Do not give up chasing the light—Gene therapy aids the blind in regaining their sight

It is said that the eyes are the windows to the soul, but there are many people with congenital blindness who cannot "see" the light. According to publicly available data from the World Health Organization, there are at least 2.2 billion people globally with impaired vision or blindness. China, in particular, has the highest number of blind individuals in the world, accounting for 18% to 20% of the total global population of blind people. Genetic eye diseases constitute approximately one-third of childhood blindness and visual impairment cases and account for 80% of cases of blindness and severe visual impairment. Congenital (inherited) eye diseases are currently the most common cause of blindness in children and adolescents, clearly posing the most significant threat to the vision of future generations.

Inherited Retinal Diseases (IRD) are a heterogeneous group of genetic disorders that affect the retina. They result in visual impairments due to dysfunction and degeneration of photoreceptors, retinal pigment epithelium, or choroid. There are over 300 pathogenic genes associated with IRD. However, current research primarily focuses on common photoreceptor IRDs and macular degeneration diseases, including Retinitis Pigmentosa, Leber Congenital Amaurosis, and Stargardt Disease.

Many rare IRDs remain incurable, and without treatment, patients suffer from severe visual impairments and blindness. Since most rare IRDs have a monogenic nature and the retina is an efficient target site for gene therapy vectors like Adeno-Associated Virus (AAV), there is a growing availability of standardized pharmacological efficacy assessment methods for ocular diseases. As such, gene therapies are quickly emerging as a new hope for patients with rare diseases of the eye, such as IRDs.

In this context, we will introduce the development of gene therapy approaches for several rare IRDs and recommend reliable preclinical animal disease models for preclinical drug efficacy assessment.

Gyrate Atrophy Of Choroid & Retina (GACR)

Gyrate atrophy of choroid & retina (GACR) is a rare autosomal recessive genetic disorder classified as an amino acid metabolism disorder. It is characterized by chorioretinal degeneration and atrophy caused by pathogenic mutations in the gene encoding mitochondrial enzyme Ornithine Aminotransferase (OAT). OAT is primarily expressed in the liver, and individuals with GACR exhibit elevated levels of ornithine in their blood and other bodily fluids. Patients experience progressive narrowing of their visual field, ultimately leading to blindness. Early symptoms typically include night blindness and peripheral choroid and retinal degeneration-induced visual field constriction. While in later stages, there may be significant central vision loss, and complete blindness can occur when the macula is affected. Currently, there is no curative treatment, but some propose that high levels of ornithine may be toxic to the delicate structure of retina, and early interventions to reduce ornithine levels might potentially prevent or delay disease progression ^[1-2].

Mice lacking OAT exhibit neonatal hypoornithinemia and lethality, which can be rescued by short-term arginine supplementation. Post-weaning, they develop hyperornithinemia similar to human GACR patients. Their retinal degeneration progresses slowly, manifested by a gradual decrease in electroretinogram (ERG) amplitude. Over time, abnormalities appear in the retinal pigment epithelium, the outer segments of the photoreceptors become shortened and disorganized, and photoreceptor cells are progressively lost. OAT-deficient mice display a disease phenotype resembling human GACR, making them excellent preclinical animal models ^[3-4].

Figure 1. Significant Reduction in Electroretinogram (ERG) Amplitude in OAT-Deficient Mice (Oat-/-) ^[3]

Gene therapy has emerged as an effective solution for many monogenic genetic diseases. In a recent study, AAV8 vectors were used to deliver liver-specific promoters to regulate the OAT gene for treatment. Following the administration of this gene therapy vector, the levels of ornithine in the blood and eyes of OAT-deficient mice decreased, electroretinograms showed improvement, and retinal structure partially recovered. These effects were sustained for at least one year ^[5]. The study demonstrated that AAV8-OAT gene therapy effectively corrected hyperornithinemia and improved both the function and structure of the retina. This provides a conceptual proof of effectiveness for liver-targeted AAV-mediated gene therapy for GACR.

Figure 2. AAV-Mediated Liver-Specific OAT Gene Therapy Improves Disease Phenotype in OAT-Deficient Mice (OatΔ) ^[5]

Usher Syndrome Type III (USH3)

Usher Syndrome, also known as Genetic Deafness-Retinitis Pigmentosa Syndrome, is a group of autosomal recessive inherited genetic disorders characterized by retinitis pigmentosa (RP) and varying degrees of hearing loss. Among this group of genetically heterogeneous disorders, Usher Syndrome Type III (USH3) is the rarest form. USH3 is characterized by progressive sensorineural hearing loss in adolescence and retinitis pigmentosa. The clarin-1 (CLRN1) gene is the only known gene associated with the pathogenesis of USH3.

Individuals with Usher Syndrome Type III typically have normal hearing at birth but usually begin to experience hearing loss during late childhood or adolescence, which worsens over time. Similar to the hearing phenotype, the ocular phenotype is also progressive. Visual symptoms often appear around puberty, with many patients initially experiencing night blindness, followed by a gradual reduction in their visual field, resulting in tunnel vision. Subsequently, they lose central vision and color perception, ultimately progressing to complete blindness ^[6].

