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2. |
- Adamson, R. E., et al.
(författare)
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In Vitro Primary Cell Culture as a Physiologically Relevant Method for Preclinical Testing of Human Oncolytic Adenovirus
- 2012
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 23:2, s. 218-230
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Tidskriftsartikel (refereegranskat)abstract
- Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.
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3. |
- Ager, S, et al.
(författare)
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Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity
- 1996
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 7:17, s. 2157-2164
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Tidskriftsartikel (refereegranskat)abstract
- Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.
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5. |
- Berglin-Enquist, Ida, et al.
(författare)
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Successful low-risk hematopoietic cell therapy in a mouse model of type 1 Gaucher disease.
- 2008
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Ingår i: - : Mary Ann Liebert Inc. ; , s. 1189-1190
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Konferensbidrag (refereegranskat)abstract
- Hematopoietic stem cell (HSC) based gene therapy offers the possibility of permanent correction for genetic disorders of the hematopoietic system. However, optimization of present protocols is required before gene therapy can be safely applied as general treatment of genetic diseases. In this study we have used a mouse model of type 1 Gaucher disease (GD) to demonstrate the feasibility of a low-risk conditioning regimen instead of standard radiation, which is associated with severe adverse effects. We first wanted to establish what level of engraftment and glucosylceramidase (GCase) activity is required to correct the pathology of the type 1 GD mouse. Our results demonstrate that a median WT cell engraftment of 7 % corresponding to GCase activity levels above 10 nmol/hr and mg protein was sufficient to reverse pathology within bone marrow (BM) and spleen in the GD mouse. Moreover, we applied non-myeloablative doses of busulphan as a pretransplant conditioning regimen and show that even WT cell engraftment in the range of 1-10% can confer a beneficial therapeutical outcome in this disease model. Taken together, our data provide encouraging evidence for the possibility to develop safe and efficient conditioning protocols for diseases that only require a low level of normal or gene corrected cells for a permanent and beneficial therapeutic outcome. ______________________________________________________________________________ Author contributions: I.B.E.: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; E.N.: Collection of data, data analysis and interpretation; J.-E.M.: Collection and assembly of data, data analysis and interpretation; M.E.: Collection and assembly of data, data analysis and interpretation; J.R.: Data analysis and interpretation, manuscript writing; S.K.: Conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.
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6. |
- Bush, Ronald A, et al.
(författare)
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Preclinical Dose-Escalation Study of Intravitreal AAV-RS1 Gene Therapy in a Mouse Model of X-linked Retinoschisis : Dose-Dependent Expression and Improved Retinal Structure and Function
- 2016
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 27:5, s. 376-389
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Tidskriftsartikel (refereegranskat)abstract
- Gene therapy for inherited retinal diseases has been shown to ameliorate functional and structural defects in both animal models and in human clinical trials. X-linked retinoschisis (XLRS) is an early-age onset macular dystrophy resulting from loss of an extracellular matrix protein (RS1). In preparation for a human clinical gene therapy trial, we conducted a dose-range efficacy study of the clinical vector, a self-complementary AAV delivering a human retinoschisin (RS1) gene under control of the RS1 promoter and an interphotoreceptor binding protein enhancer (AAV8-scRS/IRBPhRS), in the retinoschisin knockout (Rs1-KO) mouse. The therapeutic vector at 1 × 10(6) to 2.5 × 10(9) (1E6-2.5E9) vector genomes (vg)/eye or vehicle was administered to one eye of 229 male Rs1-KO mice by intravitreal injection at 22 ± 3 days postnatal age (PN). Analysis of retinal function (dark-adapted electroretinogram, ERG), structure (cavities and outer nuclear layer thickness) by in vivo retinal imaging using optical coherence tomography, and retinal immunohistochemistry (IHC) for RS1 was done 3-4 months and/or 6-9 months postinjection (PI). RS1 IHC staining was dose dependent across doses ≥1E7 vg/eye, and the threshold for significant improvement in all measures of retinal structure and function was 1E8 vg/eye. Higher doses, however, did not produce additional improvement. At all doses showing efficacy, RS1 staining in Rs1-KO mouse was less than that in wild-type mice. Improvement in the ERG and RS1 staining was unchanged or greater at 6-9 months than at 3-4 months PI. This study demonstrates that vitreal administration of AAV8 scRS/IRBPhRS produces significant improvement in retinal structure and function in the mouse model of XLRS over a vector dose range that can be extended to a human trial. It indicates that a fully normal level of RS1 expression is not necessary for a therapeutic effect.
