ALBERT

Description: We report the results of haplocompatible peripheral blood stem cell transplantation (PBSCT) utilizing CD34+ selection and T-cell depletion for 17 patients with severe combined immunodeficiency disease (SCID). Of these patients, 11 had T−B−NK+, 1 had T−B+NK+, and 5 had T−B+NK− phenotype. A total of 15 cell preparations were processed immediately after collection, in which two were shared between two twin siblings. Total viable nucleated cells (TVNC) in the original cell collections were between 5.9 and 9.13×1010 (median 7.0×1010) with 0.47–2.39% (median 0.9%) CD34+ cells. After Isolex 300i (Baxter Inc., n=14) or CliniMACS System (Miltenyi Biotec Inc., n=1) processing, a median number of 379×106 (89–970×106) TVNC were recovered, with a median viability of 98% (83–100%) and median purity of 96% (89–100%) CD34+ cells. All Isolex processed (n=14) cells were further T-cell depleted with OKT3 monoclonal antibody, yielding a median of 0.09% CD3+ cells (0.008–0.4%). One preparation that utilized the CliniMACS System yielded only 0.06% CD3+ cells, hence did not receive further T-cell depletion. Recovery of CD34+ cells after complete processing was from 13.3% to 60.2% (median 50.1%). Twelve patients (70.6%) are alive 2 months to 8.7 years post transplant. A total of five patients died from infections or transplant-related complications. Four patients suffered from autoimmune hemolytic anemia, which resulted in one death. Fourteen patients engrafted. One of three patients who did not engraft subsequently received a boost from the same donor but eventually died without engraftment, and 2 received a matched unrelated BMT with myeloablative conditioning and recovered T- and B-cell function. At last follow-up the median time for the recovery of T- and B-cell function was 8.5 months and 1 year, respectively. The dose of CD3 did not show any influence on T- or B-cell function recovery (p=0.48 and 0.09, respectively). And the dose of CD34+ cells did not influence T-cell function recovery (p=0.1), but did influence B-cell function recovery, which was statistically significant (p=0.02). The B− SCID phenotype is associated with a poorer outcome compared to the B+ SCID phenotype, with 50% and 100% survival rates, respectively. However, this result was not statistically significant (p=0.07). Of the 9 surviving patients followed for more than 2 years, most are in good general health. The body height growth curve is within the 5th and 10–25th percentiles in 3 and 4 patients, respectively. For body weight, the growth curve is within 10–25th and 50–75th percentiles in 5 and 2 patients, respectively. Five have achieved successful recovery of both T- and B-cell immunity and require no medication at last visit; however, 2 of these had graft failure following their initial haplocompatible transplant, and received a second BMT from a matched unrelated donor with conditioning. Three patients with X-linked SCID (3/5) didn’t achieve B-cell reconstitution and still require IVIG replacement therapy. Based on these results, we conclude that for SCID patients who lack an HLA-matched related donor, CD34+ selected T-cell depleted haplocompatible PBSCT is an effective treatment.

Description: Acute myelogenous leukemia (AML) is often associated with mutations in transcription factors that are essential for normal hematopoietic development and differentiation. The basic leucine zipper (bZIP) protein C/EBPalpha is mutated in 7–10% of AML. Two different classes of C/EBPalpha mutations are found in AML: 1) mutations that introduce a stop codon between the primary translational initiation site and a downstream ATG, resulting in translation of an N-terminally truncated protein (p30) 2) mutations that disrupt the basic region of the bZIP domain in the C-terminus, abolishing DNA binding. Of note, biallellic mutations are often but not always observed. It has been proposed that p30 is dominant-negative over wt C/EBPalpha (p42), blocking its transcriptional activity. We have previously shown that p30 blocks erythrocytic commitment and inhibits terminal granulocytic differentiation when expressed in human but not murine primary hematopoietic cells. To identify the molecular mechanism by which the p30 inhibits differentiation and to evaluate the role of homo- and heterodimerization between p30 and p42 in leukemogenesis, we created a p30 that carries point mutations in bZIP (p30-L12V) that disrupt dimerization. Expression of p30-L12V had no effect on the differentiation of human CD34+ cells, arguing for the requirement of dimerization. To inhibit potential heterodimerization between p30 and p42 but permit p30 homodimerization, we generated a p30-GZ, in which the leucine zipper was replaced by an artificial dimerization domain. Expression of p30-GZ induced a similar differentiation block as the normal p30, arguing against a dominant-negative function of p30 over p42 mediated by dimerization. In a second approach to understand the mechanism by which p30 inhibits differentiation, we wanted to identify gene targets of p30. For this purpose, p30 was fused to the modified ligand-binding domain of the estrogen receptor (p30-ERtm). Expression of p30-ERtm in CD34+ cells in the presence of tamoxifen showed a phenotype very similar to the one observed after the expression of p30, while in the absence of tamoxifen no effect was observed. We used this system of inducible p30 expression in human CD34+ cells to evaluate gene expression patterns. If p30 is dominant-negative and interferes with the transcriptional activation of p42, reciprocal expression patterns of target genes should be observed for wt C/EBPalpha and p30. Our data show that homodimerization of p30 is necessary and sufficient to block erythrocytic commitment and terminal granulocytic differentiation of human CD34+ cells and to induce known C/EBPalpha target genes. These data argue that the formation of p30/p42 heterodimers is not required for the effect of C/EBPalpha p30, questioning the proposed dominant-negative function.

Description: Preclinical animal models are important for evaluating the safety and therapeutic efficacy of new therapeutic modalities such as gene therapy. From the different large animal models, nonhuman primate models have emerged over the last decades as highly desirable experimental systems from both a pathophysiologic and pharmacokinetic viewpoint and the study of nonhuman primates has provided important information on the efficacy and safety of gene therapy systems in vivo prior to human trials. The common marmoset (Callithrix jacchus) has the advantage that it is a small, and thus relatively inexpensive nonhuman primate model. Currently, very little data on the transduction efficiency of foamyviral vectors for gene transfer into marmoset stem cells exists. We therefore performed a direct comparison using identically designed gammaretroviral, lentiviral and foamyviral vector constructs expressing the enhanced green fluorescent protein (EGFP) from the spleen focus forming virus (SFFV) promoter pseudotyped with either the modified human foamy virus (HFV) envelope EM140 or the G-protein of vesicular stomatitis virus (VSV-G) for the transduction of common marmoset embryonic stem cells (CMES) as well as marmoset CD34+ hematopoietic progenitor cells. Virus stocks of these vectors were prepared by polyethyleneimine-mediated transfection of 293T cells and concentrated approximately 10-fold by centrifugation for 4 hours at 10.000 g at 4°C. Three different target cell populations were transduced: common marmoset embryonic stem cells (CMES) or cryopreserved CD34-enriched cells from bone marrow of a common marmoset either after a two-day prestimulation in the presence of IL-6, FLT3L, cSCF and TPO at a concentration of 100 ng/mL each, or after overnight incubation with 100 ng/mL SCF only. Equal numbers of cells were exposed to the four different vector preparations for 14 hours in 12-well dishes coated with CH-296. The read-out was based on fluorescence microscopy of colonies plated in methyl cellulose as well as flow cytometry (FACS). Foamyviral vectors with the foamyviral envelope were the most efficient gene transfer tool for marmoset hematopoietic CD34-positive cells with stable transduction rates of over 80% as assessed by flow cytometry at both 2 or 7 days after the end of transduction and on average 88% transduction efficiency into colony forming cells (CFU-C). Transduction of CFU-C with all other vector preparations was below 60%. In CMES, initial gene transfer rates of over 80% were achieved with the VSV-G pseudotype lentiviral vector, however, expression decreased to 13% after 7 days. In contrast, the foamyviral vector pseudotyped with the foamyviral envelope decreased only from 49% to 24% after 7 days. In conclusion, we achieved stable viral gene transfer and expression in CMES cells as well as highly efficient gene transfer into common marmoset hematopoietic CD34 positive cells using foamyviral vectors. These results suggest that foamyviral vectors may be highly feasible vectors for stem cell gene transfer and thus set the stage for a more detailed analysis of this vector system in transplantation studies in this nonhuman primate model.

