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Methods in Molecular Biology (Clifton,... 2008Adeno-associated virus (AAV) is one of the most promising viral gene transfer vectors that has been shown to effect long-term gene expression and disease correction with... (Review)
Review
Adeno-associated virus (AAV) is one of the most promising viral gene transfer vectors that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The surface of the AAV capsid is an essential component that is involved in cell binding, internalization, and trafficking within the targeted cell. Prior to developing a gene therapy strategy that utilizes AAV, the serotype should be carefully considered since each capsid exhibits a unique tissue tropism and transduction efficiency. Several approaches have been undertaken in an effort to target AAV vectors to specific cell types, including utilizing natural serotypes that target a desired cellular receptor, producing pseudotyped vectors, and engineering chimeric and mosaic AAV capsids. These capsid modifications are being incorporated into vector production and purification methods that provide for the ability to scale-up the manufacturing process to support human clinical trials. Protocols for small-scale and large-scale production of AAV, as well as assays to characterize the final vector product, are presented here. The structures of AAV2, AAV4, and AAV5 have been solved by X-ray crystallography or cryo-electron microscopy (cryo-EM), and provide a basis for rational vector design in developing customized capsids for specific targeting of AAV vectors. The capsid of AAV has been shown to be remarkably stable, which is a desirable characteristic for a gene therapy vector; however, recently it has been shown that the AAV serotypes exhibit differential susceptibility to proteases. The capsid fragmentation pattern when exposed to various proteases, as well as the susceptibility of the serotypes to a series of proteases, provides a unique fingerprint for each serotype that can be used for capsid identity validation. In addition to serotype identification, protease susceptibility can also be utilized to study dynamic structural changes that must occur for the AAV capsid to perform its various functions during the virus life cycle. The use of proteases for structural studies in solution complements the crystal structural studies of the virus. A generic protocol based on proteolysis for AAV serotype identification is provided here.
Topics: Animals; Capsid; Dependovirus; Gene Transfer Techniques; Genetic Vectors; Humans
PubMed: 18369962
DOI: 10.1007/978-1-59745-210-6_2 -
Nature Communications Sep 2022Because of their small size, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a...
Because of their small size, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.
Topics: CRISPR-Cas Systems; Dependovirus; Endonucleases; Gene Editing; Genetic Therapy
PubMed: 36153319
DOI: 10.1038/s41467-022-33346-1 -
Journal of the Association For Research... Oct 2022Adeno-associated viruses (AAVs) are viral vectors that offer an excellent platform for gene therapy due to their safety profile, persistent gene expression in... (Review)
Review
Adeno-associated viruses (AAVs) are viral vectors that offer an excellent platform for gene therapy due to their safety profile, persistent gene expression in non-dividing cells, target cell specificity, lack of pathogenicity, and low immunogenicity. Recently, gene therapy for genetic hearing loss with AAV transduction has shown promise in animal models. However, AAV transduction for gene silencing or expression to prevent or manage acquired hearing loss is limited. This review provides an overview of AAV as a leading gene delivery vector for treating genetic hearing loss in animal models. We highlight the advantages and shortcomings of AAV for investigating the mechanisms and preventing acquired hearing loss. We predict that AAV-mediated gene manipulation will be able to prevent acquired hearing loss.
Topics: Animals; Dependovirus; Genetic Vectors; Genetic Therapy; Gene Transfer Techniques; Hearing Loss; Deafness; Transduction, Genetic
PubMed: 36002664
DOI: 10.1007/s10162-022-00866-y -
Frontiers in Immunology 2021Adeno-associated virus is a highly efficient DNA delivery vehicle for genome editing strategies that employ CRISPR/Cas9 and a DNA donor for homology-directed repair.... (Review)
Review
Adeno-associated virus is a highly efficient DNA delivery vehicle for genome editing strategies that employ CRISPR/Cas9 and a DNA donor for homology-directed repair. Many groups have used this strategy in development of therapies for blood and immune disorders such as sickle-cell anemia and severe-combined immunodeficiency. However, recent events have called into question the immunogenicity of AAV as a gene therapy vector and the safety profile dictated by the immune response to this vector. The target cells dictating this response and the molecular mechanisms dictating cellular response to AAV are poorly understood. Here, we will investigate the current known AAV capsid and genome interactions with cellular proteins during early stage vector transduction and how these interactions may influence innate cellular responses. We will discuss the current understanding of innate immune activation and DNA damage response to AAV, and the limitations of what is currently known. In particular, we will focus on pathway differences in cell line verses primary cells, with a focus on hematopoietic stem and progenitor cells (HSPCs) in the context of gene editing, and what we can learn from HSPC infection by other parvoviruses. Finally, we will discuss how innate immune and DNA damage response pathway activation in these highly sensitive stem cell populations may impact long-term engraftment and clinical outcomes as these gene-editing strategies move towards the clinic, with the aim to propose pathways relevant for improved hematopoietic stem cell survival and long-term engraftment after AAV-mediated genome editing.
