In vivo measurement of RBC survival in patients with sickle cell disease before or after hematopoietic stem cell transplantation.

ABSTRACT: Stable, mixed-donor-recipient chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with sickle cell disease (SCD) is sufficient for phenotypic disease reversal, and results from differences in donor/recipient-red blood cell (RBC) survival. Understanding variability and predictors of RBC survival among patients with SCD before and after HSCT is critical for gene therapy research which seeks to generate sufficient corrected hemoglobin to reduce polymerization thereby overcoming the red cell pathology of SCD. This study used biotin labeling of RBCs to determine the lifespan of RBCs in patients with SCD compared with patients who have successfully undergone curative HSCT, participants with sickle cell trait (HbAS), and healthy (HbAA) donors. Twenty participants were included in the analysis (SCD pre-HSCT: N = 6, SCD post-HSCT: N = 5, HbAS: N = 6, and HbAA: N = 3). The average RBC lifespan was significantly shorter for participants with SCD pre-HSCT (64.1 days; range, 35-91) compared with those with SCD post-HSCT (113.4 days; range, 105-119), HbAS (126.0 days; range, 119-147), and HbAA (123.7 days; range, 91-147) (P<.001). RBC lifespan correlated with various hematologic parameters and strongly correlated with the average final fraction of sickled RBCs after deoxygenation (P<.001). No adverse events were attributable to the use of biotin and related procedures. Biotin labeling of RBCs is a safe and feasible methodology to evaluate RBC survival in patients with SCD before and after HSCT. Understanding differences in RBC survival may ultimately guide gene therapy protocols to determine hemoglobin composition required to reverse the SCD phenotype as it relates directly to RBC survival. This trial was registered at www.clinicaltrials.gov as #NCT04476277.

Thyroid and Adrenal Dysfunction in Hemoglobinopathies Before and After Allogeneic Hematopoietic Cell Transplant.

Purpose: To determine the rate and clinical characteristics associated with abnormal thyroid and adrenal function in recipients of nonmyeloablative hematopoietic cell transplantation (HCT) for sickle cell disease (SCD) and beta-thalassemia. Methods: We retrospectively reviewed patients who enrolled in 4 nonmyeloablative HCT regimens with alemtuzumab and total body irradiation (TBI). Baseline and annual post-HCT data were compared, which included age, sex, sickle phenotype, thyroid panel (total T3, free T4, thyroid stimulating hormone, antithyroid antibodies), cortisol level, ACTH stimulation testing, ferritin, medications, and other relevant medical history. Results: Among 43 patients in haploidentical transplant and 84 patients in the matched related donor protocols with mostly SCD, the rate of any thyroid disorder pre-HCT was 3.1% (all subclinical hypothyroidism) and post-HCT was 29% (10 hypothyroidism, 4 Grave's disease, and 22 subclinical hypothyroidism). Ninety-two (72%) patients had ferritin >1000 ng/dL, of which 33 patients (35.8%) had thyroid dysfunction. Iron overload was noted in 6 of 10 patients with hypothyroidism and 12 of 22 patients with subclinical hypothyroidism.Sixty-one percent were on narcotics for pain control. With respect to adrenal insufficiency (AI) pre-HCT, 2 patients were maintained on corticosteroids for underlying rheumatologic disorder and 8 had AI diagnosed during pre-HCT ACTH stimulation testing (total 10, 7.9%). Post-HCT, an additional 4 (3%) developed AI from corticosteroid use for acute graft vs host disease, Evans syndrome, or hemolytic anemia. Conclusion: Although iron overload was common in SCD, thyroid dysfunction pre-HCT related to excess iron was less common. Exposure to alemtuzumab or TBI increased the rates of thyroid dysfunction post-HCT. In contrast, AI was more common pre-HCT, but no risk factor was identified. AI post-HCT was infrequent and associated with corticosteroid use for HCT-related complications.

