Abstract
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.
Highlights
In addition to its fundamental role of storing genetic information, DNA is an important component in the pathogenesis of several diseases
Actin Inhibition Assay—Supercoiled pBR322 plasmid DNA (New England Biolabs) at 29 g/ml was treated with diluted culture media from human 293 cells transfected with wild type or variant DNase I (final DNase I concentration of 1 ng/ml as measured by a human DNase I ELISA [13, 15]) preincubated with increasing amounts of G-actin for min in the presence of mM Hepes, pH 7, 100 g/ml bovine serum albumin, 1 mM MgCl2, and 2.5 mM CaCl2 at room temperature
We chose to characterize the A114F mutant, which might potentially reduce any immunogenicity, because phenylalanine is found at position 114 in several other naturally occurring human DNase I-like enzymes, at least one of which has been shown to be actin-resistant (20 –22)
Summary
In addition to its fundamental role of storing genetic information, DNA is an important component in the pathogenesis of several diseases. To improve the biochemical activity and potentially the efficacy of DNase I, we have recently assessed the importance of various residues at the DNA-binding interface and engineered hyperactive variants that utilize a more efficient functional mechanism involving processive nicking of double-stranded DNA [12, 13]. We have characterized hyperactive, actin-resistant, and combination variants of human DNase I in several biologically relevant assays to determine their relative potency and pharmacodynamics in CF sputum and human serum These results have significant implications for their therapeutic potential for treatment of both CF and SLE
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