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PubMed2001

Treatment of malignant gliomas with a replicating adenoviral vector expressing herpes simplex virus-thymidine kinase.

We evaluated the interaction between oncolytic, replication-competent adenoviral vectors and the herpes simplex virus-1 thymidine kinase (HSV1-tk) gene/ganciclovir (GCV) suicide system for the treatment of malignant gliomas. We constructed a panel of replication-competent adenoviral vectors in which the luciferase (IG.Ad5E1(+). E3Luc) or HSV1-tk gene (IG.Ad5E1(+).E3TK) replace the M(r) 19,000 glycoprotein (gp19K) coding sequence in the E3 region. IG.Ad5E1. IG.Ad5.ClipLuc and IG.AdApt.TK are E1-deleted viruses that contain the luciferase or the HSV1-tk gene in the former E1 region driven by the human cytomegalovirus promoter. IG.Ad5. Sarcoma 1800HSA.E3Luc contains an irrelevant gene in the E1 region, whereas the gp19K coding sequence in the E3 region is replaced by the luciferase gene as in the replicating virus IG.Ad5E1(+).E3Luc. For in vitro experiments, we used a panel of human glioma cell lines (U87 MG, T98G, A172, LW5, and U251), a rat gliosarcoma cell line (9 L), and human lung (A549) and prostate carcinoma (P3) cell lines. In vitro, GCV sensitivity (10 microg/ml) was studied in U87 MG cells after infection at a multiplicity of infection of 1 and 10. A s.c. U87 MG glioma xenograft model was established in NIH-bg-nu-xid mice. Tumors of 100-150 mm(3) were treated with a single injection of adenovirus 10(9) IU suspended in 100 microl of PBS, and GCV 100 mg/kg was administered i.p. twice daily for 7 days. The cytopathic effect of all three replication-competent adenoviral vectors was similar to the cytopathic effect of wild-type adenovirus 5 on all human cell lines tested, indicating that deletion of the E3 gp19K sequences did not affect the oncolytic effect of the vectors. In vitro, luciferase expression was the same for both E1-deleted vectors (IG.Ad5.ClipLuc and IG.Ad5. Sarcoma 1800HSA.E3Luc), demonstrating the strength of the internal E3 promoter even in the absence of E1A. However, in vitro expression levels obtained with replication-competent IG.Ad5E1(+). E3Luc were 3 log higher (allowing infection with a 2-3-log lower multiplicity of infection) in the human cell lines. In U87 MG glioma cells, the oncolytic effect of replication-competent IG.Ad5E1(+).E3TK was significantly enhanced by the addition of GCV and greatly exceeded the cytotoxicity of replication-incompetent IG.AdApt.TK combined with GCV. In established s.c. U87 MG glioma xenografts, a single injection of IG.Ad5E1(+).E3TK resulted in a significant slowing of tumor growth and prolonged survival compared with injection of IG.AdApt.TK. Addition of GCV slowed tumor growth, further adding to survival. In conclusion, the oncolytic effect of replicating adenoviral vectors and HSV1-tk/GCV have potent antitumor effects in gliomas. When combined, these two approaches are complementary, resulting in a significantly improved treatment outcome. In addition, replication-competent adenoviral vectors missing the E3 gp19K coding sequences, have oncolytic efficacy comparable with wild type. In combination with high expression levels obtained with the natural E3 promoter, such vectors are promising new anticancer agents.

Adenoviridae↗
PubMed2002

Targeting of adenovirus vectors to tumor cells does not enable efficient transduction of breast cancer metastases.

