1. Treating Retinitis Pigmentosa with Stem Cell Therapy

Cell therapy is a potential method for vision restoration that can be achieved through two forms: (1) replacing dysfunctional cells with effective stem cells and (2) restoring the dysfunctional cells by releasing trophic factors. Successful integration and the formation of new synaptic connections with the host are essential for transplanted cells to be functional ^[1].

1.1. Potential Models and Sources

1.1.1. The Possibilities of Embryonic and Pluripotent Stem Cells

Embryonic stem cells (ESCs) are pluripotent stem cells that can self-renew and develop into the three primary germ cells. However, ESCs can be rejected by the immune system. Induced pluripotent stem cells (iPSCs) provide an alternative source that can differentiate into any somatic cell and are generated from a somatic cell line without using human embryos. Dysfunction of the retinal pigment epithelium causes significant vision loss by affecting photoreceptors. Replacing damaged retinal pigment epithelium and photoreceptors with healthy pluripotent stem cells can delay disease progression and potentially restore vision loss ^[2][3].

In 2004, rat models demonstrated the potential of iPSC implantation to improve vision. In 2021, Surendran et al. investigated the effects of hiPSC-derived retinal cells in mice, observing improved photoreceptor progenitor and retinal pigment epithelium functions,increased pigmentation and ciliation, tight junction protein expression, and growth factor secretion. Retinal organoid transplantation in rats also showed significant visual function improvement and integration into the host. Recent studies have demonstrated the efficacy of hiPSC-RPE cells in slowing the progression of retinal degeneration in a swine model. In addition, combining RPE and retinal sheets in RSC rats can fully replace the degenerated retina, making it a potential therapy for advanced retinal degeneration.

The use of iPSCs has several advantages over using human embryos, such as ethical concerns and the ability to match patients based on blood type. However, some drawbacks should be considered. iPSCs may retain gene expression from the original cells, which can affect properties like senescence and proliferation (epigenetic memory). Furthermore, iPSCs' ability to proliferate indefinitely raises safety concerns, including the formation of teratomas.

1.2. Current Bone Marrow Stem Cells Therapies

Bone marrow stem cells (BMSCs) are a type of multipotent stem cell found in bone marrow, including mesenchymal stem cells (MSCs) and hematopoietic stem cells (CD34+ cells). BMSCs are mainly studied for their potential in treating retinitis pigmentosa (RP) due to their ability to release anti-angiogenic and neurotrophic factors, as well as immunomodulatory proteins like insulin-like growth factor-1 (IGF-1), MHC class II antigens, and Th2-related cytokines. While the ability of BMSCs to fully differentiate into photoreceptors is still under investigation, preclinical studies have shown promising results in tracking PKH26-dyed cells and observing their integration and counteraction of inflammation in mouse models. Overall, BMSCs have the advantage of being easily extracted and manipulated and have the ability to migrate towards lesion sites and trans-differentiate.

1.3. Therapies Based on Stem Cell-Derived RPE and Retinal Progentiator Cells

Stem cells have the potential to provide trophic support for photoreceptors. Early clinical trials using encapsulated RPE cells producing ciliary neurotrophic factors have shown photoreceptor protection in patients with RD. In vivo studies on mice have shown that transplanting MSCs into the vitreous can improve the survival and preservation of photoreceptors, delaying the progression of RP. Transplanting both fetal RPE cells and MSCs has significantly improved ERG results and increased the survival rate of transplanted cells. Bioengineering of cells is a promising strategy to increase the number of photoreceptor cells and promote the angiogenesis needed for repair and regeneration. Conjunctiva mesenchymal stem cells have been induced into photoreceptor-like cells in fibrin gel to form 3D scaffolds. These studies indicate that coculture transplantation is more beneficial compared to single-cell transplantation.

Retinal progenitor cells (RPCs) are multipotent stem cells derived from human fetuses between 16 to 20 weeks of gestation, and can be induced to differentiate into photoreceptors and other neuronal cells of the retina in vitro ^[4][5][6]. Transplantation of RPCs has been shown in preclinical studies to improve visual function and form functional synapses with host cells, indicating the potential to stabilize or reverse progressive vision loss ^[7][8][9]. In particular, C-Kit+ RPCs derived from organoids have shown exceptional results in forming functional synapses with host cells in mice ^[8]. Additionally, RPCs secrete trophic factors that can promote retinal survival and photoreceptor replacement ^[9].

