Therapeutic tools for familial ALS

William Camu, Elisa De La Cruz, Florence Esselin

Explorations Neurologiques et Centre de référence SLA, Univ Montpellier, CHU Gui de
Chauliac, INM, INSERM, Montpellier, France

**Correspondance to :**

Pr William Camu, Clinique du Motoneurone, Explorations Neurologiques, CHU Gui de
Chauliac, 34295 Montpellier Cedex 5, France.

Email: w-camu@chu-montpellier.fr

tel: +33467330281; fax: +33467338136

**Conflict of interest:** none
Familial ALS (FALS) accounts for 10 to 15% of ALS cases. In more than 70% of FALS patients, a causal gene is identified and animal models have been developed for a subset of them, mainly for the most frequently mutated genes. Therapeutic tools to treat those patients are dominated by gene-specific therapy and the most advanced approaches target the *SOD1* gene mutations. Either by direct delivery of antisense oligonucleotides (ASO) or using viral vectors such as adenoviruses (AAV) to deliver ASOs, gene specific therapies have shown promising results in animal models. The recent use of subpial injections of AAV9+anti SOD1 ASO now shows that the disease is completely prevented or stopped in the animal, depending on the moment of injection, e.g., before or after disease onset. However, the use of viral vectors in humans seems to be limited at least by their immunogenicity. Antibody-based therapies are also efficient to treat animal models, but to a lesser extent. Most of the experiments targeted the SOD1 protein in its misfolded conformation. This approach seems better tolerated than the AAV one, an important limit being the choice of the epitope.

Unexpectedly, some advances in treating the C9ORF72 animal model have been obtained using a modulation of microbiota, and this strategy has the great advantage to have an easy route of administration and a good safety profile. The landscape of experimental FALS treatment is rapidly evolving and results are promising. This is an important unmet need for ALS patients and several human phase I, II and III trials are ongoing.
# 1. Introduction

Familial amyotrophic lateral sclerosis (FALS) accounts for 10 to 15% of ALS cases [1, 2]. To date, a causal gene is identified in almost 70% of the FALS cases but recent works suggested that 5 to 10% of ALS cases initially considered as sporadic may also carry a pathogenic or likely pathogenic gene mutation [3,4]. The large majority of the inherited cases carry a pathological repeat expansion in the *C9ORF72* gene (C9RE). Although the percentage may vary between countries and continents (rare in Asia), it represents at least 40% of the FALS cases in France, Europe and North America [2]. The second most frequent gene responsible for FALS cases is *SOD1*, accounting for 15 to 25% of the patients. Other gene mutations have been identified, but at a much lower frequency. While *FUS* and *TARDBP* may account for 5% of FALS patients, pathogenic mutations in the numerous other genes known to be associated with FALS are only anecdotally identified. Despite these advances, the disease remains devastative without any cure, to date. However, the discovery of causal genes now offers the possibility to evaluate different strategies that specifically target mutated genes.

# 2. Therapeutic tools, preliminary aspects

To develop a therapeutic tool for genetically-determined ALS, one first step is to identify a pathogenic mutation. The description of pathogenic mutations in patients initially considered as sporadic is important and suggests that systematic genetic screening should be done in all ALS patients, with or without family history. One can consider that in the near future, developing strategies for a large and systematic genetic screening of all ALS patients by next generation sequencing (NGS) could represent the premise for developing therapeutic tools, as it will help in identifying unexpected candidates for newly developed treatments. Following the same trend, the GENIALS study (NCT04819555), which prospectively included 1000 newly diagnosed ALS patients from French ALS centers, to systematically search for C9RE
and *SOD1* gene mutations, will help to extend our knowledge about the respective proportion of genetically determined ALS cases.

The interest of finding a pathogenic mutation is to give physicians/researchers a potential therapeutic target. Such a target may be the gene, the gene expression processes, the protein itself, or the metabolic pathways involved. In ALS, a great part of research is now focusing on gene therapy. The action on some metabolic pathways may not be specific of FALS, such as antioxidant therapies or autophagy modulators and this particular aspect will thus not be treated here. Conversely, monoclonal antibodies have been developed to treat animal models and showed interesting results that warrant some attention. Modulation of the microbiome is one new approach in the field of FALS and primary results from investigations will be also described. However, in each of these therapeutic fields, only few human trials have given published results.

