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.

Various methods exist within gene-targeted therapeutics. In the context of IRD (Inherited Retinal Dystrophy), the method employed generally depends on the genetic inheritance pattern of the causative gene mutation (recessive or dominant). Recessive mutations are characterized by a dysfunctional gene or protein product and do not impact the function of the wild-type gene. Patients with these recessive mutations manifest the disease only when both copies of a particular gene carry the mutation. The prevailing approach for gene therapy in such cases aims to introduce a functional gene from an external source to supplement the missing functionality—a method commonly referred to as gene supplementation.

In cases of dominant mutations, the disease can manifest even with a single faulty gene out of the two copies. This can occur due to either:

1. 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.
2. 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.

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.

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.

1. 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.
2. 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.

The enzymes employed in genome editing come in three main categories or generations:

1. First generation – ZFN (Zinc finger nuclease): Based on various transcription factors’ DNA binding motifs, each motif recognizes three base pairs of DNA.
2. 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.
3. 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.

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.

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.