The most efficient ABCA4 dual vector system, based on the in vitro studies mentioned, the hybrid system, was repackaged in AAV8(Y733F) and subretinally injected into 1-month-old ABCA4−/− mice with 1 μL of 3 × 109 vg/mL of each half vector, and protein expression was then analyzed 4 weeks after injection by Western blot (A) or immunohistochemistry (B, C) with specific antibodies against the N-terminal (rim5B4) half of human ABCA4. As a control, the front half vector (5′ vector) only was injected (A, C) in some animals. GCL, retinal ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segments; RPE, retinal pigment epithelium.