Nanoparticle immunosuppressant for retinal applications

Abstract

Provided herein are pharmaceutical compositions that include (a) a dendrimer nanoparticle, wherein the dendrimer nanoparticle comprises Dexamethasone-21-succinate, and (b) a plurality of retinal cells. Also provided herein are methods for treating a degenerative and hereditary retinopathy that include administering any one of the pharmaceutical compositions described herein to a subject in need thereof.

Description

[0001] Atorney Docket No. 44807-0514WO1

[0002] NANOP ARTICLE IMMUNOSUPPRESSANT FOR RETINAL APPLICATIONS CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 765,241, filed on February 28, 2025, and U.S. Provisional Patent Application No. 63 / 766,266, filed on March 3, 2025. The disclosure of the prior applications are considered part of the disclosure of this application and are incorporated herein by reference in their entirety.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates to the field of biotechnology, and more specifically, to compositions and methods of delivering therapeutic agents.

[0006] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with government support under grant EY033103 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Exogenous retinal cell transplantation is being developed as a potential treatment for degenerative and hereditary retinopathies. Retinal pigment epithelial (RPE) and photoreceptor transplantation techniques have seen substantial progress, including first-in-human testing.

[0010] However, local inflammatory reactions including immune rejection have truncated treatment durability and possibly blunted maximal efficacy.

[0011] SUMMARY

[0012] Provided herein are pharmaceutical compositions that include (a) a dendrimer nanoparticle, wherein the dendrimer nanoparticle comprises Dexamethasone-21 -succinate; and (b) a plurality of retinal cells. In some embodiments, the dendrimer nanoparticle comprises a hydroxyl PAMAM dendrimer. In some embodiments, the dendrimer nanoparticle comprises a glucose dendrimer. In some embodiments, the dendrimer comprises a Cy3-dendrimer or a Cy5-Atorney Docket No. 44807-0514WO1

[0013] dendrimer. In some embodiments, the Dexamethasone-21 -succinate and hydroxyl PAMAM dendrimer are conjugated via copper-catalyzed azide-alkyne cycloaddition (CuACC) reaction.

[0014] Also provided herein are methods for treating a degenerative and hereditary retinopathy that include administering any one of the pharmaceutical compositions described herein to a subject in need thereof. In some embodiments, the administering comprises an injection of the pharmaceutical composition. In some embodiments, the injection comprises subretinal injection. In some embodiments, the pharmaceutical composition is delivered into a retinal cell of the subject, and wherein the Dexamethasone-21 -succinate is released intracellularly in the retinal cell. In some embodiments, the degenerative and hereditary retinopathy comprises Retinitis Pigmentosa (RP), Choroideremia, Stargardt Disease, Cone-rod Dystrophy (CRD), or Leber Congenital Amaurosis (LCA). In some embodiments, the subject is a human. In some embodiments, the subject is a retinal cell transplant recipient.

[0015] 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 this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0016] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0017] BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows biodistribution resulting from subretinal and intravitreal administration of dendrimer during retinal cell transplantation.

[0018] FIG. 2 shows biodistribution resulting from intraperitoneal, subconjunctiva and intravitreal administration of dendrimer during retinal cell transplantation.Atorney Docket No. 44807-0514WO1

[0019] FTG.3 shows representative OCT images of the OPN1LW-GFP retinal cells post transplantation and Kaplan-Meier plot of the donor retinal cell survival rate in retinal cell with D-Dex group, cell-only group, retinal cell with dexamethasone group, and dendrimer-only group.

[0020] FIG. 4 shows histological examination of retinas in GNAT1&2-KO recipient mice 8 weeks following transplantation.

