GC-09. Testing of small molecules for homology-directed repair stimulation at CCR5 locus during the transfection of primary human hematopoietic stem cells by CCR5-Uco-TALEN mRNA
Alyona I. Shakirova1, Kirill V. Lepik1, Albert A. Muslimov1, Vladislav S. Sergeev1, T. R. Karpov1, K. I. Anoshkin2, Marina O. Popova1, Boris Fehse1,3, Alexander D. Kulagin1
1 RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia
2 Research Center for Medical Genetics, Moscow, Russia
3 Research Department of Cell and Gene Therapy, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Dr. Alena Shakirova, phone: +7 (911) 733-51-48, e-mail: firstname.lastname@example.org
Targeted insertion of protein-coding sequences regions into the genome of hematopoietic stem cells (HSCs) mediated by engineered nucleases represents a promising platform for gene therapy of monogenic diseases. Homologous recombination (HR) represents the key mechanism for the introduction of genetic material into the double-stranded breaks (DSBs) generated by the targeted nuclease. Increasing the HR efficiency may enable the insertion of large DNA fragments into the HSC genome more efficiently upon delivery of donor repair templates by both viral and non-viral carriers. The mechanisms of DNA damage-induced innate immune response represent an important factor influencing the outcomes of DSB formation induced by engineered nucleases. The aim of this work is to evaluate the effects of adding small-molecule inhibitors of TLR9/AIM2/cGAS, STING and caspase antagonists A151, H151, and Z-VAD-FMK to cultures of primary human HSCs on the HR rate at the CCR5 locus after CCR5-Uco-TALEN mRNA transfection.
Materials and methods
Uridine-depleted CCR5-Uco-TALEN mRNA was synthesized by Trilink. Magnetic selection of CD34 + HSCs from the bone marrow of healthy donors was performed using the CD34 MicroBead kit (Miltenyi Biotec). After the activation phase of cultivation, HSCs were transfected with 25 µg/ml CCR5-Uco-TALEN mRNA using Gene Pulser Xcell (BioRad) device. After transfection, HSCs were cultured for 24 hours at 32°C in a StemMACS HSC Expansion Media XF (Miltenyi Biotech) medium. Small molecule inhibitors A151, H151 and FMK were added at concentrations 4 mkg/ml, 0.5 mkg/ml, and 25 mkg/ml respectively at 3 hours before the electroporation. The proportion of non-homologous end joining (NHEJ) events at the CCR5 locus was estimated by digital droplet PCR (ddPCR) on a QX200 System (Bio-Rad) according to the previously described protocol (Mock et al., 2015). In order to count the burden of HR-repaired CCR5 alleles the copy number of the reference gene EPOR was additionally estimated by the method of multiplex ddPCR (Schwarze et al., 2021). The difference between the copy number of the reference gene and the sum of HR-repaired and wild-type alleles was considered the proportion of alleles repaired by HR.
The total average efficiency of the CCR5 gene knockout, calculated as the sum of the NHEJ- and HR-repaired alleles, ranged from 9 to 53.5%. The addition of a FMK small molecule to the HSCs culture significantly affected not only the CCR5 gene knockout overall efficiency (53.5%), but the frequency of both NHEJ (27.2%) and HR (26.3%) as well. The addition of H151 did not affect the overall efficiency of the CCR5 gene knockout (33.5%), as well as the ratio of NHEJ (18.3%) and HR (15.2%) events in the samples. In the presence of A151 small molecule, the CCR5 knockout efficiency was significantly reduced (9%), and 95.6% of knockout alleles were the result of NHEJ events at DSB formed by CCR5-Uco-TALEN.
The small molecules contribution to the stimulation of HR-mediated DSB repair after the CCR5-Uco-TALEN mRNA transfection of primary hematopoietic stem cells was studied. Z-VAD-FMK seems to be the most promising.
K.V. Lepik thanks the Russian Foundation for Basic Research for the support, grant No. 19-29-04025mk.
TALEN, homology directed repair, STING inhibitors, HSC.