Novel peptides, compositions and methods for delivering agents into cells and tissues

Abstract

Provided herein are cell-penetrating peptides that efficiently target many cell types including retinal and corneal cells. These peptides can be used to deliver cargo molecules across the plasma membrane without the need for chemical coupling. Compositions and viral vectors containing these cell-penetrating peptides are also provided. Methods of using these peptides, compositions and viruses to deliver various agents to target cells and tissues are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 869,831, filed on July 2, 2019, which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Project No. W81XWH-16-1-0650 awarded by the U.S. Army. The Government has certain rights in this invention.

[0005] Sequence Listing

[0006] A sequence listing is attached to this application and is submitted as an ASCII text file named "166118_00949_ST25.txt", which is 2.09KB in size and was created on June 15, 2020. The sequence listing is submitted electronically with this application through EFS-Network, and the sequence listing is incorporated herein by reference in its entirety.

[0007] introduction

[0008] For most drugs, the plasma membrane is an impermeable barrier to their transfer into the cytoplasm or nucleus. To overcome this barrier, there is great interest in the development of cell-penetrating peptides (CPPs) (Guidotti et al., 2017). A variety of CPPs have been described that are capable of delivering bioactive conjugates including proteins, peptides, DNA, siRNA, and small molecule drugs into cells and tissues (Bechara and Sagan, 2013). CPPs are grouped into several classes: a) cationic, including HIVTAT, penetratin, or polyarginine; b) amphipathic, including transportan and Pep-1, and c) hydrophobic, including Pep-7 – these CPPs and their general properties have been reviewed elsewhere (Guidotti et al., 2017). Although CPPs with properties useful for delivering molecules across the plasma membrane have been identified, the efficiency of individual peptides varies depending on the type of tissue being targeted.

[0009] Despite advances in the pharmaceutical industry, eye diseases and injuries remain a significant medical problem. According to the National Institutes of Health, eye diseases that lead to blindness are one of the most common causes of disability in the United States [National Eye Institute (1999-2003). A Report of the National Eye Council, National Institutes of Health]. The eye's natural defense system complicates the delivery of drugs to ocular tissues. The blinking action cleans the ocular surface and renews the tear film, which contains immunoglobulins and antimicrobial proteins. The rapid clearance of drugs by the tear film makes local delivery of molecules to the ocular surface difficult, resulting in a loss of more than 95% of a given drug. In addition, due to the recycling of aqueous humor, the half-life of drugs that are able to penetrate into the intraocular chamber is generally short. Therefore, improving methods for delivering drugs to ocular tissues is an important medical need.

[0010] In the present application, CPPs that efficiently target the retina and cornea are provided. These CPPs are unique in that they do not require chemical coupling between the CPP and the heterologous molecule to deliver the heterologous molecule across the plasma membrane, thus providing a promising method for delivering therapeutic drugs to the retina to treat diseases and injuries.

[0011] Overview

[0012] The peptides provided herein can be used as cell-penetrating peptides, which can effectively deliver agents to cells or tissues. In some embodiments, the peptide is not coupled or linked to an agent, and is still able to cross the cell membrane and deliver the agent. The present invention provides a peptide comprising SEQ ID NO: 1 or having 90% sequence identity with SEQ ID NO: 1, wherein X in SEQ ID NO: 1 represents an optional flexible linker region. The present invention also provides a peptide having an amino acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4 or consisting of SEQ ID NO: 2 or SEQ ID NO: 4, or a peptide having 90% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4, wherein each X in SEQ ID NO: 2 represents an optional flexible linker region.

[0013] Provided are pharmaceutical compositions comprising an agent and the peptides of the invention. Also provided are polynucleotides and nucleic acid constructs encoding the peptides of the invention.

[0014] Also provided is a recombinant virus comprising a polynucleotide encoding the peptide of the invention. The peptide of the invention may be inserted into a viral capsid protein. Adeno-associated viruses (AAV) including AAV9 may be used.

[0015] In addition, the present invention provides methods of delivering an agent to a cell or tissue by contacting the cell or tissue with a disclosed composition or with an agent and a disclosed peptide or a composition comprising the peptide.

[0016] Also provided is a method of delivering an agent to cells or tissues of a subject by first formulating a medicament comprising the agent and the peptide of the present invention and then administering the medicament to the subject.

[0017] Also provided are methods of delivering a virus to a cell or tissue by contacting the cell or tissue with both the virus of the invention and a peptide.

[0018] The compositions, peptides and viral delivery mechanisms provided herein can be used to treat subjects in need of treatment for a condition or disease. The condition or disease can be selected from those including degenerative eye diseases, diseases associated with inflammation or oxidative stress, neovascularization or fibrosis, and eye damage.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Fluorescence microscopy images are shown, which show that the Nuc1 peptide penetrated retinal tissues and cells after intravitreal injection. After intravitreal injection, FAM-labeled Nuc1 localized to all layers of the retina, including GCL, INL, IS and OS (A). Immunostaining used tubulin (B), PKC (C), rod opsin (D) and glutamine synthetase (E). GCL, ganglion cell layer; ONL, outer nuclear layer; IS, inner segment; OS, outer segment. For some figures, images with higher magnification are also presented.

[0021] Figure 2 Representative fluorescence microscopy images are shown, which show that Nuc 1 promotes the penetration of recombinant proteins into retinal cells. Co-injection of mCherry / RFP and FAM-labeled Nuc1 into the vitreous of adult mice resulted in the uptake of mCherry into multiple retinal cell types, most abundant in the ONL (A). A higher magnification of the ONL (A) is presented. Cryosections of mice co-injected with mCherry and unlabeled Nuc1 were stained for tubulin of ganglion cells (B), PKC of bipolar cells (C), glutamine synthetase of Müller cells (D), or cone opsin (E). Notably, the recombinant mCherry / RFP protein did not significantly penetrate the retina when injected intravitreally alone (F), as shown by the absence of RFP signal in the central figure. GCL, ganglion cell layer; ONL, outer nuclear layer; IS, inner segment; OS, outer segment. For some figures, images with higher magnification are also presented.

[0022] Figure 3This indicates that Nuc1 promotes the delivery of functional proteins into the retina. TUNEL staining of apoptotic cells was performed in normal mice, MNU, MNU+XIAP, or MNU+XIAP+Nuc1 injected mice. Mice were challenged with PBS to determine normal levels of apoptotic cells or MNU-induced apoptosis. Co-injection of Nuc1 with XIAP significantly prevented apoptosis compared to mice injected with XIAP alone (A). In a retinal detachment model, apoptosis was also observed to be significantly inhibited after Nuc1+XIAP (B). GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer.

[0023] Figure 4 It is shown that Nuc1 promotes antibody delivery into the retina. Anti-VEGF antibodies inhibit laser-induced neovascularization (CNV), which can be enhanced by co-administering anti-VEGF antibodies with Nuc1; intravitreal injection (A) or delivery as daily topical drops for 10 days (B). The upper figure shows a fluorescence microscopy image of RPE / choroidal flat mounts stained with FITC-coupled Griffonia suffruticosa lectin I, and the area of ​​the laser spot is quantified in the scatter plot below.

[0024] Figure 5 Nuc1 enhances the efficacy of decorin in the treatment of alkali burns and choroidal neovascularization (CNV). Corneal cryosections collected from an alkali burn mouse model were stained for α-actin, a marker of fibrosis (red), and nuclei were stained with DAPI (blue) (A). Fluorescence microscopy images of isolectin and α-smooth muscle actin (α-SMA) staining in the eyes of laser-induced CNV mice treated with intravitreal injection of decorin alone or a combination of decorin and Nuc1 peptide (decorin + Nuc1) (B). Dot plots of data presented in the isolectin (left) and α-SMA (right) staining areas (B) measured in the eyes of laser-induced CNV mice treated with intravitreal injection of decorin alone or a combination of decorin and Nuc1 peptide (decorin + Nuc1) (C).

[0025] Figure 6 This indicates that Nuc1 promotes the delivery of small molecules into the retina. Although the fluorescently labeled BH4 peptide was unable to penetrate the retina, intravitreal co-injection of BH4 and Nuc1 did penetrate the retina (A). Nuc1 also significantly enhanced the penetration of fluorescently labeled dexamethasone (Dex-Fl; B). Cell nuclei were stained with DAPI (blue). GCL: ganglion cell layer; ONL: outer nuclear layer; INL: inner nuclear layer.

[0026] Figure 7Figure 1 shows that Nuc 1 enhances adeno-associated virus infection of retinal cells in vivo. Nuc1 enhances the delivery of AAV9 after subretinal injection (A) or intravitreal injection (B). The infection enhancement was uneven across the retina as measured by fundus photography (C). Frozen sections were co-stained for bipolar cells (PKC). Nuclei were stained with DAPI (blue). GCL: ganglion cell layer; ONL: outer nuclear layer; INL: inner nuclear layer.

[0027] Figure 8 Figure 3: Incorporation of Nuc1 into the AAV capsid does not significantly enhance infection. Subretinal injection of AAV-Nucl-CAG-GFP did not result in a significant increase in GFP expression relative to AAV-CAG-GFP (A). In addition, the vector did not penetrate the retina after intravitreal injection (B). In contrast, AAV-Nuc1-CAG-GFP penetrated the retina when co-injected with Nuc1 (C). Nuclei were stained with DAPI (blue). GCL: ganglion cell layer; ONL: outer nuclear layer; INL: inner nuclear layer.

[0028] Fig. 9 Results of AAV-IKV-GFP infection after subretinal injection are shown. After subretinal injection, AAV-IKV-GFP resulted in significant infection of photoreceptors. Sections were co-stained with rod opsin (A), cone opsin (B), glutamine synthetase (C), PKC (D). GCL, ganglion cell layer; ONL, outer nuclear layer. For some figures, images with higher magnification are also presented.

[0029] Fig.10 Results of AAV-IKV-GFP infection after intravitreal injection are shown. After intravitreal injection, AAV-IKV-GFP resulted in severe infection of multiple retinal cells. Sections were co-stained with cone opsin (A), rod opsin (B), PKC / bipolar cells (C), tubulin (D), or glutamine synthetase (E). GCL, ganglion cell layer; ONL, outer nuclear layer. For some figures, images with higher magnification are also presented. While the expression patterns presented in Figures AD were consistently observed; the pattern presented in Figure E was rarely found.

[0030] Fig.11Figure 3 shows that maximum transgene expression was achieved when AAV-IKV was co-injected with Nuc1. After intravitreal injection, co-injection of AAV-IKV-GFP with Nuc1 resulted in maximum transgene expression, which appeared throughout the retinal surface (A). Higher magnification images (B) indicate that photoreceptors are highly GFP-positive, including the retinal pigment epithelium and choroid. Fundus imaging of live animals also revealed that transgene expression appeared throughout the retinal surface (C). Higher exposures of the inner retina showed GFP-positive inner plexiform layer (IPL) and ganglion cell layer (GCL), including some Müller cells (D). Longer exposure images of retinal sections showed that cells in the inner nuclear layer (INL) were also positive, co-stained with PCK for bipolar cells (E). Co-injection of Nuc1 enhanced mRNA levels by approximately 4.3-fold relative to AAV-CAG-GFP. Nuc1 also enhanced the expression of AAV-IKV-GFP by approximately 8.5-fold. IKV-GFP+Nuc1 has approximately 300-fold higher mRNA content relative to AAV-GFP (F).

[0031] Fig.12 Intravitreal AAV delivery inhibits oxidative stress in the outer retina. Eyes of C57 / Bl6J mice injected with AAV-IKV-Nrf2 showed significantly reduced 8-OHdG staining relative to eyes of mice injected with AAV-IKV-GFP (A). Similarly, NRF2 knockout mice injected with AAV-IKV-Nrf2 showed significantly reduced 8-OHdG staining relative to eyes of mice injected with AAV-IKV-GFP (B). Quantification of these retinas showed a significant reduction in 8-OHdG staining in the ONL (C).

[0032] Fig.13 Intravitreal AAV delivery inhibits choroidal neovascularization and fibrosis in the outer retina. AAV-IKV-Decorin was found to be significantly more effective than AAV-IKV-Eyela or AAV-IKV-GFP in laser-induced choroidal neovascularization (CNV) in terms of neovascularization and fibrosis, as measured by isolectin and smooth muscle actin (SMA) staining, respectively.

[0033] Fig.14Topical application of decorin co-delivered with Nuc1 (decorin + Nuc1) reduces corneal opacity and alleviates corneal thickening after alkali burn. Panel A shows representative images of mouse eyes that were not exposed to alkali burn and treatment (control) or exposed to alkali burn and one of the local treatments (phosphate buffered saline (PBS), decorin alone, or decorin + Nuc1). Panel B shows transverse paraffin sections (magnification 10x and 20x) stained with hematoxylin (nuclei; blue) and eosin (extracellular matrix and cytoplasm; pink) of mouse corneas that were not exposed to alkali burn and treatment (control) or exposed to alkali burn and one of the local treatments (PBS, decorin alone, or decorin + Nuc1). Ep, epithelium; St, stroma; En, endothelium.

[0034] Figure 15 shows that topical application of decorin + Nuc1 significantly reduces neovascularization and fibrosis in the cornea of ​​mice exposed to alkali burns. Figure 15 A shows representative images of frozen sections of mouse corneas that were not exposed to alkali burns and treatments (control) or exposed to alkali burns and one of the topical treatments (PBS, decorin alone, or decorin + Nuc1). The frozen sections were stained with GSL I (isolectin; green, upper panel) or smooth muscle actin (SMA, α-actin; red, middle panel). The merged images are shown in the lower panel. Corneal cell nuclei were stained with DAPI (blue). Epi, epithelium; Str, stroma; Endo, endothelium. Figure 15 B shows a bar graph of quantitative staining. The first figure shows quantification of GSL I (isolectin) staining indicating neovascularization and angiogenesis in the corneas of each of the three treatment groups (PBS, decorin alone, and decorin + Nuc1). The second graph shows quantification of SMA (α-actin) staining, indicative of fibrosis, in corneas from each of the three treatment groups (PBS, decorin alone, and decorin + Nucl). *p<0.05 (Student's t-test)

[0035] Figure 16 shows that decorin + Nucl significantly reduces inflammatory cell infiltration in the cornea of ​​mice exposed to alkali burns. Figure A of Figure 16 shows representative images of frozen sections of mouse corneas that are not exposed to alkali burns and treatment (control) or exposed to alkali burns and one of the local treatments (PBS, decorin alone or decorin + Nucl). The frozen sections were stained with inflammatory markers CD45 (green, upper figure) and F4 / 80 (red, lower figure). Corneal cell nuclei were also stained with DAPI (blue). Epi, epithelium; Str, stroma; Endo, endothelium. Figure B of Figure 16 shows a bar graph of quantitative staining. The first figure shows the quantification of CD45 staining in the cornea of ​​each group from the three treatment groups (PBS, decorin alone and decorin + Nucl). The second figure shows the quantification of F4 / 80 staining in the cornea of ​​each group from the three treatment groups (PBS, decorin alone and decorin + Nucl).

[0036] Fig.17A Decorin + Nuc1 significantly reduced the expression of cytokines (except IL-1β) in the corneas of mice exposed to alkali burns. These graphs show the expression levels (picograms / milliliter, pg / ml) of the cytokines interleukin (IL)-1β (IL-1β), IL-6, IL-17, interferon-γ (IFN-G), and tumor necrosis factor alpha (TNF-α) in lysates extracted from the corneas of mice that were not exposed to alkali burns and treatment (control) or exposed to alkali burns and one of the topical treatments (PBS, decorin alone, or decorin + Nuc1).

[0037] Fig. 17B Representative images of mouse corneal cryosections not exposed to alkali burn and treatment (control) or exposed to alkali burn and one of the topical treatments (PBS, decorin alone, or decorin + Nucl) are shown. Cryosections were stained with an antibody against the inflammatory cytokine, transforming growth factor β (TGF-β1, red). Epi, epithelium; Str, stroma; Endo, endothelium.

