UC Irvine

Gene therapy involves delivering therapeutic nucleic acids into cells to treat disease through gene expression or silencing. Use of adeno-associated viruses (AAV) as a vector has been expanding in relevance in the field of gene therapy; with two FDA approved AAV drugs, it has proven to be safe, effective, and elicit only a partial immune response. Although AAV shows great promise in treating disease in immune privileged tissue, the prevalence of anti-AAV neutralizing antibodies (nAbs) limits AAV’s use for systemic delivery.

To avoid antibody neutralization, AAV can be encapsulated in a larger, nonviral carrier that can shield it from nAb recognition. A second nucleic acid can be co-encapsulated resulting in hybrid particles that simultaneously deliver two different genes. AAV was encapsulated in an acid-degradable shell that contains siRNA to form hybrid viral/nonviral nanoparticles. The nanoparticle shell breaks down in the acidic endosome, releasing AAV and siRNA to express one gene and silence another. The nanoparticle shell shields AAV from inhibition by nAbs and antibodies are not generated by nanoparticle delivery.

Bim is a pro-apoptosis gene commonly downregulated in cancer and Mcl-1 is a pro- survival gene that is commonly upregulated. Bim/Mcl-1 nanoparticles were formed to treat various cancers using AAV encoding Bim and siRNA targeting Mcl-1. Bim AAV and Mcl-1 siRNA were found to work synergistically when co-encapsulated in nanoparticles; targeting leukemia, lung cancer, melanoma, and breast cancer cells without affecting healthy cells. The combination of Bim/Mcl-1 nanoparticles and tyrosine kinase inhibitors (TKIs), a targeted chemotherapeutic, synergistically eradicated cancer cells. Bim/Mcl-1 nanoparticles, when delivered alone or delivered in combination with TKIs, significantly improved survival rate and delayed disease progression in a mouse leukemia model.

To fully study the range of treatment options using hybrid viral/nonviral AAV nanoparticles, alterations in the makeup of nanoparticles were studied for both targeting and size control. N- Acetylgalactosamine (GalNac) and hyaluronic acid (HA) were bound to the surface of nanoparticles to target them for liver and retinal delivery, respectively. Different formulations of nanoparticles using HA instead of RNA were created to minimize size and increase efficacy in certain disease pathologies that only require the expression of a single gene.

This dissertation provides a study on the formation of hybrid viral/nonviral AAV nanoparticles and their efficacy in cancer gene therapy, as well as the adaptation of nanoparticles for use in gene delivery applications targeting the liver and retina.