WO2025264696A2 - Restorative cell therapy for eye diseases and vision loss - Google Patents

Abstract

Disclosed herein are cells, cellular compositons, methods, and systems for restoring vision loss in a patient suffering from same. In many embodiments, the disclosed cells may be retinal ganglion cells (RGCs) expressing one or more survival-enhancing factors, for example small heat shock protein Bl, HSPB 1, which may be driven by an RGC-specific promoter. In many embodiments, expression of the survival-enchancing factor may allow RGCs transplanted into the patient's retina to more effectively repopulate the RGC population.

Description

RESTORATIVE CELL THERAPY FOR EYE DISEASES AND VISION LOSS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit of priority pursuant to 35 U.S.C. § 119(e) of U.S. provisional patent application No. 63/660,872 entitled “RESTORATIVE CELL THERAPY FOR EYE DISEASES AND VISION LOSS,” filed on 17 June, 2024, which is hereby incorporated by reference in its entirety. BACKGROUND [0002] Glaucoma causes progressive optic neuropathy and it is a major cause of irreversible blindness worldwide. It is estimated that there are approximately 80 million people worldwide afflicted with glaucoma, and nearly 11 million of those are completely blind from it. The burden of glaucoma is expected to significantly increase due to the growing aging population, and it is expected that there will be approximately 110 million people worldwide suffering from glaucoma by 2040. Among several types of glaucoma, primary open-angle glaucoma (POAG) represents almost 70-90% of glaucoma cases. It is anticipated that the prevalence of POAG in the US will grow at an annual rate of 2.8% a year over the next 10 years, from about 3.6 million cases to 4.6 million cases in 2030. Previous studies have shown that African Americans and Latinos are at a greater risk for glaucoma than Caucasians, with an increased disease prevalence, earlier disease development, and increased progression to blindness. According to a previous study, in eyes with advanced glaucomatous damage, retinal ganglion cell (RGC) loss can reach 75%. In addition, the cumulative incidence of blindness in at least 1 eye is 26.5% and bilateral blindness is 5.5% 10 years after glaucoma diagnosis. [0003] A large number of glaucoma patients may not even know that they have the disease because it has no noticeable symptoms in the early stages. By the time affected people notice visual changes, the disease will have caused considerable vision damage. The vision loss in glaucoma occurs primarily due to the death of RGCs. Elevated intraocular pressure (IOP) is a significant risk factor for RGCs death, along with other risk factors that include advanced age, race, and genetics. Current medical therapies are limited to lowering IOP by topical medications, laser therapy, and/or surgical means. [0004] Current therapies may prevent further vision loss in many glaucoma cases, but to date there is no treatment that can restore vision once it has been lost. According to a “Glaucoma Forecast and Market Analysis” report by GlobalData, there is an “urgent unmet need for drugs with neuroregenerative properties…to prevent or reverse damage to the optic nerve,” “Currently there are no drugs that are poised to fill this need…so any drug that achieves this can expect significant success due to high therapeutic importance and lack of competition.” [0005] The National Eye Institute has promoted the idea of cell therapy for glaucoma through its “Audacious Goals” initiative. To date, these cell therapies have failed. Where RGCs have been used for transplantation, they have failed to integrate into the retina and extend axons trough the optic nerve. [0006] Accordingly, there is an urgent need for new therapeutic strategies that can restore vision in glaucoma patients. SUMMARY [0007] Disclosed herein are cells, compositions, methods and systems useful for restoring vision in patients in need thereof. In many embodiments, restoration of vision may be accomplished by repopulating RGCs in the retina of the patient. Disclosed herein are methods that may include isolating RGCs from human induced pluripotent stem cell-derived retinal organoids; transducing one or more genes that may aid in survival of cells, for example the gene heat shock protein B1 (HSPB1) gene may be transduced into said RGCs in culture. The transduced RGCs may then be transplanted into the retina of a patient, wherein said transplanted RGCs integrate with the retina of the patient, for example the ganglion cell layer, where, for one example they may generate a signal in a pattern electroretinography analysis. [0008] Also disclosed are cells and cell-containing compositions for use in restoring vision in a patient with vision loss. In these embodiments, the cells may be derived from the patient with vision loss and the cells may comprise at least one nucleic acid comprising a coding region for HSPB1, and a RGC promoter, and the cell may expresses one or more markers indicative of RGC identity. In many embodiments, compositions comprising the disclosed cells may include one or more pharmaceutically acceptable carriers. In some embodiments, the disclosed engineered cells may be derived from induced pluripotent stem cells and/or from allogenic tissue. BRIEF DESCRIPTION OF THE DRAWINGS [0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [0010] FIG. 1 presents a bar graph and micrographs from a study of mice subjected to ischemic injury four weeks after intravitreal injection of AAV2 containing various HSPB isoforms. [0011] FIG. 2 shows data (micrographs and bar graphs) from expression studies of HSPB1 in iPSC-RGCs. [0012] FIG. 3A is a timeline of AAV2-HSPB1 transduction in hiPSC-derived RGCs. [0013] FIG. 3B presents representative images of hiPSC-derived RGC cultures at 7 days post-dissociation, stained for RGC markers (β-III-tubulin and RBPMS). [0014] FIG. 3C is a bar graph showing the percentage of β-III-tubulin+ or RBPMS+ RGCs relative to the total DAPI+ cells on day 7 post-dissociation. [0015] FIG. 3D shows representative immunofluorescence images of hiPSC-derived RGCs stained at days 0, +3, +7, +10, and +14 post-transduction. [0016] FIG. 4A shows RGC that were exposed to oxidative (H₂O₂) or inflammatory (CM: cytokine mixture) stress, to evaluate their effect on cell survival in the presence and absence of HSPB1 overexpression. Pictures are confocal images showing live/dead cell staining with calcein-AM (green, live cells) and ethidium homodimer (red, dead cells). [0017] FIG. 4B is a bar graph quantification of experiments in FIG. 4A. [0018] FIG. 5A are confocal images of AAV2-HSPB1-transduced or control RGCs exposed to H₂O₂ or CM and stained with cleaved caspase-3 (green, apoptosis marker) and RBPMS (magenta, RGC marker). [0019] FIG. 5B is a bar graph quatification of the experiment in FIG. 5B. [0020] FIG. 6 presents confocal immunofluorescent images of TUNEL staining (red) [0021] FIG. 7 is a bar graph showing a quantification of studies in FIG. 6. [0022] FIG. 8 is a map of one embodiment of the disclosed vector for expression of HSPB1 in RGCs, showing the RGC-specific mini-promoter, Ple345 (neurofilament, light polypeptide; NEFL). [0023] FIG. 9A is a timeline of AAV2-HSPB1 transduction in retinal organoids (ROs). [0024] FIG. 9B shows representative images of enhanced axonal outgrowth, with prominent _{neurite extensions observed in the AAV2-HSPB1 and AAV2-HSPB1+ brain-derived} neurotrophic factor (BDNF) groups. [0025] FIG. 9C is a plot of maximum branch length under each condition in Fig. 9B. [0026] FIG. 9D is a timeline of AAV2-HSPB1 transduction in ROs. [0027] FIG. 9E shows representative images of neurite outgrowth visualized using the axon marker β-III-tubulin (green). [0028] FIG. 9F is a plot of maximum branch length under each condition in Fig. 