AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice.

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

Effect of dexamethasone on glucose tolerance and fat metabolism in a diet-induced obesity mouse model.

Prolonged exposure to elevated glucocorticoid levels is known to produce insulin resistance (IR), a hallmark of diabetes mellitus. Although not fully elucidated, the underlying molecular mechanisms by which glucocorticoids induce IR may provide potential targets for pharmacological interventions. Here we characterized muscle lipid metabolism in a dexamethasone-aggravated diet-induced obesity murine model of IR. Male C57BL/6 mice on a high-fat diet for 2 months when challenged with dexamethasone showed elevated food consumption and weight gain relative to age and diet-matched animals dosed with saline only. Dexamethasone treatment impaired glucose tolerance and significantly increased the intramyocellular lipid content in the tibialis anterior muscle (TA). A good correlation (r = 0.76, P < 0.01) was found between accumulation in intramyocellular lipid content in the TA and visceral adiposity. The linoleic acid (18:2) to polyunsaturated acid ratio was increased in the dexamethasone-treated animals (+29%; P < 0.01), suggesting a possible increase in stearoyl-CoA desaturase 2 activity, as reported in Sertoli cells. The treatment was also accompanied by a reduction in the percent fraction of omega-3 and long-chain polyunsaturated fatty acids in the TA. Analysis of the low-molecular-weight metabolites from muscle extracts showed that there was no dysregulation of muscle amino acids, as has been associated with dexamethasone-induced muscle proteolysis. In conclusion, dexamethasone-induced insulin resistance in diet-induced obese mice is associated with a profound perturbation of lipid metabolism. This is particularly true in the muscle, in which an increased uptake of circulating lipids along with a conversion into diabetogenic lipids can be observed.