Dmitriy Deviatkin

Objective: CRISPR/Cas9-based RNA-guided genome-editing tools are highly effective. However, off-target mutations and cellular toxicity limit their utility. The main goal of the study is to investigate whether Cas9 exhibits toxicity towards budding yeast Saccharomyces cerevisiae. Methods: Haploid and diploid S. cerevisiae strains were engineered to express either Cas9 or both Cas9 and a single-guide RNA (sgRNA). Growth curve analysis was used to evaluate the impact of Cas9 expression on yeast viability. Results and Discussion: Haploid and diploid strains expressing both Cas9 and sgRNA exhibit lower growth rates compared to strains that do not produce either Cas9 or sgRNA, presumably due to effective double-strand breaks induction. Toxic effect of Cas9 without sgRNA was observed as well. Cas9 toxicity appears more severe in haploid yeast while diploid strain is more tolerant to Cas9 overproduction. Toxicity of Cas9 may be caused by its non-specific binding to genomic DNA or excessive protein production. Conclusions: The Cas9 endonuclease is toxic in S. cerevisiae. This may compromice the efficacy of the CRISPR/Cas9 editing system. Therefore, mitigating Cas9 toxicity should be one of the strategies for improving genome editing outcomes.

Over the past decades, non-viral DNA and RNA delivery systems have been intensively studied as an alternative to viral vectors. Despite the most significant advantage over viruses, such as the lack of immunogenicity and cytotoxicity, the widespread use of non-viral carriers in clinical practice is still limited due to the insufficient efficacy associated with the difficulties of overcoming extracellular and intracellular barriers. Overcoming barriers by non-viral carriers is facilitated by their chemical structure, surface charge, as well as developed modifications. Currently, there are many different forms of non-viral carriers for various applications. This review aimed to summarize recent developments based on the essential requirements for non-viral carriers for gene therapy.

Suicide gene therapy was suggested as a possible strategy for the treatment of uterine fibroids (UFs), which are the most common benign tumors inwomen of reproductive age. For successful suicide gene therapy, DNAtherapeutics should be specifically delivered to UF cells. Peptide carriers are promising non-viral gene delivery systems that can be easily modified with ligands and other biomolecules to overcome DNA transfer barriers. Here we designed polycondensed peptide carriers modified with a cyclic RGD moiety for targeted DNA delivery to UF cells. Molecular weights of the resultant polymers were determined, and inclusion of the ligand was confirmed by MALDI-TOF. The physicochemical properties of the polyplexes, as well as cellular DNA transport, toxicity, and transfection efficiency were studied, and the specificity of αvβ3 integrin-expressing cell transfection was proved. The modification with the ligand resulted in a three-fold increase of transfection efficiency. Modeling of the suicide gene therapy by transferring the HSV-TK suicide gene to primary cells obtained from myomatous nodes of uterine leiomyoma patients was carried out. We observed up to a 2.3-fold decrease in proliferative activity after ganciclovir treatment of the transfected cells. Pro- and anti-apoptotic gene expression analysis confirmed our findings that the developed polyplexes stimulate UF cell death in a suicide-specific manner.