- Email: [email protected]
239. Enteric Nervous System Transduction Following Intravascular Delivery of AAV9
AAV VECTOR DEVELOPMENT to transduce motor neurons in adult spinal cord, which will greatly facilitate the evaluation of treatment strategies targeting this hardto-reach population of cells in adult mice, which can be readily translated into clinical application. Currently, we are evaluating intracerebroventricular infusion of rAAV vectors using osmotic pumps, aiming to achieve enhanced global CNS transduction by delivering rAAV into CSF space.
239. Enteric Nervous System Transduction Following Intravascular Delivery of AAV9
S. E. Gombash Lampe,1 C. J. Cowley,1 F. L. Christofi,1 J. A. Fitzgerald,1 K. D. Foust.1 1 The Ohio State University, Columbus.
Gastrointestinal (GI) dysfunction is frequently reported in patients with neurologic and neuromuscular diseases. Symptoms related to GI dysmotility, such as constipation, gastroparesis and gastroesophageal reflux, impair the quality of life for patients with Parkinson’s disease, multiple sclerosis, spinal muscular atrophy, spinal cord injury, autism spectrum disorders, and dementia. Peristalsis is highly regulated by the enteric nervous system, composed of the myenteric and submucosal nerve plexuses. Therefore, development of novel therapeutics that target enteric neurons may be of great benefit in treating GI symptoms. The purpose of the current study was to characterize transduction efficiency and transgene distribution in the myenteric plexus of the complete GI tract following intravenous injection of adeno-associated virus serotype 9 expressing green fluorescent protein (scAAV9CB-GFP) in neonatal (P1) and juvenile (P21) mice. Neonatal mice were intravenously injected with scAAV9-CB-GFP (3 x 1011 vg) in the temporal face vein on P1 and juvenile mice received tail vein injections on P21 (2 x 1012 vg). Mice were euthanized at 60-90 days of age and the complete GI tract was dissected to expose the myenteric plexus. Immunohistochemical labeling of the myenteric plexus revealed GFP immunoreactivity in the esophagus, stomach, small intestine, cecum and colon in mice injected at each age. Neuronal transduction ranged from 21-57% depending on GI region analyzed. GFP colocalized with HuD positive myenteric neurons and S100 positive glial cells. Characterization of specific neuronal subtypes is ongoing and co-labeling studies thus far are consistent with multiple types of neurons in the ENS. Quantification of GFP immunoreactive neurons throughout the GI tract revealed increased transduction in P21 injected mice compared to P1 injected mice in most regions. This is in contrast to CNS data where neuronal transduction is more prominent in neonate injected animals. Segmental examinations of myenteric neurons in the colon showed no oral-aboral bias. These findings are significant because it shows AAV9 can be used as a tool to study intestinal function and for the development of GI therapeutics. Comparisons with other AAV serotypes are ongoing.
240. Efficient and Targeted Transduction of Nonhuman Primate Liver With Optimized AAV3B Vectors Through Systemic Delivery
Li Zhong, Shaoyong Li, Mengxin Li, Chen Ling, Qin Su, Ran He,1 Arun Srivastava,4 Guangping Gao.1,3 1 Gene Therapy Center, UMass Medical School, Worcester, MA; 2 Pediatrics, UMass Medical School, Worcester, MA; 3Microbio & Physiol Systems, UMass Medical School, Worcester, MA; 4 Pediatrics, University of Florida, Gainesville, FL. 1,2
1,3
1,3
4
1
Recent studies have demonstrated that rAAV3B vectors transduce human liver cancer cells as well as primary human hepatocytes efficiently in vitro. The combinations of modifications of specific surface-exposed serine (S) and threonine (T) residues on rAAV3 capsids further augment transduction efficiency in human liver cancer cells. Moreover these optimized rAAV3B vectors can achieve targeted delivery in a human liver cancer xenograft model after systemic Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
administration, suggesting that AAV3B and their derivative vectors are ideal for liver-directed gene therapy in humans. Here, we used the nonhuman primate model for liver-directed gene transfer to examine the safety and efficacy of these vectors which is critically important for their further clinical development in liver gene therapy in humans. To this end, we first investigated sero-epidemiology of AAV3B in a rhesus monkey colony and found that the pre-existing neutralized antibodies (Nab) against AAV3B are relatively low in rhesus macaques (i.e. 52% of animals with no detectable Nab at a sensitivity of 1:5). To study liver tropism of optimized AAV3B vectors in NHP liver, we dosed three young (3-4 months) male rhesus macaques with S663V+T492V modified AAV3B (AAV3B.ST)-CB-EGFP vector at 1x1013 GC/kg intravenously and analysed EGFP expression and histopathology in the liver 7 days later by fluorescence microscopy. The results revealed that the optimized AAV3B.ST vector led to efficient and specific EGFP expression in the liver (36% hepatocytes in average as compared to 25% achieved by AAV7 in NHP liver) but not in other tissues, and without apparent hepatotoxicity. To quantitatively assess transduction efficiency of AAV3B and optimized AAV3B.ST vectors in NHPs, we i.v. injected three young male rhesus macaques with wild type (WT) and ST modified AAV3B-CB-rhCG vectors at 1x1013 GC/kg respectively and monitored serum rhCG levels for 3 months. Both WT and ST modified AAV3B vectors led to efficient and sustained rhCG expression in 104 – 105 rU/ml range up to 91 days, but ST modification enhanced rhCG expression by 2 (at late stage) to 5 (at early stage) folds as compared to WT AAV3B vector. This was confimed with qRT-PCR analysis of liver rhCG mRNA levels at the 91 day end point, showing again a 2-fold increased transcription by ST modification. The biodistribution analysis of the persisting vector genomes indicated a predominant liver targeting. Clinical chemistry and histopathology examinations of the study animals showed no apparent vector-related toxicity. In summary, the optimal AAV3B.ST vector can safely and specifically delivery transgene to primate liver and resulted in a further increased transduction efficiency as compared to WT AAV3B vectors after systemic administration. Our studies should be important and informative for clinical development of optimized AAV3B vectors for liver-directed gene therapy in humans. * Equally contributed authors # Co-corresponding authors.
241. Vector Design Tour De Force: Integrating Combinatorial and Rational Approaches To Derive Novel Adeno-Associated Virus (AAV) Variants
Damien Marsic,1 Lakshmanan Govindasamy,2 Seth Currlin,1 David Markusic,1 Yu-Shan Tseng,2 Roland W. Herzog,1 Mavis AgbandjeMcKenna,2 Yuan Lu,3 Steve Ghivizzani,3 Sergei Zolotukhin.1 1 Pediatrics, University of Florida, Gainesville, FL; 2Biochemistry and Molecular Biology, University of Florida, Gainesville, FL; 3Orthopaedics & Rehabilitation, University of Florida, Gainesville, FL. AAV-derived vectors are promising tools for human gene therapy applications because of their absence of pathogenicity, episomal localization and stable transgene expression. However, significant limitations to their clinical use are their lack of specificity and their susceptibility to neutralization by human antibodies. Both of these limitations are determined by the nature of amino acid residues exposed at the surface of the capsid. Methodologies to improve existing AAV vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Although both methodologies have been successful in creating improved vectors, their utility is limited by our understanding of the AAV life cycle and by the technical boundaries of the protocols for directed evolution. The strategy adopted in this project integrates both approaches in one unified design. We have conducted a process of “virtual family S91
239. Enteric Nervous System Transduction Following Intravascular Delivery of AAV9
S. E. Gombash Lampe,1 C. J. Cowley,1 F. L. Christofi,1 J. A. Fitzgerald,1 K. D. Foust.1 1 The Ohio State University, Columbus.
Gastrointestinal (GI) dysfunction is frequently reported in patients with neurologic and neuromuscular diseases. Symptoms related to GI dysmotility, such as constipation, gastroparesis and gastroesophageal reflux, impair the quality of life for patients with Parkinson’s disease, multiple sclerosis, spinal muscular atrophy, spinal cord injury, autism spectrum disorders, and dementia. Peristalsis is highly regulated by the enteric nervous system, composed of the myenteric and submucosal nerve plexuses. Therefore, development of novel therapeutics that target enteric neurons may be of great benefit in treating GI symptoms. The purpose of the current study was to characterize transduction efficiency and transgene distribution in the myenteric plexus of the complete GI tract following intravenous injection of adeno-associated virus serotype 9 expressing green fluorescent protein (scAAV9CB-GFP) in neonatal (P1) and juvenile (P21) mice. Neonatal mice were intravenously injected with scAAV9-CB-GFP (3 x 1011 vg) in the temporal face vein on P1 and juvenile mice received tail vein injections on P21 (2 x 1012 vg). Mice were euthanized at 60-90 days of age and the complete GI tract was dissected to expose the myenteric plexus. Immunohistochemical labeling of the myenteric plexus revealed GFP immunoreactivity in the esophagus, stomach, small intestine, cecum and colon in mice injected at each age. Neuronal transduction ranged from 21-57% depending on GI region analyzed. GFP colocalized with HuD positive myenteric neurons and S100 positive glial cells. Characterization of specific neuronal subtypes is ongoing and co-labeling studies thus far are consistent with multiple types of neurons in the ENS. Quantification of GFP immunoreactive neurons throughout the GI tract revealed increased transduction in P21 injected mice compared to P1 injected mice in most regions. This is in contrast to CNS data where neuronal transduction is more prominent in neonate injected animals. Segmental examinations of myenteric neurons in the colon showed no oral-aboral bias. These findings are significant because it shows AAV9 can be used as a tool to study intestinal function and for the development of GI therapeutics. Comparisons with other AAV serotypes are ongoing.
