Animals ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Rats

Gene therapy has been considered as one of the optimal treatments. Although, at the beginning of this century, a series of unexpected side effects brought gene therapy into depression, the improved lentiviral vectors, which characterised by high efficiency transfection, stable expression in target cells and good biosafety, have been applied in clinical trials in recent years and acquired a certain clinical improvements. Nowadays gene therapy becomes an eye-catching field. This review discusses the gene therapy how blocked by lentiviral vectors, the high efficiency and biosafety of lentiviral vectors, the improvement of lentiviral vector preparation and so on.
Animals ; Genetic Therapy ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Transfection

In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.
Genetic Vectors ; HL-60 Cells ; Humans ; Lentivirus ; genetics ; Transfection

Gene therapy is a rapidly developing field. The most widely used technique for foreign gene transfer is lentiviral-mediated gene therapy. Lentiviral vector has been developed from the first generation to the third generation in terms of safety. The preparation of lentiviruses with high titer remains difficult. In this study, a Fibra-Cel sheet carrier was used as an HEK293T cell carrier matrix, and several sterile cell culture spinners were combined and cultured on a roller bottle machine to scale up the adherent cells. The virus titer was maximized by screening the factors to optimize the lentivirus titer in the third-generation lentivirus packaging process one by one. Fibra-Cel sheet vector was successfully used as the matrix of HEK293T cell adhesion to culture adherent cells at large scale. The optimal conditions for large-scale preparation of the third-generation lentivirus by bottle roller were screened and three batches of lentiviruses were produced on pilot scale. The production time of lentivirus was shortened from 120 hours to 54 hours from plasmid transfection to virus collection; in terms of cost, a rolling bottle machine was used instead of a bioreactor, leading to lower cost and no need for repeated sterilization during the whole process. The safe, effective and low-cost operation of successful production will provide a technical base for the large-scale preparation of lentivirus and thus lay a firm foundation for its clinical application.
Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; Transduction, Genetic ; Transfection

Immunotherapy is becoming an effective and less invasive strategy that can be applied to the treatment of various malignancies. Lentiviral vectors (LVs) have shown great potential in immunotherapy as they can stably integrate relatively large foreign DNA, and effectively transduce dividing and non-dividing cells. Clinical application needs high quality LVs, and therefore strict quality control of the final products is necessary to ensure their purity, efficacy and safety. The quantitative detection of LVs is among the key parts of product development and quality control. In this paper, the existing methods for quantitative detection of LVs are summarized, including fluorescence activated cell sorter (FACS), P24 enzyme-linked immuno sorbent assay (P24 ELISA), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing(TRPS) and virus counter(VC).Their advantages and disadvantages are listed, and future development and challenges are discussed.
Genetic Vectors/genetics* ; Humans ; Immunotherapy ; Lentivirus/genetics* ; Neoplasms ; Transduction, Genetic

Cell Line ; Embryonic Stem Cells ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Lentivirus ; genetics ; Transfection

OBJECTIVETo construct a lentiviral RNA interference system targeting heparanase (HPSE) based on miR30 and to test its silencing effect.

METHODSThree heparanase-shRNA structures were designed based miR30. The targeting fragments were obtained by PCR, then inserted into the vector LV PP-GFP to construct the recombinant lentiviral vector LV PP-GFP/miR-HPSE-shRNA, which was identified by PCR and sequencing. The 293T cells were co-transfect with LV PP-GFP/miR-HPSE-shRNA, pHelper 1.0 vector and pHelper 2.0 vector to produce lentiviruses, with which A375 cells were infected. Real-time fluorescence quantitative PCR and Western blot were performed to evaluate the expression of heparanase RNA and protein.

RESULTSThe lentiviral miR30-based RNAi vector targeting heparanase was constructed and confirmed by PCR and sequencing. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of both heparanase mRNA and protein in infected A375 cells were decreased significantly than those in control group.

CONCLUSIONThe lentiviral miR30-based RNAi vector targeting heparanase was been constructed successfully, which can be used for further study on RNAi-mediated oncolytic viruses.

Genetic Vectors ; Glucuronidase ; genetics ; Lentivirus ; genetics ; MicroRNAs ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

The aim of this study was to establish stable expression of human thyroid stimulating hormone receptor (TSHR) alpha-subunit (hTSHRA) on human embryonic kidney 293T (HEK 293T). HEK 293T cell lines with stable expression of hTSHRA could be used for detecting affinity between hTSHRA and potential TSHR blocking-peptide. We firstly constructed hTSHRA gene into lentiviral vectors GV218. The sequence comparison indicated that we had constructed GV218-hTSHRAA. Western blot demonstrated the 52 kD aim band of hTHSRA on over-expressed HEK 293T cells. GV218-hTSHRA constructions and pHelper were then co-transfected into HEK 293T cells to form packaging plasmid. The HEK 293T cells that stably expressed hTSHRA could also express green fluorescent pro tein. The titer of lentiviral packaging vector is 2 x 10(8) TU/mL with qPCR. The lentiviral packaging vector thereafter was transfected into HEK 293T cells again. The hTSHRA expressed on the HEK 293T cells. Human TSHRA stab ly expressed on HEK 293T upon continuously passaging. Therefore, we established hTSHRA stable expression on HEK 293T cells by constructing GV218-hTHSR lentiviral packaging vector. It is a useful tool for studying TSHR affinity with anti-thyroid peptide.
Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; Plasmids ; Receptors, Thyrotropin ; biosynthesis ; Transfection

OBJECTIVETo construct microRNA-223 overexpression and suppression lentivirus vectors and determine their effects after infecting oral squamous cell carcinoma (OSCC) cell line.

METHODSLentivirus vectors GV229 and GV232 were cut by the restriction sites of Age I and EcoR I and connected to the target gene, which contained mature microRNA-223 and microRNA-223 oligonucleotide. Real-time polymerase chain reaction (PCR) method was used to detect the microRNA-223 expression level after infecting the recombinant lentivirus vector into the OSCC cell line.

RESULTSThe successful construction of microRNA-223 recombinant lentivirus vectors was confirmed by the PCR method and DNA sequencing. HN-30 cell infected with microRNA-223 overexpression vector showed a significant increased in microRNA-223 expression, whereas HN-30 cell infected with microRNA-223 inhibitor vector suppressed microRNA-223 expression.

CONCLUSIONThe microRNA-223 overexpression and suppression lentivirus vectors are successfully constructed. These vectors could alter the expression level of microRNA-223 in OSCC cell line significantly, and provide a stable cell line for functional studies in the future.

Base Sequence ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Lentivirus ; MicroRNAs ; metabolism ; Real-Time Polymerase Chain Reaction

Cell Line, Tumor ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Lipase ; genetics ; Membrane Proteins ; genetics