- Email: [email protected]
863. Liver Gene Therapy for Hemophilia with phiC31 Integrase
IMMUNODEFICIENCIES/HEMOPHILIA to serotype, and a reduced chance of insertional mutagenesis; thus serving as an ideal tool in gene therapeutics. By utilizing our novel transgenic model we have investigated the ability of rAAV vector gene delivery to rescue mutant mice from pathologies associated with the missing gene. Two rAAV vectors encoding human ADA cDNA have been generated that are independently driven by the elongation factor-1alpha (EF-1α) and creatine kinase-6 (CK-6) promoters. We have characterized the efficiency of integration of our vector constructs, expression levels in whole blood and target tissues, the impact on animal phenotype and inflammatory protein profiles, and have optimized an injection protocol that has resulted in a significant increase in the life span of diseased mice.
862. Feasibility of Cutaneous Gene Therapy Using a Factor VIII Gene and a Marker Gene in Factor VIII-Deficient Mice by Non-Invasive Electroporation Lei Zhang,1 Amy Goldbeck,2 Hongbo Lu,2 Edward Nolan,1 Dietmar Rabussay,1 Steve Fakharzadeh.2 1 Dept. of R&D, Inovio Biomedical Corporation, San Diego, CA; 2 Dept. of Dermatology, University of Pennsylvania, Philadelphia, PA. In vivo gene delivery to skin is potentially useful for a broad range of clinical purposes. We previously reported that in vivo electroporation (EP) is a simple and effective method for intracellular delivery of naked DNA to mouse skin. Furthermore, we have demonstrated that transgene-directed epidermal expression of FVIII achieved phenotypic correction in a mouse model for hemophilia A. Given these findings, our main objectives for the present study were: (i) to test the feasibility of producing circulating FVIII in hemophilia A mice by introducing a “B” domain-deleted murine FVIII gene (mFVIII) into skin cells via EP; and (ii) to compare levels and duration of transgene expression using two types of non-invasive electrodes (caliper vs. meander). To address these objectives, we codelivered independent plasmids carrying either a mFVIII cDNA or a secreted alkaline phosphatase (SEAP) marker gene to FVIII knockout mice. Both genes were under transcriptional control of CMV promoter/enhancer sequences. FVIII knockout mice were assigned to four treatment groups (N=4/group): i) skin DNA injection only, ii) skin DNA injection + EP (caliper), iii) skin DNA injection + EP (meander), and iv) muscle DNA injection + EP (caliper). A mixture containing 200 µg mFVIII plasmid DNA and 10 µg of SEAP plasmid DNA was injected at 10 sites on dorsal skin prior to EP. Muscletreated mice received injections of the DNA mixture into the tibialis muscle of each rear leg. Blood samples were obtained by tail bleeding at days 0, 2, 4, 7, 10, 15, and at 6 weeks. FVIII activity in plasma samples was measured using the COAMATIC assay and circulating SEAP was measured by ELISA. Skin biopsies were obtained from treated sites from additional mice at days 2, 4, 7, and 15 after treatment. Total RNA extracted from skin samples was used to assess expression of mFVIII and SEAP by RT-PCR. We detected transgene-specific mRNA for both mFVIII and SEAP in treated skin. Both genes showed similar expression patterns peaking at approximately day 4. The meander electrode appeared to yield more efficient EP than the caliper based on duration of transgene expression and band signal intensity. In addition, EP enhanced levels of SEAP in the circulation of skin-treated mice by one to two orders of magnitude compared to non-EP-treated mice. Furthermore, use of the meander electrode yielded an approximately twofold higher level of circulating SEAP than the caliper. Muscle-treated mice, however, displayed SEAP levels an order of magnitude higher than skin-treated mice at the time of peak expression on day 7. In contrast, mFVIII activity was not detectable in the circulation of skin- or muscletreated mice despite expression of mFVIII mRNA and high expression of the co-delivered SEAP plasmid. These results indicate that EP S332
use of a meander electrode may be more efficient, in addition to its user-friendliness, than use of a caliper electrode. However, application of this technology to gene therapy for more complex gene products, such as FVIII, will require further optimization.
