Gene Silencing with U1 Adaptor Oligonucleotides

DESCRIPTION (provided by applicant): 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 al
ter 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 RNas
e 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 developmen
t 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 a
lso 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. W
e 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 appr
oaches, 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 eff
icacy.