Gene therapy has emerged as a transformative treatment option for individuals with sickle cell disease (SCD), with recent regulatory approvals marking a pivotal shift in clinical care. However, the complexity of patient selection, stem cell mobilization, manufacturing, conditioning, and long-term follow-up underscores the need for standardized, evidence-informed guidance. This consensus document, derived from early clinical experience, and developed by the American Society for Transplantation and Cellular Therapy and the International Society for Cell & Gene Therapy, provides practical recommendations for the clinical implementation of ex vivo gene therapies for SCD. Key areas addressed include eligibility assessment, comparative considerations with allogeneic hematopoietic cell transplantation, mobilization and apheresis strategies, conditioning with myeloablative chemotherapy, fertility preservation, psychosocial care, manufacturing quality attributes, and lifelong surveillance for late effects. Collectively, these recommendations aim to harmonize clinical practice, support shared decision-making, and promote safe, equitable, and durable delivery of gene therapy for individuals with SCD.

Background: The advent of autologous gene modified cell therapies to treat monogenic disorders has been a major step forward for the field of hematopoietic stem cell transplantation (HCT) and cellular therapies. The need for disease-specific conditioning to enable these products to provide a potential cure has required extrapolation from experience in myeloablative and non-myeloablative HCT for these disorders. Methods: In this manuscript, we review the current datasets and clinical experience using different conditioning regimens for autologous gene therapies in hemoglobinopathies, metabolic and lysosomal disorders, inborn errors of immunity (IEI) and bone marrow failure (BMF) syndromes. Results: The disease specific and unique conditioning requirements of each disorder are considered in order to achieve maximal benefit while minimizing associated toxicities. Conclusions: Standardized recommendations based on these data are made for each set of disorders to harmonize treatment. Future directions and the possibility of non-genotoxic conditioning regimens for autologous gene therapies are also discussed. Ethical Statement: The authors followed all relevant ethical considerations in writing this manuscript. (c) 2024 International Society for Cell & Gene Therapy. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Since the discovery of the structure of DNA and its transcription, it has been a scientific dream to treat monogenic disorders by rewriting and correcting the genetic code. Development of recombinant DNA technology and DNA sequencing in the 1970s laid the foundation for gene-insertion therapies, with the goal to deliver a healthy functioning copy of the mutated gene in patients. Development of retrovirus and adenovirus delivery vectors in the 1980s led to the first gene-therapy treatment of a patient with adenosine deaminase–deficient severe combined immune-deficiency in 1990. The field received a major setback in 1999 as the result of the death of a patient from a severe immune reaction in the liver related to adenovirus delivery. Development of safer vectors (lentivirus, herpes simplex and adeno-associated viruses, or AAVs), improved transduction procedures and increased safety parameters in clinical trials led to a resurgence in the field in 2000s. This decade also saw the development of gene-editing technologies like zinc finger nucleases and transcription activator-like effector nucleases and the emergence of rapid gene-sequencing methods. Discovery of clustered regularly interspaced short palindromic repeats (Crispr)-Cas9 as a simple, elegant tool for creating precise double-stranded breaks in DNA in 2012 re-energized the field and has led to major applications of the platform for gene-editing and correction of monogenic disorders. Improvement in delivery platforms using lipid nanoparticles, oligonucleotides and AAV to deliver DNA or Crispr guides-Cas9 RNAs at high frequency in the cells of different tissues has ushered in the era of “in vivo” gene-editing therapies. This has led to multiple approvals of gene therapy–based treatments by the Food and Drug Administration, for example, voretigene neparvovec-rzyl (Luxturna) for bi-allelic RPE65 mutation-associated retinal dystrophy, etranacogene dezaparvovec-drlb (Hemgenix) for hemophilia B, onasemnogene abeparvovec (Zolgensma) for spinal muscular atrophy and betibeglogene autotemcel (Zynteglo) for transfusion-dependent thalassemia, to name a few. The rapid advancements have also created major challenges, especially related to assay development for safety and potency assessments, manufacturing bottlenecks for delivery to patients and regulatory and ethical issues, specifically related to germline editing. The mini-series on gene-therapy was planned to describe the state-of-the-art, recent innovations, applications and current challenges. A major goal of this mini-series is to provide the readers with an overview of these novel therapies that are already approved or are in clinical testing. Although Crispr-Cas9 is a precise DNA-editing tool, the double-stranded break created by Crispr-Cas9 can lead to large deletions and chromosomal aberrations. Hence, base-editing, which leads to single-strand breaks and leads to single-base edits in the DNA by deamination, is considered a safer methodology. Walker Lahr, Christopher Sipe, Joseph Skeat and others describe the development of cytosine and adenosine base editors and applications of this technology as a refinement of Crispr-Cas9 editing. AAV remains the major delivery tool for the guides or DNA for gene modification. Recent advances in AAV technology, specifically related to rapid creation and analyses of AAV capsid libraries, are highlighted in the paper by Joanna Szumska and Dirk Grimm. The two manuscripts, by Laura Ugalde, Sara Fananas, Raul Torres and others and Senthil Bhoopalan, Jonathan Yen, Rachel Levine and others, provide an overview of the basics of Crispr-Cas9 technology, applications and challenges of gene therapy for treating blood disorders. The potential of these one-time, functionally curative treatments is expected to grow exponentially in the near future, as the manufacturing, safety, durability, regulatory and reimbursement challenges are addressed. Recent endeavors by Food and Drug Administration in this direction, for instance, creation of Initial Targeted Engagement for Regulatory Advice on CBER products (INTERACT) and Gene Therapy Pilot Program are also steps in the right direction. We hope this series will prove of interest to the readers from pre-clinical, translational research and clinical scientists alike.

