ALBERT

Description: Key Points Application of capturing/sequencing, copy number, and RNA analysis technologies ensures comprehensive molecular diagnosis of Fanconi anemia.

Description: We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long- lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.

Description: Hematopoietic stem cell transplantation (HSCT) is followed by profound immunodeficiency. Thymic function is necessary for de novo generation of T cells after HSCT. Circulating CD45RA+ naive T-cell levels are predictive of antigen-specific T-cell responses in the absence of graft-versus-host disease (GVHD). These T cells may not represent recent thymic emigrants, since naive T cells may maintain this phenotype if not antigen-activated. To accurately measure thymic output after HSCT and determine the factors that influence thymic function, T-cell receptor excision circles (TRECs) were examined in CD4+ and CD8+ cells from a cross-section of patients following HSCT. TREC levels rose weeks after HSCT and could be detected in patients 6 years after HSCT. TREC levels correlated with the frequency of phenotypically naive T cells, indicating that such cells were not expanded progeny of naive T cells present in the donor graft. Chronic GVHD was the most important factor that predicted low TREC levels even years after HSCT. Patients with a history of resolved GVHD had decreased numbers of TREC, compared with those with no GVHD. Because few adults had no history of GVHD, it was not possible to determine whether age alone inversely correlated with TREC levels. Recipients of cord blood grafts had no evidence of decreased TREC induced by immunosuppressive prophylaxis drugs. Compared with unrelated donor grafts, recipients of matched sibling grafts had higher TREC levels. Collectively, these data suggest that thymopoiesis is inhibited by GVHD. Larger studies will be needed to determine the independent contributions of age and preparative regimen to post-transplant thymopoietic capacity.

Description: Human hematopoietic stem cells are pluripotent, ie, capable of producing both lymphoid and myeloid progeny, and are therefore used for transplantation and gene therapy. An in vitro culture system was developed to study the multi-lineage developmental potential of a candidate human hematopoietic stem cell population, CD34+CD38− cells. CD34+CD38− cells cocultivated on the murine stromal line S17 generated predominantly CD19+ B-cell progenitors. Transfer of cells from S17 stroma to myeloid-specific conditions (“switch culture”) showed that a fraction of the immunophenotypically uncommitted CD19− cells generated on S17 stroma had myeloid potential (defined by expression of CD33 and generation of colony-forming unit-cells). Using the switch culture system, single CD34+CD38− cells were assessed for their lymphoid and myeloid potential. Nineteen of 50 (38%) clones generated from single CD34+CD38− cells possessed both B-lymphoid and myeloid potential. 94.7% of the CD34+CD38− cells with lympho-myeloid potential were late-proliferating (clonal appearance after 30 days), demonstrating that pluripotentiality is detected significantly more often in quiescent progenitors than in cytokine-responsive cells (P = .00002). The S17/switch culture system permits the in vitro assessment of the pluripotentiality of single human hematopoietic cells.

Description: SLX4, the newly identified Fanconi anemia protein, FANCP, is implicated in repairing DNA damage induced by DNA interstrand cross-linking (ICL) agents, topoisomerase I (TOP1) inhibitors, and in Holliday junction resolution. It interacts with and enhances the activity of XPF-ERCC1, MUS81-EME1, and SLX1 nucleases, but the requirement for the specific nucleases in SLX4 function is unclear. Here, by complementing a null FA-P Fanconi anemia cell line with SLX4 mutants that specifically lack the interaction with each of the nucleases, we show that the SLX4-dependent XPF-ERCC1 activity is essential for ICL repair but is dispensable for repairing TOP1 inhibitor-induced DNA lesions. Conversely, MUS81-SLX4 interaction is critical for resistance to TOP1 inhibitors but is less important for ICL repair. Mutation of SLX4 that abrogates interaction with SLX1 results in partial resistance to both cross-linking agents and TOP1 inhibitors. These results demonstrate that SLX4 modulates multiple DNA repair pathways by regulating appropriate nucleases.

