Elise Malek-Adamian
Published: 2017
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"Synthetic short double-stranded small interfering RNAs (siRNAs) have the capacity to inhibit gene expression of a gene target; unfortunately, these RNAs display unfavorable metabolic stability and pharmacokinetic properties. In order to overcome these hurdles, it is possible to chemically alter the nucleotides that comprise the siRNA therapeutic to confer increased nuclease stability as well as alter their binding properties. It is also possible to use delivery vehicles, such as nanoparticles, to help improve their delivery into target cells. Therefore, in chapter 2 the creation of novel nucleoside analogues to first tackle the issue of metabolic stability will be reported; followed by chapter 3 which described the design of oligonucleotide-modified nanoparticles to increase cell permeation and allow for targeted delivery to DRR-expressing Glioblastoma multiformes cells. As such, we report on the synthesis of 2'-F,4'-OMe and 2',4'-diOMe ribo-uridine derivatives, their incorporation into siRNA duplexes, and the physicochemical and gene silencing properties of these novel 2',4'-modified siRNAs. Both nucleosides were prepared stereoselectively in 6 steps starting from 2'-F rU and 2'-OMe rU for 2'-F,4'-OMe and 2',4'-diOMe, respectively, and subsequently converted to the corresponding phosphoramidites through conventional oligonucleotide chemistry for incorporation into oligonucleotides through standard solid-phase chemistry. NMR analysis of these nucleosides revealed that the 4' substituent imparts a bias towards the North sugar pucker conformation in both cases. Incorporation of 2'-F,4'-OMe rU into DNA strands resulted in destabilizing thermal effects in DNA:DNA hybrids and slightly destabilizing effects in DNA:RNA hybrids, but neutral effects within modified RNA strands in RNA:RNA duplexes. Furthermore, the 2'-F,4'-OMe rU modification was shown to be tolerated in both the antisense and sense strand in siRNA-targeted luciferase gene knockdown, whereas the 2',4'-diOMe rU modification was shown to be tolerated in the sense strand only. The CD analysis of these same strands also confirmed that the incorporation of these novel modifications does not affect the formation of duplex; where the A-form duplex (typical of RNA duplexes) is observed. As for the nanoparticle design, a block co-polymer is functionalized with azide and furan groups on the corona to allow for conjugation of antisense oligonucleotides and antibodies targeting upregulated receptors in glioblastoma cells. In collaboration, we were able to create a modified vehicle strand necessary for conjugation to the nanoparticle corona, upon which we were successful in hybridizing the complementary antisense oligonucleotide. This duplex was then successfully conjugated to the azide groups on the surface of the nanoparticle by copper-free Click chemistry. Single-stranded and double-stranded (hybridized) antisense oligonucleotides were transfected into live DsredDRR cells and 50% knockdown of the DRR gene was observed. " --