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

描述（由申请人提供）：蛋白质和核酸已经证明了作为研究工具和人类治疗药物的巨大价值。然而，由于大多数大分子无法自发进入细胞，外源蛋白和核酸在很大程度上仅限于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中的方法引起了生物医学界的极大兴趣，因为它们可能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赖氨酸或精氨酸，产生带超正电的蛋白质，或突变为Asp或Glu，产生带超负电的蛋白质。我们发现，一些由此产生的“增压”蛋白质可以保留其原有的折叠和功能，但实际上不受聚集的影响。值得注意的是，我们生成的带超正电的蛋白质，包括一种理论净电荷为+36的绿色荧光蛋白变体，非常有效地进入多种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预先混合时，这些增压的gfp能够非常有效地将核酸传递到所有测试的五种细胞系中。在五种细胞系中的四种中，也观察到基于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，增压蛋白也可以将感兴趣的蛋白质输送到哺乳动物细胞中，增压蛋白也可以在体内输送核酸和功能蛋白。这些发现表明，增压蛋白代表了一种有前途的新型大分子递送剂，值得进一步研究，作为干扰细胞的工具，并作为未来大分子治疗的潜在组成部分。在这里，我们建议研究增压蛋白的潜力，以解决大分子递送的关键问题。具体来说，我们寻求开发增压蛋白作为将蛋白质传递到哺乳动物细胞的有效工具，阐明和改进增压蛋白的分子特性，赋予其强大的大分子传递活动，包括从核内体逃逸的能力，发现自然存在的带超正电荷的人类蛋白，可能作为开发非免疫原性传递系统的有希望的起点。并开始第一次使用增压蛋白将功能蛋白或核酸，包括那些与治疗相关的，输送到活体动物体内的研究。

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.