## 2 Scaffold-Free Approaches  
Gene therapy has been proven to restore vision by replacing absent or abnormal genes causing monogenic retinal diseases [15; 16; 17]. Viral vectors and non-viral gene nanocarriers have been used to deliver specific genes to target cells [15]. For instance, for RPE65-associated IRDs, nowadays there is an FDA (Food and Drug Administration) and EMA (European Medicine Agency)-approved gene therapy (Luxturna (voretigene neparvovecrzyl), Spark Therapeutics) based on the delivery of a functional copy of the RPE65 gene into RPE cells [17]. Therapies targeting monogenic retinal dystrophies are the most promising. Gene augmentation therapy, however, has its own limitations. Only the recessive forms where the mutations cause a loss of function, also called haplosufficiency, such that the resulting protein is too little or absent can be currently treated with gene therapy [18].  
Another scaffold-free strategy explored to regenerate damaged cells or replace dead cells is cell therapy. Healthy cells can be injected as cell suspension or transplanted as sheets to replace pathological cells, thus preventing further degeneration and improving visual function. For instance, as a possible treatment for glaucoma, the transplantation of RGCs derived from human pluripotent stem cells and from human Muller glia cells was investigated. However, difficulties remain in integrating the transplanted RGCs into the complex neurological network of the host retina [19]. Cell transplantation in animal models has been experimented also to treat AMD. [PERSON] et al. reported that a subretinal injection of human central nervous system stem cells in pigmented dystrophic rats enhanced the proliferation of the host RPE cells, providing a new mechanism for RPE regeneration and thus preserving the viability of photoreceptors [20]. [PERSON] et al. demonstrated that the embryonic stem cell-derived RPE implanted in a pathological mouse model was able to restore visual function in the short term. After 90 days, the visual acuity started decreasing [21]. This reduced efficacy could be due to the lack of interaction between the transplanted cells and [PERSON]'s membrane, thereby not forming a functional monolayer. Phase I/II clinical trials were carried out to determine the primary endpoints of safety and tolerability of a subretinal injection of a cell suspension [22]. No serious adverse effect was encountered; however, concerns about integration efficiency and long term cell survival were raised. A possible approach to overcome this issue is represented by cell sheet engineering [23; 24]. Cell sheet engineering is based on harvesting a sheet of cells along with their extracellular matrix (ECM) without the use of enzymes [25]. Harvesting  
Figure 1: Representation of a cross-section of a human eye and a detail of the multilayer organization of the retina, Bruch’s membrane and choroid. The retinal layers include a retinal pigment epithelium (RPE), cone and rod photoreceptors, horizontal cells, bipolar cells, amacrine cells, microglia and Müller glial cells and ganglion cells (RGCs).  
cells is performed with thermoresponsive coatings that enable reversible cell detachment by switching their surface hydrophobicity. Such an approach may allow the formation of an intact RPE cell monolayer to be transplanted. Furthermore, the presence of an ECM should improve the attachment to the host tissue once implanted [26]. However, the use of cell sheet engineering for the RPE is limited by the insufficient amount of the ECM secreted by these cells [18]. In addition, when multiple structures are involved in the pathology, as in AMD, cell therapy cannot be effective in the long term. For example, [PERSON]'s membrane is also compromised in AMD; therefore, it needs to be replaced along with the RPE monolayer. Alternative strategies are needed to fabricate a mechanically strong tissue that promotes cell attachment and survival while maintaining cell functionality in the long term.