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406. In Vivo Antibody Gene Transfer Provides Protective Immunity Against Virulent SIVmac316 Challenge in Rhesus Macaques
Targeting Viral Infection through Gene Delivery 404. Long-Term Rescue of a Lethal Murine Model of Methylmalonic Acidemia Using AAV 8 Mediated Gene Therapy
Randy J. Chandler,1 Charles P. Venditti.1 Genetic Diseases Research Branch, Human Genome Research Institute National Institutes of Health, Bethesda, MD.
1
Methymalonic acidemia (MMA), a severe organic acidemia, is caused by deficient activity of the ubiquitous mitochondrial enzyme methylmalonyl-CoA mutase (MUT). MMA patients can exhibit a 100-fold or more increased methylmalonic acid levels in the plasma, urine and CSF and display a clinical phenotype of lethal metabolic decompensation, growth retardation, renal failure, pancreatitis and metabolic strokes. Elective liver and combined liver-kidney transplantation has been performed in an effort to restore MUT enzymatic activity in a tissue specific manner in some patients. Although solid organ transplantation does not completely normalize the biochemical abnormalities in recipient MMA patients, it does eliminate the risk of life threatening intermittent decompensation, affords an increase in protein tolerance and can improve growth. The amelioration of disease severity by organ replacement suggests that gene therapy might hold promise as a future treatment. To assess the potential of genetic therapy for MUT MMA, we employed a mouse model of MMA that we have recently developed. This murine model of MMA harbors a null allele that produces no detectable Mut transcript or protein and a homozygous Mut-/- phenotype that displays severe clinical manifestations. We engineered an AAV 8 vector that contains the Mut gene under the control of a combined CMV-IE enhancer-chicken beta actin promoter (AAV 8 CBA-Mut). AAV 8 CBA-Mut was injected directly into the liver of newborn Mut-/- pups. All Mut-/- mice (n=29) injected with 1 or 2x1011GC of AAV 8 CBAMut survived beyond day of life (DOL) 60. Currently, 28 out of the 29 treated Mut-/- mice are alive beyond DOL 90 with a single mouse dying for unclear reasons at DOL 92. Some treated Mut-/- mice older than 200 days are alive and without disease manifestations. While greater than 70% of the untreated mutants (n=21) perished before DOL 24 with only one mouse surviving until DOL 72. The treated Mut-/- mice are thriving and indistinguishable from their wild-type (WT) littermates. AAV 8 CBA-Mut treated Mut-/- mice achieved body weights comparable to controls (p-value=NS, 1x1011GC, D24 and D60) while untreated mutants experienced post-natal growth retardation and reached only 40% of the weight of the WT littermates or perished before DOL 90. Plasma methylmalonic acid levels in the treated mutant mice on an unrestricted diet were significantly reduced compared to uncorrected animals (p<0.0001, 1x1011GC, D24 and D60), indicating that substantial Mut enzymatic activity was restored after AAV therapy, and have remained stable. At DOL 90 the liver from a treated Mut-/- mouse had WT levels of Mut protein by Western blot analysis. The results obtained to date demonstrate that AAV 8 mediated gene delivery of Mut in the murine model of MMA not only rescues the lethal phenotype, but also allows the treated mutants to tolerate an unrestricted diet and develop normally. These experiments provide the first evidence that gene therapy has clinical utility in treatment of MMA and support the development of gene therapy for other organic acidemias.
405. Transgene Expression Persists for at Least Three Years in Nonhuman Primates Following Hepatic Transduction with Helper-Dependent Adenoviral Vectors
Nicola Brunetti-Pierri,1 Thomas Ng,2 David A. Iannitti,2 William G. Cioffi,2 Gary E. Stapleton,3 Charles Mullins,3 Donna J. Palmer,1 Arthur L. Beaudet,1 Milton J. Finegold,4 Philip Ng.1 1 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Surgery, Brown Medical School, Rhode Island; 3 Pediatric Cardiology, Baylor College of Medicine, Houston, TX; 4 Pathology, Baylor College of Medicine, Houston, TX.
