Faithful modeling of mixed-lineage leukemia in murine cells has been difficult to achieve. We show that expression of MLL-AF9 in human CD34+ cells induces acute myeloid, lymphoid, or mixed-lineage leukemia in immunodeficient mice. Some leukemia stem cells (LSC) were multipotent and could be lineage directed by altering either the growth factors or the recipient strain of mouse, highlighting the importance of microenvironmental cues. Other LSC were strictly lineage committed, demonstrating the heterogeneity of the stem cell compartment in MLL disease. Targeting the Rac signaling pathway by pharmacologic or genetic means resulted in rapid and specific apoptosis of MLL-AF9 cells, suggesting that the Rac signaling pathway may be a valid therapeutic target in MLL-rearranged AML.

Insertional mutagenesis by long terminal repeat (LTR) enhancers in gamma-retrovirus-based vectors (GVs) in clinical trials has prompted deeper investigations into vector genotoxicity. Experimentally, self-inactivating (SIN) lentivirus vectors (LVs) and GV containing internal promoters/enhancers show reduced genotoxicity, although strong ubiquitously-active enhancers dysregulate genes independent of vector type/design. Herein, we explored the genotoxicity of beta-globin (BG) locus control region (LCR), a strong long-range lineage-specific-enhancer, with/without insulator (Ins) elements in LV using primary hematopoietic progenitors to generate in vitro immortalization (IVIM) assay mutants. LCR-containing LV had approximately 200-fold lower transforming potential, compared to the conventional GV. The LCR perturbed expression of few genes in a 300 kilobase (kb) proviral vicinity but no upregulation of genes associated with cancer, including an erythroid-specific transcription factor occurred. A further twofold reduction in transforming activity was observed with insulated LCR-containing LV. Our data indicate that toxicology studies of LCR-containing LV in mice will likely not yield any insertional oncogenesis with the numbers of animals that can be practically studied.

Fragile X mental retardation proteins (FMRP) are RNA-binding proteins that interact with a subset of cellular RNAs. Several RNA-binding domains have been identified in FMRP, but the contribution of these individual domains to FMRP function in an animal model is not well understood. In this study, we have generated flies with point mutations in the KH domains of the Drosophila melanogaster fragile X gene (dfmr1) in the context of a genomic rescue fragment. The substitutions of conserved isoleucine residues within the KH domains with asparagine are thought to impair binding of RNA substrates and perhaps the ability of FMRP to assemble into mRNP complexes. The mutants were analyzed for defects in development and behavior that are associated with deletion null alleles of dfmr1. We find that these KH domain mutations result in partial loss of function or no significant loss of function for the phenotypes assayed. The phenotypes resulting from these KH domain mutants imply that the capacities of the mutant proteins to bind RNA and form functional mRNP complexes are not wholly disrupted and are consistent with biochemical models suggesting that RNA-binding domains of FMRP can function independently.T HE fragile X mental retardation protein (FMRP) is an RNA-binding protein necessary for normal neuronal development and behavior in all species where its function has been examined. A general model for FMRP function is that it regulates nucleocytoplasmic transport, subcellular localization, and translation of select RNA transcripts (reviewed by Bardoni and Mandel 2002;Jin and Warren 2003;Jin et al. 2004a;Bagni and Greenough 2005). Biochemical analyses have uncovered several RNA-binding motifs associated with FMRP function, including two KH domains (hnRNP-K homology) and an arginine and glycine-rich motif (RGG box) that are common to RNA-binding proteins (Ashley et al. 1993;Siomi et al. 1993). The highly conserved N termini of FMRPs have RNA-binding capacity as well (Adinolfi et al. 1999(Adinolfi et al. , 2003. The N-terminal 110 amino acids of FMRPs are similar to Tudor/Agenet domains and are members of an extended family that is referred to as the Tudor domain ''royal family'' (Maurer-Stroh et al. 2003). This domain family is related to methyl-substratebinding proteins that are implicated in regulation of chromatin structure and includes the chromodomain.RNA substrates for FMRP have conserved elements in primary sequence and/or higher-order structures that interact with the aforementioned RNA-binding domains. A G-quartet structure within RNA interacts with the RGG box Schaeffer et al. 2001), and the second KH domain recognizes a looploop pseudoknot RNA structure referred to as a kissing complex (Darnell et al. 2005). A stem-loop structure within BC1 RNA is reported to interact specifically with the N-terminal 217 amino acids of FMRP (Zalfa et al. 2005; but see Wang et al. 2005 for an opposing view). These studies demonstrate that individual RNA-binding domains of FMRP have distinct substrates with which they inte...

Safely achieving long-term engraftment of genetically modified hematopoietic stem cells (HSCs) that maintain therapeutic transgene expression is the benchmark for successful application of gene therapy for hemoglobinopathies. We used the pigtailed macaque HSC transplantation model to ascertain the long-term safety and stability of a γ-globin lentivirus vector. We observed stable gene-modified cells and fetal hemoglobin expression for 3 years. Retrovirus integration site (RIS) analysis spanning 6 months to 3.1 years revealed vastly disparate integration profiles, and dynamic fluctuation of hematopoietic contribution from different gene-modified HSC clones without evidence for clonal dominance. There were no perturbations of the global gene-expression profile or expression of genes within a 300 kb region of RIS, including genes surrounding the most abundantly marked clones. Overall, a 3-year long follow-up revealed no evidence of genotoxicity of the γ-globin lentivirus vector with multilineage polyclonal hematopoiesis, and HSC clonal fluctuations that were not associated with transcriptome dysregulation.

