Methods and materials for delivering vectors to localized areas within a mammal

Abstract

This document provides methods and materials for delivering vectors (e.g., non-viral vectors or viral vectors such as adeno-associated viral (AAV) vectors) to localized areas within a mammal (e.g., a human). For example, a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) can be administered directly to the retina of an eye within a mammal (e.g., a human) to deliver the vectors to the retina.

Description

[0001] METHODS AND MATERIALS FOR DELIVERING VECTORS TO LOCALIZED AREAS WITHIN A MAMMAL

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 560,269, filed on March 1, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0004] TECHNICAL FIELD

[0005] This document relates to methods and materials for delivering vectors (e.g., non-viral vectors or viral vectors such as adeno-associated viral (AAV) vectors) to localized areas within a mammal (e.g., a human). For example, a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) can be administered directly to the retina of an eye within a mammal (e.g., a human) to deliver the vectors to the retina.

[0006] BACKGROUND

[0007] Many ocular gene therapies, including voretigene neparvovec-rzyl (LUXTURNA" ) and others in clinical trials, require injection of a therapeutic solution into the subretinal space. This technique creates a localized retinal detachment, often with detachment of the fovea, in eyes with retinal degeneration. Multiple studies have also demonstrated negative effects of subretinal bleb formation such as chorioretinal atrophy, inflammation, and macular holes. See, e.g., Gange et al., Ophthalmol Retina, 6(1): p. 58-64 (2022); Bommakanti et al., Ophthalmol Retina, 8(1 ):42-48 (2024); Kolesnikova et al., Mol. Genet. Genomic Med ., 10(1 l):e2038 (2022); Lorenz et al., Ophthalmology, 131(2): 161-178 (2024); and Stingl et al., Ophthalmology, 130(7): p. 764-770 (2023).

[0008] Similarly, intraocular inflammation (uveitis) has been the primary adverse side effect associated with intravitreal injection of gene therapy in clinical trials. In four independent clinical trials for LCA due to ND4 variants, uveitis was reported in 42 of 84 eyes treated across 4 independent studies (Britten-Jones et al., Genet. Med., 24(3): 521 -534 (2022)). Intravitreal injection also fails to confine the vector regionally within the eye, allowing vector to transduce cells outside of the target zone, and possibly enter the systemic circulation. This also results in significant dilution of vector as it diffuses throughout the vitreous cavity and anterior chamber (Yu-Wai-Man et al., Sci. TransL Med., 12:573 (2020)). It has also been suggested that inner limiting membrane may pose a barrier to the diffusion of viral vectors through the neurosensory retina further limiting the ability of an intravitreally delivered vector from finding its target cell (Cukras et al., Mol. Ther., 26(9): 2282-2294 (2018)).

[0009] SUMMARY

[0010] This document provides methods and materials for delivering vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) to localized areas within the mammal (e g., a human). For example, this document provides fibrin hydrogels (e.g., injection molded fibrin hydrogels) including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) and methods for using such compositions to deliver the vectors to localized areas within a mammal (e.g., a human). In some cases, fibrin hydrogels including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) can be administered to a retina of an eye within a mammal to deliver at least some the vectors to the retina. For example, fibrin hydrogels including viral vectors (e.g., AAV vectors) that contain nucleic acid that can encode a polypeptide (e.g., a therapeutic polypeptide) can be administered to a retina of an eye within a mammal to deliver at least some of the viral vectors to the retina such that the viral vectors can transduce at least some retinal cells and the transduced cells can express the polypeptide. As demonstrated herein, fibrin hydrogels can include high concentrations of AAV vectors while maintaining the infectivity (e.g., the ability to transduce a cell) of the AAV vectors. Also as demonstrated herein, AAV vectors that contain nucleic acid that can encode a polypeptide (e.g., a therapeutic polypeptide) that are included in fibrin hydrogels provided herein can diffuse out of the fibrin hydrogel and / or can be released from the fibrin hydrogel as the fibrin hydrogel degrades and can transduce cells such that the transduced cells can express the polypeptide.

[0011] Having the ability to deliver one or more vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) to localized areas within a mammal (e.g., a human) as described herein (e.g., by administering a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) provides a unique and unrealized opportunity to use lower doses of vectors (e.g., lower doses of viral vectors such as AAV vectors). For example, the methods and materials provided herein can allow slow release of vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) from a fibrin hydrogel provided herein to localized areas within a mammal (e.g., a human) and / or can allow confinement of the vectors to the localized areas.

[0012] In general, one aspect of this document features compositions including fibrin hydrogel and from about 108vector genomes to about 1014vector genomes of viral vector per mb of the fibrin hydrogel, where at least 98 percent of the viral vector is diffusible from the fibrin hydrogel and can transduce cells. The viral vector can be an adeno-associated viral vector, a lentivirus-based vector, a herpesvirus-based vector, or an adenovirus-based vector. The viral vector can be an adeno-associated viral vector. The viral vector can include nucleic acid encoding a polypeptide heterologous to the viral vector. The fibrin hydrogel can include from about 10 mg / mLto about 80 mg / mL of the fibrinogen polypeptide. The fibrin hydrogel can include from about 0.1 U / mL of a solution to about 1200 U / mL of the solution of the thrombin polypeptide. The solution is a sodium citrate solution. The fibrin hydrogel can include an agent that can slow polymerization of the gelation mixture. The agent that can slow polymerization of the gelation mixture can be trypan blue, Evans blue, VisionBlue®, Direct Blue 53, Azovan blue, compounds having the structure listed under PubChem Compound ID number (CID) 6296, compounds having the structure listed under PubChem CID 9409, Direct Blue 2, melantherine BH, pontamine sky blue 5B, Azo Fuchsine, or Acid Red 99. The fibrin hydrogel can include an agent that can slow degradation of the fibrin hydrogel. The agent that can slow degradation of the fibrin hydrogel can be tranexamic acid, aprotinin, aminocaproic acid, or nafamostat.

[0013] In another aspect, this document features methods for delivering a viral vector to a retina of an eye within a mammal. The methods can include, or consist essentially of, administering a composition to a retina of an eye within a mammal where the composition includes fibrin hydrogel and from about 108vector genomes to about 1014vector genomes of viral vector per mL of the fibrin hydrogel, where at least 98 percent of the viral vector is diffusible from the fibrin hydrogel and can transduce cells. The viral vector can be an adeno- associated viral vector, a lentivirus-based vector, a herpesvirus-based vector, or an adenovirus-based vector. The viral vector can be an adeno-associated viral vector. The viral vector can include nucleic acid encoding a polypeptide heterologous to the viral vector. The fibrin hydrogel can include from about 10 mg / mL to about 80 mg / mL of the fibrinogen polypeptide. The fibrin hydrogel can include from about 0.1 U / mL of a solution to about 1200 U / mL of the solution of the thrombin polypeptide. The solution is a sodium citrate solution. The fibrin hydrogel can include an agent that can slow polymerization of the gelation mixture. The agent that can slow polymerization of the gelation mixture can be trypan blue, Evans blue, VisionBlue®, Direct Blue 53, Azovan blue, compounds having the structure listed under PubChem Compound ID number (CID) 6296, compounds having the structure listed under PubChem CID 9409, Direct Blue 2, melantherine BH, pontamine sky blue 5B, Azo Fuchsine, or Acid Red 99. The fibrin hydrogel can include an agent that can slow degradation of the fibrin hydrogel. The agent that can slow degradation of the fibrin hydrogel can be tranexamic acid, aprotinin, aminocaproic acid, or nafamostat. The mammal can be a human. The composition can be in the form of a fibrin hydrogel patch. The administering can include placement of the fibrin hydrogel patch onto a retinal surface of the retina. The retinal surface is an epiretinal surface. The administering can include placing a plurflourocarbon (PFO) liquid on the the fibrin hydrogel. In some cases, serum or plasma can be placed on the PFO liquid. The liquid in the vitreous cavity can be removed and replaced with a substance that the viral vector cannot diffuse through. The substance can be an air bubble, a silicon oil, or an expansile gas. At least 98 percent of the viral vector can diffuse out of the fibrin hydrogel within 5 days following the administering.

[0014] In another aspect, this document features methods for treating a mammal having an ocular disease. The methods can include, or consist essentially of, administering a composition to a retina of an eye within a mammal having an ocular disease, where the composition includes fibrin hydrogel and about 108vector genomes to about 1014vector genomes per mL of the fibrin hydrogel, where at least 98 percent of the viral vector is diffusible from the fibrin hydrogel and can transduce cells within the retina, and where the viral vector can include nucleic acid encoding a polypeptide heterologous to the viral vector. The mammal can be a human. The composition can be in the form of a fibrin hydrogel patch. The administering can include placement of the fibrin hydrogel patch onto a retinal surface of the retina. The retinal surface is an epiretinal surface. The administering can include placing a PFO liquid on the fibrin hydrogel. In some cases, serum or plasma can be placed on the PFO liquid. The liquid in the vitreous cavity can be removed and replaced with a substance that the viral vector cannot diffuse through. The substance can be an air bubble, a silicon oil, or an expansile gas. At least about 98 percent of the viral vector diffuses out of the fibrin hydrogel and transduces retinal cells within the retina, and where the transduced cells express the polypeptide. The ocular disease can be retinitis pigmentosa, choroideremia, X-linked retinoschisis, age-related macular degeneration, diabetic retinopathy, glaucoma, achromatopsia, Leber congenital amaurosis, or Batten disease. The polypeptide can be a RPE65 polypeptide, a RSI polypeptide, a MERTK polypeptide, a CERKL polypeptide, a CLN3 polypeptide, a BESTl polypeptide, or an anti-VEGF antibody.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

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

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1. Comparison of plasmin mediated degradation of modified (squares) vs off the shelf TISSEEL (circles) formulation. Data are mean+SE (p<0.05, N=4 experiments). Modified gels were completely degraded at 24 hours. Hydrogels produced using TISSEEL as supplied according to the manufacturer’ s instructions were not completely degraded even after 48 hours. Figure 2. Comparison of rate of plasmin mediated degradation using modified gels with AAV (circles) and without AAV (squares). No significant difference was noted in the rate of degradation when AAV was present in the gel.

[0019] Figures 3A-3F. Injection molded fibrin hydrogel. Figure 3 A) Components of an exemplary mold include 2 stainless steel clips, a stainless-steel bottom plate with a silicon gasket, and a slide with 200 pm deep cavity in which the gel was cast. Figure 3B) An exemplary assembled mold with a side view (b) of the assembled mold. Figure 3C) A freshly molded gel in a slide. Figure 3D) A gel blank from which various shaped punches have been removed. Figure 3E) Free punches of the fibrin hydrogel of Figure 3D. Figure 3F) Free punches of the fibrin hydrogel of Figure 3D showing the variety of sizes and shapes that were obtained.

[0020] Figures 4A-4F. AAV containing fibrin hydrogel produced using an injection molding method. Figure 4A) Immunofluorescence staining of fibrin. Figure 4B) Immunofluorescence staining of AAV. Figure 4C) A merged image of Figure 4A and Figure 4B. Figure 4D) Optical coherence tomography demonstrated that the gels were -200 pm thick and homogenous. Figure 4E) Transmission electron microscopy showed that the gel was composed of a dense arrays of fibrin microfilaments. Figure 4F) At higher magnification AAV particles were observed.

[0021] Figures 5A-5B. Diffusion of encapsulated AAV vectors from fibrin gels. Figure 5A) 3mm diameter fibrin gels containing 6 x 107viral capsids were placed in wells of a 96-well plate in PBS at 37°C and sampled at various time points. AAV in the media at the time points was assayed by ELISA. -50% of capsids were detected in the media within 48 to 96 hours. Figure 5B) AAV remaining in the gel was assayed by ELISA following degradation with Plasmin and accounted for all capsid not found in the medium.

[0022] Figure 6. Transduction of ARPE-19 with AAV encapsulated in fibrin. A 6 x 107dose of AAV-GFP or AAV-GFP in a 3mm diameter fibrin gel was incubated with ARPE-19 cells for up to 1 month. Although transduction was greater using free AAV-GFP at 2 weeks, the number of transduced cells was similar at 1 month. Note gel was still present after 1 month **p<0.01 compared to 1 hour. N=10 per timepoint in Figures 5A and 5B. Figures 7A-7F. Surgical placement of fibrin encapsulated AAV hydrogels on the epiretinal surface. Figure 7A) 25-gauge valved cannulas were placed for 3-port lens-sparing vitrectomy. Figure 7B) Following vitrectomy, a 4 mm scleral incision was fashioned with MVR blade to level of choroid, which was then cauterized using argon laser. Figure 7C) A sclerotomy was performed using a keratome knife, and the inserter loaded with an oval AAV-fibrin implant was inserted through the sclerotomy. Figure 7D) The inserter tip was placed under PFO (heavy liquid), and the implant was deployed for epiretinal placement. Figure 7E) Autologous serum was injected under the PFO and around the implant to glue it in place. Figure 7F) After 15 minutes, a soft tip cannula was used to exchange the heavy liquid for air and then SF6 gas. Note, the implant remains in place throughout the process.

