Gene Therapy Using a Self-Inactivating Lentiviral Vector Improves Clinical and Laboratory Manifestations of Wiskott-Aldrich Syndrome

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 260-260 ◽  
Author(s):  
Julia I Chu ◽  
Lauren A Henderson ◽  
Myriam Armant ◽  
Frances Male ◽  
Colleen H Dansereau ◽  
...  

Abstract The X-linked disorder Wiskott-Aldrich Syndrome (WAS), characterized by thrombocytopenia, eczema, recurrent infections, autoimmunity and cancer, is typically treated with allogeneic transplantation. Somatic gene therapy (GT) using autologous CD34+ cells is a promising treatment alternative for high-risk patients lacking matched allogeneic donors, as it avoids graft versus host disease. GT using a γ-retroviral vector with intact viral enhancers was efficacious but had a high rate of leukemia due to insertional oncogenesis. We report here preliminary results of 4 WAS patients who underwent GT using a self-inactivating lentiviral (SIN-LV) vector, in which viral enhancers have been deleted and the human WAS cDNA is controlled by a 1.6kB fragment of the human WAS promoter (w1.6_hWASP_WPRE SIN-LV). WASP expression per cell induced by this vector was lower than in normal cells when examined in murine models and human trials. We hypothesized that the SIN-LV would readily correct immune abnormalities due to selective advantage for WAS protein (WASP) expressing T cells whereas correction of myeloid and platelet abnormalities would require high vector copy number (VCN) in the infused cells. Patients with severe WAS (clinical scores 3-5) were enrolled at a median age of 32 months (17 months-8 years). Patients 1 and 3 had detectable but low WASP expression. Patients 2 and 4 carried mutations that abrogated WASP expression but had evidence of somatic reversion in T and/or NK cells. CliniMACS purified CD34+ mobilized peripheral blood or bone marrow cells were transduced with the vector and infused after busulfan (12-15mg/kg) and fludarabine (120mg/m2) conditioning. CD34+ cell doses ranged from 6.3-24.91 x 106 cells/kg. VCN of the infused cells was variable (3.37, 1.34, 0.54, 1.01 copies/cell). Busulfan exposure was myeloablative or near-myeloablative in patients 1, 3, and 4 (81.2, 77.2, 84.5 mg*h/L) and submyeloablative in patient 2 (48.8 mg*h/L). All patients are alive with median follow-up of 13.5 months (9-24 months). All patients had improvement in eczema, remain platelet transfusion independent and have had no severe bleeding events. WASP expression in T cells was increased post-GT over baseline. Selective advantage for WASP expressing T cells was apparent in patients 1, 2 and 4, who had higher VCN in T cells at 6 months post-GT (0.93-2.21) than in B (0.48-1.7) or myeloid (0.13-0.89) cells. The presence of revertants in patients 2 and 4 did not appear to interfere with T cell reconstitution. In contrast patient 3 who had the highest WASP expression at baseline and the lowest VCN of infused cells (0.5 copies/cell), had the lowest VCN in T cells at 8 months (0.1 copies/cell). Defective T cell proliferation in response to anti-CD3 stimulation, characteristic of WAS, was improved post-GT. Next generation sequencing of T cell receptors in sorted naïve, memory and regulatory T cells revealed profound abnormalities of diversity, decreased entropy and marked clonal expansions pre-GT; most of these improved at 6 months post-GT. Cytoskeletal function in myeloid cells was highly abnormal pre-GT, as shown by absence of podosome formation in monocyte-derived dendritic cells (0-1% vs. 61% in controls). Podosome formation at 6 months post GT was improved but subnormal (4-40%). Only patient 1, who received the highest cell dose, the highest VCN, and myeloablative busulfan exposure had robust platelet reconstitution (pre-GT 24 versus 110 x 109/L 6 months post-GT) and high level gene marking in myeloid cells 0.89 copies/cell. At the same timepoint, patients 2, 3 and 4 had platelet counts of 20-30 x 109/L and correspondingly lower VCN in myeloid cells (0.13-0.27 copies/cell). No severe adverse events related to GT have occurred to date, with relatively short follow-up. Integration site analysis of sorted cells showed highly polyclonal reconstitution, with distributions of integration acceptor sites as expected for the lentiviral vector backbone. In summary, GT using a SIN-LV that induces expression of WASP at levels below wild type improved the clinical and laboratory manifestations of WAS, with better reconstitution in patients receiving cells with high VCN. Disclosures Off Label Use: Off-label use of CliniMACS purified CD34+ cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 165-165
Author(s):  
Christian J. Braun ◽  
Kaan Boztug ◽  
Manfred Schmidt ◽  
Michael H Albert ◽  
Adrian Schwarzer ◽  
...  

