1. ¹Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida College of Medicine, Gainesville, FL, USA
2. ²Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA
3. ³Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL, USA
4. ⁴Genetics Institute, University of Florida College of Medicine, Gainesville, FL, USA
5. ⁵Columbia University, New York, NY, USA
6. ⁶Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA

Correspondence: Professor A Srivastava, Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida College of Medicine, Cancer and Genetics Research Complex, 1376 Mowry Road, Room 492-A, Gainesville, FL 32610-3633, USA. E-mail: aruns@peds.ufl.edu

⁷These authors contributed equally to this work.

Received 11 December 2007; Revised 11 March 2008; Accepted 11 March 2008; Published online 22 May 2008.

Abstract

Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to 5% 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 second-strand 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 16-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.