Adeno-associated virus vectors for delivery of therapeutics

Abstract

To provide methods for selectively delivering therapeutics to the eye using AAV vectors.SOLUTION: For example, the cornea can be specifically targeted using the methods described. Also provided herein are compositions comprising AAV vectors packaged with CRISPR complexes, which can be delivered directly to the eye, for example the cornea, and in particular the cornea endothelium. Diseases and conditions comprising abnormalities or deterioration of tissues in the eye, such as the cornea endothelium (e.g. FECD), can be treated using the methods and compositions described herein.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 812,017, filed February 28, 2019, and No. 62 / 831,838, filed April 10, 2019, and No. 62 / 878,865, filed July 26, 2019, each of which is incorporated herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on June 3, 2020, is named 67000-1023_WO_SL.txt and is 357,652 bytes in size.

[0003] The present invention generally relates to the use of adeno-associated virus (AAV) vectors to deliver therapeutic agents to the eye, for example, to the corneal endothelium.The present invention also relates to compositions comprising AAV vectors.Corneal dystrophies can be treated with the methods and compositions of the present invention. [Background technology]

[0004] Adeno-associated viruses (AAV) are small, replication-deficient parvoviruses. AAVs are approximately 20–24 nm long and have a density of approximately 1.40–1.41 g / cc. AAVs contain a single-stranded linear genomic DNA molecule approximately 4.7 kb in length. The single-stranded AAV genomic DNA can be either positive- or negative-stranded. AAVs contain two open reading frames, Rep and Cap, flanked by two 145-base inverted terminal repeats (ITRs). AAVs contain a single intron. Cis-acting sequences that direct viral DNA replication (Rep), encapsidation / packaging, and integration into host cell chromosomes are contained within the ITRs. Three AAV promoters, p5, p19, and p40 (named for their relative map positions), drive expression of two AAV internal open reading frames that encode the rep and cap genes. p5 and p19 are rep promoters. Two rep promoters, combined with alternative splicing of a single AAV intron, result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The rep proteins have multiple enzymatic properties involved in the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production of the three related capsid proteins. A single polyadenylation site is located at map position 95 of the AAV genome. Muzyczka has reviewed the life cycle and genetics of AAV (Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992)).

[0005] AAV infection is noncytopathic in cultured cells. Natural infection in humans and other animals is asymptomatic and subclinical (does not cause disease). AAV infects many mammalian cells and therefore has the potential to target many different tissues in vivo. In addition to dividing cells, AAV can transduce slowly dividing and nondividing cells and persist essentially for the life of those cells as transcriptionally active nuclear episomes (i.e., extrachromosomal elements). The AAV proviral genome is infectious as DNA cloned into a plasmid, allowing the construction of recombinant genomes. Furthermore, signals directing AAV replication, genome encapsidation, and integration are all contained within the ITRs of the AAV genome, so that part or all of the approximately 4.3 kb of genome encoding the replication and structural capsid proteins (rep-cap) can be replaced with heterologous DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Low temperature storage of AAV is less critical because AAV is a very stable and robust virus and easily withstands the conditions used to inactivate adenovirus (56 °C-65 °C for several hours). Also, cells infected with AAV do not tolerate superinfection. These unique properties of AAV make it useful as a vector for delivering foreign DNA to cells or subjects, for example in gene therapy.

[0006] Corneal dystrophies is a term for a heterogeneous group of non-inflammatory bilateral diseases confined to the cornea. They are grouped by the anatomical location within the cornea of ​​the pathology. Most have no symptoms outside the cornea, causing corneal opacification and affecting vision (see https: / / www.cornealdystrophyfoundation.org / what-is-corneal-dystrophy).

[0007] The cornea has three main areas affected by corneal dystrophies: the corneal epithelium, the stroma, and the endothelium. Anterior corneal dystrophies affect the corneal epithelium and its basement membrane, and the superficial corneal stroma. Stromal dystrophies affect the corneal stroma. Posterior corneal dystrophies affect the Descemet's membrane and the corneal endothelium. The most common posterior corneal dystrophies are Fuchs' endothelial dystrophy.

[0008] Recently, certain pathological conditions or diseases have been found to be associated with mutations in the TCF4 gene, which encodes the transcription factor 4 protein (TCF4). Diseases associated with mutations in the TCF4 gene include Fuchs endothelial corneal dystrophy (FECD), posterior polymorphous corneal dystrophy (PPCD), primary sclerosing cholangitis (PSC), Pitt-Hopkins syndrome, distal 18q deletion, and schizophrenia.

[0009] FECD is a condition that causes vision problems. It affects the cornea of ​​the eye, specifically the endothelium. The cornea is located at the front of the eye, and there are five basic layers of corneal tissue. The epithelium is the outermost layer of the cornea. The epithelium functions to block the passage of foreign bodies (e.g., dust, water, bacteria) to the other layers of the eye and cornea, provides a smooth surface for the absorption of oxygen and cellular nutrients from tears, and distributes these nutrients to the rest of the cornea. Epithelial cells are anchored and organized in the basement membrane of the epithelium. Directly beneath the basement membrane of the epithelium is Bowman's layer, a transparent sheet of tissue made up of collagen fibers. Beneath Bowman's membrane is the stroma. The stroma makes up about 90% of the thickness of the cornea and is made up mainly of water and collagen. A thin, strong sheet of tissue, Descemet's membrane, lies beneath the stroma. Descemet's membrane is made up of collagen fibers and is made up of endothelial cells that underlie it. The endothelium is the layer beneath Descemet's membrane.

[0010] The endothelium is a very thin, innermost layer of the cornea and is essential for keeping the cornea clear. The corneal endothelium is a single layer of amitotic cells that forms a barrier between the corneal stroma and the aqueous humor. Corneal endothelial cells function by pumping fluid away from the cornea to maintain the cornea at the correct thickness and maintain transparency. In some posterior corneal dystrophies, such as FECD, the corneal endothelium is affected and cells die during the course of this progressive disease. As these cells die, the remaining cells expand to fill the space and the layer loses its ability to function properly. This results in corneal edema and increased opacity, resulting in decreased vision. In advanced stages of the disease, blindness may result. Vision loss due to FECD is the leading cause of corneal transplants in the United States.

[0011] Because the corneal endothelium is affected by these diseases, targeting them to deliver therapeutic agents may help halt disease progression. One such methodology is adeno-associated virus (AAV), which can be packaged to deliver therapeutic agents and delivered via intracameral or intrastromal injection to contact the corneal endothelium. Proteins or nucleotide sequences are typically packaged into AAV vectors.

[0012] It has been suggested that genetic factors are associated with the development of FECD. Genetic loci known to be associated with FECD include FCD1 to FCD4, ZEB1 / TCF8, SLC4A11, LOXHD1, and COL8A2. One such genetic factor is a trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. Most of the genetic predisposition to FECD is associated with the TNR in the third intron of the TCF4 gene. A repeat length of more than 50 is generally associated with the clinical diagnosis of FECD (Wieben et al., PLOS One, 7:11, e49083 (2012)). Recently, it has been suggested that this expansion of the TNR leads to aggregation of the affected TCF4 RNA and sequestration of key RNA splicing factors (Mootha, et al., Invest. Ophthalmol. Vis. Sci., 55(1):33-42(2014); Mootha, et al., Invest. Ophthalmol. Vis. Sci., 56(3):2003-11(2015); Vasanth, et al., Invest. Ophthalmol. Vis. Sci., 56(8):4531-6(2015); Soliman et al., JAMA Ophthalmol., 133(12):1386-91(2015)). Sequestration of RNA splicing factors can result in global changes in gene expression, leading to significant alterations in cellular function and cell death (Du et al., J. Biol. Chem., 290:10, 5979-5990 (2015)).

[0013] Another genetic mutation associated with FECD occurs in the COL8A2 gene (Vedana et al., Clinical Opthalmology, 10, 321-330 (2016)). Collagen VIII, or COL8 (comprising COL8A1 and COL8A2), is regularly distributed in the Descemet's membrane of the cornea. It has been shown that the corneas of patients with mutations in COL8A2 have irregular mosaic deposits of different amounts of COL8A1 and COL8A2 in an uncoordinated manner. Three point mutations in COL8A2 lead to the intracellular accumulation of mutant COL8 peptides. These point mutations are Gln455Lys, Gln455Val, and Leu450Trp. The intracellular accumulation of mutant COL8 peptides can cause early-onset FECD, as well as the associated corneal disorder PPCD, characterized by alterations in Descemet's membrane and the endothelial layer of the cornea.

[0014] AAV vectors have been used to deliver gene editing therapeutics directly to the eye, but this has generally only been shown to the posterior portion of the eye, such as the retina. Delivery of gene editing therapeutics to the anterior portion of the eye, such as the cornea, has been less well studied and less documented. There remains a need to develop delivery technologies that can preferentially deliver therapeutics to specific regions of the eye and to specific tissues or cells, particularly to the anterior portion, such as the cornea. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] U.S. Provisional Application No. 62 / 812,017 [Patent Document 2] U.S. Provisional Application No. 62 / 831,838 [Patent Document 3] U.S. Provisional Application No. 62 / 878,865 [Non-patent literature]

[0016] [Non-Patent Document 1] Muzyczka, Current Topics in Microbiology and Immunology, 158:97 - 129(1992) [Non - Patent Document 2] Wieben et al., PLOS One, 7:11, e49083(2012) [Non - Patent Document 3] Mootha, et al., Invest. Ophthalmol. Vis. Sci., 55(1):33 - 42(2014) [Non - Patent Document 4] Mootha, et al., Invest. Ophthalmol. Vis. Sci., 56(3):2003 - 11(2015) [Non - Patent Document 5] Vasanth, et al., Invest. Ophthalmol. Vis. Sci., 56(8):4531 - 6(2015) [Non - Patent Document 6] Soliman et al., JAMA Ophthalmol., 133(12):1386 - 91(2015) [Non - Patent Document 7] Du et al., J. Biol. Chem., 290:10, 5979 - 5990(2015) [Non - Patent Document 8] Vedana et al., Clinical Opththalmology, 10, 321 - 330 (2016) [Summary of the Invention] [Problems to be Solved by the Invention]

[0017] The present invention provides a method for delivering therapeutic agents to the corneal endothelium to treat diseases such as corneal dystrophies, e.g., FECD. The method of the present invention utilizes AAV to deliver therapeutic agents directly to the eye, particularly to the corneal endothelium. In certain embodiments, AAV is packaged with a protein or with nucleotides that code for the protein to be expressed in specific cells of the eye. In other embodiments, AAV is packaged with CRISPR RNP complexes (i.e., complexes with Cas proteins) to induce directed gene editing in the eye and in specific regions or cells of the eye. In some embodiments, AAV is packaged with CRISPR gRNA complexed with nucleotide sequences that code for Cas proteins. The present invention also provides compositions comprising AAV. [Means for solving the problem]

[0018] In a particular aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) the nucleotide sequence of an AAV vector or a portion thereof; b) a nucleic acid editing system comprising at least one nucleotide sequence complementary to at least one mutant allele on a target gene associated with a corneal disease or condition, a nucleic acid capable of downregulating gene expression of at least one mutant allele on a target gene associated with a corneal disease or condition, and / or at least one nucleotide sequence or a portion thereof encoding a protein expressed in the eye.

