Use of a Hybrid Adeno-Associated Viral Vector Transposon System to Deliver the Insulin Gene to Diabetic NOD Mice

Abstract

Previously, we used a lentiviral vector to deliver furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes using intervallic infusion in full flow occlusion (FFO), with resultant reversal of diabetes, restoration of glucose tolerance and pancreatic transdifferentiation (PT), due to the expression of beta (β)-cell transcription factors (β-TFs). The present study aimed to determine whether we could similarly reverse diabetes in the non-obese diabetic (NOD) mouse using an adeno-associated viral vector (AAV) to deliver INS-FUR ± the β-TF Pdx1 to the livers of diabetic mice. The traditional AAV8, which provides episomal expression, and the hybrid AAV8/piggyBac that results in transgene integration were used. Diabetic mice that received AAV8-INS-FUR became hypoglycaemic with abnormal intraperitoneal glucose tolerance tests (IPGTTs). Expression of β-TFs was not detected in the livers. Reversal of diabetes was not achieved in mice that received AAV8-INS-FUR and AAV8-Pdx1 and IPGTTs were abnormal. Normoglycaemia and glucose tolerance were achieved in mice that received AAV8/piggyBac-INS-FUR/FFO. Definitive evidence of PT was not observed. This is the first in vivo study using the hybrid AAV8/piggyBac system to treat Type 1 diabetes (T1D). However, further development is required before the system can be used for gene therapy of T1D.

Contemporary Transposon Tools: A Review and Guide through Mechanisms and Applications of Sleeping Beauty, piggyBac and Tol2 for Genome Engineering

“…By combining the entry properties of viral vectors and the integrative properties of DNA transposons, hybrid viral-transposon vectors ( Figure 3 ) can this way maintain stable long-term expression while enabling the reduction of the viral load. Recombinant adeno-associated viral vectors (rAAVs) have been successfully used with the PB [ 142 , 143 , 144 , 145 , 146 ] and SB [ 147 ] transposon systems to enable integration of the DNA material while taking advantage of AAV tropism towards specific tissues for in vivo delivery. PB [ 146 , 148 ] and SB [ 147 , 149 , 150 , 151 , 152 , 153 , 154 ] have also been used with adenoviral (AdV) vectors, baculovirus expression vectors (BEVs), and herpes simplex virus type-1 (HSV) vectors to benefit from their large payload capacity (up to 36 kb, 50 kb, and 130 kb, respectively) and their respective intrinsic properties, namely AdV’s broad tropism, BEV’s low cytotoxicity, and HSV’s preference for the central nervous system.…”

“…Various groups have combined AAV vectors, lacking the ability to integrate their cargo, with the PB transposon system. Using this strategy, diabetes type I [ 142 ], urea cycle defects [ 143 ], cystic fibrosis [ 145 ], and progressive familial intrahepatic cholestasis type 3 [ 144 ] were corrected in pig and mouse models. Other studies on cancer therapies have reported a reduction of tumor growth in mice and increased lifespan with hybrid SB-baculovirus vectors [ 150 , 151 , 152 ].…”

Novel vectors and approaches for gene therapy in liver diseases

“… 97 , 98 The AAV-PB system has also been used successfully for insulin gene delivery in a mouse model of diabetes, resulting in normoglycaemia and glucose tolerance. 99 …”

Charting the next century of insulin replacement with cell and gene therapies

“…However, we and others have shown that a similar glucose tolerance profile was also observed in mice treated with insulin under a constitutive promoter. 79,80 Furthermore, hypoglycemia remained a risk even with glucose-responsive promoters. 79,81 The main shortcoming of present-day insulin gene therapy strategies remains the absence of insulin secretory granules, unlike that in b cells.…”

“…74,[86][87][88] Non-integrative adenovirus can last for at most a few months. 71,77,[89][90][91][92][93] While adeno-associated virus (AAV) and AAV hybrid vectors are more long-lasting, 76,80,81 they prevent re-administration as a top-up strategy due to antibodies formed against the viral vectors. Repeated dosing strategies such as using a different AAV capsid, 94 neutralization of antibodies, 95 or immune suppression 96 are being studied.…”