Induction of therapeutic levels of HbF in genome-edited primary ß0 39-thalassaemia haematopoietic stem and progenitor cells.

Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of ß-thalassaemia. The homozygous mutation c.-196 C>T in the Aγ-globin (HBG1) promoter, which causes Sardinian δß0 -thalassaemia, is able to completely rescue the ß-major thalassaemia phenotype caused by the ß0 39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from ß0 -thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the -196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous ß0 39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the -196 position has the potential to induce therapeutic levels of HbF in patients with most types of ß-thalassaemia irrespective of the ß-globin gene (HBB) mutations.

A New Intronic Variant in ECEL1 in Two Patients with Distal Arthrogryposis Type 5D.

Distal Arthrogryposis type 5D (DA5D) is characterized by congenital contractures involving the distal joints, short stature, scoliosis, ptosis, astigmatism, and dysmorphic features. It is inherited in an autosomal recessive manner, and it is a result of homozygous or compound heterozygous variants in the ECEL1 gene. Here, we report two patients of Sardinian origin harboring a new intronic homozygous variant in ECEL1 (c.1507-9G>A), which was predicted to affect mRNA splicing by activating a cryptic acceptor site. The frequency of the variant is very low in the general human population, and its presence in our families can be attributed to a founder effect. This study provides an updated review of the known causative mutations of the ECEL1 gene, enriching the allelic spectrum to include the noncoding sequence.

Implications of disease-modifying therapies for multiple sclerosis on immune cells and response to COVID-19 vaccination.

Introduction: Disease-modifying therapies (DMTs) have been shown to improve disease outcomes in multiple sclerosis (MS) patients. They may also impair the immune response to vaccines, including the SARS-CoV-2 vaccine. However, available data on both the intrinsic immune effects of DMTs and their influence on cellular response to the SARS-CoV-2 vaccine are still incomplete. Methods: Here, we evaluated the immune cell effects of 3 DMTs on the response to mRNA SARS-CoV-2 vaccination by comparing MS patients treated with one specific therapy (fingolimod, dimethyl fumarate, or natalizumab) with both healthy controls and untreated patients. We profiled 23 B-cell traits, 57 T-cell traits, and 10 cytokines, both at basal level and after stimulation with a pool of SARS-CoV-2 spike peptides, in 79 MS patients, treated with DMTs or untreated, and 32 healthy controls. Measurements were made before vaccination and at three time points after immunization. Results and Discussion: MS patients treated with fingolimod showed the strongest immune cell dysregulation characterized by a reduction in all measured lymphocyte cell classes; the patients also had increased immune cell activation at baseline, accompanied by reduced specific immune cell response to the SARS-CoV-2 vaccine. Also, anti-spike specific B cells progressively increased over the three time points after vaccination, even when antibodies measured from the same samples instead showed a decline. Our findings demonstrate that repeated booster vaccinations in MS patients are crucial to overcoming the immune cell impairment caused by DMTs and achieving an immune response to the SARS-CoV-2 vaccine comparable to that of healthy controls.

miR-365-3p mediates BCL11A and SOX6 erythroid-specific coregulation: A new player in HbF activation.

Hemoglobin switching is a complex biological process not yet fully elucidated. The mechanism regulating the suppression of fetal hemoglobin (HbF) expression is of particular interest because of the positive impact of HbF on the course of diseases such as ß-thalassemia and sickle cell disease, hereditary hemoglobin disorders that affect the health of countless individuals worldwide. Several transcription factors have been implicated in the control of HbF, of which BCL11A has emerged as a major player in HbF silencing. SOX6 has also been implicated in silencing HbF and is critical to the silencing of the mouse embryonic hemoglobins. BCL11A and SOX6 are co-expressed and physically interact in the erythroid compartment during differentiation. In this study, we observe that BCL11A knockout leads to post-transcriptional downregulation of SOX6 through activation of microRNA (miR)-365-3p. Downregulating SOX6 by transient ectopic expression of miR-365-3p or gene editing activates embryonic and fetal ß-like globin gene expression in erythroid cells. The synchronized expression of BCL11A and SOX6 is crucial for hemoglobin switching. In this study, we identified a BCL11A/miR-365-3p/SOX6 evolutionarily conserved pathway, providing insights into the regulation of the embryonic and fetal globin genes suggesting new targets for treating ß-hemoglobinopathies.

Cross-sectional analysis of the humoral response after SARS-CoV-2 vaccination in Sardinian multiple sclerosis patients, a follow-up study.

Monitoring immune responses to SARS-CoV-2 vaccination and its clinical efficacy over time in Multiple Sclerosis (MS) patients treated with disease-modifying therapies (DMTs) help to establish the optimal strategies to ensure adequate COVID-19 protection without compromising disease control offered by DMTs. Following our previous observations on the humoral response one month after two doses of BNT162b2 vaccine (T1) in MS patients differently treated, here we present a cross-sectional and longitudinal follow-up analysis six months following vaccination (T2, n=662) and one month following the first booster (T3, n=185). Consistent with results at T1, humoral responses were decreased in MS patients treated with fingolimod and anti-CD20 therapies compared with untreated patients also at the time points considered here (T2 and T3). Interestingly, a strong upregulation one month after the booster was observed in patients under every DMTs analyzed, including those treated with fingolimod and anti-CD20 therapies. Although patients taking these latter therapies had a higher rate of COVID-19 infection five months after the first booster, only mild symptoms that did not require hospitalization were reported for all the DMTs analyzed here. Based on these findings we anticipate that additional vaccine booster shots will likely further improve immune responses and COVID-19 protection in MS patients treated with any DMT.

Lentiviral globin gene therapy with reduced-intensity conditioning in adults with ß-thalassemia: a phase 1 trial.

ß-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the ß chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent ß-thalassemia who were infused with autologous CD34+ cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34+ cells, expression of the transduced ß-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34+ cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.

Effect of Different Disease-Modifying Therapies on Humoral Response to BNT162b2 Vaccine in Sardinian Multiple Sclerosis Patients.

Objectives: Vaccination against COVID-19 is highly recommended to patients affected by multiple sclerosis (MS); however, the impact of MS disease-modifying therapies (DMTs) on the immune response following vaccination has been only partially investigated. Here, we aimed to elucidate the effect of DMTs on the humoral immune response to mRNA-based anti-SARS-CoV-2 vaccines in MS patients. Methods: We obtained sera from 912 Sardinian MS patients and 63 healthy controls 30 days after the second dose of BNT162b2 vaccine and tested them for SARS-CoV-2 response using anti-Spike (S) protein-based serology. Previous SARS-CoV-2 infection was assessed by anti-Nucleocapsid (N) serology. Patients were either untreated or undergoing treatment with a total of 13 different DMTs. Differences between treatment groups comprised of at least 10 patients were assessed by generalized linear mixed-effects model. Demographic and clinical data and smoking status were analyzed as additional factors potentially influencing humoral immunity from COVID-19 vaccine. Results: MS patients treated with natalizumab, teriflunomide, azathioprine, fingolimod, ocrelizumab, and rituximab showed significantly lower humoral responses compared to untreated patients. We did not observe a statistically significant difference in response between patients treated with the other drugs (dimethyl fumarate, interferon, alemtuzumab and glatiramer acetate) and untreated patients. In addition, older age, male sex and active smoking were significantly associated with lower antibody titers against SARS-CoV-2. MS patients previously infected with SARS-CoV-2 had significantly higher humoral responses to vaccine than uninfected patients. Conclusion: Humoral response to BNT162b2 is significantly influenced by the specific DMTs followed by patients, as well as by other factors such as previous SARS-CoV-2 infection, age, sex, and smoking status. These results are important to inform targeted strategies to prevent clinically relevant COVID-19 in MS patients.