Development of Cyclic Peptides Targeting the Epidermal Growth Factor Receptor in Mesenchymal Triple-Negative Breast Cancer Subtype.

Triple-negative breast cancer (TNBC) is an aggressive malignancy characterized by the lack of expression of estrogen and progesterone receptors and amplification of human epidermal growth factor receptor 2 (HER2). Being the Epidermal Growth Factor Receptor (EGFR) highly expressed in mesenchymal TNBC and correlated with aggressive growth behavior, it represents an ideal target for anticancer drugs. Here, we have applied the phage display for selecting two highly specific peptide ligands for targeting the EGFR overexpressed in MDA-MB-231 cells, a human TNBC cell line. Molecular docking predicted the peptide-binding affinities and sites in the extracellular domain of EGFR. The binding of the FITC-conjugated peptides to human and murine TNBC cells was validated by flow cytometry. Confocal microscopy confirmed the peptide binding specificity to EGFR-positive MDA-MB-231 tumor xenograft tissues and their co-localization with the membrane EGFR. Further, the peptide stimulation did not affect the cell cycle of TNBC cells, which is of interest for their utility for tumor targeting. Our data indicate that these novel peptides are highly specific ligands for the EGFR overexpressed in TNBC cells, and thus they could be used in conjugation with nanoparticles for tumor-targeted delivery of anticancer drugs.

SARS CoV-2 spike protein-guided exosome isolation facilitates detection of potential miRNA biomarkers in COVID-19 infections.

OBJECTIVES: Nearly three years into the pandemic, SARS-CoV-2 infections are occurring in vaccinated and naturally infected populations. While humoral and cellular responses in COVID-19 are being characterized, novel immune biomarkers also being identified. Recently, an increase in angiotensin-converting enzyme 2 expressing (aka, ACE2 positive) circulating exosomes (ExoACE2) were identified in the plasma of COVID-19 patients (El-Shennawy et al.). In this pilot study, we describe a method to characterize the exosome-associated microRNA (exo-miRNA) signature in ACE2-positive and ACE2-negative exosomal populations (non-ExoACE2). METHODS: We performed a sorting protocol using the recombinant biotin-conjugated SARS CoV-2 spike protein containing the receptor binding domain (RBD) on plasma samples from six patients. Following purification, exo-miRNA were characterized for ACE2-positive and ACE2-negative exosome subpopulations by RT-PCR. RESULTS: We identified differential expression of several miRNA. Specifically let-7g-5p and hsa-miR-4454+miR-7975 were upregulated, while hsa-miR-208a-3p and has-miR-323-3p were downregulated in ExoACE2 vs. non-ExoACE2. CONCLUSIONS: The SARS CoV-2 spike-protein guided exosome isolation permits isolation of ExoACE2 exosomes. Such purification facilitates detailed characterization of potential biomarkers (e.g. exo-miRNA) for COVID-19 patients. This method could be used for future studies to further the understanding mechanisms of host response against SARS CoV-2.

Unmutated IGHV1-69 CLL Clone Displays a Distinct Gene Expression Profile by a Comparative qRT-PCR Assay.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease characterized by variable clinical courses among different patients. This notion was supported by the possible coexistence of two or more independent CLL clones within the same patients, identified by the characterization of the B cell receptor immunoglobulin (BcR IG) idiotypic sequence. By using the antigen-binding site of the BcR IG as bait, the identification and isolation of aggressive and drug-resistance leukemic B-cell clones could allow a deeper biological and molecular investigation. Indeed, by the screening of phage display libraries, we previously selected a peptide binder of the idiotypic region of CLL BCR IGs expressing the unmutated rearrangement IGHV1-69 and used it as a probe to perform a peptide-based cell sorting by flow cytometry in peripheral blood samples from patients with CLL. Since the IGHV1-69 clones persisted during the follow-up time in both patients, we explored the possibility of these clones having acquired an evolutive advantage compared to the other coexisting clones in terms of a higher expression of genes involved in the survival and apoptosis escape processes. To this end, we studied the expression patterns of a panel of genes involved in apoptosis regulation and in NF-kB-dependent pro-survival signals by comparative qRT-PCR assays. According to the results, IGHV1-69 clones showed a higher expression of pro-survival and anti-apoptotic genes as compared to the other CLL clones with different immunogenetic characteristics. Moreover, these IGHV1-69 clones did not carry any characteristic genetic lesions, indicating the relevance of our approach in performing a comprehensive molecular characterization of single tumor clones, as well as for designing new personalized therapeutic approaches for the most aggressive and persistent tumor clones.