Lovo-cel gene therapy for sickle cell disease: Treatment process evolution and outcomes in the initial groups of the HGB-206 study.

lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) comprises autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified ß-globin gene (ßA-T87Q ) to produce anti-sickling hemoglobin (HbAT87Q ). The efficacy and safety of lovo-cel for SCD are being evaluated in the ongoing phase 1/2 HGB-206 study (ClinicalTrials.gov: NCT02140554). The treatment process evolved over time, using learnings from outcomes in the initial patients to optimize lovo-cel's benefit-risk profile. Following modest expression of HbAT87Q in the initial patients (Group A, n = 7), alterations were made to the treatment process for patients subsequently enrolled in Group B (n = 2, patients B1 and B2), including improvements to cell collection and lovo-cel manufacturing. After 6 months, median Group A peripheral blood vector copy number (≥0.08 c/dg) and HbAT87Q levels (≥0.46 g/dL) were inadequate for substantial clinical effect but stable and sustained over 5.5 years; both markedly improved in Group B (patient B1: ≥0.53 c/dg and ≥2.69 g/dL; patient B2: ≥2.14 c/dg and ≥6.40 g/dL, respectively) and generated improved biologic and clinical efficacy in Group B, including higher total hemoglobin and decreased hemolysis. The safety of the lovo-cel for SCD treatment regimen largely reflected the known side effects of HSPC collection, busulfan conditioning regimen, and underlying SCD; acute myeloid leukemia was observed in two patients in Group A and deemed unlikely related to insertional oncogenesis. Changes made during development of the lovo-cel treatment process were associated with improved outcomes and provide lessons for future SCD GT studies.

Phenotypic screening of the ReFRAME drug repurposing library to discover new drugs for treating sickle cell disease.

Stem cell transplantation and genetic therapies offer potential cures for patients with sickle cell disease (SCD), but these options require advanced medical facilities and are expensive. Consequently, these treatments will not be available for many years to the majority of patients suffering from this disease. What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here, we report the results of the first phase of our phenotypic screen of the 12,657 compounds of the Scripps ReFRAME drug repurposing library using a recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from sickle trait individuals. The ReFRAME library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials. From dose-response measurements, 106 of the 12,657 compounds exhibit statistically significant antisickling at concentrations ranging from 31 nM to 10 µM. Compounds that inhibit sickling of trait cells are also effective with SCD cells. As many as 21 of the 106 antisickling compounds emerge as potential drugs. This estimate is based on a comparison of inhibitory concentrations with free concentrations of oral drugs in human serum. Moreover, the expected therapeutic potential for each level of inhibition can be predicted from measurements of sickling times for cells from individuals with sickle syndromes of varying severity. Our results should motivate others to develop one or more of these 106 compounds into drugs for treating SCD.

Sickle Cell Transplantation Evaluation of Long-term and Late Effects Registry (STELLAR) to Compare Long-term Outcomes After Hematopoietic Cell Transplantation to Those in Siblings Without Sickle Cell Disease and in Nontransplanted Individuals With Sickle Cell Disease: Design and Feasibility Study.

