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.

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.

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.

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.

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.

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.

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.

Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease.

BACKGROUND AIMS: Allogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear. METHODS: In this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients. RESULTS: In co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism. CONCLUSIONS: In summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.

Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and ß-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.

Development of a forward-oriented therapeutic lentiviral vector for hemoglobin disorders.

Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level ß-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented ß-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and ß-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders.

Busulfan Combined with Immunosuppression Allows Efficient Engraftment of Gene-Modified Cells in a Rhesus Macaque Model.

Busulfan conditioning is utilized for hematopoietic stem cell (HSC) depletion in the context of HSC gene-therapy conditioning but may result in insufficient immunosuppression. In this study, we evaluated whether additional immunosuppression is required for efficient engraftment of gene-modified cells using a rhesus HSC lentiviral gene-therapy model. We transduced half of rhesus CD34+ cells with an enhanced green fluorescent protein (GFP)-encoding vector (immunogenic) and the other half with a γ-globin-encoding vector (no predicted immunogenicity). After autologous transplantation of both transduced cell populations following myeloablative busulfan conditioning (5.5 mg/kg/day for 4 days), we observed immunological rejection of GFP-transduced cells up to 3 months post-transplant and stable engraftment of γ-globin-transduced cells in two animals, demonstrating that ablative busulfan conditioning is sufficient for engraftment of gene-modified cells producing non-immunogenic proteins but insufficient to permit engraftment of immunogenic proteins. We then added immunosuppression with abatacept and sirolimus to busulfan conditioning and observed engraftment of both GFP- and γ-globin-transduced cells in two animals, demonstrating that additional immunosuppression allows for engraftment of gene-modified cells expressing immunogenic proteins. In conclusion, myeloablative busulfan conditioning should permit engraftment of gene-modified cells producing non-immunogenic proteins, while additional immunosuppression is required to prevent immunological rejection of a neoantigen.

Vibration Controlled Transient Elastography (Fibroscan®) in sickle cell liver disease - could we strike while the liver is hard?

Vibration controlled transient elastography (VCTE) is validated for the evaluation of hepatic fibrosis in different liver diseases. Sickle cell liver disease (SCLD) results from a cumulative hepatic injury and its lifelong and progressive nature raises the need for a non-invasive tool for fibrosis evaluation. Fifty patients, aged between 23 and 59 years with sickle cell disease and suspected SCLD underwent a VCTE followed by a liver biopsy. Biopsies were evaluated for various scores of liver disease that were then correlated to VCTE score. 90% of our patients had an Ishak Fibrosis (IF) score between 0-2 (Group A-minimal to no fibrosis) and 10% of the patients had IF score between 3-6 (Group B-advanced fibrosis). The median Transient Elastography (TE) for patients in Groups A and B was 4·8 kilopascals (kPa) and 17·6 kPa, respectively. A positive correlation was shown between TE and IF score, R = 0·0·68 (P = <0·0001); a positive correlation was also shown with Histology Activity Index fibrosis score, R = 0·64 (P = <0·0001). This study emphasises the need for further studies of non-invasive tools and their utility in liver fibrosis evaluation of patients with SCLD.

Natural history of benign ethnic neutropenia in individuals of African ancestry.

BACKGROUND: Benign ethnic neutropenia (BEN), defined by neutrophil count <1.5 k/µL in the absence of other causes, is an asymptomatic condition more commonly observed in individuals of African ancestry. However, the natural history of this condition has been less well described. METHODS: Individuals with BEN were retrospectively identified by chart review or referral to hematology clinics. They were then invited to enroll in a prospective natural history study. Retrospective and prospective clinical and laboratory data were combined for descriptive analyses. FINDINGS: 46 participants, younger and older adults from 2 institutions, had BEN. Hypertension was reported in 30%, musculoskeletal disorders in 15%, and upper respiratory infection in 33% of these adults. Their leukopenia resulted from isolated neutropenia, ranging from 1000 and 1500 cells/µL. The severity of infections was mild and the frequency was similar to other healthy individuals in the ambulatory clinic. INTERPRETATION: In this group of BEN participants, their leukopenia was stable over time, and they had low rates of infections or common medical disorders, confirming the benign nature of this condition. The presence of BEN in children, younger adults, and older adults suggest a hereditary pattern for BEN.