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- 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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- 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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