Long term gene therapy of Parkinson's disease using immortalized rat glial cell line with tyrosine hydroxylase gene.

Glial cell is an ideal vehicle for gene therapy of brain diseases. However, there are many limits in using primary glial cells. Therefore, an immortalized rat glial cell line (RGLT) was established by SV40 large T-antigen (LTag) gene from the primary rat fetal glial cells. The RGLT cell was shown to be non-tumorigenic after transplantation to nude mice (up to 4 weeks) and rat striatum (up to 18 months). Rat tyrosine hydroxylase (TH) gene was transfected into RGLT cell to obtain RGLT-TH cell. The TH immunohistochemical staining and HPLC-ECD analysis demonstrated the TH expression and dopamine (DA) production in RGLT-TH cells in vitro. When implanting RGLT-TH cells into the striatum of 6-hydroxydopamine (6-OHDA) lesioned hemiparkinsonism model rats, TH immunohistochemical staining showed the TH presence in striatum and HPLC-ECD analysis held at 6 months after cell implantation showed an increase of DA content in striatum. The asymmetric rotation of rats receiving RGLT-TH cells was reduced by 50%-60% and this reduction persisted stably at least for 18 months. These results suggest that the immortalized glial cell line could serve as an ideal vehicle for therapeutic gene delivery system to achieve a long-term gene therapy of neurodegenerative diseases.

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene.

AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were >15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P<0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P<0.05, P<0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was >15000 and 214.5+/-31.3 micromol.L(-1), P<0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.

Human interleukin 10 gene therapy decreases the severity and mortality of lethal pancreatitis in rats.

BACKGROUND: Studies have proven the validity of interleukin-10 (IL-10) in the treatment of experimental pancreatitis. Prophylactic human IL-10 (hIL-10) gene treatment attenuated the severity in cerulein models. Our research aims to study whether the therapeutic hIL-10 gene could decrease both severity and mortality in a lethal pancreatic model. METHODS: Severe acute pancreatitis (SAP) was induced by sodium taurocholate. A plasmid-hIL-10 construct (pcDNA3-hIL-10) complexed with cationic liposomes was administered to SAP rats by a single intraperitoneal injection. Levels of hIL-10 in the pancreas, liver, and lungs were determined by ELISA kits. The severity of pancreatitis was assessed in terms of serum amylase, histology, and tissue tumor necrosis factor alpha (TNF-alpha). Mortality, observed for 7 days, was evaluated for gene therapy or control groups. RESULTS: After hIL-10 gene therapy, hIL-10 levels in the pancreas, liver, and lungs increased significantly and the serum amylase, tissue TNF-alpha, and histological changes in pancreas, liver, and lungs decreased markedly. Therefore, mortality was significantly reduced in the hIL-10 gene therapy group, in which 70% of rats survived in the 7-day observation, while only 10% survived in untreated groups (P < 0.05). CONCLUSION: We found that liposome/hIL-10 gene therapy decreased severity and mortality in SAP, even carried out after SAP establishment, predicting a more convenient shift to clinical applications.

Treatment of Parkinson's Disease by Genetically Modified Immortalized Fibroblasts.

Primary rat fibroblast cells were immortalized by genetic modification of SV40 large T antigen (LT(Ag)) gene and called RFLT. This cell line was non-tumorigenic after grafting into nud-mouse and rat. The LT(Ag)gene stopped expression when the cells were transplanted in rat striatum, but it resumed the expression ability after the transplanted cells were recovered from striatum and cultured in vitro.TH gene and GCH gene were transfected into RFLT, respectively, and two types of transfected cells, RFLT-TH and RFLT-GCH, were obtained. In mixed culture with these two cell lines, DA was detected with HPLC-ECD. Implanting mixture of those cells into the striatum of PD rats significantly decreased their rotational asymmetry for up to 12 weeks. The expression of TH gene was proved by TH immunohistochemical staining in the sections of rat brain. The establishment of the genetically modified immortalized cells may play role in the gene therapy of PD.

Adenovirus-Mediated th Gene Therapy of Parkinson's Disease in a Experimental Rat Model.