Background: Fanconi anemia (FA) is a rare genetic disorder characterized by defective cellular deoxyribonucleic acid (DNA) repair, associated with developmental abnormalities, progressive bone marrow failure (BMF), and a predisposition to hematologic malignancies and solid tumors. 80% of FA patients develop BMF. Although allogeneic hematopoietic stem cell transplant (allo-HSCT) is a curative treatment for BMF, its utilization and efficacy is limited by availability of suitable human leukocyte antigen (HLA)-matched donors, risk of graft-versus-host disease (GVHD) and transplant-related toxicities. Ex-vivo insertion of a functional FANCA gene into autologous FA-A CD34+ enriched hematopoietic stem and progenitor cells (HSPCs) has been shown in preclinical studies to provide a survival advantage to the gene-modified stem cells, leading to correction of BMF. Feasibility of this approach was established in the FANCOLEN-1 clinical trial (Spain), although cell doses and transduction levels varied considerably. Modifications to the collection and manufacturing processes were made in the clinical studies to enhance the dose of transduced HSPCs, with the goal of preventing progression of BMF to obviate the need of an allo-HSCT. Design and Methods: RP-L102-0418 (clinicaltrials.gov # NCT03814408) is a U.S. Phase I clinical trial evaluating the feasibility and safety of autologous CD34+ cells transduced with a lentiviral vector (LV) carrying the FANCA gene (PGK-FANCA-WPRE) in two pediatric patients with FA-A. Patients <12 years of age, with early evidence of cytopenias, but with bone marrow (BM) CD34+ count >30/µL were eligible for treatment. Peripheral blood mononuclear cells were collected via leucocytapheresis on two consecutive days after mobilization with granulocyte-colony stimulating factor (G-CSF) and Plerixafor (Mozobil). CD34+ HSPCs were enriched, placed in culture with cytokines, and transduced with PGK-FANCA-WPRE LV. The investigational drug product (DP) (RP-L102) was infused fresh into patients within 4 hours of release, without any prior conditioning regimen. Patients are being followed for 3 years post-infusion for safety assessments (replication competent lentivirus (RCL), insertion site analysis (ISA)) and to ascertain early evidence of efficacy (increasing peripheral blood vector copy number (VCN) and BM mitomycin-C (MMC) resistance), along with stabilization/correction of cytopenias. Results: Two FA-A patients (aged 5 and 6 years) were consented and enrolled on the study at Stanford University. Mobilization and apheresis procedures were performed successfully without any serious adverse events. DP was successfully manufactured using "Process B" optimization including transduction enhancers, commercial-grade vector, and modified cell processing. Because of higher transduced CD34+ and colony forming cell (CFC) doses, we anticipate early development of BM MMC resistance in the current study patients. Safety and efficacy data 4 to 6 months post-treatment, including peripheral blood VCN, blood counts and bone marrow MMC resistance, will be available at the time of presentation. Conclusions: DP has been successfully manufactured in the Phase I study (N=2) to meet the required specifications.Patients are being monitored for early efficacy assessments; 6+ months of follow-up may be required to observe the proliferative advantage of transduced HSPCs.Plans for Phase II portion of the study are in progress. Disclosures Czechowicz: Rocket Pharmaceuticals, Inc.: Research Funding. Beard:Rocket Pharmaceuticals: Employment, Equity Ownership. Law:Rocket Pharmaceuticals: Employment, Equity Ownership. Nicoletti:Rocket Pharmaceuticals, Inc.: Employment, Equity Ownership. Río:Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership.