In mice, the knockout (KO) of the Clrn1 gene results in hearing impairment and retinal degeneration. The specific ocular phenotype includes a reduction in the number of photoreceptor cells in the retina, leading to visual impairment and a narrowed visual field. These symptoms typically appear early in the postnatal stage of mice and worsen over time. Additionally, Clrn1-KO mice exhibit hair cell defects as early as the second day after birth and become deaf around P21-P25.

Researchers have been able to induce late-onset progressive hearing loss related to deterioration in the hair bundle structure in Clrn1-KO mice by introducing the Clrn1 gene containing UTR regions. In addition to visual and hearing impairments, Clrn1-KO mice may also manifest phenotypic changes in other systems, such as balance disorders and motor coordination impairments ^[7-10], similar to the human USH3 phenotype.

Figure 3. Defective Electroretinogram (ERG) Amplitude in Clrn1-Knockout Mice (Clrn1-/-) ^[8]

Similarly, as a monogenic inherited disease, gene therapy approaches targeting USH3 have been under development. Several studies have been published regarding AAV-based gene therapy for USH3 ^[9-11]. Dinculescu et al. explored the feasibility of AAV-CLRN1 gene therapy in vivo using Clrn1-KO mice. Their results demonstrated that all major classes of retinal cells expressed AAV-CLRN1 driven by a ubiquitous, constitutive chicken β-actin promoter. Exogenous CLRN1 is primarily localized to the inner segment region and outer association layer, similar to the expression pattern of endogenous protein, which is crucial for the design of future USH3 gene therapy studies.

However, subretinal delivery with full-strength viral titers resulted in significant retinal functional loss, suggesting the presence of a critical threshold for CLRN1 expression in photoreceptor cells. These findings emphasize the need to carefully select the right AAV vector dosage, promoter, and delivery methods to develop safe USH3 therapeutic treatment approaches ^[11].

Figure 4. Effects of AAV-CLRN1 Gene Therapy on Retinal Protein Expression in Clrn1-Knockout and Wild-Type Mice ^[11]

Choroideremia (CHM)

Choroideremia (CHM) is a rare X-linked recessive genetic disease with a global incidence of approximately 1 in 50,000 to 1 in 100,000 individuals. The primary symptoms of the disease include a gradual decline in central vision, narrowing of the visual field, and color vision abnormalities. Choroideremia is caused by mutations in the CHM gene, which is located on the X chromosome, making males more commonly affected than females. The CHM gene encodes the membrane skeletal protein Rab escort protein-1 (REP1), which plays a crucial role in retinal pigment epithelial cells. Loss or dysfunction of REP1 leads to the degeneration of retinal pigment epithelial cells and retinal atrophy, resulting in the development of choroideremia. Currently, there is no cure for choroideremia, and treatment options are focused on symptom management ^[12]. In animal studies, mice with systemic or retina-specific knockout (KO) of Chm/REP1 exhibit eye-related phenotypic abnormalities, including progressive degeneration of photoreceptors, defective Rab prenylation, retinal pigment epithelial cell degeneration, retinal atrophy, retinal vascular abnormalities, and visual impairment ^[13-14].

Figure 5. Retina-Specific Knockout of Chm/Rep1 Leads to Severe Degeneration of Photoreceptors ^[14]

Currently, there are at least eight clinical trials evaluating gene therapy mediated by subretinal delivery of CHM-expressing adeno-associated virus (AAV) vectors as a treatment for choroideremia (CHM). Prominent examples include Biogen's BIIB111/AAV2-REP1 and 4D Molecular Therapeutics' 4D-110 ^[15]. While BIIB111/AAV2-REP1 did not meet its primary endpoint or key secondary endpoints in the Phase 3 STAR study, clinical and preclinical data prior to the STAR study still provides valuable reference data for future gene therapy approaches to treating CHM. Insights gathered from this research contribute to shaping innovative treatments for inherited retinal diseases, including choroideremia. In preclinical studies of these drugs, Chm/Rep1 gene knockout (KO) mice remain essential models for preclinical drug efficacy evaluations. In one study, AAV2/2-CBA-REP1 gene therapy had dose-dependent effects on the a-wave and b-wave amplitudes of dark-adapted ERG in the retina of Chm/Rep1 gene KO mice. Higher doses of AAV2/2-CBA-REP1 treatment demonstrated an improvement in dark-adapted retinal function in Chm/Rep1 gene KO mice ^[16].

Figure 6. Efficacy Assessment Experiment of AAV2/2-CBA-REP1 Using Chm/Rep1 Gene Knockout Mice ^[16]

Rare Disease Research Resources - Genetically Modified Mouse Models

Genetically modified mouse models play a crucial role in research of rare disease mechanisms and preclinical drug development and evaluation. Cyagen offers thousands of independently developed genome-engineered mouse strains, including various gene knockout (KO) and conditional (e.g. tissue-specific) KO (cKO/floxed) models for rare diseases, such as OAT, CLRN1, and CHM. Additionally, our custom animal model (CAM) development and CRO services can be tailored to your research needs, accelerating your rare disease study with our comprehensive, quick-turnaround CAM/CRO solutions.

Cyagen Ophthalmic Disease CRO Services

As a comprehensive contract research organization (CRO) service provider, Cyagen recognizes ophthalmic diseases as a breakthrough point for gene therapy and has established an ophthalmic gene therapy platform to support preclinical research. Our experienced professional team can perform standardized evaluation services with our suite of state-of-the-art ophthalmic instruments for small animals.  Our ocular technologies include the Micron IV small animal retinal microscopic imaging system, full-field electroretinogram (ffERG), image-guided optical coherence tomography (OCT) system, and handheld ophthalmotonometer for mice. We can provide detection services for rodent models of eye-related diseases including diabetic retinopathy, retinoblastoma, macular degeneration, pediatric retinopathy of prematurity (ROP), choroidal neovascularization, and retinitis pigmentosa. With 16 years of gene editing model construction experience, Cyagen can provide you with an array of standardized preclinical research solutions for ophthalmic gene therapy.

HUGO-GT^TM Program

To accelerate gene therapy drug development, Cyagen has launched the Next-Generation Humanized Mouse Model Development Program. Driven by our novel whole genomic DNA  Humanized Genomic Ortholog for Gene Therapy (HUGO-GT^TM) mice, which provide full genomic coverage of pathogenic genes for efficient drug screening of gene therapy methods including ASO/CRISPR/siRNA. Building upon the Rare Disease Data Center (RDDC), a comprehensive biological data platform for accessing disease-gene-animal model-drug clinical information, Cyagen has utilized TurboKnockout technology and BAC fusion technology to go from large-scale to complete genomic replacement and humanization capabilities. This has resulted in the creation of more suitable whole-genome humanized models for research on rare congenital diseases and the development of gene therapy drugs.

In the field of ophthalmic diseases, Cyagen has independently developed whole-genome humanized mice, including the wild-type whole-genome humanized mouse hRHO, as well as related ophthalmic disease models built upon it, such as the hRHO-P23H point mutation model, and other ophthalmic models like hCEP290.

References:

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[4]Wang T, Lawler AM, Steel G, Sipila I, Milam AH, Valle D. Mice lacking ornithine-delta-aminotransferase have paradoxical neonatal hypoornithinaemia and retinal degeneration. Nat Genet. 1995 Oct;11(2):185-90.

[5]Boffa I, Polishchuk E, De Stefano L, Dell'Aquila F, Nusco E, Marrocco E, Audano M, Pedretti S, Caterino M, Bellezza I, Ruoppolo M, Mitro N, Cellini B, Auricchio A, Brunetti-Pierri N. Liver-directed gene therapy for ornithine aminotransferase deficiency. EMBO Mol Med. 2023 Apr 11;15(4):e17033.

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[7]Johnson KR, Gagnon LH, Webb LS, Peters LL, Hawes NL, Chang B, Zheng QY. Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene. Hum Mol Genet. 2003 Dec 1;12(23):3075-86.

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[10] Geng R, Geller SF, Hayashi T, Ray CA, Reh TA, Bermingham-McDonogh O, Jones SM, Wright CG, Melki S, Imanishi Y, Palczewski K, Alagramam KN, Flannery JG. Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation. Hum Mol Genet. 2009 Aug 1;18(15):2748-60.

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[12] Brambati M, Borrelli E, Sacconi R, Bandello F, Querques G. Choroideremia: Update On Clinical Features And Emerging Treatments. Clin Ophthalmol. 2019 Nov 18;13:2225-2231.

[13] Kalatzis V, Roux AF, Meunier I. Molecular Therapy for Choroideremia: Pre-clinical and Clinical Progress to Date. Mol Diagn Ther. 2021 Nov;25(6):661-675. 

[14] Tolmachova T, Anders R, Abrink M, Bugeon L, Dallman MJ, Futter CE, Ramalho JS, Tonagel F, Tanimoto N, Seeliger MW, Huxley C, Seabra MC. Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia. J Clin Invest. 2006 Feb;116(2):386-94.

[15] Tolmachova T, Wavre-Shapton ST, Barnard AR, MacLaren RE, Futter CE, Seabra MC. Retinal pigment epithelium defects accelerate photoreceptor degeneration in cell type-specific knockout mouse models of choroideremia. Invest Ophthalmol Vis Sci. 2010 Oct;51(10):4913-20.

[16] Cehajic Kapetanovic J, Barnard AR, MacLaren RE. Molecular Therapies for Choroideremia. Genes (Basel). 2019 Sep 23;10(10):738.