Abstract Abstract 165FN2 CB and KB contributed equally and should be considered aequo loco. Wiskott-Aldrich-Syndrome (WAS) is a rare and life-threatening immune-disorder characterized by autoimmunity, microthrombocytopenia, immunodeficiency and susceptibility to lymphoma. WAS is caused by mutations in the WAS gene which encodes WASP, a key regulator of actin polymerization exclusively expressed in hematopoietic cells. WASP deficiency causes defects in lymphocytes, myeloid cells, and platelets. We here report a comprehensive analysis of ten patients treated by hematopoietic stem cell gene therapy between 2006 and 2009 (median follow up time 29.6 months, range 15 to 58 months). Patients were mobilized with G-CSF alone (3/10) or G-CSF combined with anti-CXCR4/AMD3100 (7/10), conditioned with busulfan (8mg/kg body weight) and received between 2.8×106 and 24.9×106 cells/kg bw, with a median transduction efficacy of 52%. Upon transplantation of retrovirus-transduced WASP-expressing progenitor cells, the proportion of corrected platelets and lymphocytes increased steadily over time reaching 85–90% and 80–85%, respectively. Interestingly, also myeloid cells showed a continuously increasing percentage of WASP-expressing fractions (20 to 70%). Due to transplantation of insufficient numbers of WASP-transduced HSC, one patient failed to engraft. He had no evidence of corrected myeloid/hematopoietic progenitor cells and continued to suffer from life-threatening infections and autoimmunity. He was successfully treated by haploidentical HSCT. All other patients had marked improvement of their clinical status. Bleeding diatheses, susceptibility to infections, and autoimmunity resolved. Patients had evidence of significant and sustained increase in platelet counts (p=0.01) together with a reconstituted WASP expression and normalization of thrombocyte size (p<0.001). After gene therapy, we observed a normalization of the T cell compartment (T cell proliferative responses and TCR Vb spectratyping), NK cell function (assessed by in vitro cytotoxicity and formation of NK cell immunological synapse). The B cell compartment showed consistent expression of WASP. In 4 of 7 patients, IgG substitution could be discontinued. These patients and also those without initial IgG substitution mounted a specific immune response to vaccines, as evidenced by determination of specific antibody titers. One patient developed a T-cell acute lymphoid leukemia 488 days after gene therapy associated with vector integration close to the LMO2 locus. Chemotherapeutic treatment induced remission that is documented since d33 after initiation of induction therapy. Long-term follow up observation indicated that gene therapy for WAS, although not without toxicity, is feasible and provides an effective alternative treatment strategy to allogeneic HSC transplantation. Disclosures: Baum: Patent office: Patents & Royalties.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1203-1203
Author(s):  
Birgit Federmann ◽  
Martin Bornhauser ◽  
Lambros Kordelas ◽  
Dietrich W. Beelen ◽  
Gernot Stuhler ◽  
...  

