Therapy Development For Spinal Muscular Atrophy: Perspectives For Muscular Dystrophies And Neurodegenerative Disorders Part 9

SOD1

The gene encoding Cu/Zn superoxide dismutase (SOD1) was the first identified mutation that causes fALS [62, 254]. 

Superoxide dismutase (SOD) is an important antioxidant enzyme that plays a role in maintaining redox balance within cells. Research shows that there is a close relationship between SOD and memory.

First of all, SOD can eliminate free radicals in the body, reduce oxidative damage to cell membranes, and protect the structure and function of neurons. This helps improve memory performance, especially in older adults. One study found that the levels of SOD in the elderly are closely related to their cognitive abilities, with higher levels of SOD expression associated with better cognitive abilities.

Secondly, SOD can also promote the generation of neurons and improve brain plasticity. This is very important for memory retention and enhancement. A study shows that SOD can stimulate the proliferation of neural stem cells in the hippocampus area of the brain and improve memory performance.

In addition, SOD can also reduce inflammatory responses and inhibit neuronal death. This also has a positive effect on preventing cognitive deterioration and disease.

To sum up, there is a close relationship between SOD and memory. In daily life, we can protect brain cells and improve memory performance by increasing the intake of SOD, such as eating more SOD-rich foods, such as blueberries, broccoli, etc. At the same time, maintaining a healthy lifestyle, such as moderate exercise and regular work and rest, is also beneficial to promoting the generation and maintenance of SOD. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of Cistanche deserticola comes from the many active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health through a variety of pathways.

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The majority of the SOD1 mutations (18.9%) correspond to fALS, while 1.2% coincide with sALS cases [334], both exhibiting a dominant inheritance pattern [6–8]. Most of the ALS-causing SOD1 mutations do not reveal any correlation between SOD1 enzymatic activity and ALS disease severity [48, 221, 269]. 

Loss of function for Sod1 in Sod1 gene knockout mice does not cause per se defects in motor axon elongation [142] or motoneuron degeneration [244]. In addition, the presence or absence of endogenous mouse Sod1 does not affect the survival of mice expressing the human SOD1G85R transgene [36]. 

This argues for a toxic GOF although SOD1-LOF might not be completely excluded. In a recently completed placebo-controlled phase 1/2/3 clinical study, SOD1 was targeted by the ASO Tofersen/BIIB067 (NCT02623699 December 8th, 2015 until March 24th, 2021) for silencing the mutant as well as the wild-type allele [201, 202]. This was a 3-part (A, B, C) study to examine the efficacy, safety, and tolerability of BIIB067. 

BIIB067 administration resulted in an approximately 36% suppression of SOD1 in the CSF which appeared safe for SOD1-ALS patients [201]. It remained open, whether this is sufficient for long-term suppression of toxic effects of mutant SOD1, although administration appeared safe. 

Only some lumbar puncture-related side effects have been reported [201]. The first two parts (phase 1/2) were primarily not designed for the assessment of motor function. However, they revealed that this treatment possibly slows disease progression and leads to better performances in vital capacity and hand-held dynamometry tests [201]. These effects have been pursued in phase 3 started in May 2019 to evaluate clinical efficacy. 

All types of SOD1 mutations and severity levels of symptomatic ALS patients were included. To explore the therapeutic effects of BIIB067 in pre-symptomatic ALS patients, again a placebo-controlled phase 3 study (which is currently in the recruitment status) was started on May 17th, 2021 (NCT04856982). The primary objective is to evaluate the efficacy of BII067 in pre-symptomatic ALS-SOD1 patients with elevated neurofilament levels. 

The secondary objectives include the evaluation of safety, pharmacodynamics, and treatment-response biomarkers. The estimated study completion date is the second half of 2027.

C9ORF72

At least in Europe and the US, the most common genetic cause of both ALS and Frontotemporal Dementia (FTD) is a GGGGCC (G4C2) hexanucleotide repeat expansion in the open reading frame 72 (C9ORF72) gene localized on chromosome 9. 

The mutation constitutes approximately 34% of fALS and nearly 25% of familial FTD cases (C9ALS/FTD) in European populations [302, 334]. Up to 25 G4C2 repeats are found in healthy individuals, while C9ALS/FTD patients harbor hundreds to thousands of repeats [60, 245, 253]. 

The expansion is located in intron 1 which also contains the promoter region of the second transcript for C9ORF72 [161, 204, 205]. Thus, reduced expression levels of the corresponding second transcript led to decreased C9ORF72 protein levels in such patients. 

However, this intronic expansion region is also translated via a non-canonical form of protein biosynthesis (repeat-associated non-AUG [RAN] translation) [15, 212, 335]. Based on the observations of RNA-foci or aberrant RNAs as well as the production of toxic homopolymeric dipeptide repeat proteins (DPRs) through RAN translation [94, 163, 205, 212, 286, 290, 315], a GOF mechanism appears to take place. 