[0038] Fig.18Decorin + Nuc1 shows that it significantly reduces the activation of Caspase-3 in the cornea of ​​mice exposed to alkali burns. The left side shows representative images of frozen sections of mouse corneas that were not exposed to alkali burns and treatment (control) or exposed to alkali burns and one of the local treatments (PBS, decorin alone, or decorin + Nuc1). The frozen sections were stained with antibodies against activated Caspase-3 (red). Corneal cell nuclei were stained with DAPI (blue). The bar graph on the right shows the quantification of activated Caspase-3 staining in the cornea of ​​each of the 3 treatment groups (PBS, decorin alone, or decorin + Nuc1).

[0039] Fig.19 Decorin+Nuc1 was shown to significantly reduce cell death in the cornea of ​​mice exposed to alkali burn. Fig.19 A shows representative images of mouse corneal cryosections that were not exposed to alkali burn and treatment (control) or exposed to alkali burn and one of the topical treatments (PBS, decorin alone, or decorin + Nucl). Cryosections were stained with the TUNEL assay (red). Corneal cell nuclei were stained with DAPI (blue). For each treatment, the left image shows an overlay of DAPI and TUNEL staining, while the right image shows TUNEL staining alone. Epi, epithelium; Str, stroma; Endo, endothelium. Fig.19 B shows a bar graph quantifying TUNEL-positive cells in corneas from each of the three treatment groups (PBS, Decorin alone, or Decorin + Nucl).

[0040] Fig. 20 Decorin + Nuc1 is shown to significantly reduce gliosis in the cornea of ​​mice exposed to alkali burns. Representative images of frozen sections of mouse corneas that were not exposed to alkali burns and treatment (control) or exposed to alkali burns and one of the topical treatments (PBS, decorin alone, or decorin + Nuc1) are shown. Cryosections were stained for glial fibrillary acidic protein (GFAP, red). Retinal nuclei were stained with DAPI (blue). GCL, ganglion cell layer; INL / ONL, inner / outer nuclear cell layer.

[0041] Fig.21 Representative confocal microscopy images of cryosections of Ai9 mouse retinas following intravitreal injection of 4 μg Cre recombinase with and without Nuc1 (1 μg) are shown. Uptake was observed in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), and retinal pigment epithelium (RPE), indicated by tdTomato expression from the reporter transgene (red). Higher magnification images of Nuc1+Cre-treated retinas are shown on the right.

[0042] Fig. 22 Representative confocal microscopy images of C57BL / 6J mouse retinal cryosections 4 hours after intravitreal injection of Cas9-GFP (with and without Nuc1) are shown. GFP signals were observed in the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL).

[0043] Fig.23 Representative confocal microscopy images of cryosections of Ai9 mouse retina after subretinal injection of Cas9 coupled to ribonucleoprotein particles (Cas9-RNP) are shown. Six weeks after injection, Td-Tomato expression was localized to the retinal pigment epithelium (RPE) and a few photoreceptors. Fig.23 A shows a 10X magnified image, while Fig.23 B shows a 40X image.

[0044] Fig.24 Representative confocal microscopy images of cryosections of Ai9 mouse retina after intravitreal injection of Cas9 coupled to ribonucleoprotein particles (Cas9-RNP) are shown. Td-Tomato expression is localized to the ganglion cell layer (GCL) and inner nuclear layer (INL).

[0045] Details

[0046] For most drugs, the plasma membrane represents an impermeable barrier. However, a special class of proteins called cell-penetrating peptides (CPPs) can cross the intact plasma membrane and facilitate the uptake of cargo molecules. Therefore, CPPs are able to deliver bioactive conjugates including proteins, peptides, DNA, siRNA and small molecule drugs into cells and tissues (Bechara and Sagan, 2013). CPPs offer easy preparation, rapid uptake, low toxicity and immune response [Jones et al. 2005 Br J Pharmacol 145: 1093-1102]. Well-studied CPPs include the human immunodeficiency virus (HIV) Tat protein [Frankel et al. 1988 Cell 55: 1189-1193], the herpes simplex virus (HSV) VP22 protein [Phelan et al. 1998 Nat Biotechnol 16: 440-443], and the Drosophila melanogaster Antennapedia homeodomain protein [Derossi et al. 1994 J Bioi Chem 269: 10444-10450]. For example, HIV Tat has been shown to function as a CPP that can ziplock DNA and deliver it to cultured cells [Ignatovich IA et al. 2003 J Biol Chem 278: 42625-42636]. However, with the exception of HIV Tat, there is very limited information available on the performance of CPPs in neuronal tissue in vivo. Furthermore, CPPs often need to be chemically coupled to cargo molecules for delivery across the plasma membrane, and such modifications may adversely affect the function of the cargo molecules.

[0047] The present invention provides peptides for delivering agents to cells or tissues, particularly to ocular cells and tissues. The peptides of the present invention include SEQ ID NO: 1 and SEQ ID NO: 2 and other peptides provided herein. Although peptides with this ability have been described previously, for example in U.S. Patent 8,778,886, the peptides of the present invention are exceptions because they do not require chemical coupling or connection to the agent to effectively deliver the agent to cells or tissues. The examples show that one such peptide, referred to as "Nuc1" and represented by SEQ ID NO: 3, may be the most effective retinal penetrating peptide described to date. In addition, Nuc1 is capable of delivering a variety of agents to ocular tissues, including recombinant proteins, antibodies, proteoglycans, steroids, viruses, and ribonucleoproteins.

[0048] The peptides used herein are synthesized by commercial suppliers of custom peptides. Solid phase techniques can be used for peptide synthesis [Robertge et al. 995 Science 269: 202], and automated synthesis can be achieved, for example, using a 431A peptide synthesizer (Applied Biosystems, Foster City, California). It will be appreciated by those skilled in the art that, in addition to chemical synthesis, other means can be used to produce peptides, including but not limited to the use of protein and peptide expression systems.

[0049] The CPP (Nuc1) described in the Examples includes two amino acid sequences selected for their potential ability to target ocular cells. The first of these sequences, ASIKVAVSA (SEQ ID NO: 4), is derived from the longer sequence CSRARKQAASIKVAVSADR (SEQ ID NO: 8), which represents the nucleolin binding region of the basement membrane glycoprotein laminin-1. The second sequence, DKPRR (SEQ ID NO: 5), is derived from the slightly longer sequence CDKPRR (SEQ ID NO: 7), which forms the heparan sulfate binding domain of a specific subtype of vascular endothelial growth factor (VEGF165). Importantly, nucleolin and heparan sulfate are found on the surface of retinal tissue, which may explain the ability of these "targeting peptides" to enter this tissue.

[0050] The peptides of the present invention optionally include a flexible linker region. Nuc1 (SEQ ID NO: 3) includes SEQ ID NO: 4 connected to SEQ ID NO: 5 by an amino acid linker comprising two glycine residues, and is shown in the examples as acting as a cell-penetrating peptide (CPP). It will be appreciated by those skilled in the art that the linker region can be changed without affecting the function of the CPP. Therefore, the peptides provided herein are represented by SEQ ID NO: 1, wherein the Xaa amino acid in the sequence represents a flexible linker region. Nuc1 is designed to include a flexible linker region between the targeting peptide sequences (laminin-1 and VEGF165-derived peptides). "Linker" is a sequence of amino acid residues for connecting peptides (optionally connected by peptide bonds). According to the present invention, the flexible linker region comprises 1 or more amino acid residues, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more residues. In a preferred embodiment of the present invention, the linker comprises 2 residues. A "flexible" linker is an amino acid sequence that has no necessary fixed structure (secondary or tertiary structure) in solution. Therefore, such a flexible linker is free to accept multiple conformations and is composed of multiple amino acids. The linker can be provided as a linker sequence present in the targeting peptide, or by inserting one or more amino acid residues between the targeting peptides. The linker can contain any amino acid sequence that does not substantially hinder the interaction of the targeting peptide with its corresponding target molecule. Preferred amino acid residues of the flexible linker sequence include glycine, alanine, serine, threonine, lysine, arginine, glutamine and glutamic acid, but are not limited thereto. In an embodiment, the flexible linker sequence contains two glycine residues, so that the Nuc1 peptide has an amino acid sequence comprising SEQ ID NO: 3. It will be appreciated by those skilled in the art that other residues can be used and the length of the linker can be varied without adversely affecting the function of the peptide. In certain embodiments, the peptide does not contain a linker region. Typically, the linker is prepared as part of a recombinant nucleic acid encoding both the linker and the targeting peptide. The linker can also be prepared by peptide synthesis and then combined with the targeting peptide. Methods for manipulating nucleic acid and peptide synthesis are well known in the art.

[0051] In this application, the terms "protein" or "peptide" or "polypeptide" are used interchangeably to refer to an amino acid sequence. The peptides described herein are not natural, but rather engineered peptide sequences. "Amino acids" and "amino acid sequences" may include naturally occurring components and non-naturally occurring components (i.e., amino acid derivatives or amino acid analogs that replace one or more naturally occurring amino acids). In addition, amino acid sequences having one or more non-peptide bonds or peptidyl bonds between two adjacent residues are included in the definition.

[0052] With respect to the peptides described herein, the phrases "% sequence identity", "percent identity" or "% identity" refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. As explained in more detail below, such conservative substitutions generally retain the charge and hydrophobicity of the substitution site, thereby retaining the structure (and therefore the function) of the polypeptide. The percent identity of an amino acid sequence can be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). The Basic Local Alignment Algorithm Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) provides a set of commonly used and freely available sequence alignment algorithms that can be obtained from multiple sources (including NCBI, Bethesda, Maryland) on its website. The BLAST software suite includes various sequence analysis programs, including "blastp", which is used to align a known amino acid sequence with other amino acid sequences from multiple databases.

[0053] Polypeptide sequence identity can be measured over the length of an entire defined polypeptide sequence, such as defined by a specific SEQ ID number, or can be measured over a shorter length, for example, over the length of a fragment taken from a larger defined polypeptide sequence, such as a fragment of at least 10, at least 15, at least 20 or more contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein (in the tables, figures or sequence listing) can be used to describe the length over which a percentage identity can be measured.

[0054] The examples show that Nuc1 can be delivered to all layers of the retina, including ganglion cells, Müller cells, bipolar cells, and photoreceptors rods and cones ( Figure 1). Notably, Nuc1 penetrates the retina significantly better than any retinal penetrating peptide we have previously described, making Nuc1 a promising candidate for the treatment of a variety of ocular diseases. For example, transduction of photoreceptors will be applied to treat diseases such as retinitis pigmentosa, while transduction of retinal pigment epithelial cells will be applied to treat age-related macular degeneration, and transduction of ganglion cells will be applied to treat glaucoma. However, the targeted cells or tissues of the present invention are not limited to the eye, as Nuc1 can be used in other tissues, including oral, genital, cartilage (chondrocytes), liver, kidney, nerve, brain, epithelium, heart and muscle tissue. In particular, based on the properties of retinal neurons, we expect that the methods of the present invention can be extended to other neuronal tissues, such as the brain. In an embodiment, the CPP can allow the agent to be delivered to photoreceptors, retinal pigment epithelium, ganglion cells, bipolar cells, Muller cells, choroidal endothelial cells, lens epithelium, corneal endothelium, corneal stroma, trabecular meshwork or iris.

[0055] Utilizing this ability to enter cells and tissues, the CPP of the present invention is able to deliver a variety of agents to the target site. In certain embodiments, the agent is a therapeutic agent, a detectable agent, or a cytotoxic agent. In some embodiments, the agent is selected from the following: low molecular weight drugs, peptides, lipids, carbohydrates, proteins, antibodies, immunogens, gene therapy or genetic engineering constructs, viruses or viral vectors or vaccines. In other embodiments, the agent is a nucleic acid, such as cDNA, mRNA, miRNA, tRNA or small interfering RNA. In a preferred embodiment, the agent is an anti-apoptotic, anti-inflammatory, anti-angiogenic or anti-fibrotic agent. As shown in the examples, the agent can be an X-linked inhibitor of apoptosis protein, decorin, an antibody to vascular endothelial growth factor, a BH4 domain peptide from Bcl-xL, NRF2 or dexamethasone, but many other agents are obvious to those skilled in the art. In other preferred embodiments, the agent is a gene therapy agent or a gene editing agent. The agent can be a recombinant virus or a Cas9 ribonucleoprotein. For example, vascular endothelial growth factor or rhodopsin can be a suitable target for gene editing. In short, in gene therapy, genes are usually delivered to cells in the form of DNA or RNA molecules. The gene is included as part of an expression construct, which also includes a promoter / enhancer element to guide the expression of the gene in the target cell. When the gene is expressed, the resulting protein product provides the desired function in the target cell. This function can correct defects or abnormalities (mutations, abnormal expressions, etc.), or it can ensure the expression of another protein with therapeutic value. Gene therapy can be performed in vitro on cells extracted from the body, which are reintroduced after modification, or gene therapy can be performed directly in appropriate tissues in vivo. Therefore, in gene editing, genes in cells or selected tissue cells are changed to replace the cell gene copy with modified genes.

[0056] In a preferred embodiment of the present invention, the agent is not chemically coupled or connected to the CPP. Coupling can adversely affect the function of the cargo or agent attached to the CPP. Therefore, the ability of the CPP of the present invention to deliver uncoupled cargo molecules represents a significant improvement over the prior art. In addition, due to the ease of production, this ability makes CPP extremely versatile. For example, it allows the efficacy of many cargo molecules or potential agents to be tested in series. It also saves the time and money spent on producing new coupling molecules each time a potential cargo molecule is tested.

[0057] In addition, polynucleotides encoding the CPP of the present invention are provided, as well as nucleic acid constructs in which these polynucleotides are operably linked to a promoter. As used herein, the phrase "operably linked to a promoter" means that the polynucleotide is juxtaposed to the promoter so that when gene expression is initiated at the promoter, the polynucleotide is included in the resulting gene product.

[0058] The present invention also provides viruses comprising polynucleotides and nucleic acid constructs encoding CPP. In a preferred embodiment, the virus is an adeno-associated virus (AAV). AAV vectors have been shown to be a feasible and effective method for delivering genes to human tissues, particularly the eye, nervous system, and liver tissue. However, other viruses modified for gene therapy applications, including retroviruses, adenoviruses, lentiviruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, poxviruses, picornaviruses, and herpes simplex viruses, may also be used in the present invention. In the examples, an AAV2 vector with an AAV9 pseudotype capsid (AAV2 / 9) was used. However, any AAV serotype, including but not limited to AAV2 / 2, AAV2 / 8, or AAV2 / 5, may be used in the present invention [Khabou et al. 2016 Biotechnol Bioeng 113(12):2712-2724].

[0059] In some embodiments, the CPP is incorporated into the viral capsid protein. Here, the inserted peptide is preferably a shorter portion of Nuc1, which contains only the targeting peptide ASIKAVSA (SEQ ID NO: 4) derived from laminin-1. In the example, the sequence is flanked by two glycine residues, forming the sequence GASIKVAVSAG (SEQ ID NO: 6), and is cloned between amino acids 588 and 589 of the AAV serotype 9 capsid protein. The incorporation of heterologous sequences into other sites of the outer membrane or capsid protein of the AAV capsid or other viral vectors has been previously described. The glycine residues on either end are flexible linker regions that can be changed by those skilled in the art to include different amino acid residues, or more than one amino acid in length. Therefore, the peptide provided herein is represented by SEQ ID NO: 2, wherein the Xaa amino acid in the sequence represents a flexible linker region (as defined above), which can be one or more amino acids in length, and different amino acid residues can be used.

[0060] The viruses described herein may be recombinant viruses or virus-like particles (VLPs), and include gene therapy vectors or VLPs. As described above, CPP sequences may be inserted into a variety of viruses or viral vectors, and may be engineered to allow polypeptide delivery and expression in target cells. It is contemplated that the viral vectors and viruses described herein will more effectively deliver the polynucleotide of interest and allow expression of polypeptides for gene therapy purposes, compared to similar viral vectors that do not insert or express the peptides provided herein, to allow for increased delivery rates to target tissues or target tissue cells. In addition, these recombinant viruses or viral vectors expressing or incorporating CPPs may also be delivered together with soluble CPPs to further increase delivery to target cells or tissues. In one embodiment, the laminin peptide sequence of SEQ ID NO: 4 or 6 is incorporated into a viral vector, and a soluble Nuc1 (SEQ ID NO: 1 or 3) peptide is co-delivered with the virus to increase delivery of the virus and its cargo.

[0061] In a related embodiment, the peptide is flanked by different flexible linker regions comprising at least one amino acid. As shown in the Examples, including this portion of Nuc1 in the AAV capsid (referred to as AAV-IKV in the Examples) significantly improved infection of retinal cells when injected subretinal or intravitreally. Notably, when the virus (with or without SEQ ID NO: 4 or SEQ ID NO: 6 in the viral capsid) was co-injected with Nuc1, infection of the recombinant virus could be further enhanced.

[0062] The present invention provides methods for delivering an agent to a cell or tissue. These methods include contacting a cell or tissue with the agent and a CPP of the present invention. The cell can be contacted with the agent directly or indirectly in vivo, in vitro or ex vivo. Contact includes administration to a cell, tissue, mammal, patient or human. In addition, contacting a cell includes adding to a cell culture additive or topically. Other suitable methods may include introducing or administering the agent to a cell, tissue, mammal or patient using a suitable administration procedure and route as defined herein. In certain embodiments of the method, the cell or tissue is in culture. Alternatively, the cell or tissue is in vivo.

[0063] Peptide-mediated molecular delivery has been used to treat human ocular diseases and other diseases. Therefore, the present invention also provides methods for delivering agents to cells or tissues of a subject. These methods include formulating a drug comprising the agent and the CPP of the present invention and administering it to a subject. The current standard of care for treating wet age-related macular degeneration includes monthly intravitreal injections of anti-VEGF antibodies (such as ranibizumab or Avastin) (Comparison of Age-related Macular Degeneration Treatments Trials Research et al., 2012). As shown in the examples, these antibodies are co-delivered with Nuc1 to enhance their efficacy, which may reduce the antibody concentration required for an effective dose or reduce the frequency of administration. In some embodiments, intravitreal or subretinal injections can be replaced with topical administration. The addition of Nuc1 allows the agent to pass through the cornea to reach the cells, thereby achieving treatment without expensive medical visits and invasive medical procedures. This ability to enhance drug efficacy has an impact on reducing treatment costs and reducing potential toxicity due to off-target activity. Subjects include, but are not limited to, vertebrates, mammals (if appropriate), humans (if appropriate), cattle, cats, dogs, pigs or mice. Other animal infection models can also be used.

[0064] The medicament or pharmaceutical composition of the present invention may optionally be formulated with one or more suitable pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include any one or all of solvents, diluents or other liquid vehicles, dispersing aids or suspending aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders and lubricants. "Remington's Pharmaceutical Compendium" [by Gennaro, Mack, Easton, Pa., 1995] describes various different carriers used to formulate pharmaceutical compositions and known preparation techniques thereof. The medicament may also optionally contain one or more other therapeutic agents. Other therapeutic agents may be selected from the group consisting of: growth factors, anti-inflammatory agents, vasopressors, collagenase inhibitors, topical steroids, interstitial metalloproteinase inhibitors, ascorbate, angiotensin II, angiotensin calreticulin, tetracycline, fibronectin, collagen, thrombospondin, B vitamins and hyaluronic acid.

[0065] The methods provided include administering the drug using any amount and any administration route effective for treating the subject. The specific dosage is selected by the physician personally according to the patient to be treated. The dosage and administration are adjusted to provide sufficient levels of the active agent or maintain the desired effect. Other factors considered include the severity of the disease state, such as the extent of the disease, medical history; the age, weight and sex of the patient; diet, time and frequency of administration; drug combination; reaction sensitivity; and tolerance / response to treatment. For any active agent, the therapeutically effective dose can be preliminarily estimated in a cell culture assay or an animal model (usually mice, rabbits, dogs or pigs). Animal models are also used to determine the desired concentration range and route of administration.

[0066] In embodiments where the target tissue is the eye, the drug can be administered to the subject's eye by a variety of routes, including transocular, intravitreal, topical, transchoroidal, intracameral, suprachoroidal, transdermal, subretinal, intraperitoneal, subcutaneous, and intravenous. It is worth noting that the natural defenses of the eye make local delivery of drugs to the eye difficult. However, as shown in the examples, topical administration of Nuc1 can enhance the delivery of drugs to the cornea. This application is particularly useful because less than 1% of the available drug penetrates into the eye tissue after topical administration [Shell JW 1984 survey Opthalmol 29: 117-128]. In addition, alternative routes of administration include: oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, oral or nasal routes.

[0067] For ocular administration, liquid dosage forms include buffers and solubilizers, preferably diluents (e.g., water), preservatives (e.g., thymosol), and one or more biopolymers or polymers (e.g., polyethylene glycol, hydroxypropyl methylcellulose, sodium hyaluronate, sodium polyacrylate, or tamarind gum) for adjusting the solution. The dosage forms for topical or transdermal administration of the drug include drops, ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. In addition to the activating agent of the present invention, ointments, pastes, creams, and gels may also contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talcum, zinc oxide, or mixtures thereof. In addition to the agent of the present invention, powders and sprays may also contain excipients, such as talcum, silicic acid, aluminum hydroxide, calcium silicate, polyimide powder, or mixtures of these substances. Sprays may also contain conventional propellants, such as chlorofluorocarbons.

[0068] Injectable preparations (e.g., sterile injectable aqueous or oily suspensions) are prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable preparations are also sterile injectable solutions, suspensions, or emulsions in nontoxic parenterally acceptable diluents or solvents, such as 1,3-butanediol solutions. Acceptable vehicles and solvents include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids (e.g., oleic acid) are used to prepare injections. The injection preparation can be sterilized, for example, by filtering through a bacterial retention filter, or by incorporating a sterilizing agent in the form of a solid composition, which can be dissolved or dispersed in sterile water or other sterile injection media before use. In order to prolong the in vivo effect of the active agent, it is generally desirable to slow down the absorption of the agent from subcutaneous or intramuscular injection. Delayed absorption of parenterally administered active agents is achieved by dissolving or suspending the agent in an oil vehicle. Injectable depot dosage forms can be prepared by forming microencapsule matrices of the agent in biodegradable polymers such as polylactic-polyglycolic acid. Depending on the ratio of active agent to polymer and the nature of the particular polymer used, the rate of release of the active agent can be controlled. Examples of other biodegradable polymers include polyorthoesters and polyanhydrides. Injectable depot formulations can also be prepared by encapsulating the agent in liposomes or microemulsions that are compatible with human tissues.

[0069] Administration of the drug or pharmaceutical composition may be therapeutic or prophylactic. Prophylactic formulations are applied to a possible wound site, or source of a wound, such as contact lenses, contact lens cleaning and rinsing solutions, containers for contact lens storage or transport, devices for contact lens handling, eye drops, surgical rinsing solutions, ear drops, eye patches, and cosmetics for the eye area (including creams, lotions, mascara, eyeliner, and eye shadow), as well as ophthalmic instruments, surgical instruments, and hearing instruments.

[0070] In some embodiments, the subject suffers from an ocular disease, disorder or injury. Some common retinal diseases include age-related macular degeneration, retinitis pigmentosa and glaucoma, which are associated with degeneration of the retinal pigment epithelium, photoreceptors and retinal ganglion cells, respectively [Hartong, et al. 2006 Lancet 368: 1795-1809; Rattner et al. 2006 Nat Rev Neurosci 7: 860-872]. Subjects may suffer from other diseases and conditions, including retinal holes, retinal detachment, diabetic retinopathy, epiretinal membranes, macular holes, macular degeneration, exophthalmos, cataracts, CMV retinitis, retinoblastoma, diabetic macular edema, ocular hypertension, ocular migraine, retinal detachment, alkali or other chemical burns, hyphema, corneal abrasions, keratitis, keratoconus, subconjunctival hemorrhage, proliferative vitreoretinopathy, Usher's syndrome and uveitis. Subjects suffering from other infections, malnutrition, or rejection of transplanted corneas may also be treated.

[0071] The present disclosure is not limited to the specific details of the construction, component arrangement or method steps described herein. The compositions and methods disclosed herein can be carried out, practiced, used, implemented and / or formed in various ways, which are obvious to those skilled in the art based on the following disclosure. The words and terms used herein are for illustrative purposes only and should not be regarded as limiting the scope of the claims. The order indications (e.g., first, second, and third) used to refer to various structures or method steps in the description and claims are not meant to be interpreted as indicating any specific structure or step, or any specific order or configuration of such structures or steps. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be carried out in any suitable order. The use of any and all embodiments or exemplary language (e.g., "such as") provided herein is intended only to facilitate the present disclosure and does not imply any limitation on the scope of the present disclosure unless otherwise stated. Any language in the specification and the structures shown in the drawings should not be interpreted as indicating that any unclaimed elements are essential to the practice of the subject matter of the present disclosure. The use of the terms "including," "comprising," or "having" and variations thereof herein is intended to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as "including," "comprising," or "having" certain elements are also contemplated as "consisting essentially of" and "consisting of" those specified elements.

[0072] Unless otherwise specified herein, the description of the range of values ​​herein is intended only as a shorthand method of referring individually to each individual value within the range, and each individual value is incorporated into the specification as if it were individually stated herein. For example, if the concentration range is specified as 1% to 50%, it is intended that the values ​​of 2% to 40%, 10% to 30%, or 1% to 3%, etc. are explicitly listed in this specification. These are only examples of specific intent, and all possible combinations of values ​​between and including the lowest and highest values ​​listed should be considered to be explicitly mentioned in this disclosure. The use of the word "about" to describe a specific amount being described, or a range of amounts is intended to indicate that values ​​very close to the amount being described are included in the amount, for example, values ​​that may or naturally be considered due to manufacturing tolerances, instrumentation, and human errors in forming measurements. Unless otherwise specified, all percentages involving amounts are by weight.

[0073] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it should be understood that, unless otherwise stated, the reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or any other country. Any discussion of the references describes the claims of their authors, and the applicant reserves the right to question the accuracy and relevance of any document cited herein. Unless otherwise expressly stated, all references cited herein are fully incorporated by reference. If there is a difference between any definition and / or description present in the cited references, the present disclosure shall prevail.

[0074] The following examples are illustrative only and are not meant to limit the scope of the invention or the appended claims. Example

[0075] Example 1 - Novel Cell-penetrating Peptide Nuc1 for Delivery of Small and Macromolecules into Retinal Cells and Tissues

[0076] The design of cell-penetrating peptides (CPPs) can take into account the biological properties of the tissue in question. For example, a previously described peptide called POD (peptide for ocular tissue transport) was modeled against the glycosaminoglycan binding region of proteins (particularly acidic and basic fibroblast growth factors) present in large quantities in the retina (Johnson et al., 2010). The glycosaminoglycan chondroitin sulfate is known to be present in large quantities in the adult retina (Clark et al., 2011), while heparan sulfate is present in large quantities in the retina during development. Molecular modeling of protein-glycosaminoglycan interactions led to the generation of POD, which was found to be one of the most effective CPPs tested in the retina (Johnson et al., 2010). Competition studies indicated that the cell-penetrating properties of POD were found to be significantly affected by the level of heparan sulfate on the cell surface (Johnson et al., 2010), thereby identifying heparan sulfate proteoglycans on retinal tissue as candidate moieties for CPP targeting the retina. The importance of heparan sulfate for CPP function has been described for another form of CPP called Vectocell peptide (De Coupade et al., 2005).

[0077] Vascular endothelial growth factor (VEGF) plays a key role in retinal homeostasis (Jin et al., 2002; Robinson et al., 2001). The VEGFA165 isoform of VEGF contains a highly basic domain that allows this isoform of VEGF to interact with and localize in the heparan sulfate-rich extracellular matrix (Krilleke et al., 2009). A VEGFA splice variant from renal epithelial cells, VEGFA165b, is identical to VEGFA165 except for the last six amino acids. VEGFA165b and VEGFA165 bind to VEGF receptors 1 and 2 with similar affinities. However, VEGFA165b binds only weakly to heparan sulfate (Cebe Suarez et al., 2006), suggesting that the C-terminus of VEGF165 (SEQ ID NO:7 CDKPRR) is involved in heparan sulfate binding.

[0078] Laminin is a large basement membrane glycoprotein in the extracellular matrix. Laminin affects tissue development, cell differentiation, migration and adhesion (Aumailley, 2013). Laminin is a heterotrimeric protein, each containing an α chain, a β chain and a γ chain. The trimeric protein intersects to form a fork-like structure that can bind to other cell membranes and extracellular matrix molecules. To date, five α variants, four β variants and three γ variants have been identified. Different combinations of these chains produce a large number of laminin molecules. To date, approximately 16 laminin molecules have been identified in mammals. At least seven laminin chains (α3, α4, α5, β2, β3, γ2 and γ3) are localized in the matrix surrounding photoreceptors and in the first synaptic layer, where photoreceptors form synapses with retinal interneurons (Libby et al., 2000). Laminin also binds to other basement membrane components (including heparan sulfate proteoglycans) and mediates cellular interactions with the basal layer. The region near the carboxyl globule of laminin-1 (SEQ ID NO:8 CSRARKQAASIKVAVSADR) is an active site for cell adhesion (Tashiro et al., 1989). Interestingly, this region also binds nucleolin (Kibbey et al., 1995), a protein that is normally present in the nucleus and surface of rapidly dividing cells (Hovanessian et al., 2010; Koutsioumpa and Papadimitriou, 2014; Mongelard and Bouvet, 2007), but is also present in the retina, including photoreceptors (Hollander et al., 1999).

[0079] We hypothesized that a novel peptide sequence consisting of a combination of a portion of the nucleolin binding region from laminin-1 and the heparan sulfate proteoglycan binding region of VEGFA165, connected by a flexible polyglycine linker, might have cell adhesion and cell penetration properties. We therefore tested the sequence ASIKVAVSAGGDKPRR (SEQ ID NO: 3) for these properties. Although we demonstrate here that this peptide Nuc1 (SEQ ID NO: 3) might function effectively in the retina, it might also function in other tissues, such as the brain, that share extracellular matrix and cell surface properties with retinal tissue.

[0080] Materials and methods:

[0081] Peptide synthesis: The fluorescently labeled Nuc1 peptide sequence Fluo(5 / 6FAM)-ASIKVAVSAGGDKPRR[COOH] (SEQ ID NO: 3) was synthesized by Thermo Fisher Scientific with a purity of >99%. The same sequence was also synthesized without the fluorescent label.

[0082] Animals: This study was performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal Care and Use Committee (IACUC) of Tufts University. Six- to eight-week-old C57BL / 6J mice were purchased from Jackson Laboratory (Bar Harbor, ME) and housed under a 12-h light / 12-h dark cycle.

