Self-complementary scAAV vector carrying dominant negative mutation dnRhoA gene and method for treating ocular diseases by using self-complementary scAAV vector

Abstract

The present invention provides a recombinant self-complementary adeno-associated virus (scAAV) nucleic acid vector comprising a ubiquitous eukaryotic promoter, such as elongation factor 1 [alpha] (EF1 [alpha]), chicken [beta]-actin (CBA) and hybrid chicken [beta]-actin (CBh) followed by a dominant negative mutation RhoA. Furthermore, provided herein are methods of using the vectors, including reducing intraocular pressure (IOP) by intraocular injection (e.g., anterior chamber injection). Also provided herein are plasmids, recombinant scAAV particles, compositions, formulations, and other methods of use associated with these vectors.

Description

Technical Field

[0001] The present disclosure provides a recombinant self-complementary adeno-associated virus (scAAV) nucleic acid vector comprising a ubiquitous eukaryotic promoter such as elongation factor 1α (EF1α), chicken β-actin (CBA), and chimeric chicken β-actin (CBh), followed by a dominant negative mutant RhoA. Also provided herein are methods of using the vectors, including reducing intraocular pressure (IOP) by intravitreal injection (e.g., anterior chamber injection). Also provided herein are plasmids, recombinant scAAV particles, compositions, formulations, and other methods of use related to these vectors. Background Art

[0002] Research and development progress in gene therapy for ocular diseases is leading the trend, bringing hope for a cure to more patient groups. Traditional gene therapy mainly targets hereditary ocular diseases such as retinitis pigmentosa, choroideremia, Leber hereditary optic neuropathy, Leber congenital amaurosis (LCA), color blindness, and X-linked retinoschisis (XLRS). The new generation of gene therapy targets signs of chronic diseases such as wet age-related macular degeneration (AMD), diabetic retinopathy, and other chronic retinal conditions.

[0003] Glaucoma is one of the leading causes of blindness in people over 60 years old and the second leading cause of blindness globally. In the United States, the prevalence of glaucoma is approximately 19 cases per 1,000 people. This is equivalent to 22 cases per 1,000 women and 16 cases per 1,000 men (National Eye Institute. Glaucoma Data Sheet). The trabecular meshwork (TM) provides the aqueous humor outflow resistance required to maintain physiological intraocular pressure (IOP). Trabecular meshwork dysfunction leads to elevated intraocular pressure (e.g., ocular hypertension), which is the main risk factor for the development of glaucoma.

[0004] Currently, clinically available treatment options include daily use of eye drops and surgery to reduce IOP. There are various types of eye drops used clinically to treat glaucoma, and they reduce IOP through different mechanisms. Commonly used eye drops include prostaglandin analogs, β-blockers, α-agonists, carbonic anhydrase inhibitors, and Rho kinase inhibitors. Most glaucoma patients are elderly, and they usually also require additional medications to treat other diseases, so these patients have poor compliance with daily use of eye drops.

[0005] When eye drops are unable to adequately lower IOP or cause significant side effects, surgery becomes an option. Laser trabeculoplasty is one of the most common surgeries used to treat open-angle glaucoma, and it works by helping to improve aqueous humor outflow. Although laser surgery has very good safety, there are still some drawbacks and limitations, such as limited duration of effect, variable responses, the need for continuous monitoring, and transient IOP elevation. If a patient's intraocular pressure cannot be controlled with topical eye drops or laser trabeculoplasty, minimally invasive glaucoma surgery (MIGS) is recommended. MIGS reduces IOP by creating a bypass. The bypass is created by placing a trabecular meshwork bypass stent or performing trabeculotomy (or trabeculectomy), thereby improving the outflow of aqueous humor into Schlemm's canal. Surgical risks include uncertain efficacy, the need for repeat surgery, and long-term effectiveness. Given the drawbacks and limitations of current treatment options, developing a gene therapy option to lower IOP that does not require frequent (or only single) dosing would provide great benefits to glaucoma patients.

[0006] RhoA is a GTP-binding protein known to play a role in cell contractility. RhoA cycles between an active and an inactive form, and this cycling between the two conformations activates Rho-associated kinase (ROCK). The relevance of the RhoA pathway in regulating the aqueous humor outflow facility was initially reported as RhoA-mediated trabecular meshwork cell contraction. A study showed that the target of a smooth muscle cell contraction inhibitor (Y-27632) is a ROCK inhibitor that reduces cell contraction by decreasing calcium sensitivity in the vascular system. These findings, along with the established role of trabecular meshwork cell contraction in the aqueous humor outflow facility, inspired a series of studies that showed that Y-27632 also affects IOP. Since then, ROCK inhibitors have been successfully developed as new drugs for glaucoma treatment. However, the rapid metabolism of chemical ROCK inhibitors has led to the need for frequent dosing to lower IOP. To meet the unmet medical needs for treating glaucoma, it is necessary to develop novel therapies that specifically target the trabecular meshwork tissue, and more specifically, the RhoA pathway, to benefit the elderly population. Summary of the Invention

[0007] The present invention is based on the discovery that gene therapy using dominant-negative mutant RhoA (dnRhoA) can lower IOP in glaucoma patients by inhibiting ROCK signaling. A ubiquitously expressed promoter can be used to drive the expression of the mutant dnRhoA. The present invention also relates to the unexpected enhancement of dnRhoA expression when using a shorter eukaryotic promoter. Several AAV capsids, including mutant AAV2, can also be used to package the viral vectors described herein.

[0008] Accordingly, one aspect of the present invention relates to a recombinant self-complementary adeno-associated virus (scAAV) particle comprising a viral capsid protein and an scAAV nucleic acid vector containing a eukaryotic promoter and a dominant negative mutant RhoA. In certain embodiments, the dominant negative mutant RhoA comprises at least one amino acid mutation (e.g., T19N).

[0009] In certain embodiments, the ubiquitous promoter comprises a truncated EF1α, CBA, CBh, or other short eukaryotic promoter.

[0010] In certain embodiments, the recombinant scAAV particles use capsids from different viral serotypes. In certain embodiments, the viral capsid comprises one or more amino acid mutations, e.g., amino acid substitutions at one or more positions such as Y444F, Y500F, and / or Y730F on the capsid in the numbering of VP1 of AAV2 (SEQ ID NO: 5). In certain embodiments, one or more amino acid mutations in the viral capsid reduce immunogenicity, increase the expression of the dominant negative mutant RhoA transgene, and / or increase the duration of the expression of the dominant negative mutant RhoA transgene.

