Multiplex Engineered Human Lymphocytes for Therapeutic Protein Delivery

抽象的。 隐性营养不良性大疱性表皮病（RDEB）是一种严重的常染色体隐性遗传病， VIIa型胶原蛋白（COL 7A 1）基因突变。RDEB的特征是COL 7A 1（C7）蛋白缺失/缺陷 沉积导致严重起泡、粘膜组织损伤和侵袭性鳞状细胞癌。的 明尼苏达大学是这种遗传性疾病的领先治疗转诊中心。姑息治疗 是非治愈性的，并且细胞治疗选择包括自体或同种异体局部和/或全身输注 在一些实施方案中，所述细胞可以是角质形成细胞、成纤维细胞、间充质基质细胞（MSC）或造血干/祖细胞（HSPC）。 这些目前采用的治疗方案都没有解决RDEB的全部病理谱。活性 伤口区域持续存在，并且粘膜疾病对干预仍然高度难治，从而导致显著的 发病率。角质形成细胞和成纤维细胞，主要的C7产生细胞，表现出有限的迁移和持久性 局部注射后。MSC和HSPC具有广泛的循环潜力，然而，它们产生 相对低水平的C7和在皮肤或粘膜中的驻留还没有很好地建立。因此， 以开发能够进入皮肤和粘膜皮肤组织的更有效的细胞疗法。γδ T细胞 在皮肤和粘膜中丰富，并且由于它们的MHC不受限制的性质，与同种异体的 然而，它们并不自然地产生C7。我们的创新方法将采用精确的基因组 使用CRISPR/Cas9进行修饰以工程化γδ T细胞以产生高水平的内源性C7。我们 假设γδ T细胞的组织迁移特性-特别是皮肤和粘膜-以及 它们所表现出的同种异体相容性使它们独特地适合于C7蛋白的治疗性递送。目标1 我们将定义一种基因组工程策略，以赋予C7高表达、增强皮肤归巢和减少 对原代人γδ T细胞的炎性/细胞毒性能力。在目标2中，我们将评估工程师的能力， 同种异体γδ T细胞归巢皮肤和粘膜，存款C7，并改善免疫缺陷 RDEB小鼠模型。此外，我们将测试唑来膦酸盐诱导的工程化细胞的体内扩增的效果。 Vγ 9VS 2 T细胞亚群对治疗功效的影响。我们的方法是一种非常新颖和创新的同种异体策略 旨在解决目前RDEB细胞疗法的关键局限性。CRISPR/Cas9在 γδ T细胞代表了一种新的工程和蛋白质递送策略，具有RDEB的翻译潜力， 其他遗传性粘膜皮肤疾病，以及通过细胞/干细胞治疗的多种疾病 移植

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.