Use of inhibitors of VEGF-c signaling for inhibiting immune responses and increasing gene therapy efficacy

Abstract

The present invention relates to a method for inhibiting an immune response to an antigen in a subject in need thereof and a method for increasing efficacy of a gene therapy in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of VEGF-C signaling. The invention further relates to a method for generating a protective immune response to an antigen in the central nervous system (CNS) in a subject in need thereof, comprising administering said antigen to the subject by intravitreal administration. The invention also relates to a method for preventing and / or treating a CNS disease in a subject in need thereof, comprising administering a disease-associated antigen to the subject by intravitreal administration.

Description

Attorney Docket No.251609.000097 USE OF INHIBITORS OF VEGF-C SIGNALING FOR INHIBITING IMMUNE RESPONSES AND INCREASING GENE THERAPY EFFICACY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 484,385, filed February 10, 2023, the disclosure of which is herein incorporated by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 18, 2024, is named 251609_000097_SL.xml and is 6,148 bytes in size. FIELD OF THE INVENTION

[0003] The present invention relates to a method for inhibiting an immune response (e.g., a neutralizing immune response) to an antigen in a subject in need thereof and a method for increasing efficacy of a gene therapy in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of VEGF-C signaling. The invention further relates to a method for generating a protective immune response to an antigen in the central nervous system (CNS) in a subject in need thereof, comprising administering said antigen to the subject by intravitreal administration. The invention also relates to a method for preventing and / or treating a CNS disease in a subject in need thereof, comprising administering a disease-associated antigen to the subject by intravitreal administration. BACKGROUND

[0004] Gene therapy has great promise for diseases with monogenic mutations. Many strategies have been implemented and the greatest success in the field so far has been through the use of adeno-associated virus (AAV)-mediated gene delivery of genetic material. As these gene therapies have started to gain approval for clinical use, a new, unforeseen problem has limited their curative effect. AAV-mediated gene therapy has a prolonged expression kinetic, allowing for curative effects in mice. But these years-long expression turned out to be insufficient for a curative 1 168570138v1Attorney Docket No.251609.000097 effect in humans, who need expression of the corrective genes for decades. In order to continue providing expression for these genes, re-dosing patients with AAVs has been attempted, but neutralizing immune responses against the vector after its initial dosing have prevented effective expression. See, e.g., Colella et al., Molecular Therapy Methods & Clinical Development, 2018, 8:87-104; Naso et al., BioDrugs, 2017, 31:317-334; Weber, Front. Immunol., 2021, Vol. 21, Article 658399.

[0005] Lymphatic vasculature is critical in mediating immune memory responses. Lymphatic activity is mediated by Vascular Endothelial Growth Factor-C (VEGF-C). Song et al., Nature, 2020, 577:689-694.

[0006] The eye, an anatomical extension of the central nervous system (CNS), displays many molecular and cellular parallels to the brain. Emerging research demonstrates that changes in the brain are often reflected in the eye, particularly in the retina (London et al., Nature Reviews Neurology, 2013, 9, 44-53). Still, the possibility of an immunological nexus between the posterior eye and the rest of the CNS tissues remains unexplored. SUMMARY OF THE INVENTION

[0007] In one aspect, the invention provides a method for inhibiting an immune response to an antigen in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of VEGF-C signaling.

[0008] In some embodiments, the inhibitor of VEGF-C signaling is administered prior to or simultaneously with the administration of the antigen. In some embodiments, (i) the antigen is administered intracranially or intravitreally, and (ii) the inhibitor of VEGF-C signaling is administered intravitreally, intracranially or via an intra-cisterna magna injection. In some embodiments, the inhibitor of VEGF-C signaling is administered to the posterior eye. In some embodiments, the inhibitor of VEGF-C signaling is administered subretinally or suprachoroidally. In some embodiments, the antigen is administered to the subject repeatedly and the inhibitor of VEGF-C signaling is administered starting from the first or second administration of the antigen and prior to or simultaneously with each subsequent administration of the antigen.

[0009] In some embodiments, the antigen is a molecule, vector or particle. In some embodiments, the antigen is a vector. In some embodiments, the vector is a viral vector (e.g., an adeno-associated virus (AAV) vector, adenovirus vector, rhabdovirus vector, herpesvirus vector, 2 168570138v1Attorney Docket No.251609.000097 or retrovirus vector [e.g., a lentivirus vector]). In some embodiments, the antigen is a particle (e.g., a virus-like particle (VLP), polymer nanoparticle, lipid nanoparticle (LNP), metallic nanoparticle, or liposome). In some embodiments, the antigen is a molecule (e.g., a molecule comprising polyethylene glycol (PEG) or a dendrimer).

[0010] In some embodiments, the inhibitor of VEGF-C signaling inhibits VEGF-C signaling via VEGFR-3. In some embodiments, the inhibitor of VEGF-C signaling is a soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof, wherein said fragment or functional derivative is capable of binding to VEGF-C. In some embodiments, the inhibitor of VEGF-C signaling is an antibody targeting VEGF-C or VEGFR-3, or a fragment or functional derivative thereof (e.g., a single-chain fragment variable (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody). In some embodiments, the inhibitor of VEGF- C signaling is a tyrosine kinase inhibitor.

[0011] In another aspect, the invention provides a method for increasing efficacy of a gene therapy in a subject in need thereof, wherein said gene therapy involves a repeated administration of a gene therapy vector, comprising administering an effective amount of an inhibitor of VEGF- C signaling. In some embodiments, the inhibitor of VEGF-C signaling is administered prior to or simultaneously with the administration of the gene therapy vector. In some embodiments, (i) the gene therapy vector is administered intracranially or intravitreally, and (ii) the inhibitor of VEGF- C signaling is administered intravitreally. In some embodiments, the inhibitor of VEGF-C signaling is administered to the posterior eye. In some embodiments, the inhibitor of VEGF-C signaling is administered subretinally or suprachoroidally. In some embodiments, the gene therapy vector is administered to the subject repeatedly and the inhibitor of VEGF-C signaling is administered starting from the first or second administration of the gene therapy vector and prior to or simultaneously with each subsequent administration of the gene therapy vector. In some embodiments, the gene therapy vector is a viral vector (e.g., an adeno-associated virus (AAV) vector, adenovirus vector, rhabdovirus vector, herpesvirus vector, or retrovirus vector [e.g., a lentivirus vector]). In some embodiments, the inhibitor of VEGF-C signaling inhibits VEGF-C signaling via VEGFR-3. In some embodiments, the inhibitor of VEGF-C signaling is a soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof. In some embodiments, the inhibitor of VEGF-C signaling is an antibody or a fragment or functional derivative thereof (e.g., a single- chain fragment variable (scFv), a diabody, a minibody, a nanobody, a single-domain antibody 3 168570138v1Attorney Docket No.251609.000097 (sdAb), or a single heavy chain antibody). In some embodiments, the inhibitor of VEGF-C signaling is a tyrosine kinase inhibitor.

[0012] In a further aspect, the invention provides a method for generating a protective immune response to an antigen in the central nervous system (CNS) of a subject in need thereof, comprising administering an effective amount of said antigen to the subject by intravitreal administration. In some embodiments, said antigen is administered to the posterior eye of the subject.

[0013] In yet another aspect, the invention provides a method for preventing and / or treating a central nervous system (CNS) disease in a subject in need thereof, comprising administering to the subject through intravitreal administration a disease-associated antigen, wherein said antigen is administered in an amount effective to induce a protective immune response to said antigen in the CNS of the subject. In some embodiments, the disease is an intracranial disease and the antigen is administered in an amount effective to induce a protective immune response to said antigen in the brain of the subject. In some embodiments, the antigen is administered to the posterior eye of the subject. In some embodiments, the disease is an infection (e.g., a bacterial infection, viral infection, fungal infection, or parasitic infection), a cancer (e.g., a CNS cancer such as, e.g., a brain cancer, astrocytomas, ependymomas, glioblastomas, oligoastrocytomas, oligodendrogliomas, lower grade gliomas, meningomas, pituitary adenomas, craniopharyngiomas, schwannoma, choroid plexus tumors, osteoma, chondroma, giant cell tumors, and brain metastatses of non-CNS cancers), or an autoimmune disease (e.g., multiple sclerosis, anti-myelin oligodendrocyte glycoprotein (MOG) disease, neuromyelitis optica (NMO), or autoimmune encephalitis).

[0014] In some embodiments of the above two methods, the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a self-antigen, or a tumor-associated antigen.

[0015] These and other aspects of the present invention will be apparent to those of ordinary skill in the art in the following description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figs. 1A-1I depict that antigens in the posterior eye elicit immune responses in the brain. Fig.1A, Schematic of the schedule of procedures for the experiments described below. Fig. 1B, WT C57BL / 6J mice were immunized using heat-inactivated HSV-2 injection intraperitoneally (IP), intracranially (IC), intracamerally (AC), and intravitreally (IVT). Survival was monitored 4 168570138v1Attorney Docket No.251609.000097 after intracranial challenge with a lethal dose of HSV-230 days later. (Naïve, n=18; IP, n=12; IC, n=6; AC, n=6; IVT, n=18). Fig. 1C, dCLN of mice were ligated using a cauterizer. Seven days later, mice were injected IVT with heat-inactivated HSV-2. Their survival was monitored after IC challenging with a lethal dose of HSV-230 days later. (Naïve, n=5; IVT immunized, n=5; LN ligation, n=6). Fig.1D, Schematic of treatment plans. Fig.1E, Mice were IVT injected with heat- inactivated HSV-2. Four weeks later, the immunized mice were joined with naïve mice. The immunized mice or naïve mice were intracranially challenged with a lethal dose of HSV-2 after 3 weeks and their survival was monitored (Naïve, n=4; IVT n=4; IVT-Naïve L infected, n=2; IVT- Naïve R infected, n=6; Naïve-Naïve R infected, n=2). Fig. 1F, Anti-HSV specific antibody was measured by ELISA after different routes of administration (IP, n=12; IC, n=6; AC, n=10; IVT, n=10). Fig.1G, WT C57BL / 6J mice were injected heat-inactivated HSV-1 through the routes of intraperitoneal (IP), intracranial (IC), intracameral (AC), or intravitreal (IVT) administration. Their survival was monitored after intracranial challenge with a lethal dose of HSV-130 days later. (Naïve, n=15; IP, n=6; IC, n=6; AC, n=6; IVT, n=18). Fig. 1H, Similar as Fig. 1A, but streptococcus pneumonia strain TIGR4 was used (Naïve, n=8; IP, n=5; IC, n=5; AC, n=8; IVT, n=8). Fig. 1I, Mice were intracranially inoculated with 50,000 GL261-Luc cells and mice were treated with irradiated GL261-Luc through the routes of subcutaneous (SC), IC, AC, IVT administration (day 7) and with anti-PD-1(RMP1-14) antibodies (days 7, 9 and 11), and monitored for survival (Naïve, n=6; SC, n=6; IC, n=6; AC, n=6; IVT, n=12). Data are representative of two independent experiments. P-values were calculated by a two-sided log-rank Mantel—Cox test.

[0017] Figs.2A-2I depict that eyes have a compartmentalized lymphatic drainage system. Fig. 2A, Schematic of intracameral / anterior chamber (AC) and intravitreal (IVT) injection of dye. Fig. 2B, C57BL / 6J mice were injected with dye with AC or IVT injection. The percentage of dye retention in the eye was analyzed from hour 6 to day 5 post injection (AC,n=6; IVT,n=6). Fig.2C, Schematic of anatomical locations of superficial cervical LNs (sCLN) and deep cervical LNs (dCLN). Fig.2D, Dye was AC (middle) or IVT (bottom) injected into the left eye and eyes, sCLN and dCLN were collected for IVIS imaging. Representative background-subtracted heat maps of dye in the eye, sCLN and dCLN 1h later after injection. Figs. 2E-2F, Dye was AC (Fig.2E) or IVT (Fig.2F) injected into the eye and dye presence was measured using a fluorescent plate reader. The percentage of ipsilateral and contralateral lymph nodes with dye was measured 12h later (n=10 mice). Figs. 2G-2H, sCLN, dCLN or both CLN were cauterized and surgically ligated. 2 days 5 168570138v1Attorney Docket No.251609.000097 later, dye was AC (Fig.2G) or IVT (Fig.2H) injected into the eye. The percentage of dye retention in the eye was measured 12h later. Data shown as mean ± s.e.m. in Fig. 2G and Fig. 2H (sham intracameral, n=3; n=4 in all other conditions). Fig. 2I, Dye was intracamerally or intravitreally injected into the eye. sCLN and dCLN were collected and measured using a fluorescent plate reader 1h later after injection. RFU, relative fluorescent unit (AC,n=8; IVT,n=8). ****P<0.0001; ***P<0.0002, IVT sCLN; ***P<0.0003, IVT dCLN. P-values were calculated using a one-way ANOVA with multiple comparisons testing (Dunnett).

[0018] Figs. 3A-3H depict that optic nerve sheath lymphatics drain the posterior eye. Figs. 3A-3B, Sectioned immunolabeled staining of optic nerve of zebrafish with lymphatics labeled with mrc1a (white). iDISCO immunolabeling of meningeal lymphatic vessels of rabbit, pig, non-human primate and human optic nerve / chiasm (LYVE-1 and VEGFR-3). Fig.3C, Whole-mount murine optic nerve VEGFR3 staining and tracer signal on optic nerve 1h later after AC (up) or IVT (bottom) injection. White arrowheads points to co-localization of VEGFR3 and tracer. Fig. 3D, Schematic of intraci sterna magna injection (ICM) and the eye and optic nerve dissection for Fig. 3E. Fig.3E, Dye was injected ICM and fluorescent signal intensity was measured lh later in the eye and optic nerve. (Control, n=11; CSF injection, n = 11) ****P<0.0001. Fig.3F, Schematic of injection methods for Fig. 3G. Fig. 3G, VEGF-C was injected with AC, IVT or ICM administration route.2 days later, dye was injected intravitreally into the eye and the percentage of dye retention was measured 12h after dye injection. (Control, n=12; AC, n=19; IVT, n=20; CSF(2µg), n=20; CSF(6µg), n=10). Fig. 3H, Whole-mount murine optic nerve sheaths stained with LYVE1, CD31, PROX1 and VEGFR3. ****P<0.0001; ***P<0.0002. Data shown as mean ± s.e.m. P-values were calculated using a one-way ANOVA with multiple comparisons testing (Dunnett) or two-tailed unpaired Student’s t-test.

[0019] Figs.4A-4G depict that lymphatic inhibition enables repeat AAV administration. Fig. 4A, Schematic of experimental plans. Fig. 4B, In vivo fluorescent fundus imaging to visualize vessels and RFP transduction with lymph node ligation conditions. Fig. 4C. Quantification of RFP intensity from Fig.4B (Primary, n=6; Secondary, n=5; sCLN ligation, n=5; dCLN ligation, n=6) **P=0.0095 Primary vs Secondary; **P=0.0044 Secondary vs dCLN ligation. Fig. 4D, In vivo fluorescent fundus imaging to visualize vessels and RFP transduction with addition of VEGF- C or soluble VEGFR-3 (sR3). Fig. 4E, Quantification of RFP intensity from Fig. 4D (Primary, n=9; Secondary, n=10; VEGF-C, n=9; sR3, n=8) *P=0.0167 Primary vs Secondary; *P=0.0334 6 168570138v1Attorney Docket No.251609.000097 Secondary vs VEGF-C; **P=0.0096 Secondary vs sR3. Figs.4F and 4G, mice were injected with AAV-RFP through intravitreal or intracameral routes. Their dCLN, sCLN and retinas were collected 10 days later and AAV specific immune responses were quantified using an ELISpot assay. P-values were calculated using a one-way ANOVA with multiple comparisons testing (Dunnett).

