Domenico Valerio

Adenosine deaminase: characterization and expression of a gene with a remarkable promoter.

Cosmid clones containing the gene for human adenosine deaminase (ADA) were isolated. The gene is 32 kb long and split into 12 exons. The exact sizes and boundaries of the exon blocks including the transcription start sites were determined. The sequence upstream from this cap site lacks the TATA and CAAT boxes characteristic for eukaryotic promoters. Nevertheless, we have shown in a functional assay that a stretch of 135 bp immediately preceding the cap site has promoter activity. This 135‐bp DNA fragment is extremely rich in G/C residues (82%). It contains three inverted repeats that allow the formation of cruciform structures, a 10‐bp and a 16‐bp direct repeat and five G/C‐rich motifs (GGGCGGG) disposed in a strikingly symmetrical fashion. Some of these structural features were also found in the promoter region of other genes and we discuss their possible function. Knowledge of the exact positions of the intron‐exon boundaries allowed us to propose models for abnormal RNA processing that occurs in previously investigated ADA‐deficient cell lines.

One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity.

We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA‐SCID patient. In addition, wild‐type sequences could be detected at the same positions, indicating a compound heterozygosity. Studies with ADA expression clones mutagenized in vitro showed that the mutation at position 304 is responsible for ADA inactivation.

Construction and expression of an adenosine deaminase::lacZ fusion gene

A eukaryotic expression vector was constructed in which the coding nucleotide sequences (ADA) of human adenosine deaminase (ADA) were fused in frame with the coding sequences of the bacterial gene lacZ encoding beta-galactosidase (beta Gal). This ADA::lacZ fusion gene was anticipated to encode a hybrid protein that has retained the biological functions of both proteins. Transfection of mammalian cells with the fusion gene resulted in the synthesis of both ADA and beta Gal. Cells expressing the gene could therefore be detected with the histochemical staining procedure that relies on the conversion of the indicator, XGal, by beta Gal. In addition, the transfected cells could be sorted on a fluorescence-activated cell sorter with the use of a vital staining procedure described for the selection of beta Gal-producing cells. Cell lines that harbored the fusion gene were tested for ADA overexpression by exposing them to the cytotoxic adenosine analog 9-beta-D-xylofuranosyl adenine (Xyl-A), in the presence of the ADA inhibitor deoxycoformycin (dCF). Resistance to Xyl-A/dCF was observed in the lines carrying ADA::lacZ and moreover, the fraction of cells that survived a stringent selection for ADA overexpression also exhibited significantly increased levels of beta Gal, which confirmed the direct linkage between ADA and lacZ expression. The use of this and other fusion genes might be useful in the development of gene-therapy protocols where they could help to meet the demand for versatile methods to detect and select cells with newly introduced genes.

Towards Gene Therapy for Adenosine Deaminase Deficiency

Deficiency of adenosine deaminase (ADA) activity causes an autosomally inherited form of severe combined immunodeficiency (ADA¯SC1D) disease (1, 2). It has been suggested that this form of SCID is caused by a defect in T- and B-cell differentiation due to the accumulation of adenine nucleosides in the absence of functional ADA (2). The cloning of sequences encoding human ADA (3, 4, 5) opened new ways to investigate the molecular basis of ADA¯SCID disease (6–10) and allowed studies aimed at the development of gene therapy protocols for ADA¯SCID patients (11–15).

Toward Gene Therapy in Hemophilia A: Introduction of Factor VIII Expression Vectors Into Somatic Cells

To study and evaluate the potential of gene therapy for the treatment of hemophilia A, we have developed a retroviral vector system for the introduction and expression of factor VIII cDNA in somatic cells in vivo Bone-marrow cells were infected with the recombinant retrovirus. Transplantation of infected bone-marrow cells into lethally irradiated mice resulted in the formation of spleen colonies. Although the presence of the vector provirus could be detected in the genomes of the stem-cell-derived cells, no synthesis of human factor VIII or its RNA could be detected. As an alternative approach, factor-VIII-producing fibroblasts of human and murine origin were implanted into athymic nude mice, embedded in an artificial collagen matrix. In the case of human skin fibroblasts, cells isolated from the grafts 4 weeks after implantation still have the capacity to secrete factor VIII when regrown in culture. However, no human factor VIII could be detected in the plasma of the recipient mice. From these data we conclude that retroviral vectors can be used for gene transfer into somatic cells of laboratory animals in vivo. The cause of the apparent lack of expression will be discussed.

Progress in the Development of Somatic Cell Gene Therapy for Adenosine Deaminase Deficiency

The objective of somatic cell gene therapy procedures is to correct genetic disorders by inserting a functional gene into the genome of either the affected tissue or into a readily accessable tissue in which expression of the introduced gene is expected to have a therapeutic effect. The accessability, the ability to transplant upon ex vivo manipulation and the selfrenewing potential of hemopoietic stem cells present in bone marrow render the hemopoietic system one of the prime target organs for gene transfer in somatic cell gene therapy procedures. A suitable model disorder for gene therapy is adenosine deaminase (ADA) deficiency, which causes an autosomally inherited form of severe combined immunodeficiency (ADA-SCID). The disease is restricted to the hemopoietic system, where the absence of functional ADA results in a defect in T- and B-cell differentiation [1]. Genetic correction of ADA-deficient hemopoietic stem cells should therefore result in a complete and lasting cure. In addition, it is believed that genetically cured blood cells will have a selective advantage over affected cells, because selective replication of normal T cells from a histocompatible donor appears to take place upon infusion into ADA-SCID patients [2].

A Novel Approach for the Selection and Detection of Cells Transfected with Adenosine Deaminase Expression Vectors

One of the prime candidates for somatic cell gene therapy is severe combined immunodeficiency (SCID) caused by a defect in the gene coding for adenosine deaminase (ADA). The development of gene therapy protocols for this disorder would greatly benefit from the availability of powerful methods for the detection and selection of cells expressing newly indroduced ADA vectors. Here we describe an expression vector that could help to meet such demands. A fusion gene was constructed which carries the sequences encoding human ADA fused in frame to the coding region of the bacterial beta-galactosidase gene (lacZ). We showed that this ADA-lacZ fusion gene encodes a hybrid protein in which the enzymatic activities known to be associated with ADA and beta-galactosidase are both retained. Cells expressing this gene could therefore be detected and selected using the sensitive and versatile methods that exist for cells producing the lacZ gene product.