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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 -
Clinical and Translational Medicine Mar 2024Adeno-associated virus (AAV)-based therapies are recognized as one of the most potent next-generation treatments for inherited and genetic diseases. However, several... (Review)
Review
Adeno-associated virus (AAV)-based therapies are recognized as one of the most potent next-generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV-based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV-based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8 kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV-based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies.
Topics: Dependovirus; Transgenes; Genetic Therapy; Genetic Vectors
PubMed: 38488469
DOI: 10.1002/ctm2.1607 -
Nature Communications Mar 2024Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized...
Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.
Topics: Humans; Genetic Vectors; Dependovirus; Antibodies, Neutralizing; Liver; Perfusion
PubMed: 38485924
DOI: 10.1038/s41467-024-46194-y -
International Journal of Molecular... Feb 2024Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However,...
A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.
Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.
Topics: Animals; Capsid Proteins; Dependovirus; Chromatography, Liquid; Liquid Chromatography-Mass Spectrometry; Workflow; Genetic Vectors; Tandem Mass Spectrometry; Baculoviridae; Mammals
PubMed: 38474031
DOI: 10.3390/ijms25052785 -
Gene Therapy May 2024Adeno-associated viruses (AAV) are commonly used in the scientific field due to their diverse application range. However, AAV shedding, the release of virions from the...
Adeno-associated viruses (AAV) are commonly used in the scientific field due to their diverse application range. However, AAV shedding, the release of virions from the host organism, can impact the safety of AAV-based approaches. An increasing number of authorities require the characterization of vector shedding in clinical trials. Recently, shedding of transduced laboratory animals has also gained attention regarding the necessary disposal measures of their waste products. However, no explicit international regulations for AAV-shedding waste exist. Generating insights into shedding dynamics becomes increasingly relevant to help authorities develop adequate regulations. To date, knowledge of AAV vector shedding in mice is very limited. Moreover, confirmation of functional shed AAV particles in mice is missing. Therefore, we examined feces, urine, and saliva of mice after CNS injection with AAV2/8. It revealed the presence of viral DNA fragments via qPCR for up to 4 days after injection. To examine AAV functionality we performed nested PCR and could not detect full-length viral genomes in any but two collected feces samples. Furthermore, a functional infection assay did not reveal evidence of intact AAV particles. Our findings are supposed to contribute murine shedding data as a foundation to help establish still lacking adequate biosafety regulations in the context of AAV shedding.
Topics: Animals; Dependovirus; Mice; Genetic Vectors; Virus Shedding; DNA, Viral; Feces; Mice, Inbred C57BL; Saliva; Humans
PubMed: 38467879
DOI: 10.1038/s41434-024-00447-z -
The Journal of General Virology Mar 2024Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite...
Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.
Topics: Animals; Swine; Active Transport, Cell Nucleus; Dependovirus; Capsid Proteins; Chiroptera; Karyopherins; Nuclear Localization Signals; alpha Karyopherins
PubMed: 38441555
DOI: 10.1099/jgv.0.001960 -
CNS Neuroscience & Therapeutics Mar 2024Epilepsy is a widespread and chronic disease of the central nervous system caused by a variety of factors. Mitochondrial ferritin (FtMt) refers to ferritin located...
Mitochondrial ferritin alleviates ferroptosis in a kainic acid-induced mouse epilepsy model by regulating iron homeostasis: Involvement of nuclear factor erythroid 2-related factor 2.
BACKGROUND
Epilepsy is a widespread and chronic disease of the central nervous system caused by a variety of factors. Mitochondrial ferritin (FtMt) refers to ferritin located within the mitochondria that may protect neurons against oxidative stress by binding excess free iron ions in the cytoplasm. However, the potential role of FtMt in epilepsy remains unclear. We aimed to investigate whether FtMt and its related mechanisms can regulate epilepsy by modulating ferroptosis.
METHODS
Three weeks after injection of adeno-associated virus (AAV) in the skull of adult male C57BL/6 mice, kainic acid (KA) was injected into the hippocampus to induce seizures. Primary hippocampal neurons were transfected with siRNA using a glutamate-mediated epilepsy model. After specific treatments, Western blot analysis, immunofluorescence, EEG recording, transmission electron microscopy, iron staining, silver staining, and Nissl staining were performed.
RESULTS
At different time points after KA injection, the expression of FtMt protein in the hippocampus of mice showed varying degrees of increase. Knockdown of the FtMt gene by AAV resulted in an increase in intracellular free iron levels and a decrease in the function of iron transport-related proteins, promoting neuronal ferroptosis and exacerbating epileptic brain activity in the hippocampus of seizure mice. Additionally, increasing the expression level of FtMt protein was achieved by AAV-mediated upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) gene in the hippocampus of seizure mice.
CONCLUSIONS
In epilepsy, Nrf2 modulates ferroptosis by involving the expression of FtMt and may be a potential therapeutic mechanism of neuronal injury after epilepsy. Targeting this relevant process for treatment may be a therapeutic strategy to prevent epilepsy.
Topics: Male; Animals; Mice; Mice, Inbred C57BL; Kainic Acid; Ferroptosis; NF-E2-Related Factor 2; Epilepsy; Seizures; Glutamic Acid; Dependovirus; Disease Models, Animal; Ferritins; Homeostasis
PubMed: 38439636
DOI: 10.1111/cns.14663 -
Nature Communications Mar 2024Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction...
Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg/Rag2/Fah/Aavr (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.
Topics: Humans; Animals; Mice; Liver; Dependovirus; Disease Models, Animal; Genetic Therapy; Hepatocytes
PubMed: 38438373
DOI: 10.1038/s41467-024-46017-0