Description: We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)–deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34+ cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (〈 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m2). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

Description: Flt3 is a class III tyrosine kinase receptor expressed on primitive human and murine hematopoietic progenitor cells (HPC). In previous studies using stroma-free short term assays, Flt3 ligand (FL) has been shown to induce proliferation of HPC at proportions similar to or less than c-kit ligand (steel factor, SF). Using long term stromal cocultivation assays, we studied the effects of FL on proliferation and differentiation of a highly primitive and cytokine nonresponsive subpopulation of human HPC, CD34+cd38- cells. Cell Proliferation was significantly greater with FL than with SF, when used individually or in combinations with interleukin-3 (IL-3) and/or IL-6. The effect of FL was greater on bone marrow (BM) CD34+CD38- cells than the more cytokine responsive cord blood CD34+CD38- cells. Little or no effect was seen with FL on more mature CD34+CD38+ cells from either BM or cord blood. The frequency of colony-forming units (CFU) and high proliferative potential-colony forming cells (HPP-CFC) during early culture ( 〈 or = 30 days) was increased by both SF and FL to similar levels. However, in the LTC-IC period (35 to 60 days) and extended long-term culture initiating cell (ELTC-IC) period ( 〉 60 days), the frequency of CFU and HPP-CFC was significantly greater in cultures containing FL than those without FL (P 〈 .0025). Fluorescence-activated cell sorter analysis of cultures after 21 days showed a significantly higher percentage of cells remained CD34+ in the combination of FL, IL-3, IL-6, and SF (F/3/6/S) than in 3/6/S (0.78% +/- 0.52% v 0.21% +/- 0.29% respectively, mean +/- SD). Cloning efficiency of BM CD34+CD38- cells was significantly increased by the addition of FL to the combination of 3/6/S (mean 11.7% v 0.5%, P 〈 .0001). These data show that FL is able to induce proliferation of CD34+CD38-cells that are nonresponsive to other early acting cytokines and to improve the maintenance of progenitors in vitro.

Description: Background: We previously reported that haplo-HSCT after depletion of α/β T cells / CD19 B cells is a suitable option for patients with PIDs, with a high success rate (Bertaina et al., Blood 2014). However, in many patients, we observed a delay in the recovery of adaptive immunity, sometimes resulting in life-threatening or even fatal events, providing the rationale for exploring an innovative approach based on the post-transplantation infusion of donor T cells, genetically modified with the novel suicide gene iC9 (BPX-501 cells), with the aim of accelerating reconstitution of adaptive immunity. As the transduced gene contains sequences for the CD19 marker, BPX-501 cells (CD3+/CD19+) can be easily tracked in peripheral blood. Patients and study design: In this multicenter, prospective phase I-II trial (enrolling both malignant and non-malignant diseases; sponsored by Bellicum Pharmaceuticals®, ClinicalTrials.gov identifier: NCT02065869) 20 children with PIDs have been enrolled. Nine children had Severe Combined Immune Deficiency (SCID), 5 Wiskott-Aldrich syndrome (WAS), 2 Chronic Granulomatosis Disease (CGD) and one each of 4 other PIDs detailed in Table 1, which also reports relevant patient-, donor- and transplant- characteristics. All patients were transplanted after depletion of donor α/β T cells and CD19 B cells, employed to prevent graft-versus-host disease (GvHD) and post-transplant lymphoproliferative disorders (PTLD). Details on cell subsets infused with the graft are reported in Table 1. No patient was given any post-transplantation GvHD prophylaxis. The phase I portion of the trial consisted of a classical 3+3 design with 3 cohorts, with escalating doses of BPX-501 cells of 2.5x105, 5x105, and 1x106 cells/kg, respectively. Patients in the phase II portion received the highest dose identified during the phase I portion (1x106 cells/kg). Results: All patients engrafted and no secondary graft failure was recorded. The median time to neutrophil and platelet recovery was 16 (range 11-35) and 10 days (range 7-14), respectively. BPX-501 cells were infused at a median time of 15 days (range 13-56) after the allograft. Four patients were enrolled in the phase I portion of the study; one received 2.5x105 and 3 received 1x106 cells/kg. The remaining 16 children were treated in the phase II part, with a dose was 1x106 cells/kg. Five children experienced Grade I (3 patients) or Grade II (2 children) acute GvHD, which resolved with either topical or systemic steroids in 3 patients. The other 2 cases resolved after the infusion of Rimiducid (AP1903) which activated the iC9 suicide gene. The cumulative incidence of acute GvHD is shown in Figure 1A. Two of the 17 patients at risk (i.e. those with a follow-up longer than 100 days after transplantation) developed mild (skin only) chronic GvHD. The median time to discharge was 36 days (range 26-180). Eight patients (40%) experienced one episode of re-hospitalization after initial discharge. After a median follow-up of 10 months (range 50 days-20 months), all patients are alive and disease-free. Details on expansion/persistence of BPX-501 cells, as well as on recovery of CD3+ cells are shown in Figure 1B and 1C, respectively. Ig serum levels of SCID patients at 6 and 12 months after transplant are depicted in Figure 1D. The median follow-up for WAS patients is 510 days (124-531 days) and their median platelet count at 1, 3 and 12 months after haplo-HSCT was 136 (range 48-168), 204 (range 98-280) and 143 (117-365) x109/L, respectively. One SCID patient who developed BCG bacterial dissemination after the allograft resolved this infection and 3 patients (2 with SCID and one with WAS) cleared cytomegalovirus (2 children) or Adenovirus (one child) infections pre-existing at time of conditioning regimen. Conclusions: These results indicate that haplo-HSCT, after depletion of α/β T cells and B cells followed by adoptive infusion of donor BPX-501 cells, is an effective alternative for those children with PIDs in need of an urgent allograft or lacking a suitable HLA-matched donor. BPX-501 cells expand in vivo and persist over time, contributing to fasten the recovery of adaptive T-cell immunity and to clear infections. In view of the absence of fatalities and of the low rate of mild chronic GvHD, we conclude that infusion of BPX-501 T cells with the iC9 cell-suicide system may potentially render haplo-HSCT a first-line option for children with PIDs. Table 1. Table 1. Figure 1. Figure 1. Disclosures Qasim: Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership, Research Funding; Catapult: Research Funding; Calimmune: Research Funding. Slatter:Medac: Other: Travel grants. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

