Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models.

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

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.

Outcomes and long-term effects of hematopoietic stem cell transplant in sickle cell disease.

INTRODUCTION: Hematopoietic stem cell transplant (HSCT) is the only readily available curative option for sickle cell disease (SCD). Cure rates following human leukocyte antigen (HLA)-matched related donor HSCT with myeloablative or non-myeloablative conditioning are >90%. Alternative donor sources, including haploidentical donor and autologous with gene therapy, expand donor options but are limited by inferior outcomes, limited data, and/or shorter follow-up and therefore remain experimental. AREAS COVERED: Outcomes are improving with time, with donor type and conditioning regimens having the greatest impact on long-term complications. Patients with stable donor engraftment do not experience SCD-related symptoms and have stabilization or improvement of end-organ pathology; however, the long-term effects of curative strategies remain to be fully established and have significant implications in a patient's decision to seek therapy. This review covers currently published literature on HSCT outcomes, including organ-specific outcomes implicated in SCD, as well as long-term effects. EXPERT OPINION: HSCT, both allogeneic and autologous gene therapy, in the SCD population reverses the sickle phenotype, prevents further organ damage, can resolve prior organ dysfunction in both pediatric and adult patients. Data support greater success with HSCT at a younger age, thus, curative therapies should be discussed early in the patient's life.

Impact of Antibiotics on the Lung Microbiome and Lung Function in Children With Cystic Fibrosis 1 Year After Hospitalization for an Initial Pulmonary Exacerbation.

Background: Cystic fibrosis (CF) is characterized by recurrent pulmonary exacerbations (PEx) and lung function decline. PEx are frequently treated with antibiotics. However, little is known about the effects of antibiotics on the airway microbiome of persons with CF over time. The purpose of this study was to evaluate changes in the microbiome and lung function in persons with CF over 1 year following an initial study pulmonary exacerbation (iPEx). Methods: Twenty children aged ≤18 years with CF were enrolled in the study, which occurred prior to the routine administration of highly effective modulator therapy. Respiratory samples and spirometry were obtained at a minimum of quarterly visits and up to 1 year after an iPEx. Metagenomic sequencing was performed, and bacterial taxa were assigned using MetaPhlAn 2.0. Paired t test, analysis of variance, and generalized least squares regression were used to compare outcome variables. Results: The mean age of study participants at the time of the iPEx was 10.6 years. There were 3 ± 1.6 PEx treated with antibiotics per person during the study period. Bacterial richness was similar at 1 year compared to iPEx (40.3 vs 39.3, P = .852), whereas the mean Shannon diversity index was significantly higher at 1 year (2.84 vs 1.62, P < .001). The number of PEx treated with antibiotics was not associated with changes in microbial diversity but was associated with changes in lung function. Conclusions: In our 1-year prospective study, we found that microbial diversity increased despite decreases in lung function associated with repeated PEx events requiring antibiotic therapy.

Patient with Lynch syndrome with subsequent development of small bowel adenocarcinoma.

Small bowel adenocarcinoma (SBA) is a rare cancer in the general population, but the incidence increases in patients with Lynch syndrome. The present case describes a 57-year-old white woman with a history of colon cancer status posthemicolectomy and diagnosis of Lynch syndrome. Twenty years after her operation, the patient presented with vague abdominal discomfort and constipation, and underwent an exploratory laparotomy which revealed a stage 3A SBA. Genetic testing of the specimen provided evidence of microsatellite instability and faulty DNA repair supporting aetiology of Lynch syndrome. This case is unique in that SBA, if present in patients with Lynch syndrome, is usually a presenting symptom and has not been widely described in literature as an occurrence so many years after. As a result, this case highlights the importance of a low threshold for a thorough evaluation in patients with Lynch syndrome who present with signs of small bowel obstruction.

An 88-year-old woman with flushing, alopecia and hirsutism and a Sertoli-Leydig cell tumour.

Sertoli-Leydig cell tumour (SLCT) is a rare, androgen-secreting sex cord-stromal tumour of the ovary that usually occurs in young premenopausal women. The major clinical manifestations are virilisation and defeminisation. The following case describes an 88-year-old G1P1 woman, 40 years after menopause, who presented with flushing, hirsutism, voice changes and alopecia along with significantly elevated levels of testosterone. Postoperative report revealed a well-differentiated SLCT in the left ovary. This case is unique in that SLCT is a very rare cancer and even more so in an 88-year-old woman. Taking this case into consideration, it becomes reasonable to check androgen and oestrogen levels in postmenopausal women, not only in patients with signs of virilisation, but also in those with non-classical presentations, such as flushing or heat spells.