Details

Autor(en) / Beteiligte

• Herrera Ortegon, Alberto

Titel

Evaluating SELEX Methods for Targeting T Cells

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2023

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• CD8+ T cells, also known as Cytotoxic T Lymphocytes (CTLs), are critical for killing virus-infected cells or cancer cells. T cells are particularly good at fighting pathogens and diseased cells early, but they become less effective over time. This is partly due to T cell exhaustion, where prolonged exposure to antigens decreases a cell’s ability to target and destroy foreign threats. To better fight chronic diseases such as cancer or HIV, therapeutics targeting T cells have become one of the fastest-growing therapeutic areas. Antibody-based therapeutics have dominated the market, but these therapeutics are challenging to develop and engineer, have high manufacturing and modification costs, risks of immunogenicity from repeated dosing of foreign proteins, and are among the most expensive therapies created to date. Small RNA-based therapies, including RNAi-directed strategies, have been developed which can address these issues. However, methods of delivering small RNA to T cells continue to be a challenge. Traditional liposome-based methods of siRNA delivery are largely ineffective at delivering RNA therapeutics into CTLs, viral delivery poses several risks due to issues of insertion into critical parts of the genome, and electroporation requires ex vivo administration.RNA aptamers propose a novel solution to the problem of T cell directed and cell specific delivery of existing RNA therapies. Aptamers, also known as chemical antibodies, are short DNA or RNA with high affinity and specificity towards a target ligand through unique 3D interactions. Synthetic oligonucleotide aptamers present a series of benefits over antibodies. They are faster to produce, easier to synthesize, capable of being chemically modified to make them stable in the serum, and have low immunogenicity.RNA Aptamers are selected via Systematic Evolution of Ligand Exponential enrichment (SELEX), an iterative process developed to select for highly specific and high binding aptamers from an initial random library pool. The goal of this thesis is to develop and evaluate SELEX methods to create aptamers targeting CD8+ T cells for the delivery of RNA therapeutics. Three methods of SELEX were tested based on artificial and native selection systems to develop CD8+ T cell targeting aptamers through selection for aptamers that bind the CD8 receptor directly. First, we used the method of Cell SELEX where the CD8 receptor is overexpressed on a foreign cell line allowing selection to be performed with the transformed cell line and counterselection to be performed with the parental cell line. Next, we used a Hybrid Protein SELEX method where a biologically relevant CD8αβ protein was conjugated to a bead system to emulate the presentation of the CD8 receptor on a cell surface. The protein was used for selection in combination with selection against purified CD8+ T cells to ensure recovered species bound to the receptor as expressed in the native cell line. The final approach tested was a method of PBMC SELEX where CD8+ T cells were used for selection directly. In this SELEX method, a competitive binding system was tested where aptamers were selected against human PBMC followed by isolation of CD8+ T cells and collection of aptamers bound to the isolated cells. This final approach was the only approach capable of developing aptamers that bound CD8+ T cells. The long-term goal of this dissertation is to use high-binding CD8+ T cell targeting aptamers to deliver small RNA therapeutics to increase the immune system’s ability to fight chronic infections. This thesis contributes to the field of RNA therapeutics by assessing the benefits and challenges of artificial and native cell systems for developing RNA aptamers to cell membrane-bound targets.