Targeting expression to megakaryocytes and platelets by lineage-specific lentiviral vectors.

Essentials Platelet phenotypes can be modified by lentiviral transduction of hematopoietic stem cells. Megakaryocyte-specific lentiviral vectors were tested in vitro and in vivo for restricted expression. The glycoprotein 6 vector expressed almost exclusively in megakaryocytes. The platelet factor 4 vector was the strongest but with activity in hematopoietic stem cells. SUMMARY: Background Lentiviral transduction and transplantation of hematopoietic stem cells (HSCs) can be utilized to modify the phenotype of megakaryocytes and platelets. As the genetic modification in HSCs is transmitted onto all hematopoietic progenies, transgene expression from the vector should be restricted to megakaryocytes to avoid un-physiologic effects by ectopic transgene expression. This can be achieved by lentiviral vectors that control expression by lineage-specific promoters. Methods In this study, we introduced promoters of megakaryocyte/platelet-specific genes, namely human glycoprotein 6 (hGP6) and hGP9, into third generation lentiviral vectors and analyzed their functionality in vitro and in vivo in bone marrow transplantation assays. Their specificity and efficiency of expression was compared with lentiviral vectors utilizing the promoters of murine platelet factor 4 (mPf4) and hGP1BA, both with strong activity in megakaryocytes (MKs) used in earlier studies, and the ubiquitously expressing phosphoglycerate kinase (hPGK) and spleen focus forming virus (SFFV) enhancer/promoters. Results Expression from the mPf4 vector in MKs and platelets was the strongest similar to expression from the viral SFFV promoter, however, the mPf4 vector, also exhibited considerable off-target expression in hematopoietic stem and progenitor cells. In contrast, the newly generated hGP6 vector was highly specific to megakaryocytes and platelets. The specificity was also retained when reducing the promoter size to 350 bp, making it a valuable new tool for lentiviral expression in MKs/platelets. Conclusion MK-specific vectors express preferentially in the megakaryocyte lineage. These vectors can be applied to develop murine models to study megakaryocyte and platelet function, or for gene therapy targeting proteins to platelets.

Generation of iPSCs from genetically corrected Brca2 hypomorphic cells: implications in cell reprogramming and stem cell therapy.

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2(Δ) (27/) (Δ27)), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2(Δ) (27/) (Δ27) induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2(Δ) (27/) (Δ27) iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2(Δ) (27/) (Δ27) mouse embryonic fibroblasts. Gene-corrected Brca2(Δ) (27/) (Δ27) iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2(Δ) (27/) (Δ27) recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2(Δ) (27/) (Δ) (27) iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.

Chromosomal instability and telomere shortening in long-term culture of hematopoietic stem cells: insights from a cell culture model of RPS14 haploinsufficiency.

The fate of cultivated primary hematopoietic stem cells (HSCs) with respect to genetic instability and telomere attrition has not yet been described in great detail. Thus, knowledge of the genetic constitution of HSCs is important when interpreting results of HSCs in culture. While establishing a cell culture model for myelodysplastic syndrome with a deletion in 5q by performing RPS14 knockdown, we found surprising data that may be of importance for any CD34+ cell culture experiments. We performed cytogenetic analyses and telomere length measurement on transduced CD34+ cells and untransduced control cells to observe the effects of long-term culturing. Initially, CD34+ cells had a normal median telomere length of about 12 kb and showed no signs of chromosomal instability. During follow-up, the median telomere length seemed to decrease and, simultaneously, increased chromosomal instability could be observed - in modified and control cells. One culture showed a clonal monosomy 7 - independent of prior RPS14 knockdown. During further culturing, it seemed that the telomeres re-elongated, and chromosomes stabilized, while TERT expression was not elevated. In summary, irrespective of our results of RPS14 knockdown in the long-term culture of CD34+ cells, it becomes clear that cell culture artefacts inducing telomere shortening and chromosomal instability have to be taken into account and regular cytogenetic analyses should always be performed.

Efficient in vivo regulation of cytidine deaminase expression in the haematopoietic system using a doxycycline-inducible lentiviral vector system.