[0021] FIGs. 5A-5F show local biodistribution of dendrimer conjugates following administration during retinal cell transplantation. FIG. 5A shows a schematic overview of the experimental workflow for dendrimer biodistribution analysis. OPN1LW-EGFP donor retinal cells were transplanted via subretinal injection, followed by administration of D-Cy3 or D-Cy5 through intravitreal (IVT), subconjunctival (SC), or intraperitoneal (IP) injection. Eyeballs were harvested at day 10 post-transplantation. FIG. 5B shows representative retinal flat-mount images showing the distribution of D-Cy3 and D-Cy5 signals relative to OPN1 LW-EGFP grafted cells following different injection routes, with quantification comparing D-Cy3 and D-Cy5 signal levels across routes. FIG. 5C shows retinal sections illustrating the laminar distribution of D-Cy3 and D-Cy5 signals. FIG. 5D shows flat-mounted retina highlighting AOIs centered on OPN1LW-EGFP+grafted cell clusters, with higher-magnification views comparing subretinal D-Cy3 and intravitreal D-Cy5 distributions relative to grafted cells. FIG. 5E shows quantification of D-Cy3 signals delivered subretinally and D-Cy5 signals delivered intravitreally within graft-associated AOIs. FIG. 5F shows nearest-neighbor analysis demonstrated that 98.4% of subretinally delivered D-Cy3 signals were within 10 pm of OPN1LW-EGFP+grafted cells, whereas only 51.3% of intravitreally delivered D-Cy5 signals were located within this distance.

[0022]

[0023] ooi. Scale bars, 50 pm (FIGs. 5B, 5C, 5F), 200 pm (FIG. 5D).

[0024] FIGs. 6A-6E show dendrimer-dexamethasone (D-Dex) promotes grafted retinal cell survival following transplantation. FIG. 6A shows a schematic of dexamethasone conjugation to a G4 PAMAM dendrimer (D-Dex). FIG. 6B shows in vivo optical coherence tomography (OCT) images showing surviving OPN1LW-EGFP+grafted cone cells at 1-, 3-, 5-, and 8-weeks posttransplantation. FIG. 6C shows Kaplan-Meier analysis of grafted retinal cell survival across treatment groups. FIG. 6D shows representative retinal sections from recipient mice at 8 weeks post-transplantation, showing endogenous EGFP signal from transplanted OPN1LW-EGFP+cone cells. FIG. 6E shows quantification of OPN1LW-EGFP' grafted cone cells in definedAtorney Docket No. 44807-0514WO1

[0025] regions of interest (ROIs) at 8 weeks post-transplantation. ***p < 0.001. Scale bar, 50 pm (FTG.

[0026] 6D)

[0027] FIGs. 7A-7D show effects of dendrimer-dexamethasone (D-Dex) on retinal immune cell responses following cell transplantation. FIG. 7A shows representative retinal sections showing CD68 (macrophages), CD3 (T cells), endogenous EGFP from OPNI LW-EGFP grafted cone cells, and DAPI across treatment groups, including dendrimer only, cells only, cells + dexamethasone (Dex), and cells + dendrimer-dexamethasone (D-Dex). FIG. 7B shows quantification of the number of CD68+cells per region of interest (ROI), showing no significant differences among groups. FIG. 7C shows quantification of the number of CD3+cells per ROI, showing no significant differences among treatment groups. FIG. 7D shows quantification of CD68+signal area per cell in the ROI showed that the cells + D-Dex group exhibited a significantly smaller CD68+area compared with the other groups. *p < 0.05; ***p < 0.001; ns, not significant. Scale bar, 50 pm (FIG. 7D).

[0028] FIGs. 8A-8F show dendrimer-dexamethasone (D-Dex) treatment is associated with reduced CD86+and increased CD206+microglia / macrophage marker expression following cell transplantation. FIG. 8A shows representative immunofluorescence images showing IBA1, CD86, OPN1LW-EGFP, and DAPI in retinas from dendrimer only, cells only, cells + Dex, and cells + D-Dex groups. FIG. 8B shows representative images showing Ibal, CD206, OPN1LW-EGFP, and DAPI across experimental groups. FIG. 8C shows quantification of the number of Ibal+CD86+cells per ROI. FIG. 8D shows quantification of the number of Ibal+CD206+cells per ROI. FIG. 8E shows ratio of CD86+to CD206+microglia / macrophages per ROI. FIG. 8F shows correlation between the number of EGFP+grafted photoreceptors and the CD86+ / CD206+ratio, showing a significant negative correlation. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar, 50 pm (FIGs. 8A, 8B).

