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

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.

[1]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[2]  M Yarus,et al.  Diversity of oligonucleotide functions. , 1995, Annual review of biochemistry.

[3]  B. Sullenger,et al.  Developing aptamers into therapeutics. , 2000, The Journal of clinical investigation.

[4]  T. Ogihara,et al.  A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[5]  N. Dyson.  The regulation of E2F by pRB-family proteins. , 1998, Genes & development.

[6]  J. D. Thompson,et al.  Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter. , 1995, Nucleic acids research.

[7]  M. Mann,et al.  Therapeutic applications of transcription factor decoy oligonucleotides. , 2000, The Journal of clinical investigation.

[8]  T. Lee,et al.  Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication , 1990, Molecular and cellular biology.

[9]  J. Nevins,et al.  The Rb/E2F pathway and cancer. , 2001, Human molecular genetics.

[10]  C. Sherr.  Cancer Cell Cycles , 1996, Science.

[11]  T. Fitzwater,et al.  A SELEX primer. , 1996, Methods in enzymology.

[12]  M. Zuker.  On finding all suboptimal foldings of an RNA molecule. , 1989, Science.

[13]  Y. Kaneda,et al.  Fusigenic viral liposome for gene therapy in cardiovascular diseases. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J. Nevins,et al.  E2F3 activity is regulated during the cell cycle and is required for the induction of S phase. , 1998, Genes & development.

[15]  J. Nevins,et al.  Toward an understanding of the functional complexity of the E2F and retinoblastoma families. , 1998, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[16]  R. Pictet,et al.  The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. , 1997, RNA.

[17]  M. Famulok,et al.  Aptamers as tools in molecular biology and immunology. , 1999, Current topics in microbiology and immunology.

[18]  J. Polak,et al.  Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial , 1999, The Lancet.

[19]  D. Engelke,et al.  Expression of small, therapeutic RNAs in human cell nuclei , 1997, Gene Therapy.

[20]  A. Ellington,et al.  In vitro selection of aptamers: the dearth of pure reason. , 1996, Current opinion in structural biology.

[21]  Eli Gilboa,et al.  Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication , 1990, Cell.

[22]  N. Kashihara,et al.  Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo. , 1998, The Journal of clinical investigation.

[23]  J. Rossi,et al.  Ribozyme therapy for HIV infection. , 2000, Advanced drug delivery reviews.

[24]  J. Nevins,et al.  Inhibition of cell proliferation by an RNA ligand that selectively blocks E2F function , 1996, Nature Medicine.

[25]  T. Cech,et al.  Self‐splicing of the Tetrahymena intron from mRNA in mammalian cells , 1999, The EMBO journal.

[26]  N. Usman,et al.  Nuclease-resistant synthetic ribozymes: developing a new class of therapeutics. , 2000, The Journal of clinical investigation.

[27]  J. Trimarchi,et al.  E2f3 is critical for normal cellular proliferation. , 2000, Genes & development.

[28]  J. Nevins,et al.  E2F1 overexpression in quiescent fibroblasts leads to induction of cellular DNA synthesis and apoptosis , 1995, Journal of virology.

[29]  J. Nevins,et al.  Expression of transcription factor E2F1 induces quiescent cells to enter S phase , 1993, Nature.

[30]  J. Lis,et al.  RNA aptamers as effective protein antagonists in a multicellular organism. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Nevins,et al.  Functional properties of a Drosophila homolog of the E2F1 gene , 1994, Molecular and cellular biology.

[32]  Evaluating Group I Intron Catalytic Efficiency in Mammalian Cells , 1999, Molecular and Cellular Biology.

[33]  P. Romeo,et al.  Generation of long read-through transcripts in vivo and in vitro by deletion of 3' termination and processing sequences in the human tRNAimet gene. , 1984, Nucleic acids research.

[34]  G. Cassani.  New Ways of Making Drugs. RNA and Peptides by Selection and Molecular Design , 1993, Annals of the New York Academy of Sciences.