Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo

Jayandharan, Giridhara R.; Zhong, Li; Li, B; Kachniarz, Bartlomiej; Srivastava, Arun

doi:10.1038/gt.2008.89

Cited by 32 publications

(49 citation statements)

References 42 publications

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“…23 Under conditions of limited phosphorylatation of capsid following cellular entry, vector performance improves because of reduced ubiquitination and proteasomal degradation. [34][35][36]46 Here, we find that capsids engineered to minimize tyrosine phosphorylation are also less presented to CD8 1 T cells. At…”

Section: Ctl-mediated Killing Is Less Efficient Against Hepatocytes Tmentioning

confidence: 83%

Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells

Martino

Basner-Tschakarjan

Markusic

et al. 2013

Blood

146

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Section: Ctl-mediated Killing Is Less Efficient Against Hepatocytes Tmentioning

confidence: 83%

Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells

Martino

Basner-Tschakarjan

Markusic

et al. 2013

Blood

146

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“…10) significantly improved transduction of AAV vectors in the AAV2 serotype background. They showed that phosphorylation of these tyrosines was prevented by their conversion to phenylalanine and that this reduced the loss of input capsids due to proteolytic degradation (27)(28)(29). We would suggest that it may also be possible that phosphorylation of these tyrosines …”

Section: Discussionmentioning

confidence: 99%

Adeno-Associated Virus Capsid Proteins May Play a Role in Transcription and Second-Strand Synthesis of Recombinant Genomes

Salganik

Aydemir

Nam

et al. 2014

J Virol

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A group of four interacting amino acids in adeno-associated virus type 8 (AAV8) called the pH quartet has been shown to undergo a structural change when subjected to acidic pH comparable to that seen in endosomal compartments. We examined the phenotypes of mutants with mutations in these amino acids as well as several nearby residues in the background of AAV2. We found that three of the mutations in this region (Y704A, E562A, and E564A) produce normal titers of mature capsids but are extremely defective for transduction (>10 7 -fold). The remaining mutants were also defective for transduction, but the defect in these mutants (E563A, E561A, H526A, and R389A) is not as severe (3-to 22-fold). Two other mutants (Y700A and Y730A) were found to be defective for virus assembly. One of the extremely defective mutants (Y704A) was found to enter the cell, traffic to the nucleus, and uncoat its DNA nearly as efficiently as the wild type. This suggested that some step after nuclear entry and uncoating was defective. To see if the extremely defective mutants were impaired in second-strand synthesis, the Y704A, E562A, and E564A mutants containing self-complementary DNA were compared with virus containing single-stranded genomes. Two of the mutants (Y704A and E564A) showed 1-log and 3-log improvements in infectivity, respectively, while the third mutant (E562A) showed no change. This suggested that inhibition of second-strand synthesis was responsible for some but not most of the defect in these mutants. Comparison of Y704A mRNA synthesis with that of the wild-type capsid showed that accumulation of steady-state mRNA in the Y704A mutant was reduced 450-fold, even though equal genome numbers were uncoated. Our experiments have identified a novel capsid function. They suggest that AAV capsids may play a role in the initiation of both second-strand synthesis and transcription of the input genome.A deno-associated virus (AAV) is a small parvovirus of the Dependovirus genus that is currently being tested as a gene therapy vector. Although it has been studied extensively, many questions remain about some of the basic processes governing AAV infection. One such process is the role of acidification of the endosomal compartments in AAV trafficking and uncoating. Several groups have shown that blocking endosomal acidification inhibits AAV2 infection and recombinant AAV (rAAV) transduction (1-4). Furthermore, Sonntag et al. (4) have demonstrated the need for AAV to traffic through the endosomal system, since microinjection of virus into the cytoplasm fails to lead to a productive infection. One hypothesis is that trafficking through the progressively acidic endosomes leads to the externalization of the VP1 and VP2 N termini, which contain both nuclear localization signal (NLS) domains and a phospholipase A2 (PLA2) domain that have been shown to be critical for infection (4-7). To this end, there is evidence that epitopes associated with the viral protein 1 (VP1)/ VP2 N termini become exposed early in infection, during a time that the vi...

