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Lancet (London, England) May 2024Autosomal recessive deafness 9, caused by mutations of the OTOF gene, is characterised by congenital or prelingual, severe-to-complete, bilateral hearing loss. However,... (Clinical Trial)
Clinical Trial
BACKGROUND
Autosomal recessive deafness 9, caused by mutations of the OTOF gene, is characterised by congenital or prelingual, severe-to-complete, bilateral hearing loss. However, no pharmacological treatment is currently available for congenital deafness. In this Article, we report the safety and efficacy of gene therapy with an adeno-associated virus (AAV) serotype 1 carrying a human OTOF transgene (AAV1-hOTOF) as a treatment for children with autosomal recessive deafness 9.
METHODS
This single-arm, single-centre trial enrolled children (aged 1-18 years) with severe-to-complete hearing loss and confirmed mutations in both alleles of OTOF, and without bilateral cochlear implants. A single injection of AAV1-hOTOF was administered into the cochlea through the round window. The primary endpoint was dose-limiting toxicity at 6 weeks after injection. Auditory function and speech were assessed by appropriate auditory perception evaluation tools. All analyses were done according to the intention-to-treat principle. This trial is registered with Chinese Clinical Trial Registry, ChiCTR2200063181, and is ongoing.
FINDINGS
Between Oct 19, 2022, and June 9, 2023, we screened 425 participants for eligibility and enrolled six children for AAV1-hOTOF gene therapy (one received a dose of 9 × 10 vector genomes [vg] and five received 1·5 × 10 vg). All participants completed follow-up visits up to week 26. No dose-limiting toxicity or serious adverse events occurred. In total, 48 adverse events were observed; 46 (96%) were grade 1-2 and two (4%) were grade 3 (decreased neutrophil count in one participant). Five children had hearing recovery, shown by a 40-57 dB reduction in the average auditory brainstem response (ABR) thresholds at 0·5-4·0 kHz. In the participant who received the 9 × 10 vg dose, the average ABR threshold was improved from greater than 95 dB at baseline to 68 dB at 4 weeks, 53 dB at 13 weeks, and 45 dB at 26 weeks. In those who received 1·5 × 10 AAV1-hOTOF, the average ABR thresholds changed from greater than 95 dB at baseline to 48 dB, 38 dB, 40 dB, and 55 dB in four children with hearing recovery at 26 weeks. Speech perception was improved in participants who had hearing recovery.
INTERPRETATION
AAV1-hOTOF gene therapy is safe and efficacious as a novel treatment for children with autosomal recessive deafness 9.
FUNDING
National Natural Science Foundation of China, National Key R&D Program of China, Science and Technology Commission of Shanghai Municipality, and Shanghai Refreshgene Therapeutics.
Topics: Humans; Genetic Therapy; Dependovirus; Child; Male; Child, Preschool; Female; Adolescent; Infant; Genetic Vectors; Treatment Outcome; Deafness; Mutation; Membrane Proteins
PubMed: 38280389
DOI: 10.1016/S0140-6736(23)02874-X -
Nature May 2024The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances; its impairment can lead to or exacerbate various diseases of the...
The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances; its impairment can lead to or exacerbate various diseases of the central nervous system. However, the mechanisms of BBB disruption during infection and inflammatory conditions remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. ), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.
Topics: Animals; Female; Humans; Male; Mice; Basement Membrane; Blood-Brain Barrier; Brain; Caspases, Initiator; Dependovirus; Endothelial Cells; Gasdermins; Inflammation; Klebsiella pneumoniae; Lipopolysaccharide Receptors; Lipopolysaccharides; Mice, Inbred C57BL; Pyroptosis; Sepsis; Single-Cell Analysis; Tight Junctions
PubMed: 38632402
DOI: 10.1038/s41586-024-07314-2 -
Trends in Biotechnology Oct 2023Accelerating the scale up of adeno-associated virus (AAV) manufacture is highly desirable to meet the increased demand for gene therapies. However, the development of... (Review)
Review
Accelerating the scale up of adeno-associated virus (AAV) manufacture is highly desirable to meet the increased demand for gene therapies. However, the development of bioprocesses for AAV gene therapies remains time-consuming and challenging. The quality by design (QbD) approach ensures bioprocess designs that meet the desired product quality and safety profile. Rapid stress tests, developability screens, and scale-down technologies have the potential to streamline AAV product and manufacturing bioprocess development within the QbD framework. Here we review how their successful use for antibody manufacture development is translating to AAV, but also how this will depend critically on improved analytical methods and adaptation of the tools as more understanding is gained on the critical attributes of AAV required for successful therapy.
Topics: Dependovirus; Genetic Therapy; Commerce; Quality Control; Genetic Vectors
PubMed: 37127491
DOI: 10.1016/j.tibtech.2023.04.002 -
Nature Reviews. Drug Discovery Oct 2023Recent advancements in gene supplementation therapy are expanding the options for the treatment of neurological disorders. Among the available delivery vehicles,... (Review)
Review
Recent advancements in gene supplementation therapy are expanding the options for the treatment of neurological disorders. Among the available delivery vehicles, adeno-associated virus (AAV) is often the favoured vector. However, the results have been variable, with some trials dramatically altering the course of disease whereas others have shown negligible efficacy or even unforeseen toxicity. Unlike traditional drug development with small molecules, therapeutic profiles of AAV gene therapies are dependent on both the AAV capsid and the therapeutic transgene. In this rapidly evolving field, numerous clinical trials of gene supplementation for neurological disorders are ongoing. Knowledge is growing about factors that impact the translation of preclinical studies to humans, including the administration route, timing of treatment, immune responses and limitations of available model systems. The field is also developing potential solutions to mitigate adverse effects, including AAV capsid engineering and designs to regulate transgene expression. At the same time, preclinical research is addressing new frontiers of gene supplementation for neurological disorders, with a focus on mitochondrial and neurodevelopmental disorders. In this Review, we describe the current state of AAV-mediated neurological gene supplementation therapy, including critical factors for optimizing the safety and efficacy of treatments, as well as unmet needs in this field.
