Erythrocyte ankyrin promoter mutations associated with recessive hereditary spherocytosis cause significant abnormalities in ankyrin expression.

Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In several kindreds with recessive, ankyrin-deficient HS, mutations have been identified in the ankyrin promoter that have been proposed to decrease ankyrin synthesis. We analyzed the effects of two mutations, -108T to C and -108T to C in cis with -153G to A, on ankyrin expression. No difference between wild type and mutant promoters was demonstrated in transfection or gel shift assays in vitro. Transgenic mice with a wild type ankyrin promoter linked to a human (A)gamma-globin gene expressed gamma-globin in 100% of erythrocytes in a copy number-dependent, position-independent manner. Transgenic mice with the mutant -108 promoter demonstrated variegated gamma-globin expression, but showed copy number-dependent and position-independent expression similar to wild type. Severe effects in ankyrin expression were seen in mice with the linked -108/-153 mutations. Three transgenic lines had undetectable levels of (A)gamma-globin mRNA, indicating position-dependent expression, and four lines expressed significantly lower levels of (A)gamma-globin mRNA than wild type. Two of four expressing lines showed variegated gamma-globin expression, and there was no correlation between transgene copy number and RNA level, indicating copy number-independent expression. These data are the first demonstration of functional defects caused by HS-related, ankyrin gene promoter mutations.

Development of a stable retrovirus vector capable of long-term expression of gamma-globin mRNA in mouse erythrocytes.

Gene therapy for patients with hemoglobin disorders such has been hampered by the inability of retrovirus vectors to transfer globin genes and the locus control region (LCR) into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells as a result of position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Transgenic mice containing the human ankyrin (Ank) gene promoter fused to the human gamma-globin gene showed position-independent, copy number-dependent expression of a linked gamma-globin mRNA. We generated a "double-copy" Ank/A gamma-globin retrovirus vector that transferred two copies of the Ank/A gamma-globin gene into target cells. Stable gene transfer was observed in primary primary mouse progenitor cells and long-term repopulating hematopoietic stem cells. Expression of Ank/A gamma-globin mRNA in mature red blood cells was approximately 8% of the level of mouse alpha-globin mRNA. We conclude that this novel retrovirus vector may be valuable for treating a variety of hemoglobinopathies by gene therapy if the level of expression can be further increased.

Long-term expression of gamma-globin mRNA in mouse erythrocytes from retrovirus vectors containing the human gamma-globin gene fused to the ankyrin-1 promoter.

Gene therapy for patients with hemoglobin disorders has been hampered by the inability of retrovirus vectors to transfer globin genes and their cis-acting regulatory sequences into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells due to position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Recently, we have shown that the human ankyrin (Ank) gene promoter directs position-independent, copy number-dependent expression of a linked gamma-globin gene in transgenic mice. We inserted the Ank/(A)gamma-globin gene into retrovirus vectors that could transfer one or two copies of the Ank/(A)gamma-globin gene to target cells. Both vectors were stable, transferring only intact proviral sequences into primary mouse hematopoietic stem cells. Expression of Ank/(A)gamma-globin mRNA in mature red blood cells was 3% (single copy) and 8% (double copy) of the level of mouse alpha-globin mRNA. We conclude that these novel retrovirus vectors may be valuable for treating a variety of red cell disorders by gene replacement therapy including severe beta-thalassemia if the level of expression can be further increased.

A minimal ankyrin promoter linked to a human gamma-globin gene demonstrates erythroid specific copy number dependent expression with minimal position or enhancer dependence in transgenic mice.

In red blood cells ankyrin (ANK-1) provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane. We have previously demonstrated that a 271-bp 5'-flanking region of the ANK-1 gene has promoter activity in erythroid, but not non-erythroid, cell lines. To determine whether the ankyrin promoter could direct erythroid-specific expression in vivo, we analyzed transgenic mice containing the ankyrin promoter fused to the human (A)gamma-globin gene. Sixteen of 17 lines expressed the transgene in erythroid cells indicating nearly position-independent expression. We also observed a significant correlation between the level of Ank/(A)gamma-globin mRNA and transgene copy number. The level of Ank/(A)gamma mRNA averaged 11% of mouse alpha-globin mRNA per gene copy at all developmental stages. The addition of the HS2 enhancer from the beta-globin locus control region to the Ank/(A)gamma-globin transgene resulted in Ank/(A)gamma-globin mRNA expression in embryonic and fetal erythroid cells in six of eight lines but resulted in absent or dramatically reduced levels of Ank/(A)gamma-globin mRNA in adult erythroid cells in eight of eight transgenic lines. These data indicate that the minimal ankyrin promoter contains all sequences necessary and sufficient for erythroid-specific, copy number-dependent, position-independent expression of the human (A)gamma-globin gene.