[0011] Also provided herein is a method of reducing intraocular pressure in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of the recombinant scAAV particles described herein. In certain embodiments, the recombinant scAAV particles can be administered by intraocular injection, optionally into the anterior chamber of the eye (e.g., to the trabecular meshwork tissue and / or the cornea) and / or into the posterior chamber of the eye (e.g., to retinal cells, including retinal ganglion cells (RGCs) and / or retinal pigment epithelial cells (RPEs)) and / or into the iris.

[0012] Another aspect of the present invention relates to a method of treating and / or preventing an ocular disease in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of the recombinant scAAV particles described herein.

[0013] A further aspect of the present invention is the use of the recombinant scAAV particles described herein to reduce intraocular pressure in a subject in need of such treatment or to treat and / or prevent an ocular disease in a subject in need of such treatment.

[0014] Another aspect of the present invention is the use of the recombinant scAAV particles described herein to manufacture a medicament for reducing intraocular pressure in a subject in need of such treatment or for treating and / or preventing an ocular disease in a subject in need of such treatment.

[0015] These and other aspects of the present invention are set forth in greater detail in the description of the invention below. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of an expression cassette in a plasmid.

[0017] Figure 2 It is a series of fluorescence microscope images and related flow cytometry measurement charts, showing eGFP expression driven by different promoters 72 hours after transfection in pooled human trabecular meshwork (HTM) cells.

[0018] Figure 3 It is a series of fluorescence microscope images and related flow cytometry measurement charts, showing eGFP expression driven by different promoters 72 hours after transduction (multiplicity of infection is 10,000) on pooled HTM cells.

[0019] Figure 4 It is a chart for quantifying GFP-positive cells by flow cytometry measurement, showing the situation after plasmid transfection or virus infection.

[0020] Figure 5 It is a Southern blot image showing digestion of a plasmid containing the dnRhoA gene with the PvuII-HF restriction enzyme and observation on a 1% agarose gel; lane 1 is a 1 kilobase (kb) ladder (Invitrogen), lane 2 is the digestion product of pGVB-2001-015, and lane 3 is the digestion product of pGVB-2001-016.

[0021] Figure 6 It is a Southern blot image showing digestion of a plasmid containing the dnRhoA gene with the SmaI restriction enzyme and observation on a 1% agarose gel; lane 1 is a 1 kb ladder (Invitrogen), lane 2 is the digestion product of pGVB-2001-015, and lane 3 is the digestion product of pGVB-2001-016.

[0022] Figure 7 It is a chart showing ROCK activity after treating HTM cells with three different ROCK activators: sphingosine-1-phosphate (S1P) 1 μM, oleoyl L-lyso-phosphatidic acid (LPA) 10 μM, and dimethyloxalylglycine (DMOG) 0.5 mM; and a ROCK inhibitor, Y-27632 50 μM.

[0023] Figure 8is a graph showing the ROCK activity levels after non-infected HTM cells, HTM cells infected with wild-type scAAV2 capsid containing the dnRhoA gene, or HTM cells infected with Y3 mutant scAAV2 capsid containing the dnRhoA gene; HTM cells from three independent donors were tested and technical replicates of three AAV infections were analyzed; data are presented as mean ± standard error and one-way ANOVA was performed followed by Dunnett's post hoc test to compare between groups; * indicates p value < 0.033 and ** indicates p value < 0.002.

[0024] Figure 9 is a graph showing the intraocular pressure measured by Icare TonoLab tonometer before and after Ad5.BMP2 injection; each bar shows the mean IOP value of 6 eyes; statistical analysis was performed using one-way ANOVA.

[0025] Figure 10 is a graph showing the intraocular pressure measured by Icare TonoLab tonometer after scAAV2.Y3.CBh.dnRhoA injection. Detailed implementation

[0026] The present invention is explained in more detail below. This description is not intended to be a detailed catalog of all different ways of implementing the present invention, nor is it intended to exhaust all features of the present invention. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, many variations and additions to the various embodiments described herein will be apparent to those skilled in the art in light of the instant disclosure of the present invention without departing from the present invention. Accordingly, the following specification is intended to illustrate certain specific embodiments of the present invention and not to exhaust all possible permutations, combinations, and variations.

[0027] Unless the context otherwise requires, it is specifically intended that the various features of the invention described herein can be used in any combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features described herein can be excluded or omitted. For example, if the specification states that a complex contains components A, B, and C, it is specifically intended that any one of A, B, or C, or any combination thereof, can be excluded and waived, either singly or in any combination.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used herein to describe the invention are for the purpose of describing particular embodiments and are not intended to limit the invention. All publications, patent applications, patents, nucleotide sequences, amino acid sequences, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification shall prevail.

[0029] The nucleotide sequences presented herein are shown only in single-stranded form and in the 5' to 3' direction from left to right, unless otherwise specified. Nucleotides and amino acids are represented in the manner recommended by the International Union of Pure and Applied Chemistry - International Union of Biochemistry (IUPAC-IUB) Commission on Biochemical Nomenclature, or (for amino acids) in either one-letter code or three-letter code, all in accordance with 37 C.F.R. § 1.822 and established usage.

[0030] Unless otherwise noted, standard methods known to those skilled in the art can be used to produce recombinant and synthetic polypeptides, antibodies or their antigen-binding fragments, manipulate nucleic acid sequences, produce transformed cells, construct recombinant adeno-associated virus (rAAV) constructs, modify capsid proteins, express AAV rep and / or cap sequences in packaging vectors, and transiently and stably transform packaging cells. These techniques are well known to those skilled in the art. See, for example, SAMBROOK et al., Molecular Cloning: A Laboratory Manual, 4th ed. (Cold Spring Harbor, N.Y., 2012); F.M. AUSUBEL et al., Current Protocols in Molecular Biology (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0031] All publications, patent applications, patents, nucleotide sequences, amino acid sequences, and other references mentioned herein are incorporated herein by reference in their entirety.

[0032] Definition

[0033] In the description of the invention herein and in the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" are intended to include the plural forms.

[0034] As used herein, "and / or" refers to all possible combinations including one or more of the listed items, as well as the lack of a combination (i.e., "or") when interpreted as a choice.

