Jun 20, 2024

Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease involving the loss of upper and lower motor neurons, progressive muscle weakness, and eventual paralysis. Disease progression occurs rapidly after onset, and patients succumb primarily due to paralysis of diaphragmatic muscles that prevent respiration within ~3-5 years. ALS-associated mutations are more frequently sporadic (over 90%), but mutations may also be inherited, accounting for ~10% of disease cases. Mutations in several genes have been identified as functionally relevant in disease progression, including Transactivation response element DNA-binding Protein 43 kDa (TDP-43), fused in sarcoma (FUS), Cu/Zn superoxide dismutase 1 (SOD1), and Hexanucleotide repeat expansion mutations in the first intron of chromosome 9 open reading frame 72 (C9orf72).¹ 

Currently, approved therapies mostly manage symptoms associated with the disease and only one gene therapy has been approved to address the root cause of the disease. However, several new strategies are progressing through clinical trials.² 

ALS therapies at different stages of clinical studies. Retrieved from Therapies Targeting ALS-Linked Genetic Mutations | The ALS Association.² *Includes familial and sporadic ALS patients.

At the recent ASGCT meeting, the oral abstract sections of Neurologic Diseases provided a platform for scientists working with preclinical models to develop and validate new gene therapies to tackle this devastating disease.

Addressing the loss of TDP-43 with an AAV-based gene therapy

TDP-43 is a nuclear protein involved in RNA processing and specifically in the repression of nonconserved cryptic exon splicing.³ TDP-43 loss of function mutations have been associated with its nuclear clearance and aggregation in cytoplasmic inclusions and are frequently found in sporadic and in some familial ALS with frontotemporal dementia (FTD) cases.³ 

Mechanistically, TDP-43 loss of function results in the expression of cryptic exons, leading to nonsense-mediated RNA decay and truncated protein products. Significantly, these splicing errors are detectable before the onset of ALS symptoms and, as such, can serve as diagnostic biomarkers.⁴

Mechanism of TDP-43 dysfunction in ALS. “TDP-43 normally binds to UG repeats flanking cryptic exons and prevents them from being incorporated into messenger RNA (mRNA). When TDP-43 is lost from the nucleus, it fails to repress the splicing of cryptic exons. As some cryptic exons are incorporated in-frame, antibodies can be developed against cryptic exon-encoded peptides to serve as fluid biomarkers. PTC: premature termination codon.”Retrieved without modifications from Irwin et al. 2024.⁴http://creativecommons.org/licenses/by/4.0/

Elucidation of this pathology-underscoring mechanism presents opportunities beyond diagnostics by identifying various relevant ALS therapeutic targets. However, Dr. Aswathy Peethambaran Mallika, PhD, Johns Hopkins University School of Medicine, shared that because TDP-43 plays such a critical role in splicing regulation, a new strategy aims to restore TDP-43 expression and function in neurons.

First, to overcome challenges such as TDP-43 aggregation and potential toxicity from its overexpression, the team at Johns Hopkins, led by Dr. Philip Wong, has engineered a chimeric form of the TDP-43 protein by replacing its aggregation-prone C-terminal domain. This new chimeric protein enables the right nuclear localization by incorporating the TDP-43 N-terminal domain and contains a novel splicing repressor domain. Studies in various in vitro and animal models have validated the function of this new chimeric TDP-43 protein in repressing cryptic exon splicing.

Next, with this new powerful tool at hand, the team is now leveraging an AAV vector for delivery of the TDP-43 chimera to evaluate its gene therapy potential in an ALS mouse model with neuron-specific loss of TDP-43 expression. To this end, they have zeroed in on using the AAV-PHP.eB vector, which has been shown previously to direct gene expression in the brain with limited liver-targeting in mice.⁵

Lastly, systemic administration of the AAV-encoded TDP-43 chimera has so far been effective at preventing muscle atrophy and body weight loss for up to 8 months. These are relevant parameters in their model, which show significant changes in untreated animals. In agreement with these positive outcomes, histological findings confirmed a significant preservation of motor neurons following the expression of the TDP-43 chimera. Because treatment safety was also demonstrated, the findings support clinical feasibility and potential benefits.

Antibody therapy targets a pathogenesis mechanism in C9orf72 ALS FTD

Expansion mutations in the C9orf72 gene are the most common cause of ALS with FTD.⁶ The abnormally long repeat expansions of the hexanucleotide-GGGCCC within the first intron of the C9orf72 sequence give rise to neuronal nuclear RNA foci and cytoplasmic inclusions in these patients. Various mechanisms are tied to disease pathology, including reduced C9orf72 protein expression, aggregation of repeat-containing RNA species, and accumulation of toxic dipeptide repeat proteins (DPRs).¹

Hexanucleotide repeat expansion in C9orf72 gene and associated pathological mechanisms. Three mechanisms of pathology have been identified: (1) transcription inhibition results in loss of function, (2) bidirectional transcription forms sense and antisense RNA sequences that accumulate in the nucleus and trap RNA binding proteins, and (3) Repeat Associated Non-AUG dependent (RAN) translation gives rise to toxic dipeptides repeat proteins (DPRs). Retrieved from Capella et al. 2019, (Figure 2) without modifications.¹(http://creativecommons.org/licenses/by/4.0/).

DPRs result from a translation process independent of an AUG initiation codon or Repeat Associated Non-AUG dependent (RAN) translation that generates multiple sense and antisense protein products. Dr. Laura Ranum’s lab at the Center for NeuroGenetics and the Genetics Institute, University of Florida, had already generated a mouse model of C9orf72-ALS expressing the human form of the protein having the disease-causing hexanucleotide repeat expansion. As shared by Dr. Lisa Romano at ASGCT, the mouse model is incredibly valuable for her studies as it recapitulates the clinical and pathological features of the disease, including neuroinflammation, motor neuron loss, and decreased survival.⁷

Leveraging this mouse model, the team has previously tested a potential antibody therapy strategy, targeting one of the six dipeptides repeat proteins involved in pathogenesis, specifically the GA RAN protein. The anti-GA Ran antibody administration in these mice proved effective by decreasing motor neuron loss, improving behavioral deficits, and increasing survival. Significantly, the team found that the antibody was active at targeting GA RAN protein for proteasomal degradation, reducing its aggregation and that of other RAN proteins, such as GP and GR RAN proteins.

With this insight, Dr. Romano is now advancing a strategy to develop an AAV-based antibody therapy for C9orf72-ALS. Unlike direct antibody administration, AAV vector delivery can achieve sufficient and targeted antibody expression through a more feasible single-dose regimen.

Dr. Romano and colleagues have engineered various antibody formats with validated anti-GA specificity, including single-chain variable fragment (ScFV) and different full-length forms (e.g., IgG1). Evaluation in vitro has allowed them to identify antibody formats that more effectively bind GA RAN protein and reduce aggregation. Additionally, Dr. Romano performed pilot studies by dosing the C9orf72-ALS mice with AAV vectors, encoding the various anti-GA antibody formats for ultimate therapeutic candidate identification. Lastly, larger efficacy studies have enabled the team to validate their antibody candidate more thoroughly through molecular and behavioral assays.

Improving Survival, Respiratory and Motor Functions in SOD1-ALS

Mutations in the SOD1 gene account for ~12% and ~2% of familial and sporadic ALS cases, respectively. The FDA approval in 2023 of Tofersen, an antisense oligonucleotide targeting the production of SOD1, marked a significant step as the first drug targeting the root cause of ALS. Tofersen was approved based on its efficacy in reducing neurodegeneration, as evidenced by a reduction in the levels of plasma neurofilament light, a marker of neuronal injury in ALS.⁸

At ASGCT, Dr. Fang Wan, UMass Chan Medical School, discussed her work as part Dr. Guangping Gao’s team developing a new small oligonucleotide-based therapy for SOD1-ALS. This approach leverages a second-generation AAV9-based vector containing a human survival motor neuron 1 (hSMN1) promoter to deliver a set of miR-33-scaffolded shRNAs targeting SOD1 transcripts.