[0019] In another aspect, the present invention provides a method of expressing a protein in an eye of a subject in need thereof, the method comprising: a) providing one or more adeno-associated (AAV) vectors comprising a nucleotide sequence encoding the protein; b) administering the AAV vector to the eye.

[0020] In another aspect, the present invention provides a method for restoring a gene expressed in the cornea to the cornea of ​​a subject in need thereof, the method comprising: a) a nucleic acid editing system comprising at least one nucleotide sequence that is complementary to at least one mutant allele on a target gene associated with a corneal disease or condition; b) administering the delivery system to the cornea of ​​the subject. When the term "repair" is used, it is also meant to include inducing repair.

[0021] In yet another aspect, the present invention provides a method of treating a corneal disease or condition caused by a mutant allele of a gene containing a trinucleotide repeat (TNR) and / or a point mutation in a subject in need thereof, comprising: a) excising at least a portion of a trinucleotide repeat (TNR) within said gene, i) providing an AAV5, AAV6, or AAV8 vector comprising one or more nucleotide sequences encoding one or more guide RNAs that target a sequence within the TNR, 5' of the TNR, 3' of the TNR, or a combination thereof; ii) administering said vector to the cornea; and / or b) correcting a point mutation in a gene or gene product, i) providing an AAV5, AAV6, or AAV8 vector comprising one or more nucleotide sequences encoding one or more guide RNAs that target a sequence of a gene associated with a point mutation in the gene product; ii) administering said vector to the cornea, The method, wherein said one or more nucleotide sequences are preferentially expressed in the cornea following said intracameral injection.

[0022] In another aspect, the invention provides a method of treating a corneal disease or condition caused by a mutant allele of a gene containing a trinucleotide repeat (TNR) and / or a point mutation in a subject in need thereof, the method comprising: a) excising at least a portion of a trinucleotide repeat (TNR) within said gene, i) providing an AAV5, AAV6, or AAV8 vector comprising one or more nucleotide sequences encoding one or more guide RNAs that target a sequence within the TNR, 5' of the TNR, 3' of the TNR, or a combination thereof; ii) administering said vector to the cornea; and / or b) correcting a point mutation in a gene or gene product, including: i) providing an AAV5, AAV6, or AAV8 vector comprising one or more nucleotide sequences encoding one or more guide RNAs that target a sequence of a gene associated with a point mutation in the gene product; ii) administering the vector to the cornea.

[0023] In another aspect, the present invention provides a method for downregulating expression of a corneal gene in a subject in need thereof, the method comprising administering to the subject: a) a nucleotide sequence of an AAV vector or a portion thereof; b) a nucleic acid capable of downregulating gene expression of at least one mutant allele on a target gene associated with a corneal disease or condition; c) administering the delivery system, including administering the delivery system to the cornea.

[0024] In another aspect, the present invention provides a method for preferentially expressing a protein in a subject in need thereof in endothelial cells of the subject's cornea, comprising: a) providing one or more adeno-associated (AAV) vectors comprising a nucleotide sequence encoding said protein or a portion thereof; b) administering an AAV vector to the cornea.

[0025] These and other objects, advantages and features of the present invention will become apparent to those skilled in the art upon reading the details of the compositions and methods as more fully described below. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 is a diagram of the layers of the cornea (see https: / / discoveryeye.org / treatment-corneal-scratches-and-abrasions / ). [Diagram 2] FIG. 2 is a diagram of the mouse eye structure and intracameral and intravitreal injections into the eye. [Diagram 3] Figure 3 shows in vivo images of mouse eyes following intracameral delivery of AAV5-eGFP. Panels A-D show images from the OD eye ("OD" refers to Oculus Dexter, Latin for right eye). Panels E-H show images from the OS eye ("OS" refers to Oculus Sinister, Latin for left eye). Panel A provides a reference for panel B. Panel E provides a reference for panel F. Panels B and F show images showing corneal fluorescence from AAV5-eGFP. Panels C and G show fundus images, and panels D and H show images demonstrating the absence of fluorescence in the retina. Two fluorescent dots are detected in the OS retina, indicated by the arrow in panel H. [Figure 4]Figure 4 shows immunohistochemistry of the same eye as in Figure 3. AAV5-eGFP was delivered by intracameral injection. OS eyes were separated into corneal flat mounts (panel A, magnified insert shown in panel B) and retinal flat mounts (panel C, magnified insert shown in panel D). Staining shows eGFP localized to the corneal endothelium and some cells staining in the retina. OD eyes were collected whole and processed with cross sections shown in panel E. Staining shows eGFP localized to the corneal endothelium but not the retina. Magnified inserts are shown in panels F and G. Panel F shows the corneal endothelial layer. Panel G shows the retina, where exposure times had to be increased to capture positive signals not seen in panel E. (green = endogenous eGFP, red = secondary staining using primary antibody against eGFP). [Diagram 5] Figure 5 shows in vivo images of a mouse eye following intracameral delivery of AAV6-eGFP. Panel A provides a reference for panel B, which shows an image showing corneal fluorescence from AAV6-eGFP. Panel C shows a fundus image, and panel D shows an image showing retinal fluorescence. [Figure 6] Figure 6 shows immunohistochemistry of the same mouse eye as in Figure 5. Staining shows that AAV6-eGFP is present in the corneal endothelium, stroma, and ciliary body (green = endogenous eGFP, red = secondary staining using primary antibody against eGFP, blue = DAPI stained nuclei). The white rectangle in panel A indicates the magnified area shown in panel B. The left arrow in panel B indicates the corneal stromal layer is positive. The right arrow in panel B indicates the positive corneal endothelial layer. [Figure 7] Figure 7 shows in vivo images of a mouse eye following intracameral delivery of AAV8-eGFP. Panel A provides a reference for Panel B, which shows an image showing corneal fluorescence from AAV8-eGFP. Panel C shows a fundus image, and Panel D shows an image showing retinal fluorescence. [Figure 8]Figure 8 shows immunohistochemistry of the same mouse eye as in Figure 7. Staining shows that AAV8-eGFP is present in the corneal endothelium, stroma, and ciliary body (green = endogenous eGFP, red = secondary staining using primary antibody against eGFP, blue = DAPI stained nuclei). The white rectangle in panel A indicates the magnified area shown in panel B. The left arrow in panel B indicates the corneal stromal layer is positive. The right arrow in panel B indicates the positive corneal endothelial layer. [Figure 9] Figure 9 shows ELISA results for eGFP protein levels from four mice (whole eyes) per AAV serotype, including AAV5-eGFP, AAV6-eGFP, and AAV8-eGFP, delivered via the intracameral route. Two mice receiving PBS + 0.001% pluronic acid were included as controls for each AAV serotype tested. Means with SEM are shown. [Figure 10] FIG. 10 is a composite showing in vivo fluorescence images and immunochemistry results of AAV2-eGFP, AAV5-eGFP, AAV6-eGFP, AAV8-eGFP, and AAV9-eGFP after IC delivery to mouse eyes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed.

[0028] The headings are for organizational purposes only and are not intended to limit the invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Unless otherwise stated, all patents, patent applications, published applications and publications, websites, and other published materials referenced throughout this disclosure are incorporated by reference in their entirety for all purposes.Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods are described.

[0030] Corneal dystrophies is a term for a heterogeneous group of non-inflammatory bilateral diseases confined to the cornea. They are grouped by the anatomical location of the pathology within the cornea. Most have no symptoms outside the cornea and cause corneal opacification, affecting vision (see https: / / www.cornealdystrophyfoundation.org / what-is-corneal-dystrophy).

[0031] Anterior corneal dystrophies affect the corneal epithelium and its basement membrane, and the superficial corneal stroma. Stromal dystrophies affect the corneal stroma. Posterior corneal dystrophies affect Descemet's membrane and the corneal endothelium. The most common posterior corneal dystrophies are Fuchs' endothelial dystrophy.

[0032] The cornea has three main areas affected by corneal dystrophies: the corneal epithelium, stroma, and endothelium. AAV5 targets the corneal endothelium after IC delivery and can be utilized to deliver gene therapy for posterior corneal dystrophies. Both AAV6 and AAV8 can target the corneal stroma, endothelium, and ciliary body after IC delivery and can be utilized to deliver gene therapy for corneal stromal dystrophies and posterior corneal dystrophies. Because some anterior corneal dystrophies affect both the epithelium and superficial corneal stroma, AAV6 and AAV8 may deliver gene therapy to the stroma.

[0033] Table D1 shows corneal dystrophies and the specific genes associated with them ((Klintworth, 2009. Corneal dystrophies. Orphanet J. Rare Dis., 4, 7. doi:10.1186 / 1750-1172-4-7). [Table 1-1] [Table 1-2]

[0034] Table D2 is from Moore, CBT, Christie, KA, Marshall, J., & Nesbit, MA (2018) Personalized genome editing - the future of corneal dystrophies. Prog Retin Eye Res, 65, 147-165. doi:10.1016 / j.preteyeres.2018.01.004. [Table 2-1] [Table 2-2]

[0035] For example, delivery of AAV directly to the eye by intracameral injection may result in viral targeting tropism to the cornea. Delivery of AAV5 via intracameral injection results in viral targeting to the corneal endothelium rather than other ocular structures. This targeting may deliver therapeutics to affected structures while sparing other ocular structures, reducing the risk of off-target effects. Intracameral delivery of AAV6 or AAV8 has also shown targeting to the corneal endothelium. However, both AAV6 and AAV8 show tropism to the retina as well as other corneal and anterior structures when using the ubiquitous CAG promoter to deliver genes.

[0036] AAV is a small virus that consists of two open reading frames, Rep and Cap, flanked by two 145-nt inverted terminal repeats (ITRs). When used for gene therapy, the Rep and Cap open reading frames are removed and a gene of interest is inserted between the ITRs along with a promoter that drives transcription of the gene of interest.

[0037] CRISPR nucleotides (e.g., nucleotides encoding gRNA and / or Cas proteins) can be packaged between the ITRs to create viral vectors for targeted delivery of therapeutic agents. In some embodiments, CRISPR nucleotides gRNA are packaged with Cas proteins (e.g., Cas9 nuclease) to form ribonucleoprotein (RNP) complexes. However, AAV can also be packaged with nucleotides encoding other proteins. AAV is a preferred viral vector because it can infect both dividing and non-dividing cells and is associated with a lack of pathogenicity.

[0038] Therefore, AAV vectors can be used to preferentially target specific layers of the cornea. For example, AAV5 specifically targets the corneal endothelium. The specificity of AAV vectors reduces the risk of off-target effects of therapeutics delivered via AAV vectors.

[0039] In certain embodiments, the AAV vector may contain one or more nucleotide sequences that are complementary to at least one target sequence on a target gene.

[0040] In some embodiments, the AAV vector can include one or more nucleotide editing systems, including, but not limited to, CRISPR systems, siRNA, shRNA, miRNA, antisense RNA, or antagomir RNA.

[0041] In certain embodiments, AAV vectors can be used for targeted gene editing or therapy in the eye, preferably the cornea or other affected anterior structures, by delivering one or more nucleotide editing systems directly to the eye.