[0035] In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted.

[0036] In addition, the term "about", when referring in this text to a measurable value, such as the amount, dose, time, temperature, etc. of a compound or agent of the present invention, means a variation of ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the indicated amount.

[0037] The transitional phrase "consisting essentially of" as used herein shall be construed to include the recited materials or steps and those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. Thus, "consisting essentially of" as used herein shall not be construed to be equivalent to "comprising".

[0038] The term "consisting essentially of" (and its grammatical variants), when applied to a polynucleotide or polypeptide sequence of the present invention, refers to a polynucleotide or polypeptide that consists of the recited sequence (e.g., SEQ ID NO) and a total of no more than ten (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) additional nucleotides or amino acids, which are located at the 5' and / or 3' or N-terminal and / or C-terminal of the recited sequence, or between the two ends (e.g., between domains), such that the function of the polynucleotide or polypeptide is not materially altered. The total of no more than ten additional nucleotides or amino acids includes the sum of all additional nucleotides or amino acids added together. The term "materially altered", when applied to a polynucleotide of the present invention, means that the ability to express the encoded polypeptide is increased by at least about 50% or more compared to the polynucleotide consisting of the recited sequence. The term "materially altered", when applied to a polypeptide of the present invention, means that the biological activity is increased by at least about 50% or more compared to the polypeptide consisting of the recited sequence.

[0039] The terms "enhanced" or "increased" mean that the indicated parameter is increased by at least about 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.

[0040] The terms "inhibit" or "reduce" or their grammatical variants as used herein refer to a reduction or decrease in the indicated level or activity by at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In certain embodiments, the inhibition or reduction results in little or essentially undetectable activity (at most an insignificant amount of about 10% or even 5%).

[0041] As used herein, a "therapeutically effective" or "therapeutically 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 will provide some relief, alleviation, or reduction of at least one clinical symptom in a subject (e.g., reducing IOP and / or reducing ocular degeneration). One of ordinary skill in the art will understand that a therapeutic effect need not be complete or curative, so long as some benefit is provided to the subject.

[0042] As used herein, "treat," "treating," or "treatment" (or grammatically equivalent terms) means reducing or at least partially ameliorating or alleviating the severity of a subject's condition and / or alleviating, mitigating, or reducing at least one clinical symptom and / or retarding the progression of the disease.

[0043] As used herein, "prevent," "preventing," or "prevention" (and its grammatical variants) means retarding or inhibiting the onset of a disease. These terms do not mean complete abrogation of the disease and include any type of prophylactic treatment to reduce the incidence of a disease, disorder, and / or clinical symptom or retard its onset.

[0044] A "preventively effective" amount as used herein is an amount sufficient to prevent and / or retard the onset and / or reduce and / or retard the severity of onset of a disease, disorder, and / or clinical symptom in a subject, as compared to in the absence of the method of the present invention. One of ordinary skill in the art will understand that a preventive level need not be complete, so long as some benefit is provided to the subject.

[0045] The terms "protein" and "polypeptide" as used herein may be used interchangeably and include peptides and proteins, unless otherwise indicated. The term "fragment," when applied to a polypeptide, will be understood to be an amino acid sequence that is shorter in length relative to a reference polypeptide or amino acid sequence and that comprises a continuous amino acid sequence that is the same as or substantially the same as (e.g., 90%, 92%, 95%, 98%, 99% identical) the reference polypeptide or amino acid sequence. Such polypeptide fragments according to the present invention, if necessary, may be included in a larger polypeptide of which it is a constituent part. In certain embodiments, such fragments may comprise, consist essentially of, and / or consist of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more continuous amino acids of the polypeptides or amino acid sequences of the present invention.

[0046] As used herein, the terms "nucleic acid", "nucleotide sequence", and "polynucleotide" are used interchangeably and include RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides, regardless of the length of the chain. A nucleic acid can be a sense strand or an antisense strand.

[0047] As used herein, the term "gene" refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, etc. A gene may or may not be used to produce a functional protein. A gene can include coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions). A gene can be "isolated", meaning a nucleic acid that is substantially or essentially free of components that are associated with the nucleic acid in its natural state. Such components include other cellular materials, recombinant production media, and / or various chemicals used in the chemical synthesis of nucleic acids.

[0048] The terms "5' portion" and "3' portion" are relative terms used to define the spatial relationship between two or more elements. Thus, for example, a "3' portion" refers to a fragment of a polynucleotide that is located downstream of another fragment. The term "3' portion" does not mean that the fragment is necessarily located at the 3' end of the polynucleotide, or even necessarily in the 3' half of the polynucleotide, although it may be. Similarly, a "5' portion" refers to a fragment of a polynucleotide that is located upstream of another fragment. The term "5' portion" does not mean that the fragment is necessarily located at the 5' end of the polynucleotide, or even necessarily in the 5' half of the polynucleotide, although it may be.

[0049] As used herein, with respect to nucleic acids, the term "operably linked" refers to a functional linkage between two or more nucleic acid sequences. For example, a promoter sequence can be described as being "operably linked" to a heterologous nucleic acid sequence because the promoter sequence initiates and / or mediates the transcription of the heterologous nucleic acid sequence. In certain embodiments, the operably linked nucleic acid sequences are contiguous and / or in the same reading frame.

[0050] 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 a start site (i.e., a Kozak sequence) that initiates the transcription of the polypeptide. The term "coding region" can be used interchangeably with open reading frame.

[0051] As used herein, the term "optimized" or "optimized for expression" means that the viral particles have been optimized to increase the expression of genes in the viral vector. In certain embodiments, the viral particles have been optimized to increase the expression of genes in an organism (e.g., human, animal, plant, fungus, archaea or bacterium) and / or in a tissue type of the organism (e.g., ocular tissue, brain tissue, muscle tissue, etc.). In certain embodiments, the viral particles are optimized by codon optimization of the gene coding sequence in the viral vector. In certain embodiments, the viral particles are optimized by using a specific viral serotype (e.g., AAV2 or AAV5). In certain embodiments, the viral particles are optimized by mutating the amino acid sequence of the viral capsid protein. In certain embodiments, the optimized viral capsid protein contains a Y3 mutation (e.g., Y444F, Y500F and Y730F mutations). In certain embodiments, compared to the unoptimized reference particles, the optimized viral particles increase the expression level of the genes in the viral vector in an organism and / or tissue type 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%).