[0023] Figures 8A-8D. Fibrin encapsulated AAV hydrogels degrade by post-operative day 5. Figure 8A) Intra-operatively, the AAV-fibrin implant (asterisk) is noted to be flat on the epiretinal surface near the optic nerve at the completion of the case. Figure 8B) At postoperative day 5, dilated fundus examination with indirect ophthalmoscopy confirms degradation of the hydrogel with no implant visualized at the original implant site (dotted oval & asterisk). Note magnification difference between intra- and post-operative images. Figure 8C) Minimal residual fibrin hydrogel is noted on the retina surface near the implant site, consistent with degradation of the implant at post-operative day 5. The small star provides orientation across Figures 8A-8C. Figure 8D) At post-operative month 1, the view is clear with no inflammation, no cataract, and no evidence of choroidal or retinal abnormalities, including atrophy and vasculitis.

[0024] Figures 9A-9E. RPE transduction detected one month after epiretinal placement of fibrin encapsulated AAV hydrogels. Figure 9A) The enucleated surgical right eye demonstrates strong GFP signal throughout the RPE layer when the retina is removed one month after epiretinal fibrin- AAV implant placement. Figure 9B) Diffuse and uniform distribution of GFP positivity is seen throughout the posterior pole of the mounted retina of the same eye as in (Figure 9A). Figure 9C) Histological analysis of the retina in A&B reveals strong GFP signal, consistent with RPE transduction by AAV. DAPI is used for nuclear staining. Figure 9D) Using FITC fluorescence, the fellow (control) eye has no GFP signal, as expected. Figure 9E) Histology confirms the absence of GFP signal in the left eye, which received no implant / gene therapy.

[0025] Figures 10A-10N. CRA and inconsistent RPE transduction 1 month after subretinal delivery of AAV2-eGFP in pigs. Figure 10A) Intra-operative photograph shows cannula delivering AAV2 solution (bleb, arrowheads) into the subretinal space. Figure 10B) OCT through the area of subretinal injection demonstrates choriocapillaris loss (asterisk) and subretinal deposits with RPE loss (arrow). Figures 10C-10F) OCT-A en face images are shown of superficial (Figure IOC), deep (Figure 10D), outer retina (Figure 10E), and choriocapillaris (Figure 10F) vascular layers. Dotted lines outline CRA extent (right of line) within bleb. Figures 10G and 10H) OCT-A images of two different pigs demonstrate normal choriocapillaris flow outside bleb and minimal to no choriocapillaris flow within CRA area. Figure 101) Near-infrared confocal SLO imaging demonstrates increased reflectance correlating to atrophy and decreased reflectance correlating to deposits within bleb (right of line). Figures 10J and 10K) Fundus photographs of two pigs with significant CRA within bleb region (Figure 10K, arrowheads). Figures 10L and 10M) H&E of retina through area of CRA within bleb. There is RPE vacuolation (Figure 10L, black arrowhead), hyper- and hypopigmentation (Figure 10M, white arrow), and occasional RPE hypertrophy (Figure 10M, black arrow). Figure ION) GFP signal using IHC reveals inconsistent RPE transduction by PJWl-eGFP (white arrows) and transduction of photoreceptors (black arrows) within bleb.

[0026] Figures 11 A-l 1G. Fibrin hydrogel characterization by OCT and mechanical strength testing. Figures HA and 1 IB) Addition of Trypan Blue slows fibrin polymerization facilitating injection molding of large surface area gels with high concentration fibrin. The mold is 28.58 x 78.36 mm (Figure 11 A) and serves as a blank from which a punch can produce smaller configured blanks or “punches” of various dimensions (Figure 1 IB). Figure 11C) OCT demonstrates a gel with thickness of 187.1 pm. Figures 1 ID and 1 IE) Average ± SEM gel thickness of three different unpunched gels was determined using OCT imaging at the 66 designated points shown in Figure 1 IE. Figure 1 IF) Thickness of an unpunched gel determined by OCT at each of the tested 66 points; Length and width correlate to the positions tested, shown in Figure 1 IE. Figure 11G) Gels made with or without aprotinin were tested for mechanical strength. Box plots represent 25thto 75thpercentiles with vertical bars providing range, horizontal bars represent median values, and crosses represent mean value (n= 5 without aprotinin, n=rl with aprotinin).

[0027] Figures 12A-12H. Characterization of fibrin-encapsulated AAV gels. TEM (Figures 12A and 12B) and SEM (Figures 12C and 12D) show dense arrays of fibrin microfilaments without AAV (Figures 12A and 12C) and with AAV particles (Figures 12B and 12D), which measure 25-31 nm. Figure 12E) Immunofluorescence (IF) demonstrates homogeneity of fibrin and AAV particles in a gel containing 2xl09AAV viral genomes (vg). Figure 12F) Serial photographs of fibrin gels over 48-hour period demonstrating plasmin-mediated degradation with addition of AAV or addition of aprotinin. Figure 12G) Protein assay was used to measure plasmin-mediated degradation of fibrin either with AAV (black squares) or without AAV (filled circles). AAV had no effect on degradation. Addition of aprotinin delays fibrin degradation (open circles). Figure 12H) ELISA assay was used to determine the percentage of AAV released from gel or retained in gel at 1 hour, 24 hours, and 48 hours at 37°C. Data are presented as means ± SEM (n=3 experiments).

[0028] Figures 13A-13G. Retained infectivity of fibrin-encapsulated AAV2-eGFP. 7xl09dose of AAV-eGFP incorporated into a 3 mm diameter fibrin gel was incubated with confluent ARPE-19 cells and evaluated at 72 hours (Figures 13A-13C) and 1 week (Figures 13D-13F) by flow cytometry (Figures 13C and 13F), bright field (Figures 13A and 13D), and fluorescence microscopy (Figures 13B and 13E). There was increased transduction (fluorescent cells) at 1 week (Figure 13E) compared to 72 hours (Figure 13B). The fibrin gel is outlined by dotted line. Arrow in Figure 13D demonstrates partial degradation of the gel by week 1. Figure 13G) Flow cytometry demonstrates a significant increase in GFP expression between 72 hours and 1 week. Data are presented as means ± SEM. Representative flow cytometry data for a representative run are included in Figure 13C and Figure 13F. **, P<0.01.

[0029] Figures 14A-14L. Surgical placement of fibrin-encapsulated AAV on the epiretinal surface. Figure 14A) Lens-sparing 25-gauge vitrectomy is performed. Figure 14B) A 4mm scleral incision is made with blade to choroid, which is then cauterized using laser prior to full incision with keratome. Figure 14C) The inserter loaded with an oval AAV-fibrin implant is inserted. Figure 14D) The inserter tip is placed under PFO, and the implant is deployed. Figure 14E) Autologous serum is injected around the implant to glue it in place under PFO. Figure 14F) A soft tip cannula is used to exchange the PFO for air and then SF6 gas. Implant remains in place. Figure 14G) Day 5, dilated fundus examination confirms degradation of the hydrogel with no implant visualized at the original site (dotted oval & asterisk). Figure 14H) Minimal residual fibrin is noted on the retina surface at day 5, consistent with degradation of the implant. The small star provides orientation across Figures 14E, 14G, and 14H. Figure 141) Month 1 post-operatively, the view is clear with no inflammation, no cataract, and no evidence of choroidal or retinal abnormalities, including atrophy and vasculitis.

[0030] Figures 15A-15I. Strong RPE transduction detected 1 month after epiretinal placement of fibrin-encapsulated AAV hydrogels in 11 pigs. Figure 15 A) Postmortem analysis of GFP expression shows strong transduction throughout the RPE (inset) when the retina (asterisk) is reflected 1 month after epiretinal gel placement. Figures 15B and 15C) Scattered cells exhibiting GFP expression within the neurosensory retina are observed throughout the posterior pole (Figure 15B) and the periphery (Figure 15C). Figure 15D) Normal fundus appearance at 1 month indicating no clinical adverse effects. Figures 15E and 15F) Histological analysis of the retina reveals GFP expression using IF (Figure 15E) and IHC for GFP (Figure 15F), consistent with RPE transduction by AAV2. Figure 15G) The fellow (control) eye has no GFP signal. Figures 15H and 151). Nuclei are stained with 4', 6- diamidino-2-phenylindole (DAPI) in Figures 15E and 15H and methyl green in Figures 15F and 151.

[0031] Figures 16A-16L. Histologic assessment of transduction, inflammation, and gliosis across two routes of AAV administration. AAV2-eGFP was administered by epiretinal placed fibrin-encapsulated AAV gel (left column, n=\ 1) and subretinal injection (middle column, 77=5). The left eyes were untreated (right column). 1-month post-injection eyes were stained for GFP (Figures 16A-16C), IBA1 (Figures 16D-16F), or GFAP (Figures 16G-16I). Figures 16A-16C) There was consistent GFP expression in RPE (black arrowhead) in the epiretinal group. Subretinal group had photoreceptor transduction and inconsistent RPE transduction (black arrowhead). Figures 16D-16I) The epiretinal and control groups had no significant macrophage recruitment (Figures 16D and 16F) or Muller cell gliosis (Figures 16G and 161); the subretinal group had an apparent increase in Muller cell gliosis (Figure 16H) without macrophage recruitment (Figure 16E). Figures 16J-16L) IF staining for GFAP and GFP. Nuclei in Figures 16J-16L were stained with DAPI. Nuclei in Figures 16A-16I were stained with methyl green.

[0032] Figures 17A-17B. Fibrin hydrogel construction. Figure 17A) Drawing of a fully assembled fibrin casting mold with green 18-gauge cannula tip placed over injection port hole. Figure 17B) Image of injected AAV2 / fibrinogen solution into the casting mold to form a complete fibrin gel blank with excess gelation solution observed coming from the pressure release port.

[0033] Figure 18. Human macular hole surgery with fibrin hydrogel. A 5 mm diameter fibrin gel (right, arrows) was placed on the epiretinal surface overlying a chronic, recurrent macular hole, refractory to conventional surgical repair methods. Serial OCT images (left) show the large macular hole pre-operatively (top left) and the gel on the retinal surface at day 1 post- operatively. The gel appeared mostly degraded by day 5 post-operatively and was completely degraded by day 12. There was no significant clinical inflammation peri-operatively, and there were no short-term or long-term complications. Noted to be smaller after surgery, the hole had an improved configuration without intraretinal fluid and without elevated retinal edges up to 26 months post-operatively.

[0034] DETAILED DESCRIPTION

[0035] This document provides methods and materials for delivering vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) to localized areas within the mammal (e.g., a human). For example, this document provides fibrin hydrogels including vectors (e.g., non- viral vectors or viral vectors such as AAV vectors) and methods for using such compositions to deliver the vectors to localized areas within a mammal (e.g., a human). A fibrin hydrogel provided herein can include (a) one or more fibrinogen polypeptides, (b) one or more thrombin polypeptides, and (c) vectors (e.g., non-viral vectors or viral vectors such as AAV vectors). For example, a fibrin hydrogel can include a cross-linked network of fibrin formed by the polymerization of fibrinogen polypeptides in the presence of thrombin polypeptides, and can include vectors (e.g., non-viral vectors or viral vectors such as AAV vectors). A fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non- viral vectors or viral vectors such as AAV vectors) for targeted delivery) can include (e.g., can be formed from a gelation mixture including) any appropriate fibrinogen polypeptide(s). In some cases, a fibrinogen polypeptide can be a synthetic polypeptide. In some cases, a fibrinogen polypeptide can be a recombinant polypeptide. In some cases, a fibrinogen polypeptide can be obtained from (e.g., can be isolated from) a mammal, such as a mammal to be treated using a fibrin hydrogel provided herein. Examples of fibrinogen polypeptides that can be included in, or used to make, a fibrin hydrogel provided herein include, without limitation, those set forth in the National Center for Biotechnology Information (NCBI) database at accession no. accession no. M64982 (version M64982.1), accession no. X51473 (version X51473.1), and accession no. M64983 (version M64983.1).

[0036] A fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non- viral vectors or viral vectors such as AAV vectors) for targeted delivery) can include (e.g., can be formed from a gelation mixture including) any amount of fibrinogen polypeptides. In some cases, a fibrin hydrogel provided herein include from about 10 milligrams fibrinogen polypeptides per milliliter (mg / mL) to about 80 mg / mL fibrinogen polypeptides (e.g., from about 10 mg / mL to about 70 mg / mL, from about 10 mg / mL to about 60 mg / mL, from about 10 mg / mL to about 50 mg / mL, from about 10 mg / mL to about 40 mg / mL, from about 10 mg / mL to about 30 mg / mL, from about 10 mg / mL to about 20 mg / mL, from about 20 mg / mL to about 80 mg / mL, from about 30 mg / mL to about 80 mg / mL, from about 40 mg / mL to about 80 mg / mL, from about 50 mg / mL to about 80 mg / mL, from about 60 mg / mL to about 80 mg / mL, from about 70 mg / mL to about 80 mg / mL, from about 20 mg / mL to about 70 mg / mL, from about 30 mg / mL to about 60 mg / mL, from about 40 mg / mL to about 50 mg / mL, from about 20 mg / mL to about 40 mg / mL, from about 30 mg / mL to about 50 mg / mL, from about 40 mg / mL to about 60 mg / mL, or from about 50 mg / mL to about 70 mg / mL). In some cases, from about 10 mg to about 80 mg of fibrinogen polypeptides per mL of gelation mixture can be used, together with fibrinogen polypeptides, to make a fibrin hydrogel provided herein.

