- Email: [email protected]
49. In Vivo Delivery of Zinc Finger Nucleases Mediates Genome Editing To Correct Genetic Disease
PRESIDENTIAL SYMPOSIUM 47. Longterm and Sustained Knockdown of Mutant AAT along with Simultaneous Augmentation of Wildtype AAT with a Single Dual Function Vector Has Minimal Effect on Global Liver miRNA Profiles
Christian Muller,1 Qiushi Tang,1 Lina Song,1 Phillip D. Zamore,2 Terence R. Flotte.1 1 Pediatrics and Gene Therapy Center, UMass Medical School, Worcester, MA; 2Biochemistry and Molecular Pharmacology, UMass Medical School, Worcester, MA.
Alpha-1 antitrypsin (AAT) is one of the primary circulating serum anti-proteases in humans. AAT is normally produced within hepatocytes and macrophages, where hepatocyte-derived AAT forms the bulk of the physiologic reserve of AAT. Approximately 4% of the North Americans possess at least one copy of a mutant allele, known as PiZ which results from a single amino acid substitution of lysine for glutamate at position 342. This mutation leads to severe deficiency of AAT, and can result in two distinct pathologic states: a lung disease which is primarily due to the loss of antiprotease function, and a liver disease due to a toxic gain of function of the PiZ-AAT mutant protein. Since mutant AAT-PiZ exhibits a gain-of-function hepatocellular toxicity accumulating in the endoplasmic reticulum, decreasing AAT-PiZ mRNA levels (and therefore the protein) may ameliorate or even reverse the liver pathology. In addition, increased secretion of functional AAT protein will theoretically protect the lungs from neutrophil elastase and associated proteolytic enzymes. We have developed several rAAV vectors that allows us to deliver MicroRNAs (miRNAs) within a similar proviral cassette expressing AAT as used in our rAAV-AAT clinical trials. The vectors incorporate miRNA sequences targeting the AAT gene while driving the expression of hardened wildtype AAT gene, thus achieving concomitant mutant AAT knockdown in the liver with increased expression of wiltype AAT. Transgenic mice expressing the human PiZ allele were injected with control or dual function rAAV9 vectors expressing both miRNAs and a hardened AAT gene with a cMyc tag at a dose of 1x10E12 particles. Serum PiZ levels were consistently knocked down by an average of 80% from baseline levels with the knockdown being stable and persistent throughout the 13 week experiment. Cohorts receiving dual function vectors showed knockdown of PiZ AAT while secreting increased serum levels of wildtype AAT as determined by a cMyc ELISA. Liver histology in treated mice revealed significant decreased globular accumulation of misfolded PiZ AAT in hepatocytes along with a reduction in inflammatory infiltrates when compared to controls. To determine if rAAV delivered miRNAs would have an effect on global miRNA expression profiles of the liver, samples from controls and treated mice were compared using a miRNA microarray. Results from the microarray studies showed that miRNA profiles were minimally affected by artificial miRNA delivered by rAAV, with only 3 miRNAs showing statistically significant differences. The largest difference was seen in mir-1 which was actually reduced in PiZ transgenic mice receiving rAAV to normal levels seen in wildtype B6 mice. The levels of miR-122 were unaffected in all mice receiving rAAVs expressing miRNA targeting the AAT gene. In conclusion dual function rAAV vectors are effective at knocking down PIZ AAT while simultaneously augmenting wildtype AAT without disturbing endogenous miRNA liver profiles.
