Intravitreal Injection of scAAV8-hRS/IRBP-hRS Vector

scAAV8-hRS/IRBP-hRS vector was administered by intravitreal injection to pups at P5 or P6. This vector is comparable to the clinical vector in a human XLRS trial [14 (link)]. Pups were anesthetized either by 20–30 µL volume via intraperitoneal injections of ketamine/xylazine mixture containing ketamine hydrochloride (30–40 mg/kg) and xylazine (3–4 mg/kg) for 10–15 min or by isoflurane inhalation (1.5% isoflurane in 100% oxygen) for 3 to 5 min. One drop of 0.5% tetracaine topical anesthetic was applied, and pupils were dilated using a drop each of 1% tropicamide and 2.5% phenylephrine. One drop of Proparacaine hydrochloride 0.5% was applied as topical ocular anesthesia. A small scleral nick was made at the nasal-temporal ora serrata below the iris using a 30-G insulin syringe needle to gain access to the posterior cavity (vitreous chamber) of the eye. Sterile 10 µL Nanofil syringes with the 35 G beveled-tip needle on a sterile 10 µL Nanofil syringe (World Precision Instruments, Inc., Sarasota, FL, USA) was inserted into the vitreous to deliver 2e10 (2 × 10¹⁰) vector genomes per eye (vg/eye) in 1.5 μL fluid. The contralateral eye served as control. Triple antibiotic ophthalmic ointment (neomycin, polymyxin B, and bacitracin) was applied to the eye at the conclusion. Animals were kept on a warming pad until their recovery [15 (link)]