Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin

Abstract

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes mellitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2-5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity.

Figure 1

Analysis of leptin expression in vitro. Supernatant was harvested from human 293 cells and analyzed by Western blot (A) or RIA (B). (A) Western blot analysis of supernatant from cells infected with 1 × 10⁹ (lane 1), 1 × 10¹⁰ (lane 2) AAV-leptin particles, mock infected cells (lane 3) or cells transfected with 2 μg of pCMVKm201-leptin (lane 4). (B) Quantitation of expression by RIA.

Figure 2

Effect of rAAV-leptin treatment on body weight and food intake. Mice received i.m. injections of either 10¹¹ particles of rAAV-leptin (○), β-galactosidase (□), or saline (▵), and weights were monitored three times weekly. (A) Mean ± SEM of 10 mice in each group. For simplicity, only one time point is shown for each week. (B) Mice were caged in groups of five and food intake over a 24-hr period was measured. The average food consumption per mouse per day is shown. The variation between the two cages in each group was negligible.

Figure 3

Physical appearance of mice following rAAV-leptin treatment. Mice were photographed 6 weeks after being treated with rAAV-leptin (Left) or saline vehicle (Right).

Figure 4

Circulating leptin levels. Serum was collected from mice at the indicated times and leptin levels were measured using the Linco RIA kit. Values are the average of five mice ± SEM. Week 7 mice were fasted for 18 hr prior to serum collection. Mice tested at weeks 5, 9, 11, and 14 were fed ad libitum prior to serum collection. Serum was not collected from C57 mice at weeks 5 and 14. (P values for treated vs. untreated are 0.09, 0.0005, 0.007, 0.0079, and 0.0079 for weeks 5, 7, 9, 11, and 15, respectively.)

Figure 5

Effects of rAAV-leptin on glucose metabolism and insulin secretion. Untreated C57 mice or ob/ob mice treated with rAAV-leptin or saline were fasted for 18 hr and bled for determination of fasting glucose (A) and insulin (B), 6 weeks postinjection. The values presented here are the mean ± SEM of five mice. (C) Glucose tolerance was determined in saline (▵), rAAV-leptin-treated (○), or C57 (∗) mice by measuring blood glucose levels at indicated times after i.p. injection of glucose. Values are the mean ± SEM of three mice in each group. Test were performed on fasted mice, 8 weeks postinjection.