48. In Vivo AAV-Mediated Gene Repair Is Enhanced by the DNA-PK Inhibitor Vanillin
Nicole K. Paulk,1 Milton Finegold,2 Markus Grompe.1 1 Oregon Stem Cell Center, Oregon Health & Science University, Portland, OR; 2Department of Pathology, Texas Children’s Hospital, Houston, TX. Gene therapy is a promising treatment to cure many monogenic diseases; however, traditional gene therapy is best suited to treat diseases of deficient or absent gene products rather than those caused by aberrantly functioning proteins. A potential solution would be manipulating genomes in vivo using site-specific recombination to modify genetic information. Gene repair by vector-mediated homologous recombination is an attractive concept, in that it restores gene function within the context of endogenous regulatory elements, thus eliminating problems of inadequate or inappropriate expression. However, the low frequency of correction in vivo has been prohibitive. To date, the best frequency of targeted integration in vivo for any cell type is only 1%. Many diseases necessitate a higher rate of stable integration (>10%) than is currently possible to achieve. Methods to increase correction to therapeutic levels are needed. One approach to enhancing targeting is a transient block of undesired repair pathways like non-homologous end-joining (NHEJ) to promote the sole use of the homologous recombination pathway. NHEJ is the primary pathway of double strand break repair in postembryonic human cells. The natural product vanillin (3-methoxy4-hydroxybenzaldehyde), acts as a potent inhibitor of NHEJ by binding to and selectively inhibiting DNA-PK. Vanillin is a known antimutagen, anticlastogen, antioxidant, antimicrobial and displays antineoplastic activity. Vanillin’s action is highly selective to DNAPK and has no effect on other steps of NHEJ or on unrelated protein kinases. Additionally, vanillin is an FDA-approved food additive for human consumption, thus an off-label use for gene therapy is feasible. Previous work from our lab has shown conventional rAAV capable of in vivo correction of the model metabolic liver disease hereditary tyrosinemia type I (HTI). The vector contained a 4.5Kb insert containing a portion of the fumarylacetoacetate hydrolase (Fah) gene mutated in HTI. Hepatic correction frequencies of up to 1% were achieved in mice with a point mutation in Fah using normal AAV dose ranges. To increase correction frequencies to more therapeutic levels, we have administered daily vanillin IP injections at 100mg/ kg for 5-7 days following one time IV AAV injection. Preliminary results show an at least 6-fold increase in gene repair with vanillin administration over AAV alone. Further studies examining varying doses and days of administration are ongoing in adult and neonatal mice to determine optimal administration protocols. Importantly, in vivo genetic confirmation that NHEJ inhibition is the responsible factor and not a reaction to vanillin itself, are ongoing in mice doubly mutant for Fah and Ku70.
Presidential Symposium 49. In Vivo Delivery of Zinc Finger Nucleases Mediates Genome Editing To Correct Genetic Disease
Michael Holmes,2 Hojun Li,1 Virginia Haurigot,1 Yannick Doyon,2 James Li,2 Anand Bhagwat,1 Sunnie Wong,2 Xavier Anguela,1 Rajiv Sharma,1 Sam Murphy,1 Fayaz Khazi,1 Shangzhen Zhou,1 David Paschon,2 Ed Rebar,2 Philip Gregory,2 Katherine High.1,3 1 CHOP, Philadelphia, PA; 2Sangamo Biosc, Richmond, CA; 3 HHMI, Philadelphia, PA. Disease-causing gene mutations can potentially be corrected in situ within the genome through genome editing and targeted gene replacement. The development of zinc finger nucleases (ZFNs) to create targeted double-strand breaks (DSBs) has permitted efficient
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
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PRESIDENTIAL SYMPOSIUM genome editing in cultured cells. As ZFN-mediated genome editing has yet to be assessed ]in vivo, we sought to determine if in vivo delivery of ZFNs can stimulate genome editing to correct a genetic disease. We made human factor IX gene (F9)-specific ZFNs with the goal of correcting Hemophilia B (HB). One ZFN pair, targeting F9 intron 1, induced a DSB in 45% of F9 alleles in K562 cells, and when coupled with a targeting (donor) vector, mediated targeted gene insertion in 18% of alleles. For in vivo ZFN testing, we made a ROSA26 knock-in mouse with liver-specific expression of a catalytic-domain deleted human F9 mini-gene (hF9mut). Targeted insertion of w.t. F9 exons 2-8 into intron 1 would allow for splicing with the genomic exon 1 to produce w.t. factor IX (F.IX). AAV8(1e11vg) delivery of ZFNs to adult hF9mut mouse liver induced a DSB in 34-47% of alleles, as assayed by detection of repaired DSBs. As