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 ϳ20-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 ϳ100-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 ϳ400-fold over wild type for the ؉2 variant E13R/N74K. The ؉3 enzymes have ϳ4000-fold enhancement for degrading moderate levels of exogenous DNA spiked into human serum, whereas the ؉6 enzyme has ϳ30,000-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.
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