[0020] Figs. 5A-5C depict a gating strategy for different immune cell populations. Flow cytometry gating strategy for lymph node GC B cells (Fig.5A), Tfh cells (Fig.5B), and multiple population of brain immune cells (Fig.5C).

[0021] Figs. 6A-6N depict that immunization through the posterior compartment of the eye can induce an immunological protection in the brain. Fig. 6A, Similar to Fig. 1A, but B cell- deficient mice (µMT) were IVT injected with heat-inactivated HSV-2 and one group of WT IVT immunized mice were treated with CD4 depletion antibody before challenge. WT C57BL / 6J mice were injected with heat-inactivated HSV-2 through the routes of IP, IC, AC, IVT administration, respectively. The percentage of B cells and CD4+T cells in various organs were analyzed ten days later. Fig. 6B, Quantification of Tfh and GC B cells in spleens. Fig. 6C, Representative FACS plots of CD4+PD-1+CXCR5+Tfh cells and CD19+GL7+Fas+GC B cells in dCLN 10 days after different routes of immunization. Fig. 6D, Similiar to Fig. 6B, but GC B cells and Tfh cells in dCLN were analyzed. Fig.6E, B cells and CD4+T cells were measured in brain tissues. Fig.6F, Similar experiment to Fig. 1D, the serum of both IVT immunized mice and naïve mice were collected 3 weeks after parabiosis. Anti-HSV specific antibody was measured by ELISA (n=8). Fig. 6G, Similar to Fig 1C, dCLNs were ligated after priming of the mice but before the rechallenge intracranially. Vaccination was completed first. Then, 7 days before the rechallenge, dCLNs were ligated, and mice survival was monitored after IC challenge with a lethal dose of HSV-2. (Naïve, n=5; IVT immunized, n=6; LN ligation, n=9). Fig.6H, Extending findings from Fig.1D, the serum and CSF of both IVT immunized mice and naïve mice were collected 3 weeks after parabiosis. Anti-HSV specific antibody was measured by ELISA (n=6). Fig.6I, Schematic of experimental plans. CD45.2 C57BL / 6J recipient mice were primed by intraperitoneal immunization. 2 weeks later, CD45.1.2 B1-8hiB cells labelled with CellTrace™ Violet Cell Proliferation Kit were transferred intravenously into recipient mice. 18h later, the mice were immunized with 10 ^g NP–OVA though IVT or AC injection. Igλ+light chain positive B1-8hicell proliferation and germinal center (GC) formation in dCLNs and sCLNs were analyzed at day 7. 7 168570138v1Attorney Docket No.251609.000097 Fig.6J, Flow cytometry gating strategy for B1-8hiGC B cells. Fig.6K, Quantification of B1-8 GC B cells in dCLNs and sCLNs (n=7-10). Fig.6L, Schematic of experimental plans. CD45.1 OVA specific CD4+T cells labelled with CFSE were transferred intravenously into CD45.2 recipient mice.18h later, the mice were immunized with 50 ^g OVA plus 1 ^g Poly(I:C) though IVT or AC injection. FTY720 was IP injected to inhibit the circulation of primed T cells 24h after immunization. The OVA-specific CD4+T cells proliferation in dCLNs and sCLNs were analyzed 72 h after immunization. Fig. 6M, Flow cytometry gating strategy for analyzing OVA-specific CD4+T proliferation (n=5-8). Fig. 6N, Quantification of CFSE-negative OVA-specific CD4+T cells in dCLNs, sCLNs, and inguinal LNs. Data shown as mean ± s.e.m. P-values were calculated using a two-tailed unpaired Student’s t-test.

[0022] Figs. 7A-7I depict anatomical characterization of meningeal lymphatic vessels covering optic nerve. Additional images of VEGFR3 and LYVE-1 staining on optic nerves of rabbit (Figs.7A-7B), pig (Figs.7C-7D), non-human primates (Figs.7E-7G), and mice (Figs.711- 7BI). Light gray arrows indicate vascular structure with single staining for either LYVE-1 or VEGFR-3. White arrows indicate co-stained vascular structures.

[0023] Fig. 8 depicts that lymphatic inhibition enables repeated AAV transduction. Representative confocal images of AAV-RFP+ cells from whole-mount mouse retina stained with DAPI.

[0024] Fig. 9 depicts that shared ocular-brain lymphatic drainage results in unifying CNS immunity.

[0025] Figs.10A-10E depict that immunization through the posterior compartment of the eye can provide therapeutic treatment of brain tumors. Fig.10A, Mice were IC inoculated with 50,000 GL261-Luc cells and mice were treated with irradiated GL261-Luc through the routes of subcutaneous (SC), IC, AC, or IVT administration (day 7) along with anti-PD-1 antibodies (days 7, 9 and 11), and monitored for tumor growth. RLU, relative luminescence units. (SC, n= 6; IC, n=6; AC, n=6; IVT, n=6). Fig.10B, Mice were IC inoculated with 50,000 CT2A-BFP cells. The percentage of BFP+DCs in the tumor, IngLNs, meninges, dCLNs and sCLNs were analyzed 14 days after tumor injection (n=5). Figs.10C-10D, Similar experiment as Fig.10A. The percentage of EMV-2 specific CD8+T cells were analyzed in dCLN, sCLN and IngLN at day 14. Fig. 10C, Flow cytometry gating strategy for EMV-2 specific CD8+T cells. Fig. 10D, Quantification of EMV-2 specific CD8+T cells in dCLN, sCLN and IngLN. (Naïve n=5; SC, n=5; IC, n=3; AC, 8 168570138v1Attorney Docket No.251609.000097 n=3; IVT, n=3). Fig.10E, Mice were SC inoculated with 50,000 B16 cells, treated with irradiated B16 cells through the routes of SC, IC, AC, IVT administration (day 7) along with anti-PD-1 antibodies (days 7, 9 and 11), and monitored for survival. (n=10 for all groups). Data shown as mean ± s.e.m. P-values were calculated using a one-way ANOVA with multiple comparisons testing (Dunnett).

[0026] Fig. 11A-11E depict that eyes have a compartmentalized lymphatic drainage system. Fig.11A, Nanoparticles were AC or IVT injected into the eye. Serum was collected, placed on a slide and imaged on a fluorescent microscope 1h later after injection (n=10). Fig.11B, Dye was AC or IVT injected into the left eye. Eyes, sCLN and dCLN were collected for IVIS imaging. Representative background-subtracted heat maps of dye in the eye, sCLN and dCLN 1h later after injection. Fig.11C, Dye was AC, IVT, or subconjunctivally injected into the left eye. dCLNs were collected and measured using a fluorescent plate reader 1h later after injection (n=8). Figs.11D- 11E, dCLNs (Fig.11D) or sCLNs (Fig.11E) were cauterized and surgically ligated.2 days later, dye was AC or IVT injected into the eye. sCLNs (Fig.11D) or dCLNs (Fig.11E) were collected and measured using a fluorescent plate reader 1h later after injection. RFU, relative fluorescence unit. (AC, n=8; IVT, n=8). P-values were calculated using a one-way ANOVA with multiple comparisons testing (Dunnett). Data shown as mean ± s.e.m. P-values were calculated using a one- way ANOVA with multiple comparisons testing (Dunnett) or two-tailed unpaired Student’s t-test.

[0027] Fig.12 is an image of H&E staining and respective cell type gene signature projected onto spatial sequencing data (light gray arrows denote where the gene signature appears for given signature).

[0028] Figs. 13A-13B depict detailed analysis of optic nerve sheath lymphatics. Fig. 13A, Whole-mount murine ventral ear sheet stained with LYVE1, CD31, PROX1 and VEGFR3. Fig. 13B, Whole-mount of optic nerve sheaths of WT mice. Up: LYVE1, CD31, PROX1 and VEGFR3 were co-stained. Bottom: LYVE1, PDPN, PROX1 and VEGFR3 were co-stained.

[0029] Figs. 154A-14D depict detailed analysis of optic nerve sheath lymphatics with lymphatic reporter mice. Figs. 14A-14B, Whole-mount murine ventral ear sheet and optic nerve sheaths of VEGFR3-CreERT2;R26- MTMG mice that were stained with LYVE1 and PROX1 (Fig.14A, scale: 50 μm; Fig.14B, scale: 500 μm). Figs.14C-14D, Whole-mount murine ventral ear sheet and optic nerve sheaths of PROX1CreERT2;CDH5 Dre;R26-STOP-mCherry mice that were stained with LYVE1, CD31 and VEGFR3 (Fig.14C, scale: 50 μm; Fig.14D, scale: 500 μm). 9 168570138v1Attorney Docket No.251609.000097

[0030] Figs.15A-15G depict that optic nerve sheath lymphatics drain the posterior eye. Figs. 15A-15C, Anti-LYVE1 antibody was IVT injected into the vitreous humor. Optic nerve sheaths were harvested 2h later and co-stained with CD31, PROX1 and VEGFR3. Fig.15C, Dye was IVT injected into the eye or intraventricularly into the brain. CSF was collected through the cisterna magna and measured using a fluorescent plate reader 1h later after injection. Fig. 15D, Anti- LYVE1 antibody was injected IVT into the eye or ICM into the CSF. Dura was collected 2h after injection and LYVE1 antibody binding on VEGFR3 positive lymphatic vessels was analyzed by immunofluorescence. Mice without anti-LYVE1 antibody injection were used as the control. Fig. 15E, AF647-OVA was IVT injected into the vitreous humor. Fluorescein was injected intra peritoneally to label blood vessels. The kinetics of AF647-OVA drainage was tracked with Phoenix MICRON® IV imaging microscope. Fig. 15F, AF647-OVA was IVT injected into the vitreous humor. The eyes with optic nerve were harvested, and the location of AF647-OVA was analyzed at the indicated time points. Fig.15G, Zoomed in images of Fig.15F.

[0031] Figs. 16A-16C depict that lymphatic inhibition enables repeated rAAV administrations. Fig. 16A, Mice were IVT injected with rAAV-VEGF-C, rAAV-sVEGFR3 or rAAV-control. Two months later, their optic nerve sheaths were harvested and stained with LYVE1. Fig. 16B, Similar to Fig. 4C, dCLNs were ligated before the primary rAAV-RFP injection. Their RFP intensity were quantified. Fig.16C, Percentage of dye retention in the eye was measured for control, dCLN-ligated, or rAAV-sVEGFR3-injected mice. Data shown as mean ± s.e.m. P-values were calculated using a one-way ANOVA with multiple comparisons testing (Dunnett).

[0032] Figs. 17A-17B depict that lymphatic inhibition enables repeated rAAV transduction. Fig. 17A, Similar to Fig.4C, murine retinas were collected, and RFP expression was quantified using western blot. Fig.17B, Similar to Fig.4C, murine retinas were collected, and RFP expression was quantified using flow cytometry. DETAILED DESCRIPTION

[0033] Lymphatic vasculature is critical in mediating immune memory responses. Lymphatic activity is mediated by VEGF-C. The present inventors hypothesized that local and transient inhibition of lymphatic vasculature function could allow for better effectiveness of the repeat gene therapy vector administration. As lymphatic activity is mediated by VEGF-C, by using soluble 10 168570138v1Attorney Docket No.251609.000097 VEGFR-3 to sequester VEGF-C, the present inventors were able to allow for maintaining effectiveness of repeated gene therapy with adeno-associated virus (AAV) vectors.

[0034] The present invention is based on a surprising discovery that intravitreal immunization with herpes virus protects mice against intracranial disease. As demonstrated herein, this protection extended to bacteria and even tumors, allowing for therapeutic immune responses against glioblastoma via intravitreal immunization. Upon further investigation, it was shown herein that the anterior and posterior compartments of the eye have two distinct lymphatic drainage systems, the latter draining to the deep cervical lymph nodes through lymphatic vasculature in the optic nerve sheath. Additionally, this posterior lymphatic drainage, like that of meningeal lymphatics, can be modulated with VEGF-C. Conversely, inhibition of lymphatic signaling on the optic nerve overcomes a major limitation in gene therapy, by diminishing neutralizing immune response to AAV and ensuring continued efficacy after multiple doses. These results reveal a shared lymphatic circuit able to mount a unified immune response between the posterior eye and the brain, highlighting an understudied immunological feature of the eye and opening up the potential for new therapeutic strategies in ocular and central nervous system (CNS) diseases.

[0035] The present invention provides a method for inhibiting an immune response to an antigen in a subject in need thereof and a method for increasing efficacy of a gene therapy in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of VEGF-C signaling. The invention further provides a method for generating a protective immune response to an antigen in the central nervous system (CNS) in a subject in need thereof, comprising administering said antigen to the subject by intravitreal administration. The invention also provides a method for preventing and / or treating a CNS disease in a subject in need thereof, comprising administering a disease-associated antigen to the subject by intravitreal administration. In one aspect, the invention provides a method for inhibiting a neutralizing immune response to an antigen in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of VEGF-C signaling. Definitions

[0036] The terms “a,” “an,” and “the” do not denote a limitation of quantity, but rather denote the presence of “at least one” of the referenced item. 11 168570138v1Attorney Docket No.251609.000097

[0037] The term “about” when used before a numerical value indicates that the value may vary within a reasonable range, such as within ±10%, ±5% or ±1% of the stated value, and include the stated value.

[0038] The terms “patient,” “individual,” “subject,” “mammal,” and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, rabbits, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.

[0039] The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0040] The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0041] The practice of the present invention employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual.3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, 12 168570138v1Attorney Docket No.251609.000097 NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g., in U.S. Patent No.7,912,698 and U.S. Patent Appl. Pub. Nos.2011 / 0202322 and 2011 / 0307437. Methods of the Invention

[0042] In one aspect, the invention provides a method for inhibiting a neutralizing immune response to an antigen in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of VEGF-C signaling.

[0043] In some embodiments, the inhibitor of VEGF-C signaling is administered prior to or simultaneously with the administration of the antigen. In some embodiments, (i) the antigen is administered intracranially or intravitreally, and (ii) the inhibitor of VEGF-C signaling is administered intravitreally, intracranially or via an intra-cisterna magna injection. In some embodiments, the inhibitor of VEGF-C signaling is administered to the posterior eye. In some embodiments, the inhibitor of VEGF-C signaling is administered subretinally or suprachoroidally. In some embodiments, the antigen is administered to the subject repeatedly and the inhibitor of VEGF-C signaling is administered starting from the first or second administration of the antigen and prior to or simultaneously with each subsequent administration of the antigen.

[0044] In some embodiments, the antigen is a molecule, vector or particle. In some embodiments, the molecule is a protein, or a peptide. In some embodiments the protein or peptide is naturally occurring. In some embodiments the protein or peptide is synthetic. In some embodiments, the protein or peptide is synthetic. In some embodiments, the antigen is a vector. In some embodiments, the vector is a viral vector (e.g., an adeno-associated virus (AAV) vector, adenovirus vector, rhabdovirus vector, herpesvirus vector, or retrovirus vector [e.g., a lentivirus vector]). In some embodiments, the antigen is a particle (e.g., a virus-like particle (VLP), polymer nanoparticle, lipid nanoparticle (LNP), metallic nanoparticle, or liposome). In some embodiments, the antigen is a molecule (e.g., a molecule comprising polyethylene glycol (PEG) or a dendrimer).

