Proteolipid vehicles formulated with fusion-associated small transmembrane (FAST) proteins for eye delivery

Abstract

Provided herein are compositions for delivering a molecular cargo to a cell or tissue of an eye. The composition may include lipids such as a cationic lipid, a helper lipid, an ionizable lipid, a PEGylated lipid, or cholesterol. Some embodiments include a fusion-associated small transmembrane (FAST) polypeptide. Also provided are methods for using the compositions. The method may include delivering a molecular cargo to a cell or tissue of an eye.

Description

Docket No. 062548-504001 WOPROTEOLIPID VEHICLES FORMULATED WITH FUSION-ASSOCIATED SMALL TRANSMEMBRANE (FAST) PROTEINS FOR EYE DELIVERYCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 692,488, filed September 9, 2024, which application is incorporated herein by reference.SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 062548-504001 WO_seqs. xml, created August 13, 2025, which is 10,631 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.FIELD

[0003] The present disclosure generally relates to compositions for molecular cargo delivery, and methods of use. The present disclosure provides compositions such as lipid vehicles or proteolipid vehicles for delivering molecular cargos to cells or tissues of the eye.BACKGROUND

[0004] It is useful to deliver molecular cargos to cells. There is a need for improved compositions and methods of delivery to cells or tissues of interest. For example, there is a need for proteolipid vehicles (PLVs) formulated for delivery to specific cell types.SUMMARY

[0005] Disclosed herein, in some embodiments, are compositions and methods for delivering a molecular cargo to a cell or tissue of an eye. Some embodiments include an ophthalmic composition. The ophthalmic composition may include lipids; a fusion-associated small transmembrane (FAST) polypeptide; and a molecular cargo, wherein the composition is formulated for ocular administration. In some embodiments, the lipids and FAST polypeptide form a proteolipid vehicle for the molecular cargo. In some embodiments, the lipids comprise an ionizable lipid, a helper lipid, cholesterol, a cationic lipid, and polyethylene glycol (PEG)ylated lipid at a lipid ratio, such as a lipid ratio of about 45-85 moles of the ionizable lipid : about 10-50 moles of the helper lipid : about 0-30 moles of cholesterol : about 0-60 moles of the cationic lipid : about 1 -15 moles of the PEGylated lipid. Some embodiments include a composition comprising: lipids comprising a cationic lipid, a helper lipid, an ionizable lipid, polyethylene glycol (PEG) or a PEGylated lipid, and optionally cholesterol; and the molecular cargo. Disclosed herein, in some embodiments, are compositions formulated for delivery of a molecular cargo to a cell or tissue ofDocket No. 062548-504001 WO an eye, comprising: lipids comprising an ionizable lipid, a helper lipid: cholesterol, a cationic lipid, and polyethylene glycol (PEG) at a lipid ratio of about 45-85 moles of the ionizable lipid : about 10-50 moles of the helper lipid : about 0-30 moles of cholesterol : about 0-60 moles of the cationic lipid : about 1 -15 moles of the PEG; and the molecular cargo. In some embodiments, the lipid ratio comprises about 55-75 moles of the ionizable lipid : about 20-40 moles of the helper lipid : about 0-15 moles of cholesterol : about 0-30 moles of the cationic lipid : about 2-8 moles of the PEG. In some embodiments, the lipid ratio comprises about 61 -71 moles of the ionizable lipid : about 25- 35 moles of the helper lipid : 0 moles of cholesterol : 0 moles of the cationic lipid : about 3-5 moles of the PEG. In some embodiments, the lipid ratio comprises 66 moles or about 66 moles of the ionizable lipid. In some embodiments, the lipid ratio comprises 30 moles or about 30 moles of the helper lipid. In some embodiments, the lipid ratio comprises 0 moles of cholesterol. In some embodiments, the lipid ratio comprises 0 moles of the cationic lipid. In some embodiments, the lipid ratio comprises 4 moles or about 4 moles of the PEG. In some embodiments, the lipid ratio comprises about 66:30:4 moles of the ionizable lipid : helper lipid : PEG.

[0006] In some embodiments, the ionizable lipid comprises 1 ,2-dioleoyl-3- dimethylammonium-propane (DODAP or DAP), 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), or Dlin-MC3-DMA. In some embodiments, the helper lipid comprises 2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) or 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the cationic lipid comprises 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP or TAP) or 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA). In some embodiments, the PEG comprises an attached lipid, and is a PEGylated lipid. In some embodiments, the PEGylated lipid comprises 1 ,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). In some embodiments, the ionizable lipid comprises DAP, the helper lipid comprises DOPE or DSPC, and the cationic lipid comprises TAP.

[0007] Some embodiments include a solvent or buffer. In some embodiments, the solvent or buffer comprises phosphate buffered saline (PBS). Some embodiments include a pH of 7.0- 8.0. Some embodiments include the molecular cargo and the lipids at a cargoJipid ratio of 1 :1 to 15:1. Some embodiments include the molecular cargo and the lipids at a cargoJipid ratio of about 1 :5.

[0008] Some embodiments include a recombinant fusion-associated small transmembrane (FAST) polypeptide. Some embodiments include the molecular cargo and the FAST polypeptide at a cargo:FAST ratio of about 1 :1 to 15:1 . In some embodiments, the FAST polypeptide comprises a p10 FAST protein, a p13 FAST protein, a p14 FAST protein, a p15 FAST protein, a p16 FAST protein, or a p22 FAST protein, or a fragment thereof. In some embodiments, the FAST polypeptide comprises a fusion protein comprising a p14 ectodomain and a p15 endodomain. In some embodiments, the FAST polypeptide comprises a p14 transmembrane domain or a p15 transmembrane domain, or a fragment thereof. In some embodiments, the FAST polypeptide comprises an amino acid sequence at least 80% identical, at least 85% identical, atDocket No. 062548-504001 WO least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to any of SEQ ID NOs: 1 -5.

[0009] In some embodiments, the molecular cargo comprises a nucleic acid or a protein. In some embodiments, the molecular cargo comprises a deoxyribonucleic acid (DNA). In some embodiments, the DNA comprises plasmid DNA (pDNA) or an antisense oligonucleotide (ASO). In some embodiments, the molecular cargo comprises a ribonucleic acid (RNA). In some embodiments, the RNA comprises a small interfering RNA (siRNA) or a messenger RNA (mRNA).

[0010] In some embodiments, the ocular administration results in delivery of the molecular cargo to an eye cell or eye tissue. In some embodiments, the cell or tissue of the eye comprises a retina. In some embodiments, the cell or tissue of the eye comprises a retinal cell. In some embodiments, the retinal cell comprises a retinal pigment epithelium (RPE) cell. In some embodiments, the retinal cell comprises a neuronal cell. In some embodiments, the retinal cell comprises a ganglion cell. In some embodiments, the retinal cell comprises a rod cell.

[0011] In some embodiments, the cell or tissue of the eye comprises a macula.

[0012] In some embodiments, the cell or tissue of the eye comprises a cornea. In some embodiments, the cell or tissue of the eye comprises a corneal cell. In some embodiments, the corneal cell comprises corneal epithelium cell.

[0013] Some embodiments include use of the composition in a method. For example, the composition may be administered to a subject. The administration may result in the molecular cargo being delivered to a cell or tissue of an eye. Some embodiments include a method for ocular delivery of a molecular cargo, including administering a composition herein to a subject.

[0014] Some embodiments include administering the composition to an eye of a subject. In some embodiments, the administration is intravitreal. In some embodiments, the administration comprises an injection. In some embodiments, the administration results in expression of the molecular cargo in the eye. In some embodiments, the subject is in need of treatment for a condition or disease of the eye. In some embodiments, the administration treats the condition or disease of the eye. In some embodiments, the molecular cargo comprises a nucleic acid, and the administration results in expression of a protein encoded by the nucleic acid in an eye of the subject. In some embodiments, the subject is in need of treatment for an eye disorder, and the molecular cargo comprises a nucleic acid that treats the disorder. In some embodiments, the disorder comprises Stargardt’s disease, Usher syndrome, or retinitis pigmentosa.

[0015] Other features and advantages of the present disclosure will become apparent from the following detailed description. The detailed description and the specific examples while indicating embodiments of the disclosure are given by way of illustration only. These detailed descriptions and specific examples are provided for the purposes of explanation, and notDocket No. 062548-504001 WO limitation, of the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a schematic of an example cargo delivery by a proteolipid vehicle (PLV) comprising FAST protein to a cell (e.g. a cell of a subject administered the PLV, or administered a composition comprising the PLV).

[0017] FIG. 2A-2D show data relating to an example of delivery of molecular cargos to eye cells in vitro. (FIG. 2A) Fluorescence imaging showing dose-dependent GFP expression from pDNA delivered to immortalized retinal pigment epithelial cells (hTERT RPE-1), retinal pigment epithelium (RPE) cells, or human corneal cells (HCE) using a PLV formulated FAST. (FIG. 2B) Graphs showing dose-dependent knock-down of HPRT1 with siRNA delivered to hTERT RPE-1 (top) or RPE cells (bottom). (FIG. 2C) Immunofluorescence images showing GFP expression from pDNA-eGFP (top) or mRNA-eGFP (bottom) in HCEs at 24 hours and at day 6. (FIG. 2D) Graphs showing IL-2, IL-10, and IFN-y levels at 24 hours, 72 hours, and at 7 days after transfection.

[0018] FIG. 3A-3D show data relating to an example of targeted delivery to mouse eyes via intravitreal administration of an example PLV composition. (FIG. 3A) A schematic of cell types in the eye. (FIG. 3B) Expression of luciferase (Luc) from pDNA as shown by live imaging of the head. Circles outline the region of interest (ROI). (FIG. 3C) Expression of Luc from pDNA as shown by ex vivo eye imaging. (FIG. 3D) Expression of Luc from pDNA as shown by ex vivo liver imaging. No detectable expression in the liver when delivered by PLV.

[0019] FIG. 4A-4C show data relating to an example of targeted delivery of Luc pDNA to the retina via intravitreal administration of an example PLV composition to mice, as assessed by (FIG. 4A) ex vivo imaging Luc expression and quantification via total radiant efficiency; (FIG. 4B) immunohistochemistry; (FIG. 4C) immunofluorescence staining (Rhodopsin: rod cell marker; Tuj1 : neuronal cell marker).

[0020] FIG. 5A-5C show data relating to an example of targeted delivery of eGFP mRNA to the retina via intravitreal administration of an example PLV composition to mice. Expression of eGFP mRNA in the retina was measured by (FIG. 5A) ex vivo I VIS imaging; (FIG. 5B) ELISA; and (FIG. 5C) immunohistochemistry.

[0021] FIG. 6A-6C show data relating to an example of targeted delivery of CRE-mRNA to the retina via intravitreal administration of an example PLV composition to Ai9 mice. Expression of tdTomato following IVT delivery of CRE-mRNA was assessed by (FIG. 6A) ex vivo imaging; (FIG. 6B) immunohistochemistry; and (FIG. 6C) immunofluorescence staining (Rhodopsin: rod cell marker; Tuj1 : neuronal cell marker).

[0022] FIG. 7 is a graph showing knockdown of a neuronal cell target gene (gene X) in mouse eyes 14 days after administration siRNA-PLV at various doses via intravitreal administration. Expression is measured by qPCR.Docket No. 062548-504001 WO

[0023] FIG. 8A-8B show data relating to an example of delivery of nanoplasmid DNA (pDNA) by subretinal injection in mice using four different PLV formulations. (FIG. 8A) Expression of GFP as assessed by fundus imaging at day 9 post injection. (FIG. 8B) Graph showing the expression of GFP as assessed by ELISA at day 14 post injection. The left bar for each condition corresponds to quantification in the Eyecup, whereas the right bar is the measurement for the Retina.

[0024] FIG. 9A-9B show data relating to an example of delivery of nanoplasmid DNA (pDNA) by subretinal injection in African Green Monkey subjects. (FIG. 9A) Expression of GFP as assessed by fundus imaging at day 7 post injection. (FIG. 9B) Expression of GFP as assessed by immunofluorescence. PLV = Proteolipid vehicle, PR = Photoreceptor, PBS = Vehicle control, UT = Untreated, NP = Nanoplasmid. The dashed boxes demarcate regions inside (1 and 2) and outside (3) the treated area.

