Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype

Abstract

Adipose tissue plays an essential role in metabolic homeostasis and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here, we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1~4) recombinant adeno-associated viral (AAV) vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass and morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue.

Targeting Visceral Fat by Intraperitoneal Delivery of Novel AAV Serotype Vector Restricting Off-Target Transduction in Liver

“…injected to male BALB/c mice at a dose of 2 × 10 10 vg per mouse, which is similar to the doses previously used in i.v. injection, oral administration, or direct fat pad injection 7, 12. 2 weeks post-Rec2 administration, mice were subjected to in vivo bioluminescence measurement, and strong luciferase activity was observed primarily in the abdomen (Figure 1A).…”

“…A few groups have targeted adipose tissue with naturally occurring AAV serotypes by direct injection to fat depots often requiring high doses 3, 4, 5. We previously report that an engineered hybrid serotype Rec2 displays superior transduction of both brown adipose tissue (BAT) and subcutaneous WAT (SAT) compared to AAV1 or AAV8 7 . Direct injection of Rec2 vector at a moderate dose (1 × 10 9 to 1 × 10 10 vg per fat depot) is sufficient to modulate the function of the targeted fat depot (BAT or SAT) and result in systemic metabolic changes 7, 8, 9, 10.…”

“…We previously report that an engineered hybrid serotype Rec2 displays superior transduction of both brown adipose tissue (BAT) and subcutaneous WAT (SAT) compared to AAV1 or AAV8 7 . Direct injection of Rec2 vector at a moderate dose (1 × 10 9 to 1 × 10 10 vg per fat depot) is sufficient to modulate the function of the targeted fat depot (BAT or SAT) and result in systemic metabolic changes 7, 8, 9, 10. Rec2 vector is efficient for both overexpression and knockdown applications 7, 8, 9, 10, 12, 27.…”

Role of Hypothalamic VGF in Energy Balance and Metabolic Adaption to Environmental Enrichment in Mice

“…rAAV serotype 1 vectors carrying TrkB.T1, BDNF, or YFP were packaged and purified as described elsewhere (35). An engineered hybrid rAAV serotype Rec2 vector carrying Cre or YFP were packaged and purified as described previously (36).…”

“…We reported that injection of a novel engineered rAAV serotype Rec2 carrying Cre recombinase fused with GFP (Cre/GFP) to the BAT and WAT of insulin receptor flox/flox mice efficiently knocked down insulin receptor expression resulting in dysfunction of transduced fat pad and impaired whole body glucose tolerance (36). To avoid high level of Cre-associated toxicity in the hypothalamus, we performed a titer titration experiment in wild-type mice and found bilateral injection of Rec2-Cre/GFP at the dose of 1 3 10 9 vg per site showed no difference of body weight or food intake compared with mice receiving Rec2-YFP (data not shown).…”