Antisense RNA-foci are known to sequester RNA-binding proteins (RBPs), leading to LOF of RBPs in corresponding neurons [163, 168, 189]. Depletion of C9orf72 in isolated mouse motoneurons leads to alterations in axon growth and presynaptic differentiation [278]. 

This phenotype is also observed in C9ORF72 ALS patients' inducible pluripotent stem cell (iPSC)-derived motoneurons and resembles some of the alterations that are observed in cell culture models of SMA. Based on these findings, a combined therapeutic approach with silencing the G4C2-repeat-containing RNAs and simultaneous increase of C9ORF72 expression by gene therapy has been proposed for C9ORF72 patients [101]. 

The ASO BIIB078 that targets the sense-strand of C9ORF72 transcripts containing the hexanucleotide G4C2 repeat has been tested for safety and tolerability in a phase 1 clinical trial with adult C9ORF72 ALS patients (NCT03626012). 

The study is still active with an estimated completion date end of 2021. The trial already started to be followed by a phase 1 extension study (NCT04288856) to assess long-term safety, tolerability, pharmacokinetics, and effects on disease progression of BIIB078 application to previously treated C9ORF72 patients. The study is still enrolling with an estimated completion in the middle of 2023.

FUS

Mutations in FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma) are a genetic cause for rare forms of fALS and FTD [35, 162, 303, 304]. FUS mutations are present in 4% of fALS patients and less than 1% of sALS patients [61, 334] with an autosomal dominant inheritance pattern. 

The ubiquitously expressed DNA-/ RNA-binding protein FUS localizes predominantly to the nucleus under physiological conditions [333]. FUS is involved in DNA repair [313] but also acts as an RNA-binding protein in several aspects of RNA metabolism including transcriptional regulation [188, 288], alternative splicing [121, 135, 164, 252], mRNA transport [96], mRNA stability [145, 297, 327], and microRNA biogenesis [105, 213]. 

Toxic GOF and LOF due to FUS aggregation and cytoplasmic mislocalization play a role in FUS-ALS/FTD pathogenesis [150]. An ASO-based therapeutic approach has been initiated for ALS caused by a specific FUS mutation (P525L) that is associated with an aggressive form of ALS with juvenile-onset. A tree of such FUS-ALS patients has received Jacifusen, a personalized ASO [13]. 

A FUS-ALS patient who already had developed respiratory problems received this personalized ASO treatment and died one year later [14]. The preliminary results from this case implicate, that adverse effects might also emerge by knocking down the wild-type FUS transcript. FUS interacts as an RNA-binding protein with transcripts from about 5500 genes [164]. 

Thus, knocking down FUS via an ASO approach could interfere with the turnover of RNAs with long introns, many of which especially encode for synaptic proteins [164]. Downregulation of such transcripts and corresponding proteins in rodent primary neurons causes morphological alterations such as enlarged growth cones [225], shorter neurites [134, 225], abnormal dendritic spines [95, 327], and altered neurotransmission [297].
In vivo, knockdown of FUS in murine hippocampal neurons causes increased phospho-tau accumulations as well as decreased neurogenesis, and thus an FTD-like phenotype [134, 297]. These data ultimately require enhanced efforts in the exploration of therapeutics that specifically target FUS expression. 

Jacifusen is scheduled to be given to eight additional patients with FUS mutations (Figueiredo, M. (2020)- Collaboration Funds Experimental Therapy for Rare FUS-ALS, accessed 3.28.20. https://alsnewstoday.com/ 2020/03/16/jacifusen-collaboration-funds-experimentaltherapy-for-patients-with-rarefus-als/). Additional trials are planned with ION3763-CS1 which also targets this FUS mutation. 

Recruitment for this trial will start in June 2021, and there are so far no clinical data available. The relevant gene therapies for SOD1-, C9ORF72-, and FUSALS are depicted in Fig. 4.

Genetic modifiers in ALS

Ataxin-2 is a protein encoded by the ATXN2 gene. Expansion of the polyglutamine tract in the human ATXN2 gene leads to spinocerebellar ataxia type 2 (SCA2) [131, 238, 262]. SCA2 is characterized by neuronal degeneration in the cerebellum and inferior olive that causes ataxia, parkinsonism, and dementia. More than ten years ago, it was discovered that this CAG repeat expansion is associated with a higher risk for ALS [76]. 

Since 2017, it has been known that reduced levels of Ataxin-2 via ASO approaches extend the life span and diminish functional and behavioral defects in a TDP-43-ALS mouse model [21]. 

In September 2020, recruitment for a study of ALS patients with or without CAG expansion in the ATXN2 gene was started to assess the safety, tolerability, and pharmacokinetics of the Ataxin-2 ASO termed BIIB105. The estimated study completion date is February 2023. The study is listed at https://clinicaltrials.gov.

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