Description: Fanconi anemia (FA) is the most common genetic cause of bone marrow failure and is caused by inherited pathogenic variants in any of 22 genes. Of these, only FANCB is X-linked. We describe a cohort of 19 children with FANCB variants, from 16 families of the International Fanconi Anemia Registry. Those with FANCB deletion or truncation demonstrate earlier-than-average onset of bone marrow failure and more severe congenital abnormalities compared with a large series of FA individuals in published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity is associated with the extent of residual FANCD2 monoubiquitination activity. We used transcript analysis, genetic complementation, and biochemical reconstitution of FANCD2 monoubiquitination to determine the pathogenicity of each variant. Aberrant splicing and transcript destabilization were associated with 2 missense variants. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination in biochemical and/or cell-based assays tended to show earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our study reveals a genotype-phenotype correlation within the FA-B complementation group of FA, where severity is associated with level of residual FANCD2 monoubiquitination.

Description: We present cell cycling and functional evidence that the CD34+CD38- immunophenotype can be used to define a rare and primitive subpopulation of progenitor cells in umbilical cord blood. CD34+CD38- cells comprise 0.05% +/- 0.08% of the mononuclear cells present in cord blood. Cell cycle analysis with the fluorescent DNA stain 7- aminoactinomycin D showed that the percentage of CD34+ cells in cycle directly correlated with increasing CD38 expression. CD34+CD38- cord blood cells were enriched for long-term culture-initiating cells (LTCIC; cells able to generate colony-forming unit-cells [CFU-C] after 35 to 60 days of coculture with bone marrow stroma) relative to CD34+CD38- cells. In an extended LTCIC assay, CD34+CD38- cells were able to generate CFU-C between days 60 and 100, clearly distinguishing them from CD34+CD38+ cells that did not generate CFU-C beyond day 40. When plated as single cells, onset of clonal proliferation was markedly delayed in a subpopulation of CD34+CD38- cells; clones (defined as = 100 cells) appeared after 60 days of culture in 2.9% of CD34+CD38- cells. In contrast, 100% of CD34+CD38+ cells formed clones by day 21. Although the CD34+CD38- immunophenotype defines highly primitive populations in both bone marrow and cord blood, important functional differences exist between the two sources. CD34+CD38- cord blood cells have a higher cloning efficiency, proliferate more rapidly in response to cytokine stimulation, and generate approximately sevenfold more progeny than do their counterparts in bone marrow.