[0042] In certain embodiments, AAV vectors can be used for targeted gene therapy in the cornea by delivering CRISPR complexes that target genes involved in corneal dystrophies, such as Fuchs endothelial corneal dystrophy (FECD). FECD is associated with an expansion of trinucleotide repeats (TNRs) in the transcription factor 4 (TCF4) gene. Most of the genetic predisposition to FECD is associated with the TNRs in the third intron of the TCF4 gene. FECD is a condition that affects the cornea of ​​the eye, particularly the endothelium. Corneal dystrophies are also associated with mutations in the COL8A gene. Mutations in the COL8A gene result in Gln455Lys, Gln455Val, or Leu450Trp mutations in the gene product.

[0043] By delivering a CRISPR complex (gRNA and Cas protein, or nucleotides encoding the Cas protein) to the corneal endothelium, the TNR, or a portion of it, can be excised from the TCF4 gene in the corneal endothelium without affecting the TCF4 gene in other parts of the eye.

[0044] In certain embodiments, the CRISPR complex is packaged into one or more AAV vectors.The CRISPR complex may target either the TNR of the TCF4 gene or the mutant allele of the COL8A2 gene.

[0045] In certain embodiments, the AAV vector can be delivered by itself, hi other embodiments, the AAV vector can be encapsulated in a lipid nanoparticle, a liposome, a non-lipid nanoparticle, or a viral capsid for delivery.

[0046] definition In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural as well, unless the context clearly indicates otherwise.

[0047] In this application, the use of "or" means "and / or" unless stated otherwise.

[0048] As used herein, the terms "comprises" and / or "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Furthermore, to the extent the terms "comprising," "having," "has," "with," "composed," "comprised," or variations thereof are used in the detailed description or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0049] As used herein, ranges and amounts may be expressed as "about" a particular value or range. "About" is intended to include the exact amount. Thus, "about 5 percent" also means "about 5 percent" and "5 percent." "About" means within typical experimental error for the intended use or purpose.

[0050] As used herein, "treatment" refers to the delivery, administration, or application of a therapeutic agent for a disease or condition. Treatment may include curing a disease, suppressing a disease, slowing or arresting the onset of a disease, ameliorating one or more symptoms of a disease, or preventing the recurrence of one or more symptoms of a disease.

[0051] As used herein, "FECD" refers to Fuchs endothelial corneal dystrophy. FECD includes patients with the condition as well as individuals who have no symptoms but are genetically predisposed to FECD.

[0052] As used herein, "AAV" refers to adeno-associated virus. AAV is an icosahedral, non-enveloped virus that is about 20-24 nm long, has a density of about 1.40-1.41 g / cc, and contains a single-stranded linear genomic DNA molecule about 4.7 kb in length. The single-stranded AAV genomic DNA is either positive or negative stranded. In certain embodiments, the term "AAV" or "AAV vector" refers to an AAV that has been modified such that a therapeutic agent, such as, for example, a CRISPR complex, replaces the Rep and Cap open reading frames between the inverted terminal repeats (ITRs) of the AAV genome.

[0053] As used herein, "AAV serotype" means a subdivision of AAV that is identifiable by serological or DNA sequencing methods and can be distinguished by its antigenicity.

[0054] As used herein, a "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Vectors include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. The term "vector" includes autonomously replicating plasmids or viruses. "Vector" can also include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid to cells, such as, for example, polylysine compounds, liposomes, lipid nanoparticles, non-lipid nanoparticles. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, lentiviral vectors, and the like. Preferably, the vector is an AAV vector.

[0055] As used herein, "RNA" refers to a molecule that contains one or more ribonucleotide residues. A "ribonucleotide" is a nucleotide that has a hydroxyl group at the 2' position of a beta-D-ribofuranose moiety. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), essentially pure RNA, synthetic RNA, and recombinantly produced RNA. The term "RNA" also refers to modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides, such as non-naturally occurring or chemically synthesized nucleotides or deoxynucleotides.

[0056] As used herein, "inhibitory RNA" refers to a nucleic acid molecule that contains a sequence complementary to a target nucleic acid that mediates a decrease in the level or activity of the target nucleic acid. Inhibitory RNA includes, but is not limited to, interfering RNA (iRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), ribozyme, antagomir, and antisense oligonucleotide.

[0057] As used herein, "shRNA" refers to an RNA molecule that comprises an antisense region, a loop portion, and a sense region, and the sense region has complementary nucleotides that base pair with the antisense region to form a double-stranded stem.Following post-transcriptional processing, shRNA is converted into siRNA by cleavage mediated by the enzyme Dicer, which is a member of the RNaseIII family.

[0058] As used herein, "siRNA" refers to any small RNA molecule capable of inhibiting or downregulating gene expression by mediating RNA interference in a sequence-specific manner.

[0059] As used herein, "antisense RNA" or "antisense oligonucleotide" is a short synthetic piece of nucleic acid whose sequence is complementary to the mRNA that codes for a protein. Antisense RNA binds to mRNA and blocks transcription.

[0060] As used herein, "antagomir" or "antagomir-RNA" refers to a small synthetic RNA that is complementary to a specific microRNA (miRNA) target and optionally with either mispairing at the cleavage site or one or more base modifications to inhibit cleavage.

[0061] As used herein, "microRNA" or "miRNA" refers to single-stranded RNA molecules approximately 21-23 nucleotides in length that regulate gene expression. miRNA molecules are partially complementary to one or more mRNAs, and their main function is to downregulate gene expression.

[0062] As used herein, "TNR" refers to a trinucleotide repeat (i.e., multiple repeats of three base pairs). The term "TNR expansion" refers to a number of TNRs that is higher than normal. For example, about 50 or more TNRs in intron 3 of TCF4 are considered to be TNR expansions.

[0063] As used herein, "CRISPR" refers to the bacterial adaptive immune system known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats).

[0064] As used herein, "guide RNA" and "gRNA" are used interchangeably and refer to an RNA sequence directed to a target DNA sequence. gRNAs include CRISPR RNA (crRNA) and trans-activating crRNA (trRNA or tracrRNA). The crRNA and trRNA may be combined into a single RNA molecule called single guide RNA (sgRNA). Alternatively, the crRNA and trRNA may be separated in separate RNA molecules to form a dual guide RNA (dgRNA). In some embodiments, the gRNA is chemically modified to include one or more modified nucleosides or nucleotides. Modifications of nucleosides and nucleotides can include one or more of: i) alteration, e.g., substitution, of one or both of the non-linked and / or linked phosphate oxygens in the phosphodiester backbone; ii) modification, e.g., substitution, of a component of the ribose sugar, such as, for example, the 2'-hydroxyl on the ribose sugar; iii) complete replacement of the phosphate moiety with a "dephosphorylated" linker; iv) modification or substitution of naturally occurring nucleobases, including non-standard nucleobases; v) substitution or modification of the ribose-phosphate backbone; vi) modification of the 3' or 5' end of the oligonucleotide, e.g., removal, modification, or substitution of a terminal phosphate group, or conjugation of a moiety, cap, or linker; and vii) modification or substitution of the sugar.

[0065] As used herein, "guide sequence" refers to a sequence of about 20 base pairs within a crRNA or trRNA that is complementary to a target sequence. The guide sequence directs the gRNA to the target sequence for cleavage by a nuclease.

[0066] As used herein, "target sequence" refers to a sequence of nucleic acid in a subject's genomic DNA to which a gRNA directs a nuclease for cleavage of the target sequence. For example, a Cas protein is directed by a gRNA to a target sequence, where the gRNA hybridizes to the target sequence and the nuclease cleaves the target sequence. The target sequence includes both the +strand and -strand of DNA (i.e., the sequence and the reverse complement of the sequence). In some embodiments, when the guide sequence is the reverse complement of the target sequence, the guide sequence may be identical to the first 20 nucleotides of the target sequence. As used herein, "target sequence" or "target site" also refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0067] As used herein, the term "CRISPR complex" refers to a combination of a gRNA and an endonucleotide encoding a Cas protein (gRNA:Cas endonucleotide) or a combination of a gRNA and a Cas protein (gRNA:Cas protein). As used herein, "ribonucleoprotein" (RNP) refers to a gRNA:Cas protein complex. The CRISPR complex of the present invention can be directed to and cleave a target sequence within or adjacent to the TNR (5' or 3') of the TCF4 gene. The CRISPR complex can also be directed to cleave a target sequence in the COL8A gene. As used herein, "protospacer adjacent motif" or "PAM" refers to a nucleotide sequence that must be adjacent to the target nucleotide sequence. The required PAM depends on the particular CRISPR system used. For example, the CRISPR / Cas system from Streptococcus pyogenes requires that the target DNA be immediately preceded by a 5'-NGG PAM (where "N" is any nucleobase followed by two guanine nucleobases) for optimal cleavage. Streptococcus pyogenes Cas9 also recognizes 5'-NAG PAMs, but appears to be less efficient at these PAM sites. Other Cas9 orthologues (e.g., from Staphylococcus aureus) require different PAM sequences.

[0068] As used herein, "indel" refers to an insertion / deletion mutation consisting of a number of nucleotides that are either inserted or deleted at the site of a nucleic acid double strand break (DSB) in DNA.

[0069] As used herein, "excision fragment" or "excision fragment" refers to the deletion of a contiguous number of nucleotides (such as a TNR) that can occur when two or more gRNAs are used together with a Cas mRNA or Cas protein.

[0070] As used herein, "promoter" refers to a DNA sequence that is recognized by the synthetic machinery of a cell or recognized by the synthetic machinery introduced, which is required to initiate specific transcription of a polynucleotide sequence. Preferably, it is a promoter operable for an AAV vector, preferably AAV5, AAV6, and / or AAV8, and a tissue-specific promoter, preferably specific to the eye, more preferably specific to the cornea, and most preferably specific to the endothelium of the cornea. AAV promoters include, for example, the AAV p5 promoter. Promoters include, but are not limited to, CAG, SYN1, CMV, NSE, CBA, PDGF, SV40, RSV, LTR, SV40, dihydrofolate reductase promoter, beta actin promoter, PGK, EF1alpha, GRK, MT, MMTV, TY, RU486, RHO, RHOK, CBA, chimeric CMV-CBA, MLP, RSV, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, and the like. AAV in which heterologous DNA is packaged can use a promoter normally associated with the heterologous nucleic acid, or can use a promoter normally associated with the AAV vector, or can use a promoter that is not normally associated with either.

[0071] As used herein, a "constitutive promoter" is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. Examples of constitutive promoters include, but are not limited to, cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Ruth sarcoma virus (RSV) promoter, elongation factor-alpha (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, or combinations thereof.

[0072] As used herein, an "inducible promoter" is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell. Examples of inducible promoters include, but are not limited to, those that are inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the promoter may be tissue-specific, such as a promoter specific for expression in the cornea, e.g., the corneal endothelium.

[0073] As used herein, a "tissue-specific promoter" is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. Tissue-specific promoters include, but are not limited to, CMV, CBA, RHO, and RHOK.

[0074] As used herein, "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. This sequence may be the core promoter sequence, or it may also include enhancer sequences and other regulatory elements required for expression of the gene product.

[0075] As used herein, "under transcriptional control" or "operably linked" means that a promoter is in the correct position and orientation relative to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide. These include the promoter, the 3'UTR, or the 5'UTR. The promoter may be recognized by RNA polymerase III (Pol III), such as, but not limited to, U6 and H1 Pol III promoters. The Pol III promoter may be, for example, mouse or human.