Abstract Abstract 1203 Poster Board I-225 HHCT using reduced intensity conditioning (RIC) and immunomagnetic CD3/CD19 graft depletion is of low toxicity and enables fast engraftment even with grafts of low CD34 content. Immune reconstitution may be improved compared to graft CD34 selection due to the cotransplantation of facilitating cells, CD34- progenitors, dendritic cells and natural killer (NK)-cells. A prospective multicenter phase II study of HHCT using CD3/CD19-depleted grafts after RIC with fludarabine (150-200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), OKT-3 (5 mg/day, day -5 to +14) was initiated. No post grafting immunosuppression was applied if the graft contained <5×104 CD3+ cells/kg. 60 adults with median age of 45 years (range, 19-61) have been enrolled from 2002 to 2009. Diagnoses were AML (n=37), ALL (n=8), NHL (n=6), myeloma (n=4), CML (n=3), CLL (n=1) and MDS (n=1). Patients were “high risk” because of refractory disease (n=6), cytogenetics (n=5), chemosensitive relapse (n=26) or relapse after prior HCT (allo=16, auto=6, both=1). At HHCT, 30 patients were in CR and 30 in PR. Grafts contained median of 6.8×106 (range, 3.5-22) CD34+ cells/kg, 4.0×104 (range, 0.9-44) CD3+ T cells/kg and 2.8×107 (range, 0.00-37.3) CD56+ cells/kg. Engraftment was rapid with median of 12 days to >500 granulocytes/μL (range, 9-50) and 11 days to >20.000 platelets/μL (range, 7-38). Incidence of grade II-IV acute GVHD and chronic GvHD (cGvHD) was 47% and 15%, respectively. Non-relapse-mortality on day 100 was 25% and 44% for the whole study period. Current overall survival (OS) is 18 of 60 patients (30%) with median follow-up of 525 days (range, 21-1542) of patients alive resulting in a Kaplan-Meier estimate 1-year OS of 41% and 2-year OS of 24%. Kaplan-Meier estimate 1-year OS was 40 % in AML, 38 % in ALL and 67% in NHL patients. No positive impact of KIR-mismatch on survival even in the subgroup of patients with AML was observed. Patients with cGVHD had a trend toward better survival with 56% vs. 39% (p=0.09). 14 patients died because of infections. Detailed immune reconstitution was analyzed in 24 patients. NK-cell engraftment was fast with median of 248 CD16+56+CD3- cells/μl (range, 1-886) on day 20. Increased natural cytotoxicity receptors (NKP30, NKP44, NKP46) and NKG2A, but decreased NKG2D and KIR-expression was observed on NK-cells in the first 3 months. T-cells regenerated delayed with median of 191 CD3+ cells/μl (range, 38-799) on day 100. A slow reconstitution of CD8+ and CD4+ T-cells with median of 66 CD8+ (range, 8-170) vs. 70 CD4+ cells/μl (range, 12-301) on day 100 and 157 (range, 32-379) vs. 181 cells/μl (range, 19-980) on day 400 was observed. The subset of memory T-cells regenerated faster compared to naïve T-cells with median of 25 CD4+45RA+ (range, 4-109) vs. 80 CD4+45RO+ cells/μl (range, 0 to 255) and 46 (range 7-191) vs. 164 cells/μl (range, 66-323) on days 80 and 250, respectively. T-cell repertoire was skewed with oligoclonal T-cell expansion to day 100 and normalization after day 200. B-cell reconstitution was slow with median of 8 (range, 0-407) CD19+20+ cells/μl on day 100. 6 of these 24 patients received donor-lymphocyte-infusions for relapse or mixed chimerism resulting in acceleration of immune recovery in T- and NK-cells. In conclusion, HHCT using RIC and CD3/CD19 depleted grafts is of low toxicity and allows fast engraftment even with low doses of CD34 cells. Overall survival seems promising in a high risk patient cohort. Recovery of NK cells occurs early and fast. KIR receptor expression remains low in the first 3 months. T- and B-cell reconstitution is delayed but seems faster compared to published data after CD34 selection. To evaluate the treatment protocol earlier during the course of disease a new study has just been initiated. Disclosures: Off Label Use: The use of Fludarabine, Thiotepa, Melphalan and OKT-3 in the conditioning is off-label-use.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 67-67 ◽  
Author(s):  
Stephan A Grupp ◽  
Noelle V. Frey ◽  
Richard Aplenc ◽  
David M Barrett ◽  
Anne Chew ◽  
...  

