Gene therapy overexpressing Klotho for Amyotrophic Lateral Sclerosis

Sergi Verdes

Assumpció Bosch Merino Xavier Navarro Acebes

Gene therapy in the nervous system (VHIR)

Amyotrophic Lateral Sclerosis (ALS) is a very aggressive neuromuscular disease that affects around 4,000 people in Spain and leads to the inevitable death of patients, with a life expectancy of only 2 to 5 years from diagnosis. It is characterized by the progressive loss of the ability to move, speak and swallow. In advanced stages, patients require intubation, assisted ventilation and adapted communication systems to stay connected to their environment. Although available treatments, such as riluzole and edaravone, slightly slow down the progression, there is no cure for ALS, and patients are a vulnerable group with few effective therapeutic resources. The multiple pathophysiological mechanisms that lead to motor neuron (MN) degeneration and muscle denervation are complex. The key to improving function in ALS lies in preventing axonal disconnection, protecting MNs and promoting muscle reinnervation. α-Klotho (KL) is a pleiotropic protein with therapeutic potential against several of these mechanisms. KL possesses neuroprotective and myoregenerative properties thanks to its antioxidant, anti-inflammatory effects and its ability to promote myelination and protect mitochondrial structure and function. In this thesis, the therapeutic potential of KL overexpression in the central nervous system (CNS) and skeletal muscles in ALS has been investigated. First, we identified a decrease in endogenous KL levels in the CNS and skeletal muscles in models of MN degeneration and muscle atrophy. Subsequently, the protective effects of KL against glutamate-induced excitotoxicity in spinal cord cultures were investigated, revealing that KL overexpression protects MNs. Gene therapy strategies based on adeno-associated viral (AAV) vectors were used to target KL overexpression in the spinal cord and muscles of the SOD1G93A mouse model of ALS. The results show that KL overexpression in the CNS has a mild impact on neuromuscular preservation, but improves motor function in both sexes. On the other hand, increasing muscle KL secretion appears to be a better therapeutic option, as it protects MNs, reduces astrocytic and microglial reactivity, preserves neuromuscular junctions (NMJs), and prevents muscle atrophy, improving locomotor function and strength, delaying disease onset, and prolonging survival. Furthermore, even at symptomatic stages, KL overexpression in muscles slows neuromuscular and motor decline. Finally, transcriptomic and proteomic studies that we conducted in the spinal cord and gastrocnemius muscle have allowed us to identify the mechanisms underlying the neuroprotective effects of KL in ALS. Our results open a therapeutic avenue to simultaneously address different alterations associated with the disease, with the potential to improve neuromuscular function and quality of life in patients. This advance has been patented and licensed, thus creating opportunities for the development of an innovative and comprehensive treatment for ALS.

Our findings have garnered substantial scientific recognition, consistently been selected for presentation at leading gene therapy and ALS conferences, including eight international congresses—such as ASGCT, ESGCT, and ENCALS—and eleven national meetings like CIBERNED, SETGYC, SCB, and UAB conferences. Most presentations were chosen for oral sessions, underscoring the impact of our work, which has also been awarded multiple distinctions, including mobility, communication, and thesis challenge awards, as well as recognition in late-breaking news and poster highlights. Following the promising preclinical results, we engaged the UAB Technology Transfer Office (TTO), which conducted a patentability search across PatBase, Espacenet, Scifinder, and Google Scholar, confirming the novelty and industrial relevance of our invention. While some prior art exists linking KL to neuromuscular diseases, our research targets a unique isoform of KL with distinct therapeutic advantages. Both our TTO and patent attorneys (ZBM and Burns & Levinson) concluded that we could address any objections by clarifying the unique attributes of our isoform, bolstering the inventive step. This led to the successful filing of a patent (US Patent Application No. 63/330,684) for specific viral vectors loaded with the sKL gene for ALS and other neuromuscular conditions. Our patent (WO2023198828A1) is now published, with a co-ownership agreement in place among VHIR, UAB, UB, and ICREA to manage and share future revenue. The project has attracted significant financial support from several funding agencies, including AGAUR (Generalitat de Catalunya), the Ministerio de Ciencia, Innovación y Universidades, and TERAV. Of note, we received the Producte AGAUR grant, designed for projects that have completed transfer planning and developed value-generation strategies for a specific candidate product. This support and entrepreneurship program allowed us to engage potential industry partners, including SwanBio Therapeutics, Lysogene, Advent France Biotech, Kurma Partners, CCR, KU Leuven, Lilly, and ANEW Medical Inc. The substantial market potential for ALS therapies, valued at USD 713.3 million in 2023 and expected to grow to USD 1.1 billion by 2032, highlights the financial appeal and industrial relevance of this therapeutic project. In a major milestone, we successfully entered into a licensing agreement with ANEW Medical Inc. for ALS applications, along with a separate patent for Alzheimer’s disease and dementia (ES2968098T3). This marked the first time a patent was licensed during the Producte AGAUR program. Klotho Neurosciences Inc. (formerly ANEW Medical Inc.), a NASDAQ-listed entity, is advancing our research with the resources and expertise to bring Klotho-based therapies to market. The company has also established a long-term agreement with UAB to set up new research laboratories on the campus, fostering collaboration and creating several workplaces. Our research group has also signed a multi-year industry research agreement to continue investigating, including the optimization of the expression cassette, the validation in the Profilin1G¹¹⁸V mouse model of ALS, the testing of myotropic AAV vectors, and the safety assessments in larger animal models. This partnership will move into preliminary human studies and regulatory documentation, collaborating with CROs and CDMOs to advance development. Future goals include completing clinical trials and ultimately licensing the technology to a major pharmaceutical partner equipped to meet market demand.

Amyotrophic Lateral Sclerosis (ALS); Neuromuscular Disease; Motor Neuron (MN) Degeneration; Muscle Denervation; Axonal Disconnection; Muscle Reinnervation; α-Klotho (KL); Pleiotropic Protein; Neuroprotective Properties; Myoregenerative Properties; Antioxidant Effects; Anti-inflammatory Effects; Myelination Promotion; Mitochondrial Structure and Function; KL Overexpression; Central Nervous System (CNS); Skeletal Muscles; Endogenous KL Levels; Glutamate-Induced Excitotoxicity; Spinal Cord Cultures; Adeno-Associated Viral (AAV) Vectors; SOD1G93A Mouse Model; Neuromuscular Preservation; Motor Function; Muscle KL Secretion; Astrocytic Reactivity; Microglial Reactivity; Neuromuscular Junctions (NMJs) Preservation; Muscle Atrophy Prevention; Locomotor Function; Strength Improvement; Disease Onset Delay; Survival Prolongation; Symptomatic Stages; Neuromuscular Decline; Motor Decline; Transcriptomic Studies; Proteomic Studies; Neuroprotective Mechanisms; Therapeutic Avenue; Neuromuscular Function Improvement; Quality of Life Improvement; Patent; License; Innovative Treatment.