New therapeutic targets, drug development for retinitis pigmentosa

Retinitis pigmentosa (RP) is the most common inherited retinal disease. Even though more than 1.5 million people worldwide are affected, there is no cure and treatment options are limited.¹

Kevin Yang Wu, MD, DMD, of the Division of Ophthalmology, Department of Surgery, University of Sherbrooke, Quebec, Canada, discussed the gene and cell therapies under development and the novel potential drug targets for RP at the 2024 American Society of Cataract and Refractive Surgery Annual Meeting in Boston, Massachusetts. The current conventional therapies, which only slow the progression of the disease, include retinoids, vitamin A supplements, protection from sunlight, visual aids and medical and surgical interventions to treat ophthalmic comorbidities.

Gene therapies

Dr Wu pointed out that only individuals with the RPE65 gene mutation, who comprise a small population of patients (0.3%-1% of all RP cases), are candidates for treatment with voretigene neparvovec (Luxturna; Spark Therapeutics), the only available gene therapy. A recent phase 3 clinical trial, in which 31 patients with confirmed biallelic RPE65 mutations were treated with reported visual function improvement and no serious adverse events after 1 year and durability of improvement after
3 to 4 years of follow-up.^2-4

The types of gene therapies under development include viral vectors, clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) gene editing system and RNA replacement. Viral vectorsinclude adenoviruses, adenoassociated viruses (AAVs) and lentiviruses.⁵ Of these, AAVs have been receiving the most attention because of their potential and small size that facilitates the efficient targeting of the retinal layers. They also exhibit low immunogenicity, allowing prolonged expression of the target gene after single-dose treatment and safe administration of a second dose in the subretinal space.⁶ AAVs are nonpathogenic and have not yet been linked to human diseases.⁵

Challenges associated with single AAV vector use include cloning capacity being limited to nonlarge genomes, procedural invasiveness and slow onset of transgene expression.⁶ Novel AAV capsids overcome challenges by facilitating intravitreal gene therapy, which has a lower treatment burden than intraretinal injections.^7-9

The CRISPR/Cas9 DNA gene editing system is a novel tool that is widely used in gene expression and suppression methods.^10,11 The system has achieved higher rates of successful gene editing in vitro; however, a major challenge is the lower performance rates. The success of gene editing, according to Dr Wu, depends on the guide RNA design, cell target type, delivery method of viral or nonviral vectors, and related host factors. Despite this, CRISPR/Cas9 gene editing may be significant for treating inherited retinal diseases, because even with slight gene modification, the phenotype can improve.

Attention has been shifting toward RNA-guided gene editing methods,¹² as the Cas13 tool can modify genes during transcriptional activity.^13,14 Major drawbacks are the potential for nonspecific collateral RNA cleavage^15-17 and the potential neurotoxicity of Cas13 enzymes through the impediment of neuronal development in vitro.¹⁸ Overall, CRISPR/cas13 methods are a novel approach for treating retinal diseases, and further studies should assess their potential and safety.¹⁹

RNA replacement therapies deplete endogenous mutant RNAs. Noncoding RNAs—of which there are three—are the key actors in gene expression regulation: micro-RNA (miRNA), small-interfering RNA (siRNA) and short hairpin RNA (shRNA).²⁰ Briefly, miRNA regulates posttranscriptional modifications by binding the binding sites of a target messenger RNA; siRNA, a double-stranded RNA, has a similar mechanism of action to that of miRNA, but its binding capacity is highly specific and can distinguish single nucleotide disparities²¹; and shRNA, whose transcripts are structurally similar to those of miRNA, acts on DNA delivery as opposed to RNA effector molecule regulation, as siRNA does.²⁰

Finally, synthetic single-stranded RNAs (antisense oligonucleotides) have therapeutic effects in inherited retinal diseases²⁰; they bind to RNAs and promote RNA cleavage. A challenge with siRNAs is the susceptibility to degradation by serologic enzymes; in contrast, shRNAs have a sustainable effect and higher potency.²²