9E. [0029] FIG. 10A are bright field microscopy images of live cultures showing neurite outgrowth from yellow fluorescent protein (YFP)-expressing ROs at 1, 2 and 3 days after chopping. [0030] Fig. 10B is live fluorescence imaging of chopped organoid cultures 5 days after chopping confirms enhanced neurite outgrowth in BDNF, AAV2-HSPB1, and AAV2- HSPB1+BDNF-treatment conditions, as assessed by YFP expression. [0031] Fig. 10C shows immunofluorescence staining for the axonal and dendritic neuronal markers TUJ1 (β-III-tubulin) and microtubule-associated protein 2 (MAP2) was performed on day 5 post-transduction to evaluate neurite outgrowth in control, BDNF, AAV2-HSPB1, and BDNF+AAV2-HSPB1-treated retinal organoid pieces. Enhanced axonal and dendritic projections were observed in the AAV2-HSPB1 and BDNF+AAV2-HSPB1 treatment groups. [0032] Fig. 11A is a timeline of RGC transplantation in mice. [0033] Fig. 11B shows immunofluorescence micrographs two weeks after transplantation of retinal flatmounts co-stained with the human-specific marker HuN (green), and the RGC marker RBPMS (red). Co-localization of HuN(+) and RBPMS(+) signals indicates transplanted human RGCs. [0034] Fig. 11C shows representative confocal images showing cross-sections of retinas immunostained for HuN (green) and RBPMS (magenta). [0035] Fig. 11D shows representative images showing human RGCs within the ganglion cell layer (GCL) 4 weeks post-transplantation, stained with HuN(+) (green) and RBPMS(+) (red) human. [0036] Figure 12A is a timeline of RGC transplantation in mice following optic nerve crush (ONC). [0037] Fig. 12B shows retinal flatmounts immunostained for HuN (green) and RBPMS (magenta). [0038] Fig. 12C is a bar graph of transplanted human RGCs per mm² retinal area stained by assessing the co-localization of HuN and RBPMS. [0039] Fig. 12D shows representative confocal images show retinal cross-sections immunostained for HuN (green) and RBPMS (magenta). [0040] Fig. 12E is a bar graph showing the quantification of human RGCs relative to the total number of cells in the ganglion cell layer (GCL). [0041] Fig. 12F is a bar graph showing P1-N2 amplitude from pattern electroretinography (pERG) recorded on day 13 post-transplantation [0042] Fig. 12G shows representative signals from control RGC- or engineered-RGC (Eng- RGC)-transplanted mouse retinas. DETAILED DESCRIPTION [0043] Disclosed herein are compositions and methods of innovative cell therapy for the treatment of glaucoma. In many embodiments, the disclosed compositions and methods include transplantation of human induced pluripotent stem cell (iPSC)-derived RGCs that have been engineered for improved survival. [0044] HSPB1 is a small heat shock protein with anti-apoptotic properties. Applicants have shown that expression of HSPB1 prevents RGC death and axonal damage in a mouse model of glaucoma.. Applicants reliably generate human retinal organoids (ROs) from induced pluripotent stem cells (iPSCs). Applicant’s ROs allow for isolation and culturing of RGCs. In many embodiments, the Applicant’s use of RO-derived RGCs provides therapeutic compositions and methods for administering autologous cells into patients to restore vision. Use of autologous cells may decrease the likelihood of rejection of transplanted cells by the patient’s immune system. [0045] Disclosed herein are compositions and methods for generating human RGCs with enhanced survival characteristics. The disclosed cells, upon transplantation, overcome many limitations of current cell therapy strategies, and provide for restoring vision in glaucoma patients. [0046] Administration of unmodified control RGCs (i.e., not engineered to express HSPB1) into the vitreous results in less than 1% of those cells integrating into the retina. Applicant’s compositions and methods improve RGC survival, greatly improving their ability to integrate into the retina. [0047] HSPB1 expression in RGCs enhances the survival of the RGCs in vitro and in vivo. In many embodiments, the enhanced survival may aid in re-populating RGCs in diseases such as glaucoma and in conditions such as ocular hypertension. In many embodiments, the HSPB1- expressing RGC (Eng-RGC) reduces the loss of RGCs by greater than about 50%, for example, 66%. In some cases, repopulated control RGCs may be decreased by about 36%, but repopulation of Eng-RGCs may result in only a 12% loss, for example after four or more weeks, for example after six weeks. [0048] HSPB1 expression in RGCs may enhance resistance to apoptosis. In many embodiments, apoptosis may be associated with caspase-3 and/or induced with hydrogen peroxide (H₂O₂). In many embodiments, apoptosis of control iPSC-RGCs in response to H₂O₂ may be about 15.4%, whereas apoptosis of Eng-RGCs may be less than about 10%, for one example less than about 5%, for example about 4.2%. [0049] Various methods may be used to administer the disclosed engineered RGCs. In one embodiment, the administration is via intravitreal injection, which may require cells to cross the inner limiting membrane (ILM) to allow for graft homing and neurite outgrowth into the host retina. In some embodiments, intravitreal injection may be enhanced by digesting the ILM prior to administration, for example, digesting the ILM with protease treatment. [0050] There is no FDA-approved therapy to directly protect RGCs in glaucoma. The only strategy for protecting RGCs is the lowering of IOP – in some cases this can only slow the rate of RGC loss. There are no known neuroprotective agents to prevent RGC death. One study by Dr. Jeffrey Goldberg of Stanford University, used encapsulated cell therapy for neuroprotection in retinal degenerative diseases. This trial used cells modified to secrete ciliary neurotrophic factor into the vitreous cavity to promote optic nerve growth by resident RGCs. Attempts by the same group to transplant mouse RGCs into rats found appreciable integration of transplanted mouse cells into the rat retina. However, successful transplantation was less than 10% - only 6 out of 70. [0051] Allogeneic umbilical cord-derived mesenchymal stem cell therapy has also been attempted for patients with traumatic optic neuropathy. These studies showed some evidence of improved visual acuity without evidence of side effects at a 1-year follow-up. However, like the studies above, and in contrast to Applicant’s methods and compositions, this was only neuroprotective. [0052] Disclosed herein are cells, compositions, and methods for cell replacement therapy. In many embodiments, the disclosed cell therapies may be autologous or allogenic. In the case of allogenic therapies, the risk of rejection may be mitigated by various techniques known to those of skill in the art. In many embodiments, the cells may be stem cells, for example pluripotent stem cells, in many embodiments induced pluripotent stem cells. [0053] The disclosed cells may be derived from retinal organoids (ROs). In one embodiment, stem cells may be used to form ROs in vitro. In these embodiments, RGC cells may be derived from the ROs, and engineered to express one or more survival enhancing factors. Genes / HSPs [0054] Disclosed herein are survival enhancing factors or genes. In many embodiments, the genes may code for small heat shock proteins (sHSP), for example, one or more of HSPB1-6. In many embodiments, the sHSP is HSPB1. The gene may code for a protein at least about 80% identical to the disclosed protein, for one example HSPB1, for example greater than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, or 81%. [0055] “Amino acid identity,” “residue identity,” “identity,” and the like, as used herein refers to the structure of the functional group (R group) on the polypeptide backbone at a given position. Naturally occurring amino acid identities are (name/3-letter code/one-letter code): alanine/ala/A; arginine/arg/R; asparagine/asn/N; aspartic acid/asp/D; cysteine/cys/C; glutamine/gln/Q; glutamic acid/glu/E; glycine/gly/G; histidine/his/H; isoleucine/ile/I; leucine/leu/L; lysine/lys/K; methionine/met/M; phenylalanine/phe/F; proline/pro/P; serine/ser/S; threonine/thr/T; tryptophan/trp/W; tyrosine/tyr/Y; and valine/val/V. [0056] An amino acid within a molecule may be substituted to create an engineered molecule. The amino acid (aa or a.a.) residue can be replaced by a residue having similar physiochemical characteristics, that is a ‘conservative substitution’ – e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, for example, based on size, charge, polarity, hydrophobicity, chain rigidity/orientation, etc., are well known in the art of protein engineering. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. binding, specificity, and/or function of a native or reference polypeptide is achieved. [0057] While conservative substitutions within a protein, i.e. buried or non-solvent accessible residues/positions, may in some cases alter the structure of the protein or affect folding of the protein, conservative substitutions at or near the protein’s surface, i.e. exposed or solvent accessible residues/positions may cause little or no discernable change to the protein’s structure and/or function, unless the altered surface protein is necessary for an interaction with another molecule, peptide, or protein. It is well within the abilities of the skilled artisan to alter the disclosed protein sequences by introducing conservative substitutions at up to 20% of the residues/positions without disrupting or changing the protein’s structure and/or function. [0058] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu. [0059] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and understood by those of skill in the art. [0060] In many embodiments, the disclosed proteins maybe coded for by a gene, nucleic acid, or nucleic acid sequence. These terms, as is well known in the art, refer to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single- stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA, cDNA, or vector DNA. Suitable RNA can include, e.g., mRNA. [0061] DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) refer to nucleic acid molecules having a backbone of sugar moieties, which are deoxyribosyl and ribosyl moieties, respectively. The sugar moieties may be linked to bases, which are the 4 natural bases (adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U)). In RNA, the bases are A, G, C, and U. The sugar moieties may also be linked to unnatural bases such as inosine, xanthosine, 7- methylguanosine, dihydrouridine and 5-methylcytidine. Other unnatural bases may also be used, for one example, wherein natural phosphodiester linkages between sugar (deoxyribosyl/ribosyl) moieties may optionally be replaced with phosphorothioates linkages. [0062] Nucleic acids of the present disclosure may be RNA, in particular mRNA or messenger RNA, which may be processed or unprocessed single-stranded RNA copies of genes, for use in synthesizing the disclosed proteins. Cells [0063] Various cells may be engineered for use in the disclosed methods and compositions. In many embodiments, the cells are stem cells, for example induced pluripotent stem cells. The disclosed cells are able to form organoids in vitro, for example retinal organoids. In many embodiments, the disclosed organoids include RGCs, which may be separated and/or isolated from the ROs and engineered to express a survival enhancing factor. Survival enhancing factors [0064] The disclosed RGCs are engineered to express one or more survival enhancing factors. In various embodiments, the factor may be a protein, for example, small heat shock protein (sHSP). In many embodiments, the sHSP is HSPB1 (as described in Transl Vis Sci Technol. 2022 Nov; 11(11): 8). In most embodiments, the expression of sHSP is higher than in unengineered cells. In many embodiments, the factor may be delivered to RGCs via a vector, for example, an adeno-associated virus (AAV), for example, AAV2. In some embodiments, expression of the factor is specific for RGCs, for expressing the factor gene using an RGC- specific vector, for example, the RGC-specific mini Promoter, Ple345 (neurofilament, light polypeptide; NEFL). Transplantation [0065] The disclosed cells may be transplanted into a patient’s eye to repopulate the retina. In many embodiments, the cells may be transplanted from an in vitro culture where the cells are engineered to express HSPB1. Survival [0066] The disclosed methods and cells result in enhanced survival in vitro and in vivo. In one embodiment, the disclosed cells may survive after transplantation into a subject or patient’s retina. In many embodiments, the engineered cells may survive at a rate that is greater than 1%, 10%, 20%, or higher after a period of time. In many embodiments, the period of time may be more than a day, a week, a month, or a year. In many embodiments, RGCs expressing the disclosed survival enhancing factor may possess a survival rate that is greater than 1X, 2X, 3X, 4X, or 10X that of cells not expressing the survival enhancing factor. About [0067] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values, it is understood that the term “about” or “approximately” applies to each one of the numerical values in that series. Administration [0068] “Intraocular” administration refers to administering a cell or composition into an eye of a patient, for example, into the vitreous. Ameliorate or improve [0069] The term “amelioration” or “improvement” as used herein refers to any change of a disease state (for example, loss of vision or glaucoma), for the better of a patient suffering therefrom, by the administration of one or more treatments, cells, and/or compositions, according to the present disclosure, to such patient or subject in need thereof. Such an improvement may be seen as a slowing down of the progression, or a cessation of the progression, or restoration of a prior condition of the patient, a decrease in the severity of any symptom, and/or an increase in frequency or duration of disease symptom-free periods or a prevention of impairment or disability due to the disease. Disease/Condition [0070] Disclosed herein are compositions and methods useful in treating various diseases, disorders, and conditions, which may be characterized by one or more symptoms, for example, loss of vision. Effective Amount [0071] The term “effective amount” refers to an amount of cells or cell composition of the present disclosure to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease or condition, or to delay or minimize symptoms associated with a disease or condition. Further, a therapeutically effective amount with respect to the disclosed cells and compositions means that amount of therapeutic agent alone, or in combination with other therapies (for example, one or more growth factors), that provides a therapeutic benefit in the treatment or prevention of a disease or condition. Engineered [0072] “Engineered” as used herein, may refer to the aspect of having been manipulated by human intervention. Disclosed herein are engineered cells, peptides, polypeptides, proteins, molecules, nucleic acids, genes, etc. In one example, a cell is considered to be “engineered” when at least one nucleic acid sequence has been intentionally introduced by human intervention (directly or indirectly) such that the cell differs from the aspect as it exists in a patient/subject or in nature. As is common practice and is understood by those in the art, progeny of an engineered cell is typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity. Engineered cells may be further engineered by adding additional nucleic acids. In contrast, “native” or “wild-type” as used herein refers to un-engineered and/or un-modified