The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood-derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies.

CRISPR/Cas9, Fetal hemoglobin, Gene addition, High-performance liquid chromatography, Thalassemia, β-hemoglobinopathy,
Human Gene Therapy Methods
Erasmus MC: University Medical Center Rotterdam

Loucari, C.C. (Constantinos C.), Patsali, P. (Petros), van Dijk, T.B, Stephanou, C. (Coralea), Papasavva, P. (Panayiota), Zanti, M. (Maria), … Kleanthous, M. (2018). Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies. Human Gene Therapy Methods, 29(1), 60–74. doi:10.1089/hgtb.2017.190