Strategies for human gene therapy trials targeting hematopoietic stem cells (HSCs) are complicated by studies in murine models due to differences in stem cell behavior, short life-span and limited HSCs that could be transduced and transplanted when studying safety of viral vectors. Recent reports on adverse genotoxic events with integrating viral vectors in clinical trials utilizing autologous gene corrected HSCs underscores the need for safer gene transfer vectors. Non-human primates are relevant models due to similarities in the behavior of hematopoietic stem/progenitor cells, global gene expression profile, ability to assess long-term engraftment of transduced cells and safety of gene-modified HSCs, and thus could relatively accurately predict risk of vector genotoxicity. As a preclinical step towards globin gene therapy for hemoglobinopathies, we used pigtailed macaque HSC transplantation (HSCT) model to ascertain long-term safety and stable transgene expression from sGbG, a lentiviral vector (LV) encoding human γ-globin coding sequences from a β-globin promoter and locus control region (LCR). We observed upregulation of endogenous macaque fetal hemoglobin post-HSCT, which decreased to minimal levels by two years post-HSCT, a well-documented phenomenon following HSCT in humans. However, fetal hemoglobin (HbF) (comprised of macaque α and human γ-globin) expression remained steady at 12-15% even after 700 days post-HSCT. At 2.5 years post-HSCT, the HbF expression in a macaque transplanted with HSCs gene-modified with sGbG was stable in the range of 13% vs. 0.1% for control macaque; the average vector copy ranged between 0.13 and 0.28 with stable gene marking during the analysis period. In order to evaluate the LV integration site clonal population in sGbG transduced macaque repopulating cells, modified genome sequencing PCR was performed on genomic DNA from white blood cells and PCR products were sequenced. The junction sequences were mapped to the rhesus macaque genome assembly. A total of 177 unique vector insertions were retrieved at 6 months post-HSCT (early) and 102 vector insertions at 2.5 years (late) post-HSCT respectively. The relative distribution of vector insertions into chromosomes revealed a slight over-representation into Chromosome 16, both at early and late time points. Analysis of distribution of LV integrations of with respect to transcription start sites (TSS) revealed no insertions within the 2.5kb region of TSS. The frequency of insertions was concentrated near the 10-50kb window of TSS both upstream (18.6%) and downstream (15.6%) respectively. Interestingly, among the retrieved insertion sites, only 10% (17 insertions) were common at both time points, while 90% of insertions were unique at each time point, suggesting clonal fluctuations, with multiple HSC clones contributing to hematopoiesis at an early time point, and unique, HSC clones emerged at a later time point. Comparison of the top ten most frequently detected insertion sites at both time points revealed one insertion at Chromosome 16 mapping to an intron of KIAA0195 (an uncharacterized protein expressed ubiquitously), retrieved at both time points contributed to 3.27% and 9.23% of gene modified cells at early and late time points, respectively. No insertions were near MDS/EVI1, PRDM16 or HMGA2 loci. Other oncogenes and cancer associated genes were in the vicinity of some integrants; however, there was no significant clustering of insertions in gene regions. To assess the effect of insertions on flanking gene expression or putative cancer associated genes, we performed mRNAseq on whole blood RNA from sGbG macaque and two control macaques. A comparative analysis of transcript levels of >30,000 genes revealed no difference in global gene expression profile, gene insertions and genes within 300kb region of the LV insertion sites. Importantly, transcript levels of the most abundant clone observed (KIAA0195, Chr16: 70791901) and flanking genes, the tRNA splicing endonuclease subunit SEN54 and CASK interacting protein 2 differed from two control macaques analyzed by <2 fold. In summary, long-term follow-up data from a macaque that received cells gene-modified with a human γ-globin LV reveal polyclonal reconstitution of transduced cells, HSC clonal fluctuation, and a normal transcriptional profile, suggesting low risk of genotoxicity from this vector. Arumugam P, Burtner C: Equal Contribution Disclosures: No relevant conflicts of interest to declare.

The MLL gene is fused to over 30 different fusion partners by reciprocal translocations in human acute leukemias. Some fusion partners are associated almost exclusively with myeloid or lymphoid leukemias while others are found in both. The degree to which the fusion partner contributes to the lineage of the resulting leukemia remains a matter of controversy. Using a novel model system, we demonstrate that myeloid vs lymphoid differentiation of hematopoietic progenitors transformed by MLL-AF9 can be predictably driven by cytokine combinations in vitro and in vivo. The t(9;11)(p22;q23) MLL-AF9 fusion gene is commonly associated with M5 myeloid leukemia but approximately 5% of MLL-AF9 leukemia is B-lymphoid. Expression of MLL-AF9 in human CD34+ cells enables efficient modeling of acute myeloid, B-lymphoid and biphenotypic leukemia. The lineage of the resulting leukemia can be readily manipulated in vitro (by altering the growth factors) or in vivo (using B-lymphoid-biased NOD/SCID mice or myeloid-biased NOD/SCID that are transgenic for human SCF, GM-CSF and IL-3). The cytokines IL-3, IL-7 and FLT3L appear to exert the major effects on lineage fate determination in vitro. Through limiting dilution and clonality analyses, we find a complex relationship between different leukemia stem cell compartments, with some LSC demonstrating multipotentiality and others showing strict lineage commitment. Data indicate that these differences are primarily due to microenvironment effects, with the identity of the initial cell that is targeted by MLL-AF9 possibly playing a role. These results would argue against a deterministic role for the fusion partner in MLL leukemia. This human-based system should prove useful in addressing the mechanism of lineage promiscuity of MLL leukemias. It also affords us the unique ability to determine the susceptibility of the different LSC to standard chemotherapeutic compounds, in addition to identifying novel therapeutic strategies that may be effective in treating MLL leukemia.

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