Gene therapy for the thalessemia syndromes requires that the introduced globin gene mimic the function of an in situ normal globin gene both in tissue specificity and efficiency of transcription. DNA mediated gene transfer experiments have shown that the human beta-globin gene with its immediate 5' and 3' flanking sequences can be expressed in a tissue and developmental-stage specific manner. Yet high level transcription of such a gene may require sequence elements not residing in the globin structural gene and its immediate flanking sequences. Comparison of the extents of DNA deletions in the Dutch and English gamma delta beta-thalassemias suggests the possible existence of regulatory sequences upstream of the embryonic epsilon-globin gene, which can regulate the expression of the far downstream beta-globin gene. A segment of DNA at 10 Kb 5' of the epsilon-globin gene has been shown to display erythroid specific enhancer activity. It is therefore possible that transcriptional activation of the beta-globin gene may involve at least two synergistic activation steps, mediated respectively by this distant enhancer sequence, and by sequences much closer to the beta-globin gene. In DNA mediated gene transfer experiments, the presence of this enhancer element, in cis to the beta-globin or a test gene, may thus significantly enhance the transcriptional efficiency of such a gene in erythroid hosts. This proposal attempts to address this possibility. The enhancer element with other probable regulatory sequences will be spliced with a beta-globin or a test gene into enhancerless plasmids or into an enhancerless retroviral vector, which will subsequently be transiently or stably introduced into appropriate cell lines or into mouse bone marrow cells. The transcriptional efficiency of the test gene will be compared to that of the endogenous alpha-or beta-globin gene of the erythroid host cells. Identification of an erythroid specific, globin gene enhancer, understanding the molecular mechanism of its action on the beta- like globin genes, and its possible role in the coordinated activation of the alpha- and beta-like globin genes located on separate chromosomes, may all aid in elucidating the regulatory mechanism of the human beta-globin gene and thus in bringing gene therapy for the beta-thalassemias significant steps closer to reality.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL039948-01A1
Application #
3356967
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1988-07-01
Project End
1991-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
1
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Organized Research Units
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Ling, Jianhua; Baibakov, Boris; Pi, Wenhu et al. (2005) The HS2 enhancer of the beta-globin locus control region initiates synthesis of non-coding, polyadenylated RNAs independent of a cis-linked globin promoter. J Mol Biol 350:883-96
Ling, Jianhua; Ainol, Lincoyan; Zhang, Ling et al. (2004) HS2 enhancer function is blocked by a transcriptional terminator inserted between the enhancer and the promoter. J Biol Chem 279:51704-13
Ling, Jianhua; Pi, Wenhu; Yu, Xiuping et al. (2003) The ERV-9 LTR enhancer is not blocked by the HS5 insulator and synthesizes through the HS5 site non-coding, long RNAs that regulate LTR enhancer function. Nucleic Acids Res 31:4582-96
Ling, Jianhua; Pi, Wenhu; Bollag, Roni et al. (2002) The solitary long terminal repeats of ERV-9 endogenous retrovirus are conserved during primate evolution and possess enhancer activities in embryonic and hematopoietic cells. J Virol 76:2410-23
Ramchandran, R; Bengra, C; Whitney, B et al. (2000) A (GATA)(7) motif located in the 5' boundary area of the human beta-globin locus control region exhibits silencer activity in erythroid cells. Am J Hematol 65:14-24
Migliaccio, A R; Bengra, C; Ling, J et al. (2000) Stable and unstable transgene integration sites in the human genome: extinction of the Green Fluorescent Protein transgene in K562 cells. Gene 256:197-214
Long, Q; Bengra, C; Li, C et al. (1998) A long terminal repeat of the human endogenous retrovirus ERV-9 is located in the 5' boundary area of the human beta-globin locus control region. Genomics 54:542-55
Cavallesco, R; Tuan, D (1997) Modulatory subdomains of the HS2 enhancer differentially regulate enhancer activity in erythroid cells at different developmental stages. Blood Cells Mol Dis 23:8-26
Kong, S; Bohl, D; Li, C et al. (1997) Transcription of the HS2 enhancer toward a cis-linked gene is independent of the orientation, position, and distance of the enhancer relative to the gene. Mol Cell Biol 17:3955-65
Yoo, J; Herman, L E; Li, C et al. (1996) Dynamic changes in the locus control region of erythroid progenitor cells demonstrated by polymerase chain reaction. Blood 87:2558-67

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