ALBERT

Description: Key Points We describe the first successful use of gene therapy in a severely affected adult with WAS. Gene therapy is a viable strategy for adult WAS patients with severe chronic disease complications where allogeneic transplantation presents.

Description: The biologic hallmark of polycythemia vera (PV) is the formation of endogenous erythroid colonies (EECs) with an erythropoietin-independent differentiation. Recently, it has been shown that an activating mutation of JAK2 (V617F) was at the origin of PV. In this work, we studied whether the STAT5/Bcl-xL pathway could be responsible for EEC formation. A constitutively active form of STAT5 was transduced into human erythroid progenitors and induced an erythropoietin-independent terminal differentiation and EEC formation. Furthermore, Bcl-xL overexpression in erythroid progenitors was also able to induce erythroid colonies despite the absence of erythropoietin. Conversely, siRNA-mediated STAT5 and Bcl-xL knock-down in human erythroid progenitors inhibited colony-forming unit-erythroid (CFU-E) formation in the presence of Epo. Altogether, these results demonstrate that a sustained level of the sole Bcl-xL is capable of giving rise to Epo-independent erythroid colony formation and suggest that, in PV patients, JAK2V617F may induce EEC via the STAT5/Bcl-xL pathway.

Description: CD19 CAR-T cells show unprecedented responses in relapsed/refractory Acute Lymphoblastic Leukaemia, but long-term persistence appears critical for their use as a stand-alone therapy. The origin of long-term persisting CAR-T cells has yet to be defined and will be critical in designing manufacturing protocols to optimise long-term persistence. Previous data are conflicting with groups showing prolonged persistence of CAR-T cells from cell products with a predominantly effector memory (TEM) phenotype, whereas others suggesting that the dominant clones originate instead from infused stem cell memory (TSCM) and central memory (TCM) T cells. To date, it has not been possible to isolate long-term (〉 1 year) persisting CAR-T cells in patients. In the CARPALL Phase I study, the use of an improved low-affinity CD19 CAR resulted in enhanced expansion and persistence of CAR-T cells in vivo (Ghorashian et al, Nature Medicine, in press). Combining this unique experimental setting with our well-established clonal tracking platform based on high-resolution integration sites (IS) analysis has enabled us to track the fate of the infused CAR-T cells. We analysed 2 patients with long-term persistent CAR-T cells detectable by flow cytometry in peripheral blood. CAR-T cells comprised 13 and 53% circulating CD3+ cells respectively at day 14 post-infusion, 7.1 and 7.7% circulating at 1 month, 0.7 and 1.3% at 6 months and 0.1% for both at the latest follow-ups (24-28 months). Blood samples taken at early (14d, 30d) and later (6m to 28m) time points were flow-sorted for CAR+ TCM/TEM mixed population and TSCM T cells, while the corresponding infused gene-modified products were separated into three subpopulations: TSCM, TCM and TEM. The integration profile of each sorted cell populations was established using linear amplification mediated-PCR (LAM-PCR) combined with high throughput sequencing. We identified a total of 7,105 and 4,692 IS from 2 patients overtime before infusion and up to 28 months after infusion. The infused CAR-T cell population was highly polyclonal before infusion. Although the total number of CAR-T cell clones decreased substantially upon in vivo selection, we did not observe any sign of aberrant clonal drifts and diversity was preserved long-term. Early after infusion during the response peak, TSCM underwent two waves of transient oligoclonal expansion. In both patients two distinct sets of individual TSCM clones contributed to the 73% and 97% of the whole analysed TSCM population at day 14 and 74% and 99% at day 30. Conversely, the largest memory/effector clones detected at the same timepoints spanned from 4% to 21% of the total TCM/TEM population. These TSCM clones subsequently contracted and were not observed at 6-28 months after infusion suggesting that different clones are responsible for the early response and prolonged immune surveillance. After 6 months post-infusion, when the IS profile of circulating CAR-T cells was compared with selected populations from the infused product, only 1.8%-6.1% of long term clones were derived from the infused TCM population, despite this accounting for the majority of IS in the products (72.7/75.8% of clones). Conversely, in both patients the majority of IS associated with long term persistence (90.7%/55.5%) were derived from the TSCM compartment. Our preliminary results raise two hypotheses on the clonal dynamics of infused CAR T cells: 1) There is an early expansion of a defined group of clones during the first 30 days, which is more pronounced in the precursor TSCM compartment. These early waves do not seem to be originated from clones that have substantially expanded in vitro such that their clonal mark could not be retained in the batch of the infused cell product analysed. Further, these clones rapidly disappear after the early anti-tumour response. 2) The long-term population of CAR-T cells seem to have a higher relation with TSCM clones that have expanded in vitro before infusion, supporting the notion that such cells in the infused batch would be the one primarily responsible for the preservation of circulating CAR-T cells in the treated patients. This study suggests for the first time that anti-leukemic response occurs along rapid waves of clonal succession and that TSCM are primarily responsible for the long-term survival of CAR-T cells. Disclosures Ghorashian: novartis: Honoraria; UCLB: Patents & Royalties: UCLB; Celgene: Honoraria. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Thrasher:4BIOCapital: Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Amrolia:UCLB: Patents & Royalties.

