Abstract
Introduction
Fetal hemoglobin (HbF) induction is a successful strategy for treating sickle cell disease. Higher levels of total HbF% are associated with better clinical outcomes, and a more uniform distribution of HbF across individual red blood cells (RBCs) is predicted to lead to better outcomes by inhibiting hemoglobin (Hb) polymerization in a higher fraction of individual RBCs. 1 Post-transcriptional silencing of BCL11A using lentivirus expression of a shRNA embedded in a microRNA architecture (shmiR) to re-activate γ-globin expression has been safe and demonstrated high levels of total HbF expression broadly distributed in red cells in a pilot clinical study (NCT 03282656). 2 We assessed the effects of BCL11A down-regulation on Hb polymer formation at the single-RBC level in 7 subjects in this trial (BCL) using novel assays of single-RBC Hb polymer content levels and single-RBC HbF.
Methods
Hb polymer was detected in individual RBCs using a previously published microfluidic system. 3 The system detects Hb polymer based on RBC morphology and on the principle that Hb polymer lowers RBC oxygen saturation in proportion to polymer concentration. 4,5 Single-RBC HbF mass was measured by flow cytometry using a previously published protocol. 6 These two measurements of distributions of single-RBC HbF mass and single-RBC Hb polymer were then integrated at the cell-population level to estimate the threshold of single-RBC HbF needed to inhibit formation of a detectable level of polymer as function of oxygen tension. Blood samples from BCL subjects (HbF% range 23 - 44, after stabilizing post-therapy) were compared to 14 hydroxyurea (HU) responsive patients (mean HbF% = 17, range 6 - 32), including 3 highly-responsive patients (HbF% = 25, 27, 31) as well as 4 individuals with sickle cell trait (AS).
Results
At 3.7% oxygen tension typical of the human microcirculation 7 and some regions of the central nervous system, 7 the typical BCL subject had detectable levels of Hb polymer in 52% (median) of RBCs vs. 66% for HU (p = 0.02) and 6% for AS (Figure 1A). At lower oxygen tensions of 1.7% typical of the bone marrow 7 or kidney medulla 7 BCL subjects had a polymerized RBC fraction of 68%, compared to 79% for HU (p<0.001) and 30% for AS. Fractions of RBC with polymer in BCL subjects stabilized about 3-6 months after treatment. By combining measurements of single-RBC HbF mass and single-RBC polymer at the population level, we estimated the threshold HbF mass or fraction required to inhibit detectable levels of polymer as a function of oxygen tension. We compared single-RBC HbF thresholds for the BCL cohort to the three HU high responders. The mean HbF threshold for polymerization inhibition estimated for the BCL cohort was lower (18%) than that for the HU high responders (25%) but was not statistically significant (p = 0.07) in this initial analysis.
Conclusions
BCL11A down-regulation in seven clinical trial subjects is associated with favorable distributions of single-RBC polymer at multiple physiologic oxygen tensions compared to HU. Future study is needed to determine whether these favorable Hb polymer distributions are simply a result of higher total HbF or also due to lower single-RBC HbF thresholds for polymer inhibition.
References
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2. Esrick EB, Lehmann LE, Biffi A, et al. Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease. N. Engl. J. Med. 2021;384(3):205-215.
3. Di Caprio G, Schonbrun E, Gonçalves BP, et al. High-throughput assessment of hemoglobin polymer in single red blood cells from sickle cell patients under controlled oxygen tension. Proc. Natl. Acad. Sci. U. S. A. 2019;116(50):25236-25242.
4. Benesch RE, Edalji R, Kwong S, Benesch R. Oxygen affinity as an index of hemoglobin S polymerization: A new micromethod. Anal. Biochem. 1978;89(1):162-173.
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7. Keeley TP, Mann GE. Defining physiological normoxia for improved translation of cell physiology to animal models and humans. Physiol. Rev. 2019;99(1):161-234.
Esrick: bluebird bio: Consultancy. Bartolucci: INNOVHEM: Other: Co-founder; F. Hoffmann-La Roche Ltd: Consultancy; Bluebird: Consultancy, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; GBT: Consultancy; Emmaus: Consultancy; Hemanext: Consultancy; AGIOS: Consultancy; Jazz Pharma: Other: Lecture fees; Fabre Foundation: Research Funding; Addmedica: Consultancy, Other: Lecture fees, Research Funding. Williams: Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding. Higgins: Danaher Diagnostics: Consultancy; Sebia, Inc.: Honoraria.
