*4.2.1 Gene therapy*

Identification of pathogenic genes promotes the generation of animal models and elucidates the physiological functions of gene products to a certain extent, thus promoting the development of gene therapy. So far, most research has focused on saving retinal organoid disease phenotypes through gene editing of patient-specific induced pluripotent stem cells [72, 73, 87, 97, 105, 106]. However, this strategy cannot be applied to patients. Adeno-associated virus (AAV) show great promise as a gene therapy vector for a wide range of retinal diseases. For example, AAV-mediated gene augmentation has successfully treated LCA caused by RPE65 mutations [107]. AAVmediated gene therapy based on retinal organoids has also shown promising results in the laboratory [81, 82, 108]. In addition, gene therapies such as antisense morpholino and antisense oligonucleotides (AONs) have also been reported (**Table 2**).

### *4.2.2 Cell replacement therapy*

Hereditary retinal degenerative diseases such as RP, Stargardt's disease and LCA are the leading cause of incurable blindness. The vision loss associated with these diseases is caused by the death of photoreceptors in the retina. Existing treatments, including neuroprotection and gene therapy, require the presence of endogenous photoreceptors. In addition, due to the complex mechanism of retinal degeneration diseases, especially RP, it has been found that there are multiple genes with multiple mutation modes, and treatment methods focusing on a single mutation are extremely difficult technically and economically. Thus, transplant-based photoreceptor cell replacement becomes an attractive therapeutic strategy for restoring visual function and, if successful, could be applied to a wide range of retinal degenerative diseases.

Research on retinal cell transplantation dates back to 2006 [109]. Mice were able to effectively integrate rod photoreceptor precursor cells isolated from juvenile mice retinas into the ONL. These cells can further differentiate in the host retina and exhibit morphological characteristics typical of mature photoreceptors, such as inner and outer segments, while expressing molecules necessary for light transduction,


#### **Table 2.**

*Gene therapy based on retinal organoids.*

forming synaptic connections with downstream cells, generating light responses and promoting visual function [109–115]. These results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring visual acuity after retinal degeneration.

However, this cannot be applied to the treatment of retinal diseases in humans. There are ethical challenges to primary photoreceptors transplantation, but stem cell-based photoreceptors can avoid this problem. It has been shown that photoreceptor cells derived from stem cells can be integrated into mouse retinas, restoring the animal's response to light [116, 117]. This is far from enough, until the appearance of retinal organoids, retinal cell transplantation and clinical transformation have made a breakthrough.

Transplantation of retinal organoids, mainly photoreceptors, is also a process of constant exploration [118]. The safety and effectiveness of transplantation, the enrichment and purification of transplanted cells, the effects of retinal organoids at different stages of development and host retinas with different degrees of degeneration on the efficiency of transplantation, and the evaluation of cell integration and function after transplantation are all issues that need to be explored.

**Table 3** gives a brief summary of some retinal organoid transplantation cases in recent years. There are two transplantation methods: one is to digest the retinal




#### **Table 3.**

*Research on transplantation of retinal organoids.*

organ into a single cell, from which the photoreceptor cells are purified and enriched, and the transplantation is carried out in the form of cell suspension. Another method is to strip the photoreceptor layer from the retinal organ and transplant it in thin slices. This method is more difficult to operate, because it is difficult to maintain the correct shape and polarity of the retina when it is transplanted into the eye of the host, and appropriate transplantation instruments need to be designed. The implant may contain some interneurons that block the connection between photoreceptor cells and the remaining inner layer of the retina in the host, and there are eye size requirements in the host animal. The implant is usually performed in rats, cats and non-human primates. In general, we have gained a lot from the exploration of retinal organ transplantation. A number of studies have shown that transplanted cells or tissues can survive in the host eye for a long time, migrate and integrate into the correct location. Integrated cells can further differentiate and mature in vivo, presenting typical cell structures, such as internal and external segments, and expressing corresponding cell markers and synaptic markers. In some studies, the formation of synaptic connections between host and graft and improvement of host visual function were also observed. In the host, transplanted cells or tissues are electrically excitatory [136], indicating their potential for restoring visual function. Through behavioral and electrophysiological experiments, we found that the host can not only slow down the progressive visual loss to some extent, but also show a relatively significant recovery of visual function [127, 130, 131, 134, 135, 137, 138]. These are exciting results and suggest that cell replacement therapy based on retinal organoids is a promising treatment that will bring light to patients with retinal diseases.

### **4.3 Retinal organoids for drug screening**

Drug development focuses on screening, a process that requires cell models. The closer the cell model was to the physiological state, the more accurate the screening was. Therefore, organoids are undoubtedly a better choice for drug screening. Some ocular supplements, vitamin E, lutein, astaxanthin, and anthocyanin, have been reported to protect retinal photoreceptor degeneration induced by 4-hydroxytamoxifen (4-OHT) and light [139]. Few studies have successfully screened effective drugs using retinal organoids. In addition, there are some studies using retinal organoids as screening tools to explore the membrane transport effects of some microbial opsin [140]. These results suggest that retinal organoids can be used to validate the effectiveness of some therapeutic products and drug molecules.
