- Email: [email protected]
826. HSV-Mediated VEGF Gene Transfer Prevents Neuropathy in the Diabetic Mouse
GENE THERAPY FOR THE NERVOUS SYSTEM I properties in vitro and in vivo. Our in vitro data indicate that the rAAV-PPT1 vector produces enzymatically active rPPT1 that is able to cross-correct PPT1(-) cells. We have now examined the in vivo characteristics of our rAAV-PPT1 vector. In PPT(-) mice given 4 intra-cranial injections (2/hemisphere) of 0.5 μl rAAV-PPT1 (1.7 X 10 9 IU/ml) at birth, there are peaks of PPT1 activity corresponding to the injection sites. The average PPT1 specific activity was 75 nmol/mg/h, representing about 25% of normal levels (+/+ range = 250-350 nmol/mg/h). The secondary elevation of another lysosomal enzyme observed in untreated PPT(-) mice was reduced throughout much of the brain in the rAAV-PPT1 treated animals indicating a therapeutic response. Storage material observed on toluidine blue stained sections was reduced in rAAV-PPT1 treated animals. In addition, quantitative measurements showed that the levels of autofluorescent material were significantly (p < 0.05) reduced in the rAAV-PPT1 treated animals and this reduction extended at least 2mm from the injection site. There was a significant (p < 0.001) improvement in the brain weights of rAAV-PPT1 treated animals. The brain weights of normal, PPT1(-), and rAAV-PPT1 treated mice at 7 months of age average 0.403 g, 0.313 g, and 0.384 g, respectively. The treated animals also displayed significant (p < 0.05) increases in cortical volume and thickness. To determine if the treated animals displayed functional improvements, we examined their motor function using the rotarod behavioral test. At five months of age the rAAV-PPT1 treated animals showed significant (p < 0.05) improvement in their ability to remain on the rotarod during all test days and trials. Additional testing at 7 months is currently underway. Experiments to determine if the lifespan is increased and the seizure frequency is reduced in the rAAV-PPT1 treated mice are also in progress. These data indicate that AAV-mediated gene therapy at birth provides both significant biochemical and clinical improvements in the murine model of INCL.
825. The Effects of Pre-Immunization to wtAAV on Single and Multiple Administrations of rAAV in the Brain Carmen Peden,1 Corinna Burger,2 Nick Muzyczka,2 Ron Mandel.1 1 Neuroscience, University of Florida College of Medicine, Gainesville, FL; 2Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL. Epidemiological studies report the prevalence of natural immunization to wild type AAV (wtAAV) to approach 80% of the population, with 30% having antibodies capable of preventing AAV infection. Likewise, some peripheral administrations of rAAV in naïve animals are reported to generate an immune response, preventing further applications of rAAV mediated gene therapy due to production of neutralizing antibodies (nAb), and/or a cellular immune response generated against the transgene product. The bloodbrain-barrier (BBB) provides some immunologic privilege to the brain parenchyma, and the immune response to rAAV administration in the brain of a naïve animal has been minimal. However, vector administration may cause sufficient disruption of the BBB to promote an immune response in a previously immunized animal. We tested the hypothesis that intracerebral rAAV administration and re-administration would not be affected by the presence of circulating antibodies to wtAAV2. Sprague-Dawley rats received a series of three peripheral immunizations using wtAAV2 (109 particles) and an adjuvant, to promote the production of circulating nAb. Along with naïve controls, the rats received an injection of rAAV2, encoding human glial cell line-derived neurotrophic factor (hGDNF), rAAV-hGDNF, in the right striatum. On day 14, rats in the double injection group received an additional injection in the contralateral striatum of rAAV-hGDNF or vehicle. Both single and double administration groups were euthanized either 14 or 28 days S318
after their last surgery for either histologic analysis or quantification of GDNF levels. Blood was also drawn at each time point for comparison of antibody titers. ELISA quantification revealed the immunized animals produced levels of GDNF below their own baseline levels in the contralateral striatum, while the naïve animals, exhibited a seven-fold increase over baseline GDNF production. Moreover, inflammatory markers predominated in the areas of the injections in the pre-immunized cases, and in the immunized double injection groups, were accompanied by actual tissue destruction. All animals in the immunized groups developed very high titers of neutralizing antibodies. We are currently characterizing the nature of the inflammatory response. In summary, we have observed that the presence of a high neutralizing antibody titer, can significantly interfere with successful gene transfer in the brain, and the robust inflammatory response generated is sufficient to cause tissue damage in some cases. It may therefore become necessary to employ some immuno-evasive strategy in patients who exhibit pre-existing immunity to wtAAV. NM is an inventor on patents related to recombinant AAV technology and; owns equity in a gene therapy company that is commercializing AAV for; gene therapy applications.
