376. A Metabolically Responsive Insulin Transgene Delivered by AAV2/8 Pseudotyped, Self-Complementary AAV (SC-AAV2/8) Controls Glycemia in STZ-Diabetic Mice
Authors: Mirek Kozlowski, Dorota Lyszcowizc, Adam G. Campbell, Darin E. Olson, Janet E. Rubin, Peter M. Thule
Journal: Molecular Therapy (2004)

Metabolically responsive insulin gene therapy promises advances in the treatment of type 1 diabetes mellitus. We have shown that hepatic expression of a transcriptionally regulated human insulin transgene successfully treats diabetes in rodents. However, consistent with first generation adenoviral delivery transgenic insulin production is transient in vivo. In contrast, transgenes delivered by recombinant adeno-associated virus (rAAV) express in murine liver for more than a year. Despite their advantages application of AAV has been limited. Transgene expression is delayed, a factor that can complicate experiments in vivo. Moreover, producing large quantities of AAV at high titers remains costly and technically demanding. Finally, common vectors infect some tissues poorly, or express weakly due to limited second-strand synthesis. Two recent developments in rAAV technology address these issues. A novel serotype, rAAV8 was shown to infect hepatocytes 10-100 fold more efficiently than rAAV2. Separately, self-complimentary (SC)-AAV particles produced by eliminating the terminal resolution sequence (TRS) from one inverted terminal repeat (ITR) were shown to accelerate and enhance transgene expression in vivo. Here we describe the combination of SC and pseudotyping strategies to obtain long-term glycemic control in diabetic mice. We created pSC-AAV2(GlRE)3BP-1 2fur by inserting a previously described glucose and insulin responsive insulin transgene into an AAV2 transfer plasmid from which the TRS had been removed from the left-ITR. Compared to vector derived from a longer, AAV2(GlRE)3BP-1 2furGFP, or similarly short sequence, AAV2(GlRE)3BP-1 2fur, infection with SC-AAV2(GlRE)3BP-1 2fur accelerated, and more than tripled secretion of human insulin from primary hepatocytes. Co-transfection of 293 cells with pSC-AAV2(GlRE)3BP-1 2fur, and a REP2/CAP8 expressing plasmid produced pseudotyped SC-AAV2/8 vector. Twenty-three male, STZ-diabetic CD-1 (BG>200 mg/dl) mice received a portal vein injection of either SC-AAV2(GlRE)3BP-1 2fur, or increasing doses of SC-AAV2/8(GlRE)3BP-1 2fur. Despite enhanced transgene expression in vitro, SC-AAV2(GlRE)3BP-1 2fur administration (3.4-4.8×1010vg, n=4) failed to reduce hyperglycemic by 18 days. In contrast, SC-AAV2/8(GlRE)3BP-1 2fur administration lowered blood glucose in 18 of 19 diabetic mice by 7 days, beginning with doses as low as 1.3×1010vg. Six of seven mice receiving the three highest doses (5.2×1010vg n=3, 7.8×1010vg n=1, 1.3×1011vg n=3) succumbed to hypoglycemia within 4 days. Two animals receiving 2.6×1010vg (n=6) died of hypoglycemia at day 34. However, 9 of the remaining 10 mice receiving 1.3-2.6×1010vg continue to grow normally and maintain near euglycemia (105±6 mg/dl). In conclusion, we have combined SC-AAV, AAV2/8 pseudotyping techniques, and metabolically responsive hepatic insulin gene therapy to treat STZ-diabetic mice in vivo. Effective viral doses may be 10-fold less than standard, non-pseudotyped vector delivering the same transgene. On-going investigations will determine the long-stability of glycemic control in this model.