Scientist Sangamo Therapeutics Brisbane, California
This presentation illustrates two case studies that address challenges of quantifying protein expression in preclinical studies of AAV gene therapy. Measuring human alpha-1 antitrypsin (hA1AT) transgene expressed protein in cynomolgus monkey serum encountered interference from monkey endogenous A1AT even after antibody screening and assay optimization to minimize the cross-reactivity. Methods of subtracting the interfering signal or concentration provided good selectivity and linearity. The literature reported hA1AT normal range in human is used as a success criterion. Utilizing measurements from healthy human donors as a reference supported the go/no-go decision making. The second assay quantitated sticky prion protein in small volumes of mouse cerebrospinal fluid (CSF), which presents several obstacles. Optimization of sample collection, buffer and storage enhanced prion recovery and stability. Furthermore, reliable prion data was obtained from mouse CSF samples with blood contamination by normalization with hemoglobin and prion levels in CSF and blood.
Learning Objectives:
describe the solution to address endogenous protein interference in quantification of biomarker expression in animal studies of gene therapy
describe the approaches of sample collection and storage for quantitation of sticky protein in small volumes of mouse cerebrospinal fluid
illustrate the normalization method to obtain reliable protein concentration data from mouse CSF samples with blood contamination