Stem cell therapy has shown promising potential for regenerating the retina in preclinical studies, with the ability to differentiate retinal progenitor cells into photoreceptors and integrate transplanted cells into the degenerating retina. The formation of functional synapses between host and transplanted cells also suggests the potential for stabilizing or reversing progressive vision loss. While clinical trials are yet to be conducted, these findings hold great promise for the future of stem cell therapy.

2. Optogenetics: Novel Therapeutic Targets in Preclinical Phase

Retinal gene therapies have limitations that prevent them from compensating for all cases of visual function loss, such as patients without genetic diagnoses and those with advanced diseases and no remaining photoreceptors. Optogenetics is a more universal therapy that can be used in such cases, and it involves converting non-light-sensitive retinal cells into artificial photoreceptors by introducing light-sensitive proteins, or opsins, into the cells. There are different types of opsins that can be used depending on the target cell and method employed. Depolarized opsins can mimic an "on" response, while hyperpolarized opsins can be used on dormant cells. Opsin genes used for optogenetic vision restoration can be categorized into two superfamilies: microbial opsins (Type 1) and animal opsins (Type 2). These opsins differ in their light sensitivity, function, and utility for vision restoration. Type 1 opsins directly affect ion channels or pumps upon light absorption, while Type 2 opsins cause intracellular G-protein-coupled receptor signaling cascades that indirectly affect ion channels.

2.1. Microbial-Derived Opsins

Garita-Hernandez et al. conducted a study to evaluate the efficiency and toxicity of different microbial opsins in human retinal organoids derived from hiPSCs ^[10]. Various depolarizing and hyperpolarizing microbial opsins, including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws, were tested. eNpHR 3.0, ReaChR, and Jaws exhibited the highest membrane localization, while CatChR and ChrimsonR showed lower membrane localization due to protein accumulation in the cytosol at high doses, leading to UPR ^[11][12]. These findings suggested that hiPSC-derived retinal organoids are useful for predicting optogenetic protein expression patterns and their effects on human retinal contexts. Recent advances in optogenetic therapy using HEK293 triple-transfected cells and/or hiPSC-derived photoreceptors equipped with microbial opsins have shown promising results in improving vision and restoring cells in Cpfl1/Rho−/− mice and wild-type mice (C57BL/6J) ^[13].

2.2. Animal-Derived Opsins

Ganglion cells have been the focus of several large animal studies investigating vision restoration. Modified mammalian ionotropic glutamate receptor (LiGluR) was delivered to the ganglion cells of Rcd1 dogs through intravitreal injection, resulting in restored vision in visible light. The delivery of maleimide-azobenzene-glutamate0 modified LiGluR resulted in acquired properties of light-gated channels and showed evidence of derived light responses. In a similar approach, optogenetic therapy was delivered to target the outer retina in Rdc1 dogs and C57BL/6 mice, demonstrating partial improvement in vision restoration through Y-maze performance and electroretinography.

Promoters play a crucial role in optogenetics. In macaque retinas, the combination of the ubiquitous cytomegalovirus promoter with an AAV2 vector increased upregulation of ChR-Ca2+-permeable ChR, improving light sensitivity. The gamma-synuclein gene promoter region was found to increase CatCh expression in ganglionic cells. The optimization of ChRs' light sensitivity through two CoChR mutants (CoChR-L112C and CoChR-H94E/L112C/K264T) delivered with an AAV2 vector restored light sensitivity in TKO mice. Chloromonas oogama, a variant of green algae, has been shown to improve light sensitivity in blind mice and restore vision under ambient light conditions.

Optogenetic therapy for vision restoration has potential safety concerns, such as phototoxicity induced by continuous light exposure and UV light. However, a study on mice transduced with optogenetic gene mVChR1 (using an AAV vector) and exposed to continuous light for one week showed no reduction in visually evoked potentials (VEPs) amplitudes in RGCs and an absence of phototoxicity. Mutations in opsins were also explored, and the third extracellular loop in mVChR1 was found to be implicated in sodium ion selectivity, and transmembrane 6 and C-Terminal regions have a role in some channel kinetics. G-protein stimulation has also shown to restore light sensitivity. Different optogenetic variants and promoters have been studied, providing potential treatment options for patients based on their specific needs. Further testing is needed to determine amino acid sequences that determine wavelength sensitivity.

In brief, optogenetics has been shown to partially restore vision in animal models of RP, but its safety and efficacy in humans remain to be fully understood as it is still in the preclinical stage. Furthermore, using various variants of opsins and promoters may improve the outcome of optogenetics, providing more options for patients with RP.

3. Overview of Vision Restoration Therapies and Neuroprotective Agents in the Preclinical Phase for Retinitis Pigmentosa

Retinitis Pigmentosa (RP) can be slowed down in its early stages by a combination of treatments, including neuroprotective agents. These agents, such as antioxidants, anti-apoptotic agents, and neurotropic factors, are administered as a preventative measure to protect the nervous system from damage. Ciliary neurotrophic factors (CNTF), brain-derived neurotrophic factors, and fibroblasts growth factors are examples of neurotropic factors that can inhibit apoptosis and inflammatory processes, reduce oxidative stress and free radicals, and slow retinal degeneration. Among these, CNTF has been clinically proven for this purpose.

3.1. Identification of Genes Regulating Neuroprotective Pathways

Signaling pathways in mice carrying the VPP transgene, including TGF-β, G-protein activated, and VEGF-mediated pathways, have been studied. A cluster of genes was identified that strongly regulates these pathways.

The TGF-β signaling pathway was investigated in mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP) [192]. Tgfbr2 was significantly upregulated in the INL and ONL of VPP retinae and in resting and reactive Müller cells, indicating their relationship with neuronal and glial cells. RNAseq analysis also revealed an increase in the expression of genes involved in the G-protein-activated signaling family, including Edn2 and Ednrb, which were significantly upregulated in VPP retinae. Edn2 has been known to protect against photoreceptor degeneration. Vegfr2 was also significantly upregulated in VPP retinae and neuronal layers, and Vegfr1 activation has been seen to provide neuroprotection and be involved in RGC survival ^[14][15][16].

3.2. Neurotropic Factors

Yang et al. demonstrated that retinal ganglion progenitor cells (RGPCs) and photoreceptor progenitor cells (PPC) can have a significant increase in survival time when administered with a stable and biocompatible polysaccharide nanoparticle (Nps) containing ciliary neurotrophic factor (CNTF) and oncostatin M (OSM). This was shown both in vitro and in vivo in rats following intravitreal delivery. The study also revealed CNTF's capacity to upregulate protease inhibitors. However, the relatively short lifespan of the cells before degradation remains a challenge ^[17][18].

3.3. Anti-Apoptotic Agents

Taurine deoxycholic acid (TUDCA) is an anti-apoptotic agent with chemical chaperone activity that improves protein-folding capacity. In Rpgr KO mice, TUDCA intraperitoneal injection has been shown to decrease photoreceptor cell death and inhibit retinal inflammation by regulating caspase-3 ^[19][20]. TUDCA was also found to decrease apoptosis and IL-1β signaling in Lrat−/− mice ^[21]. In a recent study on homozygous P23H line-3 rats, TUDCA showed a decrease in photoreceptor degeneration and partially protected vascular damage and glial activation, while its effect on astrocytes remains inconclusive ^[22][23]. Metformin was also found to have a neuroprotective effect on the retina in a rat model of RP by upregulating caspase-3, iNOS, CD68, and glial fibrillary acidic protein expression ^[24]. Additionally, Cav1.3 L-type channels have been shown to provide short-term neuroprotection in rd10 mouse models by reducing Ca2+ influx to slow down apoptosis ^[25].

NGF is a type of neuroprotective agent that acts on central and peripheral neurons and the visual system ^[26][27]. It has been shown to downregulate Bax expression and increase Bcl-2 expression, resulting in increased neuritis outgrowth and rhodopsin expression in PC12 cell lines ^[28][29]. Overexpression of IL-21 may also activate Müller cells, but further investigation is needed ^[30]. In zebrafish, intravitreal administration of rhNGF showed a regenerative effect on photoreceptor cells, with ERK1/2 pathways showing an upregulation compared to controls during early timepoints, which led to improved ONL thickness [209]. Prototype galectin-1 (Drgal 1-L2) also plays a role in retinal development and photoreceptor regeneration ^[31]. TGFβ signaling in Müller cells and retinal neurons has a neuroprotective effect and may present novel therapeutic opportunities to halt photoreceptor deterioration. However, the upregulation of the MAP kinase pathway and the activation of pro-apoptotic genes markedly accelerated VPP-induced photoreceptor degradation in mice ^[32]. Müller cells have a vital role in support, metabolism, and modulation of neuronal excitability through transporting and releasing neurotransmitters ^[33].

In early rod and cone degeneration in r10 mouse models, Egr1 mRNA was heavily upregulated, suggesting EGR1's involvement in early stress response in Müller glia ^[34]. Additionally, EPAC2, activated by cyclic AMP in rd10 models, exhibited neuroprotective properties, and P23H rhodopsin knock-in mice treated with 4-phenylbutyric acid (PBA) showed regulation of stress markers, warranting further exploration of its neuroprotective potential ^{[35] [36]}.

3.4. Neuroprotective Strategies through Antioxidant Agents for Retinitis Pigmentosa

In RP, oxidative stress is thought to contribute to the degradation of rods and cones ^[37]. Komeima and colleagues evaluated the effects of a combination of four antioxidants (vitamin E, a SOD mimetic, vitamin C, and a-lipoic acid) on rd1 mice and found that biomarkers of oxidative damage, including carbonyl adducts and acrolein, were upregulated in the retina. However, treatment with the antioxidants significantly decreased the buildup of oxidized lipids and protected cone cells from degeneration ^[38]. Another antioxidant, N-acetylcysteine (NAC), was found to protect rod and cone photoreceptor cells in rd10 mice by restoring intracellular GSH levels when administered orally ^[39]. Additionally, oral NAC was found to have neuroprotective and immunosuppressive effects on rd10 mice ^[40].

Neuroprotection through antioxidation presents challenges due to drug dosage and early-stage disease treatment. Most studies using neuroprotective agents are conducted on mice, with few side effects. Notable treatments include docosahexaenoic acid (DHA), vitamin A, retinoids and their derivatives, calcium channel blockers, calpain inhibitors, and valproic acid.

In summary, neuroprotective agents have the potential to slow the progression of RP during early stages of the disease. Combining neuroprotective agents with other treatments such as optogenetics, stem cell therapy, and gene therapy may improve outcomes for patients with RP.

4. The Potential of Exosomes as a Therapeutic Tool for Retinitis Pigmentosa

Exosomes, small vesicles released by cells, are gaining attention as a potential therapeutic option for RP. They deliver biologically active molecules to specific cells and tissues, potentially protecting photoreceptor cells.

These extracellular vesicles, found in all biological fluids, play a role in a variety of physiological and pathological processes, such as immune response, cardiovascular diseases, central nervous system diseases, and cancer. They range in size from 30-100 nm in diameter and contain various molecules, such as lipids, nucleic acids, and proteins, which they acquire when released from the parent cell by budding. As a result, they take on some of the characteristics of the parent cell's membrane.

Preclinical studies have investigated the potential therapeutic effects of exosomes in animal models of RP. ARPE-derived exosomes (RPE-Exos) and MSC-derived exosomes (MSC-Exos) have shown promising results in the treatment of RP. RPE-Exos inhibit T-cell proliferation through TNF-α and IL-6 secretion, while MSC-Exos lower levels of pro-inflammatory cytokines and suppress photoreceptor cell apoptosis. In addition, MSC-Exos increase survival of photoreceptor cells and subsequent visual function improvement through transcriptional regulation. It was hypothesized that the mechanism by which exosomes exert their effects is linked to the presence of anti-inflammatory and neuroprotective proteins. RPE-Exos have also shown promising results in restoring visual function and inhibiting apoptosis pathway activation. Administration of mouse NPC-Exos was shown to decrease photoreceptor cell apoptosis through retinal microglia activation inhibition, as well as TNFα, ILβ, and COX-2 inhibition.

Exosomes have shown potential in protecting photoreceptor cells in individuals with RP through several mechanisms, including the transfer of growth factors and modulation of immune responses ^[41]. Exosomes offer several advantages in retinal disease treatment, including the ability to bypass blood-retinal and blood-aqueous barriers, selective targeting of specific tissues and cells, and long-term stability due to their bilipid membrane ^[41]. Exosomes are also more biocompatible than MSC therapy and have been shown to lower teratoma formation and embolization risk ^[41]. Intravitreal injection of AAV2-Exos in mice has been shown to have greater penetrability in the retina ^[42].

The development of exosomes as a therapeutic option for RP faces several challenges ^[41]. Optimization of isolation and purification procedures is necessary due to the current low productivity rate. Furthermore, more robust preclinical and clinical data are required to demonstrate the safety and effectiveness of exosomes. Critical components of exosomes remain unidentified, and the long-term effects of exosome therapy and the optimal dosing regimen need to be understood.