Stable Expression of Coagulation Factors by RPS6 Promoter / 中国实验血液学杂志

OBJECTIVE@#To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia.@*METHODS@#Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene.@*RESULTS@#The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups.@*CONCLUSION@#After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.

Overexpression of connexin 40 (Cx40) inhibits the proliferation of H9c2 cardiomyocytes in rats by cell cycle arrest / 细胞与分子免疫学杂志

Objective To establish a stable strain of H9c2 cardiomyocytes overexpressing Cx40 and preliminarily investigate the effect of lentiviral vector-mediated Cx40 protein overexpression on the proliferation of H9c2 cells and its related mechanisms. Methods The Cx40 gene fragment was cloned from H9c2 cells by PCR and linked with lentivirus vector pLVX-IRES-Puro to obtain the recombinant plasmid pLVX-Flag-Cx40. Recombinant lentiviral particles carrying Flag-Cx40 were obtained by cotransfection with packaging plasmids into HEK293T cells. A stable expression strain (H9c2-Flag-Cx40 cell) was screened from infected H9c2 cells by purinomycin. The expression of Cx40 protein was detected by Western blot analysis, and the effect of Cx40 on H9c2 cells proliferation was determined by CCK-8 assay; cell cycle changes were measured by flow cytometry; the expression of the cell cycle protein cyclin D1 was detected by qRT-PCR and Western blot analysis. Co-immunoprecipitation (Co-IP) immunoprecipitation and Western blot analysis were used to identify the binding of Cx40 and Yes associated protein (YAP) in H9c2 cells; cytoplasmic and cytosolic proteins were isolated to detect the effect of Cx40 on the localization of YAP using Western blot analysis. Results Sequencing results showed that the recombinant pLVX-Flag-Cx40 expression vector was successfully established. A stable transfected cell line containing recombinant Flag-Cx40 lentivirus (H9c2-Flag-Cx40 cell) was successfully constructed from H9c2 cells. Compared with the control group, overexpression of Cx40 significantly reduced the proliferation of H9c2 cells, arrested the cell cycle at G0/G1 and reduced cyclin D1 expression. A significant increase in YAP expression was observed in the cytoplasm of the H9c2-Flag-Cx40 stable cell line, while the expression in the nucleus was significantly reduced. Cx40 bound to YAP in the cytoplasm and prevented it from entering the nucleus to play the role of transcriptional coactivation. Conclusion Overexpression of Cx40 induces cell-cycle arrest at G0/G1 phase and inhibits the proliferation in H9c2 cells.

Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects / 中华血液学杂志

Objective: To construct chimeric antigen receptor (CAR) T cells targeting CD52 (CD52 CAR-T) and validate the effect of CD52 CAR-T cells on CD52-positive leukemia. Methods: A second-generation CD52-targeting CAR bearing 4-1BB costimulatory domain was ligated into a lentiviral vector through molecular cloning. Lentivirus was prepared and packaged by 293 T cells with a four-plasmid system. Fluorescein was used to label cell surface antigens to evaluate the phenotype of CD52 CAR-T cells after infection. Flow cytometry and ELISA were used to evaluate the specific cytotoxicity of CD52 CAR-T cells to CD52-positive cell lines in vitro. Results: ①A pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP expressing plasmid was successfully constructed and used to transduce T cells expressing a novel CD52-targeting CAR. ②On day 6, CD52-positive T cells were almost killed by CD52-targeted CAR-T post lentivirus transduction [CD52 CAR-T (4.48 ± 4.99) %, vs Vector-T (56.58±19.8) %, P=0.011]. ③T cells transduced with the CAR targeting CD52 showed low levels of apoptosis and could be expanded long-term ex vivo. ④The CD52 CAR could promote T cell differentiation into central and effector memory T cells, whereas the proportion of T cells with a CD45RA(+) effector memory phenotype were reduced. ⑤CD52 CAR-T cells could specifically kill CD52-positive HuT78-19t cells but had no killing effect on CD52-negative MOLT4-19t cells. For CD52 CAR-T cells, the percentage of residual of HuT78-19t cells was (2.66±1.60) % at an the E:T ratio of 1∶1 for 24 h, while (56.66±5.74) % of MOLT4-19t cells survived (P<0.001) . ⑥The results of a degranulation experiment confirmed that HuT78-19t cells significantly activated CD52 CAR-T cells but not MOLT4-19t cells[ (57.34±11.25) % vs (13.06± 4.23) %, P<0.001]. ⑦CD52 CAR-T cells released more cytokines when co-cultured with HuT78-19t cells than that of vector-T cells [IFN-γ: (3706±226) pg/ml, P<0.001; TNF-α: (1732±560) pg/ml, P<0.01]. Conclusions: We successfully prepared CD52 CAR-T cells with anti-leukemia effects, which might provide the foundation for further immunotherapy.