[0045] In a related aspect, the invention provides a method for increasing efficacy of a gene therapy in a subject in need thereof, wherein said gene therapy involves a repeated administration 13 168570138v1Attorney Docket No.251609.000097 of a gene therapy vector, comprising administering an effective amount of an inhibitor of VEGF- C signaling. In some embodiments, the inhibitor of VEGF-C signaling is administered prior to or simultaneously with the administration of the gene therapy vector. In some embodiments, (i) the gene therapy vector is administered intracranially or intravitreally, and (ii) the inhibitor of VEGF- C signaling is administered intravitreally. In some embodiments, the inhibitor of VEGF-C signaling is administered to the posterior eye. In some embodiments, the inhibitor of VEGF-C signaling is administered subretinally or suprachoroidally. In some embodiments, the gene therapy vector is administered to the subject repeatedly and the inhibitor of VEGF-C signaling is administered starting from the first or second administration of the gene therapy vector and prior to or simultaneously with each subsequent administration of the gene therapy vector. In some embodiments, the gene therapy vector is a viral vector (e.g., an adeno-associated virus (AAV) vector, adenovirus vector, rhabdovirus vector, herpesvirus vector, or retrovirus vector [e.g., a lentivirus vector]).

[0046] In a further aspect, the invention provides a method for generating a protective immune response to an antigen in the central nervous system (CNS) of a subject in need thereof, comprising administering an effective amount of said antigen to the subject by intravitreal administration. In some embodiments, said antigen is administered to the posterior eye of the subject.

[0047] In yet another aspect, the invention provides a method for preventing and / or treating a central nervous system (CNS) disease in a subject in need thereof, comprising administering to the subject through intravitreal administration a disease-associated antigen, wherein said antigen is administered in an amount effective to induce a protective immune response to said antigen in the CNS of the subject. In some embodiments, the disease is an intracranial disease and the antigen is administered in an amount effective to induce a protective immune response to said antigen in the brain of the subject. In some embodiments, the antigen is administered to the posterior eye of the subject. In some embodiments, the disease is an infection (e.g., a bacterial infection, viral infection, fungal infection, or parasitic infection), a cancer (e.g., a CNS cancer such as, e.g., a brain cancer, astrocytomas, ependymomas, glioblastomas, oligoastrocytomas, oligodendrogliomas, lower grade gliomas, meningomas, pituitary adenomas, craniopharyngiomas, schwannoma, choroid plexus tumors, osteoma, chondroma, giant cell tumors, and brain metastatses of non-CNS cancers), or an autoimmune disease (e.g., multiple sclerosis, anti-myelin oligodendrocyte glycoprotein (MOG) disease, neuromyelitis optica (NMO), or autoimmune encephalitis). 14 168570138v1Attorney Docket No.251609.000097

[0048] In some embodiments of the above two methods, the antigen is: a) viral antigen (e.g., an influenza virus antigen [e.g., hemagluttinin (HA), neuramimidase (NA)], a herpesvirus antigen [a herpes simplex virus (HSV) antigen], a hepatitis virus antigen [e.g., Hepatitis B surface antigen (HBsAg)], a rous associated virus antigen [e.g., RAV-1 env], an infectious bronchitis virus antigen [e.g., matrix, preplomer], a flavivirus antigen [e.g., a Japanese encephalitis virus (JEV) antigen, a Yellow Fever virus antigen, or a Dengue virus antigen], a Morbillivirus antigen [e.g., a canine distemper virus antigen, a measles virus antigen, or rinderpest virus antigen, such as HA or F], a rabies virus antigen [e.g., rabies glycoprotein G], a parvovirus antigen [e.g., a canine parvovirus antigen], a poxvirus antigen [e.g., an ectromelia antigen, a canary poxvirus antigen, or a fowl poxvirus antigen], a chicken pox virus antigen (varicella zoster antigen), an infectious bursal disease virus antigen [e.g., VP2, VP3, or VP4], a Hantaan virus antigen, a mumps virus antigen. a papilloma virus antigen, an enterovirus antigen, a rabies virus antigen, an EBV antigen [e.g., BMLF1], a CMV antigen [e.g., LCMV gp33, CMV pp65], an adenovirus antigen, a rubella antigen, a measles antigen, a MVE antigen, a JEV antigen, a ZIKV antigen, a SARS-CoV-2 antigen, an HIV antigen [e.g., gp120, gp160, p18, Gag p17 / p24, Tat, Pol, Nef, Env], a CHKV antigen, an EEE antigen, a West Nile virus antigen, a Powassan virus antigen, a poliovirus antigen, or a coxackie virus antigen), b) a bacterial antigen (e.g., a Streptococcus pneumoniae antigen, Neisseria meningitides antigen, Haemophilus influenzae antigen, Escherichia coli antigen, Mycobacterium tuberculosis antigen, Neisseria gonorrhea antigen, Haemophilus vaginalis antigen, Group B Streptococcus sp. antigen, Microplasma hominis antigen, Hemophilus ducreyi antigen, Granuloma inguinale antigen, Lymphopathia venereum antigen, Treponema pallidum antigen, Brucella abortus antigen. Brucella melitensis antigen, Brucella suis antigen, Brucella canis antigen, Campylobacter fetus antigen, Campylobacter fetus intestinalis antigen, Leptospira pomona antigen, Listeria monocytogenes antigen, Brucella ovis antigen, Chlamydia psittaci antigen, Actinobacillus equuli antigen, Salmonella abortus ovis antigen, Salmonella abortus equi antigen, Pseudomonas aeruginosa antigen, Corynebacterium equi antigen, Corynebacterium pyogenes antigen, and Actinobaccilus seminist antigen; additional non-limiting examples of bacterial antigens include, e.g., lipopolysaccharides isolated from gram-negative bacterial cell walls and staphylococcus-specific, streptococcus-specific, pneumococcus-specific (e.g., PspA; see PCT Publication No. WO 92 / 14488), Neisseria gonorrhea-specific, Borrelia-specific (e.g., OspA, OspB, OspC of Borrelia 15 168570138v1Attorney Docket No.251609.000097 associated with Lyme disease such as Borrelia burgdorferi, Borrelia afzelli, and Borrelia garinii [see, e.g., U.S. Pat. No. 5,523,089; PCT Publication Nos. WO 90 / 04411, WO 91 / 09870, WO 93 / 04175, WO 96 / 06165, WO93 / 08306; PCT / US92 / 08697; Bergstrom et al., Mol. Microbiol., 1999; 3: 479486; Johnson et al., Infect. and Immun.1992; 60: 1845-1853; Johnson et al., Vaccine 1995; 13: 1086-1094; The Sixth International Conference on Lyme Borreliosis: Progress on the Development of Lyme Disease Vaccine, Vaccine 1995; 13: 133-135]), and pseudomonas-specific proteins or peptides), c) a fungal antigen (e.g., a candida antigen [e.g., MP65 from Candida albicans], a trichophyton antigen, a ptyrosporum antigen), d) a parasitic antigen (e.g., a malaria-specific protein such as, e.g., circumsporozoite (CS) protein, Thrombospondin Related Adhesion (Anonymous) protein (TRAP), also called Sporozoite Surface Protein 2 (SSP2), LSA I, hsp70, SALSA, STARP, Hep17, MSA, RAP-1, RAP-2), e) a self-antigen (e.g., gliadin, insulin B chain, glatiramer acetate (GA), achetylcholine receptor (AChR), p205, insulin, thyroid-stimulating hormone, tyrosinase, TRP1, myelin basic protein (MBP), proteolipid protein (PLP), BV3, BV13SI, BV5S2, BV6S5, BV13SI, BV14, or BV17, or f) a tumor-associated antigen (e.g., adipophilin, AIM-2, ALDHIAI, alpha-actinin-4, alpha- fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta- catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNKIAI, CTAG1, CTAG2, cyclin D1, Cyclin-Al, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6- AML1 fusion protein, EZH2, FGFS, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pme117, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-Al, MAGE-A10, MAGE-Al2, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, MEI, Melan-A / MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, 16 168570138v1Attorney Docket No.251609.000097 PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF- betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY-ESO1, MAGE- A3, HPV E2, HPV E6, HPV E7, WT-1 antigen, ErbB receptors, Melan A [MART1], gp 100, tyrosinase, TRP-1 / gp 75, and TRP-2, MAGE-1 and MAGE-3 (in bladder, head and neck, and non- small cell carcinoma), HPV EG and E7 proteins, Mucin [MUC-1], prostate-specific antigen [PSA], carcinoembryonic antigen [CEA], MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE- 1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-1, NY-ESO-1 / LAGE-2, NA-88, GnTV, TRP2-INT2, or various neo-antigens). VEGF-C protein

[0049] As used herein, a human VEGF-C protein may comprise a sequence that is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the following polypeptide sequence: MHLLGFFSVACSLLAAALLPGPREAPAAAAAFESGLDLSDAEPDAGEATAYASKDLEEQLR SVSSVDELMTVLYPEYWKMYKCQLRKGGWQHNREQANLNSRTEETIKFAAAHYNTEIL KSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTS TSYLSKTLFEITVPLSQGPKPVTISFANHTSCRCMSKLDVYRQVHSIIRRSLPATLPQCQA ANKTCPTNYMWNNHICRCLAQEDFMFSSDAGDDSTDGFHDICGPNKELDEETCQCVCR AGLRPASCGPHKELDRNSCQCVCKNKLFPSQCGANREFDENTCQCVCKRTCPRNQPLN PGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFSQPLNPGKCACECT ESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFS (SEQ ID NO: 1; available in the UniProt database under accession number #P49767; the N-terminal signaling peptide is shown in Italics and is underlined). Inhibitors of VEGF-C signaling

[0050] Various inhibitors of VEGF-C signaling are known in the art and can be used in the methods of the present invention. See, e.g., U.S. Patent No. 9,745,558, which is incorporated herein by reference in its entirety. 17 168570138v1Attorney Docket No.251609.000097

[0051] In some embodiments of the methods of the invention, the inhibitor of VEGF-C signaling inhibits VEGF-C signaling via VEGFR-3. In some embodiments, the inhibitor of VEGF- C signaling is a soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof, which fragment or functional derivative is capable of binding to VEGF-C. In some embodiments, the sR3 or the fragment or functional derivative thereof comprises the extracellular domain (ECD) of VEGFR-3. In some embodiments, the ECD of VEGFR-3 comprises positions 30-764 of SEQ ID NO: 2 or the sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical thereto. In some embodiments, the sR3 or the fragment or functional derivative thereof comprises the first immunoglobulin-like domain of the ECD of VEGFR-3. In some embodiments, the sR3 or the fragment or functional derivative thereof comprises positions 30-127 of SEQ ID NO: 2 or the sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical thereto. In some embodiments, the sR3 or the fragment or functional derivative thereof comprises the second immunoglobulin-like domain of the ECD of VEGFR-3. In some embodiments, the sR3 or the fragment or functional derivative thereof comprises positions 151- 213 of SEQ ID NO: 2 or the sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical thereto. In some embodiments, the sR3 or the fragment or functional derivative thereof comprises the third immunoglobulin-like domain of the ECD of VEGFR-3. In some embodiments, the sR3 or the fragment or functional derivative thereof comprises positions 219-326 of SEQ ID NO: 2 or the sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical thereto. In some embodiments, the sR3 or the fragment or functional derivative thereof comprises amino acids 30- 127, 151-213, and 219-326 of SEQ ID NO: 2 or the sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical thereto. In some embodiments, the VEGFR-3-based inhibitors of the invention preferably bind human VEGF-C with a Kd of about 1 nM or less. MQRGAALCLRLWLCLGLLDGLVSGYSMTPPTLNITEESHVIDTGDSLSISCRGQHPLEW AWPGAQEAPATGDKDSEDTGVVRDCEGTDARPYCKVLLLHEVHANDTGSYVCYY KYIKARIEGTTAASSYVFVRDFEQPFINKPDTLLVNRKDAMWVPCLVSIPGLNVTLRS 18 168570138v1Attorney Docket No.251609.000097 QSSVLWPDGQEVVWDDRRGMLVSTPLLHDALYLQCETTWGDQDFLSNPFLVHITG NELYDIQLLPRKSLELLVGEKLVLNCTVWAEFNSGVTFDWDYPGKQAERGKWVP ERRSQQTHTELSSILTIHNVSQHDLGSYVCKANNGIQRFRESTEVIVHENPFISVEWL KGPILEATAGDELVKLPVKLAAYPPPEFQWYKDGKALSGRHSPHALVLKEVTEASTGTY TLALWNSAAGLRRNISLELVVNVPPQIHEKEASSPSIYSRHSRQALTCTAYGVPLPLSIQW HWRPWTPCKMFAQRSLRRRQQQDLMPQCRDWRAVTTQDAVNPIESLDTWTEFVEGKN KTVSKLVIQNANVSAMYKCVVSNKVGQDERLIYFYVTTIPDGFTIESKPSEELLEGQPVL LSCQADSYKYEHLRWYRLNLSTLHDAHGNPLLLDCKNVHLFATPLAASLEEVAPGARH ATLSLSIPRVAPEHEGHYVCEVQDRRSHDKHCHKKYLSVQALEAPRLTQNLTDLLVNVS DSLEMQCLVAGAHAPSIVWYKDERLLEEKSGVDLADSNQKLSIQRVREEDAGRYLCSV CNAKGCVNSSASVAVEGSEDKGSMEIVILVGTGVIAVFFWVLLLLIFCNMRRPAHADIK TGYLSIIMDPGEVPLEEQCEYLSYDASQWEFPRERLHLGRVLGYGAFGKVVEASAFGIH KGSSCDTVAVKMLKEGATASEHRALMSELKILIHIGNHLNVVNLLGACTKPQGPLMVIV EFCKYGNLSNFLRAKRDAFSPCAEKSPEQRGRFRAMVELARLDRRRPGSSDRVLFARFS KTEGGARRASPDQEAEDLWLSPLTMEDLVCYSFQVARGMEFLASRKCIHRDLAARNILL SESDVVKICDFGLARDIYKDPDYVRKGSARLPLKWMAPESIFDKVYTTQSDVWSFGVLL WEIFSLGASPYPGVQINEEFCQRLRDGTRMRAPELATPAIRRIMLNCWSGDPKARPAFSE LVEILGDLLQGRGLQEEEEVCMAPRSSQSSEEGSFSQVSTMALHIAQADAEDSPPSLQRH SLAARYYNWVSFPGCLARGAETRGSSRMKTFEEFPMTPTTYKGSVDNQTDSGMVLASE EFEQIESRHRQESGFSCKGPGQNVAVTRAHPDSQGRRRRPERGARGGQVFYNSEYGELS EPSEEDHCSPSARVTFFTDNSY (SEQ ID NO: 2; human VEGFR-3; sequences of domain 1, 2 and 3 which are required for inhibition of VEGF-C signaling are underlined; available in the UniProt database under accession number #P35916; the N-terminal signaling peptide is shown in Italics and is underlined).

