Multiplex Engineered Human Lymphocytes for Therapeutic Protein Delivery

抽象的。 隐性营养不良性大疱性表皮松解症(RDEB)是由 胶原蛋白VIIa(COL7A1)基因突变。RDEB的特征是缺失/缺陷的COL7A1(C7)蛋白 沉积导致严重水泡、粘膜组织损伤和侵袭性鳞癌。这个 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 非治愈性和细胞疗法的选择包括自体或异体局部和/或系统输注 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)。 目前采用的这些治疗方案都不能解决RDEB的全部病理范围。主动型 伤口区域持续存在，粘膜疾病仍然高度难于干预，对显著 发病率。角质形成细胞和成纤维细胞是产生C7的主要细胞，表现出有限的迁移和持久性 在局部注射后。MSC和HSPC具有广泛的循环潜力，但它们产生 相对较低的C7水平和在皮肤或粘膜中的滞留并不是很好的确立。因此，这是至关重要的。 开发能够接触皮肤和粘膜皮肤组织的更有效的细胞疗法。γδT细胞 在皮肤和粘膜中含量丰富，由于它们的MHC不受限制的性质与同种异体相容 转移，然而，它们不会自然产生C7。我们的创新方法将使用精确的基因组 使用CRISPR/CAS9改造γδT细胞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--特别是对皮肤和粘膜--以及 它们表现出的同种异体兼容性，使它们独特地适合于C7蛋白的治疗输送。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，并减少 对原代人γδT细胞的炎症/细胞毒能力。在目标2中，我们将评估工程设计的能力 同种异体γδT细胞进入皮肤和粘膜，沉积C7，改善免疫缺陷患者的病理 RDEB小鼠模型。进一步，我们将测试唑来膦酸盐诱导工程化细胞在体内扩张的效果。 Vγ9Vδ2T细胞亚群对疗效的影响。我们的方法是一种非常新颖和创新的同种异体移植策略 旨在解决目前RDEB细胞疗法的关键限制。CRISPR/CAS9在工程中的应用 γδT细胞代表了一种新的工程和蛋白质递送策略，具有翻译潜力， 其他遗传性黏膜皮肤病，以及多种由细胞/干细胞治疗的疾病 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.