Self-complementary AAV vectors carrying dominant-negative RhoA and methods of use for treating ocular diseases

Abstract

Provided herein are recombinant self-complementary adeno-associated virus (scAAV) nucleic acid vectors containing ubiquitous eukaryotic promoters, such as elongation factor 1 alpha (EF1α), chicken beta-actin (CBA), and hybrid chicken beta-actin (CBh), followed by a dominant-negative RhoA. Also provided herein are methods of using the vectors, including intraocular injection (e.g., intracameral injection) to reduce intraocular pressure (IOP). Also provided herein are plasmids, recombinant scAAV particles, compositions, formulations, and other methods of use related to such vectors.

Description

[Technical Field]

[0001] [Indication of priority claim] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 407,150 (filed September 15, 2022); U.S. Provisional Patent Application No. 63 / 414,996 (filed October 11, 2022); and U.S. Provisional Patent Application No. 63 / 426,725 (filed November 19, 2022); the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] [Information Regarding Electronic Submission of Sequence Listing] The Sequence Listing in XML format (Title: 1635-2_ST26.xml, Size: 16,536 bytes, Created: September 13, 2023, submitted herewith) is incorporated herein by reference in its entirety for its disclosure.

[0003] [Technical field] Provided herein are recombinant self-complementary adeno-associated virus (scAAV) nucleic acid vectors containing ubiquitous eukaryotic promoters, such as elongation factor 1 alpha (EF1α), chicken beta-actin (CBA), and hybrid chicken beta-actin (CBh), followed by a dominant-negative RhoA. Also provided herein are methods of using the vectors, including intraocular injection (e.g., intracameral injection) to reduce intraocular pressure (IOP). Also provided herein are plasmids, recombinant scAAV particles, compositions, formulations, and other methods of use related to such vectors. [Background technology]

[0004] Advances in research and development of gene therapy for ocular diseases are ushering in a trend that will bring hope for a cure sooner to a larger patient population. Traditional gene therapies focus on hereditary eye diseases, such as retinitis pigmentosa, choroideremia, Leber's hereditary optic neuropathy, Leber's congenital amaurosis (LCA), color blindness, and X-linked retinitis pigmentosa (XLRS). A new generation of gene therapies targets chronic disease indications, such as wet age-related macular degeneration (AMD), diabetic retinopathy, and other chronic retinal diseases.

[0005] Glaucoma is one of the leading causes of blindness in people over 60 years of age and the second leading cause of blindness worldwide. In the United States, the prevalence of glaucoma is approximately 19 per 1,000 people. This corresponds to approximately 22 per 1,000 women and 16 per 1,000 men (National Eye Institute). Glaucoma tables The trabecular meshwork (TM) provides resistance to aqueous humor flow necessary to maintain physiological intraocular pressure (IOP). TM dysfunction leads to elevated intraocular pressure (e.g., ocular hypertension), which is a major risk factor for the development of glaucoma.

[0006] Currently, the only clinically available treatments are daily eye drops and surgery to reduce intraocular pressure. There are several types of eye drops used to treat glaucoma, which work by different mechanisms to reduce intraocular pressure. Common eye drops include prostaglandin analogs, beta-blockers, alpha-agonists, carbonic anhydrase inhibitors, and Rho-kinase inhibitors. Because many glaucoma patients are elderly and often require additional medications for other conditions, compliance with daily eye drop use is low.

[0007] Surgery is an option when eye drops fail to sufficiently lower intraocular pressure or cause severe side effects. Laser trabeculoplasty is one of the most common procedures used to treat open-angle glaucoma by improving aqueous outflow. While laser surgery has a very favorable safety profile, several notable drawbacks and limitations remain, including limited duration of effect, variable response, the need for continuous monitoring, and transient spikes in intraocular pressure. When a patient's intraocular pressure is not controlled with topical eye drops or laser trabeculoplasty, minimally invasive glaucoma surgery (MIGS) is offered. MIGS reduces intraocular pressure by creating a bypass to the normal aqueous outflow mechanism. This bypass can be achieved by placement of a trabecular bypass stent or goniotomy (or trabeculotomy), which improves aqueous outflow into Schlemm's canal. Risks of these procedures include variable effectiveness, the need for repeat procedures, and long-term persistence. Given the drawbacks and limitations of current treatments, the development of an intraocular pressure-reducing gene therapy regimen that allows for infrequent (or single) administration could greatly benefit glaucoma patients.

[0008] RhoA is a GTP-binding protein known to be involved in cell contractility. RhoA cycles between active and inactive forms, and cycling between these two conformations activates Rho kinase (ROCK). The relevance of the RhoA pathway in regulating outflow function was first reported when RhoA mediated cell contraction in TM. One study demonstrated that a smooth muscle cell contraction inhibitor (Y-27632) targeted ROCK inhibitors and that this inhibitor reduced cell contraction by decreasing calcium sensitivity in the vasculature. These findings, along with the established role of TM cell contraction in aqueous humor outflow function, led to numerous studies demonstrating that Y-27632 also affected intraocular pressure. Since then, ROCK inhibitors have been successfully developed as new drugs for the treatment of glaucoma. However, chemical ROCK inhibitors are rapidly metabolized, leading to inconveniently frequent administration to achieve intraocular pressure reduction. To fulfill the unmet medical need for glaucoma treatment, the development of novel therapeutics that specifically target TM tissue, and more specifically the RhoA pathway, is desirable to benefit the elderly population. Summary of the Invention

[0009] The present invention is based on the discovery that gene therapy using a dominant-negative mutant of RhoA (dnRhoA) can reduce intraocular pressure in glaucoma patients by inhibiting ROCK signaling. A general-purpose promoter can be used to drive the expression of mutant dnRhoA. The present invention further relates to the surprising enhancement of dnRhoA expression when shorter eukaryotic promoters are used. Multiple AAV capsids, including mutant AAV2, can also be used to package the described viral vectors.

[0010] Thus, one aspect of the present invention relates to recombinant self-complementary adeno-associated virus (scAAV) particles comprising a viral capsid protein and an scAAV nucleic acid vector comprising a eukaryotic promoter and a dominant-negative RhoA. In some embodiments, the dominant-negative RhoA comprises at least one amino acid mutation (e.g., T19N). In some embodiments, the ubiquitous promoter comprises a truncated EF1α, CBA, CBh, or other short eukaryotic promoter.