[0029] FIGs. 9A-9D show dendrimer-dexamethasone (D-Dex) enhances retinal function and visual pathway activation. FIG. 9A shows a schematic of the visual pathway and experimental workflow, including full-field ERG (ffERG) assessment and c-Fos analysis in the lateral geniculate nucleus (LGN) using coronal sections. FIG. 9B shows ffERG quantification of scotopic b-wave, photopic b-wave, and 30 Hz flicker amplitudes in dendrimer only, cells only, cells + Dex, and cells + D-Dex groups. FIG. 9C shows representative coronal LGN sectionsAtorney Docket No. 44807-0514WO1

[0030] showing c-Fos across groups. FIG. 9D shows quantification of c-Fos-positive cells per ROI in the LGN. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar, 100 pm (FIG. 9C).

[0031] DETAILED DESCRIPTION

[0032] Conventional immune suppression for organ and tissue transportation entails systemic or local delivery. Systemic side effects can be serious, and are due to off-target exposure of the drug to organs such as bone marrow, brain, liver, and kidney. Local delivery has been pursued as an option, but there are also risks of local side effects, such as tissue atrophy. Specifically, in the eye, there is a risk of developing cataract and glaucoma due to local steroids.

[0033] Provided herein are methods of delivering a controlled release formulation of an immune suppressant, to minimize off target diffusion of the immunosuppressant molecule. In some embodiments, the immune suppressant can be concentrated into a graft or transplant itself, thus preventing diffusion to outside sites.

[0034] Provided herein are pharmaceutical compositions that include a dendrimer nanoparticle, the dendrimer nanoparticle comprising Dexamethasone-21 -succinate (D-Dex). In some embodiments, pharmaceutical compositions provided herein include a dendrimer nanoparticle, the dendrimer nanoparticle comprising Dexamethasone-21 -succinate (D-Dex) and a retinal cell (e.g., a plurality of retinal cells). In some embodiments, D-Dex administration exerts an effective subretinal immunosuppressive effect, eliciting a marked enhancement in the viability of transplanted retinal cells. Also provided herein are methods for treating a degenerative and hereditary retinopathy, the method including administering any one of the pharmaceutical compositions described herein to a subject in need thereof.

[0035] Various non-limiting aspects of these pharmaceutical compositions are described herein, and can be used in any combination without limitation. Additional aspects of various components of methods of making and using the pharmaceutical compositions are known in the art.

[0036] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0037] As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed byAtorney Docket No. 44807-0514WO1

[0038] “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0039] As used herein, a “cell” can refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.

[0040] As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0041] As used herein, the term “subject” refers an organism, typically a mammal (e g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder, or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more signs or symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0042] Pharmaceutical Compositions

[0043] Provided herein are pharmaceutical compositions that include a dendrimer nanoparticle, wherein the dendrimer nanoparticle comprises Dexamethasone-21 -succinate. As used herein, the term “dendrimer” includes, but is not limited to, a molecular architecture with an interior core, interior layers (or “generations”) of repeating units regularly attached to and extending from this initiator core, each layer having one or more branching points, and an exterior surface of terminal groups attached to the outermost generation. Dendrimers have regular dendrimeric or “starbursf ’ molecular structures. In some embodiments, nanoparticle dendrimers generally have diameters of 3 to 10 nm.Atorney Docket No. 44807-0514WO1

[0044] In some embodiments, each successive dendrimer generation can be covalently bound to the previous generation. The number of reactive groups of the core structure determines n-directionality and defines the number of structures that can be attached to form the next generation. In some embodiments, the number of branches in a dendritic structure is dependent on the branching valency of the monomeric building blocks, including the core. For example, if the core is a primary amine, the amine nitrogen would then be divalent, resulting in a 1-2 branching motif.

[0045] Exemplary dendrimers are alkylated dendrimers such as poly(amido-amine) (PAMAM), poly(ethyleneimine) (PEI), polypropyleneimine (PPI), diaminobutane amine polypropylenimine tetramine (DAB-Am 4), polypropylamine (POP AM), polylysine, polyester, iptycene, aliphatic poly(ether), aromatic polyether dendrimers, or any combination thereof. In some embodiments, a dendrimer nanoparticle comprises a hydroxyl PAMAM dendrimer. In some embodiments, a dendrimer nanoparticle comprises a glucose dendrimer. In some embodiments, a dendrimer comprises a Cy3-dendrimer or a Cy5-dendrimer.