Stem cell therapies

Cell-based therapies can replace dysfunctional cells with effective stem cells and restore dysfunctional cells by releasing trophic factors. With these therapies, success demands integration over the long term and formation of new synaptic connections with the host.²³

Embryonic stem cells are pluripotent stem cells that can self-renew through division and develop into the three primary germ cells. A disadvantage is immune system rejection. Induced pluripotent stem cells (iPSCs), an alternative to embryonic stem cells, are generated from a somatic cell line and can differentiate into any somatic cell, which facilitates stem cell extraction without the need for human embryos. Although a dysfunctional retinal pigment epithelium (RPE) affects the photoreceptors with resultant visual loss, replacing the damaged RPE and photoreceptors with healthy pluripotent stem cells can delay disease progression and potentially restore vision.^24,25

Several experimental studies have reported improved vision in animal models.^26-30 Concerns about using human embryos are eliminated and patients can be matched based on compatible blood types. Drawbacks are epigenetic memory, by which derived cells retain the gene expression of the original cells, possibly affecting senescence and proliferation. Another concern is the ability of iPSCs to proliferate indefinitely and development of teratomas.

Bone marrow stem cell (BMSC) therapies include mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs). BMSCs are mainly looked at when studying RP.³¹ Their ability to differentiate into photoreceptors is under study. Preclinical studies show BMSCs release antiangiogenic and neurotrophic factors, and immunomodulatory proteins such as insulinlike growth factor-1, class II major histocompatibility complex antigens and Th2-related cytokines.^32-34

A study of primitive MSC-derived retinal progenitor cells (RPCs) in mice showed that the cells integrated and countered inflammation.³⁵ Overall, the BMSC advantages include their ability to migrate toward lesion sites, transdifferentiation, and ease of extracting and manipulating HSCs.

Stem cell–derived RPE can be used to provide trophic support for surviving photoreceptors.³⁶ Early-phase clinical trials of encapsulated RPE cells suggested photoreceptor protection in patients with retinal degeneration.³⁷ A study that injected HSCs suggested trophic regenerative effects without electroretinographic changes.³⁸ Recently, conjunctival MSCs have been induced into photoreceptorlike cells in fibrin gel to form three-dimensional scaffolds.³⁹

This cellular bioengineering is a promising strategy to increase the number of photoreceptors and promote the retinal angiogenesis needed for repair and regeneration.³⁹ In vivo murine studies showed that MSCs transplanted into the vitreous improved the survival and preservation of photoreceptors and delayed retinal degeneration in RP.⁴⁰

Transplanting fetal RPE cells and MSCs in mice significantly improved the electroretinographic results and increased the survival rate of transplanted cells through increased expression of a protein that activates rhodopsin and rhodopsin levels, as well as a decrease in caspase-3 expression.⁴¹ These results suggest that there is greater benefit in coculture transplantation compared with a single-cell transplantation.⁴¹

RPCs, multipotent stem cells in the developing neural retina of 16- to 20-week human fetuses,⁴² can be manipulated in vitro to express photoreceptor markers and differentiate into retinal neuronal cells.^43,44 Preclinical studies have shown that RPCs differentiate into rod photoreceptors when transplanted and integrate into the degenerating retina to form synaptic connections and improve visual function.⁴⁵

A mouse study showed exceptional results in the formation of functional synapses with host cells, potentially stabilising progressive vision loss.⁴⁶ The advantages of RPCs include secretion of trophic factors to increase the likelihood of retinal survival and photoreceptor replacement.⁴⁷

Novel therapeutic targets

Various approaches are under investigation and include optogenetics, a one-for-all therapy that can be used in cases where degradation has occurred, regardless of the specific mutation present, and includes microbial- and animal-derived opsins; neuroprotective agents such as antioxidants, antiapoptotic agents, and neurotropic factors such as ciliary neurotrophic factors, brain-derived neurotropic factors, and fibroblastic growth factors; and exosomes that can be used to deliver biologically active molecules to specific cells and tissues.