A novel phage display based platform for exosome diversity characterization.

We present an innovative approach allowing the identification, isolation, and molecular characterization of disease-related exosomes based on their different antigenic reactivities. The designed strategy could be immediately translated into any disease in which exosomes are involved. The identification of specific markers and their subsequent association with exosome subtypes, together with the possibility to engineer target-guided exosome-like particles, could represent the key for the effective adoption of exosomes in clinical practice.

IBtkα Activates the ß-Catenin-Dependent Transcription of MYC through Ubiquitylation and Proteasomal Degradation of GSK3ß in Cancerous B Cells.

The IBTK gene encodes the IBtkα protein that is a substrate receptor of E3 ubiquitin ligase, Cullin 3. We have previously reported the pro-tumorigenic activity of Ibtk in MYC-dependent B-lymphomagenesis observed in Eµ-myc transgenic mice. Here, we provide mechanistic evidence of the functional interplay between IBtkα and MYC. We show that IBtkα, albeit indirectly, activates the ß-catenin-dependent transcription of the MYC gene. Of course, IBtkα associates with GSK3ß and promotes its ubiquitylation, which is associated with proteasomal degradation. This event increases the protein level of ß-catenin, a substrate of GSK3ß, and results in the transcriptional activation of the MYC and CCND1 target genes of ß-catenin, which are involved in the control of cell division and apoptosis. In particular, we found that in Burkitt's lymphoma cells, IBtkα silencing triggered the downregulation of both MYC mRNA and protein expression, as well as a strong decrease of cell survival, mainly through the induction of apoptotic events, as assessed by using flow cytometry-based cell cycle and apoptosis analysis. Collectively, our results shed further light on the complex puzzle of IBtkα interactome and highlight IBtkα as a potential novel therapeutic target to be employed in the strategy for personalized therapy of B cell lymphoma.

Metabolites Profiling of Melanoma Interstitial Fluids Reveals Uridine Diphosphate as Potent Immune Modulator Capable of Limiting Tumor Growth.

Tumor interstitial fluid (TIF) surrounds and perfuses tumors and collects ions, metabolites, proteins, and extracellular vesicles secreted by tumor and stromal cells. Specific metabolites, accumulated within the TIF, could induce metabolic alterations of immune cells and shape the tumor microenvironment. We deployed a metabolomic approach to analyze the composition of melanoma TIF and compared it to the plasma of C57BL6 mice, engrafted or not with B16-melanoma cells. Among the classes of metabolites analyzed, monophosphate and diphosphate nucleotides resulted enriched in TIF compared to plasma samples. The analysis of the effects exerted by guanosine diphosphate (GDP) and uridine diphosphate (UDP) on immune response revealed that GDP and UDP increased the percentage of CD4+CD25+FoxP3- and, on isolated CD4+ T-cells, induced the phosphorylation of ERK, STAT1, and STAT3; increased the activity of NF-κB subunits p65, p50, RelB, and p52; increased the expression of Th1/Th17 markers including IFNγ, IL17, T-bet, and RORγt; and reduced the expression of IL13, a Th2 marker. Finally, we observed that local administrations of UDP in B16-engrafted C57BL6 mice reduced tumor growth and necrotic areas. In addition, UDP-treated tumors showed a higher presence of MHCIIhi tumor-associated macrophage (TAM) and of CD3+CD8+ and CD3+CD4+ tumor-infiltrating T-lymphocytes (TILs), both markers of anti-tumor immune response. Consistent with this, intra-tumoral gene expression analysis revealed in UDP-treated tumors an increase in the expression of genes functionally linked to anti-tumor immune response. Our analysis revealed an important metabolite acting as mediator of immune response, which could potentially represent an additional tool to be used as an adjuvant in cancer immunotherapy.

