- Email: [email protected]
8. Further Characterization of U2 snRNP Mediated Restriction of AAV Vector Transduction
AAV Vector Biology variants (originating from single nucleotide polymorphisms, genetic recombination, and de novo mutations) that account for more than 50 unique amino acid sequence variants. We also detected significant inter-patient variation, as well as high inter-tissue variability within individual patients. These findings suggest that AAV exhibits the capacity for high genomic diversity and accelerated intra-host evolution. These characteristics, which are shared among many other viruses, are presumed to promote survival against the host immune response, and fitness in tissue-specific cellular environments. We also validated a selection of AAV8 variants by assessing their capacity for vector packaging and in vivo gene transfer. We found that a subset of variants has a stronger competency for transcytosis and tropism to liver and muscle than the conventional AAV8 serotype. Remarkably, one AAV8 variant can cross the blood-brain barrier and target the CNS more efficiently than AAV9 after intravenous injection of adult mice. Another noteworthy discovery is the sequence variability found in the assembly-activating protein (AAP) gene, which is encoded by an alternative open reading frame of the cap gene. The diversity within AAP may define the efficacy of AAV packaging among variants, and implicate its role in AAV evolution and fitness. By exploiting AAV-host tissues as a natural incubator for viral evolution to potentiate new AAV capsid sequences, and employing a strategy to profile intact capsid sequences on a high-throughput scale, this study highlights the unprecedented diversity and evolutionary capacity of AAV. Importantly, these new findings reshape the versatility of rAAV as the ideal biotherapy for targeting a range of normal and/or diseased tissues.
AAV Vector Biology 7. High-Throughput Sequencing of AAV Proviral Libraries from the Human Population Reveals Novel Variants with Unprecedented Intraand Inter-Tissue Diversity
Guangchao Xu1, Li Luo1, Phillip W. L. Tai2, Wanru Qin1, Yuanyuan Xiao1, Chunmei Wang1, Qin Su2, Hong Ma2, Ran He2, Yuquan Wei1, Guangping Gao2 1 State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China, 2Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA Recombinant adeno-associated viruses (rAAVs) remain the most promising gene delivery vehicle for human gene therapy. The development of novel rAAVs with high transduction efficiency and specific tropisms primarily relies on PCR cloning of natural variants from primate tissues, or the engineering of capsids via directed evolution and rational design. Faithful clinical translation of leading therapeutic AAV serotypes derived from non-human tissues remains a hot-button issue. Here, we employed high-throughput variant profiling of natural AAV proviral libraries from the human population to identify novel AAV variants with unique tissue tropisms. We screened 844 surgical specimens, encompassing a wide range of tissue types and disease states from 455 patients by robust signature PCR. We found that >80% of human tissues are AAV positive with notable serotype frequencies (AAV2/3 chimera > AAV8 > AAV2 > AAV6) distributed among different tissues. Notably, we also used qPCR primers against the conserved AAV rep sequence to quantify the abundance of pAAV proviral genomes in human tissues.Next, to ensure the highest return on identifying novel variants, we employed single-molecule, real-time (SMRT) sequencing to characterize fulllength rep and cap sequences. Our preliminary findings from a single patient tissue sample revealed more than 800 unique DNA sequence S4
8. Further Characterization of U2 snRNP Mediated Restriction of AAV Vector Transduction
Claire A. Schreiber1, Yoshihiro Izumiya2, Aravind Asokan3, Yasuhiro Ikeda1 1 Molecular Medicine, Mayo Clinic, Rochester, MN, 2Dermatology, UC Davis School of Medicine, Sacramento, CA, 3Gene Therapy Center and Department of Genetics, University of North Carolina School of Medicine, Chapel Hill, NC Improving adeno-associated virus (AAV) vector transduction efficiency is central to the development of its continued, widespread use in gene therapy. More effective AAV transduction would reduce vector doses required for efficient gene delivery, minimizing the risks associated with high dose AAV vectors. Several treatments have been reported to increase AAV vector transduction, including adenoviral co-infection. Through screening of a siRNA library, we recently identified U2 snRNP as a host restriction factor for AAV vector transduction. Disruption of U2 snRNP spliceosome and associated proteins, including PHF5A, SF3B1, SF3B2 and U2AF1, potently enhanced AAV vector transduction. Relevant to gene therapy applications of AAV vectors, meayamycin B, a powerful SF3B1 inhibitor, allowed for substantial increases in AAV vector transduction (up to 400-fold). This post-entry restriction appeared to occur after the second-strand synthesis but before transgene expression or accumulation of transgene transcripts and independently of the cellular splicing machinery. No notable changes were found in the cytoplasmic trafficking, nuclear entry, or genome release of AAV vector infection by U2 snRNP inhibition. Here, we further studied the mechanism(s) underlying the U2 snRNP-mediated block of AAV vectors. We first tested another commercially available U2 snRNP inhibitor, pladienolide B (PladB) and verified that PladB treatment also showed substantial dose dependent increases (up to 700-fold) in AAV vector transduction. We next studied the relationship between adenoviral co-infection and U2 snRNP inhibition. Adenoviral coinfection alone enhanced AAV vector transduction up to 30-fold, while adenoviral co-infection, in addition to genetic or pharmacological U2 snRNP inhibition, showed marginal additive effects, suggesting a common pathway targeted by adenoviral co-infection and U2 Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
