Optimal delivery of therapeutic genes to pancreatic islets.

Abstract

Islet transplantation is a promising therapeutic option for Type 1 Diabetic (T1D) patients, with the ability to improve glycometabolic control and in select cases achieve insulin independence. Intraportally transplanted islets must reside in the hostile environment of the liver, where they are exposed to the instant blood mediated inflammatory reaction (IBMIR), alloimmunity, recurrence of islet specific autoimmunity, a highly toxic pro-inflammatory cytokine storm (e.g. IL-1β, IFN-α, IFN-γ and TNF-α) and hypoxia due to inadequate revascularization post-transplantation. The early loss of functional islet mass (50-70%) due to apoptosis following clinical transplantation contributes to islet allograft failure. Strategies to prevent apoptosis are therefore highly desirable to enhance islet survival for transplantation. In Chapter 3, the ability of Adenoviral (Ad) and Adeno-Associated Viral (AAV)-based vectors expressing a green fluorescent protein (GFP) reporter gene to transduce isolated human and rat pancreatic islets was investigated. Specific interest was placed on tyrosine mutant AAV-based vector types, which have not been previously explored in human and rodent pancreatic islets. Ad efficiently transduced isolated human and rat pancreatic islets while AAV failed to transduce human islets and showed a varied ability to transduce rat islets. The results in this chapter demonstrate that Ad vectors are more efficient at transducing isolated islets than AAV-based vector types. Chapter 4 aimed to characterise an Ad-based vector encoding an anti-apoptotic molecule termed Insulin-like Growth Factor-II (Ad-IGF-II). Ad-IGF-II effectively transduced rat pancreatic islets without affecting islet viability or function and did not induce uncontrolled islet cell proliferation. The results in this chapter suggest that Ad-IGF-II is an effective and non-toxic vector type for use in an islet gene therapy setting. In Chapter 5 and Chapter 6, the influence of local human IGF-II over expression on rat pancreatic islet cell survival in vitro and in vivo was examined, respectively. Over expression of IGF-II in islets resulted in enhanced islet survival in vitro and in an in vivo marginal mass islet transplant model. Transplantation of IGF-II over expressing islets under the kidney capsule of diabetic NOD-SCID mice restored euglycemia in 78% of recipients, compared to 46% and 18% of untransduced and Ad-GFP transduced control islet recipients, respectively. In summary, this thesis demonstrated that compared to AAV, Ad is currently the optimal vector for use in an islet gene therapy setting. Moreover, over expression of IGF-II did not affect the viability or insulin secreting capacity of islets. Finally, the induced expression of anti-apoptotic IGF-II led to enhanced islet survival in vitro and improved transplant outcomes in an in vivo marginal mass islet transplant model, indicating that IGF-II gene transfer is a potentially powerful tool to improve islet survival post-transplantation.