[0083] Intravitreal and subretinal injections: Intraperitoneal injection of ketamine (100 mg / kg, Phoenix TMMice were anesthetized with a mixture of 1% AAV (Akorn Inc., Lake Forest, IL, USA) and xylazine (10 mg / kg, Lloyed, Shenandoah, Iowa), followed by topical application of 0.5% procaine hydrochloride (Akorn Inc., Lake Forest, IL, USA) for local corneal analgesia. Mice were kept warm during anesthesia. Intravitreal and subretinal injections were performed using a 32-gauge needle and a 5 μL glass syringe as previously described (Cashman et al., 2015). For Nuc1 uptake experiments, the amount of peptide injected is indicated in the results. To investigate the effect of Nuc1 on mCherry uptake, 1 μL of mCherry (4 μg) was injected intravitreally with or without Nuc1 as described in the results. Four hours after injection (as indicated), eyes were enucleated and fixed in 4% paraformaldehyde. A total of 1 μL of AAV (doses described in the results) was injected intravitreally and subretinally per eye with or without Nuc1. Retinal cryosectioning was performed using a Micron 550 cryostat.

[0084] Laser-induced choroidal neovascularization (CNV): Laser photocoagulation was performed as described previously. Briefly, 2.5% phenylephrine hydrochloride (Bausch & Lomb) and 1% tropicamide (Bausch & Lomb) were used to dilate the pupils of sedated mice. 2.5% hypermellose (Goniovisc) was applied to minimize corneal discomfort. Four laser spots were generated in each eye using an argon laser (532 nm, IRIS Medical Light Solutions, IRIDEM; IRIDEX) with a spot diameter of 75 μm, 330 mH, and a pulse time of 100 ms.

[0085] Lectin and α-actin staining: Seven days after photocoagulation, animals were euthanized with CO2 and eyes were removed. The eye cups were fixed and the lenses and corneas were removed. After overnight fixation in 4% paraformaldehyde in 0.1 M phosphate buffer, the retinas were removed and the sclera / choroid / RPE complexes were washed in PBS. The eye cups were blocked with 5% BSA in PBS and stained with 100 μg / L fluorescein-conjugated isolectin (Vectashield) in PBS for 1 hour. The eye cups were then washed three times in PBS for 5 minutes each and incubated with a 1:200 dilution of Cy3-conjugated mouse anti-α-SMA monoclonal antibody (C6198, Sigma) at 4°C overnight. The next day, the eye cups were washed three times with PBS for 5 minutes each time, mounted on slides, and imaged using an inverted microscope (IX51; Olympus) with relevant filters, a digital camera (Retiga 2000r-FAST; Q-Imaging), and QCapture Pro software (Q-Imaging). Images of the CNV area were captured using a fluorescence microscope (Leica). The CNV area was measured using ImageJ software (NIH).

[0086] Cell culture: Progeny HEK293 cell cultures were maintained on 15 cm plates in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS; HyClone Laboraties, South Logan, UT). Cells were passaged every 3-4 days to maintain exponential growth phase.

[0087] Vector construction: To produce AAV-IKV-GFP and AAV-Nucl-GFP viruses, the pAAV9 / rep-cap plasmid was digested with Dralll, and the 1.4 kb cap DNA fragment was gel extracted and cloned into pBSx to generate pBSx1.4. The DNA sequence of the peptide was commercially synthesized. These sequences were cloned into pBSx1.4 (containing the 1.4 kb cap region) by restriction digestion using Tthlll1 and BamHI to generate pPBSx1.48 and pBSx1.4N. Finally, pPBSxl.48 and pBSx1.4N were digested with Sbfl and BsiWI, and the 1.4 kb DNA fragment was gel extracted and cloned into pAAV2 / 9rep-cap containing inverted terminal repeats (ITRs) to generate pAAV2 / 9IKV and pAAV2 / 9Nucl.

[0088] Recombinant AAV production and purification: AAV virus was generated using a modified version of a previously described protocol (Birke et al., 2014). Transfections were performed using calcium phosphate triple transfection with the AAV plasmids described above. Briefly, 80-90% confluent HEK293 cells grown in 15 cm plates were fed DMEM-10% FBS 2 h before transfection. Sufficient plasmids were precipitated using the calcium phosphate method at a ratio of 2:1:1 (helper plasmid: cis plasmid: trans plasmid) and added dropwise to 20 plates. The medium was changed to DMEM-10% FBS 24 h after transfection. Cells and media were harvested 96 h after transfection. The collected cell pellets were lysed and 14 ml of the clarified lysate was applied to a gradient iodixanol (OptiPrep; Sigma-Aldrich, St Louis, MO) solution filled in the following order into a 40 ml quick-seal centrifuge tube (Beckman Instruments, Palo Alto, CA): 4 ml 15% iodixanol, 9 ml 25% iodixanol, 9 ml 40% iodixanol and 5 ml 54% iodixanol. The tubes were centrifuged at 18°C ​​at 69,000 rpm in a 70 Ti rotor (Beckman Instruments) to remove the 4 ml fractionated solution in the 40% iodixanol layer. The final lactated Ringer's solution and glycerol added to 5% were further diafiltered and concentrated. The fractions were then aliquoted and stored at -80°C for future use.

[0089] AAV titration: AAV-CAG-GFP plasmid was digested with Smal and gel extracted to generate a standard curve for the GFP transgene. AAV was digested with DNase1 to remove genomic DNA and further incubated with proteinase K to digest the AAV capsid. DNA was extracted and purified using the phenol-chloroform method and dissolved in TE buffer. 4 Genome copies up to 2×10 8 Standard curve of genome copy range. Perform quantitative PCR and quantify the virus according to the standard curve.

[0090] Immunohistochemistry: For retinal cell immunohistochemistry, retinal cryosections were rehydrated in PBS for 15 minutes, blocked with 6% normal goat serum in PBS or using the Mouse On Mouse kit for 1 hour, and incubated with the appropriate anti-PKC primary antibody (bipolar cells) in a humidified chamber overnight. Subsequently, sections were washed and incubated with secondary antibodies labeled with Alex-fluor 544 or 488 (Molecular Probes, Eugene, OR) to localize the corresponding antibodies in retinal sections. Sections were mounted in anti-fade medium containing DAPI (Vectashield-DAPI; Vector Laboratories, Burlingame, CA) to counterstain cell nuclei, and images were captured using a Leica confocal microscope.

[0091] result

[0092] Nuc1 penetrates retinal tissue and cells after intravitreal injection

[0093] To examine whether the Nuc1 peptide can penetrate the retina and enter retinal cells after intravitreal injection, we fluorescently labeled Nuc1 with the fluorescent dye FAM and injected a total of 1 μg of Nuc1 suspended in 1 μl of H2O into the vitreous of adult (6-week-old) C57BL / 6J mice. Four hours after injection, the eyes of these mice were removed and fixed in 4% paraformaldehyde. Retinal cryosections were generated using a Micron 550 cryostat, and the sections were imaged using a fluorescence microscope. We found that although 6-FAM could not penetrate the retina (data not shown), FAM-labeled Nuc1 was localized to all layers of the retina, including the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segments (IS), and outer segments (OS) ( Figure 1 A). These cryosections were treated with targeted tubulin ( Figure 1 B), protein kinase C ( Figure 1 C), rod opsin ( Figure 1 D) or glutamine synthetase ( Figure 1 E) Co-staining with antibodies of Nuc1 shows that Nuc1 targets ganglion cells, bipolar cells, photoreceptors, and Müller cells, respectively. Interestingly, Nuc1 penetrates the retina significantly better than any retina-penetrating peptide we have previously described, including POD (Binder et al., 2011; Johnson et al., 2010). To our knowledge, Nuc1 has superior retina-penetrating properties relative to any cell-penetrating peptide previously described for the retina.

[0094] Nuc 1 helps recombinant proteins penetrate into the retina

[0095] Large functional proteins injected into the vitreous are often unable to penetrate deep into the retina and are unable to cross the plasma membrane of retinal cells. The use of cell-penetrating peptides (CPPs) such as PODs that are physically linked to heterologous proteins can overcome this inherent limitation (Johnson et al., 2010). However, PODs and other CPPs generally require chemical conjugation to heterologous proteins for delivery across the plasma membrane, and such modifications to proteins may adversely affect protein function. We hypothesized that heterologous proteins could be delivered to retinal cells without being conjugated to Nuc1. To test this hypothesis, we co-injected 4 μg of recombinant red fluorescent protein (RFP / mCherry) and 1 μg of FAM-labeled Nuc1 into the vitreous of adult (6-week-old) C57BL / 6J mice. Four hours later, tissues were processed as described above. When injected alone, no mCherry significantly penetrated into retinal tissue or cells ( Figure 2 F), while mCherry co-injected with FAM-labeled Nuc1 showed that mCherry was clearly taken up into a variety of retinal cells ( Figure 2 A), which is most abundant in the ONL. Notably, colocalization (yellow) of FAM signal (green) and mCherry signal (red) was detected in many cells within the retinal ONL ( Figure 2 A).

[0096] When FAM-labeled Nuc1 was replaced with unlabeled Nuc1 and co-injected with mCherry, robust uptake of mCherry was again observed, demonstrating that the RFP signal was not the result of bleed-through from the FAM channel ( Figure 2 B-2E). Figure 2 B) PKC Figure 2 C), glutamine synthetase ( Figure 2 D) or cone opsin ( Figure 2 E) Co-staining of retinal sections with antibodies showing mCherry localization to ganglion cells, bipolar cells, Müller cells, and cones, respectively. Due to the small size of the mouse eye, some variability was observed between animals, however, all retinas showed the strongest signal in the ONL.

[0097] Nuc1 promotes XIAP protein delivery into the retina and attenuates apoptosis

[0098] We next investigated whether the properties of Nuc1 are applicable to heterologous proteins of potential therapeutic interest. Programmed cell death, or apoptosis, is a common pathway activated as a result of retinal degeneration in diseases such as pigmentary retinopathy (Cottet and Schorderet, 2009). Previous studies using transgenic animals or viral gene delivery have found that elevated expression of apoptosis inhibitors such as X-linked inhibitor of apoptosis protein (XIAP) attenuates retinal degeneration in a variety of animal models of retinal degeneration (Leonard et al., 2007). As an alternative to transgenic delivery, delivery of XIAP protein for the treatment of retinal degenerative diseases could be envisioned. Such a treatment approach would be similar to the current standard of care for wet age-related macular degeneration (AMD, see below), in which recombinant proteins such as aflibercept are injected intravitreally to target VEGF. Protein delivery may be more easily titrated with respect to dosing regimens relative to gene delivery. Intraperitoneal injection of N-methyl-N-nitrosourea (MNU) in mice selectively induces apoptosis in the outer nuclear layer (ONL) of the retina, and thus this model can be used to test the efficacy of apoptosis inhibitors (Petrin et al., 2003).

[0099] To test the hypothesis that purified functional recombinant human XIAP can be delivered to retinal cells via intravitreal injection and inhibit MNU-induced retinal cell apoptosis, C57BL / 6J mice (male, 6-8 weeks old) were intravitreally injected with 1.4 μg recombinant human XIAP (n=6 eyes) or XIAP co-injected with 0.4 μg Nuc1 (n=6 eyes). Four hours later, mice were injected intraperitoneally with 50 mg / lkg MNU. Some mice were injected intraperitoneally with PBS alone as a measure of background cell apoptosis in the retina. After another 24 hours, the eyes of the mice were enucleated and processed as described above. To detect apoptotic cells, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method was used on frozen sections using the In Situ Cell Death Detection Kit, TMR Red (Sigma) according to the manufacturer's instructions. Eye sections were imaged and used for quantification of TUNEL-positive cells using ImageJ (FIJI version and plugin) as previously described (Maidana et al., 2015).

[0100] As expected, intraperitoneal injection of MNU induced extensive apoptosis in the ONL relative to PBS-injected animals, as evident from the large number of TUNEL-positive cells in this group of animals ( Figure 3 Eyes injected with XIAP protein alone (intravitreal) prior to MNU injection showed a non-significant decrease in the number of TUNEL-positive cells relative to MNU alone (2.2%, p=0.9902) ( Figure 3A). In other words, XIAP itself did not significantly attenuate apoptosis in the ONL. In contrast, animals co-injected with recombinant XIAP protein and Nuc1 (intravitreal) before MNU injection showed a significant decrease in the number of TUNEL-positive cells (65.7%, p < 0.0001). This suggests that Nuc1 enables functional XIAP protein to penetrate the retina and inhibit apoptosis in the ONL. Notably, although we showed in previous studies that recombinant XIAP protein can be delivered to the retina and inhibit MNU-induced apoptosis, in this study, XIAP did not need to be chemically coupled to the delivery agent. Therefore, this study reveals a significant advance in the development of functional protein delivery to retinal cells (Talreja et al., 2018).

[0101] Having demonstrated that functional XIAP can be delivered to the retina when co-injected with Nuc1, we wished to test whether our findings are applicable to other models of retinal apoptosis. Rhegmatogenous, tractional, and exudative retinal detachments are associated with a risk of blindness. Retinal detachment results in apoptosis of retinal cells in both the outer and inner layers of the retina (Arroyo et al., 2005). We wished to test the hypothesis that the combination of Nuc1 and recombinant XIAP could inhibit retinal apoptosis in a mouse retinal detachment model. To test this hypothesis, we induced retinal detachment in mice by injecting 3 μl (10 mg / ml) of sodium hyaluronate (Healon, Advanced Medical Optics, Sweden) into the subretinal space using a 32G needle attached to a 5 μl glass syringe (Hamilton). One day after retinal detachment, animals were co-injected with 1.4 μg XIAP and 0.4 μg Nuc1 into the vitreous. After 72 hours, eyes were fixed in 4% paraformaldehyde, cryosectioned using a Micron 550 cryostat, and TUNEL staining was performed as described above. We found that when the combination of Nuc1 and XIAP was injected into the retina, the number of TUNEL-positive cells was reduced by approximately 60% relative to the Healon-only control (p=0.0061) ( Figure 3 B).

[0102] Nuc1 promotes antibody delivery to the retina

[0103] Although our above results indicate that recombinant proteins may be readily used for the treatment of potential retinal diseases, the current clinical standard of care for the treatment of wet age-related macular degeneration (AMD) includes monthly intravitreal injections of anti-VEGF antibodies (such as ranibizumab or Avastin) (Comparison of Age-related Macular Degeneration Treatments Trials Research et al., 2012). To determine whether Nuc1 can improve the delivery of antibodies to retinal cells or tissues, we examined the efficacy of anti-VEGF antibodies in a wet AMD mouse model. Laser-induced choroidal neovascularization (CNV) was generated by applying four laser spots (power of 330 mW and duration of 100 ms) around the optic nerve head. Subsequently, 0.3 ng of anti-VEGF antibody alone or 0.3 ng of anti-VEGF antibody and 1 μg of Nuc1 were injected into the vitreous of mice. The amount of anti-VEGF antibody used in these studies was determined by preliminary studies (i.e., titration of 3 μg to 0.3 μg of anti-VEGF, data not shown). The dose was calculated so that anti-VEGF antibody alone could not significantly inhibit laser-induced CNV. Seven days after laser treatment, the eyeballs were harvested and RPE / choroidal flat mounts stained with FITC-conjugated Griffonia suffragana lectin I were prepared. The laser spot was imaged by fluorescence microscopy, and the area of ​​the laser spot was quantified using imageJ software. We found that the size of the CNV spot was significantly reduced (>60%; p<0.0001) when anti-VEGF antibody was co-injected with Nuc1 compared to anti-VEGF antibody alone ( Figure 4 A). Thus, intravitreal administration of Nuc1 significantly enhances the penetration and efficacy of anti-VEGF antibodies against laser-induced CNV. This property of Nuc1 may serve to enhance or reduce the efficacy of antibodies, altering the potency of a given dose of antibody such that more or less is needed to achieve a therapeutic effect.

