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4. A Rationally Engineered Novel Capsid Variant of AAV9 for Pripheral Tissue-Detargeted and CNS-Directed Systemic Gene Delivery
AAV VECTOR DEVELOPMENT & APPLICATION 4. A Rationally Engineered Novel Capsid Variant of AAV9 for Pripheral Tissue-Detargeted and CNS-Directed Systemic Gene Delivery
5. In Vivo Directed Evolution of a Novel Adeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery from the Vitreous
Li Zhong,1,2 Shaoyong Li,1,3 Kim Van Vliet,4 Mengxin Li,1 Jun Xie,1 Jia Li,1 Qin Su,1 Ran He,1 Yu Zhang,1 Huapeng Li,1 Dan Wang,1 Jason Goetzmann,5 Terence R. Flotte,1,2 Mavis AgbandjeMcKenna,4 Guangping Gao.1,3 1 Gene Therapy Center, UMass Medical School, Worcester, MA; 2 Dept of Pediatrics, UMass Medical School, Worcester, MA; 3Dept of Microbio & Physiol Systems, UMass Medical School, Worcester, MA; 4Dept of Biochemistry and Mol Biology, University of Florida, Gainesville, FL; 5New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA.
Leah C. Byrne,1 Deniz Dalkara,1 Ryan R. Klimczak,2 Meike Visel,2 Lu Yin,3 William H. Merigan,3 John G. Flannery,1,2 David V. Schaffer.1,4 1 Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA; 2Department of Molecular and Cellular Biology, University of California Berkeley, Berkeley, CA; 3Flaum Eye Institute and the Center for Visual Science, University of Rochester, Rochester, NY; 4Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, CA.
Intravenous (i.v.) gene delivery to the CNS by rAAV is an attractive approach for treating central nervous system (CNS) disorders. Among the rAAVs that can cross the blood-brain-barrier and achieve global CNS transduction, rAAV9 is the vector with such a propensity. However, one of the caveats with systemic rAAV9mediated CNS transduction is its strong transcytosis ability, resulting in simultaneously extensive transduction of all major peripheral tissues and possible transgene-related toxicity. Here, we report a rationally engineered novel capsid variant of AAV9 for peripheral tissue-detargeted and CNS-directed systemic gene delivery. We isolated a novel natural variant of AAV9 called AAVClvD8 with a significantly reduced transcytosis property. We performed structural analysis of the ClvD8 capsid using the established AAV9 capsid structure as a template. This study revealed that other than position 647, the other mutated amino acid (a.a.) residues in Clv-D8 are located on the AAV9 capsid protrusions that surround the icosahedral 3-fold axis of AAV9 VP3 protein, which plays a role in receptor binding and cellular transduction. To map the a.a. residue(s) responsible for peripheral (primarily liver) tissue transduction by i.v. delivery of rAAV9, we made single and combinatory mutations for each of the a.a. on the AAV9 capsid that are mutated in AAVClvD8. The corresponding luciferase expressing vectors were delivered into C57BL/6 mice by i.v., intranasal (i.n.) and intramuscular (i.m.) injections to compare their luciferase transduction and vector genome biodistribution profiles with rAAV9 wild type (wt). We identified a novel capsid mutant of AAV9 called AAV9HR that generated logs lower luciferase expression and vector genome abundance (VGA) in liver by all three routes and local tissue-restricted expression and genome persistence by i.m. and i.n. delivery. AAV9.HR displayed a blood clearance pattern similar to that of rAAV9wt after i.v., i.m., and i.n. administration suggesting that its ability to cross vascular barrier from tissue to vessel was not impaired. Most importantly, following i.v. delivery of rAAV9wt and rAAV9HR.EGFP to adult C57BL/6 mice, both vectors produced comparable VGA and EGFP transduction in the CNS. However, rAAV9HR led to 135-fold less VGA and significantly diminished EGFP expression in liver and other peripheral organs as compared to rAAV9wt. The similar experiments in early postnatal mice are underway. Our study represents a significant advance in understanding vector biology of AAV9 and developing novel vectors with transduction efficiency similar to rAAV9 but improved safety profile for i.v. gene therapy of CNS disorders.