[0076] As used herein, "gene editing" or "nucleic acid editing" refers to the modification of the nucleic acid sequence of a target gene.

[0077] As used herein, "nucleic acid editing system" or "gene editing system" refers to the method that can be used to perform gene editing or nucleic acid editing.Nucleic acid editing system and gene editing system include CRISPR system and interfering RNA.

[0078] As used herein, "delivery system" refers to a material used to deliver a nucleic acid to a target cell. Such materials may include a viral vector, such as an AAV vector, and a pharma- ceutically acceptable component.

[0079] As used herein, "modulation" or "modification" includes, for example, a decrease or inhibition of expression or function of a gene or protein, as well as, for example, an increase in expression or function of a gene or protein. As used herein, "modulation" or "modification" also includes complete restoration of gene function, including replacement of a mutated portion(s) of a gene or replacement of a mutated gene with a wild-type version.

[0080] As used herein, "downregulate" or "downregulation" refers to a decrease in the expression or transcription of a target nucleotide sequence. Downregulation can be a partial or temporary decrease in the expression or transcription of a target nucleotide sequence. Downregulation can be a complete elimination of the expression or transcription of a target nucleotide sequence.

[0081] As used herein, "knockdown" refers to partial or temporary reduction in the expression or transcription of a target nucleotide sequence.This can be achieved by administering a complementary nucleotide sequence that binds to the target sequence.Knockdown can be induced by antisense oligonucleotides, siRNA, etc.

[0082] As used herein, "knockout" refers to the complete elimination of expression or transcription of a target nucleotide sequence. Knockout can be induced, for example, by using the CRISPR system to cut the target nucleotide sequence from the target gene.

[0083] As used herein, non-homologous end joining (NHEJ) is a DNA repair mechanism that is the rejoining of broken ends following cleavage of a target nucleotide sequence.

[0084] As used herein, "homologous repair / homology-directed repair (HR / HDR)" refers to DNA repair, a process of homologous recombination in which a DNA template is used to provide the homology required for accurate repair of double-strand breaks. Repair can consist of inserting a desired sequence or modifying a target sequence.

[0085] As used herein, "repair template" refers to the DNA template used in HR / HDR.

[0086] As used herein, "subject" refers to a living organism. Preferably, the subject is a mammal, such as a human, non-human primate, rodent, or companion animal, such as a dog, cat, cow, pig, etc.

[0087] Regulation of gene expression Gene expression can be regulated by administering to a subject in need thereof a composition comprising a nucleotide editing system.

[0088] In one embodiment, regulating the expression of the target gene comprises administering a composition to the subject, the composition comprises a nucleic acid editing system comprising at least one nucleotide sequence that is complementary to at least one allele on the target gene associated with corneal dystrophy.In certain embodiments, the at least one nucleotide sequence that is complementary to at least one allele on the target gene is selected from siRNA, shRNA, miRNA, antisense RNA, or antagomir RNA.

[0089] Administration of the Composition In certain embodiments, the composition is administered by itself.

[0090] In a preferred embodiment, the composition comprises an adeno-associated virus (AAV) vector, or a nucleotide sequence encoding an AAV vector, or a portion thereof.

[0091] AAV Adeno-associated viruses (AAV) are small, replication-deficient parvoviruses. AAVs are approximately 20–24 nm long and have a density of approximately 1.40–1.41 g / cc. AAVs contain a single-stranded linear genomic DNA molecule approximately 4.7 kb in length. The single-stranded AAV genomic DNA can be either positive- or negative-stranded. AAVs contain two open reading frames, Rep and Cap, flanked by two 145-base inverted terminal repeats (ITRs). AAVs contain a single intron. Cis-acting sequences that direct viral DNA replication (Rep), encapsidation / packaging, and integration into host cell chromosomes are contained within the ITRs. Three AAV promoters, p5, p19, and p40 (named for their relative map positions), drive expression of two AAV internal open reading frames that encode the rep and cap genes. p5 and p19 are rep promoters. Two rep promoters, combined with alternative splicing of a single AAV intron, result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The rep proteins have multiple enzymatic properties involved in the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production of the three related capsid proteins. A single polyadenylation site is located at map position 95 of the AAV genome. Muzyczka has reviewed the life cycle and genetics of AAV (Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992)).

[0092] AAV infection is noncytopathic in cultured cells. Natural infection in humans and other animals is asymptomatic and subclinical (does not cause disease). AAV infects many mammalian cells and therefore has the potential to target many different tissues in vivo. In addition to dividing cells, AAV can transduce slowly dividing and nondividing cells and persist essentially for the life of those cells as transcriptionally active nuclear episomes (i.e., extrachromosomal elements). The AAV proviral genome is infectious as DNA cloned into a plasmid, allowing the construction of recombinant genomes. Furthermore, signals directing AAV replication, genome encapsidation, and integration are all contained within the ITRs of the AAV genome, so that part or all of the approximately 4.3 kb of genome encoding the replication and structural capsid proteins (rep-cap) can be replaced with heterologous DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans.

[0093] Several AAV serotypes have been identified, which differ in their tropism (type of cells they infect). Serotype AAV1 has tropism for CNS, heart, retinal pigment epithelium (RPE), and skeletal muscle tissues. Serotype AAV2 has tropism for CNS, kidney, photoreceptor cells, and RPE tissues. Serotype AAV3 has tropism mainly for heart and liver. Serotype AAV4 has tropism for CNS, lung, and RPE tissues. Serotype AAV5 has tropism for CNS, lung, photoreceptor cells, and RPE tissues. Serotype AAV6 has tropism for lung and skeletal muscle tissues. Serotype AAV7 has tropism for liver and skeletal muscle tissues. Serotype AAV8 has tropism for CNS, heart, liver, pancreas, photoreceptor cells, RPE, and skeletal muscle tissues. Serotype AAV9 has tropism for CNS, heart, liver, lung, and skeletal muscle tissues. The tropism of the AAV virus may be related to the variability in the amino acid sequence of the capsid protein, which may bind to different functional receptors present on different types of cells.

[0094] Depending on the promoter contained in the heterologous DNA cassette, it may be possible to target specific tissues of the eye.Modifying capsid protein may also allow specific infectivity of specific tissues or cells.In one embodiment, AAV containing Anc80 or Anc80L65 capsid protein is used to deliver therapeutic agents directly to specific tissues of the eye. In some embodiments, the AAV viral particle is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., a wild-type AAV6 capsid or a variant AAV6 capsid such as ShH10, as described in USPG Pub. 2012 / 0164106), AAV7, AAV8, AAVrh8, AAVrh8R, AAV9 (e.g., a wild-type AAV9 capsid or a modified AAV9 capsid as described in USPG Pub. 2013 / 0323226), AAV10, AAVrh10, AAV11, AAV12, a tyrosine capsid mutant, a heparin-binding capsid mutant, an AAV2R471A capsid, an AAVAAV2 / 2-7m8 capsid, an AAV including a DJ capsid (e.g., an AAV-DJ / 8 capsid, an AAV-DJ / 9 capsid, or any other capsid described in USPG Pub. 2012 / 0066783), an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a murine AAV capsid, a rAAV2 / HBoV1 capsid, or an AAV capsid described in U.S. Pat. No. 8,283,151 or International Publication No. WO / 2003 / 042397. In some embodiments, the AAV viral particle comprises an AAV capsid that includes an amino acid substitution at one or more of positions R484, R487, K527, K532, R585, or R588, numbered based on VP1 of AAV2. In further embodiments, the AAV particle comprises capsid proteins of an AAV serotype from classes A through F. In some embodiments, the rAAV viral particle comprises an AAV serotype 2 capsid.In further embodiments, the AAV serotype 2 capsid comprises an AAV2 capsid protein comprising an R471A amino acid substitution, numbered relative to AAV2 VP1. In some embodiments, the vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, caprine AAV, bovine AAV, or mouse AAV serotype inverted terminal repeat (ITR). In some embodiments, the vector comprises an AAV serotype 2 ITR. In some embodiments, the AAV viral particle comprises one or more ITRs and capsid from the same AAV serotype. In other embodiments, the AAV viral particle comprises one or more ITRs from an AAV serotype different from the capsid of the rAAV viral particle. In some embodiments, the rAAV viral particle comprises an AAV2 capsid and the vector comprises the AAV2 ITRs. In further embodiments, the AAV2 capsid comprises an AAV2 capsid protein that comprises an R471A amino acid substitution, numbered relative to AAV2 VP1 (see U.S. Patent Publication No. 2017 / 0304465).

[0095] Recently, inclusion of the human rhodopsin kinase (hGRK1) promoter in an AAV5 vector has been shown to result in rod- and cone-specific expression in the primate retina (Boye, et al., Human Gene Therapy, 23:1101-1115 (October 2012) (DOI: 10.1089 / hum.2012.125)).

[0096] Recently, it has also been shown that AAV virions with altered capsid proteins may confer greater tissue-specific infectivity. For example, AAV6 containing a variant capsid protein shows increased infectivity of retinal cells compared to the wild-type AAV capsid protein (U.S. Pat. No. 8,663,624). A variant capsid protein containing a peptide insertion between two adjacent amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2, or the corresponding positions of the capsid protein of another AAV serotype, results in increased infectivity of retinal cells compared to wild-type AAV (U.S. Pat. No. 9,193,956).

[0097] Protein expression in the cornea To express a specific protein in the cornea, an AAV vector packaged with either an endonucleoside encoding a desired protein or an AAV vector packaged with a desired protein can be delivered or administered directly to the eye. Proteins can include, for example, CRISPR-associated (Cas) proteins, or marker proteins (e.g., green fluorescent protein (GFP or eGFP)).

[0098] In certain embodiments, the AAV vector can be delivered without being encapsulated in a particle or lipid vesicle. In other embodiments, the AAV vector can be encapsulated in a lipid nanoparticle, a liposome, a non-lipid nanoparticle, or a viral capsid for delivery.

[0099] In some embodiments, the composition and / or AAV vector can be delivered directly to the eye.The composition and / or AAV vector can be administered to the anterior chamber, the posterior chamber, the cornea, or the vitreous chamber of the eye.In a preferred embodiment, the AAV vector is administered directly to the aqueous humor of the anterior chamber that is in contact with the cornea.As defined in the present invention, more than one AAV vector can be used for the purpose of regulating gene expression, such as a dual AAV vector system.

[0100] As described above, there are several AAV serotypes, each of which has a tropism for a specific type of tissue.The AAV serotype used is not particularly limited, but AAV5, AAV6 and AAV8 serotypes are the preferred AAV vectors for targeting the cornea and anterior tissue of the eye.

[0101] To test the viral tropism of different AAV serotypes in the present invention, several serotypes were packaged with nucleotide sequences encoding green fluorescent protein (GFP or eGFP). These AAV-eGFP complexes were delivered to the eye in the anterior chamber. GFP fluorescence can be measured in vivo, indicating the localization of AAV-GFP. GFP localization can also be evaluated by performing immunohistochemistry on ocular sections. As shown by immunohistochemical staining, the viral tropism of AAV5 was localized to the corneal endothelium. The viral tropism of AAV6 was localized to the corneal endothelium, stroma and endothelium, and ciliary body, and some targeted retinal cells. The viral tropism of AAV8 was localized to the corneal endothelium and stroma, and ciliary body, and some targeted retinal cells. The viral tropism of AAV2 and AAV9 was localized to both the posterior and anterior segments of the eye after IC administration, and was more expressed in the posterior segment than the anterior segment.

