Persistence of CRISPR/Cas9 Gene Edited Hematopoietic Stem Cells Following Transplantation: A Systematic Review and Meta-Analysis of Preclinical Studies

Abstract

Gene editing blood-derived cells is an attractive approach to cure selected monogenic diseases but remains experimental. A systematic search of preclinical controlled studies is needed to determine the persistence of edited cells following reinfusion.All studies identified in our systematic search (to 20 October 2020) examining the use of CRISPR/Cas9 gene editing in blood-derived cells for transplantation were included. Meta-analysis was performed to determine the engraftment and persistence of gene edited cells. A total of 3538 preclinical studies were identified with 15 published articles meeting eligibility for meta-analysis. These in vivo animal studies examined editing of hemoglobin to correct sickle cell disease (eight studies), inducing resistance to acquired immunodeficiency syndrome (two studies), and six other monogenic disorders (single studies). CRISPR-Cas9 edited hematopoietic stem and progenitor cells demonstrated equivalent early engraftment compared to controls in meta-analysis but persistence of gene-edited cells was reduced at later time points and in secondary transplant recipients. Subgroup analysis in studies targeting the hemoglobin gene revealed a significant reduction in the persistence of geneedited cells whether homology-directed repair or nonhomologous end-joining were used. No adverse side effects were reported. Significant heterogeneity in study design and outcome reporting was observed and the potential for bias was identified in all studies. CRISPR-Cas9 gene edited cells engraft similarly to unedited hematopoietic cells. Persistence of gene edited cells, however, remains a challenge and improved methods of targeting hematopoietic stem cells are needed. Reducing heterogeneity and potential risk of bias will hasten the development of informative clinical trials.

Gene editing withoutex vivoculture evades genotoxicity in human hematopoietic stem cells

“…The frequency of long deletions or rearrangements (excluding the programmed 3.1 kb deletion or inversion) was similarly reduced in cells edited with sg1617+sg1450 without as compared to with pre-stimulation cytokine culture, with an even more profound reduction of long deletions or rearrangements, typically to frequencies below the limit of detection (Figures 4G-4H and S6I-S6J). Furthermore, we observed a reduced frequency of long deletions or rearrangements in engrafting cells in xenograft recipient bone marrow as compared to input cells following editing of sg1617+sg1450, sg1618+sg1450 and sg1618+sg1449 (Figure 4I), consistent with prior observations that gene edit distribution in engrafting HSCs may differ from that in CD34+ HSPC input cell products 28,53 . Finally we evaluated the impact of combined enhancer editing and time of pre-stimulation culture on the p53-dependent DNA damage response by measuring p21 mRNA level.…”

“…Therefore, we reasoned that editing nondividing HSPCs without ex vivo cytokine culture could achieve frequent NHEJ-mediated intended allelic outcomes, preserve HSC engraftment function, and bypass unintended genotoxicities associated with DSBs in dividing cells. Indeed gene edits like MMEJ and HDR that are depleted from immunophenotypic HSCs and G0 cells within CD34+ HSPCs are selectively reduced in engrafting cells, suggesting that nondividing HSCs possess unique DNA damage repair preferences as compared to rapidly dividing progenitors 20,28,30,53,[64][65][66] . We observed that mPB HSPCs are quiescent after CD34 selection and only enter G1 and then S phase after 24-48 hours of ex vivo cytokine culture.…”

CRISPR-Cas9 to induce fetal hemoglobin for the treatment of sickle cell disease

“…In some preclinical studies, INDEL frequency decreased at the early post-transplantation, 43 , 76 , 91 and low HbF induction and INDEL percentages were reported in some non-human primates despite having high editing rates in the infusion products. 43 To overcome possible limited HbF expression after edited cell infusion, an in vivo transduction and selection system was designed in which a helper-dependent adenovirus vector (HDAd5/35++) expressing a base editor targeted the BCL11A binding site (–113A>G) and co-expressed a P140K mutant of the human O -6-methylguanine-DNA methyltransferase ( MGMT ) gene.…”

“…Although the studies in mice indicated a significant reduction of the gene-edited cells in primary and in secondary transplants (reviewed by Maganti et al. 91 ), non-human primate studies 43 , 76 and clinical trial data demonstrated persistence of edited cells (>1 year). Of note, persistence of NHEJ repaired alleles and selective reduction of microhomology-mediated end joining (MMEJ) repaired alleles have been demonstrated in both mice 97 and rhesus macaques, 43 indicating that progenitor cells favor MMEJ repair and quiescent HSCs, a rare subpopulation within the HSPC product containing the long-term engrafting HSCs, and preferentially use NHEJ repair.…”

An improved medium formulation for efficient ex vivo gene editing, expansion and engraftment of hematopoietic stem and progenitor cells

“…There is evidence that reduction of the frequency of corrected cells in vivo is negligible early after transplantation, but becomes prominent at later stages, suggesting that the cause lies within the population of HSCs as an effect of either their limited correction, limited presence in the population infused, or their limited engraftment and self-renewing ability after manipulation. 19 Therefore, strategies to preserve HSCs during the ex vivo manufacturing process are required, to ensure their abundance in the infused cellular product and the maintenance of their long-term repopulating characteristics in vivo . Indeed, it is well known that prolonged cell culture (>48 h) and stimulation protocols, usually adopted for efficient gene transfer in HSPCs, can exert a detrimental effect on their engraftment and long-term repopulation capacity.…”