BACKGROUND: There are sparse data on the long-term and late effects of hematopoietic cell transplantation (HCT) for sickle cell disease (SCD). OBJECTIVE: This study aims to establish an international registry of long-term outcomes post-HCT for SCD and demonstrate the feasibility of recruitment at a single site in the United States. METHODS: The Sickle Cell Transplantation Evaluation of Long-Term and Late Effects Registry (STELLAR) was designed to enroll patients with SCD ≥1 year post-HCT, their siblings without SCD, and nontransplanted controls with SCD to collect web-based participant self-reports of health status and practices by using the Bone Marrow Transplant Survivor Study (BMTSS) surveys, health-related quality of life (HRQOL) using the Patient-Reported Outcomes Measurement Information System (PROMIS) Pediatric Profile-25 or Pediatric Profile-29 survey, chronic graft-versus-host disease (cGVHD) using the symptom scale survey, daily pain using an electronic pain diary, the economic impact of HCT using the financial hardship survey, sexual function using the PROMIS Sexual Function SexFSv2.0 survey, and economic productivity using the American Time Use Survey (ATUS). We also piloted retrieval of clinical data previously submitted to the Center for International Blood and Marrow Transplant Research (CIBMTR); recorded demographics, height, weight, blood pressure, waist and hip circumferences, timed up and go (TUG) test, and handgrip test; and obtained blood for metabolic screening, gonadal function, fertility potential, and biorepository of plasma, serum, RNA, and DNA. RESULTS: Of 100 eligible post-HCT patients, we enrolled 72 (72%) participants aged 9-38 (median 17) years. We also enrolled 19 siblings aged 5-32 (median 10) years and 28 nontransplanted controls with SCD aged 4-46 (median 22) years. Of the total 119 participants, 73 (61%) completed 85 sets of surveys and 41 (35%) contributed samples to the biorepository. We completed ATUS interviews of 28 (24%) participants. We successfully piloted retrieval of data submitted to the CIBMTR and expanded recruitment to multiple sites in the United States, Canada, the United Kingdom, and Nigeria. CONCLUSIONS: It is feasible to recruit subjects and conduct study procedures for STELLAR in order to determine the long-term and late effects of HCT for SCD. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/36780.

Evolution of Gene Therapy, Historical Perspective.

The earliest conceptual history of gene therapy began with the recognition of DNA as the transforming substance capable of changing the phenotypic character of a bacterium and then as the carrier of the genomic code. Early studies of oncogenic viruses that could insert into the mammalian genome led to the concept that these same viruses might be engineered to carry new genetic material into mammalian cells, including human hematopoietic stem cells (HSC). In addition to properly engineered vectors capable of efficient safe transduction of HSC, successful gene therapy required the development of efficient materials, methods, and equipment to procure, purify, and culture HSC. Increased understanding of the preparative conditioning of patients was needed to optimize the engraftment of genetically modified HSC. Testing concepts in pivotal clinical trials to assess the efficacy and determine the cause of adverse events has advanced the efficiency and safety of gene therapy. This article is a historical overview of the separate threads of discovery that joined together to comprise our current state of gene therapy targeting HSC.

Optimizing haematopoietic stem and progenitor cell apheresis collection from plerixafor-mobilized patients with sickle cell disease.

We adjusted haematopoietic stem and progenitor cell (HSPC) apheresis collection from patients with sickle cell disease (SCD) by targeting deep buffy coat collection using medium or low collection preference (CP), and by increasing anticoagulant-citrate-dextrose-solution A dosage. In 43 HSPC collections from plerixafor-mobilized adult patients with SCD, we increased the collection efficiency to 35.79% using medium CP and 82.23% using low CP. Deep buffy coat collection increased red blood cell contamination of the HSPC product, the product haematocrit was 4.7% with medium CP and 6.4% with low CP. These adjustments were well-tolerated and allowed efficient HSPC collection from SCD patients.

Fertility after Curative Therapy for Sickle Cell Disease: A Comprehensive Review to Guide Care.

Curative therapy for sickle cell disease (SCD) currently requires gonadotoxic conditioning that can impair future fertility. Fertility outcomes after curative therapy are likely affected by pre-transplant ovarian reserve or semen analysis parameters that may already be abnormal from SCD-related damage or hydroxyurea treatment. Outcomes are also likely affected by the conditioning regimen. Conditioning with myeloablative busulfan and cyclophosphamide causes serious gonadotoxicity particularly among post-pubertal females. Reduced-intensity and non-myeloablative conditioning may be acutely less gonadotoxic, but more short and long-term fertility outcome data after these approaches is needed. Fertility preservation including oocyte/embryo, ovarian tissue, sperm, and experimental testicular tissue cryopreservation should be offered to patients with SCD pursing curative therapy. Regardless of HSCT outcome, longitudinal post-HSCT fertility care is required.

