4.1. Modifying Alternative Splicing to Increase Protein Production
Spinal muscular atrophy (SMA) is an autosomal recessive, degenerative, neuromuscular disorder that causes the loss of spinal motor neurons leading to muscle wasting [58
]. SMA is one of the most common causes of infant mortality, with a carrier rate of 1:50 and in incidence of 1 in 10,000 births [86
]. SMA develops due to low levels of survival motor neuron (SMN) protein, which is caused by deletions or inactivating mutations within the SMN1
is an almost identical gene to SMN1
, which differs by a single nucleotide at the beginning of exon 7 [87
]. This variation weakens a splice site in SMN2. Therefore, most of the time (85%–90%), SMN2
gene produces a transcript which lacks exon 7. When the shortened transcript is translated, SMN2 is expressed as a truncated, unstable protein (SMN2∆7) [87
]. The higher the amount of functioning, full length SMN protein produced, the less severe the disease [89
]. Therefore, genetic therapy focused on boosting SMN2 splicing to express a full length SMN protein.
, Biogen) is the only approved treatment for SMA in the USA and Europe (2017) [91
], due to patients experiencing improved motor function, a slowing of disease progression and few side effects. The progression of nusinersen into clinical use was well received by the field, as demonstrated by a standing ovation during the 2017 RNA conference, following the announcement it recieved FDA approval.
Nusinersen is an ASO that binds to a regulatory sequence in intron 7 of the SMN2 pre-mRNA molecule, a site that is normally occupied by the heterogeneous nuclear riboprotein (hnRNP A1/2), masking the regulatory sequences required for exon 7 splicing. The binding of nusinersin to this site, displaces the hnRNP A1/2 complex, promoting the inclusion of exon 7 in the mature SMN2 mature mRNA sequence, consequently increasing the levels functional SMN protein (Figure 3
Similarly, an ASO is also approved for use in Duchenne Muscular Dystrophy (DMD). DMD is a rare, X-linked recessive disorder characterised by a progressive loss of muscle tissue [94
], caused by deletions within the dystrophin gene. Deletions in this gene generates a premature stop codon, creating a truncated product which is degraded by nonsense mediated decay. Therefore, no functional dystrophin protein is produced in these cells. ASO therapy has focused on exon 51 in the dystrophin gene, redirecting the splicing machinery away from the exon, in order to restore the open reading frame of the mature mRNA transcript. This regulation of alternative splicing will generate a milder phenotype of the disease, although this is only amenable to 13% of these patients. Drisapersen, a 2′-O
-methyl phosphorothioate oligonucleotide, was the first exon-skipping ASO therapy for DMD in clinical development [96
]. However, patient improvement in phase 3 clinical trials was not sufficient for regulatory approval [98
]. Exondys 51TM
(Eteplirsen), a morpholino ASO that selectively binds to the exon-intron splice site at the beginning of exon 51, restoring the open reading frame, which results in the production of a truncated, but functional dystrophin protein. Due to limited clinical outcomes, Eteplirsen controversially received approval from the FDA for clinical use in 2016 [99
Several other ASO drugs are currently in clinical trials underpinning the great promise of future successful treatments using this tool, e.g., for Huntington’s disease (NCT02519036), amyotrophic lateral sclerosis (ALS) (NCT02623699) and acute non-arteritic anterior ischaemic optic neuropathy (NCT02341560).
