High fetal hemoglobin (HbF) levels are associated with decreased severity and mortality in sickle cell disease (SCD) and beta thalassemia (BT). We have developed a novel gene-edited cell therapy ...using autologous hematopoietic stem and progenitor cells (HSPCs) that have been genetically modified with zinc finger nucleases (ZFNs) to reactivate HbF expression. The ZFNs target the binding motif of GATA1 (GATAA) within an intronic erythroid-specific enhancer (ESE) of BCL11A, which encodes a major transcriptional repressor of HbF. Previously, we reported successful ZFN-mediated editing of the BCL11A ESE and reactivation of HbF in both dual (granulocyte colony-stimulating factor (G-CSF) and plerixafor) and single plerixafor mobilized HSPCs(Holmes 2017, Moran 2018). Both related drug candidates, ST-400 and BIVV003, are currently in phase 1/2a clinical trials for transfusion-dependent BT (NCT03432364) and SCD (NCT03653247), respectively. Here, we performed extensive genetic and phenotypic characterization of ZFN-edited HSPCs from healthy and SCD donors.
We performed single-cell characterization of BCL11A ESE-edited HSPCs from 4 healthy donors. Briefly, individual HSPCs were sorted and cultured in erythroid differentiation medium. Genomic DNA and protein lysate were collected at day 14 and 20, respectively. In total, we successfully genotyped 961 single-cell derived colonies by next-generation sequencing. The distribution was highly skewed towards biallelic-edited cells (P<3x10-149) representing 94% of edited clones, suggesting that ZFN-expressing cells are likely to become edited at both alleles. We found that each edited allele contributed additively to an increase in HbF% of 15% (P=1x10-80) as measured by UPLC. Clones harboring GATAA-disrupting indels on both alleles displayed on average 34% more HbF% than WT clones (P=1x10-112). In contrast, clones with biallelic indels that left the motif intact displayed a more modest increase (13%, P=1x10-6). Overall, our data revealed that >90% of edited cells were biallelic, displaying on average 27-38% more HbF% despite variation in donor baseline levels.
We observed a strong enrichment of biallelic-edited homozygotes (same indel pattern at both alleles) compared to an expected random distribution (161 vs 24; P<1x10-5). These clones may harbor larger deletions not captured by sequencing, as reported previously using CRISPR/Cas9 (Kosicki 2018). To address this question, we used a combination of a small amplicon sequencing assay design covering an informative SNP and a 12kb amplicon Nextera assay. We found that 27% of initially assigned homozygote clones were bona fide homozygotes (44/161) with the remaining harboring indels not originally captured. Nevertheless, most indels remained small, with 91% of indels <50bp, and deletions and insertions >1kb together consisting of less than 1% of alleles. The largest deletion was 4kb, but no indel extended outside the enhancer region of BCL11A or altered the coding region (>26 kb away). Moreover indels >50bp were not associated with enucleation levels (P=0.77), suggesting that they did not alter erythroid function. Overall, these results are consistent with previous data showing that ZFN-mediated gene editing does not impair HSPC function in vitro based on colony forming unit (CFU) production, and that injection of BIVV003 into immune-deficient NBSGW mice results in robust long-term engraftment with no impact on the number of HSPCs or their progeny, including erythrocytes.
Finally, BCL11A ESE editing in HSPCs mobilized from one SCD donor resulted in a 3-fold HbF increase consistent across technical duplicates, without impacting CFU production or erythroid enucleation. Importantly, clonal analysis revealed a similar enrichment of biallelic editing (P=6x10-4) and additive HbF up-regulation, with biallelic edited cells reaching 28% more HbF% than unedited cells (50% vs 22%, P=7x10-5). Furthermore, enucleated cells differentiated from edited HSPCs showed attenuation of sickling under hypoxic conditions supporting the potential efficacy of BIVV003. Experiments in HSPCs from additional SCD donors are ongoing.
Overall, our data have shown that ZFN-mediated disruption of BCL11A ESE results in enriched biallelic editing with on-target small indels, reactivates HbF and reduces sickling, supporting the potential efficacy and specificity of BIVV003 as a novel cell therapy for SCD.
Lessard:Sanofi: Employment. Rimmele:Sanofi: Employment. Ling:Sanofi: Employment. Moran:Sanofi: Employment. Vieira:Sanofi: Employment. Lin:Sanofi: Employment. Hong:Sanofi: Employment. Reik:Sangamo Therapeutics: Employment. Dang:Sangamo Therapeutics: Employment. Rendo:Sanofi: Employment. Daak:Sanofi: Employment. Hicks:Sanofi: Employment.
Background: Immune thrombocytopenia (ITP) is characterized by primarily autoantibody-mediated platelet destruction and impaired platelet production resulting in thrombocytopenia and an increased risk ...of bleeding. Other manifestations include increased risk of thrombosis and diminished quality of life. Current treatment approaches are directed toward lowering the rate of platelet destruction or stimulating platelet production to prevent bleeding. Rilzabrutinib is an oral, reversible, potent Bruton tyrosine kinase inhibitor that was specifically designed to treat immune-mediated diseases and mediates its therapeutic effect through a dual mechanism of action: (1) inhibiting B-cell activation and (2) interrupting antibody-coated cell phagocytosis by Fc gamma receptor in spleen and liver. A 24-week dose-finding phase I/II study of rilzabrutinib in patients with ITP showed a 40% platelet response (⩾2 consecutive platelet counts of ⩾50 × 10 9 /L and increase from baseline ⩾20 × 10 9 /L without rescue medication use) and a well-tolerated safety profile with only grade 1/2 transient adverse events across dose levels. Objectives: Assess the efficacy and safety of oral rilzabrutinib in adult and adolescent patients with persistent or chronic ITP. Design: Rilzabrutinib 400 mg BID is being evaluated in the ongoing LUNA 3 multicenter, double-blind, placebo-controlled phase III study. Methods and analysis: The primary endpoint is durable platelet response, defined as achieving platelet counts of ⩾50 × 10 9 /L for at least two-thirds of ⩾8 available weekly scheduled platelet measurements during the last 12 weeks (including ⩾2 available measurements within the last 6 weeks) of the 24-week blinded treatment period in the absence of rescue therapy. Ethics: Ethical guidelines and informed consent are followed. Discussion: The LUNA 3 trial will further investigate rilzabrutinib’s safety and efficacy in adult and adolescent patients, with the primary goal of addressing a major objective in treating patients with ITP: durability of platelet response. Trail Registration: ClinicalTrials.gov NCT04562766: https://clinicaltrials.gov/ct2/show/NCT04562766 ; EU Clinical Trials Register EudraCT 2020-002063-60: https://www.clinicaltrialsregister.eu/ctr-search/search?query=2020-002063-60 .
•With extended treatment, responses with rilzabrutinib were durable as a monotherapy and with the use of concomitant ITP medication.•Oral rilzabrutinib was well tolerated, with no treatment-related ...serious events or deaths.
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Immune thrombocytopenia (ITP) is an autoimmune disease associated with autoantibody-mediated platelet destruction and impaired platelet production, resulting in thrombocytopenia and a predisposition to bleeding. The ongoing, global phase 1/2 study showed that rilzabrutinib, a Bruton tyrosine kinase inhibitor specifically developed to treat autoimmune disorders, could be an efficacious and well-tolerated treatment for ITP. Clinical activity, durability of response, and safety were evaluated in 16 responding patients who continued rilzabrutinib 400 mg twice daily in the long-term extension (LTE) study. At LTE entry, the median platelet count was 87 × 109/L in all patients, 68 × 109/L in those who had rilzabrutinib monotherapy (n = 5), and 156 × 109/L in patients who received concomitant ITP medication (thrombopoietin-receptor agonists and/or corticosteroids, n = 11). At a median duration of treatment of 478 days (range, 303-764), 11 of 16 patients (69%) continued to receive rilzabrutinib. A platelet count of ≥50 × 109/L was reported in 93% of patients for more than half of their monthly visits. The median percentage of LTE weeks with platelet counts ≥30 × 109/L and ≥50 × 109/L was 100% and 88%, respectively. Five patients discontinued concomitant ITP therapy and maintained median platelet counts of 106 × 109/L at 3 to 6 months after stopping concomitant ITP therapy. Adverse events related to treatment were grade 1 or 2 and transient, with no bleeding, thrombotic, or serious adverse events. With continued rilzabrutinib treatment in the LTE, platelet responses were durable and stable over time with no new safety signals. This trial is registered at www.clinicaltrials.gov as #NCT03395210 and www.clinicaltrialsregister.eu as EudraCT 2017-004012-19.
The US Food and Drug Administration has approved several highly purified ω-3 fatty acid prescription drugs for the treatment of severe hypertriglyceridemia. These differ in the amounts and forms of ...docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). This study compared the bioavailability of SC401 (1530 mg EPA-ethyl esters EEs and DHA-EEs plus Advanced Lipid Technologies
ALT
, a proprietary lipid-delivery platform to improve absorption), with. Lovaza
(3600 mg ω-3, primarily EPA-EEs and DHA-EEs) under low-fat feeding conditions.
This was a Phase I, randomized, open-label, single-dose, 2-way crossover study in healthy participants housed from day -3 to day 2 in each treatment period. Blood samples for pharmacokinetic measurements were collected before and after dosing, and safety profile and tolerability were assessed.
In unadjusted analyses, SC401 had 5% lower C
and approximately the same AUC
of EPA + DHA total lipids compared with Lovaza. When adjusted for baseline, SC401 had ~6% higher C
and 18% higher AUC
for EPA + DHA total lipids, and dose- and baseline-adjusted analyses found that SC401 had ~149% higher C
and 178% higher AUC
than Lovaza for EPA + DHA total lipids. The T
was also substantially longer with Lovaza (~10 hours) than with SC401 (~6 hours).
These results indicate that SC401, an ω-3 acid EE formulation containing ALT
achieved high bioavailability of EPA and DHA, at a lower dose (1530 mg) than Lovaza (3600 mg), under low-fat feeding conditions.
The therapeutic effects of omega‐3 fatty acids on inflammatory disorders and cardiovascular diseases are well known. The current consensus considers sickle cell disease (SCD) as a chronic ...inflammatory state with significant membrane fatty acids perturbation. This new paradigm toward the pathophysiology of the disease paved the way for novel anti‐inflammatory and anti‐aggregatory therapeutic agents. The clinical trials which investigated the role of omega‐3 fatty acid in (SCD) showed good evidence that omega‐3 fatty acids are safe and effective treatment.
Blood cell aggregation and adherence to vascular endothelium and inflammation play a central role in vaso-occlusive crisis in sickle cell disease. The antiaggregatory, antiadhesive, antiinflammatory, ...and vasodilatory omega-3 (n−3) fatty acids (DHA and EPA) are significantly reduced in patients with the disease.
The aim was to investigate the therapeutic potential of omega-3 fatty acids for patients with homozygous sickle cell disease in a randomized, placebo-controlled, double-blind trial.
One hundred forty patients recruited from a single center in Sudan were randomly assigned and received, daily, 1 (age 2–4 y), 2 (age 5–10 y), 3 (age 11–16 y), or 4 (age ≥17 y) omega-3 capsules containing 277.8 mg DHA and 39.0 mg EPA or placebo for 1 y. Of these patients, 128 were followed up and the data were obtained. The primary and secondary endpoints—rates of clinical vaso-occlusive crisis and hemolytic events, blood transfusion rate, school attendance, and blood count—were analyzed by intention-to-treat analysis (n = 140).
Omega-3 treatment reduced the median rate of clinical vaso-occlusive events (0 compared with 1.0 per year, P < 0.0001), severe anemia (3.2% compared with 16.4%; P < 0.05), blood transfusion (4.5% compared with 16.4%; P < 0.05), white blood cell count (14.4 ± 3.3 compared with 15.6 ± 4.0 ×103/μL; P < 0.05), and the OR of the inability to attend school at least once during the study period because of illness related to the disease to 0.4 (95% CI: 0.2, 0.9; P < 0.05).
The findings of this trial, which need to be verified in a large multicenter study, suggest that omega-3 fatty acids can be an effective, safe, and affordable therapy for sickle cell anemia. This trial was registered with Current Controlled Trials as ISRCTN80844630.
Rapid modernization in Saudi Arabia has led to environmental challenges like pollution. Public understanding of pollutants is crucial for public participation in Saudi government efforts to monitor ...and mitigate impacts. This cross-sectional observational study aimed to assess the awareness and perceptions of environmental pollutants among 817 adults in Saudi Arabia’s Jazan region. The online survey identified transportation and industrial emissions as widely recognized hazards, but there were gaps regarding risks like asbestos. Illegal dumping and junk houses were major concerns. Females had 1.86 times higher adjusted odds of concern about outdoor environmental risks compared to males (AOR: 1.86; 95% CI: 1.12–2.84; p = 0.004). Participants with high school education or above had significantly increased odds of concern about outdoor hazards, with 4.27 times higher odds for those with high school education (AOR: 4.27; 95% CI: 1.92–9.52; p < 0.001) and 3.51 times higher odds for those with university education or above (AOR: 3.51; 95% CI: 1.59–7.72; p = 0.002). Self-reported environmental interest was strongly associated with concern about outdoor and indoor air pollution, with 4.89 times higher adjusted odds of concern about outdoor air pollution (AOR: 4.89, 95% CI: 3.02–7.93, p < 0.001) and 2.86 times higher adjusted odds of concern about indoor air quality (AOR: 2.86, 95% CI: 2.86–4.47, p < 0.001). Overall, Jazan residents display general but incomplete awareness of health hazards, signaling a need for expanded educational efforts to improve consciousness of less visible pollutants. Effective public communication strategies built on these insights can strengthen societal environmental awareness in Saudi Arabia and promote sustainability.
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