SHIP1, but not an AML-derived SHIP1 mutant, suppresses myeloid leukemia growth in a xenotransplantation mouse model.

Constitutive activation of the PI3K/AKT signaling pathway is found in ~50-70% of AML patients. The SH2-containing inositol 5-phosphatase 1 (SHIP1) is a negative regulator of PI3K/AKT signaling in hematopoietic cells. SHIP1 knockout mice develop a myeloproliferative syndrome and concomitant deletion of SHIP1 and the tumor suppressor PTEN leads to the development of lethal B-cell lymphomas. In the study presented here, we investigated the role of SHIP1 as a tumor suppressor in myeloid leukemia cells in an in vivo xenograft transplantation model. NSG Mice transplanted with UKE-1 cells derived from a secondary AML showed a significantly extended lifespan after lentiviral-mediated overexpression of SHIP1 in comparison to the vector control cohort. In contrast, the AML-derived SHIP1Y643H mutant, which has a strongly reduced enzymatic activity showed a significant reversion of the SHIP1-induced prolongation of the survival time. In addition, the analysis of 290 AML patients revealed a correlation between expression of SHIP1 and overall survival of the AML patients. These results indicate that SHIP1 can act as a tumor suppressor in acute myeloid leukemia cells and that higher SHIP1 expression is associated with prolonged overall survival in AML patients. SHIP1 may be an interesting candidate for gene therapy.

Investigation of the Mesenchymal Stem Cell Compartment by Means of a Lentiviral Barcode Library.

The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic "barcodes" has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.

Treatment of multifocal breast cancer by systemic delivery of dual-targeted adeno-associated viral vectors.

Adeno-associated viral (AAV) vectors yield high potential for clinical gene therapy but, like for other vectors systems, they frequently do not sufficiently transduce the target tissue and their unspecific tropism prevents their application for multifocal diseases such as disseminated cancer. Targeted AAV vectors have been obtained from random AAV display peptide libraries but so far, all vector variants selected from AAV libraries upon systemic administration in vivo retained some collateral tropism, frequently the heart. Here we explored, if this impediment can be overcome by microRNA-regulated transgene cassettes as the combination of library-derived capsid targeting and micro-RNA control has not been evaluated so far. We used a tumor-targeted AAV capsid variant (ESGLSQS) selected from random AAV-display peptide libraries in vivo with remaining off-target tropism toward the heart and regulated targeted transgene expression in vivo by complementary target elements for heart-specific microRNA (miRT-1d). Although this vector still maintained its strong transduction capacity for tumor target tissue after intravenous injection, transgene expression in the heart was almost completely abrogated. This strong and completely tumor-specific transgene expression was used for therapeutic gene transfer in an aggressive multifocal, transgenic, polyoma middle T-induced, murine breast cancer model. A therapeutic suicide gene, delivered systemically by this dual-targeted AAV vector to multifocal breast cancer, significantly inhibited tumor growth after one single vector administration while avoiding side effects compared with untargeted vectors.

TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer.

Adoptive immunotherapy with T lymphocytes expressing transgenic T-cell receptors (TCRs) has shown significant clinical efficacy in various malignant diseases. However, concurrent expression of endogenous and transgenic TCRs in one and the same T cell may impair efficacy and cause safety problems owing to mispairings. The most elegant approach to address these issues is the complete shutoff of the endogenous receptor chains by genome editing. To this end, we designed TCR-α and TCR-ß-specific pairs of transcription activator-like effector nucleases (TALENs). TALENs were delivered into T cells using an optimized messenger RNA-electroporation protocol. Based thereon, we obtained precise and highly efficient knockout (KO) in Jurkat (TCR-α: 59.7 ± 4.0%, TCR-ß: 37.4 ± 7.3%) as well as primary T cells (TCR-α: 58.0 ± 15.0%, TCR-ß: 41.0 ± 17.6%). Moreover, a successive KO strategy for the endogenous TCR chains combined with subsequent transduction of the respective chains of an Influenza virus-specific model TCR led to complete reprogramming of T cells with strongly improved expression and functionality of transgenic TCRs. In conclusion, we have developed novel means for the efficient genome editing in primary T lymphocytes.

[Universal modular system for in vitro screening of potential inhibitors of HIV-1 replication].

Here we describe a system based on recombinant lentiviral vectors for the safe screening of potential anti-HIV drugs. The system allows to evaluate the sensitivity of HIVl-1 reverse transcriptase and integrase (wild-type as well as mutant forms of these enzymes detected in drug-resistant virus isolates) towards different drugs and substances, but also to screen inhibitors of other stages of HIV-1 life cycle.

Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis.

Vector-encoded fluorescent proteins (FPs) facilitate unambiguous identification or sorting of gene-modified cells by fluorescence-activated cell sorting (FACS). Exploiting this feature, we have recently developed lentiviral gene ontology (LeGO) vectors (www.LentiGO-Vectors.de) for multi-gene analysis in different target cells. In this study, we extend the LeGO principle by introducing 10 different drug-selectable FPs created by fusing one of the five selection marker (protecting against blasticidin, hygromycin, neomycin, puromycin and zeocin) and one of the five FP genes (Cerulean, eGFP, Venus, dTomato and mCherry). All tested fusion proteins allowed both fluorescence-mediated detection and drug-mediated selection of LeGO-transduced cells. Newly generated codon-optimized hygromycin- and neomycin-resistance genes showed improved expression as compared with their ancestors. New LeGO constructs were produced at titers >10(6) per ml (for non-concentrated supernatants). We show efficient combinatorial marking and selection of various cells, including mesenchymal stem cells, simultaneously transduced with different LeGO constructs. Inclusion of the cytomegalovirus early enhancer/chicken beta-actin promoter into LeGO vectors facilitated robust transgene expression in and selection of neural stem cells and their differentiated progeny. We suppose that the new drug-selectable markers combining advantages of FACS and drug selection are well suited for numerous applications and vector systems. Their inclusion into LeGO vectors opens new possibilities for (stem) cell tracking and functional multi-gene analysis.

Correction: Role of growth arrest-specific gene 6-mer axis in multiple myeloma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reduced proliferation of CD34(+) cells from patients with acute myeloid leukemia after gene transfer of INPP5D.

Acute myeloid leukemia (AML) is a malignant disease characterized by deregulated proliferation of immature myeloid cells. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is frequently detected in approximately 50-70% of AML patients. The gene INPP5D encodes the SH2-containing inositol 5-phosphatase 1 (SHIP1), which is a negative regulator of PI3K/AKT signaling. After lentiviral-mediated gene transfer of INPP5D into CD34(+) cells derived from AML patients (n=12) the granulocyte macrophage-colony stimulating factor (GM-CSF)-dependent proliferation was reduced in all samples analyzed (average 86%; range 72-93%). An enzymatically inactive form of SHIP1 (D672A) had no effect. In addition, SHIP1 reduced the autonomous proliferation of CD34(+) cells from a patient with a secondary AML who had a very high peripheral blast count (300 x 10(9) l(-1)). These data show that SHIP1 can effectively block GM-CSF-dependent and autonomous proliferation of AML cells.

Insertional mutagenesis and clonal dominance: biological and statistical considerations.

Improvements of (retroviral) gene transfer vectors, stem cell isolation and culture techniques as well as transduction protocols eventually resulted not only in the successful genetic modification of cells capable of reconstituting the haematopoietic system in various animal models, but also human beings. This was a conditio sine qua non for the successful application of gene therapy for inherited diseases as meanwhile achieved for severe combined immune deficiencies (SCID-X1, ADA-SCID) and chronic granulomatous disease (CGD). Unexpectedly, in long-term animal experiments as well as in the follow up of patients from the CGD trial, haematopoietic clones bearing insertions in certain gene loci became dominant, which was most apparent in the myeloid blood compartment. Accumulating data strongly suggest that this clonal dominance was due to some growth and/or survival advantage conferred by gene-activating or -suppressing effects of the integrated retroviral vector (insertional mutagenesis). Importantly, such induced clonal dominance seems not to lead to malignant transformation of affected cell clones inadvertently. The latter finding has become the basis for the concept of 'induced haematopoietic stem cells', a potentially powerful tool to investigate genes involved in the regulation of mechanisms underlying competitive advantages of stem cells, but also in the multi-step nature of malignant transformation. Here we discuss promises and open issues of this concept as well as the important question of common insertion sites statistics and its pitfalls.

Transportation and cryopreservation may impair haematopoietic stem cell function and engraftment of allogeneic PBSCs, but not BM.