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11. |
- Enblad, Gunilla, et al.
(författare)
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CAR T-Cell Therapy : The Role of Physical Barriers and Immunosuppression in Lymphoma
- 2015
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 26:8, s. 498-505
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Forskningsöversikt (refereegranskat)abstract
- Chimeric antigen receptor (CAR) T-cells have shown remarkable results in patients with B-cell leukemia and lymphoma. However, while CAR T-cells have shown complete responses in a majority of patients with acute lymphoblastic leukemia (ALL), lymphomas are more difficult to treat. Different CAR designs and conditioning protocols seem to affect the persistence of patient responses. However, factors that determine if patients receiving the same CARs will respond or not remain obscure. In Sweden, a phase I/IIa trial using third-generation CAR T-cells is ongoing in which we intend to compare tumor biology and immunology, in each patient, to treatment response. CAR T-cell therapy is a powerful tool to add to the treatment options for this patient group but we need to perform the necessary basic research on the multifactorial mechanisms of action to give patients the best possible option of survival. Such studies are also crucial to expand the success of CAR T-cells beyond CD19+ B-cell malignancy. This review will focus on possible barriers of treating lymphoma to define factors that need to be investigated to develop the next generation of CAR T-cell therapy.
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12. |
- Engstrand, Thomas, et al.
(författare)
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Transient production of bone morphogenetic protein 2 by allogeneic transplanted transduced cells induces bone formation
- 2000
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Ingår i: Human Gene Therapy. - 1043-0342 .- 1557-7422. ; 11:1, s. 205-211
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to evaluate the use of transplantation of genetically modified allogeneic cells as a method to induce bone formation. In this study, we infected a murine osteoprogenitor cell line with a retroviral vector containing the human bone morphogenic protein 2 (BMP2) gene. Transduced cells exhibited more alkaline phosphatase activity than cells treated with any of the tested doses of recombinant human BMP2 protein (rhBMP2). The transduced cells were suspended in a collagen solution and injected into the quadriceps muscle in immunocompetent outbred mice. Radiographic and histological examinations demonstrate abundant ectopic bone formation in 85% of the transplanted animals (n = 13). PCR and Southern blot analysis for the puromycin resistance gene revealed that the transplanted cells were detectable for up to 1 week, but not at later time points. None of the animals developed tumors. Our results suggest that allogeneic BMP2-expressing transduced cells may have therapeutic potential for enhancing new bone formation. This model also provides a simple, inexpensive, and sensitive assay for evaluating in vivo the osteoinductive potentials of secreted proteins without the requirement of protein purification or the use of immunodeficient animals.
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14. |
- Georgievska, Biljana, et al.
(författare)
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Regulated delivery of glial cell line-derived neurotrophic factor into rat striatum, using a tetracycline-dependent lentiviral vector.
- 2004
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 15:10, s. 934-944
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Tidskriftsartikel (refereegranskat)abstract
- In this study, a tetracycline-regulated lentiviral vector system, based on the tetracycline-dependent transactivator rtTA2S-M2, was developed for controlled expression of glial cell line-derived neurotrophic factor (GDNF) in the rat brain. Expression of the marker gene green fluorescent protein (GFP) and GDNF was tightly regulated in a dose-dependent manner in neural cell lines in vitro. Injection of high-titer lentiviral vectors into the rat striatum resulted in a 7-fold induction of GDNF tissue levels (1060 pg/mg tissue), when doxycycline (a tetracycline analog) was added to the drinking water. However, low levels of GDNF (150 pg/mg tissue) were also detected in animals that did not receive doxycycline, indicating a significant background leakage from the vector system in vivo. The level of basal expression was markedly reduced when a 10-fold lower dose of the tetracycline-regulated GDNF vector was injected into the striatum (3–11 pg/mg tissue), and doxycycline- induced GDNF tissue levels obtained in these animals were about 190 pg/mg tissue. Doxycycline-induced expression of GDNF resulted in a significant downregulation of the tyrosine hydroxylase (TH) protein in the intact striatum. Removal of doxycycline from the drinking water rapidly (within 3 days) turned off transgenic GDNF mRNA expression and GDNF protein levels in the tissue were completely reduced by 2 weeks, demonstrating the dynamics of the system in vivo. Accordingly, TH protein expression returned to normal by 2–8 weeks after removal of doxycycline, indicating that GDNF-induced downregulation of TH is a reversible event.
