815 Development of Non Integrating Foamy Vectors for Gene Therapy Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S312 RNA VIRUS VECTORS III the ge[.]
RNA VIRUS VECTORS III the gene transfer efficiency to actively dividing cells, in particular when vectors were applied, and 2) Overall gene delivery efficiency could be highly over-estimated, depending on post-transfection (or transduction) time periods These are crucial questions needed to be addressed for in vitro and in vivo gene delivery, but to quantitatively understand and compare various vector systems cannot be achieved by experiments alone In this study, cells were cultured to form colonies where only the cells in periphery actively spread and proliferate A computer-simulated stochastic cell growth model was utilized in order to fit to the gene expression rates collected at various post-tranfection (or transduction) time points, and then the specific gene transfer rates to actively dividing and non-dividing cells at the time of transfection (or transduction) were determined The results indicated: 1) There was a significant difference in the apparent gene transfer efficiency, (e.g., 82, 56 and 40% using HEK 293 cells and VSV-G pseudotyped MoMLV vectors) 24h after transduction, depending on the ratio of actively-dividing vs non-dividing cells and time of transduction, 2) The true values of viral transduction rates are relatively consistent (e.g., approx 75 and 0% transduction rates to actively dividing and non-dividing cells over the course of 2h exposure) This means that gene transfer efficiency based on the number of gene expressing cells varies up to several folds with post-transfection (or transduction) time as well as ratio of actively dividing vs non-dividing cells More extended examples, including transfection rates to various types of actively dividing and non-dividing cells by different kinds of gene delivery vectors (e.g., lentiviral and non-viral gene delivery to actively-dividing vs non-dividing cancer cells) will be presented Implications of the findings for in vivo gene delivery will also be discussed Respiratory syncytial virus (RSV) naturally infects the ciliated cells that line the respiratory tract, the same cells that express CFTR In addition, although RSV does induce an immune response, this response does not prevent subsequent infection suggesting that an RSV-based vector might be effectively readministered, a requirement since the apical ciliated cells not divide We have inserted the large (4.5 kb) CFTR gene into four sites in the recombinant, green fluorescent protein-expressing (rg)RSV genome to generate virus expressing increasing amounts of the CFTR protein Two of these rgRSV-CFTR viruses were capable of expressing CFTR The inclusion of the CFTR gene in the (rg)RSV genome did cause a delay in the growth of the viruses, however this was overcome by 72 hours post infection The CFTR expressed by the rgRSV-CFTR viruses was functional in primary human airway epithelial cultures derived from CF patients and infection with these viruses resulted in a minimum of 30% of the normal chloride channel function seen in non-CF primary human airway epithelial cultures Unfortunately, ciliated airway cells infected with RSV die within days post infection To create an improved RSV-based vector we have generated an RSV “replicon” by removing the three glycoprotein genes from a full-length RSV cDNA The replicon is able to replicate in cells without killing them or producing infectious virus, allowing the isolation and expansion of replicon-containing cells Providing the viral glycoproteins in trans enables the production of “one-step virus” (OSV) which is capable of delivering a replicon to fresh cells where it produces its encoded proteins, but cannot spread to additional cells We inserted the CFTR gene into the RSV replicon genome in four positions, all four of which produced the CFTR protein When CFTR-expressing replicon OSV were used to infect primary human airway epithelial cultures, replicon containing cells remained within the cultures for 12-20 days, indicating that replicon containing cells have a survival advantage over those infected with the complete virus 815 Development of Non-Integrating Foamy Vectors for Gene Therapy David R Deyle,1 Yi Li,1 David W Russell.1 Department of Medicine, Division of Hematology, University of Washington, Seattle, WA 814 Respiratory Syncytial Virus Based Gene Therapy Vectors for the Treatment of Cystic Fibrosis Anna R Kwilas,1,3 Mark A Yednak,3 Liquin Zhang,4 Peter L Collins,5 Raymond J Pickles,4 Mark E Peeples.2,3 Integrated Biomedical Science Graduate Program, The Ohio State University, Columbus, OH; 2Department of Pediatrics, The Ohio State University, Columbus, OH; 3Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 4Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 5Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population It is caused by any of a number of mutations in the CF transmembrane conductance regulator (CFTR) gene A mutation in the CFTR gene, leading to the production of a partially or completely non-functional protein, results in dehydration of mucosal