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.