Engineering and Characterization of an Enzyme Replacement Therapy for Classical Homocystinuria

<div>Homocystinuria due to loss of cystathionine betasynthase</div><div>(CBS) causes accumulation of homocysteine and</div><div>depletion of cysteine. Current treatments are suboptimal, and</div><div>thus the development of an enzyme replacement therapy based on</div><div>PEGylated human truncated CBS (PEG-CBS) has been initiated.</div><div>Attenuation of potency was observed, which necessitated a screen of</div><div>several PEG-CBS conjugates for their efficacy to correct and</div><div>maintain the plasma metabolite profile of murine homocystinuria</div><div>after repeated administrations interrupted with washouts. We found</div><div>that CBS coupling with maleimide PEG inconsistently modified the</div><div>enzyme. In contrast, the PEG-CBS conjugate with 20 kDa Nhydroxysuccinimide-</div><div>PEG showed very little loss of potency likely</div><div>due to a reproducible PEGylation resulting in species modified with five PEGs per subunit on average. We developed assays</div><div>suitable for monitoring the extent of CBS PEGylation and demonstrated a sustainable partial normalization of homocystinuria</div><div>upon continuous PEG-CBS administration via osmotic pumps. Taken together, we identified the PEG-CBS conjugate suitable for</div><div>manufacturing and clinical development.</div>