863. Liver Gene Therapy for Hemophilia with phiC31 Integrase Lauren E. Woodard, Annahita Keravala, Robert T. Hillman, Julien Sage, Michele P. Calos. 1 Genetics, Stanford University School of Medicine, Stanford, CA. The phiC31 integrase system effectively integrates plasmid DNA carrying an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, long-term expression of transgenes. The phiC31 system has been used to genetically engineer Drosophila and Xenopus and for in vivo and ex vivo gene therapy in several tissues in model organisms including mouse, rat, and rabbit. The integrase system appears to be well suited for long-term gene therapy in humans. In particular, the phiC31 system may allow long-term gene expression in hepatocytes in vivo. A simple and efficient delivery system for liver using naked DNA is available for mice by using high-pressure tail vein injection. With this delivery method and phiC31 integrase, we demonstrated lifelong, therapeutic levels of expression of human factor IX in mice. An adapted version of this DNA delivery method may be appropriate for use in patients. Our current study addressed the amount of liver perturbation and subsequent proliferation seen after the highpressure tail vein procedure. Using a BrdU analog to label proliferating hepatocytes, we showed that a sizable fraction of the hepatocytes entered S phase within two days after injection. By one week post-injection, however, hepatocytes had stopped proliferating and the livers appeared normal by histological analysis. We also determined the immunogenicity and ability to re-deliver phiC31 integrase by measurement of expression levels of a reporter gene after repeated doses. The level and duration of gene expression achievable with the phiC31 integrase system in liver makes it an attractive strategy for clinical development of gene therapies for diseases such as hemophilia. Michele Calos is a co-founder and consultant for Poetic Genetics, LLC, which has licensed the phiC31 integrase technology.
864. Characterization of Transgenic Mice OverExpressing Arylsulfatase A: Implications for Gene Therapy and Sulfatases Activation Mechanisms Alessia Capotondo,1,2 Mary A. Venneri,1 Maria P. Cosma,3 Isabella Pallavicini,1 Andrea Ballabio,3 Luigi Naldini,1,2 Alessandra Biffi.1,2 1 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milano, Italy; 2Vita Salute University, San Raffaele Scientific Institute, Milano, Italy; 3Telethon Institute of Genetics and Medicine, Napoli, Italy. We have recently shown that in order to attain therapeutic efficacy by HSC gene therapy for Metachromatic Leukodystrophy (MLD), Arylsulfatase A (ARSA) over-expression in HSC and their progeny is required. The recent identification of Sulfatase Modifying Factor 1 (SUMF1) as a common activator of sulfatases and a rate-limiting factor in the biological activation of these enzymes, raises concerns for possible adverse effects of enzyme over-expression within certain cell type (Cosma M.P. et al., Cell 2003). In fact, over-expression of one type of sulfatase may lead to reduced activity of other sulfatases by a competitive interaction with their common activator. In order to assess the safety of ARSA over-expression in vivo we generated transgenic mice over-expressing the ARSA gene by lentiviral vectors (LV). Pups were genotyped for the presence of the LV backbone by PCR, and screened for vector content (expressed in LV copies per Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
862. Feasibility of Cutaneous Gene Therapy Using a Factor VIII Gene and a Marker Gene in Factor VIII-Deficient Mice by Non-Invasive Electroporation Lei Zhang,1 Amy Goldbeck,2 Hongbo Lu,2 Edward Nolan,1 Dietmar Rabussay,1 Steve Fakharzadeh.2 1 Dept. of R&D, Inovio Biomedical Corporation, San Diego, CA; 2 Dept. of Dermatology, University of Pennsylvania, Philadelphia, PA. In vivo gene delivery to skin is potentially useful for a broad range of clinical purposes. We previously reported that in vivo electroporation (EP) is a simple and effective method for intracellular delivery of naked DNA to mouse skin. Furthermore, we have demonstrated that transgene-directed epidermal expression of FVIII achieved phenotypic correction in a mouse model for hemophilia A. Given these findings, our main objectives for the present study were: (i) to test the feasibility of producing circulating FVIII in hemophilia A mice by introducing a “B” domain-deleted murine FVIII gene (mFVIII) into skin cells via EP; and (ii) to compare levels and duration of transgene expression using two types of non-invasive electrodes (caliper vs. meander). To address these objectives, we codelivered independent plasmids carrying either a mFVIII cDNA or a secreted alkaline phosphatase (SEAP) marker gene to FVIII knockout mice. Both genes were under transcriptional control of CMV promoter/enhancer sequences. FVIII knockout mice were assigned to four treatment groups (N=4/group): i) skin DNA injection only, ii) skin DNA injection + EP (caliper), iii) skin DNA injection + EP (meander), and iv) muscle DNA injection + EP (caliper). A mixture containing 200 µg mFVIII plasmid DNA and 10 µg of SEAP plasmid DNA was injected at 10 sites on dorsal skin