SBIR-STTR Award

Our understanding of complex biological phenomena and disease progression has led to the realization that changes in the expression of genes underlie many of these processes. Developing reagents that can selectively alter the expression level of any desired gene has been a goal of both scientists and clinicians for years. Historically, the most common approach was based on antisense oligonucleotides (ASOs) that encompass a broad variety of mechanisms that have in common an oligonucleotide designed to base pair with its complementary target mRNA leading to either degradation or impaired function of the mRNA. Classically, ASOs were designed to interfere with translation of the target mRNA or induce its degradation via RNase H or more recently by ribozyme activity. Current excitement has focused on RNAi that uses a distinct mechanism where oligonucleotides trigger an endogenous pre-existing gene suppression pathway that is fundamental to cellular gene regulatory networks. In spite of its general success, some mRNAs are only modestly downregulated (2-fold) by RNAi and others are refractory. Further, certain off-target effects can arise leading to unexpected consequences, underscoring the need for additional methods. The rapid rise of the RNAi field has led to an increased appreciation, of direct relevance to the present proposal, that regulatory sequence elements in mRNA 3' ends (eg. 3'UTRs) control the expression of that gene. Here we present preliminary data on the development of a new gene silencing technology that uses oligonucleotides annealing to specific sequence regions within the 3'UTR to inhibit pre-mRNA processing. We plan to improve this new technology by systematically analyzing modified bases to increase inhibitory activity. We will also determine how robust the technology is by testing its effectiveness in a variety of human cell types as well as cells from other vertebrates and by silencing several endogenous human genes as a proof-of-principle. Finally, we will also determine whether enhanced inhibition is seen when this new technology is used in conjunction with other gene silencing technologies such as RNAi where enhancement is expected because these different methods utilize fundamentally distinct mechanisms. We believe this new technology will make a significant addition to our gene silencing toolkit and may even aid emerging oligonucleotide-based therapies, although that is beyond the scope of this proposal.

Public Health Relevance:
The commercialization of this new U1 Adaptor mediated gene silencing technology will be a significant addition to the scientific research community's "gene silencing toolkit". Because this method exploits a distinctly different mechanism compared to more common gene silencing approaches, it has the potential of enhancing these traditional technologies when used in combination with them via synergistic effects. This may aid in the development of emerging oligonucleotide-based gene silencing therapies by improving sensitivity and efficacy.

Public Health Relevance:
This Public Health Relevance is not available.

Thesaurus Terms:
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Our understanding of complex biological phenomena and disease progression has led to the realization that changes in the expression of genes underlie many of these processes. Developing reagents that can selectively alter the expression level of any desired gene has been a goal of both scientists and clinicians for years. Historically, the most common approach was based on antisense oligonucleotides (ASOs) that encompass a broad variety of mechanisms that have in common an oligonucleotide designed to base pair with its complementary target mRNA leading to either degradation or impaired function of the mRNA. Classically, ASOs were designed to interfere with translation of the target mRNA or induce its degradation via RNase H. Currently, greater excitement has focused on RNAi which uses a distinct mechanism where oligonucleotides trigger an endogenous pre-existing gene suppression pathway that is fundamental to cellular gene regulatory networks. In spite of its general success, some mRNAs are only modestly downregulated (2-fold) by RNAi and others may be refractory. Further, certain off-target effects can arise leading to unexpected consequences, underscoring the need for additional methods. The rapid rise of the RNAi field has led to an increased appreciation, of direct relevance to the present proposal, that regulatory sequence elements in mRNA 3' ends (i.e., 3'UTRs) control the expression of that gene. Here we present a progress report from a Phase 1 SBIR on the development of a new gene silencing technology which we call [QOUTA]U1 Adaptors,[QOUTA] that uses oligonucleotides annealing to specific sequence regions within the 3'UTR to inhibit pre-mRNA processing. In this Phase 2 proposal, we plan to continue the optimization work begun in Phase 1 and expand to perform high throughput analysis of U1 Adaptors to establish site selection criteria and create an algorithm and design tool to assist with application of this technology to new gene targets with ease. We further propose to test the specificity of the method using whole genome microarray analysis. We intend to focus on use of this technology in genes which appear to be difficult to suppress using RNAi methods. Finally, we propose to study use of U1 Adaptors in complex genes having more than one polyadenylation site or alternative splicing involving the terminal exon. We believe this new technology will make a significant addition to our gene silencing toolkit and may even aid emerging oligonucleotide- based therapies, although that is beyond the scope of this proposal.