[0104] Although intravitreal injection of antibodies is the current clinical standard of care for wet AMD, it is an invasive and “uncomfortable” procedure for patients and has been associated with retinal detachment and endophthalmitis. Furthermore, it requires patients (usually elderly) to visit the ophthalmologist frequently, leading to reduced patient compliance. Therefore, topical administration of drugs would be a preferred method of drug delivery. We examined whether Nuc1 could enhance the efficacy of topically applied anti-VEGF antibodies. Given the extremely limited penetration of antibodies when applied topically to the eye, we used higher doses of antibodies relative to intravitreal injections. Specifically, 1.8 μg of anti-VEGF antibody was administered alone or in combination with 4 μg of Nuc1. After laser-induced CNV, topical drops containing the antibody and Nuc1 were applied to the cornea twice daily for ten days. Topical delivery of Nuc1 significantly enhanced the efficacy of topically applied anti-VEGF antibodies, resulting in laser-induced CNV ( Figure 4 B) The size was reduced by approximately 60% relative to the antibody alone (p<0.02).Thus, Nucl significantly enhanced the efficacy of topically applied antibodies in the laser-induced wet AMD model.

[0105] Nuc1 promotes decorin protein delivery into the retina and inhibits fibrosis

[0106] To further evaluate the potential of Nuc1 as a platform for delivering proteins to ocular tissues, we examined other models of ocular disease. Corneal alkali burns lead to fibrosis, angiogenesis, and inflammation, which, if left untreated, result in significant visual loss. Decorin is a proteoglycan known to have anti-angiogenic and anti-fibrotic properties (Gubbiotti et al., 2016; Jarvelainen et al., 2015).

[0107] To test the ability of Nuc1 to assist decorin to penetrate the cornea to treat fibrosis, mice were anesthetized and an alkali burn was induced in the cornea by applying a 2 mm filter paper soaked in 1N sodium hydroxide (NaOH) to the center of the right cornea for 30 seconds. The filter paper was gently removed and the cornea was rinsed 10 times with phosphate buffered saline (PBS). Topical application of PBS, decorin (0.5 μg) or decorin (0.5 μg) + Nuc1 (0.5 μg) began 24 hours after exposure to NaOH. Treatment was applied topically once a day for seven days. After 7 days, mice were killed by inhalation of CO2. The eyes were removed and fixed in 4% paraformaldehyde. Corneal cryosections were performed using a Micron 550 cryostat and stained for the fibrosis marker α-actin. We found that topical application of decorin alone reduced α-actin staining by 33.3% relative to PBS (p < 0.025), whereas application of decorin + Nuc1 reduced α-actin staining by 46.2% relative to PBS (p < 0.0028), demonstrating that Nuc1 enhances the anti-fibrotic efficacy of decorin ( Figure 5 A) In view of these promising results, the use of Nucl to deliver decorin to the cornea was subsequently investigated in more detail for the treatment of angiogenesis, fibrosis and inflammation following corneal chemical burns (see Example 2).

[0108] Furthermore, we examined whether Nuc1 could enhance the efficacy of decorin in mice with laser-induced choroidal neovascularization (CNV), a mouse model of wet AMD described in the previous section. Immediately after laser treatment, mice received an intravitreal injection of 0.5 μg of human recombinant decorin alone or in combination with Nuc1 peptide (0.5 μg), and eyes were harvested 7 days later. The size of CNV outgrowths was measured by staining the retinal pigment epithelium (RPE) with Griffonia suffragana isolectin, and fibrosis was measured by staining for α-actin (α-SMA, Figure 5 Using unpaired T-tests, we found statistically significant reductions in CNV area (isolectin, p<0.0072) and fibrosis (α-SMA, p<0.0001) in the decorin and decorin+Nuc1 treated groups ( Figure 5 C). Notably, CNV and fibrosis were reduced by approximately 70% in the presence of Nuc1 compared to decorin alone, strongly suggesting that Nuc1 enhances the penetration and efficacy of decorin, thereby ameliorating the effects of laser-induced CNV and fibrosis.

[0109] Nuc1 promotes the delivery of peptides and small molecules into the retina

[0110] Molecules that are significantly smaller than the whole protein have potential as therapeutic agents. For example, BH4 domain peptides from Bcl-xL are known to have anti-apoptotic activity in vivo (Rong et al., 2009). However, BH4 does not appear to have any significant cell-penetrating properties. Therefore, several groups have delivered BH4 to cells by chemically linking it to the cell-penetrating peptide TAT (Donnini et al., 2009; Hotchkiss et al., 2006; Park, 2011). To investigate whether Nuc1 can enhance the uptake of BH4 in the retina without the need for chemical linking, we injected 4 μg of fluorescently labeled BH4 peptide alone or in combination with 4 μg of labeled BH4 peptide and 4 μg of Nuc1 intravitreally into 6-week-old C57BU6J mice. Although the uptake of BH4 peptide alone was limited in the retina, there was a significant qualitative increase in the uptake of the fluorescently labeled BH4 peptide when BH4 was bound to Nuc1 ( Figure 6 A).

[0111] Small molecules, including steroids, can be used as anti-inflammatory agents. However, steroids such as dexamethasone produce significant side effects after injection into the vitreous, including cataract formation or induction of increased intraocular pressure (Phulke et al., 2017; Pleyer et al., 2013; Zhang et al., 2018). We hypothesized that Nuc1 might facilitate the penetration of steroids into the tissue, allowing for the application of reduced doses of steroids without loss of efficacy. To test this hypothesis, we injected 1 μg of fluorescently labeled dexamethasone alone or in combination with 1 μg of Nuc1. We found that both dexamethasone alone and dexamethasone with Nuc1 were absorbed into the retina. However, when dexamethasone was co-injected with Nuc1, its uptake was qualitatively greater ( Figure 6 B).

[0112] Nuc 1 enhances viral infection of retinal cells in vivo

[0113] Recombinant viruses, such as adeno-associated virus (AAV), are excellent vehicles for delivering recombinant genes to the retina (Bennett, 2017). However, high titers of virus are often required to achieve “therapeutic” levels of cell transduction. Despite the immunologically isolated nature of the subretinal and vitreous cavity, immune responses to recombinant AAV in these regions have been well documented (Boyd et al., 2016; Kotterman et al., 2015; Reichel et al., 2017). Low doses of virus generally produce less immune responses. To determine whether Nuc1 could enhance the potency of AAV infection, thereby reducing the need for high or higher doses of virus, we injected recombinant AAV serotype 2 (AAV 9 pseudotyped capsid; AAV2 / 9) expressing GFP (AAV-CAG-GFP) into the eyes of adult C57 / B16J mice. As expected, subretinal delivery of AAV-CAG-GFP enabled expression of the transgene (GFP) in the retinal pigment epithelium (RPE) and photoreceptors ( Figure 7 Surprisingly, when AAV2 / 9 was co-injected with 1 μg Nuc1, transgene expression was qualitatively superior to that of AAV2 / 9 alone ( Figure 7 A).

[0114] In contrast, AAV2 / 9 did not infect either the inner or outer retinal layers when injected intravitreally ( Figure 7 B). However, we found that the addition of 1 μg Nuc1 to the AAV-CAG-GFP suspension enhanced the infectivity of both the inner and outer retinal layers, including the ONL ( Figure 7 B) This improvement in infection is not uniform across the retina, as demonstrated by fundus photography of live animals ( Figure 7 C) Observed to occur in the patch. Quantification of viral infection was performed by RT-PCR (see below).

[0115] Incorporation of Nuc 1 into AAV capsids does not significantly enhance infection

[0116] To determine whether AAV2 / 9 would be more effective if the Nuc1 sequence was incorporated into the AAV capsid coat, without the need for an external peptide, we constructed recombinant AAV2 / 9 expressing GFP that contained the Nuc1 sequence (with a glycine on each end) in the VP1 capsid of AAV9. The Nuc1 sequence was inserted between amino acids 588 and 589, as defined by (Khabou et al., 2016), generating AAV-Nuc1-CAG-GFP. This modification did not result in a significant enhancement of viral infectivity via the subretinal route relative to AAV-CAG-GFP ( Figure 8 A). In addition, AAV-Nuc1-CAG-GFP did not infect the inner or outer retinal layers after intravitreal injection ( Figure 8 B) However, when AAV-Nuc1-CAG-GFP was co-injected with Nuc1 peptide via the intravitreal route, the infection of AAV-Nuc1-CAG-GFP could be enhanced ( Figure 8 C), but this result is highly variable because some of the retinas examined had very limited GFP expression in the outer retinal layers ( Figure 8 C, GFP inset). One possible explanation for this result is competition between the Nuc1 peptide and the Nuc1 sequence in the viral capsid. In fact, a greater amount of Nuc1 resulted in a decrease in the overall AAV-Nuc1-CAG-GFP infectivity (data not shown).

[0117] Retinal-penetrating AAV

[0118] To determine whether the heparan sulfate binding region of VEGFA165 in Nuc1 interferes with viral infectivity, we constructed a version of AAV-Nuc1-CAG-GFP that lacked the heparan sulfate binding sequence, so that the virus now contained a partial sequence from laminin-1, ASIKVAVSA (SEQ ID NO: 4). This shorter sequence was flanked by glycine residues at each end, forming the sequence GASIKVAVSAG (SEQ ID NO: 6), and was similarly cloned between amino acids 588 and 589 of the AAV capsid as described above to form a virus termed AAV-IKV-GFP. We found that subretinal injection of AAV-IKV-GFP into 6-week-old C57BL / 6J mice significantly improved infection of retinal cells ( Fig. 9 A-9D). Using rod opsin ( Fig. 9 A), cone opsin ( Fig. 9 B), glutamine synthetase ( Fig. 9 C) and PKC ( Fig. 9 D) Counterstaining of AAV-IKV-GFP infected retinal sections revealed that AAV-IKV-GFP infected rods, cones, Muller cells, and bipolar cells, respectively.

[0119] Surprisingly, when AAV-IKV-GFP was injected intravitreally into 6-week-old C57BL / 6J mice and treated with cone opsin ( Fig.10 A), rod opsin ( Fig.10 B) PKC Fig.10 C) or tubulin ( Fig.10 D) Counterstaining of sections revealed that AAV-IKV-GFP infected cone photoreceptors, rod photoreceptors, bipolar cells, and ganglion cells, respectively. In some retinas, Müller cells were also observed to be positive in some areas based on co-staining with glutamine synthetase ( Fig.10 E).

[0120] We next considered whether intravitreal AAV-IKV-GFP could be further enhanced by co-administration with Nuc1. Our studies above showed that incorporation of intact Nuc1 sequences into viral capsids was inhibited when co-administered with Nuc1 peptides, likely due to competition for cellular entry. With reduced incorporation of Nuc1 sequences into the capsid, we found that intravitreal AAV-IKV-GFP had the most potent retinal infection observed to date, with robust expression throughout the retina ( Fig.11 A) Yes Fig.11 Closer inspection of the boxed area in A reveals a high density of photoreceptors, RPE, and surprisingly, choroid that are GFP positive ( Fig.11 B) This GFP expression pattern is not restricted to a specific region, as fundus photography of live animals shows that GFP is expressed across a large area of ​​the retina ( Fig.11 C) In addition, Fig.11 Longer exposures of the boxed area in B reveal that the inner plexiform layer (IPL) and ganglion cell layer (GCL) are also GFP-positive, although significantly less than the ONL or RPE ( Fig.11 D). PKC counterstaining of bipolar cells revealed that, in addition to ONL and RPE, a large number of bipolar cells were GFP positive ( Fig.11 E).

[0121] To measure the mRNA expression levels of various viral constructs injected intravitreally, we performed quantitative RT-PCR on retinal tissue. We found that Nuc1 significantly enhanced the mRNA expression levels from each virus tested. Co-injection of Nuc1 enhanced the mRNA levels by approximately 4.3-fold relative to the expression level of AAV-CAG-GFP alone. Nuc1 also enhanced the expression of AAV-IKV-GFP by approximately 8.5-fold ( Fig.11 F). AAV-IKV-GFP+Nuc1 has approximately 300-fold higher mRNA levels relative to AAV-GFP. Thus, Nuc1 enhances infection of recombinant AAV, and when combined with incorporation of a partial Nuc1 sequence into the AAV capsid, we observed the greatest relative increase in infection via the intravitreal route.

[0122] Inhibition of oxidative stress in the outer retina by intravitreal AAV delivery

[0123] NRF2 (nuclear factor erythroid 2p45-related factor 2) is a master transcription factor that regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation. NRF2 targets more than 250 genes. In the steady-state state, Kelch-like ECH-associated protein-1 (KEAP1) and Cullin 3 sequester NRF2 in the cytoplasm, where it is degraded by ubiquitination. Oxidative stress disrupts ubiquitination, and NRF2 subsequently translocates to the nucleus and binds to antioxidant response elements (AREs) in the upstream promoter regions of many antioxidant genes, thereby initiating their transcription. NRF2 expression has previously been found to have therapeutic effects in multiple animal models of retinal degeneration, including AMD, pigmentary retinopathy, glaucoma, uveitis, and diabetic retinopathy; and in many diseases of the elderly, including Alzheimer's disease, amyotrophic lateral sclerosis, and Friedrich's ataxia.

[0124] Intraperitoneal injection of MNU results in significant oxidative stress in the retina. 8-Hydroxy-2-deoxyguanosine (8-OHdG) is one of the leading manifestations of free radical-induced oxidative damage in nuclear and mitochondrial DNA, and thus this marker has been widely used as a biomarker of oxidative stress. We wished to determine whether the AAV-IKV backbone combined with Nuc1 could deliver the human Nrf2 transgene to the outer layers of the mouse retina via an intravitreal route and inhibit MNU-induced oxidative stress. To test this hypothesis, adult C57Bl / 6J mice were intravitreally injected with AAV-IKV-Nrf2 (+Nuc1) or AAV-IKV-GFP (+Nuc1) as a negative control. After 3 weeks of transgene expression, mice were injected with 50 mg / kg MNU. Eyes were harvested 24 hours later, processed as described above, and stained for the presence of 8-OHdG, GFP, or Nrf2. We found that eyes of C57 / Bl6J mice injected with AAV-IKV-Nrf2 showed a significant reduction in 8-OHdG staining relative to eyes injected with AAV-IKV-GFP ( Fig.12 Similarly, NRF2 knockout (NRF2- / -) mice injected with AAV-IKV-Nrf2 showed significantly reduced 8-OHdG staining relative to eyes injected with AAV-IKV-GFP ( Fig.12 B). Quantification of these retinas demonstrated a significant reduction in 8-OHdG staining in the ONL ( Fig.12 C).

[0125] Inhibition of choroidal neovascularization and fibrosis in the outer retinal layer by intravitreal AAV delivery

[0126] In summary, we found that decorin was able to inhibit laser-induced CNV due to its known property of inhibiting both VEGF and TGF-β in the extracellular matrix. We wished to test the hypothesis that expression of decorin in the outer retina would be highly effective relative to control GFP or the recombinant anti-VEGF molecule Eyelea (aflibercept) used to treat AMD. Recombinant IKV-AAV vectors expressing GFP, Eyelea, or decorin were co-injected intravitreally with Nuc1 in adult C57Bl / 6J mice. Three weeks later, mice were exposed to laser-induced CNV similar to the studies described above for decorin and anti-VEGF antibodies and examined one week later. Quantification of CNV by isolectin and α-smooth muscle actin (SMA) showed that AAV-IKV-decorin was significantly superior to AAV-IKV-Eylea (Aflibercept) in inhibiting CNV and fibrosis. Fig.13 ).

[0127] discuss

[0128] In this study, we describe a novel peptide termed Nuc1. The peptide was designed based on the nucleoside binding properties of laminin-1 and the heparan sulfate binding properties of VEGF165A. To our knowledge, Nuc1 is the most potent peptide described to date that penetrates the retina. Importantly, unlike most cell-penetrating peptides (Johnson et al., 2008) or aptamers (Leaderer et al., 2015, 2016; Talreja et al., 2018) previously used in the retina, Nuc1 does not require chemical coupling of the peptide to the cargo. The ability to deliver proteins into retinal cells and tissues without the need for physical attachment greatly expands the utility of CPPs for therapeutic purposes. Although physical or chemical attachments between proteins and peptides have been well described, such attachments may have adverse effects on protein function. (Zhang et al., 2018). In theory, the delivery system of the present invention is able to take any protein known to be functionally active and deliver it across the plasma membrane into cells without the need for complex chemical means.