Regulated transgene expression may reduce transgene-specific and genotoxic risks associated with gene therapy. To prove this concept, we have investigated the suitability of doxycycline (Dox)-inducible human cytidine deaminase (hCDD) overexpression from lentiviral vectors to mediate effective myeloprotection while circumventing the lymphotoxicity observed with constitutive CDD activity. Rapid Dox-mediated transgene induction associated with a 6-17-fold increase in drug resistance was observed in 32D and primary murine bone marrow (BM) cells. Moreover, robust Dox-regulated transgene expression in the entire haematopoietic system was demonstrated for primary and secondary recipients of hCDD-transduced R26-M2rtTA transgenic BM cells. Furthermore, mice were significantly protected from myelosuppressive chemotherapy as evidenced by accelerated recovery of granulocytes (1.9±0.6 vs 1.3±0.3, P=0.034) and platelets (883±194 vs 584±160 10(3) per µl, P=0.011). Minimal transgene expression in the non-induced state and no overt cellular toxicities including lymphotoxicity were detected. Thus, using a relevant murine transplant model our data provide conclusive evidence that drug-resistance transgenes can be expressed in a regulated fashion in the lymphohaematopoietic system, and that Dox-inducible systems may be used to reduce myelotoxic side effect of anticancer chemotherapy or to avoid side effects of high constitutive transgene expression.

Selection for Evi1 activation in myelomonocytic leukemia induced by hyperactive signaling through wild-type NRas.

Activation of NRas signaling is frequently found in human myeloid leukemia and can be induced by activating mutations as well as by mutations in receptors or signaling molecules upstream of NRas. To study NRas-induced leukemogenesis, we retrovirally overexpressed wild-type NRas in a murine bone marrow transplantation (BMT) model in C57BL/6J mice. Overexpression of wild-type NRas caused myelomonocytic leukemias ∼3 months after BMT in the majority of mice. A subset of mice (30%) developed malignant histiocytosis similar to mice that received mutationally activated NRas(G12D)-expressing bone marrow. Aberrant Ras signaling was demonstrated in cells expressing mutationally active or wild-type NRas, as increased activation of Erk and Akt was observed in both models. However, more NRas(G12D) were found to be in the activated, GTP-bound state in comparison with wild-type NRas. Consistent with observations reported for primary human myelomonocytic leukemia cells, Stat5 activation was also detected in murine leukemic cells. Furthermore, clonal evolution was detected in NRas wild-type-induced leukemias, including expansion of clones containing activating vector insertions in known oncogenes, such as Evi1 and Prdm16. In vitro cooperation of NRas and Evi1 improved long-term expansion of primary murine bone marrow cells. Evi1-positive cells upregulated Bcl-2 and may, therefore, provide anti-apoptotic signals that collaborate with the NRas-induced proliferative effects. As activation of Evi1 has been shown to coincide with NRAS mutations in human acute myeloid leukemia, our murine model recapitulates crucial events in human leukemogenesis.

MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy.

Endogenous microRNA (miRNA) expression can be exploited for cell type-specific transgene expression as the addition of miRNA target sequences to transgenic cDNA allows for transgene downregulation specifically in cells expressing the respective miRNAs. Here, we have investigated the potential of miRNA-150 target sequences to specifically suppress gene expression in lymphocytes and thereby prevent transgene-induced lymphotoxicity. Abundance of miRNA-150 expression specifically in differentiated B and T cells was confirmed by quantitative reverse transcriptase PCR. Mono- and bicistronic lentiviral vectors were used to investigate the effect of miRNA-150 target sequences on transgene expression in the lymphohematopoietic system. After in vitro studies demonstrated effective downregulation of transgene expression in murine B220(+) B and CD3(+) T cells, the concept was further verified in a murine transplant model. Again, marked suppression of transgene activity was observed in B220(+) B and CD4(+) or CD8(+) T cells whereas expression in CD11b(+) myeloid cells, lin(-) and lin(-)/Sca1(+) progenitors, or lin(-)/Sca1(+)/c-kit(+) stem cells remained almost unaffected. No toxicity of miRNA-150 targeting in transduced lymphohematopoietic cells was noted. Thus, our results demonstrate the suitability of miRNA-150 targeting to specifically suppress transgene expression in lymphocytes and further support the concept of miRNA targeting for cell type-specific transgene expression in gene therapy approaches.

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors.

Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.

Epidermal growth factor improves lentivirus vector gene transfer into primary mouse hepatocytes.

Partial resistance of primary mouse hepatocytes to lentiviral (LV) vector transduction poses a challenge for ex vivo gene therapy protocols in models of monogenetic liver disease. We thus sought to optimize ex vivo LV gene transfer while preserving the hepatocyte integrity for subsequent transplantation into recipient animals. We found that culture media supplemented with epidermal growth factor (EGF) and, to a lesser extent, hepatocyte growth factor (HGF) markedly improved transduction efficacy at various multiplicities of infection. Up to 87% of primary hepatocytes were transduced in the presence of 10 ng EGF, compared with ~30% in standard culture medium (SCMs). The increased number of transgene-expressing cells correlated with increased nuclear import and more integrated pro-viral copies per cell. Higher LV transduction efficacy was not associated with proliferation, as transduction capacity of gammaretroviral vectors remained low (<1%). Finally, we developed an LV transduction protocol for short-term (maximum 24 h) adherent hepatocyte cultures. LV-transduced hepatocytes showed liver repopulation capacities similar to freshly isolated hepatocytes in alb-uPA mouse recipients. Our findings highlight the importance of EGF for efficient LV transduction of primary hepatocytes in culture and should facilitate studies of LV gene transfer in mouse models of monogenetic liver disease.

Lentiviral vectors for induction of self-differentiation and conditional ablation of dendritic cells.

Development of lentiviral vectors (LVs) in the field of immunotherapy and immune regeneration will strongly rely on biosafety of the gene transfer. We demonstrated previously the feasibility of ex vivo genetic programming of mouse bone marrow precursors with LVs encoding granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), which induced autonomous differentiation of long-lived dendritic cells (DCs), referred to as self-differentiated myeloid-derived antigen-presenting-cells reactive against tumors (SMART-DCs). Here, LV biosafety was enhanced by using a DC-restricted and physiological promoter, the major histocompatibility complex (MHC) II promoter, and including co-expression of the herpes simplex virus-thymidine kinase (sr39HSV-TK) conditional suicide gene. Tricistronic vectors co-expressing sr39HSV-TK, GM-CSF and IL-4 transcriptionally regulated by the MHCII promoter or the ubiquitous cytomegalovirus (CMV) promoter were compared. Despite the different gene transfer effects, such as the kinetics, levels of transgene expression and persistency of integrated vector copies, both vectors induced highly viable SMART-DCs, which persisted for at least 70 days in vivo and could be ablated with the pro-drug Ganciclovir (GCV). SMART-DCs co-expressing the tyrosine-related protein 2 melanoma antigen administered subcutaneously generated antigen-specific, anti-melanoma protective and therapeutic responses in the mouse B16 melanoma model. GCV administration after immunotherapy did not abrogate DC vaccination efficacy. This demonstrates proof-of-principle of genetically programmed DCs that can be ablated pharmacologically.

Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16.

Insertional activation of cellular proto-oncogenes by replication-defective retroviral vectors can trigger clonal dominance and leukemogenesis in animal models and clinical trials. Here, we addressed the leukemogenic potential of vectors expressing interleukin-2 receptor common gamma-chain (IL2RG), the coding sequence required for correction of X-linked severe combined immunodeficiency. Similar to conventional gamma-retroviral vectors, self-inactivating (SIN) vectors with strong internal enhancers also triggered profound clonal imbalance, yet with a characteristic insertion preference for a window located downstream of the transcriptional start site. Controls including lentivirally transduced cells revealed that ectopic IL2RG expression was not sufficient to trigger leukemia. After serial bone marrow transplantation involving 106 C57Bl6/J mice monitored for up to 18 months, we observed leukemic progression of six distinct clones harboring gamma-retroviral long terminal repeat (LTR) or SIN vector insertions in Evi1 or Prdm16, two functionally related genes. Three leukemic clones had single vector integrations, and identical clones manifested with a remarkably similar latency and phenotype in independent recipients. We conclude that upregulation of Evi1 or Prdm16 was sufficient to initiate a leukemogenic cascade with consistent intrinsic dynamics. Our study also shows that insertional mutagenesis is required for leukemia induction by IL2RG vectors, a risk to be addressed by improved vector design.