cells, genes, proteins, nucleic acids, nucleic acid sequences, alleles, and amino acid sequences, and portions thereof – for example, cells obtained from a patient. Expression [0073] “Expression” as used herein, refers to cellular processes involved in producing, displaying (e.g., on or at a cell’s surface/outer membrane), or secreting proteins and/or nucleic acids including where applicable, but not limited to, for example, transcription, transcript processing, translation, and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of a protein and/or nucleic acid coding for that protein. Inhibition [0074] “Inhibition” “downregulation” and variations of these terms generally refer to lowering of a condition or characteristic. In some cases, the condition is cell death or apoptosis. In many embodiments, the cell death or apoptosis may decrease in engineered RGCs expressing HSPB1 compared to RGCs not engineered to express HSPB1, for example, by about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 2%, about 1% or less. Mammal [0075] The term “mammal” includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs and sheep. Patient [0076] A “patient” or “subject” includes a mammal or animal, such as a human, cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, or guinea pig. The animal can be a mammal, such as a non-primate or a primate (e.g., monkey and human). In one embodiment, a patient is a human, such as a human infant, child, adolescent, or adult of any or indeterminant sex. Prevent [0077] “Prevention” as used herein means the avoidance of the occurrence, re-occurrence, or progression of a disease, disorder, or condition as specified herein, by the administration of a cell, composition, treatment, or therapy according to the present disclosure to a subject in need thereof. In relation to the present disclosure, “prevention” may refer to preventing blindness and/or preventing additional loss of vision. Protein [0078] As used herein, the terms “protein” and “polypeptide” are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. Subject in need [0079] “Subject in need,” “patient” or those “in need of treatment” include those already with existing disease (i.e., glaucoma) or condition (vision loss) as well as those at risk of developing the disease, those at early stages of the disease, or those suffering from or developing the condition. The terms also include human and other mammalian subjects that receive either prophylactic or therapeutic treatments as disclosed herein. Treat [0080] The terms “treat,” “treating,” and “treatment” refer to eliminating, reducing, suppressing, or ameliorating, either temporarily or permanently, either partially or completely, a clinical symptom, manifestation or progression of an event, disease or condition associated with immune disorders and diseases described herein. As is recognized in the pertinent field, methods and compositions employed as therapies may reduce the severity of a given disease state but need not abolish every manifestation of the disease to be regarded as useful. Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of a condition to constitute a viable prophylactic method or agent. Simply reducing the impact of a disease (for example, as disclosed herein, glaucoma or vision loss, etc. and/or reducing the number or severity of associated symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood that the disease or condition will occur or worsen in a subject, is sufficient. One embodiment of the present disclosure is directed to a method for determining the efficacy of treatment comprising administering to a patient therapeutic treatment in an amount, duration, and repetition sufficient to induce a sustained improvement over pre-existing conditions, or a baseline indicator that reflects the severity of the particular disorder. [0081] As used herein, treat, and similar terms may refer to affecting a subject’s vision. In many cases, the disclosed treatments may halt and/or reverse vision loss, resulting in restoration of a subject’s vision. Vector [0082] “Vector” refers to a nucleic acid molecule which is capable of transporting another nucleic acid linked, typically covalently, using gene engineering methods, thereto. One type of vector is a viral vector, where an additional DNA segment can be ligated into the viral genome. Yet another type of vector is a “plasmid,” which refers to circular double-stranded DNA into which an additional DNA segment can be ligated. Another type of vector is a phage vector. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for example, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction, and thus are replicated along with the host genome. In addition, certain vectors are capable of directing expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” or simply “expression vectors.” In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form among vectors. EXAMPLES Example 1 – HSPB proteins [0083] Applicants investigated the use of small heat shock proteins for enhancing RGC survival. Specifically, Applicants tested HSPB1, HSPB4, HSPB5, and HSPB6 by expressing them in RGCs and their ability to protect RGCs from death by ocular hypertension in mice. AAV2-HSPB1 (1μL,1x10⁹ viral genomes/μL; see FIG. 8) was intravitreally injected into the right eye, and the left eye was uninjected and served as the control. After 5 weeks, retinal flatmounts were immunostained for HSPB1 (green) and RBPMS (RGCs, red; RNA-binding protein with multiple splicing is a commonly used marker of retinal ganglion cells). Scale bar = 100 μm. [0084] Applicants discovered that HSPB1 offered the best protection (FIG. 1A). The bar graph shows the number of RGCs/mm² in the mid-peripheral retina. Notably, HspB1 was observed to express in both somata and axons of RGCs. Moreover, expression protects the cells from apoptosis and axonal degeneration (FIG. 1B). [0085] These results suggested that gene therapy using AAV2-delivered HSPB1 may be useful in treating glaucoma in patients who have some RGCs remaining. However, Applicants recognized that once RGC neurons are lost, there is no treatment to recover them and vision loss becomes permanent. [0086] Therefore, for late-stage glaucoma patients, the only option is to replace the lost RGCs with new ones to regain vision. Currently, the biggest obstacle to the cell replacement strategy is the poor viability of RGCs after transplantation. To overcome this challenge and increase cell integration into the retina, Applicants engineered RGCs to survive longer using the disclosed AAV2-HSPB1 expression. Example 2 – Creation and culturing of retinal organoids [0087] Applicants have created methods and systems for culturing highly purified RGCs from dissociated 3D retinal organoids, derived from human iPSCs. Applicants have also optimized transfection of the resulting RGCs with AAV2-HSPB1 (FIG. 2A). Specifically, the disclosed RGCs treated with AAV2-HSPB1 robustly expressed HspB1 after 10 days. In addition, AAV2-HSPB1 transduced RGCs exhibited strong resistance to cell death induced by oxidative stress compared to non-transduced cells (FIG. 2B). Briefly, RGCs were isolated and cultured from iPSC-derived retinal organoids. The cells were transduced with AAV2-HSPB1 (1x10⁹ viral genomes/ml). After 10 days, the cells were immunostained for HSPB1 (green) and RBPMS (red). At 10 days post-transduction, cells were treated with 500 μM H₂O₂ for 24 hours to induce apoptosis. The TUNEL assay was used to identify/quantify apoptotic RGCs. Arrows indicate TUNEL-positive cells (red). Nuclei are stained blue with DAPI. (C) The percentage of TUNEL- positive cells are shown as mean ± SD of 3-5 independent experiments. **p<0.01, ****p<0.0001. [0088] Applicants hypothesized that the presently disclosed enhanced cells and cellular compositions may improve RGC integration and/or vision