| 1. |
- Aints, A, et al.
(författare)
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Intercellular spread of GFP-VP22
- 1999
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Ingår i: The journal of gene medicine. - 1099-498X. ; 1:4, s. 275-279
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Tidskriftsartikel (refereegranskat)
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| 2. |
- Bryder, David, et al.
(författare)
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Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansion.
- 2005
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 7:2, s. 137-144
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Tidskriftsartikel (refereegranskat)abstract
- Background Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells. Methods The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed. Results We demonstrate that conditions supporting in vitro stem cell self-renewal efficiently promote oncoretroviral-mediated gene transfer to multipotent adult bone marrow stem cells, without prior in vivo conditioning. Despite using optimized culture conditions, transduction resulted in striking losses of repopulating activity, translating into low numbers of gene marked cells in competitively repopulated mice. Subjecting transduced HSCs to an ex vivo expansion protocol following the transduction procedure could partially reverse this loss. Conclusions These studies suggest that loss of repopulating ability of transduced HSCs rather than low gene transfer efficiency might be the main problem in clinical gene therapy protocols, and that a clinically feasible ex vivo expansion approach post-transduction can markedly improve reconstitution with gene marked stem cells.
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| 3. |
- EL Andaloussi, Samir, et al.
(författare)
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Induction of splice correction by cell-penetrating peptide nucleic acids
- 2006
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1099-498X .- 1521-2254. ; 8:10, s. 1262-1273
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundDirecting splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as β-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing.MethodsHeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 °C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs.ResultsAll the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 °C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis.ConclusionsThese data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.
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| 4. |
- Fischer, Yvonne, et al.
(författare)
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A retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells.
- 2007
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 9:5, s. 335-344
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Tidskriftsartikel (refereegranskat)abstract
- Early clinical trials for gene therapy of human gliomas with retroviral packaging cells (PC) have been hampered by low transduction efficacy and lack of dissemination of PC within the tumor. In the current approach, these issues have been addressed by creating a stable packaging cell line for retroviral vectors pseudotyped with glycoproteins of lymphocytic choriomeningitis virus (LCMV) based on tumor-infiltrating progenitor cells. Methods Tumor-infiltrating progenitor cells, which had been isolated from adult rat bone marrow (BM-TIC), were modified to stably express Gag-Pol proteins of moloney murine leukemia virus (Mo-MLV) and glycoproteins of LCMV. Packaging of a retroviral vector was measured by titration experiments on human fibroblast cells as well as on mouse and human glioma cell lines. Additionally, gene transfer was tested in a rat glioma model in vivo. Results The BM-TIC-derived packaging cell line (BM-TIPC) produced retroviral vectors with titers between 2-8 x 10(3) transducing units (TU)/ml. Extended culturing of BM-TIPC over several weeks and freezing/thawing of cells did not affect vector titers. No replication-competent retrovirus was released from BM-TIPC. In a rat glioma model, BM-TIPC infiltrated the tumors extensively and with high specificity. Moreover, BM-TIPC mediated transduction of glioma cells in vivo. Conclusion This proof-of-principle study shows that primary adult progenitor cells with tumor-infiltrating capacity can be genetically modified to stably produce retroviral LCMV pseudotype vectors. These BM-TIPC may be a useful tool to enhance specificity and efficacy of gene transfer to gliomas in patients.
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| 5. |
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| 6. |
- Ginn, Samantha L., et al.
(författare)
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Gene therapy clinical trials worldwide to 2017 : An update
- 2018
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1099-498X .- 1521-2254. ; 20:5
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Forskningsöversikt (refereegranskat)abstract
- To date, almost 2600 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical activity from trial databases, official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our November 2017 update, we have entries on 2597 trials undertaken in 38 countries. We have analysed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and the genes that have been transferred. Details of the analyses presented, and our searchable database are available via The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at: . We also provide an overview of the progress being made in gene therapy clinical trials around the world, and discuss key trends since the previous review, namely the use of chimeric antigen receptor T cells for the treatment of cancer and advancements in genome editing technologies, which have the potential to transform the field moving forward.