[0046] In some embodiments, a dendrimer can have carboxylic, amine and hydroxyl terminations and can be of any generation including, but not limited to, generation 1 dendrimers (Gl), generation 2 dendrimers (G2), generation 3 dendrimers (G3), generation 4 dendrimers (G4), generation 5 dendrimers (G5), generation 6 dendrimers (G6), generation 7 dendrimers (G7), generation 8 dendrimers (G8), generation 9 dendrimers (G9), or generation 10 dendrimers (G10). In some embodiments, hybrid nanoparticles can also be formed from generations of dendrimers greater than 10.

[0047] In some embodiments, a PAMAM dendrimers can contain internal amide bonds which may enhance their biodegradability, thus improving tolerance in terms of human therapeutic applications. The surface includes polar, highly reactive primary amine groups. In some embodimens, the surfaces of the amino-functional PAMAM dendrimers are cationic and can be derivatized, either through ionic interactions with negatively charged molecules, or using many well-known reagents for covalent functionalization of primary amines.

[0048] In some embodiments, the large number of end groups on a dendrimer allows for conjugation of a wide variety of molecules. In some embodiments, a dendrimer is associated with, e.g., complexed or conjugated with, one or more of a therapeutic, targeting, prophylactic or diagnostic agent. In some embodiments, a dendrimer can be conjugated to anAtorney Docket No. 44807-0514WO1

[0049] immunosuppressant agent. In some embodiments, a pharmaceutical composition comprises Dexamethasone-21 -succinate and a hydroxyl PAMAM dendrimer, wherein the Dexamethasone-21 -succinate and a hydroxyl PAMAM dendrimer are conjugated via copper-catalyzed azidealkyne cycloaddition (CuACC) reaction.

[0050] Methods of Treating a Degenerative and Hereditary Retinopathy

[0051] Also provided herein are methods for treating a degenerative and hereditary retinopathy that include administering any one of the pharmaceutical compositions described herein to a subject in need thereof.

[0052] As used herein, the term “treatment” means to ameliorate at least one symptom of a disorder or condition. Generally, the methods of treatment include administering a therapeutically effective amount of composition that reduces at least one symptom of a disorder to a subject who is in need of, or who has been determined to be in need of such treatment.

[0053] In some embodiments, the administering of the pharmaceutical composition comprises an injection of the pharmaceutical composition. In some embodiments, the injection comprises subretinal injection. As used herein, the term “administration” typically refers to the administration of a pharmaceutical composition to a subject or system to achieve delivery of an agent that is, or is included in, the pharmaceutical composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be subretinal injection. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0054] In some embodiments, a pharmaceutical composition comprising a dendrimer nanoparticle is administered together with a retinal cell. For example, a pharmaceutical composition can comprise a dendrimer nanoparticle and a retinal cell (e.g., a plurality of retinal cells) is administered to the subject. In some embodiments, a pharmaceutical composition comprising a dendrimer nanoparticle is administered separately from administration of a retinalAtorney Docket No. 44807-0514WO1

[0055] cell. For example, a retinal cell may be transplanted to a subject and a pharmaceutical composition comprising a dendrimer nanoparticle can be administered after the retinal cell is transplanted. Additionally or alternatively, a pharmaceutical composition comprising a dendrimer nanoparticle can be administered to a subject and a retinal cell can be transplanted after the pharmaceutical composition is administered.

[0056] In some embodiments, the pharmaceutical composition is delivered into a retinal cell of a subject, and wherein the Dexamethasone-21 -succinate is released intracellularly in the retinal cell. As used herein, the term “retinal cell” includes neural retinal cells or photoreceptor cells, retinal pigment epithelial (RPE) cells, iris epithelial cells, and their precursors. In some embodiments, the retinal cells are mammalian retinal cells, e.g., human retinal cells. “Retinal pigment epithelial (RPE) cells”, as used herein refer to cells of the outermost external layer of the retina. RPE cells function to provide support for the retinal photoreceptors and are responsible for the metabolic digestion of the discarded outer segments of the neural retina. “Neural retina cells”, as used herein, refer to the layer of photoreceptor cells (i.e., rod and cone cells) underlying the RPE cell layer in the retina. Neural retina cells are modified light sensitive neurons. Such precursor cells can be used as sources of the mammalian retinal cells described herein.

