Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at lower doses

Zhong, Li; Li, Baozheng; Mah, Cathryn; Govindasamy, Lakshmanan; Agbandje‐McKenna, Mavis; Cooper, Mario; Herzog, Roland W.; Zolotukhin, Irene; Warrington, Kenneth H.; Aken, Kirsten A. Weigel-Van; Hobbs, Jacqueline A.; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun

doi:10.1073/pnas.0802866105

Cited by 504 publications

(544 citation statements)

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“…The second-generation optimized CsCl purification method described and characterized herein enables flexible purification of vectors of varying serotypes, and importantly results in consistently high vector purity and potency, and comparable in these attributes to vectors prepared for clinical studies. The use of such improved AAV purification methods for pre-clinical studies, combined with further improvements in expression cassettes, use of self-complementary AAV constructs, 23 more efficient serotypes, 8,24 and improved delivery techniques are important strategies to meet the need for high levels of therapeutic transgene expression and support successful translational research.…”

Section: Discussionmentioning

confidence: 99%

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

et al. 2009

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The purity of adeno-associated virus (AAV) vector preparations has important implications for both safety and efficacy of clinical gene transfer. Early-stage screening of candidates for AAV-based therapeutics ideally requires a purification method that is flexible and also provides vectors comparable in purity and potency to the prospective investigational product manufactured for clinical studies. The use of cesium chloride (CsCl) gradient-based protocols provides the flexibility for purification of different serotypes; however, a commonly used first-generation CsCl-based protocol was found to result in AAV vectors containing large amounts of protein and DNA impurities and low transduction efficiency in vitro and in vivo. Here, we describe and characterize an optimized, secondgeneration CsCl protocol that incorporates differential precipitation of AAV particles by polyethylene glycol, resulting in higher yield and markedly higher vector purity that correlated with better transduction efficiency observed with several AAV serotypes in multiple tissues and species. Vectors purified by the optimized CsCl protocol were found to be comparable in purity and functional activity to those prepared by more scalable, but less flexible serotype-specific purification processes developed for manufacture of clinical vectors, and are therefore ideally suited for pre-clinical studies supporting translational research.

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Section: Discussionmentioning

confidence: 99%

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

et al. 2009

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“…In brief, in stage one, two PCR extension reactions were performed in separate tubes for each mutant. One tube contained the forward PCR primer and the other contained the reverse primer (Zhong et al, 2008b). In stage two, the two reactions were mixed and a standard PCR mutagenesis assay was carried out as per the manufacturer's instructions.…”

Section: Recombinant Aav2 Vectors Plasmids Antibodies and Chemicalsmentioning

confidence: 99%

“…In stage two, the two reactions were mixed and a standard PCR mutagenesis assay was carried out as per the manufacturer's instructions. PCR primers were designed to introduce changes from tyrosine to phenylalanine residues, as well as a silent change to create a new restriction endonuclease site for screening purposes (Zhong et al, 2008b). All multiple mutant AAV2 capsid plasmids were constructed using standard subcloning strategies.…”

Section: Recombinant Aav2 Vectors Plasmids Antibodies and Chemicalsmentioning

confidence: 99%

“…Then we found that mutations of critical surface-exposed tyrosine residues on AAV2 capsids circumvent the ubiquitination step, thereby avoiding proteasome-mediated degradation and resulting in high-efficiency transduction by these vectors in human cells in vitro and murine hepatocytes in vivo (Zhong et al, 2008b). As described by Markusic et al (2010), we generated multiple combinations of tyrosine mutants of AAV2 capsids, including a triple-mutant (Y730Fþ Y500Fþ Y444F), consisting of the three best performing single tyrosine-mutants, and evaluated whether the transduction efficiency could be augmented further.…”

Section: Introductionmentioning

confidence: 99%

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High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

Jayandharan

et al. 2010

Human Gene Therapy

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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. Overview SummaryFibroblasts and mesenchymal stem cells (MSCs) are promising targets for gene and cell therapy. Recombinant adeno-associated virus 2 (AAV2) vectors are currently in use in several Phase I/II clinical trials for gene therapy. However, the existing data show that AAV2 vector-mediated transduction of fibroblasts and MSCs is inefficient. We observed that AAV2 vectors containing mutations in three surface-exposed tyrosine residues significantly increase transduction efficiency in fibroblasts, as well as in both human and murine primary MSCs. The increased transduction efficiency of these triple-mutant AAV2 vectors correlates well with improved viral intracellular trafficking in fibroblasts. These data provide strong evidence that high-efficiency gene delivery and transgene expression by AAV vectors are indeed feasible, which should facilitate the generation of induced pluripotent stem cells, as well as the use of MSCs in combinatorial gene and cell therapy.

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“…Previous studies have shown that inhibition of the EGFR-PTK mediated phosphorylation of AAV capsid proteins at tyrosine residues results in decreased ubiquitination of AAV capsids and their proteasome-mediated degradation, thereby enabling nuclear transport of AAV vectors and improved gene delivery to the cell nucleus [38] . Based on these results, Zhong et al [39] hypothesized that substitution of surface-exposed tyrosine residues on AAV capsids may lead to the design of AAV vectors that facilitate efficient delivery of genes to the nucleus of target cells at lower doses. Consistent with this hypothesis, they observed that site-directed mutagenesis of surface-exposed tyrosine residues significantly improve the transduction efficiency of AAV vectors (about 10-fold increase) in human epithelial cells in vitro.…”

Section: Molecular Engineering Of Viral Vectorsmentioning

confidence: 99%

Advances in homology directed genetic engineering of human pluripotent and adult stem cells

Ramamoorthi

Curtis²,

Asuri³

2013

WJSC

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The ability to introduce precise genomic modifications in human cells has profound implications for both basic and applied research in stem cells, ranging from identification of genes regulating stem cell self-renewal and multilineage differentiation to therapeutic gene correction and creation of in vitro models of human diseases. However, the overall efficiency of this process is challenged by several factors including inefficient gene delivery into stem cells and low rates of homology directed site-specific targeting. Recent studies report the development of novel techniques to improve gene targeting efficiencies in human stem cells; these methods include molecular engineering of viral vectors to efficiently deliver episomal genetic sequences that can participate in homology directed targeting, as well as the design of synthetic proteins that can introduce double-stranded breaks in DNA to initiate such recombination events. This review focuses on the potential of these new technologies to precisely alter the human stem cell genome and also highlights the possibilities offered by the combination of these complementary strategies.© 2013 Baishideng. All rights reserved. Key words: Human stem cells; Genetic engineering; Engineered viruses; Synthetic restriction endonucleasesCore tip: This manuscript focuses on the development of novel technologies to precisely alter the human stem cell genome and highlights their implications for both basic and applied stem cell research. Specifically, we discuss the development of two main technologies: molecular engineering of viral vectors and design of artificial endonucleases. We also discuss the merits of combining these complementary approaches and suggest other possible strategies that could be explored to further improve genetic engineering of human stem cells.

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Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at lower doses

Cited by 504 publications

References 53 publications

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

High-Efficiency Transduction of Fibroblasts and Mesenchymal Stem Cells by Tyrosine-Mutant AAV2 Vectors for Their Potential Use in Cellular Therapy

Advances in homology directed genetic engineering of human pluripotent and adult stem cells

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