AAV Vector Biology snRNP inhibition in enhanced AAV vector infection. Using a series of plasmids providing adenoviral helper functions, we have identified Ad5 E4, but not the E2A and VA RNA genes, as partially responsible for the enhanced AAV transduction by adenoviral co-infection through U2 snRNP inhibition. Since the effects of U2 snRNP inhibition are most likely on the regulation of transgene expression, we also assessed the epigenetic modifications of the AAV2 genome in the presence or absence of PladB. Through chromosome immunoprecipitation (ChIP) assays we mapped histone recruitment patterns at different regions of the AAV vector epigenome. PladB treatment reproducibly enhanced recruitment of histone H2b and H3 proteins to the AAV CMV promoter, luciferase transgene, and polyA regions up to 3-fold. Our preliminary study also suggested that U2 snRNP inhibition altered specific histone modification patterns on the AAV vector genome. We are in the process of verifying the influence of U2 snRNP inhibition, as well as AAV capsid variations, on epigenetic modifications of the AAV vector epigenome. Better understanding the underlying mechanism would provide novel insights into host-virus interactions and could lead to the rational design of next generation AAV vectors with improved transduction efficiencies and safety profiles.
9. rAAV Designs Harboring DNA Secondary Structures with High Thermal Stabilities Produce Heterogenic Viral Genome Populations
Jun Xie, Ran He, Qin Su, Phillip W. L. Tai, Hong Ma, Jia Li, Guangping Gao Horae Gene Therapy Center, UMASS Medical School, Worcester, MA It has been known for more than a decade that AAV genomes in ssAAV particles are heterogeneous, and can contain smaller than unit-length molecules to varying degrees. However, the cause underlying this phenomenon remained unknown. Our earlier work demonstrated that short DNA hairpin (shDNA) sequences designed into rAAV vectors, resulted in truncated genomes through a templateswitching mechanism during AAV genome replication. Importantly, the knowledge we have gained about shDNA-mediated genomic truncation has helped to improve shRNA-cassette design. Specifically, we have found a correlation between the thermal stability of shDNA structures with the prevalence of truncation events. By introducing point mutations into the passenger strand of shRNA sequences to create DNA bulges within shDNAs in scAAV vectors, we found that lowering the thermal stability of DNA hairpins lowered the proportion of truncated to complete genomes. In addition, we found that scAAV vectors incorporated with pri-miRNA transgenes, which contain natural bulges in their stem-loop structures, also produced fewer truncated genomes, relative to shDNA-rAAV constructs. By embedding the guide strand of small RNAs into pri-miRNA scaffolds, we have defined critical improvements to the genomic integrity of rAAV vectors expressing small RNAs. Furthermore, we developed a method, named AAV-GPSeq (AAV genome populations sequencing), to directly sequence whole vector genome populations from purified rAAVs using the PacBio platform for high-throughput sequencing. We discovered through this methodology that truncation events can originate from palindromic sequences and inverted repeats that reside in the expression cassette elements of widely used ssAAV and scAAV vector designs (i.e. transcriptional regulatory elements, transgene sequences, and post-transcriptional regulatory regions). The resulting diversity of genomic truncations produces populations of packaged virions with variable transgene efficacies, and gives us the first insights into the phenomenon of rAAV heterogeneity. By examining the genomes from rAAV vectors harboring shDNAs, pri-miRNA fragments, and palindromic/inverted repeat sequences, we now highlight the importance of DNA secondary structure on vector design and genomic heterogeneity in packaged viral vectors. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Improvements upon quality control standards are thus necessary and critical to efficacious and safe clinical uses for rAAV as a biomedicine. Further implications for our novel findings towards understanding AAV replication, and new considerations for future therapeutic rAAV vector designs will be discussed. To improve the homogeneity of clinical rAAV vectors, we are optimizing the vector production procedure, testing non-palindrome promoters, changing the codon usage in transgenes to lower the thermostability of rAAV genomes, and modifying rAAV packaging cell lines to minimize template-switching events to produce more intact rAAV genomes.
