medicine, Changsha, Hunan, China; 3Division of Hematology/ Oncology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN. Adeno-associated virus type 2 (AAV), a single-stranded DNAcontaining non-pathogenic human parvovirus, has gained attention as a useful vector for human gene therapy. We have described that a cellular protein that binds the immuno-suppressant drug FK506, termed the FK506-binding protein (FKBP52), in its phosphorylated form, interacts with the single-stranded D-sequence within the AAV inverted terminal repeats, inhibits the viral second-strand DNA synthesis, and consequently, limits high-efficiency transgene expression by AAV vectors (J. Virol., 75: 8968-8976, 2001). In the present studies, we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV-mediated transduction since FKBP52 has previously been shown to form a complex with HSP90, and since heat-shock treatment has been shown to augment AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosinedephosphorylation of FKBP52, led to stabilization of the FKBP52HSP90 complex, and resulted in approximately 6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor protein tyrosine kinase, known to phosphorylate FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. In electrophoretic mobility-shift assays, HSP90 was shown to be a part of the FKBP52 complex with AAV D-sequence. However, HSP90 by itself did not bind to AAV D-sequence even though it could be phosphorylated in vitro at both serine/threonine and tyrosine residues. HSP90 significantly enhanced the binding of unphosphorylated FKBP52 to AAV D-sequence. Since HSP90 can be dissociated from FKBP52 complex by treatment with geldanamycin, a benzoquinone ansamycin, which binds to HSP90 and disrupts its function, geldanamycin treatment resulted in greater than 22-fold increase in AAV transduction in heat-shock-treated HeLa cells compared with heat-shock alone. Similarly, treatment of HeLa cells with geldanamycin significantly increased AAV transduction, but only in cells pre-treated with tyrphostin 23. Surprisingly, deliberate overexpression of the human HSP90 gene resulted a significant decrease in AAV-mediated transduction in tyrphostin 23-treated HeLa cells, whereas stable trasnfection of anti-sense HSP90 gene expression cassette led to a significant increase AAV transduction in these cells following pre-treatment with tyrphostin 23. Taken together, these studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment of cells is unlikely to be mediated by HSP90 alone, but HSP90 appears to be a co-regulator of FKBP52 in mediating AAV second-strand DNA synthesis. A better understanding of the complex interactions between these cellular proteins is likely to be important in yielding new insights in the optimal use of recombinant AAV vectors in human gene therapy. AS owns stocks in Avigen, Inc., and has stock-options in Immusol, Inc. S13