Efficient gene delivery to human and rodent islets with double-stranded (ds) AAV-based vectors

Abstract

Transplantation of allogeneic pancreatic islets is an effective approach to treat type 1 diabetes. To bypass the need for systemic administration of immunosuppression drugs following transplantation, approaches to genetically modify allogeneic islets to express anti-inflammatory, immunosuppressive, or antiapoptotic proteins prior to transplantation are being developed. Adeno-associated viral (AAV) based vectors have been used for gene transfer to islets, but the efficiency of functional transduction is low. Recently, doublestranded (ds) or double-copy (dc) based AAV vectors have been developed that allow for more rapid and efficient AAVmediated transgene expression following transduction. Here we demonstrate that intact human and murine islets can be transduced with dsAAV2-eGFP efficiently compared to single-stranded AAV2-eGFP. Furthermore, our results demonstrate that murine islets transduced with dsAAV2-eGFP have normal islet glucose responsiveness, viability, and islet insulin content. Transplantation of the dsAAV2-eGFP transduced islet restored normal glycemia in diabetic mice without eliciting an immune response. Significant dsAAV2-mediated eGFP expression was observed in the islet grafts for at least 6 months post-transplant. Finally, we demonstrated that dsAAV serotypes 2, 6, and 8 infect human islets efficiently. Taken together, these results suggest that dsAAV based vectors are highly appropriate for gene transfer to islets to facilitate transplantation.

Persistence, Localization, and External Control of Transgene Expression After Single Injection of Adeno-Associated Virus into Injured Joints

“…Double-stranded AAV serotype 2 (AAV2) vectors were produced, purified, and vector genome (vg) titer determined as previously described (Rehman et al, 2005). AAV2s with constitutively active cytomegalovirus (CMV) promoter included AAV2-CMV-Luciferase (Luc) and AAV2-CMV reverse-tetracycline-controlled transactivator (rtTA).…”

Adeno-associated viral gene transfer of transforming growth factor-β1 to human mesenchymal stem cells improves cartilage repair

“…41 A PMAMneo plasmid containing the human TGF-b1 gene under the control of the MMTV-LTR promoter and enhanced by RSVLTR 42 (a gift from Yong Li) was amplified by RT-PCR from human liver cancer cell line HepG2 using forward primer TGF-F: 5 0 -CCATGCCGCCCTCCGGGCTGC-3 0 and reverse primer TGF-R: 5 0 -TCAGCTGCACTTGCAGGAGC-3 0 . The PCR product encoding human TGF-b1 was cloned into the HindIII and NotI sites of plasmid dsAAV-cytomegalovirus-GFP (see reference below) by blunt end ligation.…”

Widespread and Stable Pancreatic Gene Transfer by Adeno-Associated Virus Vectors via Different Routes

“…For in vitro infection of the islet isolated from untreated adult mice, the AAV vectors were directly added into the culture medium at a dose of 10,000 viral genome particles/cell. The number of cells was determined based on the estimation of about 2,000 cells per islet on the average (33,34). Examination of GFP expression.…”