[0052] In some embodiments, the inhibitor of VEGF-C signaling is a fusion protein or conjugate comprising the soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof disclosed herein, wherein the soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof, is fused and / or conjugated to one of more heterologous moieties. In some embodiments, the inhibitor of VEGF-C signaling is a fusion protein or conjugate comprising domain 1, 2, or 3 of VEGFR-3. Non-limiting examples of soluble VEGFR-3 (sR3) or fragment or functional derivative 19 168570138v1Attorney Docket No.251609.000097 thereof are provided, e.g., in U.S. Patent 9,745,558, which is incorporated herein by reference in its entirety.

[0053] In some embodiments, the one of more heterologous moieties are selected from an immunoglobulin or a functional fragment thereof, an albumin or a functional fragment thereof, an albumin-binding antibody or a functional fragment thereof, and a polyethylene glycol (PEG) polymer.

[0054] In some embodiments, the immunoglobulin or functional fragment thereof comprises an IgG Fc domain. In some embodiments, the IgG Fc domain is modified to reduce a Fc effector function. In some embodiments, the IgGFc domain comprises a mutation at residue N297. In some embodiments, the mutation at residue N297 is selected from N297Q, N297A and N297G.

[0055] In one aspect, provided herein is an isolated polynucleotide molecule encoding the inhibitor of VEGF-C signaling or functional fragment thereof of the present disclosure or the fusion proteins of the present disclosure.

[0056] In some embodiments, the soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof disclosed herein, is fused to and / or conjugated to one or more heterologous moieties, such as, but not limited to, peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, sugars, etc.

[0057] In certain embodiments, the soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof disclosed herein, are fused to and / or conjugated to a moiety that specifically binds albumin including but not limited to, a small molecule, a peptide, a polypeptide, or a lipid that bind albumin. In some embodiments, the soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof disclosed herein are fused to and / or conjugated to an immunoglobulin constant region (an Fc domain), an scFv, an Fab, a single-domain antibody (VHH), an immunoglobulin, or a heavy or light chain thereof.

[0058] In certain embodiments, the soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof disclosed herein are fused to and / or conjugated to a polymer including, but not limited to, lipid polymers, polyethylene glycol (PEG), biodegradable polymers such as PLA (poly (lactic acid)) and PLGA (poly (lactic-glycolic acid)), polysaccharides, polysaccharides, poly (propylene glycol), polyoxy ethylated polyols, polyvinyl ethers, copolymers of ethylene glycol and propylene glycolpolyvinyl alcohols, dextran, hyaluronic acid, chitin, and the like. 20 168570138v1Attorney Docket No.251609.000097

[0059] In certain embodiments, the soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof disclosed herein are fused to and / or conjugated to an IGhG domain. In certain embodiments, the IGhG sequence to be fused or conjugated to the sR3 or a fragment or functional derivative thereof of the present invention may comprise a sequence that is at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the following polypeptide sequence: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5).

[0060] In certain embodiments, the soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof disclosed herein may comprise a sequence that is at least or at most 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the following polypeptide sequence: MQPGAALNLRLWLCLGLLQGLANGYSMTPPTLNITEDSYVIDTGDSLSISCRGQHPLEW TWPGAQEVLTTGGKDSEDTRVVHDCEGTEARPYCKVLLLAQTHANNTGSYHCYYKYI KARIEGTTAASTYVFVRDFKHPFINKPDTLLVNRKDSMWVPCLVSIPGLNITLRSQSSAL HPDGQEVLWDDRRGMRVPTQLLRDALYLQCETTWGDQNFLSNLFVVHITGNELYDIQL YPKKSMELLVGEKLVLNCTVWAEFDSGVTFDWDYPGKQAERAKWVPERRSQQTHTEL SSILTIHNVSQNDLGPYVCEANNGIQRFRESTEVIVHE (SEQ ID NO: 6).

[0061] In some embodiments, the soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof disclosed herein are fused to and / or conjugated to albumin or to an Fc domain or to an IGhG domain though a linker. Examples of linkers include, but not limited to, proline-rich linkers, acidic linkers, basic linkers, cleavable linkers or rigid linkers. Examples of proline-rich linkers include, but not limited to, (Pro-Pro-Gly)3 (SEQ ID NO: 7), (Pro-Gly)5 (SEQ ID NO: 8), or (Pro-Pro-Pro-Pro-Gly)3(SEQ ID NO: 9). Examples of acidic linkers include, but not limited to, (Glu-Ser-Glu-Ser)3 (SEQ ID NO: 10), (Asp-Glu)5 (SEQ ID NO: 11), or (Glu-Asp- Glu-Asp-Glu)3 (SEQ ID NO: 12). Examples of basic linkers include, but not limited to, (Lys- Ser-Lys-Ser)3 (SEQ ID NO: 13), (Arg-Lys-Arg-Lys)3(SEQ ID NO: 14), or (Lys-Arg-Lys-Arg- Lys)3(SEQ ID NO: 15). Examples of cleavable linkers include, but not limited to, (Gly-Gly- Ser)3(SEQ ID NO: 16), 21 168570138v1Attorney Docket No.251609.000097 which is cleavable by proteases such as trypsin, chymotrypsin, or thrombin, or (Leu-Val-Pro-Arg) (SEQ ID NO: 17), which is cleavable by the protease factor Xa. Examples of rigid linkers include, but not limited to, (Azido-Lys-Arg-Pro)m (SEQ ID NO: 18) which contains the non-natural amino acid azido-lysine, which can form a triazole linkage with an alkyne- containing molecule, or (Cyclohexyl-Ala-Pro-Pro) (SEQ ID NO: 19), which contains the non-natural amino acid cyclohexyl-alanine, which can form a rigid cyclohexane structure. Additional examples of useful linkers, but not limited to, includes a linker with the amino acid sequence: GGGGSGGGGS (SEQ ID NO: 20).

[0062] In some embodiments, the inhibitor of VEGF-C signaling is an antibody or a fragment or functional derivative thereof. Non-limiting examples of such antibodies are provided, e.g., in U.S. Patent 9,562,093, which is incorporated herein by reference in its entirety. In some embodiments, said antibody or antigen-binding fragment thereof is a humanized antibody or antigen binding fragment thereof, human antibody or antigen binding fragment thereof, murine antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monovalent Fab’, divalent Fab2, F(ab)’3 fragments, single-chain fragment variable (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, single heavy chain antibody, bispecific antibody or biding fragment thereof, bi-specific T-cell engager (BiTE), trispecific antibody, or chemically modified derivatives thereof.

[0063] In some embodiments, the inhibitor of VEGF-C signaling is a tyrosine kinase inhibitor. Non-limiting examples of such inhibitors include, e.g., Sunitinib, Axitinib, Sorafenib, Vatalanib, Cediranib, Motesanib (AMG706), Pazopanib, ZD6474 (Zactima), CEP-7055, GW7S6024, GW786034, Acalabrutinib, Adavosertib, Apatinib, Avapritinib, Cabozantinib, Canertinib, Crenolanib, Damnacanthal, Entospletinib, Enterectinib, Foretinib, Fostamatinib, Gliteritinib, Glesatinib, Ibrutinib, Icotinib, Linifanib, Icotinib, Linifanib, Masitinib, Mubritinib, Nontedanib, Peficitinib, Radotinib, Savolitinib, Sitravatinib, Tesevtinib, and Vatalanib.

[0064] In some embodiments, the inhibitor of VEGF-C signaling is an antisense oligonucleotide or RNA interference (RNAi) molecule (e.g., shRNA) or a guide RNA directed gene editing system (e.g., CRISPR / Cas9). 22 168570138v1Attorney Docket No.251609.000097 EXAMPLES

[0065] The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled. Materials and Methods Mice

[0066] Six-to-ten-week-old mixed sex C57BL / 6 mice, B6.Cg-Tg(TcraTcrb)425Cbn / J (OT-II), B6.129P2(C)-Ightm2Cgn / J (B1-8) and B6.12952-IghtmICgn / J (µMT) mice were purchased from Jackson Laboratory and Charles River and subsequently bred and housed at Yale University. PROX1CreERT2;CDH5 Dre;R26-STOP-mCherry and VEGFR3-CreERT2; R26-MTMG mice were gifts from the Thomas lab. All procedures used in this study (sex-matched, age-matched) complied with federal guidelines and the institutional policies of the Yale School of Medicine Animal Care and Use Committee. Cells

[0067] GL261-Luc cells were a gift from J. Zhou (Yale Neurosurgery) and were cultured in RPMI supplemented with 10% FBS, 1% penicillin / streptomycin and 1% Sodium Pyruvate. CT2A- BFP cells were a gift from T. Mathivet (Paris Centre de Recherche Cardiovasculaire). B16 cells were a gift from N. Palm (Yale Immunobiology). Cells were negative for mycoplasma contamination. Bacteria and virus

[0068] HSV-1 KOS strain, HSV-2 strains 186syn- TK- and 186syn+were generous gifts from D. Knipe (Harvard Medical School). These viruses were propagated and titrated on Vero cells (ATCC CCL-81) as previously described (Vero cells were negative for mycoplasma contamination) (Iijima, N. et al. Dendritic cells and B cells maximize mucosal Thl memory response to herpes simplex virus. J Exp Med 205, 3041-3052 (2008))43. 23 168570138v1Attorney Docket No.251609.000097

[0069] S. pneumoniae (ATCC 6303) was grown on 10% sheep blood agar plates (BD Biosciences) overnight at 37°C (5% CO2). These colonies were then transferred to Todd Hewitt broth and grown overnight. The number of bacteria were enumerated by OD600. sVEGFR3 production

[0070] sVEGFR3 was cloned into the pEZT_Dlux vector with an N-terminal H7 signal peptide and a C-terminal Avi-Tag and hexahistidine tag. Plasmids were transiently transfected into Expi293 cells using the Expifectamine 293 transfection kit (Thermo Fisher) according to the manufacturer’s instructions. After 3-5 days, expression supernatants were subjected to Ni-NTA chromatography purification and further purified by size-exclusion chromatography (SEC650 column, Bio-rad) into a final buffer of HEPES buffered saline (HBS; 10 mM HEPES, pH 7.5, 150 mM NaCl). For protein biotinylation, proteins were expressed with a C-terminal biotin acceptor tag (AviTag)-GLNDIFEAQKIEWHE (SEQ ID NO: 21). After Ni-NTA chromatography, protein biotinylation was carried out at room temperature for 2 h with soluble BirA ligase enzyme in 0.1 mM bicine (pH 8.3), 10 mM ATP, 10 mM magnesium acetate, and 0.5 mM biotin (Avidity, BIO500). Biotinylated proteins were then purified by SEC as described above. Biotinylation efficiency was assessed using an SDS-PAGE streptavidin-shift assay. An example of a soluble VEGFR-3 (sR3) produced herein was sR3 fused to an IGhG domain though a linker.

[0071] IGhG sequence: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5).

[0072] sVEGFR3: MQPGAALNLRLWLCLGLLQGLANGYSMTPPTLNITEDSYVIDTGDSLSISCRGQHPLEW TWPGAQEVLTTGGKDSEDTRVVHDCEGTEARPYCKVLLLAQTHANNTGSYHCYYKYI KARIEGTTAASTYVFVRDFKHPFINKPDTLLVNRKDSMWVPCLVSIPGLNITLRSQSSAL HPDGQEVLWDDRRGMRVPTQLLRDALYLQCETTWGDQNFLSNLFVVHITGNELYDIQL YPKKSMELLVGEKLVLNCTVWAEFDSGVTFDWDYPGKQAERAKWVPERRSQQTHTEL SSILTIHNVSQNDLGPYVCEANNGIQRFRESTEVIVHE (SEQ ID NO: 6).

[0073] Linker: GGGGSGGGGS (SEQ ID NO: 20). 24 168570138v1Attorney Docket No.251609.000097

[0074] Alternatively, AAV was produced by co-transfecting HEK-293FT cells with AAV plasmids of sVEGFR3 and AAV9 serotype plasmid and helper plasmid PDF6. Briefly, 5.2 μg of AAV-Vectorplasmid, 8.7 μg of plasmid AAV9 serotype, and 10.4 μg of pDF6 were mixed with PEI, at room temperature for 10–15 min before adding them drop-wise into HEK-293FT cells in a 150-mm dish at 80–90% confluency. Replicates collected from multiple dishes were pooled to enhance production yield. Cells were collected 72 h post-transfection. For AAV purification, chloroform (1:10 by volume) was added and the mixture was vigorously shaken for 1 h at 37 °C. NaCl was added to a final concentration of 1 M, and the mixture was shaken until dissolved and then pelleted at 20,000g and 4 °C for 15 min. The aqueous layer was discarded while the chloroform layer was transferred to another tube. PEG 8000 was added to 10% (w / v) and shaken until dissolved. The mixture was incubated at 4 °C for 1 h and then spun at 20,000g and 4 °C for 15 min. The supernatant was discarded and the pellet resuspended in Dulbecco’s phosphate- buffered saline + MgCl2, treated with Benzonase (Sigma) and incubated at 37 °C for 30 min. Chloroform (1:1 by volume) was then added, shaken and spun down at 12,000g and 4 °C for 15 min. The aqueous layer was isolated and passed through a 100kDa MWCO (Millipore). The concentrated solution was washed with PBS and the filtration process repeated. Genomic copy number (GC) of AAV was determined by real-time qPCR using custom Taqman assays (Thermo Fisher Scientific) targeted to the engineered U6 promoter. Optic nerve tissues

[0075] Tg(mrcla:eGFP) (Jung, H. M. et al. Development of the larval lymphatic system in zebrafish. Development 144, 2070-2081 (2017))44adult male and female 1 year-old zebrafish (N=3) were kindly provided by Dr. Weinstein at the NIH (Bethesda, MD). Fish were euthanized by an overdose of MS-222 and whole heads excised and fixed in 60mM HEPES buffered 4% PFA (pH 7.4) overnight at room temperature. Samples were then decalcified in 10% w / v EDTA solution (pH 7.4) for five days at room temperature. Heads were then snap frozen in OCT and 30 µm-thick cryosections obtained. Cryosections containing the optic nerve were stained with primary antibodies mouse zns-2 (ZIRC, ZDB-ATB-081002-34) (1:50) and chicken anti-GFP (Ayes Cat# GFP-1010) (1:500) followed by secondary antibodies AF488-conjugated goat anti-mouse IgG (Jackson Immuno Research 115-545-146) and Cy5-conjugated donkey anti-chicken IgY (Jackson Immuno 703-175-155 containing DAPI (5 µg / ml) for nuclear staining. Images were acquired in a Zeiss LSM. 25 168570138v1Attorney Docket No.251609.000097

[0076] Rabbits and pigs were euthanized due to unrelated health conditions and eyes were harvested post-mortem. Eyes were removed within an hour of death and immersion fixed in 10% neutral buffered formalin. Intact eyes from two female rhesus macaques (Macaca mulatta), between 25-28 years of age and euthanized due to unrelated health conditions were harvested post- mortem. Human optic nerves / chaisms were obtained through the Yale Pathology Tissue Services through the TPD (tissue procurement and distribution) service. A miniSOP was written and approved for these samples. Eyes were removed within an hour of death and immersion fixed in 10% neutral buffered formalin. Optic nerves with meningeal sheaths were cut and processed through the iDisco protocol below.