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15. |
- Granio, Ophélia, et al.
(författare)
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Adenovirus 5-fiber 35 chimeric vector mediates efficient apical correction of the CFTR defect in cystic fibrosis primary airway epithelia.
- 2010
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Ingår i: Human gene therapy. - : Mary Ann Liebert Inc. - 1557-7422 .- 1043-0342. ; 21:3, s. 251-269
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Tidskriftsartikel (refereegranskat)abstract
- In vivo gene transfer to the human respiratory tract using Adenovirus serotype 5 (Ad5) vectors has revealed their limitations related to inefficient gene transfer, host antiviral response and innate adenoviral toxicity. In the present work, we compared the cytotoxicity and efficiency of Ad5 and a chimeric Ad5F35 vector with respect to CFTR gene transfer to cystic fibrosis (CF) and non-CF human airway epithelial cells. We found that high doses of Ad5 vector had an adverse effect on the function of exogenous and endogenous CFTR. Results obtained with Ad5 capsid mutants suggested that the RGD motifs on the penton base capsomers were responsible for the negative effect on the CFTR function. This negative interference was not a result of a lower level of biosynthesis and/or altered cellular trafficking of the CFTR protein, but rather from an indirect mechanism of functional blockage of CFTR, related to the RGD-integrin-mediated endocytic pathway of Ad5. No negative interference with CFTR was observed for Ad5F35, an Ad5-based vector pseudotyped with fibers from Ad35, a serotype which uses another cell entry pathway. In vitro, Ad5F35 vector expressing the GFP-tagged CFTR (Ad5F35-GFP-CFTR) showed a 30-fold higher efficiency of transduction and chloride channel correction in CFTR-deficient cells, compared to Ad5GFP-CFTR. Ex vivo, Ad5F35-GFP-CFTR had the capacity to transduce efficiently reconstituted airway epithelia from CF patients (CF-HAE) via the apical surface, restored the chloride channel function at relatively low vector doses, and showed a relatively stable expression of GFP-CFTR for several weeks.
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18. |
- Henning, Petra, 1974, et al.
(författare)
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Genetic modification of adenovirus 5 tropism by a novel class of ligands based on a three-helix bundle scaffold derived from staphylococcal protein A.
- 2002
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Ingår i: Human gene therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 13:12, s. 1427-39
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Tidskriftsartikel (refereegranskat)abstract
- The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.
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19. |
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21. |
- Jin, Chuan, 1986-, et al.
(författare)
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Tat‐PTD‐modified Oncolytic Adenovirus Driven by the SCG3 Promoter and ASH1 Enhancer for Neuroblastoma Therapy
- 2013
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 24:8, s. 766-775
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Tidskriftsartikel (refereegranskat)abstract
- Secretogranin III (SGC3) belongs to the granin family and is highly expressed in endocrine and neural tissues. The human SCG3 promoterhas not yet been characterized. We identified that a 0.5 kb DNA fragment upstream of the SCG3 gene can selectively drivetransgene expression in neuroblastoma cell lines. The strength of transgene expression was further increased and specificity maintained,by addition of the human achaete‐scute complex homolog 1 (ASH1) enhancer. We developed an oncolytic serotype 5‐basedadenovirus, where the SCG3 promoter and ASH1 enhancer drive E1A gene expression. The virus was further modified with a cellpenetratingpeptide (Tat‐PTD) in the virus capsid, which we have previously shown results in increased adenovirus transductionefficiency of many neuroblastoma cell lines. The virus, Ad5PTD(ASH1‐SCG3‐E1A), shows selective and efficient killing of neuroblastomacell lines in vitro, including cisplatin‐, etoposide‐ and doxorubicin‐insensitive neuroblastoma cells. Furthermore, it delays tumorgrowth and thereby prolonged survival for nude mice harboring subcutaneous human neuroblastoma xenograft. In conclusion, wereport a novel oncolytic adenovirus with potential use for neuroblastoma therapy.
