Missense mutations in the GUCA1A (Guanylate Cyclase Activator 1A) gene, encoding the Ca²⁺-binding protein GCAP1 (Guanylate Cyclase Activating Protein 1), are associated with autosomal dominant cone and cone-rod dystrophy. GCAP1 is involved in photoreceptor recovery after photoactivation, by regulating cGMP synthesis of guanylate cyclases through a Ca²⁺ feedback loop. The specific aims of my thesis were to: (1) identify novel GCAP1 mutations in human patients suffering from autosomal dominant cone and cone-rod dystrophy, and assay the biochemical properties of mutant GCAP1 in vitro; (2) generate and characterize transgenic mice expressing mutant GCAP1; and (3) develop RNA interference gene therapy for the disease in the mouse models.

Two novel GCAP1 mutations, L151F and N104K, were identified from two autosomal dominant cone dystrophy families by genetic analysis. Biochemical analysis of recombinant wild-type and mutant GCAP1 proteins expressed in insect cells indicates that the novel mutations altered the Ca²⁺-sensitivity of GCAP1 and persistently stimulate guanylate cyclases to synthesize cGMP even at the physiologic dark Ca²⁺ concentrations at which GCAP1 is normally inactive. The resulting elevated levels of cGMP and Ca ²⁺ in photoreceptors are predicted to cause retinal degeneration, providing a dominant gain-offunction mechanism leading to the disease.

Two transgenic mouse models expressing GCAP1 (L151F) and GCAP1 (N104K) were generated and presented late-onset and slowly progressive photoreceptor degeneration. They closely mimicked the human disease, suggesting that they are ideal genetically manipulated mouse models for elucidating the underlying molecular mechanisms and developing therapeutic interventions for GCAP1 linked autosomal dominant cone and cone-rod dystrophy.

Ablation of GCAPs in knockout mice did not result in any pathological effects. Therefore, we developed a strategy to knockdown both mutant and wild-type Guca1a mRNA by RNA interference. Efficient GCAP1 short-hairpin (sh)RNAs were identified via an in vitro screening system and packaged into an effective self-complementary adeno-associated viral vector, scAAV2/8, that produces shRNA in photoreceptors. A scAAV2/8-bG1shRNA was delivered to a rapidly degenerating mouse model overexpressing bGCAP1 (Y99C) to demonstrate the therapeutic benefit of the strategy. The treated eyes showed a significant rescue from photoreceptor degeneration. Thus, this study demonstrated that RNAi gene therapy is a promising therapeutic tool for dominant retinal diseases resulting from GCAP1 mutations.