Advancing Gene Therapy for Inherited Retinal Diseases
Inherited Retinal Dystrophy (IRD) refers to a set of diseases caused by genetic mutations that lead to the degeneration of vision cells and the retinal pigment epithelium (RPE). Retinitis Pigmentosa is the most prevalent, affecting an estimated 1.5 million people globally.
Our research is also geared towards the development of advanced medical treatments for rare and intractable disorders affecting the outer layer of the retina. Traditional therapies primarily manage symptom progression, leaving the root cause unaddressed. We are focusing on genome editing and HITI gene insertion as part of our innovative therapeutic strategies for Retinitis Pigmentosa, particularly those with mutations in the RHO gene. In collaboration with Hiroshima University, we have successfully optimized highly specific Zinc Finger Nucleases (ZFN) to achieve cutting efficiencies comparable to CRISPR-Cas9. We are also collaborating with Kobe Eye Center Hospital to develop accurate and efficient gene introduction techniques, offering a pathway towards more effective and lasting treatments.
Inherited Retinal Dystrophy (IRD)
Inherited Retinal Dystrophy (IRD) is a collective term for retinal disorders that exhibit progressive degeneration in the photoreceptor cells and the retinal pigment epithelium (RPE) due to genetic mutations. Retinitis Pigmentosa is the most common type of IRD, with an estimated 1.5 million patients worldwide. Genetic diagnoses from medical institutions, including the Kobe City Eye Center Hospital, have reported around 300 associated genes. Most of these genes are essential for the proper functioning of the RPE and photoreceptor cells. Consequently, mutations can cause degeneration in these cells, leading to their eventual death.
To date, no definitive treatment for Retinitis Pigmentosa has been established. The primary approach is symptomatic treatment aimed at slowing the progression of the disease. This includes medications (drugs to improve dark adaptation, blood flow, and vitamins) and assistive devices like sunglasses designed to block intense light.
Visual Impairment in Retinitis Pigmentosa and Age-Related Macular Degeneration
This figure illustrates the distinct vision impairments experienced by patients with Retinitis Pigmentosa (RP) and Age-related Macular Degeneration (AMD). Patients endure a gradual loss of vision over time. For RP patients, the loss first occurs in the peripheral vision, progressively closing in on the center. In contrast, AMD patients first experience loss of central vision, making it challenging to focus on details.
With the advancements in modern science and technology, medical treatments have evolved beyond traditional small molecule formulations to encompass diverse forms such as polymers, nucleic acids, and cells. To categorize these innovative therapies and drugs, the term “drug/medical modality” is used. Gene therapy stands out as a pioneering drug modality that leverages genes (like DNA or RNA) or targets a patient’s genetic makeup (genome). It often refers to gene-targeted therapeutics, which directly influence the causative genes of diseases. Given the improvements in gene diagnosis and medical infrastructure, there’s heightened anticipation for these therapies, especially for genetic disorders with identified causative genes. Conventional modalities (like small molecules or antibodies) had technical constraints, but now, more comprehensive therapeutic effects are expected. We are advancing the development of treatments, focusing specifically on the most common dominant genetic type of retinitis pigmentosa, particularly those with mutations in the RHO (rhodopsin) gene.
Gene Therapy for Recessive Mutations
Gene Therapy for Dominant Mutations
- Haploinsufficiency: Here, one healthy gene copy isn’t enough to maintain normal function. This condition can be addressed similarly to recessive mutations by introducing a normal gene.
- Dominant-negative (or gain-of-function) mutations: This occurs when the mutated gene negatively interferes with the normal gene’s function. Addressing this requires genome editing to repair the faulty gene.
How Mutated Rhodopsin Results in Photoreceptor Degeneration
Human rhodopsin comprises 348 amino acids. To date, genetic diagnostics have reported mutations in 110 distinct locations within the rhodopsin gene. Most of these mutated genes exhibit dominant-negative or gain-of-function characteristics. The functional changes are dependent on the mutation’s location, leading to issues like reduced light sensitivity, constant activation, or transportation defects of the rhodopsin protein. Furthermore, the inhibitory effect varies depending on the mutation, necessitating precise medication prescriptions using small molecules or other therapeutic agents. Importantly, since the rhodopsin gene can also cause the disease due to haploinsufficiency, gene therapies targeting RHO need to address both the repair of dominant-negative mutations and the replenishment of the normal RHO gene expression.
Genome editing-based therapy
Genome editing, which allows rewriting of cellular genetic information, holds great promise for treating gene mutations that manifest due to dominant inhibition. This technique targets specific sequences in DNA or its transcription product, mRNA, to introduce changes.
As part of our focus on gene therapy development, we are emphasizing treatments using genome editing. Through joint research with Hiroshima University, we have developed an efficient design method for ZFN (Zinc Finger Nucleases) with high specificity, achieving cutting efficiency in adult photoreceptor cells comparable to that of CRISPR-Cas9. Furthermore, we are also committed to developing treatment agents using HITI gene insertion. In addition, in collaboration with the Kobe Eye Center Hospital, we are advancing research aimed at establishing appropriate gene introduction methods.
Therapeutic Genome Editing Techniques
The commonly utilized genome editing approach involves “cutting” at the target sequence.
The induced cuts exploit two intrinsic cellular gene repair mechanisms to insert (recombine) genes or introduce mutations.
- Gene insertion: This approach uses the homology-directed repair (HDR) mechanism, where a repair gene is synthesized and recombined from an allele. During the cutting process, an externally provided template DNA with the correct sequence facilitates precise genome editing.
- Mutation introduction: This method employs the non-homologous end-joining (NHEJ) mechanism. NHEJ repairs the cut DNA ends by arbitrarily inserting or deleting bases (InDels). As these InDels are random, they usually result in target gene modifications, primarily knockouts.
Genome Editing Tools
The enzymes employed in genome editing come in three main categories or generations:
- First generation – ZFN (Zinc finger nuclease): Based on various transcription factors’ DNA binding motifs, each motif recognizes three base pairs of DNA.
- Second generation – TALEN (Transcription activator-like effector nuclease): Originating from bacterial DNA binding motifs, each motif recognizes a single base of DNA. Compared to ZFN, TALEN recognizes longer sequences but allows designing high specificity genome editing tools.
- Third generation – CRISPR (Clustered regularly interspaced short palindromic repeats): Founded on the bacterial immune mechanism (CRISPR/Cas system), it consists of the Cas nuclease and a single-stranded guide RNA (sgRNA) recognizing the target sequence. Unlike ZFN and TALEN that use protein motifs for base recognition, CRISPR identifies target sequences through RNA-DNA interactions. This simplifies target sequence prediction and design, significantly advancing genome editing research.
High-Efficiency Genome Editing Gene Therapy
For dominant mutations, such as those targeting rhodopsin, a gene therapy remedy needs to address both the repair of the causative mutation causing dominant inhibition and the replenishment of normal RHO gene expression. Since there are 110 potential mutation sites in the rhodopsin gene locus, it’s more efficient to insert (or replace with) the full-length normal rhodopsin gene rather than develop a separate gene therapy for each mutation. This way, one treatment can potentially address all mutations, including any new ones.
When introducing external, normal-sequence DNA, the common approach utilizes HDR-based genome editing. A challenge arises since HDR is prevalent in dividing cells but less so in non-dividing cells, and the majority of adult tissue cells are non-dividing. To address this, the HITI (Homology-independent targeted integration) method was developed. It leverages the NHEJ repair pathway, notable for its high efficiency in non-dividing cells, to accurately insert foreign genes. This makes gene insertion in specific sequences of adult cells, such as retinal cells, achievable. By positioning the normal sequence DNA just prior to the start codon of a mutated gene locus, the normal gene is expressed using the original gene’s promoter, suppressing the mutated gene’s expression. Essentially, this method simultaneously rectifies dominant mutations causing dominant inhibition and restores the expression of the normal RHO gene. We are advancing the development of gene therapy drugs targeting all rhodopsin mutations by applying this HITI method.
Genome Editing with ZFN (Zinc Finger Nuclease)
While ZFNs offer the advantage of a smaller molecular weight, making them easier to load onto vectors like AAV, designing a highly specific ZFN for a target sequence is challenging and time-consuming. However, our joint research with Hiroshima University has achieved high-throughput development of highly specific ZFNs. Leveraging Hiroshima University’s FirmCut nuclease ND1, we established ZFN development technology that achieves a cleavage efficiency comparable to CRISPR-Cas9 in adult retinal cells.
In developing therapeutic agents through HITI gene insertion, it’s vital to craft the right constructs to ensure peak gene functionality, pinpoint the optimal vector for gene delivery, and hone the insertion techniques. Drawing from insights in functional genomics and stem cell development, our research diligently focuses on designing constructs for optimized gene functionality. Collaboratively working with Synplogen Co., Ltd. in Kobe, we’re pioneering the development of high-quality adenoviral vectors. Moreover, in partnership with the Kobe Eye Center Hospital, we’re advancing our understanding and methodology of precise gene insertion techniques.