restoration in chronic glaucoma mouse models. [0089] To do this, Applicants first injected eng-iPSC-RGCs into the vitreous of healthy mouse eyes to evaluate spatial and functional integration with the inner plexiform layer which contains presynaptic amacrine and bipolar cells, and their connection with the optic nerve. Applicants also evaluated temporal cell survival after transplantation. Example 3 – HSPB1 expression in RGC [0090] Retinal ganglion cells (RGC) are the first type of neuronal cells to appear in the developing retina, in the innermost retinal layer. They are identified by the expression of markers such as Brn3a, β-III-tubulin, and RBPMS, while their specification coincides with the downregulation of proliferation markers like Ki-67, indicating cell cycle exit and neuronal commitment. Notably, this spatiotemporal pattern of RGC development is recapitulated in retinal organoids (RO) derived from human induced pluripotent stem cells (hiPSC). To confirm this, Applicants monitored the expression of RGC markers at 7-day intervals from day 35 to day 70. Immunofluorescence staining was performed using RBPMS and β-III-tubulin as pan-RGC markers, and Ki-67 to identify proliferating retinal progenitor cells. On day 35, a few cells expressed RBPMS but not Brn3a, and from day 42, both markers were expressed in the retinal organoid (not shown). Based on these results and published research by other groups, Applicants continued their study using ROs at 42 days of differentiation (D42). At this time point, ROs were dissociated and cultured on Matrigel-coated plates (FIG. 3A). To assess the purity of RGCs , we performed immunofluorescence staining at 7 days of post-dissociation for RGC markers β-III- tubulin (magenta) and RBPMS (red) (FIG. 3B). We observed that over 95% of cells were positive for both β-III-tubulin and RBPMS, confirming the validity of the model for treatment efficacy studies (FIG. 3C). [0091] Figure 3A is a timeline of AAV2-HSPB1 transduction in hiPSC-derived RGCs. In these experiments, human iPSCs were differentiated into retinal organoids (ROs) and dissociated on day 42. Briefly, dissociated cells were treated with AAV2-HSPB1 (1X10⁹ vg/mL) and collected for immunofluorescent staining on days 0, +3, +7, +10, and +14 post-transduction (Dpt: days post-transduction). Figure 3B are representative images of hiPSC-derived RGCs cultures at 7 days post-dissociation, stained for RGC markers (β-III-tubulin and RBPMS; scale bar: 100 μm. Figure 3C shows a bar graph representing the percentage of β-III-tubulin+ or RBPMS+ RGCs relative to the total DAPI+ cells on day 7 post-dissociation, showing that approximately 95% of the cells expressed both markers, wherein the bars represent the mean ± SEM, N=4. [0092] To assess the transduction efficiency of HSPB1 in RGCs, Applicants performed immunofluorescence staining using antibodies against HSPB1 and RGC markers. Cells were transduced with AAV2-HSPB17 days after dissociation and harvested at 3-, 7-, 10-, and 14-days post-transduction (dpt) (FIG. 3A). Applicants observed that by +7 dpt, there was increased overexpression of HSPB1, which was colocalized with the RGC markers β-III-tubulin and RBPMS (FIG. 3D). By +10 and +14 dpt, the expression of HSPB1 remained relatively constant compared to +7 dpt (FIG. 3D). Additionally, the gene expression level of HSPB1 was significantly increased in transduced RGCs at +7 dpt. Therefore, a 7-day timeline for AAV2- HSPB1 transduction was used in all subsequent experiments. Example 4 – HSPB1 protects RGC cells from cell damage in vitro [0093] In glaucoma, RGCs experience several types of stress, including oxidative stress, ischemia and hypoxia, neuroinflammation, and glutamate excitotoxicity. To determine whether AAV2-HSPB1 enhances cell viability under glaucomatous stress RGCs were treated with either 500 μM H₂O₂ to induce oxidative stress, or a mix of pro-inflammatory cytokines (CM, 10 ng/ml), including TNF-α, IL-1β, and IFN-γ, to induce inflammatory stress at +7 dpt. First, we conducted a live/dead assay using calcein AM (green) and ethidium homodimer-1 (EtHD-1, red) following 48 h of stress induction. Under oxidative or inflammatory stress, RGCs showed a significantly increased number of EtHD-1-positive dead cells. However, AAV2-HSPB1 transduced RGCs showed significant protection against oxidative stress and a lower trend in inflammatory stress (FIG. 4A and 4B). [0094] Figure 4A shows images from these experiments. Specifically, hiPSC-derived RGCs were transduced with AAV2-HSPB1 on day 7 post-dissociation, and apoptosis was induced _{using 500 μM H2O2 or a cytokine mixture (CM: TNF-α, IL-1β, and IFN- , 20 ng/ml each) for 48} hours. Confocal images show live/dead staining with calcein-AM (green) and ethidium homodimer (red), where the scale bar shows 50 μm. Figure 4B shows quantification of immunofluorescence image demonstrating a significant increase in cell death in the H2O2- or CM-treated groups, while AAV2-HSPB1 transduction significantly reduced cell death. Bars in the graph represent the mean ± SEM; **p<0.01; one-way ANOVA of N=4 independent biological replicates per condition. [0095] Applicants next validated the anti-apoptotic effects of HSPB1 transduction by immunofluorescence staining for cleaved caspase-3 along with RBPMS using the same treatment and stress-induction paradigm (FIG. 5A and 5B). Similar to the live/dead assay described above, glaucomatous stress significantly increased the number of cleaved caspase-3 positive RGCs. However, transduced RGCs demonstrated significantly decreased levels of cleaved caspase-3 under both oxidative and inflammatory stress conditions. [0096] Figure 5A presents representative confocal images of AAV2-HSPB1-transduced or control RGCs exposed to H2O2 or CM and stained with cleaved caspase-3 (green) and RBPMS (magenta). In these images, co-localization of the two colors indicates apoptotic RGCs. The scale bar is 50 μm. In Figure 5B, a bar graph of quantification of images shows a significant increase in cell death in hiPSC-RGCs exposed to H2O2- or CM, whereas the transduced group exhibits a protective effect. The bars indicate the mean ± SEM; ** p<0.01; ***p<0.001, One- way ANOVA of N=4 replicates, where each dot represents an individual biological replicate. [0097] Finally, TUNEL staining further confirmed our observations (FIG. 6 and 7). These results suggested that AAV2-HSPB1 successfully transduces hiPSC-derived RGCs, enhancing their cellular resilience under stress conditions. [0098] These experiments show that AAV2-HSPB1 reduces apoptosis in hiPSC-derived RGCs under oxidative and inflammatory stress. Specifically, Figure 6 shows images of hiPSC- derived RGCs transduced with AAV2-HSPB1 on day 7 post-dissociation and exposed to oxidative stress (500 μM H2O2) and a pro-inflammatory cytokine mixture (CM: TNF-α, IL-1β, and IFN-γ) for 48 hours. These confocal immunofluorescent images show TUNEL staining (red), which detects apoptotic cells. A marked increase in TUNEL-positive cells was observed in the H₂O₂ and CM groups, whereas the AAV2-HSPB1-treated group showed a significant reduction in apoptosis, indicating a protective effect on hiPSC-RGCs. Scale bar: 50 μm. Figure 7 is a bar graph showing a significant increase in TUNEL-positive cells in the H₂O₂ - and CM- treated groups, whereas the AAV2-HSPB1 transduced RGCs group exhibits a significant decrease in the number of TUNEL-positive cells. Bars in the graph indicate mean ± SEM; * p<0.05; ** p<0.01; ****p<0.0001, One-way ANOVA, of N=4 independent biological replicates per condition. Example 5 - AAV2-HSPB1 promotes neurite outgrowth [0099] As the main projection neurons of the retina, RGCs extend long axons to reach their brain targets, and this connectivity is refined during development by both spontaneous and visually driven neural activity. Thus, experiments