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International Journal of Molecular... Mar 2024The purpose of this study was to compare the retention rate of Adeno-associated viral vector (AAV) gene therapy agents within different subretinal injection systems. The...
The purpose of this study was to compare the retention rate of Adeno-associated viral vector (AAV) gene therapy agents within different subretinal injection systems. The retention of AAV serotype 2-based voretigene neparvovec (VN) and a clinical-grade AAV serotype 8 vector within four different subretinal cannulas from two different manufacturers was quantified. A standardized qPCR using the universal inverted terminal repeats as a target sequence was developed. The instruments compared were the PolyTip cannula 25 g/38 g by MedOne Surgical, Inc., Sarasota, FL, USA, and three different subretinal injection needles by DORC, Zuidland, The Netherlands (1270.EXT Extendible 41G subretinal injection needle (23G), DORC 1270.06 23G Dual bore injection cannula, DORC 27G Subretinal injection cannula). The retention rate of VN and within the DORC products (10-28%) was comparable to the retention rate (32%) found for the PolyTip cannula that is mentioned in the FDA-approved prescribing information for VN. For the AAV8 vector, the PolyTip cannula showed a retention rate of 14%, and a similar retention rate of 3-16% was found for the DORC products (test-retest variability: mean 4.5%, range 2.5-20.2%). As all the instruments tested showed comparable retention rates, they seem to be equally compatible with AAV2- and AAV8-based gene therapy agents.
Topics: Animals; Serogroup; Drug Delivery Systems; Genetic Therapy; Dependovirus; Grasshoppers; Parvovirinae
PubMed: 38612516
DOI: 10.3390/ijms25073705 -
Lab Animal May 2024Recent genome editing techniques have substantially simplified the generation of genetically modified mice. A new study combines adeno-associated viruses (AAV) and...
Recent genome editing techniques have substantially simplified the generation of genetically modified mice. A new study combines adeno-associated viruses (AAV) and electroporation to generate a robust pipeline to deliver CRISPR-Cas reagents into mouse embryos.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Electroporation; Mice; Dependovirus
PubMed: 38600182
DOI: 10.1038/s41684-024-01363-w -
Fast, accurate ranking of engineered proteins by target-binding propensity using structure modeling.Molecular Therapy : the Journal of the... Jun 2024Deep-learning-based methods for protein structure prediction have achieved unprecedented accuracy, yet their utility in the engineering of protein-based binders remains...
Deep-learning-based methods for protein structure prediction have achieved unprecedented accuracy, yet their utility in the engineering of protein-based binders remains constrained due to a gap between the ability to predict the structures of candidate proteins and the ability toprioritize proteins by their potential to bind to a target. To bridge this gap, we introduce Automated Pairwise Peptide-Receptor Analysis for Screening Engineered proteins (APPRAISE), a method for predicting the target-binding propensity of engineered proteins. After generating structural models of engineered proteins competing for binding to a target using an established structure prediction tool such as AlphaFold-Multimer or ESMFold, APPRAISE performs a rapid (under 1 CPU second per model) scoring analysis that takes into account biophysical and geometrical constraints. As proof-of-concept cases, we demonstrate that APPRAISE can accurately classify receptor-dependent vs. receptor-independent adeno-associated viral vectors and diverse classes of engineered proteins such as miniproteins targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, nanobodies targeting a G-protein-coupled receptor, and peptides that specifically bind to transferrin receptor or programmed death-ligand 1 (PD-L1). APPRAISE is accessible through a web-based notebook interface using Google Colaboratory (https://tiny.cc/APPRAISE). With its accuracy, interpretability, and generalizability, APPRAISE promises to expand the utility of protein structure prediction and accelerate protein engineering for biomedical applications.
Topics: Protein Engineering; Protein Binding; Humans; SARS-CoV-2; Models, Molecular; Spike Glycoprotein, Coronavirus; Protein Conformation; Single-Domain Antibodies; Deep Learning; COVID-19; B7-H1 Antigen; Dependovirus; Genetic Vectors
PubMed: 38582966
DOI: 10.1016/j.ymthe.2024.04.003 -
Molecular Therapy : the Journal of the... Jun 2024Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery to the nervous system, are widely used in neuroscience research, and are the basis of...
Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery to the nervous system, are widely used in neuroscience research, and are the basis of FDA-approved neuron-targeting gene therapies. Here we find that an innate immune response to the AAV genome reduces dendritic length and complexity and disrupts synaptic transmission in mouse somatosensory cortex. Dendritic loss is apparent 3 weeks after injection of experimentally relevant viral titers, is not restricted to a particular capsid serotype, transgene, promoter, or production facility, and cannot be explained by responses to surgery or transgene expression. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents and an increase in the proportion of GluA2-lacking, calcium-permeable AMPA receptors. The AAV genome is rich in unmethylated CpG DNA, which is recognized by the innate immunoreceptor Toll-like receptor 9 (TLR9), and acutely blocking TLR9 preserves dendritic complexity and AMPA receptor subunit composition in AAV-injected mice. These results reveal unexpected impacts of an immune response to the AAV genome on neuronal structure and function and identify approaches to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.
Topics: Animals; Dependovirus; Immunity, Innate; Mice; Dendrites; Synaptic Transmission; Toll-Like Receptor 9; Genetic Vectors; Receptors, AMPA; Somatosensory Cortex; Genome, Viral
PubMed: 38566414
DOI: 10.1016/j.ymthe.2024.03.036 -
Signal Transduction and Targeted Therapy Apr 2024Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene... (Review)
Review
Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.
Topics: Humans; Dependovirus; Genetic Vectors; Genetic Therapy
PubMed: 38565561
DOI: 10.1038/s41392-024-01780-w -
Viruses Mar 2024Today, adeno-associated virus (AAV)-based vectors are arguably the most promising in vivo gene delivery vehicles for durable therapeutic gene expression. Advances in... (Review)
Review
Today, adeno-associated virus (AAV)-based vectors are arguably the most promising in vivo gene delivery vehicles for durable therapeutic gene expression. Advances in molecular engineering, high-throughput screening platforms, and computational techniques have resulted in a toolbox of capsid variants with enhanced performance over parental serotypes. Despite their considerable promise and emerging clinical success, there are still obstacles hindering their broader use, including limited transduction capabilities, tissue/cell type-specific tropism and penetration into tissues through anatomical barriers, off-target tissue biodistribution, intracellular degradation, immune recognition, and a lack of translatability from preclinical models to clinical settings. Here, we first describe the transduction mechanisms of natural AAV serotypes and explore the current understanding of the systemic and cellular hurdles to efficient transduction. We then outline progress in developing designer AAV capsid variants, highlighting the seminal discoveries of variants which can transduce the central nervous system upon systemic administration, and, to a lesser extent, discuss the targeting of the peripheral nervous system, eye, ear, lung, liver, heart, and skeletal muscle, emphasizing their tissue and cell specificity and translational promise. In particular, we dive deeper into the molecular mechanisms behind their enhanced properties, with a focus on their engagement with host cell receptors previously inaccessible to natural AAV serotypes. Finally, we summarize the main findings of our review and discuss future directions.
Topics: Capsid; Dependovirus; Serogroup; Tissue Distribution; Capsid Proteins; Tropism; Genetic Vectors
PubMed: 38543807
DOI: 10.3390/v16030442 -
Viruses Mar 2024The efficacy of adeno-associated virus (AAV)-based gene therapy is dependent on effective viral transduction, which might be inhibited by preexisting immunity to AAV...
The efficacy of adeno-associated virus (AAV)-based gene therapy is dependent on effective viral transduction, which might be inhibited by preexisting immunity to AAV acquired from infection or maternal delivery. Anti-AAV neutralizing Abs (NAbs) titer is usually measured by in vitro assay and used for patient enroll; however, this assay could not evaluate NAbs' impacts on AAV pharmacology and potential harm in vivo. Here, we infused a mouse anti-AAV9 monoclonal antibody into Balb/C mice 2 h before receiving 1.2 × 10 or 3 × 10 vg/kg of rAAV9-coGAA by tail vein, a drug for our ongoing clinical trials for Pompe disease. The pharmacokinetics, pharmacodynamics, and cellular responses combined with in vitro NAb assay validated the different impacts of preexisting NAbs at different levels in vivo. Sustained GAA expression in the heart, liver, diaphragm, and quadriceps were observed. The presence of high-level NAb, a titer about 1:1000, accelerated vector clearance in blood and completely blocked transduction. The AAV-specific T cell responses tended to increase when the titer of NAb exceeded 1:200. A low-level NAbs, near 1:100, had no effect on transduction in the heart and liver as well as cellular responses, but decreased transduction in muscles slightly. Therefore, we propose to preclude patients with NAb titers > 1:100 from rAAV9-coGAA clinical trials.
