Introduction Recent advances in treatment and care have improved survival rates for children and young adults with severe blood disorders such as sickle cell disease (SCD), transfusion-dependent beta-thalassaemia (TDT) and acute leukaemia. However, their quality of life and reproductive and psychosocial outcomes are not yet well studied. For SCD and TDT, robust survival data are mainly limited to North America. Thus, there is a need to fill these knowledge gaps to guide improvements in care, address unmet clinical needs and rigorously assess the efficacy of emerging novel therapies.Methods and analysis This is an observational population-based mixed-methods study of individuals diagnosed with SCD, TDT or acute leukaemia when under the age of 18 in England, involving a data linkage component and a patient-reported outcomes measures survey. Data linkage-eligible participants will be identified from national and regional databases, including the Hospital Episode Statistics, Yorkshire Specialist Register of Cancer in Children & Young People and the National Congenital Anomaly and Rare Diseases Registration Service. Data linkage will be processed within the NHS England and the University of Leeds’ secure, trusted research environments. Data will be accessed without consent under section 251 and approval by the confidentiality advisory group. It will assess survival rates for SCD and TDT as well as clinical, educational and mental health outcomes for SCD, TDT and acute leukaemia diagnosed in childhood.Survey-eligible participants for SCD, TDT and acute leukaemia cohorts will be checked for their suitability to participate by the North of England clinical care teams. An NHS-approved survey provider will facilitate data checks with the NHS National Data Opt-Out Service. Consent is required for participation in the survey and for subsequent data linkage to existing databases. Surveys are conducted in various formats (online, paper and phone), with reminders sent after 21 days. The survey will assess quality of life and psychosocial and reproductive outcomes. Participants can withdraw at any time, and support is available via telephone helplines.Ethics and dissemination The study has received ethical and information governance approval from the Health Research Authority (Reference 24/YH/0186) and the Confidentiality Advisory Group (CAG 24/CAG/0138) to process identifiable data without consent. Study results will be available to patients, physicians, researchers, stakeholders and others through open-access publishing, results sharing via media platforms and presentations at conferences and meetings.

Sickle cell disease and β-thalassemia are common monogenic disorders that cause significant morbidity and mortality globally. The only curative treatment currently is allogeneic hematopoietic stem cell transplantation, which is unavailable to many patients due to a lack of matched donors and carries risks including graft-versus-host disease. Genome editing therapies targeting either the BCL11A erythroid enhancer or the HBG promoter are already demonstrating success in reinducing fetal hemoglobin. However, where a single locus is targeted, reliably achieving levels high enough to deliver an effective cure remains a challenge. We investigated the application of a CRISPR/Cas9 multiplex genome editing approach, in which both the BCL11A erythroid enhancer and HBG promoter are disrupted within human hematopoietic stem cells. We demonstrate superior fetal hemoglobin reinduction with this dual-editing approach without compromising engraftment or lineage differentiation potential of edited cells post-xenotransplantation. However, multiplex editing consistently resulted in the generation of chromosomal rearrangement events that persisted in vivo following transplantation into immunodeficient mice. The risk of oncogenic events resulting from such translocations therefore currently prohibits its clinical translation, but it is anticipated that, in the future, alternative editing platforms will help alleviate this risk.

AMD3100 (plerixafor) is a vital component of many clinical and preclinical transplant protocols, facilitating harvest of hematopoietic stem and progenitor cells through mobilization into the peripheral blood circulation. Repeat mobilization with AMD3100 is also necessary for many patients with suboptimal first stem cell collection or those requiring repeat transplantation. In this study we investigated the mobilization efficacy of repeated AMD3100 dosages in the nonhuman primate and humanized mouse models. In nonhuman primates, we observed effective mobilization after the first AMD3100 administration but a significantly poorer response in CD34+ and hematopoietic stem cell-enriched CD90+ cells with subsequent doses of the drug. A similar loss of efficacy with repeated administration was noted in immunodeficient mice engrafted with human CD34+ cells, in whom the total human white cell population, and particularly human hematopoietic stem and progenitor cells, mobilized significantly less effectively following a second AMD3100 administration when compared with the first dose. Together, our results are expected to inform future mobilization protocols for the purposes of peripheral blood hematopoietic stem cell extraction or for applications in which hematopoietic stem cells must be made accessible for in vivo-delivered gene targeting agents.