[0077] Mice optic nerve sheaths were dissected after mice were anesthetized and transcardially perfused with cold PBS and 4% PFA (Sigma-Aldrich) sequentially. Mice were first decapitated, and the skull was exposed by cutting off the skin and scalp. One midline cut down the skull and two transverse cuts on both sides of the skull were made. Tweezers were used to peel and remove both halves of the skull to expose the brain. The brain was lifted from the posterior, and vasculature connecting the brain to other parts of the head were cut until the brain could be lifted enough to expose the optic tract. Brain tissue was removed after cutting just anteriorly to the optic chiasm. The extracranial optic canal was unroofed by removing parts of the skull above the eyes to expose the intracanalicular and canal segments of the optic nerve. Tissue surrounding the optic nerve were carefully dissected to free the nerve, and the eye was cut off at the optic nerve head. After removing the optic nerve, the optic nerve sheaths was cut along the length of the nerve and removed for staining and whole mount imaging.

[0078] For PROX1CreERT2;CDH5 Dre;R26-STOP-mCherry and VEGFR3-CreERT2; R26- MTMG mice; the mice were intraperitoneally injected 100 ^l tamoxifen (10 mg / ml, Sigma- Aldrich, T5648) for five consecutive days and optic nerve sheaths were collected two days later as above. Then optic nerve sheaths were fixed in 1% PFA for one hour and immediately processed in a blocking solution (10% normal donkey serum, 1% bovine serum albumin, 0.3% PBS-Triton X-100) for 1h incubation at room temperature. For detection of lymphatic vessels, samples were incubated with primary antibodies overnight at 4 °C, then washed five times at room temperature in PBS with 0.5% Triton X -100, before incubation with a fluorescence-conjugated secondary antibodies diluted in PBS with 5% normal donkey serum. Lymphatic vessel images were acquired using Leica confocal microscope (Stellaris 8). The following antibodies were used: Goat anti- 26 168570138v1Attorney Docket No.251609.000097 mouse VEGFR3 (R&D, #AF743, 1:400), Rat-anti-mouse LYVE1 (R&D,#MAB2125, 1:400), Syrian Hamster anti mouse Podoplanin (BioLegend,127402, 1:500), Rabbit anti-Prox1 (Angiobio, 11-002P, 1:200), Armenian hamster anti- mouse CD31 (Gene Tex, 2H8, 1:1000). The primary antibodies were detected with appropriate AF-405, AF-488–, AF-555–, AF-647-conjugated secondary antibodies (Thermofisher / Invitrogen, 1:500) after 2 hours of incubation at room temperature. ProLong™ Gold Antifade Mountant (Invitrogen, P36930) was used for mounting the sections. Antibodies for Flow cytometry

[0079] Anti-CD3 (145-2C11, APC, 152306, 1;200), anti-CD4 (RM4-5, PerCP, 100538,1:400; RM4-5, BV605, 100548,1:400), Anti-CD8a (53-6.7, BV711, 100759, 1:400; 53-6.7, PE-Cy7, 100722,1:400), anti-CD11b (M1 / 70, BV711, 101242, 1:500), anti-CD19 (6D5, APC-Cy7, 115530, 1:400), anti-IA / IE (M5 / 114.15.2, AF488, 107616, 1:800), anti-CD44 (IM7, AF700, 103026, 1:200; BV421, 103040, 1:200), anti-CD45 (30-F11, APC-Cy7, 103116, 1:200), anti-CD45.1 (A20, BV785, 110743, 1:300), anti-CD45.2 (104, Pacific Blue, 109820, 1:200), anti-CD64 (X54-5 / 7.1, PE, 139304, 1:300), anti-CD95 (Jo2, PE-Cy7, 557653, 1:400), anti-B220 (Ra3-6B2, AF700, 103232, 1:400), anti- GL7 (GL7, FITC, 144603, 1:400), anti-NK1.1 (PK136, APC-Cy7, 108724, 1:400), anti- TCRβ (H57-597, APC-Cy7, 109220,1:200) were purchased from BD Biosciences or BioLegend. Anti-Igλ Light Chain, (JC5-1, FITC, 130-098-415, 1:400) was purchased from Miltenyibiotec.. Isolation of mononuclear cells

[0080] For brain tissues, tissues were collected and incubated in a digestion cocktail containing 1 mg m1-1collagenase D (Roche) and 30 µg m1-1DNase I (Sigma-Aldrich) in RPMI at 37 °C for 45 min. Tissues were pipetted to break tissue down and filtered through a 70-µm filter. Then, cells were mixed in 3 ml of 25% Percoll (Sigma-Aldrich) solution and centrifuged at 580g for 15 min without brake. The Percoll layer was removed and cell pellets were treated with 0.5m1 ACK buffer and spun for 5 minutes at 500g. Then the cell pellets were resuspended in FACS buffer (PBS +2% FBS+ 1mM EDTA) for staining.

[0081] When analyzing lymphocytes,, a LN or spleen was put in a 60-mm x15-mm petri dish containing 2 mL FACS buffer and was ground between 2 frosted microscope slides. When analyzing DCs, a LN or spleen was digested as above. Cell suspension was filtered through a 70- 27 168570138v1Attorney Docket No.251609.000097 µm filter and spun for 5 minutes at 500g. Then the cell pellets were resuspended in FACS buffer for staining. Flow cytometry

[0082] Preparation of single-cell suspensions from spleen, LNs and brains are described above. Nonspecific binding was blocked using a Fc receptor-blocking solution (TruStain FcXTM, 101320, BioLegend, 1:200) for 10 minutes at 4°C prior to immunostaining. Subsequently, the cells were stained with corresponding antibodies for 30 min at 4°C. Then, cells were washed to remove excess antibodies and resuspended in FACS buffer. Samples were run on an Attune NxT flow cytometer and then analyzed using FlowJo software (10.8.1, Tree Star). Enzyme-linked immunosorbent assay

[0083] CSF and serum were collected from mice as previously described9. Then, serum and CSF were diluted with PBS containing 0.1% BSA in a 1:1 ratio.96-well plates were coated with 100 µl of heat-inactivated or PFA-inactivated purified HSV-2 (104to 105pfu equivalent per 100 µl) for virus-specific immunoglobulin measurement or goat anti-mouse immunoglobulin (1:1,000; SouthernBiotech, 1010-01) and then incubated overnight at 4 °C. After that, these plates were washed with PBS-Tween 20 and blocked for 2 h with 5% FBS in PBS. Samples were then plated in the wells and incubated for at least four hours at ambient temperature. After washing in PBS- Tween 20, HRP-conjugated anti-mouse Ig antibodies (SouthernBiotech, 1010-05, 1:5000) was added in the wells for 1 h, followed by washing and adding TMB solution (eBioscience). Reactions were stopped with 1 N H2SO4and absorbance was measured at 450 nm. The total antibody titers were defined by using immunoglobulin standard (C57BL / 6 Mouse Immunoglobulin Panel; SouthernBiotech). Western blot

[0084] rAAV-RFP infected retinas or control retinas were digested with cocktail containing 1 mg ml−1collagenase D (Roche) and 30 μg ml−1DNase I (Sigma-Aldrich) in RPMI at 37 °C for 45 min. Tissues were pipetted to break tissue down and filtered through a 70-μm filter. Then, cells were lysed in RIPA buffer and boiled for 5 min with sample buffer. Western blotting was performed in a similar manner to that previously reported9. In brief, 15% gels were used and run at 10 mA per gel for 30 min and 40 mA per gel until separation of ladder. Wet transfer was performed at 120 mA per gel for 90 min on ice. RFP-Tag rabbit polyclonal antibodies (OriGene Technologies, CAT#: AP09229PU-N) were used at a concentration of 1:1,000 and incubated 28 168570138v1Attorney Docket No.251609.000097 overnight in the cold room. After washing, HRP-conjugated anti-Rabbit IgG secondary antibodies (Thermofisher, G-21234) were used at a concentration of 1:5,000 at room temperature for 2h and imaged using the ChemiDoc MP imaging system (Bio-Rad). ELISpot assay

[0085] Mice were injected with rAAV-RFP through IVT or AC routes. Their dCLNs, sCLNs and retinas were collected 10 days later. Single cell suspensions were prepared and co-cultured with splenocytes at a ratio of 1:5 with the presence of rAAV-RFP virus peptides (SNYNKSVNV (SEQ ID NO: 3) and NGRDSLVNPGPAMAS (SEQ ID NO: 4)). rAAV specific immune responses were quantified using an ELISpot assay, Mouse IFN-gamma ELISpot Kit (R&D, Catalog #: EL485) manufacturer instructions were followed for the assay. Flank tumor inoculation and treatment

[0086] Mice were anaesthetized using a mixture of ketamine (50 mg kg−1) and xylazine (5 mg kg−1), and the flank was shaved and disinfected. A 1 mL syringe with a 30G needle was used to deliver 100 μl of 500,000 B16 cells subcutaneously. Then, mice were treated with irradiated B16 cells (250,000 cells) through subcutaneous (SC), intracranial (IC), intracameral (AC), or intravitreal (IVT) administration routes (day 7) along with anti-PD-1 (RMP1-14) antibodies (days 7, 9 and 11) through the intraperitoneal (IP) route, and their survival was monitored. Adoptive transfer

[0087] To directly analyze the imsmune response (antigen-specific T and B cells) in dCLNs and sCLNs after intravitreal or anterior chamber injection, OT-II and B1-8 cells were transferred and their response was analyzed after different immunization routes.

[0088] To analyze antigen-specific B cell response, a previously reported method45was followed. In brief, CD45.2 C57BL / 6 recipient mice (6–8 weeks of age) were primed by intraperitoneal immunization with 50 mg of OVA (Sigma, A5503) precipitated in alum at a 2:1 ratio in PBS.2 weeks later, resting B cells were isolated from CD45.1.2 B1-8 mice with mouse B cell isolation kit (Stem cell, 19854). Then, the B cells were labelled with CellTrace™ Violet Cell Proliferation Kit (Thermofisher, C34557). 5 million cells were transferred intravenously into recipient mice.18h later, the mice were immunized with 10 ^g NP20–OVA (Biosearchtech, N- 5051) though intravitreal or anterior chamber injection. Igλ+B1-8hicell proliferation and germinal center formation in dCLNs and sCLNs were analyzed at day 7. 29 168570138v1Attorney Docket No.251609.000097

[0089] For antigen specific T cell response, OVA-specific CD4+T cells were isolated from CD45.1 OT-II mice with mouse CD4+T Cell Isolation Kit (Stem Cell, 19852). Then, the CD4+T cells were labelled using the CFSE Cell Proliferation Kit (ThermoFisher, C34554).5 million cells were transferred intravenously into CD45.2 recipient mice. 18h later, the mice were immunized with 2 ^l 50 ^g OVA (Sigma, A5503) plus 1 ^g Poly(I:C) (Invivogen, tlrl-picw) though IVT or AC injection.150 ^g FTY720 (Sigma, SML0700) was IP injected to inhibit the circulation of primed T cells 24h after immunization46. The OVA specific CD4+T cells proliferation in ingLNs, dCLNs and sCLNs were analyzed 72 h after immunization. Intravitreal, intracameral, intracranial and intracisterna-magna injection

[0090] Six to ten week old mice were anaesthetized through intraperitoneally injecting a mixture of ketamine (50 mg kg-1) and xylazine (5 mg kg-1). For IVT or AC injection, the eye was dilated with tropicamide ophthalmic solution. For IVT injection, a 30-gauge needle was used to puncture a hole at 1 mm posterior to the corneoscleral junction. A blunt-ended Hamilton syringe with one microliter of dye, HSV-1, HSV-2, SP, or irradiated tumor cells was inserted into the vitreous humor about 1 to 2 mm deep and administered at a rate of 1µl min-1. For AC injection, the hole was punctured close to the corneoscleral junction and a blunt-ended hamilton syringe was inserted into the anterior chamber about 1 to 2 mm deep. After IVT or AC injection, petrolatum ophthalmic ointment was applied on the eye to prevent cataract formation. Intracranial injection was similar as tumor inoculation, but 3 ^L of HSV-1, HSV-2, SP, or irradiated tumor cells were administered. Intracisterna-magna injection was performed as previously described9. The mice were kept on heating pads and continuously monitored until recovery after the injection. Virus and bacteria immunization and challenge

[0091] Mice were anaesthetized with a mixture of ketamine (50 mg kg-1) and xylazine (5 mg kg-1). For HSV immunization, 106heat-inactivated HSV-1 or HSV-2 were AC, IV, IP or IC injected.30 days later, these mice were intracranially challenged with 105HSV-1 or HSV-2 and their survival were monitored. For some experiments, CD4+T cell depleting antibody (clone GK1.5, BioXCell, #BE0003-1, 200µg for three days) was injected before rechallenge. Similar experiments were done with streptococcus pneumonia, the immunization dose was 104of heat inactivated bacteria and the challenge dose was 104of live bacteria. 30 168570138v1Attorney Docket No.251609.000097 Imaging and quantification of tracer transport

[0092] For the quantification of fluorescent intensity in the eye or LNs, dextran conjugated to either fluorescein isohiocynate (40kDa and 70kDa) or tetramethylrhodamine (40kDa and 70kDa) was injected into the anterior chamber or vitreous humor through AC or IVT injection, respectively. To analyze the kinetics of dye drainage from the eye, the eye was harvested at a serial time point and was homogenized in 150 µl PBS with beads beating (Lysing Matrix D, 116913500, MP Biomedicals). Then homogenized tissue was centrifuged at 10000g for 10min and 100 µl supernatant was collect into 96-well plate and fluorescent intensity was read with emission / excitation wavelength of 494 / 514 or 555 / 585 nm. To measure the dye drainage into LNs, sCLN and dCLN were collected 12h after dye injection. The fluorescent intensity was measured as above.

[0093] For measuring nanoparticle draining in the serum, a previously published protocol17was utilized. Briefly, serum from mice were isolated and serum was placed onto microscope slides to allow for small volume high sensitivity detection of tracer transport in the blood.

[0094] For IVIS imaging of tracer transport, the eye and sCLN and dCLN were harvested after dye injection through the route of AC and IVT. They were imaged using the IVIS Spectrum In Vivo Imaging System (PerkinElmer).

[0095] For IVT tracer transport in vivo, 0.2 ^l AF647-OVA (2mg / ml, Thermofisher, O34784) was IVT injected into the vitreous humor. 100ul sodium fluorescein (1 mg / ml, Santa Cruz, sc- 206026) was injected into blood to label blood vessel. The kinetics of AF647-OVA drainage was tracked with The Phoenix MICRON® IV imaging microscope.

[0096] For analyzing the colocalization of optic nerve sheath lymphatics with IVT tracer, 1ug anti-mouse LYVE1 antibody (R&D, MAB2125) was IVT injected into the vitreous humor. Optic nerve sheaths were harvested 2h later and stained as above.