homology-directed repair is predicted to be most efficient in S/G2 of the cell cycle, we next performed targeting experiments in neonatal mice (whose rapidly dividing hepatocytes contrast with non-dividing adult hepatocytes). Although neonatal delivery (5e10vg) resulted in ZFN expression loss over time and 60% less DSB induction efficiency than in adult mice, combining ZFN and AAV8-donor vector (2.5e11vg) resulted in targeted insertion of exons 2-8 into intron 1. No targeted insertion was detected in mice receiving Mock+donor, or ZFN-alone controls. ZFN+donor mice(n=16) averaged 120-350ng/mL circulating F.IX (27% of normal) up to 8 months post-treatment, while ZFN-alone(n=17) and Mock+donor(n=15) mice had <25ng/mL F.IX. Stable genomic insertion was confirmed as no loss of F.IX expression occurred in ZFN+donor mice following partial hepatectomy, which causes loss of expression from non-integrated episomes. Moreover, w.t. mice lacking the hF9mut knock-in (i.e. no ZFN target site) had F.IX levels <30ng/mL despite receiving both ZFN+donor vectors, suggesting the majority of F.IX expression resulted from targeted insertion at the intended locus rather than random integration. To assess the effect of ZFN-mediated gene correction on an HB impaired clotting phenotype, we crossed mouse F.IX-deficient mice with hF9mut mice. HB/hF9mut mice receiving ZFN+donor had a significantly (p=0.001) shortened clotting time (44 sec avg) in the aPTT assay compared to Mock+donor mice (60 sec avg). Importantly, this degree of phenotypic correction resulted in aPTTs which were not significantly different (p=0.09) compared to w.t. mice with normal levels of F.IX. These data are the first demonstration that in vivo ZFN delivery mediates genome editing to correct genetic disease, opening a novel paradigm of in vivo gene therapy.
50. Demonstration of Safety and Antitumoral Activity of JX-594, a Targeted Multi-Mechanistic Oncolytic Poxvirus, Following Intravenous Infusion and/or Intratumoral Injections in Patients with Refractory Metastatic Cancers
Caroline Breitbach,1 James Burke,1 Anne Moon,1 Tae-Ho Hwang,2 John C. Bell,1,3 David H. Kirn.1 1 Jennerex Inc, San Francisco; 2Pusan National University, Busan, Republic of Korea; 3Center for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada. JX-594 is a first-in-class targeted oncolytic poxvirus engineered to selectively replicate in and destroy cancer cells with cell cycle abnormalities and epidermal growth factor receptor (EGFR)/ras pathway activation. Direct oncolysis plus granulocyte macrophage – colony stimulating factor (GM-CSF) expression also stimulates tumor vascular shutdown and anti-tumoral immunity. Expression of the β-galactosidase marker transgene allows for monitoring of JX594 replication in patients. Over 95 cancer patients have been treated by intravenous (IV) and/or intratumoral (IT) injections on one of eight Phase 1 or Phase 2 trials to date, including two ongoing Phase 2 trials in patients with primary liver cancer. Treatment with JX-594 by either route of administration was well-tolerated at the 1 x 10^9 S20
plaque forming units (pfu) dose level, representing the maximum tolerated dose (MTD) following IT JX-594 injection; the MTD was not reached following IV JX-594 infusion. The most common adverse events were dose-related flu-like symptoms generally resolving within 24 hours following JX-594 administration. JX-594 was detected acutely in the serum following either IT or IV treatment. Delayed JX-594 reappearance (day 3-15) in the bloodstream after initial clearance was observed in a subset of patients and is consistent with replication at the tumor sites and release into the blood. Expression of JX-594 transgenes was observed in patients treated by IT or IV administration; GM-CSF could be detected in post-treatment serum and correlated with induction of white blood cells. Furthermore, induction of antibodies to the β-galactosidase marker protein was observed in patients treated with JX-594 IV or IT, demonstrating gene expression in vivo. Objective radiographic tumor responses (RECIST and Choi criteria) and tumor vascular disruption have been observed in patients with metastatic cancers following JX-594 treatment IT (injected and non-injected tumors) or IV; Choi (necrotic) responses have been observed in approximately two-thirds of patients treated on Phase 2 trials. Finally, multiple long-term survivors (>/= 12 months in treatment-refractory patients) have been reported on JX-594 trials; in a randomized liver cancer study, patients treated with high dose JX-594 exhibited a survival advantage when compared to the low dose group. A randomized, placebo-controlled trial of JX594 in patients with refractory primary liver cancer is planned to be initiated in 2011.