We have previously shown that injection of 1x1011 vp/kg to 1x1012 vp/kg of HDAd expressing the baboon a-fetoprotein (bAFP) into the surgically isolated liver via the portal vein of nonhuman primates results in efficient hepatic transduction and stable transgene expression of at least 665 days for baboon 13947 and at least 560 days for baboons 14907 and 12345 (Brunetti-Pierri et al, 2006 Hum Gene Ther 17:391). We have also previously shown that pseudohydrodynamic injection of 1x1011 vp/kg of HDAd resulted in efficient hepatic transduction and stable bAFP expression of at least 413 days for baboons 14200 and 14252 (Brunetti-Pierri et al, 2007 Mol Ther 15:732). Because long-term transgene expression is a critical paramater for efficacy as well as for risk:benefit assessment, we have continued to monitor the aforementioned animals and have found that transgene expression has persisted at high levels for at least 1,330 days for baboon 13947 and at least 1,169 days for baboons 14907 and 12345 (Fig). In the case of pseudo-hydrodynamic delivery of HDAd, we have found that high levels of transgene expression have persisted for at least 1,085 days for baboons 14200 and 14252 (Fig). No chronic toxicity has been observed in any of these animals at any time post-injection and we will continue to monitor the levels of transgene expression in these animals to determine the absolute duration of expression. These results clearly demonstrate very longterm expression from HDAd following hepatic transduction from a single administration in nonhuman primates and further support their potential efficacy in human clinical trials.
Targeting Viral Infection through Gene Delivery 406. In Vivo Antibody Gene Transfer Provides Protective Immunity Against Virulent SIVmac316 Challenge in Rhesus Macaques
Jiangchao Zhang,1 Bruce C. Schnepp,2 K. Reed Clark,1 Ronald C. Desrosiers,3 Mary Connell,2 Philip R. Johnson.2 1 Pediatrics, Nationwide Children’s Hospital, Columbus, OH; 2 Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA; 3 Microbiology and Molecular Genetics, Harvard Medical School, Southborough, MA. We are developing a novel HIV-1 vaccine approach utilizing antibody gene transfer to provide protective host humoral immune responses against HIV-1. Reverse immunization is predicated on the efficient delivery of antibody genes that encode broadly neutralizing HIV-1 antibodies. Following in vivo gene transfer into skeletal muscle, the individual would be supplied with a constant source of potent antiHIV-1 antibodies prior to pathogen exposure. In the current iteration, we evaluated three different rAAV1 vaccine vectors each expressing
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
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Targeting Viral Infection through Gene Delivery rhesus derived, neutralizing fusion proteins for efficacy in the SIV/ macaque challenge model. Two macaque scFv-IgG2 NAbs (4L6 and 5L7) and a rhesus rhCD4-IgG2 (N4) molecule all demonstrated potent in vitro neutralization against SIVmac316. Following vector production, nine AAV1 sero-negative animals were stratified into 3 experimental groups (n=3) and injected intramuscularly with the rAAV1 vaccine vectors at a vector dose of 2x1013 DRP per animal. The monkeys were bled every 2 weeks and serum levels of the fusion proteins determined by gp120 env binding ELISA. Sustained serum levels in the range of 100-400 ug/ml have been observed for all 6 animals receiving the NAbs 4L6 and 5L7. The N4 animals show significantly lower serum levels (5-10 ug/ml), which may be related to the use of a standard single-strand rAAV1 vector for this construct (due to size) compared to self-complementary vectors for expression of the NAbs. Six naive monkeys and the 9 vaccinated primates were challenged with SIVmac316 (MID50 = 40) by the IV route 4 weeks after vaccination. Significantly, qPCR load data revealed SIV viremia in all 6 control animals 2 weeks post SIV challenge. In stark contrast, all 3 animals in the 4L6 group have undetectable levels of SIV in their sera 6 months post-challenge. Moreover, two animals in the N4 group and 1 animal in the 5L7 group also have undetectable levels of SIV. One animal in the 5L7 group mounted an idiotypic antibody response 4 weeks after vaccination that appeared to neutralize 5L7 activity. Not unexpectedly this animal became infected with similar kinetics as the control group. The infected N4 animal had the lowest serum levels of the 3 in this group and may have become infected simply because it fell below a threshold level for protection. At present we are exploring alternative explanations for the other 5L7 animal that became infected in the face of significant circulating NAb levels. These data strongly suggest that sterilizing immunity was achieved in 6 out 9 vaccinated animals challenged with a virulent dose of SIVmac316. Additional studies are planned to evaluate strategies to enhance vaccine breadth and protection at reduced vaccine dosages. In conclusion, this study provides powerful proof-of-concept data demonstrating feasibility of reverse immunization as a viable prophylactic vaccine approach.