Patients with acute myelogenous leukemia (AML) who undergo killer immunoglobulin-like receptor (KIR)-mismatched haploidentical hematopoietic stem cell transplantation (HSCT) have improved survival. Children's Oncology Group AAML05P1 is a prospective phase 2 trial of unrelated donor (URD) HSCT in which KIR typing of donors was available to the treating physician at donor selection, aiming to determine feasibility (defined as the ability to obtain donor samples from URDs and obtain KIR data before transplantation) of prospective selection of KIR-mismatched donors and effect on outcomes. Patients age <= 30 years with high-risk AML at presentation or relapsed AML were eligible; the study accrued 90 evaluable patients. After enrollment, as many as 5 potential URD samples were KIR-typed (including gene expression) in a central laboratory and results reported to the treating physician, who made the final donor selection. Cases were categorized as KIR-matched or MR-mismatched using different published strategies. Overall survival (OS), disease-free survival (DFS), and relapse did not differ significantly by KIR mismatch status. Acute graft-versus-host disease (GVHD) was significantly lower in recipients of MR-mismatched stem cells (35% versus 60%; P= .027). We examined DFS according to time to natural killer (NK) receptor recovery after HSCT. NK p44 recovery was significantly associated with KIR mismatch and with decreased DFS and increased relapse risk in multivariate Cox analysis (P = .006 and .009, respectively). We show that prospective selection of URD according to KIR type was feasible, acute GVHD was reduced, but survival did not differ using any model of KIR mismatch. However, the study enrolled mostly matched transplants, so ligand-ligand mismatch was rare, and thus the sample size was insufficient to determine potential benefit according to this model. Cord blood recipients demonstrated a trend toward improved DFS with KIR mismatch, but the study was not powered to detect a difference in this small subset of patients. Our data suggest that recovery of NK receptor expression might influence DFS after HSCT. (C) 2019 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Ruxolitinib was recently approved as a treatment for steroid refractory acute GVHD (aGVHD) and is used in adults for the treatment of chronic GVHD (cGVHD). As a JAK-1/JAK-2 inhibitor, it reduces T-cell proliferation and trafficking and leads to a potent anti-inflammatory effect. There is limited data of its use in pediatric and adolescent/young adult (AYA) patients. We report our single center experience with ruxolitinib in this population. Data on patients treated with ruxolitinib at Lucile Packard Children's Hospital between 8/1/18 and 8/31/19 were retrospectively analyzed after institutional IRB approval. aGVHD was graded according to the Modified Glucksberg scale, while cGVHD was scored as limited or extensive based on Seattle criteria. Only patients on ruxolitinib for ≥14 days were evaluated for response, which was defined as a reduction by at least one grade in severity of aGVHD. Ability to reduce other GVHD medications, especially steroids, and/or symptomatic improvement was considered a response in cGVHD. 15 patients (median age 18, range: 11-29 years) received ruxolitinib: 5 for aGVHD and 10 for cGVHD. Standard starting dose was 5 mg twice a day. Patients in the aGVHD group were steroid refractory with ≥ Grade III GVHD and had received a median of 3 (range: 2-5) different agents prior to ruxolitinib. Patients received treatment for a median of 46 days (range: 23-332). 4 of 5 patients had a partial response (PR) with one non-responder, for an overall response rate (ORR) of 80%. All patients experienced at least one episode of infection- CMV reactivation (n=2), adenovirus (n=1), viral gastroenteritis (n=1), bacterial infections (n=3) and probable fungal infection (n=1). Dose-limiting cytopenia was the only toxicity observed in 3 patients (60%), requiring dose reduction or discontinuation. 2 patients had TRM, both related to GVHD and infections. 10 patients treated for cGVHD (6 limited, 4 extensive) had received a median of 3 (range: 2-6) different agents prior to ruxolitinib. One patient required early cessation of therapy due to a severe allergic reaction and in another patient ruxolitinib was stopped due to leukemia relapse. Of the eight patients evaluable for response, treatment duration was for a median of 195 days (range: 59-372) with an ORR of 88%. 5 (63%) patients developed infections: CMV reactivation (n=2), respiratory infections (n=4), bacterial infection (n=1). Only one patient required dose reduction due to GI side effects and cytopenias. Ruxolitinib is an effective salvage therapy for severe GVHD. It was well tolerated in our patient population, with myelosuppression as the dose limiting toxicity, especially in aGHVD patients. A high rate of infections was noted during ruxolitinib treatment and concomitant antimicrobial prophylaxis is strongly recommended.