“RP may be a difficult condition to understand and treat,” Dr Wu said. “With advances in our understanding of its molecular mechanisms, we can unlock new doors to innovative therapies that will change the way we approach RP and offer a ray of hope for those [with] this disease.”

References

1. Wu KY, Kulbay M, Toameh D, Xu AQ, Kalevar A, Tran SD. Retinitis pigmentosa: novel therapeutic targets and drug development. Pharmaceutics. 2023;15(2):685. doi:10.3390/pharmaceutics15020685

2. Russell S, Bennett J, Wellman, JA, et al. Efficacy and safety of voretigene neparvovec (AAV2-HRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet.2017;390(10097):849-860. doi:10.1016/S0140-6736(17)31868-8

3. Maguire AM, Russell S, Wellman JA, et al. Efficacy, safety, and durability of voretigene neparvovec-rzyl in RPE65 mutation-associated inherited retinal dystrophy: results of phase 1 and 3 trials. Ophthalmology.2019;126(9):1273-1285. doi:10.1016/j.ophtha.2019.06.017

4. Maguire AM, Russell S, Chung DC, et al. Durability of voretigene neparvovec for biallelic RPE65-mediated inherited retinal disease: phase 3 results at 3 and 4 years. Ophthalmology.2021;128(10):1460-1468. doi:10.1016/j.ophtha.2021.03.031

5. Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther.2021;6(1):53. doi:10.1038/s41392-021-00487-6

6. Trapani I, Puppo A, Auricchio A. Vector platforms for gene therapy of inherited retinopathies. Prog Retin Eye Res.2014;43:108-128. doi:10.1016/j.preteyeres.2014.08.001

7. Pavlou M, Schön C, Occelli LM, et al. Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders. EMBO Mol Med.2021;13(4):e13392. doi:10.15252/emmm.202013392

8. Kevany B, Suh S, Lu J, Padegimas L, Palczewski K, Miller T. Novel AAV capsids demonstrate strong retinal expression in non-human primates after intravitreal administration. Invest Ophthalmol Vis Sci.2019;60(9):2900.

9. Katada Y, Kobayashi K, Tsubota K, Kurihara T. Evaluation of AAV-DJ vector for retinal gene therapy. PeerJ.2019;7:e6317. doi:10.7717/peerj.6317

10. Leonova EI, Gainetdinov RR. CRISPR/Cas9 technology in translational biomedicine. Cell Physiol Biochem.2020;54(3):354-370. doi:10.33594/000000224

11. Doudna JA, Charpentier E. Genome editing. the new frontier of genome engineering with CRISPR-Cas9. Science.2014;346(6213):1258096. doi:10.1126/science.1258096

12. Reshetnikov VV, Chirinskaite AV, Sopova JV, Ivanov RA, Leonova EI. Cas-based systems for RNA editing in gene therapy of monogenic diseases: in vitro and in vivo application and translational potential. Front Cell Dev Biol.2022;10:903812. doi:10.3389/fcell.2022.903812

13. Hu Y, Chen Y, Xu J, et al. Metagenomic discovery of novel CRISPR-Cas13 systems. Cell Discov. 2022;8(1):107. doi:10.1038/s41421-022-00464-5

14. Botto C, Dalkara D, El-Amraoui A. Progress in gene editing tools and their potential for correcting mutations underlying hearing and vision loss. Front Genome Ed.2021;3:737632. doi:10.3389/fgeed.2021.737632

15. Wang Q, Liu X, Zhou J, et al. The CRISPR-Cas13a gene-editing system induces collateral cleavage of RNA in glioma cells. Adv Sci (Weinh).2019;6(20):1901299. doi:10.1002/advs.201901299

16. Li Y, Xu J, Guo X, et al. The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model. Genome Biol.2023;24(1):20. doi:10.1186/s13059-023-02860-w

17. Ai Y, Liang D, Wilusz JE. CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells. Nucleic Acids Res.2022;50(11):e65. doi:10.1093/nar/gkac159

18. Wu QW, Kapfhammer JP. The bacterial enzyme Cas13 interferes with neurite outgrowth from cultured cortical neurons. Toxins (Basel).2021;13(4):262. doi:10.3390/toxins13040262

19. Fry LE, McClements ME, MacLaren RE. Comparison of CRISPR-Cas13 RNA editing tools for inherited retinal disease. Invest Ophthalmol Vis Sci.2022;63(7):3845.

