Multiplex Engineered Human Lymphocytes for Therapeutic Protein Delivery

用于治疗性蛋白质递送的多重工程人类淋巴细胞

基本信息

批准号：
10285243
负责人：
Beau Richard Webber
金额：
$ 19.38万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-07 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10285243
关键词：
Address Allogenic Architecture Area Autologous Bulla CRISPR/Cas technology Cell Therapy Cell Transplantation Cell surface Cells Chronic Collagen Complementary DNA Deposition Dermal Disease Engineering Epidermolysis Bullosa Dystrophica Exhibits FDA approved Failure Fibroblasts GPR2 gene Gene Mutation Gene-Modified Genes Genetic Diseases Genome Genome engineering Goals Health Hematopoietic stem cells Hereditary Disease Histopathology Home Homing Human Human Engineering Immune Impairment In Vitro Inflammation Mediators Inflammatory Infusion procedures Inherited Injections Intervention Lymphocyte Mediating Membrane Methods Minnesota Modification Morbidity - disease rate Mucous Membrane Mus Mutation Nature Organ Palliative Care Pathologic Pathology Patients Phenotype Population Production Property Proteins Rare Diseases Reagent Recombinants Refractory Research Resolution Safety Skin Squamous cell carcinoma Stem cell transplant System T-Lymphocyte T-Lymphocyte Subsets Testing Therapeutic Therapeutic Effect Tissues Transcription Initiation Site Treatment Efficacy Universities Virus Visceral Zoledronate base biomedical referral center cellular engineering comparative cytokine cytotoxic cytotoxicity design direct application engineered T cells gene function genetic referral center genome editing graft vs host disease hematopoietic repopulating cell immunodeficient mouse model improved in vivo innovation keratinocyte knockout gene mesenchymal stromal cell migration minimal risk mouse model new technology novel overexpression promoter public health relevance receptor residence stem cells tau Proteins therapeutic protein therapy development translational approach vector wound γδ T cells

Address Allogenic Architecture Area Autologous Bulla CRISPR/Cas technology Cell Therapy Cell Transplantation Cell surface Cells Chronic Collagen Complementary DNA Deposition Dermal Disease Engineering Epidermolysis Bullosa Dystrophica Exhibits FDA approved Failure Fibroblasts GPR2 gene Gene Mutation Gene-Modified Genes Genetic Diseases Genome Genome engineering Goals Health Hematopoietic stem cells Hereditary Disease Histopathology Home Homing Human Human Engineering Immune Impairment In Vitro Inflammation Mediators Inflammatory Infusion procedures Inherited Injections Intervention Lymphocyte Mediating Membrane Methods Minnesota Modification Morbidity - disease rate Mucous Membrane Mus Mutation Nature Organ Palliative Care Pathologic Pathology Patients Phenotype Population Production Property Proteins Rare Diseases Reagent Recombinants Refractory Research Resolution Safety Skin Squamous cell carcinoma Stem cell transplant System T-Lymphocyte T-Lymphocyte Subsets Testing Therapeutic Therapeutic Effect Tissues Transcription Initiation Site Treatment Efficacy Universities Virus Visceral Zoledronate base biomedical referral center cellular engineering comparative cytokine cytotoxic cytotoxicity design direct application engineered T cells gene function genetic referral center genome editing graft vs host disease hematopoietic repopulating cell immunodeficient mouse model improved in vivo innovation keratinocyte knockout gene mesenchymal stromal cell migration minimal risk mouse model new technology novel overexpression promoter public health relevance receptor residence stem cells tau Proteins therapeutic protein therapy development translational approach vector wound γδ T cells

