| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
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| 5. |
- 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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| 6. |
- 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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| 9. |
- 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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