A recombinant adenovirus (AdCMV th) encoding tyrosine hydroxylase (TH) gene with CMV promoter was constructed and propagated. Southern blot analyses was used to identify positive plaques. Virus titer was about 1.4x10(14) pfu/L as determined by plaque forming assay. In glial cells infected with AdCMV th, the TH expression was demonstrated by immunohistochemical staining and HPLC-ECD. 678.8 ng DA was detected in the extract of 1x10(6) AdCMV th infected glial cells, but no detectable DA was found in AdCMVLacZ-infected glial cells. Injection of AdCMV th (1x10(7) pfu/rat) into the striatum of PD rats significantly improved the apomorphine-induced rotation movement(approximately 60%). The improvement in rotation movement remained up to 5 months after injection, and TH expression positive cells were found in the vicinity of injection. These results indicate that adenovirus may be a useful carrier for in vivo gene therapy in the PD patients.

Transcription Regulation of bcl-2 Gene.

Various extracelluar stimulation, including those by growth factors and cytokines, can induce the bcl-2 gene expression. Bcl-2 protein induced by this stimulation seems to be essential for cell survival. In order to understand the regulations of bcl-2 transcription, recent advances at transcriptional and post-transcriptional levels in this field will be described.

Interaction between Jak3 and Nucleosome Assembly Protein 1.

Tyrosine kinase Jak3 plays a critical role in the interleukin 2 IL-2 signaling because it not only participates the Jak-Stat pathway, but also interacts with unidentified signal transducers and regulates expression of some oncogenes such as c-fos and c-myc. Abundant evidence demonstrated that phosphorylated tyrosine was necessary for the interaction between two proteins. Therefore, in order to clarify the role of Jak3 in IL-2 signal transduction, the tyrosine-phosphorylation-involved yeast two-hybrid system was constructed and the N-terminal region JH3-JH7 of Jak3 was used as a bait to screen a peripheral blood cDNA library. About 50 double-positive colonies were obtained. Sequence analysis indicated that one of them was from nucleosome assembly protein 1 gene (Nap1), and encoded a protein of 392 amino acid residues. Two-hybrid system results demonstrated that interaction between Jak3 and Nap1 depended on the level of tyrosine phosphorylation. Furthermore, immunoprecipitation and Western blot experiments confirmed that Jak3 really interacted with Nap1 in murine pro-B lymphocyte BAF/BO3beta cells.

Ubiquitin Conjugating Enzyme Ubc9 is Involved in Protein Degradation of Redox Factor-1 (Ref-1).

Redox factor-1 (Ref-1) is a bifunctional protein playing an important role in both cellular redox regulation and DNA apurinic/apyrimidinic sites' repair. To find Ref-1interacting proteins (Rips), a yeast two-hybrid screening was performed by using Ref-1 redox domain as the 'bait', and five positive clones were obtained. One of them (Rip3) was identified to be the ubiquitin-conjugating enzyme Ubc9. Simultaneous overexpression of Ubc9 in Hela cells dramatically inhibited the enhancement of AP-1 reporter gene by Ref-1. Western blot indicated that the protein level of Ref-1 dropped down as the result of simultaneous overexpression of Ubc9. These results suggest that Ubc9 is involved in the protein degradation of Ref-1, resulting in the downregulation of Ref-1 physiological function.

A Novel Type of Apoptosis Induced by Cadmium in BA/F3beta Cells.

Apoptosis is usually accompanied by DNA fragmentation and up-regulation of reactive oxygen species, and it can be inhibited by overexpression of Bcl-2. Here, cadmium was found to induce apoptosis in BA/F3beta cells. MTT assay, Hochest 33258 staining, and transmission electron microscopy analysis were used to detect the apoptosis, however, neither DNA fragmentation nor up-regulation of reactive oxygen species were observed in this type of apoptosis. Furthermore, Bcl-2 overexpression had no effect on this type of apoptosis. In conclusion, these data suggested that cadmium induced a novel type of apoptosis in BA/F3beta cells.

Isolation and Characterzation of p160.2, a Signal Transducing Protein Downstream of Jak3.

Nonreceptor tyrosine kinase Jak3, which binds to intracellular domain of interleukin-2 receptor gamma subunit (IL-2Rgamma), plays an important role in IL-2 function. To find downstream signal transducer of Jak3, the N-terminal region JH3-JH7 of Jak3 was used as a baitg to screen a cDNA library by using the yeast two-hybrid system. One real true clone was obtained out of 10(5) cotransformants. Sequencing indicated that it encoded a p160.2 protein fragment which consisted of 399 amino acid residues. Further results showed that the p160.2 fragment merely bound to the intact JH3-JH7 through its C-terminal. Northern blot revealed that human p160.2 shared homology with monkey p160.2, but was different from mouse p160.2; and p160.2 was widely expressed in various tissues.

