Isolation, enrichment and expansion of human rod progenitor cells and extracellular vesicles derived therefrom

Abstract

Described herein are methods for generating an enriched population of progenitor rod photoreceptor cells (PRP) and EVs derived therefrom, and methods of using PRP and EVs to diagnose and treat retinal disease.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 339,252, filed May 6, 2022. The entire contents of the above application are incorporated herein by reference. Technical Field

[0003] Described herein are methods for generating enriched populations of progenitor rod photoreceptor cells (PRPs) and extracellular vesicles (EVs) derived therefrom, as well as methods for diagnosing and treating retinal diseases using PRPs and EVs. Background Art

[0004] The retinal degenerative diseases that cause the death of rod photoreceptor cells and cone photoreceptor cells are the main causes of hereditary vision loss worldwide. For example, retinitis pigmentosa is a disease in which rod photoreceptors first die, leading to the death of cone photoreceptors. Induced pluripotent stem cells or embryonic stem cells (iPSC / ESC) have been proposed as possible sources of new photoreceptors to restore vision under these conditions. The research carried out in the mouse model of retinal degeneration in the past decade has emphasized several limitations of cell replacement or neuroprotection in the retina, such as the poor integration of transplanted cells in the host retina and the high dose required for neuroprotection of the retina. Summary of the invention

[0005] Described herein is a method of providing a population of progenitor rod photoreceptor cells (PRPs). The method may include providing an initial population comprising retinal cells from a mammal, preferably a fetal mammal (optionally, the initial population may include cells from a retina or optic cup organoid generated by iPSC or ESC). Cells expressing CD73 and CD276 (and optionally Cd11b) are isolated from the initial population to provide a substantially purified population of PRPs.

[0006] In some embodiments, isolating cells expressing CD73 and CD276 (and optionally Cd11b) comprises using fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS), optionally in a microfluidic device. For example, the method can include contacting an initial population of cells with antibodies that bind to CD73 and CD276, and isolating cells that bind to both antibodies.

[0007] In some embodiments, the method further comprises culturing the initial population comprising retinal cells prior to isolating cells expressing CD73 and CD276 (and optionally Cd11b).

[0008] The method may include optionally culturing a substantially purified population of CD73+ / CD276+PRP obtained. In some embodiments, a substantially purified population of PRP is cultured in a culture medium comprising serum or serum replacement, EGF, and FGF. In some embodiments, a substantially purified population of PRP is cultured under hypoxic conditions (e.g., 3-10% O2). In some embodiments, hPRP is also CD11b+.

[0009] In some embodiments, the initial population comprising retinal cells is from a fetal mammal, such as a human.

[0010] Also provided herein is a substantially purified population of PRP produced by the methods described herein. In some embodiments, at least 75%, for example, at least 80%, 85%, 90%, or 95% of the population comprises cells expressing both CD73 and CD276 (and optionally Cd11b). Further provided is a substantially purified population of PRP for treating a subject with a condition associated with the loss of retinal rods or cone photoreceptors, as described herein. In some embodiments, the condition associated with the loss of retinal rods or cone photoreceptors is an inherited retinal degenerative disease (IRD), optionally cone-rod dystrophy, retinitis pigmentosa (e.g., LCA), or Stargardt's disease; or macular degeneration, for example, dry macular degeneration. Also provided herein are compositions comprising a PRP as described herein (e.g., at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the population comprising cells expressing both CD73 and CD276 (and optionally Cd11b)), optionally formulated in a physiologically acceptable buffer or polymer gel, e.g., as described herein, optionally in a gelatin-hyaluronic acid (HA) hydrogel, e.g., a composition comprising gelatin hydroxyphenylpropionic acid (gelatin-HPA) and hyaluronic acid-tyramine (HA-Tyr).

[0011] In addition, provided herein is a method for treating a subject with a condition associated with loss of retinal rod photoreceptors. The method may include administering to the subject (e.g., by injection) a therapeutically effective amount of PRP obtained by the methods described herein, preferably wherein at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the population comprises cells expressing both CD73 and CD276 (and optionally Cd11b), optionally in a physiologically acceptable buffer or polymer gel, e.g., as described herein, optionally in a gelatin-hyaluronic acid (HA) hydrogel, e.g., formulated in a composition comprising gelatin hydroxyphenylpropionic acid (gelatin-HPA) and hyaluronic acid-tyramine (HA-Tyr).

[0012] In addition, provided herein is a method of obtaining or providing extracellular vesicles (EVs) from rod photoreceptor progenitor cells. The method may include providing a substantially purified population of PRP as described herein; maintaining a substantially purified population of PRP in culture in a culture medium, preferably wherein the culture medium is in contact with the PRP in culture for at least 12, 18, 24, 36, 48, or 72 hours; removing the culture medium from the culture; and separating EVs from the culture medium.

[0013] In some embodiments, isolating EVs comprises ultracentrifuging the culture medium to obtain a pellet comprising EVs. An exemplary ultracentrifugation protocol comprises three steps: 1500 g for 30 min; 10,000 g for about 20 min; and 15,000 g for 30 minutes.

[0014] In some embodiments, the method further comprises lyophilizing the isolated EVs.

[0015] Also provided herein are compositions comprising EVs obtained by the methods described herein. In some embodiments, the EVs are in a polymer gel scaffold, for example, a polymer scaffold comprising polycaprolactone (PCL), polylactic-glycolic acid (PLGA), polyethylene glycol (PEG), polylactic acid (PLA), polyetherimide (PEI), PNIPAAM, or hydroxyethyl methacrylate. Also provided are compositions for treating subjects with conditions associated with the loss of retinal rods or cone photoreceptors. Additionally provided herein are methods for treating subjects with conditions associated with the loss of retinal rods or cone photoreceptors, as described herein. The method may include administering to the subject a therapeutically effective amount of PRP obtained by the methods described herein.

[0016] In some embodiments, the condition associated with loss of retinal rod or cone photoreceptors is an inherited retinal degenerative disease (IRD), optionally cone-rod dystrophy, retinitis pigmentosa (e.g., LCA), or Stargardt's disease; or macular degeneration, e.g., dry macular degeneration.

[0017] As used herein, unless otherwise indicated, the term "about" means ± 10% of the indicated number.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail.

[0019] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Summary of an exemplary isolation method for human progenitor rod photoreceptors (hPRPs). Cells were isolated from human fetal retina with CD73+ / CD276+ markers. Sorted cells showed high purity >95% for both markers as well as other specific rod expression markers. High viability was seen both after sorting and after several weeks of cell culture with culture medium as described herein. Markers shown indicate maturation and cell lineage.

[0021] Figure 2 Schematic representation of an exemplary neuroprotective human EV extracted from hPRP. Illustrative exemplary schematic showing the various encapsulated molecules and proteins in EVs, as well as transmembrane proteins that enable EV signaling and genesis.

[0022] Figure 3 Illustrative schematic diagram of an exemplary EV manufacturing process. The manufacturing process for EV extraction from hPRP as well as encapsulation and in vivo injection for potential neuroprotective effects against retinal diseases.

[0023] Figure 4.Total number of cells throughout the sorting process. Total number of cells as a function of time (weeks in culture). Cell number was measured in a T75 flask using trypan blue and a hemacytometer. The average cell number after digestion of the retina was 2 million and cultured until 10 million cells were reached. Cells were sorted after reaching this number; therefore, the total number decreased. The isolated rod progenitor cells were then cultured until a high cell number (40 million) was reached at 5 weeks.

[0024] Figure 5 .Brightfield images of rod hPRPs in T75 flasks after sorting. Cells were imaged using a Leica brightfield upright microscope, showing successful cell attachment, viability, morphology, and proliferation over a 20-day culture period.

