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

描述(由申请人提供)：蛋白质和核酸已显示出作为研究工具和人类疗法的巨大价值。然而，由于大多数大分子不能自发进入细胞，外源蛋白质和核酸在很大程度上被限制在与细胞外靶点相互作用，尽管大多数潜在的生物学和医学意义的靶点被认为是细胞内的。因此，将蛋白质和核酸输送到哺乳动物细胞的方法引起了生物医学界的极大兴趣，因为它们可以解决将蛋白质和核酸用作细胞内探针和治疗剂所面临的主要挑战。作为研究蛋白质聚集问题的一部分，我们最近将几种不同蛋白质的几乎所有非保守的表面暴露残基突变为Lys或Arg，产生超正电荷的蛋白质，或突变为Asp或Glu，产生超负电荷的蛋白质。我们发现，一些由此产生的“超负荷”蛋白质可以保持其天然的折叠和功能，但实际上是免疫聚集的。值得注意的是，我们产生的超正电荷蛋白质，包括一种净理论电荷为+36的绿色荧光蛋白变体，非常有效地进入各种类型的哺乳动物细胞，包括几个对传统转基因方法具有抵抗力的细胞系。当预先与siRNA或质粒DNA混合时，这些超负荷的GFP能够非常有效地将核酸输送到所有五个被测试的细胞系中。在五个细胞系中的四个中，也观察到了基于siRNA的基因沉默或基于质粒的基因表达。我们最新的初步结果表明，超负荷蛋白质也可以将感兴趣的蛋白质运送到哺乳动物细胞中，并且超负荷蛋白质也可以在体内运送核酸和功能蛋白质。这些发现表明，增压蛋白质代表了一类很有前途的新型高分子递送剂，值得进一步研究，作为干扰细胞的工具，并作为未来大分子疗法的潜在成分。在这里，我们建议研究超负荷蛋白质的潜力，以解决大分子递送中的关键问题。具体地说，我们寻求开发超高压蛋白质作为向哺乳动物细胞输送蛋白质的有效工具，阐明和改进超高压蛋白质的分子特性，这些超高压蛋白质具有强大的大分子输送活动，包括逃脱内小体的能力，发现自然存在的超正电人类蛋白质，可以作为开发非免疫原性输送系统的有希望的起点，并开始使用超高压蛋白质向活体动物输送功能蛋白质或核酸，包括那些具有治疗意义的蛋白质或核酸。 公共卫生相关性：将蛋白质和核酸输送到哺乳动物细胞的方法引起了生物医学界的极大兴趣，因为它们被认为解决了将蛋白质和核酸用作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的蛋白质的能力，这种蛋白质被用来将外部应用的DNA、RNA和蛋白质输送到细胞中，无论是在试管中还是在活的动物中。由此产生的方法和见解可能代表着朝着广泛使用蛋白质和核酸作为药物来解决细胞内的缺陷和生物靶标迈出了重要的一步。

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.