surfaces and an inability to remove pathogens from the respiratory tract leading to morbidity and mortality in the CF population Since CFTR was first cloned in 1989 a major goal of CF research has been to develop an effective gene therapy for this disease S312 Foamy virus (FV) is a retrovirus that has been developed for gene therapy because it has not been associated with disease, is able to infect many different hosts and tissue types, and has the largest genome of all retroviruses After infection, FVs can stably integrate into the host DNA and facilitate efficient transgene expression However, integration of exogenous DNA into the genome can cause insertional mutagenesis To avoid the issues of insertional mutagenesis, previous attempts have been made to create a functional non-integrating foamy virus (NIFV) vector similar to integrase-deficient lentiviral vectors However, these efforts have been unsuccessful To further investigate this problem, we constructed NIFV vectors by introducing mutations in the highly conserved DD35E catalytic core motif of the FV integrase gene Three independent vectors stocks containing the expression cassette MSCV-GFP were generated for two single mutant NIFV vectors packaged with point mutations D131V or D188A, a double mutant NIFV vector packaged with both D131V and D188A mutations, and an integrase-proficient FV vector Genome titers were determined by Southern blot analysis and all titers ranged from x 10^8 to x 10^10 genomes/ml, showing that NIFV vector stocks were comparable to the integrase proficient FV vector stocks All three NIFV vectors and the integrase-proficient FV vector expressed GFP in human fibroblasts and HT1080 cells two days after infection Unlike integrase proficient FV vectors that showed stable GFP expression, fibroblasts and HT1080 cells transduced with NIFV vectors had a decreasing percent of GFP+ cells over a 10 day period, reflecting a lack of integration and dilution over time in the proliferating culture We also generated NIFV and integrase-proficient FV vectors Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy PRECLINICAL AND CLINICAL APPLICATIONS OF AAV containing a CMV-nuclear localized lacZ-PGK-Neo-Ori (CnZPNO) cassette and transduced fibroblasts Again, all three NIFV vectors and the integrase-proficient FV vector expressed the transgene, nuclear localized lacZ, as evidenced by the presence of beta-galactosidasepositive foci In addition, after selecting infected fibroblasts with G418, we found that the NIFV vectors integrated at a frequency several logs lower than integrase-proficient FV vectors NIFV vectorchromosome integration junctions from G418 resistant fibroblast clones will be sequenced to determine the mechanism of integraseindependent vector insertion Since non-integrating viral genomes are known to exist in various forms, including linear, one-LTR or two-LTR circles, we are currently utilizing Southern blot analysis to identify the predominate DNA species Our NIFV vector would be especially advantageous for those applications in which short-term gene expression is required, such as expressing a factor to influence development, inducing a cellular phenotype, or expressing a genomic modifying protein (e.g Cre recombinase) Also, NIFV vectors may be useful for targeted gene repair In summary, our novel NIFV system expresses transgenes, infects different cell types, has applications for gene therapy, and most importantly, does not integrate, greatly reducing the risk of genotoxicity Preclinical and Clinical Applications of AAV 816 AAV Capsid-Specific CD8+ T Cells and Their Responsiveness to Hepatic AAV Gene Transfer Hua Li,1 Katherine High,2 Hildegund C J Ertl.1 Immunology Program, The Wistar Institute, Philadelphia, PA; Children’s Hospital of Philadelphia, Philadelphia, PA Hepatic adeno-associated virus (AAV)-serotype 2-mediated gene transfer results in sustained transgene product expression in experimental animals but not in human subjects We hypothesized that loss of transgene product expression in humans might be caused by immune memory mechanisms that become reactivated upon AAV vector transfer We tested the effect of hepatic AAV2-hF.IX or AAV8-hF.IX gene transfer on AAV capsid-specific CD8+ T cells in mice AAV capsid-specific CD8+ T cells were found to proliferate in response to hepatic AAV gene transfer indicating that degradation of the AAV particles triggered a recall response The kinetics of the recall response analyzed by adoptive transfer of CD8+ T cells into mice that had received AAV-2hF.IX or AAV-8hF.IX vectors previously showed differences in the degradation rates of AAV-2 and AAV-8 vectors Other functional properties of AAV capsid-specific CD8+ T cells were analyzed and the most remarkable finding was that in mice AAV capsid-specific CD8+ T cells require a rather high concentration of their cognate antigen in order to exhibit lysis The implication of these findings on the potential role of AAV capsid-specific CD8+ T cells in limiting hepatic AAV gene transfer in humans will be discussed 817 In Vivo Allele Specific Knockdown of Mutant Alpha One Antitrypsin Using Recombinant AAV Delivered shRNA Christian