Date of Award

12-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Bioengineering

Committee Chair/Advisor

Dr. Angela Alexander-Bryant

Committee Member

Dr. Robert Latour

Committee Member

Dr. Jessica Larsen

Committee Member

Dr. Yanzhang Wei

Abstract

Ovarian cancer is the 7th leading cause of cancer related death and the 5th most commonly diagnosed cancer among women. Primarily diagnosed in stage III or stage IV, aggressive treatment is necessary and involves surgical debulking and administration of systemic chemotherapeutics. Unfortunately, these strategies fall short in effectively treating ovarian cancer and many patients experience local disease recurrence, development of multidrug resistant tumors, regional or distant metastatic events, or a combination of the three. As such, there is a significant need for additional treatment options and methods of delivery to improve therapeutic efficacy and disease survivability.

RNA interference (RNAi) is a type of gene therapy that can cause specific knockdown of genes that are responsible for cancer cell survival, migration, invasion, and a host of other oncogenic pathways. RNAi, induced by delivery of short interfering RNA (siRNA), can cause therapeutic effects by eliminating the ability of cancer cells to translate oncogenes into the proteins required for oncogenic functioning. However, significant barriers to delivery including instability in physiological conditions, inefficient cell uptake, and degradation via endosomal entrapment and lysosomal trafficking, severely reduce the efficacy of RNAi-based therapeutics. Without a robust delivery vehicle, siRNAs cannot avoid these barriers, will be rapidly degraded, and rendered completely ineffective.

Peptide-based delivery systems have shown promise as siRNA carriers and can deliver siRNA cargo intracellularly. Fusogenic peptides, cell penetrating peptides, and targeting peptides can all be used as systems to complex with and deliver siRNA cargo. This work developed a tandem peptide system, comprised of fusogenic and targeting peptide regions, to efficiently complex with siRNAs specific for the CSNK2A1 gene, guide delivery to ovarian cancer cells, and encourage endosomal breakdown and escape of siRNAs into the cytosol. The two peptide regions are specifically designed to implement active targeting of an overexpressed cell receptor in ovarian cancer, the luteinizing-hormone-releasing-hormone receptor, as well as cause endosomal membrane fusion and destabilization through pH-responsive conformation shifts in the fusogenic amino acid sequence. This action would result in release of CSNK2A1-targeted siRNAs and consequential knockdown of the CSNK2A1 gene, resulting in reduced cancer cell ability to proliferate, migrate, and recolonize after treatment.

With this work, we show the potential for this novel peptide biomaterial to be an effective carrier of siRNA cargo by effectively avoiding barriers to RNAi therapy and nanoparticle delivery systems. Development of the amphipathic fusogenic peptide and tandem targeting-fusogenic sequences were both explored in vitro with further support for the fusogenic peptide being examined using an in vivo xenograft mouse model. Exhibiting effective delivery of bioactive siRNAs, the fusogenic peptide successfully reduced expression of CSNK2A1 mRNA and CK2α protein, causing significantly reduced ovarian cancer cell migration, colonization, and tumor volume in a mouse model. The tandem peptide enhanced ovarian cancer cell uptake and specifically targeted the luteinizing-hormone-releasing-hormone receptor. Future work will include fusogenic applications in 3D tumor spheroids, targeting-fusogenic peptide linker variations, and examining the tandem peptide in vivo to assess active targeting in a clinically relevant tumor location.

Author ORCID Identifier

0000-0003-3468-7165

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