Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy.

The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rgamma), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rgamma had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rgamma may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.

Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors.

Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 x 10(7) transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.

Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

Multilineage outgrowth of both malignant and normal hemopoietic progenitor cells from individual chronic myeloid leukemia patients in immunodeficient mice.

In this study the ability of malignant and normal progenitors in peripheral blood (PB) and bone marrow (BM) of CML patients in chronic phase to proliferate and produce mature progeny after transplantation into hereditary immunodeficient (SCID and NOD/SCID) mice was examined. Engraftment in NOD/SCID mice preconditioned by total body irradiation (TBI) alone was 10-fold higher than in SCID mice preconditioned by macrophage depletion and TBI, demonstrating that NOD/SCID mice are more suitable for engraftment of chronic phase CML cells. Low-density cells at cell doses of 10-30 x 10(6) and purified CD34+ cells at doses of approximately 0.2 x 10(6) engrafted NOD/SCID mice, with levels of 2 to 20% CD45+ cells with production of monocytes, granulocytes, erythroid cells, B-lymphocytes, CD34+ cells and variable frequencies of erythroid and myeloid colony-forming cells. As demonstrated by fluorescent in situ hybridization (FISH) analysis, purified human myeloid, B-lymphoid, erythroid and CD34+ cells from chimeric mouse BM contained Philadelphia-chromosome (Ph)-positive cells and Ph- cells in similar frequencies as primary cells from the CML patients. These results demonstrate that production of mature normal as well as malignant cells of multiple lineages were supported with similar efficiency. In contrast, all human erythroid and myeloid clonogenic cells detected in the mice were Ph-, which can be attributed to less efficient maintenance or more rapid differentiation of immature Ph+ cells in the mouse microenvironment. CML blast crisis cells also grew well in NOD/SCID mice, with 80-90% of human cells produced containing the Ph- chromosome. The availability of an in vivo assay that supports outgrowth of normal and malignant stem cells from chronic phase and blast crisis CML patients will facilitate examination of differential effects of growth factors, inhibitory cytokines and cytotoxic drugs on survival of normal and malignant stem cells in vivo and on progression of chronic phase CML towards blast crisis.

Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice.

Purified CD34(+) and CD34(+)CD38(-) human umbilical cord blood (UCB) cells were transduced with the recombinant variant of Moloney murine leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope proteins. Using a 2-day growth factor prestimulation, followed by a 2-day, fibronectin fragment CH-296-supported transduction, CD34(+) and CD34(+)CD38(-) UCB subsets were efficiently transduced using either vector. The use of the SF-EGFP/PG13 retroviral packaging cell combination consistently resulted in twofold higher levels of EGFP-expressing cells than the MFG-EGFP/Am12 combination. Transplantation of 10(5) input equivalent transduced CD34(+) or 5 x 10(3) input equivalent CD34(+)CD38(-) UCB cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages of 8% and 5%, respectively, which showed that the in vivo repopulating ability of the cells had been retained. In addition, mice engrafted after transplantation of transduced CD34(+) cells using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP with median values of 2% and 23% of human CD45(+) cells, respectively, which showed that the NOD/SCID repopulating cells were successfully transduced. EGFP+ cells were found in all human hematopoietic lineages produced in NOD/SCID mice including human progenitors with in vitro clonogenic ability. EGFP-expressing cells were also detected in the human cobblestone area-forming cell (CAFC) assay at 2 to 6 weeks of culture on the murine stromal cell line FBMD-1. During the transduction procedure the absolute numbers of CAFC week 6 increased 5- to 10-fold. The transduction efficiency of this progenitor cell subset was similar to the fraction of EGFP+ human cells in the bone marrow of the NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced CD34(+) cells, ie, 6% and 27%, respectively. The study thus shows that purified CD34(+) and highly purified CD34(+)CD38(-) UCB cells can be transduced efficiently with preservation of repopulating ability. The SF-EGFP/PG13 vector/packaging cell combination was much more effective in transducing repopulating cells than the MFG-EGFP/Am12 combination.

Transplantation of human umbilical cord blood cells in macrophage-depleted SCID mice: evidence for accessory cell involvement in expansion of immature CD34+CD38- cells.

