Lentiviral-transduced hematopoictic stem cell transplantation for cystinosis

 描述（由申请人提供）：胱氨酸病是一种代谢性遗传性疾病，其特征为胱氨酸在细胞内蓄积。受影响的个人通常表现为 近端肾小管病变，并最终进展为终末期肾衰竭。胱氨酸积累也导致多器官功能障碍。使用胱氨酸病的小鼠模型，Ctns-/-小鼠，我们表明移植表达Ctns的野生型Sca 1 + HSC导致组织胱氨酸含量的显著降低和肾脏的长期保存。在原始R 01-DK 090058授权的范围内，我们优化了方案以获得有效的HSC转导，并表明使用我们的慢病毒载体构建体pCCL-CTNS离体转导的HSC也能够降低所有组织中的胱氨酸含量并改善Ctns-/-小鼠的肾功能。在向美国食品药品监督管理局（FDA）提交了预研究者新药（IND）申请后，我们现在正在进行纳入IND所需的药理学/毒理学研究，用于自体基因修饰HSC移植治疗胱氨酸病的I期临床试验。虽然这项工作为胱氨酸病的治疗提供了新的希望，但我们建议研究这种策略未来临床应用的两个关键问题。在具体目标1中，我们将研究第二种CTNS亚型CTNSLKG是否会改善实际的基因治疗策略。胱氨酸蛋白酶-LKG存在于溶酶体和质膜中，但其功能尚不清楚。然而，已发现CTNSLKG在PTC和依赖于囊泡运输并与胱氨酸病临床特征（包括范可尼综合征）相关的其他细胞类型中高度表达。我们最近还发现，囊泡运输受损胱氨酸细胞。因此，我们的假设是，胱氨酸蛋白酶LKG参与了PTC顶膜转运蛋白的囊泡运输，通过干细胞治疗引入两种CTNS亚型将增强治疗效果，特别是肾功能。如果是这样，这些数据将作为修改随后提交给FDA的临床试验策略的基础， 也可能阐明胱氨酸病中范可尼综合征的发病机制，尽管进行了大量的科学研究，但这些年来范可尼综合征仍然是一个谜。在特定目标2中，我们将研究是否可以用自体HSC移植治疗57 kb缺失（胱氨酸病中最常见的突变）的纯合子患者。事实上，这种大的缺失也去除了编码景天庚酮激酶（SHPK）的相邻的碳水化合物激酶样（CARKL）基因。虽然该基因的缺失对疾病表型没有明显的影响，但最近已显示SHPK控制巨噬细胞分化。由于我们最近发现巨噬细胞在HSC移植后胱氨酸病的组织修复中起关键作用，因此验证CARKL基因的缺失不会影响我们的干细胞治疗策略至关重要。这项工作对于未来胱氨酸病的临床试验至关重要，但也将促进对巨噬细胞介导的组织修复以及CARKL在此过程中的作用的理解。

 DESCRIPTION (provided by applicant): Cystinosis is a metabolic hereditary disease characterized by intracellular accumulation of cystine. Affected individuals typically present with proximal tubulopathy before one year of age and eventually progress to end- stage renal failure. Cystine accumulation also leads to multi-organ dysfunction. Using the mouse model for cystinosis, the Ctns-/- mice, we showed that transplantation of wildtype Sca1+ HSC expressing Ctns resulted in dramatic reductions of tissue cystine content and long-term of kidney preservation. Within the scope of the original R01-DK090058 grant, we optimized a protocol to obtain efficient HSC transduction and showed that ex vivo transduced HSCs using our lentiviral vector construct, pCCL-CTNS, were also capable of decreasing cystine content in all tissues and improved kidney function in Ctns-/- mice. After submitting a pre-Investigator New Drug (IND) application to Food and Drug Administration (FDA), we are now conducting the pharmacology/toxicology studies required for inclusion in an IND for a phase I clinical trial for autologous gene- modified-HSC transplantation for cystinosis. While this work offers new hope for the treatment of cystinosis, we are proposing to investigate two critical questions for the future clinical application of this strategy. In Specific Aim 1, we will investigate if the second CTNS isoform, CTNSLKG, would improve the actual gene therapy strategy. Cystinosin-LKG is found in the lysosomes and at the plasma membrane but its function is unknown. However, CTNSLKG has been found highly expressed in PTCs and other cell types that depend on vesicular trafficking and that correlate with cystinosis clinical features including the Fanconi syndrome. We also recently showed that vesicular trafficking was impaired in cystinosis cells. Thus, our hypothesis is that cystinosinLKG is involved in vesicular trafficking of transporters at the apical membrane of the PTCs and that introduction of the two CTNS isoforms via stem cell therapy would augment the therapeutic impact especially the kidney function. If so, these data will serve as the basis for modifying subsequent clinical trial strategies submitted to the FDA but may also elucidate the pathogenesis of the Fanconi syndrome in cystinosis that has remained a mystery all these years despite considerable scientific investigation. In Specific Aim 2, we will investigate if the patients homozygote for the 57-kb deletion, the most common mutation in cystinosis, can be treated with autologous HSC transplantation. Indeed, this large deletion also removes the adjacent Carbohydrate kinase-like (CARKL) gene encoding the sedoheptulokinase (SHPK). While the absence of this gene does not have an obvious consequence on disease phenotype, SHPK has been recently shown to control macrophage differentiation. Since we recently showed that macrophages play a key role in tissue repair in cystinosis after HSC transplantation, it is critical to verify that the absence of CARKL gene will not impact our stem cell treatment strategy. This work s critical for the future clinical trial for cystinosis but alsowill advance the understanding on macrophage-mediated tissue repair and the role of CARKL in this process.