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Heart, Lung & Circulation Jul 2023Globally, adeno-associated virus (AAV) vectors have been increasingly used for clinical gene therapy trials. In Australia, AAV-based gene therapy is available for... (Review)
Review
Globally, adeno-associated virus (AAV) vectors have been increasingly used for clinical gene therapy trials. In Australia, AAV-based gene therapy is available for hereditary diseases such as retinal dystrophy or spinal muscular atrophy 1 (SMA1). Many preclinical studies have used AAV vectors for gene therapy in models of cardiac disease with outcomes of varying translational potential. However, major barriers to effective and safe therapeutic gene delivery to the human heart remain to be overcome. These include tropism, efficient gene transfer, mitigating off-target gene delivery and avoidance of the host immune response. Developing such an enhanced AAV vector for cardiac gene therapy is of great interest to the field of advanced cardiac therapeutics. In this review, we provide an overview of the approaches currently being employed in the search for cardiac cell-specific AAV capsids, ranging from natural AAVs selected as a result of infection and latency in the heart, to the use of cutting-edge molecular techniques to engineer and select AAVs specific for cardiac cells with the use of high-throughput methods.
Topics: Humans; Gene Transfer Techniques; Dependovirus; Viral Tropism; Genetic Vectors
PubMed: 37451880
DOI: 10.1016/j.hlc.2023.06.704 -
Neuroscience Bulletin May 2023
Topics: Dependovirus; Microglia; Cells, Cultured; Transduction, Genetic
PubMed: 36333483
DOI: 10.1007/s12264-022-00975-x -
Advances in Virus Research 2020Adeno-associated virus (AAV) is a nonenveloped, ssDNA virus in the parvovirus family, which has become one of the leading candidate vectors for human gene therapy. AAV... (Review)
Review
Adeno-associated virus (AAV) is a nonenveloped, ssDNA virus in the parvovirus family, which has become one of the leading candidate vectors for human gene therapy. AAV has been studied extensively to identify host cellular factors involved in infection, as well as to identify capsid variants that confer clinically favorable transduction profiles ex vivo and in vivo. Recent advances in technology have allowed for direct genetic approaches to be used to more comprehensively characterize host factors required for AAV infection and allowed for identification of a critical multi-serotype receptor, adeno-associated virus receptor (AAVR). In this chapter, we will discuss the interactions of AAV with its glycan and proteinaceous receptors and describe the host and viral components involved in AAV entry, which requires cellular attachment, endocytosis, trafficking to the trans-Golgi network and nuclear import. AAV serves as a paradigm for entry of nonenveloped viruses. Furthermore, we will discuss the potential of utilizing our increased understanding of virus-host interactions during AAV entry to develop better AAV-based therapeutics, with a focus on host factors and capsid interactions involved in in vivo tropism.
Topics: Animals; Dependovirus; Genetic Vectors; Humans; Parvoviridae Infections; Receptors, Virus; Virus Attachment; Virus Internalization
PubMed: 32327148
DOI: 10.1016/bs.aivir.2020.01.002 -
Journal of Biosciences Sep 2019The success of viral vectors mediated gene therapy is still hampered by immunogenicity and insufficient transgene expression. Alternatively, non-viral vectors mediated... (Review)
Review
The success of viral vectors mediated gene therapy is still hampered by immunogenicity and insufficient transgene expression. Alternatively, non-viral vectors mediated gene delivery has the advantage of low immunogenicity despite showing low transgene expression. By carefully considering the advantages of each approach, hybrid vectors are currently being developed by modifying the viral vectors using non-viral biopolymers. This review provides an overview of the hybrid vectors currently being developed.
Topics: Animals; Biopolymers; Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Transgenes
PubMed: 31502562
DOI: No ID Found -
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 -
European Journal of Pharmaceutics and... Aug 2023Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations...
Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations during development, as well as the quality of different batches and the consistency of the processes. Here, we compare biophysical methods to characterize purity and DNA content of viral capsids from five different serotypes (AAV2, AAV5, AAV6, AAV8, and AAV9). For this purpose, we apply multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to obtain the species' contents and to derive the wavelength-specific correction factors for the respective insert-size. In an orthogonal manner we perform anion exchange chromatography (AEX) and UV-spectroscopy and the three methods yield comparable results on empty/filled capsid contents with these correction factors. Whereas AEX and UV-spectroscopy can quantify empty and filled AAVs, only SV-AUC could identify the low amounts of partially filled capsids present in the samples used in this study. Finally, we employ negative-staining transmission electron microscopy and mass photometry to support the empty/filled ratios with methods that classify individual capsids. The obtained ratios are consistent throughout the orthogonal approaches as long as no other impurities and aggregates are present. Our results show that the combination of selected orthogonal methods can deliver consistent empty/filled contents on non-standard genome sizes, as well as information on other relevant critical quality attributes, such as AAV capsid concentration, genome concentration, insert size length and sample purity to characterize and compare AAV preparations.