[0011] In some embodiments, the recombinant scAAV particles use capsids from different viral serotypes. In some embodiments, the viral capsids include capsids with one or more amino acid mutations, such as amino acid substitutions at one or more of positions Y444F, Y500F, and / or Y730F, numbered according to AAV2 VP1 (SEQ ID NO: 5). In some embodiments, the one or more amino acid mutations in the viral capsid reduce immunogenicity, increase expression of a dominant-negative RhoA transgene, and / or increase the duration of expression of a dominant-negative RhoA transgene.

[0012] Also provided herein are methods of reducing intraocular pressure in a subject in need thereof, the methods comprising administering a therapeutically effective amount of a recombinant scAAV particle described herein. In some embodiments, the recombinant scAAV particle may be administered by intraocular injection, wherein the injection may be into the anterior chamber (e.g., into the trabecular meshwork tissue and / or the cornea), and / or into the posterior chamber (e.g., into retinal cells, including retinal ganglion cells (RGCs) and / or retinal pigment epithelial cells (RPE)), and / or into the iris.

[0013] Another aspect of the present invention relates to a method for treating and / or preventing an ocular disease in a subject in need thereof, said method comprising administering a therapeutically effective amount of a recombinant scAAV particle described herein.

[0014] A further aspect of the invention is the use of the recombinant scAAV particles described herein to reduce intraocular pressure in a subject in need thereof, or a method of treating and / or preventing an eye disease in a subject in need thereof.

[0015] Additional aspects of the present invention relate to the use of the recombinant scAAV particles described herein in the manufacture of a medicament for reducing intraocular pressure in a subject in need thereof, or in a method for treating and / or preventing an ocular disease in a subject in need thereof.

[0016] These and other aspects of the present invention are further detailed in the detailed description of the invention below. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is an image showing a schematic diagram of the expression cassette used in the plasmid. [Figure 2] 1 is a series of fluorescence microscopy images and corresponding flow cytometry measurement graphs showing the expression of eGFP from different promoters in pooled human trabecular meshwork (HTM) cells 72 hours after transfection. [Figure 3] FIG. 1 is a series of fluorescence microscopy images and corresponding flow cytometry graphs showing the expression of eGFP from different promoters in pooled HTM cells 72 hours after transduction (multiplicity of infection 10,000). [Figure 4] Graph showing quantification of GFP-positive cells by flow cytometry after plasmid transfection or viral infection. [Figure 5] Image of a Southern blot showing restriction enzyme digestion of a plasmid containing the dnRhoA gene with PvuII-HF restriction enzyme, viewed on a 1% agarose gel; lane 1 is a 1 kilobase (kb) ladder (Invitrogen), lane 2 is a digest of pGVB-2001-015, and lane 3 is a digest of pGVB-2001-016. [Figure 6] Image of a Southern blot showing restriction enzyme digestion of a plasmid containing the dnRhoA gene with SmaI restriction enzyme, viewed on a 1% agarose gel; lane 1 is a 1 kb ladder (Invitrogen), lane 2 is a digest of pGVB-2001-015, and lane 3 is a digest of pGVB-2001-016. [Figure 7] FIG. 1 is a graph showing ROCK activity in HTM cells after treatment with three ROCK activators: sphingosine-1-phosphate (S1P) 1 μM, oleoyl-L-lysophosphatidic acid (LPA) 10 μM, and dimethyloxalylglycine (DMOG) 0.5 mM; and one ROCK inhibitor (Y-27632, 50 μM). [Figure 8] Graph showing ROCK activity levels in uninfected HTM cells, HTM cells infected with wild-type scAAV2 capsids containing the dnRhoA gene, or HTM cells infected with Y3 mutant scAAV2 capsids containing the dnRhoA gene; HTM cells from three independent donors were tested, and three AAV-infected technical replicates were analyzed; data are presented as mean ± standard error, and between-group comparisons were performed by one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test; * indicates p-value <0.033, ** indicates p-value <0.002. [Figure 9] Graph showing intraocular pressure measured with an Icare TonoLab tonometer before and after Ad5.BMP2 injection; each bar represents the mean intraocular pressure value of six eyes; statistical analysis was performed using one-way analysis of variance (ANOVA). [Figure 10] 16 is a graph showing intraocular pressure measured by an Icare TonoLab tonometer after scAAV2.Y3.CBh.dnRhoA injection. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention is described in more detail below. This description is not intended to list in detail all the different ways in which the invention may be implemented or all the features that may be added to the invention. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a specific embodiment may be omitted from that embodiment. Furthermore, numerous modifications and additions to the various embodiments shown herein will be apparent to those skilled in the art in light of this disclosure, but they do not depart from the invention. Thus, the following specification is intended to illustrate some of the specific embodiments of the invention, but is not intended to exhaustively identify all permutations, combinations, and variations thereof.

[0019] It is specifically contemplated that the various features of the invention described herein may be used in any combination, unless the context indicates otherwise. Furthermore, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted. For example, if a composite is described herein as comprising components A, B, and C, it is specifically contemplated that any or combination of A, B, or C, alone or in any combination, may be omitted or excluded.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.The terms used to describe the invention herein are intended to describe specific embodiments only and are not intended to limit the invention.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In the event of any discrepancy in terms, the present specification shall prevail.

[0021] Nucleotide sequences are presented herein in the 5' to 3' direction, left to right, single strand only, unless otherwise indicated. Nucleotides and amino acids are designated herein either in the form recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by their one-letter or three-letter codes, both in accordance with 37 CFR § 1.822 and established usage.

[0022] Unless otherwise indicated, standard methods known to those skilled in the art can be used for producing recombinant and synthetic polypeptides, antibodies or antigen-binding fragments thereof, manipulating nucleic acid sequences, producing transformed cells, constructing recombinant AAV (rAAV) constructs, modified capsid proteins, packaging vectors expressing AAV rep and / or cap sequences, and transiently or stably transfected packaging cells. These techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 4th Ed. (Cold Spring Harbor, NY, 2012); FM AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0023] All publications, patent applications, patents, nucleotide sequences, amino acid sequences, and other references mentioned herein are incorporated by reference in their entirety.

[0024] [Definition] As used in the present description and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0025] As used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items, and the lack of combinations when interpreted in the alternative ("or").

[0026] Furthermore, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted.

[0027] Furthermore, the term "about," when used herein in reference to a measurable value, such as an amount, dosage, time, temperature, etc., of a compound or agent of the invention, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, and even ±0.1% of the specified amount.

[0028] As used herein, the transitional phrase "consisting essentially of" should be construed to include the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, as used herein, the term "consisting essentially of" should not be construed as the same as "comprising."