[0037] A fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non- viral vectors or viral vectors such as AAV vectors) for targeted delivery) can include (e.g., can be formed from a gelation mixture including) any appropriate thrombin polypeptide(s). In some cases, a thrombin polypeptide can be a synthetic polypeptide. In some cases, a thrombin polypeptide can be a recombinant polypeptide. In some cases, a thrombin polypeptide can be obtained from (e.g., can be isolated from) a mammal, such as a mammal to be treated using a fibrin hydrogel provided herein. Examples of thrombin polypeptides that can be included in, or used to make, a fibrin hydrogel provided herein include, without limitation, those set forth in the NCBI database at accession no. BD 189695 (version BD189695.1), accession no. AAGW02037995 (version AAGW02037995.1), and accession no. AF080065 (version AF080065.1).

[0038] A fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non- viral vectors or viral vectors such as AAV vectors) for targeted delivery) can include (e.g., can be formed from a gelation mixture including) any amount of thrombin polypeptides. For example, a fibrin hydrogel provided herein can include from about 0.1 unit thrombin polypeptides per mL (U / mL) of solution (e.g., a sodium citrate solution) to about 1200 U / mL thrombin polypeptides (e.g., from about 0.1 U / mL to about 1000 U / mL, from about 0.1 U / mL to about 800 U / mL, from about 0.1 U / mL to about 600 U / mL, from about 0.1 U / mL to about 400 U / mL, from about 0.1 U / mL to about 200 U / mL, from about 0.1 U / mL to about 100 U / mL, from about 0.1 U / mL to about 50 U / mL, from about 0.1 U / mL to about 25 U / mL, from about 0.1 U / mL to about 1 U / mL, from about 1 U / mL to about 1200 U / mL, from about 25 U / mL to about 1200 U / mL, from about 100 U / mL to about 1200 U / mL, from about 250 U / mL to about 1200 U / mL, from about 500 U / mL to about 1200 U / mL, from about 750 U / mL to about 1200 U / mL, from about 1 U / mL to about 1000 U / mL, from about 50 U / mL to about 750 U / mL, from about 100 U / mL to about 500 U / mL, from about 200 U / mL to about 300 U / mL, from about 0.5 U / mL to about 33 U / mL thrombin polypeptides, from about 1 U / mL to about 100 U / mL thrombin polypeptides, from about 10 U / mL to about 200 U / mL, from about 200 U / mL to about 400 U / mL, from about 300 U / mL to about 500 U / mL, from about 400 U / mL to about 600 U / mL, from about 500 U / mL to about 700 U / mL, from about 600 U / mL to about 800 U / mL, or from about 700 U / mL to about 900 U / mL). In some cases, from about 0.1 U / mL to about 1200 U / mL of thrombin polypeptides per mL of gelation mixture can be used, together with fibrinogen polypeptides, to make a fibrin hydrogel provided herein.

[0039] When one or more fibrinogen polypeptides and / or one or more thrombin polypeptide(s) are obtained from (e.g., are isolated from) a mammal (e.g., a human), such as a mammal (e.g., a human) to be treated using a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery), any appropriate method can be used to obtain the fibrinogen polypeptide(s) and / or the thrombin polypeptide(s). For example, one or more fibrinogen polypeptides and / or one or more thrombin polypeptide(s) can be isolated from blood plasma obtained from a mammal (e.g., a mammal to be treated using a fibrin hydrogel provided herein) using a precipitation technique (e.g., cryoprecipitation and ammonium sulphate precipitation).

[0040] When one or more fibrinogen polypeptides and / or one or more thrombin polypeptide(s) are obtained from (e.g., are isolated from) a mammal (e.g., a human) to treat a mammal (e.g., a human), any appropriate method can be used to obtain the fibrinogen polypeptide(s) and / or the thrombin polypeptide(s) from a member of that species of mammal being treated (e.g., a human). For example, one or more fibrinogen polypeptides and / or one or more thrombin polypeptide(s) can be isolated from blood plasma obtained from one human to create a fibrin hydrogel as described herein for treating another human.

[0041] In some cases, fibrin present in a fibrin hydrogel provided herein can be in the form of one or more fibrin tissue glues. In some cases, a fibrin tissue glue can be a human fibrin tissue glue. Examples of fibrin tissue glues that can be included in a fibrin hydrogel provided herein include, without limitation, TISSEEL, EVICEL®, and VISTASEAL™.

[0042] A fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors for targeted delivery) can include (e.g., can be formed from a gelation mixture including) any appropriate type of vectors. In some cases, a vector can be a gene therapy vector. In some cases, a vector that can be included in a fibrin hydrogel provided herein can be a viral vector. For example, a viral vector can be derived from a replication deficient virus. Examples of viral vectors that can be included in a fibrin hydrogel provided herein include, without limitation, AAV vectors (e.g., AAV2 vectors), lentivirus-based vectors, herpesvirus-based vectors, and adenovirus-based vectors. In some cases, a vector that can be included in a fibrin hydrogel provided herein can be a non-viral vector. Examples of non-viral vectors that can be included in a fibrin hydrogel provided herein include, without limitation, expression plasmids, nanoparticles, and liposomes.

[0043] In some cases, a vector that can be included in a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) can include nucleic acid that can encode a polypeptide (e g., a polypeptide that is heterologous to the vector). For example, a vector that can be included in a fibrin hydrogel provided herein can include a transgene. For example, a vector that can be included in a fibrin hydrogel provided herein can include nucleic acid that can encode a therapeutic polypeptide. In some cases, a therapeutic polypeptide can be an immunoglobulin (e.g., an antibody). Examples of therapeutic polypeptides include, without limitation, RPE65 polypeptides, RS 1 polypeptides, MERTK polypeptides, CERKL polypeptides, CLN3 polypeptides, BEST1 polypeptides, and anti-VEGF antibodies. For example, a vector that can be included in a fibrin hydrogel provided herein can include nucleic acid that can encode a reporter polypeptide (e.g., a fluorescent polypeptide such as a green fluorescent polypeptide (GFP), an alkaline phosphatase, a beta-galactosidase, or a luciferase).

[0044] In addition to nucleic acid that can encode a polypeptide (e.g., a therapeutic polypeptide), a vector (e.g., a viral vector or a non-viral vector) can contain one or more regulatory elements operably linked to the nucleic acid that can encode a polypeptide (e.g., a therapeutic polypeptide). Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid. The choice of regulatory element(s) that can be included in a vector depends on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in a vector to facilitate transcription of a nucleic acid that can encode a polypeptide (e.g., a therapeutic polypeptide). A promoter can be a naturally occurring promoter or a recombinant promoter. A promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a polypeptide in a general or tissue- specific manner. As used herein, “operably linked” refers to positioning of a regulatory element relative to a nucleic acid encoding a polypeptide in such a way as to permit or facilitate expression of the encoded polypeptide. For example, a vector can contain a promoter and nucleic acid that can encode a polypeptide (e.g., a therapeutic polypeptide). In this case, the promoter is operably linked to a nucleic acid that can encode a polypeptide (e.g., a therapeutic polypeptide) such that it drives expression of the polypeptide in cells.

[0045] A fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non- viral vectors or viral vectors such as AAV vectors)) for targeted delivery) can include (e.g., can be formed from a gelation mixture including) any appropriate amount of vectors. In some cases, a fibrin hydrogel provided herein can include from about 108vector genomes to about 1014vector genomes per volume of hydrogel (e.g., from about 108to about 1013, from about 108to about 1012, from about 108to about 1011, from about 108to about IO10, from about 108to about 109, from about 109to about 1014, from about 1010to about 1014, from about 10nto about 1014, from about 1012to about 1014, from about 1013to about 1014, from about 109to about 1013, from about 1010to about 1012, from about 109to about 1011, or from about 1010to about 1012vector genomes per volume of hydrogel).

[0046] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) can include (e.g., can be formed from a gelation mixture including) one or more additional components. For example, a fibrin hydrogel provided herein can include one or more agents that can slow polymerization of the fibrin gel (e.g., to permit injection molding or extrusion of the fibrin gel). In some cases, an agent that can slow polymerization of a fibrin hydrogel provided herein can be an azo dye or an isomer thereof. Examples of agents that can slow polymerization of a fibrin hydrogel provided herein include, without limitation, trypan blue, Evans blue, VisionBlue®, Direct Blue 53, Azovan blue, compounds having the structure listed under PubChem Compound ID number (CID) 6296, compounds having the structure listed under PubChem CID 9409, Direct Blue 2, melantherine BH, pontamine sky blue 5B, Azo Fuchsine, and Acid Red 99. In some cases, an agent that can slow polymerization of a fibrin hydrogel provided herein can be as described elsewhere (see, e.g., International Patent Application Publication No. WO 2022 / 104128). In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) can include (e.g., can be formed from a gelation mixture including) one or more agents that can slow degradation of the fibrin hydrogel (e.g., to slow degradation in vivo following administration of the fibrin hydrogel to a retina of an eye within a mammal such as a human). Examples of agents that can slow degradation of a fibrin hydrogel provided herein include, without limitation, tranexamic acid, aprotinin, aminocaproic acid, and nafamostat.

[0047] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) can be degradable (e.g., can be biodegradable). For example, a fibrin hydrogel provided herein can be designed such that the volume of the fibrin hydrogel (e.g., a fibrin hydrogel that has been delivered to a mammal) can decrease over time. In some cases, a volume of a fibrin hydrogel that has been delivered to a mammal (e.g., a human) can decrease by at least about 25% (e.g., at least about 35%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 98%, or at least about 99%) over time. In some cases, a volume of a fibrin hydrogel provided herein that has been delivered to a mammal (e.g., a human) can degrade completely over time such that essentially no fibrin hydrogel is left in the mammal.

[0048] A fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non- viral vectors or viral vectors such as AAV vectors) for targeted delivery) can be any size. In some cases, a fibrin hydrogel provided herein can have a width of from about 0.2 millimeters (mm) to about 15 mm (e.g., from about 0.2 mm to about 12 mm, from about 0.2 mm to about 10 mm, from about 0.2 mm to about 8 mm, from about 0.2 mm to about 5 mm, from about 0.2 mm to about 3 mm, from about 0.2 mm to about 1 mm, from about 0.2 mm to about 0.5 mm, from about 0.5 mm to about 15 mm, from about 1 mm to about 15 mm, from about 2 mm to about 15 mm, from about 5 mm to about 15 mm, from about 7 mm to about 15 mm, from about 10 mm to about 15 mm, from about 12 mm to about 15 mm, from about 0.5 mm to about 13 mm, from about 2 mm to about 10 mm, from about 4 mm to about 8 mm, from about 1 mm to about 3 mm, from about 2 mm to about 4 mm, from about 3 mm to about 5 mm, from about 4 mm to about 6 mm, from about 5 mm to about 7 mm, from about 6 mm to about 8 mm, from about 7 mm to about 9 mm, from about 8 mm to about 10 mm, from about 9 mm to about 11 mm, from about 10 mm to about 12 mm, or from about 11 mm to about 13 mm). For example, a fibrin hydrogel provided herein can have a width of about 1.5 mm.

[0049] In some cases, a fibrin hydrogel provided herein can have a length of from about 0.2 mm to about 80 mm (e.g., from about 0.2 mm to about 60 mm, from about 0.2 mm to about 40 mm, from about 0.2 mm to about 20 mm, from about 0.2 mm to about 15 mm, from about 0.2 mm to about 12 mm, from about 0.2 mm to about 10 mm, from about 0.2 mm to about 8 mm, from about 0.2 mm to about 5 mm, from about 0.2 mm to about 3 mm, from about 0.2 mm to about 1 mm, from about 1 mm to about 80 mm, from about 5 mm to about 80 mm, from about 10 mm to about 80 mm, from about 15 mm to about 80 mm, from about 20 mm to about 80 mm, from about 40 mm to about 80 mm, from about 60 mm to about 80 mm, from about 0.3 mm to about 60 mm, from about 0.4 mm to about 40 mm, from about 0.5 mm to about 20 mm, from about 0.7 mm to about 10 mm, from about 1 mm to about 8 mm, from about 2 mm to about 7 mm, from about 3 mm to about 6 mm, from about 4 mm to about 5 mm, from about 0.3 mm to about 0.7 mm, from about 0.4 mm to about 0.8 mm, from about 0.5 mm to about 1 mm, from about 0.8 mm to about 2 mm, from about 1 mm to about 10 mm, from about 10 mm to about 20 mm, from about 20 mm to about 30 mm, from about 30 mm to about 40 mm, from about 40 mm to about 50 mm, from about 50 mm to about 60 mm, or from about 60 mm to about 70 mm). For example, a fibrin hydrogel provided herein can have a length of about 5 mm.

