Polyethylene glycol-conjugated adenosine phosphorylase: development of alternative enzyme therapy for adenosine deaminase deficiency

Abstract

Purine nucleoside phosphorylase had previously been engineered to accept 6-amino substituted purine nucleosides by two active site substitutions, Asn243Asp; Lys244Gln. In the present study, recombinant adenosine phosphorylase (AP) has been conjugated to branched polyethylene glycol (PEG) polymers of approximately 42.5 kDa. Matrix-assisted laser desorption/ionization analysis and SDS acrylamide electrophoresis analysis indicated a subunit composition of greater than 205 kDa consistent with the conjugation of as many as four PEG molecules per AP subunit. The PEG-conjugated enzyme retained greater than 90% of the native catalytic activity. Administration of the enzyme to mice demonstrated the PEG–AP to have a 67-fold increased plasma half-life compared to the native enzyme, 65.1±2.9 h versus 57.8±1.1 min, respectively. PEG–AP was principally confined to the plasma with minimal activity detected in tissues and of these spleen had the greatest activity and essentially no activity was found in urine. PEG–AP has retained activity with inosine and its injection into PNP-deficient mice resulted in a 2.7-fold increase in urine urate. AP was also shown to protect human CEM cells in culture from the toxic effects of 2′-deoxyadenosine. These studies provide evidence for consideration of PEG–AP as an alternative enzyme therapy for the inherited deficiency of adenosine deaminase.