ALBERT

Description: Umbilical cord is an extra-embryonic-annex rich of both hematopoietic stem and progenitor cells (HSPC) and mesenchymal stem cells (MSC) and it is easily accessible. The HSPC derived from umbilical cord blood (UCB) are promising as graft for allogeneic bone marrow (BM) transplantation and as source of target cells for autologous HSPC gene correction. UCB-HSPC have several advantages compared to adult ones: a less risk of graft-versus-host disease, a higher frequency of progenitors with a greater clonogenic potential and more susceptibility to be transduced by lentiviral vectors. Nonetheless, the HSPC yield from single cord blood unit is not sufficient for these clinical approaches in adults. Therefore, ex-vivo expansion of HSPC in media supplemented by cytokines and/or in vitro culture systems with feeder layers, is a valid approach to exceed this limit. MSC are a component of BM-microenvironment that play a key role in supporting of hematopoiesis by ability to secrete soluble factors and probably by the direct cell-cell interaction too. In this work, we investigated the ability of umbilical cord extracellular matrix-MSC (Wharton's Jelly-MSC) to support the ex-vivo expansion of UBC- purified CD34+ cells. In particular, we evaluated the fold increase, and the frequency of CD34+ cell and CD34+subtypes during expansion at the following culture conditions: by direct contact with WJ-MSC layer, by exposure to the soluble factors secreted by WJ-MSC layer in transwell system. The fold expansion was compared with the CD34+ cells expanded in a customized serum-free medium. CD34+ cells were isolated by immuneselection from 8 fresh UCB. The WJ-MSC were isolated from UC cut-pieces by non-enzymatic procedure but thanks to their capacity to migrate to plastic substrate. At the confluence of 60-70% the WJ-MSC were treated with mytomicin-C to arrest the cell cycle. After 48h, the immune-selected CD34+ cells were seeded in WJ-MSC at the density of 5-10 x104 in 12 well plates by direct or indirect contact (by transwell system). CD34+cells were grown in absence of feeder layers at the same conditions. Early hematopoietic cytokines (Flt-3, TPO, SCF) were supplemented in all three conditions and freshly replaced every two days of culture. Numbers and frequency of CD34+cells were evaluated according to ISHAGE method and CD34+ subtyping was performed by four color method to investigated the co-expression of the primitive surface antigens (CD38, CD133, CD90). The frequency of CD34+ cells at day 5 of culture decreased only 10% and was about 50% after 8 days of culture in conditions. The expansion of CD34 + cells at direct contact with WJ-MSC was superior (5.5 fold increase) compared to that of the other two conditions (3 fold on average). At day 8of culture, the CD34+ cells expanded 12 fold at direct contact with feeder layer, about 7 fold in a transwell system and 6 fold in basic medium. No substantial differences in the grade of expansion was revealed in heterologous vs homologous co-cultures of HSPC/WJ-MSC. Noteworthy is that in the contact system in addition to the fluctuating CD34+ cells harvested from the medium (floating CD34+ cells), we found approximately 50% of the total CD34+ cells be adherent to WJ-MSC layer, these cells were released only after enzymatic proteolytic treatment. Subtyping the CD34+cell population growing in contact to the WJ-MSC or in the conditioned medium we found that the CD34+/CD133+cell population was maintained high (72% ±12 over the total CD34+ cells) as in unmanipulated CB-HSC. The CD34+CD38- cells decreased by 2,5 fold in both systems, as early as day 5 of culture. However, in the contact system this population was 3 times more represented in the attached CD34+ cell fraction. The CD34+/CD90+ subtype was also expanded (more than 8 fold) particularly in the attached fraction, as early as 5 days of culture and was maintained to the end WJ-MSC supported ex-vivo HSPC expansion with superior effect in a cell contact system. Two phenotypically different populations of HSPC developed in this system with an increased frequency of CD34+ cells that co-expressed markers typical of more early progenitors in the attached CD34+ cell fraction. We are assessing the significance of these differences by performing molecular and functional studies of WJ-MSC-supported HSPC. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Genetic modification of autologous hematopoietic stem and progenitor cells (HSPC) is a promising clinical intervention to cure inherited monogenic diseases. Successful gene therapy trials have already been conducted using CD34+ cells from bone marrow and from mobilized peripheral blood. In this regard, cord blood (CB) represents an attractive source of HSCs due to its high concentration of high proliferative HSPC and increased susceptibility to be transduced by lentiviral vectors. Unfortunately, the major disadvantage is the limited number of HSC in the CB collection. Consequently, ex-vivo expansion of CB-HSC is desirable to extend clinical applications. Purposes: To investigate the ability of UCB-cd34+ cells to be expanded in serum-free media supplemented with the early acting hematopoietic cytokines SCF,TPO and Flt-3 ligant (STF) and to characterize CD34+ cells subtypes, clonogenic capacity and gene expression profile during expansion. We also wanted to investigate the susceptibility of the expanded cd34+ cells to be transduced by a GFP-lentiviral vector (LV-GFP) Material and Methods: CD34+ immunoselected