ISSN 1866-8836
Клеточная терапия и трансплантация

BS-05. Efficacy of genetic modification of CCR5 locus by specific CCR5-Uco-hetTALEN nuclease in hematopoietic stem cell subfraction with potential for long-term recovery of stable hematopoiesis

Valeriia O. Laushkina, Vladislav S. Sergeev, Olga S. Epifanovskaya, Marina O. Popova, Alena I. Shakirova, Kirill V. Lepik, Alexander D. Kulagin

RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia

Contact: Valeriia O. Laushkina, e-mail:

doi 10.18620/ctt-1866-8836-2023-12-3-1-176


Gene therapy based on autologous transplantation of genetically modified human hematopoietic stem cells (HSCs) becomes an important therapeutic option for many diseases. Among HSCs, the most important fraction is rare self-renewing long-term repopulating cells (LT-HSCs). They represent 0.1-1% of the total bone marrow CD34 + HSC population and are characterized by CD34 +/CD90 + immunophenotype. These cells have the greatest potential for engraftment and lifelong hematopoietic recovery, including genetically modified cells. It makes them a rational target for HSC-based clinical gene therapy. Literature analysis showed that the efficiency of editing in LT-HSC does not exceed 50%. Since the study of the effectiveness of target knockout in the LT-HSC population is an important part of preclinical studies in the development of cellular gene therapy products based on HSC, the purpose of this study was to determine it for the CCR5 gene in LT-HSC using programmed CCR5-Uco-hetTALEN nuclease for gene therapy of HIV infection.

Materials and methods

Apheresis products with mobilized by granulocyte colony stimulating factor (G-CSF) HSC were obtained from three healthy donors. CD34+ cells selection was performed on LS positive selection columns (Milteniy Biotec) using CD34 MicroBead Kit reagents (Milteniy Biotec). The purity of cells selection and the persentage of LT-HSC were assessed by flow cytofluorimetry (FC) by phenotype CD34+/CD90+/CD45RA-/Draq7-. Before the transfection procedure, the cells were cultured in a standard medium: StemSpan™ (Stem Cell Technologies) supplemented with cytokines hTPO, hSCF, hFlt3-Ligand (Milteniy Biotec). The cells were electroporated with CCR5-Uco-hetTALEN at a total concentration of 25 μg/ml and incubated for 24 hours at 32ºC. HSCs were then cultivated in standard conditions for 5 days. After incubation, LT-HSCs were sorted from a part of cultured cells on a BD FACSAria™ III (BD Biosciences) cell sorter according to the phenotype written above. DNA was isolated using the Blood & Cells ExtractDNA Kit (Eurogen). CCR5 gene knockout efficacy was evaluated by digital drop PCR (ddPCR) using the protocol described previously (Mock et al., 2015; Schwarze et al., 2021).


According to the FC results, the purity of CD34+ cells after magnetic selection ranged from 80,4 to 93,9%. The percentage of cells with immunophenotype CD34+/CD90+/CD45RA- in samples after cell sorting was 97%. According to the data obtained from the ddPCR, the overall efficiency of CCR5 knockout in LT-HSC samples was 42.05±5.15% (from 37.73% to 47.75%). In the total CD34+ cell population, it was slightly higher – 51.46±7.32 (46.42% to 59.86%). Although the differences were not statistically significant (p=0.2).


The study shows that the knockout level of the target gene in the LT-HSC population is not statistically different from that in the total population of edited CD34+ cells when using the CCR5-Uco-hetTALEN nuclease, which is consistent with the literature. It is worth to note that towards to gene therapy for HIV infection, the LT-HSC population represents particular interest, since it can be an optimal source of transplant due to biological characteristics.


Hematopoietic stem cells, long term hematopoietic stem cells, genome editing, TALEN, CCR5.

Supplement 12-3

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doi 10.18620/ctt-1866-8836-2023-12-3-1-176

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