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Other Positive Single-Stranded RNA Viruses
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
The satellite viruses encode structural proteins required for the formation of infectious particles but depend on helper viruses for completing their replication cycles. This is especially intriguing, since many RNA viruses such as endornaviruses, hypoviruses, narnaviruses, and umbraviruses do not form virions. Krupovic et al. (2016) reasonably argued that all nucleic-acid-containing nonorganismal entities that encode their own capsid proteins are to be classified within proper viral taxa, regardless of whether or not they depend on another virus for replication.
Genetic Manipulation of Human Marrow: Gene Transfer Using Retroviruses
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
Philip Hughes, R. Keith Humphries
The presence of helper virus is a concern because it will permit viral spread beyond the initial target cell, and thus it makes lineage analysis, by comparison of integration sites, impossible to assess. It would also be unacceptable for gene therapy, since the virus could spread to undesired tissue and pose an increased risk for insertional activation, or inactivation, of initial genes. One can assay for the presence of the helper virus by using a “marker rescue” test. In the widely used S+L− assay for amphotropic helper virus,13 viral-containing medium is used to infect feline CCC81 cells that are then cocultivated with rat NRK cells. The presence of amphotropic helper virus will allow the infection and subsequent release (rescue) from the CCC81 cells of viruses carrying an oncogene that causes foci in the NRK cells. Other variations on such a marker rescue technique are tests for the ability to serially passage virus on 3T3 cells,5 passage only being possible if a replication-competent virus is present. A recent highly sensitive method for screening for the helper virus is to use the polymerase chain reaction to test infected target cells for the presence of sequences, such as the env gene, which would not be present in the absence of the helper virus.
Adeno-Associated Virus-Based Delivery Systems
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
There are few reports concerning the nature of the cell surface interaction with AAV (75). Thus the receptor if it exists is unknown. Once the virion penetrates to the nucleus, the genome is uncoated and one of two pathways is followed (see Fig. 3). If a helper virus is present, full gene expression occurs. If the cell is nonpermissive, gene expression is repressed, and the viral DNA integrates in the host genome. Typically, a cell that is latent with respect to AAV produces little to no AAV gene mRNA or protein (55,76-78). Upon coinfection with adenovirus, the adenovirus E1A gene product is produced and induces transcription from the AAV p5 and pl9 promoters. A small amount of Rep products is produced (23,79). The p5 Rep products induce the synthesis of mRNA from all three AAV promoters to yield higher levels of all AAV products (23,26). Adenovirus production is drastically reduced under these conditions (18,55,80).
Circumventing the packaging limit of AAV-mediated gene replacement therapy for neurological disorders
Published in Expert Opinion on Biological Therapy, 2022
Lara Marrone, Paolo M. Marchi, Mimoun Azzouz
Ataxia-telangiectasia (AT, MIM 208900) is a devastating genetic syndrome involving neurodegeneration, immunodeficiency and cancer predisposition due to mutations in the ATM gene (cDNA, 9.1 kb). The encoded protein kinase, ataxia telangiectasia mutated (ATM), is a master regulator of DNA double-strand breaks and stress responses. In 2008, Cortez and colleagues generated a hybrid amplicon vector in which the ATM cDNA was flanked by AAV ITRs while packaged in Herpes simplex virus 1 (HSV-1) virions [113]. This construct, which also contained the AAV rep gene, could mediate the insertion of the large ATM cDNA into the AAVS1 site of human chromosome 19. Interestingly, this HSV/AAV hybrid vector ensured functional expression of ATM cDNA in AT human cells as well as in AAVS1-transgenic Atm–/ – mouse cells. Despite this proof-of-principle, this approach harbors important limitations, such as (i) the potential contamination by helper viruses during production, (ii) the inability of the AAVS1 site to support transgene expression in some cell types, and (iii) the fact that integration in the AAVS1 site disrupts the PPP1R12 C gene, which encodes a protein whose function is still unclear. Nonetheless, this ‘targeted integration’ approach, which can deliver very large transgenes (up to 150 kb) [114], remains an interesting tool worth further exploration.
Challenges and advances in translating gene therapy for hearing disorders
Published in Expert Review of Precision Medicine and Drug Development, 2020
Hildegard Büning, Axel Schambach, Michael Morgan, Axel Rossi, Helena Wichova, Hinrich Staecker, Athanasia Warnecke, Thomas Lenarz
The biggest advantage of AdV is the large packaging capacity (up to 8 kB insert). However, a limited length of expression and the potential for eliciting (strong) immune responses that make repeated dosing less effective restrict their applications. AdV are ideal for short-term gene therapy applications, such as delivery of transcription factors to induce hair cell regeneration. Currently, AdV serotype 5 (Ad5) is the most promising vector for inner ear gene therapy [55]: Because inner ear cells express the Coxsackie adenovirus receptor, they are susceptible for AdV mediated gene therapy. Binding to the Coxsackie adenovirus receptor is aided by interactions with integrins and heparan sulfate proteoglycans [56]. In principle, a wide range of other adenoviral serotypes can be used to target the inner ear [15]. Potential advantages of rare serotypes include lack of preexisting patient immunity to these vectors and the possibility to target selected sub-populations of inner ear cells by the use of distinct receptors. For example, Ad28-based vectors transduce cochlear supporting cells, which might allow use of these vectors the portion of the Ad28 binding domain that is specific for supporting cells for gene delivery exclusively to supporting cells [15]. In comparison to first-generation AdV, gutless Ad vectors promise larger packaging capacity and lower immunogenicity, features required to address some of the larger genes involved in SNHL (Table 2) [57]. However, at present, the risk of helper virus contamination has prevented their use in human clinical trials.
An update on gene therapy for lysosomal storage disorders
Published in Expert Opinion on Biological Therapy, 2019
Murtaza S. Nagree, Simone Scalia, William M. McKillop, Jeffrey A. Medin
Ultimately, clinical studies provide the most meaningful information. Robust pre-clinical studies, well-thought-out project designs, and consistent product manufacturing are all paramount so as to ensure safety and not repeat historical mistakes. Manufacturing is undoubtedly a current roadblock in the field. An AAV-based FDA-approved investigational new drug will commonly require 1015–1016 AAV particles manufactured under current good manufacturing practice for clinical usage as well as required toxicology, safety, dose, and bio-distribution studies [148]. This presents a significant hurdle as manufacturing of AAV vectors is complex and resource-intensive with many manufacturers using adherent cell culture systems that do not scale easily to generate such a large amount of vector. The field is rapidly evolving, and developing new suspension-based cell culture systems, transfection-free production methods, non-mammalian producer cell strategies, use of improved helper viruses, and optimization of vector purification protocols in an attempt to increase titers, decrease cost, and improve ease of manufacturing [148].