[0025] FIG. 10A-10J are images showing an example delivery of pDNA to the cells of the retina following intravitreal injection of pDNA in mice. Expression of luciferase was assessed by immunofluorescence. (FIG. 10A) DAPI and Luc staining only. (FIG. 10B-10D) Images that assay the expression of Luc and the marker Calbindin identifies neuronal cells (Calbindin+). (FIG. 10B) Luc expression overlaps with Calbindin staining. (FIG. 10C) Luc expression overlaps with Calbindin staining in some ganglion cells, amacrine cells, and horizonal cells (arrows). (FIG. 10D) No Luc expression is detected in retinas treated with PBS. (FIG. 10E-10G) Images that assay the expression of Luc and the marker Tuj1 which identifies ganglion cells (Tuj1 +). (FIG. 10E) Luc expression overlap with tuj1 staining. (FIG. 10F) Luc expression overlap with tuj1 staining in some ganglion cells (3 arrows point right) and some neurites in the retina (4 arrows pointing left). (FIG. 10G) No Luc expression was detected in retinas treated with PBS. (FIG. 10H-10J) Images that assay the expression of Luc and the marker Rhodopsin that identifies rod cells (Rhodopsin+). (FIG. 10H) Luc expression overlaps with rhodopsin staining. (FIG. 101) Luc expression overlaps with rhodopsin staining in the retina (arrow). (FIG. 10J) No Luc expression was detected in retinas treated with PBS.

[0026] FIG. 11A-11C are images and graphs showing intravitreal administration of FAST- PLVs resulted in significant luciferase expression in the eye. (FIG. 11 A) Whole-body bioluminescent imaging 24 hours after intravitreal injection with 5pg mRNA-FLuc encapsulated within FAST-PLVs or MC3-LNPs into each eye. (FIG. 11 B) Ex vivo organ (eyes, liver, and spleen) bioluminescent imaging of mice from FIG. 11 A, 24 hours after injection. (FIG. 11 C) Quantification of bioluminescent signal from ex vivo organs presented in FIG. 11 B. For each organ, the bar graphs represent from left to right, PBS, PLV-PLV (5ug), and MC3-LNP (5ug). Data are represented as mean ± standard deviation, n=3 biologically independent mice per group.

[0027] FIG. 12A-12H show data related to the intravitreal administration of PLVs in nonhuman primates. (FIG. 12A) Hematoxylin and eosin (H&E) staining of eye cross-sections. (FIG. 12B) Schematic showing the anatomical location of the trabecular meshwork (TM). IFDocket No. 062548-504001 WO imaging and ELISA detected GFP expression was in the TM of NHPs injected with mRNA-GFP PLV (FIG. 12C-12D) or pDNA-GFP PLV (FIG. 12E-12F). GFP was not detected in the TM of NHPs injected with mRNA-GFP LNP, but was detected in the liver (FIG. 12G-12H).

[0028] FIG. 13 shows the clinical score of NHPs at 1 , 3, 7, or 14 days subretinal injection.

[0029] FIG. 14 shows the OCT degeneration score in NHPs at 28 days after subretinal injection or LNP-mRNA or PLV-DNA compared to a control (vehicle).

[0030] FIG. 15A-15E show data related to the subretinal administration of PLVs in nonhuman primates. (FIG. 15A) Hematoxylin and eosin (H&E) staining of eye cross-sections. (FIG. 15B) GFP and vascularization monitored by Fundus imaging on day 1 and day 6. IF imaging and ELISA detected GFP in retinal pigment epithelium cells of NHPs injected with mRNA-GFP PLV (FIG. 15C) or pDNA-GFP PLV (FIG. 15D). ABCA4-FLAG was detected in Rod cells via Antiflag antibody of NHPs injected with pDNA-ABCA4 PLV (FIG. 15E).

[0031] FIG. 16 shows immunofluorescence images of C57 mice administered with mCherry mRNA encapsulated in PLV via IVT. mCherry is detected in trabecular meshwork at 3 days post-injection.

[0032] FIG. 17 shows immunofluorescence images of Ai9 mice administered with Cre mRNA encapsulated in PLV via IVT. tdTomato is detected in the trabecular meshwork at 35 days post-injection.

[0033] FIG. 18A shows data on administering a DNA cargo encoding an anti-VEGF monoclonal antibody via IVT injection in Sprague Dawley rats. Anti-VEGF is detected at therapeutic concentrations in vitreous fluid at Day 23 post-injection (FIG. 18A). FIG. 18B shows immunofluorescence staining of PECAM (measure of neovascularization) 7 days after corneal neovascularization injury in C57 / B6 mice. A single dose of anti-VEGF pDNA in PLV delivered via sub-conjunctival (SCJ) or IVT administration was able to prevent corneal neovascularization as measure by PECAM immunofluorescence.DETAILED DESCRIPTIONI. Introduction

[0034] Non-viral delivery vectors such as lipid nanoparticles (LNPs) may be used for RNA- based gene therapy approaches (e.g., siRNA, miRNA, of mRNA) and may have cost, manufacturing, or immunogenicity advantages over viral vectors. The recent FDA approval of patisiran (Onpattro) has set the stage for more systemic non-viral nucleic acid therapies. LNPs can be formulated with cationic or ionizable lipids that neutralize the anionic charge of nucleic acids and facilitate the endosomal escape of encapsulated nucleic acids through charge-mediated lipid bilayer disruption. Onpattro utilizes the ionizable lipid DLin-MC3-DMA (MC3), which can be utilized for the delivery of mRNA, an approach that may be useful for developing vaccines. MC3 may become positively charged in the acidic endosomal compartment, facilitating endosomal escape. While ionizable lipids have substantially improved tolerability compared to cationic lipids,Docket No. 062548-504001 WO their mechanism of action may potentiate apoptotic cell death, which may translate to tolerability challenges after local delivery and dose-limiting liver toxicity following systemic delivery.

[0035] LNPs may include nanostructures that can be composed of or include a combination of different classes of lipids such as a cationic or ionizable lipid (OIL), structural lipids (e.g., a phospholipid or sterol lipid) and a PEG-conjugated lipid (PEG-lipid). These lipids can selfassemble into LNPs under controlled microfluidic mixing with an aqueous phase containing the nucleic acids. A useful component of an LNP, the GIL may be responsible for electrostatically binding with cargo such as nucleic acids, and for encapsulating the cargo. CILs may facilitate endosomal escape of the cargo. PEG-lipids can prevent aggregation, degradation, and opsonization of the LNPs, while the structural lipids can promote the stability and integrity of the nanoparticle.

[0036] As opposed to positively charged cationic lipids, the charge of ionizable lipids can be dependent upon the pH of the surrounding environment. Ionizable lipids may include three sections: an amine head group, a linker group, and hydrophobic tails. Lipids with a small head group and tails composed of unsaturated hydrocarbons may adopt a conical structure, whereas lipids with a large head group and saturated tails may adopt a cylindrical structure.

[0037] Some lipids used in LNP formulations can be immunogenic, this can be problematic for LNPs used in gene therapy, but advantageous for LNP vaccines and suggests that strategic use of different types of lipids in LNP formulations depending on clinical usage could greatly improve the safety and efficacy of the final product. Cationic lipids may activate Toll-like receptor 4, which may in turn promote a strong pro-inflammatory response with induction of Th1 type cytokines IL-2, IFNy and TNFa. Compared to neutral or anionic LNPs, intravenously injected cationic LNPs may induce an IFN-I response and elevated levels of interferon responsive gene transcripts in leukocytes. Cationic lipids may be added to protein-liposome vaccines to act as adjuvants and stimulate a stronger Th1 immune response, while avoiding overstimulation of a Th2 immune response (production of IL-5 and IL-13) that may be implicated with vaccine immunopathology. The type of cationic liposome can also affect the immune activation by liposome — DNA complexes, for example non-CpG containing Lipofectamine2000 liposomes may induce 5X more cytokine production than either DOTMA / DOPE or DOTMA / CHOL liposomes. Lipofectamine2000 liposomes containing non-CpG motif DNA may also induce IFN and IL-6 production by macrophages from TLR9 deficient mice. Some anionic liposome-protein antigen mixes may show comparable adjuvant activity to that of cationic lipids in cancer vaccine development. Anionic lipid DOPA mixed with ovalbumin may induce antigen-specific CD8(+) cytotoxic T lymphocyte responses and delay growth of OVA-expressing B16-OVA tumors in mice.

[0038] Furthermore, LNPs can stimulate complement activation-related pseudoallergy (CARPA), a hypersensitivity reaction resulting in death in severe circumstances. The use of ionizable lipids may be useful to address limitations surrounding LNP use. Intracellular nucleic acid release may be useful for optimal LNP function.Docket No. 062548-504001 WO

[0039] FAST proteins may include membrane fusion proteins encoded by nonenveloped viruses. These may include non-glycosylated proteins, which in some embodiments are not components of a virion but can be expressed inside virus-infected cells and trafficked to the plasma membrane where they can mediate cell-cell membrane fusion, generating multinucleated syncytia to promote cell-cell virus transmission. In some embodiments, FAST proteins may function at physiological pH without requiring specific cell receptors, allowing them to fuse almost all cell types. FAST proteins may share three common domains. A single transmembrane domain can serve as a reverse signal-anchor sequence to direct a bitropic Nout / Cin type I topology in the membrane. This topology may localize a small N-terminal ectodomain of about 20-40 residues external to the plasma membrane and may position longer C-terminal endodomains (e.g. 40-140 residues) in the cytoplasm.

[0040] The composition may comprise a proteolipid vehicle (PLV). Some embodiments include a PLV, which may be useful for delivering a molecular cargo, such as nucleic acids or polypeptides, to a cell, the proteolipid vehicle having a lipid nanoparticle comprising one or more ionizable lipids and one or more of a fusion-associated small transmembrane (FAST) family of proteins. A PLV formulated with a FAST protein may allow for utilization of ionizable lipids at a minimal molar ratio for neutralizing the anionic charge of the molecular cargo, which may be rather than for using the ionizable lipids for facilitating endosomal escape. Incorporation of the FAST protein into the PLV platform can enhance intracellular delivery and expression of mRNA or pDNA. PLVs may display a favorable immune profile, or be less toxic than other LNPs. PCT publication no. WO2022067446, PCT publication no. WO2012040825, and Brown et al., Safe and effective in vivo delivery of DNA and RNA using proteolipid vehicles, Cell 187, 1-19 (2024), are incorporated herein by reference in their entireties, and describe some PLVs and recombinant FAST proteins.

[0041] Disclosed herein are compositions and methods for delivering a molecular cargo to a cell or tissue, such as a cell or tissue of an eye. The composition may include lipids. The lipids may include a cationic lipid, a helper lipid, an ionizable lipid, polyethylene glycol (PEG) or a PEGylated lipid, cholesterol, or a combination thereof. The composition may include the molecular cargo.

[0042] Some embodiments relate to a method of using the composition. The method may include administering the composition. The administration may result in the molecular cargo being delivered to a cell or tissue of an eye.

[0043] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.Docket No. 062548-504001 WOII. Compositions

[0044] Disclosed herein are compositions. The composition may be useful for delivering a molecular cargo. The composition may be a pharmaceutical composition. The composition may be an ophthalmic composition. For example, the composition may be formulated for ocular administration. The composition may include lipids; a fusion-associated small transmembrane (FAST) polypeptide; and a molecular cargo, wherein the composition is formulated for ocular administration. In some embodiments, the composition includes lipids. In some embodiments, the composition includes a FAST polypeptide (which may be referred to as a FAST protein). In some embodiments, the composition includes a molecular cargo. In some embodiments, the lipids and FAST polypeptide form a proteolipid vehicle for the molecular cargo. The composition may be used in a method herein. The composition may be included as part of a kit. The composition may be generated using a manufacturing method herein.

[0045] Disclosed herein are compositions for delivering a molecular cargo, for example to a cell or tissue of an eye, comprising: lipids comprising a cationic lipid, a helper lipid, an ionizable lipid, polyethylene glycol (PEG), and optionally cholesterol; and the molecular cargo. The composition may further include a fusion-associated small transmembrane (FAST) protein. The composition may be or include a pharmaceutical composition.

[0046] Some embodiments relate to or include a PLV shown in FIG. 1. Any aspect shown in the figure may be used or included in a method or composition herein.