Abstract Background CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains into a single chimeric protein. We previously reported on CTL019 cells expressing a CAR with intracellular activation plus costimulatory domains. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including 1 sustained CR in a patient with ALL (Grupp, et al. NEJM 2013). We now report on outcomes and longer follow up from our pilot studies treating 20 pts (16 children and 4 adults) with relapsed, refractory ALL. Methods T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into pts with relapsed or refractory CD19+ ALL. 17/20 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency (TE) of 11-45%. On the adult protocol, the target dose was 5 x 109 total cells split over 3 days with a TE of 6-31%. 11 pts had relapsed ALL after a prior allogeneic SCT. T cells were collected from the pt, regardless of prior SCT status, and not from allo donors. All pts s/p allo SCT had to be 6 mos s/p SCT with no GVHD or GVHD treatment. Results 16 children median age 9.5 y (5-22y) and 4 adults median age 50y (26-60y) with CD19+ ALL were treated. One child had T cell ALL aberrantly expressing CD19. 14/16 pediatric pts had active disease or +MRD after chemotherapy on the day prior to CTL019 cell infusion, while 2 were MRD(-). 3 of 4 adults had active disease prior to lymphodepleting chemotherapy, while 1 was in morphologic CR. Lymphodepleting chemotherapy varied with most receiving a Cytoxan-containing regimen the week prior to CTL019. A median of 3.7x106 CTL019 cells/kg (0.7-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 5 pts developed fevers within 24 hrs of infusion and did not receive planned subsequent infusions of CTL019 cells. 14 patients (82%) achieved a CR, including the patient with CD19+ T ALL, 3 did not respond, and 3 are pending evaluation. 11/17 evaluable pts have ongoing BM CR with median follow up 2.6 mo (1.2-15 mo). Three patients with a CR at 1 month have subsequently relapsed, 1 with CD19(-) disease. Median follow-up as of August 1, 2013 was 2.6 mo (1-15 mo) for all pts. All responding pts developed some degree of delayed cytokine release syndrome (CRS), concurrent with peak T cell expansion, manifested by fever, with variable degrees of myalgias, nausea, anorexia. Some experienced transient hypotension and hypoxia. Detailed cytokine analysis showed marked increases from baseline values of IL6 and IFNγ (both up to 1000x), and IL2R, with mild or no significant elevation in systemic levels of TNFα or IL2. Treatment for CRS was required for hemodynamic or respiratory instability in 7/20 patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab (7 pts), together with corticosteroids in 4 pts. Although T cells collected from the 11 pts who had relapsed after allo SCT were generally 100% of donor origin, no GVHD has been seen. Persistence of CTL019 cells detected by flow cytometry and/or QPCR in pts with ongoing responses continued for 1-15 months after infusion, resulting in complete B cell aplasia during the period of CTL019 persistence. Pts have been treated with IVIg without any unusual infectious complications. One child who entered a CR subsequently developed MDS with a new trisomy 8 in ALL remission and has gone to SCT, and 1 child developed a single leukemia cutis lesion at 6 mo, still BM MRD(-). Conclusions CTL019 cells are T cells genetically engineered to express an anti-CD19 scFv coupled to CD3ζ signaling and 4-1BB costimulatory domains. These cells can undergo robust in-vivo expansion and can persist for 15 mo or longer in pts with relapsed ALL. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. This approach has promise as a salvage therapy for patients who relapse after allo-SCT, and collection of tolerized cells from the recipient appears to have a low risk of GVHD. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL. Multicenter trials are being developed to test this therapy for ALL in the phase 2 setting. Disclosures: Grupp: Novartis: Research Funding. Chew:Novartis: Patents & Royalties. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Litchman:Novartis Phamaceuticals: Employment, Equity Ownership. Rheingold:Novartis: Research Funding. Shen:Novartis Pharmaceuticals: Employment, Equity Ownership. Wood:Novartis Pharmaceuticals: Employment, Equity Ownership. June:Novartis: Patents & Royalties, Research Funding.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4793-4793
Author(s):  
Giacomo Oliveira ◽  
Maria Teresa Lupo Stanghellini ◽  
Eliana Ruggiero ◽  
Nicoletta Cieri ◽  
Mattia D'Agostino ◽  
...  

Abstract BACKGROUND: Suicide gene therapy applied to haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. RESULTS: We studied 9 adult patients who underwent haplo-HSCT and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-39.5x106) for high-risk hematologic malignancies between 1995 and 2010 (TK patients). At a median follow-up of 7,4 years (range 3.2-12.3), all patients are in complete remission. Two out of 9 patients (22%) experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs. A complete recovery of NK cells, B lymphocytes and αβ or γδ T cells was observed. The CD8+ and CD4+ T cell compartment of TK patients were characterized by level of naïve and memory cell comparable to age and sex matched healthy controls. The quantification of CD4+ CD31+ CD62L+ CD45RA+ CD95- recent thymic emigrants and measure of single joint T-cell receptor excision circles demonstrated that the normalization of the T cell compartment was supported by a completely recovered thymic output. TK-cells were detected in all patients (100%), at low levels (median=4cells/uL). Ex vivo selection of pure TK-cells after polyclonal stimulation and LNGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Of notice TK-cells could be retrieved also in patients successfully treated with GCV for GvHD, thus confirming the selective action of GCV only on proliferating TK-cells. Accordingly, GCV sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. TK-cells circulating in patients displayed a memory phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells and exhibited a low level of Ki-67 positivity, thus suggesting the maintenance of a pool of gene modified memory cells through homeostatic proliferation. The number of TK-cells circulating at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. We evaluated whether the phenotype of infused TK-cells was able to affect the long-term fate of gene-modified memory T cells. We observed that the number of infused TSCM cells positively correlated with early TK-cell expansion and with their long-term persistence, suggesting that TSCMmight play a privileged role in the generation of a long-lasting immunological memory. CONCLUSION: These data show that a complete and physiological donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 12 years in treated patients. This setting can be exploited to investigate the requirements at the basis of the generation of a long-lasting immunological memory in vivo. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures Lambiase: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Chairman and CEO Other. Bonini:MolMed S.p.A: Consultancy.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 549-549 ◽  
Author(s):  
Alexis A. Thompson ◽  
John E Rasko ◽  
Suradej Hongeng ◽  
Janet L. Kwiatkowski ◽  
Gary Schiller ◽  
...  