prior to EP. Muscletreated mice received injections of the DNA mixture into the tibialis muscle of each rear leg. Blood samples were obtained by tail bleeding at days 0, 2, 4, 7, 10, 15, and at 6 weeks. FVIII activity in plasma samples was measured using the COAMATIC assay and circulating SEAP was measured by ELISA. Skin biopsies were obtained from treated sites from additional mice at days 2, 4, 7, and 15 after treatment. Total RNA extracted from skin samples was used to assess expression of mFVIII and SEAP by RT-PCR. We detected transgene-specific mRNA for both mFVIII and SEAP in treated skin. Both genes showed similar expression patterns peaking at approximately day 4. The meander electrode appeared to yield more efficient EP than the caliper based on duration of transgene expression and band signal intensity. In addition, EP enhanced levels of SEAP in the circulation of skin-treated mice by one to two orders of magnitude compared to non-EP-treated mice. Furthermore, use of the meander electrode yielded an approximately twofold higher level of circulating SEAP than the caliper. Muscle-treated mice, however, displayed SEAP levels an order of magnitude higher than skin-treated mice at the time of peak expression on day 7. In contrast, mFVIII activity was not detectable in the circulation of skin- or muscletreated mice despite expression of mFVIII mRNA and high expression of the co-delivered SEAP plasmid. These results indicate that EP S332
use of a meander electrode may be more efficient, in addition to its user-friendliness, than use of a caliper electrode. However, application of this technology to gene therapy for more complex gene products, such as FVIII, will require further optimization.
863. Liver Gene Therapy for Hemophilia with phiC31 Integrase Lauren E. Woodard, Annahita Keravala, Robert T. Hillman, Julien Sage, Michele P. Calos. 1 Genetics, Stanford University School of Medicine, Stanford, CA. The phiC31 integrase system effectively integrates plasmid DNA carrying an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, long-term expression of transgenes. The phiC31 system has been used to genetically engineer Drosophila and Xenopus and for in vivo and ex vivo gene therapy in several tissues in model organisms including mouse, rat, and rabbit. The integrase system appears to be well suited for long-term gene therapy in humans. In particular, the phiC31 system may allow long-term gene expression in hepatocytes in vivo. A simple and efficient delivery system for liver using naked DNA is available for mice by using high-pressure tail vein injection. With this delivery method and phiC31 integrase, we demonstrated lifelong, therapeutic levels of expression of human factor IX in mice. An adapted version of this DNA delivery method may be appropriate for use in patients. Our current study addressed the amount of liver perturbation and subsequent proliferation seen after the highpressure tail vein procedure. Using a BrdU analog to label proliferating hepatocytes, we showed that a sizable fraction of the hepatocytes entered S phase within two days after injection. By one week post-injection, however, hepatocytes had stopped proliferating and the livers appeared normal by histological analysis. We also determined the immunogenicity and ability to re-deliver phiC31 integrase by measurement of expression levels of a reporter gene after repeated doses. The level and duration of gene expression achievable with the phiC31 integrase system in liver makes it an attractive strategy for clinical development of gene therapies for diseases such as hemophilia. Michele Calos is a co-founder and consultant for Poetic Genetics, LLC, which has licensed the phiC31 integrase technology.
864. Characterization of Transgenic Mice OverExpressing Arylsulfatase A: Implications for Gene Therapy and Sulfatases Activation Mechanisms Alessia Capotondo,1,2 Mary A. Venneri,1 Maria P. Cosma,3 Isabella Pallavicini,1 Andrea Ballabio,3 Luigi Naldini,1,2 Alessandra Biffi.1,2 1 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milano, Italy; 2Vita Salute University, San Raffaele Scientific Institute, Milano, Italy; 3Telethon Institute of Genetics and Medicine, Napoli, Italy. We have recently shown that in order to attain therapeutic efficacy by HSC gene therapy for Metachromatic Leukodystrophy (MLD), Arylsulfatase A (ARSA) over-expression in HSC and their progeny is required. The recent identification of Sulfatase Modifying Factor 1 (SUMF1) as a common activator of sulfatases and a rate-limiting factor in the biological activation of these enzymes, raises concerns for possible adverse effects of enzyme over-expression within certain cell type (Cosma M.P. et al., Cell 2003). In fact, over-expression of one type of sulfatase may lead to reduced activity of other sulfatases by a competitive interaction with their common activator. In order to assess the safety of ARSA over-expression in vivo we generated transgenic mice over-expressing the ARSA gene by lentiviral vectors (LV). Pups were genotyped for the presence of the LV backbone by PCR, and screened for vector content (expressed in LV copies per Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy