Addition of the Human Interferon β Scaffold Attachment Region to Retroviral Vector Backbones Increases the Level of<i>in Vivo</i>Transgene Expression among Progeny of Engrafted Human Hematopoietic Stem Cells

Murray, Lesley J.; Travis, Marilyn; Luens-Abitorabi, Karin; Olsson, Katherine; Plavec, Ivan; Forestell, Sean; Hanania, Elie G.; Hill, Beth

doi:10.1089/10430340050143453

Cited by 22 publications

(15 citation statements)

References 34 publications

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“…5 To overcome this obstacle, use of epigenetic regulatory elements such as insulators, matrix attachment regions and locus control regions have been attempted. 20,21,[30][31][32][33] In this study, we found that sea urchin ArsI protects an HIV-based vector from long-term silencing in cell type-and orientation-dependent manner. ArsI was effective in HL-60 and CCE cells, but not in other cell lines.…”

Section: Discussionmentioning

confidence: 55%

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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Section: Discussionmentioning

confidence: 55%

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

et al. 2004

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“…The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) enhances gene expression by facilitating the nuclear export of RNA transcripts (Zufferey et al, 1999). In addition, the pHRST vector contains 7 copies of the human interferon-β gene matrix attachment region (MAR) which has been shown to increase gene expression (Murray et al, 2000).…”

Section: Dual Promoter Lentiviral Vectors Do Allow Separate Control Omentioning

confidence: 99%

Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages

Hui

Mostoslavsky

Eruslanov

et al. 2008

Journal of Immunological Methods

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In-depth studies of innate immunity require efficient genetic manipulation of macrophages, which is especially difficult in primary macrophages. We have developed a lentiviral system for inducible gene expression both in macrophage cell lines and in primary macrophages. A transgenic mouse strain C3H.TgN(SRA-rtTA) that expresses reverse tetracycline transactivator (rtTA) under the control of macrophage-specific promoter, a modified human scavenger receptor A (SR-A) promoter was generated. For gene delivery, we constructed a dual-promoter lentiviral vector, in which expression of a "gene-of-interest" is driven by a doxycycline-inducible promoter and the expression of a selectable surface marker is driven by an independent constitutive promoter UBC. This vector is used for transduction of bone marrow-derived macrophage precursors. The transduced cells can be enriched to 95-99% purity using marker-specific monoclonal antibodies, expanded and differentiated into mature macrophages or myeloid dendritic cells. We also successfully used this approach for inducible protein expression in hard to transfect macrophage cell lines.Because many proteins, which are expressed by activated or infected macrophages, possess cytotoxic, anti-proliferative or pro-apoptotic activities, generation of stable macrophage cell lines that constitutively express those proteins is impossible. Our method will be especially useful to study immunity-related macrophage proteins in their physiological context during macrophage activation or infection.

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“…44 Gene transfer studies were carried out using Btk/Tec doubly deficient mice for several key reasons. First, BtkTec Ϫ/Ϫ mice exhibit a pre-B-cell developmental block essentially identical with human XLA.…”

Section: Transduction Of Btktec ؊/؊ Bm Progenitors Using a Retroviralmentioning

confidence: 99%

“…43 Briefly, the vector MSCVhuBtk-SAR (subsequently referred to as MBS; Figure 1A) was generated as follows: human ␤-interferon scaffold attachment region (SAR), 44 obtained as an 877-bp fragment after XhoI and ClaI digestion of pMND-IRES-GFP-SAR (kindly provided by Dr Donald Kohn, University of Southern California [USC]), was inserted into the ClaI site of MSCV-ires-GFP 45 by a blunt-end ligation following end-fill reaction. Full-length human BTK coding sequence (98% identical with murine Btk protein) 12 was then inserted into XhoI and HindIII sites.…”

Section: Retroviral Vectors and Producer Cell Linesmentioning

confidence: 99%

Sustained correction of B-cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral-mediated gene transfer

Tabuchi

Kato

et al. 2004

Blood

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IntroductionX-linked agammaglobulinemia (XLA) is the prototypic primary humoral immunodeficiency disorder, first described by Bruton who reported a boy with severe recurrent infections and absence of the gamma-globulin serum fraction. 1 This disorder has been of major interest for more than half a century, initiating an ongoing and fruitful search for the genetic basis of this and other primary immunodeficiency diseases.XLA is characterized by a lack of mature B cells and plasma cells and a profound deficiency of all immunoglobulin types. Most patients develop recurrent infections coincident with the loss of maternal antibodies. Pyogenic infection with encapsulated bacteria is the most common clinical manifestation. [2][3][4] Current therapy consists of regular immunoglobulin replacement and prompt attention to infection.Both XLA and a related murine immunodeficiency, X-linked immunodeficiency (Xid), result from deficiencies in Bruton tyrosine kinase (Btk). [5][6][7][8][9][10] Btk, a member of the Tec family of nonreceptor kinases, 11 is expressed throughout B-lineage development except in plasma cells. 12,13 Btk contains a Src homology 1 (SH1) catalytic domain, SH2 and SH3 protein interaction domains, and a unique amino-terminus with a pleckstrin homology (PH) domain. 14 Mutational studies indicate important functional roles for each of these domains. 15 The major developmental defect in XLA occurs at the pre-Bcell transition, resulting in an increase in pro-B cells and a marked reduction in cycling pre-B cells and all subsequent stages. [16][17][18] Few IgM ϩ B cells are detectable in the blood and the hypotrophic lymphoid tissues. 19,20 Clonal expansion at the pre-B stage is regulated by pre-B-cell receptor (pre-BCR) signaling and is essential for generating an adequate pool of immature B cells. 21 Pre-BCR engagement leads to the generation of a "signalosome" that includes activated Btk, 22 an event disrupted in XLA.Several murine models of Btk deficiency currently exist. Xid mice have a spontaneous missense mutation in the PH domain. 23 The mutant protein is inefficiently recruited to the plasma membrane and fails to enter the BCR signalosome. 24 The phenotype of both Xid mice and Btk null-knockout mice (Btk Ϫ/Ϫ ) is less severe than that of XLA. [25][26][27][28] These mice produce nearly normal numbers of peripheral B cells, but splenic B-cell development is significantly compromised. Btk-deficient transitional 2 (T2) immature B cells fail to generate the BCR-dependent pro-survival, proliferative, and differentiation signals required to produce mature B An Inside Blood analysis of this article appears in the front of this issue.Reprints: David J. Rawlings, Children's Hospital and Regional Medical Center, 307 Westlake Ave North, Suite 300, Seattle, WA 98109; e-mail: drawling@u.washington.edu.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 173...

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Addition of the Human Interferon β Scaffold Attachment Region to Retroviral Vector Backbones Increases the Level ofin VivoTransgene Expression among Progeny of Engrafted Human Hematopoietic Stem Cells

Cited by 22 publications

References 34 publications

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

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

Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages

Sustained correction of B-cell development and function in a murine model of X-linked agammaglobulinemia (XLA) using retroviral-mediated gene transfer

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