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Nature Communications Nov 2023Astrocytes, one of the most prevalent cell types in the central nervous system (CNS), are critically involved in neural function. Genetically manipulating astrocytes is...
Astrocytes, one of the most prevalent cell types in the central nervous system (CNS), are critically involved in neural function. Genetically manipulating astrocytes is an essential tool in understanding and affecting their roles. Adeno-associated viruses (AAVs) enable rapid genetic manipulation; however, astrocyte specificity of AAVs can be limited, with high off-target expression in neurons and sparsely in endothelial cells. Here, we report the development of a cassette of four copies of six miRNA targeting sequences (4x6T) which triggers transgene degradation specifically in neurons and endothelial cells. In combination with the GfaABC1D promoter, 4x6T increases astrocytic specificity of Cre with a viral reporter from <50% to >99% in multiple serotypes in mice, and confers astrocyte specificity in multiple recombinases and reporters. We also present empty vectors to add 4x6T to other cargo, independently and in Cre/Dre-dependent forms. This toolbox of AAVs allows rapid manipulation of astrocytes throughout the CNS, is compatible with different AAV serotypes, and demonstrates the efficacy of using multiplexed miRNA targeting sequences to decrease expression in multiple off-target cell populations simultaneously.
Topics: Mice; Animals; Astrocytes; MicroRNAs; Serogroup; Endothelial Cells; Genetic Vectors; Dependovirus
PubMed: 37973910
DOI: 10.1038/s41467-023-42746-w -
BioEssays : News and Reviews in... Jun 2022Recombinant adeno-associated viruses (rAAVs) are promising vectors for the delivery of various genetic constructs into eukaryotic cells. rAAVs have a number of... (Review)
Review
Recombinant adeno-associated viruses (rAAVs) are promising vectors for the delivery of various genetic constructs into eukaryotic cells. rAAVs have a number of properties that make it possible to successfully use them both in vitro and in vivo. Purification and concentration of rAAV vectors are critical for achieving high viral titer, stability, efficiency, and purity. This review systematically analyses all available purification approaches. The purification methods described in this work differ substantially from each other in mechanisms, efficiency, labor time, and cost. Researchers have to choose a purification algorithm depending on the purpose of their work. We strive to simplify the choice of the necessary and sufficient technique based on the experimental needs and available resources of the laboratory.
Topics: Dependovirus; Genetic Therapy; Genetic Vectors
PubMed: 35426143
DOI: 10.1002/bies.202200019 -
Journal of Clinical Pharmacology Dec 2022Recombinant adeno-associated virus (AAV) is currently the most widely used platform for in vivo gene therapy. Clinical pharmacology is a central field for AAV gene... (Review)
Review
Recombinant adeno-associated virus (AAV) is currently the most widely used platform for in vivo gene therapy. Clinical pharmacology is a central field for AAV gene therapy, represented by the pillars of pharmacokinetics, pharmacodynamics/efficacy, and safety. In this review, we provide a comprehensive summary of clinical pharmacology considerations for recombinant AAV. The main topics covered are biodistribution and shedding, dose-exposure-response relationship, safety, immune and stress response, and clinical dose selection strategies. We highlight how the cumulative knowledge of AAV gene therapy could help with guiding clinical trial design and assessing and mitigating risks, as well as planning and executing pharmacokinetic/pharmacodynamic /safety data analyses. In addition, we discuss the major gaps and areas of growth in clinical pharmacology understanding of recombinant AAV. These include the mechanisms of the durability of treatment response and variability in biodistribution, transduction, and immunogenicity, as well as a potential influence on AAV's safety and efficacy profiles by drug product characteristics and patient intrinsic/extrinsic factors.
Topics: Humans; Pharmacology, Clinical; Dependovirus; Tissue Distribution; Genetic Therapy
PubMed: 36461742
DOI: 10.1002/jcph.2141 -
Molecular Therapy : the Journal of the... Aug 2020
Topics: Antibodies, Neutralizing; Dependovirus; Endopeptidases; Genetic Therapy; Immunoglobulin G
PubMed: 32697940
DOI: 10.1016/j.ymthe.2020.07.015 -
Journal of Neurophysiology Jul 2016Understanding how the brain works requires understanding how different types of neurons contribute to circuit function and organism behavior. Progress on this front has... (Review)
Review
Understanding how the brain works requires understanding how different types of neurons contribute to circuit function and organism behavior. Progress on this front has been accelerated by optogenetics and chemogenetics, which provide an unprecedented level of control over distinct neuronal types in small animals. In primates, however, targeting specific types of neurons with these tools remains challenging. In this review, we discuss existing and emerging strategies for directing genetic manipulations to targeted neurons in the adult primate central nervous system. We review the literature on viral vectors for gene delivery to neurons, focusing on adeno-associated viral vectors and lentiviral vectors, their tropism for different cell types, and prospects for new variants with improved efficacy and selectivity. We discuss two projection targeting approaches for probing neural circuits: anterograde projection targeting and retrograde transport of viral vectors. We conclude with an analysis of cell type-specific promoters and other nucleotide sequences that can be used in viral vectors to target neuronal types at the transcriptional level.