Abstract Background: Hematopoietic stem cell (HSC) gene therapy has the potential to induce globin production and mitigate the need for blood transfusions in β-thalassemia major. Promising early results for 2 subjects with β0/βE -thalassemia major in the ongoing HGB-205 study suggested that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered β-globin gene (βA-T87Q) can be safe and yield robust production of βA-T87Qglobin resulting in rapid transfusion independence. The Northstar study (HGB-204), which uses the same lentivirus vector and analogous study design as study HGB-205, is multi-center and multi-national, and centralizes drug product manufacturing. Herein, we provide the initial data on subjects enrolled and treated in this study. Subjects and Methods: Transfusion-dependent subjects with β-thalassemia major undergo HSC collection via mobilized peripheral blood apheresis and CD34+ cells are selected. Estimation of the mean ex-vivo vector copy number (VCN) is obtained by quantitative PCR performed on pooled colony-forming progenitors. Subjects undergo myeloablation with intravenous busulfan, followed by infusion of transduced CD34+ cells. Subjects are monitored for hematologic engraftment, βA-T87Q -globin expression (by high performance liquid chromatography) and transfusion requirements. Integration site analysis (ISA, by linear amplification-mediated PCR and high-throughput sequencing on nucleated cells) and replication-competent lentivirus (RCL) assays are performed for safety monitoring. Results: As of 31 July 2014, 3 subjects have undergone HSC collection and ex-vivo LentiGlobin BB305 gene transfer. One subject (Subject 1102) has undergone myeloablation and drug product infusion. Outcomes data are shown in Table 1. The initial safety profile is consistent with myeloablation, without serious adverse events or gene therapy-related adverse events. This subject has increasing production of βA-T87Q-globin: the proportion of βA-T87Qglobin was 1.5%, 10.9% and 19.5% of total Hb at 1, 2 and 3 months post-infusion, respectively. This subject received pRBCs on Day +14 following drug product infusion and required no further transfusions until a single unit of pRBC was transfused on Day +96 for a Hb of 8.6 g/dL and fatigue. Two additional subjects have undergone drug product manufacture and are awaiting transplantation. Safety data related to ISA and RCL assays are pending. Abstract 549. Table 1 Preliminary results of dosing parameters and transplantation outcomes Subject Age (years) and Gender Genotype BB305 Drug Product Day of Neutrophil Engraftment Drug Product- related Adverse Events βA-T87Q-Hb at last follow-up visit /Total Hb (g/dL) VCN CD34+ cell dose (x106 per kg) 1102 18 F β0/βE 1.0/1.1a 6.5 Day +17 None 1.77/8.6 1104 21 F β0/βE 0.7/0.7a 5.4 P P P 1106 20 F β0/β0 1.5 12.3 P P P As of 31 July 2014; P, pending a If more than one drug product were manufactured, the VCN of each drug product lot is presented. Conclusion: The first subject treated on the Northstar study has safely undergone drug product infusion with autologous HSCs transduced with LentiGlobin BB305 lentiviral vector and is producing steadily increasing amounts of βA-T87Q-globin. Additional follow-up of this subject plus data on additional subjects who undergo drug product infusion will be presented at the meeting. Ex-vivo gene transfer of βA-T87Q-globin to autologous HSCs is a promising approach for the treatment of patients with β-thalassemia major. Disclosures Thompson: ApoPharma: Consultancy; Novartis: Consultancy, Research Funding; Amgen: Research Funding; Glaxo Smith Kline: Research Funding; Mast: Research Funding; Eli Lilly: Research Funding. Kwiatkowski:Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy. Schiller:Sunesis, Amgen, Pfizer, Bristol Myers Squibb: Research Funding. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Petrusich:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment. Walters:Via Cord and AllCells, Inc.: Medical Director Other.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 165-165
Author(s):  
Giacomo Oliveira ◽  
Maria Teresa Lupo Stanghellini ◽  
Nicoletta Cieri ◽  
Raffaella Greco ◽  
Maddalena Noviello ◽  
...  

Abstract Background Suicide gene therapy applied to allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. Results We studied 14 adult patients who underwent allo-HSCT (haploidentical HSCT: n=11; HLA-identical HSCT n=3) and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-2.8x108) for high-risk hematologic malignancies between 1995 and 2010. At a median follow-up of 8,7 years (range 3-17), all patients are in complete remission. Five out of 14 patients experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs. We observed a complete recovery of NK cells, comprising of mature (CD56+CD16+) and immature (CD56+CD16-) NK cells. Interestingly the proportion of B cells circulating long-term in patients was significantly higher than that observed in age-related healthy controls (p<0.0001). Full recovery of CD3, including CD4 and CD8 cell counts was observed in this long-term analysis. The youngest patients (age range: 22-34 years) showed naïve and memory frequencies similar to age-matched controls. Conversely, in oldest patients (age range: 44-66 years) the frequency of naïve T cells was inferior to age-matched healthy subjects (p=0.0038), and was compensated by a larger proportion of central memory and effector memory cells. Nevertheless, we observed a high percentage of recent thymic emigrants, suggesting a full recovery of thymic output not only in young but also in old patients. Stem memory CD4 and CD8 T cell counts were similar to that of healthy controls, independently from age. CMV-specific T cells, quantified by dextramer staining, were detected in CMV+ patients. TK-cells were detected in the majority of analyzed patients (90%), at low levels (median=0,43%±6,9%). Ex vivo selection of pure TK-cells after polyclonal stimulation and NGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. The proportion of TK-cells detectable at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first 3 months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Of notice TK-cells could be retrieved also in patients successfully treated with ganciclovir for GvHD, thus confirming the selective action of ganciclovir only on proliferating TK-cells. Accordingly, ganciclovir sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. While infused TK-cells displayed a predominant effector memory phenotype, gene modified T cells persisting long-term were enriched for central memory (CD45RA-CD62L+) and stem memory (CD45RA+CD62L+CD95+) phenotypes, suggesting the higher ability of these T cell subsets to persist and shape the immunological profile long-term in treated patients. Conclusion These data show that a complete donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 14 years in treated patients. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures: Valtolina: MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Employment. Bonini:MolMed S.p.A: Consultancy.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3906-3906
Author(s):  
A Samer Samer Al-Homsi ◽  
Frank Cirrone ◽  
Kelli Cole ◽  
Kelsey Stocker ◽  
Benedetto Bruno ◽  
...  

Abstract The introduction of post-transplant cyclophosphamide (PTCy) has circumvented the need for T-cell depletion following haploidentical stem cell transplantation (SCT). By expanding the donor pool for patients from certain ethnic minorities, this has addressed to some degree an important health care disparity issue in SCT. However, a recent registry study showed increased incidence GvHD and inferior outcomes in patients receiving haploidentical SCT with PTCy, tacrolimus and mycophenolate mofetil for GvHD prevention as opposed to matched unrelated donor SCT with PTCy-based GvHD prevention. Seeking to improve the results of GvHD prevention in the setting of haploidentical SCT, we examined a combination of PTCy, abatacept and a short course of tacrolimus (CAST). Abatacept is a recombinant soluble fusion protein composed of the extracellular domain of cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) fused to the Fc region of IgG1. Abatacept blocks CD28-CD80I86 axis and prevents T-cell co-stimulation. In early studies, abatacept has shown promising results when added to methotrexate and tacrolimus in matched and mismatched donor SCT. We initiated a phase Ib-II clinical trial for patients with hematological malignancies undergoing haploidentical SCT. Patients received G-CSF mobilized peripheral blood grafts from related haploidentical donors. GvHD prevention consisted of PTCy 50mg/kg IV on day +3 and +4 with forced hydration, abatacept 10mg/kg IV on day +5, +14 and +28 and tacrolimus. Tacrolimus was started on day +5 at 0.02mg/kg/day by continuous IV and adjusted thereafter to maintain a trough level of 5-12ng/mL. Tacrolimus taper was planned to begin on day +60 and complete by day +90 in the