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

Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey brain. One month after injection, monkeys were sacrificed, and then the presence of viral infection by expression of reporter fluorescence proteins was examined. Tissues were sectioned and stained with NeuN and GFAP antibodies for identifying neuronal cells or astrocytes, respectively, and viral reporter GFP-expressing cells were counted. We found that while lentivirus infected mostly astrocytes, AAV infected neurons at a higher rate than astrocytes. Moreover, astrocytes showed reactiveness when cells were infected by virus, likely due to virus-mediated neuroinflammation. The Sholl analysis was done to compare the hypertrophy of infected and uninfected astrocytes by virus. The lentivirus infected astrocytes showed negligible hypertrophy whereas AAV infected astrocytes showed significant changes in morphology, compared to uninfected astrocytes. In the brain of cynomolgus monkey, lentivirus shows tropism for astrocytes over neurons without much reactivity in astrocytes, whereas AAV shows tropism for neurons over glial cells with a significant reactivity in astrocytes. We conclude that AAV is best-suited for gene delivery to neurons, whereas lentivirus is the best choice for gene delivery to astrocytes in the brain of cynomolgus monkeys.
Animals, Laboratory ; Antibodies ; Astrocytes ; Brain* ; Dependovirus* ; Fluorescence ; Haplorhini ; Hypertrophy ; Lentivirus* ; Macaca fascicularis* ; Neuroglia ; Neurons ; Putamen ; Tropism*

Since induced pluripotent stem cell (iPSC) was first introduced by Yamanaka in 2006, it took only six years to win a Nobel Prize for his pioneering work. It is unusual to win a Nobel Prize for such recent research with a short history. Many scientists and clinicians are interested in iPSC for its potential application. Significant progression in this field has been made, while there remain many hurdles to overcome for application of iPSC technique in real clinics. In this review, the concept of reprogramming and the basic techniques of iPSC generation will be discussed for the reader's convenience, followed by discussion of recent progress, followed by the topics of "disease modeling" and "cell therapy" with iPSC in the second half of this article. Several examples of rheumatologic application of iPSC will be provided in the main text. If rheumatologists could understand the merits and potentials of iPSC, opportunities for innovative research and therapy can be expanded.
Arthritis, Rheumatoid ; Induced Pluripotent Stem Cells* ; Lentivirus ; Nobel Prize ; Osteoarthritis ; Pluripotent Stem Cells ; Rheumatology*

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

OBJECTIVEThe aim of this study is to optimize conditions for labeling human periodontal ligament stem cells (PDLSCs) using enhanced green fluorescent protein (eGFP) infected by lentivirus vector and to obtain PDLSCs with high stable expressed eGFP.

METHODSPDLSCs were transfected with eGFP by lentivirus vector for 48 h via different multiplicity of infection (MOI) (25, 50, 100, 200 and 400) and the infection efficiency were analyzed by both fluorescent microscope and flow cytometry. The proliferation rate of infected PDLSCs was evaluated by MTT. The infected PDLSCs were further for detection of pluripotent, differentiation ability and alkaline phosphatase (ALP) expression ability.

RESULTSThe infection efficiency for each group were 44.7%, 60.9%, 71.7%, 85.8% and 86.9% respectively. Proliferation of PDLSCs was not affected when MOI was below 200; however, at MOI 400, the proliferation ability was affected compared with control group. The pluripotent and ALP abilities of PDLSCs were not changed by the infection.

CONCLUSIONInfection for 48 h at MOI 200 is optimal for labeling PDLSCs with eGFP using lentivirus vector, and the proliferation and differentiation abilities of PDLSCs are not affected obviously.

Alkaline Phosphatase ; Cell Differentiation ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Lentivirus ; Periodontal Ligament ; Stem Cells ; Transfection