[0050] In some cases, a fibrin hydrogel provided herein can have a longest dimension (e.g., a diameter) of from about 0.2 mm to about 15 mm (e.g., from about 0.2 mm to about 12 mm, from about 0.2 mm to about 10 mm, from about 0.2 mm to about 8 mm, from about 0.2 mm to about 5 mm, from about 0.2 mm to about 3 mm, from about 0.2 mm to about 1 mm, from about 0.2 mm to about 0.5 mm, from about 0.5 mm to about 15 mm, from about 1 mm to about 15 mm, from about 2 mm to about 15 mm, from about 5 mm to about 15 mm, from about 7 mm to about 15 mm, from about 10 mm to about 15 mm, from about 12 mm to about 15 mm, from about 0.5 mm to about 13 mm, from about 2 mm to about 10 mm, from about 4 mm to about 8 mm, from about 1 mm to about 3 mm, from about 2 mm to about 4 mm, from about 3 mm to about 5 mm, from about 4 mm to about 6 mm, from about 5 mm to about 7 mm, from about 6 mm to about 8 mm, from about 7 mm to about 9 mm, from about 8 mm to about 10 mm, from about 9 mm to about 11 mm, from about 10 mm to about 12 mm, or from about 11 mm to about 13 mm). For example, a fibrin hydrogel provided herein can have a longest dimension (e.g., a diameter) of about 3 mm.

[0051] In some cases, a fibrin hydrogel provided herein can have a thickness of from about 50 pm to about 1000 pm (e.g., from about 50 pm to about 800 pm, from about 50 pm to about 600 pm, from about 50 pm to about 500 pm, from about 50 pm to about 300 pm, from about 50 pm to about 200 pm, from about 50 pm to about 100 pm, from about 100 pm to about 1000 pm, from about 300 pm to about 1000 pm, from about 500 pm to about 1000 pm, from about 700 pm to about 1000 pm, from about 900 pm to about 1000 pm, from about 100 pm to about 900 pm, from about 200 pm to about 800 pm, from about 300 pm to about 700 pm, from about 400 pm to about 600 pm, from about 100 pm to about 300 pm, from about 200 pm to about 400 pm, from about 300 pm to about 500 pm, from about 500 pm to about 700 pm, from about 600 pm to about 800 pm, or from about 700 pm to about 900 pm). For example, a fibrin hydrogel provided herein can have a thickness of about 200 pm.

[0052] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) can be shaped (e.g., can be shaped in 3 dimensions). Exemplary shapes for a fibrin hydrogel provided herein include, without limitation, circles, ovals, and squares. In some cases, a fibrin hydrogel provided herein can be shaped using a mold (e.g., during production of the fibrin hydrogel). For example, one or more fibrinogen polypeptides, one or more thrombin polypeptides, and vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) can be mixed, and, prior to polymerization, can be poured into a mold to polymerize within the mold, thereby shaping the fibrin hydrogel. In some cases, a fibrin hydrogel provided herein can be derived from a produced fibrin hydrogel. For example, any shape fibrin hydrogel can be punched from a produced fibrin hydrogel. In some cases, multiple individual fibrin hydrogels provided herein can be made (e.g., can be cut or punched) from a larger sheet of fibrin hydrogel provided herein. Any appropriate mold can be used to form a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery). In some cases, a mold used to produce a fibrin hydrogel provided herein can include a substrate and a top plate, such that the fibrin hydrogel can be formed on a surface of the substrate with the substrate shaping the lower surface of the fibrin hydrogel provides herein and with the top plate shaping the upper surface of the fibrin hydrogel provided herein. For example, a gelation mixture including (a) one or more fibrinogen polypeptides, (b) one or more thrombin polypeptides, and (c) vectors (e.g., non- viral vectors or viral vectors such as AAV vectors) can be injected into the space between a substrate and top plate (which can also function as a gasket) such that the gelation mixture polymerizes within the mold, thereby producing a fibrin hydrogel provided herein. For example, a gelation mixture including (a) one or more fibrinogen polypeptides, (b) one or more thrombin polypeptides, and (c) vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) can be placed onto a substrate and top plate can be placed on the gelation mixture such that the gelation mixture polymerizes within the mold, thereby producing a fibrin hydrogel provided herein. A substrate that can be used to form a fibrin hydrogel provided herein can include any appropriate material(s) (e.g., glass, metal (e.g., stainless- steel), polyethylene, polypropylene, and polystyrene). A top plate that can be used to form a fibrin hydrogel provided herein can include any appropriate material(s) (e.g., metal (e.g., stainless-steel) and silicon).

[0053] In some cases, a mold that can be used to form a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) also can include a gasket. For example, a gasket can be placed between a substrate and a top plate used to form a fibrin hydrogel provided herein. A gasket can be placed between a substrate and a top plate used to form a fibrin hydrogel provided herein can be any appropriate thickness. In some cases, a gasket can seal the mold against leakage (e.g., ensuring uniform distribution of the gelation mixture). In some cases, a gasket can prevent the fibrin hydrogel from adhering to the top plate. In some cases, the thickness of a gasket that can be placed between a substrate and a top plate used to form a fibrin hydrogel provided herein can be used to control the thickness tolerance of the fibrin hydrogel. An exemplary mold that can be used to form a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) can be as shown in Figures 3A and 3B.

[0054] A sheet of fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) from which individual fibrin hydrogels provided herein have been punched can be as shown in Figures 3C-3F.

[0055] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) can be a circle having a diameter of about 3 mm, and a thickness of about 0.2 mm.

[0056] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) can be a rectangle having a width of about 1.5 mm, a length of about 5 mm, and a thickness of about 0.2 mm.

[0057] In some cases, a fibrin hydrogel provided herein (e g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery) can be sterilize. For example, a fibrin hydrogel provided herein can produced under aseptic or sterile conditions.

[0058] Also provided herein are methods for making a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) for targeted delivery). A fibrin hydrogel provided herein can be made using any appropriate method. In some cases, one or more fibrinogen polypeptides and one or more thrombin polypeptides, and vectors can be mixed prior to polymerization of the fibrin hydrogel. For example, a static mixer can be used to mix (e.g., homogenous mix) one or more fibrinogen polypeptides, one or more thrombin polypeptides, and vectors to make a fibrin hydrogel provided herein. In some cases, a fibrin hydrogel provided herein can be made as described in any one of Examples 1-3.

[0059] Also provided herein are methods for using a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) for targeted delivery. For example, a fibrin hydrogel provided herein can be used to deliver vectors to one or more localized areas within a mammal (e.g., a human). In some cases, fibrin hydrogels including vectors can be administered to a retina (e.g., by epiretinal administration or subretinal administration) of an eye within a mammal (e.g., a human) to deliver at least some of the vectors to the retina. For example, fibrin hydrogels including viral vectors (e.g., AAV vectors) that contain nucleic acid that can encode a polypeptide (e.g., a therapeutic polypeptide) can be administered to a retina of an eye within a mammal (e.g., a human) to deliver at least some the viral vectors to the retina such that the viral vectors can transduce at least some retinal cells and such that the transduced cells can express the polypeptide.

[0060] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) can be used to provide vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) to one or more localized areas within a mammal (e.g., a human). For example, fibrin hydrogels including vectors can be administered to a retina of an eye within a mammal (e.g., a human) and can deliver at least some of the vectors to one or more areas peripheral to the retina such as the ora serrata.

[0061] A fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non- viral vectors or viral vectors such as AAV vectors)) can release any amount of vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) included in the fibrin hydrogel including vectors to one or more localized areas within a mammal (e.g., a human). For example, at least about 25% (e.g., at least about 35%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 98%, or at least about 99%) of vectors included in a fibrin hydrogel provided herein that has been delivered to a mammal (e.g., a human) can diffuse out of the fibrin hydrogel and / or can be released from the fibrin hydrogel as the fibrin hydrogel degrades. In some cases, essentially all of the vectors included in the fibrin hydrogel provided herein that has been delivered to a mammal (e.g., a human) can diffuse out of the fibrin hydrogel and / or can be released from the fibrin hydrogel as the fibrin hydrogel degrades.

[0062] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) can be used to provide a delayed release of vectors to one or more localized areas within a mammal (e.g., a human). For example, vectors included in the fibrin hydrogel provided herein that has been administered to a retina (e.g., by epiretinal administration or subretinal administration) of an eye within a mammal can diffuse out of the fibrin hydrogel and / or can be released from the fibrin hydrogel as the fibrin hydrogel degrades over the course of about 5 days. For example, essentially all of the vectors included in the fibrin hydrogel provided herein that has been administered to a retina of an eye within a mammal (e.g., a human) can diffuse out of the fibrin hydrogel and / or can be released from the fibrin hydrogel as the fibrin hydrogel degrades over the course of from about 48 hours to about 96 hours.

[0063] When vectors included in a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) for targeted delivery) include nucleic acid that can encode a polypeptide (e.g., a therapeutic polypeptide), the expressed polypeptide can be detected within an eye within a mammal (e.g., a human) for any appropriate amount of time following administration to the retina (e.g., by epiretinal administration or subretinal administration) of the eye of the mammal. For example, a polypeptide expressed by vectors diffused from a fibrin hydrogel provided herein and / or released from the fibrin hydrogel as the fibrin hydrogel degrades can be detected in an eye of a mammal (e.g., a human) for up at least 1 month (e.g., at least 1 month, at least 2 months, at least 3 months, or more) following administration of the fibrin hydrogel.

[0064] In some cases, a fibrin hydrogel provided herein (e g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) can be used to treat a mammal (e.g., a human) having one or more ocular diseases. For example, a fibrin hydrogel provided herein can be used to deliver vectors including nucleic acid that can encode a therapeutic polypeptide to a retina of an eye within a mammal in need thereof (e.g., a mammal, such as a human, having one or more ocular diseases). For example, a fibrin hydrogel including viral vectors (e.g., AAV vectors) that contain nucleic acid that can encode a therapeutic polypeptide can be administered to a retina of an eye within a mammal (e.g., a human) to deliver at least some of the viral vectors to the retina such that the viral vectors can transduce at least some retinal cells and such that the transduced cells can express the therapeutic polypeptide, thereby treating the mammal. A fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non- viral vectors or viral vectors such as AAV vectors)) can be used to deliver vectors to any appropriate mammal. Examples of mammals that can be administered a fibrin hydrogel provided herein include, without limitation, humans, non-human primates (e.g., monkeys), horses, bovine species, porcine species, dogs, cats, horses, cows, pigs, sheep, mice, rabbit, and rats. For example, a fibrin hydrogel provided herein can be administered to a human. In some cases, a fibrin hydrogel provided herein can be administered to a human having retinal degeneration. In some cases, a fibrin hydrogel provided herein can be administered to a human having one or more ocular diseases to treat the human.

[0065] When treating a mammal (e.g., a human) having an ocular disease as described herein (e.g., by administering a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)), the ocular disease can be any type of ocular disease. Examples of ocular diseases that can be treated as described herein (e.g., by administering a fibrin hydrogel including viral vectors such as AAV vectors for targeted delivery) include, without limitation, retinitis pigmentosa, choroideremia, X-linked retinoschisis, age-related macular degeneration, diabetic retinopathy, glaucoma, achromatopsia, Leber congenital amaurosis, and Batten disease.

[0066] In some cases, a mammal (e.g., a human) having Leber congenital amaurosis (e.g., in one or both eyes) can be treated by administering a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) that contain nucleic acid that can encode a RPE65 polypeptide, such that the vectors can express the RPE65 polypeptide, thereby treating the mammal. For example, a mammal (e.g., a human) having Leber congenital amaurosis (e.g., in one or both eyes) can be treated by administering a fibrin hydrogel including viral vectors (e.g., AAV vectors) that contain nucleic acid that can encode a RPE65 polypeptide, such that the viral vectors can transduce at least some retinal cells and such that the transduced cells can express the RPE65 polypeptide, thereby treating the mammal.

[0067] In some cases, a mammal (e.g., a human) having retinitis pigmentosa (e.g., in one or both eyes) can be treated by administering a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) that contain nucleic acid that can encode a MERTK polypeptide, such that the vectors can express the MERTK polypeptide, thereby treating the mammal. For example, a mammal (e.g., a human) having retinitis pigmentosa (e.g., in one or both eyes) can be treated by administering a fibrin hydrogel including viral vectors (e.g., AAV vectors) that contain nucleic acid that can encode a MERTK polypeptide, such that the viral vectors can transduce at least some retinal cells and such that the transduced cells can express the MERTK polypeptide, thereby treating the mammal.

[0068] In some cases, a mammal (e.g., a human) having Batten disease (e.g., in one or both eyes) can be treated by administering a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) that contain nucleic acid that can encode a CLN3polypeptide, such that the vectors can express the CLN3polypeptide, thereby treating the mammal. For example, a mammal (e.g., a human) having Batten disease (e.g., in one or both eyes) can be treated by administering a fibrin hydrogel including viral vectors (e.g., AAV vectors) that contain nucleic acid that can encode a CLN3polypeptide, such that the viral vectors can transduce at least some retinal cells and such that the transduced cells can express the CLN3polypeptide, thereby treating the mammal.

[0069] In some cases, a mammal (e.g., a human) having age-related macular degeneration (e.g., in one or both eyes) can be treated by administering a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors) that contain nucleic acid that can encode an anti-VEGF antibody, such that the vectors can express the anti-VEGF antibody, thereby treating the mammal. For example, a mammal (e.g., a human) having age- related macular degeneration (e.g., in one or both eyes) can be treated by administering a fibrin hydrogel including viral vectors (e.g., AAV vectors) that contain nucleic acid that can encode an anti-VEGF antibody, such that the viral vectors can transduce at least some retinal cells and such that the transduced cells can express an anti-VEGF antibody, thereby treating the mammal.

[0070] In some cases, methods of treating a mammal having one or more ocular diseases as described herein (e.g., by administering a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) also can include identifying the mammal as having one or more ocular diseases. Examples of methods for identifying a mammal as having one or more occur diseases include, without limitation, physical examination, genetic tests, and / or ophthalmic testing.

[0071] Any appropriate method can be used to administer a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) to a retina of an eye within a mammal (e.g., a human). In some cases, a fibrin hydrogel provided herein can be placed on (e.g., adhered to) the surface of a retina of an eye within a mammal (e.g., a human). For example, a fibrin hydrogel provided herein can be placed on (e.g., adhered to) the epiretinal surface of an eye within a mammal (e g., a human). For example, a fibrin hydrogel provided herein can be placed on (e.g., adhered to) the subretinal surface of an eye within a mammal (e.g., a human).

[0072] When a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) is placed on (e.g., adhered to) the surface of a retina of an eye within a mammal (e.g., a human), a liquid (e.g., a plurflourocarbon (PFO) liquid) can be used to hold the fibrin hydrogel in place (e.g., during surgery to place the fibrin hydrogel on the surface of the retina). In some cases, a PFO liquid can be placed on the surface of a retina of an eye within a mammal (e.g., a human) and then a fibrin hydrogel provided herein can be injected under the PFO liquid (e.g., such that the PFO liquid holds the fibrin hydrogel in place (e.g., during surgery to place the fibrin hydrogel on the surface of the retina)). In some cases, a fibrin hydrogel provided herein can be placed on (e g., adhered to) the surface of a retina of an eye within a mammal (e.g., a human) and then a PFO liquid can be placed on the fibrin hydrogel (e.g., to hold the fibrin hydrogel in place (e.g., during surgery to place the fibrin hydrogel on the surface of the retina)). In some cases, the PFO liquid can be removed and the liquid in the vitreous cavity can be removed and replaced with a substance that vectors cannot diffuse through (e.g., such that vectors that diffuse from the fibrin hydrogel provided herein are directed towards the retina). Examples of substances that vectors cannot diffuse through include, without limitation, air bubbles, silicon oils, and expansile gases (e.g., SF6).

[0073] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) can be placed on (e.g., adhered to) the surface of a retina of an eye within a mammal (e.g., a human) and then one or more (e.g., one, two, three, or more) drops of serum and / or plasma can be placed on the fibrin hydrogel (e.g., to allow for adhesion of the fibrin hydrogel to the retina).

[0074] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) can be placed on (e.g., adhered to) the surface of a retina of an eye within a mammal (e.g., a human), a PFO liquid can be used to hold the fibrin hydrogel in place (e.g., during surgery to place the fibrin hydrogel on the surface of the retina), and one or more (e.g., one, two, three, or more) drops of serum and / or plasma can be placed on the fibrin hydrogel (e.g., to allow for adhesion of the fibrin hydrogel to the retina). Subsequently, the PFO liquid can be removed and the liquid in the vitreous cavity can be removed and replaced with an air bubble.

[0075] In some cases, a fibrin hydrogel provided herein (e.g., a fibrin hydrogel including vectors (e.g., non-viral vectors or viral vectors such as AAV vectors)) can be placed on (e.g., adhered to) the surface of a retina of an eye within a mammal (e.g., a human), a PFO liquid can be placed on the fibrin hydrogel (e.g., to hold the fibrin hydrogel in place (e.g., during surgery to place the fibrin hydrogel on the surface of the retina)), and one or more (e.g., one, two, three, or more) drops of serum and / or plasma can be placed on the fibrin hydrogel (e.g., to allow for adhesion of the fibrin hydrogel to the retina). Subsequently, the PFO liquid can be removed and the liquid in the vitreous cavity can be removed and replaced with SF6.

[0076] In some cases, a fibrin hydrogel provided herein is not administered using a subretinal injection.

[0077] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0078] EXAMPLES

[0079] Example 1: Characterization of fibrin gels

[0080] This Example describes the design and generation of fibrin hydrogels that can degrade fully within a mammal. Fibrin Polymerization

[0081] TISSEEL Gel

[0082] A fibrin TISSEEL kit (Baxter NDC#00338-4302-04, 4 mL) was employed using the kits provided solutions to cast a fibrin gel with in custom 15x80x0.2 mm molds (Meddux) using a FibriJet® ratio applicator (Nordson Medical SA-1001) equipped with a 1 : 1 blending connector with a mixer (Nordson Medical SA-3678) tipped with an 18-gauge cannula (Meddux). The molds were sealed in sterile ZIPLOC® bags (Sklar Sterile 96-7766) and incubated at 37°C with 5% CO2 for 3 hours to facilitate polymerization. Subsequently, polymerized gels were transferred to a 4-well culture dish (Thermo Fisher 267061). A custom punch tool was used to extract 24 individual 5 mm x 1.5 mm samples from each gel and stored in 1.5 mL tubes (Cardinal Health Cl 300-29) at -20°C until further use.

[0083] Modified Gel

[0084] 0.01 M sodium citrate was used in place of fibrin inhibitor solution in the fibrin TISSEEL kit (Baxter NDC#00338-4302-04, 4 mL). This was employed to cast a fibrin gel in custom 15x80x0.2 mm molds (Meddux) using a FibriJet® ratio applicator (Nordson Medical SA-1001) equipped with a 1 : 1 blending connector with a mixer (Nordson Medical SA-3678) tipped with an 18-gauge cannula (Meddux). The molds were sealed in sterile ZIPLOC® bags (Sklar Sterile 96-7766) and incubated at 37°C with 5% CO2 for 3 hours to facilitate polymerization. Subsequently, polymerized gels were transferred to a 4-well culture dish (Thermo Fisher 267061). A custom punch tool was used to extract 24 individual 5 mm x 1.5 mm samples from each gel and stored in 1.5 mL tubes (Cardinal Health C 1300-29) at -20°C until further use.

[0085] Modified AA V Gel

[0086] A fibrin TISSEEL kit (Baxter NDC 00338-4302-04, 4 mL) was employed to cast a fibrin gel with 150 pl of AAV2 (8.26E12 titer) in 1.5x7 mm slide molds (Meddux) using a FibriJet® ratio applicator (Nordson Medical SA-1001) equipped with a 1 : 1 blending connector with a mixer (Nordson Medical SA-3678) tipped with an 18-gauge cannula (Meddux). The molds were sealed in sterile ZIPLOC® bags (Sklar Sterile 96-7766) and incubated at 37°C with 5% CO2 for 3 hours to facilitate polymerization. Subsequently, polymerized gels were transferred to a 4-well culture dish (Thermo Fisher 267061). A 5 mm biopsy punch was used to extract 24 individual 5 mm x 1.5 mm circular samples from each gel. Samples were stored individually in 1.5 mL tubes (Cardinal Health C1300-29) at -20°C until further use.

[0087] Fibrin Degradation Assay

[0088] Plasminogen (Biotechne 1939-SE) was reconstituted in 1 mL of H2O (Gibco A12873- 01) to create a 200 pg / mL stock solution. The stock solution was further diluted to a working concentration of 25 pg / mL using H2O. Simultaneously, tissue plasminogen activator (TP A) (Sigma T0831-100UG) was reconstituted in 1 mL H2O to achieve a concentration of 100 pg / mL. The Plasmin degrading solution was prepared by adding 25 pL of TPA to 300 pL of the plasminogen working solution (25 pg / mL).

[0089] Upon removal from -20°C, the 1.5 mL tubes containing gel punches were allowed to reach room temperature. Gels were individually placed in 1.5 mL tubes with 50 pL of plasmin degradation solution and left to degrade at room temperature. Timepoint imaging (1, 2, 4, 8, 16, 24, 36, 48 hours) was conducted after which degradation solutions were preserved for protein quantification.

[0090] Protein Quantification:

[0091] Protein quantification of degraded fibrin gel at each timepoint was performed using the BCA protein quantification kit (Abeam AB 102536) following kit instructions. Samples were placed in a clear 96-well plate (Costar 9102), and OD values were measured at 562 nm using a spectrophotometer (Spectramax).

[0092] Results:

[0093] Modified gels were degraded by plasmin substantially faster than off the shelf TISSEEL. This was statistically significant as early as 1 hour after addition of plasmin. Off the shelf TISSEEL gels at the plasmin concentration used never fully degraded even after 48 hours, while modified gels were completely degraded typically within 24 hours (Figure 1). The addition of AAV to the modified gel did not alter the gel degradation characteristics (Figure 2).

[0094] Example 2: In vivo delivery of gene therapy vectors encapsulated in fibrin gels

[0095] This Examples describes the design and generation of high concentration fibrin hydrogels that can sequester AAV vectors. The AAV vectors retained infectivity (e.g., the ability to transduce a cell) such that these gels could deliver the AAV vectors to retinal cells in the eye.

[0096] Methods

[0097] Fibrin gels were cast using fibrinogen and thrombin with addition of trypan blue and AAV2-GFP with 6.9 x IO10capsids / punch (Figure 4). Fibrin gel punches with a 3 mm diameter were transferred into Geltrex-coated 96-well plates with wells containing either a saline solution or confluent retinal pigment epithelium cells (ARPE-19, an RPE-derived immortalized cell line) in media. Punches were removed at various timepoints. AAV2 in solution (i.e., released from gels) and AAV2 retained in the gels (i.e., after plasmin degradation) were measured using a Progen AAV2 ELISA kit to determine diffusion rate. ARPE-19 cells were imaged for GFP positivity weekly after fibrin placement. Electron microscopy of the fibrin gels was performed to identify viral particles within the gel matrix. For in vivo studies, gels containing AAV encoding GFP were “punched” using a 5 x 1.5 oval punch, loaded in a subretinal implantation device, and placed in the subretinal space or epiretinal surface of a domestic pig eye (Figure 7). After 1 month, animals were sacrificed, and eyes were examined for GFP fluorescence.

[0098] Results

[0099] Maximum AAV diffusion from fibrin gel punches occurred between 48 and 96 hours (Figure 5A). Approximately 50% of the AAV particles in the gel punches was recovered in the solution, and the remaining AAV was recovered from the degraded gels (Figure 5B). In vitro RPE cell transduction with 1.7 x 109viral genomes / well was determined to be optimal with no benefit of adding higher concentration of virus. One month following RPE transduction, equivalent numbers of GFP expressing cells were observed in wells with AAV solution alone compared to fibrin encapsulated-AAV (Figure 6). In pigs, epiretinal gels were degraded within 5 days (Figure 8), and GFP was detected in RPE of an animal in which AAV was delivered via the classical sub-retinal liquid bleb (Figure 9). In an animal receiving a sub-retinal AAV containing fibrin gel, GFP fluorescence in the RPE delivered at the same dose was stronger than in the animal in which AAV was delivered by injection of subretinal fluid. A single animal receiving an epiretinal placement of the fibrin gel appeared to have some RPE transduction as well, though this animal was complicated due to a post-operative infection.

[0100] Together, these results demonstrate that fibrin encapsulated-AAV diffused out of the gel, retained infectivity, and transduced cells in vitro and in vivo.

[0101] Example 3: Exemplary method for preparing fibrin hydrogel slides

[0102] Kit Building / Supplies:

[0103]

[0104] Equipment:

[0105] Preparing Sodium Citrate Solution:

[0106] Preparing a Fibrin Hydrogel Slide:

[0107] Example 4: Retinal Gene Therapy using Epiretinal AA V-Containing Fibrin Hydrogels

[0108] The results in this Example re-present and expand on at least some of the results provided in other Examples. RESULTS

[0109] Chorioretinal atrophy and transduction issues after subretinal AA V injection

[0110] Subretinal administration of AsAMl-enhanced GFP (eGFP) solution (Fig. 10A) led to progressive CRA with profound RPE changes (Figs. 10B-10M). This was despite the subretinal blebs being performed as recommended for Luxturna® administration with slow administration over several minutes and despite the AAV titer being 2-fold less than used in clinical practice. Two of the five pigs had clinically significant atrophy affecting the entire area centralis (Figs. 10J and 10K). Fundus photos (Figs. 10J and 10K), optical coherence tomography (OCT) and OCT-angiography (OCT-A) images (Figs. 10B-10H) and scanning laser ophthalmoscope (SLO) image (Fig. 101) demonstrated the extensive CRA confined within the area of the subretinal bleb, seen as well-demarcated areas of hypopigmentation (i.e., atrophy) with scattered overlying RPE clumping (i.e., cell death). Hematoxylin and eosin (H&E) staining demonstrated RPE vacuolation, hypo- and hyperpigmentation, atrophy, and occasional hypertrophy with thinning of the choroid within these areas (Figs. 10L and 10M).