407. Zinc-Finger Nucleases Targeting Hepatitis B Virus Genomic DNA
Thomas J. Cradick,1 Andrew C. Jamieson,1 Shannon Bradshaw,2 Anton McCaffrey.1 1 Internal Medicine, University of Iowa, Iowa City, IA; 2Department of Mathematics and Computer Science, Drew University, Madison, NJ. Hepatitis B virus (HBV) is a small DNA virus chronically infecting 400 million people. It is the 9th leading cause of death worldwide. Current treatments with ribavirin and interferon are costly, have significant side effects and are effective in only 50% of patients. Covalently closed circular genomic DNA (cccDNA) is created from the HBV pre-genomic RNA by an error-prone viral reverse transcriptase. Current treatments prevent production of new viral DNA, but do not directly target cccDNA, which can serve as a reservoir for viral escape mutants. When escape mutants emerge or treatment concludes, residual cccDNA allows the virus to rebound. Curing individuals of HBV will therefore require inactivation of cccDNA. There are currently no anti-viral therapeutics that directly target viral DNA genomes for destruction. We describe a novel antiviral therapeutic strategy that uses engineered zinc-finger nucleases (ZFNs) to specifically cleave cccDNA. Zinc finger DNA binding proteins (ZFPs) can be rationally designed to specifically bind novel DNA sequences. ZFPs can be fused to the catalytic domain of nucleases to create chimeric nucleases (ZFNs) that specifically cleave target DNA sequences. As ZFNs function as dimers, DNA cleavage requires binding of two different ZFNs to sites flanking the cleavage site, thereby greatly increasing specificity. ZFN have been previously used to cleave endogenous sequences to stimulate S154
homologous recombination. ZFNs were rationally designed to target adjacent sites in HBV. We identified several functional ZFNs pairs using an in vitro cleavage assay. Cleavage of HBV model substrate by ZFN pairs was demonstrated in cultured cells using Southern blots. Repair of free DNA ends by non-homologous end joining introduces deletions or insertions. These should inactivate the virus as it contains a large number of overlapping reading frames. Two assays demonstrated that cognate ZFN pairs cleaved their target and were imperfectly repaired at the target site. This was not seen with individual ZFNs, with control ZFNs or with control binding site plasmids. Mice were hydrodynamically transfected with ZFN pairs and target substrates. Preliminary results showed modest cleavage of model substrates in mice. As in tissue culture, a repair assay showed the target substrates to be mutated at the ZFN’s target site, but not in mice containing control ZFNs or targets. Further experiments will be required to confirm these results and optimize the activity. These results demonstrate one of the first examples of ZFN activity in mice and the first example of targeting a viral genome with a ZFN. Current work focuses delivery of ZFNs using viral vectors. The data presented here represents proof-of-principle for a completely novel antiviral strategy that could be broadly applicable to treatment of DNA viruses.