Background: BCL11A is a key transcription factor that suppresses the production of fetal hemoglobin (HbF) in red blood cells (RBCs), leading to the production of adult Hb (HbA). In diseases with hemoglobin production defects such as b-thalassemia, or in sickle cell disease (SCD), HbF upregulation could ameliorate anemia and reduce transfusion requirements, such as in β-thalassemia, or reduce clinical complications, including vaso-occlusive crises (VOCs), in SCD. To induce potentially curative levels of HbF in erythrocytes, we used the ex vivo CRISPR-Cas9-based gene-editing platform to edit the erythroid enhancer region of BCL11A in hematopoietic stem and progenitor cells (HSPCs), producing CTX001. Aims: CLIMB THAL-111 (NCT03655678) and CLIMB SCD-121 (NCT03745287) are multi-center, first-in-human studies of CTX001 for transfusion-dependent b-thalassemia (TDT) and SCD, respectively. Here, we present available safety and efficacy results from all patients with at least 3 months of follow-up from both studies as of July 2020. Methods: Patients (aged 18 to 35 years) with TDT receiving packed red blood cell (pRBC) transfusions of ≥100 mL/kg/year or ≥10 units/year in the previous 2 years, and those with severe SCD, defined as ≥2 VOCs/year requiring medical care in the previous 2 years, were eligible. Peripheral CD34+ HSPCs were collected by apheresis after mobilization with G-CSF (filgrastim) and plerixafor (for TDT) or plerixafor alone (SCD). The erythroid enhancer region of BCL11A was edited in CD34+ cells using a specific CRISPR guide RNA and Cas9 nuclease. Prior to CTX001 infusion on Day +1, patients received myeloablation with 4 days of busulfan. Patients were monitored for stem cell engraftment and hematopoietic recovery, adverse events, total Hb and HbF production, hemolysis, F-cells, pRBC transfusion requirements (TDT), and VOCs (SCD) during follow-up. Results: Data are presented for patients with TDT (N=5; RBC transfusion history range: 23.5 to 61 units/year; CTX001 post-infusion follow-up through Months 15, 6, 4, 4, and 3, respectively) and with SCD (N=2; 7 VOCs/year and 7.5 VOCs/year, respectively, annualized over 2 years prior to consent; CTX001 post-infusion follow-up through Months 12 and 3, respectively). In the patients with TDT, median neutrophil engraftment occurred on Day +32 (range: +27 to +36); median platelet engraftment occurred on Day +37 (range: +34 to +52). In the patients with SCD, neutrophil engraftment occurred on Day +30 and Day +22 and platelet engraftment occurred on Day +30 and Day +33, respectively. All patients demonstrated increases in total Hb and HbF over time (Figure). Patients with TDT ceased receiving pRBC transfusions soon after CTX001 infusion, with the last pRBC transfusion occurring between 0.9 and 1.9 months after CTX001 infusion. The first patient with TDT who received CTX001 has remained transfusion-free for over 15 months. Patients with SCD have had no VOCs since CTX001 infusion. The first SCD patient who received CTX001 has remained free of VOCs for over 1 year. In all 7 patients, the safety profile after CTX001 infusion was generally consistent with busulfan myeloablation. Four serious adverse events (SAEs) related or possibly related to CTX001 were reported in 1 patient with TDT: headache, haemophagocytic lymphohistiocytosis (HLH), acute respiratory distress syndrome, and idiopathic pneumonia syndrome. All 4 of these SAEs occurred in the context of HLH and were either resolved or clinically improving at the time of this analysis. No other CTX001-related SAEs were reported in the other patients with TDT or in any patients with SCD. Conclusions: These data demonstrate that CTX001, a first-in-human, CRISPR-Cas9-modified autologous HSPC product, has resulted