Leukemia inhibitory factor improves survival of retroviral vector-infected hematopoietic stem cells in vitro, allowing efficient long-term expression of vector-encoded human adenosine deaminase in vivo.

Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector-producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF-treated marrow were stably engrafted with the progeny of provirus-bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15-50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.

Cloning and characterization of a specific receptor for mouse oncostatin M.

Oncostatin M (OSM) is a member of a family of cytokines that includes ciliary neurotrophic factor, interleukin-6, interleukin-11, cardiotrophin-1, and leukemia inhibitory factor (LIF). The receptors for these cytokines consist of a common signaling subunit, gp130, to which other subunits are added to modify ligand specificity. We report here the isolation and characterization of a cDNA encoding a subunit of the mouse OSM receptor. In NIH 3T3 cells (which endogenously express gp130, LIF receptor beta [LIFRbeta], and the protein product, c12, of the cDNA described here), mouse LIF, human LIF, and human OSM signaled through receptors containing the LIFRbeta and gp130 but not through the mouse OSM receptor. Mouse OSM, however, signaled only through a c12-gp130 complex; it did not use the LIF receptor. Binding studies demonstrated that mouse OSM associated directly with either the c12 protein or gp130. These data highlight the species-specific differences in receptor utilization and signal transduction between mouse and human OSM. In mouse cells, only mouse OSM is capable of activating the mouse OSM receptor; human OSM instead activates the LIF receptor. Therefore, these data suggest that all previous studies with human OSM in mouse systems did not elucidate the biology of OSM but, rather, reflected the biological actions of LIF.

Expression of human adenosine deaminase in murine hematopoietic cells.

Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

Ligands for the receptor tyrosine kinases hek and elk: isolation of cDNAs encoding a family of proteins.

Hek is a member of the eph subfamily of receptor tyrosine kinases whose members include elk, hek2, sek, eph and eck among others. Using a soluble form of hek consisting of the extracellular region of the receptor fused to the Fc domain of human IgG1 and an expression cloning strategy, we have isolated two different but related cDNAs from the human T-lymphoma line HSB-2 that encode ligands for hek. The cDNAs encode proteins of 238 and 201 amino acids (44% amino acid identity) that are anchored to the membrane by glycosylphosphatidylinositol (GPI)-linkage. The proteins encoded by these cDNAs are bound by hek with affinity constants of 2 x 10(8) M-1. These proteins also bind the elk tyrosine kinase receptor. These cDNAs are related to other cDNAs that we have recently isolated from a human placental library that encode ligands for both hek and elk and define an emerging family of ligands for eph-related kinases (LERKs).

Molecular characterization of mouse and rat CPP32 beta gene encoding a cysteine protease resembling interleukin-1 beta converting enzyme and CED-3.

Interleukin-1 beta converting enzyme (ICE) defines a new class of mammalian cysteine protease that shares strong homology with the Caenorhabditis elegans death gene ced-3. Both ICE and CED-3, when introduced into cultured cells, induce apoptosis, indicating that this type of cysteine protease may play an important role in the process of programmed cell death. Here, we report the cloning of a mouse and rat gene encoding a novel cysteine protease. The putative proteins encoded by these cDNAs contain the conserved sequence (QACRG) necessary for covalent linkage to the substrate as well as the three amino acids responsible for substrate binding and catalysis in ICE. Amino acid sequence analysis indicates that this rodent cysteine protease is the homolog of human CPP32 beta. Mouse CPP32 beta mRNA is highly expressed in spleen, and to a lesser degree in brain, lung, liver, and kidney. The mouse CPP32 beta genomic locus spans a region of approximately 20 kb, including seven exons and six introns. Mouse interspecific backcross mapping allowed localization of CPP32 beta to the central region of mouse chromosome 8, linked to Scvr, Lpl, Jund1 and Mlr.

LERK-2, a binding protein for the receptor-tyrosine kinase ELK, is evolutionarily conserved and expressed in a developmentally regulated pattern.

We have isolated and characterized cDNA clones that encode the rat homologue of a binding protein, LERK-2, for the receptor tyrosine kinase, elk. The cDNAs contain an open reading frame of 1527 nucleotides capable of encoding a protein 345 amino acid residues in length. The nucleotide sequence of the present clones is > 90% identical to the previously identified human LERK-2 cDNA, and the predicted proteins encoded by the rat and human clones are identical at 95% of amino acid residues. Recombinant proteins expressed from the rat cDNAs bind to elk with high affinity, similar to recombinant human LERK-2 and an endogenously-expressed rat elk-binding protein. Expression of the rat LERK-2 mRNA was detected in embryonic brain, kidney, lung, skeletal muscle, thymus, liver, and heart, and diminished in the early post-natal period. Significant LERK-2 mRNA expression in the young adult rat was restricted to the lung, kidney, heart and testes.

Ectopic overexpression of c-mpl by retroviral-mediated gene transfer suppressed megakaryopoiesis but enhanced erythropoiesis in mice.

In this report, we tested whether ectopic overexpression of a cell surface receptor cDNA could be used to explore the physiological roles of that receptor. We generated c-mpl overexpressing animals by reconstituting mice with retroviral vector-transduced bone marrow (BM) cells. We observed that platelet counts in the c-mpl overexpressing mice failed to recover to normal levels and remained at <200 x 10(6)/mL post-transplantation, while platelet numbers in the control mice returned to > 800 x 10(6)/mL by 4 weeks post-transplantation. However, platelet counts in the c-mpl overexpressing mice could be stimulated to normal levels after administration of rhMGDF. No significant changes in peripheral leukocyte counts were observed, although the number of CFU-E, GM-CFC, and CFC-multi were reduced two- to threefold in the BM of the c-mpl overexpressing mice. In addition, enhanced erythropoiesis was observed in the c-mpl overexpressing mice. The mpl receptors on erythroid cells were functional as demonstrated by tyrosine-phosphorylation of mpl receptor on RBC and by in vitro erythroid colony-formation in response to MGDF stimulation, respectively. These results suggested that ectopically expressed mpl receptors competed for ligand in vivo leading to an insufficient amount of circulating thrombopoietin (Tpo) for the development of megakaryocytic lineage. These results further suggest that, in addition to sequestering circulating Tpo, overexpression of the mpl receptor on erythroid progenitors may directly contribute to enhanced erythropoiesis in vivo. Our studies demonstrate that ectopic overexpression of a receptor by retroviral-mediated gene transfer provides an approach to explore the biological roles of novel receptors.

A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans.

Granulocyte colony-stimulating factor (G-CSF) has proven effective in the prophylaxis of chemotherapy-induced neutropenia and as a mobilizer of peripheral blood progenitor cells. The longevity of G-CSF action is limited by its removal from the body by two mechanisms. The first is thought to be mediated via receptors (receptor mediated clearance [RMC]) predominantly on neutrophils, the second process is likely the result of renal clearance. With the intention of developing a novel form of Filgrastim (r-met HuG-CSF) with a sustained duration of action in vivo, a new derivative named SD/01 has been made by association of Filgrastim with poly(ethylene glycol). The desired properties of this new agent would include a prolonged duration of action sufficient to cover a complete single course of chemotherapy. SD/01 is shown here to sustain significantly elevated neutrophil counts in hematopoietically normal mice for 5 days. In neutropenic mice effects were noted for at least 9 days, accompanying a significant reduction in the duration of chemotherapy induced neutropenia. Normal human volunteers showed higher than baseline ANC for around 9 to 10 days after a single injection of SD/01. Data from these normal volunteers also indicate that mobilization of CD34+ cells and progenitors may occur in a more timely manner and to around the same absolute numbers as with repeated daily injections of unmodified Filgrastim. These data indicate that SD/01 represents an efficacious novel form of Filgrastim with actions sustained for between one and two weeks from a single injection.

Effects of leukemia inhibitory factor (LIF) on gene transfer efficiency into murine hematolymphoid progenitors.

We have investigated the effects of the cytokine leukemia inhibitory factor (LIF) on recovery and retroviral infection of murine hematopoietic stem cells maintained in short-term culture. Up to a two-fold increase in CFU-S13 recovery was observed, from 9.7 x 10(-5) cells in untreated controls to 17.6 x 10(-5) cells when 10U/ml LIF is added to the culture medium. Intermediate concentrations of LIF (.1U/ml and 1U/ml) were not significantly different from the control. Histological analysis of spleen colonies harvested thirteen days posttransplant demonstrated that LIF does not cause a detectable alteration in the differentiative potential of CFU-S13. The efficiency of retroviral-vector infection in CFU-S13 is also improved, from 15% (24/158) in untreated controls to 91% (116/127) at a LIF concentration of 10U/ml. LIF concentrations of .1U/ml and 1U/ml increased infection efficiency to 35% (14/40) and 71% (37/51), respectively. Analysis of proviral insertion sites in spleen colonies indicated that some CFU-S13 precursors were infected in the LIF-treated marrows, but no identical pairs were detected in the controls. Finally, long-term expression of provirally-encoded human adenosine deaminase (hADA) was measured in hematopoietic tissues of bone marrow transplant recipients six months posttransplant. In all tissues analyzed (spleen, thymus, bone marrow, splenic B cells, peritoneal macrophages, and blood) differentiated progeny of LIF-treated marrows had higher levels of hADA than untreated controls. Tenfold increases in levels of hADA are detected in some tissues, but levels were variable. These experiments demonstrate that LIF directly or indirectly enhances retroviral infection efficiency of hematopoietic stem cells, and might be used to improved existing gene transfer protocols.

Mice overexpressing murine oncostatin M (OSM) exhibit changes in hematopoietic and other organs that are distinct from those of mice overexpressing human OSM or bovine OSM.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) belong to the interleukin-6 family of cytokines. The authors' previous in vitro work demonstrated that in mouse cells mouse OSM (mOSM) signals through a heterodimeric receptor complex incorporating the mOSM-specific receptor mOSMRbeta while human OSM (hOSM) and bovine OSM (bOSM) use the mouse LIF receptor mLIFRbeta rather than mOSMRbeta. These in vitro data suggest that prior studies in mouse systems with hOSM or bOSM (the usual molecules used in early studies) reflect LIF rather than OSM biology. The current work assessed whether or not this divergence in actions among these three OSMs also occurs in vivo in mouse models. Adult female (C57BL/6J x DBA/2J) F(1) mice were engineered to stably overexpress mOSM, hOSM, or bOSM by retrovirus-mediated gene transfer (n = 10 or more per group). After 4 weeks, molecular and hematologic profiles and anatomic phenotypes in multiple organs were assessed by standard techniques. Animals overexpressing either hOSM or bOSM had an identical phenotype resembling that associated with LIF activation, including significant hematologic abnormalities (anemia, neutrophilia, lymphopenia, eosinopenia, and thrombocytosis); weight loss; profound enlargement (lymph node, spleen) and/or structural reorganization (lymph node, spleen, thymus) of lymphoid organs; and severe osteosclerosis. In contrast, mice overexpressing mOSM did not develop hematologic changes, weight loss, or osteosclerosis and exhibited more modest and anatomically distinct restructuring of lymphoid organs. These data indicate that activities imputed to OSM and the mOSMRbeta signaling pathway using in vitro and in vivo mouse experimental systems are unique to mOSM.

Stimulation of retroviral vector infection of murine hematopoietic progenitors.

Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.

Analysis of the intra-epithelial lymphocyte compartment in SCID mice that received co-isogenic CD4+ T cells. Evidence that mature post-thymic CD4+ T cells can be induced to express CD8 alpha in vivo.

Severe combined immunodeficient (SCID) mice injected with co-isogenic CD4+/CD45RBhigh lymph node T cells from normal donors develop a wasting disease that is caused by hyperplasia of the intestinal epithelium. SCID mice injected with purified lymph node CD4+ T cells or CD4+/CD45RBlow T cells do not develop the disease. The IEL compartment from SCID mice injected with highly purified CD4+/CD45RBhigh T cells or CD4+ T cells contained significant numbers of T cells that expressed both CD4 and CD8 alpha, but not CD8 beta. The CDr+/CD8 alpha + T cells were unique to the IEL compartment of the small intestine and were not observed in significant numbers in the lamina propria, mesenteric lymph node, nor IEL compartment of the large intestine. By using Ly-5 mismatched donors and recipients, we determined that the CD4+/CD8 alpha + T cells were derived from the donor T cells. The expression of CD8 alpha was stable in vitro, and CD8 alpha mRNA was detected in sorted CD4+/CD8 alpha + T cells by reverse transcriptase-PCR (RT-PCR). Recombinase-activating gene (RAG)-1 and -2 mRNA was not detected in the intra-epithelial lymphocyte CD4+/CD8 alpha + T cell population. Thus, it appears that under conditions unique to the epithelial layer of the small intestine, mature post-thymic CD4+ T cells can be induced to express CD8 alpha.

Cytokine regulation of hematopoietic stem cells.

Regulation of hematopoietic stem cell proliferation and differentiation is likely to be controlled by the local concentration of both inhibitory and stimulatory cytokines. A newly described regulator of early haematopoietic cells called mast cell growth factor, stem cell factor or kit ligand (referred to here as the Sl factor) has been described. This factor is the gene product of the murine Steel locus and is the ligand for the c-kit proto-oncogene, the product of the murine W locus. The effects of Sl factor on primitive hematopoietic cells suggest that this growth factor is a major stimulator of basal hemopoiesis. Further, data indicates that Sl factor acts in synergy with virtually all of the later acting growth factors to enhance the proliferative and differentiative potential of these cells.