Topics: Capsid; Dependovirus; Genetic Vectors; Capsid Proteins; Ultracentrifugation; DNA
PubMed: 37196871
DOI: 10.1016/j.ejpb.2023.05.011 -
Oncotarget Nov 2023
Topics: Humans; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; CRISPR-Cas Systems; Dependovirus
PubMed: 38010820
DOI: 10.18632/oncotarget.28459 -
Journal of Molecular Biology Aug 2018Over 50 years after its initial description, adeno-associated virus (AAV) remains the most exciting but also most elusive study object in basic or applied virology. On... (Review)
Review
Over 50 years after its initial description, adeno-associated virus (AAV) remains the most exciting but also most elusive study object in basic or applied virology. On the one hand, its simple structure not only facilitates investigations into virus biology but, combined with the availability of numerous natural AAV variants with distinct infection efficiency and specificity, also makes AAV a preferred substrate for engineering of gene delivery vectors. On the other hand, it is striking to witness a recent flurry of reports that highlight and partially close persistent gaps in our understanding of AAV virus and vector biology. This is all the more perplexing considering that recombinant AAVs have already been used in >160 clinical trials and recently been commercialized as gene therapeutics. Here, we discuss a reason for these advances in AAV research, namely, the advent and application of powerful high-throughput technology for dissection of AAV-host interactions and optimization of AAV gene therapy vectors. As relevant examples, we focus on the discovery of (i) a "new" cellular AAV receptor, AAVR, (ii) host restriction factors for AAV entry, and (iii) AAV capsid determinants that mediate trafficking through the blood-brain barrier. While items i/ii are prototypes of extra- or intracellular AAV host factors that were identified via high-throughput screenings, item iii exemplifies the power of molecular evolution to investigate the virus itself. In the future, we anticipate that these and other key technologies will continue to accelerate the dissection of AAV biology and will yield a wealth of new designer viruses for clinical use.
Topics: Biological Transport; Capsid Proteins; Dependovirus; Genetic Engineering; Genetic Therapy; Genetic Vectors; Host Microbial Interactions; Humans; Receptors, Cell Surface
PubMed: 29782834
DOI: 10.1016/j.jmb.2018.05.022 -
Journal of Virology Sep 2021Adeno-associated viruses utilize different glycans and the AAV receptor (AAVR) for cellular attachment and entry. Directed evolution has yielded new AAV variants;...
Adeno-associated viruses utilize different glycans and the AAV receptor (AAVR) for cellular attachment and entry. Directed evolution has yielded new AAV variants; however, structure-function correlates underlying their improved transduction are generally overlooked. Here, we report that infectious cycling of structurally diverse AAV surface loop libraries yields functionally distinct variants. Newly evolved variants show enhanced cellular binding, uptake, and transduction, but through distinct mechanisms. Using glycan-based and genome-wide CRISPR knockout screens, we discover that one AAV variant acquires the ability to recognize sulfated glycosaminoglycans, while another displays receptor switching from AAVR to integrin β1 (ITGB1). A previously evolved variant, AAVhum.8, preferentially utilizes the ITGB1 receptor over AAVR. Visualization of the AAVhum.8 capsid by cryoelectron microscopy at 2.49-Å resolution localizes the newly acquired integrin recognition motif adjacent to the AAVR footprint. These observations underscore the new finding that distinct AAV surface epitopes can be evolved to exploit different cellular receptors for enhanced transduction. Understanding how viruses interact with host cells through cell surface receptors is central to discovery and development of antiviral therapeutics, vaccines, and gene transfer vectors. Here, we demonstrate that distinct epitopes on the surface of adeno-associated viruses can be evolved by infectious cycling to recognize different cell surface carbohydrates and glycoprotein receptors and solve the three-dimensional structure of one such newly evolved AAV capsid, which provides a roadmap for designing viruses with improved attributes for gene therapy applications.
Topics: Amino Acid Motifs; CRISPR-Cas Systems; Capsid; Capsid Proteins; Cell Line; Cell Membrane; Cryoelectron Microscopy; Dependovirus; Directed Molecular Evolution; Genetic Variation; Glycosaminoglycans; Humans; Integrin beta1; Polysaccharides; Receptors, Cell Surface; Receptors, Virus; Virus Internalization
PubMed: 34232726
DOI: 10.1128/JVI.00587-21 -
Hearing Research Sep 2020Adeno-associated viruses (AAVs) are preferred vectors for gene replacement therapy, as they are non-pathogenic, non-inflammatory, induce stable transgene expression in... (Review)
Review
Adeno-associated viruses (AAVs) are preferred vectors for gene replacement therapy, as they are non-pathogenic, non-inflammatory, induce stable transgene expression in terminally differentiated cells, and a series of natural and engineered capsid proteins can be employed to target the vectors to specific cells. Only one feature of AAVs is limiting: the low cargo capacity for foreign DNA, restricting their application to coding sequences of <4 kb. In the last decade, splitting larger cDNAs into two AAVs and co-transducing tissue with such dual-AAV vectors has shown to result in the expression of the full-length protein in different tissues like retina, muscle and liver. This is due to the intrinsic capability of the AAV genomes to undergo homologous recombination and/or head-to-tail multimerization in nuclei of target cells. Recently, two groups independently found that a dual-AAV approach successfully delivered the 6 kb full-length otoferlin cDNA into inner hair cells of otoferlin knock-out mice and restored hearing. These pioneering studies pave the way for gene therapeutics that use dual-AAV vectors to restore hearing in forms of deafness caused by mutations in large genes.
Topics: Animals; Dependovirus; Ear, Inner; Genetic Therapy; Genetic Vectors; Membrane Proteins; Transgenes
PubMed: 31810595
DOI: 10.1016/j.heares.2019.107857