[0111] Subretinal injection of AAV2-eGFP resulted in a significant amount of photoreceptor transduction (Fig. ION) and inconsistent RPE transduction. In all subretinal pigs, immunohistochemistry (IHC) showed photoreceptors and RPE cells that exhibited high expression of GFP immediately adjacent to cells with no transduction (Fig. ION). As expected, all transduction was confined to the bleb region (i.e., no peripheral retinal / RPE transduction) with the subretinal approach (Table 1). These safety and transduction concerns led to the development of an innovative, alternative solution to administering gene therapy for RPE transduction without subretinal bleb formation: high concentration epiretinal fibrin hydrogels.

[0112] Table 1. Overview of animal surgeries and outcomes.

[0113]

[0114] For all pigs included in this study, details of gene therapy vector, surgical route, AAV2 titer administered, post-mortem eye processing, transduction sites, and adverse events are provided. (+) indicates Yes or present but without clinical significance. (++) indicates clinically significant but treatable. (+++) indicates severe clinical implications.

[0115] Production of large fibrin hydrogels using injection molding

[0116] The goal of this study was to produce fibrin hydrogels that can be used for gene therapy delivery. The reproducibility of gel characteristics, especially shape, is important as it dictates the size of the dose delivered and influences the rate of scaffold degradation and its mechanical properties. An oval gel of 1.5 x 5.1 x 0.2 mm in size has consistently used for therapeutic application, but it was found that pressing gels (Gandhi et al., Acta Biomater, 67: 134-146 (2018)) resulted in too much variability in gel thickness. Thus, a method was developed for commercial scale production of fibrin hydrogels suitable for this application and with tight dimensional tolerances.

[0117] Trypan blue in the fibrin gelation mixture significantly slows the initial polymerization of fibrin (Gandhi et al., J. Biomed. Mater. Res. A, 109:2357-2368 (2021)). This property of trypan blue was used to manufacture fibrin hydrogels using injection molding (Figs. 11 A and 1 IB; Fig. 17). A mold was used that resulted in fibrin hydrogel sheets that were uniformly of -181 ± 1 mm (average ± standard error of mean (SEM), w=3) in thickness (Figs. 11C-1 IF) and could be used as blanks from which to punch different sized doses (Figi. 11A and 1 IB). The fibrin gel mold is shown in Fig. 17 and has two plates sealed with a silicon gasket held together by metal clips. The “top” plate was 28.57 x 78.36 mm, approximately the size of a microscope slide and had a cavity that was 15.25 x 58.42 x 0.20 mm in which the gel was formed. The top plate was machined from polycarbonate for a clear plate that would allow observation of the filling of the mold. The inlet and outlet ports of the top plate were designed and placed to permit even filling and prevent formation of pockets or air bubbles during injection of the gelation mixture, to anchor the hydrogel in place, and to prevent the gel from being pulled from the bottom plate during disassembly of the mold after polymerization.

[0118] A tapered dispensing tip with an inlet that is flush with the plate surface was used. This allowed the plate to sit flat with the gel facing up, an advantage during casting as it allowed the operator casting the gel to observe it filling. The “bottom” plate was machined from aluminum and has channels milled along both sides to hold the metal clips in place. The clips hold the entire mold assembly together. The gasket was a sheet of 1 / 32” thick silicon. Thicker gaskets allowed for greater deformation under pressure resulting in an unacceptable degree of variability in gel thickness. Earlier prototypes tested use of a circumferential sealing gasket and a plastic bottom plate; however, this too resulted in unacceptable variability in thickness and sticking of the gel to the bottom plate.

[0119] The injection molding process uses a FibriJet 11 :1 ratio applicator and blending connector. The 11 : 1 ratio permits maximal fibrinogen concentration and the blending connector results in uniform mixing of the thrombin and fibrinogen solutions as they are injected into the mold (Fig. 17). A 4 mL TISSEEL kit, prepared as outlined in the methods, has sufficient volume to mold up to three 15.25 x 58.42 x 0.2 mm gel blanks.

[0120] Mechanical characteristics of injection molded fibrin hydrogels

[0121] The ability to use a punch to generate smaller shaped pieces of the hydrogel from 15.25 x 58.42 x 0.2 mm gel blanks indicated a mechanical stiffness sufficient to permit the use of punching to subdivide the gel into uniform pieces of smaller size (Figs. 11 A and 1 IB). OCT analysis indicated that the gel had sufficient elasticity to rebound at the edges to its original thickness as the average of a punch did not differ from the average of a blank. The hydrogel elasticity / compression characteristics was next tested. Mechanical strength testing of fibrin gels was similar without and with aprotinin with Young’s modulus calculated as 0.053 ± 0.01 MPa and 0.042 ± 0.01 MPa, respectively (Fig. 11G; E=0.49).

[0122] Dimensional and structural characterization of shaped fibrin hydrogels

[0123] To determine the uniformity and characteristics of the hydrogels, several physical properties were examined. Using OCT, gel thickness and homogeneity were measured. A representative OCT image is shown in Fig. 11C. Data from 66 regions of three different gels demonstrated that gel thickness varies in the range of 173.6 ± 1.0 mm to 184.4 ± 1.1 mm (average ± SEM) in any single gel with an average thickness of 180.7 ± 1.0 mm across the 198 regions measured (Fig. 1 ID). OCT measurements were taken in 66 different regions of each gel blank (Figs. 1 IE and 1 IF), and there was no significant variability in gel homogeneity by OCT across a single blank. While occasional air bubbles were observed, they were infrequent and <1 mm in diameter. Transmission electron microscopy (TEM; Figs. 12A and 12B) was performed to visualize the structure of fibrin in cross sections of the gels with and without AAV. The gels were found to have a fibrillar structure, with fibrils randomly oriented but generally uniform in diameter, length, density, and cross-linking density. Scanning electron microscopy (SEM; Figs. 12C and 12D) indicated that the hydrogel surface architecture appeared to be composed of fibrils aligned parallel to the top surface plane with crater-like voids appearing fairly heterogeneously across the surface. In gels with AAV, some AAV particles could be observed along the surface fibers and within the craters (Fig. 12D).

[0124] Even distribution and high density of AAV particles throughout fibrin gel

[0125] AAV did not impact gel structure or fibrin density, but individual particles of approximately 25-31 nm were consistently noted in both TEM (Fig. 12B, black particles) and SEM (Fig. 12D, white particles) with a homogenous distribution; these particles were consistent with the size of AAV particles. Figure 12 TEM and SEM images are representative of three different fibrin gels, and the results suggest AAV particles evenly distribute throughout the gel and reside within the spaces between the fibrils. The presence, even distribution, and density of AAV particles throughout the gels was confirmed by performing immunofluorescence staining for AAV capsids in fibrin-AAV gels (Fig. 12E).

[0126] Modifying the gel enhances the rate of degradation

[0127] An FDA-approved tissue glue; TISSEEL™ was used as a source of fibrinogen and thrombin. The components of the TISSEEL™ kit include lyophilized fibrinogen and thrombin allowing customization of the solution used to resuspend the lyophilized components. The buffer included in a TISSEEL™ kit for resuspension of fibrinogen contains aprotinin, an anti-fibrinolytic that was difficult to remove from fibrin hydrogels. Aprotinin significantly slows the degradation of fibrin gel in vivo. The solution used to resuspend fibrinogen was replaced with 0.01 M sodium citrate, pH 6.0. This resulted in a significant decrease in the time required for in vitro degradation by plasmin of the modified gel in comparison to gels prepared using the included resuspension buffer (p<0.0001; Figs. 12F and 12G). Gels containing aprotinin degraded to only -50%, even after 48 hours, while gels cast with sodium citrate and no aprotinin were 100% degraded within 36-48 hours with or without AAV incorporated (Fig. 12F).

[0128] AAV retains infectivity after release from fibrin gels

[0129] ELISA assay was used to follow diffusion / retention of AAV from the gel at 1 hour, 24 hours, and 48 hours at 37°C. It was found that AAV slowly diffused from fibrin gels (n=7 gels) over a period of days with -55% remaining in the gel at 48 hours (Fig. 12H). This confirmed that the virus does not immediately release from the gel when placed in solution. Longer timepoints (72 hours, 96 hours, and 1 week) were attempted. However, ELISA results were unreliable at these later timepoints given the virus was not stable long-term in solution. When fibrin-encapsulated AAV gels were placed in a 96-well plate of near confluent ARPE-19 cells (Figs. 13A-13F), 5.9 ± 0.7 % of cells were transduced by 72 hours with a significant transduction increase to 27.9 ± 2.9% at 1 week (Fig. 13G, P<0.01), confirming retained RPE infectivity of fibrin-encapsulated AAV. Figure 13D shows the gel starting to degrade on top of the cells with well-defined regions of completely degraded hydrogel (arrows). Figures 13B and 13E highlight the stronger transduction of the cells immediately under the implant compared to the surrounding area.

[0130] Intra-operative procedure for fibrin-AA V epiretinal placement

[0131] A surgical procedure to place and adhere the fibrin gel to the epiretinal surface was developed. Figures 14A-14F show the main surgical steps involved in epiretinal placement of a fibrin-AAV hydrogel with follow-up imaging shown for post-operative day 5 (Figs. 14G- 141).

[0132] A 3 -port core vitrectomy with tri am cinol one-assisted separation of the posterior hyaloid was performed with partial peripheral shave. Figures 14J-14L show a custom injector developed to deploy the gel into the eye via pneumatic foot pedal assist or manual injection. The gel was visible and ready for insertion in Figures 14C and 14L. Deploying the gel under PFO (Fig. 14D) and adding autologous serum around the gel (Fig. 14E) led to consistent adherence of the gel to the epiretinal surface (Fig. 14F). Across all 11 experimental epiretinal pigs, there were no issues with inserting gels or having the gels remain stuck to the area centralis even after PFO to air exchange and then air to gas exchange. All epiretinal surgeries were performed the same, each with autologous serum, to ensure consistency during protocol development.

[0133] Epiretinal placed fibrin degrades quickly without significant fibrin-related complications

[0134] Color fundus photos, OCT, and OCTA were performed at 5-7 days, 2 weeks, and 1 month for all pigs. By day 5-7, the gas bubble had dissipated in all 11 epiretinal pigs, and the fibrin gel had degraded (Fig. 14G). In two pigs, there was very minimal fibrin residue at this timepoint in the periphery (Fig. 14H), but this was completely resolved by week 2. Overall, the pigs tolerated the surgery well with no CRA and no significant inflammation (Fig. 141). Three of the 11 pigs exhibited complications of surgery unrelated to the fibrin gel. One developed a suture abscess at week 2 at the site of the sclerotomy closure despite an unremarkable surgery, and post-operative course required two weeks of systemic and topical antibiotics. The pig never had distress, and the retina appeared healthy with no signs of clinical inflammation. A second pig had a post-vitrectomy cataract develop during the 1- month study period. This limited post-operative photos and OCT analysis. A third pig was noted to have a localized retinal detachment in the periphery at the time of euthanasia. Given there is no pars plana in the pig, peripheral retinal detachments are much more common than in the human patient, and this is the reason why a thorough vitrectomy was not performed peripherally. Of these three pigs that had surgical complications, all had strong and diffuse GFP expression in the RPE with no retinal inflammation noted during clinical or postmortem examination. The minor complications noted were likely due to a lack of hygiene and the vitrectomy procedure, not the fibrin. Pig outcomes and clinical findings of all study pigs are listed in Table 1.

[0135] Epiretinal fibrin-AA V leads to robust and diffuse transduction of the RPE

[0136] GFP expression in eyes receiving epiretinal vs. subretinal AAV-GFP was qualitatively compared. When the retina was reflected on flat mounted sections in the epiretinal group, there was diffuse expression of GFP fluorescence in hexagonal cells, consistent with RPE (Fig. 15 A). In fact, the epiretinal fibrin gels consistently transduced the RPE both centrally (Fig. 15B) andm' the periphery (Figs. 15A and 15C), demonstrating that concentrating AAV therapy on the retinal surface under gas allows the vector to pass through the inner limiting membrane (ILM) and entire retina to selectively transduce the RPE - not only around the initial gel placement. None of the epiretinal pigs had evidence of clinical inflammation (Fig. 15D, representative fundus imaging at month 1). Further, all epiretinal pigs had RPE GFP transduction confirmed with both IF (Fig. 15E) and IHC (Fig. 15F). The fellow eyes of the epiretinal pigs had no GFP expression based on fluorescence microscopy, IF, and IHC testing (Figs. 15G-15I).

[0137] Interestingly, no inconsistent transduction was found in the epiretinal pigs (Figs. 15E and 15F, and Fig. 16A). The majority of RPE cells both centrally and in the periphery were transduced, in contrast to the subretinal group that that had small stretches or even single strongly expressing cells immediately adjacent to cells that had had no GFP fluorescence (Fig. ION and Fig. 16B). The fellow eye exhibited no detectable GFP fluorescence (Fig. 16 C). The epiretinal group also had modest RPE transduction (i.e., no strong / overexpression) with no apparent photoreceptor transduction (Figs. 15E and 15F). Scattered cells exhibiting GFP expression within the neurosensory retina were observed throughout the posterior pole (Fig. 15B).

[0138] Fibrin-mediated gene therapy delivery does not incite retinal inflammation

[0139] Histological analysis using IF and IHC was performed to evaluate ionized calcium- binding adaptor molecule (IBA1; Figs. 16D-16F) and glial fibrillary acidic protein (GFAP; Figs. 16G-16I) expression in experimental and fellow eyes of both surgical cohorts. The cellular immune response in the epiretinal eyes was comparable to the fellow eyes based on minimal IB Al (Fig. 16D) and GFAP expression (Fig. 16G); IBA1 and GFAP are macrophage / microglial and Muller cell gliosis markers, respectively. Neither group exhibited IBA1 expression beyond that observed in unoperated control eyes. However, the subretinal group did exhibit greater GFAP expression through inner retinal layers than was observed in eyes receiving epiretinal implants. This suggests an increased number of activated Muller cells (Fig. 16H) in subretinal vs epiretinal treated eyes. Figure 16 also highlights, again, the consistent and modest GFP transduction of the RPE without photoreceptor transduction (Fig. 16J), all of which are in stark contrast to the subretinal group (Fig. 16K) and fellow eye (Fig. 16L) findings. For all pigs that could be examined post-operatively (i.e., no cataract), there was no clinical evidence of inflammation (z.e., no vasculitis, snowballs, hypopyon, etc.). The representative sections of the subretinal group shown in Figure 16 intentionally do not involve the area of CRA as to not confound the results. However, OCT (Fig. 10B) and histological analysis (Figs. 10L and 10M) of these areas of CRA show abnormal RPE cells (vacuolation, hypo- and hyperpigmentation, atrophy, hypertrophy) and thinned choriocapillaris in the subretinal bleb region. No OCT or histological section across the 11 pigs in the epiretinal group exhibited any evidence of outer retinal atrophy or inflammation.

[0140] No anterior segment or systemic detection of AA V post-operatively

[0141] The cornea, iris, lens, vitreous, optic nerve, and liver were collected following euthanasia and tested by quantitative polymerase chain reaction (qPCR) for AAV genomic DNA. Table 2 shows qPCR results for both eyes and liver when sample was available for testing. There was no AAV detected in any liver sample or any fellow eye sample. For experimental eyes, there was no AAV detection in iris, cornea, lens, or optic nerve samples. The vitreous samples of the epiretinal pigs had AAV detected in three experimental eyes, two of which had high levels detected. The subretinal qPCR data were limited due to fixation for histology purposes; however, two liver samples from subretinal pigs were tested with no AAV detected.

[0142] Table 2. AAV detection after epiretinal fibrin gene therapy in post-mortem pig samples.

[0143] Viral (AAV2) detection by qPCR in post-mortem right eye (surgical eye), left eye (fellow eye), and liver samples in pigs that received epiretinal implants with AAV2 vims (Epi-Imp). Not Detected: - ; Not available: NA; No template control: NTC. Limit of Detection was 27 viral genomes.

[0144] MATERIALS AND METHODS

[0145] Study Design

[0146] The objective of this research was to develop a safer and more effective method of delivering gene therapy to the RPE both centrally and peripherally without subretinal injection. Safety of fibrin gels has been demonstrated in the eye of pigs (Gandhi etal., PLoS One, 15:e0227641 (2020)). Fibrin hydrogels containing AAV2-c4 / 7’7Jwere used in 11 female domestic pigs that were 25-30 kg in weight. To compare this fibrin hydrogel approach to a conventional subretinal approach, five additional female domestic pigs of similar size underwent vitrectomy with subretinal administration of JSN2-eGFP solution without hydrogel but with similar viral titers (Table 1).

[0147] Production of Fibrin-AA V Hydrogel

[0148] All steps in production of fibrin hydrogels were performed aseptically. Fibrin hydrogels were produced using 4 mL TISSEEL™ tissue glue kits (Baxter NDC#00338- 4302-04). The fibrinogen was resuspended in sodium citrate prepared from a clinical anticoagulant solution (Fenwal / NDC-0942-9504-10) diluted to 0.01M with sterile water (Gibco) for injection rather than the included resuspension buffer which contains aprotinin and was omitted to promote rapid degradation of the gel in vivo. Thrombin was reconstituted with the thrombin solution from the same TISSEEL™ Kit, and the vials incubated in a 37°C water bath overnight. The following day, 0.6 mL of sterile tissue culture grade 0.4% trypan blue (Thermo Fisher 15250061) was added to 2 mL of the resuspended fibrinogen and thrombin solutions to slow the polymerization reaction (Gandhi el al., J. Biomed. Mater. Res. A, 109:2357-2368 (2021)). The virus solution (4xlOnvg) was added to the thrombin solution for fibrin gels containing AAV. The fibrinogen solution was then drawn into an 11 mL syringe and the thrombin solution into a 1 mL syringe. The syringes were placed in a 11 : 1 ratio FibriJet Ratio Applicator Assembly. The gelation solution was then dispensed through a FibriJet Blending Connector with Mixer (Nordson Medical) and 18G cannula into a custom mold (Meddux, CO). The molds were then incubated at 37°C for 3 hours to cure. Subsequently, molds were opened and top plates containing the polymerized gels (Fig. 11 A, Fig. 17) were transferred to a 4-well culture dish (Thermo Fisher) in sterile phosphate buffered saline (PBS). The resulting gel was 15.25 x 58.42 x 0.2 mm with final concentrations of 30 mg / mL of fibrin. Within 1 hour of curing the gel blank was cut to various sizes / shapes using punches. Oval shaped gels measuring 1.5 x 5.1 x 0.2 mm were punched for in vitro characterization (e.g., degradation assay, immunostaining) and surgical implantation in the pig, whereas 3 mm circular punches of 0.2 mm thickness were used for the in vitro assay and cell culture experiments. Exact AAV2 titers per gel punch varied for each experiment, and specific titers are included in Table 1 .

[0149] AAV2-eGFP Vectors

[0150] Recombinant AAV2 / 2 vectors expressing enhanced GFP under control of the cytomegalovirus (CMV) promoter were generated as described elsewhere (Giacalone et al.. Hum. Gene Ther., 30,:967-974 (2019); and Wiley et al., Hum. Gene Ther., 27:835-846 (2016)). Recombinant AAV2 / Quad vectors expressing GFP under control of CMV promoter were also generated and used. The vector and titer used for each pig are included in Table 1.

[0151] Fibrin Gel Degradation Protein Assay

[0152] Oval shaped gels of 1.5 x 5.1 x 0.20 mm were degraded at room temperature in 50 pL of sterile water containing 0.14 pg plasminogen (specific activity of plasmin >12500 pM / min / pg, R&D Systems) activated with 0.19 pg tissue plasminogen activator (tPA, 300 U / pg, Sigma) for timepoints between 0 and 48 hours. At the endpoint, any remaining gel was removed from the solution and photographed. The solution was stored at -20°C prior to assay for total protein using a BCA protein assay kit (Abeam) according to the manufacturer’s instructions and, adjusted to account for the plasmin and tPA in the solution, and normalized against total protein released from gels degraded to 100%.

[0153] AA V2 Enzyme-Linked Immunosorbent Assay (ELISA) for Assessment of Viral Diffusion

[0154] Fibrin gels containing IxlO9vg AAV2-eGF and measuring 3 mm in diameter were placed into individual wells of a 96-well plate with 100 pL of ELISA assay buffer / well. Gels and supernatant (z.e., containing any AAV eluted from gel) were harvested from the wells in triplicate at multiple timepoints (1, 24, and 48 hours) and then frozen and stored at -80°C. Once all timepoints were collected, the gels were degraded in 25 pL of sterile H2O containing 2.3 pg of plasminogen (>12500 pM / min / pg, R&D Systems) activated with 0.19 pg of tPA (300 U / pg, Sigma) at 37°C for 3 hours. Gel solutions were diluted to a total volume of 100 pL. Using Progen AAV2 Xpress ELISA kit (Progen Biotechnik), the gel solutions (containing any retained AAV in gel) and supernatants (containing eluted AAV) were analyzed for viral capsids present.

[0155] Cell Culture

[0156] ARPE-19 cells were maintained at 37°C in a 95% air 5% CO2 incubator in DMEM / F12 containing 10% fetal bovine serum and 1% antibiotic / antimycotic. AAV- encapsulated fibrin gels were prepared as described above and punched to 3 mm diameter. Each gel contained IxlO9vg AAV2-eGFP per punch. These punches were directly added to individual wells of a confluent 96-well plate of ARPE-19 cells. Media were changed two days after addition of the hydrogels or viral solution. The plate was monitored daily for GFP expression for 1 week. Each well of the plate was imaged using a Nikon Ti Inverted fluorescence microscope and CCD camera using Nikon Elements software.

[0157] Flow Cytometry

[0158] To determine the total GFP expressing-ARPE-19 cells at 72 hours and 1 week after hydrogel placement, flow cytometry was performed. To isolate the ARPE-19 cells, TrypLE (Gibco) solution was used to release the cells. ARPE19 cells from 3-wells were pooled and placed into 96-well U-bottom plates with DMEM / F12. The plate was then washed twice with calcium / magnesium free (CMF)-PBS and stained with ghost red dye for cell viability. The cells were washed two additional times with CMF-BPS and stored in FACS buffer (5% FBS, 0.1% Sodium Azide, CMF-PBS) in the dark until flow cytometry was performed. Flow cytometry was performed using a ZE5 flow cytometer (Bio-Rad) using SSC (488 / 10), Ghost Red (775 / 50), and eGFP (525 / 35) laser line. For the 72-hour timepoint, three different runs were performed and averaged (9 wells represented). For the 1-week timepoint, nine different runs were performed and averaged (27 wells represented). Animals

[0159] Sixteen 2 to 3 -month-old female domestic pigs (Sus scrofa domesticus) weighing 20.4-35.8 kg, were used for this study. For all animals the right eyes were designated as the experimental / operative eyes, whereas the left eyes (fellow eyes) were used only as control eyes for post-mortem imaging and histological comparison.

[0160] Vitrectomy with Epiretinal Fibrin-AA V Gel Placement

[0161] Surgeries were performed as described elsehwere (Gandhi et al., PLoS One, 15:e0227641 (2020)) with the following modifications. A l-5mm lateral canthotomy was performed on the right eye to improve exposure, and a 5 cc retrobulbar block of cefazolin (100 mg / mL) was administered into the subtenon’s space superotemporally to proptose and stabilize the eye.

[0162] Three-port triamcinolone-assisted 25-gauge pars plana vitrectomy using a Constellation Vitrectomy System (Alcon, Fort Worth, TX) and wide-angled, non-contact Biom® fundus lens (Oculus Surgical, Wetzlar, Germany) was performed by a vitreoretinal surgeon with careful separation of the posterior hyaloid in the area centralis. Eye pressure was maintained using balanced salt solution (BSS) infusion. A peripheral vitrectomy was intentionally not performed on any pig as to provide vitreous support to the peripheral retina.

[0163] For eyes receiving an epiretinal implant (H=11), 1-2 mb of perfluoro-N-octane (PFO) was injected over the central retina. A 4 mm sclerotomy was then fashioned with an MVR blade, and argon laser on the Constellation Vitrectomy System was applied to the choroid prior to entering the globe with a keratome blade. The oval shaped fibrin gel containing approximately 2xl09vg AAV2- G7’P was inserted using a custom instrument (Gandhi etal., PLoS One, 15 :e0227641 (2020); and Mano et al., Transl. Vis. Sci. Technol., 11 :24 (2022)) through the sclerotomy and beneath the PFO bubble. The gel was noted in every case to lie flat on the epiretinal surface. Autologous pig serum (100 - 200 pL), which was obtained and processed same day as the surgery, was injected around the gel while the PFO was still present. This was allowed to set for 10 minutes prior to removing the PFO by fluid-air exchange. The gel remained in place for all 11 epiretinal surgeries. Non-expansile (20%) SF6 gas was then injected. The sclerotomies were sutured with 8-0 vicryl sutures, when indicated, to ensure airtight closure of all sclerotomy sites. The canthus was sutured with 4-0 chromic gut interrupted sutured. Gentamicin 0.3% drops were topically administered to the right eye three times daily for five days post-operatively.

[0164] Color fundus photography, OCT, & OCT-angiography (OCT- A)

[0165] The pig was anesthetized using isoflurane as described elsewhere (Gandhi etal., PLoS One, 15:e0227641 (2020)) for each post-operative examination. Eye drops were instilled for dilation and topical anesthesia as above. Color fundus photos, OCT, and OCTA were performed at 5-7 days, 2 weeks, and 1 month post-operatively. Fundus photographs were obtained using a custom-made video indirect ophthalmoscope. Images were processed using Photoshop (Adobe, San Jose, CA). OCT, OCT-A, and infrared SLO were performed using the Optovue Avanti OCT Angiovue System (Visionix; North Lombard, IL).

[0166] Post-mortem Exam & Histology

[0167] Pigs were euthanized by rapid intravenous injection of a pentobarbital solution (FATAL-PLUS, Vortech; 1 mL / per 10 lbs of body weight) at 1 month post-operatively. Eyes were enucleated and immersed either in PBS for immediate imaging or in Davidson's fixative for later processing. For the eyes immersed in PBS (n=6 epiretinal and n=l subretinal), the eye was immediately dissected within 1 hour of enucleation. The cornea, lens, portion of the remaining vitreous, and optic nerve were collected and stored at -80°C. These eyes were cut to allow flat mounting for examination using a Leica M165FC fluorescence stereomicroscope. Mounted posterior segments (i.e., retina and sclera) were cut into regions of interest and immediately placed in Optimal Cutting Temperature compound (OCT compound) and frozen at -80°C.

[0168] For H&E staining, IHC, and IF, pig eyes fixed in Davidson’s fixative were processed into paraffin ( / / =4 epiretinal and n=3 subretinal) and sectioned at 5 pm. Sections stained with H&E were photographed using a Nikon E-600 microscope with a color CCD camera and NIS-Elements Software (Nikon) or scanned using an Aperio® AT2 microscope slide scanner.

[0169] IHC staining was performed as described elsewhere (Marmorstein et al., Proc. Natl. Acad. Sci. USA, 99: 13067-13072 (2002); and Marmorstein et al., Proc. Natl. Acad. Sci. U S A, 97:3248-3253 (2000)) with the following modifications: following deparaffinization, heat mediated antigen retrieval was performed using an Aptum Biologies 2100 Antigen Retriever in with R-buffer B (Leica). IHC staining was performed using goat anti-GFP (Abeam), rabbit anti-IBAl (Invitrogen), or rabbit anti-GFAP (Novus) with the VectaStain® Elite® ABC- HRP and Vector VIP kits (Vector Labs) according to the manufacturer’s instructions. Nuclei were counterstained with methyl green (Vector Laboratories).

[0170] For IF staining, deparaffinized sections were subject to heat mediated antigen retrieval as above and stained with goat anti-GFP (Abeam), rabbit anti-GFAP (Novus), and rabbit anti-IBAl (Invitrogen). Secondary antibodies, applied in sequence to avoid crossreactivity, were donkey anti-goat conjugated to Alexa Flour 488 (Abeam), followed by goat anti-rabbit conjugated to Alexa Flour 568 (Abeam). Tissue autofluorescence was mitigated using the Vector TrueView Autofluorescence Quenching kit (SP-8400) according to the manufacturer’s instructions. Nuclei were then stained with DAPI (1 : 1000).

[0171] Statistical Analyses

[0172] Statistical analyses were conducted using a two sample two-tailed Mann-Whitney U test to compare two cohorts, including the Young’s modulus between fibrin gels with and without aprotinin and the percentage of GFP -positive ARPE-19 cells based on flow cytometry at 72 hours and 1 week. Kruskal-Wallis one-way analysis of variance (ANOVA) test with Dunn’s post-hoc analysis was performed to compare average gel thickness across three different gels. All statistical tests were performed using GraphPad Prism 4.0b for Macintosh (GraphPad Software, San Diego, CA). All values of gel measurements (e.g., thickness) were reported as averages ± SEM. The fibrin gel protein degradation curves were fit to a second order polynomial (quadratic) formula and compared using least squares regression method. GraphPad Prism 4.0b for Macintosh, Adobe Photoshop, and Microsoft Excel were used to generate figures. Significance for comparisons was defined as P<0.05 for all analyses.

[0173] Gel Thickness Measurement

[0174] Immediately after gel preparation, the metal backing and silicon gasket were removed (Fig. 17). A 14 x 5 grid was drawn along the plastic slide to mark areas for OCT measurement (Fig. 11). Columns were spaced 5 mm apart and rows were spaced 4 mm apart. During imaging, the plastic slide holding the fibrin gel was placed in a clear 10 cm petri-dish with sterile PBS. Gel thickness was measured at the intersections of the grid using an OCT imaging system (Lumedica OQ Labscope; version 2.0). Uniformity of thickness was determined from B scans of the gels at all indicated locations.

[0175] Immunostaining of Gel

[0176] Oval-shaped fibrin hydrogels frozen in optimal cutting temperature compound were sectioned to 10 pm and frozen for storage; sections were held at room temperature overnight for dehydration before staining. Slides were stained with rabbit anti-AAV polyclonal antibody (Invitrogen) and a mouse monoclonal anti-Fibrin antibody 59D8 (Millipore) for 1 hour at room temperature. Slides were washed and reacted with goat anti-rabbit conjugated to AlexFlour 488 (Invitrogen) and goat anti-mouse conjugated to AlexFlour 568 (Abeam) for 1 hour at room temperature. Slides were washed and mounted with Fluoromount (Invitrogen). For controls, secondary antibodies were used without primary antibodies.

[0177] Electron Microscopy

[0178] Both SEM and TEM were as previously described elsewhere (Gandhi et al., Acta Biomater, 67: 134-146 (2018); and Gandhi et al., J. Biomed. Mater. Res. A, 109:2357-2368 (2021)).

[0179] Mechanical Testing

[0180] Mechanical testing was conducted using a spherical indenter (radius = 0.25mm) on the MicroTester G2 (CellScale, Waterloo, ON) to determine Young’s modulus at the Mayo Clinic Biomechanics Core Facility. Each gel specimen was mounted on a microscope slide and submerged in a bath of heated PBS (37°C ± 1°C) during the testing. Gels were indented up to 10% of thickness with the displacement and resulting loading force data used to fit the Hertz contact model: Where F is normal force being applied to the gel, d is displacement, R is radius of the indenter, and v is the material’s Poisson’s ratio (approximated to be 0.5). E is young’s modulus, which was solved for in MATLAB.

[0181] Vitrectomy with Subretinal Viral Solution Administration

[0182] For eyes receiving subretinal AAV2-eGF solution (w=5), vitrectomy was performed as described for the epiretinal group, but no gel was inserted. A 38-gauge polyimide subretinal cannula (38g PolyTip®, MedOne, Sarasota, FL) was used to create a subretinal bleb in the area centralis with the AAV solution (2xl09vg in 300 pL) with slow manual injection using a skilled assistant. The eye pressure was lowered to 10 mmHg during subretinal bleb formation. A fluid-air exchange was performed prior to closure as previously described. There was no PFO injection or 4 mm sclerotomy made for the subretinal group. No laser or steroid was applied for any of the experimental eyes.

[0183] Real Time-qPCR for Detection of AA V in Post-Mortem Samples

[0184] After eye enucleation, samples of the cornea, lens, vitreous, and optic nerve were collected. A sample of the liver was also harvested. Genomic DNA was extracted from these tissues using the Quick-DNA™ Microprep Plus Kit (Zymo Research) and stored at -80°C until use. qPCR assays were performed using a QuantStudio™ 5 Real-Time PCR Instrument (Applied Biosystems). The amplifications were carried out in 20 pL reaction solutions containing 10 pL 2x Taqman® Universal PCR Mastermix (ThermoFisher, 4304437) with 1 pL (250 nM) Taqman® Gene Expression eGFP Probe (ThermoFisher, Assay ID: Mr04329676_mr), each primer (900 nM; Thermofisher, 4331182), and 10 ng of genomic DNA in 9 pL of sterile water. The PCR thermal cycling conditions were: 50°C 2 min; 95 °C 10 min; 95 °C 15 sec; 60 °C 1 min for 40 cycles. Each assay was performed in quadruplicate to check for reproducibility along with a GFP positive control and no template added control. Data were analyzed using QuantStudio™ Software v 1.5.1 and compared to titrated AAV spike-ins to establish a limit of detection (Hill et al., J. Ocul. Pharmacol. Ther., 40:680-687 (2024)). Human Macular Hole Repair Using Fibrin Hydrogel

[0185] In a patient with a long-standing, large macular hole, a 23G pars plana vitrectomy was performed, and the vitreous base was shaved 360 degrees. The inner limiting membrane was removed using intraocular forceps after staining with diluted indocyanine green. PFO was then injected overlying the macula. A 5 mm sclerotomy was created superior-temporally. A 5 mm diameter fibrin gel was folded and inserted into the eye using 23G intraocular forceps and placed on the epiretinal surface under the PFO overlying the macular hole. A direct PFO to silicone oil (1,000 Centistokes) exchange was performed, and the sclerotomies were sutured closed. Serial OCT images and color fundus photography were obtained in clinic starting at post-operative day 1 to assess the gel degradation and the macular hole size and configuration over time.

[0186] OTHER EMBODIMENTS

[0187] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising fibrin hydrogel and from about 108vector genomes to about 1014vector genomes of viral vector per m of said fibrin hydrogel, wherein at least 98 percent of said viral vector is diffusible from said fibrin hydrogel and can transduce cells.

2. The composition of claim 1, wherein said viral vector is selected from the group consisting of an adeno-associated viral vector, a lentivirus-based vector, a herpesvirus-based vector, and an adenovirus-based vector.

3. The composition of claim 1, wherein said viral vector is an adeno-associated viral vector.

4. The composition of any one of claims 1-3, wherein said viral vector comprises nucleic acid encoding a polypeptide heterologous to said viral vector.

5. The composition of any one of claims 1-4, wherein said fibrin hydrogel comprises from about 10 mg / mL to about 80 mg / mL of said fibrinogen polypeptide.

6. The fibrin hydrogel of any one of claims 1-4, wherein said fibrin hydrogel comprises from about 0.1 U / mL of a solution to about 1200 U / mL of said solution of said thrombin polypeptide.

7. The composition of claim 6, wherein said solution is a sodium citrate solution.

8. The composition of any one of claims 1-7, wherein said fibrin hydrogel comprises an agent that can slow polymerization of the gelation mixture.

9. The composition of claim 8, wherein said agent is selected from the group consisting of trypan blue, Evans blue, VisionBlue®, Direct Blue 53, Azovan blue, compounds having thestructure listed under PubChem Compound ID number (CID) 6296, compounds having the structure listed under PubChem CID 9409, Direct Blue 2, melantherine BH, pontamine sky blue 5B, Azo Fuchsine, and Acid Red 99.

10. The composition of any one of claims 1-9, wherein said fibrin hydrogel comprises an agent that can slow degradation of the fibrin hydrogel.11 . The composition of claim 10, wherein said agent is selected from the group consisting of tranexamic acid, aprotinin, aminocaproic acid, and nafamostat.

12. A method for delivering a viral vector to a retina of an eye within a mammal, wherein said method comprises administering a composition of any one of claims 1-11 to said retina.

13. The method of claim 12, wherein said mammal is a human.

14. The method of any one of claims 12-13, wherein said composition is in the form of a fibrin hydrogel patch.

15. The method of claim 14, wherein said administering comprises placement of said fibrin hydrogel patch onto a retinal surface of said retina.

16. The method of claim 15, wherein said retinal surface is an epiretinal surface.

17. The method of any one of claims 15-16, wherein said administering comprises placing a plurflourocarbon (PFO) liquid on said fibrin hydrogel.

18. The method of claim 17, wherein serum or plasma is placed on said PFO liquid.

19. The method of any one of claims 12-18, where liquid in the vitreous cavity is removed and replaced with a substance that said viral vector cannot diffuse through.

20. The method of claim 19, wherein said substance is selected from the group consisting of an air bubble, a silicon oil, and an expansile gas.

21. The method of any one of claims 12-20, wherein at least 98 percent of said viral vector diffuses out of said fibrin hydrogel within 5 days following said administering.

22. A method for treating a mammal having an ocular disease, wherein said method comprises administering a composition to a retina of an eye within said mammal, wherein said composition comprises fibrin hydrogel and about 108vector genomes to about 1013vector genomes per mb of said fibrin hydrogel, wherein at least 98 percent of said viral vector is diffusible from said fibrin hydrogel and can transduce cells within said retina, and wherein said viral vector comprises nucleic acid encoding a polypeptide heterologous to said viral vector.

23. The method of claim 22, wherein said mammal is a human.

24. The method of any one of claims 22-23, wherein said composition is in the form of a fibrin hydrogel patch.

25. The method of claim 24, wherein said administering comprises placement of said fibrin hydrogel patch onto a retinal surface of said retina.

26. The method of claim 25, wherein said retinal surface is an epiretinal surface.

27. The method of any one of claims 25-26, wherein said administering comprises placing a PFO liquid on said fibrin hydrogel.

28. The method of claim 27, wherein serum or plasma is placed on said PFO liquid.

29. The method of any one of claims 22-28, where liquid in the vitreous cavity is removed and replaced with a substance that said viral vector cannot diffuse through.

30. The method of claim 29, wherein said substance is selected from the group consisting of an air bubble, a silicon oil, and an expansile gas.

31. The method of any one of claims 22-30, wherein at least about 98 percent of said viral vector diffuses out of said fibrin hydrogel and transduces retinal cells within said retina, and wherein said transduced cells express said polypeptide.

32. The method of any one of claims 22-31, wherein said ocular disease is selected from the group consisting of retinitis pigmentosa, choroideremia, X-linked retinoschisis, age- related macular degeneration, diabetic retinopathy, glaucoma, achromatopsia, Leber congenital amaurosis, and Batten disease.

33. The method of any one of claims 22-31, wherein said polypeptide is selected from the group consisting of a RPE65 polypeptide, a RSI polypeptide, a MERTK polypeptide, a CERKL polypeptide, a CLN3 polypeptide, a BEST1 polypeptide, and an anti-VEGF antibody.

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