[0025] Figure 6 .Live sorting and gating was performed with a MACSQUANT cell sorter (Miltenyi) using a TYTO sorting cartridge. Clive CD73 and CD276 gating of fetal retinal cells to isolate pure hPRP. Clear populations were visible for both CD73 and CD276 and could be isolated with high viability and purity after sorting.

[0026] Figure 7 .TYTO sorting data analyzes the input, output and negative fractions of cells. Expression of sorting markers (CD73 and CD276) of the input, output and negative fractions of cells during the sorting process. TYTO sorter and MACSQUANT flow cytometer were used to analyze the data. It shows the expression of CD73 / CD276 positive cells before and after the first sorting. The CD73 / CD276 population starts at about 30-40% in unsorted samples and rises to 95% after sorting. The data are shown as the mean + SD of 10 repetitions (10 different sortings). Statistical one-way ANOVA was performed and a significant difference (*p<0.001) was shown between input and output.

[0027] Figure 8 Real-time timing and analysis of sorted cells using TYTO. Cell sorter analysis shows trigger events, sorting rate, and percentage of positively sorted cells over a 1-hour sort. A constant >90% sorting was observed throughout the sorting process.

[0028] Fig. 9.Original sorting marker expression analyzed by real-time flow cytometry. Expression of sorting markers (CD73, CD276) with their specific fluorophores (APC, PE, VioBlue) before and after sorting. The data was analyzed using a MACSQUANT flow cytometer. It shows sorting data for two fluorophores - CD73 / CD276. Upper right panel line: scatter plot and histogram for CD73 / CD276 expression for input. Middle right panel line: scatter plot and histogram for CD73 / CD276 expression for output. Upper left panel: gating strategy and number of populations corresponding to gating. The histograms in both figures show that after sorting, the CD73 and CD276 positive cell populations increase. This is demonstrated by the higher intensity of the peaks of these markers. A minimum of 50,000 cells were used for each sample in flow cytometry.

[0029] Fig.10 .Flow cytometry data evolution of 16-week tissue during the process of sorting. The left side shows the gating strategy for cells, singles and DAPI positive events to analyze hPRPs. The right side shows the expression of purity markers (recoverin, CD73, CD276) for both unsorted and hPRPs. It was found that the expression of these 3 rod markers was extremely high and pure in hPRPs, while there was 20-40% in the unsorted population. This shows that our sorting strategy is able to capture a pure population of rod progenitor cells. A minimum of 50,000 cells were used for each sample in flow cytometry. Data are shown as the mean + SD of 5 replicates (5 different flow experiments).

[0030] Fig.11 .Flow cytometry data of hPRP and fetal retina. It shows differences in all of these markers between sorted hPRP and unsorted fetal retinal tissue (also known as hRPC). Overall, rod markers (CD73, CD276, recoverin, cone-rod homeobox (CRX)) were found to be extremely high in hPRP compared to unsorted retina. Other markers of retinal cells (cones, RGC cells, bipolar cells, horizontal cells, and amacrine cells) were found to be negative in hPRP. Finally, 15-20% of hPRP were found to be proliferative, with 50% PAX6 retinal marker expression. A minimum of 50,000 cells were used for each sample in flow cytometry.

[0031] Fig.12.Immunostaining analysis of hPRP with sorting markers. CD73, CD276 and CRX are different rod photoreceptor markers, and hPRP was stained with the same primary antibody. High expression of these 3 markers can be observed in the IHC images. The nuclear stain used to identify cells is DAPI.

[0032] Fig.13 .Immunostaining for different markers used for hPRP. Cells were stained with common rod markers (rhodopsin, recoverin) as well as retinal markers (PAX6) and proliferation markers (Ki67). Overall, all markers were found in hPRP. Other retinal markers such as calbindin (amarine cells), recoverin (photoreceptors), PKCa (bipolar) and rhodopsin (rod photoreceptors) were tested and expression varied from low to no expression. Nuclei were stained with DAPI and all images were taken at 20x magnification.

[0033] Fig.14 .Whole mounted retina of a transplanted RD1 mouse. Under normal circumstances, a P31 RD1 mouse should not have any cones or any ONL staining due to the rapid degeneration process of the specie; this is what was observed in the control samples. However, in animals that received injections of hPRP, the staining shown here was found. Cones were found to block positive staining, suggesting that injections of hPRP in the vitreous have a strong neuroprotective effect on the retina of RD1 mice.

[0034] Fig.15 .ONL presence in transplanted RD1 mice. Under normal circumstances, RD1 mice at P31 have extremely degenerated retinas, visible as thinning of the ONL or absence of the ONL. To measure the impact of hPRP injections in this rapidly degenerative mouse model, we analyzed the presence of a row of ONL nuclei in a binary manner and scored all animals with multiple sections. As seen in the figure on the left, the score grades were found to be higher for animals that received hPRP injections in the subretinal space, and even higher for vitreous injections. This suggests that injections of hPRP in the vitreous have a strong neuroprotective effect on the retina of RD1 mice.

[0035] Fig.16.Intraocular pressure (IOP) test of RD1 mice. Under normal circumstances, RD1 mice at P31 should have and IOP between 10-20 mmHg, which was observed in control eyes. The IOP was found to increase by the injection of cells in the vitreous, while no changes were observed for the control and subretinal injections. However, it was found that the IOP in the eyes injected with the vitreous returned to the baseline just 1 week after transplantation. This study demonstrates the high safety of the injection of hPRP in the eyes of rodents.

[0036] Fig.17 Comparison of extracellular vesicle proteomic cargo between our EVs and the developing retina.

[0037] Fig.18 .Summary of vesicle proteomic cargo composition.

[0038] Fig.19 Foldchange of total proteome cargo in our EVs, divided into 6 categories and subcategories.

[0039] Fig. 20 .Fold changes in protein expression comparisons between our EVs and fetal retina (using only 5x limits).

[0040] Fig.21 .Proteins unique to our EVs and absent in the fetal retina.

[0041] Figure 22A-B . Functional behavioral testing in Rho- / - mice by optokinetic testing. Mice were injected with either (cells+gel) or SHAM (pure saline) at 3 weeks, and functional retinal behavior was tested weekly with an OMR device. A. Visual acuity (VA) of mice measured as threshold of spatial frequency. B. Contrast sensitivity threshold of mice, measured at 0.2 cycle / degree VA (**p=0.005).

[0042] Figure 23A-C. Cone retinal function testing in Rho- / - mice by ERG. Mice were injected with either (cells+gel) or SHAM (pure saline) at 3 weeks, and cone retinal function was tested weekly with an ERG device. A. Photopic B-wave measured at 600 cd.s / m2 for both groups. B. Calculation of the percentage increase of cell+gel injection versus SHAM injection. C. Representative photopic B-wave traces at 6 and 7 weeks. (**p=0.005).

[0043] Figure 24A-B . Cone retinal function testing in P23H mice by ERG. Mice were injected with either (cells+gel), cells, or SHAM (pure saline) at week 4, and cone retinal function was tested weekly with an ERG device. A. Photopic B waves measured at 600 cd.s / m2 for all groups. B. Calculation of percentage increase of cell injection vs. SHAM and cell+gel injection vs. SHAM injection. (**p=0.001). DETAILED DESCRIPTION

[0044] Described herein are methods and compositions that address limitations and disadvantages associated with previous approaches to isolating, purifying, and enriching rod progenitor cells. Provided are methods for generating enriched populations of human progenitor rod photoreceptor cells (hPRPs) and EVs derived therefrom, as well as methods for diagnosing and treating retinal diseases using hPRPs and EVs.

