Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors

Song, Sihong; Embury, Jennifer E.; Laipis, Philip J.; Berns, KI; Crawford, Julie M.; Flotte, TR

doi:10.1038/sj.gt.3301422

Cited by 141 publications

(110 citation statements)

References 28 publications

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“…As can be seen in Figure 3, AAV vector-mediated EGFP expression (green) in liver sections from vector-injected mice colocalized with albumin (red) but not with CD31 (blue) expression. Thus, consistent with previously published reports, [12][13][14][15][16] AAV vectors selectively transduce hepatocytes.…”

supporting

confidence: 92%

“…[12][13][14][15][16] Interestingly, a significant fraction of the viral genomes was converted to dimer-length DNA forms (d), which migrated slower on the denaturing gel, 1 week after injection, and most of them were converted to dimerlength forms 2-and 4 weeks postinjection in wtTC-PTP-TG mice (lanes 3, 7, 11). Viral second-stand DNA synthesis could also be observed in hepatocytes from FKBP52-KO mice (lanes 4, 8, 12), albeit at a lower level at 1-and 2 weeks postinjection, the underlying basis of which remains unclear.…”

Section: Aav-mediated Transduction Of Hepatocytesmentioning

confidence: 99%

“…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 immunosuppressant drug FK506, 20,21 interacts specifically with the D-sequence within the inverted terminal repeat (ITR) of the AAV genome.…”

mentioning

confidence: 99%

“…(tail-vein or portal vein) injection of AAV selectively infects hepatocytes. [12][13][14][15][16] After 2 weeks, liver sections from three hepatic lobes were observed under a confocal scanning fluorescence microscope. These results are shown in Figure 1.…”

mentioning

confidence: 99%

“…It is evident that whereas no green fluorescence was observed in livers from mock-injected animals, AAV transduction efficiency in primary hepatocytes from mTC-PTP-TG mice was low, and similar to nontransgenic normal mice (not shown), as observed previously. [12][13][14][15][16] However, hepatocytes from wtTC-PTP-TG and FKBP52-KO mice were transduced by AAV vectors with significantly higher efficiency 2-weeks postinjection. Quantitative analyses of AAV transduction efficiency of hepatocytes from TC-PTP-TG and FKBP52-KO mice, shown in Figure 2, indicate that the transgene expression increased B15-and B4-fold 1 week postinjection; B17-and B7-fold 2 weeks postinjection; and B16-and B12-fold 4 weeks postinjection in TC-PTP-TG and FKBP52-KO mice, respectively.…”

mentioning

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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supporting

confidence: 92%

Section: Aav-mediated Transduction Of Hepatocytesmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

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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Alpha‐1‐Antitrypsin Deficiency

Lomas¹,

Perlmutter²

2010

Protein Misfolding Diseases

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Ex Vivo transduced liver progenitor cells as a platform for gene therapy in mice

Song

Witek

et al. 2004

Hepatology

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Allogeneic stem cell-based transplants may be limited by allograft rejection, as is seen with conventional organ transplantation. One way to avert such a response is to use autologous stem cells, but that may carry the risk of recurrence of the original disease, particularly in the context of a genetic defect. We investigated the potential for gene modification of autologous stem cells to avoid both problems, using recombinant adenoassociated virus vector expressing human al-antitrypsin in murine liver progenitor cells. We showed that recombinant adenoassociated virus 1 was the most efficient vector for liver progenitor cell transduction among five different serotypes of recombinant adenoassociated virus vectors. Ex vivo infected green fluorescent protein-positive liver progenitor cells from C57BL/6 mice with recombinant adenoassociated virus 1-vector-expressing human a1 antitrypsin were transplanted into the liver of monocrotaline-treated and partial-hepatectomized C57BL/6 recipients. Using green fluorescent protein as a donor marker, we were able to determine that at From the Departments of 'Pharmaceutics, 2Pathology, and 3Pediatrics, and

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Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors

Cited by 141 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

Alpha‐1‐Antitrypsin Deficiency

Ex Vivo transduced liver progenitor cells as a platform for gene therapy in mice

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