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.

采用新型人源化小鼠品系开发造血干细胞基因治疗方法，用于治疗因 IL10 受体缺陷所致的极早发炎症性肠病。

• 批准号: 9535567
• 负责人: Scott B Snapper
• 金额: $ 40.79万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-08 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9535567
• 关键词: Address; Allogenic; Anti-Inflammatory Agents; Anti-inflammatory; Autologous; B-Lymphocytes; Bone Marrow; CD34 gene; CRISPR/Cas technology; Cell Lineage; Cell physiology; Cells; Colitis; Data; Defect; Development; Differentiated Gene; Disease; Disease remission; Effectiveness; Engraftment; Event; Exhibits; Frequencies; Gene Transfer; Gene-Modified; Generations; Genes; Glean; Goals; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Homeostasis; Human; IL10RB gene; Immune; Immune System Diseases; Immune System and Related Disorders; Immunology; Immunophenotyping; Immunosuppressive Agents; In Vitro; Infant; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Interleukin-10; Intestines; Investigation; Knock-out; Lead; Lentivirus Vector; Life; Lymphoid Cell; Modality; Modeling; Mucositis; Mus; Mutation; Myelogenous; Myeloid Cells; Natural Immunity; Patients; Population; Prevention; Rare Diseases; Receptor Gene; Receptor Signaling; Regulatory T-Lymphocyte; Risk; Role; Safety; Signal Transduction; Signaling Molecule; Stem cells; Subfamily lentivirinae; T-Lymphocyte; Testing; Therapeutic; Transduction Gene; Transgenic Organisms; Transplantation; acute toxicity; alternative treatment; base; cell type; cellular transduction; congenital immunodeficiency; cytokine; design; disease phenotype; early onset; effective therapy; gene correction; gene therapy; graft vs host disease; humanized mouse; immune function; in vivo; infancy; innovation; interleukin-10 receptor; intestinal homeostasis; lentivirally transduced; loss of function mutation; macrophage; monocyte; mouse model; novel; pre-clinical; prevent; promoter; receptor; reconstitution; response; therapeutic gene; transgene expression; translational impact; vector

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.

白细胞介素-10（IL 10）或IL 10受体（IL 10 R）的功能缺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）。IL 10是一种 由多种造血细胞分泌免疫抑制细胞因子，主要是骨髓、B和T细胞 细胞，其至少部分地通过限制促炎细胞因子的分泌来减少炎症。的 IL 10受体由两条IL 10 R1和两条IL 10 R2链组成，主要在 造血细胞缺乏IL 10 R或IL 10的小鼠发展为自发性结肠炎，类似地，患者 具有IL 10 RA或IL 10 RB的功能丧失突变的患者发展为严重的VEO-IBD。最近的研究 表明造血干细胞（HSC）移植（HSCT）可能导致持续缓解的 但是同种异体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HLA匹配的供体。本研究组的初步数据表明，Il 10 rb基因 转移到IL 10 R β缺陷的骨髓前体中，然后进行HSCT到IL 10 R β敲除模型中， 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HSC中 （允许普遍存在的或谱系特异性的Il 10 rb表达）以恢复IL 10 R信号传导并提供免疫应答。 替代治疗方案。在本研究中，我们将开发和优化慢病毒载体来表达IL 10 rb， 确定这些载体在已建立的鼠基因敲除模型中的体外和体内功效和安全性。 此外，我们将利用最近开发的新的人源化小鼠模型，该模型允许使用患者模型。 衍生的CD 34 + HSC来评估体内人细胞中IL 10 R2缺陷的功能后果， 以及基因转移在患者来源的HSC中的有效性。此外，我们计划生成CRISPR- Cas9基因编辑的HSC来源于IL 10 R1和IL 10 R2缺陷的人类患者，并评估其频率 以及编辑事件和功能后果的精确性。这些研究将提供临床前 阐明校正免疫失调所需的转基因表达水平和安全性的数据 在造血细胞中表达一种信号分子。最后，我们将在两种标准IL 10 R β- 缺陷鼠以及人源化小鼠模型，移植鼠和患者来源的 IL 10 R β/IL 10 R2缺陷型HSC与谱系特异性IL 10 rb/IL 10 RB表达慢病毒载体，精确表达IL 10 R β/IL 10 R2缺陷型HSC。 需要IL 10 R β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学，并解决其复杂性。 与造血干细胞的特定成分中转基因的异源表达相关， 车厢在基因治疗（威廉姆斯）和免疫学/IBD（斯纳珀）方面的深厚专业知识将形成一个优秀的 为该适应症开发基因治疗方法奠定了基础。