Targeting of oncolytic adenoviruses to tumors can potentially increase their efficacy and safety profile after systemic application. We have developed recently a capsid-modified vector containing the adenovirus serotype 35 fiber shaft and knob inserted into an Ad5 capsid. This Ad5/35 vector infects cells via a coxsackievirus adenovirus receptor-independent pathway. Here we attempted to exploit this new tropism of Ad5/35 vectors for tumor-specific infection. In vitro, the Ad5/35 vector efficiently transduced human breast cancer cells that were refractory to infection with conventional Ad5-based vectors. Additionally, primary mouse hepatocytes were relatively refractory to Ad5/35 infection in vitro or after systemic vector application to mice. In an animal model of breast cancer metastasis, intraportal infusion of MDA-MB435 cells produced multiple hepatic metastases that were surrounded by extracellular matrix and developed blood vessels confined to the tumor stroma. Tail vein injection of a standard Ad5-based vector into tumor-bearing animals resulted in transduction of mouse hepatocytes but not metastases. However, the capsid-modified Ad5/35 vector transduced only approximately 8% of metastases. The metastases that were susceptible to Ad5/35 infection demonstrated blood vessels in close proximity to tumor nests without extracellular matrix separating endothelial and tumor cells. These findings indicate that transduction of liver metastases not only requires tumor-specific tropism but also new strategies to increase accessibility of tumor cells to systemically applied oncolytic adenoviruses.

Adenoviridae↗
PubMed2002

Reduced transduction efficiency of adenoviral vectors expressing human p53 gene by repeated transduction into glioma cells in vitro.

PURPOSE: Recombinant adenoviral vectors are widely used in clinical and experimental studies to treat malignant tumors. Recently, host immune responses have been proposed as a major limitation in using adenoviral vectors for repeated gene delivery. We demonstrate another limitation unrelated to host immunity. EXPERIMENTAL DESIGN: We repeatedly transduced an adenoviral vector expressing the human p53 gene (AxCIhp53) into U373MG, a p53-susceptible cell line, and established the AxCIhp53-resistant cell line U373R. Most U373R cells survived even after AxCIhp53 treatment due to reduced transduction efficiency. Expression levels of adenovirus receptors were estimated to investigate the cause of reduced transduction efficiency. The mutant vector was used to overcome the resistance. RESULTS: The transduction efficiency of an adenoviral vector possessing the reporter LacZ gene (AxCAZ2-F/wt) for U373R cells was 25.4-fold less than that for parent cells. The expression levels of integrins alpha(v)beta(3) and alpha(v)beta(5) were found to be decreased in U373R cells without affecting the expression levels of Coxsackievirus and adenovirus receptor. The mutant vector AxCAZ2-F/K20, with a linker and a stretch of 20 lysine residues at the COOH-terminal of the fiber protein, improved the transduction efficiency of U373R cells to 12.6-fold of that of AxCAZ2-F/wt. A mutant vector carrying the p53 gene, AxCAhp53-F/K20, dramatically induced apoptosis in U373R cells. CONCLUSIONS: Glioma cells expressing low levels of adenovirus receptors might survive and proliferate to recur after repeated adenoviral transduction, even if the adenoviral transduction is effective at first. Changing the tropism of vectors is a potent method to overcome resistance.

Adenoviridae↗
PubMed2002

Long-term transgene expression in the RPE after gene transfer with a high-capacity adenoviral vector.

PURPOSE: To analyze duration of gene expression in the retinal pigment epithelium (RPE) in immunocompetent animals after gene transfer with a high-capacity adenoviral (HC-Ad) vector. METHODS: An HC-Ad vector was constructed to express the enhanced green fluorescence protein (EGFP) from the human CMV promoter. This vector (HC-AdFK7) was used to transduce rat RPE cells in cell culture and after subretinal injection in vivo in adult immunocompetent Wistar rats. In cell culture, expression of EGFP was analyzed by fluorescence microscopy. In vivo expression was monitored by scanning laser ophthalmoscopy and stereo fluorescence microscopy. After enucleation of the eyes, immunohistochemical and morphologic analyses by fluorescence light microscopy and electron microscopy were performed. RESULTS: In vitro, RPE cells were efficiently transduced with HC-AdFK7. Expression persisted for the observation time of 8 weeks. In vivo, the RPE was efficiently transduced with low doses of HC-AdFK7. EGFP synthesis was confirmed by antibody staining and found to be stable for the complete study period of 6 months. The neuroretina was well preserved over areas of subretinal vector administration, and significant morphologic changes were not detected. There was no accumulation of inflammatory T cells or macrophages. CONCLUSIONS: In contrast to previous results with earlier generation adenoviral vectors, subretinal injection of an HC-Ad vector in immunocompetent rats resulted in long-term transgene expression without evidence of adverse immune reactions or significant toxicity, probably because of the absence of expression of the viral gene from this vector. Thus, HC-Ad vectors are suitable for the treatment of eye disorders that require durable gene expression in the RPE.

