Development of an adipose-tropic AAV capsid ablating liver tropism

Figures

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  Figure 1. Rec2 variant V7 with three amino acid residue substitution on Rec2 capsid (A) Schematic of V7 trans plasmid. (B) V7 capsid amino acid sequence with residue substitution highlighted.

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  Figure 2. Intraperitoneal administration of V7 vector selectively transduces visceral adipose tissue and de-targets liver (A) GFP fluorescence in eWAT and liver at 2 weeks post i.p. injection of Rec2-GFP or V7-GFP vectors. (B) Western blotting in a separate experiment two weeks post AAV injection. (C) GFP fluorescence in dosing experiment at 2 weeks post i.p. injection. (D) Western blotting in mice receiving high dose V7-GFP. (E) Vector genome copy numbers in mice receiving high dose V7-GFP. Doses are indicated in each graph. Data are means ± SEM. Sample size (biological replicates): Rec2-GFP 2E10 vg/mouse n = 3–8, V7-GFP 2E10 vg/mouse n = 8, V7-GFP 4E10/mouse n = 5, V7-GFP 8E10 vg/mouse n = 4.

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  Figure 3. Intravenous injection of V7 vector preferentially transduces subcutaneous white adipose tissue and brown adipose tissue while de-targets liver (A) Western blotting at 2- weeks post tail vein injection of AAV8-GFP or V7-GFP. (B) Western blotting of tissues from a mouse receiving V7-GFP (8E10 vg/mouse). Molecular weight markers are shown in kDa. (C) Vector genome copy numbers in mice receiving high dose of AAV8 or V7-GFP. Doses are indicated in each graph. Data are means ± SEM. Sample size (biological replicates): n = 3 per group.

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  Figure 4. Intraperitoneal administration of V7-LEP/ADIPOQ vector rescues the excessive weight gain and impaired glycemic control in ob/ob mice (A) Schematic of the AAV vector containing human leptin and human adiponectin linking by 2A sequence. (B) Body weight. (C) Area under the curve of body weight. (D) Weight gain. (E) Area under the curve of weight gain. (F) Glucose tolerance test at 8-week post AAV injection. (G) Area under the curve of glucose tolerance test. Data are means ± SEM. Sample size (biological replicates): ob/+ buffer n = 5, ob/ob GFP 8E10 n = 5, ob/ob LEP/ADIPOQ 4E10 n = 4, ob/ob LEP/ADIPOQ 8E10 n = 4. ∗p < 0.05, ∗∗p < 0.01 from one-way ANOVA followed by Tukey’s multiple comparison test.

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  Figure 5. Intraperitoneal administration of V7-LEP/ADIPOQ vector normalizes body temperature and body composition in ob/ob mice (A) Rectal temperature at 8-week post AAV injection. (B) Body composition by Echo MRI at 9 week post AAV injection. (C) Relative tissue mass calibrated to body weight at sacrifice 9 weeks post AAV injection. Data are means ± SEM. Sample size (biological replicates): ob/+ buffer n = 5, ob/ob GFP 8E10 n = 5, ob/ob LEP/ADIPOQ 4E10 n = 4, ob/ob LEP/ADIPOQ 8E10 n = 4. ∗p < 0.05, ∗∗p < 0.01 from One-way ANOVA followed by Tukey’s multiple comparison test.

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  Figure 6. Intraperitoneal administration of V7-LEP/ADIPOQ reverses hyperinsulinemia, hyperglycemia, and hepatic steatosis in ob/ob mice (A) Serum levels of human leptin and human adiponectin. No significant difference between the low dose and high dose. (B) Serum level of insulin. (C) Serum level of glucose. (D) Liver triglyceride content. Data are means ± SEM. Sample size (biological replicates): ob/+ buffer n = 5, ob/ob GFP 8E10 n = 5, ob/ob LEP/ADIPOQ 4E10 n = 4, ob/ob LEP/ADIPOQ 8E10 n = 4. ∗p < 0.05, ∗∗p < 0.01 from One-way ANOVA followed by Tukey’s multiple comparison test.

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  Figure 7. AAV8 capsid with F503Y/Y708D/K709I substitution loses liver-tropism (A) Schematic of AAV8 mutant trans plasmid. (B) AAV8 mutant capsid amino acid sequence with residue substitution highlighted. (C) GFP fluorescence in liver and eWAT at 2-week post i.p. injection of AAV8 or AAV8 mutant vector at 2E10 vg/mouse. Data are means ± SEM. Sample size (biological replicates): AAV8 n = 3, AAV8 mutant n = 4. ∗∗p < 0.01 from Student t test. (D) Western blotting of GFP in various tissues upon extended exposure. Molecular weight markers are shown in kDa.