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AAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation.Nature Communications Feb 2024Gene therapies provide treatment options for many diseases, but the safe and long-term control of therapeutic transgene expression remains a primary issue for clinical...
Gene therapies provide treatment options for many diseases, but the safe and long-term control of therapeutic transgene expression remains a primary issue for clinical applications. Here, we develop a muscone-induced transgene system packaged into adeno-associated virus (AAV) vectors (AAV) based on a G protein-coupled murine olfactory receptor (MOR215-1) and a synthetic cAMP-responsive promoter (P). Upon exposure to the trigger, muscone binds to MOR215-1 and activates the cAMP signaling pathway to initiate transgene expression. AAV enables remote, muscone dose- and exposure-time-dependent control of luciferase expression in the livers or lungs of mice for at least 20 weeks. Moreover, we apply this AAV to treat two chronic inflammatory diseases: nonalcoholic fatty liver disease (NAFLD) and allergic asthma, showing that inhalation of muscone-after only one injection of AAV-can achieve long-term controllable expression of therapeutic proteins (ΔhFGF21 or ΔmIL-4). Our odorant-molecule-controlled system can advance gene-based precision therapies for human diseases.
Topics: Mice; Humans; Animals; Alprostadil; Transgenes; Cycloparaffins; Odorants; Receptors, G-Protein-Coupled; Dependovirus; Genetic Vectors
PubMed: 38321056
DOI: 10.1038/s41467-024-45383-z -
International Journal of Molecular... Jan 2024Genetic disorders of the central nervous system (CNS) comprise a significant portion of disability in both children and adults. Several preclinical animal models have... (Review)
Review
Genetic disorders of the central nervous system (CNS) comprise a significant portion of disability in both children and adults. Several preclinical animal models have shown effective adeno-associated virus (AAV) mediated gene transfer for either treatment or prevention of autosomal recessive genetic disorders. Owing to the intricacy of the human CNS and the blood-brain barrier, it is difficult to deliver genes, particularly since the expression of any given gene may be required in a particular CNS structure or cell type at a specific time during development. In this review, we analyzed delivery methods for AAV-mediated gene therapy in past and current clinical trials. The delivery routes analyzed were direct intraparenchymal (IP), intracerebroventricular (ICV), intra-cisterna magna (CM), lumbar intrathecal (IT), and intravenous (IV). The results demonstrated that the dose used in these routes varies dramatically. The average total doses used were calculated and were 1.03 × 10 for IP, 5.00 × 10 for ICV, 1.26 × 10 for CM, and 3.14 × 10 for IT delivery. The dose for IV delivery varies by patient weight and is 1.13 × 10 IV for a 10 kg infant. Ultimately, the choice of intervention must weigh the risk of an invasive surgical procedure to the toxicity and immune response associated with a high dose vector.
Topics: Adult; Animals; Child; Infant; Humans; Dependovirus; Central Nervous System; Blood-Brain Barrier; Administration, Intravenous; Genetic Therapy
PubMed: 38256124
DOI: 10.3390/ijms25021050 -
International Journal of Molecular... Sep 2023GM2 gangliosidoses are a group of neurodegenerative lysosomal storage disorders that are characterized by the accumulation of GM2 gangliosides (GM2), leading to rapid...
GM2 gangliosidoses are a group of neurodegenerative lysosomal storage disorders that are characterized by the accumulation of GM2 gangliosides (GM2), leading to rapid neurological decline and death. The hydrolysis of GM2 requires the specific synthesis, processing, and combination of products of three genes-, , and -within the cell's lysosomes. Mutations in these genes result in Tay-Sachs disease, Sandhoff disease, or AB-variant GM2 gangliosidosis (ABGM2), respectively. ABGM2, the rarest of the three types, is characterized by a mutation in the gene, which encodes the GM2 activator (GM2A) protein. Being a monogenic disease, gene therapy is a plausible and likely effective method of treatment for ABGM2. This study aimed at assessing the effects of administering a one-time intravenous treatment of single-stranded Adeno-associated virus serotype 9 (ssAAV9)- viral vector at a dose of 1 × 10 vector genomes (vg) per kilogram per mouse in an ABGM2 mouse model (). was administered at 1-day (neonatal) or 6-weeks of age (adult-stage). The results demonstrated that, in comparison to mice that received a vehicle injection, the treated mice had reduced GM2 accumulation within the central nervous system and had long-term persistence of vector genomes in the brain and liver. This proof-of-concept study is a step forward towards the development of a clinically therapeutic approach for the treatment of patients with ABGM2.
Topics: Humans; Animals; Mice; Dependovirus; Serogroup; Tay-Sachs Disease; Gangliosidoses, GM2; G(M2) Activator Protein; Genetic Therapy
PubMed: 37834060
DOI: 10.3390/ijms241914611 -
Journal of Virology Dec 2023The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear...
The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene , whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.