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28. |
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31. |
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32. |
- Löfvall, Henrik, et al.
(författare)
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Hematopoietic stem cell-targeted neonatal gene therapy with a clinically applicable lentiviral vector corrects osteopetrosis in oc/oc Mice
- 2019
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 30:11, s. 1395-1404
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Tidskriftsartikel (refereegranskat)abstract
- Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by nonfunctional osteoclasts. Approximately 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. Gene therapy represents a potential alternative treatment to allogeneic stem cell transplantation for IMO. The oc/oc mouse is a model of IMO characterized by a 1,500 bp deletion in the TCIRG1 gene, severe osteopetrosis, and a life span of only 3 weeks. Here we show that the osteopetrotic phenotype in oc/oc mice can be reversed by hematopoietic stem cell-targeted gene therapy with a clinically applicable lentiviral vector expressing a wild-type form of human TCIRG1 under the mammalian promoter elongation factor 1α short (EFS-hT). oc/oc c-kit+ fetal liver cells transduced with EFS-hT were transplanted into sublethally irradiated oc/oc mice by temporal vein injection 1 day after birth. A total of 9 of 12 mice survived long term (19-25 weeks) with evidence of tooth eruption, uncharacteristic of oc/oc mice. Splenocytes were harvested 19-25 weeks after transplantation and differentiated into osteoclasts on bone slices to assess resorption and on plastic to assess TCIRG1 protein expression. Vector-corrected osteoclasts showed human TCIRG1 expression by Western blot. CTX-I release relative to that mediated by oc/oc-derived osteoclasts increased 8-239-fold. Resorption pits on bone slices were observed for osteoclasts derived from 7/9 surviving transplanted oc/oc mice. Histopathology of the bones of surviving animals showed varying degrees of rescued phenotype, the majority with almost full correction. The average vector copy number per cell in the bone marrow was 1.8 ± 0.5. Overall, 75% of transplanted mice exhibited long-term survival and marked reversal of the osteopetrotic bone phenotype. These findings represent a significant step toward the clinical application of gene therapy for IMO.
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33. |
- Magnusson, Maria K, 1972, et al.
(författare)
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A transductionally retargeted adenoviral vector for virotherapy of her2/neu-expressing prostate cancer
- 2012
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 23, s. 70-82
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Tidskriftsartikel (refereegranskat)abstract
- The efficacy of adenovirus (Ad)-based gene therapy of solid tumors, such as prostate cancer, is limited. One of the many problems is that the virus infects many different cell types in the body, resulting in high toxicity, whereas the target cancer cells are often less prone to wild-type Ad infection. Our aim was to develop genetically de-and retargeted Ad vectors to reduce off-target effects and increase target infection for prostate cancer. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody® molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3]. The ZH-containing vectors could be produced to high titers and were specific for their target, resulting in efficient infection and killing of Her2/neu-positive androgen-dependent PC346C prostate cancer cells in vitro. Here we show that the oncolytic Ad[ZH/3] vector significantly prolonged survival time and reduced serum prostate-specific antigen levels in an orthotopic prostate tumor model in nude mice to the same extent as wild-type Ad5. Our results show that Her2/neu targeting using Ad-based vectors for prostate cancer is feasible and may serve as a basis for the development of gene therapy of human prostate cancer as well as other Her2/neu-expressing cancers. © 2012 Mary Ann Liebert, Inc.
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34. |
- Maitland, Norman, et al.
(författare)
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Gene Transfer Vectors Targeted to Human Prostate Cancer : Do We Need Better Preclinical Testing Systems?
- 2010
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 21:7, s. 815-827
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Tidskriftsartikel (refereegranskat)abstract
- Destruction of cancer cells by genetically modified viral and nonviral vectors has been the aim of many research programs. The ability to target cytotoxic gene therapies to the cells of interest is an essential prerequisite, and the treatment has always had the potential to provide better and more long-lasting therapy than existing chemotherapies. However, the potency of these infectious agents requires effective testing systems, in which hypotheses can be explored both in vitro and in vivo before the establishment of clinical trials in humans. The real prospect of off-target effects should be eliminated in the preclinical stage, if current prejudices against such therapies are to be overcome. In this review we have set out, using adenoviral vectors as a commonly used example, to discuss some of the key parameters required to develop more effective testing, and to critically assess the current cellular models for the development and testing of prostate cancer biotherapy. Only by developing models that more closely mirror human tissues will we be able to translate literature publications into clinical trials and hence into acceptable alternative treatments for the most commonly diagnosed cancer in humans.
