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<p>Borane (BH<sub>3<sub>) chemistry offers unique chemical characteristics that enable
boranophosphate (BP) oligonucleotides with potential to enhance RNA therapeutic applications
such as RNA interference (RNAi) and RNA aptamers. Further, BP nucleotides are substrates
for RNA polymerases which allow the enzymatic synthesis of stereoregular boranophosphate
(BP)-RNA molecules of different lengths and properties. We expect that these BP-RNAs
will interact in a novel way with the desired target molecules because they can coordinate
with a diverse array of ligand sites in proteins or other RNA molecules. This is due
to the distinct hydrophobicity, sterospecificity, and polarity properties imparted
by the phosphorus-boron (P-B) chemical bond compared to the natural phosphorus-oxygen
(P-O) bond. </p><p>The object of this dissertation is to explore the therapeutic
applications of the BP-RNA such as siRNA, RNA aptamers, and in addition investigate
the immunogenicity of this modification. We used mouse cells to determine if BP-RNA
would activate toll-like receptor (TLR 7), which is involved in innate immune response
to foreign single stranded RNA (ssRNA). This response is undesired when applied to
oligonucleotide therapeutics such as siRNA and RNA aptamers. In terms of RNAi, it
would be an advantage to have low immunogenicity and high downregulation activity
by the siRNA. To determine the innate immune activation of the BP-RNA through the
TLR 7 we used a known activator, the human immunodeficiency virus (HIV) derived single-stranded
RNA (ssRNA40) and measured the production of cytokines as a function of the number
of modified BP-linkages. The production of cytokines IL-6 and TNFα was quantified
after the boranophosphate (BP), phosphorothioate (PS) or natural ssRNA40 were transfected
into murine macrophage Raw264.7 cells. Natural and phosphorothioate RNA (PS-RNA) have
been shown to be activators of TLR 7 receptors. In contrast, we found that fully modified
BP- ssRNA40 did not activate TLR 7. This is relevant in oligonucleotide applications
such as siRNA and RNA aptamers where off-target effects such as immune activation
after administration are not desired. </p><p>Subsequently, the low immune activation
would be an advantage when coupled to RNAi activity of the oligonucleotide. Thus,
we explored whether BP modified siRNA molecules would modulate gene expression and
if there was an effect on downregulation activity when increasing the number of BH3
modifications on the phosphate backbone. Our therapeutic model was the multi-drug
resistance 1 (MDR1) gene that expresses P-glycoprotein (P-gp), which has been notoriously
difficult to modulate. The aberrant regulation of genes such as MDR1 in cancer cells
are a major cause of chemotherapeutic treatment failure against human cancers. Hence,
controlling the expression of cancer genes with antisense technology is a possible
cancer therapy. Specifically, correcting the overexpression of p-glycoprotein using
modified siRNAs that target and degrade the P-glycoprotein mRNA produced by the MDR1
gene. We found that there is a reduction of siRNA activity with an increasing number
of BP-modifications. It appears that there is a fine balance between lack of immune
response and gene downregulation when applied to BP-siRNA. </p><p>Finally, we compared
the enrichment during the Systematic Evolution of Ligands by EXponential enrichment
(SELEX) method of two libraries, one BP-RNA (UαB) compared to a doubly-modified
RNA (2'FC & UαB), against a human thrombin. Aptamers modulate protein activity
and interfere with protein signaling by binding to the desired protein with high affinity
and specificity leading to their use in therapeutic applications where protein activity
needs to be controlled or it is anomalous. In the case of blood coagulation, thrombin
plays a central role in coagulation signaling cascade and it is a good target to use
to control blood coagulation in clinical settings. We attempted to optimize the selection
of BP- RNA aptamers through 4-8 rounds of SELEX against the protein thrombin. We
found that the selection conditions were not optimal for BP-RNA SELEX possibly due
to non-specific binding to a bovine serum albumin (BSA) in the selection buffer.</p>
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