[0097] For tracking the kinetics of dye draining in the eye, 1μl AF647-OVA (AF647-OVA (2mg / ml, Thermofisher, O34784) was IVT injected into the vitreous humor. The eyes were enucleated at the indicated time points and processed in a similar manner to that previously reported47. In brief, eyes were fixed in Hartman’s fixation buffer, and three windows on the eye were created using the previously described window technique47. Serial 10 ^m sections was obtained using a cryostat (Leica CM190) after 30% sucrose gradients. The sections were 31 168570138v1Attorney Docket No.251609.000097 dehydrated and mounted with ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, P36931) and imaged with a Leica confocal microscope (Stellaris 8). CSF collection

[0098] For CSF collection, mice were anaesthetized through intraperitoneally injecting a mixture of ketamine (50 mg kg-1) and xylazine (5 mg kg-1). The dorsal neck was shaved and sterilized. A 1 cm incision was made at the base of the skull, and the dorsal neck muscles were separated using forceps to expose the cisterna magna. A custom-pulled micropipette (0.75 / 11brl GF; Stoelting) was used to penetrate the dura to collect CSF. Brain tumor inoculation and IVIS imaging

[0099] Tumor inoculation was performed as previously described with slight modifications9. Mice were anaesthetized through IP injection using a mixture of ketamine (50 mg kg-1) and xylazine (5 mg kg-1). Mice heads were shaved and scalp were sterilized. A midline scalp incision was made and a burr hole was drilled 2 mm lateral to the sagittal suture and 0.5 mm posterior to the bregma with a 25-gauge needle. Then mice were placed in a stereotaxic frame. A 10-µl Hamilton syringe loaded with 3µl of GL261-Luc cells (105cells) was inserted into the burr hole at a depth of 4 mm from the surface of the brain and left to equilibrate for 1 min before infusion. A micro-infusion pump (World Precision Instruments) was used to infuse at 1µl min-1. The syringe was left in place for another minute after the infusion was finished. The skin was stapled and cleaned. Following intramuscular administration of analgesic (meloxicam and buprenorphine, 1 mg kg-1), mice were placed in a heated cage until full recovery. Tumor size was tracked weekly thorough IVIS imaging. Mice were anaesthetized using isoflurane and injected intraperitoneally with D-Luciferin Potassium Salt Bioluminescent Substrate (PerkinElmer, 122799, 200 μl, 30 mg ml−1). After 10 min, mice were imaged using the IVIS Spectrum In Vivo Imaging System (PerkinElmer). Analyze CT2A-BFP tumor antigen drainage tissue preparation

[0100] Intracranial CT2A-BFP tumors were analyzed 14 days after injection. Tumors, meninges, IngLNs, dCLNs, and sCLNs were harvested. Mononuclear cells were isolated and stained. Parabiosis

[0101] Parabiosis was performed as previously described with slight modifications3. Naive or IVT immunized C57BL / 6 mice with similar age and weight were anaesthetized with a mixture of 32 168570138v1Attorney Docket No.251609.000097 ketamine / xylazine (50 mg / kg and 5 mg / kg body weight, respectively). After shaving the corresponding lateral aspects of each mouse, skin was cleaned and sterilized with alcohol prep pad and Betadine surgical scrub. Matching skin incisions were made from just above the knee upwards to the olecranon and two mice were sutured together with Ethicon 5-0 coated Vicryl Suture absorbable suture. Then the skin was stapled and Neosporin + Pain Relief ointment with pain relief was applied on the incisions. During the surgery, mice were kept on heating pads and continuously monitored until recovery. Ligation of superficial or deep cervical lymph nodes

[0102] For ligation of lymph nodes, mice were anaesthetized with a mixture of ketamine / xylazine (50 mg / kg and 5 mg / kg body weight, respectively) and the rostral neck was shaved and sterilized. A 2-cm incision was made and the sCLN and dCLN were exposed, sequentially. Their afferent lymph vessels were cauterized or keep intact based on the experiment conditions. Then, the incision was closed with a 5-0 Vicryl suture and mice were subjected to the same postoperative procedures as above. rAAV transduction and imaging

[0103] WT mice were intravitreally injected with AAV (3x1011viral genomes) with PBS, VEGF-C (1µg) or sVEGFR3 (1µg). One to three months later, these mice were rechallenged with rAAV-RFP (3x1011viral genomes). Eyes were either imaged on the Phoenix Micron IV, or the eyes were harvested 3 weeks after rAAV-RFP transduction and fixed with 1% PFA overnight at 4 °C. The retina whole-mount was carefully dissected and image with a Leica confocal microscope (Stellaris 8). 3D imaging of solvent-cleared organs

[0104] iDISCO was performed as previously described (iDISCO+, http: / / www.idisco.info)22. The following antibodies were used: goat anti-mouse VEGFR3 (#AF743, R&D, 1:400), rat anti- mouse LYVE1 (R&D, MAB2125,1:400), rabbit anti-mouse LYVE1 (#11-034, AngioBio sCo,1:200), mouse anti-human VEGFR3 (SC-28297, Santa Cruz Biotechnology, 1:200), rabbit anti Human LYVE-1 (102-PA50S, Angio-proteomie, 1:200), goat anti-mouse IgG-AF647 (A21235, Invitrogen, 1:500), donkey anti-goat IgG- AF647 (A21447, Invitrogen, 1:500), goat anti- rabbit IgG-AF555 (Invitrogen, A21428, 1:500). Subsequently, the transparent optic nerves were imaged using a Leica confocal microscope (Stellaris 8). Three-dimensional (3D) rendering was completed using Imaris 8 software (Oxford Instruments). 33 168570138v1Attorney Docket No.251609.000097 Spatial Transcriptomics

[0105] The 10X Visium Spatial Gene Expression for FFPE slide (PN-1000185) and associated protocols (CG000483) from 10X Genomics were used. Mouse eyeballs were fixed47, processed and sectioned48as previously described.5 μm-in-thickness of eyeball transverse sections were cut using microtome (RM2255, Leica Biosystems) and carefully placed within the fiducial frame on the Visium slide, then sections were air dried at room temperature overnight and stored in a desiccant container before spatial transcriptomics experiment. The FFPE sections were baked, stained with H&E, and then imaged using a Keyence bz-x800 all-in-one fluorescence microscope. Then, cell permeabilization and library preparation was performed following the Visium Spatial Gene Expression FFPE User Guide using supplied reagents (10X Genomics). The generated libraries were sequenced and analyzed using Space Ranger (version 2.1.0), and data were analyzed using Seurat 4.9.9.9040. Image processing and analysis

[0106] Quantitative analysis of AAV-infected cells were performed using either FIJI or ImageJ image-processing software (NIH or Bethesda). Statistical analysis

[0107] All statistical analysis was performed using GraphPad Prism software. Data were analyzed with a two-tailed unpaired Student’s t-test or paired Student’s t-test with Prism software. Statistical significance is defined as *P < 0.05, **P < 0.01, and ***P < 0.001. Example 1. The eye is an immunological window to the brain

[0108] Herpes virus has the propensity to affect many tissues-among them are multiple neural tissues such as the dorsal root ganglia, the eyes, and the brain-responsible for being the most common cause of sporadic fatal encephalitis world-wide2. To understand protective immunity against HSV in the brain, mice were vaccinated with heat-inactivated HSV-2 through four administration routes—intraperitoneal (IP) to induce systemic immunity, intracranial (IC) to induce local brain immunity, intracameral (anterior chamber, AC) and intravitreal (posterior chamber, IVT), the latter two of which are ocular compartments that have unique anatomical connections to the nervous system (Fig.1A). After a lethal intracranial HSV-2 challenge, all mice that underwent IP immunization succumbed to the infection, while IC immunization protected ~80% of the mice (Fig. 1B), highlighting that systemic immunity is not sufficient in protecting against CNS infection. Remarkably, IVT immunization also protected the mice, but all AC- 34 168570138v1Attorney Docket No.251609.000097 immunized mice, like IP-immunized mice, succumbed to the challenge (Fig.1B), suggesting that IVT immunization drives a protective immune response in the brain.

[0109] Like other organs, it was hypothesized herein that the immune response in the eye- brain axis must be mediated by local lymph nodes. Towards investigating the cellular mechanism in protection of the brain after IVT immunization, lymphatic vessels of the deep cervical lymph nodes (dCLN) were first surgically ligated, which are critical in invoking immune responses in the brain. This completely abrogated the protection provided by IVT immunization, indicating the importance of a local CNS-lymph node circuit that drains antigen to the dCLN (Fig. 1C). Additionally, by ligating the lymphatic vasculature after priming but before the rechallenge, the present results indicated that the lymphatic vessels of dCLNs were required to mount a recall response during the challenge (Fig. 6G). The effector functions after HSV immunization are thought to be mediated by antiviral antibodies and CD4+T cells3. Consistent with these reports, it was found herein that CD4+T depleted or B cell deficient (µMT) mice were no longer protected after IVT immunization (Fig. 6A). Since efficient T follicular helper (Tfh) and germinal center (GC) B cell differentiation is required for the production of long-lived plasma cells and memory B cells that drive a superior humoral immune response upon re-exposure to an antigen4,5, antigen- specific GC B cell differentiation was quantified by adoptive transfer of B1-8hiB cells6after immunizing mice with NP-OVA through AC or IVT routes. No significant differences in Tfh cells and only small increase of GC B cells were observed in the spleen of animals, but significant increases in these cells were found in the dCLN after IC and IVT immunization (Fig.5 & Fig.6B- 6D). Although both immunization routes can efficiently induce GC B cell differentiation in superficial CLNs (sCLNs), only IVT immunization induces GC B cell differentiation in dCLNs (Fig. 6I-6K). Similarly, IVT immunization significantly increases antigen-specific CD4+T cell proliferation in dCLNs compared to AC immunization (Fig. 6L-6N). Moreover, IC and IVT immunization resulted in greater accumulation of B cells and CD4+T cells in the brain compared to IP and AC immunization (Fig.6E). These data indicate that the generation of localized CD4+T and B cell responses in the deep cervical draining lymph node by IVT immunization is required host anti-viral CNS protection.

[0110] To further address whether the IVT immunization-mediated protection was developed locally in the brain, parabiotic mice pairs were generated with various immunization states (Fig. 1D). Because of the shared blood circulation, naïve mice that paired with IVT immunized mice 35 168570138v1Attorney Docket No.251609.000097 had similar amount of anti-HSV-2 antibodies in serum (Fig.6F). Despite similar antibody levels in the serum, only the mice that were initially immunized (left) and not their parabiotic pairs (right) were protected against the intracranial challenge (Fig. 1E). Similarly, only the IVT-immunized mice, but not paired naïve mice, had detectable levels of anti-HSV-2 antibodies in cerebrospinal fluid (CSF), which indicated that direct immunization is necessary to acquire anti-HSV-2 antibodies in CSF (Fig. 6H). On the other hand, because of the shared blood circulation, naïve mice that paired with IVT-immunized mice had similar amount of anti-HSV-2 antibodies in serum (Fig. 6H). Therefore, the localization of anti-HSV antibodies was examined after different immunization routes and found that although all routes of immunization led to presence of anti- HSV-2 antibodies in serum, only the IC- and IVT-immunized mice had detectable levels of anti- HSV-2 antibodies in cerebrospinal fluid (CSF) (Fig.1F). This suggests that serum antibodies do not mediate immune protection in the CNS, and rather, immune protection occurs locally via the CSF. Together, these data demonstrate that IVT immunization mediates CNS protection through a local antibody-dependent response.

[0111] Finally, to see if this ocular-brain axis mediated CNS protection extends beyond HSV- 2, the efficacy of a similar vaccination strategy was examined against HSV-1 and Streptococcus pneumoniae, the main causative agent of bacterial meningitis7,8. Mice were immunized with heat- inactivated HSV-1 or S. pneumoniae through either IP, IC, AC or IVT route and rechallenged with a lethal dose to the same pathogen 30 days later. Similar to the results in the HSV-2 model, both IVT- and IC-immunized mice showed almost complete protection. In contrast, all the naïve, IP- and AC-immunized mice succumbed to the challenge (Fig. 1G, Fig. 1H). Even in a therapeutic setting where an intracranial tumor was first established before vaccination with a cancer cell vaccine, both IVT and IC immunization showed superior protection compared to subcutaneous (SC) and AC immunization routes that only evoke systemic immunity (Fig.1I, Fig.10A). These data show that IVT and IC immunization resulted in significantly greater proportions and numbers of antigen-specific CD8+T cells compared to AC and SC immunization in dCLNs, but not in sCLNs or inguinal LNs (IngLNs)9(Fig.10B-10D). In contrast, in a model of cutaneous melanoma, SC immunization showed better protection compared to IVT and IC immunization (Fig. 10E), indicating that IVT and IC immunization induce a localized immune response but do not protect against peripheral tumors as well as a local immunization method. Together, these data 36 168570138v1Attorney Docket No.251609.000097 demonstrate that the ocular-brain immunological connection is specifically relevant and broadly applicable to many CNS diseases. Example 2. The eye has a compartmentalized drainage system

[0112] The present immunization studies uncovered an interesting divergent immune response between the anterior chamber and vitreous of the eyes. It is thought that aqueous humor flows out from the anterior chamber through the conventional outflow pathway that includes the Schlemm’s canal and trabecular meshwork10-12. Additionally, a more recently appreciated unconventional route includes flow via the ciliary muscle, supraciliary and suprachoroidal spaces13,14. In contrast, the drainage from the posterior ocular compartment, particularly the vitreous, is unclear given the lack of traditional lymphatic vasculature in the eye and is still being explored15,16.

[0113] To investigate the different drainage systems between the anterior and posterior compartments of the eye, fluorescently labeled dextrans was injected via AC or IVT routes to quantify the kinetics of dextran retention (Fig.2A). The rate of dextran clearance in the eye after IVT administration (K=1.369) was significantly slower than after AC administration (K=4.267) (Fig.2B). Dextrans with two different fluorescence and molecular weights were tested to ensure that this phenomenon was not dye specific. It was hypothesized herein that this observation could be explained by two possibilities; either the compartments have unique routes of drainage or the rate-limiting step in the drainage of each compartment is different (i.e., diffusion through the vitreous may be the slowest step). It was reasoned herein that these potential explanations could be parsed out by observing dye localization in vivo. Similar to other organ systems, the dye injected in the compartments of the eye accumulated in local lymph nodes. Using a previously described method17, it was confirmed that both AC- and IVT-injected dye could be detected in the serum. However, significantly higher concentrations of dye were detected in the blood after AC injection compared to after IVT injection (Fig.11A). Interestingly, while AC injected dye localized to the ipsilateral superficial cervical lymph node (sCLN), as previously reported18-21, IVT injected dye localized to the dCLN in addition to the sCLN (Figs. 2C-2I, Fig. 11B). Similar to after AC injection, dye does not drain to the dCLNs after subconjunctival injection (Fig.11C), indicating that penetration into the posterior eye / vitreous is required for drainage to the dCLNs. To test the requirement of these local lymphatic structures for drainage of the aqueous and vitreous humor, the sCLN, dCLN or both cervical lymph nodes were surgically ligated. It was first confirmed that ligation of dCLNs did not result in increased efflux into the sCLNs and vice versa (Extended Data 37 168570138v1Attorney Docket No.251609.000097 Fig.11D and 11E). Consistent with the above observation, sCLN ligation, but not dCLN, resulted in significantly increased dye retention in the eye following AC injection (Fig. 2G). For IVT injection, both sCLN and dCLN ligation alone led to increased dye retention in the eye (Fig.2H). Altogether, these results demonstrate a compartmentalized drainage from the anterior chamber and the vitreous humor of the eye, with a specific route from the vitreous humor to the dCLN. Example 3. Optic nerve sheath harbors functional meningeal lymphatic vessels

[0114] The present results of macromolecule localization into distinct lymph nodes indicate that a lymphatic vessel network is likely mediating drainage from each of these compartments. Although a recent study described the presence of an ocular glymphatic system in mice, similar to that of the brain15, a functional network of lymphatic vessels mediating vitreous drainage has not been resolved. Therefore, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO)22whole-optic nerve clearing was used to obtain a global view of the lymphatic vascular network associated with the optic nerve in different vertebrate species. Spatial transcriptomics helped gather an organ-level view of lymphatic-like vasculature gene signatures in the eye and the optic nerve. In addition to the trabecular meshwork10-12and retina, which have been found to harbor macrophages expressing some lymphatic-like gene signatures23, it was found herein that the optic nerve sheath also contained lymphatic-like gene signatures (Fig. 12). In parallel, iDISCO revealed lymph vessels covering zebrafish, mouse, rabbit, pig, non-human primate and human optic nerves (Figs. 3A-3C, Fig. 7), demonstrating conservation across the evolution of these lymphatic vessels. In all mammals, vascular structures with colocalized staining of LYVE-1 and VEGFR-3 (white arrows in Fig. 7) were observed. In some cases, vasculature stained with one but not the other was observed, indicating the likely presence of LYVE1-negative collecting lymphatic vasculature24or LYVE1-expressing macrophages (light gray arrows in Fig. 7). Notably, the lymphatic vasculature was localized to membranes surrounding the nerve, not in the nerve itself, in parallel to what is observed in the brain and the dural meninge25,26.

