Biomedical subjects

Thomas G Jensen

Publications and source records attributed to Thomas G Jensen.

17 recordsLinked to original sources

PubMed2005

Down-regulation of Hsp60 expression by RNAi impairs folding of medium-chain acyl-CoA dehydrogenase wild-type and disease-associated proteins.

We have analyzed the role of the highly abundant molecular chaperone Hsp60 in the biogenesis of medium-chain acyl-CoA dehydrogenase (MCAD) using RNA interference (RNAi). MCAD is a mitochondrial enzyme involved in the fatty acid metabolism and previous studies in isolated rat mitochondria or prokaryotic expression systems have shown that Hsp60 and GroEL are involved in the folding of MCAD proteins. To elucidate the impact of Hsp60 levels for folding and assembly of MCAD proteins in intact mammalian cells, we report the design and in vivo synthesis of anti-human Hsp60 small-hairpin RNAs (shRNAs). Quantitative PCR analysis of transfected HEK-293 cells showed significant down-regulation of endogenous Hsp60 mRNA 48 h post-transfection and Western blot analysis confirmed the reduced levels of Hsp60 protein. Furthermore, expression of exogenous Myc-tagged Hsp60 was decreased in shRNA-transfected cells. Flow cytometry showed that shRNA-treatment only affects green fluorescent protein targeted to mitochondria, demonstrating that the shRNA effect is specific. In cells with reduced Hsp60 levels both the amounts of total MCAD proteins and folded MCAD were reduced for MCAD wild-type and the two disease-associated variants studied. A similar effect was observed in cells expressing mitochondrial short-chain acyl-CoA dehydrogenase. Thus, in intact human cells we demonstrate that Hsp60 is involved in the folding of MCAD variant proteins. The present system can be used to study the requirement of Hsp60 for folding of other mitochondrial proteins and to assess the role of Hsp60 for the severity of genetic defects involving these proteins.

Acyl-CoA Dehydrogenase↗

PubMed2005

Characterization of transgenic mice with the expression of phenylalanine hydroxylase and GTP cyclohydrolase I in the skin.

Phenylketonuria (PKU) is a metabolic disease causing increased levels of phenylalanine in blood and body fluids. Circulating phenylalanine is normally cleared by phenylalanine hydroxylase (PAH) expressed in the liver. The aim of this study is to exploit the skin as a 'metabolic sink' removing phenylalanine from the blood. We have previously showed that the overexpression of PAH and GTP cyclohydrolase I (GTP-CH), the rate-limiting enzyme in the synthesis of the cofactor for PAH, leads to high levels of phenylalanine clearance in primary human keratinocytes. In this study, we have investigated the 'metabolic sink' strategy in an in vivo model by developing three lines of transgenic mice expressing PAH and GTP-CH in various layers of the skin. The promoters used were keratin 14 (K14), involucrin (INV) and a truncated variant of Keratin 1 (K1). The mice were crossbred to a mouse model of human PKU, the PAH(enu2) mouse, in order to obtain mice that do not express PAH in the liver and the kidney. Transgenic mice containing the INV and K14 promoters expressed PAH and GTP-CH in the epidermis. However, the K1 promoter did not lead to detectable gene expression. Analysis of the mice showed that no phenotypic effect was observed in mice expressing PAH and GTP-CH from the INV promoter. However, low level of phenylalanine clearance was observed in mice expressing PAH and GTP-CH from the K14 promoter, suggesting that the skin can be genetically engineered to function as a 'metabolic sink'.

Animals↗

PubMed2005

Manipulation of the phenylalanine metabolism in human keratinocytes by retroviral mediated gene transfer.

Phenylketonuria (PKU) is an inherited disease causing increased levels of phenylalanine in body fluids due to deficiency of hepatic phenylalanine hydroxylase (PAH) or other enzymes involved in the phenylalanine metabolism. With the long-term goal of using gene transfer to the skin to remove phenylalanine, we have previously shown that overexpression of PAH, catalyzing the hydroxylation of phenylalanine, and GTP cyclohydrolase (GTP-CH), involved in the formation of the necessary cofactor BH4,are required. Here we investigate whether manipulation of additional steps in the phenylalanine clearance pathway can further improve the phenylalanine uptake and metabolism. Transport of phenylalanine into human keratinocytes could be increased by overexpressing the two subunits LAT1 and 4F2hc of the large neutral amino acid transporter. The PAH enzyme activity was titrated by employing mutant PAH enzymes with different specific activity and by increasing the PAH copy number in transduced keratinocytes using a repeated transduction procedure. Finally, the intracellular tyrosine concentration was lowered by overexpression of tyrosinase converting tyrosine to dopaquinone. However, measured over a 24-hour period neither of these manipulations resulted in an increased phenylalanine uptake. These results suggest that other enzymes than GTP-CH, involved in BH4 synthesis and/or regeneration, can be rate-limiting in the genetically modified keratinocytes.