### 3. Gene specific therapy

Gene therapy has primarily aimed at targeting pathogenic gene mutations, but the modulation of other genes is also now being developed. Numerous genes have been described in ALS families since 1993, date of the pioneering description of superoxide dismutase-1 gene (*SOD1*) mutations. To date the four main genes (*C9ORF72*, *SOD1*, *TARDBP* and *FUS*) account for more than 90% of known pathogenic mutations. However, research development in gene therapy is not surprisingly focused on *SOD1*, as mutations in this gene have been the basis of ALS animal models since 1994 [5].

For all four major genes responsible for inherited ALS, a toxic gain of function has been described [6]. Even though the real influence of C9RE seems more complex than just a gain of function, it has been shown in all four genes that lowering or blocking expression of the gene had experimentally favorable consequences, both in vitro and in vivo. Thus, any
molecular tool that may limit or suppress the expression of the mutation is of interest. Gene therapy is the delivery of genetic material to the cell to either silence a pathogenic gene, introduce functional copies (e.g. SMN1 in infantile spinal muscular atrophies) or introduce disease-modifying genes, such as neurotrophic factors. The silencing strategy has been the most studied approach [7]. The silencing material may be delivered directly to the central nervous system (CNS), as is the case of antisense oligonucleotides (ASO) that can be injected intrathecally in humans. Viral vectors may be used to deliver ASOs, but also RNA interference (RNAi) and gene-editing (CRISPR) strategies.

## 3.1 Direct delivery of ASO to the CNS

ASOs are single-stranded nucleotide oligomers designed to alter mRNA expression. Two main ways can be used to reach this goal. One is to target mRNA for RNase H enzyme-mediated degradation. The other is to design the ASO against transcripts to induce or modulate alternative splicing. The ASO construct should optimize stability, diffusion into the cells, and immunogenicity. Those molecules cannot cross the blood-brain barrier so that treatments should be directly delivered into the CNS. Several ways can be used for this delivery: intrathecal, intracerebroventricular, or intraparenchymal. Using an anti-SOD1 ASO antibodies, it was shown that the ASO could be detected in the anterior horn and in the supratentorial structures of the animals [8]. An 80% knockdown of SOD1 mRNA and SOD1 protein was observed and paralleled by improved survival of the animal. Using intrathecal SOD1 ASOs, phase II and III human trials have ended, but their results are not completely conclusive and will be described in another chapter of the present edition. ASOs against C9ORF72 were efficient to reduce cellular foci and proved capable of reducing C9RE toxicity both in vitro and in vivo [9,10]. Despite these encouraging results, the phase I trial in ALS patients recently gave negative results. Very recently, ION363, a non-allele-specific FUS ASO, was used to silence the FUS gene [11]. Silencing was efficient, postnatal levels of the
protein in the brain and spinal cord were reduced, and motor neuron degeneration was
delayed. A phase III trial is ongoing. Apart from causal genes implicated in inherited ALS,
*ATNX2* ASOs were also considered for treating the disease. The reasons for this were, first,
that *Ataxin2* plays a major role in the formation of stress granules, structures known to
regulate nucleocytoplasmic transport [12]. Secondly, *Ataxin2* has strong interactions with
TDP-43 and TDP43 pathology is a hallmark of ALS. Thirdly, intermediate repeat expansion
in the *ATXN2* gene is a susceptibility factor for sporadic ALS. Experimentally, anti *ATXN2*
ASOs were also able to reverse the cytoplasmic mislocalization of nuclear proteins induced
by *C9RE*. The survival of the transgenic TDP-43 mice was improved by treatment with an
*ATXN2* ASO [13]. Taken together, these results suggest that *ATNX2* ASOs are good
candidates to treat both inherited and sporadic ALS, and a human phase I trial is already
ongoing with the *ATXN2* ASO named BIIB105.

The ASO strategy has already proven efficient in spinal muscular atrophy (SMA). However, they have some important limits for ALS treatment. As they degrade with time, treatment has to be repeated. The intrathecal route of administration is not without adverse events or risks of infection. In severely disabled patients, the monthly organization of lumbar punctures may be too demanding. The most important caveat is probably the limited diffusion of ASO into the CNS. Motor neurons, and particularly those in the ventral horns, are not easily accessible and treatment effect in animal models remains limited to a moderate improvement of survival.

