Zwitterlation improves efficacy and safety of therapeutic proteins

Abstract

During the past decades, engineered proteins have been widely used as biopharmaceutical drugs (e.g., recombinant therapeutic proteins, enzymes, and monoclonal antibodies) and gene editing tools (e.g., zinc fingers, TALENs and CRISPR/Cas9). This has significantly transformed the pharmaceutical industry. Compared with conventional small molecule drugs, these biomacromolecules offer the advantages of higher specificity and potency. However, safety and efficacy concerns have been raised from different aspects. In this thesis, the author discusses the tactics to improve efficacy and safety of protein therapeutics. First, to protect the protein from body clearance and immune system, a poly(carboxybetaine) (pCB) polymer is conjugated to the protein surface. Compared with PEG conjugation, the zwitterionic coating more effectively shields protein epitopes, rendering the whole protein invisible to the body immune system. This strategy is proved to be effective in increasing protein stability, prolonging in vivo circulation half-life and reducing immunogenicity, and is successfully applied to interferon-alpha (IFN-α). Compared with PEGylated IFN-α, Zwitterlated IFN-α largely retains its in vitro efficacy. In addition, the author further discusses the tactics to improve the safety and efficacy of nuclease for gene editing. To reduce the off-target effect of CRISPR/Cas9 system, the author discusses a novel method that conjugating CRISPR/Cas9 with zwitterionic pCB polymer to address the mismatch issue at the off-target sites. The author demonstrates the conjugation of CRISPR/Cas9 system with pCB polymer can both increase the efficacy of Cas9 nuclease and reduce non-specific interactions between gRNA and DNA (off-target DNA break). Finally, we converted the strategy to the mRNA version via a new strategy for reducing off-target effect of CRISPR/Cas9 systems that relies on fusing zwitterionic peptide poly(EK) extensions to the terminus of Cas9 via genetic engineering. The poly(EK) peptide is biodegradable and potentially safer for clinical applications. This technology was realized through mRNA delivery and will provide a robust and easily used strategy to reduce “off-target” mutations on a genome-wide scale and to improve the efficiency of a wide range of CRISPR/Cas9-based biological and clinical applications.