BACKGROUND Genome engineering of human hematopoietic stem and progenitor cells (HSPCs) has the potential to cure many hematological diseases, but is limited by subtherapeutic levels of modified cells in vivo. Providing a selective homing and engraftment advantage to modified cells could overcome this, such as by increasing CXCR4 expression, a key adhesion molecule for stem cell homing. In vivo studies thus far utilized wild type (WT) CXCR4. Here we investigate overexpression of a constitutively active mutant (CAM) of CXCR4 in a novel competitive transplant model. The CAM is generated by a single amino acid substitution that activates intracellular signalling without ligand binding. METHODS G-CSF-mobilized human HSPCs were fluorophore-marked (VSVG lentiviral vector, GFP or mCherry) then electroporated with either WT or CAM CXCR4 mRNA. Equal numbers of WT and CAM CXCR4 treated cells were transplanted into NSG mice after sublethal irradiation in a double-cross competitive transplant experiment, and both short term bone marrow (BM) homing and long-term engraftment evaluated. RESULTS HSPCs overexpressing CAM CXCR4 showed improved early homing to BM compared to WT CXCR4 within 48 hours. By 18 weeks the two arms contributed equally to hematopoiesis. DISCUSSION In this pilot study, overexpression of CAM CXCR4 provided greater BM homing advantage than WT CXCR4. We successfully combined lentiviral transduction with mRNA transfection demonstrating that transient CXCR4 CAM overexpression is a strategy which can be successfully applied to genome engineered cells. This novel combinatorial competitive transplant model eliminates confounding experimental variables to allow direct comparison of 2 fluorophore-marked, mRNA-transfected arms within a single animal. Genome engineering of human hematopoietic stem and progenitor cells (HSPCs) has the potential to cure many hematological diseases, but is limited by subtherapeutic levels of modified cells in vivo. Providing a selective homing and engraftment advantage to modified cells could overcome this, such as by increasing CXCR4 expression, a key adhesion molecule for stem cell homing. In vivo studies thus far utilized wild type (WT) CXCR4. Here we investigate overexpression of a constitutively active mutant (CAM) of CXCR4 in a novel competitive transplant model. The CAM is generated by a single amino acid substitution that activates intracellular signalling without ligand binding. G-CSF-mobilized human HSPCs were fluorophore-marked (VSVG lentiviral vector, GFP or mCherry) then electroporated with either WT or CAM CXCR4 mRNA. Equal numbers of WT and CAM CXCR4 treated cells were transplanted into NSG mice after sublethal irradiation in a double-cross competitive transplant experiment, and both short term bone marrow (BM) homing and long-term engraftment evaluated. HSPCs overexpressing CAM CXCR4 showed improved early homing to BM compared to WT CXCR4 within 48 hours. By 18 weeks the two arms contributed equally to hematopoiesis. In this pilot study, overexpression of CAM CXCR4 provided greater BM homing advantage than WT CXCR4. We successfully combined lentiviral transduction with mRNA transfection demonstrating that transient CXCR4 CAM overexpression is a strategy which can be successfully applied to genome engineered cells. This novel combinatorial competitive transplant model eliminates confounding experimental variables to allow direct comparison of 2 fluorophore-marked, mRNA-transfected arms within a single animal.

Genome editing therapies represent a significant advancement in next-generation, precision medicine for the management of haematological diseases, and CRISPR/Cas9 has to date been the most successful implementation platform. From discovery in bacteria and archaea over three decades ago, through intensive basic research and pre-clinical development phases involving the modification of therapeutically relevant cell types, CRISPR/Cas9 genome editing is now being investigated in ongoing clinic trials. Despite the widespread enthusiasm brought by this new technology, significant challenges remain before genome editing can be routinely recommended and implemented in the clinic. These include risks of genotoxicity resulting from off-target DNA cleavage or chromosomal rearrangement, and suboptimal efficacy of homology-directed repair editing strategies, which thus limit therapeutic options. Practical hurdles such as high costs and inaccessibility to patients outside specialised centres must also be addressed. Future improvements in this rapidly developing field should circumvent current limitations with novel editing platforms and with the simplification of clinical protocols using in vivo delivery of editing reagents.