[0052] As used herein, the term "codon optimization" refers to the optimization of a gene coding sequence or a fragment of a gene coding sequence by replacing one or more codons present in a wild-type sequence (e.g., a wild-type sequence, including but not limited to a coding sequence encoding a RhoA protein) with codons encoding the same (synonymous) amino acid, thereby increasing expression. In this way, the protein or protein fragment encoded by the gene or gene fragment is the same, but the underlying nucleotide sequence of the gene or gene fragment or the corresponding mRNA is different. In certain embodiments, the optimization improves translation efficiency by replacing one or more rare codons (i.e., codons for which tRNAs occur relatively infrequently in the cells of a particular species) with more frequently occurring synonymous codons. For example, in human codon optimization, one or more codons in a coding sequence are replaced with codons that occur more frequently in human cells for the same amino acid. Codon optimization can also increase the expression of a gene or gene fragment by other mechanisms, thereby improving the efficiency of transcription and / or translation. Strategies include, but are not limited to, increasing the overall GC content (i.e., the percentage of guanine and cytosine in the entire 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. Ideally, a codon-optimized gene or the corresponding mRNA can be distinguished from an endogenous gene and / or the corresponding mRNA in vitro or in vivo.

[0053] As used herein, an "isolated" nucleic acid or nucleotide sequence (e.g., "isolated DNA" or "isolated RNA") refers to a nucleic acid or nucleotide sequence that has been separated from at least some of the other components of a naturally occurring organism or virus, e.g., cellular or viral structural components or other polypeptides or nucleic acids that are normally associated with the nucleic acid or nucleotide sequence.

[0054] Similarly, an "isolated" polypeptide refers to a polypeptide that has been separated from at least some of the other components of a naturally occurring organism or virus, e.g., cellular or viral structural components or other polypeptides or nucleic acids that are normally associated with the polypeptide.

[0055] As used herein, the term "modified", when applied to a polynucleotide or polypeptide sequence, refers to a sequence that is different from the wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof.

[0056] As used herein, "isolating" a viral vector means that the viral vector has been at least partially separated from at least some of the other components in the starting material.

[0057] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or protein sequences remain invariant throughout the alignment window. "Identity" can be calculated by known methods, including but not limited to: Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, New York (1988); Bioinformatics: Informatics and Genome Projects

[0058] (Smith, D.W., ed.) Academic Press, New York (1993); Analysis of Comp uter Generated Sequence Data (Griffin,

[0059] A.M. and Griffin, H.G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Introduction to Sequence Analysis

[0060] (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0061] As used herein, the terms "substantially identical" or "corresponds to" mean that two nucleic acid sequences have at least 60%, 70%,

[0062] 80% or 90% sequence identity. In certain embodiments, two nucleic acid sequences can have at least 85%, 90%,

[0063] 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0064] The "identity score" for an aligned test sequence and a reference sequence segment is the number of identical components (i.e., nucleotides or amino acids) shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence.

[0065] As used herein, the terms "percent sequence identity" or "percent identity" refer to the percentage of identical nucleotides in a reference ("query") polynucleotide molecule (or its complementary strand) and a test ("subject") polynucleotide molecule (or its complementary strand) when optimally aligned (allowing for appropriate insertions, deletions, insertions or gaps, in total not exceeding 20% of the reference sequence comparison window). In certain embodiments, "percent identity" can refer to the percentage of identical amino acids in an amino acid sequence.

[0066] Those skilled in the art are well familiar with sequence alignment to determine the best alignment in a comparison window and can perform it through tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, etc., as well as computerized implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, which tools are Wisconsin

[0067] (Accelrys Inc., Burlington, Massachusetts). The percent sequence identity is expressed as the identity score multiplied by 100. The comparison of polynucleotide sequences can be for the full-length polynucleotide sequence or a part thereof, or for a longer polynucleotide sequence. For the purposes of the present invention, "percent identity" can also be determined using BLASTX version 2.0 (for translating nucleotide sequences) and BLASTN version 2.0 (for polynucleotide sequences).

[0068] The "BestFit" or "Gap" programs can be used to determine the percent sequence identity, which programs are part of the sequence analysis software package TM (version 10; Genetics Computer Group, Inc., Madison, Wisconsin). "Gap" utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol. 48:443 - 453, 1970) to find the best alignment of two sequences to maximize the number of matches and minimize the number of gaps. "BestFit" performs an optimal alignment of the best similar segments between two sequences and inserts gaps to maximize the number of matches, using the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482 - 489, 1981, Smith et al., Nucleic Acids Res. 11:2205 - 2220, 1983).

[0069] Useful methods for determining sequence identity are also described in The Guide to Large Computers (Martin J. Bishop, ed., Academic Press, San Diego, 1994) and Carillo, H. and Lipton, D. (Applied Mathematics 48: 1073 (1988)). More specifically, preferred computer programs for determining sequence identity include, but are not limited to, the BLAST programs publicly available from the National Center for Biotechnology Information (NCBI) of the National Library of Medicine, National Institutes of Health (Bethesda, Maryland); see the BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)); BLAST programs version 2.0 or higher allow the introduction of gaps (deletions and insertions) in the alignment; for peptide sequences, BLASTX can be used to determine sequence identity; for polynucleotide sequences, BLASTN can be used to determine sequence identity.

[0070] "Vector" refers to a compound used as a tool to carry foreign genetic material into another cell, where the insert can be replicated and / or expressed in the target cell. A cloning vector containing foreign nucleic acid is called a recombinant vector. Examples of nucleic acid vectors include plasmids, viral vectors, artificial chromosomes, etc. A recombinant vector typically contains an origin of replication, a multiple cloning site, and a selectable marker. A nucleic acid sequence usually consists of an insert (recombinant nucleic acid or transgene) and a larger sequence serving as the "backbone" of the vector. The purpose of transferring genetic information to another cell by a vector is usually to isolate, replicate, or express the insert in the target cell. The process of inserting a vector into a target cell is called transformation or transfection for bacteria and eukaryotic cells, although the insertion of a viral vector is usually called transduction. The term "vector" can also be used more generally to describe an item that carries foreign genetic material into another cell, such as, but not limited to, a transformed cell or a nanoparticle.