were designed to determine whether HSPB1 can enhance the neurite outgrowth of hiPSC-derived RGCs, thereby supporting the development of cell transplantation strategies. To evaluate this, neural aggregates derived from day 42 ROs were used, a technique that exposes the inner RGCs allowing their axons to extend outward. Neurite outgrowth was visualized by immunofluorescence staining for β-III-tubulin, which labels RGC axons. Since brain-derived neurotrophic factor (BDNF) is known to enhance neuronal survival and support synaptic plasticity, Applicants compared its effects with AAV2-HSPB1 transduction. First, retinal organoids were chopped and then treated with either AAV2-HSPB1, 50 ng/mL BDNF, or a combination of both (FIG. 9A). The results show that BDNF treatment promoted moderate neurite outgrowth, while AAV2-HSPB1 or AAV2-HSPB1 along with BDNF resulted in significantly enhanced axonal extensions and increased the formation of branch junctions (FIG. 9B and 9C). [00100] In parallel experiments, ROs were transduced with AAV2-HSPB1 on day 42, and neural aggregates were generated seven days after transduction (on day 49). Aggregates were plated and cultured with or without BDNF (FIG. 9D). Neurite outgrowth was assessed after 5 days by immunofluorescence staining using β-III-tubulin. Results show a significant increase in neurite extension in both BDNF and AAV2-HSPB1 individual treatment groups compared to the untreated control group. Notably, BDNF+AAV2-HSPB1-treated organoids exhibited further extension of neurites (FIG. 9E and 9F). [00101] Figure 9A-9F presents experimental results showing that AAV2-HSPB1 promotes RGC neurite outgrowth. Specifically, FIG. 9A presents a timeline of AAV2-HSPB1 transduction after chopping ROs. For these experiments, ROs were chopped on day 42, plated on Matrigel and treated with AAV2-HSPB1 (1X10⁹ vg/ml) and/or BDNF (50 ng/ml). On day 5 post- treatment, the chopped ROs were fixed and processed for immunofluorescent staining using β- III-tubulin (green), an axonal marker. Representative images showing enhanced axonal outgrowth, are presented in Figure 9B. These images show prominent neurite extension observed in the AAV2-HSPB1 and AAV2-HSPB1+BDNF groups, where the scale bar is 100 μm. In FIG. 9C, Applicants performed quantification of the maximum branch length under each condition. The bars in Figure 9C represent the mean ± SEM of results. FIG. 9D is a timeline of AAV2-HSPB1 transduction in ROs prior to chopping. For these experiments, seven days post- transduction, ROs were chopped, plated in Matrigel, and treated with or without BDNF for 5 days. Chopped ROs were then fixed and immunostained for β-III-tubulin. FIG. 9E presents representative images from these experiments, showing neurite outgrowth visualized using the axon marker β-III-tubulin (green), where the scale bar is 100 μm. FIG. 9F shows quantification of maximum branch length under each condition. Bars represent the mean ± SEM of results. For both Figure 9C and 9F, annotations are as follows ** p<0.01; ***p<0.001; ****p<0.0001, ns=not significant, determined by one-way ANOVA. N=4, with each dot representing an individual biological replicate. [00102] To further validate these results, neurite outgrowth was monitored in aggregates derived from membrane-tagged YFP-expressing ROs by fluorescence microscopy for up to 5 days. The results indicated that treatment with AAV2-HSPB1 alone significantly enhanced neurite outgrowth starting from day 2, with progressive increases in branch length and the formation of interconnected junctions over time (Figure 10). Furthermore, a proportion of the extended neurites were labeled with MAP2, a dendrite marker, suggesting robust dendrite growth alongside axonal outgrowth in both AAV2-HSPB1 and AAV2-HSPB1 + BDNF treatment groups. Together, these results indicate that HSPB1 overexpression has significant potential to enhance neurite outgrowth on its own, and its combination with BDNF further amplifies neurite extension. This synergistic effect suggests a potential therapeutic strategy for RGC transplantation that leverages the short-term neuroprotective effects of BDNF combined with the sustained benefits of AAV2-HSPB1 for axonal elongation. [00103] Figures 10A-10C represent experimental results showing axonal and dendritic neurite outgrowth in transgenic retinal organoid pieces expressing membrane-tagged YFP. Specifically, FIG. 10A are bright field microscopy images of live cultures showing neurite outgrowth from YFP-expressing retinal organoid pieces at 1, 2 and 3 days after chopping. In these experiments, Applicants observed enhanced axonal outgrowth in BDNF, AAV2-HSPB1, and AAV2- HSPB1+BDNF-treatment conditions compared to controls, wherein the scale bar is 50 μm. FIG. 10B represents live fluorescence imaging of chopped organoid cultures 5 days after chopping. This confirms enhanced neurite outgrowth in BDNF, AAV2-HSPB1, and AAV2- HSPB1+BDNF-treatment conditions, as assessed by yellow fluorescent protein (YFP) expression. FIG. 10C are images from immunofluorescence staining for the axonal and dendritic neuronal markers TUJ1 and MAP2, which were performed on day 5 post-transduction. These experiments were designed to evaluate neurite outgrowth in control, BDNF, AAV2-HSPB1, and BDNF+AAV2- HSPB1-treated retinal organoid pieces. Applicants observed enhanced axonal and dendritic projections in the AAV2-HSPB1 and BDNF+AAV2-HSPB1 treatment groups, wherein the scale bar is 50 μm. Example 6 - Engineered RGCs exhibit improved survival after transplantation [00104] Applicants next aimed to determine whether engineering iPSC-RGCs with AAV2- HSPB1 could promote their survival following transplantation into mice. For these experiments, either AAV2-HSPB1-transduced RGCs (Eng-RGCs) or non-transduced RGCs (control RGCs) were injected into the mouse vitreous, and their survival was evaluated after 2-4 weeks. Both groups were treated with 50 ng/mL of BDNF by intravitreal injection on the day of transplantation (Fig 11A). Two weeks after injection, mice were sacrificed and retinas were immunostained for human nuclear antigen (HuN) and RBPMS. Whole retinal flatmounts showed an increase in HuN-positive signaling (green) in the host retina transplanted with Eng-RGCs compared to retinas transplanted with control RGC (FIG. 11B). Higher magnification images further confirmed the co-localization of HuN (+) and RBPMS (+) human iPSC-RGCs in the Eng-RGC transplanted group. Retinal cross-sections also showed an increase in HuN (+) and RBPMS (+) human iPSC-RGCs, localized just above the ganglion cell layer (GCL), at 2 weeks post-transplantation (FIG. 11C). Notably, at 4 weeks post-transplantation, an increased number of HuN (+) RGCs were found within the GCL, suggesting their homing and/or integration (FIG. 11D). These results indicated that Eng-RGCs not only possess enhanced survival but, surprisingly, also successfully home into the GCL compared to the non-transduced control RGC group, providing better potential synaptic connections with the retinal circuitry. [00105] Figures 11A-11D present results showing that engineered RGCs (Eng-RGCs) exhibit improved survival upon transplantation into the mouse retina. FIG. 11A is a timeline of RGC transplantation in mice. For these experiments, on day 49 of differentiation, iPSC-derived RGCs were transduced with AAV2-HSPB1 (1X10⁹ vg/mL) for 7 days to generate Eng-RGCs, which were then transplanted into mouse retinas via intravitreal injection (25,000 cells/eye). Non- transduced RGCs (control RGCs) were delivered similarly to the contralateral eyes. Retinas were harvested at 2 and 4 weeks post-transplantation (wpt). FIG. 11B shows images two weeks after transplantation. Specifically, retinal flatmounts were co-stained with the human-specific marker HuN (green), and the RGC marker RBPMS (red). Co-localization