Inherited retinal degenerations are a primary clinical focus of adeno-associated virus (AAV) mediated gene therapy. These diseases predominantly involve pathogenic mutations in photoreceptor or RPE transcripts. Current delivery methods require an injurious subretinal injection to reach the outer retina, and transduce just a fraction of the retinal area. To address the need for broadly applicable retinal gene delivery, we developed and implemented in vivo directed evolution to create AAV variants that transduce the retina after injection into the easily accessible vitreous humor. A resulting novel AAV variant mediated widespread gene delivery to photoreceptors and RPE following intravitreal administration and could thus efficiently rescue the disease phenotype of rd12 mice, a rodent model of Leber’s congenital amaurosis. Intravitreal injection in non-human primates led to transduction of photoreceptors in the fovea and in punctate regions in extrafoveal and peripheral retina. AAV can therefore be engineered to overcome formidable physical and cellular barriers within structurally complex tissues, and in this case transduce photoreceptors from the vitreous, thereby substantially expanding its therapeutic promise.
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6. Adeno-Associated Virus Capsid Motif That Influences Tissue Specific Vector Transduction In Vivo
Jayme K. Warischalk,1,2 Richard J. Samulski.1,2 1 Gene Therapy Center, University of North Carolina Chapel Hill, Chapel Hill, NC; 2Pharmacology, University of North Carolina Chapel Hill, Chapel Hill, NC.
Adeno-associated viral (AAV) vectors have garnered much promise in gene therapy applications due to their lack of pathogenicity, broad tissue tropism, and ability to confer long-term gene expression. Recent widespread clinical use has identified rate-limiting steps that affect the efficiency of vector transduction. We have recently used rational design to create a chimeric AAV2 capsid (designated AAV2.5) optimized for enhanced transduction of muscle cells following intramuscular delivery. AAV2.5 combined an AAV2 capsid with 5 amino acid mutations from the AAV1 capsid, and was proven safe, tolerable and effective in Phase 1 clinical trials. Further studies determined that of the five amino acids altered position 265 influenced vector tropism as well as immune response (Single amino acid modification of adeno-associated virus capsid changes transduction and humoral immune profiles.Li C et al.,2012). We have thus chosen to further explore the mechanism by which these mutations exert their enhanced transduction by examining their role in the capsid backbone of AAV 1, 2, 6, 8 9. Through select mutations at a single amino acid position of surface loop VR1 (variable region 1) within these capsids, we have gained the ability to modulate their tissue tropism from a phenotype that is primarily liver tropic to one that exhibits robust and in many cases exclusive transduction of cardiac and/or skeletal muscle following intravenous administration of virus. Analyses of these capsid vector backbones in other targets (e.g. ocular) suggest efficient transduction that exceeds all parental capsids. Finally, we Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
5. In Vivo Directed Evolution of a Novel Adeno-Associated Virus for Therapeutic Outer Retinal Gene Delivery from the Vitreous
Li Zhong,1,2 Shaoyong Li,1,3 Kim Van Vliet,4 Mengxin Li,1 Jun Xie,1 Jia Li,1 Qin Su,1 Ran He,1 Yu Zhang,1 Huapeng Li,1 Dan Wang,1 Jason Goetzmann,5 Terence R. Flotte,1,2 Mavis AgbandjeMcKenna,4 Guangping Gao.1,3 1 Gene Therapy Center, UMass Medical School, Worcester, MA; 2 Dept of Pediatrics, UMass Medical School, Worcester, MA; 3Dept of Microbio & Physiol Systems, UMass Medical School, Worcester, MA; 4Dept of Biochemistry and Mol Biology, University of Florida, Gainesville, FL; 5New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA.
Leah C. Byrne,1 Deniz Dalkara,1 Ryan R. Klimczak,2 Meike Visel,2 Lu Yin,3 William H. Merigan,3 John G. Flannery,1,2 David V. Schaffer.1,4 1 Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA; 2Department of Molecular and Cellular Biology, University of California Berkeley, Berkeley, CA; 3Flaum Eye Institute and the Center for Visual Science, University of Rochester, Rochester, NY; 4Department of Chemical and Biomolecular Engineering, University of California Berkeley, Berkeley, CA.