[0057] As used herein, a degenerative and hereditary retinopathy refers to a progressive, visually debilitating disease that can lead to blindness in which mutations in genes that are critical to retinal function lead to progressive photoreceptor cell death and associated vision loss. In some embodiments, a degenerative and hereditary retinopathy can include, but is not limited to, Retinitis Pigmentosa (RP), Choroideremia, Stargardt Disease, Cone-rod Dystrophy (CRD), or Leber Congenital Amaurosis (LCA).

[0058] In some embodiments, a subject is a human. In some embodiments, the subject is a retinal cell transplant recipient.

[0059] EXAMPLES

[0060] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0061] Example 1 - In vivo biodistribution of dendrimer in retinal cell transplantationAtorney Docket No. 44807-0514WO1

[0062] Dexamethasone-21 -succinate was covalently conjugated to hydroxyl PAMAM dendrimer (D-Dex) via copper-catalyzed azide-alkyne cycloaddition (CuACC) reaction. The D-Dex conjugate was designed to release Dex intracellularly within the low pH and high esterase concentration environment.

[0063] First, co-transplantation was conducted using Dendrimer-cy3 (D-Cy3) with retinal cells derived from OPN1LW-GFP mice. A total of 10A6 cells, accompanied by a 2 pL solution of D-Cy3, were administered into the subretinal space of GNAT1&2-KO mice. Then, on day 3 posttransplantation, Dendrimer-Cy5 (D-Cy5) was intravitreally injected. The mice were euthanized on day 10, and an assessment of dendrimer biodistribution was performed. Upon examination of the flatmount, a substantial accumulation of D-Cy3 was observed in close proximity to OPN1LW-GFP donor cells. Subsequent nearest neighbor analysis revealed that within the transplantation site, the majority of D-Cy3 (98.4%) was situated within a 10 pm radius or less from OPN1LW-GFP donor cells. In contrast, D-Cy5 exhibited a distribution pattern indicating a relatively lesser proximity to OPN1LW-GFP donor cells, with 51.3% located in close proximity (FIG. 1). This observation suggests that dendrimer administered via subretinal injection tends to reside in closer proximity to donor cells compared to intravitreal injection, potentially exerting a specific influence on the niche at the transplantation site.

[0064] Then, administrations of Dendrimer-Cy5 (D-Cy5) were also conducted through subconjunctival and intraperitoneal injections on day 3 post-transplantation since they obviate the intraocular manipulations, thereby mitigating potential complications associated with intraocular injections. The observations revealed a noteworthy reduction in the presence of D-Cy5 at the transplantation site following intraperitoneal and subconjunctival administrations compared to the intravitreal route (FIG.2).

[0065] Example 2 - Enhancing the viability of donor cells in retinal cell transplantation through the application of Dendrimer-Dexamethasone

[0066] Having demonstrated the capacity of dendrimer to concentrate at the transplantation site, the Dendrimer-Dexamethasone (D-Dex) was next used, administered concomitantly with OPN1LW-GFP retinal cells. Control groups included a retinal cell-only group, a retinal cell with dexamethasone group, and a dendrimer-only group. By optical coherence tomography (OCT) assessments at 1-, 3-, 5-, and 8-weeks post-transplantation, the observations disclosed that, atAtorney Docket No. 44807-0514WO1

[0067] week 1, the survival rate of OPN1LW-GFP donor retinal cells was confined to 37.5% of the transplantation eyes in retinal cell-only group. In contrast, survival rates were notably higher in the dexamethasone-only group (83.3%) and reached 100% in the D-Dex group. At the 8-week post-transplantation, the D-Dex group showed sustained donor cell survival in 57.1% of eyes, while other groups failed to achieve comparable rates (FIG.3).

[0068] Subsequently, the histology of the recipient retina was assessed at the 8-week posttransplantation and a significantly higher presence of surviving donor cone cells (0PN1LW-GFP) was found within the D-Dex group while the other three groups exhibited minimal to negligible instances of surviving donor cone cells. Furthermore, the D-Dex group demonstrated a marked reduction in macrophages (CD68+), signifying a pronounced attenuation of macrophage infiltration compared to the other experimental groups (FIG. 4).