Construction and identification of a HEK293 cell line with stable TrxR1 overexpression / 南方医科大学学报

OBJECTIVE@#To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.@*METHODS@#TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay.@*RESULTS@#TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005).@*CONCLUSION@#We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.

Optimization of β-globin Stable Expression Using the Third Generation Lentiviral Vector for β-thalassemia Therapy / 中国实验血液学杂志

OBJECTIVE@#To provide a research basis for a safe and effective cell therapy for β-thalassemia through optimization of HS4 region of the third generation lentiviral vector for stable expression of β-globin.@*METHODS@#The human β-globin HS4 region in the third generation lentiviral expression vector was optimized to construct the lenti-HBB, and the transcription and translation of β-globin gene were analyzed by RT-PCR and Western blot after the transduction of lenti-HBB in MEL cell line. Furthermore, the erythroid differentiation of CD34+ cells which were transduced lentiviral virus carrying human β-globin from normal human umbilical cord blood cells and peripheral blood cells of patients with β-thalassemia major were confirmed by colony formation assay, cell smear assay and flow cytometry. The safety and effectiveness of the optimized lenti-HBB were verified by NSG mouse in vivo test.@*RESULTS@#The human β-globin was expressed stably in the MEL cells, and CD34+ cells from health umbilical cord blood as well as PBMC from patient with β-thalassemia major transduced with lenti-HBB could be differentiated to mature red blood cells. The β-globin expression and differentiation in CD34+ cells were demonstrated successfully in the NSG mouse for about 35 months after post-transplant.@*CONCLUSION@#Stable β-globin expression through the optimization of HS4 from CD34+ in the third generation lentiviral vector is safe and effective for patients with severe β-thalassemia and other β-globin abnormal diseases.

Advances in quantification of lentiviral vectors / 生物工程学报

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.

Construction and Identification of Leukemia Cell Line Stably Expressing CD123 and CLL-1 / 中国实验血液学杂志

OBJECTIVE@#To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1.@*METHODS@#The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry.@*RESULTS@#The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×10@*CONCLUSION@#Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.

Knockdown of ZFR suppresses cell proliferation and invasion of human pancreatic cancer

BACKGROUND: Zinc finger RNA binding protein (ZFR) is involved in the regulation of growth and cancer development. However, little is known about ZFR function in pancreatic cancer. METHODS: Herein, to investigate whether ZFR is involved in tumor growth, Oncomine microarray data was firstly used to evaluate ZFR gene expression in human pancreatic tumors. Then short hairpin RNA (shRNA) targeting ZFR was designed and delivered into PANC-1 pancreatic cancer cells to knock down ZFR expression. Cell viability, cell proliferation and cell cycle analysis after ZFR knockdown were determined by MTT, colony forming and FACS, respectively. In addition, cell migration and invasion were assessed using the Transwell system. RESULTS: The expression of ZFR was significantly higher in pancreatic tumors than normal pancreas tissues by Oncomine database analysis. Knockdown of ZFR by shRNA-expressing lentivirus significantly decreased the viability and invasion ability of pancreatic cancer cells. Moreover, FACS analysis showed that knockdown of ZFR in PANC-1 cells caused a significant cell cycle arrest at G0/G1 phase. Furthermore, knockdown of ZFR decreased the levels of CDK2, CDK4, CyclinA and CyclinD1 and enhanced the expression of p27, which has evidenced by qRT-PCR and Western blot analysis. CONCLUSIONS: Knockdown of ZFR might provide a novel alternative to targeted therapy of pancreatic cancer and deserves further investigation.

Swim training and the genetic expression of adipokines in monosodium glutamate-treated obese rats

Objective The aim of this study was to evaluate the genetic expression of adipokines in the adipocytes of monosodium glutamate (MSG)-treated obese rats submitted to physical activity.Materials and methods Obesity was induced by neonatal MSG administration. Exercised rats (MSG and control) were subjected to swim training for 30 min for 10 weeks, whereas their respective controls remained sedentary. Total RNA was obtained from sections of the mesenteric adipose tissue of the rats. mRNA levels of adiponectin (Adipoq), tumor necrosis factor alpha (Tnf), peroxisome proliferator-activated receptor alpha (Ppara), and peroxisome proliferator-activated receptor gamma (Pparg) adipokines were quantified by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Results In the exercise-trained control group, the expression of Adipoq increased compared to the sedentary control, which was not observed in the MSG-obese rats. Increased levels of Tnf in MSG-obese rats were not reversed by the swim training. The expression of Ppara was higher in sedentary MSG-obese rats compared to the sedentary control. Swimming increased this adipokine expression in the exercise-trained control rats compared to the sedentary ones. mRNA levels of Pparg were higher in the sedentary MSG-rats compared to the sedentary control; however, the exercise did not influenced its expression in the groups analyzed.Conclusions In conclusion, regular physical activity was not capable to correct the expression of proinflammatory adipokines in MSG-obese rat adipocytes.

Lentivirus mediated silencing of Ubiquitin Specific Peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro

BACKGROUND: Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells. RESULTS: In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G2/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells. CONCLUSIONS: All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.

Targeting PPM1D by lentivirus-mediated RNA interference inhibits the tumorigenicity of bladder cancer cells

Protein phosphatase magnesium/manganese-dependent 1D (PPM1D) is a p53-induced phosphatase that functions as a negative regulator of stress response pathways and has oncogenic properties. However, the functional role of PPM1D in bladder cancer (BC) remains largely unknown. In the present study, lentivirus vectors carrying small hairpin RNA (shRNA) targeting PPM1D were used to explore the effects of PPM1D knockdown on BC cell proliferation and tumorigenesis. shRNA-mediated knockdown of PPM1D significantly inhibited cell growth and colony forming ability in the BC cell lines 5637 and T24. Flow cytometric analysis showed that PPM1D silencing increased the proportion of cells in the G0/G1 phase. Downregulation of PPM1D also inhibited 5637 cell tumorigenicity in nude mice. The results of the present study suggest that PPM1D plays a potentially important role in BC tumorigenicity, and lentivirus-mediated delivery of shRNA against PPM1D might be a promising therapeutic strategy for the treatment of BC.

Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

CIAPIN1 gene silencing enhances chemosensitivity in a drug-resistant animal model in vivo

Overexpression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) contributes to multidrug resistance (MDR) in breast cancer. This study aimed to evaluate the potential of CIAPIN1 gene silencing by RNA interference (RNAi) as a treatment for drug-resistant breast cancer and to investigate the effect of CIAPIN1 on the drug resistance of breast cancer in vivo. We used lentivirus-vector-based RNAi to knock down CIAPIN1 in nude mice bearing MDR breast cancer tumors and found that lentivirus-vector-mediated silencing of CIAPIN1 could efficiently and significantly inhibit tumor growth when combined with chemotherapy in vivo. Furthermore, Western blot analysis showed that both CIAPIN1 and P-glycoprotein expression were efficiently downregulated, and P53 was upregulated, after RNAi. Therefore, we concluded that lentivirus-vector-mediated RNAi targeting of CIAPIN1 is a potential approach to reverse MDR of breast cancer. In addition, CIAPIN1 may participate in MDR of breast cancer by regulating P-glycoprotein and P53 expression.

Downregulation of Orai1 expression in the airway alleviates murine allergic rhinitis

Orai1 is the key subunit of the Ca2+-release-activated Ca2+ channel. Our previous report has demonstrated that Orai1 expression in the airway was upregulated in the ovalbumin (OVA)-induced allergic rhinitis (AR) mouse models. To observe whether inhibition of Orai1 expression in the airway could suppress symptoms in a murine model of AR and to assess the impacts of this inhibition on the responses of local and systemic immunocytes, we administered recombinant lentivirus vectors that encoded shRNA against ORAI1 (lenti-ORAI1) into the nostrils of OVA-sensitized mice before the challenges, and analyzed its effect on allergic responses, as compared with the unsensitized mice and untreated AR mice. Administration of lenti-ORAI1 into the nasal cavity successfully infected cells in the epithelial layer of the nasal mucosa, and significantly decreased the frequencies of sneezing and nasal rubbing of the mice. Protein levels of leukotriene C4, OVA-specific IgE, and IL-4 in the nasal lavage fluid and serum and eosinophil cation protein in the serum were also significantly reduced by lenti-ORAI1, as were the mRNA levels of these factors in the nasal mucosa and spleen. These data suggested that administration of lenti-ORAI1 into the nasal cavity effectively decreased Orai1 expression in the nasal mucosa, alleviated AR symptoms, and partially inhibited the hyperresponsiveness of the local and systemic immune cells including T cells, B cells, mast cells and eosinophils that are involved in the pathogenesis of AR.

Effect of Small Hairpin RNA Targeting Endothelin-Converting Enzyme-1 in Monocrotaline-Induced Pulmonary Hypertensive Rats

The purpose of this study was to investigate the therapeutic effects of small hairpin RNA (shRNA) targeting endothelin-converting enzyme (ECE)-1 in monocrotaline (MCT)-induced pulmonary hypertensive rats. Ninty-four Sprague-Dawley rats were divided into three groups: control (n = 24), MCT (n = 35) and shRNA (n = 35). Four-week survival rate in the shRNA group was significantly increased compared to that in the MCT group. The shRNA group showed a significant improvement of right ventricular (RV) pressure compared with the MCT group. The MCT and shRNA groups also showed an increase in RV/(left ventricle + septum) ratio and lung/body weight. Plasma endothelin (ET)-1 concentrations in the shRNA group were lower than those in the MCT group. Medial wall thickness of pulmonary arterioles were increased after MCT injection and was significantly decreased in the shRNA group. The number of intra-acinar muscular pulmonary arteries was decreased in the shRNA group. The mRNA expressions of ET-1 and ET receptor A (ETA) were significantly decreased in the shRNA group in week 4. The protein levels of ETA were decreased in the shRNA group in week 2. The protein levels of tumor necrosis factor-alpha and vascular endothelial growth factor were decreased in the shRNA group in week 4. In conclusion, the gene silencing with lentiviral vector targeting ECE-1 could be effective against hemodynamic, histopathological and gene expression changes in pulmonary hypertension.

Lentivirus-mediated RNA interference targeting E2F-1 inhibits human gastric cancer MGC-803 cell growth in vivo

The E2F-1 transcription factor is post-translationally modified and stabilized in response to various forms of DNA damage to regulate the expression of cell-cycle and pro-apoptotic genes. The sustained overexpression of E2F-1 is a characteristic feature of gastric cancer. In this study, we investigated the role of short hairpin RNA (shRNA) targeting E2F-1 gene on human gastric cancer MGC-803 cell growth in vivo, and preliminarily revealed the mechanism. Thus, we constructed recombinant pGCSIL-GFP-shRNA-E2F-1 lentiviral vector to knock down E2F-1 expression in human gastric cancer MGC-803 cells in vivo, and studied the effect of E2F-1 shRNA on growth of MGC-803 tumor and evaluated its treatment efficacy. Our data demonstrated that in a mouse model of established gastric cancer, intratumor injection of lentiviral shRNA targeting E2F-1 definitely decreased the endogenous E2F-1 mRNA and protein expression in MGC-803 tumor, and inhibited tumor growth and promoted tumor cells apoptosis. Moreover, we found that E2F-1 shRNA increased the expression of phosphatase and tensin homolog (PTEN), activated caspase-3 and caspase-9, and suppressed nuclear factor (NF)-kappaB expression in tumor tissue as determined by reverse transcription (RT)-PCR and western blotting. In summary, shRNA targeting of E2F-1 can effectively inhibits human gastric cancer MGC-803 cell growth in vivo and may be a potential therapeutic strategy for gastric cancer.

Expression of Exogenous Human Hepatic Nuclear Factor-1alpha by a Lentiviral Vector and Its Interactions with Plasmodium falciparum Subtilisin-Like Protease 2

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors as well as by individual host responses to these determinants. In both humans and mice, liver injury follows parasite entry, persisting to the erythrocytic stage in the case of infection with the fatal strain of Plasmodium falciparum. Hepatic nuclear factor (HNF)-1alpha is a master regulator of not only the liver damage and adaptive responses but also diverse metabolic functions. In this study, we analyzed the expression of host HNF-1alpha in relation to malaria infection and evaluated its interaction with the 5'-untranslated region of subtilisin-like protease 2 (subtilase, Sub2). Recombinant human HNF-1alpha expressed by a lentiviral vector (LV HNF-1alpha) was introduced into mice. Interestingly, differences in the activity of the 5'-untranslated region of the Pf-Sub2 promoter were detected in 293T cells, and LV HNF-1alpha was observed to influence promoter activity, suggesting that host HNF-1alpha interacts with the Sub2 gene.

Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector / Transferência de genes heterólogos para células corneanas epiteliais primárias utilizando vetor lentivírus

PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP) reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80 percent confluency) prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS). We evaluated the transduction efficiency (TE) over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells). RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1 percent) and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7 percent. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

OBJETIVO: Avaliar a transferência de genes heterólogos expressando a proteí­na "Green Fluorescent Protein" (GFP) para células corneanas epiteliais primárias ex vivo utilizando vetor lentivírus. MÉTODOS: Tecido corneoescleral de coelhos foi usado para obtenção de suspensão de células corneanas epitelias. As células foram semeadas na densidade de 5×10³ células/cm² e expandidas por 5 dias até uma confluência de 70-80 por cento antes de serem transduzidas. A transferência genética foi monitorada por microscopia fluorescente e por um separador de células ativadas por fluorescência. Foram avaliadas a eficiência de transdução ao longo do tempo e o efeito dose-resposta de diferentes quantidades de partículas virais. Um grupo de células foi analisado pelo separador de células ativadas por fluorescência para avaliar a transdução de células com fenótipo de cé­lulas tronco do epitélio corneano (baseado na exclusão do corante "Hoechst dye"). RESULTADOS: Os vetores lentivírus foram efetivos na transdução de cé­lulas corneanas epiteliais primárias de coelhos ex vivo. Fotodocumentação das células vivas demonstrou células epiteliais de morfologia normal e expressando o gene fluorescente (GFP). A eficiência de transdução ao longo do tempo foi maior no quinto dia após a transdução (14,1 por cento) e demonstrou uma tendência à estabilidade a partir do oitavo dia após a transdução. O número de células transduzidas foi dose-dependente e atingiu 7 por cento com as maiores concentrações de partículas virais. Quando analisadas pelo separador de células ativadas por fluorescência para detecção de células transduzidas e também de células que excluíram o corante "Hoechst dye", foi detectado que células com fenótipo de células tronco do epitélio corneano ("side-population") também foram transduzidas. CONCLUSÕES: Os vetores lentivirais podem transferir genes heterolólogos para células corneanas epiteliais primárias expandidas ex vivo de forma eficiente. Os genes foram expressos de forma estável ao longo do tempo e puderam ser transferidos tanto para células epiteliais maduras como para presumíveis células tronco epiteliais. A eficiência de transdução foi obtida de forma dose-dependente.

Death switch for gene therapy: application to erythropoietin transgene expression

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv’-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Delivery of Factor VIII Gene into Skeletal Muscle Cells Using Lentiviral Vector

PURPOSE: This study was designed to investigate whether transduction of lentiviral vectors (LV) carrying human coagulation factor VIII (hFVIII) cDNA into skeletal muscle could increase circulating hFVIII concentrations. MATERIALS AND METHODS: A LV containing bacterial LacZ gene as a control or human FVIII gene was intramuscularly administered into the thigh muscle of 5 weeks old Sparague-Dawley rats. The plasma human FVIII concentration and neutralizing anti-FVIII antibodies were measured for up to 12 weeks in these experimental animals. RESULTS: The plasma human FVIII levels in the rats injected with LV carrying FVIII cDNA peaked at post-injection 1st week (5.19 +/- 0.14 ng/mL vs. 0.21 +/- 0.05 ng/mL in control rats , p < 0.05). Elevated hFVIII concentrations were maintained for 4 weeks (2.52 +/- 0.83 ng/mL vs. 0.17 +/- 0.08 ng/mL in control rats, p < 0.05) after a single intramuscular injection. In the Bethesda assay, neutralizing antibodies for FVIII protein were detected only in FVIII-LV injected rats by the 10th week, but not in control rats. CONCLUSION: This study suggested that a single administration of an advanced generation LV carrying the human FVIII cDNA resulted in elevation of FVIII level in immune competent rats, and that this gene transfer approach to the skeletal muscle could be an effective tool in treatment of hemophilia A.