Optimization of genetic modification of HSCs in the NHP model and creation of relevant preclinical models of human disease and therapies

我的研究小组在实验室和临床工作了32年，致力于开发针对造血干细胞和祖细胞(HSPC)的安全有效的基因疗法。在恒河猴模型中，我们专注于优化针对造血干细胞和造血祖细胞的慢病毒基因添加和基因编辑治疗，以及了解和提高现有和新基因治疗系统的安全性。 鉴于慢病毒载体随机整合的潜在遗传毒性，以及与靶向基因校正方法相比，半随机基因添加的其他缺点，我们利用恒河猴探索CRISPR/Cas9基因组编辑和最近的碱基编辑来创建疾病模型和开发针对HSPC的基因编辑疗法。我们已经优化了CRISPR/Cas9基因编辑和恒河猴CD34+HSPC的碱基编辑，最初通过CRISPR/CAS诱导的非同源末端连接修复敲除基因座，产生功能缺失的Indels，现在专注于提高HDR介导的基因校正和单突变导向的碱基编辑的安全性和有效性。我们已经成功地将经过基因编辑的细胞移植到22只动物身上，其血细胞的长期水平高达70%-90%，并具有靶向NHEJ INDELS。 我们重点研究了基因编辑对恒河猴模型中HSPC植入和长期功能的定量不利影响。结合使用定量条形码和基因编辑，我们已经证明，在NHEJ和HDR编辑的情况下，功能的HSPC数量显著减少，因此使用碱基编辑对HSPC的不利影响要小得多，这不会导致双链DNA断裂。 我们通过以CRISPR/Cas9介导的编辑来靶向DNMT3、TET2和ASXL1来产生功能突变的丢失，从而创建了一个强大的克隆造血猕猴模型。我们在三个动物中显示了TET2突变克隆的显著克隆性扩张，而DNMT2或ASXL1编辑克隆的克隆性扩张不那么明显，我们还记录了TET2突变髓系细胞的高度炎症表型，这与CHIP患者心血管疾病的风险增加有关。我们有多项正在进行的研究来研究这些动物中克隆扩张的生物学，并已表明tociluzumab的治疗逆转或减缓了由于TET2缺乏而导致的克隆扩张(Shin等人，布拉德，2022)。我们假设克隆造血伴随炎症表型可能与严重的新冠肺炎疾病相关，并使用我们的猕猴克隆造血模型对此进行了初步研究，比较了克隆造血中SARS-CoV-2感染与对照动物的结果，记录了组织中和肺部排泄中更高水平的病毒(Shin等人，2023年)。 我们还对老年动物队列中的恒河猴血细胞进行了大规模的靶向测序研究，使用深度纠错测序来寻找最初在老年人类中发现的56个克隆性造血基因。我们首次发现了克隆造血的自然动物模型，显示出与人类完全相同的基因突变，而啮齿动物模型中没有这种突变(Shin等人，布拉德，2022年)。我们将这些研究扩展到人类队列，分析新冠肺炎严重性和克隆性造血存在之间的关系，并且在迄今规模最大、最具决定性的研究中，没有显示出对新冠肺炎严重性的影响(周等人，血液，2022年)。 我们还开发了RUNX1缺乏症的基因编辑猕猴模型，以更好地了解遗传性骨髓衰竭/白血病易感综合征的生物学，并评估基因治疗在纠正表型方面的可行性，询问随着时间的推移，突变细胞和正常细胞是否在嵌合状态中占据主导地位。突变细胞占主导地位，这是对这种疾病的基因治疗的一个令人担忧的发现(Lee等人，血液，2023)。与小鼠模型相比，该模型还概括了人类RUNX1缺乏症的血小板和HSPC表型。 最近的一份报告将克隆性造血与阿尔茨海默病风险的意外降低联系在一起，并假设CH髓系细胞在进入大脑或在大脑中发挥功能以防止淀粉样蛋白或tau斑块积累的能力更强。我们利用我们的猕猴CH模型和对照条形码非CH动物，通过分析纯化的猕猴小胶质细胞CH突变与血液髓系细胞水平的比较，研究TET2或其他CH突变导致脑中小胶质细胞的更高替换率。我们没有发现HSPC来源的细胞在CH环境中对小胶质细胞替代的促进作用。机械论研究正在进行中。

My research group has worked for over 32 years in the laboratory and in the clinic to develop safe and effective gene therapies directed at hematopoietic stem and progenitor cells (HSPC). In the rhesus macaque model, shown to be the only predictive assay for human clinical results, we have focused on optimizing both lentiviral gene addition and gene editing therapies targeting hematopoietic stem and progenitor cells, and on understanding and enhancing the safety of established and new gene therapy systems. Given the potential for genotoxicity with random integration of lentiviral vectors, and other drawbacks of semi-random gene addition as compared to targeted gene correction approaches, we have utilized the rhesus macaque to explore CRISPR/Cas9 genome editing and more recently base editing to create disease models and to develop gene editing therapies targeting HSPC. We have optimized CRISPR/Cas9 gene editing and base editing of rhesus CD34+ HSPC, initially knocking out loci via CRISPR/CAs-induced non-homologous end joining repair, creating loss-of function indels, and now focusing on improving the safety and efficacy of HDR-mediated gene correction and of single mutation-directed base editing. We have successfully engrafted 22 animals with gene-edited cells, with long-term levels of up to 70-90% for blood cells with targeted NHEJ indels. We have focused on investigating the quantitative adverse impact of gene editing on the engraftment and long-term function of HSPCs in the macaque model. Using quantitative barcoding together with gene editing, we have demonstrated marked loss