[0047] In some embodiments, the composition further comprises a solvent or buffer. In some embodiments, the solvent or buffer comprises phosphate buffered saline (PBS). In some embodiments, the composition further comprises a solvent. In some embodiments, the composition further comprises a buffer. Some embodiments may include a detergent.

[0048] In some embodiments, the composition comprises a pH of about 7.0-8.0. For example, the pH may be 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about or 8.0. In some embodiments, the pH is 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or a range of pHs defined by any of 2 of the aforementioned pHs.

[0049] In some embodiments, the composition includes an N:P ratio. An N:P ratio may describe a stoichiometry between protonatable nitrogen (N) in a transfection reagent and anionic phosphate groups (P) in a nucleic acid. In some embodiments, the composition comprises a nucleic acid cargo and lipids at an N:P ratio of about 1 :1 to about 15:1. The N:P ratio may be about 1 :1 , about 2:1 , about 2.5:1 , about 3:1 , about 3.5:1 , about 4:1 , about 4.5:1 , about 5:1 , about 6:1 , about 7:1 , about 8:1 , about 9:1 , about 10:1 , about 11 :1 , about 12:1 , about 13:1 , about 14:1 , or about 15:1 , or a range defined by any 2 of the aforementioned ratios. The N:P ratio may be at least 1 :1 , at least 2:1 , at least 2.5:1 , at least 3:1 , at least 3.5:1 , at least 4:1 , at least 4.5:1 , at least 5:1 , at least 6:1 , at least 7:1 , at least 8:1 , at least 9:1 , at least 10:1 , at least 11 :1 , at least 12:1 , at least 13:1 , at least 14:1 , or at least 15:1 . The N:P ratio may be less than 1 :1 , less than 2:1 , lessDocket No. 062548-504001 WO than 2.5:1 , less than 3:1 , less than 3.5:1 , less than 4:1 , less than 4.5:1 , less than 5:1 , less than 6:1 , less than 7:1 , less than 8:1 , less than 9:1 , less than 10:1 , less than 11 :1 , less than 12:1 , less than 13:1 , less than 14:1 , or less than 15:1.

[0050] In some embodiments, the composition includes a nucleic acid cargo and a FAST protein at a FAST mucleic acid ratio. For example, 1 mg of DNA may be combined with about 25 pg of FAST protein. Such a combination may equate to a FAST:DNA ratio of about 1 :20 to 1 :50 (w / w). The FASTmucleic acid ratio may be about 1 :10 (w / w), about 1 :15 (w / w), about 1 :20 (w / w), about 1 :25 (w / w), about 1 :30 (w / w), about 1 :35 (w / w), about 1 :40 (w / w), about 1 :45 (w / w), about 1 :50 (w / w), about 1 :60 (w / w), about 1 :70 (w / w), about 1 :80 (w / w), about 1 :90 (w / w), or about 1 : 100 (w / w), or a range of any 2 of the aforementioned ratios. In some embodiments, the FASTmucleic acid ratio is at least 1 :10 (w / w), at least 1 :15 (w / w), at least 1 :20 (w / w), at least 1 :25 (w / w), at least 1 :30 (w / w), at least 1 :35 (w / w), at least 1 :40 (w / w), at least 1 :45 (w / w), at least 1 :50 (w / w), at least 1 :60 (w / w), at least 1 :70 (w / w), at least 1 :80 (w / w), at least 1 :90 (w / w), or at least 1 OO (w / w). In some embodiments, the FASTmucleic acid ratio is less than 1 :10 (w / w), less than 1 :15 (w / w), less than 1 :20 (w / w), less than 1 :25 (w / w), less than 1 :30 (w / w), less than 1 :35 (w / w), less than 1 :40 (w / w), less than 1 :45 (w / w), less than 1 :50 (w / w), less than 1 :60 (w / w), less than 1 :70 (w / w), less than 1 :80 (w / w), less than 1 :90 (w / w), or less than 1 OO (w / w). The FASTmucleic acid ratio may include a FAST:DNA ratio. The FASTmucleic acid ratio may include a FAST:RNA ratio.Lipids

[0051] Disclosed herein, in some embodiments, are compositions that include a lipid or lipids. The lipid or lipids may be used to deliver a nucleic acid encoding leptin to a cell. A composition herein, such as a vesicle, vehicle or proteolipid vehicle (PLV), may include a lipid. The composition may include lipids. The lipid or lipids may be purified. The lipid or lipids may be pharmaceutically acceptable.

[0052] Disclosed herein, in some aspects, are compositions formulated for delivery of a molecular cargo to a cell or tissue of an eye, comprising: lipids comprising an ionizable lipid, a helper lipid, cholesterol, a cationic lipid, and polyethylene glycol (PEG) at a lipid ratio of about 45- 85 moles of the ionizable lipid : about 10-50 moles of the helper lipid : about 0-30 moles of cholesterol : about 0-60 moles of the cationic lipid : about 1 -15 moles of the PEG; and the molecular cargo. In some embodiments, the lipid ratio comprises about 55-75 moles of the ionizable lipid : about 20-40 moles of the helper lipid : about 0-15 moles of cholesterol : about 0- 30 moles of the cationic lipid : about 2-8 moles of the PEG. In some embodiments, the lipid ratio comprises about 61 -71 moles of the ionizable lipid : about 25-35 moles of the helper lipid : 0 moles of cholesterol : 0 moles of the cationic lipid : about 3-5 moles of the PEG. In some embodiments, the lipid ratio comprises 66 moles or about 66 moles of the ionizable lipid. In some embodiments, the lipid ratio comprises 30 moles or about 30 moles of the helper lipid. In some embodiments, the lipid ratio comprises 0 moles of cholesterol. In some embodiments, the lipid ratio comprises 0Docket No. 062548-504001 WO moles of the cationic lipid. In some embodiments, the lipid ratio comprises 4 moles or about 4 moles of the PEG. In some embodiments, the lipid ratio comprises about 66:30:4 moles of the ionizable lipid : helper lipid : PEG.

[0053] In one aspect, provided herein is a composition for delivering a molecular cargo to a cell or tissue of an eye, comprising: lipids comprising a cationic lipid, a helper lipid, cholesterol, an ionizable lipid, and polyethylene glycol (PEG) at a lipid ratio of about 45-55 moles of the ionizable lipid : about 5-40 moles of the helper lipid : about 0-30 moles of cholesterol : about I Q- 60 moles of the cationic lipid : about 1-15 moles of the PEG; and the molecular cargo. In one aspect, provided herein is a composition. The composition may be useful for delivering a molecular cargo to a cell or tissue of an eye. The composition may include lipids. The lipids may include a cationic lipid. The lipids may include a helper lipid. The lipids may include cholesterol. The lipids may include an ionizable lipid. The composition or lipids may include polyethylene glycol (PEG), or a PEGylated lipid. The lipids may be at a lipid ratio in the composition. The lipid ratio may include about 40-60 moles of the ionizable lipid. The lipid ratio may include about 45-55 moles of the ionizable lipid. The lipid ratio may include about 5-40 moles of the helper lipid. The lipid ratio may include about 0-30 moles of cholesterol, or about 1 -30 moles of cholesterol. The lipid ratio may include about 10-60 moles of the cationic lipid. The lipid ratio may include about 1 - 15 moles of the PEG. The lipid ratio may include the molecular cargo.

[0054] In one aspect, provided herein is a composition for delivering a molecular cargo to a cell or tissue of an eye, comprising: lipids comprising a cationic lipid, a helper lipid, cholesterol, an ionizable lipid, and polyethylene glycol (PEG) at a lipid molar ratio of about 45-55% of the ionizable lipid : about 5-40% of the helper lipid : about 0-30% of cholesterol : about 10-60% of the cationic lipid : about 1 -15% of the PEG; and the molecular cargo. In some embodiments, the lipid molar ratio comprises about 45-55% of the ionizable lipid : about 10-30% of the helper lipid : about 0-20% of cholesterol : about 15-25% of the cationic lipid : about 1 -4% of the PEG. In some embodiments, the lipid ratio comprises about 50% of the ionizable lipid : about 15-28% of the helper lipid : about 0-15% of cholesterol : about 18-20% of the cationic lipid : about 2% of the PEG. In some embodiments, provided herein is a composition. In some embodiments, the composition is useful for delivering a molecular cargo. In some embodiments, the delivery is to a cell or tissue of an eye. In some embodiments, the lipids comprise a cationic lipid. In some embodiments, the lipids comprise a helper lipid. In some embodiments, the lipids comprise cholesterol. In some embodiments, the lipids comprise an ionizable lipid. In some embodiments, the composition or lipids comprise polyethylene glycol (PEG) or a PEGylated lipid. In some embodiments, the lipids comprise a lipid molar ratio of about 45-55% of the ionizable lipid. In some embodiments, the lipids comprise a lipid molar ratio of about 5-40% of the helper lipid. In some embodiments, the lipids comprise a lipid molar ratio of about 0-30% of cholesterol, or about 1 - 30% of cholesterol. In some embodiments, the lipids comprise a lipid molar ratio of about 10-60%Docket No. 062548-504001 WO of the cationic lipid. In some embodiments, the lipids comprise a lipid molar ratio of about 1 -15% of the PEG. In some embodiments, the composition comprises the molecular cargo.

[0055] Some embodiments include lipids at a lipid ratio (e.g. a molar lipid ratio), including a ratio or mole percentage of an ionizable lipid. The lipid ratio may include a number of moles of the ionizable lipid in relation to the other lipids. In some embodiments, the lipid ratio includes about 40-60 moles of the ionizable lipid. In some embodiments, the lipid ratio includes 40-60 moles of the ionizable lipid. In some embodiments, the lipid ratio includes about 45-55 moles of the ionizable lipid. In some embodiments, the lipid ratio includes 45-55 moles of the ionizable lipid. In some embodiments, the lipid ratio includes about 50 moles of the ionizable lipid. In some embodiments, the lipid ratio includes 50 moles of the ionizable lipid. In some embodiments, the lipid ratio includes 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, or 86 moles of the ionizable lipid, or a range of moles defined by any two of the aforementioned integers. In some embodiments, the lipids include 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or 86% ionizable lipid, or a range defined by any two of the aforementioned percentages of the ionizable lipid. The percentage or ratio of lipids that are ionizable lipid may be or include 1 ionizable lipid, or may be or include a combination (e.g., 2 or more) ionizable lipids. In some embodiments, the lipids comprise about 45-55% of the ionizable lipid. In some embodiments, the lipids comprise about 45% of the ionizable lipid. In some embodiments, the lipids comprise about 46% of the ionizable lipid. In some embodiments, the lipids comprise about 47% of the ionizable lipid. In some embodiments, the lipids comprise about 45% of the ionizable lipid. In some embodiments, the lipids comprise about 48% of the ionizable lipid. In some embodiments, the lipids comprise about 49% of the ionizable lipid. In some embodiments, the lipids comprise about 50% of the ionizable lipid. In some embodiments, the lipids comprise about 51% of the ionizable lipid. In some embodiments, the lipids comprise about 52% of the ionizable lipid. In some embodiments, the lipids comprise about 53% of the ionizable lipid. In some embodiments, the lipids comprise about 54% of the ionizable lipid. In some embodiments, the lipids comprise about 55% of the ionizable lipid. In some embodiments, the lipids comprise about 66% of the ionizable lipid. The lipid ratio or the lipid molar ratio may include at least one of the aforementioned percentages or amounts of ionizable lipid. The lipid ratio or the lipid molar ratio may include less than one of the aforementioned percentages or amounts of ionizable lipid.

[0056] Ionizable lipids may include lipids that can carry a charge depending on the pH. Examples of ionizable lipids include but are not limited to: 1 ,2-dioleoyl-3-dimethylammonium- propane (DODAP or DAP), 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), or Dlin-MC3-DMA. In some embodiments, the ionizable lipid comprises 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP or DAP), 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), or Dlin-MC3-DMA. In someDocket No. 062548-504001 WO embodiments, the ionizable lipid comprises DODAP. In some embodiments, the lipids comprise about 50% of DODAP. In some embodiments, the ionizable lipid comprises DODMA. In some embodiments, the ionizable lipid comprises Dlin-MC3-DMA.

