Invader LNAs As Novel Gene Specific Therapeutics
Compounds that sequence-specifically bind target regions in chromosomal DNA in living cells hold tremendous potential as therapeutics, general research tools and diagnostic probes. DNA-targeting probes can modulate expression of genes involved in diseases, facilitate correction of detrimental mutations by targeted delivery of DNA-modifying agents or allow direct fluorescent labeling of chromosomal DNA. Progress in this area has been achieved with triplex forming oligonucleotides, minor groove binding polyamides, and peptide nucleic acids, but major methodological limitations have impeded development of drug candidates. Accordingly, there remains an extensive need for a DNA-targeting strategy which exhibits a) fast, high affinity binding, b) gene- specific targeting (> 15 bp) of chromosomal DNA without sequence restrictions at physiologically relevant ionic strengths, c) excellent biostability, d) uncomplicated DNA-like handling and e) straightforward design. We propose Invader Locked Nucleic Acids (LNAs) as a solution to this grand challenge. This strategy is based on our discovery that: a) oligonucleotides modified with intercalator-functionalized 2'-amino-¿-L-LNA monomers exhibit unsurpassed affinity toward DNA targets, b) duplex probes with 'monomer zippers' are exceedingly unstable, which c) generates a thermodynamic driving force for duplex probes to dissociate and bind double stranded DNA targets. The objective of this proposal is to maximize the DNA-targeting affinity of Invader LNA and to test the hypothesis that optimized Invader LNA can target chromosomal DNA and modulate gene expression in living cells. Specifically, we propose to a) optimize DNA-targeting of Invader LNA by chemical structural engineering of the monomer zippers (energetic hotspots) and the probe architecture, and b) demonstrate binding and modulation of gene expression by optimized Invader LNA in specific gene promoter regions using cultured cell models - these gene promoter regions will be studied in co-transfected plasmids and in the context of endogenous chromosomal DNA. The proposed experiments will clearly establish the feasibility of Invader LNAs as a paradigm changing DNA-targeting strategy. The successful development of Invader LNA as a reliable, biostable and easily handled probe technology will allow: a) strong, sequence- unrestricted and site-specific binding to accessible target regions of dsDNA under physiological conditions, b) modulation of gene expression, and/or c) targeted delivery of functional entities (DNA-modifying agents, donor DNA, chain cutters, optical labels or drugs) and will result in a marked acceleration in the development of drug candidates and tools for site-specific manipulation of DNA and have far-reaching implications in medicine, biology, and biotechnology. PUBLIC HEALTH RELEVANCE: The availability of oligonucleotide-based probes, which a) allow reliable and site-specific binding under physiological conditions to accessible target regions of any sequence in plasmid or chromosomal DNA and b) facilitate efficient modulation of gene expression, will herald a new era in the treatment of gene-based pathologies. Every disease or malady that is the result of mutation or dis-regulated gene expression may be treatable by carefully designed and targeted invader LNAs.