of functional HSPC numbers with both NHEJ but even more markedly HDR editing, and thus far less adverse impact on HSPCs with base editing, which does not result in double stranded DNA breaks. We have created a robust macaque model of clonal hematopoiesis by targeting DNMT3, TET2 and ASXL1 with CRISPR/Cas9 mediated editing to create loss of function mutations. We have shown marked clonal expansion of TET2 mutated clones in three animals, and less marked expansion of DNMT2 or ASXL1 edited clones, and we have documented a highly inflammatory phenotype for TET2 mutant myeloid cells, relevant to the increased risk of cardiovascular disease in CHIP patients. We have multiple ongoing studies to investigate the biology of clonal expansion in these animals, and have shown that treatment with tociluzumab reverses or slows clonal expansion due to TET2 deficiency in this model (Shin et al, Blood, 2022). We hypothesized that clonal hematopoiesis accompanied by an inflammatory phenotype could be associated with severe COVID-19 disease, and carried out pilot studies investigating this using our macaque clonal hematopoiesis model, comparing outcomes of SARS-CoV-2 infection in clonal hematopoiesis versus control animals, documenting higher levels of virus in tissue and shed in the lungs (Shin et al, 2023). We have also carried out a large scale targeted sequencing study of rhesus macaque blood cells from cohorts of aged animals, use deep error-corrected sequencing to look at 56 clonal hematopoiesis genes initially identified in aging humans. We have uncovered for the first time a natural animal model of clonal hematopoiesis, showing exactly the same genes mutated as in humans, in contrast to lack of such mutations in rodent models (Shin et al, Blood, 2022). We extended these studies to human cohorts in terms of analyzing the relationship between COVID-19 severity and the presence of clonal hematopoiesis, and in the largest and most definitive study to date, did not demonstrate an impact on COVID-19 severity (Zhou et al, Blood, 2022). We have also developed a gene editing macaque model for RUNX1 deficiency in order to better understand the biology of the inherited marrow failure/leukemia predisposition syndrome and to assess the feasibility of gene therapies in correcting the phenotype, asking whether mutant vs normal cells predominate over time in a chimeric state. Mutant cells predominate, a concerning finding for gene therapies of this condition (Lee et al, Blood, 2023). This model also recapitulates the platelet and HSPC phenotype of human RUNX1 deficiency, in contrast to murine models. A recent report linked clonal hematopoiesis to surprisingly a decreased risk of Alzheimer's disease, and postulated that CH myeloid cells were more potent in entering or functioning in the brain to prevent accumulation of amyloid or tau plaques. We have utilized our macaque CH model and control barcoded non-CH animals to investigate TET2 or other CH mutations results in higher replacement of microglial cells in the brain by analyzing purified macaque microglial cells for CH mutations compared to levels in blood myeloid cells. We have not found enhancement of microglial replacement by HSPC-derived cells in the setting of CH. Mechanistic studies are ongoing.