Degree

Doctor of Philosophy (PhD)

Department

Nutrition and Food Sciences

Document Type

Dissertation

Abstract

Abstract

Background:

As of 2018, the United States National Institutes of Health estimate that over half a billion people worldwide are affected by autoimmune disorders. Though these conditions are prevalent, treatment options remain relatively poor, relying primarily on various forms of immunosuppression which carry potentially severe side effects and often lose effectiveness overtime. Given this, new forms of therapy are needed. We propose small-interfering RNA (siRNA) for hypervariable regions of the T-cell receptor β-chain gene (TCRb) as a highly targeted, novel means of therapy for the treatment of autoimmune disorders.

Objectives:

To develop methods to produce siRNA targeting hypervariable regions of the TCRb gene. Additionally, to test said siRNA for its effects on TCRb, CD3, and CD137 expression, as well as cell viability on an ex vivo model.

Methods:

Both human Jurkat and human CD8+ anti-HPV T-cells were purchased and their mRNA isolated. A novel method for the production of siRNA was then employed to create an siRNA “cocktail” for particular hypervariable regions within TCRb. Additionally, a conventional method was employed to create siRNA for single computationally determined regions of TCRb. Synthesized siRNA was subsequently coated with lipofectamine to facilitate transfection. The coated siRNAs were then applied to respective patient cells at 10 pmol for 24h. Gene knockdown was quantified by qPCR, while cell viability was quantified by MTS assay and trypan blue count.

Results:

While all siRNA species demonstrated a significant level of knockdown on TCRb, CD3, and CD137 (P < 0.05), the siRNA “cocktail” showed the highest level of TCRb knockdown (P = 0.0406). Moreover, siRNA developed in this manner had an extremely small effect on T-cells lacking affinity for different antigens (low off targeting). The effect of each treatment on cell viability was not statistically significant (For all comparisons, P > 0.2).

Conclusion:

These results demonstrate not only the high potency of the described siRNAs on gene knockdown, but the low levels of off-targeting they exhibit. Based on this, we believe that both the previously described siRNA cocktail as well as computationally created siRNAs could potentially be utilized as effective therapeutics in the treatment of autoimmunity.

Date

1-27-2020

Committee Chair

Losso, Jack

Available for download on Sunday, February 14, 2021

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