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“…Augmented transgene expression from ssAAV2 vectors also occurs in transgenic mice overexpressing TC-PTP (Qing et al, 2003), and in mice deficient in FKBP52 (Zhong et al, 2004b). Subsequently, we have also developed scAAV-TC-PTP and scAAV-PP5 vectors (Zhong et al, 2004a;Zhao et al, 2007;Jayandharan et al, 2008). We reasoned that if scAAV-TC-PTP and scAAV-PP5 vectors are admixed with a conventional ssAAV vector before transduction, the rapid and simultaneous expression of TC-PTP and PP5 from scAAV vectors, which do not require viral second-strand DNA synthesis, would completely dephosphorylate FKBP52 at both tyrosine and serine=threonine residues, respectively.…”

mentioning

confidence: 99%

“…This would lead to more efficient second-strand DNA synthesis of the ssAAV vector, resulting in high-efficiency transgene expression. Indeed, this coadministration strategy led to an *16-fold increase in the transduction efficiency of ssAAV2 vectors in primary murine hepatocytes in vivo ( Jayandharan et al, 2008).…”

mentioning

confidence: 99%

Optimized Adeno-Associated Virus (AAV)–Protein Phosphatase-5 Helper Viruses for Efficient Liver Transduction by Single-Stranded AAV Vectors: Therapeutic Expression of Factor IX at Reduced Vector Doses

et al. 2010

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Our studies have shown that coinjection of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors carrying the enhanced green fluorescent protein (EGFP) gene with self-complementary (sc) AAV2-T cell protein tyrosine phosphatase (TC-PTP) and scAAV2-protein phosphatase-5 (PP5) vectors resulted in an *16-fold increase in EGFP expression in primary murine hepatocytes in vivo [Jayandharan, G.R., Zhong, L., Li, B., Kachniarz, B., and Srivastava, A. (2008). Gene Ther. 15, 1287-1293. In the present studies, this strategy was further optimized to achieve transgene expression at reduced vector=helper virus doses. These included the use of scAAV helper viruses containing (1) hepatocyte-specific promoters, (2) tyrosine-mutant AAV2 capsids, and (3) additional AAV serotype vectors known to efficiently transduce hepatocytes. The hepatocyte-specific transthyretin (TTR) promoter was *6-to 7-fold more efficient than the Rous sarcoma virus (RSV) promoter; tyrosinemutant AAV2 capsids were *6-to 11-fold more efficient than the wild-type AAV2 capsids; and the AAV8 serotype helper virus was *16-fold more efficient than AAV2 serotype helper virus. With these modifications, the vector dose of the helper virus could be further reduced by *50-fold. Last, coadministration of scAAV8-PP5 helper virus increased coagulation factor IX expression from an ssAAV2 vector by *7-to 10-fold, thereby achieving therapeutic levels at lower vector doses. No adverse effect on hepatocytes was observed under any of these experimental conditions. The strategy presented here should be adaptable to any ssAAV transgene cassette and, specifically, liver-directed applications of ssAAV2 vectors containing larger genes that cannot be encapsidated in scAAV vectors. Overview SummaryAlthough recombinant adeno-associated viral (AAV) vectors containing single-stranded genomes have been used in clinical trials, the single-stranded DNAs are transcriptionally inactive, and relatively large vector doses are needed to achieve expression of therapeutic genes. We have developed optimized recombinant selfcomplementary AAV vectors carrying the protein phosphatase-5 gene (scAAV-PP5) as helper viruses, coadministration of which leads to efficient transgene expression from conventional single-stranded AAV (ssAAV) vectors at reduced doses. These optimized helper viruses should prove useful in achieving high levels of expression at lower doses of therapeutic genes from ssAAV vectors containing large genes, such as factor VIII, which exceed the packaging capacity of scAAV vectors, for the potential gene therapy of hemophilia A.

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Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo

Cited by 32 publications

References 42 publications

Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells

Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells

Adeno-Associated Virus Capsid Proteins May Play a Role in Transcription and Second-Strand Synthesis of Recombinant Genomes

Optimized Adeno-Associated Virus (AAV)–Protein Phosphatase-5 Helper Viruses for Efficient Liver Transduction by Single-Stranded AAV Vectors: Therapeutic Expression of Factor IX at Reduced Vector Doses

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