826. HSV-Mediated VEGF Gene Transfer Prevents Neuropathy in the Diabetic Mouse Munmun Chattopadhyay,1 David Krisky,2 Darren Wolfe,2 James Goss,13 Marina Mata,13 Joseph C. Glorioso,2 David J. Fink.1,2,3 1 Neurology, University of Pittsburgh, Pittsburgh, PA, United States; 2Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States; 3GRECC, Pittsburgh VA Healthcare System, Pittsburgh, PA, United States. Peripheral neuropathy is a common complication of diabetes for which there is no available treatment. In animal models neurotrophic factors can prevent the progression of nerve damage but these potent peptides cannot be administered to patients in adequate doses by systemic routes. We have previously demonstrated that gene transfer achieved by subcutaneous inoculation of a herpes simplex virus (HSV)-based vector expressing neurotrophin-3 or nerve growth factor (NGF) can prevent pyridoxine-induced neuropathy in the rat, and that HSV-mediated gene transfer of NGF can prevent diabetic neuropathy in the mouse. In the study we examined whether HSVmediated gene transfer of the vascular endothelial growth factor (VEGF) could be used to prevent progression of neuropathy in diabetic mice. A VEGF-expressing vector, T0VEGF, was created in a T0ZHG backbone HSV defective for the viral genes ICP4, ICP27, ICP22, and UL41 by cotransfection with a PacI linearized plasmid (p41:0VEGF) containing VEGF165 under the control of the HSV ICP0 promoter element. Expression of VEGF protein was confirmed by Western blot and bioassay, and release of VEGF from transfected primary DRG neurons in culture was determined by ELISA. Diabetes was induced in male Swiss-Webster mice (6-8 weeks old) by the injection of streptozotocin (STZ, 100mg/kg twice over 48 hours). Two weeks after STZ, subsets of mice were inoculated subcutaneously in the plantar surface of the right hind foot with of 10 μl containing 1 × 107 pfu of either vector THVEGF or QOZHG (expressing lacZ). Diabetes induced by STZ resulted in a sensory neuropathy manifest by a decrease in the foot sensory nerve amplitude (FSA; control = 22.0 ± 1.8 μV, diabetic = 12.5 ± 1.3 μV). Transduction of dorsal root ganglia in vivo with vector THVEGF by footpad inoculation 2 weeks after STZ administration protected against the decrease in FSA measured 4 weeks after vector (THVEGF = 31 ± 1.6 μV and QOZHG = 14.1 ± 1.7 μV). Peripheral innervation of sweat glands was evaluated by measuring the number of sweat droplets in the hind paw after stimulation by subcutaneous injection Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
GENE THERAPY OF MUSCLE DISEASES of pilocarpine. Diabetic mice showed a significant reduction in pilocarpine-induced sweating (control = 82.6 ± 4.6 drops, STZ = 20.3 ± 1.2 drops). Diabetic animals inoculated with THVEGF showed a substantial preservation of this peripheral autonomic function (THVEGF = 52 ±7.2 drops, QOZHG = 20.6 ±2.4 drops) indicating restoration of nerve fibers. This was confirmed by the immunostaining of the nerve fibers in the footpad. These results suggest that gene transfer with VEGF-producing herpes-based vector prevents the progression of diabetic neuropathy in the mouse model.
