Gene therapy
The objective of the gene therapy of beta-thalassemia is a substitution, in the erythroid progenitor cells of the patients, of the damaged beta-globin gene with a functional beta-globin gene.
Fig.1 shows the different steps followed for a stable insertion of a functional gene within the genome of cells isolated from patients. In this case, the gene to be used is inserted within the genome of a retrovirus, suitably modified in order to render the viral particle able to infect target cells, insert the carried genetic material in the genome, but unable to extend infection to other cells.
The most critical point of this experimental protocol is the development of suitable vector able to penetrate target cells. The second point is related to the fact that the "therapeutic gene" must be inserted within the genome of target cells; therefore, the vector genome should contain all the sequences involved in integration of its genome within the host genome. All these features are a biological feature of certain viruses, the retroviruses (Fig.2): they are able to penetrate within host cells, insert their genomic DNA within the genome of infected cells, and synthesize all the components necessary to generate a activety infective progeny. The problem with the use of retrovirus is that they could be potentially dangerous for human health. Fig.3 shows the genome of a retrovirus; among the indicated genes, those coding gag, pol and env are eliminated to the aim of generating a vector able of integration within the host genome, but unable to generate infective particles. In place of these sequences, the retroviral vector contains the "therapeutic gene".
While genetic approaches aiming to increase normal beta-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants, low-level expression, position effects and transcriptional silencing are a problem and hampered the effectiveness of viral transduction of the human beta-globin gene when it was linked to minimal regulatory sequences.
Of great interest is the recent report by May et al. (May C., Rivella S., Callegari J., Heller G., Gaensler KM., Luzzatto L., Sadelain M.: "Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin", Nature, 406, 82-6, 2000). In this paper, the authors demonstrated that a stable introduction of a functional beta-globin gene in haematopoietic stem cells could be a powerful approach to treat beta-thalassaemia and sickle-cell disease. They showed that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine alpha-globin and two human betaA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In beta-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.
The results obtained by Sadelain's group are important because for the first time the gene able to correct a genetic function has been inserted together with all the sequences regulating the production of the beta-globin in erythroid cells. Usually these sequences, belonging to the LCR (Locus Control Region) are not directly linked to the globin genes. The scientific importance of these studies is that only a minimal region of the LCR has been used, however sufficient to direct a correct expression of the beta-globin gene.
These results are very promising; however, the way that will bring gene therapy to be applied to man is still long. The basic point is that the results obtained on experimental mice have still to be demonstrated to be applied to man.
