New Approaches for the Delivery of Proteins and Nucleic Acids Into Cells

描述（由申请人提供）：蛋白质和核酸已被证明作为研究工具和人类治疗剂具有巨大价值。然而，由于大多数大分子不能自发进入细胞，外源蛋白和核酸在很大程度上限于与细胞外靶标相互作用，即使大多数具有潜在生物学和医学意义的靶标被认为是细胞内的。因此，将蛋白质和核酸递送到哺乳动物细胞中的方法引起了生物医学界的极大兴趣，因为它们可以解决使用蛋白质和核酸作为细胞内探针和治疗剂所面临的主要突出挑战。 作为研究蛋白质聚集问题的努力的一部分，我们最近将几种不同蛋白质的几乎所有非保守表面暴露残基突变为Lys或Arg，产生带超正电荷的蛋白质，或突变为Asp或Glu，产生带超负电荷的蛋白质。我们发现，一些由此产生的“增压”蛋白质可以保留其天然的折叠和功能，但几乎不受聚集的影响。值得注意的是，我们产生的带超正电荷的蛋白质，包括一种理论净电荷为+36绿色荧光蛋白的变体，非常有效地进入不同类型的哺乳动物细胞，包括几种对传统转染方法有抗性的细胞系。当与siRNA或质粒DNA预混合时，这些增压的GFP能够非常有效地将核酸递送到测试的所有五种细胞系中。在五个细胞系中的四个中，还观察到基于siRNA的基因沉默或基于质粒的基因表达。我们最近的初步结果表明，超荷蛋白质也可以将感兴趣的蛋白质递送到哺乳动物细胞中，并且超荷蛋白质也可以在体内递送核酸和功能蛋白质。这些研究结果表明，超荷蛋白代表了一类有前途的新的大分子递送剂，值得进一步研究，作为工具扰动细胞和作为未来的大分子治疗的潜在组成部分。 在这里，我们建议研究超荷蛋白质的潜力，以解决大分子递送中的关键问题。具体而言，我们寻求开发超电荷蛋白作为将蛋白递送到哺乳动物细胞中的有效工具，以阐明和改善赋予其有效的大分子递送活性（包括从内体逃逸的能力）的超电荷蛋白的分子性质，以发现天然存在的超正电荷人蛋白，其可作为开发非免疫原性递送系统的有希望的起点，并开始使用超电荷蛋白质将功能性蛋白质或核酸（包括与治疗相关的那些）递送到活动物体内的第一项研究。 公共卫生相关性：将蛋白质和核酸递送到哺乳动物细胞中的方法受到生物医学界的极大关注，因为它们被认为解决了使用蛋白质和核酸作为细胞内探针和治疗剂所面临的主要突出挑战。我们建议研究和开发我们最近发现的一类新蛋白质的能力，“超荷蛋白质”，用于在试管和活体动物中将外部应用的DNA，RNA和蛋白质递送到细胞中。由此产生的方法和见解可能代表了广泛使用蛋白质和核酸作为药物来解决细胞内缺陷和生物靶点的重要一步。

DESCRIPTION (provided by applicant): Proteins and nucleic acids have demonstrated great value as research tools and as human therapeutics. Due to the inability of most macromolecules to spontaneously enter cells, however, exogenous proteins and nucleic acids are largely restricted to interacting with extracellular targets even though most targets of potential biological and medical interest are thought to be intracellular. Methods to deliver proteins and nucleic acids into mammalian cells are therefore of considerable interest to the biomedical community because they may address the major outstanding challenge facing the use of proteins and nucleic acids as intracellular probes and therapeutic agents. As part of an effort to study the problem of protein aggregation, we recently mutated virtually all non-conserved surface exposed residues of several different proteins to Lys or Arg, creating superpositively charged proteins, or to Asp or Glu, creating supernegatively charged proteins. We discovered that some of the resulting "supercharged" proteins can retain their native folding and function, but are virtually immune to aggregation. Remarkably, the superpositively charged proteins we generated, including a variant of green fluorescent protein with a net theoretical charge of +36, very potently enter diverse types of mammalian cells, including several cell lines resistant to traditional transfection methods. When pre-mixed with siRNA or plasmid DNA, these supercharged GFPs were able to deliver nucleic acids very efficiently into all five cell lines tested. In four of the five cell lines, siRNA-based gene silencing or plasmid-based gene expression was also observed. Our very recent preliminary results suggest that supercharged proteins can also deliver proteins of interest into mammalian cells, and that supercharged proteins can also deliver nucleic acids and functional proteins in vivo. These findings suggest that supercharged proteins represent a promising new class of macromolecule delivery agents that merit further study as tools for perturbing cells and as a potential component of future macromolecular therapeutics. Here we propose to study the potential of supercharged proteins to address key problems in macromolecule delivery. Specifically, we seek to develop supercharged proteins as effective tools for delivering proteins into mammalian cells, to elucidate and improve the molecular properties of supercharged proteins that confer their potent macromolecule delivery activities including the ability to escape from endosomes, to discover naturally occurring superpositively charged human proteins that may serve as promising starting points for the development of non-immunogenic delivery systems, and to begin the first studies using supercharged proteins to deliver functional proteins or nucleic acids, including those that are therapeutically relevant, into live animals. PUBLIC HEALTH RELEVANCE: Methods to deliver proteins and nucleic acids into mammalian cells are of considerable interest to the biomedical community because they are thought to address the major outstanding challenge facing the use of proteins and nucleic acids as intracellular probes and therapeutic agents. We propose to study and develop the ability of a new class of proteins that we recently discovered, "supercharged proteins", to be used to deliver into cells externally applied DNA, RNA, and proteins both in the test tube and in living animals. The resulting methods and insights may represent a significant step towards the widespread use of proteins and nucleic acids as drugs to address deficiencies and biological targets inside cells.