Amino Acid Transport System L↗

PubMed2004

Site-specific strand bias in gene correction using single-stranded oligonucleotides.

Targeted gene editing mediated by chimeric RNA-DNA oligonucleotides (RDOs) or single-stranded oligo-deoxyribonucleotides (ssODNs) has been demonstrated in a wide variety of cell types both in vitro and in vivo. In this study we investigated the correlation between the polarity of the used oligonucleotides and the obtained correction frequency in targeted ssODN-mediated correction of two G>A mutations (introduced at positions 659 and 1567, respectively) in an episomal beta-galactosidase gene. At position 659 the highest correction efficiency was observed using an ssODN complementary to the transcribed strand of the target gene. In contrast, at position 1567 the highest correction frequency was observed using an ssODN complementary to the nontranscribed strand of the target gene. It has been reported that site-specific gene editing mediated by ssODNs targeting the nontranscribed strand of the target gene results in a higher gene editing frequency, and it has been suggested that steric hindrance or displacement of ssODNs by traversing transcription complexes prevents efficient targeting of the transcribed strand. However, the results of the present study demonstrate that occupancy by transcriptional complexes alone does not dictate strand bias in ssODN-mediated gene editing, and that the sequences surrounding the targeted nucleotide may profoundly influence strand bias. This finding has important implications for the design of optimal ssODNs for targeted editing of a given nucleotide sequence.

Animals↗

PubMed2004

Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1.

UNLABELLED: Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. INTRODUCTION: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. MATERIALS AND METHODS: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. RESULTS: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPARgamma2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor -1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. CONCLUSION: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.

Adipocytes↗

PubMed2004

Strategies for long-term gene expression in the skin to treat metabolic disorders.

Due to its accessibility, size and contact with the blood circulation, the skin is an attractive target for somatic gene therapy. Permanent cutaneous expression can be achieved by genetic manipulation of epidermal keratinocytes ex vivo followed by transplantation or by local injection of viral vectors. Furthermore, progress is being made to develop topical gene transfer methods leading to permanent gene expression. There is experimental evidence showing that genetically engineered skin can produce and secrete medically relevant proteins to the circulation and also produce enzymes that can clear metabolites accumulating in various diseases. Thus, cutaneous gene transfer approaches may be relevant not only for local skin diseases, but also for certain systemic disorders.

Animals↗

PubMed2003

Normalization of growth in hypophysectomized mice using hydrodynamic transfer of the human growth hormone gene.

Nonviral gene transfer was investigated as a potential treatment of growth hormone deficiency (GHD) using hypophysectomized mice as a model. After a single hydrodynamic administration of naked plasmid DNA containing the human growth hormone (hGH) gene controlled by an ubiquitin promoter, sustained elevation of circulating hGH was observed the entire observation period (68 days), with a concomitant normalization of circulating insulin-like growth factor I (IGF-I) and IGF-binding protein-3. Furthermore, longitudinal growth was corrected in terms of normalization of tibia length, tail length, and body weight gain. Liver, spleen, and lung weights were normalized, whereas heart weight was normalized partly. hGH mRNA was expressed exclusively in liver tissue. In conclusion, we showed that nonviral hGH gene transfer normalizes longitudinal growth in hypophysectomized mice, indicating that this method potentially could be relevant as a new therapeutic tool in the clinical handling of GHD.

Animals↗

PubMed2003

Expression of short-chain acyl-CoA dehydrogenase (SCAD) proteins in the liver of SCAD deficient mice after hydrodynamic gene transfer.

