Construction and use of regulatable adenovectors expressing the Yamanaka genes (OSKM) for implementing regenerative medicine in the aging brain

CHIAVELLINI, PRISCILA; MARIANNE LEHMANN; MOREL GR; CANATELLI-MALLAT, MARTINA; GOYA RG

Toronto

Congreso; 13th Canadian Neuroscience meeting; 2019

IBRO

Biological rejuvenation by partial cell reprogramming is an emerging avenue of research. In this context, regulatable pluripotency gene expression systems are the most widely used at present. We constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein (GFP) and oct4, sox2, klf4 and c-myc (OSKM) genes. The OSKM genes are arranged as a bicistronic tandem (hSTEMCCA tandem) which is under the control of a Tet-Off bidirectional promoter that also controls the expression of the GFP gene. Separately, a constitutive cassette expresses the regulatory protein tTA. Vector DNA was transfected in HEK293 Cre cells, which were additionally infected with the helper adenovector H14, unable to package its DNA due to the Cre recombinase produced by the HEK293 Cre cells. The newly-generated vector was expanded by 5 iterated co-infections of the above cells and the adenovector purified by ultracentrifugation in a CsCl gradient. The titer of our preparation was 1.2 x 1012 physical viral particles/ml. As expected, GFP fluorescence in vector-transduced rat fibroblast cultures declined with the dose of doxycycline (DOX) present in the medium. Immunocytochemical analysis of transduced cells confirmed the expression of the 4 Yamanaka genes. Additionally, three days after vector injection in the hypothalamus of rats, a significant level of fluorescence was observed in the region. Addition of 2 mg/ml DOX to the drinking water reduced GFP expression. This adenovector constitutes a promising tool for implementing non-integrative partial cell reprogramming.