3.2 Viral vectors and ASO delivery

One important advantage of viral vectors is to induce permanent changes so as to avoid
repeated administration of treatment. For CNS delivery of genetic material, the adenoviruses
(AAVs) are the most commonly used. These viruses directly target CNS cells and cross the
blood-brain barrier so that, after a single injection of AAV10 in the tail of the rat, almost 70%
of the motor neurons are infected [14]. Experimentally, the ASO is introduced into the AAV
and the virus is subsequently injected into the animal. Different ways of administration have been used to try to optimize the effect on survival. With AAV10+anti *SOD1* ASO, a 90% improvement of survival was obtained while it was only 80% with the AAV9+anti *SOD1* ASO [14,15]. Despite this major effect on survival, as the routes of injection allow only 70% of the motor neurons to be targeted, it was not surprising that the disease was not stopped. To optimize the rate of infected cells, a new, sub-pial, technique was proposed. After sub-pial injections at the cervical level of the spinal cord, a 100% labeling of the motor neurons could be reached [16]. The effect of the treatment with the AAV9+anti *SOD1* ASO was unique. When the treatment was injected at day 120, e.g. before disease onset, none of the mice developed ALS. When it was injected at day 350, once the disease had started, ALS was completed blocked in the animal, which was, to our knowledge, the first time such an effect was reached with an experimental treatment in an ALS model.

Viral vectors have some limitations. The mode of injection is one important limit; however, its management seems to be only a matter of technique. The major concern with AAVs, compared to ASOs alone, is their immunogenicity. If children with SMA have been successfully treated with an AAV carrying the *SMN1* gene, without limited serious adverse effects, the administration of AAV+ASO in the adult may result in immunoallergic reactions with meningoencephalitis [17]. This could potentially be solved by either using a less immunogenic virus, yet to be discovered, or by a concomitant immunosuppressant treatment to prevent the immunoallergic reaction. One other important point, compared to the use of ASOs alone is that once the injection has been made, its effects can hardly be stopped if adverse events occur.

### 3.3 AAVs and other gene silencing techniques

RNAi and CRISPR are two other approaches that may benefit from the use of viral vectors. RNAi silences genes by acting directly on mRNA and leads to its degradation. Two types of
RNAi are used, the plasmidic construct shRNA and miRNA. miRNAs are single-stranded non-coding RNAs of about 22 nucleotides; their side effects are more limited than those of shRNA. Both techniques for silencing the *SOD1* gene have successfully improved survival of animal models. There are no trials, to date, in ALS, but a phase I/II trial with AAV-RNAi is ongoing in Huntington's disease. The use of such silencers for *C9ORF72* remains very experimental.

CRISPR-Cas9 is devoted to gene editing. It targets specific sequences and enables to either delete DNA material or introduce sequences into the genome, potentially allowing to suppress pathogenic mutations. One can thus imagine removing mutations from the *SOD1* gene or limiting C9RE. In ALS, experiments are scarce, but one study targeting the *SOD1* gene with an AAV9 vector showed a 25% improvement of mice survival after injection into the facial vein of the newborn *SOD1* transgenic animal, and disease onset was delayed [18]. More recently with a similar vector and the CRISPR construct, survival was improved by 55% after intracerebroventricular injection in newborn mice. CRISPR-Cas9 has also been used in C9RE models. In induced pluripotent stem cells (iPSC) derived from patients with C9RE, the induction of deletions in the hexanucleotide region resulted in blocking the production of dipeptide repeats, one important hallmark of the C9RE toxic process [19].

### 3.4 AAVs and gene delivery

A series of molecules, mostly small peptides, are known to support motor neuron survival, and are called motor neuron neurotrophic factors. Among them, BDNF, GDNF, IGF1 or VEGF have been implicated in ALS pathogenesis. It was thus tempting to use AAVs to introduce such genes into the CNS to modulate survival in animal models. The interest of the technique is that it does not depend on the type of mutation responsible for ALS cases and it could also be applied in sporadic cases. Several works used the *IGF1* gene with different AAVs and different routes of administration with no real positive result. Little or no effect
was also noted with other factors such as GDNF or HGF. Conversely, AAV9+VEGF
administered intrathecally, improved survival and strength of the mice and inhibited
neuroinflammation [20].

4. Antibody-based therapy in FALS

SOD1 gene mutations have been demonstrated to induce SOD1 misfolding that propagates with a prion-like mechanism [21]. There is, to date, a consensus on the occurrence and the role of the misfolding in SOD1 mutated patients. However, whether a similar mechanism is implicated in sporadic and inherited ALS cases, whichever the mutation, remains controversial. Besides blocking misfolded SOD1 (misSOD1) by an action on the SOD1 gene as previously described, another option is to design antibodies against misSOD1 itself. Initial works on antibody-based therapy have used full length monoclonal antibodies (MAb) against misSOD1. With intraventricular injections of the C4F6 anti-misSOD1 MAb, survival of SOD1 transgenic mice was improved and weight loss was delayed [22]. Several MAbs against misSOD1 have now been generated, that target different amino acid sequences. The D3H5 MAb gave similar results as C4F6, while the A5C3 was not efficient [23,24]. Conversely, the anti-K76-V82 Mab gave interesting results with not only an improvement of animal survival but also reduced SOD1 pathology with reduction of SOD1 aggregates and gliosis. [25].