[0102] The results show that AAV5, AAV6, and AAV8 exhibit selective tropism for corneal tissue. If selective targeting to the corneal endothelium is desired, the use of AAV5 is preferred. The use of other AAV serotypes with less tissue selectivity (e.g., AAV2) may result in undesirable off-target effects.

[0103] In certain embodiments, the AAV vector can be delivered by itself, hi other embodiments, the AAV vector can be encapsulated in a lipid nanoparticle, a liposome, a non-lipid nanoparticle, or a viral capsid for delivery.

[0104] In some embodiments, AAV vector can be delivered directly to eye.AAV vector can be administered to anterior chamber, posterior chamber, cornea or vitreous chamber of eye.In certain embodiments, AAV vector can be administered to corneal stroma, corneal limbus, corneal epithelial surface or corneal endothelial membrane.In a preferred embodiment, AAV vector is administered directly to the aqueous humor of anterior chamber, which is in direct contact with corneal endothelium.

[0105] Gene targeting using CRISPR complexes In certain embodiments, the CRISPR complex is used to modify a specific nucleotide sequence of a gene's DNA. The specific nucleotide sequence of a gene's DNA is the "target sequence."

[0106] The CRISPR complex is a combination of a gRNA and an endonuclease that encodes a Cas protein (gRNA:Cas endonuclease) or a combination of a gRNA and a Cas protein (gRNA:Cas protein).

[0107] gRNA contains an RNA sequence directed to a target DNA sequence. gRNA includes CRISPR RNA (crRNA) and trans-activating crRNA (trRNA or tracrRNA). The crRNA and trRNA may be combined into a single RNA molecule called single guide RNA (sgRNA). Alternatively, the crRNA and trRNA may be separated in separate RNA molecules to form a dual guide RNA (dgRNA). gRNA can target either the + or - strand of DNA.

[0108] The gRNA guides the Cas component (i.e., the endonuclease encoding the Cas protein or the Cas protein) to the target sequence. The gRNA is complementary to and hybridizes with the target sequence, or the reverse complement of the target sequence. In some embodiments, the gRNA sequence is 100% complementary or identical to the target sequence. Preferably, the degree of complementarity or identity between the guide sequence of the gRNA and its corresponding target sequence is at least about 50% or more. For example, the degree of complementarity or identity can be about 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0109] In some embodiments, the gRNA is chemically modified and contains one or more modified nucleosides or nucleotides. The nucleoside and nucleotide modifications can include one or more of: i) modification, e.g., substitution, of one or both of the non-linking phosphate oxygens in the phosphodiester backbone (e.g., phosphorothioate or borane phosphate linkages) and / or of the linking phosphate oxygens; ii) modification, e.g., substitution, of the components of the ribose sugar, e.g., 2'-O-methyl and / or 2'-fluoro and / or 4-thio modifications; iii) complete replacement of the phosphate moiety with a "dephospho" linker; iv) modification or replacement of naturally occurring nucleobases, including non-standard nucleobases; v) substitution or modification of the ribose-phosphate backbone; vi) modification of the 3' or 5' end of the oligonucleotide, e.g., removal, modification, or replacement of the terminal phosphate group, or conjugation of a moiety, cap, or linker; vii) modification or replacement of the sugar; and viii) locked or unlocked nucleic acids. Other modifications include pseudouridine, 2-thiouridine, 4-thiouridine, 5-azacytidine, 5-hydroxyuridine, 5-aminouridine 5-methyluridine, 2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine 5-methylcytidine N-4-methycytidine, 2-thiocytidine, 5-azacytidine 5-hydroxycytidine, 5-aminocytidine , N-4-methylpseudouridine, 2-thiopseudoisocytidine, 5-hydroxypseudouridine, 5-aminopseudouridine, 5-methylpseudoisocytidine, N-6-methyladenosine, 7-deazaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.

[0110] In some embodiments, the Cas component comprises a type I, type II, or type III component. In certain embodiments, the Cas component is a nuclease. In some embodiments, the Cas nuclease is Cas9 or Cpf1. Preferably, the Cas nuclease is Cas9. In some embodiments, the gene editing molecule is a Cas protein (e.g., Cpf1, CasX, CasY, C2C2, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Casl0d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (Cas A), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, or homologs or modified versions thereof. In some embodiments, the Cas protein is a Cas9 protein (e.g., wild-type Cas9, Cas9 nickase, dead Cas9 (dCas9), or split Cas9). In some embodiments, the Cas9 protein is a Streptococcus pyogenes Cas9 protein or a Staphylococcus aureus Cas9 protein.

[0111] When guided to a target sequence, the Cas nuclease cleaves the target sequence. This generates a double-stranded break in DNA, or a single-stranded break if a nickase enzyme is used. The double-stranded break in DNA can be repaired via non-homologous end joining (NHEJ), which is the religation of the broken ends. NHEJ can generate indel mutations. Alternatively, DNA can be repaired via homology-directed repair (HR) or homology-directed repair (HDR). HR and HDR generate precisely defined alterations at the target locus in the presence of an exogenously introduced repair template. In certain embodiments, the repair template comprises a nucleotide sequence encoding the desired mutation on the target gene, and the nucleotide sequence is inserted into the target locus of the gene.

[0112] Some of the sequences disclosed herein include the following list (see International Publication No. WO 2017 / 185054): SEQ ID NOs: 1-93 are target sequences on the 5' side of the TNR in intron 3 of the TCF4 gene. SEQ ID NOs: 94-190 are target sequences on the 3' side of the TNR in intron 3 of the TCF4 gene. SEQ ID NOs: 191-1063 are target sequences of the wild-type COL8A2 gene. SEQ ID NOs: 1064-1069 are target sequences of the COL8A2Gln455Lys mutation. SEQ ID NOs: 1070-1075 are target sequences of the COL8A2Gln455al mutation. SEQ ID NOs: 1076-1084 are target sequences of the COL8A2Leu450Trp mutation.

[0113] Table A shows sequences SEQ ID NOs: 1085-1088 (see exemplary sequences in International Publication No. WO 2017 / 185054).

[0114] Guide RNA and Cas components (i.e., CRISPR complex) are packaged in AAV vector for delivery to subject.In certain embodiments, AAV vector can be delivered by itself.In other embodiments, AAV vector can be encapsulated in lipid nanoparticle, liposome, non-lipid nanoparticle or viral capsid for delivery.

[0115] In some embodiments, AAV vector can be delivered directly to eye.AAV vector can be administered to anterior chamber, posterior chamber, cornea or vitreous chamber of eye.In certain embodiments, AAV vector can be administered to corneal stroma, corneal limbus, corneal epithelial surface or corneal endothelial membrane.In a preferred embodiment, AAV vector is administered directly to the aqueous humor of anterior chamber, which is in direct contact with corneal endothelium.

[0116] TCF4 gene targeting The TCF4 gene is located on chromosome 18. The cytogenetic locus is 18q21.2 (the long arm of chromosome 18 at position 21.2). The molecular location is from base pairs 55,222,311 to 55,635,993 on chromosome 18 (Homo sapiens Annotation Release 109,GRCh38.p12 (NCBI)).

[0117] The target sequence may be within or adjacent to the TNR of the TCF4 gene. The Cas nuclease is guided to the target sequence. In some embodiments, the Cas nuclease may be guided to a target sequence within the TNR of the TCF4 gene. In other embodiments, the Cas nuclease may be guided to a target sequence adjacent to the TNR. For example, the Cas nuclease may be directed to a target sequence 5' of the TNR. Or, the Cas nuclease may be directed to a target sequence 3' of the TNR. In some embodiments, the Cas protein may be directed to two target sequences adjacent to the TNR by two or more gRNAs. In some embodiments, the Cas nuclease may be directed to two target sequences by two or more gRNAs, where one is within the TNR of the TCF4 gene and the other is adjacent to the TNR of the TCF4 gene. The target sequence of the TCF4 gene is selected from SEQ ID NOs: 1-190. SEQ ID NOs: 1 to 93 are target sequences on the 5' side of the TNR in intron 3 of the TCF4 gene. SEQ ID NOs: 94 to 190 are target sequences on the 3' side of the TNR in intron 3 of the TCF4 gene. The guide sequences of the TCF4 gene are selected from SEQ ID NOs: 1089 to 1278. (See sequence listing)

[0118] One or more gRNAs comprise a guide sequence that is complementary to a target sequence of the TCF4 gene, or a reverse complement of a target sequence of the TCF4 gene. In some embodiments, the gRNA sequence is 100% complementary or identical to the target sequence. Preferably, the degree of complementarity or identity between the guide sequence of the gRNA and its corresponding target sequence is at least about 50% or more. For example, the degree of complementarity or identity can be about 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0119] In some embodiments, one gRNA is used. In other embodiments, a combination of two or more gRNAs is used. In certain embodiments, to excise the TNR of the TCF4 gene, a gRNA targeting the sequence 5' of the TNR is used in combination with a gRNA targeting the sequence 3' of the TNR. In some embodiments, a gRNA complementary to a target sequence selected from SEQ ID NOs: 1-93 is used with a gRNA complementary to a target sequence selected from SEQ ID NOs: 94-190. Table 1 shows the target sequences and corresponding guide sequences (from Example 1 of WO 2017 / 185054). Table 2 shows the combination of guide sequences (from Example 1 of WO 2017 / 185054). (See exemplary sequences in WO 2017 / 185054)

[0120] In embodiments in which the CRISPR complex comprises an endonucleotide encoding a protein, the endonucleotide may be operably linked to one or more transcriptional or translational control sequences. In certain embodiments, the endonucleotide is operably linked to one or more promoters. The promoter may be constitutive, inducible, or tissue-specific. Examples of constitutive promoters include, but are not limited to, cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Ruth sarcoma virus (RSV) promoter, elongation factor-alpha (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, or combinations thereof. Examples of inducible promoters include, but are not limited to, those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the promoter may be tissue-specific, such as a promoter specific for expression in the cornea, e.g., corneal endothelium.

[0121] In some embodiments, the nucleotide sequence encoding the gRNA can be operably linked to at least one transcription or translation control sequence. These include a promoter, a 3'UTR, or a 5'UTR. The promoter can be recognized by RNA polymerase III (Pol III), such as, but not limited to, U6 and H1 Pol III promoters. The Pol III promoter can be, for example, mouse or human.

[0122] In certain embodiments, one or more gRNAs are packaged in AAV vector in combination with any endonucleotide sequence that codes for Cas protein or Cas protein (e.g., Cas9) (i.e., CRISPR complex).The AAV serotype used is not particularly limited.Preferably, the AAV vector is of AAV5, AAV6 or AAV8 serotype.

[0123] AAV-CRISPR complex can be directly delivered to eye via intracameral or intraparenchymal injection.In certain embodiments, AAV vector can be delivered by itself.In other embodiments, AAV vector can be encapsulated in lipid nanoparticles, liposomes, non-lipid nanoparticles, or viral capsids for delivery.