ZNF521 Enhances MLL-AF9-Dependent Hematopoietic Stem Cell Transformation in Acute Myeloid Leukemias by Altering the Gene Expression Landscape.

Leukemias derived from the MLL-AF9 rearrangement rely on dysfunctional transcriptional networks. ZNF521, a transcription co-factor implicated in the control of hematopoiesis, has been proposed to sustain leukemic transformation in collaboration with other oncogenes. Here, we demonstrate that ZNF521 mRNA levels correlate with specific genetic aberrations: in particular, the highest expression is observed in AMLs bearing MLL rearrangements, while the lowest is detected in AMLs with FLT3-ITD, NPM1, or CEBPα double mutations. In cord blood-derived CD34+ cells, enforced expression of ZNF521 provides a significant proliferative advantage and enhances MLL-AF9 effects on the induction of proliferation and the expansion of leukemic progenitor cells. Transcriptome analysis of primary CD34+ cultures displayed subsets of genes up-regulated by MLL-AF9 or ZNF521 single transgene overexpression as well as in MLL-AF9/ZNF521 combinations, at either the early or late time points of an in vitro leukemogenesis model. The silencing of ZNF521 in the MLL-AF9 + THP-1 cell line coherently results in an impairment of growth and clonogenicity, recapitulating the effects observed in primary cells. Taken together, these results underscore a role for ZNF521 in sustaining the self-renewal of the immature AML compartment, most likely through the perturbation of the gene expression landscape, which ultimately favors the expansion of MLL-AF9-transformed leukemic clones.

Regulatory Role of microRNAs Targeting the Transcription Co-Factor ZNF521 in Normal Tissues and Cancers.

Powerful bioinformatics tools have provided a wealth of novel miRNA-transcription factor networks crucial in controlling gene regulation. In this review, we focus on the biological functions of miRNAs targeting ZNF521, explaining the molecular mechanisms by which the dysregulation of this axis contributes to malignancy. ZNF521 is a stem cell-associated co-transcription factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells. The aberrant expression of ZNF521 transcripts, frequently associated with miRNA deregulation, has been detected in several tumors including pancreatic, hepatocellular, gastric, bladder transitional cell carcinomas as well as in breast and ovarian cancers. miRNA expression profiling tools are currently identifying a multitude of miRNAs, involved together with oncogenes and TFs in the regulation of oncogenesis, including ZNF521, which may be candidates for diagnostic and prognostic biomarkers of cancer.

Predominant VH1-69 IgBCR Clones Show Higher Expression of CD5 in Heterogeneous Chronic Lymphocytic Leukemia Populations.

The immunoglobulin B cell receptor (IgBCR) expressed by chronic lymphocytic leukemia (CLL) B cells plays a pivotal role in tumorigenesis, supporting neoplastic transformation, survival, and expansion of tumor clones. We demonstrated that in the same patient, two or more CLL clones could coexist, recognized by the expression of different variable regions of the heavy chain of IgBCR, composing the antigen-binding site. In this regard, phage display screening could be considered the easier and most advantageous methodology for the identification of small peptide molecules able to mimic the natural antigen of the tumor IgBCRs. These molecules, properly functionalized, could be used as a probe to specifically identify and isolate single CLL subpopulations, for a deeper analysis in terms of drug resistance, phenotype, and gene expression. Furthermore, CLL cells express another surface membrane receptor, the CD5, which is commonly expressed by normal T cells. Piece of evidence supports a possible contribution of CD5 to the selection and maintenance of autoreactivity in B cells and the constitutive expression of CD5 on CLL cells could induce pro-survival stimuli. In this brief research report, we describe a peptide-based single-cell sorting using as bait the IgBCR of tumor cells; in the next step, we performed a quantitative analysis of CD5 expression by qRT-PCR related to the expressed IgBCR. Our approach could open a new perspective for the identification, isolation, and investigation of all subsets of IgBCR-related CLL clones, with particular attention to the more aggressive clones.

Phage-Displayed Peptides for Targeting Tyrosine Kinase Membrane Receptors in Cancer Therapy.

Receptor tyrosine kinases (RTKs) regulate critical physiological processes, such as cell growth, survival, motility, and metabolism. Abnormal activation of RTKs and relative downstream signaling is implicated in cancer pathogenesis. Phage display allows the rapid selection of peptide ligands of membrane receptors. These peptides can target in vitro and in vivo tumor cells and represent a novel therapeutic approach for cancer therapy. Further, they are more convenient compared to antibodies, being less expensive and non-immunogenic. In this review, we describe the state-of-the-art of phage display for development of peptide ligands of tyrosine kinase membrane receptors and discuss their potential applications for tumor-targeted therapy.

Nasal Polyposis: Insights in Epithelial-Mesenchymal Transition and Differentiation of Polyp Mesenchymal Stem Cells.

Chronic rhinosinusitis is a common inflammatory disease of paranasal sinuses, which causes rhinorrhea, nasal congestion, and hyposmia. The genetic predisposition or the exposure to irritants can sustain the inflammatory response and the development of nasal polyposis. Nasal polyps are benign and teardrop-shaped growths that project in the nasal cavities, and originate from the ethmoid sinuses. This inflammatory process is associated with high expression of IL-4, IL-5 and IL-13 and IgE. Antibodies targeting these cytokines or receptors represent a therapeutic strategy in the treatment of nasal polyposis in combination with corticosteroids. The molecular pathogenesis of nasal polyps in chronic rhinosinusitis (CRS) patients is associated with remodeling transition, a process in which epithelial cells lose their typical phenotype, acquiring a mesenchymal-like aspect. TGFß/SMAD, ERK, and Wnt/ß-catenin pathways are altered during the nasal tissue remodeling. miRNA and inhibitor molecules targeting these signaling pathways are able to interfere with the process; which could lead to alternative therapies. Nasal polyps are an alternative source of mesenchymal stem cells, which can be isolated from surgical biopsies. A molecular understanding of the biology of PO-MSCs will contribute to the delineating inflammatory process underlying the development of nasal polyps.

Zoledronic acid inhibits the growth of leukemic MLL-AF9 transformed hematopoietic cells.

A leukemic in vitro model produced by transducing Cord Blood derived-hematopoietic CD34+ cells with the MLL-AF9 translocation resulting in the oncogenic fusion protein, is used to assess for sensitivity to Zoledronic acid. These cells are practically immortalized and are of myeloid origin. Proliferation, clonogenic and stromal co-culture assays showed that the MLL-AF9 cells were considerably more sensitive to Zoledronic acid than normal hematopoietic CD34+ cells or MS-5 stromal cells. The MLL-AF9 cells were notably more inhibited by Zoledronic acid when cultured as colonies in 3 dimensions, requiring cell-cell contacts compared to suspension expansion cultures. This is coherent with the mechanism of action of Zoledronic acid inhibiting farnesyl diphosphate synthase which results in a block in prenylation of GTPases such that their role in the membrane is compromised for cell-cell contacts. Zoledronic acid can be proposed to target the MLL-AF9 leukemic stem cells before they emerge from the hematopoietic niche, which being in proximity to bone osteoclasts where Zoledronic acid is sequestered can be predicted to result in sufficient levels to result in an anti-leukemic action.

The stem cell-associated transcription co-factor, ZNF521, interacts with GLI1 and GLI2 and enhances the activity of the Sonic hedgehog pathway.

ZNF521 is a transcription co-factor with recognized regulatory functions in haematopoietic, osteo-adipogenic and neural progenitor cells. Among its diverse activities, ZNF521 has been implicated in the regulation of medulloblastoma (MB) cells, where the Hedgehog (HH) pathway, has a key role in the development of normal cerebellum and of a substantial fraction of MBs. Here a functional cross-talk is shown for ZNF521 with the HH pathway, where it interacts with GLI1 and GLI2, the major HH transcriptional effectors and enhances the activity of HH signalling. In particular, ZNF521 cooperates with GLI1 and GLI2 in the transcriptional activation of GLI (glioma-associated transcription factor)-responsive promoters. This synergism is dependent on the presence of the N-terminal, NuRD-binding motif in ZNF521, and is sensitive to HDAC (histone deacetylase) and GLI inhibitors. Taken together, these results highlight the role of ZNF521, and its interaction with the NuRD complex, in determining the HH response at the level of transcription. This may be of particular relevance in HH-driven diseases, especially regarding the MBs belonging to the SHH (sonic HH) subgroup where a high expression of ZNF521 is correlated with that of HH pathway components.

ZNF521 Represses Osteoblastic Differentiation in Human Adipose-Derived Stem Cells.

Human adipose-derived stem cells (hADSCs) are multipotent mesenchymal cells that can differentiate into adipocytes, chondrocytes, and osteocytes. During osteoblastogenesis, the osteoprogenitor cells differentiate into mature osteoblasts and synthesize bone matrix components. Zinc finger protein 521 (ZNF521/Zfp521) is a transcription co-factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells, where it has been shown to inhibit adipogenic differentiation. The present study is aimed at determining the effects of ZNF521 on the osteoblastic differentiation of hADSCs to clarify whether it can influence their osteogenic commitment. The enforced expression or silencing of ZNF521 in hADSCs was achieved by lentiviral vector transduction. Cells were cultured in a commercial osteogenic medium for up to 20 days. The ZNF521 enforced expression significantly reduced osteoblast development as assessed by the morphological and molecular criteria, resulting in reduced levels of collagen I, alkaline phosphatase, osterix, osteopontin, and calcium deposits. Conversely, ZNF521 silencing, in response to osteoblastic stimuli, induced a significant increase in early molecular markers of osteogenesis and, at later stages, a remarkable enhancement of matrix mineralization. Together with our previous findings, these results show that ZNF521 inhibits both adipocytic and osteoblastic maturation in hADSCs and suggest that its expression may contribute to maintaining the immature properties of hADSCs.

Turning Stem Cells Bad: Generation of Clinically Relevant Models of Human Acute Myeloid Leukemia through Gene Delivery- or Genome Editing-Based Approaches.

Acute myeloid leukemia (AML), the most common acute leukemia in the adult, is believed to arise as a consequence of multiple molecular events that confer on primitive hematopoietic progenitors unlimited self-renewal potential and cause defective differentiation. A number of genetic aberrations, among which a variety of gene fusions, have been implicated in the development of a transformed phenotype through the generation of dysfunctional molecules that disrupt key regulatory mechanisms controlling survival, proliferation, and differentiation in normal stem and progenitor cells. Such genetic aberrations can be recreated experimentally to a large extent, to render normal hematopoietic stem cells "bad", analogous to the leukemic stem cells. Here, we wish to provide a brief outline of the complementary experimental approaches, largely based on gene delivery and more recently on gene editing, employed over the last two decades to gain insights into the molecular mechanisms underlying AML development and progression and on the prospects that their applications offer for the discovery and validation of innovative therapies.

