GC-05. Improving the efficiency of long targeted insertions into the human cell genome using the TALEN editing platform and non-viral delivery techniques for gene therapy of socially significant diseases

Summary

Gene therapy based on transplantation of genetically modified hematopoietic stem and progenitor cells (HSPCs) using engineered nucleases (ZFN, TALEN, CRISPR/Cas9) has a strong potential for the treatment of a number of human monogenic diseases. The basis of this treatment method is the insertion of a healthy copy of a DNA segment or an entire transgene into the locus of a double-strand break formed by a nuclease, delivered as a donor template in various forms. It is known that an important factor affecting the insertion efficiency is the structure and length of the donor DNA template introduced into the cell. Among various forms (single- or double-stranded DNA, circular or linear, viral or non-viral, etc.), linear double-stranded linear DNA is of interest for preclinical development of gene therapy cell products due to potential efficiency and robust production. The limiting factor in their use is toxicity to primary human cells, which depends on the structural parameters of the templates [Paludan et al., 2013; Kath et al., 2022]. The aim of this work is to optimize the structure of the donor template introduced into the cell in the form of a linear double-stranded DNA used for homologous repair of TALEN-mediated targeted DNA breaks in order to increase the efficiency of editing/inserting therapeutic transgenes in human cell lines, which are the closest models of HSPC.

Materials and methods

Synthesis of variants of donor linear DNA templates was performed by PCR from plasmid DNA encoding the GFP transgene and homology domains to the CCR5 locus at the site of the formation of a 400 bp long double-stranded break. Transfection of K562 cells was carried out at a concentration of 3.3*10⁶ cells/mL according to a previously developed two-stage electroporation protocol using a Gene Pulser Xcell System electroporator (Bio-Rad, USA). The mRNA concentration during transfection was 25 µg/mL. The concentration of donor templates was 20 μg/mL. The following modifications of donor templates were studied: the addition of a sequence that inhibits sensors of intracellular immune response to exogenous DNA or damage to genomic DNA (TLR9/AIM2/cGAS) – A151 to the sequence of the donor template, as well as stabilization of the ends of the donor template with phosphorothioate bonds. The efficiency of transgene insertion and cell viability were assessed by flow cytometry.

Results

A two-stage transfection protocol was developed, which provides up to 85% knockout efficiency of the target CCR5 gene with cell survival from 30.4 to 46.2%. The transfection efficiency of the donor template in the form of a double-stranded linear DNA molecule was 7.1-23.6%. According to flow cytometry data, the efficiency of transgene insertion by the sixth day after transfection was 6.9-13.2%, depending on the type of modification of the ends of the donor template, and was maximal for the donor template without modifications. In comparison with the previously obtained preliminary data and control samples in the current experiment using plasmid DNA as a donor template, the efficiency of transgene insertion was higher when using linear DNA templates – 2.6% vs. 13.2%.

Conclusion

The development of linear double-stranded DNA templates as donors for the repair of precised DNA breaks holds promise for the development of gene therapy cell products for the treatment of monogenic human diseases.

The authors are grateful for financial support from the Russian Foundation for Basic Research, grant No. 19-29-04025mk.

Keywords

Gene therapy, nonviral, gene editing, TALEN platform, CCR5 gene.