Using a combination of three AAVs, each encoding for one fragment of the laminin-α2 protein flanked by short split intein resulted in the complete reconstitution of the Laminin-α2 and an improvement of the histopathological features of the dy2j dystrophic mouse model.
Abstract
Several therapies using adeno-associated virus (AAVs) as gene delivery tool have received marketing approval in the past. However, potential applications of AAVs are limited by their restricted gene packaging capacities (<4,7kb). Many monogenic diseases are caused by large complex genes with hundreds of pathogenic variants that exceed AAV capacity. Merosin-deficient Congenital Muscular dystrophy type 1A is a severe monogenic recessive disease caused by the absence of functional copies of the LAMA2 gene. To date, no treatment options are available, with the most promising approach being gene replacement therapy to provide a functional copy. The LAMA2 coding sequence, spanning 9,3 kb, encodes for the laminin-α2, a subunit of the trimeric protein Laminin-211 found in the basement membrane of skeletal muscle cells and Schwan cells. Efforts have been focused on gene or protein replacement along with basement membrane engineering. Here, we exploit the ability of inteins to reconstitute full-length protein in a scarless manner. Using a combination of three AAVs, each encoding for one fragment of the laminin-α2 protein flanked by short split intein, resulted in the complete reconstitution of the Laminin-α2 and an improvement of the histopathological features of the dy2j dystrophic mouse model.
Cockayne Syndrome (CS) is an autosomal recessive, progressive developmental and neurodegenerative disease. Approximately 30% of cases are caused by mutations in the ERCC8/CSA gene. Patients with CS present with cutaneous photosensitivity, growth failure, shorter life span, and a progressive degeneration of the central nervous system. Loss-of-function mutations in CSA result in deficiencies in the transcription-coupled nucleotide excision repair (TC-NER). Currently, no therapies are available for these patients. Adeno-associated virus (AAV)-mediated gene therapy offers an opportunity to address this unmet need. We designed a new AAV vector encoding human CSA under a ubiquitous promoter. We tested the therapeutic efficacy of this AAV9-CSA vector by neonatal intracerebroventicular injection in the Csa-/-;Xpa-/- mouse model. Treatment with AAV9-CSA resulted in a significant increase in lifespan, and broad distribution of human CSA in the brain and heart, without evidence of vector-related toxicity. Despite clear therapeutic benefit, we also observed neuroradiological abnormalities, and neuropathologic alterations, including hypomyelination, astrocytosis, and microgliosis, as well as likely life-limiting transcriptomic alterations in liver at endpoint. Nonetheless, the success of these experiments paves the way for the first clinical translation of an AAV gene therapy for CS patients into humans.
A. R. Batista, Aine C. Scholand, William S. Callahan et al.· Journal of Clinical Investig...· 0 citations
Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle disease. DMD results from mutations preventing the production of the subsarcolemmal protein dystrophin, a protein critical for muscle stability. In the absence of dystrophin, muscle fibers are damaged and degenerate, leading to an inflamed and fibrotic muscle phenotype. DMD patients have a life expectancy of 20 to 40 years old and succumb to cardiac and respiratory failure. Adeno-associated virus (AAV) is a promising vector to deliver gene therapies (such as truncated dystrophin proteins, i.e., microdystrophin) to the muscle of DMD patients, due to its safety profile and broad tissue tropism. Currently, naturally occurring AAV variants including serotypes 8, 9, and rh74 are used in clinical trials, and an AAVrh74-based microdystrophin therapy (Elevidys) was approved in 2023 to treat DMD. Despite regulatory success, limited results are available from patients treated with Elevidys and in other clinical trials, and many questions remain about the biology of AAV vectors in muscle. Individual muscle cells, or myofibers, have different phenotypes (myofiber type), closely linked to their genetically regulated expression of different isoforms of the contractile protein myosin heavy chain. Based on previous literature, we hypothesized that AAV serotypes 8 and 9 may have a selective tropism for certain myofiber types, or restricted tropism in others. Fast-twitch myofiber types are susceptible to greater injury in DMD, and thus, benefit greater from microdystrophin gene transfer. To determine the myofiber type preference AAV serotypes 8 and 9 we performed histological analysis of reporter gene and microdystrophin expression in archival tissues from preclinical studies in dystrophic canines (cDMD model). We did not detect a myofiber type preference. However, the results from our histological analysis indicated that microdystrophin expression is maintained long-term (8-40 months following systemic delivery); and that micro-dystrophin expressing fibers were protected from atrophy. These results offer important insight for clinical development of microdystrophin gene therapies. Selecting the best AAV variants for muscle-directed gene therapy is a critical step in developing effective treatments. Currently, there is no way to evaluate systemic delivery of AAV vectors to human muscle. To overcome this barrier, we adapted a human skeletal muscle xenograft model, to use as a platform for AAV delivery. We characterized this model using 222 transplanted muscles from human donors, and developed rigorous methods to evaluate muscle transplant quality, and AAV transduction of the muscles. We showed for the first time, that human muscles transplanted in immune-deficient mice are efficiently transduced by AAV vectors at following intravenous injection of the host mice. We first used this platform to compare the three AAV serotypes in clinical trials (8, 9, and rh74). We found that all three have similar delivery efficiency (i.e., infection) in human muscles, but only AAV9 led to high-level mRNA production. These results not only present a novel finding on the superior efficiency of AAV9 but also highlight a new way to evaluate AAV vectors in a preclinical context. Other human transplantation models have been used to discover next-generation AAV serotypes for liver-directed gene therapy. We expect that the human muscle xenograft model has similar potential for discovery of serotypes with enhanced efficiency in human muscle.
Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disorder marked by lack of dystrophin expression. Symptoms include loss of ambulation, respiratory problems, and cardiac complications with heart failure being the leading cause of death. Dystrophin transduces force from the actin cytoskeleton to the extracellular matrix to protect cells during muscle contraction. Restoration of dystrophin expression by gene transfer holds promise in addressing the root cause of disease. We compared the changes to transcriptional profiles after gene transfer by adeno-associated virus or lentivirus to examine whether viral treatment alone impacts cell homeostasis. We delivered GFP to cardiomyocytes differentiated from induced pluripotent stem cells (iPSCs) with DMD mutations. Global transcriptional profiling revealed a downregulation of metabolic genes after lentiviral transduction compared to untreated controls. In both AAV and lentivirus-treated DMD iPSC-cardiomyocytes, we observed an activation of the p53 DNA damage response in addition to a downregulation of cell cycle genes, suggesting stress-induced G2/M checkpoint arrest following viral delivery. These findings demonstrate that gene therapy mediated by viral vectors activates cell stress pathways. Interventions to mitigate these stress responses may be necessary for safe and effective gene transfer in diseased cells.
Elaine C. Lai, Abiageal R. Keegan, A. Eguchi· bioRxiv· 0 citations
This study offers the most compelling clinical evidence so far that gene replacement therapy can safely restore signi fi cant hearing in children with OTOF-related deafness, with bene fi ts lasting up to 2.5 years, setting a new standard for treating inherited sensory disorders.
B. Pattnaik, Jose-Alein Sahel· Signal Transduction and Targ...· 0 citations
Hemophilia is an X-linked hereditary bleeding disorder caused by loss-of-function mutations in the genes encoding coagulation factors, leading to excessive bleeding and potentially being life-threatening. Currently, regular treatment for hemophilia is the infusion of recombinant blood coagulation factors. This approach is not only costly but can also give rise to complications such as the development of neutralizing antibodies (Nabs), which impede the therapeutic outcome. Hemophilia is a monogenic disease, making gene therapy a promising curative measure that includes gene addition and gene editing approaches. Adeno-associated virus (AAV) vectors have gained massive attention as the premier delivery vehicle for clinical gene therapy due to their diverse tissue tropisms dictated by the natural and engineered AAV capsids, their excellent safety profile since AAVs do not cause any known human diseases, low immunogenicity, and long-lasting gene expression. Several AAV-based gene addition therapies have been approved for hemophilia B and hemophilia A, and dozens of similar AAV-based clinical trials are underway. Gene editing technologies, like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), have shown immense potential in the treatment of genetic diseases. CRISPR/Cas9-mediated transgene integration provides critical advantages over regular gene insertion therapy by enabling site-specific, targeted genomic integration rather than random integration. Clinical trials focusing on AAV-CRISPR/Cas9-mediated knock-in of human genes represent the frontier of
in vivo
genetic medicine, and one such prominent ongoing clinical trial for hemophilia B is designed to insert the human Factor IX gene into hepatocytes. This review outlines recent advances and feasible strategies of CRISPR/Cas9-based genome editing for hemophilia therapy, highlights the potential applications of next-generation gene editing and vector delivery technologies, and offers new insights for the advantages, limitations, and future directions for these novel treatment modalities.
Xiao-Yu Zhang, Kai-Di Xu, Shi-Hui Zou et al.· Frontiers in Genome Editing· 0 citations
Givinostat significantly improved exon skipping and dystrophin restoration in human DMD myoblasts carrying an exon 52 deletion, and combining givinostat with ASOs to enhance dystrophin restoration and muscle preservation in DMD.
Xaysongkhame Phongsavanh, C. Gastaldi, Astrid Mottais et al.· Molecular Therapy: Nucleic A...· 0 citations
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