[0045] Human rod photoreceptor progenitor cells (hPRP)

[0046] Provided herein are purified or enriched populations of rod photoreceptor precursor cells, such as human progenitor rod photoreceptor cells (hPRPs), methods of producing these cells, and uses of the cells for treating ocular disorders, such as retinal degenerative diseases and other diseases. Described are methods of isolating, purifying, and amplifying rod photoreceptor progenitor cells using microfluidics-based cell sorting, including scalable GMP-capable protocols for producing, for example, human progenitor rod photoreceptors, providing the ability to create universal, allogeneic rod photoreceptor cells for preserving and restoring vision, such as Figure 1 as shown in .

[0047] As shown herein, the combination of CD73 / CD276 is useful as a surface marker for marking and isolating rod photoreceptors. It was found that CD276 was upregulated in rod photoreceptor progenitor cells and could be released from the cell membrane and on the cell surface. We used it to mark rod photoreceptor progenitor cells (CD276 is only expressed by rod photoreceptor progenitor cells alone). CD73 is also known as 5'-nucleotidase ecto (NT5E); exemplary sequences of CD73 are provided in GenBank as Acc.Nos.NP_002517.1 (5'-nucleotidase isoform 1 preproprotein) and NP_001191742.1 (5'-nucleotidase isoform 2 preproprotein). Exemplary sequences of CD276 are provided in GenBank as Acc. Nos. NP_001019907.1 (CD276 antigen isoform a precursor); NP_001316557.1 and NP_079516.1 (CD276 antigen isoform b precursor); and NP_001316558.1 (CD276 antigen isoform c).

[0048] As described herein, fetal retinal cells (e.g., at 10-16 weeks in human embryonic age (gestational age) or similar ages in the development of other mammals such as mice), optionally cultured under hypoxic conditions (3-10% O2), optionally cultured with a nutrient mixture containing serum (such as fetal bovine serum (FBS)) or serum replacement (such as PHYSIOLOGIX Xeno-Free Serum Replacement (Nucleus Biologics) or KNOCKOUT Serum Replacement (Thermo Fisher)), EGF, and FGF, provide a time frame during retinal development when expression of rod progenitor cells is highest; differentiated iPSCs and ESCs can also be used. When iPSCs / ESCs are used, the ESCs / iPSCs are grown into optic cups or retinal organoids, and the organoids are used as a starting point for sorting using the methods described herein, for example, based on the presence of CD73 and CD276 (e.g., at days 40 to 60). See, e.g., Nakano et al., Cell Stem Cell. 2012 Jun 14; 10(6):771-785; Kuwahara et al., Methods Mol Biol. 2017; 1597:17-29; Eiraku et al., Nature. 2011 Apr 7; 472(7341):51-6; Reh and Fischer, Methods Enzymol. 2006; 419:52-73; Fathi et al., Front Neurosci. 2021 Apr 20; 15:668857; and Achberger et al., Adv Drug Deliv Rev. 2019 Feb 1; 140:33-50.

[0049] The present technology can be used for the isolation of rod progenitor cells, optionally using a microfluidic device such as the MACSQUANT TYTO sterile sorting chamber microfluidic cell sorting system (Miltenyi), to provide an enriched population of cells with higher purity and viability than previously reported. The microfluidic device allows the capture of cells labeled with two positive markers (CD73 / CD276) and / or the deletion of populations of unwanted cells (all the remaining CD73- and / or CD276- cells). The present method can be used to obtain a population of cells in which at least 75%, for example, at least 80%, 85%, 90%, or 95% of the cells are positive for both (expression) CD73 and CD276. In this experiment, the expression of the confirmatory markers recovery protein, CRX, CD73, and CD276 showed that approximately 95% of the cells were positive for these markers. In addition, the use of the culture conditions described herein to maintain the isolated cells allows the cells to proliferate to obtain a large amount of hPRP. When transplanted into mouse eyes, hPRP cells showed an extremely high ability to provide neuroprotection in a degenerative model. To the best of the inventors' knowledge, this is the first description of a method for isolating and culturing highly pure rod progenitor cells capable of providing high neuroprotection to the retina.

[0050] hPRP cells can be differentiated to provide a population of human rod photoreceptor cells. Many protocols are known; an exemplary protocol includes culturing cells in DMEM / F12 3:1+KSR / FBS with 0.1% 2-ME (b-mercaptoethanol), 0.2% IGF-1 (IGF1 recombinant human protein), 2% B27 supplement, 1% taurine, and 1 μM 9-cis retinal. The culture medium is changed approximately every 2 days, and the cells mature into rod photoreceptors in about 60 to 90 days.

[0051] Extracellular vesicles (EVs) derived from human progenitor rod photoreceptor cells (hRP).

[0052] EV and microvesicles are lipid-encapsulated cell fragments with diameters ranging from 50nm to 2mm; they can be derived from almost any cell type, including embryonic stem cells, hematopoietic stem cells, neurons, and malignant cells. EVs typically have a diameter of 30-130nm and are formed by the endosomal sorting complex (ESCRT) required for transport. Microvesicles are heterogeneous in size, with a diameter of 100-2000nm. Although EVs fall within the size range of microvesicles, microvesicles differ in formation, secretion, and content. The formation of microvesicles involves interactions between the phospholipid proteins of the cytoskeleton and plasma membrane. EV and microvesicles have been shown to participate in cell-cell communication via the transfer of proteins, mRNA, miRNA, and DNA, for example, as Figure 2 EVs and microvesicles encapsulate factors that represent the genotype and phenotype of the cells of origin.

[0053] Provided herein are EVs derived from human progenitor rod photoreceptor cells (hPRPs) isolated using the methods described herein. Some or all of the proteins shown in Table 1 are preferably present in EVs for use in the present methods and compositions, optionally including at least one, two, three, four, five, or all seven of the following: thioredoxin (TXN, also known as RDCVF; NP_001231867.1); EGF-containing fibrin extracellular matrix protein 1 (EFEMP1; NP_001034437.1); glutathione S-transferase Mu 1 isoform 1 (GSTM1; NP_000552.2 or NP_666533.1); sarcoglycan delta (SGCD; NP_000328.2, NP_758447.1, or NP_001121681.1); staphylococcal nuclease and tudor domain-containing 1 (GSTM1; NP_000552.2 or NP_666533.1); domain containing 1) (SND1; NP_055205.2), ADAM metallopeptidase domain 9 (ADAM9; NP_003807.1); and ezrin (EZR; NP_003370.2). Thus EVs may include specific proteins containing one, two, three, four, five, six, or all seven of TXN, EFEMP1, GSTM1, SGCD, SND1, ADAM9, and EZR. Without wishing to be bound by theory, it is believed that these proteins provide a therapeutic effect. A summary and schematic diagram of exemplary EVs are provided in Figure 2 . Antibody-directed targeting of EVs increases the specificity of delivering neuroprotective proteins, miRNAs, and other molecular cargoes in EVs to photoreceptors. This can be used to promote the delivery of appropriate concentrations of neuroprotective cargoes and internalization by photoreceptors to activate recovery and regeneration processes. EV targeting for the delivery of therapeutic agents has been successfully demonstrated in non-ocular tissues (see, e.g., Chen et al., Front. Cell Dev. Biol., 08 October 2021. doi.org / 10.3389 / fcell.2021.751079).

[0054] hPRP cells, for example, continuously secrete EVs into the culture medium throughout the manufacturing and culture process. EVs can be obtained from hPRP cells using known methods. Exemplary manufacturing methods are described in Figure 3In this example, EVs secreted by hPRP cells are obtained from their culture medium. The culture medium is frozen and then ultracentrifuged to separate the EVs and discard debris, dead cells and other undesirable components. The final precipitate is resuspended in PBS, freeze-dried and stored, keeping the EV structure intact. Finally, the EVs are resuspended in an aqueous carrier (optionally containing PBS or a hydrogel carrier) and injected into the vitreous of the eye. The culture medium contains the selected and secreted EVs.