[0129] We demonstrate that heterologous proteins retain function after being delivered to the retina by Nuc1. For example, recombinant XIAP inhibits MNU-induced retinal apoptosis and retinal apoptosis induced by retinal detachment. Furthermore, when Nuc1 was applied to the cornea after chemical burn, it enhanced the efficacy of anti-fibrotic proteins such as decorin. This observation was unexpected given that Nuc1 was specifically designed to target the retina. Therefore, Nuc1 may have effects in tissues other than the retina and cornea, but this remains to be determined.

[0130] Delivery of antibodies by intravitreal injection is the current clinical standard of care (Comparison of Age-related Macular Degeneration Treatments Trials Research et al., 2012). We demonstrate that the efficacy of low or lower doses of antibodies can be enhanced when co-delivered with Nuc1. This has implications for both “cost of goods” as well as reducing potential toxicity of the drug due to off-target activity. For example, systemic leakage of anti-VEGF antibodies from the vitreous is detrimental to patients (Christoforidis et al., 2017; Hwang et al., 2012; Michalska-Malecka et al., 2016). Reducing the dose of antibody required to achieve efficacy in the vitreous could reduce such systemic side effects. Steroids represent another drug whose therapeutic use is hampered by side effects. Dexamethasone causes cataract formation and increased intraocular pressure when injected intravitreally (Zhang et al., 2018). These side effects may be mitigated when combined with Nuc1 due to the reduction in the dose required to be effective; however, this remains to be determined.

[0131] Nuc1 is also able to enhance the uptake of recombinant AAV by retinal cells, particularly photoreceptors. AAV and other viruses produce immune responses when injected at high doses (Boyd et al., 2016; Reichel et al., 2017). The ability to enhance infection reduces the total viral dose required to achieve a therapeutic effect for gene therapy applications. In addition, the ability to incorporate Nuc1 sequences, or shorter sequences derived from them, into AAV capsids simplifies the production of recombinant AAV for use as “drugs.” We did not observe a significant increase in AAV infection when Nuc1 peptides were co-injected with AAV containing Nuc1 sequences, likely due to competition for cell surface receptors required for infection. In our studies, the greatest infection was achieved with Nuc1 peptides combined with AAV containing laminin-1 sequences in the AAV capsid. Previous attempts to enhance AAV infection via the intravitreal route have generally required biopanning with complex selection procedures (Dalkara et al., 2013). Our more “design-oriented” approach is significantly simpler and has been shown to be at least as effective. While the safety of these vectors remains to be determined, the ability to enhance infection via subretinal or intravitreal routes significantly enhances the field of ocular gene therapy. Based on the nature of retinal neurons, we anticipate that the approach described in this manuscript may be extended to other neuronal tissues, such as the brain, but this remains to be determined.

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[0179] Example 2 - Local delivery of decorin using the cell-penetrating peptide Nucl enhances recovery of alkali burn-induced corneal injury in mice

[0180] Corneal injuries account for approximately 4% of all cases of complete vision loss. Approximately 80% of ocular injuries are chemical in nature, with alkaline and acidic agents responsible for approximately 11–22% of ocular injuries and 4% of all occupational injuries [1–3]. The corneal epithelium functions as a barrier between the external environment and the interior of the eye [4]. When the corneal surface is damaged, multiple responses are activated, including fibrosis, angiogenesis, and inflammation, to heal the injury and protect the eye from further damage. However, if excessive, these responses themselves may cause further damage [5]. Acute inflammation and neovascularization of the anterior segment of the eye can lead to abnormal healing of the epithelial tissue and corneal scarring. Due to their lipophilic nature, alkaline agents rapidly penetrate ocular tissues, causing necrosis and ischemia [6]. Patients with corneal damage caused by alkali burns are at increased risk for glaucoma and irreversible vision loss.

[0181] Alkali burns lead to infiltration of corneal macrophages and leukocytes, as well as upregulation of proinflammatory cytokines such as IL-1β, TNF-α, IL-6, and vascular endothelial growth factor A (VEGF-A). An imbalance of pro- and anti-angiogenic molecules in the cornea triggers neovascularization [7, 8]. Epithelial regeneration through cell proliferation and apoptosis of stromal keratocytes occurs near the burn / injury site [9]. In addition to corneal damage, alkali burns have been reported to cause apoptosis of retinal ganglion cells and optic nerve damage.

[0182] The eyelids are the first line of ocular defense, protecting the eye from allergens, foreign particles, and pathogens. The blinking action cleanses the ocular surface and renews the tear film, which is composed of immunoglobulins A and G as well as antimicrobial proteins such as lysozyme, β-lysine, and metal chelators [10, 11]. However, one of the challenges associated with the topical delivery of molecules to the ocular surface is that the tear film rapidly clears drugs from the surface, resulting in more than 95% drug loss. Drugs that are able to penetrate the inner compartment generally have a short half-life due to the recirculation of the aqueous humor. To our knowledge, there are no reports on the topical delivery of proteins to the cornea for the treatment of alkali burns.

[0183] Decorin is a small, leucine-rich proteoglycan that plays an important role in regulating cell proliferation, survival, and differentiation by regulating multiple growth factors in the cornea, such as TGF-β.[12-16] Decorin also plays a key role in maintaining corneal transparency by inhibiting scarring and angiogenesis. Mutations in decorin have been shown to be associated with congenital mesenchymal dystrophy.

[17]

[0184] Overexpression of decorin has been shown to significantly reduce fibrosis in in vivo models of brain and spinal cord injury. In this study, we examined the effects of local delivery of decorin alone and in combination with the cell-penetrating peptide Nuc1 on fibrosis, angiogenesis, apoptosis, and inflammation levels in a mouse model of corneal alkali burn.

[0185] Materials and methods:

[0186] Peptide synthesis: Nuc1 peptide, sequence ASIKVAVSAGGDKPRR (SEQ ID NO: 3) was synthesized by Thermo Fisher Scientific with a purity of >99%.

[0187] Animals: 6- to 8-week-old C57BL / 6J mice were purchased from Jackson Laboratory (Bar Harbor, ME) and housed under a 12-h light / 12-h dark cycle. This study was performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal Care and Use Committee (IACUC) of Tufts University.

[0188] Animal injury model: 0.1 mg / g body weight of ketamine (Phoenix TM Mice were anesthetized with a mixture of 1% dapoxetine (Akorn Inc., St Joseph, MO) and 0.1 mg / g body weight of xylazine (Lloyed, Shenandoah, Iowa), followed by topical application of 0.5% procaine hydrochloride (Akorn Inc., Lake Forest, IL, USA) for local corneal analgesia. The mice were kept warm during anesthesia. Alkali burns were induced in the cornea by applying a 2 mm filter paper soaked in 1N sodium hydroxide (NaOH) on the center of the right cornea for 30 seconds. The filter paper was gently removed from the cornea and the cornea was rinsed 10 times with phosphate buffered saline (PBS) to remove residual NaOH. The left eye was not exposed to NaOH and served as a control. Topical application of PBS, recombinant core protein polysaccharide (0.5 μg) + Nuc1 (0.5 μg) was started after 24 hours of exposure to NaOH. The treatment was applied topically once a day for seven days. Eye images were captured using a digital camera on the 7th day. After 7 days, the mice were killed by CO2 inhalation and then cervical dislocation. Eyes were enucleated and fixed in 4% paraformaldehyde. Corneal cryosectioning was performed using a Micron 550 cryostat.

[0189] Immunohistochemistry: Cryosections were air-dried for 10 minutes and washed in PBS for 5 minutes. They were then incubated in 6% normal goat serum and PBS-Triton for 1 hour at room temperature for permeabilization and blocking. Slides were incubated with FITC-conjugated isolectin or with primary antibodies against one of the following substances: α-smooth muscle actin (SMA), glial fibrillary acid protein (GFAP), CD45, transforming growth factor β (TGF-β1), F4 / 80, transforming growth factor β (TGF-β1) (Abcam; ab92486) or activated Caspase-3. Incubation was performed overnight at 4°C in a humidified chamber. For samples treated with antibodies, detection was performed by incubation with Cy3-conjugated goat anti-rabbit / anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, Pennsylvania) for 1 hour at room temperature. Slides were washed three times with PBS and mounted in Vectashield antifade mounting medium containing DAPI to counterstain cell nuclei. Stained sections were imaged using an Olympus IX51 microscope and appropriate filters. Images were acquired using a Retiga 2000r camera. The intensity of antibody-specific staining was quantified using ImageJ software.

[0190] Histopathology: Eyes were harvested for histological examination 7 days after treatment and fixed in Hartman's fixative. After 48 hours, the specimens were dehydrated by an alcohol step and embedded in paraffin. 5 mm sections were cut in different planes (including the optic nerve area) and stained with hematoxylin-eosin (H&E). Imaging of the stained sections was performed using an Olympus BX51 microscope and a Retiga 2000r camera.

[0191] Multiplex ELISA: Follow the manufacturer's

[0192] Description, multiplex ELISA was performed using Bio-plex pro mouse cytokine Th17 A 6 plex group I (Bio Rad, M6000007NY) to detect IL-6, IL-17, IL-10, IFN-G, TNF-α and IL-1β. Briefly, alkali burn was induced as described above. Mice were sacrificed on day 7 and corneas were extracted. The corneas were divided into small pieces and stored in Bioplex cell lysis buffer at -80°C. For each sample, two corneas were mixed together and 50 μl of lysis buffer was used for each sample in duplicate. Samples were run using Bioplex manager MP software and data were analyzed using Bioplex manager 6.0.

[0193] TUNEL assay: To detect cell death, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was performed on corneal cryosections using the in situ cell death detection kit, TMRRed (Sigma) according to the manufacturer's instructions. Sections were imaged as described above, and images were used to quantify TUNEL-positive cells using ImageJ (FIJI version and plugin) as previously described

[18] .

[0194] Statistical analysis: Experimental values ​​are expressed as mean ± SEM. One-way ANOVA test was used to analyze the statistical differences between more than two groups, and unpaired T test was used to analyze the differences between two groups. A p value less than or equal to 0.05 was considered statistically significant.

[0195] result

[0196] Nuc1 enhances the ability of decorin to reduce corneal opacity and cellular infiltration

[0197] To test the effects of topical application of decorin alone and decorin combined with Nuc1 (Decorin + Nuc1) on corneal opacity, mice were exposed to alkali burn as described in Methods. 24 hours after alkali burn, decorin alone, decorin + Nuc1, or PBS were applied once daily for 7 days. On day 7, mice were sacrificed for corneal imaging ( Fig.14 A). The corneas of control mice not exposed to alkali burn or drug treatment were observed to be smooth and transparent ( Fig.14 A). In contrast, the corneas of alkali burn-exposed mice treated with PBS for 7 days were observed to have diffuse "cloudiness" over the entire corneal surface. In addition, the cornea had an irregular surface with prominent blood vessels ( Fig.14 A). Mice exposed to alkali burn followed by topical application of decorin alone showed corneal appearance similar to that of mice treated with PBS, with prominent opacities, irregular surfaces, and blood vessels ( Fig.14 A). However, mice exposed to alkali burn and subsequently applied with decorin + Nuc1 exhibited smoother corneal surfaces and significantly reduced corneal opacity, with no obvious blood vessels, relative to PBS-treated mice exposed to alkali burn ( Fig.14 A). Clinical opacity scores were performed on mice according to the method described by Anderson et al.

[19] . According to the scoring system, mice treated with PBS after alkali burn had a score of 4 (completely opaque, pupil not visible), mice treated with decorin had a score of 2.8 (opaque, pupil barely detectable), and mice treated with decorin + Nuc1 had a score of 1.5 (slightly opaque, iris and pupil still detectable).

[0198] Transverse sections of the corneas from alkali-burn-exposed mice treated with each of the above methods were stained with hematoxylin and eosin (H+E; Fig.14 B). Corneas of alkali burn-exposed mice treated with PBS showed thinner corneas and cell loss of the epithelial cell layer relative to corneas of untreated control mice ( Fig.14 B). In addition, there was a clear cellular infiltration in the cornea and under the cornea. Increased epithelial thickness was observed in the corneas of alkali burn-exposed mice treated with decorin relative to corneas treated with PBS ( Fig.14 B). However, corneas treated with decorin showed significant cavitation. In contrast, in the corneas of alkali-burn-exposed mice treated with decorin + Nuc1, the upper cell layer of the epithelium was repaired significantly, and the integrity of the corneal surface was increased ( Fig.14 B).

[0199] Decorin + Nuc1 significantly reduces neovascularization and fibrosis in alkali-burned cornea

[0200] After alkali burn, the cornea is susceptible to neovascularization and fibrosis. Upregulation of transforming growth factor β (TGF-β) promotes the migration of corneal epithelial cells and keratocytes to the alkali burn site, where keratocytes differentiate into myofibroblasts

[20] . Uncontrolled and sustained activation of myofibroblasts leads to pathological fibrosis. We documented the presence of vascular endothelial cells and the expression of the myofibroblast marker α-smooth muscle actin (SMA) in the cornea after alkali burn

[21] . To determine the effects of decorin alone or decorin + Nuc1 on alkali burn-induced corneal neovascularization and fibrosis, mice were exposed to alkali burn and treated as described above. Seven days after treatment, mice were sacrificed and their corneas were harvested for cryosectioning and stained with FITC-conjugated Griffonia suffruticosa lectin I (GSL I) / isolectin and antibodies against α-smooth muscle actin (SMA). In the corneas of untreated control mice, little or no staining for isolectin (green) or SMA (red) was observed ( Fig.15A ). However, corneas of alkali-exposed mice treated with PBS showed extensive isolectin and SMA staining throughout all corneal layers, suggesting extensive neovascularization and fibrosis. Topical application of decorin to alkali-burn-exposed corneas showed a significant reduction in isolectin staining in the corneal epithelium, stroma, and endothelium, as well as in the aqueous humor ( Fig.15A ), indicating reduced neovascularization. In corneas treated with decorin, some reduction in SMA staining was evident, especially in the posterior cornea and aqueous humor. However, topical application of decorin + Nuc1 to corneas exposed to alkali burns showed an almost complete disappearance of isolectin and SMA staining in the stroma, endothelium, and aqueous humor, with a significant reduction in both staining in the epithelium ( Fig.15A). This suggests that decorin + Nuc1 mediates highly significant neovascularization and protection from fibrosis in corneas exposed to alkali burns.

[0201] Quantification of isolectin staining of the cornea ( FIG. 15B ) showed that, although topical decorin alone significantly reduced isolectin staining in the cornea, the amount of GSL I staining was not significantly different from that of PBS-treated corneas. However, topical application of decorin + Nucl to corneas exposed to alkali burns showed a significant reduction of isolectin staining by 83.9% relative to PBS-treated corneas (p < 0.042), indicating a significant reduction in neovascularization. Quantification of SMA staining of the cornea showed that topical decorin alone significantly reduced SMA staining by 33.25% relative to PBS-treated corneas (p < 0.025). However, topical application of Nucl + decorin to corneas exposed to alkali burns resulted in a more significant reduction in SMA staining, i.e., 46.22% (p < 0.0028) relative to PBS-treated corneas ( FIG. 15B ), indicating that decorin + Nucl has an enhanced fibrosis protective ability relative to decorin alone.

[0202] Decorin+Nuc1 significantly reduces inflammatory cell infiltration in the cornea of ​​the alkali burn model

[0203] Next, we examined inflammation in the corneas of alkali burn-exposed mice following treatment with PBS, decorin alone, or decorin + Nuc1. Transverse sections of the corneas were stained for CD45 and F4 / 80 to determine the presence of leukocytes, particularly macrophages. As expected, there was little or no CD45 or F4 / 80 antibody staining in the corneas of untreated control mice ( Fig.16A In mouse corneas exposed to alkali burn and subsequently treated with PBS, there is extensive staining of CD45 (green channel) and F4 / 80 (red channel) antibodies throughout the stroma ( Fig.16A), indicating a massive infiltration of inflammatory cells in these corneas. Corneas of mice exposed to alkali burns and then treated topically with decorin alone or decorin + Nucl showed reduced CD45 and F4 / 80 staining in the anterior stroma (close to the epithelium). Quantification of CD45 staining in the corneas showed a significant 39.6% reduction in the number of CD45-positive cells in corneas treated with decorin alone relative to corneas treated with PBS (p < 0.0147) (Figure 16B). Corneas topically treated with decorin + Nucl also showed a significant 59.8% reduction in CD45 staining relative to corneas treated with PBS (p < 0.0017) (Figure 16B). However, there was no significant difference in CD45 staining between corneas treated with decorin alone and corneas treated with decorin + Nucl (Figure 16B). Quantification of F4 / 80 staining in the corneas showed that the staining of the corneas treated with decorin + Nucl was significantly reduced by 57.9% (p < 0.0052) relative to the corneas treated with PBS (Figure 16B), indicating that macrophage infiltration in these corneas was significantly reduced. In contrast, there was no significant reduction in F4 / 80 staining in the corneas treated with decorin alone relative to the corneas treated with PBS (Figure 16B). This data indicates that the effect of topical application of decorin + Nucl on the improvement of inflammatory cell infiltration in the corneas of alkali burn-exposed mice is more effective than topical application of decorin alone, especially for macrophages.

[0204] The inflammatory markers CD45 and F4 / 80 play an important role in the production of cytokines / chemokines at the site of injury, which is responsible for further tissue destruction and scar formation. Therefore, we further determined the expression levels of Th17 cytokines using the Bioplex assay. Corneal lysates were collected from mice exposed to alkali burns 7 days after treatment, and the levels of IL-1β, IL-6, tumor necrosis factor α (TNF-α), interferon γ (IFN-G), IL-17, and IL-10 were determined. Compared with the corneas of untreated control mice that were not exposed to alkali burns, increases in five cytokines (TNF-α, IL-1β, IL-6, IL-17, and IFN-G) were observed in the corneas of mice exposed to alkali burns and treated with PBS ( Fig.17A ). In both control and treatment groups, the levels of the cytokine IL-10 were below the detection limit of the assay (data not shown). We observed decreased expression of TNF-α, IFN-G, IL-17, and IL-6 after treatment with decorin alone or decorin + Nuc1 relative to PBS-treated mice ( Fig.17A). Notably, mice treated with decorin + Nuc1 showed a greater decrease in expression compared to mice treated with decorin alone. Interestingly, increased IL-1β expression was observed in the corneas of mice treated with decorin alone and decorin + Nuc1 ( Fig.17A ).

[0205] Alkali burns also lead to the production of the inflammatory cytokine, transforming growth factor β (TGF-β1), which plays an important role in the differentiation of keratocytes into myofibroblasts and aggravates the damage of corneal tissue by inducing further expression of cytokines, thereby forming an inflammatory feedback loop. Using antibody staining against TGF-β1, we observed that after PBS treatment of corneal alkali burns in mice, TGF-β1 was significantly expressed in the corneal epithelium, stroma, and endothelium ( Fig. 17B Although decorin treatment alone resulted in decreased TGF-β1 expression, TGF-β1 levels were significantly lower in mice treated with decorin + Nuc1 compared with decorin alone ( Fig. 17B ).

[0206] Nuc1 decorin + Nuc1 significantly reduces cell death in the cornea of ​​an alkali burn mouse model

[0207] Caspase-3-mediated apoptosis has been shown to occur in the cornea of ​​mice exposed to alkali burn. To determine whether decorin alone or decorin + Nuc1 has an effect on alkali burn-induced cell death, transverse cryosections of mouse corneas collected on day 7 after treatment were stained for activated caspase-3 ( Fig.18 There was no detectable activated Caspase-3 in the corneas of untreated control mice that were not exposed to alkali burn ( Fig.18 ). However, activated Caspase-3 staining was observed in all layers (epithelium, stroma, and endothelium) of the cornea of ​​alkali burn-exposed mice treated with PBS ( Fig.18 Compared with corneas from PBS-treated mice, activated Caspase-3 staining was significantly reduced in the corneal stroma and endothelium, and possibly in the epithelium, in mice exposed to alkali burn and treated topically with decorin alone ( Fig.18 ). However, in the corneas of mice exposed to alkali burn and treated topically with decorin + Nuc1, activated Caspase-3 staining in all three layers of the cornea (including the epithelium) was significantly reduced relative to PBS-treated mice. Quantification of the fluorescent signal of activated Caspase-3 staining in corneas from each treatment group and the "untreated" group showed that activated Caspase-3 staining was significantly reduced in the corneas treated with decorin alone (51.5%) or decorin + Nuc1 (74.6%, p < 0.0001, Fig.18) treated with decorin significantly reduced activated Caspase-3 staining in the corneas of mice. The amount of activated Caspase-3 staining in the corneas treated with decorin + Nuc1 was also significantly reduced (47.6%, p = 0.0001) compared to the corneas treated with decorin alone ( Fig.18 ).

[0208] Gross cell death was also assessed by TUNEL staining of corneal cryosections ( Fig.19 A). Little or no TUNEL staining was observed in untreated corneas of control mice not exposed to alkali burn ( Fig.19 A). TUNEL staining, indicative of cell death, was observed in the stroma and epithelium of the cornea of ​​mice exposed to alkali burn and subsequently treated with PBS ( Fig.19 A). TUNEL staining in the corneal stroma of mice exposed to alkali burn and treated with decorin alone was significantly reduced ( Fig.19 In the corneas of mice exposed to alkali burn and treated with decorin + Nuc1, there was little or no TUNEL staining in any corneal layer, indicating that cell death in the corneas of these mice was almost completely prevented ( Fig.19 B). Quantification of TUNEL staining in these corneas indicated that TUNEL staining in the corneas of mice exposed to alkali burn and treated with decorin alone was significantly reduced (44.8%, p<0.0882) relative to PBS-treated mice ( Fig.19 B). However, a greater and significant reduction in TUNEL staining was observed in corneas exposed to alkali burn and treated with decorin + Nucl relative to the PBS treated group (90.45%, p < 0.006) ( Fig.19 B).

[0209] Decorin+Nuc1 significantly reduces gliosis in the cornea of ​​an alkali-burn mouse model

[0210] Gliosis is the activation of Müller glia in the retina and is a rapid stress response to alkali injury. Increased expression of glial fibrillary acidic protein (GFAP) is a hallmark of gliosis. Previous studies have shown that an increase in the proinflammatory cytokine TNF-A in response to alkali burns leads to the induction of retinal gliosis

[22] . To determine whether topical treatment of the cornea of ​​mice exposed to alkali burns with decorin alone or decorin + Nuc1 results in protection of the retina from glial cell activation, we stained transverse cryosections of the retinas of mice subjected to corneal alkali burns and treated topically with PBS, decorin alone, or decorin + Nuc1 for 7 days for GFAP ( Fig. 20). The GFAP staining pattern observed in the retinas of control mice not exposed to alkali burn was consistent with the absence of activated glial cells ( Fig. 20 In control retinas, GFAP was observed only in astrocytes and Müller cell end feet in the anterior retina. Retinas from mice exposed to alkali burn and treated with PBS were observed to have a GFAP staining pattern consistent with Müller glial activation ( Fig. 20 ), GFAP was observed throughout the Müller cells, extending from the ganglion cell layer (GCL) through the inner nuclear layer and into the outer nuclear layer (INL / ONL). Retinas from mice exposed to alkali burn and treated with decorin alone also had a GFAP staining pattern consistent with Müller cell activation, but the staining was reduced relative to retinas from mice treated with PBS ( Fig. 20 ). In these retinas, GFAP staining was observed extending from the GCL through the Müller cells into the inner plexiform layer, but not in the INL or ONL of these retinas. However, in the retinas of mice exposed to alkali burn and treated with decorin + Nuc, GFAP staining was significantly reduced relative to retinas of mice treated with decorin alone. In these retinas, staining was mostly confined to the Müller cell end feet in the GCL, with occasional Müller cell staining in the inner plexiform layer ( Fig. 20 ). These data suggest that both decorin alone and decorin+Nuc1 mediate the reduction of retinal gliosis in mice exposed to alkali burn. However, we cannot exclude the possibility that decorin alone and decorin+Nuc1 directly affect retinal Müller cell activation.

[0211] discuss

[0212] Improving the retention and efficacy of drugs delivered to the eye is key to improving the bioefficiency and therapeutic response of topical therapies. Due to the high turnover rate of the precorneal tear film, the rapid clearance of aqueous drugs is a major challenge as it reduces the efficacy of drugs delivered to the cornea. Intensive topical drug administration routes or invasive drug delivery methods (including subconjunctival or intravitreal injections) are often accompanied by complications. In cases where the drug is ineffective, surgery is often required to treat or remove the resulting corneal scar, increasing the risk of morbidity and the duration of patient discomfort after treatment.

[0213] Ocular burns are often associated with vision-threatening complications and represent a clinical challenge for ophthalmologists. Current medical treatment is aimed at controlling inflammation through the prudent use of corticosteroids or prophylactic antibiotics. The alkali burn mouse model is well documented and provides a robust, clinically relevant means to evaluate the anti-scarring and anti-fibrotic effects of recombinant human decorin. Topical administration of decorin protein (with or without the cell-penetrating peptide Nuc1) resulted in a significant reduction in corneal opacity levels after 7 days of topical treatment. This reduction in corneal opacity may have long-term benefits for patients, thereby preserving vision. Interestingly, we observed that this treatment not only prevented fibrosis but was also associated with a significant reduction in inflammation, thereby preventing retinal gliosis. Our results are consistent with previous studies that indicate that the production of the proinflammatory cytokine TNF-α in the cornea after alkali burns leads to gliosis [22–24]. This reduction in inflammation is also beneficial because it protects the epithelial and stromal layers of the cornea by inhibiting the activation of the caspase-3-dependent apoptotic pathway. Topical application of decorin was also able to modulate the pathology associated with alkali burns, possibly due to its ability to penetrate the disrupted microenvironment of the injured cornea. Furthermore, the combination of decorin and Nuc1 resulted in a significant reduction in haze, opacity, fibrosis, and inflammation after alkali burns. Our results suggest that Nuc1 may enhance the intrinsic ability of decorin protein to accelerate the healing process by increasing its retention time and thus its therapeutic efficiency.

[0214] Alkali burns result in multiple damages to corneal structures, including epithelial thinning, stromal edema, and deposition of cellular infiltrates. Together with the tear film and apical mucosa, the epithelium is the first line of defense of the eye. Topical application of decorin together with the peptide Nuc1 restored epithelial morphology and reduced stromal edema as well as the total thickness of the cornea. In addition, previous studies have reported that decorin regulates several growth factors and signaling pathways (SMAD2 and SMAD3 via TGF-β) in corneal fibroblasts, thereby attenuating scarring during fibrosis. Since alkali burns are acute in nature, endogenous decorin may not be able to neutralize overactive TGF-β molecules, allowing activation of downstream fibrotic cascades.

[0215] Alkali burns cause extensive anterior segment inflammation leading to extensive damage to the inner and outer retinal layers as well as the optic nerve. A recent report documented that patients with alkali burns who underwent Boston keratoprosthesis implantation presented with elevated intraocular pressure (i.e., glaucoma). Despite aggressive treatment measures and surgery, the eyes of burn patients deteriorated more rapidly compared with non-burned implanted patients. The mechanism of retinal damage after alkali burns has not been elucidated, but a few studies have suggested the possibility of posterior alkali diffusion. There are also reports suggesting that retinal damage is caused by the production of inflammatory cytokines, particularly TNF-α, rather than simply diffusion

[25] . In addition, the proinflammatory cytokines TNF-α and IL-1β contribute to inflammation in several infectious and non-infectious ocular pathologies. IL-6 also plays an important role in corneal inflammatory diseases, as IL-6R antagonists reduce corneal inflammation and neovascularization

[25] . Increased inflammation creates a feedback loop that leads to further upregulation of proinflammatory genes

[26] . The inflammatory environment disrupts the blood-retina barrier and leads to the infiltration of immune cells from the blood, mainly neutrophils and macrophages

[26] . These infiltrating immune cells activate resident immune cells in the eye. In addition, synaptic signaling from Müller glia

[27] triggers the production of cytokines in the retina, causing cellular stress in the anterior and posterior segments. Previous studies have reported that apoptosis in the INL and ONL of the mouse and rabbit retina caused by increased anterior chamber pH causes inflammation in uveitis [27, 28]. In our study, we observed robust gliosis in the retina and apoptosis in the cornea seven days after alkali burn injury, which was accompanied by increased levels of inflammatory cytokines TNF-α, IL-6, and IFN-G in corneal lysates. Topical application of decorin helps to reduce inflammation, thereby controlling gliosis and apoptosis. Although these results are encouraging, the multiple protective effects of decorin in the treatment of alkali burns need further investigation.

[0216] In conclusion, although extensive preclinical safety studies are necessary, we have demonstrated that topical delivery of the antifibrotic protein decorin combined with Nuc1 may be safe and that Nuc1 can penetrate the inflamed cornea and deliver the cargo protein into the corneal tissue. Decorin combined with Nuc1 can effectively rescue the corneal surface after alkali burns. Alkali burns belong to the category of severe ocular trauma, and currently there are few treatment options. In our study, decorin retained its therapeutic potential and remained in contact with the ocular surface for a long enough time that it was able to significantly reduce corneal scarring. Therefore, the topical application of decorin combined with Nuc1 is a promising treatment option for alkali burns due to its ability to combat ocular fibrosis, inflammation, and cell death.

[0217] Example 3 - Nuc1 enhances retinal delivery of gene editing agents

[0218] To complement gene therapy and protein therapy, there is great interest in the field of gene editing. A commonly used protein for gene editing is Cas9. Compared with gene therapy, long-term expression of transgenes is desired, and off-target effects can be avoided by transient expression of gene editing proteins (such as Cas9). In addition, Cas9 and its functional related substances are bacterial proteins, so if they are expressed indefinitely in human cells, they are immunogenic and toxic. Gene therapy methods in which transgene expression is regulated in vivo are complicated. Therefore, a more practical solution to the above problems is to deliver gene editing proteins transiently into the cell nucleus. Once those exogenously delivered proteins have completed their gene editing functions, they should be naturally degraded in the cell in a manner that is common to essentially all intracellular proteins. In order for this approach to be successful, cell barriers must be overcome to allow Cas9 to be delivered to target cells. We hypothesize that CPP Nuc1 can be used for this purpose.

[0219] Nuc 1 enhances retinal uptake of Cre recombinase and promotes gene editing

[0220] To test this hypothesis, we first investigated whether Nuc1 could promote or enhance the uptake of Cre recombinase protein in Ai9 mice. In these mice, when Cre recombinase excises the floxed stop codon, the tdTomato reporter transgene expression cassette is activated. We injected 4 micrograms of Cre recombinase protein alone or in combination with 1 microgram of Nuc1 intravitreally into adult (approximately 6 weeks old) Ai9 mice. We found that although tdTomato expression was detectable in Ai9 mice injected with Cre recombinase alone, a larger number of cells and more types of cells showed reporter expression when Cre recombinase was co-injected with Nuc1. Specifically, there were significantly more tdTomato-positive Müller cells ( Fig.21 ). In fact, there were almost no Miller cells expressing tdTomato with Cre recombinase alone. Therefore, our data demonstrate that Nuc1 enhances the uptake of exogenously delivered Cre recombinase and that the Cre recombinase delivered by Nuc1 is functional in the nucleus. The observation that Cre recombinase can enter cells on its own is surprising. However, the above assay is highly sensitive due to the fact that one gene editing event is greatly amplified by the accumulation of protein in the cell through continuous production. Therefore, the ability of Nuc1 to enhance the uptake of Cre recombinase is functionally important.

[0221] Nuc 1 promotes retinal uptake of Cas9 and facilitates gene editing

[0222] The molecular weight of Cre recombinase is approximately 38Kd, which is much smaller than Cas9 (molecular weight approximately 160Kd). To determine whether gene editing can be achieved using a significantly larger protein, we injected Cas9-GFP fusion protein into C57BL / 6J mice intravitreally. Cas9-GFP itself cannot penetrate the retina and is localized to the inner limiting membrane ( Fig. 22 This indicates that while smaller proteins such as Cre recombinase can enter the retina on their own, the larger, more therapeutically relevant Cas9-GFP protein cannot. However, when co-injected with Nuc1, Cas9-GFP localized to multiple cell types in the retina, including the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). Fig. 22 ).

[0223] Next, we wished to address whether Nuc1 could enhance the uptake of Cas9 coupled to ribonucleoprotein particles (RNPs) in the retinas of Ai9 mice. Therefore, we co-injected Nuc1 and Cas9 complexed with RNPs targeting the floxed stop codon in the tdTomato reporter gene described above intravitreally or subretinally. After subretinal injection, tdTomato was prominently expressed in the retinal pigment epithelium (RPE, Fig.23 A-23B), indicating successful targeting of the RNP. However, when Cas9-RNP was injected without Nuc1, there was no tdTomato expression, confirming that Nuc1 is required for efficient delivery of Cas9-RNP in the retina. At higher concentrations, intravitreal injection of Nuc1 and Cas9-RNP also initiated tdTomato expression in cells. The cell types that showed reporter gene expression in different parts of the retina with intravitreal injection were variable, but included Müller cells and photoreceptors ( Fig.24 ).

[0224] References

[0225] 1. Griffith, GL, et al., Treatment of corneal chemical alkali burns with a cross / inked thiolated hyaluronic acid film. Burns, 2018.44(5): p.1179-1186.

[0226] 2.Clare,G.,et al.,Amniotic membrane transplantation for acute ocularburns.Cochrane Database Syst Rev,2012(9):p.CD009379.

[0227] 3.Behndig,A.,et al.,A case of unilateral acid burn.Acta OphthalmolScand,2003.81(5):p.526-9.

[0228] 4.Bron,A.J.,The architecture of the corneal stroma.Br J Ophthalmol,2001.85(4):p.379-81.

[0229] 5.Strober,W.,I.J.Fuss,and R.S.Blumberg,The immunology of mucosa!models of inflammation.Annu Rev Immunol,2002.20:p.495-549.

[0230] 6.Fish,R.and R.S.Davidson,Management of ocular thermal and chemicalinjuries,including amniotic membrane therapy.Curr Opin Ophthalmol,2010.21(4):p.317-21.

[0231] 7.Tzeng,H.E.,et al.,Interleukin-6 induces vascular endothelial growthfactor expression and promotes angiogenesis through apoptosis signal-regulating kinase I in human osteosarcoma.Biochem Pharmacol,2013.85(4):p.531-40.

[0232] 8.Shin,Y.J.,et al.,Chemical injury-induced corneal opacity andneovascularization reduced by rapamycin via TGF-beta l / ERK pathwaysregulation.Invest Ophthalmol Vis Sci,2013.54(7):p.4452-8.

[0233] 9.Dua,H.S.,et al.,Limbal epithelial crypts:a novel anatomicalstructure and a putative limbal stem cell niche.Br J Ophthalmol,2005.89(5):p.529-32.

[0234] 10.Gasymov,O.K.,et al.,Interaction of tear lipocalin with lysozymeand lactolerrin.Biochem Biophys Res Commun,1999.265(2):p.322-5.

[0235] 11.Glasgow,B.J.,et al.,Tear lipocalins:potential lipid scavengers forthe corneal surface.Invest Ophthalmol Vis Sci,1999.40(13):p.3100-7.

[0236] 12.Mohan,R.R.,et al.,Decorin biology,expression,function and therapyin the cornea.Curr Mol Med,2011.11(2):p.110-28.

[0237] 13.Du,S.,et al.,Decorin inhibits angiogenic potential of choroid-retinal endothelial cells by downregulating hypoxia-induced Met,Rac1,HIF-lalpha and VEGF expression in cocultured retinal pigment epithelial cells.ExpEye Res,2013.116:p.151-60.

[0238] 14.Grant,D.S.,et al.,Decorin suppresses tumor cell-mediatedangiogenesis.Onco gene,2002.21(31):p.4765-77.

[0239] 15.Mohan,R.R,et al.,Targeted decorin gene therapy delivered withadeno-associated virus effectively retards corneal neovascularization invivo.PLoS One,2011.6(10):p.e26432.

[0240] 16.Iozzo,R.V.,et al.,Decorinantagonizes / GF receptor I(IGF-IR)functionby interfering with IGF-IR activity and attenuating downstream signaling.JBiol Chem,2011.286(40):p.34712-21.

[0241] 17.Bredrup,C.,et al.,Congenital stromal dystrophy of the corneacaused by a mutation in the decorin gene.Invest Ophthalmol Vis Sci,2005.46(2):p.420-6.

[0242] 18.Maidana,D.E.,et al.,A Novel lmageJ Macro for Automated Cell DeathQuantitation in the Retina.Invest Ophthalmol Vis Sci,2015.56(11):p.6701-8.

[0243] 19.Anderson,C.,Q.Zhou,and S.Wang,An alkali-bum injury model ofcorneal neovascularization in the mouse.J Vis Exp,2014(86).

[0244] 20.Chowdhury,S.,et al.,Pirfenidone nanoparticles improve cornealwound healing and prevent scarring following alkali burn.PLoS One,2013.8(8):p.e70528.

[0245] 21.Chen,W.S.,et al.,Galectin-3 Inhibition by a Small-MoleculeInhibitor Reduces Both Pathological Corneal NeovascularizationandFibrosis.Invest Ophthalmol Vis Sci,2017.58(1):p.9-20.

[0246] 22.Paschalis,E.I.,etal.,The Role of Microglia and PeripheralMonocytes in Retinal Damage after Corneal Chemical Injury.Am J Pathol,2018.188(7):p.1580-1596.

[0247] 23.Paranthan,R.R.,et al.,A robust model for simultaneously inducingcorneal neovascularization and retinal gliosis in the mouse eye.Mal Vis,2011.17:p.1901-8.

[0248] 24.Zhou,C.,et al.,Sustained Subconjunctival Delivery of InfliximabProtects the Cornea and Retina Following Alkali Burn to the Eye.InvestOphthalmol Vis Sci,2017.58(1):p.96-105.

[0249] 25.Chen,Y.,et al.,MK2 inhibitor reduces alkali bum-inducedinflammation in rat cornea.Sci Rep,2016.6:p.28145.

[0250] 26.Ogura,S.,et al.,Sustained inflammation after pericyte depletioninduces irreversible blood-retina barrier breakdown.JCl Insight,2017.2(3):p.e90905.

[0251] 27.Wizeman,J.W.and R.Mohan,Expression of peptidyiarginine deiminase 4in an alkali injury model of retinal gliosis.Biochem Biophys Res Commun,2017.487(1):p.134-139.

[0252] 28.Kumar, B., SMCashman, and R.Kumar-Singh, Complement-MediatedActivation of the NLRP3 Jnflammasome and Its Inhibition by AAV-MediatedDelivery of CD59 in a Model of Uveitis. Mol Ther, 2018.26(6): p.1568-1580. Sequence Listing <110> R. Kumar-Singh <120> Novel peptides, compositions and methods for delivering agents into cells and tissues <130> 166118.00904 <160> 8 <170> Patent version 3.5 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Generalized Nuc1 peptides with variable linkers <220> <221> MISC_FEATURE <222> (10)..(10) <223> X is an optional flexible joint <400> 1 Ala Ser Ile Lys Val Ala Val Ser Ala Xaa Asp Lys Pro Arg Arg 1 5 10 15 <210> 2 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Generalized KV peptides flanked by variable linkers <220> <221> MISC_FEATURE <222> (1) <223> X is an optional flexible joint <220> <221> MISC_FEATURE <222> (11)..(11) <223> X is an optional flexible joint <400> 2 Xaa Ala Ser Ile Lys Val Ala Val Ser Ala Xaa 1 5 10 <210> 3 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Nuc1 peptide <400> 3 Ala Ser Ile Lys Val Ala Val Ser Ala Gly Gly Asp Lys Pro Arg Arg 1 5 10 15 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <400> 4 Ala Ser Ile Lys Val Ala Val Ser Ala 1 5 <210> 5 <211> 5 <212> PRT <213> Homo sapiens <400> 5 Asp Lys Pro Arg Arg 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Partial sequence of laminin-1 flanked by glycine linkers <400> 6 Gly Ala Ser Ile Lys Val Ala Val Ser Ala Gly 1 5 10 <210> 7 <211> 6 <212> PRT <213> Homo sapiens <400> 7 Cys Asp Lys Pro Arg Arg 1 5 <210> 8 <211> 19 <212> PRT <213> Homo sapiens <400> 8 Cys Ser Arg Ala Arg Lys Gln Ala Ala Ser Ile Lys Val Ala Val Ser 1 5 10 15 Ala Asp Arg

Claims

1. A cell-penetrating peptide, comprising SEQ ID NO: 5 connected to the C-terminus of SEQ ID NO: 4 via a flexible linker.

2. The cell-penetrating peptide according to claim 1, wherein the cell-penetrating peptide consists of SEQ ID NO:

3.

3. A pharmaceutical composition comprising the cell-penetrating peptide according to claim 1 or claim 2 and a cargo molecule.

4. The pharmaceutical composition of claim 3, wherein the cargo molecule is selected from the group consisting of peptides, recombinant proteins, antibodies, proteoglycans, steroids, viruses, nucleic acids, ribonucleoproteins, small molecule therapeutics, and detectable labels.

5. The pharmaceutical composition according to claim 3, wherein the cargo molecule is selected from the group consisting of anti-apoptotic agents, anti-inflammatory agents, anti-angiogenic agents and anti-fibrotic agents.

6. The pharmaceutical composition according to claim 3, wherein the cargo molecule is selected from the group consisting of X-linked inhibitor of apoptosis protein, decorin, anti-vascular endothelial growth factor antibody, BH4 domain peptide from Bcl-xL, NRF2 and dexamethasone.

7. The pharmaceutical composition according to claim 3, wherein the cargo molecule is a gene therapy agent or a gene editing agent.

8. The pharmaceutical composition according to claim 7, wherein the cargo molecule is selected from the group consisting of gene therapy vectors, Cre and Cas9 proteins.

9. The pharmaceutical composition of claim 3, wherein the cargo molecule is a viral vector.

10. A polynucleotide encoding the cell-penetrating peptide according to claim 1 or claim 2.

11. A polynucleotide encoding a viral capsid protein, wherein the cell-penetrating peptide according to claim 1 or claim 2 is inserted into the viral capsid protein.

12. A nucleic acid construct comprising the polynucleotide of claim 10, wherein the polynucleotide is operably linked to a promoter.

13. An adeno-associated virus (AAV), comprising a modified viral capsid protein, wherein the modified viral capsid protein comprises SEQ ID NO:

6.

14. The virus of claim 13, wherein the virus has an AAV9 capsid.

15. The virus of claim 13 or claim 14, further comprising a polynucleotide encoding a polypeptide cargo molecule.

16. The virus of claim 15, wherein the polypeptide cargo molecule is selected from the group consisting of decorin and NRF2.

17. The virus of claim 15, wherein the polypeptide cargo molecule is selected from the group consisting of an anti-apoptotic agent, an anti-inflammatory agent, an anti-angiogenic agent, an anti-fibrotic agent, a gene therapy agent, and a gene editing agent.

18. An in vitro delivery method for cells or tissues, the method comprising contacting the cells or tissues with the cell-penetrating peptide of claim 1 or claim 2, wherein the cell-penetrating peptide enters the cells or tissues, wherein the cells or tissues are retinal or corneal.

19. The method of claim 18, further comprising contacting the cell or tissue with a cargo molecule, wherein the cell penetrating peptide increases delivery of the cargo molecule to the cell or tissue or allows transduction of the cargo molecule into the cell or tissue as compared to delivery to the cell in the absence of the cell penetrating peptide.

20. The method of claim 18 or claim 19, wherein the cell penetrating peptide is not conjugated or physically linked to the cargo molecule.

21. A method for delivering a cargo molecule to a cell or tissue in vitro, the method comprising contacting the cell or tissue with a pharmaceutical composition as described in any one of claims 3-9 or a virus as described in any one of claims 13-17, wherein the cargo molecule is delivered or transduced into the cell or tissue, and wherein the cell or tissue is retinal or corneal.

22. Use of the cell-penetrating peptide of claim 1 or claim 2, the pharmaceutical composition of any one of claims 3-9, the nucleic acid construct of claim 12, or the virus of any one of claims 13-17 in the preparation of a medicament for delivering a cargo molecule to a cell or tissue of a subject, wherein the cell or the tissue is retinal or corneal.

23. The use according to claim 22, wherein the medicament is formulated for administration by a route selected from the group consisting of intravitreal, transchoroidal, intracameral, suprachoroidal, transdermal, subretinal, intraperitoneal, subcutaneous and intravenous routes.

24. The use according to claim 22, wherein the medicament is formulated for administration via the ocular route.

25. The use according to claim 22, wherein the medicament is formulated for administration by a topical route.

26. The use according to claim 22, wherein the medicament is used for ocular degenerative diseases or ocular injuries.

27. The use according to claim 26, wherein the ocular degenerative disease is age-related macular degeneration, retinitis pigmentosa, uveitis, glaucoma or diabetic retinopathy.

28. The use according to claim 26, wherein the eye injury is an alkali burn or a retinal detachment.

29. The use according to any one of claims 23-25, wherein the cell penetrating peptide increases the delivery of the cargo molecule to the cell or the tissue compared to the cargo molecule alone.

30. A method of delivering a virus to a cell or tissue in vitro, the method comprising contacting the cell or tissue with a virus as described in any one of claims 13-17, wherein the virus is delivered or transduced into the cell or tissue at an increased rate compared to a cell or tissue contacted with a virus that does not comprise the modified viral capsid protein, wherein the cell or tissue is retinal or corneal.

31. The method of claim 30, further comprising contacting the cell or tissue with a cell-penetrating peptide and the virus, wherein the cell-penetrating peptide consists of SEQ ID NO: 5 connected to the C-terminus of SEQ ID NO: 4 via a flexible linker.

32. The method of claim 31, wherein the cell penetrating peptide consists of SEQ ID NO:

3.

33. Use of the virus of any one of claims 13-17 in the preparation of a medicament for delivering the virus to a cell or tissue of a subject, wherein the cell or tissue is retinal or corneal.

34. The use according to claim 33, wherein the medicament is for treating ocular degenerative diseases or ocular injuries.

35. Use of a combination comprising (a) a cell-penetrating peptide as described in claim 1 or 2 and (b) a virus as described in any one of claims 13-17 in the preparation of a medicament for delivering a cargo molecule to a tissue or cell of a subject, wherein the cell or the tissue is retinal or corneal.

36. The use according to claim 35, wherein the combination comprises (a) the cell-penetrating peptide according to claim 2 and (b) the virus according to claim 13.

37. The use according to claim 35, wherein the medicament is for treating ocular degenerative diseases or ocular injuries.

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