Lentiviral-transduced hematopoictic stem cell transplantation for cystinosis

 描述(申请人提供)：胱氨酸病是一种代谢遗传性疾病，其特征是细胞内胱氨酸积聚。受影响的个人通常表现为 近端肾小管病变在一岁之前，最终发展为终末期肾功能衰竭。胱氨酸积聚也会导致多器官功能障碍。在CTNS-/-小鼠模型上，我们发现表达CTNS的野生型Sca1+HSC移植后，组织胱氨酸含量显著降低，肾脏保存时间延长。在最初的R01-DK090058拨款范围内，我们优化了一种获得高效HSC转导的方案，并表明使用我们的慢病毒载体构建的PCCL-CTNS体外转导的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。在向食品和药物管理局(FDA)提交了Pre-Investigator新药(IND)申请后，我们现在正在进行药理学/毒理学研究，以纳入IND用于自体基因修饰的造血干细胞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带来了新的希望，但我们建议为这一策略的未来临床应用研究两个关键问题。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。Cystinosin-LKG存在于溶酶体和质膜中，但其功能尚不清楚。然而，CTNSLKG已被发现在PTC和其他依赖于囊泡运输的细胞类型中高表达，并与包括Fanconi综合征在内的胱氨酸病的临床特征相关。我们最近还发现，在胱氨酸病细胞中，囊泡运输受到了损害。因此，我们的假设是，CystinosinLKG参与了PTCs顶膜转运蛋白的囊泡运输，通过干细胞治疗引入两种CTNS亚型将增加治疗效果，特别是对肾功能的影响。如果是这样的话，这些数据将作为修改提交给FDA的后续临床试验策略的基础，但 也可能阐明尽管进行了大量科学研究，但多年来一直是一个谜的胱氨酸病的范科尼综合征的发病机制。在特定的目标2中，我们将调查57-kb缺失纯合子的患者是否可以通过自体HSC移植来治疗。事实上，这种大的缺失也移除了邻近的碳水化合物激酶样蛋白(CARKL)基因，该基因编码了七碳七糖蛋白激酶(SHPK)。虽然该基因的缺失对疾病表型没有明显的影响，但SHPK最近被证明控制巨噬细胞的分化。由于我们最近发现巨噬细胞在HSC移植后的组织修复中起着关键作用，因此验证CARKL基因的缺失不会影响我们的干细胞治疗策略是至关重要的。S的这项工作对于未来的胱氨酸病的临床试验至关重要，但也将加深对巨噬细胞介导的组织修复以及CARKL在这一过程中作用的理解。

 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.