[0055] EV or hPRP can be suspended in, for example, a physiologically acceptable buffer such as phosphate buffered saline (PBS), or in a biocompatible polymer such as a hydrogel. Since EVs injected only in phosphate buffered saline (PBS) can be targeted by the host immune system, thereby reducing their effectiveness in inducing regeneration, polymer scaffolds containing biocompatible biomaterials can be used to encapsulate EVs before delivery; hPRP can also be formulated in polymer gels for administration. Suitable biodegradable and biocompatible polymer materials can include one or more of gelatin, chondroitin sulfate, hyaluronic acid, alginate, collagen, and chitosan-based scaffolds, for example, gelatin-hyaluronic acid gels, for example, compositions comprising gelatin hydroxyphenylpropionic acid (gelatin-HPA) and hyaluronic acid-tyramine (HA-Tyr) (see, for example, WO2021113515 and Dromel et al., NPJ Regen Med. 2021Dec 20; 6(1): 85). Such materials provide nontoxic and biocompatible degradation products, while self-triggering higher immune responses than synthetic polymers. Other materials that can be used include but are not limited to polycaprolactone (PCL), polylactic-glycolic acid (PLGA), polyethylene glycol (PEG), polylactic acid (PLA), polyetherimide (PEI), PNIPAAM or hydroxyethyl methacrylate. See, for example, US20140234381 and US20110004304.

[0056] How to use

[0057] EVs and hPRPs produced as described herein can be used to treat retinal degenerative diseases, e.g., diseases associated with loss of photoreceptor rods or cones such as macular degeneration, e.g., dry macular degeneration; retinitis pigmentosa; and other inherited retinal diseases, including Stargardt disease, Leber congenital amaurosis (LCA), or cone-rod dystrophy (CRD); in particular, the present method can be used in situations where the rods are lost first. For example, certain retinal diseases affected by the loss of cones (e.g., secondary loss of cones in retinitis pigmentosa (RP) leading to blindness) can be treated using hPRPs as described herein or EVs isolated from hPRP cultures; in some embodiments, EVs contain rod-derived cone viability factor (RdCVF), which is an inactive thioredoxin ( ) secreted by rod photoreceptors that protects cones from degeneration. et al., Cell. 2015 May 7; 161(4):817-32). Degeneration of retinal photoreceptors is also seen in the late stages of dry AMD, also known as geographic atrophy (GA). In some embodiments, the method may include administering a population of hPRP to replace cells lost to the degenerative disease. Inherited retinal diseases treatable by the methods described herein include Bardet-Biedl syndrome (Bardet-Biedl syndrome). syndrome, autosomal recessive; chorioretinal atrophy or degeneration, autosomal dominant; cone or cone-rod dystrophy, autosomal dominant; cone or cone-rod dystrophy, autosomal recessive; cone or cone-rod dystrophy, X-linked; congenital stationary night blindness, autosomal dominant; congenital stationary night blindness, autosomal recessive; congenital stationary night blindness, X-linked; Leber congenital amaurosis, autosomal dominant; Leber congenital amaurosis, autosomal recessive; macular degeneration, autosomal dominant; macular degeneration, autosomal recessive; developmental disorders of the retina of the eye, autosomal dominant; optic atrophy, autosomal dominant; optic atrophy, autosomal recessive; optic atrophy, X-linked; retinitis pigmentosa, autosomal dominant; retinitis pigmentosa, autosomal recessive; retinitis pigmentosa, X-linked; X-linked; Usher syndrome syndrome), autosomal recessive; autosomal dominant retinopathy; autosomal recessive retinopathy; mitochondrial retinopathy; and X-linked retinopathy, as well as retinopathy associated with syndrome / systemic disease. See, e.g., RetNet, the Retinal Information Network, available at sph.uth.edu / retnet / home.htm.

[0058] The method generally comprises administering a therapeutically effective amount of hPRP or EV as described herein, for example, by intraocular administration, for example, by subconjunctival, intracameral, or intravitreal injection; see, for example, Amo et al., Prog Retin Eye Res. 2017 Mar; 57: 134-185; Yamada and Olsen, Dev Ophthalmol. 2016; 55: 71-83. For example, purified and lyophilized EVs can be stored at -80°C and can be resuspended before administration, for example in an operating room, for example, by intravitreal injection, for example with a 31 gauge needle. EVs can be resuspended in a physiologically acceptable buffer such as PBS, or in a polymer scaffold as described herein. Dissociated hPRP cells, e.g., in suspension, in a hydrogel (e.g., as described herein, e.g., a gelatin-hyaluronic acid gel, e.g., a composition comprising gelatin hydroxyphenylpropionic acid (gelatin-HPA) and hyaluronic acid-tyramine (HA-Tyr) (see, e.g., WO2021113515 and Dromel et al., NPJ Regen Med. 2021 Dec 20; 6(1):85), or in a retrievable permeable capsule loaded with stem cells, can be delivered, e.g., via retinal delivery following pars plana vitrectomy (PPV) or via a transscleral approach without vitrectomy (Hinkle et al., Stem Cell Research & Therapy volume 12, Article number: 538 (2021); Falkner-Radler et al., The British journal ofophthalmology. 2011;95(3):370-5; Bhattacharya et al., Curr Mol Biol Rep. 2017 Sep;3(3):172-182.

[0059] In rodent studies, approximately 3 ug of lyophilized EVs were delivered with 3 uL per injection. The concentration, amount, and frequency of dosing to be used clinically can be determined based on animal and clinical trial data.

[0060] Example

[0061] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0062] Materials and methods

[0063] The following materials and methods were used in the examples below.

[0064] Cell culture and isolation process of rods

[0065] Human fetus arrival: Fetal eyes arrived within 24 hours in a 2°C chamber. Dissection was started directly to maintain high viability.

[0066] Dissection and dissociation: Dissection of the retina was performed on all eyes. The retina was then dissociated in papain for 30 min in an incubator. The cell suspension was then centrifuged and seeded in T25 or T75 flasks coated with fibronectin (depending on the number of cells).

[0067] Culture 1-2 weeks: Cells were cultured in 2D layers for 1-2 weeks in hypoxic conditions (5% O2) using FDA-approved culture medium (animal product-free; described below). After reaching confluence (approximately 10 million cells), cells were then passaged and centrifuged to begin sorting.

[0068] Sorting with CD73 / CD276 (PE and APC): Cells were stained with CD73-PE and CD276-APC for 30 min at 2°C. After washing and preparation of the cell sorter, cells were sorted by gating on approximately 20-30% of the positive population. Pre- and post-sorting analysis was performed.

[0069] Cultivation to high yield: The final cell line is cultured for 4 weeks using the same medium and flasks to obtain approximately 40 million cells for experiments or for creating cell banks.

[0070] Culture medium

[0071] DMEM / F12-500ml

[0072] Knockout serum (KSR) - 55ml - 10%

[0073] 100x l-Glutamax 5.5.ml-1%

[0074] 100x Non-essential Amino Acids - 5.5ml - 1%

[0075] Sodium Pyruvate - 5ml - 1%

[0076] B-Mercaptoethanol - 5 μl - 0.001%

[0077] rhEGF(peprotech)-1000ul(10μg / 1ml)-0.2%

[0078] rhFGF(peprotech)-500ul(10μg / ml)-0.1%

[0079] Eyecup collection

[0080] 1. Place the eyeball in buffer (HBSS / PBS) on a petri dish and carefully unhook the retina by removing the lens and vitreous humor without traces of RPE or ciliary body.

