Enzyme Delivery With Supercharged Proteins to Address Citrullinemia

描述（由申请人提供）：在体外和体内将大分子递送到哺乳动物细胞中已经实现了新的研究领域，并提供了强大的新治疗选择的潜力。最近的研究已经产生了许多交付平台，但这些解决方案仍然受到范围，效力和安全性的限制。Liu Group最近报道了一种工程改造的带超级正电荷的绿色荧光蛋白（+36 GFP），其具有在体外将核酸递送到多种哺乳动物细胞系中以及在体外和体内有效递送蛋白而无毒性的能力。这些结果使我们确定了一个子集的天然存在的人类超荷电蛋白（SCP）具有类似的有效，但以前未知的细胞穿透和蛋白质递送特性。这些天然存在的人超荷电蛋白中的几种，包括HBEGF、c-Jun和N-DEK，显示出在体外和活的成年小鼠中将功能性Cre重组酶递送到哺乳动物细胞中，这表明天然存在的人SCP可能代表一类新的生物递送剂，其具有多种重要性质，例如电荷、结构、分子量、免疫原性、稳定性和体内半衰期。在此基础上，我建议应用SCP将人氨基琥珀酸合成酶I（AS 1）导入哺乳动物细胞模型和I型瓜氨酸血症小鼠模型。AS 1催化瓜氨酸和天冬氨酸向氨基琥珀酸的ATP依赖性转化。AS 1的有害突变是I型瓜氨酸血症的主要原因，其特征是瓜氨酸和氨的血清水平极高。瓜氨酸血症患者必须保持高度限制的低蛋白饮食，通常需要肝脏移植。静脉注射编码AS 1的腺病毒可延长AS 1缺陷小鼠幼仔的生命，从约30小时延长至约16天。然而，腺病毒治疗在产生中和抗体后失去疗效，并且还与其他风险因素相关。在这里，我建议系统地解决的可能性，直接交付的AS 1到肝细胞使用SCP可以挽救瓜氨酸血症细胞在细胞培养和体内的代谢缺陷。

DESCRIPTION (provided by applicant): The delivery of macromolecules into mammalian cells in vitro and in vivo has enabled new areas of research and offers the potential for powerful new treatment options. Recent research has generated many delivery platforms but these solutions remain limited by scope, potency, and safety. The Liu Group recently reported an engineered superpositively charged green fluorescent protein (+36 GFP) with the ability to deliver nucleic acids into a variety of mammalian cell lines in vitro and to potently deliver protein in vitro and n vivo without toxicity. These results led us to identify a subset of naturally occurring human supercharged proteins (SCPs) with similarly potent but previously unknown cell-penetrating and protein delivery properties. Several of these naturally occurring human supercharged proteins, including HBEGF, c-Jun, and N-DEK, were shown to deliver functional Cre recombinase into mammalian cells in vitro and in live adult mice, suggesting that naturally occurring human SCPs may represent a new class of biological delivery agents with a diversity of important properties such as charge, structure, molecular weight, immunogenicity, stability, and in vivo half-life. Building on this foundation, I propose to apply SCPs to deliver human argininosuccinate synthetase I (AS1) into mammalian cell models and mouse models of Type I citrullinemia. AS1 catalyzes the ATP- dependent conversion of citrulline and aspartate to argininosuccinic acid. Deleterious mutations to AS1 are the major cause of Type I citrullinemia, which is characterized by extremely high serum levels of citrulline and ammonia. Citrullinemia patients must be kept on a highly restricted low-protein diet and generally require a liver transplant. Intravenous injectio of adenovirus encoding AS1 extends life in AS1-deficient mouse pups from ~30 hours to ~16 days. Adenoviral treatment, however, loses efficacy upon development of neutralizing antibodies and is associated with other risk factors as well. Here I propose to systematically address the possibility that the direct delivery of AS1 into hepatocytes using SCPs can rescue the metabolic deficiency in citrullinemia cells both in cell culture and in vivo.