51. A Phase I Dose-Escalating Study of AAV1 – γ-Sarcoglycan Gene Therapy for Limb Girdle Muscular Dystrophy Type 2C
Serge Herson,1 Fayçal Hentati,2 Aude Rigolet,1 Norma B. Romero,3 Anthony Behin,3 France Leturcq,4 Pascal Laforet,3 Thierry Maisonobe,3 Rim Amouri,2 Hafedh Haddad,5 Muriel Audit,6 MarieFrançoise Rosier-Montus,5 Bernard Gjata,5 Carole Masurier,5 François M. Lemoine,7 Pierre Carlier,4 Jean-Yves Hogrel,4 Bruno Eymard,4 Lee Sweeney,8 Richard Mulligan,9 David Klatzmann,7 Mustapha Cheraï,7 Didier Caizergues,5 Thomas Voït,4 Olivier Benveniste.1 1 Internal Medicine, Université Pierre et Marie Curie, Assistance Publique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France; 2Department of Molecular Neurobiology and Neuropathology, National Institute of Neurology, Tunis, Tunisia; 3 Institute of Myology, Université Pierre et Marie Curie, Assistance Publique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France; 4Laboratoire de Biochimie Génétique, Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Paris, France; 5 Généthon, Evry, France; 6GenoSafe, Evry, France; 7UMR 7211, CNRS, Université Pierre et Marie Curie, Assistance PubliqueHôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France; 8 Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA; 9Harvard Gene Therapy Initiative, Department of Genetics, Harvard Medical School, Boston, MA. OBJECTIVE: Safety of dose-escalating AAV1 – γ-sarcoglycan gene therapy (Phase I) for Limb Girdle Muscular Dystrophy type 2C. BACKGROUND: Gamma-sarcoglycanopathy or limb girdle muscular dystrophy type 2C (LGMD 2C) is an as yet untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene (γSGC). DESIGN/METHODS: Nine non-ambulant LGMD2C patients (2 M, 7 F, age 27 y [16 to 38]), with a del525T homozygous mutation on the γSGC gene and absence of γSGC immunostaining in muscle biopsy were enrolled. They were divided into 3 groups to receive 3 escalating doses of an AAV1 vector expressing human γSGC gene under the control of the desmin promoter, by local intramuscular injection into the extensor carpi radialis muscle. The first group received a single injection of 3e+9 viral genome (vg) in 100 µl, the Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
Christian Muller,1 Qiushi Tang,1 Lina Song,1 Phillip D. Zamore,2 Terence R. Flotte.1 1 Pediatrics and Gene Therapy Center, UMass Medical School, Worcester, MA; 2Biochemistry and Molecular Pharmacology, UMass Medical School, Worcester, MA.
Alpha-1 antitrypsin (AAT) is one of the primary circulating serum anti-proteases in humans. AAT is normally produced within hepatocytes and macrophages, where hepatocyte-derived AAT forms the bulk of the physiologic reserve of AAT. Approximately 4% of the North Americans possess at least one copy of a mutant allele, known as PiZ which results from a single amino acid substitution of lysine for glutamate at position 342. This mutation leads to severe deficiency of AAT, and can result in two distinct pathologic states: a lung disease which is primarily due to the loss of antiprotease function, and a liver disease due to a toxic gain of function of the PiZ-AAT mutant protein. Since mutant AAT-PiZ exhibits a gain-of-function hepatocellular toxicity accumulating in the endoplasmic reticulum, decreasing AAT-PiZ mRNA levels (and therefore the protein) may ameliorate or even reverse the liver pathology. In addition, increased secretion of functional AAT protein will theoretically protect the lungs from neutrophil elastase and associated proteolytic enzymes. We have developed several rAAV vectors that allows us to deliver MicroRNAs (miRNAs) within a similar proviral cassette expressing AAT as used in our rAAV-AAT clinical trials. The vectors incorporate miRNA sequences targeting the AAT gene while driving the expression of hardened wildtype AAT gene, thus achieving concomitant mutant AAT knockdown in the liver with increased expression of wiltype AAT. Transgenic mice expressing the human PiZ allele were injected with control or dual function rAAV9 vectors expressing both miRNAs and a hardened AAT gene with a cMyc tag at a dose of 1x10E12 particles. Serum PiZ levels were consistently knocked down by an average of 80% from baseline levels with the knockdown being stable and persistent throughout the 13 week experiment. Cohorts receiving dual function vectors showed knockdown of PiZ AAT while secreting increased serum levels of wildtype AAT as determined by a cMyc ELISA. Liver histology in treated mice revealed significant decreased globular accumulation of misfolded PiZ AAT in hepatocytes along with a reduction in inflammatory infiltrates when compared to controls. To determine if rAAV delivered miRNAs would have an effect on global miRNA expression profiles of the liver, samples from controls and treated mice were compared using a miRNA microarray. Results from the microarray studies showed that miRNA profiles were minimally affected by artificial miRNA delivered by rAAV, with only 3 miRNAs showing statistically significant differences. The largest difference was seen in mir-1 which was actually reduced in PiZ transgenic mice receiving rAAV to normal levels seen in wildtype B6 mice. The levels of miR-122 were unaffected in all mice receiving rAAVs expressing miRNA targeting the AAT gene. In conclusion dual function rAAV vectors are effective at knocking down PIZ AAT while simultaneously augmenting wildtype AAT without disturbing endogenous miRNA liver profiles.
48. In Vivo AAV-Mediated Gene Repair Is Enhanced by the DNA-PK Inhibitor Vanillin
Nicole K. Paulk,1 Milton Finegold,2 Markus Grompe.1 1 Oregon Stem Cell Center, Oregon Health & Science University, Portland, OR; 2Department of Pathology, Texas Children’s Hospital, Houston, TX. Gene therapy is a promising treatment to cure many monogenic diseases; however, traditional gene therapy is best suited to treat diseases of deficient or absent gene products rather than those caused by aberrantly functioning proteins. A potential solution would be manipulating genomes in vivo using site-specific recombination to modify genetic information. Gene repair by vector-mediated homologous recombination is an attractive concept, in that it restores gene function within the context of endogenous regulatory elements, thus eliminating problems of inadequate or inappropriate expression. However, the low frequency of correction in vivo has been prohibitive. To date, the best frequency of targeted integration in vivo for any cell type is only 1%. Many diseases necessitate a higher rate of stable integration (>10%) than is currently possible to achieve. Methods to increase correction to therapeutic levels are needed. One approach to enhancing targeting is a transient block of undesired repair pathways like non-homologous end-joining (NHEJ) to promote the sole use of the homologous recombination pathway. NHEJ is the primary pathway of double strand break repair in postembryonic human cells. The natural product vanillin (3-methoxy4-hydroxybenzaldehyde), acts as a potent inhibitor of NHEJ by binding to and selectively inhibiting DNA-PK. Vanillin is a known antimutagen, anticlastogen, antioxidant, antimicrobial and displays antineoplastic activity. Vanillin’s action is highly selective to DNAPK and has no effect on other steps of NHEJ or on unrelated protein kinases. Additionally, vanillin is an FDA-approved food additive for human consumption, thus an off-label use for gene therapy is feasible. Previous work from our lab has shown conventional rAAV capable of in vivo correction of the model metabolic liver disease hereditary tyrosinemia type I (HTI). The vector contained a 4.5Kb insert containing a portion of the fumarylacetoacetate hydrolase (Fah) gene mutated in HTI. Hepatic correction frequencies of up to 1% were achieved in mice with a point mutation in Fah using normal AAV dose ranges. To increase correction frequencies to more therapeutic levels, we have administered daily vanillin IP injections at 100mg/ kg for 5-7 days following one time IV AAV injection. Preliminary results show an at least 6-fold increase in gene repair with vanillin administration over AAV alone. Further studies examining varying doses and days of administration are ongoing in adult and neonatal mice to determine optimal administration protocols. Importantly, in vivo genetic confirmation that NHEJ inhibition is the responsible factor and not a reaction to vanillin itself, are ongoing in mice doubly mutant for Fah and Ku70.
