The Design, Synthesis, and Evaluation of Zwitterionic and Cationic Lipids for In Vivo RNA Delivery and Non-Viral CRISPR/Cas Gene Editing
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The delivery of nucleic acids is an emerging therapeutic modality in clinical development for the treatment of many genetic diseases. The use of RNA interference (RNAi) as a therapeutic is an exciting and rapidly developing field that offers a promising alternative to small molecule drugs for the treatment of dysregulatory diseases, including cancer. Small interfering RNA (siRNA) can be designed against any mRNA target, and upon loading into the RNA-induced silencing complex (RISC) can enable sequence-specific target recognition and degradation. Meanwhile, messenger RNA is currently being utilized for protein replacement therapy and for the development of vaccines by expressing viral antigens on dendritic cells. However, because RNA molecules are unable to passively diffuse across plasma membranes due to a high molecular weight (~13 kDa for siRNA, >300 kDa for mRNA), hydrophilicity and strong anionic charge, while also being unstable and highly immunogenic when injected systemically, nucleic acid therapeutics require carriers for effective delivery. To date, many successful carriers have been designed using amphiphilic lipid-like compounds containing amine-rich cores, but the challenges of efficient endosomal release and delivery to organs outside of the liver remain major hurdles in the field of RNA therapeutics. This dissertation reports the design, synthesis and characterization of two new classes of lipids with unique chemical structures and in vivo RNA delivery capabilities to the lung: zwitterionic amino lipids (ZALs) and cationic sulfonamide amino lipids (CSALs). ZALs contain an amine rich core, hydrophobic tails introduced via conjugate addition or epoxide opening, and a zwitterionic sulfobetaine head group. ZALs were designed with a combination of cationic and zwitterionic lipid properties, to help stabilize and effectively deliver long RNA molecules. A lead compound, ZA3-Ep10, was effective for in vivo messenger RNA delivery and the first reported demonstration of in vivo non-viral gene editing by delivering mRNA components encoding the CRISPR/Cas gene editing platform. CSALs contain a unique chemical scaffold containing an internal quaternary ammonium group and a sulfonamide linker. A rational investigation of structure-activity relationships revealed that CSALs containing an acetate sidearm, a dimethyl amino head group and higher hydrophobic content were effective in delivery siRNA to human cancer cells in vitro. CSALs also demonstrated lung localization upon systemic delivery in vivo while also demonstrating the ability to redirect liver targeting ionizable lipid nanoparticles to the lung. These new classes of materials demonstrate the importance of structural consideration in material design for the development of nucleic acid therapeutics, while also providing structural templates for developing carriers for effective delivery to tissues outside of the liver.
Gene Transfer Techniques