[Gene therapy of SCID-X1]. / Gentherapie der SCID-X1.

X-linked severe combined immunodeficiency (SCID-X1) is an inherited disease caused by inactivating mutations in the gene encoding the interleukin 2 receptor common gamma chain (IL2RG), which is located on the X-chromosome. Affected boys fail to develop two major effector cell types of the immune system (T cells and NK cells) and suffer from a functional B cell defect. Although drugs such as antibiotics can offer partial protection, the boys normally die in the first year of life in the absence of a curative therapy. For a third of the children, bone marrow transplantation from a fully matched donor is available and can cure the disease without major side effects. Mismatched bone marrow transplantation, however, is complicated by severe and potentially lethal side effects. Over the past decade, scientists worldwide have developed new treatments by introducing a correct copy of the IL2RG-cDNA. Gene therapy was highly effective when applied in young children. However, in a few patients the IL2RG-gene vector has unfortunately caused leukaemia. Activation of cellular proto-oncogenes by accidental integration of the gene vector has been identified as the underlying mechanism. In future clinical trials, improved vector technology in combination with other protocol modifications may reduce the risk of this side effect.

Self-inactivating retroviral vectors with improved RNA processing.

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5' untranslated region (5'UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3'UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3' long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Psi). The latter concept is new and allows the use of an intron in the 5'UTR, taking advantage of retroviral splice sites surrounding Psi. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 10(6) infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis.

Delta-Notch signalling regulates cell-fate choices in a variety of tissues during development. We report the expression of Delta4 (D14) in arterial endothelium during mouse embryogenesis and in the endothelium of tumor blood vessels. The expression of D14 in the mouse begins at 8 dpc in the dorsal aortae, umbilical artery and the heart. Subsequent expression is restricted to smaller vessels and capillaries and is reduced in most adult tissues. However, it is high in the vasculature of xenograft human tumors in the mouse, in endogenous human tumors and is regulated by hypoxia. These data implicate D14 and the Notch signalling pathway in angiogenesis and suggest possible new targets for antiangiogenic tumor therapy.

Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers.

One of the current challenges in gene therapy is to construct a vector that will target specific tissues. Targeting expression to endothelium is of particular interest in the treatment of several pathologies. We have shown previously that defined regions of the E-selectin and KDR promoters confer endothelial cell specific expression following retroviral delivery. However, the levels of expression were low. In an attempt to increase expression but to preserve the tissue specificity we have examined hypoxic and cytokine-inducible enhancer elements in combination with the KDR and E-selectin promoters. Both enhancers should be active in the tumour environment, boosting expression and giving additional specificity of gene expression in the tumour endothelium. The hypoxia response element (HRE) of the murine phosphoglycerate kinase-1 (PGK-1) promoter was used as a hypoxic enhancer and the tandem-binding site for NFKB from the murine vascular cell adhesion molecule-1 (VCAM-1) promoter as a cytokine-inducible enhancer. The HRE conferred hypoxia inducibility to the KDR and E-selectin promoters. Endothelial specificity of expression was retained with the KDR but not the E-selectin promoter. The NFKB-binding site conferred responsiveness to TNF-alpha to the KDR promoter, however the level of induction was less than that achieved with the HRE. Retrovirus combining both enhancer elements transferred inducibility by hypoxia and TNF-alpha, and reached the highest expression levels upon stimulation. These results confirm that heterologous enhancer elements may operate on a single endothelial cell specific promoter. These findings make the use of inducible enhancers a promising strategy for increasing tissue specific gene expression.

