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A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

ABSTRACT: Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a feasible approach to circumvent these issues and sustain a durable functional β-cell mass in vivo. However, efficient and safe delivery of nucleic acids to intact islet remains a challenging task. Here we describe a simple and easy-to-use lentiviral transduction protocol that allows the transduction of approximately 80 % of mouse and human islet cells while preserving islet architecture, metabolic function and glucose-dependent stimulation of insulin secretion. Our protocol will facilitate to fully determine the potential of gene expression modulation of therapeutically promising targets in entire pancreatic islets for xenotransplantation purposes.

SUBMITTER: Jimenez-Moreno CM

PROVIDER: S-EPMC5411998 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

Jimenez-Moreno Carmen Maria CM Herrera-Gomez Irene de Gracia IG Lopez-Noriega Livia L Lorenzo Petra Isabel PI Cobo-Vuilleumier Nadia N Fuente-Martin Esther E Mellado-Gil Jose Manuel JM Parnaud Geraldine G Bosco Domenico D Gauthier Benoit Raymond BR Martin-Montalvo Alejandro A

Current gene therapy 20150101 4

Successful normalization of blood glucose in patients transplanted with pancreatic islets isolated from cadaveric donors established the proof-of-concept that Type 1 Diabetes Mellitus is a curable disease. Nonetheless, major caveats to the widespread use of this cell therapy approach have been the shortage of islets combined with the low viability and functional rates subsequent to transplantation. Gene therapy targeted to enhance survival and performance prior to transplantation could offer a f ...[more]

PMID: 26122098

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Project description:Highlights • Lentiviral vectors have a limited half-life, and in order to reach the target, the distance between the target cell and the vector should be minimized.• Microfluidic systems provide a micro-scaled environment in which lentiviral vectors have a high chance of reaching the target cell.• Using microfluidic systems decreases the transduction time.• Compared to standard cell culture plates, microfluidic systems require considerably lower culture media and reagents for a transduction procedure.• Microfluidic system has proven to be very beneficial in enhancing lentiviral transduction. Background HIV-1-derived lentiviral vectors (LVs) are capable of transducing human cells by integrating the transgene into the host genome. In order to do that, LVs should have enough time and space to interact with the surface of the target cells. Herein, we used a microfluidic system to facilitate the transduction of BCP-ALL cells. Methods and Results We used a SU-8 mold to fabricate a PDMS microfluidic chip containing three channels with a 50 μm height and a surface matching 96-well plates. In order to produce LVs, we used HEK293T cells to package the second generation of LVs. First, we evaluated the cell recovery from the microfluidic chip. Cell recovery assessment showcased that 3 h and 6 h of incubation in microfluidic channels containing 100,000 NALM-6 (BCP-ALL) cells with 2μL of culture media yielded 87±7.2% and 80.6 ± 10% of cell recovery, respectively. Afterward, the effects of LV-induced toxicity were evaluated using 10–30% LV concentrations in time frames ranging from 3 h to 24 h. In 96-well plates, it took 12–24 h for the viruses with 20% and 30% concentrations to affect the cell survival significantly. These effects were intensified in the microfluidic system implying that microfluidic is capable of enhancing LV transduction. Based on the evidence of cell recovery and cell survival we chose 6 h of incubation with 20% LV. Conclusion The results from EGFP expression showcased that a microfluidic system could increase the LV transduction in BCP-ALL cells by almost 9-folds. All in all, the microfluidic system seems to be a great armamentarium in optimizing LV-based transduction. Graphical abstract Image, graphical abstract

Project description:RNA interference (RNAi) is widely used in gene knockdown analysis and as a tool to screen host genes involved in viral infection. Owing to the limitations of transducing cells with synthetic small interfering RNAs (siRNAs), lentiviral short hairpin RNA (shRNA) vectors are more widely used. However, we found that stable transduction with lentiviral shRNA vectors inhibited hepatitis C virus (HCV) propagation in human hepatoma cells. We found by microRNA (miRNA) microarray analysis that this inhibition was induced by the alteration of host miRNA expression. In addition to one miRNA (miR-196b-5p) previously reported to be involved in HCV infection, other miRNAs (miR-216a-5p, -216b-5p, 217, and -30b-5p) were found to influence HCV infection in this study. Further studies suggested that this effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration site of the lentiviral vector might determine the change in miRNA expression. Moreover, the upregulation of JUN contributed to the dysregulation of miR-216a-5p, -216b-5p, and -217 in stably transduced cells. Although the changes in miRNA expression were beneficial for inhibiting HCV infection in our study, this off-target effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.IMPORTANCE We found that stable transduction with lentiviral shRNA was able to nonspecifically inhibit HCV infection by the dysregulation of host miRNAs. Previous studies showed that the overexpression of shRNAs oversaturated the host miRNA pathways to inhibit HCV infection. In contrast, the miRNA machinery was not affected in our study. Knockout studies suggested that the nonspecific effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration sites in the host genome determined the changes in miRNAs. Stable transduction with lentiviral vectors was able to increase the expression of JUN, which in turn upregulated miR-216a-5p, miR-216b-5p, and miR-217. miR-216a-5p and miR-216b-5p might inhibit HCV by suppressing the host autophagic machinery. Our study suggested a novel nonspecific effect of lentiviral vectors, and this side effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.

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A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

Publications

A Simple High Efficiency Intra-Islet Transduction Protocol Using Lentiviral Vectors.

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