- Email: [email protected]
251. Defining Conserved Structural Components of the AAV Capsid That Enable Tissue-Specific Transduction Following Systemic Administration
Evolving Better Parvoviral Vectors for Gene Therapy day +5; N=13). Time to platelet counts >20,000/µl without platelet transfusions was 14-54 days. Median VCN in DP and peripheral blood leukocytes (PBL) and proportions of PBLs and CD14+ myeloid cells expressing ALDP as detected by flow cytometric analysis are below. Integration site analysis shows polyclonal reconstitution in all subjects, with no indication of vector mediated clonal skewing to date. Neurologic outcomes (NFS and major functional disabilities [MFD]) were assessed at each study visit. MRI results (Loes Score and gadolinium enhancement) were periodically reviewed centrally. MRI data were available on 8 subjects with ≥6m follow up. All 8 had resolution of gadolinium enhancement on brain MRI. Median change in Loes Score was 1 (0-6), and no patient demonstrated progression of NFS. Two SAEs were assessed possibly related to Lenti-D drug product (DP), BK virus cystitis and tachycardia. To date, the safety profile of Lenti-D DP appears consistent with myeloablative conditioning. These early results demonstrate feasibility of centralized manufacturing for a HSC gene therapy trial and short-term safety and efficacy of gene therapy in CALD. Assessment of long-term outcomes and safety continues. Median (IQR) VCN and ALDP expression over time % PBLs expressing VCN ALDP PreDP: 1.0 (0.6, 1.6), 0.9 (0.5, 1.5), treatment N=17 N=17 PBL: 0.5 (0.3, 13.5 (9.3, 20.1), 6 months 1.0), N=13 N=12 12 PBL: 0.7 (0.4, 22.9 (18.8, 29.5), months 0.8), N=9 N=5
% CD14+ expresing ALDP 1.0 (0.7, 1.6), N=11 26.4 (14.0, 36.0), N=10 32.5 (22.3, 45.2), N=6
Evolving Better Parvoviral Vectors for Gene Therapy 251. Defining Conserved Structural Components of the AAV Capsid That Enable Tissue-Specific Transduction Following Systemic Administration
Jayme K. Warischalk1,2, Samuel C. Eaton2, R. Jude Samulski1,2 1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 2Dept. of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC We have previously correlated the destabilization of hydrogen bond networks within recombinant adeno-associated viral (rAAV) capsid surface loop variable region 1 (VR1) to transduction efficiency following intramuscular injection. To investigate the utility of this finding for systemic applications, the capsid structures of rAAV serotypes 1 through 9 were computationally analyzed and VR1 residues participating in hydrogen bonding were individually deleted. Mutant capsids were assessed for tissue tropism and transduction efficiency following intravenous administration into mice. Ex vivo luciferase assays revealed increased muscle transduction and targeting in five of the nine serotypes (rAAV1, rAAV6, rAAV7, rAAV8, rAAV9); cardiac and skeletal muscle transduction were enhanced by up to 170-fold and 9027-fold over parental serotypes, while hepatic transduction was decreased up to 427-fold. VR1 stability also appeared critical to the remaining serotypes, as VR1 deletion mutations rendered rAAV2 and rAAV3b defective in transduction, and rAAV4 and rAAV5 defective in virion production. Intriguingly, amino acid insertions into VR1 produced an opposing phenotype: rAAV1, rAAV2, rAAV3b, and rAAV6 capsids could instead be targeted to transduce the liver with high efficiency, increasing hepatic transduction up to 132-fold. Finally, VR1 mutation synergized with
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
established tyrosine-to-phenylalanine mutations to further increase transduction efficiency while maintaining preferential cardiac/skeletal muscle targeting.
252. Using Cre-Dependent In Vivo Selection to Identify AAV Variants That Enable Efficient and Widespread Gene Transfer to the Adult Central Nervous System
Benjamin E. Deverman1, Piers L. Pravdo1, Bryan P. Simpson1, Sripriya Ravindra Kumar1, Yicheng Luo1, Ken Y. Chan1, Abhik Banerjee1, Wei-Li Wu1, Bin Yang1, Nina Huber2, Sergiu P. Pasca2, Viviana Gradinaru1 1 Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 2Department of Psychiatry and Behavioral Sciences, Stanford, Stanford, CA Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting the development of novel AAV capsids with more desirable transduction characteristics. We have developed a capsid selection method, called Cre-recombination-based AAV targeted evolution (CREATE), that enables the identification of AAV capsids that more efficiently transduce defined cell populations in vivo (Deverman et al. in press, Nature Biotechnology). We generated AAV capsid libraries and used CREATE to identify variants that cross the blood brain barrier and efficiently and widely transduce astrocytes in the mouse central nervous system (CNS) after intravenous injection. One variant, AAVPHP.B, transfers genes throughout the adult CNS with an efficiency that is 40- to 92-fold greater (depending on the CNS region) than that of the current standard, AAV9. It transduces the majority of astrocytes and neurons across multiple CNS regions, and in vitro, it transduces human neurons and astrocytes more efficiently than does AAV9. We are now evolving AAV-PHP.B for even greater transduction of specific CNS cell types as a means to both develop more effective vectors and to gain insight into the mechanism of enhanced transduction. Our identification of AAV-PHP.B and several other enhanced vectors after only two rounds of selection establishes CREATE as a powerful method to customize AAV vectors for biomedical applications.