Presidential Symposium 49. In Vivo Delivery of Zinc Finger Nucleases Mediates Genome Editing To Correct Genetic Disease
Michael Holmes,2 Hojun Li,1 Virginia Haurigot,1 Yannick Doyon,2 James Li,2 Anand Bhagwat,1 Sunnie Wong,2 Xavier Anguela,1 Rajiv Sharma,1 Sam Murphy,1 Fayaz Khazi,1 Shangzhen Zhou,1 David Paschon,2 Ed Rebar,2 Philip Gregory,2 Katherine High.1,3 1 CHOP, Philadelphia, PA; 2Sangamo Biosc, Richmond, CA; 3 HHMI, Philadelphia, PA. Disease-causing gene mutations can potentially be corrected in situ within the genome through genome editing and targeted gene replacement. The development of zinc finger nucleases (ZFNs) to create targeted double-strand breaks (DSBs) has permitted efficient
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
S19
PRESIDENTIAL SYMPOSIUM genome editing in cultured cells. As ZFN-mediated genome editing has yet to be assessed ]in vivo, we sought to determine if in vivo delivery of ZFNs can stimulate genome editing to correct a genetic disease. We made human factor IX gene (F9)-specific ZFNs with the goal of correcting Hemophilia B (HB). One ZFN pair, targeting F9 intron 1, induced a DSB in 45% of F9 alleles in K562 cells, and when coupled with a targeting (donor) vector, mediated targeted gene insertion in 18% of alleles. For in vivo ZFN testing, we made a ROSA26 knock-in mouse with liver-specific expression of a catalytic-domain deleted human F9 mini-gene (hF9mut). Targeted insertion of w.t. F9 exons 2-8 into intron 1 would allow for splicing with the genomic exon 1 to produce w.t. factor IX (F.IX). AAV8(1e11vg) delivery of ZFNs to adult hF9mut mouse liver induced a DSB in 34-47% of alleles, as assayed by detection of repaired DSBs. As homology-directed repair is predicted to be most efficient in S/G2 of the cell cycle, we next performed targeting experiments in neonatal mice (whose rapidly dividing hepatocytes contrast with non-dividing adult hepatocytes). Although neonatal delivery (5e10vg) resulted in ZFN expression loss over time and 60% less DSB induction efficiency than in adult mice, combining ZFN and AAV8-donor vector (2.5e11vg) resulted in targeted insertion of exons 2-8 into intron 1. No targeted insertion was detected in mice receiving Mock+donor, or ZFN-alone controls. ZFN+donor mice(n=16) averaged 120-350ng/mL circulating F.IX (27% of normal) up to 8 months post-treatment, while ZFN-alone(n=17) and Mock+donor(n=15) mice had <25ng/mL F.IX. Stable genomic insertion was confirmed as no loss of F.IX expression occurred in ZFN+donor mice following partial hepatectomy, which causes loss of expression from non-integrated episomes. Moreover, w.t. mice lacking the hF9mut knock-in (i.e. no ZFN target site) had F.IX levels <30ng/mL despite receiving both ZFN+donor vectors, suggesting the majority of F.IX expression resulted from targeted insertion at the intended locus rather than random integration. To assess the effect of ZFN-mediated gene correction on an HB impaired clotting phenotype, we crossed mouse F.IX-deficient mice with hF9mut mice. HB/hF9mut mice receiving ZFN+donor had a significantly (p=0.001) shortened clotting time (44 sec avg) in the aPTT assay compared to Mock+donor mice (60 sec avg). Importantly, this degree of phenotypic correction resulted in aPTTs which were not significantly different (p=0.09) compared to w.t. mice with normal levels of F.IX. These data are the first demonstration that in vivo ZFN delivery mediates genome editing to correct genetic disease, opening a novel paradigm of in vivo gene therapy.
