Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV (‘Berlin patient’). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

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The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3b (GSK-3b) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27 Kip1 and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3b signaling and leads to an increased transit time through the G1 phase of the cell cycle.

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The recently discovered CRISPR/Cas9 system is widely used in basic research and is a useful tool for disease modeling and gene editing therapies. However, long-term expression of DNA-modifying enzymes can be associated with cytotoxicity and is particularly unwanted in clinical gene editing strategies. Because current transient expression methods may still suffer from cytotoxicity and/or low efficiency, we developed non-integrating retrovirus-based CRISPR/Cas9 all-in-one particles for targeted gene knockout. By redirecting the gammaretroviral packaging machinery, we transiently delivered Streptococcus pyogenes Cas9 (SpCas9) mRNA and single-guide RNA transcripts into various (including primary) cell types. Spatiotemporal co-delivery of CRISPR/Cas9 components resulted in efficient disruption of a surrogate reporter gene, as well as functional knockout of endogenous human genes CXCR4 and TP53. Although acting in a hit-and-run fashion, knockout efficiencies of our transient particles corresponded to 52%–80% of those obtained from constitutively active integrating vectors. Stable SpCas9 overexpression at high doses in murine NIH3T3 cells caused a substantial G0/G1 arrest accompanied by reduced cell growth and metabolic activity, which was prevented by transient SpCas9 transfer. In summary, the non-integrating retrovirus-based vector particles introduced here allow efficient and dose-controlled delivery of CRISPR/Cas9 components into target cells.

The t(12;21) translocation, generating the TEL/AML1 fusion protein, is the most common genetic lesion in childhood cancer. Using a bone marrow transplantation model, we demonstrate that TEL/AML1 expression impinges on normal hematopoietic differentiation, leading to the in vivo accumulation and persistence of an early progenitor compartment with a Sca1 þ /Kit hi /CD11b þ phenotype and an increased self-renewal capacity, as documented by replating assays in vitro. Differentiation of these cells is not blocked, but the frequency of mature blood cells arising from TEL/AML1-transduced progenitors is low. Impaired differentiation is prominently observed in the pro-B-cell compartment, resulting in an proportional increase in early progenitors in vivo, consistent with the t(12;21) ALL phenotype. Despite the accumulation of both multipotent and B-cell progenitors in vivo, no leukemia induction was observed during an observation period of over 1 year. These results are consistent with findings in twins with concordant ALL, showing that TEL/AML1 generates a preleukemic clone in utero that persists for several years in a clinically covert fashion. Furthermore, our studies showed that the pointed domain of TEL/AML1, which recruits transcriptional repressors and directs oligomerization with either TEL/ AML1 or wild-type TEL, was essential for the observed differentiation impairment and could not be replaced with another oligomerization domain.

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 factorindependent 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) - IntroductionAcute myelogenous leukemia (AML) is a complex disease that is classified by histochemical, cytochemical, and genetic markers. Genetic alterations affecting members of the core binding factor (CBF), a heterodimeric transcription factor, are some of the most common events in AML and define a large group commonly designated CBF-AML, 1 which includes patients with either t(8;21) or inv(16). Evidence from experimental studies supports the hypothesis that these fusion proteins impair myeloid differentiation and/or expand a hematopoietic stem/progenitor cell pool, but are insufficient to cause leukemia. [2][3][4][5][6] Secondary genetic alterations, such as mutations in receptor tyrosine kinases (RTKs), are thus required for induction of an overt AML. Although the KIT receptor is expressed on the majority of AML blasts, mutations in the KIT gene are exclusively associated with CBF-AML. [7][8][9][10][11] In addition to mutations affecting D816 within the KIT tyrosine kinase domain 2 (TK2), complex insertion/deletion mutations in exon 8 affecting the extracellular (EC) domain are also observed in CBF-AML, with an especially high prevalence (26%) in inv(16) patients, and correlate with a higher risk of relapse. 8,9 In addition to EC mutations, a mutation in the transmembrane (TM) domain (V530I) has also been described in CBF-AML. 9,11 Previous studies have shown that the KIT D816V mutation induces growth factorindependent growth of myeloid and mast cells and is insensitive to the RTK inhibitor imatinib mesylate (STI571, Gleevec [manufactured by Novartis, Basel, Switzerland]). [12][13][14] The purpose of this study was to test the functional activity of the EC and TM KIT mutations associated with CBF-AML, to determine their sensitivity to imatinib, and to delineate the activated signaling pathways. Study designSite-directed mutagenesis was used to create KIT mutants from either murine or human KIT cDNAs, as indicated in Table 1. KIT mutants were cloned into the SF91 retroviral vector coexpressing the yellow fluorescence protein (YFP), and were used to infect FDC-P1 cells in the presence of inter...

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p-T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant-negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time-dependent augmentation of AKT S473 and GSK-3alpha S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK-3alpha was not dependent on PI3K activity. PKR inhibition augmented levels of p-S473 AKT and p-S21/9 GSK-3alpha/beta in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK-3alpha or beta phosphorylation in the presence of the AKT inhibitor, A443654. Pre-treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2alpha, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2alpha phosphorylation. The effects of PKR inhibition on AKT and GSK-3 phosphorylation were found to be, in part, PP2A-dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK-3.