Alemtuzumab clearance, lymphocyte count, and T-cell chimerism after hematopoietic stem cell transplant in sickle cell disease.

STUDY OBJECTIVE: Alemtuzumab is a monoclonal antibody that targets the cell surface antigen CD52 on lymphocytes. Although it is used for the treatment of hematologic malignancies, such as chronic lymphocytic leukemia, and incorporated into many hematopoietic stem cell transplant (HSCT) conditioning regimens, few studies have evaluated the pharmacology of alemtuzumab in adult patients with sickle cell disease (SCD). We therefore examined the pharmacokinetics (PK) and pharmacodynamics (PD) of alemtuzumab in adults with SCD who received a matched related donor HSCT to determine if the clearance of alemtuzumab affects transplant outcomes. DESIGN: PK and PD analysis of patient data from a single-center clinical trial. SETTING: Clinical research center. PATIENTS: Twenty-two adult patients with SCD who received one of two nonmyeloablative allogeneic HSCT regimens: alemtuzumab and total body irradiation (Alem-TBI) or pentostatin, cyclophosphamide, alemtuzumab, and total body irradiation (Pento-Cy-Alem-TBI). MEASUREMENTS AND MAIN RESULTS: Alemtuzumab serum concentrations, absolute lymphocyte counts, T-cell (CD3), and myeloid (CD14/15) chimerism were collected at distinct time points and analyzed. A semi-mechanistic PK population model was built to understand inter-individual differences in pharmacology. Alemtuzumab was detectable up to 28 days post-HSCT. The mean alemtuzumab level 7 days after transplant for patients on Alem-TBI was 818 ng/ml, significantly lower than the mean level of 1502 ng/ml for patients on Pento-Cy-Alem-TBI (p < 0.001), but this difference decreased as time progressed. The clearance of alemtuzumab was linear, and the half-life was longer in the Pento-Cy-Alem-TBI group (average half-life = 61.1 h) compared to the Alem-TBI group (average half-life = 44.1 h) (p < 0.001). The CD3 chimerism at 2 and 4 months after transplant positively correlated with alemtuzumab levels collected on day 14 after transplant (R2  = 0.40 and p = 0.004 at 2 months, R2  = 0.36 and p = 0.005 at 4 months), but this significance was lost by 6 months after HSCT. No correlation was seen between alemtuzumab levels and CD14/15 chimerism. CONCLUSION: Between 2 and 4 months after transplant, higher alemtuzumab levels measured 14 days after transplant correlated with patients having better engraftment, suggesting more lymphodepletion may be needed to reduce graft failure in these two non-myeloablative matched related donor HSCT regimens.

Increased Rates of Rhabdomyolysis in Male Hematopoietic Cell Transplantation Recipients Taking Sirolimus and Trimethoprim/Sulfamethoxazole.