The vector was originally developed and tested in a spinal muscular atrophy mouse model to drive a more physiological expression of hSMN1.⁹ Gao and Xie’s team at UMass Chan Medical School found efficacy and safety benefits from using the native promoter, as opposed to the stronger cytomegalovirus enhancer/chicken β-actin (CMVen/CB) promoter implemented in the FDA approved therapy, Onasemnogene abeparvovec (Zolgensma®), for spinal muscular atrophy (SMA).⁸ 

Second-generation AAV vector improved human survival motor neuron 1 (hSMN1) transgene expression. Incorporation of the native human SMN1 promoter into the AAV9 vector drives a more physiological level of protein expression, benefiting outcomes, such as life span and motor function while reducing toxicity. Retrieved without modifications from Xie et al. 2024.⁹ (http://creativecommons.org/licenses/by/4.0/).

Additionally, Gao and Xie’s previous work had demonstrated benefits of using an artificial miRNA scaffold (i.e., miR-33) to deliver shRNAs. This approach was shown to improve the integrity of the recombinant AAV vector genome and consequently that of shRNA sequences been delivered and overall supported efficient and accurate gene silencing.¹⁰

These previous findings have provided strong validated tools for developing a new strategy targeting SOD1. The resulting vector, hSMN1-dual-amiR has been tested preclinically by Dr. Fang Wan in a mouse model of SOD1-ALS (hSOD1^G93A), delaying the onset of ALS and significantly improving survival. Concomitantly, reduced expression of SOD1 through this new strategy led to improved motor and respiratory functions.

Reference

 1. Cappella M, Ciotti C, Cohen-Tannoudji M, Biferi MG. Gene Therapy for ALS-A Perspective. Int J Mol Sci. 2019 Sep 6;20(18):4388. https://doi.org/10.3390%2Fijms20184388

 2. ALS.org (n.d.). Therapies Targeting ALS-Linked Genetic Mutations | The ALS Association. The ALS Association. Retrieved May 22, 2024, from https://www.als.org/research/als-research-topics/genetics/therapies-targeting-als-linked-genetic

 3. Ling JP, Pletnikova O, Troncoso JC, Wong PC. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science. 2015 Aug 7;349(6248):650-5. https://doi.org/10.1126/science.aab0983

 4. Irwin KE, et al., A fluid biomarker reveals loss of TDP-43 splicing repression in presymptomatic ALS-FTD. Nat Med. 2024 Feb;30(2):382-393. https://doi.org/10.1038%2Fs41591-023-02788-5

 5. Goertsen D, et al., AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset. Nat Neurosci. 2022 Jan;25(1):106-115. https://doi.org/10.1038/s41593-021-00969-4

 6. Gijselinck I, Cruts M, Van Broeckhoven C. The Genetics of C9orf72 Expansions. Cold Spring Harb Perspect Med. 2018 Apr 2;8(4):a026757. https://doi.org/10.1101%2Fcshperspect.a026757

 7. Nguyen L, et al., Antibody Therapy Targeting RAN Proteins Rescues C9 ALS/FTD Phenotypes in C9orf72 Mouse Model. Neuron. 2020 Feb 19;105(4):645-662.e11. https://doi.org/10.1016/j.neuron.2019.11.007

 8. FDA.gov (2023, April 25). FDA approves treatment of amyotrophic lateral sclerosis associated with a mutation in the SOD1 gene. U.S. Food and Drug Administration. Retrieved May 22, 2024, from https://www.fda.gov/drugs/news-events-human-drugs/fda-approves-treatment-amyotrophic-lateral-sclerosis-associated-mutation-sod1-gene

 9. Xie Q, et al., Improved gene therapy for spinal muscular atrophy in mice using codon-optimized hSMN1 transgene and hSMN1 gene-derived promotor. EMBO Mol Med. 2024 Apr;16(4):945-965. https://doi.org/10.1038/s44321-024-00037-x

 10. Xie J, Tai PWL, Brown A, Gong S, Zhu S, Wang Y, Li C, Colpan C, Su Q, He R, Ma H, Li J, Ye H, Ko J, Zamore PD, Gao G. Effective and Accurate Gene Silencing by a Recombinant AAV-Compatible MicroRNA Scaffold. Mol Ther. 2020 Feb 5;28(2):422-430. https://doi.org/10.1016/j.ymthe.2019.11.018