Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo

McCarty, Douglas M.; Fu, Huiling; Monahan, Paul E.; Toulson, Charles E.; Naik, Pankaja; Samulski, R. Jude

doi:10.1038/sj.gt.3302134

Cited by 481 publications

(391 citation statements)

References 20 publications

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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with a TC-PTP expression plasmid in vitro, and in primary hepatocytes in TC-PTP-TG and FKBP52-KO mice in vivo (unpublished results).…”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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“…In an attempt to overcome time limitations we used wt AdV coinfection, a potential weakness in pseudotype evaluations ex vivo. The use of newly developed self-complementary AAV vectors may provide more insight into vector tropism in future studies [35]. However, we can speculate that if AAV2/2 shows tropism for colonic epithelial cells in animal models in vivo, it could also be used for gene delivery to human colonic mucosa.…”

Section: Discussionmentioning

confidence: 99%

Gene Delivery to Intestinal Epithelial Cells In vitro and In vivo with Recombinant Adeno-Associated Virus Types 1, 2 and 5

et al. 2007

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Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intraperitoneal (IP) injection and superior mesenteric artery (SMA) injection routes. Tropism and efficiency were determined by microscopy, flow cytometry, immunohistochemistry and PCR. Caco2 cells were more permissive to AAV transduction. Human colon epithelial cells in organ culture were more effectively transduced by AAV2/2. SMA injection provided the most effective means of vector gene transfer to small intestine and colonic epithelial cells in vivo. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript applicable for the investigation of IBD pathogenesis and novel therapeutic options, but also revealed the need for further studies to identify more efficient pseudotypes.

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“…A deletion in the D-region of one of the ITRs of the proviral plasmids leads to efficient packaging of double-stranded rAAV, which are usually referred to as 'self-complementary' rAAV or sc-rAAV. 26 We have made extensive use of pseudotyping and of the scvariant in our development of rAAV vectors for pain as described in detail below.…”

Section: Capsid Choice (Pseudotyping) and The Self-complementary Raavmentioning

confidence: 99%

AAV for pain: steps towards clinical translation

Beutler

Reinhardt

2009

Gene Ther

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Recombinant adeno-associated virus (rAAV) vectors consisting of self-complementary genomes and packaged in certain capsids can target primary sensory neurons efficiently and can control neuropathic pain long term by expressing opioid or non-opioid analgesic genes. This review examines the therapeutic potential of the approach in five sections: Pain control in oncology (including a discussion of cancer centers as translational pain research environment); vector biology; safety considerations and immunological lessons learned from rAAV clinical trials of other disorders; development of intrathecal rAAV therapy in rodent models of pain; and preclinical steps towards clinical translation of rAAV for pain. In the field of analgesic drug development, clinical validation of new approaches identified in rodents is currently a critical limiting step. Small-molecule therapeutics suitable as conventional drugs to probe novel targets in clinical trials are often unavailable. In this context, gene therapy could fill an important gap in the drug development process facilitating first-into-human trials of untested targeted treatments, each instantiated as a therapeutic gene.

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Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo

Cited by 481 publications

References 20 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

Gene Delivery to Intestinal Epithelial Cells In vitro and In vivo with Recombinant Adeno-Associated Virus Types 1, 2 and 5

AAV for pain: steps towards clinical translation

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