10. High-Efficiency Transduction of Primary Human CD34+ Hematopoietic Stem/Progenitor Cells by AAV6 Serotype Vectors: Strategies for Overcoming Donor Variation and Implications in Genome Editing
Zifei Yin1, Kanit Bukhai2, George Aslanidi3, Chen Ling3, Mengqun Tan4, Mervin Yoder5, Philippe Leboulch2, Emmanuel Payen2, Arun Srivastava3 1 Traditional Chinese Medicine, Second Military Medical University, Shanghai, China, 2Institute of Emerging Diseases and Innovative Therapies, University of Paris-Sud, Paris, France, 3Pediatrics, University of Florida College of Medicine, Gainesville, FL, 4Physiology, Central South University, Changsha, China, 5Pediatrics, Indiana University School of Medicine, Indianapolis, IN We first reported that of the 10 most commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). More recently, two independent groups also reported successful transduction of primary human CD34+ cells using the wild-type (WT) AAV6 vectors (Sci. Transl. Med., 7: 307ra156, 2015; Nat. Biotechnol., 33: 1256-1263, 2015), except that multiplicities of infection (MOIs) ranging from 100,000-200,000 vgs/cell were used to achieve ~40-55% transduction efficiency. Furthermore, the transduction efficiency of the WT AAV6 vector varies greatly in HSPCs from different donors, ranging between ~6-87%. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705F+Y731F+T491V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, ‘cross-transduction’. The combined use of these strategies resulted in transduction efficiency exceeding 90% at an MOI of 20,000 vgs/cell in primary human cord blood-derived HSPCs at day 4 (Fig. 1A). scAAV6 vectors were more efficient than ssAAV6 vectors, but at high cell density, there was a modest enhancement in EGFP-positivity even with ssAAV6 vectors. However, 14 days post-transduction, virtually no EGFP-positive cells could be detected (Fig. 1B), suggesting the loss of vector genomes, and hence, the lack of stable integration of vector genomes in HSPCs. Our studies have significant implications in the optimal use of capsidoptimized AAV6 vectors in genome editing in HSPCs. *These authors contributed equally to this work #Co-corresponding authors
S5
AAV Vector Biology 7. High-Throughput Sequencing of AAV Proviral Libraries from the Human Population Reveals Novel Variants with Unprecedented Intraand Inter-Tissue Diversity
Guangchao Xu1, Li Luo1, Phillip W. L. Tai2, Wanru Qin1, Yuanyuan Xiao1, Chunmei Wang1, Qin Su2, Hong Ma2, Ran He2, Yuquan Wei1, Guangping Gao2 1 State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China, 2Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA Recombinant adeno-associated viruses (rAAVs) remain the most promising gene delivery vehicle for human gene therapy. The development of novel rAAVs with high transduction efficiency and specific tropisms primarily relies on PCR cloning of natural variants from primate tissues, or the engineering of capsids via directed evolution and rational design. Faithful clinical translation of leading therapeutic AAV serotypes derived from non-human tissues remains a hot-button issue. Here, we employed high-throughput variant profiling of natural AAV proviral libraries from the human population to identify novel AAV variants with unique tissue tropisms. We screened 844 surgical specimens, encompassing a wide range of tissue types and disease states from 455 patients by robust signature PCR. We found that >80% of human tissues are AAV positive with notable serotype frequencies (AAV2/3 chimera > AAV8 > AAV2 > AAV6) distributed among different tissues. Notably, we also used qPCR primers against the conserved AAV rep sequence to quantify the abundance of pAAV proviral genomes in human tissues.Next, to ensure the highest return on identifying novel variants, we employed single-molecule, real-time (SMRT) sequencing to characterize fulllength rep and cap sequences. Our preliminary findings from a single patient tissue sample revealed more than 800 unique DNA sequence S4
8. Further Characterization of U2 snRNP Mediated Restriction of AAV Vector Transduction
Claire A. Schreiber1, Yoshihiro Izumiya2, Aravind Asokan3, Yasuhiro Ikeda1 1 Molecular Medicine, Mayo Clinic, Rochester, MN, 2Dermatology, UC Davis School of Medicine, Sacramento, CA, 3Gene Therapy Center and Department of Genetics, University of North Carolina School of Medicine, Chapel Hill, NC Improving adeno-associated virus (AAV) vector transduction efficiency is central to the development of its continued, widespread use in gene therapy. More effective AAV transduction would reduce vector doses required for efficient gene delivery, minimizing the risks associated with high dose AAV vectors. Several treatments have been reported to increase AAV vector transduction, including adenoviral co-infection. Through screening of a siRNA library, we recently identified U2 snRNP as a host restriction factor for AAV vector transduction. Disruption of U2 snRNP spliceosome and associated proteins, including PHF5A, SF3B1, SF3B2 and U2AF1, potently enhanced AAV vector transduction. Relevant to gene therapy applications of AAV vectors, meayamycin B, a powerful SF3B1 inhibitor, allowed for substantial increases in AAV vector transduction (up to 400-fold). This post-entry restriction appeared to occur after the second-strand synthesis but before transgene expression or accumulation of transgene transcripts and independently of the cellular splicing machinery. No notable changes were found in the cytoplasmic trafficking, nuclear entry, or genome release of AAV vector infection by U2 snRNP inhibition. Here, we further studied the mechanism(s) underlying the U2 snRNP-mediated block of AAV vectors. We first tested another commercially available U2 snRNP inhibitor, pladienolide B (PladB) and verified that PladB treatment also showed substantial dose dependent increases (up to 700-fold) in AAV vector transduction. We next studied the relationship between adenoviral co-infection and U2 snRNP inhibition. Adenoviral coinfection alone enhanced AAV vector transduction up to 30-fold, while adenoviral co-infection, in addition to genetic or pharmacological U2 snRNP inhibition, showed marginal additive effects, suggesting a common pathway targeted by adenoviral co-infection and U2 Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
AAV Vector Biology snRNP inhibition in enhanced AAV vector infection. Using a series of plasmids providing adenoviral helper functions, we have identified Ad5 E4, but not the E2A and VA RNA genes, as partially responsible for the enhanced AAV transduction by adenoviral co-infection through U2 snRNP inhibition. Since the effects of U2 snRNP inhibition are most likely on the regulation of transgene expression, we also assessed the epigenetic modifications of the AAV2 genome in the presence or absence of PladB. Through chromosome immunoprecipitation (ChIP) assays we mapped histone recruitment patterns at different regions of the AAV vector epigenome. PladB treatment reproducibly enhanced recruitment of histone H2b and H3 proteins to the AAV CMV promoter, luciferase transgene, and polyA regions up to 3-fold. Our preliminary study also suggested that U2 snRNP inhibition altered specific histone modification patterns on the AAV vector genome. We are in the process of verifying the influence of U2 snRNP inhibition, as well as AAV capsid variations, on epigenetic modifications of the AAV vector epigenome. Better understanding the underlying mechanism would provide novel insights into host-virus interactions and could lead to the rational design of next generation AAV vectors with improved transduction efficiencies and safety profiles.