Mueller,1 Qiushi Tang,1 Brian O’Sullivan Murphy,1 Sofia Braag,1 Terence R Flotte.1 UMass Gene Therapy Center, UMass Medical School, Worcester, MA Alpha-1 antitrypsin (AAT) deficiency is one of the most common genetic diseases in North America, with a carrier frequency of approximately 4% in the US population Homozygosity for the most common mutation caused by a single base pair change (Glu342Lys, PI*Z) leads to the synthesis of a mutant protein, which accumulates and polymerizes within hepatocytes rather than being efficiently secreted This lack of secretion causes severe serum deficiency Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy predisposing patients to chronic lung disease 12 to 15% of patients with PI*ZZ also develop liver disease, which can be severe, even in infancy This is thought to be due to toxic effects of the accumulated mutant Z-AAT within the hepatocyte Thus, an approach to reduce AAT-deficient liver disease will likely require some mechanism to decrease the amount of Z-AAT within hepatocytes Here we describe studies of allele specific small interfering RNAs (siRNAs) designed to down regulate PI*Z specific AAT within hepatocytes Two different siRNA sequences were identified and cloned into a recombinant adeno-associated virus (rAAV) backbone as U6 driven shRNA, one with the single base pair change at position 10 (p10) and one with the change at position 16 (p16) of the targeting siRNA molecule Each construct was able to reduce Z-AAT levels, but experiments on wildtype PiM AAT expressing cell culture models showed the greatest PiZ specific reduction with p10, whereas the p16 construct also significantly down regulated wildtype AAT The rAAV-U6p10 was then packaged into AAV8 capsids and used in vivo to transduce the livers of human Z-AAT over-expressing transgenic mice These studies show a decrease in total human AAT after weeks, and a clearing of Z-AAT accumulation by immunohistochemistry in the livers The rAAV8-U6-p10 vector may hold promise as a potential therapy for patients with AAT liver disease 818 Proteasome Inhibitors Enhance Gene Delivery by Adeno-Associated Virus Vectors Carrying Large Genomes in Hemophilia Mouse and Dog Models Junjiang Sun,1 Paul E Monahan,1 R Jude Samulski,1 Clinton D Lothrop, Jr Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Biochemistry and Molecular Genetics, School of Medicine, University of Alabama, Birmingham, AL The use of adeno-associated virus (AAV) vectors for correction of many clinically relevant congenital deficiencies has been hampered by the relatively small size of the AAV genome (4680 nt) and inefficient AAV vector delivery of genes that are larger than the wild type (wt) size We previously demonstrated in vitro that proteasome inhibitor (PI) treatment concurrent with AAV delivery improves transduction using larger than wt expression cassettes that exceed the size of the wtAAV genome We have extended these studies using AAV2 or vectors carrying a 5.6 kb factor VIII cassette in hemophilia A mice treated with or without proteasome inhibitor PI enhancement of expression was observed that was increased on average 6-fold (AAV2) and 3-fold (AAV8); enhanced expression persisted throughout the year of observation In addition, a limiting dose of x 1013 vg/kg AAV8.canine FVIII was delivered via portal vein to two hemophilia A dogs after receiving PI I.V.; two hemophilic dogs received vector without PI No toxicity was observed; specifically, there were no abnormalities of liver transaminases, blood platelet, white blood cell or other cell counts, or development of FVIII inhibitory antibodies The Whole Blood Clotting Time (WBCT), which is normally prolonged to greater than 20 minutes in hemophilic dogs, corrected to the normal range (6-10 minutes) by the first timepoint at one week in the dogs receiving PI Over one year after vector delivery dogs receiving AAV8cFVIII alone had a mean WBCT of 13.6 minutes, and experienced bleeding episodes apiece, a bleeding rate not different from untreated hemophilic dogs followed in parallel Dogs receiving vector with PI had a mean WBCT of 9.0 minutes, and correction of the hemophilic phenotype was evidenced by zero bleeding episodes Because male gender may be associated with higher hepatic expression from AAV vectors, a second litter of dogs was subsequently treated with two females receiving AAV + PI and one male receiving vector alone After five months follow-up, the PI-treated females show the same pattern of improved hemostatic correction The first litter has now been followed for 27 months remaining asymptomatic with S313 ... recombinase) Also, NIFV vectors may be useful for targeted gene repair In summary, our novel NIFV system expresses transgenes, infects different cell types, has applications for gene therapy, and most... Jr Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Biochemistry and Molecular Genetics, School of Medicine, University of Alabama, Birmingham, AL The use of. .. (AAV) vectors for correction of many clinically relevant congenital deficiencies has been hampered by the relatively small size of the AAV genome (4680 nt) and inefficient AAV vector delivery of genes