Delphine Trochet

Bernard Prudhon

Stéphane Vassilopoulos

Marc Bitoun
email: m.bitoun@institut-myologie.org

Therapy for dominant inherited diseases by Allele-Specific RNA Interference: Successes and Pitfalls

Keywords: Allele-specific silencing, Dominant inherited diseases, Pitfalls, RNA interference, Single nucleotide substitution, Gene-based therapy

RNA interference (RNAi) is a conserved mechanism for post-transcriptional gene silencing mediated by messenger RNA (mRNA) degradation. RNAi is commonly induced by synthetic siRNA or shRNA which recognizes the targeted mRNA by base pairing and leads to target-mRNA degradation. RNAi may discriminate between two sequences only differing by one nucleotide conferring a high specificity of RNAi for its target mRNA. This property was used to develop a particular therapeutic strategy called "allele-specific-RNA interference" devoted to silence the mutated allele of genes causing dominant inherited diseases without affecting the normal allele. Therapeutic benefit was now demonstrated in cells from patients and animal models, and promising results of the first phase Ib clinical trial using siRNA-based allele-specific therapy were reported in Pachyonychia Congenita, an inherited skin disorder due to dominant mutations in the Keratin 6 gene. Our purpose is to review the successes of this strategy aiming to treat dominant inherited diseases and to highlight the pitfalls to avoid.

RNA interference (RNAi) is a post-transcriptional mechanism of gene silencing first characterized in plants as an anti-virus or anti-transgene defense mechanism [START_REF] Fire | Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[END_REF]. Thereafter, RNAi was shown to induce gene-selective inactivation in a wide variety of eukaryote cells, including mammalian cells [2 , 3]. RNAi is classically induced by chemically synthetized small interfering RNA (siRNA) of 19-21 nucleotides with two 3' overhanging nucleotides or by short hairpin RNA (shRNA) synthetized from RNA polymerase III promoters and converted into siRNA by the endogenous cell machinery. The siRNA recognizes the targeted messenger RNA (mRNA) by base pairing of the seed region located from the second to the seventh nucleotide of the siRNA antisense strand referred to as "guide strand". The annealing directs the cleavage of the mRNA after the tenth nucleotide of the siRNA guide strand [START_REF] Elbashir | Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate[END_REF] in the RNA-induced silencing complex (RISC). A fascinating aspect of RNAi stems from its high specificity for the targeted mRNA sequence. This property led to the development of the allelespecific RNAi (AS-RNAi) as a therapeutic strategy for dominant inherited diseases by targeting the mutated allele without affecting the wild type (WT) allele. Proof of concept for a therapeutic use of AS-RNAi was first established in 2002 against dominant mutations of oncogenes or tumor suppressors [START_REF] Martinez | Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways[END_REF][START_REF] Brummelkamp | Stable suppression of tumorigenicity by virus-mediated RNA interference[END_REF] (Table S1) and, thereafter, rapidly achieved for monogenic dominant inherited diseases [START_REF] Gonzalez-Alegre | Toward therapy for DYT1 dystonia: allelespecific silencing of mutant TorsinA[END_REF][START_REF] Miller | Allele-specific silencing of dominant disease genes[END_REF][START_REF] Abdelgany | Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference[END_REF][START_REF] Ding | Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis[END_REF]. During the last decade, therapeutic benefit was also demonstrated in patient-derived cells and animal models, and promising results of the first clinical trial were reported [START_REF] Leachman | First-inhuman mutation-targeted siRNA phase Ib trial of an inherited skin disorder[END_REF]. In this review, we aim at reviewing the state of the art of this strategy applied to treating dominant inherited diseases. The review of the literature also highlights potential limitations and pitfalls to avoid.