4.2. Inhibiting Protein Production to Reduce Amyloidosis
In an exciting breakthrough for RNA-based therapeutics, the first short interfering RNA drug, Onpattro (patisiran) (Alnylam Pharmaceuticals), was granted approval for clinical use by the US FDA and within the EU in 2018 [100
]. Onpattro is a new treatment option for patients with hereditary transthyretin-mediated (hATTR) amyloidosis [102
], a rare, progressive, neurodegenerative and life threatening disease, with a median survival time of 4.7 years following diagnosis [104
]. hATTR amyloidosis is caused by mutations in the transthyrethin (TTR)
gene, which causes the TTR protein to misfold. The misfolded protein aggregates into amyloid fibrils which accumulates in multiple organs [105
], causing heterogeneous clinical presentations which include polyneuropathy and cardiomyopathy [106
]. TTR is mainly expressed in the liver and transports thyroxine and retinol-binding protein [108
]. Liver transplantation is the standard of care for hATTR amyloidosis. However, this treatment is limited to availability of donors and comes with risks of immunosuppression in the patients [109
Other treatment strategies which stabilise the TTR protein and prevent amyloid formation, have also been studied in hATTR amyloidosis patients. Difusinal, a nonsteroidal anti-inflammatory drug, increased survival time. However, patients suffered with serious side effects and, therefore, difusinal was not approved for clinical use. Tafamidis, a chaperone that stabilises the correctly folded TTR protein, delays degeneration of the neurons in hATTR amyloidosis and patients had an improved quality of life. Tafamidis is approved in Europe for treatment of hATTR amyloidosis.
Due to the limited success of stabilising the TTR protein, an alternative therapeutic strategy is to inhibit gene expression of TTR. Over 120 genetic variants of the TTR gene are associated with hATTR amyloidosis [110
], which makes an siRNA a viable option for drug development as it can be designed to target all TTR transcripts, regardless of any specific mutation [111
]. Onpattro is a lipid nanoparticle formulation of siRNA, delivered by intravenous injection, which is targeted to the hepatocytes. The siRNA binds the TTR transcript at a conserved site in the 3′ UTR of the TTR transcript, this triggers the cleavage, and subsequent degradation of the WT and mutated TTR transcripts (Figure 3
B). After Onpattro treatment, synthesis of TTR protein is reduced in a dose-proportional manner (as measured by serum TTR levels), preventing further amyloidosis and promoting the clearance of the fibrils already present in the cells [112
]. Clinical trials concluded that Onpattro resulted in significant improvement in patient’s quality of life and in clinical outcomes with 56% of patients showing an improvement after 18 months of treatment, in comparison to 4% of patients showing improvement with placebo treatment [111
]. Onpattro also showed a consistent safety profile [103
Another oligonucleotide-based drug for hTTR amyloidosis was also approved for clinical use in October 2018 in the EU and the US [115
] as patients experienced delayed neuropathic disease progression after treatment. Tegsedi (inotersen) (Ionis Pharmaceuticals/Akcea Therapeutics) is a chemically modified 20 mer antisense oligonucleotide (ASO), which is complementary to a conserved region of the 3’UTR of the TTR transcripts present in both the wild type and disease-causing variants. Tegsedi’s mode of action is distinct from Onpattro, as chemically synthesized ASOs bind in a 1:1 stoichiometry to a complementary section of mRNA, causing degradation of the bound transcript by RNAse H [117
]. RNAi, however, is catalysed by the Ago2 protein, and, therefore, one siRNA can cleave a large number of target mRNAs [118
]. However, in comparison to siRNAs, ASOs enter the target cell of interest with a higher efficiency.
While some siRNA drugs are successfully advancing through the drug development pipeline, others have been withdrawn from further development. An example of this is Bevasiranib (OPKO Health), a 5-methoxy (CH3
0) modified siRNA duplex designed to target vascular endothelial growth factor (VEGF), to treat diabetic macular edema or age-related macular degeneration (AMD) [119
]. Bevasiranib reached phase 3 clinical trials. However, this study was halted due to poor performance. Studies with altered drug regimens and combination therapies are reportedly in the preparation stages.
Similarly, AGN211745 (Allergan and Sirna Therapeutics) entered clinical trials targeting VEGF receptor I (VEGFRI) to treat AMD [121
]. However, this study was stopped before completion, despite acceptable safety reports of the drug, as patient improvement was less than expected.