Engineering and Characterization of an Enzyme Replacement Therapy for Classical Homocystinuria

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