cells from 10 UCB were grown for 8 days in customized serum-free medium formulated for HSC expansion, supplemented with STF cytokines. Numbers end frequency of CD34+cells and co-expression of the primitive surface antigens (CD38, CD133, CD90) was evaluated during expansion. Colonies developed in methylcellulose were scored for enumeration ad typing. LV-GFP transduction efficiency was evaluated in CD34+ cells cultured for 4 days in expansion medium plus STF and for 24 hrs in X-vivo10 medium with STF±IL-3 cytokines; the last condition slightly expands CD34+ cells (1.3 fold) and are currently used for HSPC-lentivector transduction in gene therapy clinical trials. The transduction efficiency was evaluated by measuring the percentage of GFP+ cells in the bulk and in colonies developed in methylcellulose and the VCN/cell by Q-PCR. Gene expression profiles were analyzed by human whole genome Agilent microarray Technology to detect differentially expressed genes between expanded, ex-vivo medium cultured and un-cultured cells. Results: We found an average of 8 fold-increase CD34+cells at day 4 and of 22 fold- increase at day 8 of culture. The frequency of CD34+ was maintained at day 4 and declined of about 50% at day 8. CD34+/CD38- early progenitors doublet as early as day 4, differences in CD34+/CD133+ and CD34+/CD90+cells were not significant. The number of CFU slightly increased during expansion while the relative frequency of colonies type did not significantly changed. Four days expanded CD34+ cells were transduced more efficiently than those grown in ex-vivo medium even in presence of IL-3 added to the STF cytokine cocktail. Comprehensive gene expression profile analysis highlighted about 4000 genes differentially expressed in CD34+ cells expanded for 4 and for 8 days compared to that of the un-cultured cells. Conversely, the expression profiles analysis did not show any clear separation between different cell culture methods (expansion vs ex-vivo medium). Specifically, the number of differentially expressed genes in common between the different culture conditions compared with the un-cultured cells was statistically significant. Unsurprisingly, the common up-regulated genes were related to the cell cycle. The likeness between the gene expression profiles of the different culture conditions was also validated by the identification of a significantly small number of differentially expressed genes between them. Conclusions: UCB-CD34+ cells can be efficiently expanded and transduced in serum free conditions. The expanded cells exhibited phenotypic marchers typical of early progenitors and developed colonies in number and in type similar to the unmanipulated cells and exhibited whole gene expression profile that is consisted with that of CD34+ cells exposed for the short term culture conditions currently used in gene therapy trial mediated by lentiviral vectors. Results from this study open a window on the future possibility of using homologous UCB-HSC as target for gene correction in patients diagnosed for a genetic disorder in prenatal time. The genetically modified cells would be stored and used for gene therapy in the same individual in pediatric age. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic stem cell engineering is a promising therapy to cure b-thalassemia, in particular for patients who lack a suitable BM donor for allogeneic transplantation. Since the engrafted gene-corrected stem cells will not have any selective advantage over the unmodified ones, the effectiveness of the therapy in this setting largely depends on the infusion of high numbers of gene-modified cells and on the conditioning regimen. The quality of the infused cells is also crucial for the clinical outcome and the duration of the therapeutic effect. HSPCs mobilization, particularly when G-CSF and plerixafor are used in combination, has been proved to be the optimal approach to harvest a large number of CD34+cells in patients with hematological malignancies and in healthy volunteers. However adult heavily-transfused thalassemia patients have intrinsic characteristics that may adversely affect both the safety and the efficacy of mobilization. We conducted a clinical trial to investigate the safety and effectiveness of mobilizing HSPCs with G-CSF+plerixafor in adult patients affected by β-thalassemia major with the aim to reach a cell dose of ≥8x106 CD34+cells/Kg. We studied the kinetic of CD34+cells during mobilization and performed a comprehensive characterization of their molecular and functional properties. All patients completed the mobilization according to the protocol (G-CSF 10 μg/kg/day for four days, followed by plerixafor 240 μg/kg in the evening on day 4) and no serious adverse events occurred. Leukapheresis was done 10-12 hours after plerixafor (on day 5). Three of the four patients reached the target cell dose or more in single-apheresis collections, even one patient where a significant dose reduction of G-CSF was halved due to early hyperleukocytosis. For one patients the number of cells collected in the first apheresis was slightly below the established target and therefore, according to the protocol, she was subjected to a second apheresis on day 6, after an additional dose of plerixafor. The total yield from the combined apheresis in this patient was 13.0 CD34+cells /Kg. CD34+ cell yields per single apheresis in our patients were comparable to those in healthy donors (12 pts) mobilized in our hospital with G-CSF alone. A significant increase in the