Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach.
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ABSTRACT:
SUBMITTER: Labisch JJ
PROVIDER: S-EPMC8281989 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectiveEfficient gene transfer to the vascular wall via intravenous vector injection would be useful for experimental vascular biology and gene therapy. Initial studies of lentiviral vector tropism suggested that intravenously injected vectors do not transduce murine vascular tissue; however, there are also reports of highly efficient aortic transduction after jugular vein injection of high-titer lentiviral vectors. We sought to reproduce these results. Approach and Results: We injected high-titer preparations of GFP (green fluorescent protein)-expressing lentiviral vector into jugular veins of 8 mice; 6 mice received vehicle only. Four days later, samples of aorta (thoracic and abdominal), liver, spleen, and other tissues were harvested and processed for quantitative polymerase chain reaction detection of vector DNA and immunohistochemical detection of GFP. Our vector DNA assay did not detect transduction of any of the 16 aortic segments. This finding excludes an aortic transduction efficiency of >0.02 vector copies per cell. In contrast, vector DNA was detected in all 8 spleen and liver extracts (median, 0.8 and 0.1 vector copies per cell, respectively; P<0.001 versus vehicle controls). Quantitative polymerase chain reaction signals from DNA extracted from heart, lung, kidney, skeletal muscle, and femoral artery did not differ from background polymerase chain reaction signals from DNA extracted from tissues of vehicle-injected mice (P≥0.7 for all). Immunohistochemistry revealed GFP in scattered cells in spleen and liver, not in aorta.ConclusionsInjection of high-titer lentiviral vectors via the jugular vein transduces cells in the spleen and liver but does not efficiently transduce the aorta. Graphic Abstract: A graphic abstract is available for this article.
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Project description:Lentiviral vectors (LV) have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented.
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Project description:The growing number of drug-resistant bacterial infections worldwide is driving renewed interest in phage therapy. Based on the use of a personalized cocktail composed of highly specific bacterial viruses, this therapy relies on a range of tests on agar media to determine the most active phage on a given bacterial target (phage susceptibility testing), or to isolate new lytic phages from an environmental sample (enrichment of phage banks). However, these culture-based techniques are still solely interpreted through direct visual detection of plaques. The main objective of this work is to investigate computer-assisted methods in order to ease and accelerate diagnosis in phage therapy but also to study phage plaque growth kinetics. For this purpose, we designed a custom wide-field lensless imaging device, which allows continuous monitoring over a very large area sensor (3.3 cm2). Here we report bacterial susceptibility to Staphylococcus aureus phage in 3 hr and estimation of infectious titer in 8 hr 20 min. These are much shorter time-to-results than the 12 to 24 hours traditionally needed, since naked eye observation and counting of phage plaques is still the most widely used technique for susceptibility testing prior to phage therapy. Moreover, the continuous monitoring of the samples enables the study of plaque growth kinetics, which enables a deeper understanding of the interaction between phage and bacteria. Finally, thanks to the 4.3 μm resolution, we detect phage-resistant bacterial microcolonies of Klebsiella pneumoniae inside the boundaries of phage plaques and thus show that our prototype is also a suitable device to track phage resistance. Lensless imaging is therefore an all-in-one method that could easily be implemented in cost-effective and compact devices in phage laboratories to help with phage therapy diagnosis.
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Project description:Viral vectors provide an efficient means for modification of eukaryotic cells, and their use is now commonplace in academic laboratories and industry for both research and clinical gene therapy applications. Lentiviral vectors, derived from the human immunodeficiency virus, have been extensively investigated and optimized over the past two decades. Third-generation, self-inactivating lentiviral vectors have recently been used in multiple clinical trials to introduce genes into hematopoietic stem cells to correct primary immunodeficiencies and hemoglobinopathies. These vectors have also been used to introduce genes into mature T cells to generate immunity to cancer through the delivery of chimeric antigen receptors (CARs) or cloned T-cell receptors. CAR T-cell therapies engineered using lentiviral vectors have demonstrated noteworthy clinical success in patients with B-cell malignancies leading to regulatory approval of the first genetically engineered cellular therapy using lentiviral vectors. In this review, we discuss several aspects of lentiviral vectors that will be of interest to clinicians, including an overview of lentiviral vector development, the current uses of viral vectors as therapy for primary immunodeficiencies and cancers, large-scale manufacturing of lentiviral vectors, and long-term follow-up of patients treated with gene therapy products.
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Project description:Lentiviral vectors have markedly enhanced gene therapy efficiency in treating congenital diseases, but its long-term safety remains controversial. Most gene therapies for congenital eye diseases need to be carried out at early ages, yet the assessment of related risks to ocular development posed by lentiviral vectors is challenging. Utilizing single-cell transcriptomic profiling on human retinal organoids, this study explored the impact of lentiviral vectors on the retinal development and found that lentiviral vectors can cause retinal precursor cells to shift toward photoreceptor fate through the up-regulation of key fate-determining genes such as PRDM1. Further investigation demonstrated that the intron and intergenic region of PRDM1 was bound by PHLDA1, which was also upregulated by lentiviral vector exposure. Importantly, knockdown of PHLDA1 successfully suppressed the lentivirus-induced differentiation bias of photoreceptor cells. The findings also suggest that while lentiviral vectors may disrupt the fate determination of retinal precursor cells, posing risks in early-stage retinal gene therapy, these risks could potentially be reduced by inhibiting the PHLDA1-PRDM1 signaling axis.
2024-08-28 | GSE251752 | GEO