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Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01 Page 27 of 35

Other challenges for the future of synthetic ASO and siRNA drugs originate from the rapid advance of
alternative techniques such as viral gene therapy for delivering a shRNA, which leads to the same effect as
ASO and siRNA in terms of steric blocker for exon skipping or translation inhibition, and for inducing
mRNA cleavage/degradation. Indeed, at the present time, most proofs of concept for RNA drugs have been
obtained at the laboratory scale by specifically designed AAV or lentivirus vectors. However, this gene
therapy approach is less flexible than synthetic ASOs and siRNA because no conditional expression system
has been clinically approved at the present time, which renders hazardous the control of gene therapy
treatment termination. Moreover, synthetic compounds might be preferable in terms of production costs,
and finally, synthetic RNA drugs can be administered repeatedly without initiating any immune response,
whereas distinct viral serotypes must be used for multiple-dose administration.

Genome editing is a rapidly evolving technology. Targeted DNA double-strand breaks (DSBs) using
CRISPR-Cas9 have revolutionized genetic intervention by enabling efficient and accurate genome editing in
a broad range of eukaryotic systems. Multiple applications are actively investigated, such as targeted
knockout of dominant negative pathological genes or viral genomes (either integrated or episomal).

[4]
In 2021, a clinical trial involving the in vivo use of CRISPR-Cas9 in humans was disclosed . The in vivo
gene-editing therapeutic agent NTLA-2001 consists of lipid nanoparticles LNP containing a single guide
RNA (sgRNA) that targets the human TTR gene. A human-codon-optimized mRNA sequence of
Streptococcus pyogenes Cas9 protein was added to the LNP formulation. Preclinical results of IV NTLA-
2001 were impressive, with a durable 95% drop in TTR concentration in monkeys. The genome-editing
drug was then administered to patients with hereditary ATTR amyloidosis with polyneuropathy. The
treatment was associated with only mild adverse events and led to a decrease in serum TTR protein
concentration resulting from the targeted knockout of the TTR gene.

Multiple other genome editing preclinical studies are promising, for instance, in myotonic dystrophy
[171]
targeting the DMPK gene [see Figure 12] . Since LNPs can efficiently deliver CRISPR-Cas9 to the liver,
many ASO and siRNA indications might eventually also be treated by genome-editing technology.
However, more time is necessary to assess the safety of this approach in the long term, especially concerning
off-target effects, genotoxicity, and germ-line modification, since CRISPR-Cas9 makes cuts in the genomic
DNA in contrary to ASOs and siRNA, which target mRNA. Also, genome editing represents an irreversible
therapy through a one-shot knockout of the targeted gene. Thus, the pros and cons of this technology must
be weighted for each specific indication in comparison to reversible synthetic RNA drugs.

CONCLUSION AND PERSPECTIVES
The examples detailed here illustrate the wide and versatile capacities of antisense therapies in muscular and
neuromuscular disorders. A large additional number of these diseases could potentially benefit from RNA
drugs. Without being exhaustive, one can mention several other diseases where specific silencing has shown
benefit in preclinical models. In facioscapulohumeral muscular dystrophy (FSHD), the knockdown of the
FSHD region gene 1 (FRG1) was achieved using miRNAs delivered using an AAV vector system . The
[172]
AAV2/9-mediated delivery of an shRNA targeting the Pmp22 mRNA and injected in the sciatic nerve
[173]
prevented the development of pathological features in a rat model of Charcot-Marie-Tooth disease 1 A .
An allele-specific RNA interference using an AAV9 has been described in a Charcot-Marie-Tooth disease
type 2D mouse model. RNAi sequences targeting the dominant mutant of glycyl-tRNA synthetase (GARS)
mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This
prevented neuropathy in mice treated at birth. However, delaying the treatment until after disease onset
drastically reduced the benefit of gene therapy, and the therapeutic effect decreased with the delay in