Gene therapy using hematopoietic stem cells (HSCs) as the target cell population has great potential to improve treatment of a wide range of inherited and acquired blood diseases. Replication-defective retroviruses have been the vehicles of choice for gene delivery and expression in HSCs because of their ability to stably integrate into the genome of target cells. For more than a decade, our laboratory has been designing and optimizing retroviral vectors for gene transfer studies of HSC biology. In particular, our MSCV (murine stem cell virus) retroviral vector has proven to be highly efficient at delivering functional genes to the murine hematopoietic system. For this reason, the MSCV platform was chosen for use in two HSC gene therapy trials currently underway in the United States. To date, however, the outcomes of most clinical trials with retroviral vectors have been disappointing. This is believed to be due in part to low surface density of the amphotropic envelope receptor and the fact that retroviral vectors such as MSCV, which are derived from oncoretroviruses, can only integrate into cells undergoing mitosis. Thus it has been proposed that pantropic vectors developed from the lentivirus, human immunodeficiency virus (HJV), which can readily transfer genes into various types of stationary cells, may be more suitable for gene delivery to HSCs, which reside almost exclusively in the G0/G1 phase of the cell cycle. Even if efficient lentivirus-based gene transfer in HSCs is achieved, accumulated data indicate that in vivo transgene expression is frequently subject to transcriptional silencing and position effects. We propose therefore to develop next-generation HIV-based lentiviral vectors expressly for human HSC gene transfer applications. Our hypothesis is that utilization of transcriptional regulatory elements permissive for expression in HSCs in conjunction with chromatin insulator sequences and scaffold/matrix attachment regions will lead to maintenance of high-level transgene expression in HSCs and their differentiated progeny. To this end, the performance of next-generation lentiviral vectors utilizing the MSCV long terminal repeat as an internal promoter and harboring the chicken b-globin 5' constitutive hypersensitive site (5' HS4) insulator and/or the human interferon-b scaffold attachment region (IFN-SAR) will be assessed in human hematopoietic repopulating cells using a surrogate non-obese diabetic/severe combined immunodeficient (NOD/SCID) xenograft assay and in a murine hemophilia A model.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL065519-03
Application #
6638689
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Qasba, Pankaj
Project Start
2001-06-15
Project End
2004-06-30
Budget Start
2003-06-01
Budget End
2004-06-30
Support Year
3
Fiscal Year
2003
Total Cost
$346,950
Indirect Cost
Name
American National Red Cross
Department
Type
DUNS #
003255213
City
Washington
State
DC
Country
United States
Zip Code
20006
Hawley, Teresa S; Riz, Irene; Yang, Wenjing et al. (2013) Identification of an ABCB1 (P-glycoprotein)-positive carfilzomib-resistant myeloma subpopulation by the pluripotent stem cell fluorescent dye CDy1. Am J Hematol 88:265-72
Hawley, Robert G; Chen, Yuzhong; Riz, Irene et al. (2012) An Integrated Bioinformatics and Computational Biology Approach Identifies New BH3-Only Protein Candidates. Open Biol J 5:6-16
Ramezani, Ali; Zweier-Renn, Lynnsey A; Hawley, Robert G (2011) Factor VIII delivered by haematopoietic stem cell-derived B cells corrects the phenotype of haemophilia A mice. Thromb Haemost 105:676-87
Riz, Irene; Hawley, Teresa S; Hawley, Robert G (2011) Lentiviral fluorescent protein expression vectors for biotinylation proteomics. Methods Mol Biol 699:431-47
Riz, Irene; Zweier-Renn, Lynnsey A; Toma, Ian et al. (2011) Apoptotic role of IKK in T-ALL therapeutic response. Mol Cancer Res 9:979-84
Ramezani, Ali; Hawley, Robert G (2010) Strategies to insulate lentiviral vector-expressed transgenes. Methods Mol Biol 614:77-100
Riz, Irene; Hawley, Teresa S; Luu, Truong V et al. (2010) TLX1 and NOTCH coregulate transcription in T cell acute lymphoblastic leukemia cells. Mol Cancer 9:181
Zweier-Renn, Lynnsey A; Hawley, Teresa S; Burkett, Sandra et al. (2010) Hematopoietic immortalizing function of the NKL-subclass homeobox gene TLX1. Genes Chromosomes Cancer 49:119-31
Riz, Irene; Lee, Hyo Jung; Baxter, Kristin K et al. (2009) Transcriptional activation by TLX1/HOX11 involves Gro/TLE corepressors. Biochem Biophys Res Commun 380:361-5
Ramezani, Ali; Hawley, Robert G (2009) Correction of murine hemophilia A following nonmyeloablative transplantation of hematopoietic stem cells engineered to encode an enhanced human factor VIII variant using a safety-augmented retroviral vector. Blood 114:526-34

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