NFP37 SOMATIC GENE THERAPY Divulgation Abstracts phase B

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FILIPOWICZ,Aleksandra	Kantonsspital Basel, Dept. Forschung, Experimentelle Hämatologie, Hebelstr. 20, CH - 4031 Basel, tel 061 265 2332; fax 061 265 2350 e-mail: Aleksandra.Wodnar-Filipowicz@unibas.ch
Title:	Lentivirus-mediated gene transfer into hematopoietic precursor cells from unbilical cord blood and fetal liver
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DIVULGATION TEXT AT SUBMISSION (1998):

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Genetic modification of hematopoietic stem cells is a promising approach for the treatment of inherited disorders of the hematopoietic system, provided the relevant gene can be stably expressed in cells with capacity to self-renew and differentiate to the affected hematopoietic lineages. Recently, lentiviral vectors, based on the human immunodeficiency virus type 1 (HIV-1), have been developed and shown to be efficient for the delivery and expression of genes in non- dividing cells, including primitive hematopoietic precursors.

Umbilical cord blood (CB) is an established source of hematopoietic stem cells for allogeneic transplantation. Moreover, autologous CB has been used for gene therapy in children with adenosine deaminase deficiency. With advances in prenatal diagnosis, congenital defects can now be identified in the first trimester of pregnancy, calling for the earliest possible therapeutic intervention to prevent the manifestation of disease. This could be achieved by stem cell transplantation in utero before the development of any tissue damage. Allogeneic transplantation has been successful in fetuses with congenital immunodeficiency syndromes profiting from a selective survival advantage of genetically corrected lymphoid progenitors. However, engraftment has not been possible in other hereditary disorders, such as hemoglobinopathies or storage diseases, likely due to immune intolerance and competition by the host bone marrow environment. The limitations and risks associated with immunological barriers in acceptance of allogeneic grafts might be circumvented by gene therapy with autologous genetically corrected CB cells transplanted in utero. While postnatal gene therapy is already possible for some diseases, therapy with autologous CB stem cells harvested during pregnancy and genetically modified ex vivo has not yet been attempted.

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We have recently demonstrated that fetal CB obtained from human fetuses in the second and early third trimester of pregnancy is rich in hematopoietic precursors with high self-renewal capacity (Wyrsch et al, Exp Hematol 27:1338-1345, 1999). Here, we describe the use of a lentivirus-based gene transfer vector for efficient transduction of human CD34⁺ progenitor cells from CB of early gestational stages with the GFP marker gene. The results of our experiments show that CB progenitors obtained in the second and early third trimester can be targeted for gene transfer by lentiviral vectors and may, therefore, prove useful for autologous gene therapy of genetic diseases amenable to prenatal stem cell transplantation. The Figure illustrates hematopoietic colonies expressing GFP, used a marker transgene for lentiviral vector-based transduction of primitive LTC- ICs from human CB.

Use of lentiviral vectors for gene transfer in a clinical setting will require further modifications in vector design. These will include choice of promoters or inclusion of regulatory regions preventing a gradual downregulation of expression of a transferred gene. Also design of vectors with exclude the possibility of recombination of infective viral particles belongs to important issues of optimizing the biological safety of this system prior to clinical application. Gene therapy in utero is a novel concept; its clinical realization will depend on careful consideration of risks and benefits to the mother and child; the medical and ethical aspects of this novel treatment option in humans are currently under consideration.

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