Date of Award

Spring 5-17-2014

Degree Type

Dissertation

Degree Name

PhD. Chemistry

Department

Chemistry

Advisor

David Sabatino, Ph.D.

Committee Member

Allan D. Blake, Ph.D.

Committee Member

John R. Sowa, Jr., Ph.D.

Committee Member

Nicholas H. Snow, Ph.D.

Keywords

Branch siRNA, Hyperbranch siRNA, HepG2 cancer cells, RNAi, siRNA transfection, GRP78 silencing, Branch point amidite, Apoptosis, Cancer targeted gene therapy

Abstract

The cancer epidemic continues to afflict millions of humans world-wide each year and despite a renewed hope with the development of new and improved forms of therapy, a cure for cancer remains an elusive goal. This is partly related to the rise of resilient forms of tumors that have evolved with resistance towards conventional chemotherapy and radiation treatments. Moreover, these non-specific therapeutic regimens are highly toxic, leading to severe immunosuppressive effects which poisons the body and compromises the road towards remission. In an effort to mitigate these limitations, cancer-targeting approaches are currently experiencing a renaissance in the translation of new medicines from pre-clinical to bedside use. Notably, gene therapy has recently gained widespread traction in cancer research in the advent of the first RNA interference (RNAi) application in humans. RNAi solicits the use of a double-stranded RNA substrate, aptly named short-interfering RNA (siRNA), which binds to and triggers the degradation of a targeted complementary mRNA strand within the catalytic site of the RNA-Induced Silencing Complex (RISC). In this manner, malignant mRNA expression is silenced, thereby inhibiting the translation of proteins that can lead to the production of pathological disorders such as cancer. In spite of their utility, several challenges still remain towards the development of a fruitful cancer-targeting gene therapy approach.

Here, a new class of siRNA motifs is presented to increase substrate efficacy in the RNAi application. Our biological target is a member of the heat shock family of chaperone proteins, the Glucose Regulated Protein of 78 kilodaltons (GRP78) which signals tumor initiation, proliferation and resistance towards chemotherapy. Moreover, GRP78 is overexpressed and cell surface localized on a wide range of resilient tumor types but not on healthy cells, making it a viable bio-marker for the development of the proposed cancer-targeting gene therapy approach.

Significantly, an efficient solid-phase synthesis method is described for the production of linear, V-shape, Y-branch and hyperbranch GRP78-silencing siRNAs. The novel V-shape, Y-branch and hyperbranch motifs were then studied by CD spectroscopy and thermal denaturation experiments. CD spectroscopy was used to characterize the requisite A-type double-stranded RNA helix for RNAi application; whereas thermal denaturation experiments were used to validate siRNA hybrid stabilities. With stable siRNA hybrids in hand, their biological activity was assessed in HepG2 hepatoblastoma cells, which constitutes a morbid form of pediatric liver cancer and a valid tumor model for studying our GRP78-targeting strategy. The GRP78 silencing activity of the putative branch and hyperbranch siRNAs is discussed and related to its underlying mechanisms for inducing apoptosis in cancer. Biological studies confirmed potent suppression of GRP78 expression (50-60%) while compromising cancer cell viability by ~20%. The development of an effective cancer-targeting gene therapy approach is highlighted by preliminary results that showcase the utility of a cancer-targeting peptide (CTP) to condense and deliver siRNA within cancer cells for therapeutic treatment. The latter forms the basis of our cancer-targeting gene therapy approach. Thus, branched and hyperbranched siRNAs may serve as potent siRNA candidates in cancer gene therapy applications.

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