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项目摘要

Abstract. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe autosomal recessive disease caused by collagen type VIIa (COL7A1) gene mutations. RDEB is characterized by absent/defective COL7A1 (C7) protein deposition causing severe blistering, mucosal tissue damage, and aggressive squamous cell carcinoma. The University of Minnesota serves as a leading treatment referral center for this inherited disorder. Palliative care is non-curative and cellular therapy options include autologous or allogeneic local and/or systemic infusion of keratinocytes, fibroblasts, mesenchymal stromal cells (MSC), or hematopoietic stem/progenitor cells (HSPC). None of these currently employed treatment options resolve the full pathological spectrum of RDEB. Active wound areas persist, and mucosal disease remains highly refractory to intervention contributing to significant morbidity. Keratinocytes and fibroblasts, the primary C7 producing cells, show limited migration and persistence following localized injection. MSC and HSPC have broad circulatory potential, however, they produce comparatively low levels of C7 and residence in the skin or mucosa is not well established. Thus, it is essential to develop more efficacious cellular therapies capable of accessing skin and mucocutaneous tissues. γδ T cells are abundant within skin and mucosa, and due to their MHC-unrestricted nature are compatible with allogeneic transfer, however they do not naturally produce C7. Our innovative approach will employ precision genome modification using CRISPR/Cas9 to engineer γδ T cells to produce high levels of endogenous C7. We hypothesize that the tissue migratory properties of γδ T cells—particularly to the skin and mucosa—as well as their demonstrated allo-compatibility, make them uniquely suited for therapeutic delivery of C7 protein. In Aim 1 we will define a genome engineering strategy to confer high C7 expression, enhanced skin homing, and reduced inflammatory/cytotoxic capacity to primary human γδ T cells. In Aim 2 we will evaluate the ability of engineered allogeneic γδ T cells to home to skin and mucosa, deposit C7, and ameliorate pathology in an immunodeficient mouse model of RDEB. Further, we will test the effect of zoledronate induced in vivo expansion of the engineered Vγ9Vδ2 T cell subset on therapeutic efficacy. Our approach is a highly novel and innovative allogeneic strategy designed to address key limitations of current cellular therapies for RDEB. The application of CRISPR/Cas9 in γδ T cells represents a novel engineering and protein delivery strategy with translational potential for RDEB, other inherited mucocutaneous disorders, and a multitude of diverse disorders treated by cell/stem cell transplant.

抽象的。隐性营养不良表皮溶解囊泡（RDEB）是一种严重的常染色体隐性疾病胶原型VIIA（COL7A1）基因突变。 RDEB的特征是没有/缺陷COL7A1（C7）蛋白沉积会导致严重的起泡，粘膜组织损伤和侵袭性的鳞状细胞癌。明尼苏达大学是该遗传疾病的领先治疗转诊中心。姑息治疗非曲线和细胞疗法的选择包括自体或同种异体局部和/或全身输注角质形成细胞，成纤维细胞，间充质基质细胞（MSC）或造血干/祖细胞（HSPC）。这些目前使用的治疗方案都无法解决RDEB的完整病理谱。积极的伤口区域持续存在，粘膜疾病仍然对干预措施高度难治性发病率。角质形成细胞和成纤维细胞（主要C7产生细胞）显示出有限的迁移和持久性局部注射后。 MSC和HSPC具有广泛的电路潜力，但是它们会产生 C7水平相对较低，并且在皮肤或粘膜中的住所尚未得到很好的确定。那是必不可少的为了开发能够进入皮肤和粘膜组织的更有效的细胞疗法。 γδT细胞在皮肤和粘膜中丰富，并且由于其MHC无限制性质与同种异体兼容转移，但是它们不会自然产生C7。我们的创新方法将采用精确基因组使用CRISPR/CAS9对γδT细胞进行修改以产生高水平的内源性C7。我们假设γδT细胞的组织迁移特性（尤其是皮肤和粘膜）以及它们表现出的同性兼容性，使其非常适合C7蛋白的治疗递送。在目标1中我们将定义一种基因组工程策略，以赋予高C7表达，增强皮肤归巢和减少原代人γδT细胞的炎症/细胞毒性能力。在AIM 2中，我们将评估工程的能力同种异体γδT细胞可在免疫缺陷型中放在皮肤和粘膜，沉积C7和改善病理 RDEB的鼠标模型。此外，我们将测试工程化的唑来膦酸盐诱导的诱导的效果 vγ9VΔ2T细胞子集在热效率上。我们的方法是一种非常新颖而创新的同种异体策略旨在解决RDEB当前细胞疗法的关键局限性。 CRISPR/CAS9在 γδT细胞代表了一种新型的工程和蛋白质输送策略，具有RDEB的翻译潜力，其他遗传性粘膜状疾病和细胞/干细胞治疗的多种潜水员疾病移植。