Advancing Therapeutic Gene Editing through Development of Novel Delivery Modalities and Gene Editing Strategies

Abstract

Since the landmark discovery of CRISPR-Cas9 system in 2012, the field of gene editing has rapidly expanded its toolkit. Advances such as base editors (BEs) and prime editors (PEs) now enable precise and permanent DNA modifications. These technologies hold promise not only for correcting a broad spectrum of genetic diseases, but also for innovative applications in common diseases, such as cancer and autoimmune disorders. However, realizing the therapeutic potential of gene editing technologies requires overcoming several key challenges. In this thesis, I describe the development of novel delivery modalities and gene editing strategies to advance the therapeutic applications of CRISPR-based precision gene editing technologies. In Chapter 1, I review the current state of gene editing technologies, highlighting CRISPR-based precision gene editing tools, existing delivery modalities with a focus on in vivo delivery, and key preclinical and ongoing clinical applications. In Chapter 2, I describe the development of an engineered virus-like particle (eVLP) system for the in vivo delivery of PE components. PE is a versatile gene editor capable of introducing any base substitution as well as small insertions and deletions, but its large size and complex guide RNA (gRNA) structure pose challenges for efficient delivery. eVLPs represent a promising delivery modality that combine the advantages of viral and non-viral delivery modalities. Through iterative rounds of PE protein and gRNA engineering, along with eVLP construct optimization, we generated PE-eVLPs with substantially improved cargo packaging, thereby enhancing potency. In this chapter, I describe the engineering campaigns that improved the PE-eVLP potency and demonstrate their applications in mouse models for efficient transient delivery of PE cargo in vivo. In Chapter 3, I describe the development of a gene editing strategy to target prion disease, a fatal and rapidly progressing neurodegenerative disease with no effective treatments. Reducing endogenous prion protein has been proposed as a promising therapeutic hypothesis to delay disease onset or progression. To this end, we developed a base editing approach to permanently install a premature stop codon in the gene encoding prion protein, thereby reducing endogenous prion protein levels. Through delivery of base editing components via adeno-associated virus (AAV), we achieved significant lifespan extension in a mouse model of prion disease. In this chapter, I describe the development of base editing strategy as a potential treatment for prion disease. In Chapter 4, I describe the progress in developing a base editing strategy to treat Hutchinson-Gilford Progeria Syndrome (hereafter referred to as Progeria). In 2021, our lab reported a base editing strategy delivered by a dual-AAV system that corrected the pathogenic mutation causing Progeria, achieving efficient editing in key tissues such as the heart and descending aorta, and extending lifespan in a mouse model of Progeria. To advance this strategy toward clinical translation, we developed a base editing system that can be delivered with a single AAV, which may improve both in vivo efficacy and manufacturability. In this chapter, I describe the development and characterization of this single-AAV strategy for Progeria. Finally, in Chapter 5, I conclude by discussing the future directions of each project and the broader outlook for therapeutic gene editing.