Generation and transduction optimization of a baculoviral vector encoding the antimitotic gene meis1 as a preliminary in vitro model to evaluate possible cell cycle regulators

LÓPEZ, AYELÉN EMILCE; HALEK, JULIA MARÍA; BELAICH, MARIANO NICOLÁS; SIMONIN, ALEJANDRO; CUNIBERTI, LUIS; LOCATELLI, PAOLA; BAUZÁ, MARÍA DEL ROSARIO; CROTTOGINI, ALBERTO; OLEA, FERNANDA DANIELA

Rosario

Congreso; Reunión anual de la Sociedad Argentina de Fisiología; 2019

Sociedad Argentina de Fisiología

Introduction: Ischemic heart disease, including acute myocardial infarction, is the main cause of death worldwide. Considering the poor results of clinical trials on cardiac regeneration, there is a growing interest in developing gene therapies aimed at inducing the adult cardiomyocyte mitosis, an approach that would warrant efficient electromechanical coupling of new cells to the myocardial sincitium. Among the strategies to deliver genes to the heart is the use of viral vectors. Genetically engineered baculovirus, an originally insect-infecting virus, has been shown to efficiently infect mammalian cells while conferring a good safety profile. Our aim was to design a set of baculoviral vectors to overexpress the cell cycle inhibitor meis1 and 2 shRNAs targeting this gene, and evaluate the effects on the proliferation rate in the rat myoblast cell line H9c2. Material and methods: Plasmids containing the genetic constructions were commercially obtained and employed to generate the baculoviral particles (Bv) through Bac-to-bac system: BacMeis1-GFP; BacshRNA A-mCherry and BacshRNA B-BFP. H9c2 cells were transduced at different multiplicities of infection (MOI) with a Bv encoding GFP (BacGFP).Transduction efficiency (TE) was measured by flow cytometry. Then the peak of expression of meis1 was assessed at 24, 36, 48 and 72 hours by RT-qPCR. The effect of meis1 overexpression on cell proliferation was evaluated by MTS assay at 5 days post-transduction (PT).Results: TE was 26,41 %, 49,72 % and 53,6 % for MOI 100, 300 and 900, respectively. The peak expression of meis1 was observed between 36 and 48 hours PT reaching a 6-fold increase vs. meis1 basal levels. MTS proliferation assay revealed a decrease in cell division rate at 5 days PT (area under the curve: 3±0.099 BacMeis1-GFP vs. 3.2±0.031 BacGFP, p