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Biomedical subjects

M Cotten

Publications and source records attributed to M Cotten.

At least 55 records · Page 3Linked to original sources
PubMed1995

Receptor-mediated gene transfer into human T lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex.

Retrovirus-mediated gene transfer is currently the method of choice for the transfection of human T lymphocytes for applications in gene therapy. Use of retroviral vectors, however, is hampered by limits on the size of the genetic material to be transferred, the requirement of dividing target cells, and by potential safety questions. Synthetic peptide-enhanced or adenovirus-enhanced receptor-mediated transferrinfection of DNA (SPET and AVET, respectively) is a powerful method for the introduction of genetic material into mammalian cells. Although transferrin has proven to be a useful ligand for gene transfer in many cell types, gene expression in T cells with transferrin/DNA complexes is usually not satisfactory. To improve gene transfer to T cells, antibodies directed against the CD3-T cell receptor complex were tested for their ability to function as ligands for DNA delivery. In T cell lines, up to 50% of the cells expressed a beta-galactosidase reporter gene using anti-CD3 gene transfer complexes. Applying optimized conditions, prestimulated primary peripheral blood lymphocytes were also transfected successfully, although at a lesser efficiency (5%).

Antibodies↗
PubMed1995

Generation of high-titer retroviral vectors following receptor-mediated, adenovirus-augmented transfection.

A new procedure is described for the generation of high-titer, helper-free retrovirus vectors employing receptor-mediated, adenovirus-augmented transfection into a standard packaging cell line. Viral titers are increased 30-fold to 100-fold in transiently (> 10(5) infectious units per mL) and stable (> 10(7) infectious units per mL) transfected cells as compared with either CaPO4-mediated transfection or retroviral infection of a packaging cell line. Further, expression of the transduced genes was drastically increased in the transfected cells, but, as expected, there was no difference in transduction efficiency and gene expression in the infected target cells. The increases in viral titers were most likely due to the high number of stable, integrated copies of the vector plasmid DNA in the resulting packaging lines following G418 selection. In addition, experiments generating recombinant retroviruses from non-packaging cell lines are presented. The results suggest that this procedure may be of use to generate high-titer retrovirus vectors in packaging cell lines as well as in primary cells, thus providing a technical basis for in vivo gene transfer upon transplantation of these cells into various organs.

3T3 Cells↗
PubMed1994

Psoralen treatment of adenovirus particles eliminates virus replication and transcription while maintaining the endosomolytic activity of the virus capsid.

Adenovirus entry into its host cell transiently permeabilizes the cell allowing the coentry of reagents such as DNA. We compare here adenovirus inactivation with beta-propiolactone and several psoralen derivatives, seeking reagents that disrupt the viral genome without impairing the viral entry functions. No virus replication can be detected after 8-methoxypsoralen (8-MOP) modification. Viral transcription is not detectable by Northern analysis, and reverse transcriptase/PCR analysis demonstrates at least a 1000-fold decrease in viral transcription after 8-MOP treatment. Using [3H]8-MOP, the psoralen is found to enter the virus capsid and react throughout the viral genome, with approximately one psoralen modification per 100 bp of viral DNA. This inactivated adenovirus allows us to deliver DNA to target cells without interference from adenovirus gene expression or replication. Furthermore, we can now study the host cell response to adenovirus entry without the complications of adenovirus gene expression.

Adenoviridae↗
PubMed1994

High-level expression of various apolipoprotein(a) isoforms by "transferrinfection": the role of kringle IV sequences in the extracellular association with low-density lipoprotein.

Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor- and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7, 9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or CHO cells. All constructions contained kringle IV-36, which includes the only unpaired cysteine residue (Cys-4057) in apo(a). r-Apo(a) was synthesized as a precursor and secreted as mature apolipoprotein into the medium. When medium containing r-apo(a) with 9, 15, or 18 kringle IV repeats was mixed with normal human plasma LDL, stable complexes formed that had a bouyant density typical of Lp(a). Association was substantially decreased if Cys-4057 on r-apo(a) was replaced by Arg by site-directed mutagenesis or if Cys-4057 was chemically modified. Lack of association was also observed with r-apo(a) containing only 3, 5, or 7 kringle IV repeats without "unique kringle IV sequences", although Cys-4057 was present in all of these constructions. Synthesis and secretion of r-apo(a) was not dependent on its sialic acid content. r-Apo(a) was expressed even more efficiently in sialylation-defective CHO cells than in wild-type CHO cells. In transfected CHO cells defective in the addition of N-acetylglucosamine, apo(a) secretion was found to be decreased by 50%. Extracellular association with LDL was not affected by the carbohydrate moiety of r-apo(a), indicating a protein-protein interaction between r-apo(a) and apoB. These results show that, besides kringle IV-36, other kringle IV sequences are necessary for the extracellular association of r-apo(a) with LDL. Changes in the carbohydrate moiety of apo(a), however, do not affect complex formation.

Animals↗
PubMed1994

In vivo production of human factor VII in mice after intrasplenic implantation of primary fibroblasts transfected by receptor-mediated, adenovirus-augmented gene delivery.

Hemophilia A is caused by defects in the factor VIII gene. This results in life-threatening hemorrhages and severe arthropathies. Today, hemophiliacs are treated with human blood-derived factor VIII. In the future, it may be possible to use gene therapy to avoid long-term complications of conventional therapy and to improve the quality of life. However, initial gene therapy models using retroviral vectors and nonviral gene transfer techniques to introduce factor VIII gene constructs have been hampered by low expression levels of factor VIII. We show here that high expression levels of the B-domain-deleted human factor VIII in primary mouse fibroblasts and myoblasts are obtained by using receptor-mediated, adenovirus-augmented gene delivery (transferrinfection). We demonstrate that, presumably owing to the high molecular weight of factor VIII or its metabolic instability, secretion into the blood and attainment of therapeutic in vivo levels of factor VIII is achieved only if transfected autologous primary fibroblasts or myoblasts are delivered to the liver or spleen, but not if myoblasts are implanted into muscle, a strategy known to be successful for factor IX delivery.

Adenoviridae↗
PubMed1994

Efficient foreign gene expression in Epstein-Barr virus-transformed human B-cells.

Epstein-Barr virus (EBV) is a herpesvirus that transforms B-cells (B-LCL) and has undergone intense scrutiny owing to its association with Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas. B-LCL have also proven useful in the study of human immunology. We describe a novel system for inducing efficient foreign gene expression in B-LCL using biotinylated adenovirus as an endosome-disrupting agent. Plasmid DNA is coupled to the exterior of viral particles by streptavidin-polylysine chimeric proteins. Up to 67% of B-LCL may be induced to express foreign genes in vitro in transient expression systems, and gene expression lasts for at least 17 days. We have expressed firefly luciferase, beta-galactosidase (beta-gal), chloramphenicol acetyltransferase, HIV gag, and env genes, as well as infectious HIV, and the EBV-specific BZLF gene in B-LCL with this system. In vivo delivery of a beta-gal reporter gene to B-LCL was documented in a SCID mouse model. Potential applications include study of genetic regulation of EBV infection and transformation events, study of potential gene therapies for EBV-related B-cell tumors, and production of antigen-presenting cells for use in immunologic assays. Because of the high percentage of cells transformed and the length of foreign gene expression, the possibility of examining foreign gene expression in transient assays, without selection for clonal populations, exists.

Animals↗
PubMed1994

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells.

The mucin-type carbohydrate Tn cryptantigen (GalNAc alpha 1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase. Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach. We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and beta-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis. Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen. Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection. Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression. When the beta-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a chromogenic substrate. The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor. We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells. This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques. Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocols.

Adenoviridae↗
PubMed1994

Genetic modification of cells by receptor-mediated adenovirus-augmented gene delivery: a new approach for immunotherapy of cancer.

Most concepts of gene therapy of cancer are based on the generation of an enhanced immune response against the cancer by means of vaccination with gene-modified cancer cells. We have investigated the applicability of a new gene transfer technique which uses the receptor-mediated endocytosis pathway and the endosome disruption activity of adenovirus for the generation of a cancer vaccine consisting of interleukin-2 (IL-2)-transfected, irradiated murine melanoma cells (clone M-3). This technique resulted in very high IL-2 expression (in the range of 30,000 Units IL-2/10(6) cells/24 hrs) in the transfected cells without the need to selection of stably expressing cell clones. We found that this high IL-2 expression of the melanoma cells correlates with high efficacy of the vaccine. Immunization of animals with this vaccine elicits a systemic T-cell-mediated immune response which protects from tumor development after implantation of highly tumorigenic doses of wild-type melanoma cells.

Adenoviridae↗
PubMed1994

Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary human cells in the presence of adenovirus.

Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells.

Adenoviridae↗
PubMed1993

Somatic gene therapy for cancer: the utility of transferrinfection in generating 'tumor vaccines'.

The last few years have seen the development of a branch of somatic gene therapy which aims at strengthening the immune surveillance of the body, leading to eradication of disseminated cancer tumor cells and occult micrometastases after surgical removal of the primary tumor. Such a tumor vaccination protocol calls for cultivation of the primary tumor tissue and the insertion of one of three types of genes into the isolated cultured tumor cells followed by irradiation of the transfected or transduced cells to render them incapable of further proliferation. The cells so treated constitute the 'tumor vaccine'. A review of the literature suggests that for mouse models, in the initial period after inoculation, rejection of the tumor cells is usually effected by non-T-cell immunity, whereas the long-term systemic immune response is based on cytotoxic T-cells. High expression of the gene inserted into the tumor cells may be critical for the success of the vaccination procedure. Examples are given which indicate that transferrinfection, a procedure to introduce genes by adenovirus-augmented receptor-mediated endocytosis, meets some important prerequisites for successful application of this type of gene therapy.

Animals↗
PubMed1993

Non-viral approaches to gene therapy.

Several advances in non-viral gene transfer technology have been reported over the past year. Cationic lipids have been successfully used to deliver genes in vivo, providing a clear alternative to recombinant viruses. In addition, investigators have demonstrated that direct application of DNA via injection or particle bombardment can be used for vaccination. Analysis of the mechanisms employed by viruses to invade cells has demonstrated a crucial role for membrane-active proteins or peptides in the entry process. Several non-viral systems that include membrane-active elements are now available.

Animals↗
PubMed1993

Direct in vivo gene transfer to airway epithelium employing adenovirus-polylysine-DNA complexes.

Adenovirus-polylysine-DNA complexes were evaluated for their capacity to accomplish direct in vivo gene transfer to airway epithelium employing a rodent model. Binary complexes containing transferrin or adenovirus, or combination complexes containing both transferrin and adenovirus, were evaluated. The highest in vitro gene transfer efficiency in primary cultures of airway epithelial cells was accomplished by the combination complexes. This result was paralleled in vivo. Transient gene expression of up to 1 week was observed with localization of the transduced cells to the region of the small airways. These results establish the feasibility of this type of approach for gene therapy applications.

Adenoviridae↗
PubMed1993

Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants.

Delivery of genes via receptor-mediated endocytosis is severely limited by the poor exit of endocytosed DNA from the endosome. A large enhancement in delivery efficiency has been obtained by including human adenovirus particles in the delivery system. This enhancement is probably a function of the natural adenovirus entry mechanism, which must include passage through or disruption of the endosomal membrane. In an effort to identify safer virus particles useful in this application, we have tested the chicken adenovirus CELO virus for its ability to augment receptor-mediated gene delivery. We report here that CELO virus possesses pH-dependent, liposome disruption activity similar to that of human adenovirus type 5. Furthermore, the chicken adenovirus can be used to augment receptor-mediated gene delivery to levels comparable to those found for the human adenovirus when it is physically linked to polylysine ligand-condensed DNA particles. The chicken adenovirus has the advantage of being produced inexpensively in embryonated eggs, and the virus is naturally replication defective in mammalian cells, even in the presence of wild-type human adenovirus.

Adenoviruses, Human↗
PubMed1992

Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle.

Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or beta-galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (ii) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (iii) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-polylysine-mediated gene transfer.

Amino Acid Sequence↗
PubMed1992

High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles.

One limit to successful receptor-mediated gene delivery is the exit of the endocytosed material from the endosome. We demonstrate here the delivery of marker genes to tissue culture cells using a modification of the receptor-mediated gene delivery technique that exploits the endosomolytic activity of defective adenovirus particles. In particular, greater than 90% of the transfected-cell population is found to express a beta-galactosidase gene, and, most importantly, this high level of expression can be obtained with psoralen-inactivated virus particles. Furthermore, because the delivered gene is not carried within the genome of the adenovirus particle, the size constraints are relieved, and we can, therefore, show the delivery of a 48-kilobase cosmid DNA molecule.

Adenoviruses, Human↗
PubMed1992

Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes.

We are developing efficient methods for gene transfer into tissue culture cells. We have previously shown that coupling of a chimeric adenovirus with polylysine allowed the construction of an adenovirus-polylysine-reporter-gene complex that transferred the transporter gene with great efficiency into HeLa cells. We have now explored simpler, biochemical means for coupling adenovirus to DNA/polylysine complexes and show that such complexes yield virtually 100% transfection in tissue culture cell lines. In these methods adenovirus is coupled to polylysine, either enzymatically through the action of transglutaminase or biochemically by biotinylating adenovirus and streptavidinylating the polylysine moiety. Combination complexes containing DNA, adenovirus-polylysine, and transferrin-polylysine have the capacity to transfer the reporter gene into adenovirus-receptor- and/or transferrin-receptor-rich cells.

Adenoviruses, Human↗