absence of GvHD. All patients received standard supportive care including levofloxacin until neutrophil engraftment, posaconazole until day +75, acyclovir for 1 year and, if CMV positive by serology, letermovir until day +100. Pneumocystis Jiroveci prophylaxis was started after neutrophil engraftment and continued until 6 months post-transplant. G-CSF was administered routinely until neutrophil engraftment. Since September 2020, 19 patients were enrolled. Three patients are too early in their post-transplant course and were excluded from this analysis. Patients' characteristics are summarized in the table. All but 2 patients received cryopreserved products. Median times to ANC and platelet engraftment were 18.5 days (14-30) and 28.5 (16-61). All 16 patients achieved full whole blood donor chimerism by day +30. There was no secondary graft failure. With a median follow-up was 149.5 days (41-308) with 10 patients having &gt;120 days and 8 &gt;180 days of follow-up, 4 patients developed skin acute GvHD (all grade I). No patient developed grade II-IV acute GvHD. Two patients developed skin chronic GvHD (limited, both moderate). Both cases were diagnosed following COVID-19 vaccination. Fifteen patients completed tacrolimus taper by day +90. Two patients received systemic steroids, one for treatment of cGvHD. The remaining patients required no further immunosuppressive therapy beyond day +90. CMV activation rate was 25%. One patient had EBV reactivation and required preemptive therapy with 2 weekly rituximab doses. There were no cases of adenovirus, HHV-6 virus or BK virus reactivation. Four patients developed renal insufficiency (3 in the setting of acute sepsis and 1 with thrombotic microangiopathy, which resolved after tapering off tacrolimus. One patient with adult T-cell leukemia/lymphoma relapsed and died. All other patients are alive and well. In summary, our preliminary results suggest that CAST with shortened course of tacrolimus is feasible and seems to offer very promising outcomes with low rates of acute GvHD. The study is accruing actively and the results of a larger cohort with longer follow-up will be presented at the meeting. If confirmed, by improving the outcomes of haploidentical SCT, this regimen may further address a health care disparity issue, offering almost every patient in need of allogeneic SCT an alternative donor option with equal outcomes. Figure 1 Figure 1. Disclosures Al-Homsi: Daichii Sanyko: Consultancy; Celyad: Other: Advisory Board. Abdul-Hay: Abbvie: Consultancy; Servier: Other: Advisory Board, Speakers Bureau; Jazz: Other: Advisory Board, Speakers Bureau; Takeda: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Abatacept - off label use as GvHD prevention Cyclophosphamide - off label use as GvHD prevention


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 243-243 ◽  
Author(s):  
Howard M. Lederman ◽  
Margaret A. Connolly ◽  
Ram Kalpatthi ◽  
Russell E. Ware ◽  
Winfred C. Wang ◽  
...  

Abstract Abstract 243 Susceptibility to encapsulated bacteria, particularly Streptococcus pneumonia, is well known in sickle cell disease (SCD). Hydroxyurea (HU) is commonly used in adults and children with SCD, but, little is known about the effects of HU on immune function in SCD and recommendations for immunization are lacking. As HU reversibly inhibits ribonucleotide reductase, causing cell cycle arrest at the G1-S interface, we postulated that HU might delay the transition from naïve to memory T cells, resulting in a delay in immunologic maturation, with deleterious effects on vaccine responses; therefore, T cell subsets, including conversion of naïve to memory T cells and antibody (Ab) responses to pneumococcal (Pnu), measles, mumps and rubella (MMR) vaccinations were studied during the BABY HUG Trial of HU for infants and toddlers with SCD. Methods: Blood was collected for measurement of: T cell subsets at entry (9–18 mos of age), age 24 mo and exit after 2 yrs of treatment; Pnu Ab levels were measured (by EIA after preabsorption with C-polysaccharide) at entry, before and 2–8 wks after 23-valent polysaccharide (PS) PCV23 vaccine at age 24 mo, and exit; and MMR antibodies at entry, 1–10 weeks after MMR at age 12 mo, age 24 mo and exit. Results: Of the 193 subjects in BABY HUG, T cell subsets were available for 91 HU and 88 placebo (PL) subjects. At entry, there were no significant differences in absolute lymphocyte count (ALC), or % or absolute T cell subsets between the HU and PL groups. At age 24 mo, both groups had an age-related decline in ALC, with significant differences between the HU and PL groups in ALC (5740 vs. 7323/mm3; p=0.003), absolute CD4 cells (1912 vs. 2247/mm3; p=0.022) and memory CD4 cells (392 vs. 487/mm3; p=0.003). The same pattern was seen at exit for ALC (4836 vs. 