Intravenous (i.v.) gene delivery to the CNS by rAAV is an attractive approach for treating central nervous system (CNS) disorders. Among the rAAVs that can cross the blood-brain-barrier and achieve global CNS transduction, rAAV9 is the vector with such a propensity. However, one of the caveats with systemic rAAV9mediated CNS transduction is its strong transcytosis ability, resulting in simultaneously extensive transduction of all major peripheral tissues and possible transgene-related toxicity. Here, we report a rationally engineered novel capsid variant of AAV9 for peripheral tissue-detargeted and CNS-directed systemic gene delivery. We isolated a novel natural variant of AAV9 called AAVClvD8 with a significantly reduced transcytosis property. We performed structural analysis of the ClvD8 capsid using the established AAV9 capsid structure as a template. This study revealed that other than position 647, the other mutated amino acid (a.a.) residues in Clv-D8 are located on the AAV9 capsid protrusions that surround the icosahedral 3-fold axis of AAV9 VP3 protein, which plays a role in receptor binding and cellular transduction. To map the a.a. residue(s) responsible for peripheral (primarily liver) tissue transduction by i.v. delivery of rAAV9, we made single and combinatory mutations for each of the a.a. on the AAV9 capsid that are mutated in AAVClvD8. The corresponding luciferase expressing vectors were delivered into C57BL/6 mice by i.v., intranasal (i.n.) and intramuscular (i.m.) injections to compare their luciferase transduction and vector genome biodistribution profiles with rAAV9 wild type (wt). We identified a novel capsid mutant of AAV9 called AAV9HR that generated logs lower luciferase expression and vector genome abundance (VGA) in liver by all three routes and local tissue-restricted expression and genome persistence by i.m. and i.n. delivery. AAV9.HR displayed a blood clearance pattern similar to that of rAAV9wt after i.v., i.m., and i.n. administration suggesting that its ability to cross vascular barrier from tissue to vessel was not impaired. Most importantly, following i.v. delivery of rAAV9wt and rAAV9HR.EGFP to adult C57BL/6 mice, both vectors produced comparable VGA and EGFP transduction in the CNS. However, rAAV9HR led to 135-fold less VGA and significantly diminished EGFP expression in liver and other peripheral organs as compared to rAAV9wt. The similar experiments in early postnatal mice are underway. Our study represents a significant advance in understanding vector biology of AAV9 and developing novel vectors with transduction efficiency similar to rAAV9 but improved safety profile for i.v. gene therapy of CNS disorders.
Inherited retinal degenerations are a primary clinical focus of adeno-associated virus (AAV) mediated gene therapy. These diseases predominantly involve pathogenic mutations in photoreceptor or RPE transcripts. Current delivery methods require an injurious subretinal injection to reach the outer retina, and transduce just a fraction of the retinal area. To address the need for broadly applicable retinal gene delivery, we developed and implemented in vivo directed evolution to create AAV variants that transduce the retina after injection into the easily accessible vitreous humor. A resulting novel AAV variant mediated widespread gene delivery to photoreceptors and RPE following intravitreal administration and could thus efficiently rescue the disease phenotype of rd12 mice, a rodent model of Leber’s congenital amaurosis. Intravitreal injection in non-human primates led to transduction of photoreceptors in the fovea and in punctate regions in extrafoveal and peripheral retina. AAV can therefore be engineered to overcome formidable physical and cellular barriers within structurally complex tissues, and in this case transduce photoreceptors from the vitreous, thereby substantially expanding its therapeutic promise.
S2
6. Adeno-Associated Virus Capsid Motif That Influences Tissue Specific Vector Transduction In Vivo
Jayme K. Warischalk,1,2 Richard J. Samulski.1,2 1 Gene Therapy Center, University of North Carolina Chapel Hill, Chapel Hill, NC; 2Pharmacology, University of North Carolina Chapel Hill, Chapel Hill, NC.
Adeno-associated viral (AAV) vectors have garnered much promise in gene therapy applications due to their lack of pathogenicity, broad tissue tropism, and ability to confer long-term gene expression. Recent widespread clinical use has identified rate-limiting steps that affect the efficiency of vector transduction. We have recently used rational design to create a chimeric AAV2 capsid (designated AAV2.5) optimized for enhanced transduction of muscle cells following intramuscular delivery. AAV2.5 combined an AAV2 capsid with 5 amino acid mutations from the AAV1 capsid, and was proven safe, tolerable and effective in Phase 1 clinical trials. Further studies determined that of the five amino acids altered position 265 influenced vector tropism as well as immune response (Single amino acid modification of adeno-associated virus capsid changes transduction and humoral immune profiles.Li C et al.,2012). We have thus chosen to further explore the mechanism by which these mutations exert their enhanced transduction by examining their role in the capsid backbone of AAV 1, 2, 6, 8 9. Through select mutations at a single amino acid position of surface loop VR1 (variable region 1) within these capsids, we have gained the ability to modulate their tissue tropism from a phenotype that is primarily liver tropic to one that exhibits robust and in many cases exclusive transduction of cardiac and/or skeletal muscle following intravenous administration of virus. Analyses of these capsid vector backbones in other targets (e.g. ocular) suggest efficient transduction that exceeds all parental capsids. Finally, we Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy