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Lentivirus-mediated globin gene tran...

Weill Medical College of Cornell University.

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Lentivirus-mediated globin gene transfer for the treatment of severe hemoglobinopathies.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Lentivirus-mediated globin gene transfer for the treatment of severe hemoglobinopathies./
Author:	Lisowski, Leszek.
Description:	419 p.
Notes:	Adviser: Michel Sadelain.
Contained By:	Dissertation Abstracts International69-04B.
Subject:	Biology, Genetics. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoeng/servlet/advanced?query=3312025
ISBN:	9780549609674

Lentivirus-mediated globin gene transfer for the treatment of severe hemoglobinopathies.
Lisowski, Leszek.

Lentivirus-mediated globin gene transfer for the treatment of severe hemoglobinopathies. - 419 p.

Adviser: Michel Sadelain.

Thesis (Ph.D.)--Weill Medical College of Cornell University, 2008.

The beta-thalassemias are inherited autosomal recessive anemias, which are caused by over 200 mutations that diminish or abolish expression of the beta-globin gene. In the most severe forms in homozygotes or compound heterozygotes, the anemia is fatal within the first years of life in the absence of lifelong transfusion therapy. Globin gene transfer in autologous hematopoietic stem cells is a promising therapeutic option for subjects with beta-thalassemia major. In this approach, high level, erythroid-specific transgene expression should correct ineffective erythropoiesis following the delivery of ideally 1 or 2 copies per cell of the transferred human beta-globin gene. In order to increase the output of the vector encoded human globin gene, I have undertaken a systematic analysis of the contribution of the beta-globin promoter, HS1 and HS4 elements of the beta-globin locus control region (LCR) to the specificity, inducibility, long-term in vivo expression and therapeutic potential of globin lentiviral vectors. To this end, I created a panel of lentiviral vectors harboring different lengths of globin promoter, HS1 and HS4 elements of the LCR. These vectors were tested in vitro in murine erythroleukemia (MEL) cells and in beta-thalassemic mice in vivo. Transgene expression was measured at the mRNA level using quantitative primer extension and at the protein level measuring total and chimeric (mouse alpha2 - human beta2) hemoglobin. Expression was normalized to vector copy number obtained from southern blot and/or TaqMan analysis of the genomic DNA extracted from cells or total peripheral blood of treated animals. I show here the major roles played by the beta-globin promoter, HS1 and HS4 in vivo in beta-thalassemic mice. The 265bp promoter together with LCR containing HS2, 3 and 4 drives the highest globin expression in vitro in MEL cells but not in vivo. Promoters ranging from 130 to 1555bp vary globin expression over a 2.5-fold range. Partial deletions of HS4 reduce globin expression by 44% and, most strikingly, addition of HS1 increases beta-globin transgene expression by 52% leading to substantial improvement in correcting anemia in beta-thalassemic mice. Vector encoded globin transgene was also expressed in vitro in human Hel (Human Erythroleukemia) and K562 (Human myelogenous leukemia) cell lines as well as in human embryonic stem cells derived erythroid cells. This analysis underscores the importance of carefully analyzing the size and relative positioning of transcriptional control elements within tissue-specific vectors and the need to carefully quantify vector expression in animal models before undertaking clinical studies.

ISBN: 9780549609674Subjects--Topical Terms:

1017730
Biology, Genetics.

Lentivirus-mediated globin gene transfer for the treatment of severe hemoglobinopathies.
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245 1 0 $a Lentivirus-mediated globin gene transfer for the treatment of severe hemoglobinopathies.
300 $a 419 p.
500 $a Adviser: Michel Sadelain.
500 $a Source: Dissertation Abstracts International, Volume: 69-04, Section: B, page: 2077.
502 $a Thesis (Ph.D.)--Weill Medical College of Cornell University, 2008.
520 $a The beta-thalassemias are inherited autosomal recessive anemias, which are caused by over 200 mutations that diminish or abolish expression of the beta-globin gene. In the most severe forms in homozygotes or compound heterozygotes, the anemia is fatal within the first years of life in the absence of lifelong transfusion therapy. Globin gene transfer in autologous hematopoietic stem cells is a promising therapeutic option for subjects with beta-thalassemia major. In this approach, high level, erythroid-specific transgene expression should correct ineffective erythropoiesis following the delivery of ideally 1 or 2 copies per cell of the transferred human beta-globin gene. In order to increase the output of the vector encoded human globin gene, I have undertaken a systematic analysis of the contribution of the beta-globin promoter, HS1 and HS4 elements of the beta-globin locus control region (LCR) to the specificity, inducibility, long-term in vivo expression and therapeutic potential of globin lentiviral vectors. To this end, I created a panel of lentiviral vectors harboring different lengths of globin promoter, HS1 and HS4 elements of the LCR. These vectors were tested in vitro in murine erythroleukemia (MEL) cells and in beta-thalassemic mice in vivo. Transgene expression was measured at the mRNA level using quantitative primer extension and at the protein level measuring total and chimeric (mouse alpha2 - human beta2) hemoglobin. Expression was normalized to vector copy number obtained from southern blot and/or TaqMan analysis of the genomic DNA extracted from cells or total peripheral blood of treated animals. I show here the major roles played by the beta-globin promoter, HS1 and HS4 in vivo in beta-thalassemic mice. The 265bp promoter together with LCR containing HS2, 3 and 4 drives the highest globin expression in vitro in MEL cells but not in vivo. Promoters ranging from 130 to 1555bp vary globin expression over a 2.5-fold range. Partial deletions of HS4 reduce globin expression by 44% and, most strikingly, addition of HS1 increases beta-globin transgene expression by 52% leading to substantial improvement in correcting anemia in beta-thalassemic mice. Vector encoded globin transgene was also expressed in vitro in human Hel (Human Erythroleukemia) and K562 (Human myelogenous leukemia) cell lines as well as in human embryonic stem cells derived erythroid cells. This analysis underscores the importance of carefully analyzing the size and relative positioning of transcriptional control elements within tissue-specific vectors and the need to carefully quantify vector expression in animal models before undertaking clinical studies.
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