ZNF423: A New Player in Estrogen Receptor-Positive Breast Cancer.

Preventive therapy can target hormone-responsive breast cancer (BC) by treatment with selective estrogen receptor modulators (SERMs) and reduce the incidence of BC. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) with relevant predictive values, SNPs in the ZNF423 gene were associated with decreased risk of BC during SERM therapy, and SNPs in the Cathepsin O gene with an increased risk. ZNF423, which was not previously associated with BC is a multifunctional transcription factor known to have a role in development, neurogenesis, and adipogenesis and is implicated in other types of cancer. ZNF423 is transcriptionally controlled by the homolog ZNF521, early B cell factor transcription factor, epigenetic silencing of the promoter by CpG island hyper-methylation, and also by ZNF423 itself in an auto-regulatory loop. In BC cells, ZNF423 expression is found to be induced by estrogen, dependent on the binding of the estrogen receptor and calmodulin-like 3 to SNPs in ZNP423 intronic sites in proximity to consensus estrogen response elements. ZNF423 has also been shown to play a mechanistic role by trans-activating the tumor suppressor BRCA1 and thus modulating the DNA damage response. Even though recent extensive trial studies did not classify these SNPs with the highest predictive values, for inclusion in polygenic SNP analysis, the mechanism unveiled in these studies has introduced ZNF423 as a factor important in the control of the estrogen response. Here, we aim at providing an overview of ZNF423 expression and functional role in human malignancies, with a specific focus on its implication in hormone-responsive BC.

ZNF521 Has an Inhibitory Effect on the Adipogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors present in the bone marrow stroma and in subcutaneous abdominal fat, an abundant and easily accessible source of MSCs with the ability to differentiate along multiple lineage pathways. The stem cell-associated transcription co-factor Zinc Finger Protein 521 (ZNF521/zfp521) has been implicated in the control of the homeostasis of hematopoietic, neural and osteo-adipogenic progenitors. Here we document through the analysis of a panel of human adipose-derived stem cells (hADSCs), that ZNF521 strongly inhibits the generation of mature adipocytes. Enforced overexpression of ZNF521 in these cells resulted in a significant delay and reduction in adipocyte differentiation upon exposure to inducers of adipogenesis. Of particular relevance, ZNF521 was able to inhibit the expression of ZNF423, recently identified as an essential commitment factor necessary for the generation of pre-adipocytes. Conversely, silencing of ZNF521 was found to significantly enhance the adipogenic differentiation of hADSCs. Inhibition of adipogenesis by ZNF521 was at least in part due to inhibition of EBF1. Taken together, these results confirm a role for ZNF521 as a key negative regulator of adipocyte differentiation of hADSCs.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications. The availability of strategies for safe ex vivo expansion of CB-derived HSCs (CB-HSCs) may allow to extend the use of these cells in adult patients and to avoid the risk of insufficient engraftment or delayed hematopoietic recovery.Here we describe a system for the ex vivo expansion of CB-HSCs based on their transient exposure to a recombinant TAT-BMI-1 chimeric protein. BMI-1 belongs to the Polycomb family of epigenetic modifiers and is recognized as a central regulator of HSC self-renewal. Recombinant TAT-BMI-1 produced in bacteria was able to enter the target cells via the HIV TAT-derived protein transduction peptide covalently attached to BMI-1, and conserved its biological activity. Treatment of CB-CD34+ cells for 3 days with repeated addition of 10 nM purified TAT-BMI-1 significantly enhanced total cell expansion as well as that of primitive hematopoietic progenitors in culture. Importantly, TAT-BMI-1-treated CB-CD34+ cells displayed a consistently higher rate of multi-lineage long-term repopulating activity in primary and secondary xenotransplants in immunocompromised mice. Thus, recombinant TAT-BMI-1 may represent a novel, effective reagent for ex vivo expansion of CB-HSC for therapeutic purposes.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.