Sergei Zolotukhin, Ph.D.
Cancer and Genetics Research Center
Cancer and Genetics Research Complex, Rm 230G
Research Project 1
About 1 billion people in the world are overweight or obese, compared to 850 million who are underweight. In spite of a heavy burden imposed by the obesity epidemic, and a massive effort by various pharmaceutical companies, no safe and reliable weight-reducing drug is yet available. While most research has highlighted the importance of environmental and behavioral modifications in treating obesity, few studies address the possibility of genetic intervention. The long-term goal of this project is to develop gene-based therapeutics to alleviate obesity and attendant type 2 diabetes.
This laboratory has initiated research to explore the novel concept of whether gene therapy could be successfully applied to treat a multitrait disorder such as diet-induced obesity (DIO). To validate this concept, we utilized an unconventional adult transgenic rat model where the gene under investigation is induced or repressed in a sustained fashion using viral vectors targeting metabolically active tissues. We have tested seven independent gene targets in longitudinal in vivo experiments concluding that: 1) it is possible to reduce whole body adiposity and improve glucose homeostasis by targeting single genes in key metabolic pathways; and 2) one-time therapeutic intervention could prevent the development of DIO in young animals and partially correct pre-existent obesity in older animals.
The current research focuses on one particular gene encoding adipocyte-specific hormone adiponectin, which proved to be the most promising among the targets tested. We hypothesize that adiponectin gene therapy targeting the liver reduces systemic adiposity and improves glucose homeostasis acting both locally at the hepatocellular level and systemically at the level of brain/periphery nutrient sensing. Our studies are focusing on: 1) Elucidating the underlying mechanisms responsible for the reduction in systemic adiposity in DIO rats treated with adiponectin gene therapy; 2) Determining whether enhanced adiponectin signaling in skeletal muscle or liver of DIO rats retards age-related changes and extends the maximum life span in treated animals; and 3) Studying transgenerational effect of adiponectin gene therapy by testing the relative contribution of environmental and genetic factors in the phenotype of DIO dam’s F1/F2 offspring.
The results of these studies will determine if gene therapy is a viable long-term therapeutic strategy to control BW and reduce insulin resistance while establishing adiponectin as a gene target-of-choice in treating obesity and diabetes caused by environmental (DIO) factors. In addition, the data will validate a novel prevention strategy of therapeutic intervention prior to pregnancy.
Research Project 2
Recombinant Adeno-associated virus (rAAV) vectors have emerged as one of the most versatile gene therapy delivery vehicles. To be used as therapeutic agents, rAAV vectors have to meet stringent criteria of high titer, purity, and lack of replication competent AAV. In addition, to support the high titer demands of clinical trials the manufacturing protocol has to be scalable. To produce rAAV, several approaches have been developed over the years. One strategy utilizes insect cells co-infected with three baculovirus expression vectors (BEVs). While extremely promising, the original protocol had not been widely adopted due to several inherent and built-in shortcomings. One of such includes a requirement for the triple co-infection with helpers Bac-Rep, Bac-VP, and Bac-GOI (gene of interest flanked by AAV inverted terminal repeats).
Our laboratory develops the novel system of rAAV production in insect cells. The system takes advantage of DNA regulatory elements from both wt Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and wt AAV2. The endpoint design consists of only two components: 1) stable Sf9-based cell line incorporating integrated copies of rep and cap genes, and 2) Bac-GOI. Rep and cap genes are designed to remain silent until the cell is infected with Bac-GOI helper providing both rAAV transgene cassette and immediate-early (IE-1) transcriptional transregulator required to initiate the expression of AAV helper genes. The described arrangement provides identical levels of Rep and Cap proteins in all cells while allowing for a higher MOI with Bac-GOI thus ensuring significantly higher yields of rAAV.
Additional publications can be found in PubMed.
- Aslanidi G, Kroutov V, Philipsberg G, Lamb K, Campbell-Thompson M, Walter GA, Kurenov S, Aguirre JI, Keller P, Hankenson K, MacDougald OA, and Zolotukhin S Ectopic expression of Wnt10b decreases adiposity and improves glucose homeostasis in obese rats. Am. J. Physiol. Endocrin. & Metabolism, 2007 Sep;293(3):E726-36.
- Kohlbrenner E, Aslanidi G, Nash K, Shklyaev S, Campbell-Thompson M, Byrne BJ, Snyder RO, Muzyczka N, Warrington KH, Jr.1, and Zolotukhin S. Successful Production of Pseudotyped rAAV Vectors Using a Modified Baculovirus Expression System. Molec Ther. 2005 Dec;12(6):1217-25.
- Prima V, Tennant M, Gorbatyuk OS, Muzyczka N, Scarpace PJ, Zolotukhin S. Differential modulation of energy balance by leptin, ciliary neurotrophic factor, and leukemia inhibitory factor gene delivery: microarray deoxyribonucleic acid-chip analysis of gene expression. Endocrinology. 2004 Apr;145(4):2035-45.
- Shklyaev S, Aslanidi G, Tennant M, Prima V, Kohlbrenner E, Kroutov V, Campbell-Thompson M, Crawford J, Shek EW, Scarpace PJ, Zolotukhin S. Sustained peripheral expression of transgene adiponectin offsets the development of diet-induced obesity in rats. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14217-22.