Hematopoietic cell transplantation (HCT) offers long-term cure against early morbidity and mortality of hemoglobinopathies, such as sickle cell disease (SCD) and beta-thalassemia. Following HCT, sirolimus is an immunosuppressant used to prevent graft-versus-host disease (GVHD) while receiving trimethoprim-sulfamethoxazole (TMP-SMX) for Pneumocystis jirovecii prophylaxis and other antimicrobial agents (including acyclovir). One rare adverse event associated with both drugs is rhabdomyolysis, defined as creatine kinase (CK) elevation at least 5 to 10 times the upper limit of normal. This study was conducted to evaluate the rate of and risk factors for developing rhabdomyolysis in the post-HCT setting. Across 4 haploidentical and matched related donor (MRD) nonmyeloablative protocols, CK levels were prospectively monitored and patients were retrospectively identified for rhabdomyolysis. The rhabdomyolysis was graded based on the severity of CK elevation and other organ injury. At diagnosis, patients were queried for concurrent medication use (ie, sirolimus, TMP-SMX, acyclovir, or statins), sex, age, donor genotype, and time from transplantation. Among 127 patients with mostly SCD, rhabdomyolysis occurred in 22 (17%), including 2 recipients of haploidentical donor HCT and 20 recipients of MRD HCT. The time to the development of rhabdomyolysis was 61 and 73 days for the 2 recipients of haploidentical HCT and a median of 73 days for the MRD HCT recipients. Among the 22 patients who developed rhabdomyolysis, 20 (91%) were receiving sirolimus (2 haploidentical HCT recipients and 18 MRD HCT recipients), and 14 (64%) were also receiving TMP-SMX (all in the MRD HCT group). Seventy-five percent of the haploidentical donors and 69% of the MRDs had sickle cell trait. All but 2 patients with rhabdomyolysis were male. No patients who developed rhabdomyolysis were receiving statins at any point. Higher-than-expected rates of rhabdomyolysis were found post-transplantation for patients with SCD and beta-thalassemia. Contributing risk factors included immunosuppression with sirolimus, TMP-SMX, male sex, and sickle trait donor. These factors differ from the excessive muscle strain or injury, seizures, infections, or HMG-CoA inhibitors typically identified in non-HCT recipients.

Disease severity impacts plerixafor-mobilized stem cell collection in patients with sickle cell disease.

Recent studies suggest that plerixafor mobilization and apheresis in patients with sickle cell disease (SCD) is safe and can allow collection of sufficient CD34+ hematopoietic stem cell (HSC) collection for clinical gene therapy applications. However, the quantities of plerixafor-mobilized CD34+ cells vary between different SCD patients for unknown reasons. Twenty-three participants with SCD underwent plerixafor mobilization followed by apheresis, processing, and HSC enrichment under a phase 1 safety and efficacy study conducted at 2 institutions. Linear regression or Spearman's correlation test was used to assess the relationships between various hematologic and clinical parameters with total CD34+ cells/kg collected. Median CD34+ cells/kg after 2 or fewer mobilization and apheresis cycles was 4.0 × 106 (range, 1.5-12.0). Similar to what is observed generally, CD34+ yield correlated negatively with age (P < .001) and positively with baseline (P = .003) and preapheresis blood CD34+ cells/µL (P < .001), and baseline white blood cell (P = .01) and platelet counts (P = .03). Uniquely for SCD, CD34+ cell yields correlated positively with the number of days hydroxyurea was held (for up to 5 weeks, P = .01) and negatively with markers of disease severity, including hospitalization frequency within the preceding year (P = .01) and the number of medications taken for chronic pain (P = .002). Unique SCD-specific technical challenges in apheresis were also associated with reduced CD34+ cell collection efficiency and purification. Here, we describe factors that impact plerixafor mobilization success in patients with SCD, confirming known factors as described in other populations in addition to reporting previously unknown disease specific factors in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT03226691.

Non-myeloablative human leukocyte antigen-matched related donor transplantation in sickle cell disease: outcomes from three independent centres.

Non-myeloablative haematopoietic progenitor cell transplantation (HPCT) from matched related donors (MRD) has been increasingly utilized in sickle cell disease (SCD). A total of 122 patients received 300 cGy of total body irradiation (TBI), alemtuzumab, unmanipulated filgrastim-mobilized peripheral blood HPC and sirolimus. The median follow-up was four years; median age at HPCT was 29 years. Median neutrophil and platelet engraftment occurred on day 22 and 19 respectively; 41 patients required no platelet transfusions. Overall and sickle-free survival at one and five years were 93% and 85% respectively. Age, sex, pre-HPCT sickle complications, ferritin and infused HPC numbers were similar between graft failure and engrafted patients. Mean donor myeloid chimaerism at one and five years post HPCT were 84% and 88%, and CD3 was 48% and 53% respectively. Two patients developed grade 1 and 2 skin graft-versus-host disease (GVHD) with no chronic GVHD. Median days of recipients taking immunosuppression were 489; 83% of engrafted patients have discontinued immunosuppression. Haemoglobin, haemolytic parameters and hepatic iron levels improved post HPCT. Pulmonary function testing, hepatic histology and neurovascular imaging remained stable, suggesting cessation of further sickle-related injury. Fourteen patients had children. In this largest group of adult SCD patients, this regimen was highly efficacious, well-tolerated despite compromised organ functions pre HPCT, and without clinically significant GVHD.

Computer Algorithm-Based Hydroxyurea Dosing Facilitates Titration to Maximum Tolerated Dose in Sickle Cell Anemia.

Adults with sickle cell disease (SCD) experience acute and chronic complications and die prematurely. When taken at maximum tolerated dose (MTD), hydroxyurea prolongs survival; however, it has not consistently reversed organ dysfunction. Patients also frequently do not take hydroxyurea, at least in part because of physician discomfort with prescribing hydroxyurea. We sought to develop a computer program that could easily titrate hydroxyurea to MTD. This was a single-arm, open-label pilot study. Fifteen patients with homozygous SCD were enrolled in the protocol, and 10 patients were followed at baseline and then for 1 year after hydroxyurea initiation or dose titration. Fetal hemoglobin significantly increased in all 10 patients from 8.3% to 25.1% (P < .001). Nine patients were titrated to MTD in an average of 7.9 months, and the tenth patient's hydroxyurea dose was increased to 33 mg/kg/day. Computer program dosing recommendations were the same as manual dosing decisions made using the same algorithm for all patients and at all times. We also evaluated markers of cardiopulmonary, liver and renal damage. Although cardiopulmonary function did not significantly improve, direct bilirubin and alanine aminotransferase levels significantly decreased (P < .001 and P < .01, respectively). Last, although kidney function did not improve, degree of proteinuria was significantly reduced (P < .05). We have developed a computer program that reliably titrates hydroxyurea to MTD. A larger study is indicated to test the program either as a computer program or a downloadable application.

The impact of pre-existing HLA and red blood cell antibodies on transfusion support and engraftment in sickle cell disease after nonmyeloablative hematopoietic stem cell transplantation from HLA-matched sibling donors: A prospective, single-center, observational study.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is curative for patients with sickle cell disease (SCD). Prior to HSCT, patients with SCD commonly receive RBC transfusions with some becoming RBC or HLA alloimmunized. This alloimmunization may impact post-HSCT transfusion requirements and donor engraftment. METHODS: The study population included patients with SCD transplanted on a single-center nonmyeloablative, HLA-matched sibling HSCT trial at the National Heart, Lung, and Blood Institute (NHLBI) who had a pre-HSCT sample available for HLA class I antibody testing. We evaluated transfusion requirements and engraftment outcomes comparing patients with and without pre-existing HLA and RBC antibodies. FINDINGS: Of 36 patients studied, 10 (28%) had HLA class I antibodies and 11 (31%) had a history of RBC alloantibodies. Up to day +45 post-HSCT, patients with HLA antibodies received more platelet transfusions (median 2.5 vs 1, p = 0.042) and those with RBC alloantibodies received more RBC units (median 7 vs 4, p = 0.0059) compared to respective non-alloimmunized patients. HLA alloimmunization was not associated with neutrophil engraftment, donor chimerism, or graft rejection. However, RBC alloimmunization correlated with a decreased donor T cell chimerism at 1 year (median 24% vs 55%, p = 0.035). INTERPRETATION: Pre-existing HLA and RBC alloantibodies are clinically significant for patients undergoing HLA-matched nonmyeloablative HSCT. Testing for both HLA and RBC antibodies is important to help estimate transfusion needs peri­HSCT. The association of lower donor T cell chimerism and pre-existing RBC alloantibodies needs further investigation. FUNDING: NIH Clinical Center and NHLBI Intramural Research Program (Z99 CL999999, HL006007-11) and the Thrasher Research Fund.