[0069] Example 3 - Dendrimer conjugates exhibit route-dependent retinal biodistribution following cell transplantation

[0070] To determine the preferred delivery route for dendrimer-based therapeutics in the context of retinal cell transplantation, dendrimer biodistribution was evaluated. Retinal cells isolated from OPNILW-EGFP / Nrl- / - mice (P3-P6) were co-transpl anted with dendrimer-Cy3 (D-Cy3) into the subretinal (SR) space of Gnatl- / -; Gnat2- / - double-knockout mice. Subsequently, dendrimer-Cy5 (D-Cy5) was administered via intravitreal (IVT), subconjunctival (SC), or intraperitoneal (IP) injection. Retinas were harvested and analyzed 10 days post-transplantation (FIG. 5A)

[0071] On day 10 post-transplantation, regions of interest (ROIs) were defined around clusters of surviving OPN1LW-EGFP+grafted retinal cells, and the associated D-Cy3 and D-Cy5 signals were identified. Quantification of dendrimer signal within these ROIs revealed significantly higher colocalization of SR delivered D-Cy 3 -positive puncta at sites of grafted cells (median 204,748, interquartile range (IQR) 184,520-225,143) compared with all other delivery routes (p < 0.001). IVT delivery of D-Cy5 resulted in a lower but still substantial dendrimer signal (median 132,202; IQR, 124,813-143,302), which was significantly greater than that observed following SC (median 35,467; IQR, 18,012-39,743) or IP(median 6,818; IQR, 4,613-9,645) administration (p < 0.01). SC delivery produced a significantly higher dendrimer signal than IP delivery (p < 0.05) (FIG.5B).Atorney Docket No. 44807-0514WO1

[0072] Next, dendrimer distribution was assessed across retinal layers. Laminar analysis of retinal sections demonstrated that SR delivered D-Cy3 was preferentially distributed in the outer retina, overlapping with transplanted photoreceptor locations, with 97.95% of the signal remaining within the subretinal space. In contrast, IVT delivered D-Cy5 exhibited broader retinal distribution, with 34.77% localized to the subretinal space, 29.05% within the outer nuclear and outer plexiform layers (ONL / OPL), and 36.18% distributed across the ganglion cell layer / retinal nerve fiber layer (GCL / RNFL), inner plexiform layer (IPL), and inner nuclear layer (INL).

[0073] Following SC and IP administration, D-Cy5 signals were detected predominantly in the subretinal space (83.41% and 65.05%, respectively), with smaller fractions localized to inner retinal layers (9.87% and 21.45% in GCL / RNFL, IPL, and INL), suggesting a role of retinal vascular-mediated distribution pattern (FIG. 5C).

[0074] Dendrimer proximity to grafted cells was considered to be an important parameter, as spatial localization may reflect targeted accumulation and biological interaction. To evaluate dendrimer proximity, the two delivery routes that yielded the highest dendrimer signal were focused on, namely SR and IVT. Retinal flat-mount imaging demonstrated that SR delivered D-Cy3 particles generally clustered near OPN1LW-EGFP+cell aggregates, whereas IVT delivered D-Cy5 appeared diffusely distributed throughout the retina (FIG. 5D). Quantitative analysis showed that 75.5% of dendrimer signal associated with grafted cells originated from D-Cy3 delivered via the SR route, compared with 24.5% from IVT delivered D-Cy5 (p < 0.05, FIG. 5E). These data indicated that SR injection enhanced targeting of dendrimer to transplanted cells. Nearest-neighbor analysis further revealed that 98.4% of SR delivered D-Cy3 signals were located within 10 pm of OPN1LW-EGFP+grafted cells, whereas only 51.3% of IVT delivered D-Cy5 signals met this criterion (FIG. 5F). Together, these results demonstrate that SR delivery provides superior spatial targeting of dendrimer-based therapeutics to transplanted retinal cells compared to other delivery routes.