Cyclic angiogenesis and blood vessel regression in the ovary: blood vessel regression during luteolysis involves endothelial cell detachment and vessel occlusion.

Angiogenesis occurs as a cyclically regulated process in the ovary and the uterus. After ovulation, there is massive sprouting of blood vessels in the growing corpus luteum (CL) during the first third of the ovarian cycle. During luteolysis and for several weeks thereafter, all newly formed vessels regress. Here we have systematically analyzed regression of blood vessels during luteolysis to identify mechanisms of blood vessel regression. Blood vessel counts are highest in the midcycle CL and drop rapidly after the onset of luteolysis. After a rapid phase of tissue dissociation, blood vessel regression proceeds slowly over several weeks in the residual CL. Endothelial cells in regressing vessels acquire a distinctly rounded and condensed phenotype. Ultrastructural analysis of blood vessel regression processes in the cyclic CL suggests two major mechanisms of blood vessel regression: a) detachment of rounded endothelial cells from their basement membrane, leaving areas devoid of covering endothelial cell monolayer, and b) contraction and occlusion of arterioles and small arteries with pronounced proliferation of smooth muscle cells. In situ detection of nucleosomal fragmentation products demonstrates numerous apoptotic luteal cells, but only a few apoptotic endothelial cells in the regressing CL. Induction of apoptosis in cultured endothelial cell monolayers by RGD peptides demonstrated that endothelial cells detach from their adhesive surface before fully becoming positive for nucleosomal fragmentation products. These data indicate that cyclic angiogenic processes in the ovary offer a suitable experimental system to analyze mechanisms of blood vessel growth and regression, and suggest that detachment of endothelial cells before apoptosis as well as contractive occlusion of blood vessels may be critical determinants of blood vessel regression.

Ovarian angiogenesis. Phenotypic characterization of endothelial cells in a physiological model of blood vessel growth and regression.

Angiogenesis occurs during embryogenesis and is a down-regulated process in the healthy adult that is almost exclusively linked to pathological conditions such as tumor growth, wound healing, and inflammation. Physiological angiogenic processes in the adult are restricted to the female reproductive system where they occur cyclically during the ovarian and uterine cycle as well as during pregnancy. By systematically analyzing the phenotypic changes of endothelial cells during bovine corpus luteum (CL) formation and regression, we have established a physiological model of blood vessel growth and regression. Quantitation of vessel density, percentage of vessels with lumen, and ratio of Bandeiraea simplicifolia-I to von Willebrand Factor-positive endothelial cells were established as parameters of angiogenesis. Sprouting endothelial cells invade the growing CL and continue to grow throughout the first third of the ovarian cycle. Thereafter the mature CL is characterized by a dense network of vessels with gradually decreasing vessel density. During luteolysis and for several weeks thereafter (regressing and residual CL) all newly formed vessels regress, which is accompanied by gradual foreshortening and rounding of endothelial cells and subsequent detachment. Based on histochemical detection of nucleosomal fragmentation products physiological blood vessel regression in the cyclic CL does not appear to involve endothelial cell apoptosis. Lectin histochemical analysis revealed a distinct alteration of endothelial cell glycoconjugate expression during ovarian angiogenesis comparable with the distinct pattern of hyperglycosylation of cultured migrating endothelial cells (up-regulation of binding sites for Lycopersicon esculentum lectin, wheat germ agglutinin, neuraminidase-treated peanut agglutinin, and Ricinus communis agglutinin-I on sprouting ECs). Northern blot analysis of glycosyltransferases during the different stages of angiogenesis revealed an up-regulation of beta-galactoside alpha 2,6-sialyltransferase and alpha 1,3-galactosyltransferase mRNA expression during the angiogenic stages of CL formation. These data establish the ovarian angiogenesis model as a suitable experimental system to study the functional and phenotypic properties of endothelial cells in sprouting and regressing blood vessels and provide additional evidence for the importance of endothelial cell surface glycoconjugates during angiogenesis.