Inherited retinal diseases (IRDs) represent a diverse group of genetic disorders leading to progressive degeneration of the retina due to mutations in over 280 genes. This review focuses on the various methodologies for the preclinical characterization and evaluation of adeno-associated virus (AAV)-mediated gene therapy as a potential treatment option for IRDs, particularly focusing on gene therapies targeting mutations, such as those in the RPE65 and FAM161A genes. AAV vectors, such as AAV2 and AAV5, have been utilized to deliver therapeutic genes, showing promise in preserving vision and enhancing photoreceptor function in animal models. Despite their advantages—including high production efficiency, low pathogenicity, and minimal immunogenicity—AAV-mediated therapies face limitations such as immune responses beyond the retina, vector size constraints, and challenges in large-scale manufacturing. This review systematically compares different experimental models used to investigate AAV-mediated therapies, such as mouse models, human retinal explants (HREs), and induced pluripotent stem cell (iPSC)-derived retinal organoids. Mouse models are advantageous for genetic manipulation and detailed investigations of disease mechanisms; however, anatomical differences between mice and humans may limit the translational applicability of results. HREs offer valuable insights into human retinal pathophysiology but face challenges such as tissue degradation and lack of systemic physiological effects. Retinal organoids, on the other hand, provide a robust platform that closely mimics human retinal development, thereby enabling more comprehensive studies on disease mechanisms and therapeutic strategies, including AAV-based interventions. Specific outcomes targeted in these studies include vision preservation and functional improvements of retinas damaged by genetic mutations. This review highlights the strengths and weaknesses of each experimental model and advocates for their combined use in developing targeted gene therapies for IRDs. As research advances, optimizing AAV vector design and delivery methods will be critical for enhancing therapeutic efficacy and improving clinical outcomes for patients with IRDs.

Genome instability in induced pluripotent stem cells (IPSC) poses a significant challenge for their use in research and medicine. Cataloging and precisely describing all the identified aberrations that arise during cell reprogramming, expansion, and differentiation is essential for improving approaches to instability prevention and ensuring genetic quality control. We report the karyotypic analysis of 65 cell lines derived from skin fibroblasts, urinal sediment, and peripheral blood mononuclear cells of 33 individuals, 82% of whom suffer from monogenic genetic disorders not associated with genetic instability. Trisomy of chromosomes 20 and 8 was revealed recurrently, while the 1q arm was the most frequently affected region involved in interstitial duplications and unbalanced translocations with chromosomes 15 and 18. The localization of rearrangement breakpoints identified by SNP arrays within the large DCC gene and histone gene clusters links genetic instability in IPSCs to replication-stress-induced chromosome breakage at common and early replicating fragile sites.

Maturity-onset diabetes of the young type 10 (MODY10) is a monogenic diabetes subtype caused by heterozygous mutations in the insulin gene (INS), leading to defective proinsulin processing, endoplasmic reticulum (ER) stress, and β-cell dysfunction. Current management relies on sulfonylureas or insulin therapy, but these fail to address the underlying genetic defect. Recent research has elucidated the molecular mechanisms of MODY10, including ER stress induced by proinsulin misfolding, activation of the unfolded protein response (UPR), and β-cell apoptosis. Emerging therapies such as Adeno-Associated Virus (AAV)-mediated gene delivery to induce the glucose-responsive hepatic insulin expression, plasmid-based single-chain insulin analogs, and cell-based therapies show promise in preclinical studies. However, critical challenges remain, including immune responses to AAV vectors, incomplete correction of dominant-negative mutant effects, and the need for long-term safety data. This review summarizes current knowledge on MODY10 genetics, pathophysiology, and therapeutic innovations, while identifying key gaps for future research to enable precision medicine approaches.

Cone dystrophy with supernormal rod response (CDSRR) is associated with pathogenic variants of the KCNV2 gene that result in severe symptoms, including color vision defects, decreased visual acuity, and specific changes in electroretinogram responses. Two iPSC lines were obtained from two patients in the same family with different types of mutations in the KCNV2 gene. These lines could serve as a useful model for studying the pathogenetic mechanism and treatment development for CDSRR. PBMCs from donors have been reprogrammed into iPSC lines. Derived clones were characterized with mutation sequencing, analysis of common pluripotency-associated markers at the protein levels, and in vitro differentiation studies.

Background/Objectives: Cone dystrophy with supernormal rod response (CDSRR) is a rare autosomal recessive retinal disorder characterized by a delayed and markedly decreased photoreceptor response. In this article, we aim to describe the clinical course and associated molecular findings in children with cone dystrophy with supernormal rod response associated with recessive mutations in the KCNV2 gene, which encodes a subunit (Kv8.2) of the voltage-gated potassium channel. Methods: The genetic testing of two patients included the next-generation sequencing of a retinal dystrophy panel and direct Sanger sequencing to confirm KCNV2 gene variants, in addition to an electroretinogram (ERG) and spectral domain optical coherence tomography (SD-OCT). Results: Cone dystrophy with supernormal rod response is associated with identified variants in the KCNV2 gene. The genetic analysis of the first case identified a compound heterozygous mutation in the KCNV2 gene, including a de novo nonsense duplication at cDNA position 1109, which led to the premature termination of the p.Lys371Ter codon in the second extracellular domain of the protein. Two patients showed changes in the full-field electroretinogram, especially in the first case, which demonstrated a close to supernormal total electroretinogram amplitude. This study increased the range of the KCNV2 mutation database, added an unreported de novo substitution pattern to KCNV2 gene variants, and linked it to the evaluated clinical studies. Conclusions: The initial clinical manifestations were varied, but both patients presented with hypermetropia and slight exotropia. The ERG findings are characteristic of KCNV2 mutations, and patients exhibited an increased b-wave latency in DA3.0 ERG (combined rod–cone response).

Optogenetic gene therapy may employ recombinant adeno-associated virus (AAV) vectors for specific preclinical applications in the treatment of inherited retinal diseases. Human retinal explants as an ex vivo model assist determine cellular tropism and the level of subsequent gene expression followed by delivery of viral vectors. Extracts of retinal tissue can be obtained and cultured for a certain time; thus, it is possible to transduce and confirm the effectiveness of the virus using various methods, including immunohistochemical staining. Consequently, we described method for harvesting and culturing human retinal fragments from divergent parts of the retina that differ anatomically and molecularly. The fragments were fixed, sectioned and stained. A dissociation method has also been described to obtain a retinal cell suspension that can be used in other experiments such as flow cytometry or mRNA/protein level expression assays.