Employing novel humanized murine strains to develop hematopoietic stem cell gene therapeutic approaches for very early-onset inflammatory bowel disease due to IL10-receptor deficiency.

白介素10(IL10)或IL10受体(IL10R)功能丧失突变导致危及生命的 免疫缺陷表现为严重的极早发型炎症性肠病(VEO-IBD)。IL10是一种 由多种造血细胞分泌的免疫抑制细胞因子，主要是髓系细胞、B细胞和T细胞 细胞，减少炎症，至少部分是通过限制促炎细胞因子的分泌。这个 IL10受体由两条IL10R1和两条IL10R2链组成，主要表达于 造血细胞。缺乏IL10R或IL10的小鼠会发生自发性结肠炎，类似的，患者 如果IL10RA或IL10RB发生功能缺失突变，则会发展成严重的VEO-IBD。最近的研究表明 研究表明，造血干细胞移植(HSCT)可导致慢性粒细胞白血病的持续缓解 疾病，但异基因造血干细胞移植具有显著的急性毒性和移植物抗宿主病(GVHD)风险 并且需要合适的人类白细胞抗原相合的供者。我们课题组的初步数据表明，Il10rb基因 将IL10Rβ缺陷的骨髓前体细胞转入IL10Rβ基因敲除模型后进行造血干细胞移植 预防IBD的发展。我们建议通过慢病毒载体将基因转移到自体造血干细胞中。 (允许普遍存在或特定于谱系的IL10rb表达)来恢复IL10R信号并提供 另一种治疗方案。在本方案中，我们将开发和优化慢病毒载体来表达IL10rb， 在已建立的小鼠基因敲除模型中，确定这些载体在体外和体内的有效性和安全性。 此外，我们将利用最近开发的新的人性化小鼠模型，允许患者使用 CD34+造血干细胞在体内评估IL10R2缺乏对人细胞功能的影响 以及在患者来源的造血干细胞中进行基因转移的有效性。此外，我们计划生成CRISPR- 从IL10R1和IL10R2缺陷者来源的Cas9基因编辑的HSC及其频率评估 以及编辑事件和功能结果的精确度。这些研究将为临床前提供 澄清纠正免疫失调所需的转基因表达水平和安全性的数据 在造血细胞中表达信号分子。最后，我们将在两个标准IL10Rβ中确定- 缺陷小鼠和人源化小鼠模型，移植小鼠和患者来源的 IL10Rβ/IL10R2基因缺陷的HSCs与表达IL10Rb/IL10RB基因的慢病毒载体 需要白介素10Rβ表达以纠正免疫功能障碍的细胞群。这些研究将加强我们的 了解这种罕见疾病的基本细胞病理生物学，并解决其复杂性 与转基因在特定的造血成分中的异源表达有关 车厢。在基因治疗(Williams)和免疫学/IBD(Snapper)方面的深厚专业知识将形成优秀的 为这一适应症开发基因治疗方法奠定了基础。

Loss-of-function mutations in interleukin-10 (IL10) or the IL10 receptor (IL10R) lead to a life-threatening immune deficiency presenting as severe very early-onset inflammatory bowel disease (VEO-IBD). IL10 is an immunosuppressive cytokine that is secreted by a variety of hematopoietic cells, primarily myeloid, B and T cells, which reduces inflammation, at least in part by limiting the secretion of pro-inflammatory cytokines. The receptor for IL10 is composed of two IL10R1 and two IL10R2 chains and is expressed predominantly on hematopoietic cells. Mice deficient for either IL10R or IL10 develop spontaneous colitis and similarly, patients with loss-of-function mutations of either the IL10RA or IL10RB develop severe VEO-IBD. Recent studies have shown that hematopoietic stem cell (HSC) transplantation (HSCT) may lead to a sustained remission of the disease, but allogeneic HSCT carries significant risks of acute toxicities and graft-versus-host-disease (GVHD) and requires a suitable HLA-matched donor. Preliminary data from our group has indicated that Il10rb gene transfer into IL10Rβ-deficient bone marrow precursors followed by HSCT into IL10Rβ knockout models can prevent the development of IBD. We propose to use gene transfer into autologous HSCs via lentivirus vectors (permitting either ubiquitous or lineage-specific Il10rb expression) to restore IL10R signaling and provide an alternative treatment option. In this proposal, we will develop and optimize lentivirus vectors to express Il10rb, determine the in vitro and in vivo efficacy and safety of these vectors in established murine knockout models. In addition, we will utilize novel humanized mouse models recently developed that permits the use of patient derived CD34+ HSCs to assess both the functional consequence of IL10R2 deficiency in human cells in vivo as well as the effectiveness of gene transfer in patient-derived HSCs. Moreover, we plan to generate CRISPR- Cas9 gene-edited HSCs derived from IL10R1- and IL10R2-deficient human patients and assess the frequency and precision of editing events and functional consequences. These investigations will provide the preclinical data clarifying the transgenic expression levels needed for correcting the immune dysregulation and the safety of expressing a signaling molecule in hematopoietic cells. Finally, we will determine in both standard IL10Rβ- deficient murine as well as humanized mouse models, transplanted with murine- and patient-derived IL10Rβ/IL10R2-deficient HSCs with lineage-specific Il10rb/IL10RB-expressing lentivirus vectors, the precise cell populations that require IL10Rβ expression to correct immune dysfunction. These studies will enhance our understanding of the basic cellular pathobiology of this rare disease and also address the complexities associated with heterologous expression of transgene in specific components of the hematopoietic compartment. Deep expertise in gene therapy (Williams) and immunology/IBD (Snapper) will form an excellent basis to develop a gene therapy approach for this indication.