[0081] 2. The retina was suspended in 10 ml of papain solution at 37°C for 30 min to dissociate the tissue into a single cell suspension.

[0082] 3. Add 20 ml of HBSS to the test tube and centrifuge the sample at 2000 rpm for 5 min.

[0083] 4. Aspirate the buffer and resuspend the pellet in 1 ml of culture medium and place in a T-75 flask for further expansion.

[0084] Preparing cells for sorting

[0085] 5. Remove cells from confluent flasks using trypsin (1:6 in HBSS).

[0086] 6. Incubate the cells with trypsin at 37°C for 3 to 4 minutes.

[0087] 7. Collect the cells in a 50 ml tube and add about 40 ml of culture medium containing KSR to the cell suspension.

[0088] 8. Centrifuge the suspension at 1200 rpm for 5 min at 15°C.

[0089] 9. Aspirate the supernatant and resuspend the cell pellet in 200 μl of Miltenyi running buffer (Cat. No. 130-107-207).

[0090] Staining for pure rod photoreceptor sorting

[0091] 10. Place the cells on ice and incubate in CD73 PE (Cat. No. 130-095-183, Miltenyi), CD276 antibody, anti-human, (Cat. No. 130-099-998) and incubate for 30 min.

[0092] 11. After incubation, resuspend cells in 10 ml of HBSS and filter using a 30 um filter (Miltenyi).

[0093] 12. The samples were then centrifuged at 300 g for 5 min to wash away the antibodies.

[0094] 13. Meanwhile, prepare the TYTO sorting chamber by injecting 1 ml of filtered TYTO buffer in the input chamber. Pressurize the sorting chamber to move the buffer into the positive and negative chambers.

[0095] 14. Remove the buffer from the input chamber to complete the sorting chamber preparation.

[0096] 15. The supernatant was then discarded and the cells were resuspended in 5 to 10 ml (4 to 6 million cells in 5 ml, more than 5 million cells in 10 ml) of TYTO buffer depending on the cell number and injected into the input chamber of the TYTO sorting chamber for sorting.

[0097] 16. Before running the TYTO sort, place 100 μl of the cell suspension in an Eppendorf for MACSQUANT analysis.

[0098] Post-sorting culture

[0099] 1. Using a fine tip pipette, remove the cells from the positive chamber and rinse the chamber with HBSS.

[0100] 2. Place 50ul of cell suspension in an Eppendorf for post-sorting MACSQUANT analysis.

[0101] 3. The remaining cell suspension was washed with 15 ml of HBSS by centrifugation at 300x for 5 min.

[0102] 4. Resuspend the cell pellet in culture medium and plate onto fibronectin-coated T-25 or T-75 flasks.

[0103] 5. Incubate at 37°C in hypoxic conditions (5% O2).

[0104] 6. The flasks were maintained for 48 hours without medium change.

[0105] 7. Replace the complete medium 2 days after incubation.

[0106] 8. Thereafter, fresh medium was added every 2 days until confluence was achieved.

[0107] 9. Trypsinize confluent flasks (should be confluent in 7 to 10 days) and plate cells on a t-75 for further expansion.

[0108] 10. Change the medium daily until confluence is reached in 3 to 4 days.

[0109] Cell sorting and analysis

[0110] Sorting strategy

[0111] The cells analyzed with MACSQUANT before running TYTO were set as controls for running the sort. The selected markers CD73 and CD276 were analyzed with MACSQUANT by adjusting the voltage to pull the desired population in the highest quadrant of intensity. After executing this first gating strategy, the TYTO sorting chamber was placed in the machine and the microfluidic flow was started.

[0112] The cells are first pushed through a negative chamber in order to stabilize the pressure (about 150hPa), but also to stabilize the flow of cells in front of the lasers (40ms between two cells). In order for the machine to know its speed, the cells must be dyed in at least two different fluorescent dyes, this way the machine can calculate the time it takes for the cell to go from the first laser to the second laser, and therefore measure its speed. Once these two variables are stable, the lasers can be started, and the fluorescence data begins to appear, showing about 3000-5000 cells that have been pushed in front of the lasers. Each cell passes in front of three lasers, and its fluorescence intensity is measured and reported on a graph.

[0113] The same gating strategy used in the control on MACSQUANT is reported on the real-time TYTO sorting machine, and sorting is started. However, in order to correctly capture each cell, the delay of valve speed and action must be correctly set. The backscattering of each cell colony (BSB and BSV) flowing through the microfluidic device is fitted, and the threshold for both BSB and BSV is set to 10^2-5.10^2. The threshold and gated channels of PE and VioBlue are concentrated on the high fluorescence colony, about 10^2 to 10^3. The gate time is set at 40ms, with a pressure of 150hPa. These settings can be changed according to cell size, shape, granularity, intensity, rigidity and concentration, and these settings can be changed by analyzing cell staining and colony before starting sorting. In general, the goal is to modify parameters to obtain a high fluorescence colony of about 10^2 to 10^3 in both PE and VioBlue lasers that can be sorted.

[0114] During the entire sorting process (usually about 2 hours), the percentage of sorted, gated and positive cells is measured and reported as a function of time. At the end of sorting, the sorting chamber is removed from the machine and the cells are taken back to cell culture for the next step.

[0115] Flow cytometry

[0116] hPRPs (5 x 10^5 / mL in culture medium) were trypsinized and the collected cell pellets were phenotyped and then analyzed using flow cytometry assay.

[0117] Flow cytometry was performed using a MACSQUANT flow cytometer (Miltenyi, San Diego) (100). Cells were collected and fixed with paraformaldehyde for 15 min at 4°C. Cells were then washed in wash buffer (BD Biosciences) and incubated in blocking buffer (Pharmingen staining buffer with 2% goat serum) for 30 min at room temperature. Blocked cells were seeded onto flat-bottom 96-well culture dishes (treated, sterile, polystyrene, Thomas Scientific) and stained with conjugated primary antibodies (DAPI-Vioblue, CD73-PE, CD276-APC, cone arrestin-FITC, blue opsin-FITC, rhodopsin-FITC, CRX-APC, recoverin-APC, calbindin-FITC, PkCa-FITC, Brna3a-FITC, Ki67-APC, Thy1.2-APC, vimentin-FITC, Cynoxin-APC, PAX6-APC, NRL-APC, Cmyc-FITC) overnight at room temperature. Primary antibodies were diluted in 200 uL of antibody buffer (TBS, 0.3% Triton X-100 and 1% goat serum). After cell washing 3 times 15min, the second antibody is anti-rabbit and anti-mouse of goat origin, and is diluted 1:200 in antibody buffer (Jackson Immunoresearch Laboratory). Second antibody is applied and placed at room temperature for 3h. Use MACSQUANT flow cytometer to measure light scattering and fluorescence signal (record 2x 10^5 events) from each hole. Use MACSQUANTify software (Miltenyi) analysis result. Single cell colony of each primary anti-DAPI positive is gated. Compared with blank and species-specific isotype control, estimate the ratio of positive cells in gated colony.

[0118] Immunohistochemistry (IHC):

[0119] Live hPRPs grown on slides and cryosections from Long Evans left eyes were fixed with 4% paraformaldehyde (Irvine Scientific) in 0.1 M PBS for 20 min at room temperature. These fixed cells and sections were blocked and permeabilized with blocking solution [Tris-buffered saline (TBS), 0.3% Triton X-100, and 3% goat serum (Jackson Immunoresearch Laboratories, West Grove, PA)] for 15 min. The samples were then incubated with 0.1 M The samples were rinsed twice with TBS buffer for 15 min each, mounted on poly-lysine microscope slides, and incubated with primary antibodies (DAPI-Vioblue, CD73-APC, CD276-Vioblue, rhodopsin-Cy3, recoverin-FITC, PKCa-Cy3, CRX-APC, Ki67-Cy3, PAX6-FITC) at a concentration determined in the laboratory at 4°C overnight. The next day, the samples were rinsed 3 times with TBS for 15 min. Secondary antibodies (anti-mouse and anti-rabbit from goat) were applied for 1 hour at room temperature. The samples were then washed with TBS for the last time before being mounted on poly-lysine microscope slides with lox viscosity slide mountant. Digital images were obtained with an Epifluroscent microscope using a 20x objective. Electronic image files were managed using Matlab software.

