Table 2. Testing ONs in therapeutic strategies against polyQ diseases.
| Disease/Gene | Silencing agent | Model | Main results | Ref. Year |
|---|---|---|---|---|
| Non-allele-selective targeting | ||||
| SCA1/ATXN1 | shRNA in AAV1 vector | Transgenic mouse model (human ataxin-1 with 82Q) | ICB injection resulted in a reduction in the accumulation of mutant ataxin-1 and an improvement of motor coordination | (162) 2004 |
| HD/HTT | shRNA in AAV1 vector | Transgenic mouse model (HD-N171–82Q) | Reduced inclusions in stratum, correction of disease phenotype after IST injection | (163) 2005 |
| HD/HTT | siRNA | Transgenic mouse model R6/2 | IVT infusion of siRNAs in the liposome complex reduced huntingtin inclusions, improved motor coordination and prolonged lifespan | (167) 2005 |
| HD/HTT | shRNA in AAV5 vector | Transgenic mouse model R6/1 | Reduced NIIs and disease phenotype ameliorated after IST injection | (164) 2005 |
| HD/HTT | shRNA in AV vector | Transgenic mouse model R6/2 and model induced by the AAV vector containing the human HTT fragment | IST injection resulted in a reduction in huntingtin inclusions | (165) 2007 |
| HD/HTT | Cholesterol-conjugated siRNA | Model induced by the AAV vector containing the human HTT fragment | Inhibition of neurodegeneration and improvement in motor coordination by IST injection | (168) 2007 |
| HD/HTT | miRNA-based shRNA in AAV2/1 vector | Knock-in mouse model CAG140 and a transgenic mouse model (N171–82Q) | miRNA-based constructs were found to be safer than typical shRNAs after IST injection and caused decreased striatal toxicity, improvement in motor coordination and a prolonged life span | (166,247) 2008 2009 |
| HD/HTT | shRNA in LV vector | Mouse and rat models induced by the injection of the LV vector containing a human HTT fragment (with 82Q) | The silencing of mHTT only or together with normal Htt resulted in a decrease of huntingtin inclusions after IST injections | (172) 2009 |
| SCA3/ATXN3 | shRNA in LV vector | Rat model induced by the injection of the LV vector containing human ATXN3 (with 72Q) | Silencing of both, normal endogenous Atxn3 and mutant transgene, by IST injections of vectors resulted in neuropathology reduction and did not cause any significant side effects | (161) 2010 |
| HD/HTT | siRNA | Non-human primates | Convection-enhanced delivery of siHtt by IST (putamen) injection resulted in the widespread distribution throughout the striatum and efficient huntingtin silencing | (175) 2011 |
| HD/HTT | miRNA-based shRNA in AAV2/1 vector | Non-human primates | HTT suppression after IST (putamen) injection did not cause any neuronal degeneration or behavioral abnormalities, and no immune response to AAV vector was detected | (174) 2011 |
| HD/HTT | shRNA in AAV vector | Non-human primates | The efficient downregulation of normal htt in the striatum after IST (putamen) injection, without adverse behavioral effects and histopathological changes in brain tissue | (176) 2012 |
| HD/HTT | AON | Transgenic mouse models: YAC128, BACHD, R6/2 and non-human primates | RNase H-activating AONs, delivered by IVT infusion, mediated HTT transgene silencing throughout the CNS and improvement in motor coordination | (169) 2012 |
| SCA1/ATXN1 | Artificial miRNA in AAV vector | B05 mouse model | The efficient knockdown of a transgene in the cerebellum, after ICB injections of vectors, improved neuropathology and rescued the behavioral phenotype | (149) 2013 |
| SCA3/ATXN3 | Artificial miRNA mimics in AAV2/1 vector | Transgenic mouse model (SCA3/MJD84.2) | Effective downregulation of ataxin-3 in the cerebellum after ICB injections of vectors, but no corrected phenotype was reported | (170,171) 2013 |
| HD/HTT | shRNA in AAV9 vector | Transgenic mouse models: BACHD and N171-82Q | Systemic delivery of viral vectors by intra-jugular vein injection reduced mHTT expression in brain and peripheral tissues, prevented inclusion formation in key brain regions and prevented severe weight loss | (274) 2014 |
| HD/HTT | miRNA-based shRNA in AAV2/1 vector | Transgenic mouse model YAC128 | Reduction of striatal huntingtin aggregates and improvements in behavioral deficits after IST injections | (173) 2014 |
| Polymorphism site-targeting | ||||
| SCA3/ATXN3 | siRNA, shRNA in AV vector | Cos-7 and HeLa cells transfected with plasmids containing a SNP site | Selective suppression of mutant ataxin-3 by targeting a SNP site located just after a CAG repeat tract | (183) 2003 |
| SCA3/ATXN3 | siRNA | HEK293T cells transfected with plasmids containing a SNP site | Inhibition of mutant ataxin-3 by targeting a SNP site located just after a CAG repeat tract | (184) 2004 |
| HD/HTT | siRNA | HeLa cells transfected with plasmids containing a SNP site | Five SNPs targeted, mismatch position 16 described as providing high discrimination | (190) 2006 |
| HD/HTT | siRNA | Patient-derived fibroblast cell lines | One SNP targeted and allele-selective siRNA selected | (191) 2008 |
| SCA3/ATXN3 | shRNA in LV vector | Rat model induced by the injection of the LV vector with human ATXN3 | Selective silencing of mutant transgene, after IST injections of vectors, resulted in a decrease of neuropathological abnormalities | (186) 2008 |
| HD/HTT | siRNA | Patient-derived fibroblast cell lines | Selective suppression of mutant huntingtin by targeting a polymorphic site of a 3-nt deletion | (301) 2009 |
| HD/HTT | siRNA | NIH3T cells transfected with plasmids containing a SNP site, patient-derived fibroblast cell lines | Three SNPs targeted and allele-selective siRNAs selected | (192) 2009 |
| HD/HTT | siRNA | Patient-derived fibroblast cell lines | Three SNPs targeted, five allele-selective siRNAs were estimated to be useful for three-quarters of HD patients | (193) 2009 |
| SCA7/ATXN7 | shRNA, pri-miRNA-based hairpins | HEK293T cells transfected with plasmids containing a SNP site | pri-miRNA mimics targeted mutant ATXN7 transcript efficiently and decreased mutant protein aggregation | (188) 2009 |
| HD/HTT | siRNA | HeLa cells transfected with plasmids containing a SNP site and patient-derived lymphoblast cell lines | Four SNPs targeted and allele-selective siRNAs selected | (194) 2010 |
| HD/HTT | AON | Patient-derived fibroblast cell lines, cultured primary neurons and BACHD and YAC128 mouse models | Effective and selective (∼5-fold) silencing of HTT transgene in a model containing a targeted SNP variant using AON modified with PS, MOE, S-cEt and delivered by IST injections | (195) 2011 |
| SCA3/ATXN3, HD/HTT, SCA1/ATXN1 | siRNA | Patient-derived fibroblast cell lines | For each SNP targeted allele-selective siRNAs were identified that preferentially inhibited the expression of the mutant allele | (179) 2012 |
| SCA3/ATXN3 | shRNA in LV vector | Transgenic mouse model (expressing truncated ataxin-3 with 69Q in Purkinje cells) | Reduction in Purkinje cell pathology and improvement in motor coordination after allele-selective transgene silencing using vectors delivered by ICB injections | (187) 2013 |
| HD/HTT | AON | Transgenic mouse model Hu97/18 | AON modified with PS, S-cEt and 2'MOE, showed >100-fold selectivity in the silencing of mutant HTT and efficient and selective huntingtin suppression in CNS after delivery by IVT infusion | (196) 2013 |
| CAG repeat-targeting | ||||
| SBMA/AR | dsRNA (81 bp) | Drosophila S2 cells and HEK293T cells | Non-allele-selective silencing of normal and mutant AR transgenes | (302) 2002 |
| SCA1/ATXN1 | shRNA in AAV vector | HEK293T or PC6–3 neuronal cells transfected with plasmids encoding normal and mutant ataxin-1 | shCAG, targeting CAG repeat region was used as a positive control of silencing, both alleles were silenced efficiently | (162) 2004 |
| HD/HTT, SCA3/ATXN3 | AON | Patient-derived fibroblast cell lines | Selective inhibition of mutant huntingtin and ataxin-3 with PNA- and LNA-modified AONs (selectivity fold > 6) | (201,303) 2009 |
| HD/HTT | AON | Patient-derived fibroblast cell lines | Selectivity of mutant HTT silencing even >6-fold for AONs with modifications LNA, cEt, carba-LNA, cEt-PS, MOE-2'F-PS | (202) 2010 |
| HD/HTT | siRNA | Patient-derived fibroblast cell lines | Selectivity of mutant HTT silencing > 40-fold for siRNA containing mismatches with the target sequence | (199) 2010 |
| HD/HTT | siRNA | Patient-derived fibroblast cell lines | Selective inhibition of mutant huntingtin using CAG/CUG duplexes with specific mutations | (200) 2011 |
| SCA3/ATXN3 | AON, siRNA | Patient-derived fibroblast cell lines | Selectivity of mutant ataxin-3 silencing 5-fold for PNA modified AON and 16-fold for siRNAs | (204) 2011 |
| HD/HTT | AON | Patient-derived fibroblast cell lines | AONs modified with LNA and containing oligospermine conjugate for efficient delivery | (304) 2011 |
| HD/HTT | AON | Patient-derived fibroblast cell lines | High allele selectivity was demonstrated for morpholino-modified AON | (179) 2012 |
| HD/HTT | ss-siRNA chemically modified | Patient-derived fibroblast cell lines and knock-in mouse model HdhQ150/Q7 | Selectivity of mutant HTT silencing >30-fold in cell culture, specific HTT silencing in mouse brain by ss-siRNAs delivered by IVT infusion | (206) 2012 |
| HD/HTT, SCA3/ATXN3 | siRNA with abasic substitutions | Patient-derived fibroblast cell lines | RNA duplexes containing centrally located abasic residues (and additional 2'OMe and PS modifications) showed allele selectivity in HTT and ATXN3 silencing | (305) 2013 |
| HD/HTT, SCA3/ATXN3 | siRNA with UNA substitutions | Patient-derived fibroblast cell lines | siRNAs containing single UNA-modified nucleotides in antisense strand showed >40 and >10-fold selectivity for mutant HTT and mutant ATXN3, respectively | (306) 2013 |
| SCA3/ATXN3 | ss-siRNA chemically modified | Patient-derived fibroblast cell lines | Selectivity of mutant ATXN3 silencing >35-fold for selected ON | (207) 2013 |
| HD/HTT | sd-siRNA | Patient-derived fibroblast cell lines | Guide strand-only siRNAs showed high selectivity, >60-fold, in HTT silencing | (208) 2013 |
The table is divided into the three parts, depending on the strategy type, which include the targeting of the following: the sequence harboring the polymorphism site, CAG expansion or another region of the transcript sequence. In each part of the table, the studies are placed in the order from the oldest studies to the most recent. The specific ON-based tools and models used in the studies are listed, together with brief descriptions of the main results. The selectivity of the silencing is based on the ratio of IC50 for inhibiting the normal allele versus IC50 for the mutant allele. For description of ON administration in vivo, the abbreviations are used: IST, intrastriatal; IVT, intraventricular; ITH, intrathecal; ICB, intracerebellar.