Page 34 - Read Online

Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01 Page 25 of 35

differences with the endogenous human gene, thus allowing it to be discriminated and untargeted by the
therapeutic silencing ASO, siRNA, or shRNA. Additionally, codon optimization might also contribute to
ensuring a high expression level of the wild-type protein.

Oculopharyngeal muscular dystrophy (OPMD) is a rare muscle disease characterized by an onset of
weakness in the pharyngeal and eyelid muscles. Inherited in an autosomal dominant mode, the disease is
found on all continents, with several clusters identified in Quebec and Israel. The clinical diagnosis is
confirmed by a genetic test which in most cases shows an 11-18 expansion of GCA or GCG triplets in the
gene of Poly(A) Binding Protein Nuclear 1 (PABPN1) on chromosome 14. The disease is caused by the
extension of the polyalanine tract in the PABPN1 protein leading to the gain-of-function formation of
intranuclear inclusions or aggregates in the muscle of OPMD patients, which are the hallmark of the
[157]
disease . Although PABPN1 is ubiquitously expressed and l contributes to control gene expression in all
tissues, playing key roles in post-transcriptional processing of RNA, the PABPN1 variant pathological
phenotype is restricted to a limited set of skeletal muscles affected in OPMD. The exact pathophysiological
process leading to the localized pathology and the precise role of intranuclear aggregates are still unclear .
[158]
In a gene therapy approach, two AAV vectors were co-delivered, the first expressing a cassette including a
triple shRNA under the control of RNA polymerase III promoter and the second expressing human codon-
optimized PABPN1 under the control of a skeletal and cardiac muscle-specific promoter. This treatment
significantly reduced the number of myonuclei containing PABPN1-positive insoluble intranuclear
inclusions, showed significant improvements in several histopathological features (muscle regeneration,
fibrosis, and muscle force), and almost completely normalized the transcriptome muscles of A17 mice to
that of wild-type mice .
[159]

Subsequent work managed to condense the two AAV vectors into one and reported reversion of already
established insoluble aggregates and partial muscle rescue from atrophy, which are both crucially important
since, in most cases, OPMD patients already have an established disease when diagnosed. Also reported
were the prevention of the formation of muscle fibrosis and stabilization of muscle strength to healthy
[160]
muscle levels . Recent progress towards a clinical trial described a unique single bifunctional construct
under the control of a muscle-specific promoter for the co-expression of both the codon-optimized
PABPN1 protein and two siRNAs against PABPN1 modeled into microRNA (miRNA) backbones. A single
intramuscular injection of the AAV9 vector in a murine model resulted in the inhibition of mutant
PABPN1 and in PABPN1 replacement, leading to restoration at a normal level of muscle strength and other
[161]
muscle parameters .

Challenges faced by the synthetic ASO and siRNA technology
RNA drugs face several challenges and bottlenecks. The first one concerns pharmacodynamic/
pharmacokinetic (PK/PD) properties. Except for the liver, delivery of RNA drugs to other organs and
tissues is still not ideally performed. Lack of proper delivery of sufficient amount into muscles of the
Eteplirsen morpholino ASO might be responsible for its suboptimal performance [7,87] , and CNS delivery of
RNA drugs still necessitates intrathecal, intraventricular or intracerebral administration, which is clinically
demanding and associated with infection risk. For muscular and neuromuscular indications, a novel
muscle-targeting platform obtained by conjugation of siRNAs with anti-CD71 Fab' fragment has been
reported [134,162] . This conjugate led to one-month durable gene-silencing in the heart and skeletal muscle after
IV administration in normal mice, and to significant gene-silencing when injected intramuscularly. In a
mouse model of peripheral artery disease, the intramuscular administration of an anti-myostatin siRNA
resulted in significant silencing of myostatin in muscle and led to the recovery of the running performance.