[0071] As used herein, the term "promoter" refers to a polynucleotide sequence to which a polymerase (DNA or RNA) or related transcription factor can bind to initiate transcription. In certain embodiments, the promoter can be a promoter sequence from a virus (e.g., CMV, AAV, etc.), a prokaryote (e.g., E. coli), or a eukaryote (e.g., human, chicken, mouse, yeast, etc.) genome. In certain embodiments, the eukaryotic promoter has low immunogenicity. For example, a viral vector containing the eukaryotic promoter has 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 certain embodiments, the eukaryotic promoter has stable expression in a subject. For example, the expression from a viral vector containing the eukaryotic promoter has a variance of about 1% to about 40% (e.g., about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or about 40%) starting from the baseline expression level after administration to the subject. In certain embodiments, the promoter can be a short promoter, e.g., having a sequence length of about 100 nucleotides to about 2,000 nucleotides (e.g., a length of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, or about 1,200 nucleotides). In certain embodiments, the short promoter is about 500 to about 1,000 nucleotides in length (e.g., about 500, 600, 700, 800, 900, or about 1,000 nucleotides). In certain embodiments, the promoter is a CMV5, CBA, or EF1α promoter (e.g., a truncated EF1α promoter). In certain embodiments, the EF1α promoter is about 1,000 to about 1,400 nucleotides in length (e.g., about 1,000, 1,100, 1,200, 1,300, or about 1,400 nucleotides). In certain embodiments, the promoter can be a hybrid promoter, e.g., where two promoter sequences from one or more different organisms are operably linked together. In certain embodiments, the hybrid promoter is a CBh promoter (e.g., a CMV and chicken β-actin hybrid promoter). In certain embodiments, the CBA and / or CBh promoter is about 400 to about 1,000 nucleotides in length (e.g., about 400, 500, 600, 700, 800, 900, or about 1,000 nucleotides).

[0072] The "subject" of the present invention can include any desired animal. In certain embodiments, the subject can be, for example, a mammal, a reptile, a bird, an amphibian, or a fish. Mammalian subjects can include, but are not limited to, laboratory animals (e.g., rats, mice, guinea pigs, rabbits, primates, etc.), farm or commercial animals (e.g., cows, pigs, horses, goats, donkeys, sheep, etc.), or domestic animals (e.g., cats, dogs, ferrets, hamsters, gerbils, etc.). In certain embodiments, the mammalian subject can be a primate, or a non-human primate (e.g., chimpanzee, baboon, macaque (e.g., rhesus macaque, cynomolgus macaque, stump-tailed macaque, pig-tailed macaque), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc.). In certain embodiments, the mammalian subject can be a human.

[0073] The "subject in need of treatment" of the present invention can be any subject known or suspected of having an increased risk of developing an eye disease (e.g., glaucoma, AMD (e.g., dry AMD or wet AMD), diabetic retinopathy, and / or retinal tear), and / or intraocular hypertension (increased IOP).

[0074] Compositions and Preparations The present disclosure provides recombinant scAAV vectors and methods of using the same to treat eye diseases (e.g., glaucoma) and / or intraocular hypertension. Accordingly, the present disclosure provides recombinant scAAV particles, compositions comprising the recombinant scAAV particles, and methods of treating eye diseases (e.g., glaucoma) and / or intraocular hypertension. In certain embodiments, the viral vectors described herein can be used with any AAV capsid, whether or not containing mutations, for manufacturing AAV particles for treating eye diseases (e.g., glaucoma) and / or intraocular hypertension, or any combination thereof. The present disclosure also provides pharmaceutical formulations and dosages of viral injection solutions for intravitreal injection. Another aspect of the present disclosure is the common injection routes for treating eye diseases (e.g., glaucoma) and / or intraocular hypertension, or any combination thereof. Other aspects of the present disclosure relate to the therapeutic effects of the viral vectors in glaucoma patients.

[0075] The current inventors of the present invention have been optimizing delivery vectors, their cargoes, and the underlying mechanisms for regulating gene expression of the cargoes. For the delivery vector, the inventors sought a vector with a long trabecular meshwork duration, low immunogenicity, and selected and mutated capsid viruses to increase gene transfer efficiency. For the cargo, an effective dominant negative mutant RhoA was identified to reduce IOP in a glaucoma animal model. For the promoter, a hybrid chicken β-actin (CBh) promoter was selected to drive transgene expression. Collectively, the viral vector of the present invention may 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 manufactured and characterized in vitro, and its efficacy was subsequently tested in a rat glaucoma model.

[0076] In certain embodiments, the AAV particles can have an altered VP1 capsid protein, an altered VP2 capsid protein, an altered VP3 capsid protein, or any combination thereof. In certain embodiments of the aspects and embodiments described herein, the AAV viral particles comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV12, AAV2R471A, bovine AAV, and / or murine AAV. In certain embodiments, the AAV viral particles comprise an AAV capsid containing one or more amino acid substitutions at positions Y444F, Y500F, and / or Y730F, according to the VP1 numbering of AAV2 (SEQ ID NO: 5). In certain embodiments, the vector comprises an inverted terminal repeat (ITR) of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV12, AAV2R471A, bovine AAV, and / or murine AAV. In certain embodiments, the vector comprises a mutated AAV ITR, resulting in the formation of self-complementary AAV. In certain embodiments, the AAV viral particles comprise one or more ITRs, and the capsid is derived from the same AAV serotype. In other embodiments, the recombinant scAAV viral particles comprise an AAV2 capsid, and the vector comprises an AAV2 ITR.

[0077] In certain embodiments, various formulations can be used to facilitate transduction of the virus in ocular tissues. For example, for administration of an injectable aqueous solution of recombinant scAAV particles, if desired, the solution can be appropriately buffered and the liquid diluent is first made isotonic with sufficient saline or glucose. Thus, in certain embodiments, the viral injection solution can contain a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with the recombinant scAAV particles. By "pharmaceutically acceptable" is meant that the material is not toxic or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects. Such pharmaceutical carriers can be sterile liquids (e.g., water, oil, saline solution, aqueous glucose-containing solution, and / or glycerol solution), suspending agents, preservatives (e.g., methyl, ethyl, and / or propyl-hydroxybenzoates), and pH regulators (such as inorganic acids, organic acids, and / or bases). In certain embodiments, the carrier includes a buffered saline solution (e.g., phosphate-buffered saline, HEPES-buffered saline, etc.). In certain embodiments, USP-level carriers and excipients can be used for delivery of recombinant scAAV particles to human subjects. Such compositions can further optionally contain liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, and / or nanoparticles, or can otherwise be formulated for delivery to the cells, tissues, organs, or body of a subject in need thereof.