408. Controlling HIV In Vivo – Establishment of HIV Resistant CD4 T Cells by Engineered Zinc Finger Protein Nucleases
Elena E. Perez,2 Jianbin Wang,1 Jeffrey C. Miller,1 Kenneth A. Kim,1 Olga Liu,2 Dmitry Guschin,1 Edward J. Rebar,1 Bruce L. Levine,2 Richard G. Carroll,2 Dale Ando,1 Philip D. Gregory,1 James L. Riley,2 Michael C. Holmes,1 Carl H. June.2 1 Sangamo BioSciences, Inc., Richmond, CA; 2Dept. of Pathology and Laboratory Medicine, Univ. of Pennsylavania, Philadelphia, PA. Background: HIV requires the CD4 co-receptors CCR5 or CXCR4 to infect its target cells. Elimination of HIV co-receptors would prevent viral entry and thus is an attractive therapy for HIV patients, yet no current methods enable therapeutically relevant disruption of a chosen human gene. We have developed zinc-finger protein nucleases (ZFNs) targeting the CCR5 gene to create a double strand break (DSB) at a predetermined site. Natural DNA repair pathways can subsequently be usurped to imperfectly repair the DSB resulting in the permanent disruption of the target gene. Methods: Engineered zinc finger DNA binding proteins designed to recognize the CCR5 gene were fused to the FokI catalytic domain to create ZFNs in which the DNA binding specificity of the ZFP determines DSB location. The CCR5 ZFNs were transiently expressed in human cells using a chimeric Ad5/F35 Adenoviral vector for in vivo assessment of function. Functional disruption was measured by genotype via a PCR-based assay and phenotype by HIV challenge assays both in vitro and in vivo. Results: Cell-based assays revealed the CCR5-ZFNs generated DSBs in vivo leading to efficient target gene disruption (>50%) in primary human CD4 T-cells. ZFN-treated primary CD4 T cells and transformed CD4 cell lines were shown to be specifically resistant to R5-tropic HIV infection, resulting in enrichment of ZFNgenerated CCR5 null cells upon long-term exposure both in vitro and in vivo. Importantly, ZFN-modified CD4 T-cells preferentially expanded in the presence of HIV in an in vivo xenotransplantation model using NOD/SCID/common g -/- mice. Conclusion: These data demonstrate that ZFN-treated cells can be permanently modified to prevent CCR5-dependent HIV infection. Modified CD4 T-cells selectively expanded in the presence of HIV in vivo and functioned normally in response to stimulation suggesting that these cells may be able to reconstitute immune function in patients with HIV/AIDS via maintenance of an HIV-resistant CD4 T-cell population. These Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Randy J. Chandler,1 Charles P. Venditti.1 Genetic Diseases Research Branch, Human Genome Research Institute National Institutes of Health, Bethesda, MD.
1
Methymalonic acidemia (MMA), a severe organic acidemia, is caused by deficient activity of the ubiquitous mitochondrial enzyme methylmalonyl-CoA mutase (MUT). MMA patients can exhibit a 100-fold or more increased methylmalonic acid levels in the plasma, urine and CSF and display a clinical phenotype of lethal metabolic decompensation, growth retardation, renal failure, pancreatitis and metabolic strokes. Elective liver and combined liver-kidney transplantation has been performed in an effort to restore MUT enzymatic activity in a tissue specific manner in some patients. Although solid organ transplantation does not completely normalize the biochemical abnormalities in recipient MMA patients, it does eliminate the risk of life threatening intermittent decompensation, affords an increase in protein tolerance and can improve growth. The amelioration of disease severity by organ replacement suggests that gene therapy might hold promise as a future treatment. To assess the potential of genetic therapy for MUT MMA, we employed a mouse model of MMA that we have recently developed. This murine model of MMA harbors a null allele that produces no detectable Mut transcript or protein and a homozygous Mut-/- phenotype that displays severe clinical manifestations. We engineered an AAV 8 vector that contains the Mut gene under the control of a combined CMV-IE enhancer-chicken beta actin promoter (AAV 8 CBA-Mut). AAV 8 CBA-Mut was injected directly into the liver of newborn Mut-/- pups. All Mut-/- mice (n=29) injected with 1 or 2x1011GC of AAV 8 CBAMut survived beyond day of life (DOL) 60. Currently, 28 out of the 29 treated Mut-/- mice are alive beyond DOL 90 with a single mouse dying for unclear reasons at DOL 92. Some treated Mut-/- mice older than 200 days are alive and without disease manifestations. While greater than 70% of the untreated mutants (n=21) perished before DOL 24 with only one mouse surviving until DOL 72. The treated Mut-/- mice are thriving and indistinguishable from their wild-type (WT) littermates. AAV 8 CBA-Mut treated Mut-/- mice achieved body weights comparable to controls (p-value=NS, 1x1011GC, D24 and D60) while untreated mutants experienced post-natal growth retardation and reached only 40% of the weight of the WT littermates or perished before DOL 90. Plasma methylmalonic acid levels in the treated mutant mice on an unrestricted diet were significantly reduced compared to uncorrected animals (p<0.0001, 1x1011GC, D24 and D60), indicating that substantial Mut enzymatic activity was restored after AAV therapy, and have remained stable. At DOL 90 the liver from a treated Mut-/- mouse had WT levels of Mut protein by Western blot analysis. The results obtained to date demonstrate that AAV 8 mediated gene delivery of Mut in the murine model of MMA not only rescues the lethal phenotype, but also allows the treated mutants to tolerate an unrestricted diet and develop normally. These experiments provide the first evidence that gene therapy has clinical utility in treatment of MMA and support the development of gene therapy for other organic acidemias.
