Therapy Development For Spinal Muscular Atrophy: Perspectives For Muscular Dystrophies And Neurodegenerative Disorders Part 10

ASO treatments for muscular dystrophies

Duchenne's muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD), the two forms of X-linked muscular dystrophy are caused by mutations in the Dystrophin gene. DMD occurs with an incidence of about 1:5000, whereas BMD affects children with an incidence of 1:30,000 [155]. 

Duchenne muscular dystrophy is a rare genetic disease that causes muscle weakness, fatigue, stiffness, and other symptoms. However, most patients can still maintain their lives and exercise abilities through drug treatment and rehabilitation training, and their relationship with memory may not be greatly affected.

Many people with Duchenne muscular dystrophy can demonstrate excellent memory skills in school, work, and life. Research shows that this has a lot to do with their self-management abilities and coping strategies. Because the disease causes muscle fatigue and weakness, patients often need to be more organized about their time and activities to make the most of their most active hours. This ability also exercises their self-control and self-management abilities, which in turn helps improve their memory and learning abilities.

Therefore, although Duchenne muscular dystrophy will have a certain impact on people's physical health, it does not mean that patients will inevitably lag behind ordinary people in terms of memory and learning abilities. On the contrary, they tend to have stronger advantages in self-management and coping strategies, which also helps them achieve better results in study and workplace competition. Therefore, we should treat this disease and patients with a positive attitude, and give them more support and encouragement, so that they can have a healthier and fulfilling life. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche deserticola can also promote the growth and repair of nerve cells, thus enhancing the connectivity and function of neural networks. These effects can help improve memory, learning, and thinking speed, and may also prevent the development of cognitive dysfunction and neurodegenerative diseases.

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The severe form, DMD, usually starts before 4  years. Affected boys lose ambulation around 12 years of age and get ventilation by the age of 18 [119]. The average life span today is 20–30 years due to advances in cardiac and respiratory care. 

In contrast to SMA, the spectrum of mutations in DMD is broad and ranges from point mutations to deletions as well as small insertions to large duplications [24]. The dystrophin gene is one of the largest human genes containing 79 exons and approximately 2.4 million base pairs [2, 24, 41]. The primary role of the dystrophin protein is to link the actin-cytoskeleton with the extracellular matrix in cardiac and skeletal muscles by forming interactions with the subsarcolemmal actin and the large oligomeric dystrophin–glycoprotein complex (DPG). 

This regulates the proper functioning of muscle fibers. Defects of the DPG result in muscle weakness due to contraction-induced damage, necrosis, and inflammation, and a replacement of functional myofibers by fibrous and fatty connective tissue [29]. 

Disease severity depends very much on residual functions of the truncated dystrophin protein, which is derived from the mutant gene. Interestingly, mild forms of the disease have not only been detected in patients with point mutations which have only minor consequences for protein structure and function but also in BMD patients in which multiple exon-encoded domains are not transcribed, resulting in a highly truncated dystrophin mRNA of only 8.8  kb [79]. 

This observation has paved the way to defne essential subdomains within the dystrophin protein and exons within transcripts which are functionally important and need to be present for mitigating disease severity. Similarly, these findings are the basis for the design of artificial mini-dystrophin genes which could be used for gene therapy through viral vectors that can only carry cDNAs of limited length [52].

Exon skipping

Retention or preservation of the open reading frame (ORF) is an option to restrict the physiological consequences of dystrophin loss in DMD patients with nonsense mutations. Retention of the ORF can be mediated via ASO delivery. 

The binding of the ASOs to the dystrophin pre-mRNA transcript induces deletion/skipping of certain exon(s) and can thus restore the ORF [72]. The resulting shorter ORF produces a phenotype similar to that described for the milder form of DMD-the BMD. Tree ASOs with phosphorodiamidate morpholino oligomer (PMO) backbone which are used for exon skipping-elegize (exon 51), Goodison (exon 53), and viltolarsen (exon 53)-have been approved by the FDA. 

However, the ASOs also show some limitations [75, 306] [1]. Since DMD patients have variants in many different exons that cause multiple reading frame disruptions, such single-exon treatments are only applicable to a subset of DMD patients. 