Hydrodynamic administration of naked DNA was investigated as a method for in vivo expression of variant proteins involved in metabolic diseases, using short-chain acyl-CoA dehydrogenase (SCAD) deficient mice (BALB/cByJ) as a model. Human SCAD wild-type (WT) and two disease-associated SCAD variant proteins (R147W and G185S) were expressed in mouse liver by means of single injections of SCAD cDNA under the control of a ubiquitin promoter. SCAD expression was detected two days after injection. The activity decreased after the first week but continued to be detectable for at least 31 days after injection. Analysis of SCAD WT, R147W, and G185S proteins in liver cells showed that all three SCAD proteins were processed to the mature protein in mitochondria. Concomitantly, the SCAD activity in BALB/cByJ mice injected with SCAD WT, G185S, and R147W cDNA was 30, 39, and 13%, respectively, of the level in normal mice. A tendency to a reduction in the level of butyrylcarnitine in blood was observed although only approximately 5% of the liver cells expressed the SCAD protein. Thus, hydrodynamic gene transfer allows for functional testing of SCAD variant proteins in vivo.

Animals↗

PubMed2003

Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene. Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3' end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN). No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms. Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells.

Base Sequence↗

PubMed2003

Epidermolysis bullosa simplex keratinocytes with extended lifespan established by ectopic expression of telomerase.

As part of a strategy to develop somatic gene therapy of epidermolysis bullosa simplex (EBS) we have established patient keratinocytes with expanded lifespan by ectopic expression of the human telomerase gene (hTert). The presence of an active telomerase enzyme was demonstrated by the telomerase repeat amplification protocol (TRAP). The hTert(+) cells have a normal karyotype and the cells have, until now, undergone more than 80 population doublings (PDs) after hTert retroviral transduction while control cells exhibited senescence-associated proliferation arrest after 8 PDs. In organotypic culture the hTert(+) cells are capable of forming a stratified epidermis illustrating their preserved ability to differentiate.

Cell Line, Transformed↗

PubMed2003

Platelet derived growth factor (PDGF) responsive epidermis formed from human keratinocytes transduced with the PDGF beta receptor gene.

Platelet-derived growth factor is a major proliferative and migratory stimulus for connective tissue cells during the initiation of skin repair processes. In response to injury, locally produced platelet-derived growth factor is secreted by a diversity of cutaneous cell types whereas target activity is confined to cells of mesenchymal origin, e.g. dermal fibroblasts and smooth muscle cells. Although epidermal cells contribute to cutaneous platelet-derived growth factor activity by their ample capacity to secrete platelet-derived growth factor ligand, normal epidermal keratinocytes are not known to express any member of the platelet-derived growth factor receptor family. In order to study if epidermis may be genetically transformed to a platelet-derived growth factor sensitive compartment we aimed to introduce the gene encoding human platelet-derived growth factor receptor beta (PDGF beta R) into epidermal keratinocytes using a retrovirus-derived vector. Successful gene transfer to primary cells was confirmed by immunofluorescence staining, southern blotting, and ligand-induced receptor autophosphorylation. By culturing a mixture of PDGF beta R-transduced and unmodified keratinocytes at the air-liquid interface on devitalized dermis, we were able to establish a multilayered epithelium showing histologic similarities to that evolved from native keratinocytes or keratinocytes transduced with the reporter gene encoding enhanced green fluorescent protein. Receptor-modified epidermal tissue cultured for 6 days and examined by immunofluorescence microscopy was shown to contain PDGF beta R-expressing keratinocytes distributed in all layers of living epidermis. By continued tissue culture in serum-containing medium, the epidermis became increasingly cornified although receptor-positive cells were still observed within the viable basal compartment. Stimulation of PDGF beta R-transduced epidermis with recombinant platelet-derived growth factor BB had a mitogenic effect as reflected by an increased frequency of Ki-67 positive keratinocytes. The study demonstrates that transgene expression of human PDGF beta R can be achieved in epidermal keratinocytes by retroviral transduction, and that ligand activation of such gene-modified skin equivalent enhances cell proliferation. In perspective, viral PDGF beta R gene transfer to keratinocytes may be a useful approach in studies of receptor tyrosine kinase mediated skin repair and epithelialization.

3T3 Cells↗

PubMed2003

Production of retroviral vectors in primary human keratinocytes after DNA-mediated gene transfer leads to prolonged gene expression.

