Viral gene delivery is showing great promise for treating retinal disease. Whilst subretinal vector delivery has mainly been used to date, intravitreal delivery has potential advantages if low retinal transduction efficiency could be overcome. To this end we investigated the effects of co-injection of glycosaminoglycan degrading enzymes, singly or in combination, with AAV2 as a method of increasing retinal transduction. Experiments using healthy mice demonstrated that these enzymes enhance retinal transduction. We found that heparinase III produced the greatest individual effect, and this was enhanced further by combining with hyaluronan lyase. In addition, this optimised AAV2-enzyme combination led to a marked improvement in transduction in retinas with an advanced retinal degeneration compared to AAV2 alone. Safety studies measuring retinal function by flash electroretinography indicated that retinal function was unaffected in the acute period and at least 12 months post enzyme treatment, while pupillometry confirmed that retinal ganglion cell activity was unaffected. Retinal morphology was not altered by the enzyme injection. Collectively these data confirm the efficacy and safety of this intravitreal approach in enhancing retinal transduction efficiency by AAV in rodents. Translating this method into other species such as non-human primates or for clinical applications will have challenges and require further studies.