[0124] In some embodiments, AAV vector can be delivered directly to eye.AAV vector can be administered to anterior chamber, posterior chamber, cornea or vitreous chamber of eye.In certain embodiments, AAV vector can be administered to corneal stroma, corneal limbus, corneal epithelial surface or corneal endothelial membrane.In a preferred embodiment, AAV vector is administered directly to the aqueous humor of anterior chamber, which is in direct contact with corneal endothelium.

[0125] COL8A2 gene targeting Mutations in the COL8A2 gene, and therefore in the gene product, can also be treated with the methods and compositions described herein by developing CRISPR complexes that target specific sequences in the COL8A2 gene that cause the mutation.

[0126] In some embodiments, the CRISPR complex can be used to excise the target mutant nucleotide sequence on the COL8A2 gene, excising the nucleotide sequence of the DNA encoding the mutant gene product. The DNA can then be repaired in a process of NHEJ, leading to the generation of indels and loss of the mutant allele. In other embodiments, the use of the CRISPR complex can be performed with either an exogenous template for HR / HDR, or an endogenous normal allele as a template for HR / HDR, resulting in the correction of the nucleic acid mutation that results in an amino acid mutation in the alpha2 subunit of COL8. Mutations that can be corrected include the Gln455Lys mutation caused by the c.1364C>A nucleotide change, the Gln455Val mutation caused by the c.1363-1364CA>GT nucleotide change, or the Leu450Trp mutation caused by the c.1349T>G nucleotide change.

[0127] The target sequence of the COL8A2 gene can be selected using the NCBI reference sequence NM_005202.3 of transcript variant 1 of the COL8A2 gene. This sequence does not contain mutations at positions 455 and 450 of the amino acid sequence of the COL8 gene product and can be considered as the "wild type" COL8A2 gene sequence. The target sequence can be selected from Chr1:36097532-36100270 (hg38). The target sequence of the COL8A2 gene can be selected from SEQ ID NO: 191-1063. The target sequences of the wild type COL8A2 gene are shown in Table 3. Guide sequences complementary to these target sequences can be developed to target the COL8A2 gene.

[0128] Target sequences for mutant alleles can also be developed based on the differences in the nucleotide sequences of the mutant alleles. Table 4 shows target sequences specific for the Gln155Lys mutation caused by the c.1364C>A nucleotide change (SEQ ID NO: 1064-1069). Table 5 shows target sequences specific for the Gln455Val mutation caused by the c.1363-1364CA>GT nucleotide change (SEQ ID NO: 1070-1075). Table 6 shows target sequences specific for the Leu450Trp mutation caused by the c.1349T>G nucleotide change (SEQ ID NO: 1076-1084). Mutant alleles can be targeted using gRNAs that contain a guide sequence complementary to the target sequence or a guide sequence complementary to the reverse complement of the target sequence.

[0129] In certain embodiments, one or more gRNAs are packaged in AAV vector in combination with either the endonucleotide sequence encoding Cas protein or Cas protein (e.g., Cas9) (i.e., CRISPR complex).The AAV serotype used is not particularly limited.Preferably, the AAV vector is of AAV5, AAV6 or AAV8 serotype.

[0130] AAV-CRISPR complex can be directly delivered to eye via intracameral or intraparenchymal injection.In certain embodiments, AAV vector can be delivered by itself.In other embodiments, AAV vector can be encapsulated in lipid nanoparticles, liposomes, non-lipid nanoparticles, or viral capsids for delivery.

[0131] In some embodiments, AAV vector can be delivered directly to eye.AAV vector can be administered to anterior chamber, posterior chamber or cornea.In certain embodiments, AAV vector can be administered to corneal stroma, corneal limbus, corneal epithelial surface or corneal endothelial membrane.In a preferred embodiment, AAV vector is administered directly to the aqueous humor of anterior chamber, which is in direct contact with corneal endothelium.

[0132] Exemplary embodiments In certain embodiments, the mutant allele is encoded by a target sequence on the target gene.

[0133] In some embodiments, at least one nucleotide sequence that is complementary to at least one mutant allele on the target gene hybridizes to a target sequence on the target gene in a cell of a subject.

[0134] In a specific embodiment, the target gene is TCF4 or COL8A2.

[0135] In some embodiments, at least one target sequence is selected from the group consisting of SEQ ID NOs: 1-1084.

[0136] In some embodiments, at least one target sequence is specific to the TCF4 gene, and the target sequence is selected from SEQ ID NOs: 1-190.

[0137] In some embodiments, the target sequence is specific to the COL8A2 gene, and the target sequence is selected from SEQ ID NOs: 191-1084.

[0138] In some embodiments, the nucleic acid editing system is a CRISPR system, siRNA, shRNA, miRNA, antisense RNA, or antagomir RNA.

[0139] In some embodiments, the nucleic acid editing system is a CRISPR system.

[0140] In some embodiments, the nucleic acid editing system is a CRISPR-Cas system.

[0141] In some embodiments, the CRISPR-Cas system comprises a nucleotide sequence encoding a CRISPR-associated (Cas) gene and a nucleotide sequence encoding a guide RNA (gRNA).

[0142] In some embodiments, the Cas gene encodes a Cas protein.

[0143] In some embodiments, the Cas protein encoded by the Cas gene is a Cas nuclease.

[0144] In some embodiments, the Cas nuclease is Cas9.

[0145] In some embodiments, guide RNAs include CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA or trRNA).

[0146] In some embodiments, the guide RNA is a single guide RNA (sgRNA), where both the crRNA and the tracrRNA are combined on one guide RNA molecule.

[0147] In some embodiments, the guide RNA is a double guide RNA (dgRNA), where the crRNA and tracrRNA are on separate RNA molecules that are used simultaneously but are not combined.

[0148] In some embodiments, the CRISPR-Cas system is a CRISPR-Cas9 system.

[0149] In some embodiments, the crRNA and tracrRNA form a complex with a nucleotide sequence encoding a Cas9 nuclease.

[0150] In some embodiments, the nucleotide sequence complementary to at least one mutant allele is a gRNA.

[0151] In some embodiments, at least one guide RNA comprises a crRNA sequence that is complementary to at least one target sequence selected from SEQ ID NOs: 1-1084.

[0152] In some embodiments, at least one guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 1089-1278.

[0153] In some embodiments, the delivery system, vector, gene editing system, or composition further comprises a repair template.

[0154] In some embodiments, the repair template is selected from the group consisting of a DNA repair template, an mRNA repair template, an siRNA repair template, an miRNA repair template, and an antisense oligonucleotide repair template.

[0155] In some embodiments, the AAV vector serotype is selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrhlO.

[0156] In some embodiments, the AAV vector serotype is AAV5, AAV6, or AAV8.

[0157] In some embodiments, the AAV vector serotype is AAV5.

[0158] In some embodiments, the AAV vector serotype is AAV6.

[0159] In some embodiments, the AAV vector serotype is AAV8.

[0160] In some embodiments, the delivery system, vector, nucleotide or gene editing system, or composition further comprises a promoter.

[0161] In some embodiments, the promoter is optimized for use with an AAV5, AAV6 or AAV8 vector.

[0162] In some embodiments, the promoter is tissue-specific and active in the eye when operably linked to nucleotides whose sequence is complementary to at least one mutant allele on the AAV vector or target gene.

[0163] In some embodiments, the tissue-specific promoter is active in the cornea or other anterior ocular tissue.

[0164] In some embodiments, the tissue-specific promoter is active in the corneal endothelium.

[0165] In some embodiments, the target gene is preferentially expressed in the anterior segment of the eye. Preferably, the target gene is preferentially expressed in the cornea, and most preferably, in the endothelium of the cornea.

[0166] In some embodiments, the delivery system, vector, nucleotide or gene editing system, or composition is preferentially expressed in the anterior part of the eye after IC injection.Preferably, the delivery system, vector, nucleotide or gene editing system, or composition is preferentially expressed in the cornea after IC injection, and most preferably, in the corneal endothelium.

[0167] In some embodiments, the delivery system, vector, nucleotide or gene editing system, or composition is suitable for treating an ocular disease or condition.

[0168] In some embodiments, the ocular disease or condition is a corneal disease or condition.

[0169] In some embodiments, the corneal disease or condition is a superficial corneal dystrophy, anterior corneal dystrophy, corneal stromal dystrophy, or posterior corneal dystrophy.

[0170] In some embodiments, the corneal disease or condition is a posterior corneal dystrophy.

[0171] In some embodiments, the posterior corneal dystrophies are Fuchs endothelial corneal dystrophy (FECD; both early and late onset), posterior polymorphous dystrophy (PPCD; types 1, 2, and 3), congenital endothelial dystrophies (types 1 and 2), and X-linked endothelial corneal dystrophies.

[0172] In some embodiments, the corneal dystrophy is FECD. EXAMPLES

[0173] Example 1. Methods for preparation and administration of AAV vectors AAV vectors Wild-type AAV2, AAV5, AAV6, AAV8, and AAV9 vectors were generated by methods known in the art. Each AAV encodes eGFP under the ubiquitous CAG promoter. Each AAV was supplied at 1e13vg / mL in a PBS+0.001% pluronic acid formulation.

[0174] Intracameral (IC) injection Adult male C57BL / 6J mice (10–11 weeks old) were purchased from Jackson Laboratories. All animal procedures and handling were performed in accordance with the ARVO Statement for Animal Use and Regeneron Pharmaceuticals IACUC-reviewed protocols. Mice were anesthetized with a ketamine / xylazine mixture via intraperitoneal injection. Eyes were rinsed with sterile saline, followed by a drop of tropicamide (to dilate the pupil) and a drop of proparacaine (to numb the cornea). A Drummond Scientific Nanoject II microinjection device fitted with a pulled glass needle (beveled with sandpaper) was used to load the needle with AAV solution and inject the AAV solution into each anterior chamber. The glass needle was injected through the cornea parallel to the iris into the aqueous humor of the anterior chamber. A small amount of aqueous humor was allowed to leak out. An air bubble was pushed into the cornea, followed by 1.5 μL of AAV solution containing 1.5e10vg. After injection, the needle was held still for 30 seconds and then quickly and smoothly withdrawn. Both the OD (right eye) and OS (left eye) of each animal were injected. Control animals received an injection of PBS + 0.001% pluronic acid instead of the AAV solution. Mice were placed on their ventral side (to prevent leakage and pooling) to recover from anesthesia, and ointment was applied to each eye to prevent corneal drying and abrasion.

[0175] Intravitreal injection Adult male C57BL / 6J mice (10–11 weeks old) were purchased from Jackson Laboratories. All animal procedures and handling were performed in accordance with the ARVO Statement for Animal Use and Regeneron Pharmaceuticals IACUC-reviewed protocols. Mice were anesthetized with a ketamine / xylazine mixture via intraperitoneal injection. Eyes were rinsed with sterile saline followed by one drop of tropicamide (to dilate the pupil) and one drop of proparacaine (to numb the cornea). A Drummond Scientific Nanoject II microinjection device with a pulled glass needle (beveled with sandpaper) was used to load the AAV solution into the needle and inject the AAV solution into the vitreous humor of the vitreous chamber. The glass needle was injected into the vitreous chamber through the sclera at the limbus. 1.5 μL of AAV solution containing 1.5e10 vg was injected into the vitreous chamber using the microinjection device. The needle was withdrawn in a quick, smooth motion. Each animal was injected both OD (right eye) and OS (left eye). Control animals received an injection of PBS + 0.001% pluronic acid instead of the AAV solution. Mice were placed on their ventral side to recover from anesthesia (to prevent leakage and pooling) and ointment was applied to each eye to prevent corneal drying and abrasion.

