ENGINEERING ACTIN-RESISTANT HUMAN DNASE-I FOR TREATMENT OF CYSTIC-FIBROSIS

Human deoxyribonuclease I (DNase I), an enzyme recently approved for t reatment of cystic fibrosis (CF), has been engineered to create two cl asses of mutants: actin-resistant variants, which still catalyze DNA h ydrolysis but are no longer inhibited by globular actin (G-actin) and active site variants, which no longer catalyze DNA hydrolysis but stil l bind G-actin, Actin-resistant variants with the least affinity for a ctin, as measured by an actin binding ELISA and actin inhibition of [P -33] DNA hydrolysis, resulted from the introduction of charged, alipha tic, or aromatic residues at Ala-114 or charged residues on the centra l hydrophobic actin binding interface at Tyr-65 or Val-67, In CF sputu m, the actin-resistant variants D53R, Y65A, Y65R, or V67K were 10- to 50-fold more potent than wild type in reducing viscoelasticity as dete rmined in sputum compaction assays. The reduced viscoelasticity correl ated with reduced DNA length as measured by pulsed-field gel electroph oresis, In contrast, the active site variants H252A or H134A had no ef fect on altering either viscoelasticity or DNA length in CF sputum, Th e data from both the active site and actin-resistant variants demonstr ate that the reduction of viscoelasticity by DNase I results from DNA hydrolysis and not from depolymerization of filamentous actin (F-actin ). The increased potency of the actin-resistant variants indicates tha t G-actin is a significant inhibitor of DNase I in CF sputum, These re sults further suggest that actin-resistant DNase I variants may have i mproved efficacy in CF patients.