Recent data suggest that the practice of using frozen allogeneic grafts is becoming increasingly common among transplant centres. Therefore, we retrospectively analysed 31 frozen allogeneic PBSC and 8 BM grafts by flow cytometry with regard to their CD34+ content, membrane integrity (7-AAD) and stem cell-specific enzyme activity (aldehyde dehydrogenase, ALDH) in relation to individual transplantation results. Membrane integrity of CD34+ cells was significantly impaired in cryopreserved PBSC but not in BM compared to unfrozen allografts. In 9 out of 31 frozen PBSC (but none of the BM) grafts numbers of SSC(lo)ALDH(br) cells per kg body weight (BW) were significantly reduced while in the same grafts the numbers of CD34+ cells per kg BW were close to normal. Overall, 9 out of 33 patients (27%) who received unrelated PBSC allografts cryopreserved after transportation did not achieve engraftment. For comparison, primary graft failure was observed in our centre in only 7 out of 493 recipients (1.4%) of fresh allogeneic PBSC grafts. Moreover, we did not see any graft failure in patients receiving frozen/thawed BM or autologous PBSC transplants. We, therefore, conclude that PBSC grafts become much more sensitive to cryopreservation after transport and/or storage. Importantly, the engraftment potential of frozen HSC grafts may reliably be predicted by measuring ALDH activity.

Minimal residual disease diagnostics in myeloid malignancies in the post transplant period.

Allogeneic SCT is important in myelodysplastic syndrome, the BCR-ABL-negative chronic myeloproliferative diseases (CMPDs) and in poor-risk AML. Techniques to monitor the minimal residual disease, for example, by PCR or immunophenotyping gain increasing importance in the post transplantation period as basis for improved and earlier therapeutic interventions in impending relapse. Recent markers such as the NPM1 mutations in AML or the JAK2V617F mutation in the CMPD can be exactly quantified by real-time PCR and were evaluated for their prognostic value in the post transplantation phase and for their utility to plan adoptive immunotherapy in case of molecular relapse. With respect to chimerism, new and very sensitive methods were introduced, for example, quantitative assessment of genetic polymorphisms by real-time PCR, but also methods here are still highly individualized. Only in CML, where SCT focuses now on poor-risk cases or cases of tyrosine kinase inhibitor failure, follow-up schedules are standardized. Standardization of the different diagnostic techniques and of the intervals in the post transplantation period is urgently needed also in other myeloid malignancies and should be focus of future studies.

Remarkable leukemogenic potency and quality of a constitutively active neurotrophin receptor, deltaTrkA.

Neurotrophins and their receptors play a key role in neurogenesis and survival. However, we and others have recently obtained evidence for a potential involvement of this receptor system in leukemia. To investigate mechanisms underlying the leukemogenic potential of activated neurotrophin receptor signaling, we analyzed in vivo leukemogenesis mediated by deltaTrkA, a mutant of TRKA (tropomyosin-related kinase A) isolated from a patient with acute myeloid leukemia (AML). Retroviral expression of deltaTrkA in myeloid 32D cells induced AML in syngeneic C3H/Hej mice (n=11/11, latency approximately 4 weeks). C57Bl/6J mice transplanted with deltaTrkA-transduced primary lineage negative (Lin-) bone marrow cells died of a transient polyclonal AML (n=7/15, latency of <12 days). Serial transplantation of AML cells did not re-induce this disease but rather acute lymphoblastic leukemia (ALL, latency >78 days). All primary recipients surviving the early AML developed clonal ALL or myeloid leukemia (latency >72 days) that required additional genetic lesions. PI3K and mTOR-raptor were identified as the crucial mediators of leukemic transformation, whereas STAT and MAP kinase signaling pathways were not activated. Thus, our findings reveal potent and unique transforming properties of altered neurotrophin receptor signaling in leukemogenesis, and encourage further analyses of neurotrophin receptors and downstream signaling events in hematological malignancies.

Impact of molecular residual disease post allografting in myelofibrosis patients.

We screened 136 patients with myelofibrosis and a median age of 58 years who underwent allogeneic stem cell transplantation (AHSCT) for molecular residual disease for JAKV617F (n=101), thrombopoietin receptor gene (MPL) (n=4) or calreticulin (CALR) (n=31) mutation in peripheral blood on day +100 and +180 after AHSCT. After a median follow-up of 78 months, the 5-year estimated overall survival was 60% (95% confidence interval (CI): 50-70%) and the cumulative incidence of relapse at 5 years was 26% (95% CI: 18-34%) for the entire study population. The percentage of molecular clearance on day 100 was higher in CALR-mutated patients (92%) in comparison with MPL- (75%) and JAKV617F-mutated patients (67%). Patients with detectable mutation at day +100 or at day +180 had a significant higher risk of clinical relapse at 5 years than molecular-negative patients (62% vs 10%, P<0.001) and 70% vs 10%, P<0.001, respectively) irrespectively of the underlying mutation. In a multivariate analysis, high-risk diseases status (hazard ratio (HR) 2.5; 95% CI: 1.18-5.25, P=0.016) and detectable MRD at day 180 (HR 8.36, 95% CI: 2.76-25.30, P<0.001) were significant factors for a higher risk of relapse.