[0176] Example 2. Evaluation of the specificity of proteins targeted to various tissues of the eye. In vivo imaging In vivo imaging was performed at baseline before injection and at post-injection time points using a Heidelberg Spectralis HRA+OCT (Heidelberg Engineering, Inc, Germany). Mice were anesthetized and a drop of tropicamide was applied to the eye to dilate the pupil, followed by a drop of proparacaine to numb the cornea. At each time point, infrared and fluorescence images to detect AAV-eGFP fluorescence were taken at the posterior retinal fundus (+25 diopter small animal imaging lens) and anterior cornea (anterior segment module). The FA modality of the Heidelberg Spectralis HRA+OCT was used to detect eGFP protein fluorescence resulting from AAV-eGFP injection.

[0177] immunohistochemistry Mice that received AAV-eGFP injections, such as AAV5-eGFP, AAV6-eGFP, and AAV8-eGFP, via intraocular injection were euthanized for enucleation. Control mice that received intraocular injections of PBS + 0.001% pluronic acid were euthanized for enucleation. Each eye was enucleated and fixed in 4% PFA overnight at 4°C. Eyes were washed with PBS and then incubated in 30% sucrose at 4°C for a minimum of 3 days. Eyes were then embedded in OCT embedding compound and a subset of samples was sent for sectioning by Histoserv Inc (Maryland). To amplify areas of AAV-eGFP localization, a primary antibody for eGFP was incubated overnight at 4°C on slides containing sectioned mouse eye tissue. A secondary antibody was conjugated to Alexa-Fluor594 (red) to distinguish from endogenous eGFP non-amplified signal in green. DAPI (blue) was added to the slides to label nuclei and aid in the identification of cell types and regions.

[0178] Slides were imaged using a Keyence microscope (Keyence Corporation of America, Illinois, USA). Areas of green and / or red fluorescence were assessed for both anatomical eye regions and cellular localization.

[0179] eGFP protein measurement Four mice (whole eyes) for each of the AAV serotypes, including AAV5-eGFP, AAV6-eGFP, and AAV8-eGFP, delivered by intraocular injection, were euthanized for enucleation. Two mice that received PBS + 0.001% pluronic acid were included as controls for each AAV serotype tested and were euthanized for enucleation. Each eye was kept separate and processed as an individual sample. Eyes were immersed in 1X cell extraction buffer PTR (provided in the ELISA kit) and homogenized using a tissue lyser with stainless steel beads. Samples were centrifuged and protein containing lysates were collected. Total protein measurements were measured using a BCA kit (Pierce BCA Protein Assay Kit, ThermoFisher). Samples were assayed in triplicate for eGFP protein expression using the GFP SimpleStep ELISA kit (Abcam). eGFP expression per eye was calculated as ng / μg of total protein isolated from the eye.

[0180] Transduction efficiency and tropism vary depending on the AAV serotype used. In vivo imaging on a Heidelberg Spectralis was used to determine the region of AAV transduction after IC administration. AAV2, AAV6, AAV8, and AAV9 were found to target both the posterior and anterior segments of the eye after IC administration, with AAV2, AAV6, and AAV9 showing the strongest eGFP expression in the anterior segment, whereas AAV5 was shown to target only the anterior eye tissues. The data also show that AAV5, AAV6, and AAV8 have a strong tropism for the anterior region after IC injection. Furthermore, IC injection can also deliver AAV to posterior tissues, as shown by the strong tropism of AAV2 and AAV9 to the posterior region after IC injection.

[0181] Example 3. Correction of genetic mutations in corneal endothelial cells Correction of a targeted gene mutation, such as a TCF4 or COL8A2 mutation, in corneal endothelial cells is achieved by administering a composition comprising a nucleic acid editing system that includes a CRISPR / Cas complex.

[0182] The CRISPR / Cas complex is complementary to the portion of the target gene containing the mutation and contains a guide sequence directed to the target DNA sequence and an endonuclease that encodes a Cas nuclease.

[0183] The CRISPR / Cas complex is guided to the target sequence, and the Cas nuclease cleaves the target sequence. Gene insertion mutations are corrected by cleaving the target sequence and repairing the DNA break. Gene mutations, which are nucleotide changes, are corrected by cleaving the nucleotide sequence of the mutated sequence and repairing the DNA with a repair template containing the nucleotide sequence of the wild-type gene.

[0184] CRISPR / Cas complexes are preferably packaged in AAV vectors, such as AAV5, AAV6, or AAV8. AAV vectors are generated by methods known in the art. Each AAV encodes a target sequence under the ubiquitous CAG promoter. Each AAV is provided at 1e13g / mL in PBS+0.001% pluronic acid formulation.

[0185] AAV vectors packaged in CRISPR / Cas complexes are administered directly into the anterior chamber via intracameral injection. Mice carrying such mutations are anesthetized with a ketamine / xylazine mixture by intraperitoneal injection. The eyes are rinsed with sterile saline, followed by a drop of tropicamide (to dilate the pupil) and a drop of proparacaine (to numb the cornea). Using a Drummond Scientific Nanoject II microinjection device fitted with a pulled glass needle (beveled with sandpaper), the needle is filled with AAV solution and used to inject the AAV solution into each anterior chamber. The glass needle is injected through the cornea parallel to the iris into the aqueous humor of the anterior chamber. A small amount of aqueous humor may leak out. Push an air bubble into the cornea, followed by 1.5 μL of AAV solution containing 1.5e10vg. After injection, the needle is held stationary for 30 seconds before being quickly and smoothly withdrawn. Inject into both the OD (right eye) and OS (left eye) of each animal. Control animals receive an injection of PBS + 0.001% pluronic acid instead of the AAV solution. Place the mouse on its ventral side to recover from anesthesia (to prevent leakage or pooling) and apply ointment to each eye to prevent corneal drying and abrasion.

[0186] Correction of gene expression is confirmed by dissecting corneas from treated and control mouse eyes (and isolating endothelial cells from said corneas) and performing DNA and / or RNA nucleic acid sequencing.

[0187] Example 4 - Downregulation of gene expression in corneal endothelial cells. Gene expression is downregulated by administering a composition comprising at least one inhibitory nucleotide sequence complementary to at least one allele on a target gene, selected from siRNA, shRNA, miRNA, antisense RNA, or antagomir RNA. The target gene is a corneal mutant gene, such as TCF4 or COL8A2. The inhibitory RNA is present in the composition by itself or as part of a CRISPR / Cas complex.

[0188] The inhibitory RNA is packaged in an AAV vector as in Example 3. The inhibitory RNA is preferably packaged in an AAV vector, such as AAV5, AAV6 or AAV8. The AAV vector is generated by methods known in the art. Each AAV encodes a target sequence under the ubiquitous CAG promoter. Each AAV is supplied at 1e13g / mL in a PBS+0.001% pluronic acid formulation.

[0189] Similar to Example 3, AAV vectors packaged with inhibitory RNA are administered directly into the anterior chamber via intracameral injection. Mice are anesthetized with a ketamine / xylazine mixture via intraperitoneal injection. Eyes are rinsed with sterile saline followed by a drop of tropicamide (to dilate the pupil) and a drop of proparacaine (to numb the cornea). Using a Drummond Scientific Nanoject II microinjection device fitted with a pulled glass needle (beveled with sandpaper), the needle is filled with AAV solution and used to inject the AAV solution into each anterior chamber. The glass needle is injected through the cornea parallel to the iris into the aqueous humor of the anterior chamber. A small amount of aqueous humor may leak out. An air bubble is pushed into the cornea followed by 1.5 μL of AAV solution containing 1.5e10vg. After injection, the needle is held stationary for 30 seconds before being quickly and smoothly withdrawn. Inject into both the OD (right eye) and OS (left eye) of each animal. Control animals receive an injection of PBS + 0.001% pluronic acid instead of the AAV solution. Place the mouse on its ventral side to recover from anesthesia (to prevent leakage or pooling) and apply ointment to each eye to prevent corneal drying and abrasion.

[0190] Downregulation of gene expression is confirmed by dissecting corneas from treated and control mouse eyes and measuring the amount of protein encoded by the gene in the samples via Western blot. Successful downregulation of gene expression will result in reduced levels of the encoded protein in corneal tissue from treated versus control mice.

[0191] Although the present invention has been described in detail, including preferred embodiments thereof, it will be understood that those skilled in the art, upon consideration of this disclosure, may make modifications and / or improvements thereto within the scope and spirit of the invention. Exemplary sequences from WO2017 / 185054 [Table 3] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12] [Table 4-13] [Table 4-14]

Table 5-1

Table 5-2

Table 5-3

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 6-28

Table 6-29

Table 6-30

Table 6-31

Table 6-32

Table 6-33

Table 6-34

Table 6-35

Table 6-36

Table 6-37

Table 6-38

Table 7

Table 8

Table 9-1

Table 9-2

Claims

1. a) a nucleotide sequence of an AAV vector or a portion thereof; b) a nucleic acid editing system comprising at least one nucleotide sequence complementary to at least one mutant allele on a target gene associated with a corneal disease or condition, a nucleic acid capable of downregulating gene expression of at least one mutant allele on a target gene associated with a corneal disease or condition, and / or at least one nucleotide sequence encoding a protein expressed in the eye, or a portion thereof.

2. The composition of claim 1 , wherein the protein is preferentially expressed in the anterior segment of the eye relative to other ocular tissues or cells.

3. The composition of claim 1 , wherein the protein is preferentially expressed in the cornea compared to other ocular tissues or cells.

4. The composition of claim 4 , wherein the protein is a transcription factor, a collagen, a nuclease, or a fluorescent protein.

5. The composition of claim 4, wherein the protein is transcription factor 4 (TCF4).

6. The composition of claim 1, wherein the nucleic acid editing system is a CRISPR system.

7. The composition of claim 1, wherein the nucleic acid editing system is a CRISPR-Cas system.

8. The composition of claim 1, wherein the CRISPR-Cas system comprises a nucleotide sequence encoding a CRISPR-associated (Cas) gene and a nucleotide sequence encoding a guide RNA (gRNA).

9. The composition of claim 1, wherein the CRISPR-Cas system is a CRISPR-Cas9 system.

10. 2. The composition of claim 1, wherein the nucleotide sequence complementary to at least one mutant allele is a gRNA.

11. The composition of claim 1, wherein the at least one nucleotide sequence complementary to at least one mutant allele on a target gene is selected from an siRNA, an shRNA, an miRNA, an antisense RNA, or an antagomir RNA.

12. The composition of claim 1 , wherein the mutant allele is encoded by a target sequence on the target gene.

13. The composition of claim 1 , wherein the at least one nucleotide sequence complementary to at least one mutant allele on a target gene hybridizes to a target sequence on the target gene in a cell of a subject.

14. The composition of claim 1 , wherein the target gene is TCF4 or COL8A2.

15. The composition of claim 1, wherein at least one target sequence is selected from the group consisting of SEQ ID NOs: 1-1084.

16. The composition of claim 1, wherein at least one target sequence is specific to the TCF4 gene, and said target sequence is selected from SEQ ID NOs: 1-190.