5764/mm3; p=0.015), absolute CD4 cells (1510 vs. 1747/mm3; p=0.043) and memory CD4 cells (360 vs. 444/mm3; p=0.003). IgG Ab levels to Pnu antigens were measured at entry [after 3 doses of PS/protein conjugate (PC) PCV7 containing Pnu PS type 26 (6B), but not 51 (7F)], before and after immunization with PS PCV23 vaccine at 24 mo, and at exit, with no significant differences between the HU and PL groups in mean Ab levels at any time point for either serotype (Fig 1&2). For those immunized with MMR prior to study treatment, there were no differences in % immune for measles, mumps, or rubella between the HU and PL groups at any time point. For 40 HU and 38 PL subjects immunized after starting study treatment, there was a smaller % of HU-treated children with protective response to measles vaccine 1 – 10 wks after immunization [HU 71.4% (10/14) vs. PL 100% (24/24); p=0.014]. Response to mumps [HU 78.6% (11/14) vs. PL 91.7% (22/24)] and rubella [HU 66.7% (10/15) vs. PL 92% (23/25)] vaccines showed similar trends, but no statistical significance. The HU vs. PL difference in failure of response to at least one vaccine virus was of similar significance (p = 0.021) to that of measles alone. By age 24 mo, there were no significant differences in response to MMR (89.7 to 100%). Mean Ab levels to measles and rubella were also significantly lower in the HU group 1–10 wks after immunization (p=0.005 and p=0.011, respectively). Conclusions: HU given to infants and young children with SCD causes a reduction in ALC, CD4 T cell and memory T cell counts. Additional monitoring of HU-treated children with SCD for these and related T cell parameters is warranted until the biological effects on immunity are known with certainty. Response to Pnu vaccine was not affected by HU therapy, suggesting that there is no increased risk for Pnu sepsis in infants and children with SCD treated with HU, consistent with the clinical results in the BABY HUG trial. Although the sample was small, a delay in achieving protective measles Ab levels was seen in the HU group, and possibly for mumps and rubella. This delay cannot be estimated precisely, due to study design, but Ab levels to all 3 viruses were similar at exit, indicating that effective immunization can be achieved despite HU use. For endemic or local epidemics of measles, mumps, rubella or other pathogens requiring immunization, adherence to recommended accelerated immunization schedules will be especially important for children with SCD treated with HU. Disclosures: Off Label Use: Off-label use of hydroxyurea in infants and children. Casella:Adventrx: Consultancy, Research Funding.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1759-1759 ◽  
Author(s):  
Franck Lemiale ◽  
Mario Pereira ◽  
Laurent Humeau ◽  
Boro Dropulic

Abstract Recently, we initiated the first ex vivo HIV-based gene therapy trial in humans with HIV+ CD4+ T cells. In this protocol, a modified lentiviral vector carrying an anti-HIV payload is used to modify CD4+ T cells isolated from HIV-infected patients by apheresis and CD8 negative selection. The T cells are activated in the presence of vector and expanded using immobilized CD3/CD28 antibodies, and then infused back into the patient. T cell receptor (TCR) repertoire analysis has value for safety monitoring of adoptive T cell transfers in the detection of aberrant clonal expansions or deletions. In this study, the TCR Vβ repertoire was assessed using a flow cytometry based assay at various time points in the selection/transduction/expansion process of CD4+ T cells. PBMC isolated from whole blood of HIV+ patients were CD4-selected using a CD8 negative selection, followed by enrichment by CD3 antibody. CD4+ purified cells were transduced with the lentiviral vector, VRX496, in the presence of retronectin, and then co-cultured with CD3/CD28 coated M450 Dynabeads for ten days. The TCR Vβ repertoire was assessed in throughout the process using a FACS-based assay that employs a panel of 20 monoclonal antibodies recognizing most of the 24 Vβ families in PBMC and CD4+ T cells. Repertoires from subjects with normal polyclonal TCR profiles were conserved, as shown by the absence of any significant change in any Vβ family. Moreover, the transduction/expansion of CD4+ T cells from a patient with a previously skewed TCR profile allowed the improvement of the TCR Vβ repertoire. Finally, no significant difference was observed in the repertoire of cells transduced with VRX496 versus mock-transduced cells. These data demonstrate stability of the repertoire diversity and thus provide important support information in favor of the safety of a gene therapy approach involving lentiviral vector mediated modification and expansion of CD4+ T-cells.


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