[0057] Some embodiments include lipids at a lipid ratio (e.g., a molar lipid ratio), including a ratio or mole percentage of a helper lipid. The lipid ratio may include a number of moles of the helper lipid in relation to the other lipids. In some embodiments, the lipid ratio includes about 5-40 moles of the helper lipid. In some embodiments, the lipid ratio includes 5-40 moles of the helper lipid. In some embodiments, the lipid ratio includes about 10-30 moles of the helper lipid. In some embodiments, the lipid ratio includes 10-30 moles of the helper lipid. In some embodiments, the lipid ratio includes about 15-28 moles of the helper lipid. In some embodiments, the lipid ratio includes 15-28 moles of the helper lipid. In some embodiments, the lipid ratio includes about 15 moles of the helper lipid. In some embodiments, the lipid ratio includes 15 moles of the helper lipid. In some embodiments, the lipid ratio includes about 20 moles of the helper lipid. In some embodiments, the lipid ratio includes 28 moles of the helper lipid. In some embodiments, the lipid ratio includes 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 moles of the helper lipid, or a range of moles defined by any two of the aforementioned integers. In some embodiments, the lipids include 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% helper lipid, or a range defined by any two of the aforementioned percentages of the helper lipid. The percentage or ratio of lipids that are helper lipid may be or include 1 helper lipid, or may be or include a combination (e.g., 2 or more) helper lipids. In some embodiments, the lipids comprise about 5-40% of the helper lipid. In some embodiments, the lipids comprise about 5% of the helper lipid. In some embodiments, the lipids comprise about 6% of the helper lipid. In some embodiments, the lipids comprise about 7% of the helper lipid. In some embodiments, the lipids comprise about 8% of the helper lipid. In some embodiments, the lipids comprise about 9% of the helper lipid. In some embodiments, the lipids comprise about 10% of the helper lipid. In some embodiments, the lipids comprise about 11% of the helper lipid. In some embodiments, the lipids comprise about 12% of the helper lipid. In some embodiments, the lipids comprise about 13% of the helper lipid. In some embodiments, the lipids comprise about 14% of the helper lipid. In some embodiments, the lipids comprise about 15% of the helper lipid. In some embodiments, the lipids comprise about 16% of the helper lipid. In some embodiments, the lipids comprise about 17% of the helper lipid. In some embodiments, the lipids comprise about 18% of the helper lipid. In some embodiments, the lipids comprise about 19% of the helper lipid. In some embodiments, the lipids comprise about 20% of the helper lipid. In some embodiments, the lipids comprise about 21% of the helper lipid. In some embodiments, the lipids comprise about 22% of the helper lipid. In some embodiments, the lipids comprise about 23% of the helper lipid. In some embodiments, the lipids comprise about 24% of the helper lipid. In some embodiments, the lipids comprise about 25% ofDocket No. 062548-504001 WO the helper lipid. In some embodiments, the lipids comprise about 26% of the helper lipid. In some embodiments, the lipids comprise about 27% of the helper lipid. In some embodiments, the lipids comprise about 28% of the helper lipid. In some embodiments, the lipids comprise about 29% of the helper lipid. In some embodiments, the lipids comprise about 30% of the helper lipid. In some embodiments, the lipids comprise about 31% of the helper lipid. In some embodiments, the lipids comprise about 32% of the helper lipid. In some embodiments, the lipids comprise about 33% of the helper lipid. In some embodiments, the lipids comprise about 34% of the helper lipid. In some embodiments, the lipids comprise about 35% of the helper lipid. In some embodiments, the lipids comprise about 36% of the helper lipid. In some embodiments, the lipids comprise about 37% of the helper lipid. In some embodiments, the lipids comprise about 38% of the helper lipid. In some embodiments, the lipids comprise about 39% of the helper lipid. In some embodiments, the lipids comprise about 40% of the helper lipid. The lipid ratio or the lipid molar ratio may include at least one of the aforementioned percentages or amounts of the helper lipid. The lipid ratio or the lipid molar ratio may include less than one of the aforementioned percentages or amounts of the helper lipid.

[0058] In some embodiments, the helper lipid comprises 2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the helper lipid comprises DOPE. In some embodiments, the helper lipid comprises DSPC. In some embodiments, the lipids comprise about 28% of DOPE. In some embodiments, the lipids comprise about 20% of DOPE. In some embodiments, the lipids comprise about 15% of DSPC.

[0059] Some embodiments include lipids at a lipid ratio (e.g., a molar lipid ratio), including a ratio or mole percentage of cholesterol. The lipid ratio may include a number of moles of cholesterol in relation to the other lipids. In some embodiments, the lipid ratio includes about 0-30 moles of the cholesterol. In some embodiments, the lipid ratio includes 0-30 moles of the cholesterol. In some embodiments, the lipid ratio includes about 0-20 moles of the cholesterol. In some embodiments, the lipid ratio includes 0-20 moles of the cholesterol. In some embodiments, the lipid ratio includes about 0-15 moles of the cholesterol. In some embodiments, the lipid ratio includes 0-15 moles of the cholesterol. In some embodiments, the lipid ratio includes about 10-15 moles of the cholesterol. In some embodiments, the lipid ratio includes 10-15 moles of the cholesterol. In some embodiments, the lipid ratio includes about 0 moles of the cholesterol. In some embodiments, the lipid ratio includes 0 moles of the cholesterol. In some embodiments, the lipid ratio includes about 10 moles of the cholesterol. In some embodiments, the lipid ratio includes 10 moles of the cholesterol. In some embodiments, the lipid ratio includes about 15 moles of the cholesterol. In some embodiments, the lipid ratio includes 15 moles of the cholesterol. In some embodiments, the lipid ratio includes 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 moles of the cholesterol, or a range of moles defined by any two of the aforementioned integers. In some embodiments, the lipids include 0%,Docket No. 062548-504001 WO 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% cholesterol, or a range defined by any two of the aforementioned percentages of the cholesterol. The percentage or ratio of lipids that are cholesterol may be or include 1 cholesterol, or may be or include a combination (e.g. 2 or more) cholesterols. In some embodiments, the lipids comprise about 0-30% of cholesterol. In some embodiments, the lipids comprise about 0-20% of cholesterol. In some embodiments, the lipids comprise about 0-10% of cholesterol. In some embodiments, the lipids comprise about 0% of cholesterol. In some embodiments, the lipids comprise about 1% of cholesterol. In some embodiments, the lipids comprise about 2% of cholesterol. In some embodiments, the lipids comprise about 3% of cholesterol. In some embodiments, the lipids comprise about 4% of cholesterol. In some embodiments, the lipids comprise about 5% of cholesterol. In some embodiments, the lipids comprise about 6% of cholesterol. In some embodiments, the lipids comprise about 7% of cholesterol. In some embodiments, the lipids comprise about 8% of cholesterol. In some embodiments, the lipids comprise about 9% of cholesterol. In some embodiments, the lipids comprise about 10% of cholesterol. In some embodiments, the lipids comprise about 11% of cholesterol. In some embodiments, the lipids comprise about 12% of cholesterol. In some embodiments, the lipids comprise about 13% of cholesterol. In some embodiments, the lipids comprise about 14% of cholesterol. In some embodiments, the lipids comprise about 15% of cholesterol. In some embodiments, the lipids comprise about 16% of cholesterol. In some embodiments, the lipids comprise about 17% of cholesterol. In some embodiments, the lipids comprise about 18% of cholesterol. In some embodiments, the lipids comprise about 19% of cholesterol. In some embodiments, the lipids comprise about 20% of cholesterol. The lipid ratio or the lipid molar ratio may include at least one of the aforementioned percentages or amounts of the cholesterol. The lipid ratio or the lipid molar ratio may include less than one of the aforementioned percentages or amounts of the cholesterol.

[0060] Some embodiments include lipids at a lipid ratio (e.g., a molar lipid ratio), including a ratio or mole percentage of a cationic lipid. The lipid ratio may include a number of moles of the cationic lipid in relation to the other lipids. In some embodiments, the lipid ratio includes about 10- 60 moles of the cationic lipid. In some embodiments, the lipid ratio includes 10-60 moles of the cationic lipid. In some embodiments, the lipid ratio includes about 15-25 moles of the cationic lipid. In some embodiments, the lipid ratio includes 15-25 moles of the cationic lipid. In some embodiments, the lipid ratio includes about 18-20 moles of the cationic lipid. In some embodiments, the lipid ratio includes 18-20 moles of the cationic lipid. In some embodiments, the lipid ratio includes about 18 moles of the cationic lipid. In some embodiments, the lipid ratio includes 18 moles of the cationic lipid. In some embodiments, the lipid ratio includes about 20 moles of the cationic lipid. In some embodiments, the lipid ratio includes 20 moles of the cationic lipid. In some embodiments, the lipid ratio includes 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47,Docket No. 062548-504001 WO 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 moles of the cationic lipid, or a range of moles defined by any two of the aforementioned integers. In some embodiments, the lipids include 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% cationic lipid, or a range defined by any two of the aforementioned percentages of the cationic lipid. The percentage or ratio of lipids that are cationic lipid may be or include 1 cationic lipid, or may be or include a combination (e.g., 2 or more) cationic lipids. In some embodiments, the lipids comprise about 10-60% of the cationic lipid. In some embodiments, the lipids comprise about 15- 25% of the cationic lipid. In some embodiments, the lipids comprise about 18-20% of the cationic lipid. In some embodiments, the lipids comprise about 15% of the cationic lipid. In some embodiments, the lipids comprise about 16% of the cationic lipid. In some embodiments, the lipids comprise about 17% of the cationic lipid. In some embodiments, the lipids comprise about 18% of the cationic lipid. In some embodiments, the lipids comprise about 19% of the cationic lipid. In some embodiments, the lipids comprise about 20% of the cationic lipid. In some embodiments, the lipids comprise about 21% of the cationic lipid. In some embodiments, the lipids comprise about 22% of the cationic lipid. In some embodiments, the lipids comprise about 23% of the cationic lipid. In some embodiments, the lipids comprise about 24% of the cationic lipid. In some embodiments, the lipids comprise about 25% of the cationic lipid. The lipid ratio or the lipid molar ratio may include at least one of the aforementioned percentages or amounts of the cationic lipid. The lipid ratio or the lipid molar ratio may include less than one of the aforementioned percentages or amounts of the cationic lipid.

[0061] Examples of cationic lipids include but are not limited to 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP or TAP) and 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA). In some embodiments, the cationic lipid comprises 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP or TAP) or 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA). In some embodiments, the cationic lipid comprises DOTAP. In some embodiments, the cationic lipid comprises DOTMA. In some embodiments, the lipids comprise DOTAP. In some embodiments, the lipids comprise about 20% DOTAP. In some embodiments, the lipids comprise about 18% DOTAP.

[0062] Some embodiments include a PEGylated lipid. For example, a lipid may include PEG. For example, the lipids may be PEGylated, or may include a combination of PEGylated lipids and at least one other type of lipid. A PEGylated lipid may include a PEG length or PEG size. For example, the PEGylated lipid may include 0.5-2 kDa PEG. In some embodiments, the PEG of a PEGylated lipid has a molecular mass of about 0.1 kDa, about 0.2 kDa, about 0.3 kDa, about 0.4 kDa, about 0.5 kDa, about 0.6 kDa, about 0.7 kDa, about 0.8 kDa, about 0.9 kDa, about 0.10 kDa, about 0.11 kDa, about 0.12 kDa, about 0.13 kDa, about 0.14 kDa, about 0.15 kDa, about 0.16 kDa, about 0.17 kDa, about 0.18 kDa, about 0.19 kDa, about 0.20 kDa, about 0.21Docket No. 062548-504001 WO kDa, about 0.22 kDa, about 0.23 kDa, about 0.24 kDa, or about 0.25 kDa, or a range of masses defined by any 2 of the aforementioned masses. In some embodiments, the PEG of a PEGylated lipid has a molecular mass of at least 0.1 kDa, at least 0.2 kDa, at least 0.3 kDa, at least 0.4 kDa, at least 0.5 kDa, at least 0.6 kDa, at least 0.7 kDa, at least 0.8 kDa, at least 0.9 kDa, at least 0.10 kDa, at least 0.11 kDa, at least 0.12 kDa, at least 0.13 kDa, at least 0.14 kDa, at least 0.15 kDa, at least 0.16 kDa, at least 0.17 kDa, at least 0.18 kDa, at least 0.19 kDa, at least 0.20 kDa, at least 0.21 kDa, at least 0.22 kDa, at least 0.23 kDa, at least 0.24 kDa, or at least 0.25 kDa. In some embodiments, the PEG of a PEGylated lipid has a molecular mass of less than 0.1 kDa, less than 0.2 kDa, less than 0.3 kDa, less than 0.4 kDa, less than 0.5 kDa, less than 0.6 kDa, less than 0.7 kDa, less than 0.8 kDa, less than 0.9 kDa, less than 0.10 kDa, less than 0.11 kDa, less than 0.12 kDa, less than 0.13 kDa, less than 0.14 kDa, less than 0.15 kDa, less than 0.16 kDa, less than 0.17 kDa, less than 0.18 kDa, less than 0.19 kDa, less than 0.20 kDa, less than 0.21 kDa, less than 0.22 kDa, less than 0.23 kDa, less than 0.24 kDa, or less than 0.25 kDa.