827. Math1 Gene Transfer Generates New Cochlear Hair Cells in Mature Guinea Pigs In Vivo Kohei Kawamoto, Shin-Ichi Ishimoto, Ryosei Minoda, Douglas Brough, Yehoash Raphael. Hair cell loss in the mammalian cochlea is irreversible and results in permanent hearing loss. Math1, the basic helix-loop-helix transcription factor homologue of the Drosophila atonal gene, is a positive regulator of hair cell differentiation during cochlear development. We now demonstrate that in vivo inoculation of adenovirus vector with Math1 gene insert into cochlear endolymph results in Math1 over-expression in non-sensory cells of the mature guinea pig cochlea. Math1 over-expression leads to the appearance of immature hair cells in the organ of Corti and new hair cells adjacent to the organ of Corti. Some of the new hair cells attract neurons. Thus, non-sensory cells in the mature cochlea retain the competence to generate new hair cells upon over-expression of Math1 in vivo, suggesting potential applications of Math1 gene therapy for producing new hair cells in cochleae with depleted hair cell populations.
828. Rescue of Vision through Adult and Fetal AAV Vector Administration to RPE65-/- Mice Enrico M. Surace,1 Nadine S. Dejneka,1 Tomas S. Aleman,1 Artur V. Cideciyan,1 Ernest M. Glover,1 Waixing Tang,1 Tonia S. Rex,1 Albert M. Maguire,1 Samuel G. Jacobson,1 Jean Bennett.1 1 Department of Ophthalmology, F.M Kirby Center, Scheie Eye Institute, Philadelphia, PA, United States. Leber congenital amaurosis (LCA), the most common inherited form of blindness in children, accounts for nearly 5% of all retinal dystrophies. Seven causative genes have been identified, accounting for 50% of all LCA cases. RPE65 mutations are responsible for 710% of LCA. This gene encodes a highly conserved 61kDA protein that is primarily expressed in the microsomal membrane fraction of retinal pigment epithelium (RPE) and in cone photoreceptors. Studies in Rpe65-/- mice reveal that the protein is critical to the visual cycle: mice deficient in RPE65 cannot produce 11-cis-retinal, the chromophore necessary for generating rhodopsin in photoreceptors. Significant progress has already been made in developing gene-basedtherapies for LCA animal models using AAV2/2. In this study we determined the impact of delivering human RPE65 to RPE cells with an AAV vector that has a strong tropism for these cells and also examined the effect of age on delivery. An AAV2/1 hybrid vector was generated with an AAV2 genome encoding the human RPE65 cDNA (AAV2/1-CMV-hRPE65). The virus was administered to Rpe65-/- mice subretinally, in adults (between 1 and 4.5 months), or in fetuses [embryonic day 13 (E13)]. Delivery of AAV2/1-CMVhRPE65 in adults resulted in efficient transduction of RPE and some cones cells. Visual function in treated eyes showed lower thresholds by electroretinography and pupillometry than in control eyes. Following subretinal fetal injections (E13), RPE65 protein was detected in a wide area of the RPE and there was evidence of improved visual function, suggesting that an in utero approach may also be a feasible intervention for the Rpe65-/- phenotype. Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