405. Transgene Expression Persists for at Least Three Years in Nonhuman Primates Following Hepatic Transduction with Helper-Dependent Adenoviral Vectors
Nicola Brunetti-Pierri,1 Thomas Ng,2 David A. Iannitti,2 William G. Cioffi,2 Gary E. Stapleton,3 Charles Mullins,3 Donna J. Palmer,1 Arthur L. Beaudet,1 Milton J. Finegold,4 Philip Ng.1 1 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Surgery, Brown Medical School, Rhode Island; 3 Pediatric Cardiology, Baylor College of Medicine, Houston, TX; 4 Pathology, Baylor College of Medicine, Houston, TX.
We have previously shown that injection of 1x1011 vp/kg to 1x1012 vp/kg of HDAd expressing the baboon a-fetoprotein (bAFP) into the surgically isolated liver via the portal vein of nonhuman primates results in efficient hepatic transduction and stable transgene expression of at least 665 days for baboon 13947 and at least 560 days for baboons 14907 and 12345 (Brunetti-Pierri et al, 2006 Hum Gene Ther 17:391). We have also previously shown that pseudohydrodynamic injection of 1x1011 vp/kg of HDAd resulted in efficient hepatic transduction and stable bAFP expression of at least 413 days for baboons 14200 and 14252 (Brunetti-Pierri et al, 2007 Mol Ther 15:732). Because long-term transgene expression is a critical paramater for efficacy as well as for risk:benefit assessment, we have continued to monitor the aforementioned animals and have found that transgene expression has persisted at high levels for at least 1,330 days for baboon 13947 and at least 1,169 days for baboons 14907 and 12345 (Fig). In the case of pseudo-hydrodynamic delivery of HDAd, we have found that high levels of transgene expression have persisted for at least 1,085 days for baboons 14200 and 14252 (Fig). No chronic toxicity has been observed in any of these animals at any time post-injection and we will continue to monitor the levels of transgene expression in these animals to determine the absolute duration of expression. These results clearly demonstrate very longterm expression from HDAd following hepatic transduction from a single administration in nonhuman primates and further support their potential efficacy in human clinical trials.