[0063] Some embodiments include lipids at a lipid ratio (e.g., a molar lipid ratio), including a ratio or mole percentage of PEG (e.g., associated with a lipid) or a PEGylated lipid. The lipid ratio may include a number of moles of the PEGylated lipid in relation to the other lipids. In some embodiments, the lipid ratio includes about 1-15 moles of the PEGylated lipid. In some embodiments, the lipid ratio includes 1-15 moles of the PEGylated lipid. In some embodiments, the lipid ratio includes about 1-4 moles of the PEGylated lipid. In some embodiments, the lipid ratio includes 1 -4 moles of the PEGylated lipid. In some embodiments, the lipid ratio includes about 2 moles of the PEGylated lipid. In some embodiments, the lipid ratio includes 2 moles of the PEGylated lipid. In some embodiments, the lipid ratio includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 moles of the PEGylated lipid, or a range of moles defined by any two of the aforementioned integers. In some embodiments, the lipids include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEGylated lipid, or a range defined by any two of the aforementioned percentages of the PEGylated lipid. The percentage or ratio of lipids that are PEGylated lipid may be or include 1 PEGylated lipid, or may be or include a combination (e.g., 2 or more) PEGylated lipids. In some embodiments, the lipids comprise about 1 -15% of the PEG. In some embodiments, the lipids comprise about 1 -8% of the PEG. In some embodiments, the lipids comprise about 1 -4% of the PEG. In some embodiments, the lipids comprise about 1% of the PEG. In some embodiments, the lipids comprise about 2% of the PEG. In some embodiments, the lipids comprise about 3% of the PEG. In some embodiments, the lipids comprise about 4% of the PEG. The lipid ratio or the lipid molar ratio may include at least one of the aforementioned percentages or amounts of the PEGylated lipid. The lipid ratio or the lipid molar ratio may include less than one of the aforementioned percentages or amounts of the PEGylated lipid.

[0064] In some embodiments, the PEG comprises an attached lipid. In some embodiments, the PEG is a PEGylated lipid. In some embodiments, the PEGylated lipidDocket No. 062548-504001 WO comprises 1 ,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). In some embodiments, the lipids comprise about 2% of DMG-PEG.

[0065] In some embodiments, the lipid ratio comprises about 66:30:4 moles of the ionizable lipid : helper lipid : PEG. In some embodiments, the lipid ratio comprises 66:30:4 moles of the ionizable lipid : helper lipid : PEG. In some embodiments, the lipid percentages are about 66:30:4 moles of the ionizable lipid : helper lipid : PEG. In some embodiments, the lipid percentages are 66:30:4 moles of the ionizable lipid : helper lipid : PEG.

[0066] A lipid may include a neutral lipid. The lipids may include neutral lipids. The lipid or lipids may be ionizable. The lipid or lipids may be ionized. A lipid may include a charged lipid. The lipids may include charged lipids. A charged lipid or lipid may be negatively charged. A charged lipid or lipid may be positively charged.

[0067] A composition herein may include a vehicle. A composition herein may include a proteo-lipid vehicle. A composition herein may include a vesicle. A vehicle may be or include a vesicle. A vehicle may be or include a proteo-lipid vehicle. A vesicle may be or include a proteo- lipid vesicle. A proteo-lipid vehicle may be or include a proteo-lipid vesicle.FAST proteins

[0068] Disclosed herein, in some embodiments, are compositions that include a fusion- associated small transmembrane (FAST) polypeptide. A FAST polypeptide may also be referred to as a FAST protein. A FAST protein may be combined with a lipid or lipids, e.g. as part of a composition. A FAST protein may be used to deliver a nucleic acid encoding leptin to a cell. A composition herein, such as a vesicle, vehicle or proteo-lipid vehicle (PLV), may include a FAST protein. The FAST protein may be purified. The FAST protein may be pharmaceutically acceptable.

[0069] In some embodiments, the composition further comprises a FAST polypeptide. In some embodiments, the composition comprises a recombinant FAST polypeptide. FAST proteins can share three common domains: A single transmembrane (TM) domain; an ectodomain; and an endodomain. The recombinant FAST polypeptide can be synthesized to combine the domains from different FAST proteins. The recombinant FAST polypeptide can comprise fragments of domains from the FAST proteins. The recombinant FAST polypeptide may include 1 , 2, or 3 of the domains. The recombinant FAST protein may include multiple domains from the same FAST protein, or may include multiple domains from different FAST proteins.

[0070] In some embodiments, the recombinant FAST polypeptide comprises a p10 FAST protein, a p13 FAST protein, a p14 FAST protein, a p15 FAST protein, a p16 FAST protein, or a p22 FAST protein, or a fragment thereof. In some embodiments, the FAST polypeptide comprises a p14 FAST protein, or a fragment thereof. In some embodiments, the FAST polypeptide comprises a p15 FAST protein, or a fragment thereof.Docket No. 062548-504001 WO

[0071] In some embodiments, the FAST polypeptide comprises a p14 ectodomain. In some embodiments, the FAST polypeptide comprises a p15 ectodomain. In some embodiments, the FAST polypeptide comprises a p14 TM domain. In some embodiments, the FAST polypeptide comprises a p15 TM domain. In some embodiments, the FAST polypeptide comprises a p14 endodomain. In some embodiments, the FAST polypeptide comprises a p15 endodomain. In some embodiments, the FAST polypeptide comprises a fusion protein comprising a p14 ectodomain and a p15 endodomain. In some embodiments, the FAST polypeptide comprises a p14 transmembrane domain or a p15 transmembrane domain, or a fragment thereof. The FAST polypeptide may include a p14 ectodomain sequence, a p14 transmembrane domain sequence, and a p15 endodomain sequence. The FAST polypeptide may include a p14 ectodomain sequence, a p15 transmembrane domain sequence, and a p15 endodomain sequence.

[0072] The recombinant FAST polypeptide can comprise functional variants of the domains. For example, substitutions within the p14 ectodomain such as V9I, P13A, G14A and E15A may maintain fusion activity. As such, variation in the wild-type p14 ectodomain sequence can occur while maintaining functionality of the recombinant polypeptide.

[0073] The precise boundaries of the domains can vary. For example, the last two residues of a p14 ectodomain (e.g., amino acid chain WE) may be part of the transmembrane domain. The recombinant FAST polypeptide can comprise conservative amino acid substitutions.

[0074] In some embodiments, the recombinant FAST polypeptide comprises a sequence in Table 1. The sequence of the recombinant FAST polypeptide may be at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence in Table 1 . In some embodiments, the sequence of the recombinant FAST polypeptide is less than 75%, less than 80%, less than 85%, less than 86%, less than 87%, less than 88%, less than 89%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identical to a sequence in Table 1 . The recombinant FAST polypeptide may include a combination of sequences from Table 1 or variants herein.Table 1. Example FAST protein sequencesDocket No. 062548-504001 WO

[0075] The FAST protein may be or include the amino acid sequence of SEQ ID NO: 1 .In some embodiments, the FAST protein includes or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 . The FAST protein may be or include the amino acid sequence of SEQ ID NO: 2. In some embodiments, the FAST protein includes or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. The FAST protein may be or include the amino acid sequence of SEQ ID NO: 3. In some embodiments, the FAST protein includes or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. The FAST protein may be or include the amino acid sequence of SEQ ID NO: 4. In some embodiments, the FAST protein includes or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. The FAST protein may be or include the amino acid sequence of SEQ ID NO: 5. In some embodiments, the FAST protein includes or consists of a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5.Docket No. 062548-504001 WO

[0076] The different domains can be linked by one or more linkers. A linker can, for example, provide a structural function. The linker can comprise a peptide linker. An example of a linker includes glycine.

[0077] In some embodiments, the recombinant FAST polypeptide comprises an ectodomain. In some embodiments, the recombinant FAST polypeptide comprises a p14 ectodomain. In some embodiments, the recombinant FAST polypeptide comprises a p14 ectodomain or a fragment thereof. In some embodiments, the p14 ectodomain comprises an amino acid sequence of at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the p14 ectodomain comprises an amino acid sequence of at least 85% sequence identity to SEQ ID NO: 6. In some embodiments, the p14 ectodomain comprises an amino acid sequence of at least 90% sequence identity to SEQ ID NO: 6. In some embodiments, the p14 ectodomain comprises an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, the p14 ectodomain comprises an amino acid sequence of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the p14 ectodomain comprises an amino acid sequence of SEQ ID NO: 6.

[0078] In some embodiments, the recombinant FAST polypeptide comprises a transmembrane domain (TMD). In some embodiments, the recombinant FAST polypeptide comprises a p14 transmembrane domain. In some embodiments, the recombinant FAST polypeptide comprises a p14 transmembrane domain or fragment thereof. In some embodiments, the recombinant FAST polypeptide comprises a p15 transmembrane domain. In some embodiments, the recombinant FAST polypeptide comprises a p15 transmembrane domain or fragment thereof. In some embodiments, the TMD comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the TMD comprises an amino acid sequence of at least 85% sequence identity to SEQ ID NO: 7. In some embodiments, the TMD comprises an amino acid sequence of at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the TMD comprises an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the TMD comprises an amino acid sequence at least 80%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to SEQ ID NO: 7. In some embodiments, the TMD comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the TMD comprises an amino acid sequence of at least 85% sequence identity to SEQ ID NO: 8. In some embodiments, the TMD comprises an amino acid sequence of at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, the TMD comprises an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, the TMD comprises an amino acid sequence at least 80%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identical to SEQ ID NO: 8.Docket No. 062548-504001 WO

[0079] In some embodiments, the recombinant FAST polypeptide comprises a p15 endodomain or a fragment thereof. In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 85% sequence identity to SEQ ID NO: 9. In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, the p15 endodomain comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9.

[0080] In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, the p15 endodomain comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the p15 endodomain comprises an amino acid sequence of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10.

[0081] The recombinant FAST polypeptide can comprise a tag such as an affinity tag. For example, the tag may include a HIS tag, a cMyc tag, or a FLAG tag. The tag may be useful for purification.

[0082] The recombinant FAST polypeptide may be prepared using in vivo or in vitro recombinant protein expression systems. In vivo expression systems comprise a host cell and a vector for expression in the host cell. The host is not particularly limited as long as the cell can express the recombinant FAST polypeptide. Prokaryotic and eukaryotic expression systems are available commercially. The selection of suitable system for expression of the recombinant FAST polypeptide is within the skill of one of ordinary skill in the art. The recombinant FAST polypeptide can, for example, be produced using an insect derived cell line (e.g., Sf9) and the Baculovirus expression system. After expression, the recombinant FAST polypeptide can be extracted and purified by methods known to one of ordinary skill in the art, such as affinity chromatography, sizeexclusion chromatography, hydrophobic interaction chromatography, ion-exchange chromatography.

[0083] The recombinant FAST polypeptide may also be produced using a cell-free expression system. Such a system can comprise a nucleic acid (mRNA or DNA) that encodes the recombinant FAST polypeptide and a cell extract comprising molecules of the transcriptional andDocket No. 062548-504001 WO translational molecular machineries, such as RNA polymerases, ribosomes, tRNAs and amino acids, enzymatic cofactors etc. Cell-free protein expression systems are available commercially and can be used.Cargo

[0084] The molecular cargo can be any cargo that can be encapsulated by the PLV for delivery to a cell or tissue of an eye. For example, the molecular cargo can comprise plasmid DNA (pDNA), mRNA, siRNA, miRNA, self-amplifying mRNA (SAM), genetic adjuvants, promoters, molecular gene editing tools, peptides, epitopes, antigens, small drug molecules, biological molecules, structural macromolecules, and / or therapeutic macromolecules.

[0085] In some embodiments, the molecular cargo comprises a nucleic acid. The nucleic sacid may encode a protein such as a therapeutic protein. The nucleic acid may encode another nucleic acid. For example, the nucleic acid may include a deoxyribonucleic acid (DNA), and encode a ribonucleic acid (RNA). The nucleic acid can be a DNA, a ribonucleic acid (RNA), or a combination thereof. The nucleic acid can be single-stranded or double-stranded. The nucleic acid may be modified. The nucleic acid may be a therapeutic nucleic acid. For example, the nucleic acid may comprise an siRNA or other nucleic acid that inhibits a target.

[0086] In some embodiments, the molecular cargo comprises a deoxyribonucleic acid (DNA). In some embodiments, the DNA comprises plasmid DNA (pDNA) and / or an antisense oligonucleotide (ASO). The DNA may be or include pDNA. The DNA may be or include an ASO.

[0087] In some embodiments, the molecular cargo comprises a ribonucleic acid (RNA). In some embodiments, the RNA comprises a small interfering RNA (siRNA) or a messenger RNA (mRNA). In some embodiments, the RNA is or includes an siRNA. In some embodiments, the RNA is or includes mRNA. In some embodiments, the RNA comprises a modification.

[0088] In some embodiments, the molecular cargo comprises a protein. Some examples of proteins include signaling proteins, enzymes, kinases, receptors, or structural proteins. The protein may be a therapeutic protein. The protein may be anti-inflammatory. The protein may be a protein normally expressed in the eye.III. Methods

[0089] The composition disclosed herein can be used to deliver a molecular cargo to a cell or tissue of an eye. Accordingly, some aspects include a method for delivering a molecular cargo to a cell or tissue of an eye. The method may comprise providing a composition disclosed herein. Some embodiments include administering a composition herein to a subject, wherein the composition delivers a molecular cargo to a cell or tissue of an eye. The administration may treat the subject. The administration may treat a disease. The administration may treat an eye disease in the subject.Docket No. 062548-504001 WO

[0090] The administration may be ocular. In some embodiments, the ocular administration results in delivery of the molecular cargo to an eye cell or eye tissue. The cargo may include a nucleic acid such as plasmid DNA or mRNA, which may encode a protein. The protein may be reduced in a diseased eye cell. In such cases, delivery of the nucleic acid to an eye cell may increase or restore protein levels. The increase or restoration may be measured and shown to be increased relative to a baseline measurement such as a baseline measurement before a treatment. The cargo may include a nucleic acid such as an ASO or siRNA, which may target another nucleic acid encoding a protein. The protein may be overexpressed in a diseased eye cell. In such cases, delivery of the nucleic acid to an eye cell may decrease or restore normal protein levels. The decrease or restoration may be measured and shown to be decreased relative to a baseline measurement such as a baseline measurement before a treatment.

[0091] An administration of a composition herein may be to an eye. Administration may result in delivery to an eye tissue such as a corneal or retina. In some embodiments, the composition is delivered to a cornea. In some embodiments, the composition is delivered to a retina. Administration may result in delivery to an eye cell such as a corneal cell, retinal cell, neuronal cell, rod cell, or ganglion cell. In some embodiments, the composition is delivered to a corneal cell. In some embodiments, the composition is delivered to a retinal cell. In some embodiments, the composition is delivered to a neuronal cell. In some embodiments, the composition is delivered to a rod cell. In some embodiments, the composition is delivered to a ganglion cell.

[0092] In some embodiments, the cell or tissue of the eye is a cornea. In some embodiments, the cell or tissue of the eye is a corneal epithelium cell. In some embodiments, the cell or tissue of the eye is a retina. In some embodiments, the cell or tissue of the eye is a retinal cell. In some embodiments, the cell or tissue of the eye is a neuronal cell of the retina. In some embodiments, the cell or tissue of the eye is a ganglion cell of the retina. In some embodiments, the cell or tissue of the eye is a rod cell of the retina. In some embodiments, the cell or tissue of the eye is the macula part of the eye.

[0093] Delivering the molecular cargo can result in expression of the molecular cargo. Expression of the molecular cargo may be assessed by various methods, such as ELISA, RNAScope, immunohistochemistry, and immunofluorescence.

[0094] In some embodiments, delivering the molecular cargo results in expression of the molecular cargo in the cell or tissue of the eye at a level of at least about 1 .5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold greater than expression in a non-eye cell or tissue. In some embodiments, delivering the molecular cargo results in expression of the molecular cargo in the cell or tissue of the eye at a level of less than about 2-fold, less than about 3-fold, less than about 4-fold, less than about 5-fold, less than aboutDocket No. 062548-504001 WO 6-fold, less than about 7-fold, less than about 8-fold, less than about 9-fold, less than about 10- fold, less than about 15-fold, less than about 20-fold, less than about 50-fold, or less than about 100-fold greater than expression in a non-eye cell or tissue. In some embodiments, the non-eye cell or tissue is liver.

[0095] The cell or tissue of the eye can be from any animal, for example, a mammal. The mammal can for example be a human or a non-human primate. In some embodiments, the cell or tissue of the eye is a cell or tissue of a mammalian eye. In some embodiments, the cell or tissue of the eye is a cell or tissue of a human eye.

[0096] The composition disclosed herein may be used for delivery by a variety of methods. For example, the composition may be for in vivo, ex vivo, and / or in vitro delivery. In vivo delivery can comprise, for example, injection. The composition may be formulated to comprise a pharmaceutically acceptable carrier.

[0097] In some embodiments, the delivery comprises in vivo delivery. In some embodiments, the delivery comprises ex vivo delivery. In some embodiments, the delivery comprises in vitro delivery.

[0098] The composition disclosed herein may be used with any suitable route of administration. The administration may include an injection. The administration may be at or near a target cell or target tissue, such as at or near a subject’s eye. The administration may be intravitreal or subretinal. In some embodiments, the method comprises administering the composition by intravitreal injection. In some embodiments, the method comprises administering the composition by subretinal injection.

[0099] The composition disclosed herein may be used to deliver a genetic medicine to treat conditions or diseases relating to the eye, such as Stargardt’s disease, Usher syndrome, and retinitis pigmentosa. As used herein, the term “genetic medicine” means any nucleic acid that can be used to treat or prevent a condition or disease in a subject. The nucleic acid may be single stranded or double stranded, including DNA, RNA, and DNA / RNA hybrid molecules, and encompasses modifications and analogues.

[0100] Some embodiments include administering the composition to an eye of a subject, where the composition includes a molecular cargo. In some embodiments, the molecular cargo comprises a nucleic acid, and the administration results in expression of a protein encoded by the nucleic acid in an eye of the subject. The administration may include an effective amount, for example an amount of the composition effective to treat a disease such as an eye disease or disorder. In some embodiments, the subject is in need of treatment for an eye disorder, and the molecular cargo comprises a nucleic acid that treats the disorder. Examples of some disorders that may be treated include Stargardt’s disease, Usher syndrome, or retinitis pigmentosa.Docket No. 062548-504001 WOIV. Manufacturing

[0101] The composition disclosed herein may be manufactured by a method comprising: providing an aqueous phase comprising the molecular cargo and the fusion-associated small transmembrane (FAST) polypeptide; providing an organic phase comprising the lipids; and mixing the aqueous phase and the organic phase together, thereby forming a mixed phase comprising a molecular cargo-proteolipid vehicle.

[0102] The organic phase can be generated by, for example, combining the ionizable lipid, the helper lipid, the cationic lipid, the PEGylated lipid, and optionally the cholesterol together to form combined lipids; dehydrating the combined lipids; and rehydrating the combined lipids in an organic solvent, such as ethanol.

[0103] The lipids of the organic phase can be sourced commercially, or synthesized to the desired specifications.

[0104] Mixing the aqueous phase and the organic phase can cause the molecular cargo to be encapsulated by a proteolipid vehicle (PLV) comprising the lipids and the recombinant FAST polypeptide, forming the molecular cargo-proteolipid vehicle.

[0105] The aqueous phase and the organic phase can be mixed by any suitable method. For example, the mixing can comprise microfluidic mixing. The method can be performed using a microfluidic device.

[0106] Microfluidic mixing comprises the manipulation of liquids at micro-scale dimensions using a microfluidic device. Microfluidic devices for lipid nanoparticle production include for example T- or Y-shaped microfluidic devices, sheath-flow (3 inlets)-type microfluidic devices, chaotic mixer devices, planar asymmetric split-and-recombine micromixers, and microfluidic devices with baffle structures (iLiNP).

[0107] Microfluidic mixing can be performed using commercial microfluidic mixing instruments, such as NanoAssemblr® (Precision NanoSystems, Vancouver, BC).

[0108] Mixing of the aqueous phase and the organic phase can comprise the use of peristaltic pump. For example, a peristaltic pump can be used to control the flow rate of the aqueous phase and the organic phase during microfluidic mixing. Controlling the flow rate can, for example, allow production of lipid nanoparticles of specific size ranges. The peristaltic pump can comprise a T-junction or a Y-junction.

[0109] Other methods for making lipid nanoparticles, such as methods that comprise high- pressure homogenization or microemulsion, may also be used.

[0110] Mixing the aqueous phase and the organic phase can cause formation of a mixed phase comprising the molecular cargo-proteolipid vehicles.

[0111] The method can further comprise exchanging a solution of the mixed phase for a buffer. Any buffer that preserves the integrity of the molecular cargo-proteolipid vehicles can be used. The buffer can be buffered by, for example, phosphate and / or acetate. For example, theDocket No. 062548-504001 WO buffer can comprise phosphate buffered saline (PBS). The buffer can be a buffer suitable for administration, for example, by injection.

[0112] Any suitable method of solution exchange, including but not limited to dialysis and filtration, may be used. Types of filtration suitable for solution exchange of the mixed phase include, for example, tangential flow filtration (TFF), and dead-end filtration.

[0113] The method can further comprise concentrating the mixed phase, to form a concentrated product enriched for the molecular cargo-proteolipid vehicles.

[0114] Suitable methods for concentrating the mixed phase include but are not limited to evaporation, and filtration. Types of filtration suitable for concentration of the mixed phase include, for example, tangential flow filtration (TFF), and dead-end filtration.

[0115] Concentration of the mixed phase can occur before, during and / or after exchanging of the solution of the mixed phase. For example, tangential flow filtration can serve to both exchange the solution and concentrate the mixed phase. The mixed phase can also be concentrated first, for example, in the solution where the molecular cargo-proteolipid vehicles are formed, followed by exchanging the solution for the buffer.

[0116] The mixed phase, the solution-exchanged mixed phase, and the concentrated product can be sterilized, for example, by filter sterilization.V. Kits

[0117] In yet another aspect, provided herein is a kit for delivering a molecular cargo to a cell or tissue of an eye. The kit may comprise lipids. The lipids may comprise an ionizable lipid. The lipids may comprise a helper lipid. The lipids may comprise cholesterol. The lipids may comprise a cationic lipid. The lipids may comprise polyethylene glycol (PEG) or a PEGylated lipid. The lipids may be provided separately in the kit. The lipids may be provided together in a container at a lipid ratio. The kit may comprise a recombinant fusion-associated small transmembrane (FAST) polypeptide. The kit may comprise a molecular cargo. The kit may comprise an instruction manual. The kit may comprise other components and / or reagents. Some embodiments include reagents for producing any aspect or combination of aspects used or included in a composition or method herein.VI. Definitions

[0118] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure pertains.

[0119] As used herein, the singular forms “a”, “an” and “the” include the plural forms unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. These terms can convey that any combination is specifically contemplated. Solely for illustrativeDocket No. 062548-504001 WO purposes, the expression “A, B, and / or C” can mean A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.

[0120] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, and words having similar meanings such as "including", "having", “containing” and their derivatives, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, and do not exclude the presence of additional or unrecited features, elements, components, groups, integers and / or steps.

[0121] The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unrecited features, elements, components, groups, integers and / or steps.