in increases in HbF and total Hb in the first 7 patients infused. All patients infused with CTX001 demonstrated hematopoietic engraftment with a post-infusion safety profile generally consistent with myeloablation. All 5 patients with TDT have been transfusion-free since ~2 months after CTX001 infusion and the 2 patients with severe SCD have had no VOCs during follow-up after CTX001 infusion. These early data demonstrate that CTX001 is a potential functional cure for the treatment of TDT and SCD. Data will be updated for the presentation. Data from these ongoing studies were submitted on behalf of the CLIMB THAL-111 and CLIMB SCD-121 Investigators. Figure Disclosures Frangoul: Vertex Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Bobruff:CRISPR Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cappellini:BMS: Honoraria; CRISPR Therapeutics, Novartis, Vifor Pharma: Membership on an entity's Board of Directors or advisory committees; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fernandez:CRISPR Therapeutics: Current Employment, Current equity holder in publicly-traded company. Grupp:Juno/BMS: Other; Cellectis: Other; TCR2: Other: SAB; Servier: Research Funding; Janssen/JnJ: Consultancy; CBMG: Consultancy; Humanigen: Consultancy; GlaxoSmithKline: Consultancy; Roche: Consultancy; CRISPR Therapeutics/Vertex Pharmaceuticals: Other; Allogene: Other; Kite/Gilead: Research Funding; Novartis: Consultancy, Other: SSC, Research Funding; Adaptimmune: Other: SAB; Jazz: Other: SSC. Handgretinger:Amgen: Honoraria. Ho:CRISPR Therapeutics: Current Employment, Current equity holder in publicly-traded company. Imren:Vertex Pharmaceuticals Incorporated: Current Employment, Current equity holder in publicly-traded company. Kattamis:Agios: Consultancy; Vertex: Membership on an entity's Board of Directors or advisory committees; Ionis: Membership on an entity's Board of Directors or advisory committees; Genesis Pharma SA: Membership on an entity's Board of Directors or advisory committees; Vifor: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apopharma/Chiesi: Honoraria, Speakers Bureau. Lekstrom-Himes:Vertex Pharmaceuticals Incorporated: Current Employment, Current equity holder in publicly-traded company. Locatelli:Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceeutical: Speakers Bureau. Lu:Vertex Pharmaceuticals Incorporated: Current Employment, Current equity holder in publicly-traded company. de Montalembert:Bluebird bio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vertex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Addmedica: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mulcahey:Vertex Pharmaceuticals Incorporated: Current Employment, Current equity holder in publicly-traded company. Shanbhag:Vertex Pharmaceuticals Incorporated: Current Employment, Current equity holder in publicly-traded company. Sheth:Agios: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; La Jolla: Research Funding; Acceleron: Consultancy; Bluebird Bio: Consultancy; Novartis: Consultancy, Research Funding; DisperSol Technologies: Research Funding; Terumo: Research Funding; Vertex Pharmaceuticals/CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees. Soni:CRISPR Therapeutics: Current Employment, Current equity holder in private company. Steinberg:Vertex Pharmaceuticals/CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Fulcrum Therapeutics: Membership on an entity's Board of Directors or advisory committees; DSMB: Membership on an entity's Board of Directors or advisory committees; Imara: Membership on an entity's Board of Directors or advisory committees. Weinstein:CRISPR Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wu:Bayer: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.

Gene modification of hematopoietic stem cells is increasingly becoming popular as a therapeutic approach, given the recent approvals and the number of new applications for clinical trials targeting monogenetic and immunodeficiency disorders. Technological advances in stem cell selection, culture, transduction and gene editing now allow for efficient ex vivo genetic manipulation of stem cells. Gene-addition techniques using viral vectors (mainly retrovirus- and lentivirus-based) and gene editing using various targeted nuclease platforms (e.g., Zinc finger, TALEN and Crispr/Cas9) are being applied to the treatment of multiple genetic and immunodeficiency disorders. Herein, the current state of the art in manufacturing and critical assays that are required for ex vivo manipulation of stem cells are addressed. Important quality control and safety assays that need to be planned early in the process development phase of these products for regulatory approval are also highlighted.

Background Survival outcomes remain dismal in infants undergoing HSCT. Regimen related toxicity, organ dysfunction and infections remain the major causes of TRM in infants undergoing HSCT. Better strategies for preventing infections and organ toxicity are needed to improve survival. Feeding human milk (maternal or third party) may be an important strategy in infants to prevent infections and reduce toxicity due to its beneficial effect on pro-inflammatory cytokines, local immunity and gut microbiome. There is no data in pediatric HSCT practice regarding effect of breast feeding/human milk on survival outcomes. We conducted a survey study to assess current practices among the pediatric transplant centers regarding use of human milk during HSCT and the reasons, if any for restricting it. Methods We conducted a survey of PBMTC transplant centers in US and Canada regarding the utilization of human milk. The study was IRB approved at Stanford University and only information regarding institutional practices was collected. One response from each institution was included in the analysis and descriptive statistics was used to analyze the data. Results Twenty seven centers answered the survey questions. Only three institutions allowed unrestricted breast feeding, while two did not allow any form of human milk during HSCT. Majority (81%) of the centers allowed human milk during HSCT with some restrictions. The most common restriction was the diagnosis of immune-deficiency (SCID) due to the fear of CMV transmission; but only 11 (50%) of these centers tested mothers for their CMV status. Two centers irradiated breast milk prior to feeding. Only 2 centers had a program for providing pre-tested, pasteurized human milk from third party donors to infants undergoing HSCT. Only one center had an active research program looking at use of human milk in the HSCT setting. Conclusions Though there are established immunological and microbiological benefits of human milk, its utilization is variable among pediatric HSCT centers. Majority of the institutions restrict breast feeding in infants with the diagnosis of SCID, but only half of these centers test mothers for their CMV status. Use of pre-tested, third party human milk during HSCT is very limited. More research regarding the benefits and use of human milk in infants undergoing HSCT is needed to establish standardized guidelines.

Allogeneic hematopoietic stem cell transplantation (HSCT) can cure transfusion-dependent thalassemia (TDT). In a multicenter trial we investigated the efficacy of reduced-intensity conditioning (RIC) before unrelated donor (URD) HSCT in children with TDT. Thirty-three children, ages 1 to 17 years, received bone marrow (BM) or umbilical cord blood (UCB) allografts. Median time to neutrophil engraftment was 13 days (range, 10 to 25) and 24 days (range, 18 to 49) and platelet engraftment 23 days (range, 12 to 46) and 50 days (range, 31 to 234) after BM and UCB allografts, respectively. With a median follow-up of 58 months (range, 7 to 79), overall and thalassemia-free survival was 82% (95% CI, .64% to .92%) and 79% (95% CI, ,6% to .9%), respectively. The cumulative incidence of grades II to IV acute graft-versus-host disease (GVHD) after BM and UCB allografts was 24% and 44%; the 2-year cumulative incidence of chronic extensive GVHD was 29% and 21%, respectively; 71% of BM and 91% of UCB recipients discontinued systemic immunosuppression by 2 years. Six patients who had Pesaro risk class 2 (n = 5) and class 3 (n =1) died of GVHD (n = 3), viral pneumonitis (n = 2) and pulmonary hemorrhage (n = 1). Outcomes after this RIC compared favorably with URD HSCT outcomes for TDT and supported engraftment in 32 of 33 patients. Efforts to reduce GVHD and infectious complications are being pursued further. (C) 2018 American Society for Blood and Marrow Transplantation.

Survival for high-risk neuroblastoma patients is still suboptimal. Although stem cell transplantation (SCT) is used, there is no consensus as to which conditioning regimen has the greatest efficacy and fewest toxicities. We assessed the incidence of and risk for hepatic veno-occlusive disease (VOD) for neuroblastoma patients who underwent autologous SCT with busulfan and melphalan (BuMel) at eight centers following Children’s Oncology Group (COG)-based induction chemotherapy. Data regarding the patients, SCT characteristics, busulfan steady-state concentrations, incidence of VOD, and survival were evaluated. VOD was defined using the modified Seattle criteria. Possible factors associated with VOD (age, busulfan-pharmacokinetic parameters, history of hepatic dysfunction, and day of neutrophil engraftment) were evaluated. Seventy five patients were included and 23 children (31%) developed VOD at a median of 19 days after SCT (range 14–27 days). VOD was the cause of death in 4 patients (5%). In a multivariable analysis, young age (OR 1.7 (95% CI: 1.16–2.56; p = 0.012)) and early day of neutrophil engraftment (OR 1.4 (95% CI: 1.08–2.14; p = 0.041) were associated with the development of VOD. Initial or cumulative busulfan steady-state concentration were not associated with VOD. We found that despite the use of intravenous busulfan with adjusted serum levels, the incidence of VOD remains high in pediatric neuroblastoma patients.

