In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice

Abstract

Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes.

FIG. 1.

Transduction of WAT after local administration of AAV vectors. A: Immunostaining against GFP (green) in sections of eWAT 2 weeks after the intraeWAT administration of 2 × 10¹¹ vg of AAV-CAG-GFP vectors of serotypes 6, 7, 8, or 9. Blue, nuclei. Original magnification ×100 (upper panel) and ×200 (lower panel). B: GFP content in eWAT treated with 2 × 10¹¹ vg of AAV-CAG-GFP vectors of serotypes 1, 6, 7, 8, or 9 at 2 weeks postinjection (n = 5 mice/group). C: Immunostaining against GFP (brown) in sections of iWAT 2 weeks after the intraiWAT administration of 2 × 10¹¹ vg of AAV8 or AAV9-CAG-GFP vectors. Original magnification ×100. D: GFP expression levels in iWAT 2 weeks postinjection of 2 × 10¹¹ vg of AAV8 or AAV9-CAG-GFP (n = 6). AU, arbitrary units. E: HK2 expression levels in adipocytes isolated 2 weeks after intraeWAT administration of 2 × 10¹¹ vg of AAV9-CMV-HK2 or AAV9-CMV-null vectors (n = 5). F: In vitro basal and insulin-stimulated 2DG uptake by adipocytes isolated 2 weeks after intraeWAT administration of 2 × 10¹¹ vg of AAV9-CMV-HK2 or AAV9-CMV-null vectors (n = 5). G: In vivo 2DG uptake by eWAT, iBAT, and heart 2 weeks after the intraeWAT injection of 1.4 × 10¹² vg of AAV9-mini/aP2-null or AAV9-mini/aP2-HK2 vectors (n = 7). H: Follow-up of circulating levels of human SeAP in animals administered intraeWAT with 2 × 10¹² vg of AAV9-mini/aP2-SeAP or saline (n = 3). Values shown are means ± SEM. RLU, relative light units. *P < 0.05, **P < 0.01, and ***P < 0.001; # P < 0.05 vs. AAV9-CMV-null at the same insulin concentration.

FIG. 2.

Transduction of BAT after intraiBAT administration of AAV vectors. A: Immunostaining against GFP (brown) in sections of iBAT after intraiBAT administration of 2 × 10⁹ vg of AAV8 or AAV9-CAG-GFP. Original magnification ×200 and ×400 (insets). B: RFP expression levels in iBAT after administration of 10¹⁰ vg of AAV-CMV-RFP vectors of serotypes 1, 2, 5, 7, 8, or 9 (n = 3–5 mice/group). AU, arbitrary units. C: Immunostaining against GFP (brown) in sections of iBAT administered with 2 × 10¹¹ vg of AAV8 or AAV9-mini/UCP1-GFP. Original magnification ×200 and ×400 (insets). D: In vivo 2DG uptake by iBAT, eWAT, and heart after intraiBAT administration of 7 × 10¹⁰ vg of AAV8-mini/UCP1-HK2 (n = 6) or AAV8-mini/UCP1-null vectors (n = 10). E: VEGF164 and PECAM1 expression levels in iBAT after intraiBAT administration of 2 × 10¹¹ vg of AAV9-mini/UCP1-VEGF164 or AAV9-mini/UCP1-null vectors (n = 5). F: Immunostaining against CD105 (brown) in iBAT from the same cohorts. Original magnification ×400 and ×1,000 (insets). All analyses were performed 2 weeks after vector administration. Values shown are means ± SEM. *P < 0.05.

FIG. 3.

Transduction of WAT and BAT after systemic administration of AAV vectors to lean mice. Tail vein injection was used to deliver 5 × 10¹² vg of AAV8- or AAV9-CAG-GFP vectors to lean mice, and samples were analyzed 2 weeks after vector administration. A: Immunostaining against GFP (green) in eWAT. Blue, nuclei. Original magnification ×100 (left panels) and ×200 (right panels). B: Immunostaining against GFP (in brown) in iBAT sections. Original magnification ×200 and ×400 (insets). Relative GFP expression levels in iWAT, retroperitoneal WAT (rWAT), mesenteric WAT (mWAT), eWAT, and iBAT fat depots after intravascular administration of vectors to ICR mice (C) and C57Bl6 mice (D) (ICR: n = 3 for AAV8 and n = 5 for AAV9; C57Bl6: n = 4). Values shown are means ± SEM. AU, arbitrary units.

FIG. 4.

Transduction of WAT and BAT after systemic administration of AAV vectors to obese diabetic mice. Immunostaining against GFP (brown) in eWAT, iWAT, and iBAT sections after intravascular administration of 3 × 10¹² vg of AAV8- or AAV9-CAG-GFP vectors to ob/ob (n = 4) (A) and db/db (n = 4)mice (C). Original magnification ×200. GFP expression in inguinal (iWAT), retroperitoneal (rWAT), mesenteric (mWAT), eWAT, and iBAT depots from the same cohorts of ob/ob (B) and db/db mice (D). All analyses were performed 2 weeks after vector delivery. Values shown are means ± SEM. *P < 0.05 vs. AAV9. AU, arbitrary units.

FIG. 5.

Assessment of adipose tissue inflammation after systemic administration of AAV vectors. Flow cytometric quantification of the number of macrophages (mΦ), T lymphocytes (T cells), and natural killer cells (NK) in eWAT (A) and iBAT (B) 1 month after intravascular administration of 3 × 10¹² vg of AAV8 or AAV9-CAG-GFP to C57Bl6 mice (n = 3–4). C: Relative expression of adipokines and proinflammatory cytokines in eWAT at 2 weeks after intravascular administration of 2 × 10¹² vg of AAV8 or AAV9-CAG-null vectors to C57Bl6 mice (n = 7–8). Lep, leptina; Adipoq, adiponectin; Retn, resistin. D: Serum adiponectin levels in the same cohorts. Values shown are means ± SEM.

FIG. 6.

Specific transduction of adipocytes after systemic administration of AAV vectors. A: Immunostaining against GFP (brown) in iBAT 2 weeks after systemic delivery of 2 × 10¹² vg of AAV9-mini/aP2-GFP or AAV9-mini/UCP1-GFP vectors. The arrowheads indicate transduced brown adipocytes. Original magnification ×200 and ×400 (insets). VEGF164 (B) and PECAM1 (C) expression levels in iBAT 1 month after the intravascular administration of 8 × 10¹² vg of AAV9-mini/UCP1-VEGF164 or AAV9-mini/UCP1-null vectors (n = 5). Immunostaining against CD105 (brown) (D) and α-SMA (brown) (E) in the same cohorts as in B and C. The red arrowheads indicate vessels. Original magnification ×400 and ×1,000 (insets). Values shown are means ± SEM. *P < 0.05; **P < 0.01.

FIG. 7.

Efficient adipocyte transduction and de-targeting of transgene expression from liver and heart with mirT sequences. GFP immunostaining (brown) in eWAT, iWAT, iBAT, liver, and heart at 2 weeks after intravascular administration of 10¹² vg of AAV9-CAG-GFP, AAV9-CAG-GFP-miRT122, AAV9-CAG-GFP-miRT1, or AAV9-CAG-GFP-doublemiRT vectors. Original magnification ×100 (liver and heart), ×200 (eWAT and iWAT), and ×400 (iBAT and insets).