Regardless of the gene’s size and nature of the disease-causing variant, gene editing tools provide unprecedented opportunities for effective and permanent treatment of both dominant and recessively inherited, but also X-linked forms of vision loss. At the forefront of this revolutionary approach, we are actively developing and testing novel editing strategies to target diverse pathogenic variants in known Inherited Retinal Disease genes. In a first proof-of-concept study, we successfully corrected a pathogenic deep intronic variant in ABCA4, a gene linked to Stargardt disease and other cone-rod dystrophies.

• De Angeli P, Reuter P, Hauser S, Schöls L, Stingl K, Wissinger B, Kohl S. Effective splicing restoration of a deep-intronic ABCA4 variant in cone photoreceptor precursor cells by CRISPR/SpCas9 approaches. Mol Ther Nucleic Acids. 2022 Jul 31;29:511-524. doi: 10.1016/j.omtn.2022.07.023. eCollection 2022 Sep

Our current projects are focused on therapeutically viable editing strategies, primarily harnessing the potential of our enhanced-deletion editing platform. Key areas of interest include achieving mutation-independent rescue for autosomal dominant inherited retinal diseases (such as RHO), unbiased exploration for the most promising gRNA/Cas combinations for rescuing common variants causing missplicing in the ABCA4 gene, and addressing the common USH2A variant c.7595-2144A>G. Three different mutation types contribute to X-linked Blue cone monochromatism (BCM). Point mutations, most commonly the C203R inactivating missense mutation, exon 3 interchange haplotype mutations resulting in missplicing, and large deletions. In a recent study, we were able to characterized the extent of these deletions in >70 families and identified 42 distinct structural variants ranging from 142 bp to 207 kb. By the deletion mapping we also were able to refine the upstream locus control region to an essential enhancer element of 358 bp.

• Wissinger et al. The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying Blue Cone Monochromacy: Evidence for the instability of gene clusters with increased copy number. PNAS June 27, 2022; 119 (27) e2115538119; doi.org/10.1073/pnas.2115538119

For autosomal recessively inherited Achromatopsia (Rod monochromatism), a subject of intense research for many years, we have been able to identify all disease-associated genes - CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6. Finding pathogenic variants in ATF6 in patients affected by Achromatopsia was intriguing, as all other achromatopsia genes are exclusively expressed in the cone photoreceptor, and encode for proteins of the phototransduction cascade. In contrast, ATF6 is expressed in every cell of the body and is known for its function in endoplasmatic reticulum stress regulation and the unfolded protein response. Why and how pathogenic variants in ATF6 exclusively causes a cone photoreceptor defect is to date unknown.

• Kohl et al. Mutations in the unfolded protein response regulator ATF6 cause the cone dysfunction disorder achromatopsia. Nat Genet. 2015;47:757ff. DOI: 10.1038/ng.3319

In this context, we have amassed the world's most extensive collection of patient DNA for this rare disorder, comprising over 1000 patients and their families, achieved through international collaboration. We have produced comprehensive papers detailing the mutation spectrum of the Achromatopsia associated genes, enabling the assessment of the prevalence of these genes, along with the prevalence of specific common recurrent mutations. Deep genetic characterization identified yet hidden variations in CNGB3 including copy number variations and deep intronic mutations as an important disease cause solving a considerable portion of yet unexplained cases.

• Mayer et al. CNGB3 mutation spectrum including copy number variations in 552 achromatopsia patients. Hum Mutat. 2017;38:1579ff. DOI: 10.1002/humu.23311
• Weisschuh et al. Mutations in the gene PDE6C encoding the catalytic subunit of the cone photoreceptor phosphodiesterase in patients with achromatopsia. Hum Mutat. 2018;39:1366ff. DOI: 10.1002/humu.23606
• Felden et al. Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene. Hum Mutat. 2019;40:1145ff. DOI: 10.1002/humu.23768
• Weisschuh et al. Deep-intronic variants in CNGB3 cause achromatopsia by pseudoexon activation. Hum Mutat. 2020;41:255ff. DOI: 10.1002/humu.23920
• Solaki et al. Comprehensive variant spectrum of the CNGA3 gene in patients affected by achromatopsia. Hum Mutat. 2022 Jul;43(7):832-858. doi: 10.1002/humu.24371

Today, in the era of high-throughput genetic sequencing for both diagnostic and research purpose, and first gene therapeutic agents for inherited retinal dystrophies evolving, standardized variant classification has become essential. It is crucial to verify that the identified variant(s) in a given patient, and especially the target of gene therapy, definitively are the genetic cause of the patient's disease. Guidelines for variant interpretation have been developed (i.e., ACMG/AMP classification) and are further standardized on a subject-, gene- or variant-specific basis. In this context, functional assessment of variants, especially for missense, in-frame deletion or insertion or late nonsense variants, and variants suspected or predicted to impact splicing need further attention. In our research efforts, we have pioneered the development of minigene splice assays, employing them to scrutinize variants that are suspected to induce missplicing. This enabled us to assess the potential impact of these variants on splicing, confirming the pathogenic effect of the majority of tested variants and allowing for variant re-classification.

• Reuter et al. Systematic analysis of CNGA3 splice variants identifies different mechanisms of aberrant splicing. Sci Rep. 2023 Feb 18;13(1):2896. doi: 10.1038/s41598-023-29452-9. PMID: 36801918
• Bodenbender, et al. Biallelic Variants in TULP1 Are Associated with Heterogeneous Phenotypes of Retinal Dystrophy. Int J Mol Sci. 2023 Feb; 24(3): 2709. DOI: 10.3390/ijms24032709 Reith, et al.
• A Novel, Apparently Silent Variant in MFSD8 Causes Neuronal Ceroid Lipofuscinosis with Marked Intrafamilial Variability. Int J Mol Sci. 2022 Feb; 23(4): 2271. DOI: 10.3390/ijms23042271
• Weisschuh, et al. Mutations at a split codon in the GTPase-encoding domain of OPA1 cause dominant optic atrophy through different molecular mechanisms. Hum Mol Genet. 2022 Mar 1; 31(5): 761–774. DOI: 10.1093/hmg/ddab286
• Weisschuh et al. Clinical Characteristics of POC1B-Associated Retinopathy and Assignment of Pathogenicity to Novel Deep Intronic and Non-Canonical Splice Site Variants. Int J Mol Sci. 2021 May 20;22(10):5396. DOI: 10.3390/ijms22105396

To analyze the effect of putatively disease-associated missense (and in-frame deletion) variants on CNG channel function, we have developed an in vitro aequorin-based luminescence bioassay. The analysis of 150 variants in CNGA3 has already been completed, and the system is now transferred to test such variants in CNGB3, and subsequently CNGA1 and CNGB1. Solaki et al. Functional evaluation allows ACMG/AMP-based re-classification of CNGA3 variants associated with achromatopsia. Genetics in Medicine. doi.org/10.1016/j.gim.2023.100979 Culminating out of all this research, we were able to coordinate and participate in the first-in-man phase I/II clinical safety study for gene supplementation therapy of CNGA3-associated achromatopsia (http://www.rd-cure.de/). In the preceding natural history study we assessed and described the phenotype of 32 CNGA3-associated Achromatopsia patients, of which 9 were finally treated in the clinical trial by subretinal injection. 

• Zobor, RD-Cure Consortium, et al. The Clinical Phenotype of CNGA3-Related Achromatopsia: Pretreatment Characterization in Preparation of a Gene Replacement Therapy Trial. Invest Ophthalmol Vis Sci. 2017;58:821ff. doi: 10.1167/iovs.16-20427.
• Fischer, RD-Cure Consortium, et al.. Safety and Vision Outcomes of Subretinal Gene Therapy Targeting Cone Photoreceptors in Achromatopsia: A Nonrandomized Controlled Trial. JAMA Ophthalmol. 2020;138:643ff. DOI: 10.1001/jamaophthalmol.2020.1032
• Reichel, RD-Cure Consortium, et al.. Three-year results of phase I retinal gene therapy trial for CNGA3-mutated achromatopsia: results of a non randomised controlled trial. Br J Ophthalmol. 2021 May 18. Online ahead of print. DOI: 10.1136/bjophthalmol-2021-319067

A considerable number of cases with a clinical diagnosis of Leber hereditary optic neuropathy (LHON) used to remain genetically undefined. Only recently it has been shown that autosomal recessive mutations in DNAJC30 account for a large proportion of these cases. We were able to replicate this in a large cohort of genetically unsolved LHON and optic atrophy cases, and provide evidence that the missense variant c.152A>G;p.(Tyr51Cys) accounts for 90% of disease-associated alleles in this autosomal recessively inherited LHON cohort, and we confirmed a strong founder effect for this missense variant in the European population. In addition, clinical investigation of these patients with arLHON revealed a younger age of onset, a more frequent bilateral onset and an increased clinically relevant recovery compared with LHON associated with disease-causing variants in the mitochondrial DNA.

• Kieninger et al. DNAJC30 disease-causing gene variants in a large Central European cohort of patients with suspected Leber's hereditary optic neuropathy and optic atrophy. J Med Genet. 2022 Jan 28. Online ahead of print. DOI: 10.1136/jmedgenet-2021-108235