Towards Transformative Therapies for Sickle Cell Disease

Pain in sickle cell disease (SCD) is more often chronic than previously thought, though acute pain is still the hallmark of the disease. Both are undertreated. In adults and even in children, the quantity and severity of SCD pain may be vastly underestimated, because most of the “iceberg” of chronic SCD pain is “submerged” at home. The implications of this iceberg phenomenon are significant for pain treatment. Earlier, clinicians focused on adequate treatment of acute pain in SCD. It is still a challenge. Appropriate acute care triage of SCD crises to a high rather than low priority, coupled with individualized pain plans, can ensure timely, more effective acute therapy, and likely save hospitalizations. But a comprehensive focus requires remittive and palliative therapies for chronic pain treated at home. Three issues dominate the chronic pain debate. First, should opioids be the mainstay of chronic palliative SCD therapy? Second, does preventing sickling, preventing vasculopathy from SCD, preventing organ failure, or even curing SCD with bone marrow transplant, result in improvement of acute or chronic subjective pain? Third, do we have enough information to justify use of therapies intended for neuropathic or central pain, present in diseases as diverse as irritable bowel syndrome, temporo-mandibular joint syndrome, fibromyalgia, chronic pelvic pain, and rheumatoid arthritis?

Transformative SCD pain therapy would feature an array of interventions for acute and chronic SCD pain, and a full classification of pain phenotypes which qualified patients for acute and chronic pain palliative and remittive interventions.

Human induced pluripotent stem cells (hiPSCs) hold promise for both disease modeling and the development of novel therapeutic treatments for sickle cell anemia (SCA). Such models are practical systems to screen new drug therapies and to examine the effects of gene editing. hiPSCs can theoretically produce all cell types including erythroid cells. However, in vitro modeling of SCA with reprogrammed cells has been limited by their inability to differentiate into beta globin-expressing, enucleated erythroid cells. Here, we propose strategies to produce improved in vitro models of SCA using these cell types. We derived hiPSCs from sickle cell patients with hemoglobin SS disease seen at our hematology clinic at Boston Children’s Hospital. Using a cocktail of transcription factors promoting self-renewal and multi-potentiality expressed under the control of a doxycycline-regulated promoter (Erg, HoxA9, RORa, Sox, Myb) we generated conditionally immortalized hematopoietic cell lines that serve as a renewable source of robust erythroid progenitors in vitro. Concurrently, we further improved the in vitro differentiation protocols described to generate 30-40% beta-globin-expressing, erythroid cells with an enucleation rate of 20-50%. In future studies, we hope to further improve in vitro beta globin expression via genetic/epigenetic modulation of the globin loci. Such a sickling platform will permit the employment of hiPSCs to test the therapeutic hypothesis that genetic manipulation of BCL11A, a master regulator of fetal hemoglobin (HbF) expression, will ameliorate sickling. The generation of hiPSC-SCA models will be critical in broadening the current understanding of the molecular mechanisms of this disease, the development of improved pharmacological treatments and a future of autologous cell therapy for the cure of SCA.

Erythrocyte adhesion contributes to sickle cell disease severity. We have developed a standardized microfluidic flow-based adhesion assay to measure erythrocyte adhesion indices and longitudinal variability. Blood samples were collected from heterozygote (AS; n=4) and normal (AA; n=10) controls and SCD subjects (n=35; 13-48 yrs, avg 25.9yrs) at steady state as verified by an electronic patient reported outcome (ePRO) tool. Blood was drawn every 3 weeks for 3 months (AA and AS controls)/6 months (SCD patients). The standardized adhesion assay was performed by flowing 30mL of whole blood over immobilized VCAM-1, followed by quantifying the adherent cells (adhesion index; cells/mm2). Correlations between steady state adhesion and clinical laboratory parameters were assessed using the Spearman rank correlation coefficient. Steady state adhesion varied from sample-to-sample (n=292; mean = 353.5 ± 236.3; median = 294 cells/mm2). Steady state adhesion had statistically significant positive correlations with white blood cell count (WBC; r=0.239; p<0.0001), C-reactive protein (CRP; r=0.254; p<0.0001), platelet count (r=0.124; p=0.0357), and reticulocyte percentage (r=0.5108; p<0.0001), and statistically significant negative correlations with hematocrit (r=-0.1599; p=0.0067) and fetal hemoglobin (r=-0.485; p<0.0001). Finally, we demonstrated a statistically significant correlation between the historical disease severity of each patient and steady state adhesion. Steady-state adhesion indices and longitudinal adhesion variability can serve as a basis to further study adhesion as a biomarker for sickle cell disease severity, a platform to assess preclinical response to SCD therapy, and monitor response to therapy.

The transition from the predominant production of fetal hemoglobin (HbF) to adult hemoglobin shortly after the time of birth in humans has been extensively studied, given that elevated production of HbF can ameliorate clinical symptoms in both sickle cell disease and β-thalassemia. A number of factors regulating HbF production have been identified through unbiased human genetic studies of both common and rare variation, including BCL11A, KLF1, and MYB. However, the mechanisms underlying the developmental regulation of HbF expression and the regulation of factors such as BCL11A in this process is unknown. In this presentation, we will discuss recent work from our group using both human genetic observations and functional studies in human erythroid cells, which has allowed us to identify new pathways regulating this process. Specifically, we will discuss new insight into the regulation of BCL11A protein expression during human ontogeny. While BCL11A is robustly expressed in adult erythroid cells, it is expressed at significantly lower levels in cord blood and fetal liver erythroid cells from humans. We have investigated the underlying mechanisms by which these differences arise during human development. This new insight into the regulation of BCL11A production during human hematopoiesis suggests new therapeutic avenues to reduce BCL11A expression and thereby induce HbF synthesis in patients with sickle cell disease and β-thalassemia.