Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo

Wang, Z; Hi, Ma; Li, Jian; Sun, Lingyi; Zhang, J; Xiao, Xianmin

doi:10.1038/sj.gt.3302133

Cited by 372 publications

(354 citation statements)

References 28 publications

(38 reference statements)

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“…It is possible, but not likely, that phosphorylated forms of FKBP52 inhibit AAV DNA strand-annealing. Our data, presented here, are consistent with recently published reports 28,29 as well as those obtained by Samulski and co-workers using singlepolarity AAV vectors, which transduce murine muscle cells efficiently, even in the complete absence of DNA strand-annealing (RJ Samulski, personal communication). Furthermore, using the same single-polarity AAV vectors, we have also documented high-efficiency transduction in HeLa cells stably transfected with a TC-PTP expression plasmid in vitro, and in primary hepatocytes in TC-PTP-TG and FKBP52-KO mice in vivo (unpublished results).…”

Section: Aav-mediated Transduction Of Hepatocytessupporting

confidence: 93%

“…Since TC-PTP catalyzes tyrosine dephosphorylation of FKBP52, it is conceivable that the use of recently described self-complementary AAV (scAAV) vectors [27][28][29] carrying the TC-PTP gene would be exploitable to augment the transduction efficiency of conventional AAV vectors provided that deliberate expression of TC-PTP is not deleterious in primary cells. We have thus far not detected any toxicity in our TC-PTP-TG mice, 9 who remain fertile and healthy more than 1 year of age.…”

Section: Aav-mediated Transduction Of Hepatocytes L Zhong Et Almentioning

confidence: 99%

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Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo

Zhong

Yang

et al. 2004

Gene Ther

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Adeno-associated virus 2 (AAV) vectors are currently in use in Phase I/II clinical trials for gene therapy of cystic fibrosis and hemophilia B. Although 100% of murine hepatocytes can be targeted by AAV vectors, the transgene expression is limited to B5% of hepatocytes. Since the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strandannealing accounts for the lack of efficient transgene expression. We and others, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis attributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, when present in phosphorylated forms, inhibits the viral second-strand DNA synthesis, and consequently, limits transgene expression in nonhepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice overexpressing the cellular T-cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We demonstrate here that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We also document efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the ratelimiting step in the efficient transduction of hepatocytes, which should have implications in the optimal use of recombinant AAV vectors in human gene therapy. Gene Therapy (2004) The adeno-associated virus 2 (AAV) genome is a singlestranded DNA, which is transcriptionally inactive. Since single-stranded viral genomes of either polarity are encapsidated with equal frequency, 1 it has been suggested that following infection, DNA strand-annealing renders the viral genomes transcriptionally active. 2,3 Others and we, on the other hand, have suggested that viral second-strand DNA synthesis is the rate-limiting step in AAV-mediated transgene expression. [4][5][6][7][8][9][10][11] We previously observed that following intravenous (i.v.) administration into the tail-vein, AAV vectors selectively target the liver in mice, but less than 5% of hepatocytes express the transgene. 12 These studies were subsequently corroborated by other investigators. [13][14][15][16] However, it is not entirely clear why the remainder of the 95% of hepatocytes do not allow transgene expression despite being AAV DNA positive. 13,[17][18][19] In our previous studies, we have identified that a 52 kDa cellular protein, FKBP52, which binds the immunosupp...

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Section: Aav-mediated Transduction Of Hepatocytessupporting

confidence: 93%

Section: Aav-mediated Transduction Of Hepatocytes L Zhong Et Almentioning

confidence: 99%

Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo

Zhong

Yang

et al. 2004

Gene Ther

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“…53 For production of AAV2 vectors with detargeted capsids, pDG (R484E;R585E) containing double mutations of capsid protein amino acids (R484E; R585E) involved in heparin binding of AAV2 was co-transfected with pUF2CMV-Luc. 38 For generation of the targeted d-sarcoglycan vectors, we used pMT187VNSTRLPXX2, pDGDVP and pdsCMV-MLC0.26-SGCD, a derivate of pdsAAV-CMV-GFP 54 with the murine d-sarcoglycan cDNA introduced with BsrGI/NheI under the control of a MluI/HindIII fragment of the CMV-enhanced myosin light chain (CMVenhMLC) promoter. 38 For construction of control d-sarcoglycan or luciferase vectors with AAV9 capsids, we used p5E18-VD2-9 55 (kindly provided by Drs Guangping Gao and Jim Wilson) instead of pMT187VNSTRLPXX2.…”

Section: Production and Titration Of Aav Particlesmentioning

confidence: 99%

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

et al. 2010

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Selection of targeted vectors from virus display peptide libraries is a versatile and efficient approach to improve vector specificity and efficiency. This strategy has been used to target various cell types in vitro. Here, we report the screening of an adeno-associated virus type 2 (AAV2) display peptide library in vivo to select vectors specifically homing to heart tissue after systemic application in mice. Selected library clones indicated superior specificity of gene transfer compared with wild-type AAV2, AAV9 and a heparin binding-deficient AAV2 mutant. Such targeted vectors were able to reconstitute expression of d-sarcoglycan in the heart of adult d-sarcoglycan knockout mice after systemic gene transfer in vivo, attesting to the therapeutic potential of this approach.

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“…3,4 Thus, failure to undergo viral second-strand synthesis remains a predominant rate-limiting step in the observed low efficiency of singlestranded AAV (ssAAV) vector-mediated transgene expression both in vitro and in vivo. [5][6][7][8][9][10][11][12][13][14] The use of selfcomplementary AAV (scAAV) vectors that bypass the requirement for viral second-strand DNA synthesis can circumvent this problem. 13 However, their widespread use is limited by their limited packaging capacity (B3.3 kb), 15 which is significantly less than that of conventional ssAAV vectors (B6 kb).…”

mentioning

confidence: 99%

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

Jayandharan

Zhong

et al. 2008

Gene Ther

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Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to B5% of murine hepatocytes. Viral second-strand DNA synthesis continues to be a rate-limiting step for efficient transduction by the single-stranded AAV (ssAAV) vectors. This is due, in part, to the presence of a cellular chaperone (FK506-binding) protein, FKBP52, phosphorylated forms of which interact with the D-sequence in the inverted terminal repeats of AAV2 genome and inhibit the viral second-strand DNA synthesis. Our previous studies have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), and at serine/threonine residues by protein phosphatase 5 (PP5) enhances viral secondstrand DNA synthesis and consequently, the transgene expression. We have also reported that coinfection with a self-complementary AAV (scAAV)-TC-PTP vector results in up to sixfold increase in the transduction efficiency of conventional ssAAV2 vectors in primary murine hepatocytes in vivo. We reasoned that coinfection with scAAV-TC-PTP and scAAV-PP5 vectors may lead to a further increase in the transduction efficiency of ssAAV2 vectors. We demonstrate here that this strategy does indeed lead to B16-fold increase in the transduction efficiency of conventional ssAAV vectors in primary murine hepatocytes in vivo following tail-vein injections. Neither scAAV2-TC-PTP nor scAAV2-PP5 vectors alone or together had any adverse effect on the hepatocytes. Thus, this coinfection strategy may be useful for achieving expression from recombinant ssAAV2 vectors containing larger genes, such as coagulation factor VIII, which exceed the packaging capacity of scAAV vectors, for the potential gene therapy of hemophilia A.

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Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo

Cited by 372 publications

References 28 publications

Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo

Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

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

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