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| 7. |
- Gronevik, E, et al.
(författare)
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Gene expression and immune response kinetics using electroporation-mediated DNA delivery to muscle
- 2005
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 7:2, s. 218-227
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Tidskriftsartikel (refereegranskat)abstract
- Background Injection of DNA encoding exogenic proteins into muscle tissue combined with electroporation often results in a transient increase of the encoded protein concentration in the muscle and the blood. The reduction is normally due to an immune response against the exogenic protein but other factors may also be involved. How various electroporation parameters affect the concentration kinetics of syngenic and exogenic proteins is studied in relation to immune response and muscle damage after electroporation-mediated DNA transfer to muscle. Methods Electroporation was applied to mouse quadriceps muscles after injection of DNA encoding either secreted alkaline phosphatase (SEAP) or a mouse IgG molecule. Protein concentrations in blood or muscle and antibody responses were measured for a period up to 3 months. Tissue inflammation and muscle cell damage were studied on muscle cross-sections and assessed by measuring the concentrations of creatine phosphokinase (CPK) in blood. Results Mice with the highest SEAP concentration in blood at day 7 also had the highest rate of decrease afterwards, the strongest antibody responses against SEAP and the highest acute levels of CPK in blood. DNA-transfected muscle fibers were significantly reduced in number from days 7 to 14. Mononuclear cells surrounded the reporter gene expressing muscle fibers, thus indicating a cellular immune response. When using DNA encoding a syngenic protein the protein concentration in blood was relatively stabile over a 3-month period, but showed different kinetics for various electroporation parameters. Conclusions Our findings suggest that the optimal electroporation. parameters for DNA vaccination may be different from the optimal parameters for long-term expression of genes encoding syngenic proteins.
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| 8. |
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| 9. |
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| 10. |
- Horvath, Lazlo, et al.
(författare)
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Knock down of GAD67 protein levels normalizes neuronal activity in a rat model of Parkinson's disease.
- 2011
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 13:3, s. 188-197
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Dopamine depletion of the striatum is one of the hallmarks of Parkinson's disease. The loss of dopamine upregulates GAD67 expression in the striatal projection neurons and causes other changes in the activity of the basal ganglia circuit. METHODS: To normalize the GAD67 expression in the striatum after dopamine depletion we developed several lentiviral vectors that express RNAi directed against GAD67 mRNA. The vectors were injected into the striatum of hemiparkinsonian rats and the level of GAD67 protein as well as a marker of neuronal activity, mtCO1, was analyzed using Western blots. RESULTS: Unilateral lesions of the dopamine neurons in substantia nigra resulted in an increased level of GAD67 protein in the ipsilateral striatum. Furthermore, we detected significantly higher levels of mtCO1, after dopamine depletion in the striatum. Using a lentiviral vectors with a synthetic miRNA scaffold to deliver RNAi we were able to normalize the GAD67 protein levels in the parkinsonian rat striatum. In addition, we were able to normalize the increased neural activity, which resulted from the loss of dopamine as measured by the marker mtCO1. CONCLUSIONS: We conclude that RNAi directed against GAD67 may be a valid approach to correct the dysregulation of the basal ganglia circuit in a rat model of Parkinson's disease. The possibility to correct for a loss of dopamine using non-dopamimetic tools is interesting as it may be more directed towards the casual mechanisms of the motor symptoms. Copyright © 2011 John Wiley & Sons, Ltd.
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| 11. |
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| 12. |
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| 13. |
- Köping-Hoggard, M, et al.
(författare)
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Relationship between the physical shape and the efficiency of oligomeric chitosan as a gene delivery system in vitro and in vivo
- 2003
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 5:2, s. 130-141
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Tidskriftsartikel (refereegranskat)abstract
- Background Chitosans of high molecular weights have emerged as efficient. nonviral gene delivery systems, but the properties and efficiency of well-defined low molecular weight chitosans ((.) 5 kDa) have not been studied. We therefore characterized DNA complexes Of Such low molecular weight chitosans and related their physical shape and stability to their efficiency as gene delivery systems in vitro and in vivo.Methods Individual complexes between six different chitosan oligomers (6-, 8-, 10-, 12-, 14- and 24-mers) and fluorescence-labeled T4 DNA were visualized and classified into six physical shapes using video-enhanced fluorescence microscopy. The effects of chitosan chain length, charge ratio (+/-) and solvent properties (pH and ionic strength) on the stability and structure of the complexes were studied. Gene expression in vitro and in vivo were studied using a luciferas reporter gene.Results Free DNA appeared as extended coils. Chitosan complexes had, a variety of physical shapes depending on the experimental conditions. in general, the fraction of complexes that had nonaggregated, globular structures increased with increasing chain length of the chitosan oligomer, increasing charge ratio and reduction of pH (from 6.5 to 3.5). A further increase in charge ratio for globular complexes or a further reduction in pH (to 2.5) increased the fraction of aggregates, indicating a window where pharmaceutically desirable globules are obtained. Gene transfection efficiencies in vitro and in vivo were related to the physical shape and stability of the complexes. Only the 24-mer formed stable complexes that gave a high level of gene expression comparable to that of high molecular weight ultrapure chitosan (UPC) in vitro and in vivo.Conclusions Chitosan oligomers form complexes with DNA in a structure-dependent manner. We conclude that the 24-mer, which has more desirable physical prope ties than UPC, is more attractive as a gene delivery system than the conventional high molecular weight chitosans. Copyright (C) 2002 John Wiley Sons, Ltd.
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| 14. |
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| 15. |
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| 16. |
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| 17. |
- Navarro-Galve, B, et al.
(författare)
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Gene marking of human neural stem/precursor cells using green fluorescent proteins
- 2005
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 7:1, s. 18-29
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Tidskriftsartikel (refereegranskat)abstract
- Background Ex vivo gene therapy and cell replacement in the nervous system may provide therapeutic opportunities for neurodegenerative disorders. The development of optimal gene marking procedures for human neural stem cells (hNSCs) is crucial for the success of these strategies, in order to provide a correct understanding of the biology of transplanted cells. Methods hNSCs were modified to express various members of the green fluorescent protein family of proteins. Both DNA and retroviral expression vectors were used. Cells were analyzed for transgene expression under transient and stable expression schemes, and in the presence or absence of drug selection, by fluorescence microscopy, histochemistry, immunocytochemistry, immunoblotting, RT-PCR and flow cytometry. Genetically marked cells were analyzed in vivo after intrastriatal transplantation in neonatal rats. Results Using the same experimental procedures, we have compared Aequorea victoria enhanced green fluorescent protein (Av-eGFP) and Renilla raniformis GFP (Rh-GFP, h- from humanized) for the purpose of gene marking of hNSCs. Our findings revealed practical problems for the derivation of stable Av-eGFP-expressing hNSCs, whereas Rh-GFP could be well expressed. In a second phase of the study, stable Rh-GFP-expressing clonal hNSCs were derived. Rh-GFP did not interfere with the differentiation potential of the cells, and expression levels were identical between division and differentiation conditions. Thirdly, in vivo, we have confirmed the usefulness of Rh-GFP for the study of the transplant performance of hNSCs, and demonstrated that Rh-GFP does not interfere with multipotency and differentiation. Conclusions Searching for suitable and useful reporter genes, we have found that Rh-GFP works efficiently for the purpose of stable gene marking of and is highly useful in vivo. The nature, properties, and possible side hNSCs, effects of marker genes are discussed, since these are important parameters to consider in gene marking studies involving hNSCs.
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| 18. |
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| 19. |
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| 20. |
- Relander, Thomas, et al.