mean peripheral blood CD34+ cells (12.1± 8.2 fold), was unanimously observed after plerixafor addiction. The frequencies of the more primitive CD34+cell subtypes (CD34+CD38- and CD34+CD38-133+) as well as the clonogenic capacity tested in short term in vitro assay were found significantly increased too. Comprehensive microarray analysis of genes expressed in the CD34+ cells purified from the same patient upon mobilization with G-CSF alone (G/CD34+cell) and with G-CSF+plerixafor (G+pl/CD34+cell) highlighted a different HSCs repertoire. According to the mechanism of plerixafor mobilization, CXCR-4 gene expression was found 5-fold higher in G+pl/CD34+cells. CXCR-4 gene is known to be expressed on the surface of more primitive CD34+ HSCs with long-term repopulating potential and plays a central role in the regulation of adhesion of them to native niche in the BM. A substantial number of genes with previously shown implication in mechanisms of homing and engraftment (CXCR4, CD82, DPP4, ROBO4), or genes linked to stress resistance (CXCL4, SOD2, IL8, PPBP) as well as several chemokines genes involved in cell mobility (CXCL2, CXC3, CXCR2) were also found to be up-regulated in G+pl/CD34+cells. Overall, the yields, the primitive signatures of CD34+cells indicate the G-CSF+plerixafor mobilized peripheral blood as optimal graft that should favor HSPCs engraftment after transplantation. This findings has therapeutic implications not only for b-thalassemia but also for other hematopoietic stem cell gene therapy applications. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F1200015000. Disclosures No relevant conflicts of interest to declare.

Description: Retroviral vectors are currently the most suitable vehicles for therapeutic gene transfer in hematopoietic stem cells. However, these vectors are known to integrate rather randomly throughout the genome, suffering the so called chromosomal position effects (PE). Such a critical occurrence most probably depends upon the ability of heterochromatin to spread in the inserted vector sequences. Moreover, the use of transgenes imply genotoxicity effects, since the cis-regulatory sequences harbored by the vector can disturb the proper transcription of the resident genes neighboring the integration site, potentially leading to malignant transformation. Due to their enhancer blocker activity, the incorporation of chromatin insulators in flanking position to the transferred unit can reduce the mentioned dangerous effects. Moreover, by acting as barriers to the spread of heterochromatin, chromatin insulators can also mitigate vector silencing. We have previously shown that the sea urchin sns5 chromatin insulator activity is conserved in mouse and human erythroid milieu: it blocks the βglobin-LCR-HS2 enhancer/globin promoter interaction when placed between them. In addition, when placed in flanking location of a γ-retrovirus vector, sns5 impedes PE variegation and improves vector-specific expression following integration in the erythroid genome. Importantly, by binding both erythroid-specific and ubiquitous factors, sns5 favors the accumulation inside the provirus locus of epigenetic marks commonly associated to an euchromatic state (Acuto S. et al., BCMD 2005; D'Apolito D. et al., 2009; Di Caro D. et al., J Mol Biol 2004; Cavalieri V. et al., NAR 2009). In this study we extend these findings, demonstrating that sns5 works as chromatin insulator also when placed in flanking position of a GFP transgene contained in a lentivirus vector (LV-GFP). A large panel of mouse erythroleukemic clones (MELC) was generated after transduction with uninsulated and sns5 -insulated LV-GFP. Individual clones were screened for single vector integrants (by Q-PCR), and for GFP-expression (by cytofluorimetry). Our results shown that the inclusion of the sns5 element in a forward orientation increased the fraction of vector expressing cells (89% for the insulated vector vs 42% for the uninsulated ones). The clonal variegation of expression, assessed as frequency of clones that showed a percentage of GFP-negative cells in the progeny, decreased in clones transduced with the insulated vectors (7.4% vs 13,9%). It has been suggested that chromatin insulators could shape the architecture of topologically independent chromosome domains. High resolution mapping of chromosomal domains in drosophila and higher eukaryotes highlighted that chromatin insulators play a critical role in shaping the architectural genome organization both in a local chromosome environment and in long range chromosomal interaction. Intriguingly, by using the Chromosome Conformation Capture (3C) technology, we demonstrated that the sns5 -flanked LV-GFP integrated at a single copy in the erythroid cell genome is organized into an independent chromatin loop at the integration site. Worth to mention, no looping was detected in the absence of sns5, indicating that the two flanking copies of sns5 are specifically involved in the reorganization of the chromatin structure at the provirus locus. In conclusion our results not only confirm the conserved and striking boundary function of sns5, but also provide a new clue concerning the molecular mechanism that allows this function to occur. On these basis, our findings reassure the use of sns5 to improve both efficacy and safety of lentiviral vectors for gene therapy. This work was funded by the Assessorato Regionale della Salute, Regione Siciliana (PO FESR 4.1.1.1 RIMEDRI) Disclosures No relevant conflicts of interest to declare.