Targeting Viral Infection through Gene Delivery 406. In Vivo Antibody Gene Transfer Provides Protective Immunity Against Virulent SIVmac316 Challenge in Rhesus Macaques
Jiangchao Zhang,1 Bruce C. Schnepp,2 K. Reed Clark,1 Ronald C. Desrosiers,3 Mary Connell,2 Philip R. Johnson.2 1 Pediatrics, Nationwide Children’s Hospital, Columbus, OH; 2 Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA; 3 Microbiology and Molecular Genetics, Harvard Medical School, Southborough, MA. We are developing a novel HIV-1 vaccine approach utilizing antibody gene transfer to provide protective host humoral immune responses against HIV-1. Reverse immunization is predicated on the efficient delivery of antibody genes that encode broadly neutralizing HIV-1 antibodies. Following in vivo gene transfer into skeletal muscle, the individual would be supplied with a constant source of potent antiHIV-1 antibodies prior to pathogen exposure. In the current iteration, we evaluated three different rAAV1 vaccine vectors each expressing
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
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Targeting Viral Infection through Gene Delivery rhesus derived, neutralizing fusion proteins for efficacy in the SIV/ macaque challenge model. Two macaque scFv-IgG2 NAbs (4L6 and 5L7) and a rhesus rhCD4-IgG2 (N4) molecule all demonstrated potent in vitro neutralization against SIVmac316. Following vector production, nine AAV1 sero-negative animals were stratified into 3 experimental groups (n=3) and injected intramuscularly with the rAAV1 vaccine vectors at a vector dose of 2x1013 DRP per animal. The monkeys were bled every 2 weeks and serum levels of the fusion proteins determined by gp120 env binding ELISA. Sustained serum levels in the range of 100-400 ug/ml have been observed for all 6 animals receiving the NAbs 4L6 and 5L7. The N4 animals show significantly lower serum levels (5-10 ug/ml), which may be related to the use of a standard single-strand rAAV1 vector for this construct (due to size) compared to self-complementary vectors for expression of the NAbs. Six naive monkeys and the 9 vaccinated primates were challenged with SIVmac316 (MID50 = 40) by the IV route 4 weeks after vaccination. Significantly, qPCR load data revealed SIV viremia in all 6 control animals 2 weeks post SIV challenge. In stark contrast, all 3 animals in the 4L6 group have undetectable levels of SIV in their sera 6 months post-challenge. Moreover, two animals in the N4 group and 1 animal in the 5L7 group also have undetectable levels of SIV. One animal in the 5L7 group mounted an idiotypic antibody response 4 weeks after vaccination that appeared to neutralize 5L7 activity. Not unexpectedly this animal became infected with similar kinetics as the control group. The infected N4 animal had the lowest serum levels of the 3 in this group and may have become infected simply because it fell below a threshold level for protection. At present we are exploring alternative explanations for the other 5L7 animal that became infected in the face of significant circulating NAb levels. These data strongly suggest that sterilizing immunity was achieved in 6 out 9 vaccinated animals challenged with a virulent dose of SIVmac316. Additional studies are planned to evaluate strategies to enhance vaccine breadth and protection at reduced vaccine dosages. In conclusion, this study provides powerful proof-of-concept data demonstrating feasibility of reverse immunization as a viable prophylactic vaccine approach.
407. Zinc-Finger Nucleases Targeting Hepatitis B Virus Genomic DNA
Thomas J. Cradick,1 Andrew C. Jamieson,1 Shannon Bradshaw,2 Anton McCaffrey.1 1 Internal Medicine, University of Iowa, Iowa City, IA; 2Department of Mathematics and Computer Science, Drew University, Madison, NJ. Hepatitis B virus (HBV) is a small DNA virus chronically infecting 400 million people. It is the 9th leading cause of death worldwide. Current treatments with ribavirin and interferon are costly, have significant side effects and are effective in only 50% of patients. Covalently closed circular genomic DNA (cccDNA) is created from the HBV pre-genomic RNA by an error-prone viral reverse transcriptase. Current treatments prevent production of new viral DNA, but do not directly target cccDNA, which can serve as a reservoir for viral escape mutants. When escape mutants emerge or treatment concludes, residual cccDNA allows the virus to rebound. Curing individuals of HBV will therefore require inactivation of cccDNA. There are currently no anti-viral therapeutics that directly target viral DNA genomes for destruction. We describe a novel antiviral therapeutic strategy that uses engineered zinc-finger nucleases (ZFNs) to specifically cleave cccDNA. Zinc finger DNA binding proteins (ZFPs) can be rationally designed to specifically bind novel DNA sequences. ZFPs can be fused to the catalytic domain of nucleases to create chimeric nucleases (ZFNs) that specifically cleave target DNA sequences. As ZFNs function as dimers, DNA cleavage requires binding of two different ZFNs to sites flanking the cleavage site, thereby greatly increasing specificity. ZFN have been previously used to cleave endogenous sequences to stimulate S154
