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Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01 Page 23 of 35

Figure 12. Different ASO- and siRNA-based strategies against Myotonic Dystrophy Type 1. A: MBNL proteins are sequestered by
nuclear foci formed by the mRNA from the poly CTG DMPK variant. B: Poly ACG ASO or siRNA lead to DMPK mRNA cleavage and
nuclear foci degradation. Alternatively, they can also function as a steric blocker of MBNL binding to the foci, thus releasing the
necessary amount of MBNL to restore the adult phenotype splicing pattern. C, D: Since the miRNA (miR)-23b has been shown to
repress MBNL expression, the administration of antagomir-23b antagonizes the miRNA (miR)23b which increases MBNL expression,
thus compensating for the sequestration of MBNL by variant DMPK mRNA. E: The lack of free MBNL induces mis-splicing toward the
fetal phenotype of various proteins. F: Splice correctors ASOs can restore the adult form of the mis-spliced proteins.

reasons including toxicity linked to the off-target effect of some of such specific ASO or siRNA, and cost
considerations. In addition, this strategy is not usable for genetic diseases linked to gene duplication or to
triplet repeat expansion such as Huntington’s disease (CAG repeat expansion) or spinocerebellar ataxia
subtypes (CAG or CTG repeats), in which the available variant sequences that could be targeted are present
on both the wild-type and the mutated gene.

An ASO or siRNA targeting a non-variant sequence on the pathological gene is the most natural way to
provide an RNA drug that could ideally treat all patients, independently of their specific variant genotype.
However, this leads to complete repression of both the wild-type and dominant-negative variant alleles.

The above strategies cannot be applied if at least a minimal expression of the wild-type protein is required,
and then allele-specific silencing must be sought . Allele-specific silencing can be achieved by targeting
[140]
one or several single nucleotide polymorphisms (SNP) associated with a variant allele in the patient
population. This approach is only feasible if common specific SNPs can be identified in a high percentage of
the various mutated alleles in the diseased population. This strategy has been followed in Huntington’s
disease (HD) and spinocerebellar ataxia [141,142] .

Epidemiological studies on the HD patient population and targetable SNPs suggest that 80%-85% of HD
patients could be treated with panels of 2 to 5 SNP heterozygosities, meaning that only the expanded variant
allele possesses these 2 to 5 SNPs in these individuals [141,143,144] . Exciting recent preclinical results obtained
with a brain-targeted di-siRNA have been reported with fully chemically modified, therapeutically
translatable siRNAs targeting SNP heterozygosities specific to Huntingtin variants. It allowed a 50-fold
discriminative power on the huntingtin variant genetic allele in a cell-based assay on human neurons
derived from human Huntington chorea patient iPSCs. This optimized si-RNA was obtained by repeated
targeted screening and chemical optimization. Selective silencing of the mutant huntingtin HTT allele