Human adenosine deaminase expression in mice.

A replication defective retroviral vector containing a human adenosine deaminase (hADA) cDNA was produced by GP + E-86 packaging cells at high titer. We report long-term expression of hADA in the hematopoietic tissues of mice transplanted with bone marrow cells infected by in vitro co-cultivation with vector producing cells. Western analysis using an hADA-specific antibody allowed detection of the protein in the peripheral blood of all 37 transplanted mice for at least 9 weeks. Sixty-eight percent of the animals continued to express hADA in one or more of their hematopoietic tissues for the experimental period, and hADA was found in both spleen colonies and tissues of secondary recipients. There was provirus integration and expression in myeloid, erythroid, and lymphoid cell lineages, indicating extensive repopulation by the progeny of infected stem cells. The vector did not contain a selectable marker, and the infected stem cells did not have a competitive in vivo advantage. Nevertheless, we observed consistent gene transfer into hematopoietic stem cells and long-term expression of a human gene product in their progeny.

Murine leukemia inhibitory factor enhances retroviral-vector infection efficiency of hematopoietic progenitors.

We have investigated the in vitro effects of the cytokine leukemia inhibitory factor (LIF) on normal murine hematopoietic progenitors by measuring recovery and retroviral vector infection efficiency of 13-day posttransplant, spleen-colony-forming cell (CFU-S 13) in short-term culture. Up to a twofold increase in CFU-S13 recovery was observed, from 9.7 x 10(-5) cells in untreated controls to 17.8 to 19.5 x 10(-5) cells, depending on the concentration of LIF. Histologic analysis of spleen colonies from control and LIF-treated marrows demonstrated that there was no detectable alteration in the differentiative potential of CFU-S13. The efficiency of CFU-S13 infection was increased from 15% in untreated controls to 84% to 91% in LIF-treated marrows. Analysis of proviral integration sites in spleen colonies indicated that some CFU-S13 precursors were infected in the LIF-treated marrows.

The receptor tyrosine kinase-related gene (ryk) demonstrates lineage and stage-specific expression in hematopoietic cells.

We attempted to isolate novel receptor tyrosine kinase, which may play a role in hematopoietic development by screening for expressed sequences with conserved tyrosine kinase catalytic domains. Among the known tyrosine kinases identified in this screen, we found a gene with characteristics of a receptor tyrosine kinase but unusual motifs in the catalytic domain. This gene is identical to ryk described independently by other investigators. Chromosomal fluorescence in situ hybridization localization of human ryk was clarified by using monochromosomal hybrids and placing it as a single locus in 3q22. Although Northern analysis reveals widespread expression in adult mouse tissues, we have found that ryk expression is not ubiquitous. Expression increased in bone marrow cells from mice treated with 5-fluorouracil. Northern analysis on cell lines indicates expression in CD3-, CD4-, CD8- T cells (at a low level), pre-T cells, thymic epithelial cells, and mature myeloid cells, but not myeloid precursors or B cell precursors. Expression analysis with the use of RT-PCR on mouse bone marrow cells separated on the basis of cell surface markers (B220, CD4, CD8, Gr-1, Mac-1) reveals that this receptor is expressed in differentiated cells (Lin+) but is not expressed in the precursor cells (Lin-). Flow cytometric analysis with a monospecific anti-Ryk Ab demonstrates that Ryk+ cells constitute 36.7% and Lin+/Ryk+ cells constitute 33.7% of low density bone marrow cells whereas Ryk+ cells represent only 0.3% of the Lin- population. We conclude that ryk expression is regulated during hematopoietic development by lineage commitment and stage of maturation.

Expression vectors for human adenosine deaminase gene therapy.

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one fo these vectors (p delta NN2ADA), human ADA was detected in 60-85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

Ligands for EPH-related tyrosine kinase receptors are developmentally regulated in the CNS.

Elk is a member of the eph family of receptor-like tyrosine kinases. Although its function is unknown, elk is postulated to play a role in nervous system development. Using Northern analysis, we examined the developmental regulation of RNAs encoding elk, and several ligands for the eph family of RTKs, the LERKs. Expression of elk, LERK-1, and LERK-2 RNAs is high in all regions examined in the embryonic and postnatal rat brain and decreases to low levels with age. One exception is the adult olfactory bulb which continues to express a moderate level of LERK-2. In contrast, moderate LERK-4 expression was limited to the developing hippocampus and cerebral cortex. These data indicate that elk and some of the LERKs may play a role in nervous system development, maintenance, and/or regeneration.