Q. Nguyen1, R. G. Witt1, C. Eikani1, B. Wang1, T. C. Mackenzie1 1University Of California – San Francisco,San Francisco, CA, USA
Introduction:
Sly Syndrome, or Mucopolysaccharidosis Type 7 (MPS7), is an autosomal recessive disorder in which missing β-glucuronidase (GUSB) results in systemic cellular dysfunction and in utero demise. Postnatal enzyme replacement therapy (ERT) is unable to treat neurologic phenotype because the blood brain barrier is closed after birth. Furthermore, patients can develop an immune response to the missing protein after repeated doses, which limits the treatment efficacy. In this study, we investigate whether administration of the enzyme in utero, prior to formation of the blood brain barrier and to maturation of the immune system will result in brain penetration and induce tolerance.
Methods:
We bred heterozygous MPS7 mice and injected the resultant fetuses at E14.5 with recombinant human GUSB or control phosphate-buffered saline (PBS) through maternal midline laparotomy. To assess for tissue enzyme levels, we harvested brain tissue at E18-P1 and performed chromogenic GUSB enzyme activity assays. To compare against postnatal treatment, we also injected adult MPS7 mice with enzyme and harvested brain tissue 4 days after injection. To assess for long-term immune response to enzyme, we performed serial post-natal enzyme injections, then challenged mice with GUSB plus Complete Freund’s Adjuvant (CFA) at 6-8 weeks of age and then measured antibody production using ELISA for IgG1, IgG2b, IgG2c, and IgG3. Mice challenged with CFA continued to receive serial enzyme booster injections.
Results:
For the brain penetration studies, we administered enzyme or PBS in utero (n=9), or postnatally (n=6). Enzyme replacement resulted in detectable brain enzyme activity after in utero but not after postnatal treatment (Fig. 1A). For the tolerance studies, we detected a significant decrease in IgG1 antibody production with IUERT compared to both the untreated and post-natal ERT groups (n=91) (Fig. 1B). During the course of the ELISA studies, we found that some mice expired spontaneously after receiving booster injections following the CFA challenge, likely secondary to anaphylaxis, but that in utero exposure to the enzyme was protective against treatment-related death (Fig. 1C).
Conclusion:
We have found that IUERT delivers enzyme to brain tissue, and also successfully induces long-term tolerance to the missing enzyme. Future studies will examine the subtype of brain cells that take up the enzyme as well as quantify levels of IgE which likely contribute to the anaphylaxis. These findings have broad applicability for using the fetal environment to induce tolerance and treat neurologic disorders, thus opening the field of fetal surgery to include emerging molecular therapies.
