Wenqin Ma

High-efficiency Transduction and Correction of Murine Hemophilia B Using AAV2 Vectors Devoid of Multiple Surface-exposed Tyrosines

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30-80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.

Human Hepatocyte Growth Factor Receptor Is a Cellular Coreceptor for Adeno-Associated Virus Serotype 3

Adeno-associated viruses (AAVs) use a variety of cellular receptors/coreceptors to gain entry into cells. A number of AAV serotypes are now available, and the cognate receptors/coreceptors for only a handful of those have been identified thus far. Of the 10 commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells in general. However, in our more recent studies, we observed that AAV3 vectors transduced human liver cancer cells remarkably well, which led to the hypothesis that AAV3 uses hepatocyte growth factor receptor (HGFR) as a cellular coreceptor for viral entry. AAV3 infection of human liver cancer cell lines was strongly inhibited by hepatocyte growth factor, HGFR-specific small interfering RNA, and anti-HGFR antibody, which corroborated this hypothesis. However, AAV3 vectors failed to transduce murine hepatocytes, both in vitro and in vivo, suggesting that AAV3 specifically uses human HGFR, but not murine HGFR, as a cellular coreceptor for transduction. AAV3 may prove to be a useful vector for targeting human liver cancers for the potential gene therapy.

High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.

Adeno-associated virus 2 (AAV2) vectors transduce fibroblasts and mesenchymal stem cells (MSCs) inefficiently, which limits their potential widespread applicability in combinatorial gene and cell therapy. We have reported that AAV2 vectors fail to traffic efficiently to the nucleus in murine fibroblasts. We have also reported that site-directed mutagenesis of surface-exposed tyrosine residues on viral capsids leads to improved intracellular trafficking of the mutant vectors, and the transduction efficiency of the single tyrosine-mutant vectors is ∼10-fold higher in human cells. In the current studies, we evaluated the transduction efficiency of single as well as multiple tyrosine-mutant AAV2 vectors in murine fibroblasts. Our results indicate that the Y444F mutant vectors transduce these cells most efficiently among the seven single-mutant vectors, with >30-fold increase in transgene expression compared with the wild-type vectors. When the Y444F mutation is combined with additional mutations (Y500F and Y730F), the transduction efficiency of the triple-mutant vectors is increased by ∼130-fold and the viral intracellular trafficking is also significant improved. Similarly, the triple-mutant vectors are capable of transducing up to 80-90% of bone marrow-derived primary murine as well as human MSCs. Thus, high-efficiency transduction of fibroblasts with reprogramming genes to generate induced pluripotent stem cells, and the MSCs for delivering therapeutic genes, should now be feasible with the tyrosine-mutant AAV vectors.

Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo

BACKGROUND AIMS Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. METHODS We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. RESULTS We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. CONCLUSIONS These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno- Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo. However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3. In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

Enhanced Transgene Expression from Recombinant Single-Stranded D-Sequence-Substituted Adeno-Associated Virus Vectors in Human Cell Lines In Vitro and in Murine Hepatocytes In Vivo

ABSTRACT We have previously reported that the removal of a 20-nucleotide sequence, termed the D sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome significantly impairs rescue, replication, and encapsidation of the viral genomes (X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Mol Biol 250:573–580, 1995; X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Virol 70:1668–1677, 1996). Here we describe that replacement of only one D sequence in either ITR restores each of these functions, but DNA strands of only single polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded DNA (ssDNA), which is transcriptionally inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription suppressor sequence in one of the D sequences, which shares homology with the binding site for the cellular NF-κB-repressing factor (NRF). The removal of this D sequence from, and replacement with a sequence containing putative binding sites for transcription factors in, single-stranded AAV (ssAAV) vectors significantly augments transgene expression both in human cell lines in vitro and in murine hepatocytes in vivo. The development of these genome-modified ssAAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy. IMPORTANCE The results of the studies described here not only have provided novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV), that causes no known disease but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased levels of gene expression. Thus, these studies have significant implications for the potential use of these novel AAV vectors in human gene therapy.

A Simple Method to Increase the Transduction Efficiency of Single-Stranded Adeno-Associated Virus Vectors In Vitro and In Vivo

We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors.

The Adeno-Associated Virus Genome Packaging Puzzle

Chen Ling1,2, Yuan Wang1-3, Yuan Lu4, Lina Wang1-3, Giridhara R Jayandharan5, George V Aslanidi1,2, Baozheng Li1,2, Binbin Cheng1,3, Wenqin Ma1,2, Thomas Lentz6, Changquan Ling3, Xiao Xiao6,7, R Jude Samulski6, Nicholas Muzyczka2,8,9 and Arun Srivastava1,2,8-10* 1Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA 2Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA 3Department of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai, China 4Department of Orthopedics & Rehabilitative Medicine, University of Florida College of Medicine, Gainesville, FL, USA 5Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur, India 6Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 7Division of Molecular Pharmaceutics, University of North Carolina School of Pharmacy, Chapel Hill, NC, USA 8Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, USA 9Genetics Institute, University of Florida College of Medicine, Gainesville, FL, USA 10Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL, USA *Corresponding author: Arun Srivastava, Division of Cellular & Molecular Therapy, Cancer and Genetics Research Complex, 2033 Mowry Road, Room 492-A, Gainesville, FL 32611-3633, USA, Tel: (352) 273-8259; Fax: (352) 273-8342; E-mail: aruns@peds.ufl.edu

91. Development of Optimized [“Opt”] AAV Vectors by Combining Capsid-Modified NextGen and Genome-Modified GenX AAV Vectors for High-Efficiency Transduction at Further Reduced Doses

There is little doubt that AAV did not evolve for the purposes of delivery of therapeutic genes. The use of first generation AAV vectors, albeit successful, is unlikely to reach its full potential. We have described the development of capsid-modified next generation [NextGen] AAV vectors for both AAV2 (Proc Natl Acad Sci USA, 105: 7827-7832, 2008; Mol Ther., 18: 2048-2056, 2010; Vaccine, 30: 3908-3917, 2012; PLoS One, 8: e59142, 2013) and AAV3 (Gene Ther., 19: 375-384, 2012; Hum Gene Ther., 25: 1023-1034, 2014) serotypes, in which specific surface-exposed tyrosine (Y), serine (S), threonine (T), and lysine (K) residues on viral capsids were modified to achieve high-efficiency transduction at lower doses Fig. 1AFig. 1A). We have also described the development of genome-modified generation X [GenX] AAV vectors (J Virol., 89: 952-961, 2015), in which the transcriptionally-inactive, single-stranded AAV genome was modified to achieve improved transgene expression (Fig. 1BFig. 1B). Thus, we reasoned that encapsidation of GenX AAV genomes into NextGen AAV capsid might lead to further increased transduction at further reduced vector doses. To this end, the following sets of ssAAV2 as well as ssAAV3 serotype vectors containing the firefly luciferase (Fluc) reporter gene were generated: (i) wild-type (WT) genome and Y444+500+730F+T492V AAV2 quadruple-mutant (QM) capsid (WT-Fluc-AAV2/QM); (ii) two GenX genomes and AAV2 QM capsid (LC1-Fluc-AAV2/QM and LC2-Fluc-AAV2/QM); (iii) WT genome and S663V+T492V AAV3 double-mutant (DM) capsid (WT-Fluc-AAV3/DM); and (iv) two GenX genomes and AAV3 DM capsid (LC1-Fluc-AAV3/DM and LC2-Fluc-AAV3/DM). The combination of the modified-genomes with the capsid-mutants led to ~5-6-fold increase with both AAV2 and AAV3 serotype vectors following transduction of a human hepatocellular carcinoma (HCC) cell line, Huh7, at an MOI of 1,000 vgs/cell under identical conditions in vitro. When male C57BL/6 mice were injected via tail-vein with WT-Fluc-AAV2/QM, LC1-Fluc-AAV2/QM, and LC2-Fluc-AAV2/QM vectors at a relatively low dose of 5×109 vgs/mouse, the AAV2 QM capsid-mutant vectors led to ~6-10-fold increase in transgene expression in the liver. Similarly, when WT-Fluc-AAV3/DM, LC1-Fluc-AAV3/DM, and LC2-Fluc-AAV3/DM vectors were injected via tail-vein in NSG mice xenografted with human liver tumors at relatively low dose of 3×109 vgs/mouse, led to ~5-8-fold increase with the combination of modified-genomes with the AAV3 DM capsid-mutant vectors, which was restricted to human liver tumors. Taken together, these data document that the combination of NextGen capsids and GenX genomes leads to the generation of optimized [Opt] AAV serotype vectors (Fig. 1CFig. 1C), which transduce cells and tissues more efficiently, both in vitro and in vivo, at significantly reduced doses. These studies have significant implications in the potential use of the Opt AAV serotype vectors in human gene therapy.View Large Image | Download PowerPoint Slide