Sickle cell disease and β-thalassemia are among the most common monogenic disorders globally, causing significant morbidity and early mortality. The only curative option available is allogeneic hematopoietic stem cell transplantation, which is limited by a lack of matched donors and risks, including graft versus host disease and secondary malignancy. Retroviral gene transfer is being explored in clinical trials, but an alternative approach is more targeted CRISPR/Cas9 genome editing, to recapitulate naturally occurring hereditary persistence of fetal hemoglobin (HPFH) which ameliorates disease. HbF reinduction is achieved by disrupting either transcription factor binding sites within the HBG promoter, or an erythroid enhancer sequence within the HbF repressor, BCL11A. Results from preclinical studies have suggested that HbF levels may remain suboptimal when each locus is targeted individually. We thus investigated the feasibility of a dual editing approach, targeting both loci simultaneously or sequentially, to maximize HbF production. G-CSF-mobilized human peripheral blood hematopoietic stem and progenitor cells (HSPCs) underwent CRISPR/Cas9 genome editing at either HBG promoter, BCL11A erythroid enhancer, or both loci targeted either simultaneously or sequentially. Immunodeficient mice were transplanted with edited cells. HSPCs cultured in differentiation media demonstrated comparable levels of editing at each locus in the single edited arms (by TIDE).The dual editing approach did not impair editing efficiency at each site when conducted sequentially and only slightly reduced efficiency in the simultaneously-edited reactions. HbF reinduction was also greatest in sequentially double-edited reactions, with HbF/HbA ratio as high as 3.9 times that seen in mock reactions by flow cytometry and 4.1 times by HPLC. In single-edited arms, HbF reinduction was slightly greater with HBG promotor than BCL11A erythroid enhancer editing. Two separate chromosomal translocation events encompassing both loci were detected in each double-edited arm and quantified using digital droplet PCR. Both were more frequent in simultaneous (mean of 1.0% and 0.6%) than sequential reactions (means <0.2%, n=3, p=0.0420 and p=0.0296). Analysis of single BFU-E colonies grown on methocult media revealed markedly different indel patterns at the 2 target loci. Concurrent hemoglobin fraction analysis of these clonal populations by HPLC demonstrated greatest HbF proportion in double-edited colonies. Where ≥60% editing was reported at both loci mean HbF was 71.3%, compared to 47.1% with ≥60% editing at HBG only, and 29.6% with ≥60% editing at BCL11A only (p=0.0373). Following transplant into mice, engraftment and lineage differentiation were comparable among all experimental arms. At necropsy, bone marrow populations of human CD45+ cells, CD34+CD38low HSPCs and, within these, HSC-enriched CD90+CD45RA- subpopulation, were present at comparable levels indicating that the dual editing approach did not impair engraftment. Translocation events were detected, albeit at frequencies of less than 0.25%, in the bone marrows of all 4 mice transplanted with simultaneously dual-edited cells and in 5 of 6 mice transplanted with sequentially dual-edited cells. Bone marrow cells cultured ex vivo demonstrated greatest HbF from mice transplanted with sequentially double-edited cells. In summary, we present evidence of maximized HbF reinduction with sequentially applied multiplex genome editing at BCL11A erythroid enhancer and HBG promoter, with no impairment of engraftment or differentiation. However, chromosomal translocation events were consistently seen in double-edited reactions, even when edits at each locus were applied sequentially, and persisted in vivo after engraftment, thus rendering this approach inappropriate for clinical applications. However, multiplex editing and targeting both the BCL11A erythroid enhancer and HBG promoter to maximize fetal hemoglobin induction may be a promising strategy for alternative platforms such as base editors which are expected to greatly reduce or completely eliminate the occurrence of translocations. Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Enochian: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for > 1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34(+)CD90(+)CD45RA(-), allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.

BackgroundANCA-associated vasculitides (AAV) are chronic autoimmune diseases, which can present with life-threatening multi-system involvement. Despite the use of rituximab and other available modern biologic therapies some patients with AAV develop severe and refractory courses of disease.ObjectivesThe aim of this study was to analyse outcomes of autologous hematopoietic stem cell transplantation (HSCT) for refractory AAVMethodsAdults receiving HSCT for AAV and whose data were registered within the EBMT Autoimmune Disease Working Party (ADWP) were identified retrospectively through the EBMT database. Treating physicians were surveyed to produce a retrospective evaluation of outcomes.Results7 patients underwent HSCT primarily for AAV between 1999–2014 in 6 centres across Europe. 5 females and 2 males were transplanted; 6 had a diagnosis of granulomatosis with polyangiitis (GPA) and 1 eosoniphilic granulomatosis with polyangiitis (EGPA). Median age was 39 years (range 32–55 years). Patients had received 4–6 prior lines of therapy, including cyclophosphamide (CYC, with median cumulative dose of 80 g) and steroids in every case, and rituximab in 4 cases. Stem cell source was peripheral blood in every case; CD34-selection was performed in 4 cases, mean CD34+ cell dose was 4.2 × 106/kg (range 0.6–7.9 × 106/kg). Conditioning regimen was CYC/ATG in 5 patients and CYC in 2 patients. Median follow-up was 86 months (range 1–204 months). Transplant-related mortality (TRM) occurred in 2 causes. All but one patient went into remission but 3 later relapsed at 6, 12 and 36 months, respectively, and required further treatment for disease control. At time of last clinical follow-up, 3 patients had drug-dependent partial response; 1 had drug-dependent complete remission and 1 had drug-free complete remission.ConclusionsOutcomes of HSCT for these heavily pre-treated AAV patients were variable. Only 1 patient achieved drug-free complete remission and TRM was observed in a quarter. Nevertheless, HSCT had the potential to stabilise AAV in patients who initially failed to respond to conventional therapies. These data do not support HSCT for advanced stage ANCA-positive vasculitis, although it may have a place as salvage treatment in otherwise refractory patients. As for other autoimmune diseases, HSCT may provide better outcomes when performed at early stage of disease. Overall, HSCT should only performed in clinical trial settings in experienced centres.Disclosure of InterestNone declared

