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Batten Disease is a rare, neurodegenerative disorder that causes vision loss, cognitive and behavioral abnormalities, motor impairment, and early death. Mutations in one of 13 ceroid lipofuscin neuronal (CLN) genes can cause varying subtypes, of Batten Disease. Vision loss is a common first presenting symptom in several Batten Disease forms which quickly progresses to complete blindness. We previously established gene replacement strategies for CLN6 and CLN3 Batten Disease using adeno-associated viral vector serotype 9 (AAV9) delivered into the cerebrospinal fluid (CSF). In accordance with previous studies, I confirmed that AAV9 retinal transduction is achieved after CSF administration in mice and non-human primates, which might allow partial rescue of the vision phenotype. However, it is unclear if all necessary cell-types required for the prevention of vision loss are targeted via this delivery route. Hence, we propose that the best strategy to prevent vision loss would be to design an optimized intraocular gene replacement approach that can be combined with the current in-clinic AAV9 CSF-based gene therapies. In my thesis work, I characterized, for the first time, the expression profile of CLN3, CLN6, and CLN8 in the murine, primate, and porcine retina via single-cell RNA sequencing. We found that CLN transcripts are broadly expressed and therefore the success of an intraocular therapy would be maximized by targeting several of these cell types for gene replacement. In the first part of my thesis work, I compared the transduction profiles of AAV9 and Anc80L65, a newly engineered retina-tropic vector, following subretinal and intravitreal administration in wildtype mice. Through the development of a novel AI retinal quantification method, we found overall similar transduction profiles between the two capsids. In addition, preliminary results measuring both AAV9 and Anc80 capsid cross-reactivity with AAV2 in human serum indicates Anc80 may not be as immunologically distinct as previously thought. Based on this data, it seemed more advantageous to use the same already existing AAV9 gene therapy vector for the ocular treatment as well. Overall, the intravitreal administration route allowed for transduction in a greater number of retinal cell-types than a subretinal approach, however probably still not with the required efficiency for complete visual rescue. Therefore, the second aspect of my thesis work was to develop a novel intravitreal strategy that was able to increase AAV9 transduction to the murine and minipig retina and improve targeting of CLN expressing cell-types. We used the neuraminidase enzyme, which was shown to improve AAV9 transduction in other tissues previously to remove sialic acid residues covering the AAV9 entry receptor and facilitate increased cell entry. We administered neuraminidase both prior to or in combination with AAV9. Preliminary proof-of-concept studies in the murine and minipig model indeed confirmed improved targeting of the inner retinal layer cells, but also suggests that optimization of vector and neuraminidase dose are still required before this approach could be used in clinic. Overall, these findings support the potential efficacy of our proposed intravitreal/CSF combination gene replacement strategy. The results from this thesis work can be used to improve current Batten gene therapies to prevent vision loss and they can also help to inform the design of future gene replacement strategies for other retinal diseases.