[0115] To improve sensitivity of the stains that were performed, the optic nerve sheath was dissected completely out from the optic nerve. Without perfusion fixation of mice, the optic nerve sheath retracted upon enucleation, resulting in bunching of the lymphatic vasculature towards the proximal end of the nerve (Fig. 7I). To address this, perfusion fixation prior to enucleation was performed, preserving the anatomy of the lymphatic network along the nerve (Fig.7H). Following IVT injection, but not AC injection, the tracer was visualized in VEGFR-3-positive vasculature 38 168570138v1Attorney Docket No.251609.000097 along the optic nerve (Fig. 3C). Using the well-characterized ear lymphatic system as a control (Fig. 13A), the presence of optic nerve sheath lymphatics was validated by using additional markers: VEGFR3, PROX1, LYVE1, CD31 and Podoplanin (Fig. 3C and Fig. 13B). To further characterize these vasculatures using a genetic approach, two different reporter mice were utilized enabling highly specific staining of lymphatics: VEGFR3-CreERT2;R26-MTMG mice and PROX1CreERT2;CDH5 Dre;R26-STOP-mCherry mice (Figs. 14A-14D). Altogether, these parallel methods confirmed that lymphatics indeed exist in the optic nerve sheath.

[0116] It was then sought to evaluate the functionality of these lymphatics. Following IVT injection, but not AC injection, of a fluorescent tracer, the tracer was visualized in VEGFR-3- positive vasculature along the optic nerve. Consistently, IVT-injected LYVE-1 antibodies could effectively label optic nerve sheath lymphatics (Fig.15A, 15B), suggesting that macromolecules like proteins can freely penetrate into these lymphatics from the vitreous. The leading hypothesis of how molecules travel into these lymphatics was based on an understanding of the dural lymphatics of the brain25,26. It was hypothesized herein that molecules from the eye pass through the CSF into the optic nerve sheath lymphatics, similar to how molecules move from the brain to the dural lymphatics. To test this, the distribution of dye and antibodies after IVT injection was examined. A trace amount of dye was detected in the CSF after IVT injection compared to after brain intraventricular injection (Fig.15C). Furthermore, antibodies injected intravitreally were not able to sufficiently label the dural lymphatics. These data indicated that IVT-injected dye likely entered the local CSF space around the optic nerve and did not drain to the dCLNs through conventional brain dural meninges (Fig. 15D). To parse out how macromolecules reach the lymphatic vasculature around the optic nerve sheath, it was tracked how molecules injected into the vitreous left the eye in vivo. After IVT AF647-OVA injection, it was observed that there was directional flow towards the optic nerve head (Fig. 15E), showing that the pressure gradient between intraocular and intracranial spaces drives efflux of ocular fluid through the optic nerve. Examination at a higher resolution after sectioning the eye revealed that the tracer was largely concentrated at the optic nerve head (Fig. 15F), with some diffusion through the retinas. The diffusion through the retina showed a pattern similar to the previously described glymphatic patterns of the retinas, a paracellular perpendicular streak across the retina (Fig.15G)15.

[0117] To validate the ability of these lymphatics to drain the vitreous, VEGF-C, a lymphatic stimulator27,28, was delivered through three different compartmentalized routes: AC (targets 39 168570138v1Attorney Docket No.251609.000097 lymphatics of anterior eye), IVT (targets posterior eye, optic nerve and possibly anterior eye), or intracisterna magna injection (ICM; targets optic nerve) (Fig.3F). First, using fluorescent tracers, it was confirmed that ICM-injection only has access to the optic nerve and not the eye15,29(Figs. 3D, 3E). Two days after VEGF-C delivery, fluorescent-dextran was injected intravitreally and retention was analyzed 12h later. AC administration of VEGF-C did not change dextran drainage from the vitreous, indicating that the anterior lymphatic stimulation is not sufficient in driving vitreous drainage. In contrast, IVT and ICM administration of VEGF-C increased dextran drainage from the vitreous, the latter in a dose dependent manner (Fig.3G), supporting the observation that the posterior compartment of the eye drains to the dCLN through lymphatic vasculature on the optic nerve sheath. To further confirm the role of VEGF-C in modulating optic nerve sheath lymphatics, rAAV-VEGF-C or sVEGFR-3 (sR3) was also delivered through the IVT route and found that expression of VEGF-C or sVEGFR-3 resulted in significant increases or decreases in optic nerve sheath lymphatic vasculature, respectively (Fig. 16A). In summary, these data highlight the existence of a functional lymphatic vessel network on the optic nerve sheath that connects the posterior eye to a unique set of lymph nodes different from the rest of the eye and shared with the rest of the CNS. Example 4. Harnessing ocular lymphatics to boost gene therapy efficacy

[0118] Having confirmed the immunological significance of this posterior ocular drainage, the potential therapeutic applications that harness this finding was investigated next. Recombinant AAVs (rAAV) are the most common in vivo gene delivery vectors due to their safe and effective transduction30. Additionally, using AAV2 as a vector to treat patients with RPE65-mediated inherited retinal dystrophy was the first gene therapy approved by FDA31. However, despite its modified immunological properties, acquired immunity against rAAV limits the efficacy of its repeated administration32, 33. The rAAV-specific CD8+T cell induction after AC and IVT administration in lymph nodes and the retinas was characterized by utilizing an ELISpot assay. Although both AC and IVT injection induced a similar amount of rAAV-specific CD8+T cells in the sCLNs, IVT injection specifically induced more rAAV-specific CD8+T cells in the dCLNs and the retinas, which were not observed after the AC injection (Figs. 4F and 4G). It was hypothesized herein that lymphatic drainage on the optic nerve contributes to immunological response to rAAV, especially for retina-based therapeutic approaches, thereby preventing its utility. It was examined herein whether ablation of ocular lymphatic vessels could enable 40 168570138v1Attorney Docket No.251609.000097 prolonged therapeutic efficacy after repeated rAAV injection into the eye. To test this, the primary dose of rAAV was administered first and 1-3 months later a second dose of RFP-expressing rAAV (AAV-RFP) was administered. As a baseline control, naïve mice were given AAV-RFP at the time of the second dose (Fig. 4A). Consistent with the observation regarding local CNS immunity, AAV-RFP expression in the secondary infection was significantly lower than after primary infection (Figs.4B, 4C). The importance of ocular lymphatic vessels was confirmed by performing a similar experiment with sCLN and dCLN ligation, which demonstrated that only dCLN ligation was sufficient in mitigating the immune response against rAAV (Figs. 4B, 4C) and recovering secondary rAAV-RFP transduction efficiency. Additionally, it was confirmed that the ligation procedure did not affect the primary challenge (Fig.16A). Because surgically ligating the lymph nodes would not be a therapeutically viable option for treatment, an alternative method to inhibit the molecular activity of lymphatics was utilized: IVT administration of sR3, which was found to inhibit lymphatic drainage to a similar extent as surgical ligation (Fig.16C). It was observed herein that immune response against rAAV was further exacerbated by the presence of VEGF-C, which is known to increase immune responses by increasing lymphatic drainage9,28. Conversely, inhibiting VEGF-C signaling during the primary rAAV transduction using a soluble VEGFR-3 (sR3) allowed for the second dose to be just as effective as the primary infection (Figs.4D, Fig.8, 17A-17B). These experiments establish the role of posterior ocular lymphatics in modulating the efficacy of AAV-based gene therapies and suggest a platform to overcome current barriers limiting repeated gene therapy. Discussion

[0119] It was demonstrated herein that the posterior compartment of the eye has a unique lymphatic drainage system that anatomically engages with the CNS meningeal lymphatic network at the deep cervical lymph nodes. Using spatial transcriptomics, multiple imaging techniques, genetic manipulation in vivo, and tracer-injection experiments, the present inventors mapped a large lymphatic vasculature network in the optic nerve sheath and described its role in draining the vitreous to the deep cervical lymph nodes. These results are in accordance with the nexus between the eye and CNS’ and further extend this concept by demonstrating a shared immune surveillance mechanism between the two sites (Fig.9).

[0120] Previously, both the eye and brain, which are devoid of traditional lymphatic vessels, were considered immune-privileged organs because foreign tissue grafts can survive after 41 168570138v1Attorney Docket No.251609.000097 implantation36,3718,38-40. The present study revealed that antigens delivered to the posterior eye through IVT vaccination can drain to dCLN and initiate a protective immune response in the brain. IVT vaccination conferred immunologic protection to the brain following IVT vaccination by directing the recruitment of local CD4+T cells, B cells, and antibodies to the CNS compartment.

[0121] Why this compartmentalization has evolved proposes an interesting question. One possibility is that the centralization of the CNS and the emergence of lymphatic networks in vertebrates drove the polarization of the two lymphatic drainages in the eye. A clue to this may be in the lateralization that was observed during drainage to the sCLNs but not dCLNs, which is likely a consequence of the anatomical separation of skin, muscle and tissue surrounding the orbit. Contrarily, bilateral drainage to the dCLNs from the posterior eye occurs through a network of CSF draining lymphatics. The anterior lymphatics acts in synchrony with the cornea / conjunctiva, a generalized mucosal surface with constant exposure to outside invaders that may never have access to the brain. The optic nerve lymphatics described herein connect the posterior eye (retina) with the brain, allowing coordinated immune responses that may prepare the brain for imminent threats. The segregation of eye immune responses into anterior and posterior lymphatic networks may predate mammals and future studies in other vertebrates may help illuminate when and why this innovation first emerged. Although the use of different disease models and therapeutic interventions in the present studies show the universality of the findings of the present disclosure, it is likely that different pathogens or diseases may skew towards specific arms of the adaptive immune response. Of note, in experiments by the inventors of the present disclosure it is not that the sCLNs lack antigens from the CNS; rather drainage into the sCLNs is not sufficient to mount an immune response in the CNS. One possibility is that sCLNs and dCLNs may differ in their ability to generate effective brain-homing T cells or brain-resident T cells41,42.

[0122] The present findings imply that immune tolerance to foreign antigens in the posterior eye and brain is driven by active immune suppression, both passive and active, and not lack of an immune response. Thus, the posterior drainage system of the eye may play a pivotal role in modulating immune activation and suppression for various diseases involving the CNS. Current conventional methods to treat or diagnose diseases of the CNS either are invasive or fail to initiate local immunity. In cases with suspected increased mass effect in the CNS due to infection or malignancy, it can often be dangerous to access the CSF through methods such as a lumbar 42 168570138v1Attorney Docket No.251609.000097 puncture. The present studies propose the vitreous as a relatively non-invasive and more accessible method to invoke CNS immunity.

[0123] These concepts were utilized to illustrate that disruption of the posterior lymphatic vessels can negate immune response to rAAV delivered to the posterior eye and thus restore therapeutic benefit. Contrary to the notion of immune privilege, AAV therapy for ocular diseases faces significant hurdles due to the cellular and humoral immune response that it initiates33. The present study illustrates that disruption of the posterior lymphatic vessels can negate immune response to AAV delivered to the posterior eye and thus restore therapeutic benefit. These findings further confirm that this optic nerve drainage system identified in the present study is responsible for the immunological outcome in response to antigens in the vitreous humor. The results also signify that the posterior lymphatic drainage system of the eye is a versatile and accessible target for manipulating immune responses in many different contexts.

[0124] Altogether, the present study establishes that the connection between the eye and the brain is not only a neuronal one but also a shared immune circuit that coordinates immunity from the periphery to the CNS. 43 168570138v1Attorney Docket No.251609.000097 References

[0125] 1. London, A., Benhar, I. & Schwartz, M. The retina as a window to the brain—from eye research to CNS disorders. Nature Reviews Neurology 9, 44-53, doi:10.1038 / nrneuro1.2012.227 (2013).

[0126] 2. Marcocci, M. E. et al. Herpes Simplex Virus-1 in the Brain: The Dark Side of a Sneaky Infection. Trends in Microbiology 28, 808-820, doi:10.1016 / j.tim.2020.03.003 (2020).

[0127] 3. Iijima, N. & Iwasaki, A. Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help. Nature 533, 552-556, doi:10.1038 / nature 17979 (2016).

[0128] 4. Victora, G. D. & Nussenzweig, M. C. Germinal Centers. Annu Rev Immunol 40, 413442, doi:10.1146 / annurev-immuno1-120419-022408 (2022).

[0129] 5. Cyster, J. G. & Allen, C. D. C. B Cell Responses: Cell Interaction Dynamics and Decisions. Cell 177, 524-540, doi:10.1016 / j.ce11.2019.03.016 (2019).

[0130] 6. Shih, T.-A. Y., Roederer, M. & Nussenzweig, M. C. Role of antigen receptor affinity in T cell–independent antibody responses in vivo. Nat Immunol 3, 399-406, doi:10.1038 / ni776 (2002).

[0131] 7. Koedel, U., Scheld, W. M. & Pfister, H.-W. Pathogenesis and pathophysiology of pneumococcal meningitis. The Lancet Infectious Diseases 2, 721-736, doi:10.1016 / S1473- 3099(02)00450-4 (2002).

[0132] 8. Iovino, F., Seinen, J., Henriques-Normark, B. & van Dijl, J. M. How Does Streptococcus pneumoniae Invade the Brain? Trends in Microbiology 24, 307-315, doi:10.1016 / j.tim.2015.12.012 (2016).

[0133] 9. Song, E. et al. VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours. Nature 577, 689-694, doi:10.1038 / s41586-019-1912-x (2020).

[0134] 10. Thomson, B. R. et al. A lymphatic defect causes ocular hypertension and glaucoma in mice. The Journal of clinical investigation 124, 4320-4324, doi:10.1172 / JCI77162 (2014).

[0135] 11. Park, D.-Y. et al. Lymphatic regulator PROX1 determines Schlemm’s canal integrity and identity. The Journal of clinical investigation 124, 3960-3974, doi:10.1172 / JCI75392 (2014).

[0136] 12. Aspelund, A. et al. The Schlemm’s canal is a VEGF-C / VEGFR-3–responsive lymphatic-like vessel. The Journal of clinical investigation 124, 3975-3986, doi:10.1172 / JCI75395 (2014). 44 168570138v1Attorney Docket No.251609.000097

[0137] 13. Goel, M., Picciani, R. G., Lee, R. K. & Bhattacharya, S. K. Aqueous humor dynamics: a review. Open Ophthalmol J 4, 52-59, doi:10.2174 / 1874364101004010052 (2010).