of HuN(+) and RBPMS(+) signals indicates transplanted human RGCs. FIG. 11C are representative confocal images showing cross-sections of retinas immunostained for HuN (green) and RBPMS (magenta). The Eng-RGC transplanted group shows a higher number of HuN(+) and RBPMS(+) cells (indicated by yellow arrows) compared to the control RGC group, suggesting enhanced Eng-RGC survival at 2 weeks post-transplantation. FIG. 11D presents representative confocal images at 4 weeks post-transplantation, showing that HuN(+) (green) and RBPMS(+) (red) human RGCs were observed within the ganglion cell layer (GCL). Example 7 - Engineered RGC transplantation partially preserves retinal function following optic nerve crush. [00106] Applicants next explored the potential of using Eng-RGCs for cell therapy. In these experiments membrane-tagged-YFP-expressing RGCs engineered with AAV2-HSPB1 were transplanted into the retina of mice subjected to optic nerve crush (ONC). Specifically, Eng- RGCs or control RGCs were intravitreally delivered one week after ONC, when host mouse RGCs had decreased by 50%. Two weeks post-transplantation, mice were euthanized, and the surviving RGCs were quantified (FIG. 12A). At this point, robust YFP expression was detected in Eng-RGC transplanted retinas compared to control RGC-transplanted retinas. By three weeks after ONC, both host mouse RGCs (magenta) and transplanted YFP (+) cells were markedly decreased, whereas the Eng-RGC transplanted group displayed persistent YFP expression (FIG. 12B). Quantification analysis showed no significant differences in transplanted RGC survival rates between control and ONC conditions, although greater variability was observed in the ONC group. Notably, Eng-RGCs exhibited significantly higher survival compared to control RGCs (FIG. 12C). Analysis of retinal cross-sections further confirmed a greater survival of Eng-RGCs in the GCL compared to control human RGCs (FIG. 12D and 12E). Moreover, pattern electroretinography (pERG) was used to assess the functional integration of transplanted RGCs at day 13 post-transplantation under ONC conditions (FIG.12F-12G).pERG is a commonly used electrophysiological technique to assess RGC function, which results from their stimulation by presynaptic cells inresponse to a light stimulus and therefore indicates both the functional capacity of RGCs and their integration within the retinal circuitry. Three weeks after ONC, the P1 amplitude was severely reduced, and control RGC-transplanted retinas showed no improvement. In contrast, Eng-RGC-transplanted retinas demonstrated significantly higher P1 amplitudes compared to control RGC-transplanted retinas. These results suggest that Eng-RGCs partially but significantly improve RGC function under ONC conditions. [00107] Figures 12A-12G present results frome experiments showing that transplanted Eng- RGCs exhibit enhanced survival in a mouse model of optic nerve crush. FIG. 12A is a timeline of these experiments of RGC transplantation in mice following optic nerve crush (ONC). Specifically, mice were subjected to ONC, and one week later, either control RGCs or Eng- RGCs were transplanted into the vitreous. Two weeks post-transplantation (wpt), retinas were collected for analysis. FIG.12B shows retinal flatmounts immunostained for HuN (green) and RBPMS (magenta). FIG. 12C is a bar graph of surviving transplanted human RGCs. The RGCs were counted by assessing the cells having co-localized HuN and RBPMS signals per mm² retinal area. FIG. 12D are representative confocal images showing retinal cross-sections immunostained for HuN (green) and RBPMS (magenta). Although ONC led to a marked reduction in endogenous RBPMS (+) RGCs, the Eng-RGC transplanted group exhibited higher number of surviving human RGCs than the control RGC-transplanted group. FIG. 12E is a bar graph showing the quantification of human RGCs relative to the total number of cells in the ganglion cell layer (GCL) from N=4 independent biological replicates per condition. Figures 12F and 12G presents results from pattern electroretinography (pERG) recorded on day 13 post- transplantation. The P1/N2 amplitude ratio is shown in the bar graph of FIG. 12F, and representative signals from control RGC- or Eng-RGC-transplanted mouse retinas are shown in FIG. 12G. Each dot in FIG. 12F represents an independent biological replicate, andthe bars indicate the mean ± SEM of results; * p<0.05; ****p<0.0001, ns= not significant. These results demonstrate that Eng-RGCs significantly improve RGC function following ONC. [00108] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the detailed description is to be regarded as illustrative in nature and not restrictive. [00109] All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference as if each was included at its citation, in its entirety. In case of conflict between reference and specification, the present specification, including definitions, will control. [00110] Although the present disclosure has been described with a certain degree of particularity, it is understood the disclosure has been made by way of example, and changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims. [00111] Although the present disclosure has been described with a certain degree of particularity, it is understood that the disclosure has been made by way of example, and changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims. [00112] The description of certain embodiments included herein is merely exemplary in nature and is in no way intended to limit the scope of the disclosure or its applications or uses. In the included detailed description of embodiments of the present systems and methods, reference is made to the accompanying drawings which form a part hereof, and which are shown by way of illustration specific to embodiments in which the described systems and methods may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice presently disclosed systems and methods, and it is to be understood that other embodiments may be utilized, and that structural and logical changes may be made without departing from the spirit and scope of the disclosure. Moreover, for the purpose of clarity, detailed descriptions of certain features will not be discussed when they would be apparent to those with skill in the art so as not to obscure the description of embodiments of the disclosure. The included detailed description is therefore not to be taken in a limiting sense, and the scope of the disclosure is defined only by the appended claims. [00113] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present disclosure and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. [00114] As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular. [00115] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. [00116] It is to be appreciated that any one of the examples, embodiments or processes described herein may be combined with one or more other examples, embodiments and/or processes or be separated and/or performed amongst separate devices or device portions in accordance with the present systems, devices and methods. [00117] Finally, the above discussion is intended to be merely illustrative of the present system and should not be construed as limiting the appended claims to any particular embodiment or group of embodiments. Thus, while the present system has been described in particular detail with reference to exemplary embodiments, it should also be appreciated that numerous modifications and alternative embodiments may be devised by those having ordinary skill in the art without departing from the broader and intended spirit and scope of the present system as set forth in the claims that follow. Accordingly, the specification and drawings are to be regarded in an illustrative manner and are not intended to limit the scope of the appended claims.

