Fundamentals of Anti-Sickling Therapies - Sickle cell disease affects 100,000 Americans and millions world-wide: a mutation in the β-globin gene gives rise to a hemoglobin variant (sickle hemoglobin, or HbS) that is prone to self-assembly. The resulting protein fibers distort red blood cells and cause the disease’s pathology. Two gene therapies have recently been approved for treatment of this disorder; both are based on the observation that co-expression of fetal hemoglobin (HbF) reduces the formation of sickle fibers. However, we lack a mechanistic understanding of how HbF achieves its anti-sickling effects; simple models in which HbF is suggested to reduce fiber nucleation by diluting HbS fail to explain HbF’s observed actions. Hence, advances in gene delivery have outpaced our basic knowledge of how a given gene product is functioning. This gap in understanding limits both the informed optimization of these new gene therapies, as well as rational approaches to designing potential novel combination treatments. In an alternate approach, high-throughput screening has been used to identify large numbers of potential anti-sickling compounds, drawn from pools of FDA-approved drugs or agents already assessed in clinical trials. However, this promise dims in light of our ignorance of how well their success in the screening translates into actual anti-sickling effects. Again, this knowledge gap will limit efforts at rational optimization. We have identified three critical aspects of HbS pathophysiology for which our lack of understanding poses a significant barrier to rational development and optimization of therapeutic approaches. Aim 1. Developing a mechanistic understanding of how HbF modulates HbS polymerization. The issue: HbF copolymerization with HbS is not understood and poorly characterized. Our hypothesis: Sickle fibers can accommodate vacancies within the polymer that allow HbF inclusion. Our approach: We will measure copolymerization to test our model for HbF incorporation into fibers. Aim 2. Reconciling in vitro and in-cell polymerization. The issue: Fiber formation rates in cells expressing both HbF and HbS disagree with predictions from solution. Our hypothesis: Hb concentration in HbF-containing cells is lower than expected, slowing fiber formation. Our approach: We will measure intracellular concentration and fiber-formation rates simultaneously in live cells. Aim 3. Probing the mechanism of action for newly identified anti-sickling compounds. The issue: Initial high-throughput screens failed to rule out hits that succeed by diminishing oxygen delivery. Our hypothesis: Some compounds flagged as hits in a cell-based assay actually alter O2 affinity, and as such will perform poorly in vivo. Our approach: We will measure binding curves, solubility, and assembly kinetics under low pO2. This work will, in effect, provide the missing “owner’s manual” for the gene and drug therapies that offer so much promise to those suffering from sickle cell disease.
