Abstract:
:Rat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (IIFur) and/or containing a histidine B10 to aspartic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The altered cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creates a more stable form of insulin leading to an increase in insulin accumulation. Myoblasts transfected with a proinsulin cDNA construct mutated at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mutation at the B10 position (pCMV.B10) accumulated 852 +/- 16, 150 +/- 13, and 883 +/- 39 microU (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the culture medium within 2 hr post-transfection. Significant (pro)insulin release continued for 1 week and gradually diminished over a month. Approximately 50% of the proinsulin released from rat myoblasts transfected with pCMV.IFur.IIFur.B10 was completely processed into mature insulin based on densitometric analysis of autoradiographs of gels containing immunoprecipitated 35S-Cys-labeled (pro)insulin. However, only a trace of the proinsulin encoded by pCMV.B10 was processed. In an isolated rat adipocyte [14C]glucose oxidation assay, insulin released from myoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmid expression was studied by transfecting myoblasts with the beta-galactosidase (beta-Gal) gene in pCMV, allowing them to divide and fuse into multinucleated myotubes, followed by staining for beta-Gal. Approximately 80% of myotubes expressed beta-Gal. The results indicate that proinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblasts a viable target cell for gene therapy of IDDM.
journal_name
Hum Gene Therjournal_title
Human gene therapyauthors
Simonson GD,Groskreutz DJ,Gorman CM,MacDonald MJdoi
10.1089/hum.1996.7.1-71subject
Has Abstractpub_date
1996-01-01 00:00:00pages
71-8issue
1eissn
1043-0342issn
1557-7422journal_volume
7pub_type
杂志文章abstract::We report a novel method for targeting adenovirus-mediated gene delivery. By irradiating mammalian cells prior to adenoviral transduction, adenoviral gene transfer is greatly improved and the adenoviral genome integrates into cellular DNA. In this work, human and rodent cell lines were irradiated and subsequently tran...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.1997.8.9-1025
更新日期:1997-06-10 00:00:00
abstract::La Crosse virus (LACV)-mediated encephalitis is the most frequently reported arboviral disease in the United States, but to date no vaccine against this virus is available. We have established a new animal model, genetically targeted mice lacking a functional interferon type I receptor (IFNAR-1). These mice show an ag...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/10430349950017653
更新日期:1999-07-01 00:00:00
abstract::Gene therapy for heart diseases requires availability of an efficient vector for gene transfer into myocardium. Recombinant adenovirus expressing the Escherichia coli beta-galactosidase (beta-Gal) gene was shown to infect rat cardiocytes efficiently in vivo. However, a time course of gene expression showed that transg...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.1995.6.10-1265
更新日期:1995-10-01 00:00:00
abstract::We report on an antitumor treatment involving electrogene therapy (EGT), a newly developed in vivo gene transfer method using electroporation. We carried out in vivo EGT in a subcutaneous model of CT26 colon carcinoma cells, using plasmid DNAs encoding interleukin 12 (IL-12) subunits. For this purpose, we developed tw...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/104303401750270922
更新日期:2001-07-01 00:00:00
abstract::Overexpression of human manganese-containing superoxide dismutase (MnSOD) activity has been demonstrated to suppress malignancy in human melanoma and breast carcinoma cells in vitro and in vivo. To study its effects on human oral squamous carcinoma cells, stable transfection and expression of MnSOD in SCC-25 cells hav...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.1997.8.5-585
更新日期:1997-03-20 00:00:00
abstract::The addition of replication-defective recombinant adenovirus to plasmid transfection (termed here "adenofection") has been shown to increase plasmid transgene expression in limited studies. Similarly, the addition of cationic liposomes to adenovirus increases adenovirus-mediated gene transduction (termed here "lipoduc...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/10430349950017059
更新日期:1999-09-20 00:00:00
abstract::Ornithine transcarbamylase deficiency (OTCD) is an inborn error of urea synthesis that has been considered as a model for liver-directed gene therapy. Current treatment has failed to avert a high mortality or morbidity from hyperammonemic coma. Restoration of enzyme activity in the liver should suffice to normalize me...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/10430340152712719
更新日期:2002-01-01 00:00:00
abstract::The combination of immunization strategies with gene therapy methods constitutes a powerful tool for the purpose of genetic immunization. The cutaneous microenvironment, rich in professional antigen-presenting cells and accessory cells capable of initiating and controlling the intensity of specific immune responses, m...