240. Efficient and Targeted Transduction of Nonhuman Primate Liver With Optimized AAV3B Vectors Through Systemic Delivery
Li Zhong, Shaoyong Li, Mengxin Li, Chen Ling, Qin Su, Ran He,1 Arun Srivastava,4 Guangping Gao.1,3 1 Gene Therapy Center, UMass Medical School, Worcester, MA; 2 Pediatrics, UMass Medical School, Worcester, MA; 3Microbio & Physiol Systems, UMass Medical School, Worcester, MA; 4 Pediatrics, University of Florida, Gainesville, FL. 1,2
1,3
1,3
4
1
Recent studies have demonstrated that rAAV3B vectors transduce human liver cancer cells as well as primary human hepatocytes efficiently in vitro. The combinations of modifications of specific surface-exposed serine (S) and threonine (T) residues on rAAV3 capsids further augment transduction efficiency in human liver cancer cells. Moreover these optimized rAAV3B vectors can achieve targeted delivery in a human liver cancer xenograft model after systemic Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
administration, suggesting that AAV3B and their derivative vectors are ideal for liver-directed gene therapy in humans. Here, we used the nonhuman primate model for liver-directed gene transfer to examine the safety and efficacy of these vectors which is critically important for their further clinical development in liver gene therapy in humans. To this end, we first investigated sero-epidemiology of AAV3B in a rhesus monkey colony and found that the pre-existing neutralized antibodies (Nab) against AAV3B are relatively low in rhesus macaques (i.e. 52% of animals with no detectable Nab at a sensitivity of 1:5). To study liver tropism of optimized AAV3B vectors in NHP liver, we dosed three young (3-4 months) male rhesus macaques with S663V+T492V modified AAV3B (AAV3B.ST)-CB-EGFP vector at 1x1013 GC/kg intravenously and analysed EGFP expression and histopathology in the liver 7 days later by fluorescence microscopy. The results revealed that the optimized AAV3B.ST vector led to efficient and specific EGFP expression in the liver (36% hepatocytes in average as compared to 25% achieved by AAV7 in NHP liver) but not in other tissues, and without apparent hepatotoxicity. To quantitatively assess transduction efficiency of AAV3B and optimized AAV3B.ST vectors in NHPs, we i.v. injected three young male rhesus macaques with wild type (WT) and ST modified AAV3B-CB-rhCG vectors at 1x1013 GC/kg respectively and monitored serum rhCG levels for 3 months. Both WT and ST modified AAV3B vectors led to efficient and sustained rhCG expression in 104 – 105 rU/ml range up to 91 days, but ST modification enhanced rhCG expression by 2 (at late stage) to 5 (at early stage) folds as compared to WT AAV3B vector. This was confimed with qRT-PCR analysis of liver rhCG mRNA levels at the 91 day end point, showing again a 2-fold increased transcription by ST modification. The biodistribution analysis of the persisting vector genomes indicated a predominant liver targeting. Clinical chemistry and histopathology examinations of the study animals showed no apparent vector-related toxicity. In summary, the optimal AAV3B.ST vector can safely and specifically delivery transgene to primate liver and resulted in a further increased transduction efficiency as compared to WT AAV3B vectors after systemic administration. Our studies should be important and informative for clinical development of optimized AAV3B vectors for liver-directed gene therapy in humans. * Equally contributed authors # Co-corresponding authors.
241. Vector Design Tour De Force: Integrating Combinatorial and Rational Approaches To Derive Novel Adeno-Associated Virus (AAV) Variants
Damien Marsic,1 Lakshmanan Govindasamy,2 Seth Currlin,1 David Markusic,1 Yu-Shan Tseng,2 Roland W. Herzog,1 Mavis AgbandjeMcKenna,2 Yuan Lu,3 Steve Ghivizzani,3 Sergei Zolotukhin.1 1 Pediatrics, University of Florida, Gainesville, FL; 2Biochemistry and Molecular Biology, University of Florida, Gainesville, FL; 3Orthopaedics & Rehabilitation, University of Florida, Gainesville, FL. AAV-derived vectors are promising tools for human gene therapy applications because of their absence of pathogenicity, episomal localization and stable transgene expression. However, significant limitations to their clinical use are their lack of specificity and their susceptibility to neutralization by human antibodies. Both of these limitations are determined by the nature of amino acid residues exposed at the surface of the capsid. Methodologies to improve existing AAV vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Although both methodologies have been successful in creating improved vectors, their utility is limited by our understanding of the AAV life cycle and by the technical boundaries of the protocols for directed evolution. The strategy adopted in this project integrates both approaches in one unified design. We have conducted a process of “virtual family S91