IMPROVED POTENCY OF HYPERACTIVE AND ACTIN-RESISTANT HUMAN DNASE-I VARIANTS FOR TREATMENT OF CYSTIC-FIBROSIS AND SYSTEMIC LUPUS-ERYTHEMATOSUS

The ability of recombinant human DNase I (DNase I) to degrade DNA to l ower molecular weight fragments is the basis for its therapeutic use i n cystic fibrosis (CF) patients and its potential use as a treatment f or systemic lupus erythematosus (SLE), To increase the potency of huma n DNase I, we have generated and characterized three classes of mutant s: (a) hyperactive variants, which have from one to six additional pos itively charged residues (+1 to +6) and digest DNA much more efficient ly relative to wild type, (b) actin-resistant variants, which are no l onger inhibited by G-actin, a potent inhibitor of DNase I, and (c) com bination variants that are both hyperactive and actin-resistant. For D NA scission in CF sputum where the DNA concentration and length are la rge, we measured a similar to 20-fold increase in potency relative to wild type for the +3 hyperactive variant Q9R/E13R/N74K or the actin-re sistant variant A114F; the hyperactive and actin-resistant combination variant was similar to 100-fold more potent than wild type DNase I. F or digesting lower concentrations of DNA complexed to anti-DNA antibod ies in human serum, we found a maximal enhancement of similar to 400-f old over wild type for the +2 variant E13R/N74K. The +3 enzymes have s imilar to 4000-fold enhancement for degrading moderate levels of exoge nous DNA spiked into human serum, whereas the +6 enzyme has similar to 30,000-fold increased activity for digesting the extremely low levels of endogenous DNA found in serum. The actin resistance property of th e combination mutants further enhances the degree of potency in human serum. Thus, the human DNase I variants we have engineered for improve d biochemical and pharmacodynamic properties have greater therapeutic potential for treatment of both CF and SLE.