[0078] In certain embodiments, a composition comprising any one of the recombinant scAAV particles described herein comprises a balanced salt solution (BSS) supplemented with from about 0.001% to about 0.035% Tween 20 (polysorbate 20) (e.g., about 0.001%, 0.007%, 0.014%, 0.021%, 0.028%, or about 0.035% or any range therein). In certain embodiments, a composition comprising any one of the recombinant scAAV particles described herein comprises from 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 therein), from 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 therein), pH 8.0, supplemented with from 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 therein).

[0079] Method of Use

[0080] The methods of the invention are useful in both veterinary and medical applications.

[0081] The dose of the AAV particles of the invention depends on the mode of administration, the disease or condition to be treated, the condition of the individual subject, the viral vector, and the gene to be delivered, and can be determined by conventional means. Exemplary doses for achieving a therapeutic effect are at least about 10^5, 10^6, 10^7, 10^8, 10^9, 10^10, 10^11, 10^12, 10^13, 10^14, 10^15 transduction units or more, preferably about 10^8 - 10^13 transduction units, and more preferably 10^11 transduction units.

[0082] As used herein, the term "administering" or "administration" refers to introducing or delivering the compositions of the invention to a subject to perform their intended function (e.g., reducing the IOP of a subject or treating glaucoma). In certain embodiments, the recombinant scAAV viral vector can be administered by intraocular injection. As used herein, the term "intraocular injection" refers to any convenient route of injection by which the recombinant scAAV vector can be delivered to ocular tissue (e.g., any part of the eye). In certain embodiments, the intraocular injection is into the anterior part of the eye (e.g., the anterior chamber), e.g., by anterior chamber injection. In certain embodiments, the intraocular injection is into the posterior part of the eye (e.g., the posterior chamber), e.g., into the vitreous of the eye, e.g., by vitreous injection. In certain embodiments, the intraocular injection is into the cornea of the eye, e.g., by intrastromal corneal injection. In certain embodiments, the intraocular injection is into the subretinal space of the eye (e.g., the suprachoroidal space), e.g., by suprachoroidal injection. Other convenient routes of administration include, but are not limited to, intravenous, intraarterial, periocular, subconjunctival, and subfascial injection, topical administration (e.g., topical ocular administration), and intranasal administration.

[0083] The viral vectors of the present invention may be effective in treating or preventing neurological disorders. In certain embodiments, the viral vectors described herein can provide IOP-dependent or IOP-independent neurotrophic protection to retinal cells (such as retinal ganglion cells). In certain embodiments, the viral vectors described herein prevent the progression of glaucoma by reducing IOP and / or providing direct neurotrophic protection. In certain embodiments, the viral vector prevents the progression of glaucoma by simultaneously providing direct neurotrophic protection and reducing intraocular pressure. This therapy provides an unexpected dual benefit in the treatment of glaucoma. In certain embodiments, the viral vector can prevent the progression of glaucoma without reducing IOP. Thus, this treatment method may provide an unexpected benefit, namely, an IOP-independent neurotrophic protection effect. In certain embodiments, the viral vectors described herein can accelerate wound healing after corneal injury. Thus, this treatment method provides an unexpected benefit of corneal protection. After the present invention has been described, the same invention will be further explained in the following examples, which are included herein for illustrative purposes only and are not intended to limit the present invention.

[0084] SEQ ID NO: 1

[0085] Promoter CMV5

[0086]

[0087] SEQ ID NO: 2

[0088] Truncated EF1α promoter

[0089]

[0090] SEQ ID NO: 3

[0091] Promoter hybrid chicken β-actin (CBh)

[0092] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0093] SEQ ID NO: 4

[0094] Amino acid sequence of dominant negative mutant RhoA (T19N)

[0095] MAAIRKKLVIVGDGACGKNCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAALQARRGKKKSGCLVL

[0096] SEQ ID NO: 5

[0097] Amino acid sequence of AAV2 VP1

[0098] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0099] SEQ ID NO: 6

[0100] Amino acid sequence of wild-type RhoA

[0101] MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAALQARRGKKKSGCLVL

[0102] SEQ ID NO: 7

[0103] scAAV2.CBh.dnRhoA vector sequence containing the CBh promoter and the dnRhoA gene

[0104]

[0105] Example

[0106] Example 1: Comparing promoters for driving GFP expression in HTM cells

[0107] To identify suitable promoters in HTM cells, the efficiency of several promoters for driving green fluorescent protein (GFP) expression was compared. Four commonly used promoters were selected to construct plasmids ( Figure 1 ): CMV5 (SEQ ID NO: 1), EF1α (SEQ ID NO: 2), and CBh (SEQ ID NO: 3). The first step was to test the expression of the new plasmids in HTM cells by transfection with Lipofectamine (ThermoFisher). HTM cells (from two individual donors) were seeded into 24-well plates one day before transfection to reach 70 - 80% confluence. 0.5 μg of plasmid was transfected into the cells using Lipofectamine 3000 transfection reagent (ThermoFisher) to examine promoter efficiency. After the cells had grown for 72 hours, they were observed by fluorescence microscopy and the proportion of GFP-positive cells was quantified by flow cytometry. Overall, the GFP expression levels in HTM cells recorded by fluorescence microscopy and flow cytometry were similar between different promoter groups ( Figure 2 and Figure 4 ).

[0108] The promoter efficiency was further tested in HTM cells by AAV virus transduction. The plasmids used in the previous transfection experiments were used to package AAV particles. Since the plasmids all contain delta-ITR, the viral DNA is self-complementary. AAV2 with the Y3 mutation (Y444F, Y500F, and Y730F) was selected as the capsid. Then, HTM cells were seeded into 24-well plates. The cells were infected at 10,000 MOI. The cells were examined 3 days after growth. The proportion of GFP-positive cells recorded by flow cytometry showed different results between different promoter groups. The proportion of GFP-positive cells in the cell group infected with the vector containing the CBh promoter was much higher compared to the cell groups infected with the vectors containing the CMV5 and EF1α promoters ( Figure 3 and Figure 4 ).

