NOVEL APPROACHES TO INCREASE y-GLOBIN Expression in Sickle Cell Disease

Children'S Hospital Of Philadelphia, Philadelphia PA

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Abstract

Sickle cell disease is caused by a point mutation the beta-globin gene. Under hypoxic conditions, hemoglobin[unreadable] containing mutant beta-globin protein (HbS) forms insoluble polymers, leading to defects in red cell shape,[unreadable] flexibility and adhesion. Elevated levels of y-globin reduce hemoglobin polymerization and improve the[unreadable] clinical manifestation of the disease. Therefore, treatment of patients is aimed at raising expression of the y[unreadable] globin gene, which is, normally silent in adult life. This application proposes to explore two approaches to raise[unreadable] y-globin levels. Specific Aim I focuses on the use of synthetic DNA ligands, called polyamides, to raise the y[unreadable] to beta-globin ratio. Polyamides are low molecular weight, cell permeable polymers consisting of pyrrole and[unreadable] imidazole derivatives that brad to predetermined DNA sequences with specificities and affinities approaching[unreadable] those of natural DNA binding proteins. In collaboration with Dr. Peter Dervan whose laboratory is at the[unreadable] forefront in developing polyamides, we synthesized and purified several polyamides designed to inhibit[unreadable] transcription factor binding to essential reomalatory sites in the b-globin gene promoter. We will examine these[unreadable] polyamides in primary human bone marrow and umbilical cord erythroid cells for their effectiveness in[unreadable] inhibiting beta-globin expression in vivo. In addition, we will design new polyamides with the goal to activate y[unreadable] globin gene expression directlv. Studies in Specific Aim II will test a novel viral vector for its capacity to[unreadable] express a transgene in a fashion that is resistant to gene silencing. One major limitation of viral vectors is:[unreadable] that they are often subject to gene silencing, especially in hematopoietic (HS) and embryonic stem (ES) ceils[unreadable] and their differentiated progeny. Virally induced gene silencing is associated with deacetylation of histones,[unreadable] and treatment of cells with deacetvlase inhibitors leads to reactivation of viral gene expression. We have[unreadable] produced a viral vector that contains a modified histone acetvltransferase that binds specifically to the viral[unreadable] DNA sequence. The proposed studies wil! test this vector s ability to express a transgene at high levels and in a[unreadable] fashion resistant to gene silencing in HS and ES cells. The long term goal of this Specific Aim is to use this[unreadable] vector for y-globin gene expression in HS cells of test animals and ultimately patients with sickle cell disease.