Adenoviridae↗
PubMed2002

What can SV40-derived vectors do for gene therapy?

The limited success of gene therapy as an approach to treating human disease largely reflects the limitations of the gene delivery vectors that have been used. Poor titers, low transduction efficiency, waning transgene expression and immunogenicity have remained obstacles in the field. As a consequence, much research in normal, immunocompetent animals has not demonstrated therapeutic levels of gene delivery, and results from most human clinical trials have been predictably discouraging. Recombinant gene transfer vectors derived from SV40 virus (rSV40) are potentially of great interest for those working in gene therapy, since these vectors are not subject to many of the problems that have limited gene delivery using other vector systems. rSV40 is made at a very high titer and infects - and so transduces - almost all nucleated cell types very efficiently, regardless of lineage or whether they are resting or dividing; they integrate and are not susceptible to transgene silencing; and they elicit no detectable immune response on the part of normal animals and so can be used to deliver multiple transgenes over time and in sequence. The recent development of 'gutless' rSV40 vectors has expanded the range of potential therapeutic transgenes that can be delivered with this system and added flexibility to the expression configurations that can be accommodated. All of these functional characteristics of SV40-derived vectors have their bases in the biology of SV40 and similar viruses, and have important implications for the potential utility of rSV40 vectors in gene therapeutics. Like all viral gene delivery systems, these vectors have their idiosyncrasies and limitations. They also allow gene delivery that bypasses many of the difficulties that have plagued the field from its inception.

Animals↗
PubMed2002

Localization of lens intrinsic membrane protein MP19 and mutant protein MP19(To3) using fluorescent expression vectors.

PURPOSE: [corrected] MP19 is the second most abundant major intrinsic protein of the lens fiber cell membrane. A specific heritable mutation at amino acid 15 in the MP19 protein, termed MP19To3, results in total cataract and microphthalmia in the mouse. The goals of this study were to determine the specific localization of MP19 in the cell membrane and to determine whether the mutant MP19To3 protein migrates to the cell membrane in a similar fashion to normal MP19. METHODS: MP19 and MP19To3 cDNAs were cloned into two different sets of expression vectors. The first set was composed of two vectors, pEGFP-N1 and pDsRed2-N1. The first vector expressed green fluorescent protein and the second expressed a red fluorescent protein when transfected into mammalian cells. The two lens membrane protein cDNAs were separately cloned into the vectors so that the cDNA was at the 5'-end of the fluorescent protein coding DNA. These vectors expressed each of the lens proteins fused to the fluorescent protein upon transfection into mammalian cell cultures. The second vector set was a single vector, pcDNA4/TO which must be induced in the transfected cells by tetracycline in order to express the cloned cDNAs. Each of the membrane cDNAs coupled to the fluorescent protein coding region was cut out of the first vector set and cloned into pcDNA4/TO and stable clones were isolated. Each of the prepared plasmids was transfected into human and chick embryo lens epithelial cells and human T-RexTM-293 cells. The fluorescent cells were viewed using confocal and episcopic-fluorescence microscopy. RESULTS: Each of the transfected plasmids expressed fluorescent protein in all three cell lines. MP19 was observed to transport to the cell membrane. When compared to the distribution of another, separate fusion protein consisting of a signal peptide that targets to cell membranes fused to EGFP, MP19 did not distribute uniformly on the membrane, but appeared to localize into "spots" or pools of fluorescent material around the cell membrane. In contrast, MP19To3 protein appeared to not distribute to the cell membrane; it instead appeared to collect in a particular subcellular compartment within the cell. CONCLUSIONS: The distribution of MP19 and MP19To3 in the cell appeared to be quite distinct. MP19 was observed to distribute to the cell membrane while MP19To3 did not. The fact that the MP19To3 did not traffic to the membrane, instead appearing to be trapped within a subcellular compartment within the cell sheds further light on the cause of the cataract and microphthalmia observed in the MP19To3 mutation, and further sheds information on the pathway of MP19 transport to the cell membrane.

Animals↗
PubMed2001

[High efficiency gene transfer into breast cancer cells by pseudotyped retroviral vector].

OBJECTIVE: To investigate the efficiency of gene transfer mediated by pseudotyped retroviral vector in breast cancer, and to provide an useful vector for breast cancer gene therapy. METHODS: We used a MuLV vector pseudotyped with the vesicular stomatitis virus glycoprotein(VSV-G) envelope to transduce breast cancer cells MDA-MB-435, To compare the transfer efficiency with MuLV. RESULTS: The transfer efficiency is (92 +/- 12)% by using MuLV/VSV-G vector, (24 +/- 5)% by MuLV vector. The transfer efficiency of MuLV/VSV-G is 3.8 times that of MuLV vector. CONCLUSION: Our results suggest that pseudotyped retroviral vector MuLV/VSV-G is a kind of high efficiency gene transfer vector in breast cancer cells.

Breast Neoplasms↗
PubMed2003

Recombinant adeno-associated virus: formulation challenges and strategies for a gene therapy vector.

Recombinant adeno-associated virus (AAV)-based vectors capable of expressing therapeutic gene products in vivo have shown significant promise for human gene therapy. One challenge facing the field is the development of vector formulations to achieve optimal vector safety, stability and efficacy. Formulation challenges for AAV vectors can be divided into those relating to maintaining vector activity during purification and storage, and those relating to efficient target tissue transduction in vivo. AAV vectors are potentially susceptible to loss of activity through aggregation, proteolysis and oxidation, as well as through non-specific binding to product contact materials used for vector purification and storage. These deleterious changes need to be thoroughly characterized, and the conditions and excipients to prevent them need to be identified. For in vivo administration, major vector formulation challenges include optimization of efficiency and specificity of target tissue transduction, and the ability to overcome host immune responses.

Adenoviridae↗
PubMed2002

[Research on the preparation of bacterial artificial chromosome (BAC) vector DNA].

Bacterial artificial chromosome (BAC) library plays a pivotal role in genomics studies. A crucial step in BAC library construction is preparation of BAC vector DNA. Preparation of highly purified vector DNA is affected by a series of factors including digestion of restriction enzyme and dephosphorylation of linearized vector DNA. In our study, the BAC vector pECBAC1 was digested by the restriction enzyme of BamHI and dephosphorylated by HK phosphatase respectively. In order to improve the ligation capability of vector DNA, gel purification of linearized vector DNA was also conducted in our study. At the same time, we did a series of experiments to get high quality of vector DNA for construction of BAC library. They included the optimal concentration of restriction enzyme, optimal digestion time, the type of phosphatase and gel purification of linearized vector DNA.

Bacteriophage T4↗
PubMed2003

[Development of gene therapy for hematopoietic stem cell using viral vectors].

Hematopoietic stem cells (HSC) are attractive targets for gene therapy of inherited and acquired disorders in hematopoietic system in that they possess the properties of self-renewal, proliferation, and multi-lineage differentiation. For successful gene therapy, the viral vector-mediated gene addition strategy has two essential prerequisites: 1) the efficient transfer of therapeutic gene into HSC; 2) the long-term and stable expression of the transgene at therapeutic levels. The oncoretrovirus-derived vectors are best understood and most widely investigated. Recent successful cases of gene therapy for severe combined immunodeficiency due to adenosine deaminase or gamma c chain deficiencies have provided strong evidences that retrovirus-mediated gene transfer into HSC will work in clinical treatment. While these results are encouraging, some obstacles remain to be circumvented including low efficiency of gene transfer and gene silencing in retroviral vector system. The therapeutic gene can be efficiently introduced into HSC by HIV-1-based lentiviral vector due to its capability to infect the quiescent cells. A variety of preclinical studies are now conducted and a number of valuable results highlight the efficacy of lentiviral-mediated gene transfer into HSC. However, the potential value of lentiviral vectors in human gene therapy remains to be demonstrated. Adeno-associated virus vector is an alternative to retroviral and lentiviral vectors. This review summarizes the characteristics of integrating vectors, the improved HSC transduction protocols, and the optimized gene expression strategies and outlines the important advances of preclinical and clinical trials in hematopoietic stem cell gene therapy.

Adenoviridae↗
PubMed2000

[Construction of BPI23-Fc gamma 1 recombinant protein prokaryotic expression vector and the expression and biological assessment of BPI23-Fc gamma 1 recombinant protein].

OBJECTIVE: To construct pBV-BPI600-Fc gamma 1(700) recombinant expression vector, to transform into Escherichia coli DH5 alpha, and to induce the expression of BPI23-Fc gamma 1 anti-bacterial recombinant protein. METHODS: Genes which encode BPI23 and Fc gamma 1 were amplified by RT-PCR from mRNA that was extracted from HL-60 cell and normal human leukocytes; Recombinant cloning vector and recombinant expression vector were constructed. pBV-BPI600-Fc gamma 1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5 alpha and BPI23-Fc gamma 1 recombinant protein was expressed by temperature induced method. RESULTS: (1) Expected amplified products BPI600 bp and Fc gamma 1(700) bp were obtained by RT-PCR. (2) pUC18-BPI180, pUC18-BPI420, and pUC18-Fc gamma 1(700) recombinant cloning vector were successfully constructed, and sequences were identical with the reported ones. (3) pBV-BPI600-Fc gamma 1(700) recombinant expression vector was successfully constructed, and results of the enzyme digestion analysis were identical with expected ones. (4) pBV-BPI600-Fc gamma 1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5 alpha and BPI23-Fc gamma 1 recombinant protein was expressed by temperature-induced method, and its expression level was accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fc gamma 1 recombinant protein had anti-bacterial activity and biological functions of complement fixation, opsonization. CONCLUSION: pBV-BPI600-Fc gamma 1(700) recombinant expression vector was successfully constructed, and BPI23-Fc gamma 1 recombinant protein with BPI and IgGFc double biological activity was expressed in Escherichia coli.

Antimicrobial Cationic Peptides↗
PubMed2003

[Construction and identification of a tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor].

OBJECTIVE: To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor. METHODS: The fragment of the enhanced green fluorescent protein (EGFP) gene was PCR amplified from the pEGFP-N1 plasmid and cloned into the multiple cloning site of pLNCX vector, and the recombinant was named as pLNCX-EGFP. The fragment of human telomerase reserve transcriptase gene promoter was amplified from the human genome by using the human telomerase reserve transcriptase gene specific primers, and cloned into the pLNCX-EGFP vector, where the cytomegalovirus promoter was previously removed using restriction enzymes, in sense orientation relative to the green fluorescent protein coding sequence. Then the expression vector pLNT-EGFP under the control of the human telomerase reserve transcriptase gene promoter, containing green fluorescent protein reporter gene, was successfully constructed. To detect the transcriptional activity of the human telomerase reserve transcriptase gene promoter, transient transfection of this specific expression vector into HLF cell lines with high telomerase activity and WI38 cell lines without telomerase activity was performed. RESULTS: The expression vector proven by restriction enzymes digestion and sequencing was in correspondence with the design. The results of transient transfection showed that the pLNT-EGFP vector could highly expressed green fluorescent protein reporter gene in telomerase-positive cells, but not in telomerase-negative cells. CONCLUSION: A tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor has been successfully constructed.

Cytomegalovirus↗
PubMed2003

[Construction of eukaryotic expression vector of hMTH1 gene antisense RNA].

OBJECTIVE: To construct pEGFP-C1-T vector, an eukaryotic expression plasmid of hMTH1 gene antisense RNA. METHODS: The conservative region of hMTH1 gene was amplified by RT-PCR after total RNA being extracted from human embryo lung fibroblast (HLF) and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the conservative region of hMTH1 gene was inserted into pEGFP-C1 vector reversedly and pEGFP-C1-T vector was constructed. The efficiency of antisense inhibition was verified by Western blotting after cell transfection. RESULTS: 423 bp fragment including conservative region of hMTH1 gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGFP-C1-T vector was successfully constructed by means of recombinant DNA technology. Additionally pEGFP-C1-T vector could efficiently decrease hMTH1 protein level by 46%. CONCLUSION: The efficient expression vector of hMTH1 gene antisense RNA, pEGFP-C1-T has been constructed successfully.

DNA Repair Enzymes↗
PubMed2003

[Construction and biological activities of CTLA4Ig adenovirus vectors].

AIM: To construct CTLA4Ig adenovirus vectors (AdCTLA4Ig) by homologous recombination and study their activity, and to employ the vectors to induce cardiac transplantation tolerance by gene therapy. METHODS: CTLA4Ig gene was cloned to pCA13 adenovirus shuttle plasmid by recombination strategy. Construction of CTLA4Ig adenovirus vectors was performed by homologous recombination of pCA13 plasmid containing CTLA4Ig gene with adenovirus helper plasmid, followed by packaged with 293 cells. Expression and secretion of CTLA4Ig was confirmed by RT-PCR, SDS-PAGE and Western blot. The inhibitory effect of supernate of 293 cells infected with AdCTLA4Ig on MLR in-vitro was observed. A biological activity of CTLA4Ig adenovirus vectors was determined by AdCTLA4Ig gene therapy in rats in-vivo. RESULTS: The Construction of CTLA4Ig adenovirus vector was successful. It was confirmed that the supernatant of 293 cells infected with AdCTLA4Ig could inhibit MLR in-vitro. It was also showed that CTLA4Ig adenovirus vectors could induce transplantation tolerance and prolong allograft survival when they were administrated in rats in-vivo. CONCLUSION: CTLA4Ig adenovirus vectors successfully constructed can infect 293 package cells and secrete CTLA4Ig. The CTLA4Ig protein can inhibit T cell activation. The CTLA4Ig adenovirus vectors can be employed to gene therapy in-vivo, and induce transplantation tolerance.

Abatacept↗
PubMed2004

Construction and identification of eukaryotic expression vector of human full-length PLCgamma1 gene.

OBJECTIVE: To construct the eukaryotic expression vector of human full-length PLCgamma1 gene for further study of the role of PLCgamma1 in cancer invasion. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) technique was used to amplify human full-length PLCgamma1 gene from MG63 cells with a pair of specific primers containing the restriction sites for HindIII and NotI. After purification, the product of RT-PCR was digested with HindIII and NotI before insertion into the corresponding sites of eukaryotic expression vector pLNCX2, yielding the recombinant plasmid pLNCX2/PLCgamma1. PCR, restriction endonuclease analysis and DNA sequencing were performed to identify the recombinant eukaryotic expression vector pLNCX2/PLCgamma1. RT-PCR and Western blotting were used to detect the expression of the PLCgamma1 gene in LoVo cells after transient transfection via Lipofectamine TM 2000. RESULTS: A 3 878-bp full-length PLCgamma1 gene fragment was successfully amplified by RT-PCR and inserted into eukaryotic expression vector pLNCX2. After digestion by HindIII and NotI, the recombinant eukaryotic expression vector pLNCX2/PLCgamma1 yielded a 3 878-bp fragment (PLCgamma1 gene) and a 6 100 bp fragment (vector). HindIII-BglII digestion was also done to verify the correctness of the recombinant plasmid, resulting in the identification of the fragments as expected. Sequencing analysis further confirmed the results. In addition, RT-PCR and Western blotting verified that the PLCgamma1 could overexpress in LoVo cells after transfection with recombinant eukaryotic expression vector pLNCX2/PLCgamma1. CONCLUSION: The recombinant eukaryotic expression vector pLNCX2/PLCgamma1 has been constructed successfully.

Base Sequence↗
PubMed2005

Replication-defective genomic HSV gene therapy vectors: design, production and CNS applications.

Herpes simplex virus (HSV) is a neurotropic double-stranded DNA virus. In an unmodified form it is a human pathogen, causing recurrent cold sores, keratitis and, rarely, severe encephalitis. Elimination of pathogenic functions results in the generation of a valuable gene transfer vector for neurological applications. Replication-defective genomic HSV-based vectors are highly infectious, and efficiently transduce and express transgenes in a broad range of both dividing and non-dividing cells. Clinically relevant yields of clinical grade vector can be produced by growth in cell lines that complement the viral functions that are deleted in the vectors to eliminate pathogenicity. The viral genome is over 150 kb in length and many of the viral genes may be deleted without compromising viral growth in vitro, and therefore large or multiple transgenes can be accommodated within the vectors. The wild-type virus adopts a lifelong latent state in neurons of sensory ganglia. This property can be exploited in the generation of vectors to allow long-term transgene expression in neurons. In this review, we summarize recent progress in the areas of vector development and vector production, and in developing gene transfer therapeutics to treat malignant glioma.

Animals↗
PubMed2005

Barriers to gene delivery using synthetic vectors.

Progress has been made in the development of different types of nucleic acids such as DNA and siRNA with the potential to form the basis of new treatments for genetic and acquired disorders. The lack of suitable vectors for the delivery of nucleic acids, however, represents a major hurdle to their continued development and therapeutic application. Synthetic vectors based on polycations are promising vectors for gene delivery as they are relatively safe and can be modified by the incorporation of ligands for targeting to specific cell types. However, the levels of gene expression mediated by synthetic vectors are low compared to viral vectors. The aim of this chapter is to give an overview of the main barriers that have been identified as limiting gene transfer using polycation-based synthetic vectors. The chapter is divided into two sections to focus on both extracellular and intracellular barriers. We describe novel strategies that are being used to develop increasingly sophisticated vectors in an attempt to overcome these barriers. For instance, we describe approaches to prolong the plasma circulation of polyplexes by the incorporation onto their surface of hydrophilic polymers such as polyethylene glycol (PEG) and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). In addition, strategies to improve transfer of nucleic acids from the outside of the cell to the nucleus are described to overcome barriers such as escape from endocytic vesicles and translocation across the nuclear membrane. Furthermore, we highlight new types of vectors based on reducible polycations that are triggered by the intracellular environment to facilitate efficient cytoplasmic release of nucleic acids.

Active Transport, Cell Nucleus↗
PubMed2006

[Construction of TRAIL gene eukaryotic expression vector modulated by hTERT gene core promoter and its effect on apoptosis of ovarian cancer cells].

AIM: To construct the TNF-related apoptosis inducing ligand(TRAIL) gene eukaryotic expression vector modulated by human telomerase reserse transcriptase (hTERT) gene core promoter and to study its effect on apoptosis of ovarian cancer cells. METHODS: Genomic RNA was extracted from human placenta tissues and the fragment of TRAIL was obtained by RT-PCR. The amplified gene fragment was subsequently cloned into hTERTpromoter-pIRES2-EGFP vector and CMV promoter-pIRES2-EGFP vector after sequencing. The hTERTpromoter-pIRES2-EGFP-TRAIL and CMV promoter-pIRES2-EGFP-TRAIL eukaryotic expression vectors were constructed respectively. The recombinant plasmids were transfected into ovarian carcinoma cell line, SKOV3, and the levels of mRNA were determined by RT-PCR. The cell cycle and apoptosis rate of SKOV3 cells were determined by FCM. RESULTS: The constructed two recombinant vectors were verified by restriction enzyme digestion analysis and DNA sequencing. After being transfected with two recombinant vectors, the growth of SKOV3 cells was strongly inhibited and apoptotic features appeared. CONCLUSION: The recombinant eukaryotic expression vector has been constructed successfully. TRAIL gene driven by hTERTpromoter can be obviously expressed in ovarian carcinoma SKOV3 cells, suggesting that the specific expression vector modulated by hTERT core gene promoter may be a novel and promising approach to the tumor treatment.

Apoptosis↗