Mutant huntingtin (HTT) protein causes Huntington’s Disease (HD), an incurable neurological disorder. Silencing mutant HTT using nucleic acids would eliminate the root cause of HD. Developing nucleic acid drugs is challenging, and an ideal clinical approach to gene silencing would combine the simplicity of single-stranded antisense oligonucleotides with the efficiency of RNAi. Here we describe RNAi by single-stranded silencing RNAs (ss-siRNAs). ss-siRNAs are potent (>100-fold more than unmodified RNA) and allele-selective (>30-fold) inhibitors of mutant HTT expression in cells derived from HD patients. Strategic placement of mismatched bases mimics micro-RNA recognition and optimizes discrimination between mutant and wild-type alleles. ss-siRNAs require argonaute protein and function through the RNAi pathway. Intraventricular infusion of ss-siRNA produced selective silencing of the mutant HTT allele throughout the brain in a mouse HD model. These data demonstrate that chemically modified ss-siRNAs function through the RNAi pathway and provide allele-selective compounds for clinical development.

Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.

Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado–Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.

In the 2nd sentence of the fi rst paragraph on page 756 the index of the units of the dissociation constant appears incorrectly. The units should read nM. The sentence is corrected hereby to read: "A calculated apparent dissociation constant (K D) of 4 × 10-9 M was obtained, while in the case of Plain and AptaMIP NPs Acrydite, the KD values were, respectively, 13.7 × 10-9 M and 26.4 × 10-9 M."

Oligonucleotides and their derivatives are a proven chemical strategy for modulating gene expression. However, their negative charge remains a challenge for delivery and target recognition inside cells. Here we show that oligonucleotide-oligospermine conjugates (Zip nucleic acids or ZNAs) can help overcome these shortcomings by serving as effective antisense and antigene agents. Conjugates containing DNA and locked nucleic acid (LNA) oligonucleotides are active and oligospermine conjugation facilitates carrier-free cell uptake at nanomolar concentrations. Conjugates targeting the CAG triplet repeat within huntingtin (HTT) mRNA selectively inhibit expression of mutant huntingtin protein. Conjugates targeting the promoter of progesterone receptor (PR) function as antigene agents to block PR expression. These observations support further investigation of ZNA conjugates as gene silencing agents.

Abasic substitutions within DNA or RNA are tools for evaluating the impact of absent nucleobases. Because of the importance of abasic sites in genetic damage, most research has involved DNA. Little information is available on the impact of abasic substitutions within RNA or on RNA interference (RNAi). Here, we examine the effect of abasic substitutions on RNAi and allele-selective gene silencing. Huntington's disease (HD) and Machado Joseph Disease (MJD) are severe neurological disorders that currently have no cure. HD and MJD are caused by an expansion of CAG repeats within one mRNA allele encoding huntingtin (HTT) and ataxin-3 (ATX-3) proteins. Agents that silence mutant HTT or ATX-3 expression would remove the cause of HD or MJD and provide an option for therapeutic development. We describe flexible syntheses for abasic substitutions and show that abasic RNA duplexes allele-selectively inhibit both mutant HTT and mutant ATX-3. Inhibition involves the RNAi protein argonaute 2, even though the abasic substitution disrupts the catalytic cleavage of RNA target by argonaute 2. Several different abasic duplexes achieve potent and selective inhibition, providing a broad platform for subsequent development. These findings introduce abasic substitutions as a tool for tailoring RNA duplexes for gene silencing.

Mutant huntingtin (HTT) protein is the cause of Huntington's disease (HD), an incurable neurological disorder. Almost all patients are heterozygous for mutant HTT and approaches that reduce levels of mutant HTT while leaving expression of wild-type HTT intact might be ideal options for therapeutic development. We have developed several allele-selective strategies for silencing HTT, including single-stranded silencing RNAs (ss-siRNAs). ss-siRNAs are oligonucleotides containing chemical modifications that permit action through the RNA interference (RNAi) pathway. Modified ss-siRNAs chosen to test the effects of varying oligomer length, lipid modification, the introduction of mismatched bases, and variation of chemical modification. We find that several modified ss-siRNA are potent and allele-selective inhibitors of HTT expression. An ss-siRNA with three mismatched bases relative to the CAG repeat was an allele-selective inhibitor of HTT expression in the HdhQ175 mouse model. Multiple allele-selective ss-siRNAs provide a wide platform of modifications to draw on for further optimization and therapeutic development. Our data provide insights into how ss-siRNAs can be modified to improve their properties and facilitate the discovery of the lead compounds necessary for further development.

Methods for the quantification of antisense oligonucleotides (AONs) provide insightful information on biodistribution and intracellular trafficking. However, the established methods have not provided information on the absolute number of molecules in subcellular compartments or about how many AONs are needed for target gene reduction for unconjugated AONs. We have developed a new method for nuclear AON quantification that enables us to determine the absolute number of AONs per nucleus without relying on AON conjugates such as fluorophores that may alter AON distribution. This study describes an alternative and label-free method using subcellular fractionation, nucleus counting, and locked nucleic acid (LNA) sandwich enzyme-linked immunosorbent assay to quantify absolute numbers of oligonucleotides in nuclei. Our findings show compound variability (diversity) by which 247,000-693,000 LNAs/nuclei results in similar target reduction for different compounds. This method can be applied to any antisense drug discovery platform providing information on specific and clinically relevant AONs. Finally, this method can directly compare nuclear entry of AON with target gene knockdown for any compound design and nucleobase sequence, gene target, and phosphorothioate stereochemistry.