[0120] In vivo assessment of cell and EV effects

[0121] in vivo transplantation

[0122] Fifteen rd-1 (C3H / HeOuJ) (3 weeks old, approximately 15 g in weight) were used as recipients in the experiment. Transplantation was performed on non-immunosuppressed mice. Mice were sedated by intraperitoneal injection of ketamine (100-200 mg / kg) for anesthesia and xylazine (20 mg / kg). The eyes were first anesthetized with topical ophthalmic proparacacine (0.5%), and then Genteal was used to keep the lens moist during surgery.

[0123] Recipient mice were injected with hPRP or Rod-Exo single cell injections in the subretinal space and in the vitreous. Conjunctival incisions and small sclerotomies were made using fine disposable scalpels. Cells were injected into the subretinal space using a glass pipette (inner diameter, 150um) attached to a 50-uL Hamilton syringe via a polyethylene tube. hPRP was injected into the retinal bleb as a single cell suspension in PBS. All samples contained approximately 1x 10^5 cells, and the injection volume in all repeats was 2uL. Similarly, a 31g needle was used to perform intravitreal injections with cells in 3uL of PBS. For subretinal injections, a glass coverslip applied to the eye was used to check for the presence of blebs. If a shiny bleb was seen under a dissecting surgical microscope, the subretinal space injection was considered successful. Triple antibiotics (Bac / Neo / Poly) were given topically at the end of surgery to prevent further infection. The mice were then placed in their cages for a 21-day study.

[0124] The study protocol was reviewed and approved by the Schepens Eye Research Institute Animal Facility and conformed to the Association for Research in Vision Ophthalmology statement for the use of animals in ophthalmic and vision research.

[0125] Tissue processing

[0126] Mice were sacrificed by CO2 asphyxiation for 2 min at 8, 15 and 21 days post-transplantation. Eyes were enucleated and placed in 4% paraformaldehyde for 24 hours. Tissues were then saturated with increasing concentrations of sucrose (5%, 10%, 20%) in Sorensen's phosphate buffer. Eyes were stored in 30% sucrose overnight or until dissection. Tissues were embedded in frozen section gelatin medium overnight and sectioned on a cryostat at 15 μm thickness.

[0127] For subretinal injections, during the sectioning process, every fifth section was stained with TRA-1-85-FITC and STEM121-FITC (human cell markers), rhodopsin-PE and recoverin-APC (host photoreceptor markers) and DAPI-Vioblue (nuclei) and examined for the presence of hPRP by epi-fluorescence. Sections were observed for engraftment and cell survival in both the subretinal and intravitreal spaces of mice under a Lecia Sp8 confocal microscope.

[0128] For intravitreal injections, during the sectioning process, every 6th section was stained with H&E and examined under brightfield microscopy for retinal morphology and architecture, as well as photoreceptor layer thickness. Blind counts of the outer nuclear layer of photoreceptors were performed to measure the possible neuroprotective effects of hPRP. Frozen vials of culture medium were kept at -80°C before use. Frozen vials of culture medium were analyzed by Dr. Redenti's laboratory prior to isolation and lyophilization of EVs, where complete proteomic and miRNA analysis was performed. A general protocol for EV isolation and analysis was performed.

[0129] Rod-Exo Manufacturing Process

[0130] Manufacturing process Figure 3 In summary. EVs secreted by hPRP cells are obtained from their culture medium. The culture medium is frozen and then ultracentrifuged to separate EVs, and debris, dead cells, and other undesirable components are discarded. The final precipitate is resuspended in PBS, lyophilized and stored, keeping the EV structure intact. Finally, the EVs are resuspended in water and injected into the vitreous of the eye. hPRP secretes EVs throughout the manufacturing and culture process. These EVs are released in the culture medium. After immunoselection (sorting) and after 2 passages, we save and freeze approximately 40-50 mL tubes of this culture medium. Those culture medium vials contain selected and secreted EVs.

[0131] Isolation of rod-exosomes from culture medium

[0132] The frozen medium from the fetal hPRP culture (for fetal-derived EVs) was thawed in a water bath (37°C). This was followed by a series of ultracentrifugations of the samples. The first sample was spun at 300g for 10 min to discard cells from the source. This was followed by a 2000g spin for 10 min to discard dead cells, followed by a 10,000g spin for 30 min to discard all cell debris. Finally, two ultracentrifugations were performed to sediment the EVs (the only remaining component of the supernatant): both ultracentrifugations were at 150,000g for 70 minutes. Finally, the final supernatant was discarded, and the EV precipitate was obtained. The EVs were then measured, analyzed, and used for testing and experiments.

[0133] Freeze-drying and storage of rod-exosomes

[0134] The separated and precipitated EVs were resuspended in a final volume (50 mL) containing PBS. Lyophilization was performed by controlled freeze drying at -80°C to obtain the final powdered product. The final powder was stored at -80°C and stored in vials, containing 1 g of powdered EVs.

[0135] Functional testing of EVs and proteins

[0136] Lyophilized EVs were resuspended in PBS and tested with Western blot, Bradford assay, Nano sight, proteomic analysis, and ELISA to check the concentration of proteins, the concentration of EVs, protein functionality, and the presence of therapeutic proteins. The tests and analyses were compared with pre-lyophilized EVs, using Western blot for imaging and analysis of the presence of specific proteins; NANO SIGHT for analysis and measurement of EV concentrations; Bradford assay for measurement of total protein concentration; ELISA for measurement of protein functionality; and proteomic analysis for comprehensive analysis of fold changes in proteins.

[0137] In vivo testing

[0138] In this experiment (Example 3), 7 mice with the genotype rho- / -, 3 weeks old and weighing about 120g, and P23 homozygous mice, 4 weeks old and weighing about 117g, were used. The mice were not given any immunosuppressive drugs before transplantation. To induce anesthesia, mice were injected with ketamine (40-80mg / kg) and xylazine (10mg / kg). Proparacaine (0.5%) was applied topically to anesthetize the eyes, followed by Genteal to maintain the humidity of the lens during surgery. Recipient mice were injected with HPRP (human progenitor rod photoreceptors), gel + HPRP and sham groups (sham) in the intravitreal space. The gel consists of gelatin-hyaluronic acid functionalized with tyramine side chains used at a concentration of 5.5% (w / v) (Dromel et al., NPJ Regen Med. 2021 Dec 20; 6 (1): 85). 0.1U / ml of HRP and 1mM of H2O2 cross-linked polymers were used. Cells and gel were injected into the vitreous cavity using a glass pipette (inner diameter, 100 um) attached to a 50-uL Hamilton syringe via polyethylene tubing. All samples contained approximately 75,000 cells, and the gel injection volume in all replicates was 1 uL. Triple antibiotics (Bac / Neo / Poly) were given topically at the end of surgery to prevent further infection. The mice were then placed in their cages and returned to their normal habitat. The study protocol was reviewed and approved by the Sigpens Eye Institute Animal Facility and complies with the Association for Research in Vision Ophthalmology's statement on the use of animals in ophthalmic and visual research.