50. Demonstration of Safety and Antitumoral Activity of JX-594, a Targeted Multi-Mechanistic Oncolytic Poxvirus, Following Intravenous Infusion and/or Intratumoral Injections in Patients with Refractory Metastatic Cancers
Caroline Breitbach,1 James Burke,1 Anne Moon,1 Tae-Ho Hwang,2 John C. Bell,1,3 David H. Kirn.1 1 Jennerex Inc, San Francisco; 2Pusan National University, Busan, Republic of Korea; 3Center for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada. JX-594 is a first-in-class targeted oncolytic poxvirus engineered to selectively replicate in and destroy cancer cells with cell cycle abnormalities and epidermal growth factor receptor (EGFR)/ras pathway activation. Direct oncolysis plus granulocyte macrophage – colony stimulating factor (GM-CSF) expression also stimulates tumor vascular shutdown and anti-tumoral immunity. Expression of the β-galactosidase marker transgene allows for monitoring of JX594 replication in patients. Over 95 cancer patients have been treated by intravenous (IV) and/or intratumoral (IT) injections on one of eight Phase 1 or Phase 2 trials to date, including two ongoing Phase 2 trials in patients with primary liver cancer. Treatment with JX-594 by either route of administration was well-tolerated at the 1 x 10^9 S20
plaque forming units (pfu) dose level, representing the maximum tolerated dose (MTD) following IT JX-594 injection; the MTD was not reached following IV JX-594 infusion. The most common adverse events were dose-related flu-like symptoms generally resolving within 24 hours following JX-594 administration. JX-594 was detected acutely in the serum following either IT or IV treatment. Delayed JX-594 reappearance (day 3-15) in the bloodstream after initial clearance was observed in a subset of patients and is consistent with replication at the tumor sites and release into the blood. Expression of JX-594 transgenes was observed in patients treated by IT or IV administration; GM-CSF could be detected in post-treatment serum and correlated with induction of white blood cells. Furthermore, induction of antibodies to the β-galactosidase marker protein was observed in patients treated with JX-594 IV or IT, demonstrating gene expression in vivo. Objective radiographic tumor responses (RECIST and Choi criteria) and tumor vascular disruption have been observed in patients with metastatic cancers following JX-594 treatment IT (injected and non-injected tumors) or IV; Choi (necrotic) responses have been observed in approximately two-thirds of patients treated on Phase 2 trials. Finally, multiple long-term survivors (>/= 12 months in treatment-refractory patients) have been reported on JX-594 trials; in a randomized liver cancer study, patients treated with high dose JX-594 exhibited a survival advantage when compared to the low dose group. A randomized, placebo-controlled trial of JX594 in patients with refractory primary liver cancer is planned to be initiated in 2011.
51. A Phase I Dose-Escalating Study of AAV1 – γ-Sarcoglycan Gene Therapy for Limb Girdle Muscular Dystrophy Type 2C
Serge Herson,1 Fayçal Hentati,2 Aude Rigolet,1 Norma B. Romero,3 Anthony Behin,3 France Leturcq,4 Pascal Laforet,3 Thierry Maisonobe,3 Rim Amouri,2 Hafedh Haddad,5 Muriel Audit,6 MarieFrançoise Rosier-Montus,5 Bernard Gjata,5 Carole Masurier,5 François M. Lemoine,7 Pierre Carlier,4 Jean-Yves Hogrel,4 Bruno Eymard,4 Lee Sweeney,8 Richard Mulligan,9 David Klatzmann,7 Mustapha Cheraï,7 Didier Caizergues,5 Thomas Voït,4 Olivier Benveniste.1 1 Internal Medicine, Université Pierre et Marie Curie, Assistance Publique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France; 2Department of Molecular Neurobiology and Neuropathology, National Institute of Neurology, Tunis, Tunisia; 3 Institute of Myology, Université Pierre et Marie Curie, Assistance Publique-Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France; 4Laboratoire de Biochimie Génétique, Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Paris, France; 5 Généthon, Evry, France; 6GenoSafe, Evry, France; 7UMR 7211, CNRS, Université Pierre et Marie Curie, Assistance PubliqueHôpitaux de Paris, Hôpital Pitié-Salpêtrière, Paris, France; 8 Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA; 9Harvard Gene Therapy Initiative, Department of Genetics, Harvard Medical School, Boston, MA. OBJECTIVE: Safety of dose-escalating AAV1 – γ-sarcoglycan gene therapy (Phase I) for Limb Girdle Muscular Dystrophy type 2C. BACKGROUND: Gamma-sarcoglycanopathy or limb girdle muscular dystrophy type 2C (LGMD 2C) is an as yet untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene (γSGC). DESIGN/METHODS: Nine non-ambulant LGMD2C patients (2 M, 7 F, age 27 y [16 to 38]), with a del525T homozygous mutation on the γSGC gene and absence of γSGC immunostaining in muscle biopsy were enrolled. They were divided into 3 groups to receive 3 escalating doses of an AAV1 vector expressing human γSGC gene under the control of the desmin promoter, by local intramuscular injection into the extensor carpi radialis muscle. The first group received a single injection of 3e+9 viral genome (vg) in 100 µl, the Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy