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Page 22 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

Figure 11. Tri-GalNac chemical group addressing RNAi to the hepatocyte asialoglycoprotein receptor ASGPR. The tri-GalNac is
covalently linked to the 3’ end of the siRNA sense strand, so as not to interfere with capture by RISC of the complementary antisense
strand necessary for mRNA cleavage.

number of repeats correlates with disease severity .
[128]

Variant DMPK mRNA form hairpin structures made from CUG repeats and assembling in well-
characterized nuclear foci. The molecular mechanism of DM1 pathology results from the toxicity of these
nuclear foci, which bind and sequester proteins of the family muscleblind-like (MBNL), thus interfering
with their natural splicing functions [129,130] . The mis-splicing reactions due to insufficient availability of the
MBNL protein in the nucleus induce a fetal-like pattern in adult DM1 cells, which concerns a multiplicity of
proteins, such as muscle chlorine channel, insulin receptor, cardiac troponin T, bypass integrator 1, skeletal
rapid troponin T, dystrophin (DMD) and cardiac sodium channel SCN5A. This spliceopathy leads to
[131]
myotonia and other clinical symptoms .

ASO and siRNA strategies have been intensively studied on DM1 cellular models such as patient fibroblasts
and mouse or drosophila models containing up to 1000 CTG triplets in DMPK gene, and in clinical trials
with a gapmer ASO containing constrained ethyl (cEt) locked nucleic acid (LNA) [132,133] . A recent clinical
trial was initiated in 2022 with DYNE-101, a muscle membrane antigen-binding fragment antibody (Fab)
conjugated to an ASO to enable targeted muscle tissue delivery [134,135] .

The multiple strategies displayed in Figure 12 illustrate that DM1 represents a model disease case where
multiple RNA drug interventions can be envisioned, and this either separately or in combination. Since
MBNL proteins are sequestered by poly CTG DMPK nuclear foci, the use of ASO or siRNA carrying a poly
ACG triplet might lead to DMPK nuclear foci cleavage and degradation. Alternatively, an ASO can also
function as a steric blocker of MBNL binding, thus releasing the necessary amount of MBNL for restoring
adult phenotype splicing pattern [Figure 12A and B] [130,131,136] . A second strategy is to target the miRNA
(miR)-23b, which has been shown to negatively control MBNL expression. Thus, the administration of a
blocker ASO complementary to (miR)23b would up-regulate MBNL expression and potentially compensate
[137]
for the sequestration of MBNL by variant DMPK mRNA [Figure 12C and D] . Finally, since MBNL
sequestering and lack of availability induces mis-splicing towards the fetal phenotype of various proteins,
splice correctors ASOs, similar to the one described in Figures 2 and 9 for SMA, represent an appealing
strategy to restore the expression of the adult form of the critical mis-spliced proteins [Figure 12E and F].
Such a downstream approach has been successfully reported using a morpholino ASO to restore the muscle
chloride channel. This was achieved in electroporated muscle fibers or using ultrasound-enhanced
[138]
delivery of morpholino with bubble liposomes .
[139]
Allele-specific silencing for centronuclear myopathy
In gain-of-function dominant-negative disorders, an ideal strategy would be to specifically target for ASO or
siRNA degradation the variant sequence on the pre-RNA and mRNA. However, this would require a
personalized ASO or siRNA drug for each variant. This might not be feasible for several pharmacological