[0139] Eye test

[0140] The OptoMotry designed by Cerebral Mechanics Inc. for rodents was used for optokinetic tracking (OKT) in a non-invasive manner. We placed mice on a platform surrounded by four LCD screens in a light-proof box and presented them with visual stimuli via the screens. In order to evaluate VA, vertical sine wave gratings of different spatial frequencies were presented to the mice. The gratings were displayed at a fixed distance from the animals, and the minimum spatial frequency that triggered a tracking response was recorded as the VA threshold. VA was measured separately for each eye, with an average test time of 20 minutes for each animal. For contrast sensitivity, tracking motion was identified as slow, steady head movement along the direction of the rotating grating. The spatial frequency threshold was measured by testing mice at various spatial frequencies between 0.064 and 0.514 cycles / degrees, with an average test time of 20 minutes for each animal. The OptoMotry device uses a proprietary algorithm that adjusts the test stimulus based on the tracking reflex of mice. The contrast threshold was measured at a spatial frequency of 0.2 cycles / degrees and calculated as the inverse of the Michelson contrast. The inverse of the contrast threshold was then plotted for analysis.

[0141] Electroretinogram

[0142] To prepare for the ERG test, the animals were anesthetized and their pupils were dilated using 1% tropicamide eye drops, followed by a drop of 1% proparacaine instilled on the corneal surface. To prevent dehydration, a drop of Genteal (corneal lubricant) was applied to the cornea of ​​the untreated eye. In the treated eye, a drop of 0.9% sterile saline was applied to the cornea to allow electrical contact with the gold wire loop recording electrode. A 25-gauge platinum needle was inserted subcutaneously in the forehead to serve as a reference electrode, and another needle was inserted subcutaneously near the tail to serve as a ground electrode. The ERG test was completed by a series of flash intensities generated by a Ganzfeld controlled by Diagnosys Espion3. Both dark (weak light) and photopic (normal light) responses were tested. During the test, a brief flash was presented to the eye to stimulate the retina. The electrical response generated by the retina was recorded by the gold wire loop electrode on the cornea and transmitted to Diagnosys Espion3. The response was analyzed to determine the retinal function of the animal. This procedure can help diagnose and monitor a variety of retinal diseases and conditions.

[0143] Example 1. Production of hPRP

[0144] Fetal retinas were cultured in culture as described above and sorted based on the expression of CD73+ and CD276+ using a Miltenyi TYTO cell sorter. The number and morphology of sorted cells can be seen in Figure 4and 5 .

[0145] 4.2 Isolation of hPRP from fetal tissue

[0146] Figure 6 Shown is the real-time gating strategy and sorting of hPRPs from fetal retina using both PE and VioBlue lasers for CD73 and CD276 positive cells, respectively. Gates are set around the correct populations to start sorting and isolate pure hPRPs. After sorting, all populations (input, positive and negative fractions) are analyzed. Figure 7 The percentage of positive hPRPs in each population is shown, reaching more than 95% in the positive compartments of TYTO.

[0147] To confirm the success of the entire sorting run, analysis of the trigger rate, sorting rate, and percentage of successfully sorted cells were monitored and analyzed during the entire process. Figure 8 The evolution of these variables during the 1 h sorting period is shown. Finally, the final population of hPRPs was analyzed in a MACSQUANT flow cytometer to precisely measure the number of positive cells (CD73+ / CD276+) captured in the positive chamber compared to the input from the fetal retina ( Fig. 9 ).

[0148] 4.3 Characterization of hPRP

[0149] After sorting and 4 weeks of culture, hPRPs were analyzed for their phenotype using both flow cytometry and immunohistochemical staining ( Figure 10-13 ). Fig.10 Key markers (CD73, CD276 and recoverin) for high purity of rods in hPRP were presented. These markers were found to be above 95% in all cell populations after culture and sorting. Analysis of multiple retinal markers was performed by flow cytometry to demonstrate the presence of rods and the absence of all other retinal cells ( Fig.11 The presence of high expression of rod markers confirmed the purity of hRPR, while all other retinal cell markers (RGC cells, bipolar cells, amacrine cells, horizontal cells, cone cells, and Muller cell markers) were found to be negative, ensuring high rod purity. To confirm these results, hPRPs were stained with the same markers and imaged under a fluorescent microscope to demonstrate their expression ( Figure 12-13 ). High expression of rod and photoreceptor markers was found, while other retinal cell markers were absent in hPRP.

[0150] 4.4 In vivo injection of hPRP into the vitreous and subretinal space

[0151] To demonstrate the role of hPRP in neuroprotection of the retina. Cells were injected into the vitreous and subretinal space of rd1 mice (a well-characterized mouse model of retinal degeneration). Cells were injected at P25, and animals were sacrificed and analyzed 7 and 21 days after transplantation. To demonstrate neuroprotection, the retina was stained with a cone arrestin marker (in red) to measure the number of host cones remaining in the retina (which degenerate at a controlled rate). Fig.14 Whole-field flat mounts of these retinas are shown, showing the presence of a large number of cones at day 21, indicating a high neuroprotective effect from the injection of hPRP. The retinas were also dissected to measure the outer nuclear layer (ONL) size, confirming the presence of photoreceptors in the degenerated rd1 mice. After the measurements ( Fig.15 ), a greater number of cells were found in the ONL of mice that received hPRP both in the subretinal space and in the vitreous compared with the control group. Finally, to test the safety of hPRP injections, intraocular pressure (IOP) was measured throughout the experiment, as Fig.16 A normal increase in IOP was found a few days after the intravitreal injection and returned to the normal range (10-20 mmHg) after 5 days with no increase during the rest of the experimental period.

[0152] Sorting process for purification of rod precursor photoreceptor cells (CD73 + / CD276 + ) produced a 95% pure population of viable rod photoreceptor cells (showing uniform proliferation) with a Ki-67 index of approximately 20%.

[0153] These cells can be used for cell replacement, drug discovery and screening or other applications that may require photoreceptors or their precursors.

[0154] Example 2. Isolation and Characterization of Rod-EVs

[0155] Proteomic analysis of EVs was performed and fold changes in proteins were compared with those in 14-week fetal retina. Fig.17 As seen in the Venn diagram of Figure 3, 31 proteins were found to be unique to our EVs, whereas 361 proteins were found in both our EVs and the fetal retina.

[0156] Fig.18 The entire proteome cargo found and analyzed in our EVs is summarized. The proteins were divided into six main groups:

[0157] 1. Retina-specific: proteins involved in the development and regeneration of the retina (cones, photoreceptors, ganglion cells, bipolar cells)

[0158] 2. Cytoprotection: Proteins involved in neuroprotection of cells

[0159] 3. CNS development: proteins involved in the development of nerve cells (neurons, axons, synapses)

[0160] 4. Immune response: proteins involved in triggering immune responses and immune reactions. (Cytokines, macrophages)

[0161] 5. Common Cellular Processes: Proteins found in all cells, involved in normal cell activities (adhesion, proliferation, ...)

[0162] EV-specific: proteins found on the surface of all EVs

[0163] Table 1 shows the main and most significant proteins found in each category, along with a description of their protein roles, genes associated with the proteins, and their activities.

[0164] Table 1. Vesicle proteomic cargo composition

[0165]

[0166]

[0167] To simplify the analysis of all proteins, subcategories were created within each of the six different groups established previously. The role and activity of each protein was analyzed using the protein database (Uniprot) and the gene database (GeneCard). Fig.19 The classification of the entire proteome cargo of our EVs is shown as the fold changes of all proteins in all subcategories. Notably, 42 proteins were found for retina-specific development, 80 proteins for cytoprotection, 51 proteins for CNS development, and only 26 proteins for immune response.