Topics: Humans; Capsid Proteins; Dependovirus; DNA; Epithelium; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Transduction, Genetic; Bronchi; Active Transport, Cell Nucleus; Gene Editing
PubMed: 37966249
DOI: 10.1128/jvi.01330-23 -
International Journal of Molecular... Jul 2023Lesions in the human anterior cruciate ligament (ACL) are frequent, unsolved clinical issues due to the limited self-healing ability of the ACL and lack of treatments...
Lesions in the human anterior cruciate ligament (ACL) are frequent, unsolved clinical issues due to the limited self-healing ability of the ACL and lack of treatments supporting full, durable ACL repair. Gene therapy guided through the use of biomaterials may steadily activate the processes of repair in sites of ACL injury. The goal of the present study was to test the hypothesis that functionalized poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can effectively deliver recombinant adeno-associated virus (rAAV) vectors as a means of overexpressing two reparative factors (transforming growth factor beta-TGF-β and basic fibroblast growth factor-FGF-2) in primary human ACL fibroblasts. Effective, durable rAAV reporter red fluorescent protein and candidate TGF-β and FGF-2 gene overexpression was achieved in the cells for at least 21 days, especially when pNaSS-grafted PCL films were used versus control conditions, such as ungrafted films and systems lacking vectors or films (between 1.8- and 5.2-fold differences), showing interactive regulation of growth factor production. The expression of TGF-β and FGF-2 from rAAV via PCL films safely enhanced extracellular matrix depositions of type-I/-III collagen, proteoglycans/decorin, and tenascin-C (between 1.4- and 4.5-fold differences) in the cells over time with increased levels of expression of the specific transcription factors Mohawk and scleraxis (between 1.7- and 3.7-fold differences) and without the activation of the inflammatory mediators IL-1β and TNF-α, most particularly with pNaSS-grafted PCL films relative to the controls. This work shows the value of combining rAAV gene therapy with functionalized PCL films to enhance ACL repair.
Topics: Humans; Transforming Growth Factor beta; Dependovirus; Anterior Cruciate Ligament; Fibroblast Growth Factor 2; Fibroblasts
PubMed: 37446318
DOI: 10.3390/ijms241311140 -
International Journal of Molecular... Jul 2023Duchenne muscular dystrophy (DMD) is a yet incurable rare genetic disease that affects the skeletal and cardiac muscles, leading to progressive muscle wasting and...
Duchenne muscular dystrophy (DMD) is a yet incurable rare genetic disease that affects the skeletal and cardiac muscles, leading to progressive muscle wasting and premature death. DMD is caused by the lack of dystrophin, a muscle protein essential for the biochemical support and integrity of muscle fibers. Gene replacement strategies for Duchenne muscular dystrophy (DMD) employing the adeno-associated virus (AAV) face the challenge imposed by the limited packaging capacity of AAV, only allowing the accommodation of a short version of dystrophin (µDys) that is still far removed from correcting human disease. The need to develop strategies leading to the expression of a best performing dystrophin variant led to only few studies reporting on the use of dual vectors, but none reported on a method to assess in vivo transgene reconstitution efficiency, the degree of which directly affects the use of safe AAV dosing. We report here on the generation of a dual AAV vector approach for the expression of a larger dystrophin version (quasidystrophin) based on homologous recombination, and the development of a methodology employing a strategic droplet digital PCR design, to determine the recombination efficiency as well as the occurrence of unwanted concatemerization events or aberrant expression from the single vectors. We demonstrated that, upon systemic delivery in the dystrophic D2.B10-Dmd/J (DBA2mdx) mice, our dual AAV approach led to high transgene reconstitution efficiency and negligible Inverted Terminal Repeats (ITR)-dependent concatemerization, with consequent remarkable protein restoration in muscles and improvement of muscle pathology. This evidence supports the suitability of our system for gene therapy application and the potential of this methodology to assess and improve the feasibility for therapeutic translation of multiple vector approaches.
Topics: Mice; Humans; Animals; Muscular Dystrophy, Duchenne; Dystrophin; Dependovirus; Muscle, Skeletal; Mice, Inbred mdx; Genetic Vectors
PubMed: 37511179
DOI: 10.3390/ijms241411421 -
Frontiers in Immunology 2023During gene therapy trials, immune responses against adeno-associated virus (AAV) vectors are monitored by antibody assays that detect the humoral and T-cell mediated...