253. Expanded Packaging Capacity of AAV by Lumenal Charge Alteration Matthew Tiffany, Mark A. Kay Stanford University, Stanford, CA
Adeno-associated virus (AAV) is widely believed to be the safest viral vector for gene therapy. Moreover, the array of AAV serotypes available allows transduction of different tissues in vivo. However, one major limitation of AAV is the relatively small DNA packaging size (4,700 nt). Existing serotypes have been over-packaged with limited success and varying reproducibility. The consensus appears to be that AAV can be over packaged by ~10%, but with a concomitant reduction in both viral titers and in vivo transduction. We have taken a novel approach to increase the packaging limitations of AAV by focusing on the lumen of AAV where vector DNA interacts with the capsid. We have created a series of AAV capsid variants that alter the capsid lumenal charge using AAVDJ as the scaffold. The variants add positively charged lysine and arginine residues at lumenally exposed sites within the capsid that, we believe, may allow for interaction and condensation of vector DNA increasing the packaging capacity and/or stabilizing the capsid in an over-packaged state. We present in vitro characterization of the library of capsid variants and demonstrate that variants with intermediate charge (+4 to +7 over wildtype) can be produced at high titer when over-packaged up to 6.2 kb. In addition, these variants can transduce S99
% CD14+ expresing ALDP 1.0 (0.7, 1.6), N=11 26.4 (14.0, 36.0), N=10 32.5 (22.3, 45.2), N=6
Evolving Better Parvoviral Vectors for Gene Therapy 251. Defining Conserved Structural Components of the AAV Capsid That Enable Tissue-Specific Transduction Following Systemic Administration
Jayme K. Warischalk1,2, Samuel C. Eaton2, R. Jude Samulski1,2 1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 2Dept. of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC We have previously correlated the destabilization of hydrogen bond networks within recombinant adeno-associated viral (rAAV) capsid surface loop variable region 1 (VR1) to transduction efficiency following intramuscular injection. To investigate the utility of this finding for systemic applications, the capsid structures of rAAV serotypes 1 through 9 were computationally analyzed and VR1 residues participating in hydrogen bonding were individually deleted. Mutant capsids were assessed for tissue tropism and transduction efficiency following intravenous administration into mice. Ex vivo luciferase assays revealed increased muscle transduction and targeting in five of the nine serotypes (rAAV1, rAAV6, rAAV7, rAAV8, rAAV9); cardiac and skeletal muscle transduction were enhanced by up to 170-fold and 9027-fold over parental serotypes, while hepatic transduction was decreased up to 427-fold. VR1 stability also appeared critical to the remaining serotypes, as VR1 deletion mutations rendered rAAV2 and rAAV3b defective in transduction, and rAAV4 and rAAV5 defective in virion production. Intriguingly, amino acid insertions into VR1 produced an opposing phenotype: rAAV1, rAAV2, rAAV3b, and rAAV6 capsids could instead be targeted to transduce the liver with high efficiency, increasing hepatic transduction up to 132-fold. Finally, VR1 mutation synergized with
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
established tyrosine-to-phenylalanine mutations to further increase transduction efficiency while maintaining preferential cardiac/skeletal muscle targeting.
252. Using Cre-Dependent In Vivo Selection to Identify AAV Variants That Enable Efficient and Widespread Gene Transfer to the Adult Central Nervous System
Benjamin E. Deverman1, Piers L. Pravdo1, Bryan P. Simpson1, Sripriya Ravindra Kumar1, Yicheng Luo1, Ken Y. Chan1, Abhik Banerjee1, Wei-Li Wu1, Bin Yang1, Nina Huber2, Sergiu P. Pasca2, Viviana Gradinaru1 1 Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 2Department of Psychiatry and Behavioral Sciences, Stanford, Stanford, CA Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting the development of novel AAV capsids with more desirable transduction characteristics. We have developed a capsid selection method, called Cre-recombination-based AAV targeted evolution (CREATE), that enables the identification of AAV capsids that more efficiently transduce defined cell populations in vivo (Deverman et al. in press, Nature Biotechnology). We generated AAV capsid libraries and used CREATE to identify variants that cross the blood brain barrier and efficiently and widely transduce astrocytes in the mouse central nervous system (CNS) after intravenous injection. One variant, AAVPHP.B, transfers genes throughout the adult CNS with an efficiency that is 40- to 92-fold greater (depending on the CNS region) than that of the current standard, AAV9. It transduces the majority of astrocytes and neurons across multiple CNS regions, and in vitro, it transduces human neurons and astrocytes more efficiently than does AAV9. We are now evolving AAV-PHP.B for even greater transduction of specific CNS cell types as a means to both develop more effective vectors and to gain insight into the mechanism of enhanced transduction. Our identification of AAV-PHP.B and several other enhanced vectors after only two rounds of selection establishes CREATE as a powerful method to customize AAV vectors for biomedical applications.
253. Expanded Packaging Capacity of AAV by Lumenal Charge Alteration Matthew Tiffany, Mark A. Kay Stanford University, Stanford, CA
Adeno-associated virus (AAV) is widely believed to be the safest viral vector for gene therapy. Moreover, the array of AAV serotypes available allows transduction of different tissues in vivo. However, one major limitation of AAV is the relatively small DNA packaging size (4,700 nt). Existing serotypes have been over-packaged with limited success and varying reproducibility. The consensus appears to be that AAV can be over packaged by ~10%, but with a concomitant reduction in both viral titers and in vivo transduction. We have taken a novel approach to increase the packaging limitations of AAV by focusing on the lumen of AAV where vector DNA interacts with the capsid. We have created a series of AAV capsid variants that alter the capsid lumenal charge using AAVDJ as the scaffold. The variants add positively charged lysine and arginine residues at lumenally exposed sites within the capsid that, we believe, may allow for interaction and condensation of vector DNA increasing the packaging capacity and/or stabilizing the capsid in an over-packaged state. We present in vitro characterization of the library of capsid variants and demonstrate that variants with intermediate charge (+4 to +7 over wildtype) can be produced at high titer when over-packaged up to 6.2 kb. In addition, these variants can transduce S99