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Page 24 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

(> 85%) was detected throughout the brain in an HD mouse model with this technology [62,145] .

Autosomal dominant centronuclear myopathy (AD-CNM, ORPHA 169 189) is a rare congenital myopathy
characterized by numerous centrally placed nuclei on muscle biopsy. Clinical features are those of
congenital myopathy: hypotonia, distal/proximal muscle weakness, rib cage deformities sometimes
associated with respiratory insufficiency, ptosis, ophthalmoparesis, and weakness of the muscles of facial
expression with dysmorphic facial features.

AD-CNM results from heterozygous mutations in the DNM2 gene, which encodes dynamin 2, and to date,
37 mutations (mainly missense) have been identified [146,147] . Dominant DNM2 mutations also cause rare
cases of Charcot-Marie-Tooth peripheral neuropathy (CMT) and hereditary spastic paraplegia . The
[149]
[148]
DNM2 protein belongs to the superfamily of large guanosine triphosphatases (GTPases) and is involved in
endocytosis and intracellular vesicle trafficking through its role in the deformation of biological membranes,
particularly in muscle cell T-tubule biogenesis [146,150] . The role of DNM2 as a regulator of actin and
microtubule cytoskeletons has also been reported. A potential AD-CNM pathophysiological mechanism is
the formation of abnormally stable polymers by mutant Dynamin 2 .
[151]
A single copy of the wild-type allele in heterozygous knockout mice expressing 50% Dnm2 displays a
healthy wild-type phenotype . Moreover, homozygous DNM2 mutation reported in 3 consanguineous
[152]
patients leads to a lethal congenital syndrome associating akinesia, joint contractures, hypotonia, and
skeletal abnormalities, together with brain and retinal hemorrhages . These and other data point to the
[153]
necessity of an allele-specific siRNA drug targeting only the dynamin 2 variant, in order to maintain 50% of
dynamin 2 production.

The proof of concept of silencing the variant mRNA without affecting the wild-type transcript has been
obtained in a mouse model with an shRNA gene delivered by an AAV virus . Complete rescue of the
[146]
muscle phenotype was maintained for at least 1 year after a single injection of the shRNA AAV, leading to a
maintained reduction of the variantDnm2 transcript. This study also suggested a new potential
pathophysiological mechanism linked to mutant protein accumulation with age, which does not occur in
wild-type animals, and which can be prevented by the shRNA treatment.

Further progress was obtained by screening a library of siRNAs targeted to specific heterozygous SNPs
associated with the DNM2 dominant-negative variant gene in the AD-CNM patient population. About 75%
of patients could be covered by four different siRNAs targeting the four SNPs most frequently associated in
a heterozygote manner with the AD-CNM patient population.

One allele-specific siRNA strategy is now in a clinical trial for the treatment of pachyonychia congenital, a
rare skin disorder linked to a dominant negative mutation in keratin 6a [154,155] . More considerations on the
allele-specific siRNA technology have been developed recently .
[156]

Combined RNA drug silencing and replacement gene therapy for dominant-negative
oculopharyngeal muscular dystrophy
For the gain-of-function disorders related to triplet expansion which have been discussed in the preceding
section, an alternative approach has been proposed which does not require the presence of specific SNP on
the heterozygote variant allele. The strategy is based on the simultaneous knockdown of both variant and
wild-type endogenous mRNAs, together with the administration of a gene encoding the wild-type protein.
The replacement gene sequence must be codon-optimized using the genetic code degeneration to carry