[0109] Example 2: Construction of scAAV2.dnRhoA

[0110] To generate recombinant scAAV viral particles, a cargo sequence (e.g., dominant-negative mutant RhoA) was inserted into the ITRs (wild-type and deleted, ITR-ΔITR) to create specially designed plasmids named pGVB-2001-15 and pGVB-2001-16. The EF1α and CBh promoters were used in the pGVB-2001-15 and pGVB-2001-16 plasmids, respectively. The amino acid sequence of RhoA is shown in SEQ ID NO: 6, and the dominant-negative mutation is a T19N mutation as shown in the dnRhoA sequence (SEQ ID NO: 4). The plasmid containing dnRhoA was verified by digestion with PvuII-HF restriction enzyme and then subjected to an endotoxin-free maxiprep ( Figure 5 ). The integrity of the ITR region was verified by SmaI digestion ( Figure 6 ). Then, scAAV2 was produced by triple transfection of the plasmid into HEK293 cells. The viral preparation was purified by iodixanol gradient ultracentrifugation. The gradient was prepared by sequentially adding: 6 mL of 15% iodixanol, 6 mL of 25% iodixanol, 5 mL of 40% iodixanol, and 5 mL of 54% iodixanol. 7 - 8 mL of the lysate was added to the top of the gradient, and the tube was then filled with phosphate-buffered saline (PBS).

[0111] The samples were centrifuged at a centrifugal force of 350,000 x g at 18 °C for 2 hours. After centrifugation, a 1-inch long, 18-gauge needle attached to a 10-mL syringe was used to pierce the tube 3 - 5 mm below the 40 / 54 interface. Approximately 1 mL of the 54% interface was collected, and 4 mL of the 40% interface was collected without disturbing the proteinaceous material at the 40 / 25 interface.

[0112] After ultracentrifugation purification, 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 PBS containing 0.1% Pluronic F-68 to the filter and incubating for 10 minutes at room temperature. After incubation, the 0.1% Pluronic solution was discarded. Then 15 mL of PBS containing 0.01% Pluronic was added to the filter and centrifuged at 2000 x g for 5 minutes, and the filtrate was discarded. Finally, 15 mL of PBS containing 0.001% Pluronic and 200 mM NaCl was added to the filter and centrifuged at 2000 x g for 5 minutes, and the filtrate was discarded. After preparing the filter, the sample was diluted 1:2 with the formulation buffer (PBS containing 0.001% Pluronic F-68). The sample was added to the filter and centrifuged at 2000 x g for 5 minutes. After discarding the filtrate, more sample could be added and centrifuged. After concentrating all the samples on the filter, the formulation buffer was added and centrifugation was repeated until 50 mL of the formulation buffer passed through the filter. Then centrifugation was continued at short intervals of 2 - 3 minutes until the desired volume was reached.

[0113] 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 to activate ROCK. Overexpression of dnRhoA competes with endogenous RhoA for binding to ROCK, thereby inhibiting the activation of ROCK. Measuring the activity of ROCK in cells can be developed into a cell-based assay for testing the function of scAAV2.CBh.dnRhoA.

[0114] Rho family members are key regulatory components of signaling pathways that direct cell movement, adhesion, and cell division by rearranging the actin cytoskeleton. Rho is activated by extracellular signals such as lysophosphatidic acid (LPA). The actions of Rho are mediated by downstream Rho effectors. One such effector is ROCK. ROCK mediates Rho signaling and rearranges the actin cytoskeleton by phosphorylating several substrates that contribute to actin filament assembly and contractility. For example, ROCK inactivates myosin phosphatase by specifically phosphorylating the Thr696 site on myosin phosphatase target subunit 1 (MYPT1), resulting in an increased phosphorylation level of the 20 kDa myosin light chain.

[0115] The ROCK activity detection kit is an enzyme immunoassay kit for detecting the phosphorylation of Thr696 site on specific MYPT1 by ROCK. A strip microplate is pre-coated with recombinant MYPT1. After incubating the substrate wells with ROCK samples, phosphorylated MYPT1 is detected by anti-phospho-MYPT1 (Thr696) antibody.

[0116] Human trabecular meshwork cells were seeded into 12-well plates at a density of 500,000 cells per well. After 48 hours of seeding, the cells were harvested with cell lysis buffer. Before incubating with the pre-coated strip wells, the cell lysates were treated with a ROCK activator or a ROCK inhibitor. Then the ROCK activity was measured according to the kit protocol. Three chemicals were used to activate ROCK: S1P (sphingosine-1-phosphate, 1 μM), LPA (oleoyl L-lyso-phosphatidic acid, 10 μM), and DMOG (dimethyloxalylglycine, 0.5 mM). One chemical was used to inhibit ROCK activity: Y-27632 (50 μM). The data were expressed as relative ROCK activity and normalized to the untreated cell lysates. As Figure 7 shown, all chemical activators increased the ROCK activity, and LPA was the strongest activator. The chemical inhibitor decreased the ROCK activity, as expected.

[0117] In the following experiment, human trabecular meshwork cells were seeded into 12-well plates at a density of 500,000 cells per well. Infections with scAAV2.Y3.CBh.dnRhoA or scAAV2.CBh.dnRhoA were performed at an MOI of 10,000 to compare the wild-type AAV2 capsid and the Y3 mutant AAV2 capsid. The cells were harvested 2 days after infection. The ROCK activity was measured. Compared with the cells infected with the wild-type capsid, the decrease in ROCK activity in the cells infected with the Y3 mutant capsid was statistically significant, while the decrease in ROCK activity in the cells infected with the wild-type capsid was less obvious ( Figure 8 ).

[0118] Example 4: Measuring RhoA expression after transduction of HTM cells with scAAV2.CBh.dnRhoA In the following experiment, human trabecular meshwork cells were seeded into 12-well plates at a density of 500,000 cells per well. Infections with scAAV2.Y3.CBh.dnRhoA were performed at an MOI of 10,000. The cells were harvested 2 days after infection to extract total RNA. The RNA content was measured by NanoDrop. RT-PCR was performed to measure the expression of total RhoA.