journal_title:Human gene therapy
pub_type: 杂志文章,评审
doi:10.1089/104303400750035852
更新日期:2000-11-01 00:00:00
abstract::Esophageal cancer is characterized by rapid clinical progression and poor prognosis, due to early-stage invasion of adjacent tissues and metastasis. Tissue factor pathway inhibitor-2 (TFPI-2) has been implicated as a metastasis-associated gene in many types of tumors. Here we describe the potential involvement of TFPI...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.2008.129
更新日期:2009-01-01 00:00:00
abstract::Diabetes mellitus is associated with increased risk of heart failure. It has been previously demonstrated in mice that a single injection of adeno-associated virus 8 encoding urocortin 2 (AAV8.UCn2) increases glucose disposal in models of insulin resistance and improves the function of the failing heart. The present s...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.2018.150
更新日期:2019-06-01 00:00:00
abstract::Lentiviral vectors are promising tools for gene transfer into the central nervous system. We have characterized in detail transduction with human immunodeficiency virus type 1 (HIV-1)-derived vectors encoding enhanced green fluorescent protein (eGFP) in the adult mouse brain. Different brain regions such as the striat...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/10430340252899019
更新日期:2002-05-01 00:00:00
abstract::Interleukin-12 (IL-12) is a cytokine that exhibits pleiotropic effects on lymphocytes and natural killer cells and has been shown to have promise for the immunotherapy of cancer. The combination of the immune costimulatory molecule B7.1 and IL-12 has been shown to be synergistic for T cell activation. By transfecting ...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.1997.8.9-1125
更新日期:1997-06-10 00:00:00
abstract::The first report of in vivo gene delivery to the retina dates back to 1987 when a retroviral vector was injected intraocularly in newborn mice. Later came the observation that retinal cells could be successfully transduced using adenoviral and then adeno-associated and lentiviral vectors. By 2000, it had become clear ...
journal_title:Human gene therapy
pub_type: 杂志文章,评审
doi:10.1089/hum.2017.164
更新日期:2017-11-01 00:00:00
abstract::Production of clinical-grade gammaretroviral vectors for ex vivo gene delivery requires a scalable process that can rapidly generate large amounts of vector supernatant, clear large numbers of residual packaging cells with minimal decreases in vector titer, and satisfy all current regulatory guidelines regarding produ...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.2010.064
更新日期:2011-01-01 00:00:00
abstract::Here we show potent inhibition of HIV-1 replication in a human T cell line and primary human CD4(+) cells by expressing a single antiviral protein. Nullbasic is a mutant form of the HIV-1 Tat protein that was previously shown to strongly inhibit HIV-1 replication in nonhematopoietic cell lines by targeting three steps...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.2012.176
更新日期:2013-03-01 00:00:00
abstract::DNA expression vectors may be administered to patients like conventional medicines to have a finite and controlled duration of action. The clinical application of these medicines will require a precise understanding of the kinetics of the administered gene, the mRNA transcript, and the gene product. The apparent kinet...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.1994.5.6-679
更新日期:1994-06-01 00:00:00
abstract::One of the major obstacles to pulmonary-directed gene therapy using adenoviral vectors is the induction of inflammation. We investigated whether the adenoviral particles that constitute the initial inoculum can serve as an inflammatory stimulus, independent of their ability to express genes that they contain. Viral pa...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.1995.6.12-1553
更新日期:1995-12-01 00:00:00
abstract::The use of viral thymidine kinase (TK) gene coupled with the administration of ganciclovir to render cancer cell death has been studied extensively. Many of these experiments utilized retrovirus to transfer the TK gene under the control of a nonspecific promoter. Because nonspecific expression of the viral TK gene may...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.1996.7.4-463
更新日期:1996-03-01 00:00:00
abstract::The efficiency of gene therapy strategies against cancer is limited by the poor distribution of the vectors in the malignant tissues. To solve this problem, a new generation of tumor-specific, conditionally replicative adenoviruses is being developed. To direct the replication of the virus to breast cancer, we have co...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/10430340050143435
