Massachusetts Institute of Technology

Irving M London

Philippe LeBoulch

Jane-Jane Chen

Dorothy Y Tuan

William B Solomon

Gene therapy methods and compositions for high level expression of anti-sickling globin proteins in erythroid cells for treating Sickle Cell disease are described.

Compositions and methods for treating sickle cell disease

The cDNA which encodes heme-regulated eIF-2.alpha. kinase (HRI) has been cloned from a lambda Zap II cDNA library of rabbit reticulocytes. The rabbit HRI cDNA is highly homologous to human HRI and hybridizes to the human HRI DNA under moderately stringent conditions. The rabbit HRI cDNA contains 2729 amino acids. In vitro translation of HRI mRNA transcribed from HRI cDNA yields a 90 kDa polypeptide with eIF-2.alpha. kinase activity. Since HRI is a potent inhibitor of protein synthesis, it is anti-proliferative in nature. In addition, the unusually high degree of homology of HRI to three protein kinases involved in the regulation of cell division suggests that HRI plays a direct role in the regulation of cell division. Since regulation of protein synthesis is vital for cell growth and differentiation, the cDNA can be inserted into cells to manipulate proliferation and differentiation, especially of cells that are proliferating in an uncontrolled manner or characterized by arrested differentiation, such as some of the types of cancers. Initiation of protein synthesis can also be regulated by another eIF-2.alpha. kinase which is activated by double-stranded RNA (dsI) which represents an interferon-mediated response to viral infection. Deletion mutants of HRI cDNA can be constructed that are insensitive to regulation by heme, which should be more effective than native HRI in its anti-viral and anti-proliferative action.

DNA encoding the heme-regulated eukaryotic initiation factor 2.alpha.

A process and means for the design and the optimization of retroviral vectors transducing human .beta.-globin gene and .beta.-Locus Control Region (.beta.-LCR) derivatives, hereafter referred to as [.beta.-globin/LCR] retroviruses, which successfully meet the following criteria required for gene therapy applications: (1) stability of proviral transmission (low frequency of rearrangements similar to retroviral vectors considered stable in the art) upon infection of cell-lines and murine bone marrow cells, (2) improved viral titer, thereby allowing successful infection of bone marrow cells, and (3) high erythroid expression of the transduced human .beta.-globin gene, are described, along with specific constructs.

Retroviral vectors for transducing .beta.-globin gene and .beta.-locus

The cDNA which encodes heme-regulated eIF-2.alpha. kinase (HRI) has been cloned from a lambda Zap II cDNa of rabbit reticulocytes. The rabbit HRI cDNA is highly homologous to human HRI and hybridizes to the human HRI DNA under moderately stringent conditions. The rabbit HRI cDNA contains 2729 amino acids. In vitro translation of HRI mRNA transcribed from HRI cDNA yields a 90 kDa polypeptide with eIF-2.alpha. kinase activity. Since HRI is a potent inhibitor or protein synthesis, it is anti-proliferative in nature. In addition, the unusually high degree of homology of HRI to three protein kinases involved in the regulation of cell division suggests that HRI plays a direct role in the regulation of cell division. Since regulation of protein synthesis is vital of cell growth and differentiation, the cDNA can be inserted into cells to manipulate proliferation and differentiation, especially of cells that are proliferating in an uncontrolled manner of characterized by arrested differentiation, such as some of the types of cancers. Deletion mutants of HRI cDNA can be constructed that are insensitive to regulation by heme, which should be more effective than native HRI in its anti-viral and anti-proliferative action.

A human erythroid specific enhancer element is described. The enhancer element can be used to enhance transcription of a structural gene in erythroid cells. Methods for gene therapy employing the enhancer element are also disclosed.

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Cambridge, MA, United States of America

Irving M London