Lentivirus Vector Gene Expression during ES Cell-Derived Hematopoietic Development In Vitro

Hamaguchi, Isao; Woods, Niels‐Bjarne; Panagopoulos, Ioannis; Andersson, Elisabet; Mikkola, Hanna; Fahlman, Cecilia; Zufferey, Romain; Carlsson, Leif; Trono, Didier; Karlsson, Stefán

doi:10.1128/jvi.74.22.10778-10784.2000

Cited by 100 publications

(61 citation statements)

References 48 publications

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“…23,26 Superior persistence of gene expression in differentiating embryonic stem cells has been demonstrated for lentiviral vectors with an internal promoter in comparison to retroviral LTR vectors. [35][36][37] Our data, in line with previous observations of Stewart et al, 38 suggest a hypothesis that improved performance in primitive embryonic cells is mainly dependent on the use of an appropriate promoter in an internal position of a retroviral vector. …”

Section: Discussionsupporting

confidence: 82%

Self-inactivating retroviral vectors with improved RNA processing

et al. 2004

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Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5 0 untranslated region (5 0 UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3 0 UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3 0 long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (C). The latter concept is new and allows the use of an intron in the 5 0 UTR, taking advantage of retroviral splice sites surrounding C. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 10 6 infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

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Section: Discussionsupporting

confidence: 82%

Self-inactivating retroviral vectors with improved RNA processing

et al. 2004

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“…39,40 This indicates significant differences in epigenetic transgene regulation between primitive embryonic and adult stem cells, or, in general, between pregastrulation and postgastrulation cells. 41 It will be interesting to determine whether the type of control regions present in the vector, which lacked any element known to affect chromatin modulation besides a strong retroviral enhancer/promoter, 26 contributed to the promising outcome after immunoselection.…”

Section: Discussionmentioning

confidence: 99%

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell

Schiedlmeier

Düllmann

et al. 2002

Leukemia

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Many applications of hematopoietic gene therapy require selection for clones with active transgene expression. However, it was unclear whether the clonal progeny of a retrovirally transduced hematopoietic stem cell would be capable of maintaining transgene expression through serial repopulation and multilineage differentiation. Such investigations require simultaneous analyses of clonality, multilineage activity and transgene copy numbers. Using a mouse model, the present study demonstrates that a single hematopoietic stem cell expressing a marker gene from one or two insertions of a simple retroviral vector actively maintains multilineage transgene expression in the vast majority (80-99%) of bone marrow and peripheral blood cells. Gene expression persisted through serial transplantations for at least 97 weeks post gene transfer and was observed in the lymphoid (B, T and NK cells), myeloid (CD11b + , Gr-1 + ), erythroid (Ter119 + , mature red blood cells) and megakaryocytic (as indicated by platelets) progeny. Therefore, a single immunoselection for hematopoietic stem cells expressing the transgene in vivo was sufficient to establish a completely chimeric hematopoiesis. These observations imply that the retroviral vectors used in this study contain cis-elements that mediate expression through massive clonal expansion and multilineage differentiation, provided the insertion occurred in genetic loci permissive for expression in hematopoietic stem cells.

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“…25,26 Therefore, we studied whether ArsI can protect lentiviral vectors from differentiation-associated silencing. As a differentiation model, we induced granulocytic differentiation of HL-60 cells by dimethylsulfoxide (DMSO) treatment.…”

Section: Effects Of Insulators On Differentiation-associated Silencinmentioning

confidence: 99%

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

et al. 2004

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Suppressed expression of transgenes in vivo is the major obstacle in the gene therapy. For the long-term expression, we utilized a chromatin insulator from sea urchin arylsulfatase (Ars) gene locus (Ars insulator, ArsI), which has been shown to epigenetically regulate gene expression across species. ArsI was able to prevent silencing of the transgene in a myeloid cell line, HL-60, and a murine embryonic stem cell line, CCE, in an orientation-dependent manner, but not in Huh-7, K562 and MCF-7 cells, indicating that the effect of ArsI on gene silencing was cell type dependent. Although anti-silencing effect of ArsI was almost equivalent to that of chicken b-globin insulator, incorporation of ArsI into lentiviral vector had little effect on the virus titer compared with chicken b-globin insulator. Clonal analysis of transduced HL-60 cells revealed that ArsI protects the lentiviral vector from position effects regardless of its orientation. Furthermore, chromatin immunoprecipitation assays revealed that a high acetylation level was observed in the promoter of the insulated vector, whereas that of ArsI was independent of its anti-silencing capacity. In addition to it having little deteriorative effect on the virus titer, the identified anti-silencing effect of ArsI suggested its possibility for application in gene therapy.

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Lentivirus Vector Gene Expression during ES Cell-Derived Hematopoietic Development In Vitro

Abstract: The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES

Cited by 100 publications

References 48 publications

Self-inactivating retroviral vectors with improved RNA processing

Self-inactivating retroviral vectors with improved RNA processing

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

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