The ability of
recombinant human DNase I (
DNase I) to degrade
DNA to lower molecular weight fragments is the basis for its
therapeutic use in
cystic fibrosis (CF) patients and its potential use as a treatment for
systemic lupus erythematosus (SLE). To increase the potency of human
DNase I, we have generated and characterized three classes of mutants: (a) hyperactive variants, which have from one to six additional positively charged residues (+1 to +6) and digest
DNA much more efficiently relative to wild type, (b) actin-resistant variants, which are no longer inhibited by
G-actin, a potent inhibitor of
DNase I, and (c) combination variants that are both hyperactive and actin-resistant. For
DNA scission in CF sputum where the
DNA concentration and length are large, we measured a approximately 20-fold increase in potency relative to wild type for the +3 hyperactive variant Q9R/E13R/N74K or the actin-resistant variant A114F; the hyperactive and actin-resistant combination variant was approximately 100-fold more potent than wild type
DNase I. For digesting lower concentrations of
DNA complexed to
anti-DNA antibodies in human serum, we found a maximal enhancement of approximately 400-fold over wild type for the +2 variant E13R/N74K. The +3
enzymes have approximately 4000-fold enhancement for degrading moderate levels of exogenous
DNA spiked into human serum, whereas the +6
enzyme has approximately 30,000-fold increased activity for digesting the extremely low levels of endogenous
DNA found in serum. The actin resistance property of the combination mutants further enhances the degree of potency in human serum. Thus, the human
DNase I variants we have engineered for improved biochemical and pharmacodynamic properties have greater therapeutic potential for treatment of both CF and SLE.