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Human Gene Therapy Methods Dec 2019Ongoing development of recombinant vectors based on adeno-associated virus (rAAV) is providing an increasingly powerful and widely used toolkit for gene transfer and... (Review)
Review
Ongoing development of recombinant vectors based on adeno-associated virus (rAAV) is providing an increasingly powerful and widely used toolkit for gene transfer and genome editing applications. While conceptually simple, the system harbors considerable complexity that presents many potential pitfalls for the inexperienced user. The short inverted terminal repeats (ITRs) can prove to be particularly problematic during vector engineering due to inherent instability necessitating diligent quality control measures during vector manufacture. This is especially important from a clinical standpoint when consistent purity and potency are paramount, and all components of the system are rigorously scrutinized by regulatory agencies. Despite the discovery over 30 years ago that the AAV ITRs are the only -acting elements of the virus required for vector production, there is a scarcity of reviews specifically focused on these complex elements. This review provides an overview of the ITR with the dual purpose of acting as a user's guide in the application of AAV vector technology and as a roadmap for ongoing vector development and optimization.
Topics: Dependovirus; Genetic Vectors; High-Throughput Nucleotide Sequencing; Humans; Nucleic Acid Conformation; Terminal Repeat Sequences
PubMed: 31752513
DOI: 10.1089/hgtb.2019.276 -
Gene Therapy Aug 2021Dysfunction and/or reduced activity in the tongue muscles contributes to conditions such as dysphagia, dysarthria, and sleep disordered breathing. Current treatments are... (Review)
Review
Dysfunction and/or reduced activity in the tongue muscles contributes to conditions such as dysphagia, dysarthria, and sleep disordered breathing. Current treatments are often inadequate, and the tongue is a readily accessible target for therapeutic gene delivery. In this regard, gene therapy specifically targeting the tongue motor system offers two general strategies for treating lingual disorders. First, correcting tongue myofiber and/or hypoglossal (XII) motoneuron pathology in genetic neuromuscular disorders may be readily achieved by intralingual delivery of viral vectors. The retrograde movement of viral vectors such as adeno-associated virus (AAV) enables targeted distribution to XII motoneurons via intralingual viral delivery. Second, conditions with impaired or reduced tongue muscle activation can potentially be treated using viral-driven chemo- or optogenetic approaches to activate or inhibit XII motoneurons and/or tongue myofibers. Further considerations that are highly relevant to lingual gene therapy include (1) the diversity of the motoneurons which control the tongue, (2) the patterns of XII nerve branching, and (3) the complexity of tongue muscle anatomy and biomechanics. Preclinical studies show considerable promise for lingual directed gene therapy in neuromuscular disease, but the potential of such approaches is largely untapped.
Topics: Dependovirus; Gene Transfer Techniques; Genetic Therapy; Hypoglossal Nerve; Motor Neurons
PubMed: 33574581
DOI: 10.1038/s41434-021-00225-1 -
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 -
Cellular and Molecular Life Sciences :... Feb 2022Since the revolutionary discovery of the CRISPR-Cas technology for programmable genome editing, its range of applications has been extended by multiple biotechnological... (Review)
Review
Since the revolutionary discovery of the CRISPR-Cas technology for programmable genome editing, its range of applications has been extended by multiple biotechnological tools that go far beyond its original function as "genetic scissors". One of these further developments of the CRISPR-Cas system allows genes to be activated in a targeted and efficient manner. These gene-activating CRISPR-Cas modules (CRISPRa) are based on a programmable recruitment of transcription factors to specific loci and offer several key advantages that make them particularly attractive for therapeutic applications. These advantages include inter alia low off-target effects, independence of the target gene size as well as the potential to develop gene- and mutation-independent therapeutic strategies. Herein, I will give an overview on the currently available CRISPRa modules and discuss recent developments, future potentials and limitations of this approach with a focus on therapeutic applications and in vivo delivery.
Topics: CRISPR-Cas Systems; Dependovirus; Gene Editing; Genetic Therapy; Humans; Transcriptional Activation
PubMed: 35152318
DOI: 10.1007/s00018-022-04175-8 -
Expert Opinion on Biological Therapy Sep 2022
Topics: Dependovirus; Genetic Vectors; Humans
PubMed: 35373689
DOI: 10.1080/14712598.2022.2060737 -
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 Feb 2017
Topics: Capsid; Dependovirus; Genetic Vectors; Humans
PubMed: 28186848
DOI: 10.1089/hum.2017.29039.trf -
Medicine Jun 2023Adeno-associated virus (AAV) plays a vital role in ocular gene therapy and has been widely studied since 1996. This study summarizes and explores the publication outputs...
BACKGROUND
Adeno-associated virus (AAV) plays a vital role in ocular gene therapy and has been widely studied since 1996. This study summarizes and explores the publication outputs and future research trends of AAV-based ocular gene therapy.
METHODS
Publications and data about AAV-based ocular gene therapy were downloaded from the Web of Science Core Collection or ClinicalTrials.gov database. The publications and data were analyzed by Microsoft Excel, CiteSpace, VOS viewer, and a free online platform (http://bibliometric.com).
RESULTS
Totally 832 publications from the Web of Science Core Collection relevant to AAV-based ocular gene therapy were published from 1996 to 2022. These publications were contributed by research institutes from 42 countries or regions. The US contributed the most publications among these countries or regions, notably the University of Florida. Hauswirth WW was the most productive author. "Efficacy" and "safety" are the main focus areas for future research according to the references and keywords analysis. Eighty clinical trials examined AAV-based ocular gene therapy were registered on ClinicalTrials.Gov. Institutes from the US and European did the dominant number or the large proportion of the trials.
CONCLUSIONS
The research focus of the AAV-based ocular gene therapy has transitioned from the study in biological theory to clinical trialing. The AAV-based gene therapy is not limited to inherited retinal diseases but various ocular diseases.
Topics: Humans; Dependovirus; Face; Retina; Bibliometrics; Genetic Therapy
PubMed: 37327269
DOI: 10.1097/MD.0000000000034043 -
Nucleic Acid Therapeutics Dec 2015Altering endogenous genes in cells is an integral tool of modern cell biology. The ease-of-use of the CRISPR/Cas9 system to introduce genomic DNA breaks at specific... (Review)
Review
Altering endogenous genes in cells is an integral tool of modern cell biology. The ease-of-use of the CRISPR/Cas9 system to introduce genomic DNA breaks at specific sites in vivo has led to its rapid and wide adoption. In the absence of a DNA template, the lesion is repaired by nonhomologous end joining resolving as internal deletions. However, in the presence of a homologous DNA template, homology-directed repair occurs with variable efficiencies. Recent work has demonstrated that highly efficient gene targeting can be induced by combining CRISPR/Cas9 targeting of genomic loci with recombinant adeno-associated virus (rAAV) to provide a single-stranded homologous DNA template. Here we review the current state of CRISPR/Cas-based gene editing and provide a practical guide to applying the CRISPR/Cas and rAAV system for highly efficient, time- and cost-effective gene targeting.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Dependovirus; Genetic Engineering; Genetic Vectors; Recombination, Genetic
PubMed: 26540648
DOI: 10.1089/nat.2015.0545 -
Human Gene Therapy Apr 2022
Topics: Dependovirus; Genetic Therapy; Genetic Vectors; Uncertainty
PubMed: 35442070
DOI: 10.1089/hum.2022.29207.wxi