Description: The RUNX1 gene encodes an alpha subunit of the core-binding factor (CBF), an important heterodimeric transcription factor in hematopoietic ontogeny and development, and is one of the most frequently disrupted genes in acute leukemia. In addition to its involvement in several translocations, the RUNX1 gene is often subject to deletions or point mutations in acute myelogenous leukemia (AML). Interestingly, in addition to complete loss-of-function mutations, many of the alterations involve missense point mutations within the Runt domain that disrupt DNA binding activity (DB-mutants). In vitro assays have suggested that these DB mutants have a dominant-negative (DN) activity, presumably due to their ability to bind and sequester CBF beta but inability to bind DNA. A strict correlation between the type of mutation and its monoallelic or biallelic incidence is not apparent even though a DN mutant should only affect one allele while a loss of function mutation should affect both alleles. It has been hypothesized that loss of one allele (haploinsufficiency) is sufficient for loss of tumor suppressor activity but the relative high incidence of specific DB mutations suggests a more complex scenario. We thus sought to determine if expression of DB mutants in murine bone marrow (BM) resulted in a similar phenotype as the loss of Runx1, or if these mutations are associated with a gain-of-function. Two RUNX1-DB mutants were thus evaluated using the established retroviral transduction/transplantation mouse model. Between 3 and 6 months after transplantation, peripheral blood, spleen and BM cells were analyzed. Long-term repopulating cells expressing RUNX1 DB-mutants were able to contribute normally to both myeloid and lymphoid compartments, although a disproportionate increase in the B-cell compartment was observed in 3 out of 10 mice. Surprisingly, and inconsistent with a DN activity, disruption of normal T-cell or megakaryocytic development was not observed in the mice, in contrast to Runx1−/+ mice. Significantly, however, replating assays in vitro demonstrated that RUNX1-DB mutants lead to a significant increase in self-renewal activity, in contrast to BM cells of floxed Runx1 mice expressing the Cre recombinase, which showed a less dramatic effect on self-renewal. Colonies derived from CFU-Cs expressing RUNX1-DB mutants were composed of dysplastic granulocytic and monocytic cells, with an increasing number of immature blasts after multiple replatings (〉7), whereas residual colonies from Runx1fl/− BM receiving CRE showed a different morphology with more mature cells. Thus our data suggest that RUNX1-DB mutants do not act in a dominant negative fashion to inhibit normal RUNX1 function, but impart a gain-of-function that results in impaired myeloid differentiation and increased self-renewal potential, consistent with its association with AML.

Description: Multiple genetic alterations are required to induce acute myelogenous leukemia (AML). Mutations in the extracellular domain of the KIT receptor are almost exclusively found in patients with AML carrying translocations or inversions affecting members of the core binding factor (CBF) gene family and correlate with a high risk of relapse. We demonstrate that these complex insertion and deletion mutations lead to constitutive activation of the KIT receptor, which induces factor-independent growth of interleukin-3 (IL-3)–dependent cells. Mutation of the evolutionary conserved amino acid D419 within the extracellular domain was sufficient to constitutively activate the KIT receptor, although high expression levels were required. Dose-dependent growth inhibition and apoptosis were observed using either the protein tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) or by blocking the phosphoinositide-3-kinase (PI3K)–AKT pathway. Our data show that the addition of kinase inhibitors to conventional chemotherapy might be a new therapeutic option for CBF-AML expressing mutant KIT.