[0029] The term "consisting essentially of" (and grammatical variations) when applied to polynucleotide or polypeptide sequences of the invention refers to a polynucleotide or polypeptide consisting of both a recited sequence (e.g., SEQ ID NO:) and a total of 10 or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids at the 5' and / or 3' ends, or at the N- and / or C-termini of the recited sequence or between the two ends (e.g., between domains), where the function of the polynucleotide or polypeptide is not substantially altered. A total of 10 or fewer additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added. The term "materially altered," when applied to a polynucleotide of the invention, refers to an increase or decrease in the ability to express the encoded polypeptide by at least about 50% or more compared to the expression level of a polynucleotide consisting of the recited sequence. The term "substantially altered," when applied to a polypeptide of the invention, refers to an increase or decrease in biological activity by at least about 50% or more compared to the activity of a polypeptide consisting of the recited sequence.

[0030] The term "enhance" or "increase" refers to at least about a 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, or even 15-fold increase in a particular parameter.

[0031] As used herein, the terms "inhibit" or "reduce," or grammatical variations thereof, refer to a decrease or reduction in a particular level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, or more. In specific embodiments, the inhibition or reduction results in little or essentially no detectable activity (at most a trace amount, e.g., less than about 10%, or even 5%).

[0032] As used herein, a "therapeutically effective" or "treatment effective" amount is an amount that provides some improvement or benefit to a subject. In other words, a "therapeutically effective" or "therapeutically effective" amount is an amount that provides some relief, alleviation, or reduction in at least one clinical symptom in a subject (e.g., reducing intraocular pressure and / or inhibiting ocular degeneration). Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

[0033] The terms "treat," "treating," or "treatment of" (or grammatical equivalents) mean to lessen the severity of, or at least partially improve or alleviate, a subject's condition, and / or to reduce, alleviate, or diminish at least one clinical symptom, and / or to slow the progression of the condition.

[0034] As used herein, the terms "prevent," "prevents," or "prevention" (and their grammatical equivalents) mean to delay or inhibit the onset of a disease. These terms do not require the complete elimination of a disease, but rather encompass any type of prophylactic treatment to reduce the incidence of or delay the onset of a condition.

[0035] As used herein, a "prevention effective" amount is an amount sufficient to prevent and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject, and / or an amount sufficient to reduce the severity and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject, compared to what would occur in the absence of the method of the invention. One of skill in the art will understand that the degree of prevention need not be complete, as long as some benefit is provided to the subject.

[0036] As used herein, the terms "protein" and "polypeptide" are used interchangeably and include both peptides and proteins, unless otherwise specified.

[0037] The term "fragment," when applied to a polypeptide, refers to an amino acid sequence that is shortened in length compared to a reference polypeptide or amino acid sequence, and is understood to comprise, consist essentially of, and / or consist of an amino acid sequence of contiguous amino acids that is identical or nearly identical (e.g., having 90%, 92%, 95%, 98%, 99% identity) to the reference polypeptide or amino acid sequence. Such polypeptide fragments according to the invention may, where appropriate, be contained within a larger polypeptide of which it is a component. In some embodiments, such fragments may comprise, consist essentially of, and / or consist of a peptide having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more contiguous amino acids of a polypeptide or amino acid sequence according to the invention.

[0038] As used herein, "nucleic acid," "nucleotide sequence," and "polynucleotide" are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The terms polynucleotide, nucleotide sequence, or nucleic acid refer to a chain of nucleotides regardless of the length of the chain. A nucleic acid can be a sense strand or an antisense strand.

[0039] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, etc. A gene may or may not be usable to produce a functional protein. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, 5' and 3' untranslated regions). When a gene is "isolated," it refers to a nucleic acid that is substantially or essentially free from components normally found associated with that nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant products, and / or various chemicals used to chemically synthesize the nucleic acid.

[0040] The terms "5' portion" and "3' portion" are relative terms that define the spatial relationship between two or more elements. Thus, for example, the "3' portion" of a polynucleotide refers to a segment of the polynucleotide that is located downstream of another segment. The term "3' portion" is not intended to indicate that a segment is necessarily at the 3' end of the polynucleotide, or even necessarily in the 3' half of the polynucleotide, although this may be the case. Similarly, the "5' portion" of a polynucleotide refers to a segment of the polynucleotide that is located upstream of another segment. The term "5' portion" is not intended to indicate that a segment is necessarily at the 5' end of the polynucleotide, or even necessarily in the 5' half of the polynucleotide, although this may be the case.

[0041] As used herein with respect to nucleic acids, the term "operably linked" refers to a functional linkage between two or more nucleic acids. For example, a promoter sequence is described as "operably linked" to a heterologous nucleic acid sequence because the promoter sequence initiates and / or mediates transcription of the heterologous nucleic acid sequence. In some embodiments, operably linked nucleic acid sequences are contiguous and / or in the same reading frame.

[0042] As used herein, the term "open reading frame (ORF)" refers to a portion of a polynucleotide (e.g., a gene) that encodes a polypeptide and includes an initiation site (i.e., a Kozak sequence) from which transcription of the polypeptide begins. The term "coding region" may be used interchangeably with open reading frame.

[0043] As used herein, the terms "optimized" or "optimized for expression" refer to viral particles that have been optimized to increase gene expression in a viral vector. In some embodiments, the viral particles are optimized to increase gene expression in an organism (e.g., an animal such as a human, an animal, a plant, a fungus, an archaea, or a bacterium) and / or are optimized for gene expression in a tissue type of the organism (e.g., eye tissue, brain tissue, muscle tissue, etc.). In some embodiments, the viral particles are optimized by codon optimization of the gene coding sequence in the viral vector. In some embodiments, the viral particles are optimized by using a specific viral serotype (e.g., AAV2 or AAV5). In some embodiments, the viral particles are optimized by mutating the amino acid sequence of the viral capsid protein. In some embodiments, the optimized viral capsid protein includes a Y3 mutation (e.g., Y444F, Y500F, and Y730F mutation). In some embodiments, the optimized viral particles increase expression of a gene from the viral vector by about 5% to about 1000% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or about 1000%) in an organism and / or tissue type compared to a reference particle (a non-optimized particle).