In vivo expansion and multilineage outgrowth of human immature hematopoietic cell subsets from umbilical cord blood (UCB) were studied by transplantation into hereditary immunodeficient (SCID) mice. The mice were preconditioned with Cl2MDP-liposomes to deplete macrophages and 3.5 Gy total body irradiation (TBI). As measured by immunophenotyping, this procedure resulted in high levels of human CD45(+) cells in SCID mouse bone marrow (BM) 5 weeks after transplantation, similar to the levels of human cells observed in NOD/SCID mice preconditioned with TBI. Grafts containing approximately 10(7) unfractionated cells, approximately 10(5) purified CD34+ cells, or 5 x 10(3) purified CD34+CD38- cells yielded equivalent numbers of human CD45+ cells in the SCID mouse BM, which contained human CD34+ cells, monocytes, granulocytes, erythroid cells, and B lymphocytes at different stages of maturation. Low numbers of human GpA+ erythroid cells and CD41+ platelets were observed in the peripheral blood of engrafted mice. CD34+CD38+ cells (5 x 10(4)/mouse) failed to engraft, whereas CD34- cells (10(7)/mouse) displayed only low levels of chimerism, mainly due to mature T lymphocytes. Transplantation of graded numbers of UCB cells resulted in a proportional increase of the percentages of CD45+ and CD34+ cells produced in SCID mouse BM. In contrast, the number of immature, CD34+CD38- cells produced in vivo showed a second-order relation to CD34+ graft size, and mice engrafted with purified CD34+CD38- grafts produced 10-fold fewer CD34+ cells without detectable CD34+CD38- cells than mice transplanted with equivalent numbers of unfractionated or purified CD34+ cells. These results indicate that SCID repopulating CD34+CD38- cells require CD34+CD38+ accessory cell support for survival and expansion of immature cells, but not for production of mature multilineage progeny in SCID mouse BM. These accessory cells are present in the purified, nonrepopulating CD34+CD38+ subset as was directly proven by the ability of this fraction to restore the maintenance and expansion of immature CD34+CD38- cells in vivo when cotransplanted with purified CD34+CD38- grafts. The possibility to distinguish between maintenance and outgrowth of immature repopulating cells in SCID mice will facilitate further studies on the regulatory functions of accessory cells, growth factors, and other stimuli. Such information will be essential to design efficient stem cell expansion procedures for clinical use.

Facilitated engraftment of human hematopoietic cells in severe combined immunodeficient mice following a single injection of Cl2MDP liposomes.

Transplantation of normal and malignant human hematopoietic cells into severe combined immunodeficient (SCID) mice allows for evaluation of long-term growth abilities of these cells and provides a preclinical model for therapeutic interventions. However, large numbers of cells are required for successful engraftment in preirradiated mice due to residual graft resistance, that may be mediated by cells from the mononuclear phagocytic system. Intravenous (i.v.) injection of liposomes containing dichloromethylene diphosphonate (Cl2MDP) may eliminate mouse macrophages in spleen and liver. In this study outgrowth of acute myeloid leukemia (AML) cells and umbilical cord blood (UCB) cells in SCID mice conditioned with a single i.v. injection of Cl2MDP liposomes in addition to sublethal total body irradiation (TBI) was compared to outgrowth of these cells in SCID mice that had received TBI alone. A two- to 10-fold increase in outgrowth of AML cells was observed in four cases of AML. Administration of 10(7) UCB cells reproducibly engrafted SCID mice that had been conditioned with Cl2MDP liposomes and TBI, whereas human cells were not detected in mice conditioned with TBI alone. As few as 2 x 10(4) purified CD34+ UCB cells engrafted in all mice treated with Cl2MDP liposomes. In SCID mice treated with macrophage depletion unexpected graft failures were not observed. Histological examination of the spleen showed that TBI and Cl2MDP liposomes i.v. resulted in a transient elimination of all macrophage subsets in the spleen, whereas TBI had a minor effect. Cl2MDP liposomes were easy to use and their application was not associated with appreciable side-effects. Cl2MDP liposome pretreatment in combination with TBI allows for reproducible outgrowth of high numbers of human hematopoietic cells in SCID mice.