[0075] Example 4 - Dendrimer-dexamethasone enhances long-term survival of transplanted retinal cells

[0076] To evaluate whether dendrimer-dexamethasone (D-Dex) enhanced grafted retinal cell survival, dexamethasone-21 -succinate was covalently conjugated to hydroxyl-terminated PAMAM dendrimers (FIG. 6A) and the persistence of detectable transplanted cells wasAtorney Docket No. 44807-0514WO1

[0077] assessed. Retinal cells derived from OPNILW-EGFP / Nrl- / - mice (P3-P6) with D-Dex were cotransplanted into the subretinal space of Gnatl- / -; Gnat2- / - double-knockout mice (n=12). Control groups included dendrimer only (n=6), retinal cells only (n=6), and retinal cells with free dexamethasone (Dex) (n=8).

[0078] Using serial optical coherence tomography (OCT) imaging at 1, 3, 5, and 8 weeks posttransplantation, more durable subretinal graft persistence was observed in the D-Dex-treated group. In contrast, progressive graft loss was detected in the dendrimer-only and cells-only group (FIG. 6B). Kaplan-Meier survival analysis further supported these observations. Survival declined most rapidly in the cells-only group, with complete loss of OPN1LW-EGFP signal by 5 weeks post-transplantation, indicating substantial transplanted cell loss at early time points (FIG.

[0079] 6C). Cells treated with Dex showed a delayed but continued decline in survival, with complete loss of OPN1LW-EGFP signals by 8 weeks. To validate the imaging studies using histology, histological analysis was then performed at 8 weeks post-transplantation. There was a substantially higher number of OPN1LW-EGFP+grafted cone cells in the subretinal space of mice co-administered with D-Dex (47.3 ± 30.9). In contrast, significantly fewer surviving transplanted cells were observed in the cells-only (0.4 ± 0.5) and cells + dexamethasone (3.7 ± 3.5) groups (p < 0.001) (FIGs. 6D, 6E).

[0080] Example 5 - D-Dex modulates microglia / macrophage polarization

[0081] To investigate whether D-Dex alters immune cell responses following transplantation, macrophage and T-cell markers were examined in recipient retinas. Immunofluorescence analysis showed that the distribution of CD68+macrophages and CD3+T cells was comparable across all treatment groups (FIG. 7A). Quantitative analysis revealed no significant differences in the number of CD68+or CD3+cells among the dendrimer-only, cells-only, cells + Dex, and cells + D-Dex groups (FIGs. 7B-7C).

[0082] However, the cells + D-Dex group exhibited a significantly reduced CD68+staining per cell (signal area 89.4 ± 49.5 pm2) compared to the dendrimer-only (146.2 ± 45.5 pm2, p < 0.05), cells-only (159.1 ± 67.8 pm2, p < 0.01), and cells + Dex (213.0 ± 73.8 pm2, p < 0.001) groups, suggesting that D-Dex may attenuate macrophage activation rather than reducing their recruitment (FIG. 7D).Atorney Docket No. 44807-0514WO1

[0083] Given the observed reduction in CD68+activation with D-Dex treatment, microglia / macrophage polarization states were then assessed. Immunofluorescence staining showed decreased expression of the pro-inflammatory marker CD86 and increased expression of the anti-inflammatory marker CD206 in the cells + D-Dex group compared to other conditions (FIGs. 8A-8B). Quantitative analysis demonstrated a significant reduction in IBA1+CD86+cells and a corresponding increase in IBA1+CD206+cells following D-Dex treatment (FIGs. 8C-8D).

[0084] Accordingly, the CD86+ / CD206+ratio was significantly lower in the cells + D-Dex group (0.3 ± 0.2) compared to the dendrimer-only (3.5 ± 3.4, p < 0.05), cells-only (5.5 ± 3.1, p < 0.001), and cells + Dex (4.0 ± 2.0, p < 0.05) groups (FIG. 8E), indicating a shift toward an antiinflammatory, M2-like polarization state.

[0085] A negative association was found between the CD86+ / CD206+ratio and the quantity of surviving EGFP+transplanted photoreceptors (R = -0.61, p = 0.0024) (FIG. 8F). These data suggest that D-Dex-mediated immunomodulation shifts microglia / macrophage polarization toward a CD206+anti-inflammatory state, thereby improving donor cell survival.