Clofarabine is a purine nucleoside analog with immunosuppressive and anti-leukemic activity and its inclusion in reduced intensity regimens could potentially improve outcomes. We performed a prospective Phase I study of clofarabine combined with 2 Gy total body irradiation (TBI) as a non-myeloablative (NMA) preparative regimen for allogeneic stem cell transplantation in pediatric patients, who were considered at high risk of mortality from standard myeloablative regimens. The main goal of the study was to delineate the maximum feasible dose (MFD) of clofarabine in combination with 2 Gy TBI. Eighteen patients, 1-21 years of age and in complete remission were enrolled in the two stratamatched related donor (RD) and unrelated donor (UD) and evaluated for day100 dose limiting events (DLE-non-engraftment, non-relapse mortality (NRM), and severe renal insufficiency) after receiving clofarabine at the starting dose level of 40 mg/m 2 . All 6 patients (3 in each stratum) engrafted with no day100 DLE seen in the first cohort. The dose was increased to 52 mg/m 2 in the next and an expanded cohort (total 12 patients) and no DLE was observed at day100 and at 1-year study endpoint. The regimen was well tolerated with transient transaminitis, gastrointestinal and skin reactions as the common reversible toxicities observed with clofarabine. 52 mg/m 2 of clofarabine was deemed the MFD. Disease relapse led to mortality in 6 (33%) patients during follow-up with 1-year event free survival and overall survival of 60% (95% CI, 34-79) and 71% (95% CI, 44-87), respectively.

Sickle cell disease results from a homozygous missense mutation in the β-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector–mediated addition of an antisickling β-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling β-globin remained high (approximately 50% of β-like–globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526.)

Basic science studies have provided new insights into the pathophysiology of β-thalassemias. Studies of genotypic and phenotypic heterogeneity among patients and better understanding of control of erythropoiesis have provided new targets for designing novel agents that can be tailored to individual patient needs. JAK-2 kinase inhibitors and agents targeting the GDF-11/SMAD pathway are in clinical trials. Recent understanding of the control of switch of HbF to HbA during infancy has provided new targets for development of drugs and gene-editing strategies. Advancement in vector design, purification and transduction of human stem cells have led to multiple gene therapy clinical trials that are exploring the clinical benefits, safety and durability of these approaches. HSCT remains the only curative approach at present and continued improvements in unrelated and haplo-identical transplants is helping to expand the donor pool. Even though the future looks promising, carefully designed clinical trials in adults and children, with suitable end-points are still needed to confirm the efficacy, toxicity of these new agents, and improvements in quality of life of patients with β-thalassemia.

Clofarabine is a purine nucleoside analog with immunosuppressive and antileukemic activity and its inclusion in reduced-intensity regimens could potentially improve outcomes. We performed a prospective phase I study of clofarabine combined with 2 Gy total body irradiation (TBI) as a nonmyeloablative preparative regimen for allogeneic stem cell transplantation in pediatric patients who were considered at high risk of mortality from standard myeloablative regimens. The main goal of the study was to delineate the maximum feasible dose (MFD) of clofarabine in combination with 2 Gy TBI. Eighteen patients, 1 to 21 years of age and in complete remission, were enrolled in 2 strata (matched related donor and unrelated donor) and evaluated for day100 dose-limiting events (DLE) (nonengraftment, nonrelapse mortality [NRM], and severe renal insufficiency) after receiving clofarabine at the starting dose level of 40 mg/m(2). All 6 patients (3 in each stratum) engrafted with no day 100 DLE seen in the first cohort. The dose was increased to 52 mg/m2 in the next and an expanded cohort (total of 12 patients) and no DLE were observed at day 100 and at the 1-year study endpoint. The regimen was well tolerated with transient transaminitis and gastrointestinal and skin reactions as the common reversible toxicities observed with clofarabine. The dose of 52 mg/m2 of clofarabine was deemed the MFD. Disease relapse led to mortality in 6 (33%) patients during follow-up with 1-year event-free survival and overall survival of 60% (95% confidence interval [CI], 34 to 79) and 71% (95% CI, 44 to 87), respectively. This regimen leads to successful engraftment using both related and unrelated donors with exceptionally low rates of NRM. (C) 2017 American Society for Blood and Marrow Transplantation.

Unrelated donor (URD) hematopoietic cell transplantation (HCT) in children with sickle cell disease (SCD) is associated with a high incidence of rejection and graft-versus-host disease (GVHD). We report on the first 4 patients with severe SCD who underwent URD HCT using a novel myeloablative and immunosuppressive regimen composed of busulfan, fludarabine, and antithymocyte globulin with a single dose of post-transplant cyclophosphamide along with tacrolimus and mycophenolate mofetil for GVHD prophylaxis. Three patients engrafted and remain disease-free after a median follow-up period of 2.5 years. One patient had primary graft failure attributed to low stem cell content of the graft. Of interest, none of the engrafted patients developed acute or chronic GVHD. This preparative regimen along with the use of post-transplant cyclophosphamide offers a promising approach for unrelated donor transplants in patients with SCD and needs further corroboration in larger number of patients.