Isolation of stem cell-specific cDNAs from hematopoietic stem cell populations.

We have begun to isolate gene sequences that are specifically expressed in hematopoietic stem cells (HSCs). There are at least three fundamental requirements for the isolation of HSC-specific transcripts. First, highly enriched populations of HSCs, and an HSC-depleted cell population for comparison must be isolated. Secondly, the gene isolation procedures must be adapted to accommodate the small amounts of RNA obtained from purified HSCs. Finally, a defined screening strategy must be developed to focus on sequences to be examined in more detail. In this report, we describe the characterization of populations of HSCs that are highly enriched (Lin- c-kitHI) or depleted (Lin- c-kitNEG) of HSCs. We compared two methods for gene isolation, differential display polymerase chain reaction (DD-PCR) and subtractive hybridization (SH), and found that the latter was more powerful and efficient in our hands. Lastly we describe the strategy that we have developed to screen clones for further study.

A human beta-spectrin gene promoter directs high level expression in erythroid but not muscle or neural cells.

beta-Spectrin is an erythrocyte membrane protein that is defective in many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. It is expressed not only in erythroid tissues but also in muscle and brain. We wished to determine the regulatory elements that determine the tissue-specific expression of the beta-spectrin gene. We mapped the 5'-end of the beta-spectrin erythroid cDNA and cloned the 5'-flanking genomic DNA containing the putative beta-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, a beta-spectrin gene erythroid promoter with two binding sites for GATA-1 and one site for CACCC-related proteins was identified. All three binding sites were required for full promoter activity; one of the GATA-1 motifs and the CACCC-binding motif were essential for activity. The beta-spectrin gene promoter was able to be transactivated in heterologous cells by forced expression of GATA-1. In transgenic mice, a reporter gene directed by the beta-spectrin promoter was expressed in erythroid tissues at all stages of development. Only weak expression of the reporter gene was detected in muscle and brain tissue, suggesting that additional regulatory elements are required for high level expression of the beta-spectrin gene in these tissues.

Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice.

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.

Improved amphotropic retrovirus-mediated gene transfer into hematopoietic stem cells.

The efficiency of amphotropic retrovirus-mediated gene transfer into human Hematopoietic Stem Cells (HSC) is less than 1%. This has impeded gene therapy for hematopoietic diseases. In this study we demonstrate that populations of mouse and human HSC contain low to undetectable levels of the amphotropic virus receptor mRNA (ampho R mRNA), and are resistant to transduction with amphotropic retroviral vectors. In a subpopulation of mouse HSC expressing 7-fold higher levels of ampho R mRNA, transduction with amphotropic retrovirus vectors was 30-fold higher. We conclude that retrovirus transduction of HSC correlates with ampho R mRNA levels. Our results predict that alternative sources of HSC or retroviruses will be required for human gene therapy of hematopoietic diseases. One alternative source of stem cells is from individuals treated with cytokines. We have previously shown that mice treated with G-CSF and SCF have an immediate increase in peripheral blood HSC immediately after treatment, followed by a 10-fold increase in bone marrow HSC 14 days after treatment. In this report we show that when rhesus monkey bone marrow cells collected 14 days after G-CSF and SCF treatment were transduced with amphotropic retroviruses, gene transfer levels were approximately 10%, which was easily detected by Southern blot analysis. We conclude that the increased gene transfer may be the result of increased expression of the amphotropic retrovirus receptor, increased numbers of cycling HSC or both.

Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.

In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells to the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells saving the most primitive CD34+/CD38- or CD34+/CD38dim phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.

The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.

The low level of amphotropic retrovirus-mediated gene transfer into human hematopoietic stem cells (HSC) has been a major impediment to gene therapy for hematopoietic diseases. In the present study, we have examined amphotropic retrovirus receptor (amphoR) and ecotropic retrovirus receptor mRNA expression in highly purified populations of mouse and human HSC. Murine HSC with low to undetectable levels of amphoR mRNA and relatively high levels of ecotropic retrovirus receptor mRNA were studied. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, ecotropic provirus sequences were detected in 10 of 13 long-term repopulated animals, while amphotropic proviral sequences were detected in only one recipient. A second distinct population of murine HSC were isolated that express 3-fold higher levels of amphoR mRNA. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus sequences, a significant increase in the amphotropic retrovirus transduction (P = 0.018). These results indicate that, among the heterogeneous populations of HSC present in adult mouse bone marrow, the subpopulation with the highest level of amphoR mRNA is more efficiently transduced by amphotropic retrovirus. In a related study, we found low levels of human amphoR mRNA in purified populations of human HSC (CD34+ CD38-) and higher levels in committed progenitor cells (CD34+ CD38+). We conclude that the amphoR mRNA level in HSC correlates with amphotropic retrovirus transduction efficiency.