Cloning of Glial Cell Line-derived Neurotrophic Factor Gene and Its Expression in Eukaryotic Cells.

Glial cell line-derived neurotrophic factor gene (gdnf) was obtained by RT-PCR method. Its pro-duct in COS7 cells showed neurotrophic and protective effect on the dopaminergic neurons. Then gdnf gene was cloned into retrovirus vector pLNCX and packaged with PA317. Infectious particles thus obtained were used to infect rat myoblast cell line L-6TG. A cell clone L-6TG/gdnf was obtained after G418 selection. It will be applied to the ex vivo gene therapy study of Parkinson's disease.

Preliminary Study on Gene Therapy of PD Monkey Using Microcapsulated Rat Transgenetic Myoblasts.

Rat myoblast cells transfected with tyrosine hydroxylase gene were microcapsulated using the ALG/PLL system. These microcapsulated cells could survive grow and produce tyrosine hydroxylase(TH)for at least two months in vitro. After the implantation of these microcapsules into monkey striatum microcapsulated cells still survived one month more. No obvious gliosis was seen around implanted microcapsules. Then the microcapsules were transplanted into the striatum of PD monkeys and their typical PD rotation numbers were significantly decreased.

Expression of Brain-derived Neurotrophic Factor (BDNF) Gene in Rat Myoblast Cells.

Brain-derived neurotrophic factor (BDNF) gene was cloned into retrovirous vector plasmid pLNCX to form the pLNC/BDNF. After packaging in PA317 cell line, the infectious particles were used to infect rat myoblast cell line L-6TG. After selection with G418, the L-6TG/BDNF cells were collected. Results from Southern blot show that bdnf gene has successfully integrated into the L-6TG genomic DNA. The expression of bdnf gene has been proved by Northern blot and Dot blot. The content of BDNF in the culture medium of L-6TG/BDNF was about 25 ng.10(-6) cells per 24 h per ml.

Specific Expression of Suicide Gene in Liver Cancer Cells Mediated by Adenovirus.

Shuttle plasmid containing HSV-tk gene and regulatory sequence of the afp gene was constructed and recombined with the right arm of adenovirus DNA. The recombinant adenovirus vector was named pAdrAFPTK. Meanwhile, an AdCMVTK was constructed as control in which the tk gene was controlled under CMV promoter. PCR and Southern blot analyses were used to identify positive plaques. Virus titer was about 1x10(15) pfu/L determined by plaque forming assay. The AFP-positive cells or AFP-negative cells were infected with AdCMVTK or AdrAFPTK and then treated with GCV, respectively. Cytotoxic effects were assayed with MTT method. The IC(50) of GCV for both HeLa cells or BRL-3A cells (both were AFP-negative cells) and HepG2 cells (AFP-positive cells) were 1.3 &mgr;mol/L, 2&mgr;mol/L and <1&mgr;mol/L, respectively, after infection with AdCMVTK (m.o.i.=100). However, in the cases of infection with AdrAFPTK (m.o.i.=100), IC(50) were 1 000 &mgr;mol/L, >1 000 &mgr;mol/L and <1&mgr;mol/L for HeLa cells, BRL-3A cells and HepG2 cells, respectively. Results showed that this vector possessed advantages of high title, high infectivity coming from adenovirus and the character of cell type-specificity gene expression. The AdrAFPTK/GCV system may become a new, potent and specific approach for the gene therapy of the primary hepatoma.

Construction of Two AFP-positive Hepatoma Cell-specific Gene Regulatory Sequences.

Two chimerical regulation sequences for gene expression, one (called ATrPS) harboring an enhancer of human alpha1-antitrypsin gene (AT) and a promoter and silencer(rPS)of rat afp gene, and another (called rAFP) consisting of enhancer III of rat afp gene and its rPS, were constructed, respectively. Then, two CAT expression vectors, rAFP-pCAT and ATrPS-pCAT, were constructed in which the cat reporter gene was put under the control of these elements, respectively. CAT activities could were detected in the AFP positive liver cancer cells and also in those liver cancer cells, liver cells or non-liver cells whose AFP were negative. Results showed that for both constructs, the CAT activities could be found in all AFP positive hepatoma cells, however, while this activity can not be detected in all AFP negative cells. Similar results were observed in primary cell cultures. The results showed that our regulation elements of gene expression really possessed cell-type specificity for AFP positive cells. The cell-type specificity remains when the length of rAFP was cut short as to 0.67 kb; and the activity seemed higher. These regulatory sequences of gene expression may be used in gene therapy for primary liver cancer.

The Construction of an Adenovirus Vector Containing Cytosine Deaminase Gene and Its Application.

A replication-defective recombinant adenovirus vector containing CMV promoter and Escherichia coli cytosine deaminase (cd) gene (AdCMVCD) was constructed. It was shown by Southern blotting and RT-PCR that cd gene had been inserted into AdCMVCD and was expressed in the infected cells. The recombinant virus was purified by gradient centrifugation in CsCl and its titer was 1x10(15) pfu/L. HeLa and C6 cells infected with AdCMVCD(m.o.i=100) became sensitive to the prodrug 5FC, and the number of viable cells decreased to less than 20% of the control after treatment with 100 &mgr;mol/L 5FC. Significant bystander effect was also observed. When cells infected with AdCMVCD were mixed with wild type cells at a ratio of 3.3 : 96.7, more than 60% of the cells could be killed in the presence of 50 &mgr;mol/L 5FC. These results suggest that the AdCMVCD/5FC system may provide a new approach for gene therapy of cancers.

Use of Carcinoembryonic Antigen Gene Promoter in Colorectal Carcinoma-specific Suicidal Gene Therapy.

The possibility of tumor-specific suicidal gene therapy of colorectalcarcinoma was investigated using carcinoembryonic antigen (CEA) -positive human colorectal carcinoma cell line LoVo and CEA-negative HeLa cell line as a model. After confirming cellular specificity of cea promoter by CAT assay, eukaryotic expression plasmid pCEACD was constructed in which the expresstion of E. coli cytosine deaminase (CD) gene was under the control of cea promoter. The expression of CD gene increased the sensitivity of LoVo cells to 5-fluorocytosine (5FC) by 750 fold, while the sensitivity of HeLa cells to 5FC was increased by only 7.5 fold. These results suggest that the expression of CD gene driven by cea promoter specifically killed CEA-positive colorectal carcinoma cells. Transmission electron microscopy and DNA fragmentation assay demonstrate that CD/5FC system induced apoptosis in LoVo cells.

Expression of Cytosine Deaminase Gene in Human Colon Carcinoma Cells by Recombinant Retroviral Vector.

The recombinant retroviral vector pLCDSN containing E. coli cytosine deaminase gene was constructed. After packaging with PA317 cell line, the infectious particles were used to infect human colon carcinoma cell line LoVo. A single clone harbouring EC-CD gene was picked after G418 selection. There was no significant difference in cell growth curve or morphology between the LoVo/LCDSN and LoVo cells. Both of them were very sensitive to 5-FU in vitro (IC(50), approximately 0.5 &mgr;mol/L). However, the expression of the CD gene did increase the sensitivity of these cells to the nontoxic prodrug, 5-FC, decreasing the IC(50) for 5-FC from 22 000 &mgr;mol/L for parental LoVo cells to 13 &mgr;mol/L for LoVo/LCDSN cells. Obvious by side effect was also observed. When cells transduced with CD gene were mixed with wild type cells at a ratio of 30:70, above 80% of the cancer cells could be killed after treatment with a nontoxic concentration of 5-FC.

A New Computer-imitated Model of Spatial Interaction between Interleukin-2 and Interleukin-2 Receptor Complex.

It is very meaningful to construct the spatial model of ligand-receptor interaction between interleukin-2 (IL-2) and interleukin-2 receptor (IL-2R) complex for the further studies in the structure-function relationship of IL-2 or IL-2R. In our study of the IL-2 molecule, we have found that Glu62 residue takes part in the binding to IL-2R alpha subunit and have confirmed that Glu126 residue in the residue bound to the IL-2R gamma subunit. In consideration with these discoveries and the progress in IL-2R subunits study, we have suggested a new model of the spatial interaction between IL-2 and IL-2R.

The Influence of Amino Acid Substitutions on the Function of Interleukin-2.

The effects of some residues in IL-2 on its activity have been studied by site-directed mutagenesis. After changing 39 Met or 43 Lys to Pro, neither the CD spectra (s) nor the biological activities of these mutants were changed, implying that the 39 Met and 43 Lys were not involved in the reported alpha-helical structure. However after changing 52 Glu, 53 Leu or 54 Lys to Pro respectively the CD spectra were markedly changed and the activities were decreased significantly, showing that these three amino acids in IL-2 were integrated in the alpha-helix structure and very important for their biological functions. The significance of the above results was discussed.