UC Davis

Despite the fact that ischemic cardiovascular diseases are the main cause of morbidity and mortality globally, therapies currently utilized have consistently fallen short of establishing perfusion in affected tissues to the level that healthy tissues experience. Therefore, the quality of life of those that experience cardiovascular disease face a decreased quality of life as the disease pathogenesis worsens over time. Therapeutic angiogenesis seeks to establish blood flow in affected tissues through the generation of new blood vessels. Through the use of biomaterial systems, angiogenesis activity can be localized to the region of interest through the controlled presentation of proangiogenic agents, proteins, and advanced biological systems such as gene therapies.

Hypoxia inducible factor 1 alpha (HIF-1α) is the proangiogenic master regulator that governs endogenous hypoxic responses of mammalian cells of all lineages – controlling the expression of hundreds of downstream genes involved in angiogenesis and tissue homeostasis. Targeting therapeutic activation at the HIF-1α level allows for the generation of factors at all levels of the angiogenesis process – from sprouting and neovessel formation to blood vessel maturation and long term stabilization. Within this thesis, the governing hypothesis that guides the following work is that the gene editing nuclease SpCas9 can be targeted to the HIF-1α regulator HIF prolyl-hydroxylase (HIF-PH) encoding region EGLN1 which, when edited, will lead to the long-term upregulation of HIF-1α for a lasting proangiogenic response. A virus like particle (VLP) system to mediate the delivery of SpCas9 ribonucleoprotein complexes (RNPs) to targeted cell populations was likewise characterized and investigated, and a macroscale alginate hydrogel delivery system to control the presentation of these VLPs and to maintain their activity was also investigated.

The initial work of this dissertation investigated the potential of in silico generated single-guide RNA (sgRNA) to introduce indel mutations localized to human EGLN1. Three candidate sgRNAs were screened for their genomic knockout (KO) capabilities and their ability to upregulate both HIF-1α and its downstream proangiogenic effectors in human cells. Even minor (3.4%) editing activity in the EGLN1 target region leads to dramatic increases in intracellular HIF-1α in edited cell populations in both normoxic and hypoxic oxygen tensions (7.8x and 18.2x, respectively). In addition, downstream proangiogenic effectors such as vascular endothelial growth factor (VEGF-A) secretion was significantly increased in response to editing under normoxic and hypoxic conditions (31.9x and 14.9x, respectively). Increases in tissue homeostatic factor VEGF-C and significant increases in several relative proangiogenic transcripts were also seen.

A VLP carrier derived from a lentivector (LV) gene therapy system was characterized on a biomolecular level and its capabilities of delivering functional SpCas9 RNPs to human cells investigated. Individual VLPs were determined to encapsulate between 369 and 1,610 SpCas9 RNPs and were demonstrated to deliver significant amounts of SpCas9 protein to human cells. Additionally, EGLN1 gene editing in cultures exposed to VLPs was along the same order of editing in cultures in which both SpCas9 and sgRNA were supplied in excess – demonstrating strong on-target editing potential. Additionally, data presented here suggests that transgene packing of VLPs may be different from that of LV systems – motivating further characterization of oligonucleotide packaging capacity of VLP structures specifically to better define their applicability in a broad variety of gene editing endeavors.

Towards the goal of identifying an alginate hydrogel biomaterial system to localize VLP editing activity in situ, controlled presentation of VLPs and analysis of their subsequent activity post-encapsulation was conducted in vitro. A Box-Behnken design of experiments model was used to generate candidate alginate hydrogel groups with varied input factors that would impact final hydrogel physical properties. Groups that presented mesh structures that would allow for large scale release of VLPs were selected and screened for nanoparticle, LV, and VLP release and activity assays. Further investigations regarding both crosslinking mechanisms and divalent crosslinker concentration were conducted to determine impact on internal hydrogel nanostructure formation. Release assays suggest that VLP physical structures are different from LV particles on which they were based, and activity assays determined that VLPs were able to effectively deliver RNP payloads after prolonged biomaterial encapsulation while LVs were unable to maintain their biological activity.

Taken together, the data presented in this thesis suggests that EGLN1 KO initiates a robust proangiogenic response, VLPs can effectively deliver functional SpCas9 RNPs, and that VLP activity is maintained even after prolonged periods of encapsulation.