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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 -
Communications Biology May 2023The adeno-associated virus (AAV) is a potent vector for in vivo gene transduction and local therapeutic applications of AAVs, such as for skin ulcers, are expected....
The adeno-associated virus (AAV) is a potent vector for in vivo gene transduction and local therapeutic applications of AAVs, such as for skin ulcers, are expected. Localization of gene expression is important for the safety and efficiency of genetic therapies. We hypothesized that gene expression could be localized by designing biomaterials using poly(ethylene glycol) (PEG) as a carrier. Here we show one of the designed PEG carriers effectively localized gene expression on the ulcer surface and reduced off-target effects in the deep skin layer and the liver, as a representative organ to assess distant off-target effects, using a mouse skin ulcer model. The dissolution dynamics resulted in localization of the AAV gene transduction. The designed PEG carrier may be useful for in vivo gene therapies using AAVs, especially for localized expression.
Topics: Polyethylene Glycols; Dependovirus; Genetic Vectors; Genetic Therapy; Biocompatible Materials
PubMed: 37193797
DOI: 10.1038/s42003-023-04851-w -
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 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 -
Molecular Therapy : the Journal of the... Jul 2008Recombinant adeno-associated virus (rAAV) holds promise as a gene therapy vector for a multitude of genetic disorders such as hemophilia, cystic fibrosis, and the... (Review)
Review
Recombinant adeno-associated virus (rAAV) holds promise as a gene therapy vector for a multitude of genetic disorders such as hemophilia, cystic fibrosis, and the muscular dystrophies. Given the variety of applications and tissue types toward which these vectors may be targeted, an understanding of rAAV transduction is crucial for the effective application of therapy. rAAV transduction mechanisms have been the subject of much study, resulting in a body of knowledge relating to events from virus-cell attachment through to vector genome conformation in the target cell nucleus. Instead of utilizing one mechanism in each phase of vector transduction, rAAV appears to employ multiple possible pathways toward transgene expression, in part dependent on rAAV serotype, dose, and target cell type. Once inside the nucleus, the rAAV genome exists in a predominantly episomal form; therefore, nondividing cells tend to be most stably transduced. However, rAAV has a low frequency of integration into the host cell genome, often in or near genes, and can be associated with host genome mutations. This review describes the current understanding of the mechanisms and rate-limiting steps involved in rAAV transduction.
Topics: Animals; DNA, Recombinant; Dependovirus; Genetic Therapy; Genetic Vectors; Humans; Mice; Transduction, Genetic; Virus Integration; Virus Internalization
PubMed: 18500252
DOI: 10.1038/mt.2008.103 -
Viruses Sep 2023The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with...
The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with optimized yield and transduction efficiency. The Baculovirus/Sf9 system is a promising platform for high yield production; however, a major drawback to using an invertebrate cell line compared to a mammalian system is a generally altered AAV capsid stoichiometry resulting in lower biological potency. Here, we introduce a term of the structural and biological "fitness" of an AAV capsid as a function of two interdependent parameters: (1) packaging efficiency (yield), and (2) transduction efficiency (infectivity). Both parameters are critically dependent on AAV capsid structural proteins VP1/2/3 stoichiometry. To identify an optimal AAV capsid composition, we developed a novel Directed Evolution (DE) protocol for assessing the structural and biological fitness of Sf9-manufactured rAAV for any given serotype. The approach involves the packaging of a combinatorial capsid library in insect Sf9 cells, followed by a library screening for high infectivity in human Cre-recombinase-expressing C12 cells. One single DE selection round, complemented by Next-Generation Sequencing (NGS) and guided by in silico analysis, identifies a small subset of VP1 translation initiation sites (known as Kozak sequence) encoding "fit" AAV capsids characterized by a high production yield and superior transduction efficiencies.
Topics: Animals; Humans; Genetic Vectors; Dependovirus; Capsid Proteins; Cell Line; Capsid; Mammals
PubMed: 37896760
DOI: 10.3390/v15101983 -
Trends in Pharmacological Sciences Jun 2021As gene therapy enters mainstream medicine, it is more important than ever to have a grasp of exactly how to leverage it for maximum benefit. The development of new... (Review)
Review
As gene therapy enters mainstream medicine, it is more important than ever to have a grasp of exactly how to leverage it for maximum benefit. The development of new targeting strategies and tools makes treating patients with genetic diseases possible. Many Mendelian disorders are amenable to gene replacement or correction. These often affect post-mitotic tissues, meaning that a single stably expressing therapy can be applied. Recent years have seen the development of a large number of novel viral vectors for delivering specific therapies. These new vectors - predominately recombinant adeno-associated virus (AAV) variants - target nervous tissues with differing efficiencies. This review gives an overview of current gene therapies in the brain, ear, and eye, and describes the optimal approaches, depending on cell type and transgene. Overall, this work aims to serve as a primer for gene therapy in the central nervous and sensory systems.
Topics: Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Humans; Nerve Tissue; Transgenes
PubMed: 33863599
DOI: 10.1016/j.tips.2021.03.004 -
Human Gene Therapy Methods Feb 2016Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are... (Review)
Review
Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then-through a variety of mechanisms-result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction.
Topics: Animals; Capsid Proteins; DNA, Viral; Dependovirus; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Genome Size; Genome, Viral; Humans; Transgenes; Virus Assembly
PubMed: 26757051
DOI: 10.1089/hgtb.2015.140