GENE THERAPY OF MUSCLE DISEASES 829. Microdystrophin Gene Therapy of Cardiomyopathy in Mdx Mice Yongping Yue,1 Zhenbo Li,2 Scott Q. Harper,3 Robin L. Davisson,2 Jeffrey S. Chamberlain,3 Dongsheng Duan.1 1 Molecular Microbiology & Immunology, University of Missouri, Columbia, MO; 2Anatomy & Cell Biology, University of Iowa, Iowa City, IA; 3Neurology, University of Washington, Seattle, WA. Majority Duchenne muscular dystrophy (DMD) patients develop cardiomyopathy and many also die from cardiac failure. Encouraging gene therapy results have been achieved in the skeletal muscle of a mouse DMD model (mdx), yet few gene therapy attempts have been made in treating dystrophic cardiomyopathy. Unlike limb muscles, the anatomic location and the physiologic function of the heart present a formidable challenge to gene transfer. To overcome this hurdle, we have developed a novel heart gene transfer method in newborn mdx mice. Anesthesia was achieved in 46 s (n=114) by placing newborn mice in a custom-designed ice-chamber. Recombinant adeno-associated virus (AAV) was delivered to the cardiac cavity with a glass microinjector. Injection was completed in 93 s (n=112). To optimize the method, we studied the effect of cold-shock time on recovery. Without injection, it took 528 s (n=7) and 453 s (n=16) for mice to recover from a 2 and a 5 min cold-shock respectively. In AAV injected mice, the overall recovery time was not significantly different from controls (2 min, 484 s, n=77; 5 min, 492 s, n=86). However, there was a slightly higher survival rate in the 2 min group (96.43%) compared to the 5 min group (94.64%). The ubiquitous CMV and the muscle specific CK6 promoters have been used in mdx limb muscle gene therapy. To determine the most suitable promoter for cardiac muscle, we compared expression of AV.CMV.LacZ and AV.CK6.LacZ. Despite a similar level of expression in the limb muscle, the CMV promoter led to substantially higher expression in the cardiac muscle (2.96x106RU/mg, n=10) than the CK6 promoter (7.23x104RU/mg, n=10) at 1 m post-infection (pi). This result highlighted the importance of selecting appropriate expression cassettes for DMD gene therapy. DMD therapy needs longterm expression. To evaluate if neonatal gene transfer lead stable expression in the heart, we compared AV.RSV.AP expression at 1 and 6 m pi. Quantitative study revealed a 3-fold increase in AP+ heart cells at 6 m pi (238±38/section, n=6) compared to that at 1 m pi (82±24/section, n=6). It is possible that gene delivery to neonatal mice may have targeted some progenitor cells. Microdystrophin genes are highly truncated versions of the dystrophin gene. They hold tremendous potentials in DMD therapy. Since cardiomyopathy is an independent process, it is critical to evaluate these microgenes in the heart. One of the most important consequences of losing dystrophin is the disappearance of the dystrophin glycoprotein complex (DGC). The DGC mediates mechanical interactions between the cytoskeleton and the extra-cellular matrix. Since the C-terminal domain is completely deleted in microgenes, we investigated whether AAV-mediated microdystrophin expression could restore the DGC in the heart. Consistent with reporter gene transfer, AAV-mediated ΔR4-R23/Δ71-78 microgene transfer in neonatal mdx hearts resulted in profound gene expression in outer- and inner-layers of mdx hearts at 10 m pi (n=9). Importantly, immunofluorescent staining confirmed the successful restoration of other DGC components such as βsarcoglycan and β-dystroglycan in heart cells. Our data have significant implications in the development of DMD heart gene therapy.
