In utero and postnatal mouse injections

Intravenous in utero injections were performed in a similar fashion as previously published and as follows³⁴. Specifically, fetuses of time-dated R26^mTmG/mTmG × Balb/c (to generate R26^mTmG/+ fetuses) and Idua-W392X mice were injected on gestational day (E) 15.5. Under isoflurane anesthesia and after providing local anesthetic (0.25% bupivacaine subcutaneously), a midline laparotomy was performed, and the uterine horns were exposed. The vitelline vein—which runs along the uterine wall and enters the portal circulation resulting in first-pass effect to liver and systemic delivery via the ductus venosus—was identified under a dissecting microscope and 15 μL of phosphate buffered saline (PBS) or total virus (7.5 μL of each split-intein AAV vector) was injected per fetus using a 100-μm beveled glass micropipette. Based on the viral titers (Supplementary Table ²), this resulted in the injection of 6.5 × 10¹⁰ total genome copies for Idua-W392X fetuses injected with AAV.ABE.Idua and 2 × 10⁹ genome copies for R26^mTmG/+ fetuses injected with AAV.SpCas9.mTmG. C57BL/6 injections of AAV9 GFP and mCherry were conducted similarly with the delivery of 15 μL of total virus at a 1:1 ratio with a total injection of 2.5 × 10¹⁰ total genome copies. A successful injection was confirmed by temporary clearance of blood from the vein and absence of injectate extravasation. The uterus was returned to the abdominal cavity and the laparotomy incision was closed in two layers with 4-0 Vicryl suture.

Viral injections into adult Idua-W392X mice were performed at 10 weeks of age via the retroorbital vein under isoflurane anesthesia. A total volume of 300 μL of virus (150 μL of each split-intein AAV vector) was injected such that ~1.3 × 10¹² genome copies were injected per mouse. Based on the average weight of 10-week-old Idua-W392X mice (20 g) and E15.5 fetuses (1 g), both adult and fetal Idua-W392X mice received ~6.5 × 10¹⁰ total genome copies/gram.