Adeno-associated virus 2-mediated gene transfer: role of a cellular serine/threonine protein phosphatase in augmenting transduction efficiency

Abstract

We have documented that a cellular chaperone protein, FKBP52, when phosphorylated at tyrosine and/or serine/ threonine (Ser/Thr) residues, interacts with the D-sequence in the inverted terminal repeats of the adeno-associated virus 2 (AAV) genome, inhibits the viral second-strand DNA synthesis, and leads to inefficient transgene expression from recombinant AAV vectors in certain cell types. We have also demonstrated that FKBP52 is dephosphorylated at tyrosine residues by T-cell protein tyrosine phosphatase (TC-PTP), and that deliberate overexpression of TC-PTP leads to more efficient viral second-strand DNA synthesis, and increased transgene expression. However, the identity of the putative Ser/Thr protein phosphatase that dephosphorylates FKBP52 at Ser/Thr residues has remained elusive. Using known inhibitors of Ser/Thr phosphatases, we have now identified protein phosphatase 5 (PP5) to be a candidate enzyme.Deliberate overexpression of PP5 in 293 cells, which does not influence cellular growth, leads to B5-fold increase in the transduction efficiency of conventional single-stranded AAV vectors, but no significant enhancement in the transduction efficiency of self-complementary AAV vectors, suggesting that PP5 plays a role in AAV second-strand DNA synthesis. Electrophoretic mobility-shift assays show that in cells overexpressing PP5, the extent of the complex formation between FKBP52 and the AAV D-sequence is significantly reduced. These studies suggest that PP5-mediated dephosphorylation of FKBP52 at Ser/Thr residues augments viral second-strand DNA synthesis and enhances AAV transduction efficiency, which has implications in the optimal use of these vectors in human gene therapy.

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

“…FKBP52 (F), phosphorylated at tyrosine residues (red symbol) and serine/threonine (yellow symbol), which strongly inhibits the second-strand DNA synthesis of a conventional ssAAV vector, is dephosphorylated at tyrosine residues by T-cell protein tyrosine phosphatase (TC-PTP; blue semi-oval), and at serine/threonine residues by protein phosphatase 5 (PP5; green semi-oval) expressed from scAAV-TC-PTP and scAAV-PP5 vectors, which allows more efficient viral second-strand DNA synthesis of conventional ssAAV vector, and consequently, leads to more efficient transgene expression. 12 Consistent with our hypothesis, we observed an additive effect in the increase in EGFP expression in HeLa, KB and 293 cells when both TC-PTP and PP5 expression plasmids were cotransfected (Figure 2b). In anticipation of extending these studies to an in vivo animal model, we next wished to deliver TC-PTP and PP5 genes via scAAV vectors.…”

“…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).…”

“…8,9,11,[17][18][19] We have also documented that FKBP52 is dephosphorylated at Tyr residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), and at Ser/Thr residues by protein phosphatase 5 (PP5). 9,12 Dephosphorylated FKBP52 can no longer bind to the Dsequence, which in turn, facilitates the viral secondstrand DNA synthesis, and augments the transgene expression from ssAAV2 vectors. We have demonstrated augmentation in ssAAV vector-mediated transgene expression in transgenic mice overexpressing TC-PTP, 9 and in mice deficient in FKBP52.…”

“…However, the observed increases were only modest, approximately 5-to 6-fold. 11,12 In an attempt to further augment the transduction efficiency of ssAAV vectors, we reasoned that coinfection with AAV-TC-PTP and AAV-PP5 vectors together may exert, at the very least, an additive effect. As both TC-PTP and PP5 cDNA sizes (B2.5 kb) are within the packaging capacity of scAAV vectors 15 and AAV2 remains the best characterized serotype for gene transfer protocols, 20,21 we examined whether coinfection with scAAV2-TC-PTP+scAAV2-PP5 vectors might augment the transduction efficiency of conventional ssAAV2 vectors.…”

“…The scAAV2 plasmids containing the RSV promoter-driven murine TC-PTP (scAAV2-TC-PTP) and the RSV promoter-driven human PP5 (scAAV2-PP5) were constructed by standard cloning methods as described previously. 11,12 Highly purified stocks of recombinant scAAV2-TC-PTP and scAAV2-PP5 vectors were generated by the triple-plasmid transfection protocol as described previously. 22 The physical particle titers of recombinant vector stocks were determined by quantitative DNA slot-blot analyses.…”