310. Development of Generation X Recombinant AAV Vectors for Human Gene Therapy

We have previously reported the development of capsid-modified next generation (NextGen) AAV serotype vectors that transduce cells and tissues more efficiently at reduced vector doses (Proc. Natl. Acad. Sci., USA, 105: 7827-7832, 2008). More recently, we have also described the development of genome-modified generation X (GenX) AAV vectors that also transduce cells and tissues more efficiently (J. Virol, 89: 952-961, 2015). The recombinant AAV genome contains inverted terminal repeats (ITRs) of 145 nucleotides at both ends. In these studies, a 20-nucleotide sequence, termed the D-sequence, was replaced with a substitute sequence, which led to enhanced transgene expression in human cell lines in vitro an in murine hepatocytes in vivo (J. Virol., 89: 952-961, 2015). In our present studies, we observed that a sequence, GGTTCCT, at the end of the D-sequence, shares partial homology to the consensus glucocorticoid receptor-binding element (GRE) site, 5’-GGTACANNNTGTT/CCT-3’. The TGTTCT half-site is an essential core element, which has been reported to be sufficient to relay glucocorticoid signaling. In electrophoretic mobility-shift assays (EMSAs), we documented that purified GR protein could specifically bind to double-stranded D-sequence oligonucleotides, suggesting that the D-sequence potentially functions as a ½ GRE site. Based on these results, we hypothesized that replacement of the D-sequence with a full GRE binding-site in the ITR might further increase transgene expression from these GenX AAV genomes. To this end, recombinant AAV vectors were generated in which the D-sequence was replaced with a fully functional GRE site. Insertion of a full GRE binding-site in the ITR significantly increased the transgene expression from these GenX AAV genomes following encapsidation in the wild-type (WT) AAV2 capsid vectors in human cell lines in vitro, and the extent of the transgene expression was further increased by dexamethasone-treatment. When 1×1010 vgs of recombinant AAV2 vectors containing the Gaussia luciferase (Gluc) reporter gene in the unmodified AAV genome, or those containing the GRE sequence, were administered via tail-vein into C57BL6/J mice, the transduction efficiency of the AAV-GRE vectors was ~8-fold higher in murine hepatocytes in vivo up to 9-weeks post-vector administration. More interestingly, when AAV-GRE genomes containing the enhanced green fluorescence protein (EGFP) reporter gene were encapsidated in the optimal NextGen AAV capsid-modified quadruple-mutant (Y444F+Y500F+Y730F+T491V) AAV2 vectors, the transduction efficiency of these vectors was further increased by ~8-fold in murine hepatocytes in vivo at a dose as low as 5×108 vgs/mouse. Taken together, the availability of these novel GenX AAV vectors containing the D-sequence substitution, and the fully functional GRE site insertion, to achieve high-efficiency transgene expression, has implications in the use of these vectors in human gene therapy.