Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Vector”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,297 records · Page 72Linked to original sources
PubMed1997

Vaccine Trials for the Clinician: Prospects for Viral and Non-Viral Vectors.

Recent progress in tumor genetics, tumor biology, and tumor immunology has renewed interest in the development of tumor vaccines. Unlike the previous generation of vaccines that consisted of the patient's own tumor cells in some form, the new vaccines contain defined peptides or genes with a known function. In order to induce a potent and long-lasting cell-mediated antitumor response, viral as well as nonviral vectors have been explored as vehicles for gene delivery. Both types of vectors have shown encouraging results in animal models. However, because of the many possible vectors that have been designed, it may be too early to say which type of vector is most efficient in the human. Clearly viral vectors have a higher transduction efficiency than most nonviral delivery systems. A drawback is that viral vectors may be toxic or immunogenic. Current research focuses on enhancing the targeting and specificity of both viral and nonviral vectors and control of transgene expression levels. Clinical studies using a variety of both viral and nonviral vectors have begun, and the results are forthcoming.

Journal Article↗
PubMed2000

Dihydrotestosterone as a selective cellular/nuclear localization vector for anti-gene peptide nucleic acid in prostatic carcinoma cells.

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed to enter the cell, bind to the complementary polynucleotide sequence and inhibit DNA transcription and mRNA translation. Although PNAs have a very limited ability in penetrating nuclei of living cells, there are indications that covalent linkage of the PNA to appropriate vectors, e.g., a nuclear localization signal, permits access to the genome. Here we test the ability of dihydrotestosterone (T) covalently linked to PNA to act as a vector for targeting c-myc DNA to prostatic cancer cell nuclei. LNCaP cells, which express the androgen receptor gene, and DU145 cells, in which the androgen receptor gene is silent, offer a model to test this biologically active hormone as a cell-specific vector. T vector was covalently linked to the NH2-terminal position of a PNA complementary to a unique sequence of c-myc oncogene (PNAmyc-T). To localize PNAmyc-T and vector-free PNA within the cells, a rhodamine (R) group was attached at the COOH-terminal position (PNAmyc-R, PNAmyc-TR); cellular uptake was monitored by confocal fluorescence microscopy. PNAmyc-R was detected only in the cytoplasm of both prostatic cell lines, whereas PNAmyc-TR was localized in nuclei as well as in cytoplasm of LNCaP cells. In contrast, PNAmyc-TR uptake in DU145 cells was minimal and exclusively cytoplasmic. In LNCaP cells, MYC protein remained unchanged by exposure to vector-free PNAmyc, whereas a significant and persistent decrease was induced by PNAmyc-T. In DU145 cells, MYC expression was unaltered by PNAmyc with or without the T vector. Our data show that the T vector facilitates cell-selective nuclear localization of PNA and its consequent inhibition of c-myc expression. These findings suggest a strategy for targeting of cell-specific anti-gene therapy in prostatic carcinoma.

Antisense Elements (Genetics)↗
PubMed1998

Gene therapy for primary and metastatic pancreatic cancer with intraperitoneal retroviral vector bearing the wild-type p53 gene.

BACKGROUND: Metastatic pancreatic cancer is uniformly fatal because no effective chemotherapy is available. Mutations in the p53 tumor suppressor gene are found in up to 70% of pancreatic adenocarcinomas. We examined the efficacy of a retroviral vector containing the wild-type p53 gene on metastatic pancreatic cancer in a nude mouse model. METHODS: Bxpc3 human pancreatic cancer cells were transduced with either a retroviral p53 vector or an LXSN empty vector. Cells were examined for incorporation of tritiated thymidine to determine the effect of p53 retroviral transduction on DNA synthesis, and a TACS2 assay for apoptosis was performed. The functional activity of p53 in transduced cells was assessed by Western blot analysis with an antibody to WAF1/p21. In vivo effects of intraperitoneal injections of the p53 vector were examined in a nude mouse model of peritoneal carcinomatosis. RESULTS: Cells treated with the p53 vector exhibited a 59% to 85.5% reduction in cell number compared with the control cells (P < .05). p53-treated cells demonstrated decreased incorporation of tritiated thymidine (12.7% +/- 0.7% vs 17.5% +/- 1.4%; P = .002), increased staining for apoptosis, and increased expression of the WAF1/p21 protein. Treatment of nude mice with the retroviral p53 vector resulted in a significant inhibition of growth of the primary pancreatic tumor, as well as the peritoneal tumor deposits, compared with the LXSN control vector. CONCLUSIONS: Intraperitoneal delivery of a retroviral p53 vector may provide a novel treatment approach for peritoneal carcinomatosis from pancreatic cancer.

Animals↗
PubMed2000

Modular retro-vectors for transgenic and therapeutic use.

Retroviruses have been used for many years as vectors for human gene therapy as well as for making transgenic animals. However, the efficient insertion of genes by retroviruses is often complicated by transcriptional inactivation of the retroviral long terminal repeats (LTRs) and by the production of replication-competent retroviruses (RCR). Solutions to these and other difficulties are being found in modular vectors, in which the desirable features of different vector systems are combined. Examples of synergistic vectors include virosomes (liposome/virus delivery), adeno-retro vectors, and MLV/VL30 chimeras. As gene delivery systems become increasingly complex, methodology is also needed for precise assembly of modular vectors. Gene self-assembly (GENSA) technology permits seamless vector construction and simultaneous, multifragment assembly.

Animals↗
PubMed2000

A family of multifunctional thiamine-repressible expression vectors for fission yeast.

A series of thiamine-repressible shuttle vectors has been constructed to allow a more efficient DNA manipulation in Schizosaccharomyces pombe. These high-copy-number vectors with regulatable expression (pJR) are based on the backbone of the pREP-3X, pREP-41X and pREP-81X plasmids. The pJR vectors are all uniform in structure, containing: (a) sequences for replication (ori) and selection (AmpR) in Escherichia coli; (b) the f1 ori sequence of the phage f1 for packaging of ssDNA, making them suitable for site-directed mutagenesis; and (c) the ars1 sequence for replication in S. pombe. The pJR vectors differ among them in: (a) the selectable marker (Saccharomyces cerevisiae LEU 2 gene, which complements S. pombe leu1- gene and S. pombe ura4+ and his3+ genes); (b) the thiamine-repressible nmt1 promoter (3X, 41X and 81X with extremely high, moderate or low transcription efficiency, respectively); and (c) the multiple cloning site (two multiple cloning sites, with 12 restriction sites each). The expression level of the pJR vectors has been analysed using the beta-galactosidase gene as reporter. Three levels of expression for each nmt1 promoter version, with any selectable marker and for either repressed or induced conditions, have been found. The expression is dependent on the distance to the initiation codon, varying from 0.001 to 15 times the activity characterized for the pREP plasmids. Also, the gene expression has been found to be extremely sensitive to the nucleotide sequence prior to the initiation codon, being up to 50-fold higher with an A/T sequence than with a G/C sequence. Finally, the beta-galactosidase mRNA levels were found to be similar in each nmt1 series, suggesting a translational effect on gene expression. As a result, any of these 18 new vectors allow performing gene expression in fission yeast, as well as a more versatile cloning, sequencing and mutagenesis, directly in the plasmid without the need for subcloning into intermediary vectors.

Base Sequence↗
PubMed2001

Flow cytometric assessment of transduction efficiency and cytotoxicity of herpes simplex virus type 1-based amplicon vectors.

BACKGROUND: In this study, we compared herpes simplex virus type 1 (HSV-1) amplicon vector stocks prepared by transient cotransfection with two different BAC-cloned packaging-defective HSV-1 helper genomes, fHSVDeltapacDelta27 and fHSVDeltapac, with respect to transduction efficiency and cytotoxicity. Both fHSVDeltapacDelta27 and fHSVDeltapac are packaging defective because the pac signals have been deleted; fHSVDeltapacDelta27 contains an additional deletion in the HSV-1 ICP27 gene, which increases the safety of the system. METHODS: HSV-1 amplicon pHSVGFP under the control of the HSV-1 immediate-early (IE) 4/5 promotor was packaged into virus particles by transient cotransfection with either fHSVDeltapacDelta27 or fHSVDeltapac DNA. Cultures were infected with the two different vector stocks and examined under the fluorescence microscope and analyzed by flow cytometry over a 5-day period to assess transduction efficiency and cytotoxicity. RESULTS: Both vector stocks, pHSVGFP[fHSVDeltapacDelta27] and pHSVGFP[fHSVDeltapac], efficiently transduced the target cells. Interestingly, the highest mean fluorescence intensities were measured at 1 day after infection, whereas the number of GFP-fluorescent cells reached a peak at day 3 after infection. At day 3 after infection, a slight increase in the number of dead cells was observed in those cultures transduced with high doses of vector stock. Between days 3 and 4 after infection, the number of dead cells increased dramatically in all the cultures, transduced and nontransduced. Only the cultures infected with a high dose of pHSVGFP[fHSVDeltapac] displayed a significant further increase in the number of dead cells between days 4 and 5 postinfection. CONCLUSIONS: Flow cytometry allowed comparison of transduction efficiency and cytotoxicity mediated by the two different amplicon vector stocks. Cultures infected with pHSVGFP[fHSVDeltapacDelta27] were more viable than those infected with pHSVGFP[fHSVDeltapac](P < 0.05). The practical implications of this study are at the level of vector design. Flow cytometry has proven a fast and reliable approach to assess the quality of potential gene transfer vectors prior to their use in (pre) clinical trials.

Animals↗
PubMed2003

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.

Cells, Cultured↗
PubMed1987

Expression of retroviral vectors in transgenic mice obtained by embryo infection.

Pre-implantation embryos were infected with the retroviral vector MMCV-neo, which carries the neomycin resistance (neo) gene and the v-myc gene. Three transgenic substrains (M-TKneo 1-3) were derived which stably transmit a single intact copy of the vector. In all of the substrains, expression of the neo gene from the internal thymidine kinase (TK) promoter was detected, with two of the substrains expressing the gene in all tissues analysed. In the third substrain, the vector had integrated on the X chromosome and neo expression varied between different tissues. A second series of transgenic mice were obtained with the retroviral vector SAX, in which the human adenosine deaminase cDNA (ADA) is under the control of an internal SV40 promoter. Four substrains (M-SAX 1-4) were analysed; however, no expression of the ADA cDNA was detected. In all mice, no expression was found of the genes under the control of the viral 5' long terminal repeats (LTRs). In the M-TKneo substrains the vector was hypomethylated irrespective of its expression whereas in the M-SAX mice the vector was hypermethylated. These results demonstrate for the first time that the TK promoter can apparently express a gene in all tissues of adult mice and that retroviral vectors with internal promoters may provide an alternative to DNA injection for the efficient expression of genes in transgenic mice.

Animals↗
PubMed2004

Biocompatibility of adenoviral vectors in poly(vinyl chloride) tubing catheters with presence or absence of plasticizer di-2-ethylhexyl phthalate.

Adenoviral (Ad) vectors feature attractive characteristics for gene therapy of a wide variety of diseases. In many cases, the Ad vector must be administered using catheters and other plastic medical devices. Although poly(vinyl chloride) is one of the most frequently used catheter materials, it is relatively rigid and requires the addition of a plasticizer such as di-2-ethylhexyl phthalate (DEHP) to increase its flexibility. In this study, we demonstrated that exposure to a DEHP-containing catheter decreased the infectivity of Ad vectors but not the total particle number of the vector. Loss of Ad vector infectivity was directly related to the time of exposure to the DEHP-containing catheter, but it was not due to simple leaching of the chemical from the plastic. The loss of Ad vector infectivity could be prevented by preflushing the tube with albumin. Careful consideration of the compatibility between gene therapy vectors and medical delivery devices will be critical to the success of human gene therapy applications.

Adenoviridae↗
PubMedDate not supplied

EBV replicon vector system enhances transgene expression in vivo: applications to cancer gene therapy.

BACKGROUND: A major limitation of current cancer gene therapies is low and transient expression of the therapeutic gene. For long-term expression of transgenes in vivo, an Epstein-Barr virus (EBV) replicon vector has been developed. The present study examines the effect of the EBV replicon vector system and its application to a suicide gene therapy for melanoma in mice. METHODS: An EBV replicon vector system, pEBc, consisting of EBV nuclear antigen-1 (EBNA-1) and the origin of latent viral DNA replication, oriP, was used to express either the luciferase gene or the herpes simplex virus (HSV) thymidine kinase (TK) gene. The expression vector was introduced in vivo into melanoma tumor masses in mice by means of HVJ-cationic liposomes. The time-course of gene expression and the anticancer effect of the EBV replicon vector were investigated in comparison with pcLuc, which lacks the EBV components. RESULTS: Luciferase expression was sustained in both cultured cells and melanoma masses by pEBc but not by pcLuc. The luciferase expression level in melanoma masses was higher by pEBcLuc than by pcLuc, although Southern blot analysis showed the number of copies of pEBcLuc retained in the melanoma masses to be fewer than that of pcLuc. The effectiveness of EBV replicon vector on suicide gene therapy of melanoma in mice was also demonstrated. CONCLUSION: The EBV replicon vector appears useful for cancer gene therapy. Analysis of the transgene in tumors suggests that the EBV replicon system may be responsible for efficient transcription but not retention of the transgene.

Animals↗
PubMedDate not supplied

Sustained delivery of therapeutic concentrations of human clotting factor IX--a comparison of adenoviral and AAV vectors administered in utero.

BACKGROUND: Prenatal somatic gene therapy has been considered for genetic disorders presenting with morbidity at birth. Haemophilia is associated with an increased risk of catastrophic perinatal bleeding complications such as intracranial haemorrhage, which could be prevented by gene transfer in utero. Prenatal gene therapy may be more promising than postnatal treatment, as the fetus may be more amenable to uptake and integration of therapeutic DNA and the immaturity of its immune system may permit life-long immune tolerance of the transgenic protein, thus avoiding the dominant problem in haemophilia treatment, the formation of inhibitory antibodies. METHODS: Adenovirus serotype 5-derived or AAV serotype 2-derived vectors carrying human clotting factor IX (hfIX) cDNA or a reporter gene were administered intramuscularly, intraperitoneally or intravascularly to late-gestation mouse fetuses. Both vector types were evaluated with respect to the kinetics of hfIX delivery to the systemic circulation and possible immune responses against the vector or the transgene product. RESULTS: Mice treated in utero by intramuscular injection of an adenoviral vector carrying hfIX cDNA exhibited high-level gene expression at birth and therapeutic--albeit continuously decreasing--plasma concentrations of hfIX over the entire 6 months of the study. Adenoviral vector spread to multiple organs was detected by polymerase chain reaction (PCR). Intramuscular, intraperitoneal or intravascular application of AAV vectors carrying hfIX cDNA led to much lower plasma concentrations of hfIX shortly after birth, which appeared to decline during the first month of life but stabilized in some of the mice at detectable levels. No signs of immune responses were found, either against the different viral vectors or against hfIX. CONCLUSION: This study demonstrates for the first time that sustained systemic delivery of a therapeutic protein can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy in utero and provides a basis for considering this concept as a preventive therapeutic strategy for haemophilia and perhaps also for other plasma protein deficiencies.

Adenoviridae↗
PubMedDate not supplied

Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy.

BACKGROUND: A central triple-stranded DNA structure created during HIV-1 reverse transcription, the central flap, acts as a cis-active nuclear import determinant of the HIV-1 DNA genome. Insertion of the sequences responsible for formation of the central DNA flap into an HIV-1-derived vector strongly enhances its transduction efficiency. METHODS: HIV-1 vectors with or without inclusion of the DNA flap and encoding the same melanoma polyepitope were constructed to transduce dendritic cells (DCs) and to evaluate their capacity for induction of melanoma-specific cytotoxic T-lymphocyte (CTL) responses ex vivo and in vivo. RESULTS: HIV-1 vectors including the DNA flap transduced up to 100% of immature mouse and human DCs. Inoculation of HLA-A2.1 transgenic mice with this flap vector elicited vigorous and multi-specific long-term anti-melanoma CTL responses, whereas the parental vector lacking the flap sequence was less efficient. Furthermore, human DCs transduced ex vivo with the recombinant DNA flap vector displayed efficient multi-specific primary human CTL responses against melanoma. CONCLUSION: Lentiviral vectors including the DNA flap should be powerful tools both for active immunization and for the ex vivo priming of CTL for tumor immunotherapy.

Animals↗
PubMedDate not supplied

The tet-off system is more effective than the tet-on system for regulating transgene expression in a single adenovirus vector.

BACKGROUND: Control of transgene expression in mammalian cells is desirable for gene therapy and the study of gene function in basic research. This study evaluates the functionality of single adenovirus (Ad) vectors containing a tetracycline-controllable expression system (tet-off or tet-on system). METHODS: An Ad-mediated binary transgene expression system was generated containing a tet-off or a tet-on system, which introduced the gene of interest with a tetracycline-regulatable promoter and the tetracycline-responsive transcriptional activator gene into the E1 and E3 deletion regions, respectively. The functionality of the Ad-mediated tet-off and tet-on systems was compared under various conditions in vitro and in vivo. RESULTS: The Ad vector containing the tet-off system provided negative control of gene expression ranging from 20- to 500-fold, depending on the cell type and condition. In contrast, the Ad vector containing the tet-on system increased gene expression by only 2- to 28-fold and required about two-log orders higher concentration of an inducer (doxycycline) to switch on the gene expression, compared with the Ad vector containing the tet-off system. CONCLUSION: Ad vectors containing the tet-off system are a better choice for regulated gene expression than Ad vectors containing the tet-on system. Single Ad vectors containing the tetracycline-controllable expression system will greatly facilitate in vitro and in vivo analyses of gene function and may be useful for gene therapy.

Adenoviridae↗
PubMedDate not supplied

Feline immunodeficiency virus vectors. Gene transfer to mouse retina following intravitreal injection.

BACKGROUND: Transduction of the murine retinal pigmented epithelium (RPE) with adenovirus vectors requires technically difficult and invasive subretinal injections. This study tested the hypothesis that recombinant vectors based on feline immunodeficiency virus (FIV) could access the retina following intravitreal injection. METHODS: FIV vectors expressing E. coli beta-galactosidase (FIVbetagal) were injected alone, or in combination with adenovirus vectors expressing eGFP, into the vitreous of normal mice and eyes evaluated for transgene expression. In further studies, the utility of FIV-mediated gene transfer to correct lysosomal storage defects in the anterior and posterior chambers of eyes was tested using recombinant FIV vectors expressing beta-glucuronidase. FIVbetagluc vectors were injected into beta-glucuronidase-deficient mice, an animal model of mucopolysacharridoses type VII. RESULTS: The results of this study show that similar to adenovirus, both corneal endothelium and cells of the iris could be transduced following intravitreal injection of FIVbetagal. However, in contrast to adenovirus, intravitreal injection of FIVbetagal also resulted in transduction of the RPE. Immunohistochemistry following an intravitreal injection of an AdeGFP (adenovirus expressing green fluorescent protein) and FIVbetagal mixture confirmed that both viruses mediated transduction of corneal endothelium and cells of the iris, while only FIVbetagal transduced cells in the retina. Using the beta-glucuronidase-deficient mouse, the therapeutic efficacy of intravitreal injection of FIVbetagluc (FIV expressing beta-glucuronidase) was tested. Intravitreal injection of FIVbetagluc to the eyes of beta-glucuronidase-deficient mice resulted in rapid reduction (within 2 weeks) of the lysosomal storage defect within the RPE, corneal endothelium, and the non-pigmented epithelium of the ciliary process. Transgene expression and correction of the lysosomal storage defect remained for at least 12 weeks, the latest time point tested. CONCLUSION: These studies demonstrate that intravitreal injection of FIV-based vectors can mediate efficient and lasting transduction of cells in the cornea, iris, and retina.

Animals↗
PubMedDate not supplied

Gutted adenoviral vector growth using E1/E2b/E3-deleted helper viruses.

BACKGROUND: Helper-dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high-titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo. METHODS: Replication-defective helper viruses were generated that are deleted for Ad E1, E2b and E3 genes, but which contain loxP sites flanking the packaging signal. Complementing Ad packaging cell lines (C7-cre cells) were also generated by transfecting 293 cells with the Ad E2b genes encoding DNA polymerase and pre-terminal protein, and with a cre-recombinase plasmid. RESULTS: We show that C7-cre cells allow efficient production of gutted Ad using deltaE1 + deltaE2b + deltaE3 helper viruses whose growth can be limited by cre-loxP-mediated excision of the packaging signal. Gutted Ad vectors carrying approximately 28 kb cassettes expressing full-length dystrophin were prepared at high titers, similar to those obtained with E2b+ helpers, with a resulting helper contamination of <1%. CONCLUSIONS: These new packaging cell lines and helper viruses offer several significant advantages for gutted Ad vector production. They allow gutted virus amplification using a reduced number of passages, which should reduce the chances of selecting rearranged products. Furthermore, the residual helper contamination in gutted vector preparations should be less able to elicit immunological reactions upon delivery to tissues, since E2b-deleted vectors display a profound reduction in viral gene expression.

Adenoviridae↗
PubMed2003

Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome.

BACKGROUND: As an alternative method to the conventional therapies for Hunter's syndrome, which is a lethal lysosomal storage disorder, we have developed gene delivery vehicles using a series of retroviral vectors. The objective of this study was to develop a safe and efficient retroviral vector and to optimize conditions for efficient transduction of human bone marrow CD34+ stem cells using our vector. METHODS: We constructed three types of MLV-based retroviral vectors expressing iduronate-2-sulfatase (IDS) which is deficient in patients suffering from Hunter's syndrome: MIN-IDS and MIM-IDS, which express IDS along with bacterial neo and human MDR genes, respectively, and MT-IDS lacking any selectable marker. Respective producer lines were derived from the packaging line, PG13, and compared for viral titer and levels of gene expression. After comparing, the retroviral vector, MT-IDS, was used to transduce human bone marrow CD34+ stem cells on fibronectin under various MOIs. RESULTS: In comparison, MT-IDS not only produced the highest viral titer (close to 10(7) cfu/ml), but also showed the highest level of gene expression in various transduction assays and RNA analysis. When 1.5 x 10(5) human CD34+ bone marrow cells were transduced with MT-IDS under the most optimal MOIs, about 80% of total colony forming units were shown to contain the IDS cDNA. CONCLUSIONS: Minimum-sized retroviral vector that contains no selective marker as well as a viral coding sequences could drive a high level of gene expression, be produced efficiently from the producer line, and enter hematopoietic cells at a high frequency. Our data suggest the great potential for using MT-based vector(s) in a gene therapy trial for Hunter's syndrome utilizing human CD34+ stem cells as target cells.

Bone Marrow Cells↗
PubMed2003

Functional delivery of large genomic DNA to human cells with a peptide-lipid vector.

BACKGROUND: Nonviral gene transfer vectors have the potential to deliver much larger DNA constructs than current viral vectors but suffer from a low transfection efficiency. The LID vector, composed of Lipofectin (L), an integrin-targeting peptide (I) and DNA (D), is a highly efficient synthetic vector, both in vitro and in vivo, which may allow the transfer of genomic loci for gene therapy. METHODS: Transfection efficiencies were quantitated using the green fluorescent protein (GFP) reporter. Expression of a large genomic locus (NBS1 [Nijmegen breakage syndrome], encoding nibrin) was assessed by immunofluorescence. RESULTS: We report a systematic study of the parameters influencing delivery of BAC-based plasmids ranging in size from 12 to 242 kb using the LID vector. We showed 60% of cells were transfected with the smaller plasmids while plasmids up to 242 kb were consistently delivered to over 10% of cells. The number of transfected cells was related to number of plasmids in the transfection complex independent of plasmid size. Atomic force microscopy showed that LID particle size increased with plasmid size consistent with one plasmid molecule per particle. When LID vectors were used to deliver the NBS1 gene as a 143 kb construct to primary NBS cells, at least 57% of cells expressing GFP also expressed functional nibrin. CONCLUSIONS: We show that LID vectors represent a promising tool for the transfer of complete genomic loci.

Cell Cycle Proteins↗
PubMed2003

Long-term transgene expression in cardiac and skeletal muscle following fetal administration of adenoviral or adeno-associated viral vectors in mice.

BACKGROUND: In utero gene transfer may provide advantages for the correction of congenital genetic disorders. In the present study we compare the ability of adenovirus (AdCMVLacZ), and two serotypes of adeno-associated virus (AAVCMVLacZ serotypes 2 and 2/5), to target cardiac and skeletal muscle after prenatal systemic or intramuscular injection in mice and assess the immune response to the vectors. METHODS: Day 14 gestation fetal mice underwent direct intraperitoneal or intramuscular injection of AdCMVLacZ, and AAVCMVLacZ serotypes 2 and 2/5 vectors. Tissues were processed for beta-galactosidase expression in frozen or high-resolution thin plastic sections at early and late time points. Neutralizing antibodies to Ad and AAV were analyzed in separate fetal experimental and neonatal or adult control groups after administration and re-administration of the vectors. RESULTS: A single injection of each vector in utero resulted in sustained expression of beta-galactosidase transgene in skeletal and cardiac muscle. Transgene expression was detected for the length of the study, i.e. 86, 58, and 31 weeks after birth for AdCMVLacZ, and AAVCMVLacZ serotypes 2 and 2/5, respectively. High-level expression in the myocardium was observed independent of the vector or route of administration. Neutralizing antibody responses to AAV and Ad antigens were reduced and long-term expression in muscle was not ablated on postnatal re-administration of vector. CONCLUSIONS: Sustained, high-level cardiac and skeletal muscle transgene expression can be obtained after prenatal gene transfer with each of these vectors. The potential for immune response to viral antigens is altered, but not entirely ablated after in utero exposure.

Adenoviridae↗