Engineering minimally immunogenic cargos and delivery modalities for gene therapy

Abstract

Since the discovery of CRISPR-Cas9 systems, gene therapies have revolutionized the field of molecular biology by introducing functional genes into cells to correct genetic defects or diseases. To date, several gene therapies are pending approval for use in the clinic and have shown promise in the treatment of a variety of genetic disorders including retinal dystrophy, hemophilia, lysosomal storage disorders and certain types of cancer. However, there are several challenges to using CRISPR-Cas9 in the clinic, including the efficiency and specificity of the gene editing process, the potential for off-target effects, and the immunogenicity of the CRISPR-Cas9 system. One of the main challenges of gene therapies is the immunogenicity of the (1) therapeutic vector and (2) cargo. Existing delivery systems trigger immune responses, rendering therapies ineffective and pose considerable risks to the patient population. Even the cargos, Cas nucleases, have been shown to generate humoral and cellular immunity in the general population. Thus, there is a need for minimally immunogenic cargos and delivery modalities to advance gene therapy to the clinic.

The goal of this thesis is to design and optimize minimally immunogenic (1) vehicles and (2) cargos for translational gene therapy delivery. (1) For the development of gene therapy delivery vectors, previous work has identified endogenous proteins that can form capsids and package nucleic acid. In this work, I focus on the PNMA or Paraneoplastic MA-containing protein family to engineer a delivery system that can form capsids, package nucleic acid, and deliver functional, minimally immunogenic cargo to target cells. (2) For the development of non-immunogenic gene therapy cargos, I engineer existing gene therapy cargos, such as SaCas9 and AsCas12a, to be minimally immunogenic while retaining native functionality.

This work overall highlights the promise of protein engineering to minimize immunogenicity of delivery systems and gene editing nucleases while optimizing for their functionality in vivo. I hope this work will be expanded and grow to serve as a foundation for personalized gene therapy medicine.