[0119] Example 5: Testing the efficacy of scAAV2.CBh.dnRhoA using a glaucoma transgenic model

[0120] The efficacy of scAAV2.Y3.CBh.dnRhoA was tested in a glaucoma rat model transduced with Ad.BMP2. Intraocular pressure was elevated by increasing the resistance of trabecular meshwork tissue to aqueous humor outflow. This increased resistance can be caused by a variety of dysfunctional trabecular meshwork cells and mechanisms. However, it is generally believed that disruption of the extracellular matrix (ECM) tissue structure of trabecular meshwork cells is the most common source of increased outflow resistance. Bone morphogenetic protein 2 (BMP2) belongs to the TGFβ superfamily. BMP2 itself has the full potential to initiate bone formation and induce the differentiation of pluripotent mesenchymal progenitor cells into the osteogenic lineage. Similarly, BMP2 induces primary HTM cells to exhibit osteoblast-like characteristics in vitro. Overexpression of the BMP2 gene by an adenovirus vector can increase alkaline phosphatase (ALP) activity in primary HTM cells, which promotes the release of free phosphate and contributes to the formation of hydroxyapatite crystals (which is part of the mineralization process). Overexpression of the BMP2 gene in trabecular meshwork tissue is sufficient to elevate intraocular pressure and create an animal model similar to intraocular hypertension or glaucoma.

[0121] Before Ad5.BMP2 injection, the baseline intraocular pressure of the rats was measured using a TonoLab tonometer (Icare). Under anesthesia, approximately 5 μl of Ad5.BMP2 virus (titer 1.8 X 10^10 pfu / ml) was injected into the anterior chamber of the rats' eyes. Then, the intraocular pressure was monitored until day 28 after injection. The baseline intraocular pressure of the rats' eyes was approximately 12 mmHg. After Ad5.BMP2 injection, the average 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.

[0122] Once the intraocular pressure elevation was established, scAAV2.Y3.CBh.dnRhoA was injected to reduce the intraocular pressure. Only eyes with an intraocular pressure elevation of more than 50% compared to the baseline were selected for AAV injection. Then, the intraocular pressure was continuously monitored until day 14 after AAV injection. Only 7 days after AAV injection, a reduction in intraocular pressure of approximately 50% was observed. Then, the intraocular pressure was maintained at a level close to the baseline until day 14( Figure 10 ).

[0123] The foregoing is a description of the invention and should not be construed as a limitation thereof. The invention is defined by the following claims, and equivalents of the claims should also be included therein.

Claims

1. A recombinant self-complementary adeno-associated virus (scAAV) particle, comprising: a) an AAV capsid protein; and b) an scAAV viral genome comprising a eukaryotic promoter operably linked to a polynucleotide encoding a dominant negative mutant RhoA.

2. The recombinant scAAV particle according to claim 1, wherein the dominant negative mutant RhoA comprises at least one amino acid mutation.

3. The recombinant scAAV particle according to claim 2, wherein the at least one amino acid mutation is a threonine-to-aspartic acid mutation (T19N) at position 19 of SEQ ID NO:

6.

4. The recombinant scAAV particle according to any one of claims 1 to 3, wherein the eukaryotic promoter is a short eukaryotic promoter (e.g., about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or about 1200 nucleotides in length), optionally, the short eukaryotic promoter is a truncated elongation factor 1α (EF1α), chicken β- actin (CBA), or hybrid chicken β-actin (CBh) promoter.

5. The recombinant scAAV particle according to any one of claims 1 to 4, wherein the polynucleotide encoding the dominant negative mutant RhoA is codon-optimized for human expression (e.g., the optimized particle expresses the polynucleotide encoding the dominant negative mutant RhoA in humans about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60％、80％、90％、100％、125％、150％、175％、200％、350％、400％、 450%, or about 500% more than the non-optimized particle).

6. The recombinant scAAV particle according to any one of claims 1 to 5, wherein the capsid protein is optimized for expression in ocular tissue (e.g., the optimized particle expresses the polynucleotide encoding the dominant negative mutant RhoA in ocular tissue about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 350%, 400%, 450%, or about 500% more than the non-optimized particle).

7. The recombinant scAAV particle according to any one of claims 1 to 6, wherein the particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV12, AAV2R471A, bovine AAV, or murine AAV serotype.

8. The recombinant scAAV particle according to any one of claims 1 to 7, wherein the capsid protein comprises one or more amino acid mutations.

9. The recombinant scAAV particle according to claim 8, wherein the one or more amino acid mutations include Y444F, Y500F, and / or Y730F amino acid mutations, numbered according to the VP1 of AAV2 (SEQ ID NO: 5).

10. The recombinant scAAV particle according to claim 8, wherein the one or more amino acid mutations reduce immunogenicity and / or increase the expression of the dominant negative mutant RhoA.

11. The recombinant scAAV particle according to claim 6, wherein the ocular tissue includes the trabecular meshwork, iris, cornea, and / or retina.

12. A method of reducing intraocular pressure (IOP) in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of the recombinant scAAV particle according to any one of claims 1 to 11 to the subject, thereby reducing the intraocular pressure of the subject.

13. The method according to claim 12, wherein the administration comprises intraocular injection.

14. The method according to claim 13, wherein the intraocular injection comprises injection into the anterior chamber of the eye, optionally into the trabecular meshwork tissue and / or cornea.

15. The method according to claim 14, wherein the intraocular injection is an anterior chamber injection.

16. The method according to claim 13, wherein the intraocular injection comprises injection into the posterior chamber of the eye, optionally into retinal cells (e.g., retinal ganglion cells and / or retinal pigment epithelial cells).

17. A method of treating and / or preventing an ocular disease in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of the recombinant scAAV particle according to any one of claims 1 to 11 to the subject, thereby treating and / or preventing the ocular disease of the subject.

18. The method according to claim 17, wherein the ocular disease is associated with elevated intraocular pressure.

19. The method according to claim 18, wherein the ocular disease is glaucoma, age-related macular degeneration (AMD), wet AMD, diabetic retinopathy, and / or retinal tear.

20. The method according to any one of claims 17 to 19, wherein the administration comprises intraocular injection.

21. The method according to claim 20, wherein the intraocular injection comprises injection into the anterior chamber of the eye, optionally into the trabecular meshwork tissue and / or cornea.

22. The method according to claim 21, wherein the intraocular injection comprises injection into the posterior chamber of the eye, optionally into retinal cells (e.g., retinal ganglion cells and / or retinal pigment epithelial cells).

Images