[0168] The first comparison of proteomic cargo was performed between our EVs and 14-week fetal retinal EVs. This comparison allowed us to characterize specific beneficial therapeutic agents, but more importantly immune triggering proteins. Fig. 20As seen in the previous study, most common cellular processes and EVs had similar expression in the fetal retina and our EVs. When comparing the fetal retina and our EVs, average levels of retina-specific proteins were found. However, neuroprotective proteins were extremely upregulated in our EVs compared to the fetal retina. Most importantly were proteins that are well known to be involved in many failure scenarios in cell therapy (TGFB1) and immune response (JCHAIN). In our analysis, we found that these proteins were expressed 10,000-fold less in our EVs compared to the fetal retina. Notably, other immune-triggering proteins were found at the same levels in the fetal retina and our EVs.

[0169] Final analysis performed on the proteomic cargo of our EVs revealed that the proteins in question were present only in our EVs but not in the fetal retina. Fig.21 As seen in the EVs, 31 proteins were found not present in the fetal retina but were highly expressed in our EVs. Notably, no immune triggering proteins were found to be unique to our EVs or absent in the fetal retina, suggesting that those immune triggering proteins are common proteins found in most stem cell therapies.

[0170] A comprehensive list of proteins found in EV (402) and analyzed as described above is provided in Table 2.

[0171] Table 2. List of all proteins found in hPRP-exosomes with fold changes

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] Example 3. In vivo evaluation of rod-EVs

[0184] Rho- / - mice are knockout mice that lack the gene for rhodopsin, a light-sensitive protein found in the rods of the retina. Rhodopsin is essential for vision under low light conditions. Therefore, Rho- / - mice have impaired vision in dim light, and their rods do not function properly. They are often used in studies related to retinal degeneration. On the other hand, P23h homozygous mice have a mutation in the rhodopsin gene that causes them to produce a defective form of the rhodopsin protein. This mutation is similar to the mutation found in humans with autosomal dominant retinitis pigmentosa (RP). P23h homozygous mice have progressive retinal degeneration and vision loss, similar to RP patients. They are used in studies related to understanding the molecular mechanisms of RP and testing potential treatments. Therefore, although both rho- / - and P23h homozygous mice are used in vision-related studies, their genetic modifications and related characteristics are different.

[0185] Due to this mutation, rod photoreceptors malfunction and begin to degenerate as early as 3 weeks after birth. This is followed by the death of cones starting at 5-6 weeks after birth. For neuroprotection of the retina, we injected 4 week old mice with Technocytes+Gel (a gel composed of gelatin and hyaluronic acid combined at a concentration of 5.5% w / v and cross-linked using HRP and H2O2) to potentially prevent and slow the degeneration of these mice, with a focus on cone neuroprotection. We tested the functionality and retinal behavior of these mice weekly after the injections. Functional behavioral testing was performed using optokinetic tests, Figure 22A-B , to measure both visual acuity and contrast sensitivity. We confirmed that, as shown in previous papers (Xiao et al., Invest Ophthalmol Vis Sci. 2019 Oct 1; 60(13): 4196-4204), visual acuity in Rho- / - did not decrease before 12 weeks, independent of the injection group. However, we observed a decrease in contrast sensitivity in the SHAM group 2 weeks after injection. Cell + gel treatment was able to prevent this decline and delay it for at least 1 month, as seen in the significant differences at weeks 8 and 9. Although this is an ongoing study, we expect to obtain high persistence in the neuroprotective functional behavior of these mice until at least 12-14 weeks.

[0186] These results were confirmed by testing of cone function using electroretinography (ERG), as seen in Figures 23A-C. We focused on the photopic B wave, which is a representative of cone function in the retina. As seen in Figure 23A, a significant increase in cone function was observed at 7 and 8 weeks post-injection, which was also measured using percent increase (Figure 23B) and confirmed with ERG tracking (Figure 23A-C). Fig.23C ).

[0187] Overall, this ongoing research demonstrates that our gel+cells can neuroprotect cone function in the retina of degenerating Rho- / - mice.

[0188] As explained in the methods section, P23H has a genetic mutation with misfolding of the rhodopsin protein. We performed similar studies on Rho- / -, knowing that P23H has a faster degenerative profile. Although no scotopic waves were observed in P23H, we were able to measure photopic B waves in 3 different groups (SHAM, cells, cells+gel) using ERG, as shown in Figure 24A-B Compared with the SHAM group, significant improvements in photopic B waves were observed for both the cell group and the cell+gel group 1 week after injection.

[0189] These results show that this treatment is still effective even in cases of advanced retinal degeneration.

[0190] Other Implementations

[0191] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. A method of providing a population of progenitor rod photoreceptor cells (PRPs), the method comprising: providing a starting population comprising retinal cells from a mammal, preferably a fetal mammal; isolating cells expressing CD73 and CD276 from said initial population to provide a substantially purified population of PRP; and The substantially purified population is optionally cultured.

2. The method of claim 1, wherein isolating cells expressing CD73 and CD276 comprises using fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS), optionally in a microfluidic device.

3. The method of claim 1 or 2, further comprising culturing the initial population comprising retinal cells prior to isolating cells expressing CD73 and CD276.

4. The method of claims 1 to 3, wherein the substantially purified population of PRP is cultured in a medium comprising serum or serum replacement, EGF, and FGF.

5. The method of claims 1 to 4, wherein the substantially purified population of PRP is cultured under hypoxic conditions.

6. The method of claims 1 to 5, wherein the initial population comprising retinal cells is from a fetal mammal.

7. The method according to claims 1 to 6, wherein the mammal is a human.

8. A substantially purified population of PRP produced by the method according to claims 1 to 7, preferably wherein at least 75% of the population comprises cells expressing CD73 and CD276.

9. The substantially purified population of PRP according to claim 8, for use in treating a subject having a condition associated with loss of retinal rod or cone photoreceptors.

10. A method of treating a subject having a condition associated with loss of retinal rod photoreceptors, the method comprising administering to the subject a therapeutically effective amount of PRP obtained by the method according to claims 1 to 7, preferably wherein at least 75% of the population comprises cells expressing CD73 and CD276.

11. A method of providing extracellular vesicles (EVs) from rod photoreceptor progenitor cells, the method comprising: Providing a substantially purified population of PRP according to claim 8; maintaining a substantially purified population of said PRP in culture in a culture medium, preferably wherein said culture medium is contacted with said PRP in culture for at least 12, 18, 24, 36, 48, or 72 hours; removing the culture medium from the culture; and EVs are isolated from the culture medium.

12. The method of claim 11, wherein isolating EVs comprises ultracentrifuging the culture medium to obtain a precipitate containing EVs.

13. The method of claim 11 or 12, further comprising freeze-drying the isolated EVs.

14. A composition comprising EVs obtained by the method according to claims 11 to 13.

15. The composition of claim 14, wherein the EV is in a polymer scaffold.

16. The composition of claim 15, wherein the polymer scaffold comprises polycaprolactone (PCL), polylactic-glycolic acid (PLGA), polyethylene glycol (PEG), polylactic acid (PLA), polyetherimide (PEI), PNIPAAM, or hydroxyethyl methacrylate.

17. A composition according to claims 14-16 for use in treating a subject having a condition associated with loss of retinal rod or cone photoreceptors.

18. A method of treating a subject having a condition associated with loss of retinal rod or cone photoreceptors, the method comprising administering to the subject a therapeutically effective amount of PRP obtained by the method according to claims 11 to 13.

19. According to any one of claims 9, 10, 17 or 18, wherein the condition associated with loss of retinal rod or cone photoreceptors is an inherited retinal degenerative disease (IRD), optionally cone-rod dystrophy, retinitis pigmentosa (e.g., LCA), or Stargardt's disease; or macular degeneration, e.g., dry macular degeneration.

Images