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Molecular Therapy : the Journal of the... Oct 2021Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal...
Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal vector, its single-stranded DNA genome is prone to intra- and inter-molecular recombination leading to rearrangements and integration into the host cell genome. Here, we ascertained the integration frequency of rAAV in human hepatocytes transduced either ex vivo or in vivo and subsequently expanded in a mouse model of xenogeneic liver regeneration. Chromosomal rAAV integration events and vector integrity were determined using the capture-PacBio sequencing approach, a long-read next-generation sequencing method that has not previously been used for this purpose. Chromosomal integrations were found at a surprisingly high frequency of 1%-3% both in vitro and in vivo. Importantly, most of the inserted rAAV sequences were heavily rearranged and were accompanied by deletions of the host genomic sequence at the integration site.
Topics: Animals; Cells, Cultured; Chromosomes; Dependovirus; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Hepatocytes; Humans; Liver Regeneration; Mice; Transduction, Genetic; Virus Integration
PubMed: 34461297
DOI: 10.1016/j.ymthe.2021.08.031 -
Current Opinion in Virology Oct 2014Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue... (Review)
Review
Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue tropism, and limited viral host response has made it an attractive vector system for gene therapy. The viral protein capsid, the primary interface with the host, is the main determinant for these phenotypes, is highly variable, and is most subject to pressures during replication. Here, we explore the evolutionary path of AAV and other parvoviruses in respect to these phenotypes, as well as directed evolution and engineering strategies that have exploited the lessons learned from natural selection in order to address remaining limitations of AAV as a therapeutic gene transfer platform.
Topics: Biological Evolution; Dependovirus; Directed Molecular Evolution; Genetic Therapy; Genetic Vectors; Humans; Selection, Genetic; Viral Tropism
PubMed: 25128609
DOI: 10.1016/j.coviro.2014.07.008 -
Current Opinion in Biotechnology Apr 2022Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical... (Review)
Review
Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical breakthroughs. As of April 2021, there are two rAAV-based gene therapies approved and more than two-hundred active clinical trials (approximately thirty in Phase III). However, the expected increase in demand for rAAV vectors still poses several challenges. Discussed herein are key aspects related to R&D needs and Chemistry, Manufacturing and Control (CMC) efforts required to attend this growing demand. Authors provide their perspective on strategic topics for rAAV-based therapies success: scalability and productivity; improved safety; increased process understanding combined with development of orthogonal bioanalytics that are able to identify, monitor and control Critical Quality Attributes (CQAs) during bioprocessing.
Topics: Dependovirus; Genetic Therapy; Genetic Vectors
PubMed: 35007989
DOI: 10.1016/j.copbio.2021.12.009 -
Cell Reports Nov 2023Promoters are essential tools for basic and translational neuroscience research. An ideal promoter should possess the shortest possible DNA sequence with cell-type...
Promoters are essential tools for basic and translational neuroscience research. An ideal promoter should possess the shortest possible DNA sequence with cell-type selectivity. However, whether ultra-compact promoters can offer neuron-specific expression is unclear. Here, we report the development of an extremely short promoter that enables selective gene expression in neurons, but not glial cells, in the brain. The promoter sequence originates from the human CALM1 gene and is only 120 bp in size. The CALM1 promoter (pCALM1) embedded in an adeno-associated virus (AAV) genome directed broad reporter expression in excitatory and inhibitory neurons in mouse and monkey brains. Moreover, pCALM1, when inserted into an all-in-one AAV vector expressing SpCas9 and sgRNA, drives constitutive and conditional in vivo gene editing in neurons and elicits functional alterations. These data demonstrate the ability of pCALM1 to conduct restricted neuronal gene expression, illustrating the feasibility of ultra-miniature promoters for targeting brain-cell subtypes.
Topics: Mice; Animals; Humans; RNA, Guide, CRISPR-Cas Systems; Neurons; Brain; Neuroglia; Genetic Therapy; Genetic Vectors; Dependovirus
PubMed: 37910509
DOI: 10.1016/j.celrep.2023.113348 -
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 -
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 -
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