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Optimizing aptamer activity for gene therapy applications using expression cassette SELEX
Robert E Martell1, Joseph R Nevins, Bruce A Sullenger
1Geriatric Research and Education Clinical Center, Durham VA Medical Center, Durham, North Carolina 27710, USA.
Summary
Researchers developed a new method to express functional RNA aptamers for gene therapy. This technique ensures aptamers fold correctly, enabling effective inhibition of E2F transcription factors involved in cell proliferation.
Area of Science:
- Molecular Biology
- Gene Therapy
- Biochemistry
Background:
- RNA aptamers are valuable tools for targeting proteins, but their expression in gene therapy is hindered by folding issues.
- Previous attempts to express E2F aptamers in tRNA cassettes resulted in misfolded, inactive molecules in mammalian cells.
Purpose of the Study:
- To develop a novel expression strategy for functional RNA aptamers in gene therapy applications.
- To overcome conformational constraints that inhibit aptamer folding and function when expressed within cellular contexts.
Main Methods:
- A novel expression cassette SELEX (Systematic Evolution of Ligands by Exponential Enrichment) strategy was employed.
- Sequences flanking the E2F aptamer were randomized and selected for high-affinity binding to E2F1.
- Chimeric tRNA-E2F aptamers were generated and tested for expression, binding affinity, and functional activity in human 293 cells.
Main Results:
- The expression cassette SELEX strategy yielded RNA aptamers with high affinity for E2F (IC50 of 15 nM).
- These chimeric tRNA-E2F aptamers demonstrated high expression levels in mammalian cells.
- The functional aptamers inhibited E2F-mediated transactivation by up to 80%.
Conclusions:
- The developed expression cassette SELEX method effectively produces functional RNA aptamers for gene therapy.
- This approach facilitates the use of aptamers by ensuring proper folding and high expression levels.
- This strategy holds significant promise for advancing aptamer-based gene therapies targeting various diseases.