ISSN 1866-8836
Клеточная терапия и трансплантация

AW-01. Transplantation of human hematopoietic stem cells with CCR5 knock out by CCR5-Uco-TALEN nuclease to NBSGW mice: pilot study

Yaroslava V. Komarova1, Alena I. Shakirova1, Vladislav S. Sergeev1, Svetlana A. Osipova2, Ekaterina V. Shchelina2, Anatoliya V. Onopchenko2, Marina L. Vasutina2, Olga G. Bredneva2, Kirill S. Yakovlev3, Yana R. Orshanskaya3, Konstantin V. Sivak3, Timofey E. Karpov1, Albert R. Muslimov1, Marina O. Popova1, Kirill V. Lepik1, Alexander D. Kulagin1

1 RM Gorbacheva Research Institute, Pavlov University, St. Petersburg, Russia
2 VA Almazov National Medical Research Centre, St. Petersburg, Russia
3 AA Smorodintsev Research Institute of Influenza, St. Petersburg, Russia

Contact: Dr. Kirill V. Lepik, phone: +7 (911) 783-95-08, e-mail:

doi 10.18620/ctt-1866-8836-2022-11-3-1-132


The method of hematopoietic stem cells (HSCs) transplantation to immunodeficient mice is an important component of the pre-clinical development of medicinal cell products. The traditional in vivo model used to study human hematopoiesis is NSG mice line, but currently the NBSGW is gaining interest [Adigbli, 2021]. Unlike NSG, NBSGW mice do not require pre-transplant conditioning due to the presence of a mutation of c-Kit, the SCF receptor, providing an advantage for transplanted cells. In the current study, for the first time in Russia, NBSGW line was used to study the engraftment and biodistribution of modified human HSCs after in vitro editing of the CCR5 gene mediated by TALEN nuclease. Our objective was to assess the engraftment and biodistribution after transplantation of hHSCs with CCR5 knockout mediated by TALEN nuclease.

Materials and methods

The production of CD34+ HSCs with CCR5 gene knockout cell product prototype was performed according to described protocol [Shakirova Mol. Ther.2022 30;4S1]; prototype samples were cryopreserved. The procedure of transplantation, observation of animals and biomaterial harvesting was performed in the Almazov National Medical Research Centre animal facility. The study included six NBSGW mice (females) obtained from Jackson Laboratory (USA), the median age was 9(8-10) weeks. Pre-transplant conditioning was not performed. On day 0, three mice were transplanted with CD34+ cells at a dose of 1×106, two mice at a dose of 2×106, one control animal was mock-transplanted. Animals were kept in an SPF environment, under daily supervision, weekly veterinary examination and weight control. On day 56, mice were euthanized: samples of blood, bone marrow (BM) and spleen cells were used for donor chimerism assessment by immunophenotyping (FACS) (7AAD/hCD45). The proportion of CCR5 alleles knock out was analyzed by ddPCR [Mock et al., 2015]. A histological examination of tissue samples of BM, spleen, heart, liver, kidneys, intestines, muscles and skin was performed.


During the observation period, no lethal cases were registered. A weekly weight gain was recorded within the species norm. A chimerism level assessed by FACS was 3.8%-38.7% in BM with the maximum values in animal receiving higher HSC doses. In the blood and spleen, the level of chimerism reached 0.2%-3.8% and 0.5%-1.0%, respectively. The frequency of CCR5 gene knockout in spleen cells was 18.6% (13.4%-21.9%), in BM – 19.8%(5.6%-30.1%), in blood samples the alleles with knockout were not found. The histological structure of the skin and muscles, myocardium, intestine, liver, and kidney samples corresponded to the species norm. In the presented samples of the spleen of all animals, aplasia of the white pulp, extramedullary hematopoiesis were detected without significant differences. The histological structure of BM of the control animal was normal. Animals of the experimental group had foci of hematopoiesis formed by non-species-typical medium- and large-sized cells. Histological examination showed no signs of neoplasia.


In this pilot study, the method of transplantation of human HSCs to NBSGW mice was developed. In the absence of pre-transplant conditioning, the engraftment of human HSCs was demonstrated. The potential for multilinear recovery of hematopoiesis and biodistribution of HSCs after the CCR5 gene editing procedure was confirmed.


The authors are appreciated for financial support from Russian Foundation for Fundamental Studies, grant №19-29-04025мк.


Transplantation, hematopoietic stem cells, genome editing, TALEN, CCR5, model, NBSGW mice, NSG mice.

Volume 11, Number 3

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doi 10.18620/ctt-1866-8836-2022-11-3-1-132

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