SOD1 protein can also be targeted using nanobodies (Nbs). Nbs are antibodies with a single
monomer variable fragment. They are thus very small sized, 15kDa versus 150kDa for a
classical antibody, facilitating production and therapeutic use. Nbs can be subcloned into an
AAV and injected intravenously to the animal. The Nb B1 was tested in G93A transgenic
mice, after subcloning into AAV9, and improved survival as well as SOD1 pathology in the
mice, lowering motor neurons loss, gliosis and SOD1 aggregates [26]. Another Nb, W20,
targeting toxic SOD1 oligomers, and administered intranasally, significantly improved motor
neuron survival, reduced SOD1 oligomer levels and inhibited gliosis and neuroinflammation
in the spinal cord and brain stem of the animal [27].

C9RE, the most frequent cause of genetically-determined ALS, has mainly been studied
through its dipeptide repeats (DPR). The expansion is transcribed into sense and antisense
RNAs further translated into DPR. Among the DPRs, proline-arginine (PR), glycine-arginine
(GR), and glycine-alanine (GA) are the most neurotoxic; they increase the frequency of DNA
double strand breaks. Most antibody-based studies on DPRs in animal ALS models have
focused on poly-GA. The use of the anti-GA 5F2 reduced poly-GA levels, aggregation in the
cultured neurons, and the TDP-43 pathology secondary to poly-GA aggregation [28].

Antibodies and Nbs targeting TDP-43, a hallmark of ALS pathology, have also been used in
vivo and in vitro. Studies are more limited than those targeting SOD1. Results are also
limited, with nonetheless an encouraging limitation of TDP-43 proteinopathy in neurons [29].

The use of antibodies to treat FALS has some advantages, such as the possibility to use
intravenous injections or intranasal administration and a good safety profile. However, in
animal models the results of the treatments are less promising than with gene therapy. The
treatment also needs to be repeated. The choice of the target seems important and it is likely
that this antibody-based strategy will be largely improved in the next years. However,
regarding the strategy against misSOD1, some unexpected difficulties may occur. While there
is consensus about the induction of a misSOD1 in SOD1-mutated patients, works have shown
several patterns of immunoreactivity in the neural cells of patients with different mutations
[30]. This may suggest that there are several different conformations of the misfolded protein.
This would imply that a given antibody against misSOD1 will not target all the misSOD1 and
may not be proposed to all patients.

5. Modulation of the microbiota
Study of the microbiota is an emerging research field in neurodegenerative disorders.

Modulation of the microbiota has been shown to influence different metabolic pathways, most of them being implicated in ALS pathogenesis, such as neuroinflammation, immunity or the production of neuroprotective molecules [31]. As early as 2004, it was demonstrated that the administration of a component of Gram-negative bacteria, the lipopolysaccharide, induced a significant worsening of *SOD1* transgenic mice. More recently, two pioneering works have been published in *Nature* that may represent important steps for the modulation of the microbiota in ALS. In the first work, the authors were able to improve or worsen the *SOD1* transgenic mice using different types of gut bacteria [32]. Worsening of ALS was obtained using *Ruminococcus* and *Parabacterioides*, belonging to a microbiota group implicated in acetate and hydrogen genesis. The disease in the mice was slowed by *Akkermansia muciniphila*, which is a gut bacterium known to induce a rise of nicotinamide levels, a neuroprotective compound. The second work focused on the *C9RE* mice model of ALS, and studied the influence of the gut bacteria implicated in the modulation of immunity and inflammation [33]. The rationale of this approach was linked to the product of the *C9ORF72* gene, known to inhibit the anti-inflammatory influence of the microbiota. The hypothesis was that C9RE induces a deregulation of the microbiota resulting in increased inflammation that subsequently worsens ALS. By reducing proinflammatory gut bacteria, they slowed the disease in transgenic mice. In the animal, biological parameters of inflammation were lowered and protective immunity was improved. For these authors, it could be the reason why some patients with C9RE do not develop the disease or may have a significantly delayed onset.

## 6. Conclusion

Among the different approaches being considered to treat FALS patients, gene-specific therapy appears as the most promising. The cure of the animal, with subpial injections of
AAV9+anti-SOD1 ASO represents a major advance in the field. Progress has to be made in the different treatment strategies, but it is tempting to think that most of the therapies for FALS will potentially benefit sporadic cases too.

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