homologous recombination. ZFNs were rationally designed to target adjacent sites in HBV. We identified several functional ZFNs pairs using an in vitro cleavage assay. Cleavage of HBV model substrate by ZFN pairs was demonstrated in cultured cells using Southern blots. Repair of free DNA ends by non-homologous end joining introduces deletions or insertions. These should inactivate the virus as it contains a large number of overlapping reading frames. Two assays demonstrated that cognate ZFN pairs cleaved their target and were imperfectly repaired at the target site. This was not seen with individual ZFNs, with control ZFNs or with control binding site plasmids. Mice were hydrodynamically transfected with ZFN pairs and target substrates. Preliminary results showed modest cleavage of model substrates in mice. As in tissue culture, a repair assay showed the target substrates to be mutated at the ZFN’s target site, but not in mice containing control ZFNs or targets. Further experiments will be required to confirm these results and optimize the activity. These results demonstrate one of the first examples of ZFN activity in mice and the first example of targeting a viral genome with a ZFN. Current work focuses delivery of ZFNs using viral vectors. The data presented here represents proof-of-principle for a completely novel antiviral strategy that could be broadly applicable to treatment of DNA viruses.
408. Controlling HIV In Vivo – Establishment of HIV Resistant CD4 T Cells by Engineered Zinc Finger Protein Nucleases
Elena E. Perez,2 Jianbin Wang,1 Jeffrey C. Miller,1 Kenneth A. Kim,1 Olga Liu,2 Dmitry Guschin,1 Edward J. Rebar,1 Bruce L. Levine,2 Richard G. Carroll,2 Dale Ando,1 Philip D. Gregory,1 James L. Riley,2 Michael C. Holmes,1 Carl H. June.2 1 Sangamo BioSciences, Inc., Richmond, CA; 2Dept. of Pathology and Laboratory Medicine, Univ. of Pennsylavania, Philadelphia, PA. Background: HIV requires the CD4 co-receptors CCR5 or CXCR4 to infect its target cells. Elimination of HIV co-receptors would prevent viral entry and thus is an attractive therapy for HIV patients, yet no current methods enable therapeutically relevant disruption of a chosen human gene. We have developed zinc-finger protein nucleases (ZFNs) targeting the CCR5 gene to create a double strand break (DSB) at a predetermined site. Natural DNA repair pathways can subsequently be usurped to imperfectly repair the DSB resulting in the permanent disruption of the target gene. Methods: Engineered zinc finger DNA binding proteins designed to recognize the CCR5 gene were fused to the FokI catalytic domain to create ZFNs in which the DNA binding specificity of the ZFP determines DSB location. The CCR5 ZFNs were transiently expressed in human cells using a chimeric Ad5/F35 Adenoviral vector for in vivo assessment of function. Functional disruption was measured by genotype via a PCR-based assay and phenotype by HIV challenge assays both in vitro and in vivo. Results: Cell-based assays revealed the CCR5-ZFNs generated DSBs in vivo leading to efficient target gene disruption (>50%) in primary human CD4 T-cells. ZFN-treated primary CD4 T cells and transformed CD4 cell lines were shown to be specifically resistant to R5-tropic HIV infection, resulting in enrichment of ZFNgenerated CCR5 null cells upon long-term exposure both in vitro and in vivo. Importantly, ZFN-modified CD4 T-cells preferentially expanded in the presence of HIV in an in vivo xenotransplantation model using NOD/SCID/common g -/- mice. Conclusion: These data demonstrate that ZFN-treated cells can be permanently modified to prevent CCR5-dependent HIV infection. Modified CD4 T-cells selectively expanded in the presence of HIV in vivo and functioned normally in response to stimulation suggesting that these cells may be able to reconstitute immune function in patients with HIV/AIDS via maintenance of an HIV-resistant CD4 T-cell population. These Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy