PubMed Health⌕ Search

Biomedical subjects

M Finer

Publications and source records attributed to M Finer.

9 recordsLinked to original sources
PubMed2001

Improved adenoviral vector for vascular gene therapy : beneficial effects on vascular function and inflammation.

First-generation, E1-deleted adenoviral vectors (E1-AV) can transduce the vascular endothelium with high efficiency, but their use is limited by the resulting acute endothelial injury and the long-term development of intimal hyperplasia. To reduce the impact of viral proteins on the gene-modified cells, a second-generation adenoviral vector with an additional pair of deletions in the E4 region was developed. To determine whether this E1/E4-AV vector would be useful for vascular gene transfer, we directly compared the efficiency of gene transfer to uninjured rabbit carotid arteries using either an E1/E4-AV or an E1-AV vector encoding beta-galactosidase. Both vectors efficiently transduced vascular endothelium; however, the E1/E4-AV vector gene-modified vessels showed higher beta-galactosidase expression 10 days after gene transfer. Importantly, the E1/E4-AV vector produced substantially less endothelial cell activation, less inflammation, and reduced neointimal hyperplasia compared with the E1-AV vector-treated vessels. The E1-AV vector-transduced vessels also demonstrated significantly impaired endothelium-dependent relaxation whereas the E1/E4-AV vector did not impact vasomotor function, even at doses of virus in 5-fold excess of the amount required for >90% transduction of the endothelium. We conclude that the E1/E4-AV vector is superior to the E1-AV vector for vascular gene therapy because of the prolonged transgene expression, reduced vascular inflammation, reduced intimal hyperplasia, and maintenance of normal vasomotor function.

Adenoviridae↗
PubMed2001

Gene therapy: progress and challenges.

Gene therapy is the delivery of new genetic material into a patient's somatic cells for the treatment of disease and is made possible through the development of viral and non-viral gene transfer vectors. In the first five years of gene therapy, clinical studies failed to yield efficacy data with the vectors available at that time. The lack of consistent clinical benefit prompted the United States National Institute of Health Recombinant DNA Advisory Committee to evaluate gene therapy research and conclude that substantial improvements in gene transfer vectors were needed in the areas of vector safety and control of the level and duration of gene expression, and to increase the understanding of the biological interaction of gene transfer vectors with the host. We will describe the progress in development of gene delivery technology, focusing on improvements in vector safety, analysis of vector biodistribution and GMP manufacturing of viral and non-viral gene transfer systems over the last six years since the report. Whereas 5 years ago, investigators tested every vector for every potential disease indication, the accumulated database now enables investigators to select a single vector based upon it's known performance in a wide number of animal models and human clinical studies. We will also highlight several directions investigators have taken to improve the safety and efficacy of gene therapy vectors.

Adenoviridae↗
PubMed2000

Novel chimeric p16 and p27 molecules with increased antiproliferative activity for vascular disease gene therapy.

We describe the construction and characterization of a series of novel cyclin-dependent kinase inhibitors with increased antiproliferative activity for use in the genetic treatment of hyperproliferative cell disorders, such as angioplasty-induced restenosis. These inhibitors were generated through the fusion of truncated versions of the p27 gene to the full-length p16 gene. Biochemically, the p27-p16 chimeric molecules were of comparable potency to the parental p27 in inhibiting the activities of several cyclin-dependent kinases in vitro. Replication-deficient adenoviruses encoding the parental p16, p27 genes, or their derivatives were created to assess the potency of the novel cyclin-dependent kinase inhibitor chimeric molecules to inhibit vascular smooth muscle cell proliferation, which is the seminal event in the restenosis process. One of the p27-p16 chimeric molecules, W9, was observed to be the most potent inhibitor of human primary smooth muscle and endothelial cell proliferation when compared to the p16, p27, p27 derivatives or several alternative p27-p16 chimeric molecules. Overexpression of the W9 chimeric molecule in human coronary artery smooth muscle cells induced human coronary artery smooth muscle cell growth arrest in G1 but did not induce cell apoptosis. Recombinant adenoviral vectors that express this W9 chimeric cyclin-dependent kinase inhibitor molecule constitute a novel potent antiproliferative agent for the treatment of restenosis.

Adenoviridae↗
PubMed1999

The mechanism of delayed selfing in Collinsia verna (Scrophulariaceae).

Collinsia verna, blue-eyed Mary, has floral attributes of an outcrossing species, yet most flowers readily self-pollinate under greenhouse conditions. Here we describe the mechanism of self-pollination in C. verna via changes in relative positions of the stigma and anthers and late timing of receptivity, resulting in delayed selfing. Each flower contains four anthers that dehisce sequentially over ∼1 wk. Pollen that is not collected by pollinators accumulates in the keel petal and retains high viability (>80% pollen germination) up to the time of corolla abscission. The stigmatic surface does not become receptive until after the third anther dehisces. This overlap in the sexual phases is concurrent with a change in herkogamy during floral development. In most flowers (70%), the stigma has moved to the front of the keel and is positioned near the anthers when the third anther dehisces. Under field conditions, fruiting success of plants within pollinator exclosures was ∼75% of the fruiting success in open-pollinated plants (33% fruiting success via autogamy vs. 44% fruiting success, respectively). Collinsia verna plants in pollinator exclosures exhibit variation in autogamy rates within natural populations (range 0-80%). In addition, only half of naturally pollinated, receptive flowers examined had pollen tubes growing in their styles. In contrast, shortly after corolla abscission, nearly all flowers examined (96%) had pollen tubes in their styles. Thus we find that in C. verna, autogamy occurs late in floral development, which has the potential to provide substantial reproductive assurance, and that individuals vary in their ability to set fruit through this mechanism. We suggest that delayed selfing mechanisms may be overlooked in other species and that variable pollinator availability may play a significant role in the maintenance of mixed mating in species with delayed selfing, such as C. verna.

Journal Article↗
PubMed1982

Isolation and characterization of rat skeletal muscle and cytoplasmic actin genes.

Southern blots of rat genomic DNA indicate the existence of at least 12 EcoRI DNA fragments containing actin gene sequences. By using specific probes and stringent conditions of hybridization, it was found that only one of these fragments contains sequences of the skeletal muscle alpha-actin gene. Recombinant bacteriophages originating from eight different actin genes were isolated from rat genomic DNA libraries. One of them, Act 15, contains the skeletal muscle actin gene. Another clone, Act I, contains a gene coding for a cytoplasmic actin, identified tentatively as the beta-actin gene. Both genes have a large intron very close to the 5' end of their transcribed region, followed by several small introns. DNA sequence analysis and comparison with the available data on actin genes in other organisms indicated an interesting relationship between the positions of introns and the evolutionary relatedness. Several intron sites are conserved from at least the echinoderms to the vertebrates; others appear to be present in some actin genes and not in others.

Actins↗
PubMed1980

Cleavage and circularization of single-stranded DNA: a novel enzymatic activity of phi X174 A* protein.

Purified phi X gene A* protein cleaves phi X single stranded DNA. The cleavage appears to be stoichiometric, whereby a gene A* protein molecule cleaves a phosphodiester bond and binds to the DNA fragment. The size of the cleavage product was inversely proportional to the ratio of A* protein to DNA in the reaction mixture. The cleavage of the DNA resulted in the formation of an A* protein - ssDNA complex identified on SDS-polyacrylamide gels and by banding in CsCl. An A* protein-ssDNA complex was isolated by gel filtration and shown to be active in a ligating reaction in which the two ends of the DNA fragment were joined to form a covalently closed circle. The joining reaction required Mg++ ions and was accompanied by the release of the protein from the DNA.

Bacteriophage phi X 174↗
PubMedDate not supplied

Large-scale manufacturing of safe and efficient retrovirus packaging lines for use in immunotherapy protocols.

BACKGROUND: The use of gene modified T lymphocytes for immunotherapy in a cancer or AIDS clinical trial requires an efficient, safe ex vivo method for modification of these cells at manufacturing scale. Since retroviruses have been shown to be a moderately effective means of stably integrating therapeutic genes into T lymphocytes, we wanted to create packaging and producer cell lines that would produce replication competent retrovirus (RCR)-free supernatants, at large scale (> 200 l), and transduce with high efficiency. METHODS: cDNA expression plasmids containing only coding sequences for gagpol or env were built and sequentially transfected into human 293 cells. Packaging and producer clones were characterized for stability, titer and RCR. A producer clone delivering chimeric immune receptors was scaled-up and supernatants used to transduce patient T lymphocytes for clinical studies. PCR and RT-PCR assays were utilized to evaluate the transmission of HERV-H sequences. Relative infectivity of producer clones pseudotyped with different envelopes was determined by transduction and RT assays. RESULTS: RCR-free, human 293 split-genome packaging lines, pseudotyped with amphotropic, xenotropic, or 10A1 envelopes, were created. A CC49 zeta producer clone was scaled-up to 5 x 54 l lots and supernatants used to safely and efficiently transduce patient T lymphocytes with minimal ex vivo manipulation. While 293 cells express HERV-H mRNA, the transmission frequency in our packaging clones was less than 1 HERV-H sequence per 5 x 10(5) proviral integrations. Additionally, 10A1 and xenotropic packaging lines had higher infectivities than the amphotropic clone. CONCLUSION: These packaging lines represent the safest configuration for the large-scale production of retroviral vectors, and are capable of producing high titer, RCR-free retroviral vector for large scale clinical use. While all three clones efficiently transduce human T lymphocytes, the 10A1 clone has the highest infectivity. These packaging cell lines will be valuable for use in human gene therapy protocols.

Animals↗