Description: Key Points This study provides proof of concept that SAP gene transfer into HSCs can correct the multiple immune defects seen in XLP1.

Description: Our mathematical model of integration site data in clinical gene therapy supported the existence of long-term lymphoid progenitors capable of surviving independently from hematopoietic stem cells. To date, no experimental setting has been available to validate this prediction. We here report evidence of a population of lymphoid progenitors capable of independently maintaining T and NK cell production for 15 years in humans. The gene therapy patients of this study lack vector-positive myeloid/B cells indicating absence of engineered stem cells but retain gene marking in both T and NK. Decades after treatment, we can still detect and analyse transduced naïve T cells whose production is likely maintained by a population of long-term lymphoid progenitors. By tracking insertional clonal markers overtime, we suggest that these progenitors can support both T and NK cell production. Identification of these long-term lymphoid progenitors could be utilised for the development of next generation gene- and cancer-immunotherapies.

Description: Historically, survival and activity of individual human hematopoietic progenitor subtypes have been studied exclusively via transplantation in permissive mouse models. Despite being a relevant investigational tool, such animal models do not recapitulate the human hematopoietic milieu. Specifically, the production of human T and NK cells in these mice is severely impaired therefore, as of today, it has been impossible to measure with enough confidence the in vivo dynamics of human T/NK lymphoid progenitors. Hematopoietic stem cell gene therapy (GT) using integrating viral vectors has opened a unique opportunity to trace the fate of transplanted cells for the first time directly in vivo in humans by means of integration sites (IS) clonal tracking. Our mathematical modelling of IS data in clinical GT supported the existence of a population of long-term lymphoid progenitors (LtLP) capable of surviving independently from hematopoietic stem cells (HSC). However, to date, no experimental setting has been available to validate such statistical prediction neither in the mouse nor in humans. We here report the first formal evidence, directly in vivo in humans, that a population of bipotent lymphoid progenitors is capable of surviving and maintaining de novo T/NK production for at least 15 years after loss of transplanted HSC. In 5 SCID-X1 patients treated with gammaretroviral vector HSC-GT we observed that genetically engineered myeloid and B cells have stayed constantly below detection limits starting from 6 months after GT. On the contrary T and NK cells remained vector positive for up to 19 years (average vector copies/cell equal 2.1 and 2.3 respectively). We initially thought that such data would be consistent with permanent loss of engineered HSC and survival of vector-marked long-living circulating mature lymphocytes. However, strikingly and unexpectedly, by a comprehensive immunophenotyping of T cells overtime, we detected vector-positive short-living CD45RA+CD62L+CD95- naïve T cells (Tn) in the circulation of these patients. We first obtained functional validation of the identity of such Tn cells by IFN-y productionassays and we confirmed positive thymic activity by measurement of TREC content. High throughput sequencing of 1,193 T-cell receptor (TCR) rearrangements in FACS-sorted T cell subtypes confirmed that these engineered Tn cells had normal TCR diversity and that new rearrangements were detectable overtime. Similarly, Vbeta repertoire measured by spectratyping displayed normal profiles in all patients. These results suggested that a de novo T-cells production is maintained in these patients by a putative LtLP population in absence of supply by gene-corrected HSC. Using Tn data as surrogate markers of LtLP clonal dynamics and composition, we collected and analysed 12,756 unique IS from 5 populations including Tn cells in a window of 10.1 to 14.9 years after the observed loss of transplanted HSC. Clonal diversity was stable in all T subtypes and IS sharing across subpopulations and timepoints was high and consistent with active in vivo output by LtLP and differentiation of Tn into memory/effector T cells. Analyzing nature and recapture probability of IS we observed that IS in LtLP are significantly enriched in genes involved in lymphocyte survival/activation and we could estimate that T cell production is currently sustained by 2,092-6,056 individual engineered LtLP clones. Moreover, by studying 651 IS collected from CD3-CD56+ purified NK cells we observed that up to 41.2% of them were shared with independently analyzed Tn suggesting that the LtLP active in these patients have a dual T/NK restricted potential. Lastly, we detected and tracked overtime 52 clones bearing IS in MECOM, CCND2 and LMO2 including the IS originally associated with vector-induced leukemia in one patient. Our data show absence of any sustained clonal expansions within the engineered LtLP population for up 19 years after GT, the longest follow up available to date in clinical HSC-GT. In conclusion, our data provide the first formal evidence in vivo in human that a de novo production of genetically engineered T and NK cells can be physiologically maintained by a population of LtLP surviving up to 15 years after loss of transplanted HSC (manuscript submitted). Identification and exploitation of such human LtLP population might be crucial for the development of next generation GT and cancer immunotherapy approaches. Disclosures Baricordi: AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Thrasher:Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees.