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Neuroscience Letters Aug 2019Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In... (Review)
Review
Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In neuroscience, recombinant viruses such as lentiviruses and adeno-associated viruses (AAVs) have been successfully used to target various cell types in the brain, although effective transduction of microglia is rare. In this review, we provide a short background of lentiviruses and AAVs, and strategies for designing recombinant viral vectors. Then, we will summarize recent literature on successful microglial transductions in vitro and in vivo, and discuss the current challenges. Finally, we provide guidelines for reporting the efficiency and specificity of viral targeting in microglia, which will enable the microglial research community to assess and improve methodologies for future studies.
Topics: Animals; Dependovirus; Gene Targeting; Genetic Vectors; HIV-1; Humans; Lentivirus; Microglia; Transduction, Genetic
PubMed: 31158432
DOI: 10.1016/j.neulet.2019.134310 -
Viruses Jun 2020The adeno-associated virus (AAV) is a small, nonpathogenic parvovirus, which depends on helper factors to replicate. Those helper factors can be provided by coinfecting... (Review)
Review
The adeno-associated virus (AAV) is a small, nonpathogenic parvovirus, which depends on helper factors to replicate. Those helper factors can be provided by coinfecting helper viruses such as adenoviruses, herpesviruses, or papillomaviruses. We review the basic biology of AAV and its most-studied helper viruses, adenovirus type 5 (AdV5) and herpes simplex virus type 1 (HSV-1). We further outline the direct and indirect interactions of AAV with those and additional helper viruses.
Topics: Adenoviridae; Coinfection; Dependovirus; Helper Viruses; Herpesvirus 1, Human; Humans; Parvoviridae Infections; Viral Proteins; Virus Replication
PubMed: 32575422
DOI: 10.3390/v12060662 -
BioDrugs : Clinical Immunotherapeutics,... May 2023Recombinant adeno-associated viruses (AAVs) have emerged as promising gene delivery vehicles resulting in three US Food and Drug Administration (FDA) and one European... (Review)
Review
Recombinant adeno-associated viruses (AAVs) have emerged as promising gene delivery vehicles resulting in three US Food and Drug Administration (FDA) and one European Medicines Agency (EMA)-approved AAV-based gene therapies. Despite being a leading platform for therapeutic gene transfer in several clinical trials, host immune responses against the AAV vector and transgene have hampered their widespread application. Multiple factors, including vector design, dose, and route of administration, contribute to the overall immunogenicity of AAVs. The immune responses against the AAV capsid and transgene involve an initial innate sensing. The innate immune response subsequently triggers an adaptive immune response to elicit a robust and specific response against the AAV vector. AAV gene therapy clinical trials and preclinical studies provide important information about the immune-mediated toxicities associated with AAV, yet studies suggest preclinical models fail to precisely predict the outcome of gene delivery in humans. This review discusses the contribution of the innate and adaptive immune response against AAVs, highlighting the challenges and potential strategies to mitigate these responses, thereby enhancing the therapeutic potential of AAV gene therapy.
Topics: Humans; Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Immunity, Innate
PubMed: 36862289
DOI: 10.1007/s40259-023-00585-7 -
Trends in Microbiology May 2022Adeno-associated virus (AAV) is the leading vector in emerging treatments of inherited diseases. Higher transduction efficiencies and cellular specificity are required... (Review)
Review
Adeno-associated virus (AAV) is the leading vector in emerging treatments of inherited diseases. Higher transduction efficiencies and cellular specificity are required for broader clinical application, motivating investigations of virus-host molecular interactions during cell entry. High-throughput methods are identifying host proteins more comprehensively, with subsequent molecular studies revealing unanticipated complexity and serotype specificity. Cryogenic electron microscopy (cryo-EM) provides a path towards structural details of these sometimes heterogeneous virus-host complexes, and is poised to illuminate more fully the steps in entry. Here presented, is progress in understanding the distinct steps of glycan attachment, and receptor-mediated entry/trafficking. Comparison with structures of antibody complexes provides new insights on immune neutralization with implications for the design of improved gene therapy vectors.
Topics: Dependovirus; Genetic Vectors; Polysaccharides; Receptors, Cell Surface; Serogroup; Virus Internalization
PubMed: 34711462
DOI: 10.1016/j.tim.2021.09.005 -
Journal of Pharmaceutical Sciences Jul 2021Adeno-associated virus (AAV) has emerged as a leading platform for gene delivery for treating various diseases due to its excellent safety profile and efficient... (Review)
Review
Adeno-associated virus (AAV) has emerged as a leading platform for gene delivery for treating various diseases due to its excellent safety profile and efficient transduction to various target tissues. However, the large-scale production and long-term storage of viral vectors is not efficient resulting in lower yields, moderate purity, and shorter shelf-life compared to recombinant protein therapeutics. This review provides a comprehensive analysis of upstream, downstream and formulation unit operation challenges encountered during AAV vector manufacturing, and discusses how desired product quality attributes can be maintained throughout product shelf-life by understanding the degradation mechanisms and formulation strategies. The mechanisms of various physical and chemical instabilities that the viral vector may encounter during its production and shelf-life because of various stressed conditions such as thermal, shear, freeze-thaw, and light exposure are highlighted. The role of buffer, pH, excipients, and impurities on the stability of viral vectors is also discussed. As such, the aim of this review is to outline the tools and a potential roadmap for improving the quality of AAV-based drug products by stressing the need for a mechanistic understanding of the involved processes.
Topics: Dependovirus; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors
PubMed: 33812887
DOI: 10.1016/j.xphs.2021.03.024 -
Molecular Therapy : the Journal of the... Aug 2022On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, featuring leading... (Review)
Review
On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, featuring leading experts in preclinical and clinical AAV gene therapy, to further contextualize and understand this phenomenon. Recombinant AAV (rAAV) vectors are used to develop therapies for many conditions given their ability to transduce multiple cell types, resulting in long-term expression of transgenes. Although most rAAV DNA typically remains episomal, some rAAV DNA becomes integrated into genomic DNA at a low frequency, and rAAV insertional mutagenesis has been shown to lead to tumorigenesis in neonatal mice. Currently, the risk of rAAV-mediated oncogenesis in humans is theoretical because no confirmed genotoxic events have been reported to date. However, because insertional mutagenesis has been reported in a small number of murine studies, there is a need to characterize this genotoxicity to inform research, regulatory needs, and patient care. The purpose of this white paper is to review the evidence of rAAV-related host genome integration in animal models and possible risks of insertional mutagenesis in patients. In addition, technical considerations, regulatory guidance, and bioethics are discussed.
Topics: Animals; Dependovirus; Genetic Vectors; Humans; Mice; Mutagenesis, Insertional; Plasmids; Transgenes; Virus Integration
PubMed: 35690906
DOI: 10.1016/j.ymthe.2022.06.004 -
European Journal of Pharmaceutics and... Sep 2023Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and... (Review)
Review
Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and physico-chemical properties, delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge. Adeno-associated viruses (AAV) have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA (ssDNA) genomes over the past decades. More than a hundred products have been tested in clinical settings and three products have received market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the-art rAAV formulations and can further serve as a map for rational formulation development activities in the future.
Topics: Dependovirus; Genetic Vectors; Genetic Therapy
PubMed: 37423416
DOI: 10.1016/j.ejpb.2023.07.002 -
Human Gene Therapy Nov 2022Recombinant adeno-associated viruses (rAAVs) are currently the most prominently investigated vector platform for human gene therapy. The rAAV capsid serves as a potent...
Recombinant adeno-associated viruses (rAAVs) are currently the most prominently investigated vector platform for human gene therapy. The rAAV capsid serves as a potent and efficient vehicle for delivering genetic payloads into the host cell, while the vector genome determines the function and effectiveness of these biotherapies. However, current production schemes yield vectors that may consist of heterogeneous populations, compromising their potencies. The development of next-generation sequencing methods within the past few years have helped investigators profile the diversity and relative abundances of heterogenous species in vector preparations. Specifically, long-read sequencing methods, like single molecule real-time (SMRT) sequencing, have been used to uncover truncations, chimeric genomes, and inverted terminal repeat (ITR) mutations in vectors. Unfortunately, these sequencing platforms may be inaccessible to investigators with limited resources, require large amounts of input material, or may require long wait times for sequencing and analyses. Recent advances with nanopore sequencing have helped to bridge the gap for quick and relatively inexpensive long-read sequencing needs. However, their limitations and sample biases are not well-defined for sequencing rAAV. In this study, we explored the capacity for nanopore sequencing to directly interrogate rAAV content to obtain full-length resolution of encapsidated genomes. We found that the nanopore platform can cover the entirety of rAAV genomes from ITR to ITR without the need for pre-fragmentation. However, the accuracy for base calling was low, resulting in a high degree of miscalled bases and false indels. These false indels led to read-length compression; thus, assessing heterogeneity based on read length is not advisable with current nanopore technologies. Nonetheless, nanopore sequencing was able to correctly identify truncation hotspots in single-strand and self-complementary vectors similar to SMRT sequencing. In summary, nanopore sequencing can serve as a rapid and low-cost alternative for proofing AAV vectors.
Topics: Humans; Genetic Vectors; Nanopores; Nanopore Sequencing; Dependovirus; Terminal Repeat Sequences
PubMed: 36178359
DOI: 10.1089/hum.2022.143 -
Cell Apr 2020The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new... (Review)
Review
The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.
Topics: Animals; CRISPR-Cas Systems; Dependovirus; Gene Editing; Genetic Therapy; Genetic Vectors; Humans
PubMed: 32243786
DOI: 10.1016/j.cell.2020.03.023 -
Human Gene Therapy Apr 2022In the past two decades, adeno-associated virus (AAV) vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and...
In the past two decades, adeno-associated virus (AAV) vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and clinical trials. In addition, AAV vectors have been extensively studied for their safety and efficacy. In particular, the presence of empty AAV capsids and particles containing "inaccurate" vector genomes in preparations has been a subject of concern. Several methods exist to separate empty capsids from full particles; but thus far, no single technique can produce vectors that are free of empty or partial (non-unit length) capsids. Unfortunately, the exact genome compositions of full, intermediate, and empty capsids remain largely unknown. In this work, we used AAV-genome population sequencing to explore the compositions of DNase-resistant, encapsidated vector genomes produced by two common production pipelines: plasmid transfection in human embryonic kidney cells (pTx/HEK293) and baculovirus expression vectors in insect cells (rBV/Sf9). Intriguingly, our results show that vectors originating from the same construct design that were manufactured by the rBV/Sf9 system produced a higher degree of truncated and unresolved species than those generated by pTx/HEK293 production. We also demonstrate that empty particles purified by cesium chloride gradient ultracentrifugation are not truly empty but are instead packaged with genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). Our data suggest that the frequency of these "mutated" ITRs correlates with the abundance of inaccurate genomes in all fractions. These surprising findings shed new light on vector efficacy, safety, and how clinical vectors should be quantified and evaluated.
Topics: Animals; Baculoviridae; Dependovirus; Genetic Vectors; HEK293 Cells; Humans; Insecta
PubMed: 35293222
DOI: 10.1089/hum.2022.050