A Single-Administration AAV Gene Therapy Expressing an Affinity-Detuned Feline AIM (m-fAIM) for Chronic Kidney Disease in the Domestic Cat: A Theoretical Development Program
This work presents a theoretical development program for a single-administration adeno-associated virus (AAV) gene therapy designed to treat chronic kidney disease (CKD) in the domestic cat. The approach centers on the delivery of an affinity-detuned variant of feline Apoptosis Inhibitor of Macrophage (m-fAIM), engineered to correct a species-specific molecular defect: feline AIM binds IgM approximately 1000-fold more strongly than murine AIM, owing to a cluster of charged arginine residues (R299, R300, R338) on the SRCR3 domain. This abnormally high-affinity interaction is hypothesized to prevent AIM release from the IgM pentamer into urine, blunting its protective role in tubular debris clearance and contributing to feline CKD pathogenesis. This research investigates affinity-detuned feline AIM/CD5L as a potential therapeutic strategy for chronic kidney disease in domestic cats. Building on a previously defined gene-therapy concept, the study focuses on the molecular and genomic qualification of engineered feline AIM variants rather than on demonstrating therapeutic efficacy. The work integrates the experimentally deposited feline AIM/CD5L sequence with the current Felis catus reference genome to establish a precise and traceable relationship between therapeutic protein residues, coding sequence, transcript identity, and endogenous genomic coordinates. Particular attention is given to three engineered positions within AIM—R299, R300, and R338—which define the human-aligned m-fAIM_hz variant and the murine-aligned m-fAIM_mz variant. The study performs transcript-aware and codon-resolved mapping of these residues, compares the nucleotide complexity required to generate each engineered allele, and evaluates sequence-level properties relevant to protein expression and developability. The introduction of Cys300 in the hz variant is specifically investigated as a potential structural and biochemical liability requiring experimental validation. A major component of the research examines whether the endogenous feline CD5L locus is accessible to CRISPR-based precision editing. The complete contemporary CD5L locus is analyzed for canonical SpCas9 target sites, and candidate regions surrounding the therapeutic codons are compared with an independently generated CHOPCHOP dataset obtained using a historical feline genome assembly. Sequence-level reconciliation is used to connect the historical and current coordinate systems and to determine whether the same therapeutic regions are independently identified as CRISPR-accessible. The project also critically evaluates structural and computational evidence, distinguishing experimentally determined structures, sequence-consistent predictive models, raw computational outputs, and artifacts that cannot support biological interpretation. Genome-wide off-target specificity is treated separately from local targetability and is not considered established without a validated whole-genome analysis. Overall, the research transforms the affinity-detuned feline AIM concept from a protein-engineering hypothesis into a reference-grounded, transcript-aware, codon-resolved, and independently cross-checked molecular framework. It establishes the genomic identity and computational accessibility of the relevant therapeutic sites while leaving protein function, editing efficiency, genome-wide safety, and therapeutic efficacy as experimental questions to be addressed in future studies.