Prolonged stability and controlled expression of gene constructs transferred directly to human skin improve the possibility of using this tissue in somatic gene therapy. We aim to develop a simple transfection method resulting in retroviral mediated gene transfer to keratinocyte stem cells in situ. We here show that after DNA-mediated gene transfer into primary human keratinocytes it is possible to achieve production of retroviral vectors, leading to the transduction of co-cultured keratinocytes and prolonged reporter gene expression. The method is a first step in a strategy to generate retroviral producer cells in situ in the skin furthermore the method can be used for rapid analysis of the possible effects of transgenes in cultured human keratinocytes without preparatory retroviral vector production in packaging cell lines.

Cell Culture Techniques↗

PubMed2002

Mechanisms underlying targeted gene correction using chimeric RNA/DNA and single-stranded DNA oligonucleotides.

Chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides have been developed for site-specific correction of episomal and chromosomal target genes. The gene repair approach relies on specific hybridization of the oligonucleotides to the target gene generating a mismatch with the targeted point mutation. Restored gene function is anticipated to occur through activation of endogenous repair systems that recognize the created mismatch. We present an overview of the gene correction results obtained in several target genes by employing various oligonucleotide designs and a discussion of the possible mechanisms underlying the gene correction techniques. Experimental data suggest that modified single-stranded oligonucleotides form intermediate three-stranded heteroduplexes involving the human RecA homologue, hRad51, whereas chimeric RNA/DNA oligonucleotides may participate in three or four-stranded intermediate structures. Protein factors such as hRad52, hRad54, hRPA, and p53 may modulate the heteroduplex formation and participate in the activation of the endogenous mismatch repair and/or nucleotide excision repair pathway(s). The efficiency of the gene correction process may furthermore be influenced by the differential recognition of mismatches by repair enzymes and possible sequence context effects.

Adenosine Triphosphatases↗

PubMed2002

Physiological effects of human growth hormone produced after hydrodynamic gene transfer of a plasmid vector containing the human ubiquitin promotor.

Human growth hormone (hGH) deficiency causes retarded growth and abnormalities in body fat distribution and abundance, muscle growth, and bone mineralization. While injection of recombinant hGH may reverse or ameliorate symptoms of deficiency, therapy aiming at in vivo synthesis of hGH is still of considerable clinical interest. Hydrodynamic injection of a simple plasmid vector containing a human ubiquitin promotor and the hGH gene were found to result in high and prolonged expression. Synthesis of hGH was achieved both in NOD/SCID mice and in three different inbred strains of immune competent mice. Although hGH antibodies were produced in immunocompetent mice, physiological effects of the protein were documented by increase in body weight, increased serum levels of insulin-like growth factor I and relative increased weight of the internal organs. Nonviral gene therapy may be regarded as a potential future way of reconstituting hGH expression in deficient individuals.

Animals↗

PubMed2002

Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria.

Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH) and increased levels of phenylalanine. PAH requires the cofactor BH(4) to function and the rate-limiting step in the synthesis of BH(4) is GTP cyclohydrolase I (GTP-CH). The skin is a potential target tissue for PKU gene therapy. We have previously shown that overexpression of PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH(4) supplementation [Gene Ther. 7 (2000) 1971]. Here, we investigate the capacity of fibroblasts, another cell type from the skin, to metabolize phenylalanine. After retroviral gene transfer of PAH and GTP-CH both normal and PKU patient fibroblasts were able to metabolize phenylalanine, however, in lower amounts compared to genetically modified keratinocytes. Further comparative analyses between keratinocytes and fibroblasts revealed a higher copy number of transgenes in keratinocytes and also a higher metabolic capacity.

Blotting, Southern↗

PubMed2002

Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells.

Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells. The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.

Animals↗

PubMed2002

Skin genetically engineered as a bioreactor or a 'metabolic sink'.

Genetically manipulated human keratinocytes can produce and secrete medically relevant proteins to the circulation. Genetically modified skin may also function as a 'metabolic sink' detoxifying the body of metabolites which accumulate in certain metabolic diseases. At the National Institutes of Health (NIH), Bethesda, Md., a clinical trial investigating the treatment of an ocular disease using the skin as a 'metabolic sink' for ornithine accumulating in gyrate atrophy patients is being prepared. The trial will involve the transplantation of a small patch of autologous keratinocytes, transduced ex vivo, onto the thighs of patients with gyrate atrophy. We are now investigating other diseases where this technology may be applicable such as in the treatment of hyperphenylalaninemia or hypercholesterolemia.

Bioreactors↗