[0044] As used herein, the term "codon-optimized" refers to a gene coding sequence or a fragment of a gene coding sequence that has been optimized to increase expression by replacing one or more codons normally present in the coding sequence (e.g., in a wild-type sequence, including the coding sequence for a RhoA protein) with codons for the same (synonymous) amino acid. Thus, the proteins or protein fragments encoded by the genes or gene fragments are identical, but the underlying nucleobase sequences of the genes or gene fragments, or the corresponding mRNAs, are different. In some embodiments, optimization replaces one or more rare codons (i.e., codons corresponding to tRNAs that occur relatively infrequently in cells from a particular species) with more frequently occurring synonymous codons to improve translation efficiency. For example, in human codon optimization, one or more codons in a coding sequence are replaced with codons that correspond to the same amino acid and occur more frequently in human cells. Codon optimization can also increase expression of a gene or gene fragment through other mechanisms that may improve transcription and / or translation efficiency. Strategies include, but are not limited to, increasing the total GC content (i.e., the percentage of guanine and cytosine throughout the coding sequence), decreasing the CpG content (i.e., the number of CG or GC dinucleotides in the coding sequence), removing cryptic splice donor or acceptor sites, and / or adding or removing ribosome entry and / or start sites, such as Kozak sequences. Desirably, codon-optimized genes or gene fragments exhibit improved protein expression, e.g., the proteins or protein fragments encoded thereby are expressed at detectably higher levels in cells compared to the expression levels of the protein or protein fragment provided by the wild-type gene or gene fragment in an otherwise similar cell. Codon optimization also provides the ability to distinguish codon-optimized genes and / or corresponding mRNAs from endogenous genes and / or corresponding mRNAs in vitro or in vivo.

[0045] As used herein, an "isolated" nucleic acid or nucleotide sequence (e.g., "isolated DNA" or "isolated RNA") means that the nucleic acid or nucleotide sequence is separated from or substantially free of at least some of the other components of the naturally occurring organism or virus (e.g., structural components of a cell or virus or other polypeptides or nucleic acids that are normally found in association with the nucleic acid or nucleotide sequence).

[0046] Similarly, an "isolated" polypeptide means that the polypeptide is separated from or substantially free of at least some of the other components of the naturally occurring organism or virus (e.g., structural components of a cell or virus, or other polypeptides or nucleic acids that are normally found in association with the polypeptide).

[0047] As used herein, the term "modified," when applied to a polynucleotide or polypeptide sequence, refers to a sequence that differs from the wild-type sequence by one or more deletions, additions, substitutions, or any combination thereof.

[0048] As used herein, "isolating" a viral vector (or grammatical equivalents) means that the viral vector is at least partially separated from at least some of the other components in the starting material.

[0049] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout the alignment window of components (e.g., nucleotides or amino acids). "Identity" can be readily calculated by known methods, including, but not limited to, those described in: Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0050] As used herein, the terms "substantially identical" or "corresponding to" mean that two nucleic acid sequences have at least 60%, 70%, 80%, or 90% sequence identity. In some embodiments, two nucleic acid sequences may have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0051] The "identity fraction" of an aligned segment of a test sequence and a reference sequence is the number of identical elements shared by the two aligned sequences divided by the total number of elements in the reference sequence segment (i.e., the entire reference sequence or a smaller, defined portion of the reference sequence).

[0052] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of matching nucleotides when the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) is compared with a test ("subject") polynucleotide molecule (or its complementary strand) and the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20% of the reference sequence over the comparison window). In some embodiments, "percent identity" may refer to the percentage of matching amino acids in an amino acid sequence.

[0053] Optimal sequence alignment for aligning a comparison window is well known to those skilled in the art and can be performed by tools such as the Smith and Waterman local homology algorithm, the Needleman and Wunsch homology alignment algorithm, the Pearson and Lipman similarity search method, and, optionally, computer implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, available as part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, Massachusetts). Percent sequence identity is expressed as the percentage identity multiplied by 100. Comparison of one or more polynucleotide sequences can be performed on full-length polynucleotide sequences or portions thereof, or on longer polynucleotide sequences. For purposes of the present invention, "percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0054] Percent sequence identity can be determined using the "BestFit" or "Gap" programs in the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc., Madison, Wis.). "Gap" utilizes the Needleman and Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 48:443-453, 1970) to align two sequences, maximizing the number of matches and minimizing the number of gaps. "BestFit" uses the Smith and Waterman local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482-489, 1981; Smith et al., Nucleic Acids Res. 11:2205-2220, 1983) to optimally align the most similar segments between two sequences and insert gaps to maximize the number of matches.

[0055] Useful methods for determining sequence identity are also described in: Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo, H., and Lipton, D., (Applied Math 48:1073(1988)). More specifically, preferred computer programs for determining sequence identity include, but are not limited to, the Basic Local Alignment Search Tool (BLAST) program, which is publicly available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health, Bethesda, MD 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol. 215:403-410 (1990)); BLAST program version 2.0 and above allows for the introduction of gaps (deletions and insertions) into the alignment; for peptide sequences, BLASTX can be used to determine sequence identity; and for polynucleotide sequences, BLASTN can be used to determine sequence identity.

[0056] A "vector" refers to a compound used as a vehicle to transport foreign genetic material into another cell for replication and / or expression therein. A cloning vector containing a foreign nucleic acid is called a recombinant vector. Examples of nucleic acid vectors are plasmids, viral vectors, cosmids, expression cassettes, and artificial chromosomes. Recombinant vectors typically contain an origin of replication, a multiple cloning site, and a selectable marker. The nucleic acid sequence typically consists of an insert (recombinant nucleic acid or transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector to transfer genetic information to another cell is usually to isolate, amplify, or express the insert in the target cell. An expression vector (expression construct or expression cassette) is used to express foreign genes in the target cell and generally contains a promoter sequence that drives the expression of the foreign gene / ORF. Insertion of a vector into a target cell is called transformation or transfection in bacterial and eukaryotic cells, while insertion of a viral vector is often called transduction. The term "vector" may also be used generally to refer to anything that serves to carry foreign genetic material into another cell (e.g., but not limited to, a transformed cell or a nanoparticle).