Claims

CLAIMS We claim: 1. A method of restoring vision in patients with eye disease such as glaucoma, comprising: isolating retinal ganglion cells (RGC) from human retinal organoids; transducing into said RGC a gene for enhancing survival of the RGC; transplanting said transduced RGCs into a retina of a patient, wherein said transplanted RGCs integrate into the retina of the patient.

2. The method of claim 1, wherein the gene is a heat shock protein (HSP).

3. The method of claim 1 or claim 2, wherein the gene is heat shock protein B1 (HSPB1).

4. The method of any one of claims 1-3, wherein transplanting comprises administration of the RGC via intravitreal injection.

5. The method of any one of claims 1-4, wherein integration into the retina is into the ganglion cell layer (GCL).

6. The method of any one of claims 1-5, wherein the transduced, integrated RGCs contribute to better signal response in pattern electroretinography measurements.

7. The method of any one of claims 1-6, wherein transduced, integrated RGCs show enhanced resistance to one or more of increased ocular pressure, apoptosis, and oxidative stress, relative to RGC that have not been transduced.

8. The method of any one of claims 1-7, wherein transduced, integrated RGCs are derived from induced pluripotent stem cells (iPSCs).

9. The method of any one of claims 1-8, wherein transduced, integrated RGCs are selected from allogenic or autologous cells.

10. An engineered cell for restoring vision in a patient with vision loss, comprising: a cell derived from the patient; comprising a nucleic acid comprising a coding region for at gene that enhances survival of a retinal ganglion cell (RGC), a nucleic acid comprising an RGC-specific promoter, wherein the cell expresses one or more markers indicative of RGC identity.

11. The engineered cell of claim 10, wherein the gene is a heat shock protein (HSP).

12. The engineered cell of claim 10 or claim 11, wherein the gene is heat shock protein B1 (HSPB1).

13. The engineered cell of any one of claims 10-12, wherein the cell is an engineered RGC.

14. The engineered cell of any one of claims 10-13, wherein the engineered cell shows enhanced resistance to one or more of increased ocular pressure, apoptosis, and oxidative stress, relative to an RGC that is not engineered to include the gene.

15. The engineered cell of any one of claims 10-14, wherein engineered cell is derived from an induced pluripotent stem cell (iPSC).

16. A composition for restoring vision in a patient with vision loss, comprising: a pharmaceutically acceptable carrier; at least one cell engineered to comprise at least one nucleic acid comprising a coding region for a gene that enhances survival.

17. The composition of claim 16, wherein the nucleic acid comprises a retinal ganglion cell (RGC) specific promoter, and the cell expresses one or more markers indicative of RGC identity.

18. The composition of claim 16 or claim 17, wherein the gene is a heat shock protein (HSP).

19. The composition of any one of claims 16-18, wherein the gene is heat shock protein B1 (HSPB1).

20. The composition of any one of claims 16-19, wherein the cell has enhanced resistance to one or more of increased ocular pressure, apoptosis, and oxidative stress, relative to cells lacking the nucleic acid.

21. The composition of any one of claims 16-20, wherein the engineered cell is derived from induced pluripotent stem cells (iPSCs).

22. The composition of any one of claims 16-21, wherein the cell is an allogenic or autologous cell.

23. The composition of any one of claims 16-22, for administration to a patient via intravitreal injection.

24. The composition of claim 23, wherein after administration, the cell integrates into a ganglion cell layer (GCL) of the patient’s retina.

6. The method of any one of claims 1-5, wherein the transduced, integrated RGCs generate a signal in a pattern electroretinography experiment. 7. The method of any one of claims 1-6, wherein