Natno Biotechl: Hematopoietic stem cell gene editing strategy update! More efficient and more applicable.

!---- Hematopoietic Stem Cell (HSCs) Targeted Gene Editing is a promising and promising treatment for a wide range of diseasesIt can achieve in situ correction, functional restoration and physiological expression control of mutant allele by targeting genome editingProgrammable nucleases, such as CRISPR/Cas, are genetically edited by introducing site-specific DNA double-stranded fractures (DSBs) into the genomeDSB repair can be achieved through high-fidelity homologous directional repair (HDR), using an external DNA template for gene correction or targeted integrationHowever, the inefficiency of HDR in HSCs and the unknown effects of this process on cloning composition and transplant dynamics hinder clinical applicationThe team of San Raffaele Telethon Institute for Gene Therapy, a pioneer in gene therapy, Luigi Naldini, from Milan, Italy, and Pietro Geeenovese of Harvard Medical School, in collaboration with the Harvard Medical School," published in nature Biotechnology magazine, a research paper entitled GeneEdeed of the long human-hematopoie goals stem seroeed dydd ding The authors used a new strategy called BAR-Seq to apply to cloning tracking of editing cells, and the results showed that although implanted editing cloning retained the ability to multiseries and self-renewal, the new editing strategy activated p53 and greatly reduced the HSC clone library in blood cell chimeric miceThe composition of the multi-clone graft was restored by instantaneous suppression of p53Through the instantaneous expression of adenovirus Ad5-E4orf6/7 protein, the authors enabled the Ad5-E4orf6/7 protein to collect cell cycle control factor E2F on its target gene, promote the cell cycle process, increase the operation of hDR mechanism, and thus improve HDR efficiency and force it to progress to the S/G2 cell cycle stageThe combination of E4orf6/7 expression and p53 inhibition improves HDR editing efficiency by up to 50% without interfering with the reproliferation and self-renewal of edited HSCsThere are two obstacles to previous scenarios: the p53 response is too strong, and the other is that HDR is limitedThe author's new approach overcomes these two obstacles, and the cloning of edited hematopoietic stem/progenitor cells (HSPCs) proves multi-clone recombination, and retains the self-renewal ability and pluripotentity of individualed HSCs, increasing confidence in future clinical applicationsThe significant reduction in the number of HSPC clones is a good explanation for the lower implant rate of human implants compared to untreated cellsAlthough the strong activation of the p53 pathway and its downstream pathways such as p21, p14 and p16 indicates the occurrence of harmful processes such as permanent growth stagnation, aging and apoptosis, the mechanisms remain to be fully exploredAt present, the author's new method is limited to the dominant cloning in the edited cell graft, and it is not possible to study the static and short-lived progenitor cellsHDR occurs mainly in S/G2, and the authors' data also show that cell cycle regulation is the speed limit step of HDR editing in HSPCsAfter p53 inhibition, the increase in HDR caused by Ad5-E4orf6/7 is more pronounced, possibly because the negative feedback mediated p21 and p14 is triggered by offsetting E2F activation, which may also explain why Ad5-E4orf6/7 did not increase the implantation of standard editing cellsP53-/- cells come with selective advantages for the harmful effects of p53 activation, but these cells are rare Instant suppression of edit-induced p53 responses will reduce the risk of selecting p53 mutant cloning and single/oligarch amplification The oligoclone component may delay hematopoietic recovery and limit the size, long-term stability, and safety of engineered cell grafts The use of mRNA transient expression p53 inhibitors and Ad5-E4orf6/7 can eliminate the risk of genomic integration of potential transformation factors The study later unexpectedly revealed another benefit of Ad5-E4orf6/7 treatment, namely, the reduction of certain immune response/chemopation factor genes, which may help immune escape from the proviral virus This phenomenon may also reduce the risk of antigen presentation and recruitment of immune effectagents in HSPC, which still contains bacterial and viral-derived immunogens (such as Cas nuclease and AAV shell proteins) shortly after editing Overall, the increase in the clone library and the percentage of edited HSPC obtained by this new enhancement scheme is clinically beneficial The higher the proportion of HDR-edited cells in cell products, the less competition there will be with unedited and residual HSPc in the host, and the better the chimericness is achieved, resulting in better efficacy These benefits provide a good balance between the inherent risks of first human clinical trials (e.g primary immunodeficiency) Therefore, HSPC gene editing is likely to eventually lead to very effective clinical treatment