[0057] As used herein, the term "promoter" refers to a polynucleotide sequence to which a polymerase (DNA or RNA) or associated transcription factors bind and initiate transcription. In some embodiments, a promoter may be a promoter sequence derived from the genome of a virus (e.g., CMV, AAV, etc.), a prokaryote (e.g., Escherichia coli), or a eukaryote (e.g., human, chicken, mouse, yeast, etc.). In some embodiments, eukaryotic promoters have low immunogenicity; for example, viral vectors comprising eukaryotic promoters have an anti-drug antibody (ADA) response rate of about 1% to about 30% (e.g., about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or about 30%). In some embodiments, the eukaryotic promoter has stable expression in a subject, for example, expression from a viral vector containing a eukaryotic promoter has a fluctuation in expression of about 1% to about 40% (e.g., about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or about 40%) from the baseline expression level after administration to the subject. In some embodiments, the promoter can be a short promoter, for example, having a sequence length of about 100 nucleotides to about 2000 nucleotides (e.g., about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or about 1200 nucleotides in length). In some embodiments, the short promoter is about 500 to about 1000 nucleotides in length (e.g., about 500, 600, 700, 800, 900, or about 1000 nucleotides in length). In some embodiments, the promoter is a CMV5, CBA, or EF1α promoter (e.g., a truncated EF1α promoter). In some embodiments, the EF1α promoter is about 1000 to about 1400 nucleotides in length (e.g., about 1000, 1100, 1200, 1300, or about 1400 nucleotides).In some embodiments, the promoter may be a hybrid promoter, e.g., a promoter in which two promoter sequences from one or more different organisms are operably linked to each other. In some embodiments, the hybrid promoter is a CBh promoter (e.g., a hybrid promoter of CMV and chicken β-actin). In some embodiments, the CBA and / or CBh promoter is about 400 to about 1000 nucleotides in length (e.g., about 400, 500, 600, 700, 800, 900, or about 1000 nucleotides).

[0058] A "subject" of the present invention may include any animal in need thereof. In some embodiments, the subject may be, for example, a mammal, reptile, bird, amphibian, or fish. Mammalian subjects may include, but are not limited to, laboratory animals (e.g., rats, mice, guinea pigs, rabbits, primates, etc.), livestock or commercial animals (e.g., cows, pigs, horses, goats, donkeys, sheep, etc.), or domestic animals (e.g., cats, dogs, ferrets, gerbils, hamsters, etc.). In some embodiments, the mammalian subject may be a primate or a non-human primate (e.g., chimpanzees, baboons, macaques (e.g., rhesus monkeys, cynomolgus monkeys, long-tailed monkeys, pig-tailed monkeys), monkeys (e.g., squirrel monkeys, owl monkeys, etc.), marmosets, gorillas, etc.). In some embodiments, the mammalian subject may be a human.

[0059] A "subject in need" of the methods of the present invention can be any subject known to be or suspected of being at increased risk of developing an ocular disease (e.g., glaucoma, AMD (e.g., dry AMD or wet AMD), diabetic retinopathy, and / or retinal breaks), and / or ocular hypertension (elevated pressure inside the eye).

[0060] Compositions and Formulations Provided herein are recombinant scAAV vectors and methods for treating ocular diseases (e.g., glaucoma) and / or ocular hypertension using the same. Accordingly, the present disclosure provides recombinant scAAV particles, compositions comprising the recombinant scAAV particles, and methods for treating ocular diseases (e.g., glaucoma) and / or ocular hypertension. In some embodiments, the disclosed viral vectors can be used in combination with any AAV capsid (with or without mutations) to manufacture AAV particles for treating ocular diseases (e.g., glaucoma) and / or ocular hypertension, or any combination thereof. Further provided herein are pharmaceutical formulations and dosages of viral injectates that can be used for intraocular injection. Another aspect of the present disclosure provided herein is the injection route commonly used in treating ocular diseases (e.g., glaucoma) and / or ocular hypertension, or any combination thereof. Another aspect of the present disclosure relates to the therapeutic efficacy of viral vectors in glaucoma patients.

[0061] We have optimized the delivery vector, its cargo, and potential mechanisms for regulating cargo gene expression. For the delivery vehicle, we sought a serotype-selected mutant capsid virus with long TM duration, low immunogenicity, and improved gene transfer efficiency. For the cargo, we identified a dominant-negative RhoA mutation that was efficient in reducing intraocular pressure in an animal model of glaucoma. For the promoter, we selected a hybrid chicken β-actin (CBh) promoter to drive transgene expression. Taken together, the viral vector of the present invention can contain a RhoA gene containing a dominant-negative mutation (e.g., SEQ ID NO: 4) and a ubiquitous promoter such as CBh (SEQ ID NO: 3). The viral vector (SEQ ID NO: 7) was produced and characterized in vitro, and its efficacy was subsequently tested in a rat glaucoma model.

[0062] In some embodiments, the AAV particle may have an altered VP1 capsid protein, an altered VP2 capsid protein, an altered VP3 capsid protein, or any combination thereof. In some embodiments of the above aspects and embodiments, the AAV viral particle comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, a mutant capsid containing a tyrosine, a mutant capsid with a heparin-binding motif, capsid AAV2R471A, capsid AAV2 / 2, and / or capsid AAV DJ. In some embodiments, the AAV viral particle comprises an AAV capsid containing an amino acid substitution at one or more of the following positions: Y444F, Y500F, and / or Y730F, numbered according to AAV2 VP1 (SEQ ID NO: 5). In some embodiments, the vector comprises an inverted terminal repeat (ITR) of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, bovine AAV, and / or murine AAV. In some embodiments, the vector comprises a mutated AAV ITR that allows the formation of self-complementary AAV. In some embodiments, the AAV viral particle comprises one or more ITRs and a capsid derived from the same AAV serotype. In other embodiments, the AAV viral particle comprises one or more ITRs derived from an AAV serotype that is different from the serotype of the recombinant scAAV viral particle capsid. In some embodiments, the recombinant scAAV viral particle comprises an AAV2 capsid, wherein the vector comprises AAV2 ITRs.

[0063] In some embodiments, various formulations can be used to facilitate viral transduction in ocular tissues. For example, when administering an injectable aqueous solution of recombinant scAAV particles, the solution is appropriately buffered, if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. Thus, in some embodiments, a viral infusion can include a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with recombinant scAAV particles. "Pharmaceutically acceptable" means a substance that is non-toxic or free of other undesirable properties. That is, the substance can be administered to a subject without causing any undesired biological effects. Such pharmaceutical carriers can be sterile liquids (e.g., water, oil, saline, aqueous dextrose, and / or glycerol solutions), suspending agents, preservatives (e.g., methyl, ethyl, and / or propylhydroxybenzoates), and pH adjusters (e.g., inorganic acids, organic acids, and / or bases). In some embodiments, the carrier comprises a buffered saline solution (e.g., phosphate buffered saline, HEPES buffered saline, etc.). In some embodiments, United States Pharmacopeia (USP) grade carriers and excipients may be used to deliver recombinant scAAV particles to a human subject. Such compositions may further comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and / or nanoparticles, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof.

[0064] In some embodiments, a composition comprising any one of the recombinant scAAV particles disclosed herein comprises a balanced salt solution (BSS) supplemented with about 0.001% to about 0.035% (e.g., about 0.001%, 0.007%, 0.014%, 0.021%, 0.028%, or about 0.035%, or any range thereof) of Tween 20 (polysorbate 20). In some embodiments, a composition comprising any one of the recombinant scAAV particles disclosed herein comprises about 25 mM to about 500 mM sodium citrate (e.g., about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, or about 500 mM, or any range thereof), about 1 mM to about 50 mM Tris (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 mM, or any range thereof, pH 8.0), and further comprises about 0.0001% to about 0.005% Pluronic F-68 (e.g., about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, or about 0.005%, or any range thereof) is added.

[0065] [How to use] The methods of the present invention have use in both veterinary and medical applications.

[0066] The dosage of the AAV particles of the present invention depends on the mode of administration, the disease or condition being treated, the condition of the individual subject, the viral vector, and the gene being delivered, and can be determined by conventional methods. An exemplary dose to achieve a therapeutic effect is at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15The viral titer is at least 10 transduction units, preferably at about 10 8 ~10 13 transduction units, more preferably 10 11 It is a transduction unit.

[0067] The terms "administering" or "administration" of a composition of the invention to a subject include any route of introducing or delivering an agent to a subject to perform its intended function (e.g., to reduce intraocular pressure or treat glaucoma in a subject). In some embodiments, a recombinant scAAV viral vector can be administered via intraocular injection. As used herein, the term "intraocular injection" refers to any suitable injection route that can result in delivery of a recombinant scAAV vector to an ocular tissue (e.g., any part of the eye). In some embodiments, intraocular injection is injection into the anterior segment of the eye (e.g., the anterior chamber), e.g., by intracameral injection. In some embodiments, intraocular injection is injection into the posterior segment of the eye (e.g., the posterior chamber), e.g., by intravitreal injection. In some embodiments, intraocular injection is injection into the cornea of ​​the eye, e.g., by intrastromal corneal injection. In some embodiments, the intraocular injection is subretinal injection, e.g., via subretinal injection. In some embodiments, the intraocular injection is subretinal injection, e.g., via suprachoroidal injection. Other suitable routes of administration include, but are not limited to, intravenous, intraarterial, periocular, subconjunctival, and subtenon injection, topical administration (e.g., topical administration to the eye), and intranasal administration.

[0068] The viral vectors may be effective in treating or preventing neurological dysfunction. In some embodiments, the viral vectors described herein may provide intraocular pressure-independent or intraocular pressure-dependent neuroprotective effects to retinal cells, such as RGCs. In some embodiments, the viral vectors described herein prevent the progression of glaucoma by lowering intraocular pressure and / or by providing direct neuroprotection. In certain embodiments, the viral vectors may prevent the progression of glaucoma by both providing direct neuroprotection and by lowering intraocular pressure. This treatment provides an unexpected dual advantage in the treatment of glaucoma. In some embodiments, the viral vectors described herein may prevent the progression of glaucoma without lowering intraocular pressure. Thus, this treatment method may provide an unexpected advantage in intraocular pressure-independent neuroprotective effects.

[0069] In some embodiments, the viral vectors described herein can promote corneal wound healing after injury, thus providing an unexpected advantage in corneal protection.

[0070] Having described the invention, it is further illustrated in the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention.

[0071] SEQ ID NO: 1 Promoter CMV5

[0072] SEQ ID NO: 2 Promoter truncated EF1α

[0073] SEQ ID NO: 3 Promoter Hybrid chicken β-actin (CBh) CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATGGGACTTTCCATTGACGTCATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTAC CATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGG GGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0074] SEQ ID NO:4 Amino acid sequence of dominant negative RhoA(T19N) MAAIRKKLVIVGDGACGK N CLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAALQARRGKKKSGCLVL

[0075] SEQ ID NO:5 Amino acid sequence of AAV2 VP1 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTN VDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0076] SEQ ID NO:6 Amino acid sequence of wild-type RhoA MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAALQARRGKKKSGCLVL

[0077] SEQ ID NO:7 Sequence of the scAAV2.CBh.dnRhoA vector, containing the CBh promoter and dnRhoA gene. [Example]

[0078] Example 1: Comparison of promoters for driving GFP expression in HTM cells To identify a suitable promoter in HTM cells, the efficiency of several promoters in driving green fluorescent protein (GFP) expression was compared. Four commonly used promoters were selected to construct the plasmids (Figure 1): CMV5 (SEQ ID NO: 1), EF1α (SEQ ID NO: 2), and CBh (SEQ ID NO: 3). As a first step, expression of the new plasmid in HTM cells was tested by Lipofectamine (ThermoFisher) transfection. HTM cells (pooled from two individual donors) were seeded into 24-well plates the day before transfection and allowed to reach 70-80% confluency. Cells were transfected with 0.5 μg of plasmid to confirm promoter efficiency using Lipofectamine 3000 transfection reagent (ThermoFisher). Cells were cultured for 72 hours and then imaged by fluorescence microscopy. Cells were then examined by flow cytometry, and the percentage of GFP-positive cells was quantified. Overall, the levels of GFP expression in HTM cells, as recorded by fluorescence microscopy and flow cytometry, were similar among the different promoter groups (Figures 2 and 4).

[0079] The promoter efficiency was further tested in the form of AAV viral transduction in HTM cells. AAV particles were packaged with the plasmids used in the previous transfection experiments. Because both plasmids contained the ΔITR, the viral DNA was self-complementary. AAV2 with Y3 mutations (Y444F, Y500F, and Y730F) was selected as the capsid. HTM cells were then seeded into 24-well plates. Cells were infected at an MOI of 10,000 and examined after 3 days of culture. The percentage of GFP-positive cells recorded by flow cytometry showed different results between the groups containing different promoters. Cells infected with the CBh promoter-containing vector had a much higher percentage of GFP-positive cells than those infected with the CMV5 and EF1α promoter-containing vectors (Figures 3 and 4).