[0138] 14. Johnson, M., McLaren, J. W. & Overby, D. R. Unconventional aqueous humor outflow: A review. Experimental Eye Research 158, 94-111, doi:https: / / doi.org / 10.1016 / j.exer.2016.01.017 (2017).

[0139] 15. Wang, X. et al. An ocular glymphatic clearance system removes beta-amyloid from the rodent eye. Science translational medicine 12, doi:10.1126 / scitranslmed.aaw3210 (2020).

[0140] 16. Kasi, A., Liu, C., Faiq, M. A. & Chan, K. C. Glymphatic imaging and modulation of the optic nerve. Neural Regen Res 17, 937-947, doi:10.4103 / 1673-5374.324829 (2022).

[0141] 17. Tietjen, G. T., DiRito, J., Pober, J. S. & Saltzman, W. M. Quantitative microscopy- based measurements of circulating nanoparticle concentration using microliter blood volumes. Nanomedicine 13, 1863-1867, doi:10.1016 / j.nano.2017.04.003 (2017).

[0142] 18. Camelo, S., Kezic, J., Shanley, A., Rigby, P. & McMenamin, P. G. Antigen from the Anterior Chamber of the Eye Travels in a Soluble Form to Secondary Lymphoid Organs via Lymphatic and Vascular Routes. Investigative Ophthalmology & Visual Science 47, 1039-1046, doi:10.1167 / iovs.05-1041 (2006).

[0143] 19. Tam, A. L. C., Gupta, N., Zhang, Z. & Yücel, Y. H. Quantum dots trace lymphatic drainage from the mouse eye. Nanotechnology 22, 425101, doi:10.1088 / 0957-4484 / 22 / 42 / 425101 (2011).

[0144] 20. Lee, J. Y. et al. Structural Confirmation of Lymphatic Outflow from Subconjunctival Blebs of Live Humans. Ophthalmology Science 1, 100080, doi:https: / / doi.org / 10.1016 / j.xops.2021.100080 (2021).

[0145] 21. Yücel, Y. H. et al. Active Lymphatic Drainage From the Eye Measured by Noninvasive Photoacoustic Imaging of Near-Infrared Nanoparticles. Investigative Ophthalmology & Visual Science 59, 2699-2707, doi:10.1167 / iovs.17-22850 (2018).

[0146] 22. Renier, N. et al. iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell 159, 896-910, doi:https: / / doi.org / 10.1016 / j.cell.2014.10.010 (2014).

[0147] 23. Xu, H., Chen, M., Reid, D. M. & Forrester, J. V. LYVE-1–Positive Macrophages Are Present in Normal Murine Eyes. Investigative Ophthalmology & Visual Science 48, 2162- 2171, doi:10.1167 / iovs.06-0783 (2007). 45 168570138v1Attorney Docket No.251609.000097

[0148] 24. Makinen, T. et al. PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev 19, 397-410, doi:10.1101 / gad.330105 (2005).

[0149] 25. Louveau, A. et al. Structural and functional features of central nervous system lymphatic vessels. Nature 523, 337-341, doi:10.1038 / nature14432 (2015).

[0150] 26. Aspelund, A. et al. A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med 212, 991-999, doi:10.1084 / jem.20142290 (2015).

[0151] 27. Breslin, J. W. et al. Vascular endothelial growth factor-C stimulates the lymphatic pump by a VEGF receptor-3-dependent mechanism. American Journal of Physiology-Heart and Circulatory Physiology 293, H709-H718, doi:10.1152 / ajpheart.00102.2007 (2007).

[0152] 28. Jeltsch, M. et al. Hyperplasia of Lymphatic Vessels in VEGF-C Transgenic Mice. Science 276, 1423-1425, doi:10.1126 / science.276.5317.1423 (1997).

[0153] 29. Mathieu, E. et al. Evidence for Cerebrospinal Fluid Entry Into the Optic Nerve via a Glymphatic Pathway. Investigative Ophthalmology & Visual Science 58, 4784-4791, doi:10.1167 / iovs.17-22290 (2017).

[0154] 30. Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov 18, 358-378, doi:10.1038 / s41573-019-0012-9 (2019).

[0155] 31. Russell, S. et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. The Lancet 390, 849-860, doi:https: / / doi.org / 10.1016 / S0140-6736(17)31868-8 (2017).

[0156] 32. Colella, P., Ronzitti, G. & Mingozzi, F. Emerging Issues in AAV-Mediated In Vivo Gene Therapy. Mol Ther Methods Clin Dev 8, 87-104, doi:10.1016 / j.omtm.2017.11.007 (2018).

[0157] 33. Mingozzi, F. & High, K. A. Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122, 23-36, doi:10.1182 / blood-2013-01-306647 (2013).

[0158] 34. Killer, H. E., Laeng, H. R. & Groscurth, P. Lymphatic capillaries in the meninges of the human optic nerve. J Neuroophthalmol 19, 222-228 (1999).

[0159] 35. Sherman, D. D. et al. Identification of orbital lymphatics: enzyme histochemical light microscopic and electron microscopic studies. Ophthalmic Plast Reconstr Surg 9, 153-169 (1993).

[0160] 36. Streilein, J. W. Ocular immune privilege: therapeutic opportunities from an experiment of nature. Nat Rev Immunol 3, 879-889, doi:10.1038 / nri1224 (2003). 46 168570138v1Attorney Docket No.251609.000097

[0161] 37. Medawar, P. B. Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br J Exp Pathol 29, 58-69 (1948).

[0162] 38. Camelo, S., Shanley, A., Voon, A. S. P. & McMenamin, P. G. The Distribution of Antigen in Lymphoid Tissues following Its Injection into the Anterior Chamber of the Rat Eye. The Journal of Immunology 172, 5388, doi:10.4049 / jimmunol.172.9.5388 (2004).

[0163] 39. Boonman, Z. F. H. M. et al. Intraocular Tumor Antigen Drains Specifically to Submandibular Lymph Nodes, Resulting in an Abortive Cytotoxic T Cell Reaction. The Journal of Immunology 172, 1567, doi:10.4049 / jimmunol.172.3.1567 (2004).

[0164] 40. Sonoda, K.-H. et al. The analysis of systemic tolerance elicited by antigen inoculation into the vitreous cavity: vitreous cavity-associated immune deviation. Immunology 116, 390-399, doi:https: / / doi.org / 10.1111 / j.1365-2567.2005.02239.x (2005).

[0165] 41. Ayasoufi, K. et al. Brain resident memory T cells rapidly expand and initiate neuroinflammatory responses following CNS viral infection. Brain, Behavior, and Immunity 112, 51-76, doi:https: / / doi.org / 10.1016 / j.bbi.2023.05.009 (2023).

[0166] 42. Steinbach, K. et al. Brain-resident memory T cells represent an autonomous cytotoxic barrier to viral infection. J Exp Med 213, 1571-1587, doi:10.1084 / jem.20151916 (2016).

[0167] 43. Iijima, N. et al. Dendritic cells and B cells maximize mucosal Th1 memory response to herpes simplex virus. J Exp Med 205, 3041-3052, doi:10.1084 / jem.20082039 (2008).

[0168] 44. Jung, H. M. et al. Development of the larval lymphatic system in zebrafish. Development 144, 2070-2081, doi:10.1242 / dev.145755 (2017).

[0169] 45. Gitlin, A. D., Shulman, Z. & Nussenzweig, M. C. Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 509, 637-640, doi:10.1038 / nature13300 (2014).

[0170] 46. Ciabattini, A. et al. Distribution of Primed T Cells and Antigen-Loaded Antigen Presenting Cells Following Intranasal Immunization in Mice. PloS one 6, e19346, doi:10.1371 / journal.pone.0019346 (2011).

[0171] 47. Pang, J. et al. Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing. STAR Protocols 2, 100879, doi:https: / / doi.org / 10.1016 / j.xpro.2021.100879 (2021). 47 168570138v1Attorney Docket No.251609.000097

[0172] 48. Pang, J. et al. A freeze-substitution approach with solvent-based glyoxal fixative to prevent distortion of ocular structures. Journal of Histotechnology 45, 172-181, doi:10.1080 / 01478885.2022.2111744 (2022). * * *

[0173] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. It is further to be understood that all values are approximate, and are provided for description.

[0174] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes. 48 168570138v1

Claims

Attorney Docket No.251609.000097 CLAIMS 1. A method for inhibiting an immune response to an antigen in a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of VEGF-C signaling.

2. The method of claim 1, wherein the inhibitor of VEGF-C signaling is administered prior to or simultaneously with the administration of the antigen.

3. The method of claim 1 or claim 2, wherein (i) the antigen is administered intracranially or intravitreally, and (ii) the inhibitor of VEGF-C signaling is administered intravitreally, intracranially or via an intra-cisterna magna injection.

4. The method of claim 3, wherein the inhibitor of VEGF-C signaling is administered to the posterior eye.

5. The method of claim 3, wherein the inhibitor of VEGF-C signaling is administered subretinally or suprachoroidally.

6. The method of any one of claims 1-5, wherein the antigen is administered to the subject repeatedly and the inhibitor of VEGF-C signaling is administered starting from the first or second administration of the antigen and prior to or simultaneously with each subsequent administration of the antigen.

7. The method of any one of claims 1-6, wherein the antigen is a molecule, vector, or particle.

8. The method of claim 7, wherein the antigen is a vector.

9. The method of claim 8, wherein the vector is a viral vector.

10. The method of claim 9, wherein the viral vector is an adeno-associated virus (AAV) vector, adenovirus vector, rhabdovirus vector, herpesvirus vector, or retrovirus vector. 49 168570138v1Attorney Docket No.251609.000097 11. The method of claim 10, wherein the retrovirus vector is a lentivirus vector.

12. The method of claim 7, wherein the antigen is a particle.

13. The method of claim 12, wherein the particle is a virus-like particle (VLP), polymer nanoparticle, lipid nanoparticle (LNP), metallic nanoparticle, or liposome.

14. The method of claim 7, wherein the antigen is a molecule.

15. The method of claim 14, wherein the molecule comprises polyethylene glycol (PEG).

16. The method of claim 14, wherein the molecule is a dendrimer.

17. The method of any one of claims 1-16, wherein the inhibitor of VEGF-C signaling inhibits VEGF-C signaling via VEGFR-3.

18. The method of any one of claims 1-17, wherein the inhibitor of VEGF-C signaling is a soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof, wherein said fragment or functional derivative of sR3 is capable of binding to VEGF-C.

19. The method of any one of claims 1-16, wherein the inhibitor of VEGF-C signaling is an antibody targeting VEGF-C or VEGFR-3, or a fragment or functional derivative thereof.

20. The method of claim 19, wherein the antibody or the fragment or functional derivative thereof is a single-chain fragment variable (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

21. The method of any one of claims 1-16, wherein the inhibitor of VEGF-C signaling is a tyrosine kinase inhibitor.

22. A method for increasing efficacy of a gene therapy in a subject in need thereof, wherein said gene therapy involves a repeated administration of a gene therapy vector, comprising administering an effective amount of an inhibitor of VEGF-C signaling. 50 168570138v1Attorney Docket No.251609.000097 23. The method of claim 22, wherein the inhibitor of VEGF-C signaling is administered prior to or simultaneously with the administration of the gene therapy vector.

24. The method of claim 22 or claim 23, wherein (i) the gene therapy vector is administered intracranially or intravitreally, and (ii) the inhibitor of VEGF-C signaling is administered intravitreally.

25. The method of claim 24, wherein the inhibitor of VEGF-C signaling is administered to the posterior eye.

26. The method of claim 24, wherein the inhibitor of VEGF-C signaling is administered subretinally or suprachoroidally.

27. The method of any one of claims 22-26, wherein the gene therapy vector is administered to the subject repeatedly and the inhibitor of VEGF-C signaling is administered starting from the first or second administration of the gene therapy vector and prior to or simultaneously with each subsequent administration of the gene therapy vector.

28. The method of any one of claims 22-27, wherein the gene therapy vector is a viral vector.

29. The method of claim 28, wherein the viral vector is an adeno-associated virus (AAV) vector, adenovirus vector, rhabdovirus vector, herpesvirus vector, or retrovirus vector.

30. The method of claim 29, wherein the retrovirus vector is a lentivirus vector.

31. The method of any one of claims 22-30, wherein the inhibitor of VEGF-C signaling inhibits VEGF-C signaling via VEGFR-3.

32. The method of any one of claims 22-31, wherein the inhibitor of VEGF-C signaling is a soluble VEGFR-3 (sR3) or a fragment or functional derivative thereof, wherein said fragment or functional derivative of sR3 is capable of binding to VEGF-C. 51 168570138v1Attorney Docket No.251609.000097 33. The method of any one of claims 22-31, wherein the inhibitor of VEGF-C signaling is an antibody or a fragment or functional derivative thereof.

34. The method of claim 33, wherein the antibody or the fragment or functional derivative thereof is a single-chain fragment variable (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody.

35. The method of any one of claims 22-34, wherein the inhibitor of VEGF-C signaling is a tyrosine kinase inhibitor.

36. A method for generating a protective immune response to an antigen in the central nervous system (CNS) of a subject in need thereof, comprising administering an effective amount of said antigen to the subject by intravitreal administration.

37. The method of claim 36, wherein the antigen is administered to the posterior eye of the subject.

38. A method for preventing and / or treating a central nervous system (CNS) disease in a subject in need thereof, comprising administering to the subject through intravitreal administration a disease-associated antigen, wherein said antigen is administered in an amount effective to induce a protective immune response to said antigen in the CNS of the subject.

39. The method of claim 38, wherein the disease is an intracranial disease and the antigen is administered in an amount effective to induce a protective immune response to said antigen in the brain of the subject.

40. The method of claim 38 or claim 39, wherein the antigen is administered to the posterior eye of the subject.

41. The method of any one of claim 38-40, wherein the disease is an infection, cancer, or autoimmune disease. 52 168570138v1Attorney Docket No.251609.000097 42. The method of claim 41, wherein the disease is an infection.

43. The method of claim 42, wherein the infection is a bacterial infection, viral infection, fungal infection, or parasitic infection.

44. The method of claim 41, wherein the disease is a cancer.

45. The method of claim 44, wherein the cancer is a CNS cancer.

46. The method of claim 45, wherein the CNS cancer is a brain cancer.

47. The method of claim 45, wherein the CNS cancer is selected from astrocytomas, ependymomas, glioblastomas, oligoastrocytomas, oligodendrogliomas, lower grade gliomas, meningomas, pituitary adenomas, craniopharyngiomas, schwannoma, choroid plexus tumors, Osteoma, Chondroma, Giant cell tumors, and brain metastatses of non-CNS cancers.

48. The method of claim 41, wherein the disease is an autoimmune disease.

49. The method of claim 48, wherein the autoimmune disease is multiple sclerosis, anti-myelin oligodendrocyte glycoprotein (MOG) disease, neuromyelitis optica (NMO), or autoimmune encephalitis.

50. The method of any one of claims 35-49, wherein the antigen is a viral antigen, bacterial antigen, fungal antigen, parasitic antigen, self-antigen, or tumor-associated antigen.

51. The method of claim 50, wherein the antigen is a viral antigen.

52. The method of claim 50, wherein the antigen is a bacterial antigen.

53. The method of claim 50, wherein the antigen is a tumor-associated antigen. 53 168570138v1