Topics: Humans; Dependovirus; Genetic Therapy; Transgenes; Models, Biological; Nervous System Diseases; Genetic Vectors
PubMed: 37658167
DOI: 10.1038/s41573-023-00766-7 -
Nature Oct 2023The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype-phenotype relationships in complex tissues in a...
The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype-phenotype relationships in complex tissues in a high-throughput manner. Here we introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening, termed AAV-Perturb-seq, a tuneable and broadly applicable method for transcriptional linkage analysis as well as high-throughput and high-resolution phenotyping of genetic perturbations in vivo. We applied AAV-Perturb-seq using gene editing and transcriptional inhibition to systematically dissect the phenotypic landscape underlying 22q11.2 deletion syndrome genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and previously undescribed pathways orchestrating neuronal functions in vivo that explain approximately 40% of the transcriptional changes observed in a 22q11.2-deletion mouse model. Our findings suggest that the 22q11.2-deletion syndrome transcriptional phenotype found in mature neurons may in part be due to the broad dysregulation of a class of genes associated with disease susceptibility that are important for dysfunctional RNA processing and synaptic function. Our study establishes a flexible and scalable direct in vivo method to facilitate causal understanding of biological and disease mechanisms with potential applications to identify genetic interventions and therapeutic targets for treating disease.
Topics: Animals; Humans; Mice; Dependovirus; Gene Editing; Genetic Association Studies; Neurons; Phenotype; Prefrontal Cortex; Transcription, Genetic; Single-Cell Analysis; CRISPR-Cas Systems; DiGeorge Syndrome; Disease Models, Animal; RNA Processing, Post-Transcriptional; Synapses; Genetic Predisposition to Disease
PubMed: 37730998
DOI: 10.1038/s41586-023-06570-y -
Molecular Therapy : the Journal of the... Nov 2023High-dose systemic gene therapy with adeno-associated virus (AAV) is in clinical trials to treat various inherited diseases. Despite remarkable success in spinal... (Review)
Review
High-dose systemic gene therapy with adeno-associated virus (AAV) is in clinical trials to treat various inherited diseases. Despite remarkable success in spinal muscular atrophy and promising results in other diseases, fatality has been observed due to liver, kidney, heart, or lung failure. Innate and adaptive immune responses to the vector play a critical role in the toxicity. Host factors also contribute to patient death. This mini-review summarizes clinical findings and calls for concerted efforts from all stakeholders to better understand the mechanisms underlying lethality in AAV gene therapy and to develop effective strategies to prevent/treat high-dose systemic AAV-gene-therapy-induced immunotoxicity.
Topics: Humans; Dystrophin; Dependovirus; Genetic Therapy; Immunity, Humoral; Muscular Dystrophy, Duchenne; Genetic Vectors
PubMed: 37822079
DOI: 10.1016/j.ymthe.2023.10.015 -
The AAPS Journal Jul 2023Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in... (Review)
Review
Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.
Topics: Humans; Dependovirus; Tissue Distribution; Genetic Therapy; Drug Development; Polymerase Chain Reaction
PubMed: 37523051
DOI: 10.1208/s12248-023-00842-1 -
Circulation Aug 2023Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a...
BACKGROUND
Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance.
METHODS
We developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. We compared the gene delivery and therapeutic efficacy of EV-AAVs with an equal titer of free AAVs in the presence of NAbs, both in vitro and in vivo. In addition, we investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging.
RESULTS
Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAbs, both to human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9-sarcoplasmic reticulum calcium ATPase 2a to infarcted hearts in preimmunized mice significantly improved ejection fraction and fractional shortening compared with AAV9-sarcoplasmic reticulum calcium ATPase 2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Trafficking studies using human induced pluripotent stem cell-derived cells in vitro and mouse hearts in vivo showed significantly higher expression of EV-AAV6/9-delivered genes in cardiomyocytes compared with noncardiomyocytes, even with comparable cellular uptake. Using cellular subfraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake.
CONCLUSIONS
Together, using 5 different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared with free AAVs in the presence of NAbs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure.
Topics: Humans; Mice; Animals; Dependovirus; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Genetic Vectors; Induced Pluripotent Stem Cells; Antibodies, Neutralizing; Extracellular Vesicles
PubMed: 37409482
DOI: 10.1161/CIRCULATIONAHA.122.063759 -
European Journal of Pharmaceutics and... Sep 2023Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and... (Review)
Review
Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and physico-chemical properties, delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge. Adeno-associated viruses (AAV) have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA (ssDNA) genomes over the past decades. More than a hundred products have been tested in clinical settings and three products have received market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the-art rAAV formulations and can further serve as a map for rational formulation development activities in the future.
Topics: Dependovirus; Genetic Vectors; Genetic Therapy
PubMed: 37423416
DOI: 10.1016/j.ejpb.2023.07.002 -
Nature Methods Jul 2023The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However,...
The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.
Topics: Mice; Animals; Genetic Vectors; Gene Transfer Techniques; Mice, Transgenic; Genetic Therapy; Transgenes; Dependovirus; Transduction, Genetic
PubMed: 37291262
DOI: 10.1038/s41592-023-01896-x