Public Health Relevance:
The commercialization of this new U1 Adaptor mediated gene silencing technology will be a significant addition to the scientific research community's [QOUTA]gene silencing toolkit[QOUTA]. Because this method exploits a distinctly different mechanism compared to more common gene silencing approaches, it has the potential of enhancing these traditional technologies when used in combination with them via additive effects. This may aid in the development of emerging oligonucleotide-based gene silencing therapies by improving sensitivity and efficacy.

Thesaurus Terms:
3' Untranslated Regions; 3'utr; Adoption; Affect; Algorithms; Alternate Splicing; Alternative Splicing; Anti-Sense Oligonucleotides; Antisense Agent; Antisense Oligonucleotides; Base Pairing; Biologic Phenomena; Biological Phenomena; Biology; Chemicals; Chemistry; Communities; Complex; Cultured Cells; Dna Chips; Dna Microarray; Dna Microarray Chip; Dna Microchips; Data Set; Dataset; Development; Disease Progression; Endoribonuclease H; Endoribonuclease H (Calf Thymus); Exons; Fingerprint; Gwas; Gene Action Regulation; Gene Expression; Gene Expression Inhibitor; Gene Expression Regulation; Gene Inactivation; Gene Regulation; Gene Regulation Process; Gene Silencing; Gene Targeting; Genes; Genes, Regulator; Genome; Goals; Human; Human, General; In Element; Indium; Intervening Sequences; Introns; Investigation; Length; Man (Taxonomy); Man, Modern; Measures; Mediating; Messenger Rna; Method Loinc Axis 6; Methodology; Methods; Methods And Techniques; Methods, Other; Microarray Analysis; Microarray-Based Analysis; Modification; Oligo; Oligonucleotides; Oligonucleotides, Antisense; Pathway Interactions; Pattern; Phase; Polyadenylation; Position; Positioning Attribute; Post-Transcriptional Gene Silencing; Post-Transcriptional Gene Silencings; Posttranscriptional Gene Silencing; Posttranscriptional Gene Silencings; Pre-Mrna; Precursor Rna; Process; Programs (Pt); Programs [publication Type]; Progress Reports; Publications; Publishing; Quelling; Rna Interference; Rna Polyadenylation; Rna Silencing; Rna Silencings; Rna Splicing, Alternative; Rna, Messenger; Rna, Messenger, Precursors; Rna, Small Interfering; Rnai; Rnase H; Reagent; Refractory; Regulator Genes; Research; Resistance; Ribonuclease H; Ribonuclease H, Calf Thymus; Sbir; Sbirs (R43/44); Science Of Chemistry; Scientific Publication; Scientist; Selection Criteria; Sequence-Specific Posttranscriptional Gene Silencing; Site; Small Business Innovation Research; Small Business Innovation Research Grant; Small Interfering Rna; Specificity; Tail; Targetings, Gene; Techniques; Technology; Testing; To Specify; Transcript; Transcriptional Regulatory Elements; Transfection; Translations; U1 Rna; U1 Small Nuclear Rna; U1 Snrna; Utrs; Untranslated Regions; Walking; Work; Base; Commercialization; Design; Designing; Experiment; Experimental Research; Experimental Study; Genome Wide Association Scan; Genome Wide Association Studies; Genome Wide Association Study; Genome-Wide; Genome-Wide Analysis; Genome-Wide Scan; Genomewide Association Scan; Genomewide Association Studies; Genomewide Association Study; Genomewide Scan; High Throughput Analysis; Improved; Knock-Down; Mrna; Mrna Precursor; Microarray Technology; New Technology; New Therapeutics; Next Generation Therapeutics; Novel; Novel Therapeutics; Pathway; Potency Testing; Premrna; Programs; Public Health Relevance; Regulatory Gene; Research Study; Resistant; Response; Sirna; Success; Tool; Trans Acting Element; Whole Genome Association Studies; Whole Genome Association Study