Topics: Animals; Dependovirus; Gene Transfer Techniques; Genetic Vectors; Lentivirus; Neural Pathways; Neurons; Optogenetics; Primates; Viral Tropism
PubMed: 27052579
DOI: 10.1152/jn.00087.2016 -
Current Opinion in Biotechnology Jun 2022Gene therapy is designed to cure various diseases resulting from genetic defects. Currently, recombinant adeno-associated viral vectors (rAAV) are the vehicles of choice... (Review)
Review
Gene therapy is designed to cure various diseases resulting from genetic defects. Currently, recombinant adeno-associated viral vectors (rAAV) are the vehicles of choice for therapeutic gene delivery in vivo. To date, manufacturing sufficient rAAV product to meet rapidly expanding clinical demand remains a bottleneck in the industry. In the past decade, multiple production platforms have been rapidly implemented with encouraging improvements in productivity and scalability. In this review, we discuss the advantages and limitations of the most popular production platforms in the industry with a focus on the cell culture process scale-up.
Topics: Cell Culture Techniques; Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors
PubMed: 35398708
DOI: 10.1016/j.copbio.2022.102721 -
Journal of Virology Jul 2023Parvoviruses are among the smallest and superficially simplest animal viruses, infecting a broad range of hosts, including humans, and causing some deadly infections. In... (Review)
Review
The Structures and Functions of Parvovirus Capsids and Missing Pieces: the Viral DNA and Its Packaging, Asymmetrical Features, Nonprotein Components, and Receptor or Antibody Binding and Interactions.
Parvoviruses are among the smallest and superficially simplest animal viruses, infecting a broad range of hosts, including humans, and causing some deadly infections. In 1990, the first atomic structure of the canine parvovirus (CPV) capsid revealed a 26-nm-diameter T=1 particle made up of two or three versions of a single protein, and packaging about 5,100 nucleotides of single-stranded DNA. Our structural and functional understanding of parvovirus capsids and their ligands has increased as imaging and molecular techniques have advanced, and capsid structures for most groups within the family have now been determined. Despite those advances, significant questions remain unanswered about the functioning of those viral capsids and their roles in release, transmission, or cellular infection. In addition, the interactions of capsids with host receptors, antibodies, or other biological components are also still incompletely understood. The parvovirus capsid's apparent simplicity likely conceals important functions carried out by small, transient, or asymmetric structures. Here, we highlight some remaining open questions that may need to be answered to provide a more thorough understanding of how these viruses carry out their various functions. The many different members of the family share a capsid architecture, and while many functions are likely similar, others may differ in detail. Many of those parvoviruses have not been experimentally examined in detail (or at all in some cases), so we, therefore, focus this minireview on the widely studied protoparvoviruses, as well as the most thoroughly investigated examples of adeno-associated viruses.
Topics: Animals; Humans; Capsid; Capsid Proteins; DNA, Viral; Parvoviridae; Parvoviridae Infections; Dependovirus
PubMed: 37367301
DOI: 10.1128/jvi.00161-23 -
Journal of Molecular Medicine (Berlin,... Aug 2021Gene therapy of genetically determined diseases, including some pathologies of the respiratory system, requires an efficient method for transgene delivery. Recombinant... (Review)
Review
Gene therapy of genetically determined diseases, including some pathologies of the respiratory system, requires an efficient method for transgene delivery. Recombinant adeno-associated viral (rAAV) vectors are well studied and employed in gene therapy, as they are relatively simple and low immunogenic and able to efficiently transduce eukaryotic cells. To date, many natural and artificial (with modified capsids) AAV serotypes have been isolated, demonstrating preferential tropism toward different tissues and cells in accordance with the prevalent receptors on the cell surface. However, rAAV-mediated delivery is not strictly specific due to wide tropism of some viral serotypes. Thus, the development of the methods allowing modulating specificity of these vectors could be beneficial in some cases. This review describes various approaches for retargeting rAAV to respiratory cells, for example, using different types of capsid modifications and regulation of a transgene expression by tissue-specific promoters. Part of the review is devoted to the issues of transduction of stem and progenitor lung cells using AAV, which is a complicated task today.
Topics: Animals; Dependovirus; Disease Management; Disease Susceptibility; Gene Expression; Gene Expression Regulation; Gene Transfer Techniques; Genetic Engineering; Genetic Therapy; Genetic Vectors; Humans; Lung Diseases; Organ Specificity; Promoter Regions, Genetic; Stem Cells; Transduction, Genetic; Transgenes
PubMed: 34021360
DOI: 10.1007/s00109-021-02086-y -
Nature Reviews. Drug Discovery Oct 2022
Topics: Dependovirus; Genetic Vectors; Humans
PubMed: 36056263
DOI: 10.1038/d41573-022-00148-5 -
Human Gene Therapy Apr 2017AAV has been studied for 55 years and has been developed as a vector for about 35 years. By now, there is a fairly good idea of the dimensions of what would be useful to... (Review)
Review
AAV has been studied for 55 years and has been developed as a vector for about 35 years. By now, there is a fairly good idea of the dimensions of what would be useful to know to employ AAV optimally as a vector, but there are still many unanswered questions within the system. As with all biological systems, each good experiment raises further questions to answer. This article provides an overview of those areas in which unknown information can be identified and of those questions that have not yet been recognized. Some of these are touched on in the six review articles in this issue of Human Gene Therapy.
Topics: Dependovirus; Genetic Therapy; Genetic Vectors; Humans
PubMed: 28335618
DOI: 10.1089/hum.2017.048