Allogeneic stem-cell transplantation in patients with refractory acute leukemia: a long-term follow-up.

We examined retrospectively 44 patients with refractory acute leukemia (acute myeloid leukemia (AML)/acute lymphoblastic leukemia=25/19) who underwent allogeneic transplantation at our center between 11/1990 and 04/2004. The median leukemic blasts was 25% and age 28 years (range, 3-56). Twenty-one patients had untreated relapse, 13 failed reinduction, eight in partial remission and two aplastic. Conditioning was myeloablative using cyclophosphamide, busulfan, total-body irradiation and etoposide (Bu/Cy/VP, n=22; TBI/Cy/VP, n=17; others, n=5) followed by marrow or peripheral blood transplant (n=23/21) from unrelated or related donors (n=28/16). All patients had graft-versus-host disease (GVHD) prophylaxis with cyclosporin and methotrexate. One patient experienced late graft failure. Severe acute-GVHD and chronic-GVHD appeared in eight and 14 patients, respectively. Thirteen patients (30%) remain alive after a median of 25.3 months (range, 2.4-134.1); with 31 deaths, mostly from relapse (n=15) and infections (n=12). Overall survival (OS) and progression-free survival (PFS) at 5 years was 28 and 26%, respectively. OS and PFS were significantly better with blasts < or =20% and time to transplant < or =1 year while transplant-related mortality was less with the use of TBI. We conclude that patients with refractory leukemia can benefit from allogeneic BMT, especially with < or =20% marrow blast.

Retroviral vector insertions in T-lymphocytes used for suicide gene therapy occur in gene groups with specific molecular functions.

Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.

Aldehyde dehydrogenase activity as a marker for the quality of hematopoietic stem cell transplants.

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH), hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon, we investigated the usefulness of ALDH activity for characterizing HSC graft quality, particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time, and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly, in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells, this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism, whereas all other patients showed early complete donor chimerism. In conclusion, we suggest to measure ALDH activity as a surrogate marker for HSC activity, and to transport and store PBSC under controlled cooling conditions.

Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

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 3beta (GSK-3beta) (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-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

The inositol 5-phosphatase SHIP is expressed as 145 and 135 kDa proteins in blood and bone marrow cells in vivo, whereas carboxyl-truncated forms of SHIP are generated by proteolytic cleavage in vitro.

The inositol polyphosphate 5-phosphatase SHIP plays an important role in negative signalling in B cells and mast cells and in the down-regulation of cytokine receptor-mediated signals in myeloid cells. SHIP is expressed as a 145 kDa full-length protein and an isoform of 135 kDa due to alternative splicing. Additional smaller forms of SHIP which are truncated at the carboxy terminus have been described in bone marrow and peripheral blood mononuclear cells (PBMC). Our data demonstrate that human bone marrow cells and PBMC from healthy donors and patients with acute myeloid leukemia express the 145 kDa form of SHIP and low amounts of a 135 kDa form of SHIP in vivo whereas C-terminal-truncated SHIP proteins are generated by a PMSF-sensitive protease during the preparation of cell lysates in vitro. We have further characterized this protease and identified a proteolytic cleavage site in the human SHIP protein C-terminal to tryptophan residue 941. These data support a physiological role for the 145 and 135 kDa forms of SHIP in bone marrow and peripheral blood cells from normal donors and patients with acute myeloid leukemia.

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell.

Many applications of hematopoietic gene therapy require selection for clones with active transgene expression. However, it was unclear whether the clonal progeny of a retrovirally transduced hematopoietic stem cell would be capable of maintaining transgene expression through serial repopulation and multilineage differentiation. Such investigations require simultaneous analyses of clonality, multilineage activity and transgene copy numbers. Using a mouse model, the present study demonstrates that a single hematopoietic stem cell expressing a marker gene from one or two insertions of a simple retroviral vector actively maintains multilineage transgene expression in the vast majority (80-99%) of bone marrow and peripheral blood cells. Gene expression persisted through serial transplantations for at least 97 weeks post gene transfer and was observed in the lymphoid (B, T and NK cells), myeloid (CD11b(+), Gr-1(+)), erythroid (Ter119(+), mature red blood cells) and megakaryocytic (as indicated by platelets) progeny. Therefore, a single immunoselection for hematopoietic stem cells expressing the transgene in vivo was sufficient to establish a completely chimeric hematopoiesis. These observations imply that the retroviral vectors used in this study contain cis-elements that mediate expression through massive clonal expansion and multilineage differentiation, provided the insertion occurred in genetic loci permissive for expression in hematopoietic stem cells.