Topics: Animals; CRISPR-Cas Systems; Capsid; Capsid Proteins; DNA Damage; Dependovirus; Gene Editing; Genetic Therapy; Genetic Vectors; Genome, Viral; Humans; Immunity, Innate; Mice
PubMed: 34122418
DOI: 10.3389/fimmu.2021.660302 -
Molecular Therapy : the Journal of the... Apr 2023
Topics: Genetic Therapy; Gene Transfer Techniques; Immunity; Dependovirus; Genetic Vectors
PubMed: 36924775
DOI: 10.1016/j.ymthe.2023.02.020 -
Current Opinion in Biotechnology Oct 2021Recombinant adeno-associated virus (rAAV) is an increasingly important gene therapy vector, but its properties present unique challenges to critical quality attribute... (Review)
Review
Recombinant adeno-associated virus (rAAV) is an increasingly important gene therapy vector, but its properties present unique challenges to critical quality attribute (CQA) identification and analytics development. Advances in, and ongoing hurdles to, characterizing rAAV proteins, nucleic acids, and vector potency are discussed in this review. For nucleic acids and vector potency, current analytical techniques for defined CQAs would benefit from further optimization, while for proteins, more complete characterization and mapping of properties to safety and efficacy is needed to finalize CQAs. The benefits of leveraging reference vectors to validate analytics and CQA ranges are also proposed. Once defined, CQA specifications can be used to establish target parameters for and inform the development of next generation rAAV processes.
Topics: Dependovirus; Genetic Therapy; Genetic Vectors; Reference Standards
PubMed: 34273809
DOI: 10.1016/j.copbio.2021.06.025 -
Molecular Therapy : the Journal of the... Mar 2016In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination... (Review)
Review
In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process--referred to as AAV-mediated gene targeting--has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.
Topics: Animals; Dependovirus; Endonucleases; Gene Editing; Gene Targeting; Gene Transfer Techniques; Genetic Engineering; Genetic Vectors; Genome; Genomics; Humans; Research; Virus Integration
PubMed: 26373345
DOI: 10.1038/mt.2015.151 -
Molecular Therapy : the Journal of the... Dec 2021
Topics: Dependovirus; Genetic Vectors; HeLa Cells; Humans; Virus Integration
PubMed: 34758291
DOI: 10.1016/j.ymthe.2021.10.024 -
Molecular Therapy : the Journal of the... Apr 2003
Topics: Dependovirus; Gene Targeting; Genetic Vectors; Humans; Models, Biological; Transduction, Genetic
PubMed: 12727103
DOI: 10.1016/s1525-0016(03)00072-8 -
Viruses Apr 2019Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic...
Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic diseases. In this study, the unique N-terminus of AAV capsid viral protein 1 (VP1u), containing a canonical group XIII PLA enzyme domain, was observed to also exhibit proteolytic activity. This protease activity can target casein and gelatin, two standard substrates used for testing protease function but does not self-cleave in the context of the capsid or target globular proteins, for example, bovine serum albumin (BSA). However, heated BSA is susceptible to VP1u-mediated cleavage, suggesting that disordered proteins are substrates for this protease function. The protease activity is partially inhibited by divalent cation chelators ethylenediaminetetraacetic acid (EDTA) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and human alpha-2-macroglobulin (A2M), a non-specific protease inhibitor. Interestingly, both the bovine pancreatic (group VIIA) and bee venom (group III) PLA enzymes also exhibit protease function against casein. This indicates that PLA groups, including VP1u, have a protease function. Amino acid substitution of the PLA catalytic motif (HD/AN) in the AAV2 VP1u resulted in attenuation of protease activity, suggesting that the protease and PLA active sites are related. However, the amino acid substitution of histidine H38, which is not involved in PLA function, to alanine, also affects protease activity, suggesting that the active site/mechanism of the PLA and protease function are not identical.
Topics: Amino Acid Sequence; Animals; Calcium; Capsid Proteins; Dependovirus; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Intrinsically Disordered Proteins; Models, Molecular; Peptide Hydrolases; Protease Inhibitors; Protein Conformation; Protein Interaction Domains and Motifs; Proteolysis; Substrate Specificity; Viral Proteins; Virion
PubMed: 31035643
DOI: 10.3390/v11050399