[0086] Example 6 - D-Dex enhances cone-mediated retinal and visual pathway function following transplantation

[0087] To determine whether enhanced grafted cell survival following D-Dex treatment translated into an improvement in visual pathway function, full-field electroretinography (ffERG) (FIG. 9 A) was performed. Cone-specific functional improvements were assayed because a cone-rich donor cell suspension derived from the OPN1LW-EGFP / Nrl7" mouse line was transplanted. Photopic b-wave amplitudes were significantly higher in the cells + D-Dex group (median 29.4 pV; IQR, 25.9-31.8 pV) compared with the dendrimer-only (median 14.0 pV [IQR 10.4-16.6 pV]; p < 0.001), cells-only (median 16.7 pV [IQR 16.1-19.1 pV]; p < 0.01), and cells + Dex (median 21.0 pV [IQR 13.2-28.2 pV]; p < 0.05) groups. Similarly, 30-Hz flicker responses were significantly increased in the cells + D-Dex group (median 9.4 pV; IQR, 8.2-12.9 pV) relative to the dendrimer-only (median 5.3 pV [IQR 3.1-9.3 pV]; p < 0.001), cells-only (median 5.9 pV [IQR 4.2-7.7 pV]; p < 0.01), and cells + Dex (median 6.0 pV [IQR 4.7-7.3 pV]; p < 0.05) groups (FIG. 9B). There were no differences in scotopic amplitudes between groups. These results are consistent with improved cone photoreceptor function in the Gnatl- / -; Gnat2- / - doubleknockout mice.Attorney Docket No. 44807-0514WO1

[0088] To determine whether improved retinal function was associated with enhanced downstream visual pathway activation, c-Fos expression was analyzed in the lateral geniculate nucleus (LGN), a primary relay center between the retina and visual cortex. Immunofluorescence staining of coronal LGN sections showed a significantly higher number of c-Fos+cells in the LGN of the cells + D-Dex group (21.8 ± 7.7) compared with the dendrimer-only (6.8 ± 4.3, p < 0.001), cells-only (10.2 ± 4.3, p < 0.001), and cells + Dex (12.0 ± 3.9, p < 0.001) groups, indicating enhanced activation of the downstream visual pathway (FIGs. 9C-9D). These findings are consistent with the observed improvements in cone-mediated visual pathway function.

Claims

Atorney Docket No. 44807-0514WO1WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising:(a) a dendrimer nanoparticle, wherein the dendrimer nanoparticle comprises Dexamethasone-21 -succinate; and(b) a plurality of retinal cells.

2. The pharmaceutical composition of claim 1, wherein the dendrimer nanoparticle comprises a hydroxyl PAMAM dendrimer.

3. The pharmaceutical composition of claim 1, wherein the dendrimer nanoparticle comprises a glucose dendrimer.

4. The pharmaceutical composition of claim 1 , wherein the dendrimer comprises a Cy3-dendrimer or a Cy5-dendrimer.

5. The pharmaceutical composition of claim 2, wherein the Dexamethasone-21 -succinate and hydroxyl PAMAM dendrimer are conjugated via copper-catalyzed azide-alkyne cycloaddition (CuACC) reaction.

6. A method for treating a degenerative and hereditary retinopathy, the method comprising:administering the pharmaceutical composition of any one of claims 1-5 to a subject in need thereof.

7. The method of claim 6, wherein the administering comprises an injection of the pharmaceutical composition.

8. The method of claim 7, wherein the injection comprises subretinal injection.

9. The method of any one of claims 6-8, wherein the pharmaceutical composition is delivered into a retinal cell of the subject, and wherein the Dexamethasone-21 -succinate is released intracellularly in the retinal cell.Attorney Docket No. 44807-0514WO110. The method of any one of claims 6-9, wherein the degenerative and hereditary retinopathy comprises Retinitis Pigmentosa (RP), Choroideremia, Stargardt Disease, Cone-rod Dystrophy (CRD), or Leber Congenital Amaurosis (LCA).

11. The method of any one of claims 6-10, wherein the subject is a human.

12. The method of claim 11, wherein the subject is a retinal cell transplant recipient.

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