alexa Optimizing aptamer activity for gene therapy applications using expression cassette SELEX.
Engineering

Engineering

Journal of Biosensors & Bioelectronics

Author(s): Martell RE, Nevins JR, Sullenger BA

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Abstract RNA aptamers against a variety of clinically relevant target proteins have been generated. For example, we previously isolated an RNA aptamer that inhibits the function of the E2F family of transcription factors that play a critical role in the control of cell proliferation. However, the development of this and other aptamers for gene therapy applications has been complicated by the fact that expression of RNA aptamers in the context of flanking sequences can inhibit the ability of an aptamer to fold into its functional conformation. Insertion of the E2F aptamer into a tRNA expression cassette resulted in the production of high levels of chimeric tRNA that contains a misfolded and inactive aptamer in transfected mammalian cells. To overcome this problem, we randomized the sequence flanking the aptamer and selected for chimeric tRNAs that retained high affinity binding to E2F1. This expression cassette SELEX strategy yielded RNAs that bind E2F with high affinity (IC50 of 15 nM) and which can be expressed at high levels in mammalian cells. Moreover, these chimeric tRNA-E2F aptamers are functional and can inhibit E2F-mediated transactivation by up to 80\% in human 293 cells. Expression cassette SELEX should greatly facilitate the use of aptamers for a variety of gene therapy applications.
This article was published in Mol Ther and referenced in Journal of Biosensors & Bioelectronics

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