Multi-exon skipping has been proposed to overcome the limited scope of single-exon skipping by targeting DMD patients with variant exons [16]. A cocktail of ASOs targeting the variant hotspots of exons 45–55 can efficiently skip these exons in both immortalized DMD patient muscle cells and mouse models [73, 167]. 

However, a mixture of several ASOs also has a higher risk for off-target effects. This needs to be tested in clinical trials. If this funding is successfully translated to the clinic, it could potentially be useful for more than 65% of DMD patients [74].

Genome editing

Genome editing with CRISPR-Cas9 appears as an attractive option for ORF restoration of the dystrophin gene. Application of CRISPR-Cas9 does not require re-injections because DNA instead of pre-mRNA is targeted. This approach could also be useful for treating patients with duplications in certain exons of the DMD gene since it allows the removal of extra exons and other gene insertions. 

The use of a multiplexed guide-RNA (gRNA) targeting the variant-prone exons 45–55 or 47–58, has been shown to restore dystrophin expression [306] in cultured patient-derived myoblasts. When these myoblasts were implanted into mice, expression was maintained [226].
However, because Cas9 induces double-strand breaks (DSBs) by the gRNA in a targeted manner, of-target DNA-cutting remains an issue. Currently, there are no clinical trials using genome editing approaches for DMD [75].

Perspectives of AAV‑based gene therapies beyond SMA

In the case of mutation-based LOF, relevant therapeutic approaches need to re-establish gene function. In the case of SMA, the scAAV9-SMN1 gene therapy provided proof that this approach is feasible for the treatment of neurodegenerative disease. 

The dose of 1.1× 1014 vg/kg body weight appeared sufficient to transduce the gene into a clinically relevant number of motoneurons and to keep adverse effects such as severe acute liver injury at a low level. 

An increase in liver transaminase levels has been discussed as a consequence of a massive immune response against viral particles [195]. Unfortunately, in the case of another neuromuscular disorder-X-linked myotubular myopathy-the systemic delivery of a high dose of AAV8 particles containing the cDNA for myotubularin-1 was fatal. Two of six patients who received a dose of 2× 1014 mg/kg or more died of progressive liver dysfunction followed by sepsis; it is presumed that AAVs directly damage liver cells [117]. 

Tus, AAVs are on one side effective gene transfer vehicles but on the other side bear the disadvantage of severe inflammatory reactions, particularly in systemic treatments with high doses. Strategies have been proposed to identify patients at risk for severe side effects [57, 58], and to overcome this problem by modulating the immune reaction towards a dampened response. 

This could be achieved either by depleting immunoglobulins via plasmapheresis; or even more specifically, by use of the IgG cleaving metalloproteases IdeS or IdeZ [63]. Such approaches to reduce AAV-autoantibodies could help to reduce the side effects of AAV-based gene therapies for disorders such as DMD where systemic treatment with high numbers of virus particles is necessary.
Furthermore, it can be beneficial for therapies for adult-onset disorders when patients are expected to have developed high AAV-antibody titers due to multiple previous exposures to such viruses. Another strategy could be local injection, either by intrathecal application of the recombinant viruses or injection into the cisterna manga. 

This approach is currently followed with an AAV1-based gene therapy trial to increase the expression of Progranulin in patients with frontotemporal dementia with granulin mutations [120] (Press release January 28th, 2021: Passage Bio-Passage Bio Receives FDA Clearance of IND Application for PBFT02 Gene Therapy Candidate for Treatment of Patients with Frontotemporal Dementia with Granulin Mutations). 

It will show how the immune system reacts when AAV particles are injected into the CSF, how many brain cells can take up the viral particles to produce the transgene, and which levels of transgene expression are necessary for a clinically relevant effect. 

In a study reported by Mueller et al., two ALS patients were treated with a single intrathecal infusion of AAVrh10 containing microRNAs to target SOD1. Downregulation of SOD1 transcripts and protein was identified in spinal cord autopsy samples via Western blot in one of these patients. 

The same patient showed transient improvements in the strength of his right leg but no change in vital capacity, whereas the second patient maintained a stable vital capacity over the 12-month observation period [214]. 

The authors proposed that intrathecal infusion of AAV-delivered microRNAs for SOD1 might have the potential for sustained beneficial effects, but possibly require immunosuppression.

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