(författare)
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Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes
- 2002
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 4:2, s. 122-132
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Tidskriftsartikel (refereegranskat)abstract
- Background The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34(+)/CD38(low) and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes. Methods CB cells were transduced on Retronectin using an MSCV-based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13-MGIN (GALV) 293GPG-MGIN (VSV-G) or AM12-MGIN (amphotropic). Results Sorted CD34(+)/CD38(low) cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP-positive cells was 61.8+/-6.6% (PG13-MGIN), 26.9+/-3.5% (293GPG-MGIN), and 39.3+/-4.8% (AM12-MGIN). For transplantation experiments, CD34(+) cells were prestimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5 x 10(5) cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum-free medium and transduction of CD34(+) cells using VSV-G-pseudotyped vectors under serum-free conditions was very inefficient. In contrast, transduction with PG13-MGIN under serum-free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3+/-6.6%), and efficient gene transfer to SRCs (46.2+/-4.8%). Conclusions The best conditions for transduction and engraftment of CB SRCs were obtained with GALV-pseudo typed vectors using serum-free conditions. Copyright (C) 2002 John Wiley Sons, Ltd.
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| 21. |
- Rosenqvist, Nina, et al.
(författare)
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Activation of silenced transgene expression in neural precursor cell lines by inhibitors of histone deacetytation
- 2002
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 4:3, s. 248-257
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Tidskriftsartikel (refereegranskat)abstract
- Background Ex vivo gene therapy in the central nervous system (CNS) holds great promise for diseases such as the neurodegenerative disorders. However, achieving stable, long-term transgene expression in grafted cells has proven problematic. This study reports the establishment of an in vitro model of transgene down-regulation in cells grafted to the CNS using the immortalized neural progenitor cell lines HiB5 and RN33B. Methods Neural cell lines were transduced at 33 C with different GFP constructs, both viral and non-viral, containing either viral or non-viral promoters. Cell differentiation in vitro was obtained by culturing the cells at 37 C in serum-free defined media, which halts cell division, and GFP-expression was analysed by FACS. As early as day 3 of culture at 37degreesC, the transgene expression decreased markedly in most cell lines. To validate the assay, the same clones were grafted to the adult rat striatum and the down-regulation of GFP-expression was evaluated. Results The temporal pattern of down-regulation was found to be similar in vitro and in vivo. Using this assay, it was shown that addition of inhibitors of histone deacetylation, but not an inhibitor of DNA methylation, reversed the silencing of GFP in quiescent neural progenitors by up to 308% of control values. Conclusion These results suggest that the same mechanisms controlling gene transcription of the host cell's genome are active in controlling transgene expression and that this should be taken into account when constructing vectors for gene therapy. The assay reported in this study could be used as a screening method to evaluate new vectors. Copyright (C) 2002 John Wiley Sons, Ltd.
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| 22. |
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| 23. |
- Svahn, MG, et al.
(författare)
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Adding functional entities to plasmids
- 2004
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Ingår i: The journal of gene medicine. - : Wiley. - 1099-498X .- 1521-2254. ; 66 Suppl 1, s. S36-S44
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Tidskriftsartikel (refereegranskat)
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| 24. |
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| 25. |
- Tolstrup Nielsen, Troels, et al.
(författare)
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Neuron-specific RNA interference using lentiviral vectors
- 2009
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 11:7, s. 559-569
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Tidskriftsartikel (refereegranskat)abstract
- Background Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result Of the Strict requirement for precise transcriptional initiation and termination. Recently, pol 11 promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. Methods In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell Culture and in the brain. Results We demonstrate robust kockdown of green fluorescent protein using lentiviral vectors driving RNAi from the ubiquitously-expressing promoter of the cytomegalovirus (CMV) and, in addition, we show for the first time neuron-specific knockdown in the brain using a neuron-specific promoter. Furthermore, we show that the expression pattern of the presumed ubiquitously-expressing CMV promoter changes over time from being expressed initially in neurons and glial cells to being expressed almost exclusively in neurons in later stages. Conclusions In the present study, we developed vectors for cell-specific RNAi for use in the brain. This offers the possibility of specifically targeting RNAi to a subset of cells in a complex tissue and may prove to be of great importance in the design of future gene therapeutic paradigms. Copyright (C) 2009 John Wiley & Sons, Ltd.
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