更新日期:2000-09-20 00:00:00
abstract::Development of multidrug resistance (MDR) is the major obstacle to successful cancer chemotherapy. We have developed Daudi human lymphoma cells that are 20-fold more resistant than the parent cell line to vincristine (VCR) by infecting cells with pHaMDR1/A retroviral vector (Daudi/MDR20). Three DNA sequences of anti-M...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/10430349950018175
更新日期:1999-05-01 00:00:00
abstract::Targeted delivery of intravenously administered genetically altered cells or stem cells is still in an early stage of investigation. We developed a method of delivering iron oxide (ferumoxide)-labeled mesenchymal stem cells (MSCs) to a targeted area in an animal model by applying an external magnet. Rats with or witho...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/104303404322959506
更新日期:2004-04-01 00:00:00
abstract::Mutations in the gene encoding the peroxisomal ATP-binding cassette transporter (ABCD1) cause elevations in very long-chain fatty acids (VLCFAs) and the neurodegenerative disease adrenoleukodystrophy (ALD). In most adults, this manifests as the spinal cord axonopathy adrenomyeloneuropathy (AMN). A challenge in virus-b...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.2018.079
更新日期:2019-05-01 00:00:00
abstract::Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a devastating disease caused by mutations in TYMP, which encodes thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction results in systemic thymidine and deoxyuridine overload, which interferes with mitochondrial DNA replication. Preclinical stu...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.2018.217
更新日期:2019-08-01 00:00:00
abstract::We studied hematopoietic progenitors from fetal baboon blood, marrow, and liver at four time points (125, 140, 160, and 175 days) during the third trimester (gestation approximately 180 days) to determine if fetal baboons might be an appropriate model for in utero gene therapy of hematopoietic stem cells (HSCs). Cells...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/10430349950018742
更新日期:1999-03-01 00:00:00
abstract::The expanding use of adenoviral vectors for gene therapy has brought about the need for new analytical tools. We have developed an anion-exchange high-performance liquid chromatography method to analyze recombinant adenovirus serotype 5 samples. Before this assay, available analytical methods consisted of either long-...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.1997.8.4-453
更新日期:1997-03-01 00:00:00
abstract::Recombinant adeno-associated virus (rAAV) is a prototypical gene therapy vector characterized by excellent safety profiles, wide host range, and the ability to transduce differentiated cells. Numerous rAAV-based vectors providing efficient and sustained expression of transgenes in target tissues have been developed fo...
journal_title:Human gene therapy
pub_type: 杂志文章,评审
doi:10.1089/hum.2005.16.551
更新日期:2005-05-01 00:00:00
abstract::Nonviral jet injection is an applicable technology for in vivo gene transfer of naked DNA. However, little is known about the biodistribution and clearance of jet-injected DNA, or about its localization within tissue and cells. Therefore, in this study we analyzed the intratumoral and systemic biodistribution of jet-i...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/hum.2006.17.611
更新日期:2006-06-01 00:00:00
abstract::Guidelines for testing gene therapy products for ecotropic replication-competent retrovirus (Eco-RCR) have not been delineated as they have for amphotropic viruses. To evaluate biologic assays that can detect these viruses, we compared an S(+)/L(-) assay and a marker rescue assay designed specifically for Eco-RCR dete...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/104303402760293619
更新日期:2002-09-20 00:00:00
abstract::Recombinant adenoviruses have great potential as gene delivery systems because of their ability to infect a wide range of target cells. However, systemic delivery of viral vectors to tissues other than liver and spleen has been inefficient because of the rapid clearance of the circulating virus by the liver. In the pr...
journal_title:Human gene therapy
pub_type: 杂志文章
doi:10.1089/10430340050015806
更新日期:2000-03-01 00:00:00
abstract::Recombinant adeno-associated virus serotype 2 (rAAV2)-based human gene therapy for cystic fibrosis has progressed through a series of preclinical studies and phase I and II clinical trials. This agent has shown an encouraging safety profile, consistent levels of DNA transfer, and positive evidence of short-term clinic...
journal_title:Human gene therapy
pub_type: 临床试验,杂志文章
doi:10.1089/hum.2005.16.921
更新日期:2005-08-01 00:00:00