[0122] Further, terms of degree such as "substantially", "about" and "approximately" as used herein mean within an acceptable error range for the particular value, or a reasonable amount of deviation of the modified term, as determined by one of ordinary skill in the art. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0123] More specifically, the term “about” means plus or minus 0.1 to 20%, 5-20%, or 10- 20%, 10%-15%, preferably 5-10%, most preferably about 5% of the number to which reference is being made. The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0124] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” may be used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0125] A “nucleic acid” may include single stranded or double stranded polymers of nucleotide monomers, including DNA, RNA, or DNA / RNA hybrid molecules, or may encompass modifications or analogues.

[0126] The terms “polypeptide” and “protein” are used interchangeably and refer to a polymer of at least two amino acids. The amino acids may be natural amino acids, or analogues or derivatives thereof. The term encompasses modifications.

[0127] The term “functional fragment” in the context of a protein or polypeptide can mean a fragment that is capable of having one or more activities of a reference protein or polypeptide.

[0128] A sequence herein may encode or include a conservative amino acid substitution. As used herein, the term “conservative amino acid substitution” refers to a substitution of an aminoDocket No. 062548-504001 WO acid residue with another amino acid residue without abolishing the protein's desired properties. The substitution can be with an amino acid of the same class. These classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, such as aspartic acid and glutamic acid; and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and cysteine. A conservative amino acid substitution also include substitution with a chemically derivatized residue or non-natural amino acid so long as the substitution does not abolish the protein's desired properties.

[0129] The term "sequence identity" as used herein refers to the percentage of sequence identity between two amino acid sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. The sequences can be of the same length or of different lengths. Gaps may be artificially introduced into the sequence to attain proper alignment. Once the optimal alignment has been set up, the degree of identity is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions. As would be understood by a person skilled in the art, the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. Various sequence analysis software and online programs can be used to determine sequence identity, such as BLAST.

[0130] In determining a sequence identity, thymine (T) and uracil (U) may be interchangeable. T and U may be interchangeable when describing an oligonucleotide. In some embodiments, Ts and Us are interchangeable depending on whether the oligonucleotide is an RNA or DNA, where RNA includes U and DNA includes T. Where a T is provided as a part of DNA, a U may be envisaged as part of an RNA, and vice versa.

[0131] Any discrepancies between the written description and a sequence listing submitted herewith may typically be resolved in favor of the written description.

[0132] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0133] The term “treat”, “treating” or similar terms may include achieving a beneficial or desired result, which can comprise (1) diminishing the extent of or reversing a disease or condition; (2) slowing, delaying, or arresting the progression of a disease or condition; (3) ameliorating or alleviating one or more symptoms of a disease or condition.

[0134] The term “prevent”, “preventing” or the like, in the context of a disease or condition, may include avoiding the development of a disease or condition and / or the onset of one or more symptoms.

[0135] The term “effective amount” may include an amount that is sufficient to elicit a desired effect or response, such as to reduce weight gain or to maintain weight loss.Docket No. 062548-504001 WO

[0136] As used herein, the term “administering”, “administer”, or the like may include introducing the PLV of the present disclosure and / or a molecule, as the case may be, to a subject by a variety of methods. The term can encompass external and internal administration. Internal administration can refer to, for example, where a molecule is generated inside the subject’s body. For example, administering a polypeptide may encompass administering a nucleic acid encoding the polypeptide, and the polypeptide is expressed from the nucleic acid inside a cell of the subject. The term can encompass sequential administration, e.g. introducing the compound at a regular interval.

[0137] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[0138] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.EXAMPLESExample 1 : In vitro delivery of molecular cargos to retinal and corneal cells

[0139] Plasmid DNA (pDNA) for GFP expression was delivered to hTERT-immortalized retinal pigment epithelial cells (hTERT RPE-1 ), isolated primary mouse retinal pigment epithelium (RPE) cells, and human corneal epithelium (HCE) cells using a PLV formulated with FAST. 0.1 ug, 1 ug, or 2ug of the PLV was used. FIG. 2A shows GFP was expressed at a dose-dependent manner. The FAST protein used in Examples 1-10 had the amino acid sequence of SEQ ID NO: 1 , which includes a p14 ectodomain, a p14 transmembrane domain, and a p15 endodomain.

[0140] siRNA targeting the HPRT1 gene was delivered to hTERT-immortalized retinal pigment epithelial cells (hTERT RPE-1) isolated primary mouse retinal pigment epithelium (RPE) cells at different doses using a PLV formulated with FAST. FIG. 2B shows dose-dependent knockdown of the expression of HPRT 1 .

[0141] pDNA or mRNA for GFP expression was delivered to EpiSkin HCEs using a PLV formulated with FAST. GFP expression was measured by immunofluorescence at hour 24 and at day 6 after delivery. FIG. 2C shows that with pDNA, durable GFP expression can be achieved. In vitro delivery resulted in minimal pro-inflammatory response as measured by levels of IL-2, IL-10 and IFN-y (FIG. 2D).Example 2: In vivo delivery to the retina by IVT injection

[0142] Different cargos were delivered to the eye by intravitreal injection in mice (FIG. 3A). Expression was measured by in vivo imaging, ex vivo imaging, immunohistochemistry and immunofluorescence staining of retinal sections, and qPCR for gene silencing.Plasmid DNADocket No. 062548-504001 WO

[0143] Following IVT injection of plasmid DNA for luciferase expression using PLVs or LNPs, Luc expression was measured by live imaging of the head, ex vivo imaging of the liver and ex vivo imaging of the eye. FIG. 3B-3C show both PLV and LNP deliveries allowed Luc expression in the head and eye. However, LNP resulted in delivery to the liver and abnormal livers (FIG. 3D).

[0144] Targeted delivery of Luc pDNA to the retina via intravitreal administration of an example PLV composition to mice resulted in Luc expression as assessed by ex vivo imaging (FIG. 4A) and immunohistochemistry (FIG. 4B). Immunofluorescence staining shows Luc expression in Rod cells and neuronal cells of the retina (FIG. 4C). mRNA

[0145] PLV delivery of mRNA for expression of GFP was investigated. Mice were injected with 10ug of mRNA eGFP (5uL IVT at 2mg / mL) encapsulated by the PLV. 16 hours post-delivery, eyes were harvested and ex vivo imaging was performed with I VIS imaging system (FIG. 5A). Total GFP expression in the eye was quantified by GFP ELISA (FIG. 5B). Eyes were fixed with 10% NBF for immunohistochemistry using GFP antibody (FIG. 5C). FIG. 5A-5C show robust expression from mRNA throughout the retina.

[0146] PLV formulated Ore mRNA was delivered to Ai9 mice by IVT. FIG. 6A-6C show robust activation of tdTomato following IVT delivery. tdTomato recombination and expression in the retina was measured by whole eye imaging (FIG. 6A). IHC confirmed tdTomato expression throughout the retina (FIG. 6B). Finally, co-localization of tdTomato and rod and neuronal cell markers confirm delivery to these cell types (FIG. 6C). siRNA

[0147] siRNA targeting a neuronal cell target gene (gene X) was delivered to mouse eyes by IVT administration. Expression of the gene 14 days post-injection was measured by qPCR. FIG. 7 shows knockdown of the gene at various doses.Example 3: Delivery of pDNA by subretinal delivery

[0148] Wild-type mice were subject to one of four different PLV formulations encapsulating a GFP nanoplasmid cargo (GFP-pDNA), administered via subretinal injection (Table 1). The formulations in the table included varying amounts of lipids, and were similar to the lipid formulations in other examples provided herein such as in Example 5.Table 1. Formulations for subretinal injectionDocket No. 062548-504001 WO

[0149] Expression was monitored by Fundus imaging on Day 9 (FIG. 8A) and GFP ELISA of eyecup and retina at Day 14 post subretinal injection (FIG. 8B). GFP ELISA shows that expression in the eyecup and retina (non-photoreceptor cells) was robust 14 days after subretinal injection (FIG. 8B). Expression levels achieved with different formulations as measured by GFP ELISA correlate with Fundus imaging results.Example 4: Additional immunofluorescence imaging shows expression of luciferase in the cells of the retina following IVT

[0150] FIG. 10A-10J show immunofluorescence staining with markers for different cell types and Luc in the retina following intravitreal injection of pDNA-Luc in mice. Expression of Luc colocalized with markers for neuronal cells (Calbindin+), ganglion cells (Tuj1+), and rod cells (rhodopsin+). Luc expression overlapped with Calbindin, Tuj1 , and rhodopsin staining in the retina.Example 5: Additional luciferase expression data showing delivery of cargo to the eye

[0151] Delivery of mRNA-FLuc in FAST-PLV by intravitreal injection was compared to delivery of MC3-LNP by intravitreal injection. The MC3-LNP formulation was DLin-MC3- DMA / DSPC / Cholesterol / DMG-PEG2000 with the molar ratio 50:10:38.5:1.5. The MC3-LNP formulation was a lipid nanoparticle (LNP) formulation, and did not include a FAST protein. MC3- LNP formulation This formulation was provided for comparison to proteo-lipid vehicles (PLVs) that included FAST proteins. Intravitreal injection was conducted by inserting a 34G Hamilton syringe into the limbus of the eye and injecting 2pL of test article into the vitreous humor.

[0152] Intravitreal administration of FAST-PLVs resulted in significant luciferase expression in the eye (FIG. 11A-11C), with ex vivo imaging demonstrating that the bulk of expression coming from the back of the eye where the retina is located (FIG. 11 B). Significant off- target liver and spleen luciferase expression was observed in mice injected with MC3-LNPs (FIG. 11A-11C). Taken together, these results demonstrate that FAST-PLV formulations drove robust expression in the local area or organ subsequent to localized administration of mRNA to the eye. In contrast, current clinically-approved LNP formulations resulted in substantial off-target liver and spleen expression.Lipid Formulation

[0153] A lipid formulation designated 41 N was made by combining the ionizable lipid (DODAP), helper lipid (DOPE) and PEGylated lipid (DMG-PEG2000) in the following lipid molar ratio: 41 N (66:30:4). The lipids were heated in a 37°C water bath for 1 min, vortexed for 10 seconds each, then combined and vortexed for 10 seconds. The combined lipid mixture was dehydrated in a rotavapor at 60 rpm for 2 hours, under vacuum, then rehydrated with 14 mL 100%Docket No. 062548-504001 WO ethanol, and sonicated (Branson 2510 Sonicator) at 37°C, set to sonication of 60. The lipid formulation was aliquoted in 500 pL batches and stored at -20°C.

[0154] An optimized FAST-PLV formulation was finalized using the 41 N lipid formulation and a 5:1 molar ratio of DODAP to pDNA.FAST-PLV Construction

[0155] FAST-PLVs were made with lipid formulation 41 N. The NanoAssemblr Benchtop microfluidics mixing instrument (Precision NanoSystems, Vancouver, 974 BC, NIT0013, and NA- 1 .5-88, respectively) was used to mix the organic and aqueous solutions and make the PLVs. The organic solution consisted of lipid formulation. The aqueous solution consisted of nucleic acid cargo, 5 nM FAST protein, and 10 mM acetate buffer (pH 4.0). The Benchtop NanoAssemblr running protocol consisted of a total flow rate of 12 mL / min and a 3:1 aqueous to organic flow rate ratio. PLVs were dialyzed in 8000 MWCO dialysis tubing (BioDesign, D102) clipped at one end. The loaded tubing was rinsed with 5 mL of double distilled water and dialyzed in 500 mL of Dialysis Buffer (ENT 1844) with gentle stirring (60 rpm) at ambient temperature for 1 hour and was repeated twice with fresh Dialysis Buffer. PLVs were concentrated using a 100 kDa Ultra filter (Amicon, UFC810096) according to the manufacturer’s instructions. PLVs were filter sterilized through 0.2 pm Acrodisc Supor filters (Amicon, UFC910008).Example 6: Intravitreal (IVT) PLV injection to deliver cargo to the non-human primate (NHP) eye

[0156] African green monkeys {Chlorocebus sabaeus) were undergone baseline screening by physical exams and ocular exams consisting of tonometry, slit lamp biomicroscopy, fundoscopy, fundus color and fluorescence imaging, confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomograph (OCT). Animals with normal findings were enrolled and assigned to treatment groups. Baseline screening and subsequent procedures were performed under sedation with intramuscular ketamine (8mg / kg) and xylazine (1 .6 mg / kg) to effect, and pupil dilation with topical 10% phenylephrine, 1% tropicamide and / or 1% cyclopentolate.

[0157] For IVT administration, animals were subjected to one of three different encapsulation and GFP-encoding cargo combinations, administered via IVT injection at the doses listed in Table 2.IVT dosing

[0158] Topical proparacaine 0.5% were administered, allowing a minimum of 30 seconds to take effect. An eye speculum was placed prior to disinfecting the ocular surface with 5% Betadine solution followed by a sterile 0.9% saline rinse. INVT injections were performed according to the treatment assignment using a 31 -gauge 5 / 16-inch needle / syringe (Ulticare VetRx U-100, or equivalent) inserted inferior temporal at the level of the ora serrata ~2 mm posterior toDocket No. 062548-504001 WO the limbus. Following injection, a topical suspension tobramycin and dexamethasone or (or equivalent) was administered.Table 2. Formulations for intravitreal injection in NHP

[0159] At day 6 after IVT injection, hematoxylin and eosin (H&E) staining of the cross section of eyes (FIG. 12A) showed that IVT injection of PLVs was well tolerated in the NHP Retina. Immunofluorescence (IF) imaging detected GFP expression in the trabecular meshwork (TM) with the mRNA-GFP PLV and pDNA-GFP PLV groups but not in the control buffer group (FIG. 12B- 12C and FIG. 12E). Detection of GFP protein was confirmed using ELISA (FIG. 12D and FIG. 12F). IF imaging did not detect GFP expression in the TM with the mRNA-GFP LNP group (FIG. 12G). ELISA results confirmed the absence of GFP in the TM, but GFP was detected in the liver (FIG. 12H).Example 7: Tolerability of subretinal (SR) injection in non-human primate primates (NHPs)

[0160] Clinical scores of NHP at 1 , 3, 7, or 14 days after subretinal injection show that SR was well-tolerated in NHPs (FIG. 13). The Optical Coherence Tomography (OCT) degeneration at day 28 was comparable between NHPs injected with cargo encapsulated by PLV or LNP and the control (vehicle; FIG. 14).

[0161] OCT was performed on both eyes using a Heidelberg Spectralis HRA+OCT (or OCTA) with HEYEX image capture and analysis software. A posterior pole volume scan centered on the macular utilizing the auto re-scan follow-up imaging function referencing the baseline images were performed. Images were qualitatively assessed to report abnormalities.Example 8: Subretinal (SR) PLV injection to deliver cargo to the non-human primate (NHP) eye

[0162] African green monkeys {Chlorocebus sabaeus) were subjected to one of three different encapsulation and cargo combinations, administered via SR injection and at the doses listed in Table 3.

[0163] Subretinal Dosing: Topical 1% atropine or atropine gel was administered to each eye 1 -3 days prior to subretinal dosing. Monkeys received subretinal injections in accordance with Table3. Topical proparacaine 0.5% was administered, allowing a minimum of 30 seconds to take effect. The ocular surface, lid margins and periorbital skin were disinfected with 5% Betadine followed by a sterile 0.9% saline rinse or balanced salt solution (BSS). A sterile eye drape and lid speculum were placed. Two 25-gauge vitrectomy ports (Alcon valved entry system 1 -CT, orDocket No. 062548-504001 WO equivalent) were placed via trocar at the level of the ora serrata in the superotemporal and inferotemporal quadrants. A contact vitrectomy lens was centered on the cornea employing 0.9% saline (or BSS) as a coupling agent. With the surgeon positioned temporally, a 25-gauge light pipe was inserted through the left (or right) vitrectomy port into the vitreous cavity for intraocular illumination, maintaining the tip in the anterior vitreous. A subretinal cannula (MedOne 25 / 38g part number 3247, DORC SR 1270. EXT with 23g / 41g needle, or equivalent), attached to a microfluid injector coupled to an Alcon Constellation Vision System was introduced through the right (or left) vitrectomy port and advanced through the vitreous. The 38-gauge (Or 41 -guage) flexible microtip was advanced to gently touch the retinal surface, targeting a point in the superotemporal region within approximately 2-disc diameters of the optic nerve head (ONH). Upon observing slight blanching of the retinal surface at the point of contact, the test article was injected at a steady pressure of 4-6 mmHg (pressure threshold was set to 14mmHg). When an initial bleb was raised, a target volume of test article was administered at the set pressure at the completion of which the cannula tip was retained in place for several seconds prior to being retracted, taking care not to tear the elevated retinal surface. All instrumentation were removed from the eye. The sclerotomies were self-sealing and topical antibiotic suspension (tobramycin and dexamethasone or equivalent) were instilled in the eye.Table 3. Formulations for subretinal injection in NHP

[0164] At day 6 after SR injection, H&E staining of eye cross sections (FIG. 15A) showed that SR injection of PLVs was well tolerated in the NHP Retina. Fluorescence fundus imaging showed robust expression of mRNA and DNA cargo with normal vascularization in the NHP (FIG. 15B).

[0165] IF imaging of NHPs injected with mRNA-GFP PLV at day 6 detected GFP signal localized to retinal pigment epithelium (RPE) cells (FIG. 15C). This was confirmed with ELISA.

[0166] IF imaging of NHPs injected with pDNA-GFP PLV at day 6 detected GFP signal localized to retinal pigment epithelium (RPE) cells (FIG. 15D). This was confirmed with ELISA.

[0167] IF imaging of NHPs injected with mRNA-ABCA4 PLV at day 6 detected ABCA4 signal localized to Rod cells which are positive for Rhodopsin (FIG. 15E).Example 9: mCherry and tdTomato expression post-IVT injection in mice

[0168] mCherry mRNA was encapsulated in PLV and administered to C57 mice via IVT injection. mCherry signal was detected in the trabecular meshwork (TM) by immunofluorescenceDocket No. 062548-504001 WO (IF) imaging 3 days post-injection (FIG. 16). H&E of the adjacent slice indicates good tolerability to the PLV.

[0169] Ore mRNA was encapsulated in PLV and administered to Ai9 mice via IVT injection. tdTomato signal was detected in the TM by IF imaging 35 days post-injection (FIG. 17). H&E of the adjacent slice indicates good tolerability to the PLV.Example 10: DNA-based monoclonal antibody therapy in the eye

[0170] A DNA cargo encoding an anti-VEGF monoclonal antibody (mAb) was encapsulated in PLV and delivered via IVT injection in Sprague Dawley rats. Anti-VEGF reached therapeutic concentrations in vitreous fluid at Day 23 after an IVT injection (FIG. 18A).

[0171] A single dose of anti-VEGF pDNA in PLV was delivered either via sub-conjunctival (SCJ) or IVT administration at day 0. Corneal neovascularization (alkali) injury was induced at day 21. At day 28 (seven days after injury), C57 / B6 mice pre-treated with anti-VEGF pDNA in PLV showed a significant decrease in PECAM staining for neovascularization (FIG. 18B).

[0172] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0173] While the present application has been described with reference to certain embodiments, it is to be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

Claims

Docket No. 062548-504001 WOCLAIMS1. An ophthalmic composition, comprising: lipids; a fusion-associated small transmembrane (FAST) polypeptide; and a molecular cargo; wherein the composition is formulated for ocular administration.

2. The composition of claim 1 , wherein the lipids and FAST polypeptide form a proteolipid vehicle for the molecular cargo.

3. The composition of claim 1 , wherein the lipids comprise an ionizable lipid, a helper lipid, cholesterol, a cationic lipid, and polyethylene glycol (PEG)ylated lipid at a lipid ratio of about 45-85 moles of the ionizable lipid : about 10-50 moles of the helper lipid : about 0-30 moles of cholesterol : about 0-60 moles of the cationic lipid : about 1 -15 moles of the PEGylated lipid.

4. The composition of claim 3, wherein the lipid ratio comprises about 55-75 moles of the ionizable lipid : about 20-40 moles of the helper lipid : about 0-15 moles of cholesterol : about 0-30 moles of the cationic lipid : about 2-8 moles of the PEGylated lipid.

5. The composition of claim 3, wherein the lipid ratio comprises about 61 -71 moles of the ionizable lipid : about 25-35 moles of the helper lipid : 0 moles of cholesterol : 0 moles of the cationic lipid : about 3-5 moles of the PEGylated lipid.

6. The composition of claim 3, wherein the lipid ratio comprises 66 moles or about 66 moles of the ionizable lipid.

7. The composition of claim 3, wherein the lipid ratio comprises 30 moles or about 30 moles of the helper lipid.

8. The composition of claim 3, wherein the lipid ratio comprises 0 moles of cholesterol.

9. The composition of claim 3, wherein the lipid ratio comprises 0 moles of the cationic lipid.

10. The composition of claim 3, wherein the lipid ratio comprises 4 moles or about 4 moles of the PEGylated lipid.11 . The composition of claim 3, wherein the lipid ratio comprises about 66:30:4 moles of the ionizable lipid : helper lipid : PEGylated lipid.

12. The composition of claim 3, wherein the ionizable lipid comprises 1 , 2-dioleoy I-3- dimethylammonium-propane (DODAP or DAP), 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), or Dlin-MC3-DMA.

13. The composition of claim 3, wherein the helper lipid comprises 2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE) or 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).Docket No. 062548-504001 WO14. The composition of claim 3, wherein the cationic lipid comprises 1 , 2-dioleoy I-3- trimethylammonium-propane (DOTAP or TAP) or 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).

15. The composition of claim 3, wherein the PEGylated lipid comprises 1 ,2- dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG).

16. The composition of claim 3, wherein the ionizable lipid comprises DAP, the helper lipid comprises DOPE or DSPC, and the cationic lipid comprises TAP.

17. The composition of claim 3, further comprising a solvent or buffer.

18. The composition of claim 17, wherein the solvent or buffer comprises phosphate buffered saline (PBS).

19. The composition of claim 1 , comprising a pH of about 7.0-8.0.

20. The composition of claim 1 , comprising an N:P ratio of about 1 :1 to about 15:1 .21 . The composition of claim 20, wherein the N:P ratio is about 1 :5.

22. The composition of claim 1 , comprising the FAST polypeptide and the molecular cargo at a FAST :cargo ratio of about 1 :10 (w / w) to about 1 :100 (w / w).

23. The composition of claim 1 , wherein the FAST polypeptide comprises a p10 FAST protein, a p13 FAST protein, a p14 FAST protein, a p15 FAST protein, a p16 FAST protein, or a p22 FAST protein, or a fragment thereof.

24. The composition of claim 1 , wherein the FAST polypeptide comprises a fusion protein comprising a p14 ectodomain and a p15 endodomain.

25. The composition of claim 1 , wherein the FAST polypeptide comprises a p14 transmembrane domain or a p15 transmembrane domain, or a fragment thereof.

26. The composition of claim 1 , wherein the FAST polypeptide comprises an amino acid sequence at least 80% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to any of SEQ ID NOs: 1 -5.

27. The composition of claim 1 , wherein the molecular cargo comprises a nucleic acid or a protein.

28. The composition of claim 1 , wherein the molecular cargo comprises a deoxyribonucleic acid (DNA).

29. The composition of claim 28, wherein the DNA comprises plasmid DNA (pDNA) or an antisense oligonucleotide (ASO).

30. The composition of claim 1 , wherein the molecular cargo comprises a ribonucleic acid (RNA).31 . The composition of claim 30, wherein the RNA comprises a small interfering RNA (siRNA) or a messenger RNA (mRNA).Docket No. 062548-504001 WO32. The composition of claim 1 , wherein the ocular administration results in delivery of the molecular cargo to an eye cell or eye tissue.

33. The composition of claim 32, wherein the eye tissue comprises a retina or cornea.

34. The composition of claim 32, wherein the eye cell comprises a retinal cell, a corneal cell, a neuronal cell, a rod cell, or a ganglion cell.

35. A method for ocular delivery of a molecular cargo, comprising administering the composition of any one of claims 1 -34 to a subject.

36. The method of claim 35, wherein the administration is intravitreal or subretinal.

37. The method of claim 36, wherein the administration comprises an injection.

38. The method of claim 35, wherein the molecular cargo comprises a nucleic acid, and the administration results in expression of a protein encoded by the nucleic acid in an eye of the subject.

39. The method of claim 35, wherein the subject is in need of treatment for an eye disorder, and the molecular cargo comprises a nucleic acid that treats the disorder.

40. The method of claim 39, wherein the disorder comprises Stargardt’s disease, Usher syndrome, or retinitis pigmentosa.

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