20. Gemayel MC, Bhatwadekar AD, Ciulla T. RNA therapeutics for retinal diseases. Expert Opin Biol Ther.2021;21(5):603-613. doi:10.1080/14712598.2021.1856365

21. Bajan S, Hutvagner G. RNA-based therapeutics: from antisense oligonucleotides to MiRNAs. Cells.2020;9(1):137. doi:10.3390/cells9010137

22. Knott SRV, Maceli A, Erard N, al. A computational algorithm to predict ShRNA potency. Mol Cell.2014;56(6):796-807. doi:10.1016/j.molcel.2014.10.025

23. Jindal N, Banik A, Prabhakar S, Vaiphie K, Anand A. Alteration of neurotrophic factors after transplantation of bone marrow derived lin-ve stem cell in NMDA-induced mouse model of retinal degeneration. J Cell Biochem.2017;118(7):1699-1711. doi:10.1002/jcb.25827

24. Kashani AH, Uang J, Mert M, et al. Surgical method for implantation of a biosynthetic retinal pigment epithelium monolayer for geographic atrophy: experience from a phase 1/2a study. Ophthalmol Retina.2020;4(3):264-273. doi:10.1016/j.oret.2019.09.017

25. Mandai M, Fujii M, Hashiguchi T, et al. iPSC-derived retina transplants improve vision in Rd1 end-stage retinal-degeneration mice. Stem Cell Reports. 2017;8(1):69-83. doi:10.1016/j.stemcr.2016.12.008

26. Salas A, Duarri A, Fontrodona L, et al. Cell therapy with hiPSC-derived RPE cells and RPCs prevents visual function loss in a rat model of retinal degeneration. Mol Ther Methods Clin Dev.2021;20:688-702. doi:10.1016/j.omtm.2021.02.006

27. Surendran H, Nandakumar S, Reddy KVB, et al. Transplantation of retinal pigment epithelium and photoreceptors generated concomitantly via small molecule-mediated differentiation rescues visual function in rodent models of retinal degeneration. Stem Cell Res Ther.2021;12(1):70. doi:10.1186/s13287-021-02134-x

28. Lin B, McLelland BT, Aramant RB, et al. Retina organoid transplants develop photoreceptors and improve visual function in RCS rats with RPE dysfunction. Invest Ophthalmol Vis Sci.2020;61(11):34. doi:10.1167/iovs.61.11.34

29. Duarri A, Rodríguez-Bocanegra E, Martínez-Navarrete G, et al. Transplantation of human induced pluripotent stem cell-derived retinal pigment epithelium in a swine model of geographic atrophy. Int J Mol Sci.2021;22(19):10497. doi:10.3390/ijms221910497

30. Thomas BB, Lin B, Martinez-Camarillo JC, et al. Co-grafts of human embryonic stem cell derived retina organoids and retinal pigment epithelium for retinal reconstruction in immunodeficient retinal degenerate Royal College of Surgeons rats. Front Neurosci.2021;15:752958. doi:10.3389/fnins.2021.752958

31. Ferroni L, Gardin C, Tocco I, et al. Potential for neural differentiation of mesenchymal stem cells. Adv Biochem Eng Biotechnol.2013;129:89-115. doi:10.1007/10_2012_152

32. Bassi ÊJ, Almeida DC, Moraes-Vieira PMM, Câmara NO. Exploring the role of soluble factors associated with immune regulatory properties of mesenchymal stem cells. Stem Cell Rev Rep.2012;8(2):329-342. doi:10.1007/s12015-011-9311-1

33. Soleymaninejadian E, Pramanik K, Samadian E. Immunomodulatory properties of mesenchymal stem cells: cytokines and factors. Am J Reprod Immunol.2012;67(1):1-8. doi:10.1111/j.1600-0897.2011.01069.x

34. Ding SLS, Kumar S, Mok PL. Cellular reparative mechanisms of mesenchymal stem cells for retinal diseases. Int J Mol Sci.2017;18(8):1406. doi:10.3390/ijms18081406

35. Brown C, Agosta P, McKee C, et al. Human primitive mesenchymal stem cell-derived retinal progenitor cells improved neuroprotection, neurogenesis, and vision in rd12 mouse model of retinitis pigmentosa. Stem Cell Res Ther.2022;13(1):148. doi:10.1186/s13287-022-02828-w

36. Kolomeyer AM, Zarbin MA. Trophic factors in the pathogenesis and therapy for retinal degenerative diseases. Surv Ophthalmol.2014;59(2):134-165. doi:10.1016/j.survophthal.2013.09.004

37. Talcott KE, Ratnam K, Sundquist SM, et al. Longitudinal study of cone photoreceptors during retinal degeneration and in response to ciliary neurotrophic factor treatment. Invest Ophthalmol Vis Sci.2011;52(5):2219-2226. doi:10.1167/iovs.10-6479

38. Moisseiev E, Smit-McBride Z, Oltjen S, et al. Intravitreal administration of human bone marrow CD34+ stem cells in a murine model of retinal degeneration. Invest Ophthalmol Vis Sci.2016;57(10):4125-4135. doi:10.1167/iovs.16-19252

39. Soleimannejad M, Ebrahimi-Barough S, Nadri S, et al. Retina tissue engineering by conjunctiva mesenchymal stem cells encapsulated in fibrin gel: hypotheses on novel approach to retinal diseases treatment. Med Hypotheses.2017;101:75-77. doi:10.1016/j.mehy.2017.02.019

40. Zhang J, Li P, Zhao G, et al. Mesenchymal stem cell-derived extracellular vesicles protect retina in a mouse model of retinitis pigmentosa by anti-inflammation through MiR-146a-Nr4a3 axis. Stem Cell Res Ther. 2022;13(1):394. doi:10.1186/s13287-022-03100-x

41. Pan T, Shen H, Yuan, S, et al. Combined transplantation with human mesenchymal stem cells improves retinal rescue effect of human fetal RPE cells in retinal degeneration mouse model. Invest Ophthalmol Vis Sci.2020;61(8):9. doi:10.1167/iovs.61.8.9

42. Wang NK, Tosi J, Kasanuki JM, et al. Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa. Transplantation.2010;89(8):911-919. doi:10.1097/TP.0b013e3181d45a61

43. Klassen HJ, Ng TF, Kurimoto Y, et al. Multipotent retinal progenitors express developmental markers, differentiate into retinal neurons, and preserve light-mediated behavior. Invest Ophthalmol Vis Sci.2004;45(11):4167-4173. doi:10.1167/iovs.04-0511

44. Qiu G, Seiler MJ, Thomas BB, Wu K, Radosevich M, Sadda SR. Revisiting nest in expression in retinal progenitor cells in vitro and after transplantation in vivo. Exp Eye Res.2007;84(6):1047-1059. doi:10.1016/j.exer.2007.01.014

1. MacLaren RE, Pearson RA, MacNeil A, et al. Retinal repair by transplantation of photoreceptor precursors. Nature.2006;444(7116):203-207. doi:10.1038/nature05161

2. He XY, Zhao CJ, Xu H, et al. Synaptic repair and vision restoration in advanced degenerating eyes by transplantation of retinal progenitor cells. Stem Cell Reports.2021;16(7):1805-1817. doi:10.1016/j.stemcr.2021.06.002

3. Stern JH, Tian Y, Funderburgh J, et al. Regenerating eye tissues to preserve and restore vision. Cell Stem Cell.2018;23(3):453. doi:10.1016/j.stem.2018.08.014

Kevin Yang Wu, MD, DMD | E: yang.wu@usherbrooke.ca

Wu has no financial interests related to this topic.