Topics: Animals; Mice; Humans; Antibodies, Neutralizing; Glycogen Storage Disease Type II; Genetic Therapy; Liver; Disease Models, Animal; Dependovirus; Genetic Vectors; Antibodies, Viral
PubMed: 38543765
DOI: 10.3390/v16030400 -
Physiological Reports Mar 2024Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the...
Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure-overloaded heart. CF-specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno-associated virus serotype 9 (AAV9)-hTCF21 system provide a powerful tool for understanding CF biology in vivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell-specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs in vivo. Here, we report a simplified CF-specific rat transgenic model using an AAV9-hTCF21 system that achieved a CF-specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF-specific transgenic models for studying cardiovascular diseases in vivo.
Topics: Mice; Animals; Rats; Humans; Myocytes, Cardiac; Dependovirus; Cardiomyopathies; Heart Diseases; Mice, Knockout; Fibroblasts; Basic Helix-Loop-Helix Transcription Factors
PubMed: 38538007
DOI: 10.14814/phy2.15989 -
ELife Mar 2024Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production...
Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2 gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production.
Topics: Genetic Vectors; Mutation; Capsid Proteins; Capsid; Mutagenesis; Dependovirus
PubMed: 38536879
DOI: 10.7554/eLife.87730 -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... Feb 2024To investigate the protective effect of NDUFA13 protein against acute liver injury and liver fibrosis in mice and explore the possible mechanisms.
OBJECTIVE
To investigate the protective effect of NDUFA13 protein against acute liver injury and liver fibrosis in mice and explore the possible mechanisms.
METHODS
BALB/C mice (7 to 8 weeks old) were divided into normal group, CCl group, CCl+AAV-NC group and CCl+AAV-NDU13 group (=18). Mouse models of liver fibrosis were established by intraperitoneal injection of CCl twice a week for 3, 5 or 7 weeks, and the recombinant virus AAV8-TBG-NC or AAV8-TBG-NDUFA13 was injected via the tail vein 7-10 days prior to CCl injection. After the treatments, pathological changes in the liver of the mice were observed using HE and Masson staining. Hepatic expression levels of NDUFA13 and α-SMA were detected with Western blotting, and the coexpression of NDUFA13 and NLRP3, TNF-α and IL-1β, and α-SMA and collagen Ⅲ was analyzed with immunofluorescence assay.
RESULTS
HE and Masson staining showed deranged liver architecture, necrotic hepatocytes and obvious inflammatory infiltration and collagen fiber deposition in mice with CCl injection ( < 0.001). NDUFA13 expression markedly decreased in CCl-treated mice ( < 0.001), while a significant reduction in inflammatory aggregation and fibrosis was observed in mice with AAV-mediated NDUFA13 overexpression ( < 0.001). In CCl+AAV-NDU13 group, immunofluorescence assay revealed markedly weakened activation of NLRP3 inflammasomes ( < 0.001), significantly decreased TNF-α and IL-1β secretion ( < 0.001), and inhibited hepatic stellate cell activation ( < 0.05) and collagen formation in the liver ( < 0.001).
CONCLUSION
Mitochondrial NDUFA13 overexpression in hepatocytes protects against CCl- induced liver fibrosis in mice by inhibiting activation of NLRP3 signaling.
Topics: Mice; Animals; Dependovirus; Tumor Necrosis Factor-alpha; NLR Family, Pyrin Domain-Containing 3 Protein; Mice, Inbred BALB C; Liver; Liver Cirrhosis; Hepatocytes; Collagen; Hepatic Stellate Cells; Carbon Tetrachloride
PubMed: 38501404
DOI: 10.12122/j.issn.1673-4254.2024.02.01