The Use of Supercharged Coiled-Coil Proteins in Combination with Lipid Carriers For Efficient SiRna Delivery

Abstract

Protein engineering techniques have been used to address a wide variety of therapeutic challenges and proteins are often used as the core functional components of functional engineered assemblies. When combined with other material types, such as liposomal carriers, higher order nucleic acid delivery vehicles can be created with a broad range of potential applications. Proteins have the benefit of being biocompatible, as well as modifiable at the amino acid sequence level to impart new and useful functionality. Proteins also utilize the process of biological self-assembly to predictably form higher order structures with themselves and other material types, a property which becomes particularly useful for the design of non-viral gene delivery vectors. Rational protein design principles can be used to optimize binding to nucleic acids such as siRNA, as well as the ability to overcome biological challenges such as endosomal escape. In this work, supercharged coiled-coil proteins are combined with lipid reagents to develop an siRNA delivery vector known as a lipoproteoplex (LPP). The development of the LPP is carried out via three specific aims: 1) optimization of the protein component of the LPP using rational protein design, 2) extensive characterization of the LPP to determine physical properties and behavior in cellular systems, and 3) optimization of the lipid component to improve the safety profile of the LPP. Work in Aim 1 characterizes a supercharged coiled-coil protein library to understand the relationship between protein secondary structure, siRNA binding capacity, and transfection activity when formulated with a cationic liposome. Results reveal that the alpha-helicity of each protein in the library drives its siRNA binding and transfection activity as part of the LPP, but only if the protein does not have a high propensity for self-aggregation. A protein variant known as N8 is found to be better at delivering siRNA than the rest of the protein library and is taken forward for further characterization. Aim 2 focuses on characterizing the size, charge, and morphology of the LPP. Functional data is also gathered by using a diabetic skin wound model in which the Keap1/Nrf2 anti-inflammatory pathway is targeted via delivery of siKeap1. This is done to understand how the LPP is taken into cells and which properties of the LPP contribute to efficient siRNA transfection and ultimately a desired therapeutic outcome. Results reveal that the LPP forms cationic nanoparticles with a spherical morphology, and the protein component is responsible increasing endosomal escape of the siRNA cargo into the cytoplasm following endocytosis. Functional data shows that delivery of siKeap1 using the LPP can improve wound healing times both in vitro and in vivo. Lastly, work in Aim 3 focuses on formulating the LPP with the N8 protein and an exosome as the lipid component to form a vehicle known as an E-LPP. The size and charge of the E-LPP is characterized as well as the ability of the N8 protein to facilitate siRNA loading into the E-LPP. The E-LPP forms cationic nanoparticles and the presence of N8 improves siRNA loading when sonication and passive incubation are used. The E-LPP is found to have comparable transfection activity to a LPP formulated with a cationic liposome, but the E-LPP is found to be much less toxic. Together, these studies highlight the development of the LPP platform and its ability to be functionalized with different protein sequences and lipid types. This work will have a clinical impact since it will contribute to the development of a modular non-viral siRNA delivery platform which can be adapted to address monogenic disorders. The first use case of this vehicle will be for the treatment of chronic skin wounds in diabetic patients that will provide physicians with a powerful alternative to current wound care strategies. Ultimately, the nucleic acid cargo of the LPP can be customized to treat a wide range of pathologies with a favorable safety profile and the potential for repeat dosing.