During gene therapy trials, immune responses against adeno-associated virus (AAV) vectors are monitored by antibody assays that detect the humoral and T-cell mediated cellular responses to AAV vectors. T cell assays commonly utilize the collection of patients' peripheral blood mononuclear cells (PBMCs) and stimulation with AAV-derived overlapping peptides. We recently described that spontaneous deamidation coincides with T cell epitopes in AAV capsids and that spontaneous deamidation may enhance or decrease immunogenicity in some individuals. This raised the concern for false negative results of antibody detection and PBMC immune monitoring assays because these assays use wild-type (WT) AAV or WT peptides for T cell re-stimulation and these peptides may not re-activate T cells that were stimulated with deamidated AAV capsid. To investigate this concern, we modeled the scenario by expanding T cells with deamidated peptides and evaluated the cross-reactivity of expanded T cells to WT peptides. In the majority of samples, cells that were expanded with deamidated peptides and restimulated with WT peptide had significantly lowered IL-2 and IFN-γ production. Spiking the four deamidated peptides to the WT peptide pool used for re-stimulation, restored the signal and corrected the performance of the assay. We also evaluated the impact of deamidation on anti AAV binding antibodies and did not observe a major impact on seroprevalence detection of AAV9. These data indicate that a high level of deamidation in AAV therapy may result in underestimation or even failure to detect immune responses against WT peptides during cellular immune monitoring.
Topics: Humans; T-Lymphocytes; Leukocytes, Mononuclear; Monitoring, Immunologic; Seroepidemiologic Studies; Dependovirus; Peptides
PubMed: 37469509
DOI: 10.3389/fimmu.2023.1211529 -
Nature Communications Aug 2023Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their...
Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in the primate brain. In the present study, we created a novel vector of which capsid was composed of capsid proteins derived from both of the AAV serotypes 1 and 2 (AAV1 and AAV2). Following the injection into the frontal cortex of macaque monkeys, this mosaic vector, termed AAV2.1 vector, was found to exhibit the excellence in transgene expression (for AAV1 vector) and neuron specificity (for AAV2 vector) simultaneously. To explore its applicability to chemogenetic manipulation and in vivo calcium imaging, the AAV2.1 vector expressing excitatory DREADDs or GCaMP was injected into the striatum or the visual cortex of macaque monkeys, respectively. Our results have defined that such vectors secure intense and stable expression of the target proteins and yield conspicuous modulation and imaging of neuronal activity.
Topics: Animals; Dependovirus; Transduction, Genetic; Genetic Vectors; Brain; Transgenes; Primates; Parvovirinae; Capsid Proteins; Neurons
PubMed: 37553329
DOI: 10.1038/s41467-023-40436-1 -
Cell Reports. Medicine May 2024Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that...
Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that macrophage migration inhibitory factor (MIF), whose levels are extremely low in spinal MNs, inhibits mutant SOD1 misfolding and toxicity. In this study, we show that a single peripheral injection of adeno-associated virus (AAV) delivering MIF into adult SOD1 mice significantly improves their motor function, delays disease progression, and extends survival. Moreover, MIF treatment reduces neuroinflammation and misfolded SOD1 accumulation, rescues MNs, and corrects dysregulated pathways as observed by proteomics and transcriptomics. Furthermore, we reveal low MIF levels in human induced pluripotent stem cell-derived MNs from familial ALS patients with different genetic mutations, as well as in post mortem tissues of sporadic ALS patients. Our findings indicate that peripheral MIF administration may provide a potential therapeutic mechanism for modulating misfolded SOD1 in vivo and disease outcome in ALS patients.
Topics: Macrophage Migration-Inhibitory Factors; Amyotrophic Lateral Sclerosis; Animals; Humans; Motor Neurons; Superoxide Dismutase-1; Mice; Induced Pluripotent Stem Cells; Intramolecular Oxidoreductases; Mice, Transgenic; Dependovirus; Disease Models, Animal; Male; Mutation; Female; Protein Folding
PubMed: 38703766
DOI: 10.1016/j.xcrm.2024.101546 -
Gene Therapy May 2024Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure...
Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure impacts gene therapy eligibility. The aim of this multicenter epidemiologic study was to determine the prevalence and persistence of preexisting immunity against AAV2, AAV5, and AAV8, in adult participants with hemophilia A or B. Blood samples were collected at baseline and annually for ≤3 years at trial sites in Austria, France, Germany, Italy, Spain, and the United States. At baseline, AAV8, AAV2, and AAV5 neutralizing antibodies (NAbs) were present in 46.9%, 53.1%, and 53.4% of participants, respectively; these values remained stable at Years 1 and 2. Co-prevalence of NAbs to at least two serotypes and all three serotypes was present at baseline for ~40% and 38.2% of participants, respectively. For each serotype, ~10% of participants who tested negative for NAbs at baseline were seropositive at Year 1. At baseline, 38.3% of participants had detectable cell mediated immunity by ELISpot, although no correlations were observed with the humoral response. In conclusion, participants with hemophilia may have significant preexisting immunity to AAV capsids. Insights from this study may assist in understanding capsid-based immunity trends in participants considering AAV vector-based gene therapy.
Topics: Humans; Dependovirus; Male; Hemophilia A; Adult; Longitudinal Studies; Antibodies, Viral; Antibodies, Neutralizing; Genetic Therapy; Adaptive Immunity; Genetic Vectors; Middle Aged; Prevalence; Young Adult
PubMed: 38355967
DOI: 10.1038/s41434-024-00441-5