9. rAAV Designs Harboring DNA Secondary Structures with High Thermal Stabilities Produce Heterogenic Viral Genome Populations
Jun Xie, Ran He, Qin Su, Phillip W. L. Tai, Hong Ma, Jia Li, Guangping Gao Horae Gene Therapy Center, UMASS Medical School, Worcester, MA It has been known for more than a decade that AAV genomes in ssAAV particles are heterogeneous, and can contain smaller than unit-length molecules to varying degrees. However, the cause underlying this phenomenon remained unknown. Our earlier work demonstrated that short DNA hairpin (shDNA) sequences designed into rAAV vectors, resulted in truncated genomes through a templateswitching mechanism during AAV genome replication. Importantly, the knowledge we have gained about shDNA-mediated genomic truncation has helped to improve shRNA-cassette design. Specifically, we have found a correlation between the thermal stability of shDNA structures with the prevalence of truncation events. By introducing point mutations into the passenger strand of shRNA sequences to create DNA bulges within shDNAs in scAAV vectors, we found that lowering the thermal stability of DNA hairpins lowered the proportion of truncated to complete genomes. In addition, we found that scAAV vectors incorporated with pri-miRNA transgenes, which contain natural bulges in their stem-loop structures, also produced fewer truncated genomes, relative to shDNA-rAAV constructs. By embedding the guide strand of small RNAs into pri-miRNA scaffolds, we have defined critical improvements to the genomic integrity of rAAV vectors expressing small RNAs. Furthermore, we developed a method, named AAV-GPSeq (AAV genome populations sequencing), to directly sequence whole vector genome populations from purified rAAVs using the PacBio platform for high-throughput sequencing. We discovered through this methodology that truncation events can originate from palindromic sequences and inverted repeats that reside in the expression cassette elements of widely used ssAAV and scAAV vector designs (i.e. transcriptional regulatory elements, transgene sequences, and post-transcriptional regulatory regions). The resulting diversity of genomic truncations produces populations of packaged virions with variable transgene efficacies, and gives us the first insights into the phenomenon of rAAV heterogeneity. By examining the genomes from rAAV vectors harboring shDNAs, pri-miRNA fragments, and palindromic/inverted repeat sequences, we now highlight the importance of DNA secondary structure on vector design and genomic heterogeneity in packaged viral vectors. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Improvements upon quality control standards are thus necessary and critical to efficacious and safe clinical uses for rAAV as a biomedicine. Further implications for our novel findings towards understanding AAV replication, and new considerations for future therapeutic rAAV vector designs will be discussed. To improve the homogeneity of clinical rAAV vectors, we are optimizing the vector production procedure, testing non-palindrome promoters, changing the codon usage in transgenes to lower the thermostability of rAAV genomes, and modifying rAAV packaging cell lines to minimize template-switching events to produce more intact rAAV genomes.
10. High-Efficiency Transduction of Primary Human CD34+ Hematopoietic Stem/Progenitor Cells by AAV6 Serotype Vectors: Strategies for Overcoming Donor Variation and Implications in Genome Editing
Zifei Yin1, Kanit Bukhai2, George Aslanidi3, Chen Ling3, Mengqun Tan4, Mervin Yoder5, Philippe Leboulch2, Emmanuel Payen2, Arun Srivastava3 1 Traditional Chinese Medicine, Second Military Medical University, Shanghai, China, 2Institute of Emerging Diseases and Innovative Therapies, University of Paris-Sud, Paris, France, 3Pediatrics, University of Florida College of Medicine, Gainesville, FL, 4Physiology, Central South University, Changsha, China, 5Pediatrics, Indiana University School of Medicine, Indianapolis, IN We first reported that of the 10 most commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs), both in vitro and in murine xenograft models in vivo (Cytotherapy, 15: 986-998, 2013; PLoS One, 8(3): e58757, 2013). More recently, two independent groups also reported successful transduction of primary human CD34+ cells using the wild-type (WT) AAV6 vectors (Sci. Transl. Med., 7: 307ra156, 2015; Nat. Biotechnol., 33: 1256-1263, 2015), except that multiplicities of infection (MOIs) ranging from 100,000-200,000 vgs/cell were used to achieve ~40-55% transduction efficiency. Furthermore, the transduction efficiency of the WT AAV6 vector varies greatly in HSPCs from different donors, ranging between ~6-87%. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705F+Y731F+T491V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, ‘cross-transduction’. The combined use of these strategies resulted in transduction efficiency exceeding 90% at an MOI of 20,000 vgs/cell in primary human cord blood-derived HSPCs at day 4 (Fig. 1A). scAAV6 vectors were more efficient than ssAAV6 vectors, but at high cell density, there was a modest enhancement in EGFP-positivity even with ssAAV6 vectors. However, 14 days post-transduction, virtually no EGFP-positive cells could be detected (Fig. 1B), suggesting the loss of vector genomes, and hence, the lack of stable integration of vector genomes in HSPCs. Our studies have significant implications in the optimal use of capsidoptimized AAV6 vectors in genome editing in HSPCs. *These authors contributed equally to this work #Co-corresponding authors
S5