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825. The Effects of Pre-Immunization to wtAAV on Single and Multiple Administrations of rAAV in the Brain Carmen Peden,1 Corinna Burger,2 Nick Muzyczka,2 Ron Mandel.1 1 Neuroscience, University of Florida College of Medicine, Gainesville, FL; 2Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL. Epidemiological studies report the prevalence of natural immunization to wild type AAV (wtAAV) to approach 80% of the population, with 30% having antibodies capable of preventing AAV infection. Likewise, some peripheral administrations of rAAV in naïve animals are reported to generate an immune response, preventing further applications of rAAV mediated gene therapy due to production of neutralizing antibodies (nAb), and/or a cellular immune response generated against the transgene product. The bloodbrain-barrier (BBB) provides some immunologic privilege to the brain parenchyma, and the immune response to rAAV administration in the brain of a naïve animal has been minimal. However, vector administration may cause sufficient disruption of the BBB to promote an immune response in a previously immunized animal. We tested the hypothesis that intracerebral rAAV administration and re-administration would not be affected by the presence of circulating antibodies to wtAAV2. Sprague-Dawley rats received a series of three peripheral immunizations using wtAAV2 (109 particles) and an adjuvant, to promote the production of circulating nAb. Along with naïve controls, the rats received an injection of rAAV2, encoding human glial cell line-derived neurotrophic factor (hGDNF), rAAV-hGDNF, in the right striatum. On day 14, rats in the double injection group received an additional injection in the contralateral striatum of rAAV-hGDNF or vehicle. Both single and double administration groups were euthanized either 14 or 28 days S318
after their last surgery for either histologic analysis or quantification of GDNF levels. Blood was also drawn at each time point for comparison of antibody titers. ELISA quantification revealed the immunized animals produced levels of GDNF below their own baseline levels in the contralateral striatum, while the naïve animals, exhibited a seven-fold increase over baseline GDNF production. Moreover, inflammatory markers predominated in the areas of the injections in the pre-immunized cases, and in the immunized double injection groups, were accompanied by actual tissue destruction. All animals in the immunized groups developed very high titers of neutralizing antibodies. We are currently characterizing the nature of the inflammatory response. In summary, we have observed that the presence of a high neutralizing antibody titer, can significantly interfere with successful gene transfer in the brain, and the robust inflammatory response generated is sufficient to cause tissue damage in some cases. It may therefore become necessary to employ some immuno-evasive strategy in patients who exhibit pre-existing immunity to wtAAV. NM is an inventor on patents related to recombinant AAV technology and; owns equity in a gene therapy company that is commercializing AAV for; gene therapy applications.
826. HSV-Mediated VEGF Gene Transfer Prevents Neuropathy in the Diabetic Mouse Munmun Chattopadhyay,1 David Krisky,2 Darren Wolfe,2 James Goss,13 Marina Mata,13 Joseph C. Glorioso,2 David J. Fink.1,2,3 1 Neurology, University of Pittsburgh, Pittsburgh, PA, United States; 2Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States; 3GRECC, Pittsburgh VA Healthcare System, Pittsburgh, PA, United States. Peripheral neuropathy is a common complication of diabetes for which there is no available treatment. In animal models neurotrophic factors can prevent the progression of nerve damage but these potent peptides cannot be administered to patients in adequate doses by systemic routes. We have previously demonstrated that gene transfer achieved by subcutaneous inoculation of a herpes simplex virus (HSV)-based vector expressing neurotrophin-3 or nerve growth factor (NGF) can prevent pyridoxine-induced neuropathy in the rat, and that HSV-mediated gene transfer of NGF can prevent diabetic neuropathy in the mouse. In the study we examined whether HSVmediated gene transfer of the vascular endothelial growth factor (VEGF) could be used to prevent progression of neuropathy in diabetic mice. A VEGF-expressing vector, T0VEGF, was created in a T0ZHG backbone HSV defective for the viral genes ICP4, ICP27, ICP22, and UL41 by cotransfection with a PacI linearized plasmid (p41:0VEGF) containing VEGF165 under the control of the HSV ICP0 promoter element. Expression of VEGF protein was confirmed by Western blot and bioassay, and release of VEGF from transfected primary DRG neurons in culture was determined by ELISA. Diabetes was induced in male Swiss-Webster mice (6-8 weeks old) by the injection of streptozotocin (STZ, 100mg/kg twice over 48 hours). Two weeks after STZ, subsets of mice were inoculated subcutaneously in the plantar surface of the right hind foot with of 10 μl containing 1 × 107 pfu of either vector THVEGF or QOZHG (expressing lacZ). Diabetes induced by STZ resulted in a sensory neuropathy manifest by a decrease in the foot sensory nerve amplitude (FSA; control = 22.0 ± 1.8 μV, diabetic = 12.5 ± 1.3 μV). Transduction of dorsal root ganglia in vivo with vector THVEGF by footpad inoculation 2 weeks after STZ administration protected against the decrease in FSA measured 4 weeks after vector (THVEGF = 31 ± 1.6 μV and QOZHG = 14.1 ± 1.7 μV). Peripheral innervation of sweat glands was evaluated by measuring the number of sweat droplets in the hind paw after stimulation by subcutaneous injection Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
GENE THERAPY OF MUSCLE DISEASES of pilocarpine. Diabetic mice showed a significant reduction in pilocarpine-induced sweating (control = 82.6 ± 4.6 drops, STZ = 20.3 ± 1.2 drops). Diabetic animals inoculated with THVEGF showed a substantial preservation of this peripheral autonomic function (THVEGF = 52 ±7.2 drops, QOZHG = 20.6 ±2.4 drops) indicating restoration of nerve fibers. This was confirmed by the immunostaining of the nerve fibers in the footpad. These results suggest that gene transfer with VEGF-producing herpes-based vector prevents the progression of diabetic neuropathy in the mouse model.