Objective: To assess impact of a topical decontamination regimen on rates of catheter-related bloodstream infections (CRBSI) in intensively-treated haematology patients. Methods: A historically-controlled cohort study was used to evaluate the effect of applying chlorhexidine or Octenisan (R) body washes and nasal Prontoderm (R) ointment for 5 days around the time of Hickman line insertion on the incidence of CRBSI and infection-free catheter time. Lines inserted during a 24 month period prior to implementation of the decolonisation regimen were compared with those inserted during a 12 month period after the intervention was applied. Results: During the post-intervention period, 163 lines were inserted in 147 patients, compared to 303 lines in 242 patients in the pre-intervention period. CRBSI rates in treated and untreated patients respectively were 6.8 and 35.0 cases per 10,000 line-days by 21 days (p = 0.009), and 14.4 and 26.0 cases respectively per 10,000 line-days by 180 days (p = 0.025). The incidence rate of Staphylococcus aureus CRBSI in treated and untreated patients were 0.0 and 4.6 cases per 10,000 line-days respectively (p = 0.012). Multivariable Cox regression estimated an 81% probability (95% confidence interval 74%-85%) that a treated line develops a CRBSI later than an untreated line by 21 days post-insertion. Conclusion: Implementation of this safe and effective topical decontamination regimen enhances routine CRBSI-prevention measures for haematology patients requiring central venous line insertion. (C) 2017 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

OBJECTIVES:Modern management of myeloma has significantly improved survival, with increasing numbers of patients living beyond a decade. However, little is known about the long-term cardiovascular and respiratory status of intensively treated and multiply relapsed survivors. METHODS:We performed detailed cardiovascular and respiratory evaluations in patients with intensively treated, advanced but stable myeloma. All patients had received at least two lines of treatment, including at least one haematopoietic stem cell transplantation procedure, but had stable, controlled disease and were off active treatment at the time of evaluation. RESULTS:Thirty-two patients with a median duration of 6 years (range 2-12) from original diagnosis of myeloma and three lines (range 2-6) of treatment were evaluated. Despite normal physical examination in the majority, there was a high prevalence of sub-clinical cardiac and respiratory dysfunction, reflected by abnormalities of electrocardiography (45%), echocardiography (50%), serum N-terminal pro-B-type natriuretic peptide level (NT-pro-BNP, 50%), and pulmonary function testing (45%). NT-pro-BNP level correlated negatively with quality of life (P = 0.012) and positively with serum ferritin (P = 0.027). Dyspnoea score correlated with BMI (P = 0.001). Risk factors for cardiovascular disease (obesity, hypertension, hyperlipidaemia, and hyperinsulinaemia) were common. DISCUSSION:Even in the absence of overt clinical features, the majority of intensively treated long-term survivors of myeloma have established cardiovascular and/or respiratory dysfunction, above levels expected in the general population of a similar age. CONCLUSION:This study supports routine screening and lifestyle modification combined with primary and secondary preventive strategies to reduce cardiovascular and respiratory disease and to preserve quality of life in transplanted myeloma patients.

A recent paper by Bate et al (‘PEPtalk: postexposure prophylaxis against varicella in children with cancer’)1 reports national data regarding practice in postexposure prophylaxis (PEP) of chickenpox in immunocompromised children. We provide additional depth of detail regarding practice in a large tertiary paediatric haematology unit in England by presenting a service evaluation of postexposure chickenpox prophylaxis in children with leukaemia. We also highlight the probability of underestimated cases of PEP and clinical chickenpox in the paper by Bate et al . While chickenpox in healthy children is normally mild and self-limiting, in immunocompromised …

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