[0080] Example 2: Construction of scAAV2.dnRhoA To generate recombinant scAAV viral particles, cargo sequences (e.g., dominant-negative RhoA) were inserted into the ITRs (wild-type and deleted, ITR-ΔITR) to create specially designed plasmids designated pGVB-2001-15 and pGVB-2001-16. The pGVB-2001-15 and pGVB-2001-16 plasmids used the EF1α and CBh promoters, respectively. The amino acid sequence of RhoA is shown in SEQ ID NO: 6, and the dominant-negative mutation is the T19N mutation shown in the dnRhoA sequence in SEQ ID NO: 4. The dnRhoA-containing plasmids were confirmed by restriction enzyme digestion with PvuII-HF before performing endotoxin-free maxiprep (Figure 5). The integrity of the ITR region was confirmed by SmaI digestion (Figure 6). Next, scAAV2 was produced by triple transfection of the plasmids into HEK293 cells. The viral preparation was purified by ultracentrifugation through an iodixanol gradient. The gradient was layered as follows: 6 mL of 15% iodixanol, 6 mL of 25% iodixanol, 5 mL of 40% iodixanol, and 5 mL of 54% iodixanol. Seven to eight mL of lysate was added to the top of the gradient, and the tube was filled with phosphate-buffered saline (PBS).

[0081] The samples were centrifuged at 350,000 × g for 2 hours at 18°C. After centrifugation, the tubes were punctured 3-5 mm below the 40% / 54% interface with a 1-inch, 18-gauge needle attached to a 10 mL syringe. Approximately 1 mL of the 54% interface was withdrawn, and 4 mL of the 40% interface was withdrawn without disturbing the proteinaceous material at the 40% / 25% interface.

[0082] After purification by ultracentrifugation, the virus was concentrated and buffer exchanged using an Amicon Ultra-15 50 kDa centrifugal filter. The filter was prepared by adding 15 mL of 0.1% Pluronic F-68 solution in PBS and incubated at room temperature for 10 minutes. After incubation, the 0.1% Pluronic solution was discarded. Next, 15 mL of 0.01% Pluronic F-68 solution in PBS was added to the filter, which was centrifuged at 2000 x g for 5 minutes, with the flow-through discarded. Finally, 15 mL of 0.001% Pluronic F-68 solution in PBS was added to the filter, which was centrifuged at 2000 x g for 5 minutes, with the flow-through discarded. After the filter was prepared, the sample was diluted 1:2 with formulation buffer (0.001% Pluronic F-68 solution in PBS). The sample was added to the filter and centrifuged at 2000 x g for 5 minutes. The flow-through was discarded, but additional sample could be added and centrifuged again. Once all sample was concentrated onto the filter, formulation buffer was added and centrifugation was repeated until 50 mL of formulation buffer had passed through the filter. Centrifugation was then continued in short intervals of 2–3 min until the desired volume was reached.

[0083] Example 3: Testing the function of scAAV2.CBh.dnRhoA using a cell-based assay RhoA is a GTP-binding protein that cycles between active and inactive forms and activates ROCK. Overexpression of dnRhoA competes with endogenous RhoA to bind to ROCK, thereby inhibiting ROCK activation. Measuring ROCK activity in cells can be developed as a cell-based assay to test the function of scAAV2.CBh.dnRhoA.

[0084] Members of the Rho family are essential regulatory components of signaling pathways that direct cell motility, adhesion, and cytokinesis through reorganization of the actin cytoskeleton. Rho is activated by extracellular signals, such as lysophosphatidic acid (LPA). Rho action is mediated by downstream Rho effectors. One of these effectors is ROCK. ROCK mediates Rho signaling and reorganizes the actin cytoskeleton through phosphorylation of multiple substrates, contributing to actin filament assembly and contractility. For example, ROCK inactivates myosin phosphatase through specific phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr696, resulting in an increase in the phosphorylated content of the 20-kDa myosin light chain.

[0085] The ROCK activity assay kit is an enzyme immunoassay developed to detect the specific phosphorylation of MYPT1 at Thr696 by ROCK. A strip-well microtiter plate is pre-coated with recombinant MYPT1. After incubating the substrate wells with ROCK samples, phosphorylated MYPT1 is detected with an anti-phosphorylated MYPT1 (Thr696) antibody.

[0086] Human trabecular meshwork cells were seeded in 12-well plates at a density of 500,000 cells per well. 48 hours after seeding, cells were harvested using cell lysis buffer. Cell lysates were treated with ROCK activators or ROCK inhibitors and then incubated with pre-coated strip wells. ROCK activity was then measured according to the kit's protocol. Three compounds were used to activate ROCK: S1P (sphingosine-1-phosphate, 1 μM), LPA (oleoyl-L-lysophosphatidic acid, 10 μM), and DMOG (dimethyloxalylglycine, 0.5 mM). One compound was used to inhibit ROCK activity: Y-27632 (50 μM). Data were presented as relative ROCK activity, normalized to cell lysates treated without drug. As shown in Figure 7, all chemical activators increased ROCK activity, with LPA being the most potent activator. Chemical inhibitors decreased ROCK activity, as expected.

[0087] In the next experiment, human trabecular meshwork cells were seeded in 12-well plates at a density of 500,000 cells per well. Infection with scAAV2.Y3.CBh.dnRhoA or scAAV2.CBh.dnRhoA was performed at an MOI of 10,000 to compare wild-type and Y3-mutant AAV2 capsids. Cells were harvested 2 days postinfection. ROCK activity was measured. The reduction in ROCK activity in cells infected with virus carrying the Y3-mutant capsid was statistically significant, whereas the reduction in cells infected with virus carrying the wild-type capsid was less pronounced (Figure 8).

[0088] Example 4: Measurement of RhoA expression in HTM cells after transduction with scAAV2.CBh.dnRhoA In the following experiments, human trabecular meshwork cells were seeded in 12-well plates at a density of 500,000 cells per well. Infection with scAAV2.Y3.CBh.dnRhoA was performed at an MOI of 10,000. Cells were harvested 2 days postinfection for total RNA extraction. RNA content was measured by nanodrop. RT-PCR was performed to measure total RhoA expression.

[0089] Example 5: Testing the efficacy of scAAV2.CBh.dnRhoA using a transgene model of glaucoma The efficacy of scAAV2.Y3.CBh.dnRhoA was tested in a rat model of glaucoma with elevated intraocular pressure (IOP) achieved by Ad.BMP2 transduction. Elevated intraocular pressure is the result of increased resistance to aqueous humor outflow through the trabecular meshwork. This increased resistance can be caused by various dysfunctional trabecular meshwork cells and mechanisms. However, it is widely accepted that the most common cause of increased outflow resistance is a disruption of the extracellular matrix (ECM) of the trabecular meshwork. Bone morphogenetic protein 2 (BMP2) belongs to the TGFβ protein superfamily. BMP2 alone has the full potential to initiate bone formation and induces the differentiation of multipotent mesenchymal progenitor cells into the osteogenic lineage. Similarly, BMP2 induces osteogenic-like properties in primary HTM cells in vitro. Overexpression of the BMP2 gene in primary HTM cells transduced with an adenoviral vector increased alkaline phosphatase (ALP) activity. It is an enzyme that promotes the free phosphate and contributes to the formation of calcium phosphate precipitates (hydroxyapatite crystals), which are part of the mineralization process. Overexpression of the BMP2 gene in trabecular meshwork tissue is sufficient to increase intraocular pressure and generate animal models resembling ocular hypertension or glaucoma.

[0090] The baseline intraocular pressure of rats was measured using a TonoLab tonometer (Icare) before Ad5.BMP2 injection. Approximately 5 μL of Ad5.BMP2 virus (titer 1.8 × 10) was injected. 10pfu / mL) was injected into the anterior chamber of rats under anesthesia. Intraocular pressure was then monitored up to 28 days after injection. The baseline intraocular pressure of the rat eyes was approximately 12 mmHg. After Ad5.BMP2 injection, the mean intraocular pressure rapidly increased to approximately 25 mmHg and then stabilized at approximately 15 mmHg (Figure 9). The increase in intraocular pressure was significant at all measurement time points.

[0091] After elevated intraocular pressure was established, scAAV2.Y3.CBh.dnRhoA was injected to reduce intraocular pressure. Only eyes that showed a greater than 50% increase in intraocular pressure compared to baseline were selected for AAV injection. IOP was then continuously monitored up to day 14 after AAV injection. Approximately 50% reduction in intraocular pressure was observed 7 days after AAV injection. Thereafter, intraocular pressure remained at levels close to baseline until day 14 (Figure 10).

[0092] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The present invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

1. Recombinant self-complementary adeno-associated virus (scAAV) particles, which are: a) AAV capsid protein; and b) A scAAV viral genome comprising a eukaryotic promoter operably linked to a polynucleotide encoding dominant inhibitory RhoA; including, Recombinant self-complementary adeno-associated virus particles.

2. Recombinant scAAV particles as described in claim 1, The dominant inhibitory RhoA comprises at least one amino acid mutation. Recombinant scAAV particles.

3. Recombinant scAAV particles as described in claim 2, The aforementioned at least one amino acid mutation is a mutation from threonine to asparagine (T19N) at amino acid position 19 of SEQ ID NO:

6. Recombinant scAAV particles.

4. Recombinant scAAV particles as described in claim 1, The eukaryotic promoter is a short eukaryotic promoter (for example, one with a length of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or about 1200 nucleotides), The aforementioned short eukaryotic promoter may be a truncated elongation factor 1-α (EF1α), chicken β-actin (CBA), or hybrid chicken β-actin (CBh) promoter. Recombinant scAAV particles.

5. Recombinant scAAV particles as described in claim 1, The polynucleotide encoding the dominant inhibitory RhoA is codon-optimized for expression in humans (for example, optimized particles express about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 350%, 400%, 450%, or about 500% more of the polynucleotide encoding the dominant inhibitory RhoA in humans compared to unoptimized particles). Recombinant scAAV particles.

6. Recombinant scAAV particles as described in claim 1, The capsid protein is optimized for expression in ocular tissue (for example, optimized particles express about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 350%, 400%, 450%, or about 500% more of the polynucleotide encoding the dominant inhibitory RhoA in ocular tissue compared to unoptimized particles). Recombinant scAAV particles.

7. Recombinant scAAV particles as described in claim 1, The particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV12, AAV2R471A, bovine AAV, or mouse AAV serotype. Recombinant scAAV particles.

8. Recombinant scAAV particles as described in claim 1, The capsid protein contains one or more amino acid mutations. Recombinant scAAV particles.

9. Recombinant scAAV particles as described in claim 8, The one or more amino acid mutations include the amino acid mutations Y444F, Y500F, and / or Y730F, numbered according to VP1 (Sequence ID 5) of AAV2. Recombinant scAAV particles.

10. Recombinant scAAV particles as described in claim 8, The one or more amino acid mutations reduce immunogenicity and / or increase the expression of the dominant inhibitory RhoA. Recombinant scAAV particles.

11. Recombinant scAAV particles as described in claim 6, The ocular tissue includes the trabecular meshwork, iris, cornea, and / or retina. Recombinant scAAV particles.

12. A pharmaceutical composition for use in a method to lower intraocular pressure (IOP) in a subject requiring the same, comprising a therapeutically effective amount of recombinant scAAV particles described in any one of claims 1 to 11, The method comprises the step of administering a therapeutically effective amount of the pharmaceutical composition to the subject, thereby lowering the intraocular pressure in the subject. Pharmaceutical composition.

13. A pharmaceutical composition according to claim 12, The aforementioned administration step includes intraocular injection. Pharmaceutical composition.

14. A pharmaceutical composition according to claim 13, The intraocular injection may include injection into the anterior chamber, and may also include injection into the trabecular meshwork and / or the cornea. Pharmaceutical composition.

15. A pharmaceutical composition according to claim 14, The aforementioned intraocular injection is an anterior chamber injection. Pharmaceutical composition.

16. A pharmaceutical composition according to claim 13, The intraocular injection may include injection into the posterior chamber and may also include injection into retinal cells (e.g., retinal ganglion cells and / or retinal pigment epithelial cells). Pharmaceutical composition.

17. A pharmaceutical composition for use in a method for treating and / or preventing an eye disease in a subject requiring the use of recombinant scAAV particles as described in any one of claims 1 to 11, The method comprises the step of administering a therapeutically effective amount of the pharmaceutical composition to the subject, thereby treating and / or preventing the eye disease in the subject. Pharmaceutical composition.

18. A pharmaceutical composition according to claim 17, The aforementioned eye disease is associated with elevated intraocular pressure. Pharmaceutical composition.

19. A pharmaceutical composition according to claim 18, The aforementioned eye diseases are glaucoma, age-related macular degeneration (AMD), exudative AMD, diabetic retinopathy, and / or retinal tears. Pharmaceutical composition.

20. A pharmaceutical composition according to claim 17, The aforementioned administration step includes intraocular injection. Pharmaceutical composition.

21. A pharmaceutical composition according to claim 20, The intraocular injection may include injection into the anterior chamber, and may also include injection into the trabecular meshwork and / or the cornea. Pharmaceutical composition.

22. A pharmaceutical composition according to claim 21, The intraocular injection may include injection into the posterior chamber and may also include injection into retinal cells (e.g., retinal ganglion cells and / or retinal pigment epithelial cells). Pharmaceutical composition.

Images