827. Math1 Gene Transfer Generates New Cochlear Hair Cells in Mature Guinea Pigs In Vivo Kohei Kawamoto, Shin-Ichi Ishimoto, Ryosei Minoda, Douglas Brough, Yehoash Raphael. Hair cell loss in the mammalian cochlea is irreversible and results in permanent hearing loss. Math1, the basic helix-loop-helix transcription factor homologue of the Drosophila atonal gene, is a positive regulator of hair cell differentiation during cochlear development. We now demonstrate that in vivo inoculation of adenovirus vector with Math1 gene insert into cochlear endolymph results in Math1 over-expression in non-sensory cells of the mature guinea pig cochlea. Math1 over-expression leads to the appearance of immature hair cells in the organ of Corti and new hair cells adjacent to the organ of Corti. Some of the new hair cells attract neurons. Thus, non-sensory cells in the mature cochlea retain the competence to generate new hair cells upon over-expression of Math1 in vivo, suggesting potential applications of Math1 gene therapy for producing new hair cells in cochleae with depleted hair cell populations.
828. Rescue of Vision through Adult and Fetal AAV Vector Administration to RPE65-/- Mice Enrico M. Surace,1 Nadine S. Dejneka,1 Tomas S. Aleman,1 Artur V. Cideciyan,1 Ernest M. Glover,1 Waixing Tang,1 Tonia S. Rex,1 Albert M. Maguire,1 Samuel G. Jacobson,1 Jean Bennett.1 1 Department of Ophthalmology, F.M Kirby Center, Scheie Eye Institute, Philadelphia, PA, United States. Leber congenital amaurosis (LCA), the most common inherited form of blindness in children, accounts for nearly 5% of all retinal dystrophies. Seven causative genes have been identified, accounting for 50% of all LCA cases. RPE65 mutations are responsible for 710% of LCA. This gene encodes a highly conserved 61kDA protein that is primarily expressed in the microsomal membrane fraction of retinal pigment epithelium (RPE) and in cone photoreceptors. Studies in Rpe65-/- mice reveal that the protein is critical to the visual cycle: mice deficient in RPE65 cannot produce 11-cis-retinal, the chromophore necessary for generating rhodopsin in photoreceptors. Significant progress has already been made in developing gene-basedtherapies for LCA animal models using AAV2/2. In this study we determined the impact of delivering human RPE65 to RPE cells with an AAV vector that has a strong tropism for these cells and also examined the effect of age on delivery. An AAV2/1 hybrid vector was generated with an AAV2 genome encoding the human RPE65 cDNA (AAV2/1-CMV-hRPE65). The virus was administered to Rpe65-/- mice subretinally, in adults (between 1 and 4.5 months), or in fetuses [embryonic day 13 (E13)]. Delivery of AAV2/1-CMVhRPE65 in adults resulted in efficient transduction of RPE and some cones cells. Visual function in treated eyes showed lower thresholds by electroretinography and pupillometry than in control eyes. Following subretinal fetal injections (E13), RPE65 protein was detected in a wide area of the RPE and there was evidence of improved visual function, suggesting that an in utero approach may also be a feasible intervention for the Rpe65-/- phenotype. Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
GENE THERAPY OF MUSCLE DISEASES 829. Microdystrophin Gene Therapy of Cardiomyopathy in Mdx Mice Yongping Yue,1 Zhenbo Li,2 Scott Q. Harper,3 Robin L. Davisson,2 Jeffrey S. Chamberlain,3 Dongsheng Duan.1 1 Molecular Microbiology & Immunology, University of Missouri, Columbia, MO; 2Anatomy & Cell Biology, University of Iowa, Iowa City, IA; 3Neurology, University of Washington, Seattle, WA. Majority Duchenne muscular dystrophy (DMD) patients develop cardiomyopathy and many also die from cardiac failure. Encouraging gene therapy results have been achieved in the skeletal muscle of a mouse DMD model (mdx), yet few gene therapy attempts have been made in treating dystrophic cardiomyopathy. Unlike limb muscles, the anatomic location and the physiologic function of the heart present a formidable challenge to gene transfer. To overcome this hurdle, we have developed a novel heart gene transfer method in newborn mdx mice. Anesthesia was achieved in 46 s (n=114) by placing newborn mice in a custom-designed ice-chamber. Recombinant adeno-associated virus (AAV) was delivered to the cardiac cavity with a glass microinjector. Injection was completed in 93 s (n=112). To optimize the method, we studied the effect of cold-shock time on recovery. Without injection, it took 528 s (n=7) and 453 s (n=16) for mice to recover from a 2 and a 5 min cold-shock respectively. In AAV injected mice, the overall recovery time was not significantly different from controls (2 min, 484 s, n=77; 5 min, 492 s, n=86). However, there was a slightly higher survival rate in the 2 min group (96.43%) compared to the 5 min group (94.64%). The ubiquitous CMV and the muscle specific CK6 promoters have been used in mdx limb muscle gene therapy. To determine the most suitable promoter for cardiac muscle, we compared expression of AV.CMV.LacZ and AV.CK6.LacZ. Despite a similar level of expression in the limb muscle, the CMV promoter led to substantially higher expression in the cardiac muscle (2.96x106RU/mg, n=10) than the CK6 promoter (7.23x104RU/mg, n=10) at 1 m post-infection (pi). This result highlighted the importance of selecting appropriate expression cassettes for DMD gene therapy. DMD therapy needs longterm expression. To evaluate if neonatal gene transfer lead stable expression in the heart, we compared AV.RSV.AP expression at 1 and 6 m pi. Quantitative study revealed a 3-fold increase in AP+ heart cells at 6 m pi (238±38/section, n=6) compared to that at 1 m pi (82±24/section, n=6). It is possible that gene delivery to neonatal mice may have targeted some progenitor cells. Microdystrophin genes are highly truncated versions of the dystrophin gene. They hold tremendous potentials in DMD therapy. Since cardiomyopathy is an independent process, it is critical to evaluate these microgenes in the heart. One of the most important consequences of losing dystrophin is the disappearance of the dystrophin glycoprotein complex (DGC). The DGC mediates mechanical interactions between the cytoskeleton and the extra-cellular matrix. Since the C-terminal domain is completely deleted in microgenes, we investigated whether AAV-mediated microdystrophin expression could restore the DGC in the heart. Consistent with reporter gene transfer, AAV-mediated ΔR4-R23/Δ71-78 microgene transfer in neonatal mdx hearts resulted in profound gene expression in outer- and inner-layers of mdx hearts at 10 m pi (n=9). Importantly, immunofluorescent staining confirmed the successful restoration of other DGC components such as βsarcoglycan and β-dystroglycan in heart cells. Our data have significant implications in the development of DMD heart gene therapy.
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