Enzyme Delivery With Supercharged Proteins to Address Citrullinemia

描述(申请人提供)：在体外和体内将大分子输送到哺乳动物细胞中使新的研究领域成为可能，并为强大的新治疗方案提供了可能性。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。刘组最近报道了一种经过工程设计的超正电荷绿色荧光蛋白(+36GFP)，它能够在体外将核酸输送到各种哺乳动物细胞系中，并在体外和体内有效地输送蛋白质，而不会产生毒性。这些结果使我们确定了自然产生的人类超负荷蛋白(SCP)的一个子集，这些蛋白具有类似的效力但以前未知的细胞穿透和蛋白质输送特性。其中几种自然产生的人类超负荷蛋白，包括HBEGF、c-jun和N-dek，被证明在体外和成年小鼠体内都能将Cre重组酶功能转移到哺乳动物细胞中，这表明自然产生的人SCP可能是一类具有多种重要性质的生物递送剂，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SCPS将人精氨酸琥珀酸合成酶I(AS1)导入哺乳动物细胞模型和I型瓜氨酸血症小鼠模型。AS1催化依赖于ATP的瓜氨酸和天冬氨酸转化为精氨酸琥珀酸。AS1的有害突变是I型瓜氨酸血症的主要原因，I型瓜氨酸血症的特征是血清中极高的瓜氨酸和氨水平。瓜氨酸血症患者必须严格限制低蛋白饮食，通常需要肝移植。静脉注射编码AS1的腺病毒可以延长AS1缺陷小鼠的寿命，从~30小时延长到~16天。然而，腺病毒治疗一旦产生中和抗体就会失去疗效，而且还与其他危险因素有关。在这里，我建议系统地解决利用SCP将AS1直接导入肝细胞可以挽救细胞培养和体内的瓜氨酸血症细胞的代谢缺陷的可能性。

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.