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35. |
- Majhen, Dragomira, et al.
(författare)
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Adenovirus-based vaccines for fighting infectious diseases and cancer : progress in the field
- 2014
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert. - 1043-0342 .- 1557-7422. ; 25:4, s. 301-317
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Forskningsöversikt (refereegranskat)abstract
- The field of adenovirology is undergoing rapid change in response to increasing appreciation of the potential advantages of adenoviruses as the basis for new vaccines and as vectors for gene and cancer therapy. Substantial knowledge and understanding of adenoviruses at a molecular level has made their manipulation for use as vaccines and therapeutics relatively straightforward in comparison with other viral vectors. In this review we summarize the structure and life cycle of the adenovirus and focus on the use of adenovirus-based vectors in vaccines against infectious diseases and cancers. Strategies to overcome the problem of preexisting antiadenovirus immunity, which can hamper the immunogenicity of adenovirus-based vaccines, are discussed. When armed with tumor-associated antigens, replication-deficient and oncolytic adenoviruses can efficiently activate an antitumor immune response. We present concepts on how to use adenoviruses as therapeutic cancer vaccines and consider some of the strategies used to further improve antitumor immune responses. Studies that explore the prospect of adenoviruses as vaccines against infectious diseases and cancer are underway, and here we give an overview of the latest developments.
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39. |
- Moscatelli, Ilana, et al.
(författare)
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Targeting NSG Mice Engrafting Cells with a Clinically Applicable Lentiviral Vector Corrects Osteoclasts in Infantile Malignant Osteopetrosis
- 2018
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 29:8, s. 938-949
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Tidskriftsartikel (refereegranskat)abstract
- Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by nonfunctional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to develop a clinically applicable lentiviral vector expressing TCIRG1 to correct osteoclast function in IMO. Two mammalian promoters were compared: elongation factor 1α short (EFS) promoter and chimeric myeloid promoter (ChimP). EFS promoter was chosen for continued experiments, as it performed better. IMO osteoclasts corrected in vitro by a TCIRG1-expressing lentiviral vector driven by EFS (EFS-T) restored Ca2+ release to 92% and the levels of the bone degradation product CTX-I to 95% in the media compared to control osteoclasts. IMO CD34+ cells from five patients transduced with EFS-T were transplanted into NSG mice. Bone marrow was harvested 9–19 weeks after transplantation, and human CD34+ cells were selected, expanded, and seeded on bone slices. Vector-corrected IMO osteoclasts had completely restored Ca2+ release. CTX-I levels in the media were 33% compared to normal osteoclasts. Thus, in summary, evidence is provided that transduction of IMO CD34+ cells with the clinically applicable EFS-T vector leads to full rescue of osteoclasts in vitro and partial rescue of osteoclasts generated from NSG mice engrafting hematopoietic cells. This supports the continued clinical development of gene therapy for IMO.
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43. |
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44. |
- Sandberg, Linda, et al.
(författare)
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Replication-competent Ad11p vector (RCAd11p) efficiently transduces and replicates in hormone-refractory metastatic prostate cancer cells
- 2009
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 20:4, s. 361-373
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Tidskriftsartikel (refereegranskat)abstract
- Selective replication-competent adenovirus serotype 5 vectors have been used for prostate cancer therapy. Unfortunately, gene transfer is inefficient because hormone-refractory metastatic prostate cancer cells have minimal coxsackievirus-adenovirus receptor expression. Vectors based on species B adenoviruses are attractive tools for use in human gene therapy because the viruses have low seroprevalence and they have efficient transduction capacity. Most species B adenoviruses use ubiquitously expressed complement-regulatory CD46 protein as a cellular receptor. Here we report the transduction efficacy and oncolytic capacity of a replication-competent Ad11p (RCAd11p) vector in human prostate cancer cells. Green fluorescent protein was efficiently expressed in a dose-dependent manner in PC-3 and DU 145 cells derived from metastasis of prostate cancer to bone and brain, respectively. However, transduction was less effective in LNCaP cells derived from prostate cancer metastasis to lymph nodes. The oncolytic capacity of the RCAd11p vector was 100 times higher in PC-3 cells than in the two other cell lines. The oncolysis was independent of the level of expression of p53 in the cells or on the absence of E1B55k expression in the vector. In vivo experiments revealed significant growth inhibition of PC-3 tumors in the xenograft mouse group treated with RCAd11p vector or Ad11pwt in comparison with the untreated control group. Thus, we have demonstrated that RCAd11p vector intrinsically possesses oncolytic properties, which were active in targeting tumor cells. Consequently, the novel RCAd11p vector has great potential for the treatment of incurable metastatic prostate disease.
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45. |
- Schenk, Ellen, et al.
(författare)
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Clinical Adenoviral Gene Therapy for Prostate Cancer
- 2010
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 21:7, s. 807-813
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Tidskriftsartikel (refereegranskat)abstract
- Prostate cancer is at present the most common malignancy in men in the Western world. When localized to the prostate, this disease can be treated by curative therapy such as surgery and radiotherapy. However, a substantial number of patients experience a recurrence, resulting in spreading of tumor cells to other parts of the body. In this advanced stage of the disease only palliative treatment is available. Therefore, there is a clear clinical need for new treatment modalities that can, on the one hand, enhance the cure rate of primary therapy for localized prostate cancer and, on the other hand, improve the treatment of metastasized disease. Gene therapy is now being explored in the clinic as a treatment option for the various stages of prostate cancer. Current clinical experiences are based predominantly on trials with adenoviral vectors. As the first of a trilogy of reviews on the state of the art and future prospects of gene therapy in prostate cancer, this review focuses on the clinical experiences and progress of adenovirus-mediated gene therapy for this disease.
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46. |
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48. |
- Sundarasetty, Bala Sai, et al.
(författare)
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Lentivirus-Induced Dendritic Cells for Immunization Against High-Risk WT1(+) Acute Myeloid Leukemia
- 2013
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 24:2, s. 220-237
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Tidskriftsartikel (refereegranskat)abstract
- Wilms' tumor 1 antigen (WT1) is overexpressed in acute myeloid leukemia (AML), a high-risk neoplasm warranting development of novel immunotherapeutic approaches. Unfortunately, clinical immunotherapeutic use of WT1 peptides against AML has been inconclusive. With the rationale of stimulating multiantigenic responses against WT1, we genetically programmed long-lasting dendritic cells capable of producing and processing endogenous WT1 epitopes. A tricistronic lentiviral vector co-expressing a truncated form of WT1 (lacking the DNA-binding domain), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) was used to transduce human monocytes ex vivo. Overnight transduction induced self-differentiation of monocytes into immunophenotypically stable "SmartDC/tWT1" (GM-CSF+, IL-4(+), tWT1(+), IL-6(+), IL-8(+), TNF-alpha(+), MCP-1(+), HLA-DR+, CD86(+), CCR2(+), CCR5(+)) that were viable for 3 weeks in vitro. SmartDC/tWT1 were produced with peripheral blood mononuclear cells (PBMC) obtained from an FLT3-ITD+ AML patient and surplus material from a donor lymphocyte infusion (DLI) and used to expand CD8(+) T cells in vitro. Expanded cytotoxic T lymphocytes (CTLs) showed antigen-specific reactivity against WT1 and against WT1(+) leukemia cells. SmartDC/tWT1 injected s.c. into Nod.Rag1(-/-).IL2r gamma c(-/-) mice were viable in vivo for more than three weeks. Migration of human T cells (huCTLs) to the immunization site was demonstrated following adoptive transfer of huCTLs into mice immunized with SmartDC/tWT1. Furthermore, SmartDC/tWT1 immunization plus adoptive transfer of T cells reactive against WT1 into mice resulted in growth arrest of a WT1(+) tumor. Gene array analyses of SmartDC/tWT1 demonstrated upregulation of several genes related to innate immunity. Thus, SmartDC/tWT1 can be produced in a single day of ex vivo gene transfer, are highly viable in vivo, and have great potential for use as immunotherapy against malignant transformation overexpressing WT1.
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