Background: Young people with high risk AML have a poor prognosis. AML patients receiving killer immunoglobulin-like receptor (KIR) mismatched haploidentical HSCT have reported improved survival due to reduced TRM and relapse. COG AAML05P1 is a prospective phase 2 trial of unrelated donor HSCT in which KIR typing of donors was available to the treating physician at the time of donor selection. The aim of the study was to determine whether prospective selection of KIR mismatched donors is feasible and improves outcomes. In addition, NK-cell reconstitution post-HSCT and impact on outcomes was assessed in those with KIR compatible and incompatible grafts. Methods: The study accrued 90 (66 marrow; 22 UCB, 2 peripheral blood) evaluable patients between January 2008 and May 2014. Patients 30 years old with high risk AML at presentation or relapsed AML were eligible. After enrollment, in collaboration with the National Marrow Donor Program, as many as 5 potential unrelated donor samples were KIR typed (including gene expression) in a central laboratory (Wing Leung, St Jude ChildrenÕs Hospital) and results reported to the treating physician, who made the final donor selection using their own best clinical judgement. Transplant was performed using standard conditioning and GVHD prophylaxis, including in vivo T-cell depletion with ATG. Results: Cases were categorized as KIR matched or mismatched using different strategies, published in prior analyses. Impact of mismatch on disease-free survival (DFS) is summarized in Table 1.Table 1DFS at 2 Years (2 SEs) in Those Receiving KIR Matched and Mismatched Unrelated Donor HSCT Defined by Different ModelsModelDFS: Marrow TransplantsDFS: UCB TransplantsDFS: All PatientsDFS Match Versus MismatchP-ValueKIR MatchKIR MismatchKIR MatchKIR MismatchKIR MatchKIR MismatchReceptor ligand match40 ± 22%43 ± 16%40 ± 44%89 ± 21%40 ± 20%50 ± 15%.687Ligand/ligand mismatch37 ± 15%0 ± 0%57 ± 37%100 ± 0%38 ± 14%67 ± 39%.183Missing ligand43 ± 22%31 ± 15%29 ± 34%77 ± 23%39 ± 18%45 ± 13%.981HaplotypeA/B + B/BA/AA/B + B/BA/AA/B + B/BA/AA/B + B/B versus A/AP-valueKIR haplotype (centromeric)51 ± 19%33 ± 17%38 ± 34%88 ± 23%48 ± 17%43 ± 16%.529KIR haplotype (telomeric)41 ± 23%42 ± 16%56 ± 33%71 ± 34%46 ± 19%45 ± 15%.600 Open table in a new tab Overall survival and acute GVHD also did not differ significantly by KIR mismatch. We also examined DFS according to time to first NK receptor recovery after HSCT. NKp44 recovery was significantly associated with decreased DFS in a univariate Cox analysis (P = .001). Conclusions: Prospective selection of unrelated donors according to KIR type was feasible. DFS and GVHD did not differ in recipients of matched and mismatched bone marrow grafts using any model of KIR mismatch. Cord blood recipients had superior DFS with KIR mismatch, although sample size was too small for statistical significance. Our data suggest that recovery of NK receptor expression might influence DFS after HSCT.

The effect of whole body gamma irradiation (WBI) in single fraction was studied, as well as its influence on the secretion of various biochemical markers and cellular component that could be used as acute radiation lung injury marker. Sprague dawley rats were treated with WBI (60Co) of radiation dose from 1 Gy to 5 Gy (dose rate - 0.95 Gy/min). Bronchoalveolar lavage fluid was retrieved from all animals in control and radiation treated groups up to 72 h post radiation. Bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH ), acid phosphatase (AP ), alkaline phosphatase (ALP ), cell count and total protein. Intragroup and intergroup comparison of BALF parameters at different radiation doses showed significant difference. LDH was significantly increased as the dose increased from 1Gy to 5Gy (P = 0.00) after 2 h with effect size of difference (r > 0.3). ALP was significantly altered after 3Gy and 4Gy (P < 0.05). AP was significantly altered at 2Gy-5Gy (p < 0.05). Total protein level changed significantly from 1Gy to 5Gy (P < 0.00). Cellular content of BALF showed significant changes after radiation exposure. BALF parameters like LDH, AP, ALP, neutrophils, lymphocytes, total leukocyte count and total protein were sensitive to radiation exposure and their levels vary significantly up to 72 h after single whole body radiation exposure in Sprague dawley rats. It can be concluded that the biochemical indices in BALF have more wide application in evaluation of acute radiation induced lung injury.