4. Optimization of Globin Lentiviral Vector Design for the Treatment of β-Thalassemia
Authors: Leszek Lisowski, Stefano Rivella, Michel Sadelain
Journal: Molecular Therapy (2005)

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The stable introduction of a functional globin gene into human hematopoietic stem cells (HSC) is a potentially curative treatment for the |[beta]|-thalassemias and sickle-cell diseases. To this end, we are investigating the transcriptional requirements for expressing the highest possible levels of the human |[beta]|-globin gene in murine and primate erythroid cells.

The endogenous |[beta]|-globin gene is controlled by two proximal enhancers and distal regulatory elements known as the |[beta]|-globin locus control region (LCR), which spans 7 DNase hypersensitive sites (HS) located 50|[ndash]|80kb upstream of the human |[beta]|-globin gene. We have previously shown that the juxtaposition of HS 2, 3 and 4 with the |[beta]|-globin gene promoter from position |[minus]|615 and the 3|[prime]| |[beta]|-globin enhancer yield tissue specific and therapeutic |[beta]|-globin expression in thalassemic mice. Furthermore, addition of the LCR reduces the position effect and transcriptional inactivation.

To identify the best possible vector design before investigating globin gene transfer in phase I clinical studies, we are further investigating the role of these elements as well as insulators and lentiviral vector elements.

To investigate the role of HS1/HS4 in LCR function as well as the activity of different length |[beta]|-globin promoters on globin transgene expression we have generated a panel of constructs with specific modifications in the |[beta]|-globin promoter, HS1 and/or HS4 regions of the LCR. These constructs were transduced into murine erythroleukemia (MEL) cells to assess tissue specificity, stage specificity and average expression level of the vector-encoded human |[beta]|-globin gene.

In contrast to one previously published report, the extended promoter (1.6kb) proved to be less active in MEL cells. Conversely, the shortening the promoter from |[minus]|615 to |[minus]|265, and removal of the putative silencer element believed to be present in that region, might have enhanced |[beta]|-globin expression. The HS4 region of the LCR, containing GATA-1 and AP-1 binding sites, significantly contributes to expression of the human |[beta]|-globin gene in transgenic mice. AT rich flanking regions of HS4 might encompass scaffold/matrix-associated regions, which may play an important role in LCR controlled |[beta]|-globin gene expression. To test this hypothesis we deleted either the 3|[prime]| or 5|[prime]| AT rich flanking regions of HS4. Deletion of the 3|[prime]| flanking region did not affect the expression level, however truncation of the 5|[prime]|AT rich region decreased |[beta]|-globin gene expression in MEL cells.

Furthermore we have assessed the role of HS1 on |[beta]|-globin expression. HS1 has been reported to contain a GATA-1 binding site and provides position-independent expression on a linked human |[beta]|-globin gene in transgenic mice, without affecting the level of expression. Addition of HS1 did not affect the average globin gene expression in MEL cells, and we are further investigating the role of this element on |[beta]|-globin gene expression in vivo.

In light of our data we have selected four lentiviral vectors containing the human |[beta]|-globin gene under the control of |[beta]|-globin promoter with or without the silencer element as well as full HS4 elements with or without the HS1 element. We are currently studying these vectors in an animal model of |[beta]|-Thalassemia.