AS-RNAi, a versatile strategy for different types of dominant mutations

The specificity of RNAi-inducing molecules (siRNA and shRNA) led to the development of AS-RNAi for different types of mutations responsible for dominant inherited diseases (Fig. 1). The majority of the AS-RNAi was developed for silencing of mutated alleles differing from the WT alleles by a single nucleotide substitution (Tables 1,2 and S1), by targeting directly pathogenic missense mutations or disease-associated single nucleotide polymorphisms (da-SNP) in particular cases of triplet repeat diseases. However, AS-RNAi technology was used in several other molecular contexts (Fig. 1 and Table S2). Efficient AS-RNAi was achieved for targeting threenucleotide deletions [START_REF] Gonzalez-Alegre | Toward therapy for DYT1 dystonia: allelespecific silencing of mutant TorsinA[END_REF][START_REF] Gonzalez-Alegre | Silencing primary dystonia: lentiviral-mediated RNA interference therapy for DYT1 dystonia[END_REF][START_REF] Zhang | Allele-specific silencing of mutant Huntington's disease gene[END_REF], multiple nucleotide deletions [START_REF] Takahashi | Specific inhibition of tumor cells by oncogenic EGFR specific silencing by RNA interference[END_REF] or substitutions [START_REF] Ohnishi | Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles[END_REF][START_REF] Feng | Allele-specific silencing of Alzheimer's disease genes: the amyloid precursor protein genes with Swedish or London mutations[END_REF][START_REF] Miller | Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles[END_REF][START_REF] Rousseau | Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta[END_REF][START_REF] Loy | Allele-specific gene silencing in two mouse models of autosomal dominant skeletal myopathy[END_REF][START_REF] Shukla | RNA interference and inhibition of MEK-ERK signaling prevent abnormal skeletal phenotypes in a mouse model of craniosynostosis[END_REF][START_REF] Rodriguez-Lebron | Allele-specific RNAi mitigates phenotypic progression in a transgenic model of Alzheimer's disease[END_REF] and diseaseassociated splicing isoforms [START_REF] Fan | RNA interference against a glioma-derived allele of EGFR induces blockade at G2M[END_REF][START_REF] Pendaries | siRNA-mediated allele-specific inhibition of mutant type VII collagen in dominant dystrophic epidermolysis bullosa[END_REF][START_REF] Bolduc | siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy[END_REF][START_REF] Tsou | Splice isoform-specific suppression of the Cav2.1 variant underlying spinocerebellar ataxia type 6[END_REF]. If the nucleotide repeats responsible for triplet repeat diseases are not considered as suitable targets for AS-siRNA [START_REF] Miller | Allele-specific silencing of dominant disease genes[END_REF] because identical target sequences are present in normal and mutated alleles, Li et al. [START_REF] Li | Sequence-dependent and independent inhibition specific for mutant ataxin-3 by small interfering RNA[END_REF] reported effective AS-RNAi against the CAG expansion of the disease-related allele of Ataxin 3 (ATXN3) using siRNA covering the extremity of the expanded region and its flanking sequence. In that case, WT and mutated sequences differ only by the number of repeated motifs while the target sequence of the siRNA is the same in both transcripts. The different repeat sequence lengths may probably induce structural changes allowing allele-specificity in this particular case. More recently, such AS-silencing was achieved using RNA duplexes targeting exclusively the repeated sequence [START_REF] Fiszer | Inhibition of mutant huntingtin expression by RNA duplex targeting expanded CAG repeats[END_REF][START_REF] Hu | Allele-selective inhibition of huntingtin expression by switching to an miRNAlike RNAi mechanism[END_REF][START_REF] Hu | Allele-selective inhibition of ataxin-3 (ATX3) expression by antisense oligomers and duplex RNAs[END_REF] but probably by a "miRNA mimicking mechanism" which does not involve mRNA cleavage. Regardless, these results suggest that allelespecific silencing may be envisaged in diseases with nucleotide expansion even in absence of da-SNP.

Nevertheless, we will largely focus this review on the AS-RNAi targeting single nucleotide substitutions.

How to develop AS-RNAi targeting single nucleotide substitutions causing dominant inherited diseases

For targeting single nucleotide substitutions, a perfect AS-siRNA has to achieve effective knock-down of the mutated allele without affecting the normal allele. With this objective, AS-siRNA is designed to fully match the mutated sequence but harbors one mismatch against the WT. The position of the mutated nucleotide in the siRNA, the nature of the mismatch against the WT sequence, and the flanking sequences may influence efficiency and specificity of silencing. Because all these parameters are largely dependent on sequence and structure of a given mRNA, no definitive rules have been established to date for the design of AS-siRNA and development of efficient molecules has relied on empirical testing. Using 19-base-pair siRNA, 19 possible single-mismatched siRNA exist relative to the position of the mutated nucleotide (Fig. 1). Several studies have reported the results from systematic screening for the 19 possible siRNA (Fig. S1) [START_REF] Schwarz | Designing siRNA that distinguish between genes that differ by a single nucleotide[END_REF][START_REF] Hickerson | Single-nucleotidespecific siRNA targeting in a dominant-negative skin model[END_REF][START_REF] Sierant | Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference[END_REF][START_REF] Liao | Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy[END_REF][START_REF] Pedrioli | Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder[END_REF][START_REF] Muller | Allele-specific siRNA knockdown as a personalized treatment strategy for vascular Ehlers-Danlos syndrome in human fibroblasts[END_REF][START_REF] Atkinson | Development of allelespecific therapeutic siRNA for keratin 5 mutations in epidermolysis bullosa simplex[END_REF][START_REF] Allen | Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy[END_REF][START_REF] Courtney | Development of allele-specific gene-silencing siRNAs for TGFBI Arg124Cys in lattice corneal dystrophy type I[END_REF]. From these studies, central mismatches (from positions 8 to 14) were associated with AS-RNAi with highest specificity at position 10. These data highlight the crucial role of the central region of the siRNA and the fact that AS-RNAi appears more efficient when the siRNA is designed to block the cleavage of the WT allele rather than its annealing. The conclusions from systematic screening may be refined by overall analysis of the 87 single-mismatched siRNA or shRNA reported in the Table S1. The central region proves to be a master region to develop AS-siRNA with the highest specificity reached at position 10 (19 out of 87; i.e. 22% of the reported AS-siRNA), followed by positions 9 and 11 (16% each). By adding siRNA mismatched at the position 16, these four siRNA (9, 10, 11, and 16) represent 63% of the already reported efficient molecules. Not only the position but also the nature of the mismatch between siRNA and WT sequences may influence AS-RNAi efficacy with theoretically purine:purine (pu:pu) mismatches introducing the largest possible destabilization compared to pyrimidine:pyrimidine (py:py) or purine:pyrimidine (pu:py) mismatches. By definition, in this particular case, the nature of the mismatch is fixed by the mutated nucleotide. It is noteworthy that siRNA with single mismatches at positions 9, 10, 11, and 16 have been shown to discriminate mutated and WT alleles whatever the nature of the mismatch (Table S1).

With the objective to increase allele-specificity, a second mismatch was introduced in the siRNA or shRNA.

In that case, the RNAi-inducing molecule harbors one mismatch relative to the mutated sequence but two against the WT. Twelve successful examples of double-mismatched siRNA are indicated in the Table S1 using 19-base pair siRNA including 10 cases in which the first mismatch is located at position 9 or 10. In these cases, the most efficient positions for the second mismatch are 12, 13 and 14 and the majority (6 out of 10) introduces a purine:purine mismatch. Nevertheless, introducing a second mismatch does not always increase AS-RNAi efficiency compared to single-mismatched siRNA [START_REF] Scholefield | Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype[END_REF][START_REF] Sibley | Identification of allele-specific RNAi effectors targeting genetic forms of Parkinson's disease[END_REF][START_REF] Sapru | Silencing of human alpha-synuclein in vitro and in rat brain using lentiviral-mediated RNAi[END_REF][START_REF] Nishimura | Allele-specific knockdown of ALS-associated mutant TDP-43 in neural stem cells derived from induced pluripotent stem cells[END_REF][START_REF] Noguchi | Allele-specific Gene Silencing of Mutant mRNA Restores Cellular Function in Ullrich Congenital Muscular Dystrophy Fibroblasts[END_REF].

Huang et al. analyzed silencing properties of siRNAs on 230 reporter constructs and confirmed that some positions are more sensitive than others to target a given mismatch [START_REF] Huang | Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs[END_REF]. This study and the successful examples now available for 31 distinct mRNA (Table S1) suggest a two-step strategy in order to develop AS-RNAi. Single mismatched siRNA at positions 9, 10, 11, and 16 should be privileged as a first screening strategy whatever the nature of the mismatch. In case of low allele-specificity, a second purine:purine mismatch may be introduced at positions 12, 13, or 14 in addition to the first mismatch at positions 9, and 10. These 10 different siRNA sequences (out of the 667 possible single and double-mismatched siRNA) may facilitate the development of AS-RNAi as they represent 65% of the efficient AS-siRNA already reported. This strategy may be associated with the recently developed formulas for calculating allele-discrimination [START_REF] Takahashi | A novel measurement of allele discrimination for assessment of allele-specific silencing by RNA interference[END_REF] in order to optimize determination of allele-specific siRNA.

Proof of concept of AS-RNAi in patient-derived cells and animal models

Phenotype reversion in patient-derived cells and/or animal models is a crucial step for preclinical development. This was achieved in vitro for numerous disease-related genes mainly in patient-derived fibroblasts (Table 1). Interestingly, induced pluripotent stem cells (iPSCs) derived towards neuronal cells as an Amyotrophic lateral sclerosis model [START_REF] Nishimura | Allele-specific knockdown of ALS-associated mutant TDP-43 in neural stem cells derived from induced pluripotent stem cells[END_REF] or toward cardiomyocytes as a Long QT syndrome model [START_REF] Matsa | Allele-specific RNA interference rescues the long-QT syndrome phenotype in human-induced pluripotency stem cell cardiomyocytes[END_REF] were recently used to study AS-RNAi in functionally relevant disease models. Validation of AS-RNAi in pertinent disease-related cells appears particularly important in absence of primary cultures from the affected tissue and iPSC represent probably a model of choice.

In vivo AS-RNAi was reported for the first time in 2003 by Ding and collaborators after co-injection of WT and mutated SOD1 constructs with shRNA in mice [START_REF] Ding | Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis[END_REF]. Beyond the first in vivo proof of concept, this pioneer study demonstrated the rapid silencing of the mutated allele evidenced 24 hours post-transfection. Afterwards, different types of animal models (transgenic mice or vector-mediated overexpression of mutated transcripts)

were developed in order to study the feasibility of AS-RNAi in vivo (Table 2). These models confirmed the rapid down-regulation of the mutated alleles [START_REF] Hickerson | Single-nucleotidespecific siRNA targeting in a dominant-negative skin model[END_REF][START_REF] Pedrioli | Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder[END_REF] and highlighted several important features of in vivo ASsilencing. First, early treatment in pre-symptomatic animals is able to prevent the appearance and/or the progression of the disease [START_REF] Rodriguez-Lebron | Allele-specific RNAi mitigates phenotypic progression in a transgenic model of Alzheimer's disease[END_REF][START_REF] Alves | Allele-specific RNA silencing of mutant ataxin-3 mediates neuroprotection in a rat model of Machado-Joseph disease[END_REF][START_REF] Drouet | Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells[END_REF][START_REF] Xia | Allele-specific RNAi selectively silences mutant SOD1 and achieves significant therapeutic benefit in vivo[END_REF][START_REF] Nobrega | RNA interference mitigates motor and neuropathological deficits in a cerebellar mouse model of machado-joseph disease[END_REF]. Second, rescue of phenotype is also possible when treatment is started in symptomatic mice [START_REF] Nobrega | Silencing mutant ataxin-3 rescues motor deficits and neuropathology in Machado-Joseph disease transgenic mice[END_REF]. Third, the therapeutic effect is rapid as evidenced after 2-4 weeks of treatment [START_REF] Nobrega | RNA interference mitigates motor and neuropathological deficits in a cerebellar mouse model of machado-joseph disease[END_REF][START_REF] Nobrega | Silencing mutant ataxin-3 rescues motor deficits and neuropathology in Machado-Joseph disease transgenic mice[END_REF] without evident long-term toxicity [START_REF] Rodriguez-Lebron | Allele-specific RNAi mitigates phenotypic progression in a transgenic model of Alzheimer's disease[END_REF]. Fourth, a low therapeutic threshold was demonstrated as incomplete reduction of the mutated allele was sufficient for therapeutic benefit [START_REF] Drouet | Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells[END_REF][START_REF] Xia | Allele-specific RNAi selectively silences mutant SOD1 and achieves significant therapeutic benefit in vivo[END_REF]. The last point may be of importance for particular mutations for which highly efficient AS-siRNA could not be easily designed.

In addition to these helpful experimental in vivo models, therapeutic potential of AS-RNAi was also investigated in Knock-in mouse models expressing disease-causing mutations in the endogenous mouse genes to recapitulate more closely the human heterozygous conditions. This was achieved in a mouse model of the Apert syndrome expressing a Fgfr2 (Fibroblast growth factor receptor type 2) mutation [START_REF] Shukla | RNA interference and inhibition of MEK-ERK signaling prevent abnormal skeletal phenotypes in a mouse model of craniosynostosis[END_REF], in two models of neuromuscular disorders due to Ryr1 (Ryanodine receptor type 1) mutations [START_REF] Loy | Allele-specific gene silencing in two mouse models of autosomal dominant skeletal myopathy[END_REF], and in one model of Hypertrophic cardiomyopathy due to a Myh6 (Myosin heavy chain 6) mutation [START_REF] Jiang | Allele-Specific Silencing of Mutant Myh6 Transcripts in Mice Suppresses Hypertrophic Cardiomyopathy[END_REF]. Among these, the Knockin-Myh6 mice represent the only case of mutation due to a single nucleotide substitution (in the three other models, the mutations come from double or triple substitutions). AS-RNAi in these knock-in models confirmed the safety, the rapidity, and efficacy of this therapeutic strategy to rescue or prevent the phenotype. A low therapeutic threshold was evidenced in the Myh6 model as a reduction of 28.5% of the mutated allele benefited the mice whereas a reduction of 50% led to a partial rescue in one Ryr1 model. In addition, AS-RNAi only delayed the cardiac phenotype but was unable to rescue an established cardiomyopathy in the Myh6 mice and a dissipation of protective effect over time was noticed [START_REF] Jiang | Allele-Specific Silencing of Mutant Myh6 Transcripts in Mice Suppresses Hypertrophic Cardiomyopathy[END_REF]. These findings that may be gene-and/or diseasespecific reveal the importance of functional validation in pertinent Knock-in mouse models for each targeted dominant disease.

Clinical trials in humans

Pachyonychia congenita (PC) is a highly disabling autosomal dominant skin disorder due to mutations in the Keratin 6 gene (KRT6A). The clinical manifestations of PC include painful plantar calluses which have been targeted successfully by AS-RNAi in a clinical trial reported in 2010 [START_REF] Leachman | First-inhuman mutation-targeted siRNA phase Ib trial of an inherited skin disorder[END_REF]. Treatment consisted of 17 weeks of intra-callus injections performed twice-weekly with dose escalation from 0.1 mg to 17 mg of siRNA against the mutated Krt6a mRNA. Clinical improvement with callus regression was noticed from 10 weeks after the first injection and was maintained during a relatively long period of 14 weeks, i.e. 7 weeks after the last injection.

Despite the spatially and temporally limited effects, which highlight the importance of the delivery method for future trials, this first report of AS-RNAi in human are promising for PC and other dominant inherited diseases.

Pitfall and limitation

Several specific aspects of the AS-RNAi technology, which may limit development of future effective treatments for dominant inherited diseases, require consideration. 1) Probably the most important point is that the expected result of this approach is to retain only 50% expression from the spared WT allele of the gene of interest . Consequently, absence of haploinsufficiency needs to be clearly established. In this context, in vitro knock-down studies, investigation of heterozygous knock-out animal models when available and analysis of the spectrum of gene mutations and genotype-phenotype correlation in patients are indispensable.

2) Another serious limitation is the restriction in the choice for the targeted sequence in the region of the mRNA harboring the mutation. This may limit efficiency of RNAi as all the regions of a given mRNA are not similarly sensitive to RNAi and may restrict efficacy and allele-specific silencing. In other words, AS-RNAi will not be possible in all cases. However, this problem may be overcome if targetable SNP is present on the mutated allele.

3) It is important to take into account the possibility that the AS-RNAi molecule can inhibit the translation of the WT allele whereas the quantified WT mRNA level remains unchanged [START_REF] Ohnishi | Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles[END_REF]. This may be due to a "microRNA effect" affecting the translation as already described for mismatched siRNA [START_REF] Saxena | Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells[END_REF]. Consequently, allele-specific properties should not be established exclusively by measuring the mRNA expression level but need to be combined to protein expression and protein activity when possible. 4) A loss of allele-specificity with increased dose of RNAi-inducing molecule [START_REF] Scholefield | Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts[END_REF] or an arrest of protective effect over time may appear [START_REF] Jiang | Allele-Specific Silencing of Mutant Myh6 Transcripts in Mice Suppresses Hypertrophic Cardiomyopathy[END_REF]. This highlights the importance of the choice of the vector, mode of delivery and tight control of the delivered amount of future therapeutic molecules in vivo. In this context, miRNA-based hairpins have been used in order to express AS-RNAi-inducing molecules [START_REF] Tsou | Splice isoform-specific suppression of the Cav2.1 variant underlying spinocerebellar ataxia type 6[END_REF][START_REF] Scholefield | Design of RNAi hairpins for mutation-specific silencing of ataxin-7 and correction of a SCA7 phenotype[END_REF]. This approach may offer an attractive alternative for in vivo tissue-specific expression compared to siRNA or shRNA and to avoid potential toxicity of shRNA expression in vivo [START_REF] Mcbride | Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi[END_REF][START_REF] Grimm | Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways[END_REF].

5)

In order to facilitate the screening for AS-siRNA in vitro, artificial assay systems with reporter genes have been developed. Using this screening procedure, siRNA maintaining their allele specificity against the bona fide mRNA target were identified [START_REF] Ohnishi | Assessment of allele-specific gene silencing by RNA interference with mutant and wild-type reporter alleles[END_REF][START_REF] Schwarz | Designing siRNA that distinguish between genes that differ by a single nucleotide[END_REF][START_REF] Liao | Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy[END_REF][START_REF] Atkinson | Development of allelespecific therapeutic siRNA for keratin 5 mutations in epidermolysis bullosa simplex[END_REF][START_REF] Dykxhoorn | Determinants of specific RNA interferencemediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism[END_REF][START_REF] Lombardi | A majority of Huntington's disease patients may be treatable by individualized allele-specific RNA interference[END_REF]. However, significant discrepancies have been shown comparing reporter systems and full-length mRNA or even between two different reporter systems [34-37, 39, 43, 59]. Consequently, screening for AS-siRNA on the full-length natural target (endogenous or overexpressed) should be privileged.

6) The best AS-siRNA sequences identified in vitro are often incorporated into plasmids or viral vectors to be expressed in vivo as shRNA. The shRNAs are known to retain the efficacy and allele specificity of the original siRNA [START_REF] Miller | Allele-specific silencing of dominant disease genes[END_REF][START_REF] Ding | Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis[END_REF] but some divergences may exist between siRNA and the corresponding shRNA [START_REF] Abdelgany | Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference[END_REF][START_REF] Gonzalez-Alegre | Silencing primary dystonia: lentiviral-mediated RNA interference therapy for DYT1 dystonia[END_REF][START_REF] Rousseau | Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta[END_REF]. Indeed, shRNA are processed by the endogenous Dicer RNAse to generate siRNA. Given that Dicer cleavage site in the shRNA loop is difficult to predict that may introduce one or two-nucleotide differences between the expected and the produced siRNA. Consequently, even if several examples showed that AS-siRNA and AS-shRNA are similarly efficient, it would be important to validate shRNA constructs in vitro before starting in vivo studies in animals. 7) By definition, the AS-RNAi strategy is a mutation-specific approach. This may be interesting for mutation hot spots concentrated in a reduced number of nucleotides allowing development of common molecular tools for a large cohort of patients. In contrast, this may require personalized therapy by developing specific tools for mutations affecting few patients. 8) Another limitation comes from the study of Jiang and collaborators [START_REF] Jiang | Allele-Specific Silencing of Mutant Myh6 Transcripts in Mice Suppresses Hypertrophic Cardiomyopathy[END_REF] which stress the necessity to start treatment at a pre-symptomatic stage. In such cases, AS-RNAi could be limited to familial forms of diseases receiving an early genetic diagnosis. Development of preclinical studies in knock-in animal models appears crucial to determine the optimal timing of treatment. 9) Interferon response classically attributed to long double-stranded RNA has not been reported in AS-RNAi to date. Nevertheless, some siRNA [START_REF] Hornung | Sequencespecific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7[END_REF] and shRNA [START_REF] Bridge | Induction of an interferon response by RNAi vectors in mammalian cells[END_REF] have been shown to induce this response. Consequently, monitoring a possible interferon response should be included, especially for in vivo studies, to avoid misinterpretation of treatment efficacy.

Concluding remarks

Despite of the possible limitations indicated above, the AS-RNAi technology emerged during the last decade as a powerful strategy for dominant inherited diseases. However, some aspects still need to be resolved especially those concerning in vivo delivery. Similar to all the siRNA-based therapeutics, using the best vector for delivering the optimal dose will probably be the key bottleneck when systemic delivery is required for widespread dissemination or in contrast for limited expression in one affected tissue. Also, the long-term consequences of chronic stimulation of the RNAi pathway (potential toxicity or immunological side effects as reviewed in [START_REF] Olejniczak | Recent advances in understanding of the immunological off-target effects of siRNA[END_REF]) as well as off-target effects need to be carefully investigated. Continuous improvements to overcome these limitations and increase efficacy of RNAi-based therapeutics, including chemical modifications, are occurring and will benefit the AS-RNAi strategy. Furthermore, development of preclinical testing in animal models of dominant inherited diseases are now crucial in order to deepen the specificities of the AS-RNAi approach in vivo such as long-term maintenance of allele-specificity. Nevertheless, the proof of concept for AS-RNAi therapy now available for numerous dominant inherited diseases strongly suggests that the promising result of the first clinical trial for Pachyonychia Congenita paves the way for future successful clinical trials. No benefit for onset and progression of motor deficits probably due to insufficient neuronal transduction [START_REF] Towne | Systemic AAV6 delivery mediating RNA interference against SOD1: neuromuscular transduction does not alter disease progression in fALS mice[END_REF] Myh6: Myosin heavy chain 6, HCM: Hypertrophic cardiomyopathy, ATXN3: Ataxin 3, MJD/SCA3: Machado-Joseph disease/Spinocerebellar ataxia 3, SOD1: Superoxide dismutase 1, ALS: Amyotrophic lateral sclerosis, HTT: Huntingtin, HD: Huntington's disease, KRT9: Keratin 9, EPPK: Epidermolytic palmoplantar keratoderma, KRT6A: Keratin 6a, PC: Pachyonychia congenita, da-SNP: disease associated-Single Nucleotide Polymorphism.

ATXN3

MJD

Figure 1 :

 1 Figure 1: AS-RNAi targetable mutations. For targeting a single nucleotide substitution, the 19 possible siRNAs are indicated relative to the position of the mutated nucleotide. This specific nucleotide determines the position of the mismatch between the siRNA and the WT mRNA. P10 indicates the siRNA mismatched at position 10. nt: nucleotide. Specific nucleotides of the mutated mRNA are indicated in blue.

  -N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, HTT: Huntingtin, HD: Huntington's disease, ATXN1: Ataxin 1, ATXN3: Ataxin 3, ATXN7: Ataxin 7, SCA1: Spinocerebellar ataxia 1, SCA3: Spinocerebellar ataxia 3, SCA7: Spinocerebellar ataxia 7, TARDBP: TAR-DNA Binding protein (TDB-43), ALS: Amyotrophic lateral sclerosis, PSEN1: Presenilin 1, AD: Alzheimer's disease, ACVR1: Activin A receptor type 1, FOP: Fibrodysplasia ossificans progressive, COL1A2: Collagen type I alpha 2 chain, OI: Osteogenesis imperfecta, COL3A1: Collagen type 3 alpha 1, vEDS: Vascular type of the Elhers-Danlos syndrome, COL6A1: Collagen type VI alpha 1, UCMD: Ullrich congenital muscular dystrophy, KCNH2: Potassium channel voltage-gated subfamily H member 2, LQT2: Long QT syndrome type 2, KRT12: Keratin 12, MECD: Meesmann epithelial corneal dystrophy, TGFBI: TGFβ-induced gene, LCDI: Lattice corneal dystrophy type I, da-SNP: disease associated-Single Nucleotide Polymorphism, iPSC: induced pluripotent stem cell, MSC: mesenchymal stem cells.

  : cells transfected with WT or mutated transcripts, b: co-transfection of WT and mutants in vitro, c: cell-free assay or reporter assay in vitro, e: Transgenic mice expressing WT or mutated transcripts, h: cell lines endogenously expressing WT or mutant, i: overexpression of mutated transcript in vivo, j: patient-derived cells, k: knock-in mouse model. TOR1A: Torsin A, DYT1: Dystonia. HTT: Huntingtin, HD: Huntington's disease, EGFR: Epidermal growth factor receptor, APP: Amyloid precursor protein, AD: Alzheimer's disease, COL1A1: 1 chain of type I collagen, OI: Osteogenesis imperfecta, RYR1: Ryanodyne receptor type 1, MH: Malignant hyperthermia, CCD: Central core disease, FGFR2: Fibroblast growth factor receptor 2, CS: Craniosynostosis, COL7A1: 1 chain of type VII collagen, DDEB: Dominant dystrophic epidermolys bullosa, COL6A3: 3 chain of type VI collagen, UCMD: Ullrich congenital muscular dystrophy, CACNA1A: Cav2.1 voltage-gated calcium channel, SCA6: Spinocerebellar ataxia type 6, ATXN3: Ataxin 3, MJD: Machado-Joseph disease.

Table 1 : Single nucleotide substitutions targeted by Allele-specific RNAi in patient-derived cells

 1

	Gene	Disease	Targeted	Model	Phenotype	References
			change		reversion
	ACVR1	FOP	p.R206H	Primary MSC	yes	[63]
	ACVR1	FOP	p.R206H	Lymphoblastoid	yes	[64]
			p.G356D	cells
	ATXN1	SCA1	da-SNP	Fibroblast	-	[65]
	ATXN3	SCA3	da-SNP	Fibroblast	-	[65]
	ATXN7	SCA7	da-SNP	Fibroblast	yes	[54]
	COL1A2	OI	da-SNP	Bone-derived	-	[66]
				cells
	COL3A1	vEDS	p.G252V	Fibroblast	yes	[35]
	COL6A1	UCMD	p.G284R	Fibroblast	yes	[43]
	GNE	Sialuria	p.R266Q	Fibroblast	yes	[67]
	HTT	HD	da-SNP	Fibroblast	-	[68]
	HTT	HD	da-SNP	Fibroblast	-	[58]
	HTT	HD	da-SNP	Lymphoblastoid	-	[69]
				cells
	HTT	HD	da-SNP	Fibroblast	-	[65]
	HTT	HD	da-SNP	Neural stem cells	yes	[48]
	KCNH2	LQT2	p.A561T	iPSC-derived	yes	[46]
				cardiomyocyte
	KRT12	MECD	p.L132P	Corneal epithelial	-	[70]
				cells
	PSEN1	AD	p.L392V	Fibroblast	yes	[32]
	TARDBP	ALS	p.M337V	iPSC-derived
				neural stem cell

Table 2 : Single nucleotide substitutions targeted by Allele-specific RNAi in animal models

 2

	Gene	Disease Targeted	Model	Comment	Phenotype	Ref.
			change			reversion
	ATXN3	MJD/	da-SNP	Lentivirus-mediated	Single injection in	Reduce the	[47]
		SCA3		expression of	striatum	formation of
				mutant and shRNA		histological lesions
				in rat brain		8 weeks post-
						injection
	ATXN3	MJD/	da-SNP	Lentivirus-mediated	Single	Prevent	[50]
		SCA3		expression of human	intracerebellar	pathological
				full length mutant	injection in pre	lesions, neuronal
				ATXN3 and AS-	symptomatic mice	loss, and motor
				shRNA		impairments 10
						weeks post-
						injection
	ATXN3	MJD/	da-SNP	Lentivirus-mediated	Single	Decrease in	[51]
		SCA3		expression of	intracerebellar	histological lesions,
				shRNA in	injection in pre	prevent neuronal
				transgenic mice	symptomatic mice.	loss and alleviate
				expressing the		motor impairments
				human mutant		10 weeks post-
						injection
	HTT	HD	da-SNP	Lentivirus-mediated	Single injection in	Decrease in	[48]
				expression of	striatum. Mice	formation of
				shRNA in	injected with	histological signs in
				transgenic mice	shRNA against the	rats 8 weeks post-
				expressing full-	da-SNP or the WT	injection
				length human	nucleotide
				mutant or in rat
				expressing human
				reporter construct
	KRT6A	PC	p.N171K	Bioluminescence	Single intradermal	-	[31]
				mouse model	injection of siRNA
					and reporter
					constructs in
					footpad. Live
					imaging at 24, 48
					and 72h. Rapid
					down expression
					(24h post injection)
	KRT9	EPPK	p.R163Q	Bioluminescence	Single intradermal	-	[34]
				mouse model	injection of siRNA
					and reporter
					constructs in
					footpad. Live
					imaging at 24 hours
					post-injection
	Myh6	HCM	p.R403Q	MHC 403/+ knock-in	Single injection in	No reversion 2	[52]
				mice	thoracic cavity of	months post-
					siRNA-expressing	injection in
					AAV9	symptomatic mice.
						Prevent
						cardiomyopathy in
						presymptomatic
						mice (8 weeks post-
						injection in 1-day-
						old mice). Arrest of

Table 2 : AS-RNAi targeting multiple nucleotide changes responsible for dominant diseases.

 2

		/	da-SNP	siRNA	7/8	a	7 pu:pu		yes	[8]
		SCA3					8 pu:pu			[26]
					11		11 pu:pu
	ATXN3	MJD/	da-SNP	shRNA	11	a,i	pu:pu		yes	[47]
		SCA3
	ATXN3	MJD/	da-SNP	shRNA	11	e	pu:pu		yes	[51]
		SCA3
	ATXN3	MJD/	da-SNP	shRNA	11	i	pu:pu		Yes,	[50]
		SCA3
	ATXN3	MJD/	da-SNP	siRNA	9, 10	j	pu:pu		-	[65]
		SCA3
	ATXN7	SCA7	da-SNP	siRNA	16	j	pu:pu		yes	[54]
	ATXN7	SCA7	da-SNP	shRNA/miRNA	16	b	pu:py		yes	[39]
	CHRNA1	SCCMS	p.S226F	siRNA	10	a	pu:py		-	[9]
	CHRNA1	SCCMS	p.S226F	shRNA	9	a	pu:py		-	[9]
	COL1A2	OI	da-SNP	siRNA	7	j	pu:py		-	[66]
	COL3A1	vEDS	p.G252V	siRNA	10	j	pu:pu	Syst.	yes	[35]
								Screen.
	COL6A1	UCMD	p.G284R	siRNA	8, 8/13	j	8 pu:py, 13		yes	[43]
							py:py
	GNE	Sialuria	p.R266Q	siRNA	10	j	pu:py		yes	[67]
	HBB	SCA	p.E6V	siRNA	10	a, f	pu:pu		-	[57]
	HBV-P	HBV	p.M204I	siRNA	16	f	pu:pu		-	[72]
	HTT	HD	da-SNP	siRNA	4	j	pu:pu		-	[68]
	HTT	HD	da-SNP	siRNA	10	j	pu:py		-	[58]
	HTT	HD	da-SNP	siRNA	10	j	pu:py		-	[69]

Acknowledgements

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Association Institut de Myologie (AIM), the Université Pierre et Marie Curie-Paris6 (UPMC) and the Centre National de la Recherche Scientifique (CNRS). DT is recipient of an AIM fellowship. All authors wrote the review.

Conflict of interest

There is no conflict of interest to disclose.

Supplementary