17. 2. The composition of claim 1, wherein the target sequence is specific to the COL8A2 gene, and the target sequence is selected from SEQ ID NOs: 191-1084.

18. 2. The composition of claim 1, wherein at least one guide RNA comprises a crRNA sequence that is complementary to at least one target sequence selected from SEQ ID NOs: 1-1084.

19. 2. The composition of claim 1, wherein at least one guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 1089-1278.

20. The composition of claim 1 , wherein the editing system further comprises a repair template.

21. 21. The composition of claim 20, wherein the repair template is selected from the group consisting of a DNA repair template, an mRNA repair template, an siRNA repair template, an miRNA repair template, and an antisense oligonucleotide repair template.

22. 2. The composition of claim 1, wherein the AAV vector serotype is selected from the group consisting of AAV5, AAV6, and AAV8.

23. The composition of claim 1 , wherein the AAV vector serotype is AAV5.

24. The composition of claim 1 , wherein the AAV vector serotype is AAV6.

25. The composition of claim 1, wherein the AAV vector serotype is AAV8.

26. The composition of claim 1 , wherein the composition further comprises a promoter.

27. 25. The composition of claim 24, wherein the promoter is optimized for use with an AAV5, AAV6, or AAV8 vector.

28. 28. The composition of claim 27, wherein the promoter is tissue specific and active in the eye when operably linked to the AAV vector or a nucleotide sequence that is complementary to at least one mutant allele on a target gene.

29. The composition of claim 28 , wherein the tissue-specific promoter is active in the cornea.

30. The composition of claim 28 , wherein the tissue-specific promoter is active in the corneal endothelium.

31. 2. The composition of claim 1, wherein the target gene is preferentially expressed in the anterior segment of the eye following intracameral (IC) injection.

32. The composition of claim 1 , wherein the disease or condition of the cornea is a superficial corneal dystrophy, anterior corneal dystrophy, corneal stromal dystrophy, or posterior corneal dystrophy.

33. The composition of claim 1 , wherein the disease or condition of the cornea is a posterior corneal dystrophy.

34. 33. The composition of claim 32, wherein the posterior corneal dystrophies are selected from the group consisting of Fuchs endothelial corneal dystrophy (FECD; both early and late onset), posterior polymorphous dystrophies (PPCD; types 1, 2, and 3), congenital endothelial dystrophies (types 1 and 2), and X-linked endothelial corneal dystrophies.

35. 35. The composition of claim 34, wherein the corneal dystrophy is FECD.

36. The composition of claim 1 , wherein the protein is preferentially expressed in the posterior segment of the eye relative to other ocular tissues or cells.

37. 37. The composition of claim 36, wherein the AAV vector serotype is selected from the group consisting of AAV2 and AAV9.

38. 1. A method for expressing a protein in the eye of a subject in need thereof, comprising: a) providing one or more adeno-associated (AAV) vectors comprising a nucleotide sequence encoding said protein; b) administering the AAV vector to the eye.

39. 39. The method of claim 38, wherein the protein is preferentially expressed in the anterior portion of the eye compared to other portions of the eye.

40. 40. The method of claim 39, wherein the protein is preferentially expressed in the cornea compared to other tissues or cells of the eye.

41. 41. The method of claim 40, wherein the protein is preferentially expressed in endothelial cells of the cornea as compared to other tissues or cells of the eye.

42. 42. The method of claim 41 , wherein the AAV vector serotype is selected from the group consisting of AAV5, AAV6, and AAV8.

43. 43. The method of claim 42, wherein the AAV vector serotype is AAV5.

44. 43. The method of claim 42, wherein the AAV vector serotype is AAV6.

45. 43. The method of claim 42, wherein the AAV vector serotype is AAV8.

46. 39. The method of claim 38, wherein the protein is a Cas protein.

47. 47. The method of claim 46, wherein the protein is Cas9.

48. 39. The method of claim 38, wherein the protein is a transcription factor, a collagen, a nuclease, or a fluorescent protein.

49. 39. The method of claim 38, wherein the protein is transcription factor 4 (TCF4).

50. 39. The method of claim 38, wherein the vector further comprises a repair template.

51. 51. The method of claim 50, wherein the repair template is selected from the group consisting of a DNA repair template, an mRNA repair template, an siRNA repair template, an miRNA repair template, and an antisense oligonucleotide repair template.

52. 51. The method of claim 50, wherein the vector further comprises a promoter.

53. 53. The method of claim 52, wherein the promoter is optimized for use with an AAV5, AAV6, or AAV8 vector.

54. 53. The method of claim 52, wherein the promoter is tissue specific and is active in the eye when operably linked to the AAV vector or a nucleotide sequence that is complementary to at least one mutant allele on a target gene.

55. 53. The method of claim 52, wherein the tissue-specific promoter is active in the cornea.

56. 53. The method of claim 52, wherein the tissue-specific promoter is active in the corneal endothelium.

57. 39. The method of claim 38, wherein the vector is administered to the subject via injection into the eye.

58. 58. The method of claim 57, wherein the vector is administered to the subject via injection into the anterior segment of the eye.

59. 58. The method of claim 57, wherein the vector is administered to the corneal stroma, limbus, epithelial surface of the cornea, or endothelial membrane of the cornea.

60. 58. The method of claim 57, wherein the vector is administered to the subject via intracameral (IC) injection.

61. 39. The method of claim 38, wherein the protein is preferentially expressed in the cornea compared to other ocular tissues or cells following IC injection.

62. 39. The method of claim 38, wherein the protein is preferentially expressed in corneal endothelial cells as compared to ocular tissues or cells following IC injection.

63. 39. The method of claim 38, suitable for treating an eye disease or condition.

64. 64. The method of claim 63, wherein the disease or condition of the eye is a disease or condition of the cornea.

65. 65. The method of claim 64, wherein the disease or condition of the cornea is a superficial corneal dystrophy, anterior corneal dystrophy, corneal stromal dystrophy, or posterior corneal dystrophy.

66. 66. The method of claim 65, wherein the disease or condition of the cornea is a posterior corneal dystrophy.

67. 67. The method of claim 66, wherein the posterior corneal dystrophies are Fuchs endothelial corneal dystrophy (FECD; both early and late onset), posterior polymorphous dystrophy (PPCD; types 1, 2, and 3), congenital endothelial dystrophies (types 1 and 2), and X-linked endothelial corneal dystrophies.

68. 68. The method of claim 67, wherein the corneal dystrophy is FECD.

69. 39. The method of claim 38, wherein the protein is preferentially expressed in the posterior portion of the eye as compared to other portions of the eye.

70. 70. The method of claim 69, wherein the AAV vector serotype is selected from the group consisting of AAV2 and AAV9.

71. 1. A method for repairing a gene expressed in the cornea in a subject in need thereof, comprising: a) providing a delivery system comprising a nucleic acid editing system comprising at least one nucleotide sequence that is complementary to at least one mutant allele on a target gene associated with said corneal disease or condition; b) administering the delivery system to the cornea of ​​the subject.

72. The method of claim 71, wherein the nucleic acid editing system is a CRISPR-Cas system.

73. 73. The method of claim 72, wherein the CRISPR-Cas system is a CRISPR-Cas9 system.

74. 72. The method of Claim 71, wherein said nucleotide sequence complementary to at least one mutant allele is a gRNA.

75. 72. The method of claim 71, wherein the at least one nucleotide sequence complementary to at least one mutant allele on a target gene is selected from an siRNA, an shRNA, an miRNA, an antisense RNA, or an antagomir RNA.

76. 72. The method of claim 71, wherein the mutant allele is encoded by a target sequence on the target gene.

77. 72. The method of claim 71, wherein the at least one nucleotide sequence complementary to at least one mutant allele on a target gene hybridizes to a target sequence on the target gene in a cell of the subject.

78. 72. The method of claim 71, wherein the target gene is TCF4 or COL8A2.

79. 72. The method of claim 71, wherein at least one target sequence is selected from the group consisting of SEQ ID NOs: 1-1084.

80. 72. The method of claim 71, wherein at least one target sequence is specific to the TCF4 gene, and said target sequence is selected from SEQ ID NOs: 1-190.

81. 72. The method of claim 71, wherein the target sequence is specific to the COL8A2 gene, and said target sequence is selected from SEQ ID NOs: 191-1084.

82. 72. The method of claim 71, wherein the delivery system further comprises a repair template.

83. 83. The method of claim 82, wherein the repair template is selected from the group consisting of a DNA repair template, an mRNA repair template, an siRNA repair template, an miRNA repair template, and an antisense oligonucleotide repair template.

84. 75. The method of Claim 74, wherein the gRNA hybridizes to a target sequence of the target gene in a cell of the subject.

85. 72. The method of claim 71, wherein the delivery system further comprises a promoter.

86. 86. The method of claim 85, wherein the promoter is optimized for use with an AAV5, AAV6, or AAV8 vector.

87. 86. The method of claim 85, wherein the promoter is tissue-specific and active in the eye when operably linked to a nucleotide sequence that is complementary to at least one mutant allele on the AAV vector or target gene.

88. 88. The method of claim 87, wherein the tissue-specific promoter is active in the cornea.

89. 88. The method of claim 87, wherein the tissue-specific promoter is active in the corneal endothelium.

90. 72. The method of claim 71, wherein the delivery system is administered to the subject via injection into the eye.

91. 72. The method of claim 71, wherein the delivery system is administered to the subject via injection into the anterior segment of the eye.

92. 72. The method of claim 71, wherein the delivery system is administered to the corneal stroma, the limbus, the epithelial surface of the cornea, or the endothelial membrane of the cornea.

93. 72. The method of claim 71, wherein the delivery system is administered to the subject via intracameral injection.

94. 72. The method of claim 71, suitable for treating an ophthalmic disease or condition.

95. 95. The method of claim 94, wherein the disease or condition of the eye is a disease or condition of the cornea.

96. 96. The method of claim 95, wherein the disease or condition of the cornea is a superficial corneal dystrophy, anterior corneal dystrophy, corneal stromal dystrophy, or posterior corneal dystrophy.

97. 97. The method of claim 96, wherein the disease or condition of the cornea is a posterior corneal dystrophy.

98. 98. The method of claim 97, wherein the posterior corneal dystrophies are selected from the group consisting of Fuchs endothelial corneal dystrophy (FECD; both early and late onset), posterior polymorphous dystrophies (PPCD; types 1, 2, and 3), congenital endothelial dystrophies (types 1 and 2), and X-linked endothelial corneal dystrophies.

99. 99. The method of claim 98, wherein the corneal dystrophy is FECD.

100. 1. A method of treating a corneal disease or condition caused by a mutant allele of a gene containing a trinucleotide repeat (TNR) and / or a point mutation in a subject in need thereof, comprising: a) excising at least a portion of said trinucleotide repeat (TNR) within said gene, i) providing an AAV5, AAV6, or AAV8 vector comprising one or more nucleotide sequences encoding one or more CRISPR guide RNAs that target a sequence within the TNR, 5' of the TNR, 3' of the TNR, or a combination thereof; ii) administering said vector to said cornea; and / or b) correcting a point mutation in said gene or gene product, i) providing an AAV5, AAV6, or AAV8 vector comprising one or more nucleotide sequences encoding one or more CRISPR guide RNAs targeting a sequence of said gene associated with a point mutation in said gene product; ii) administering the vector to the cornea. The method, wherein said one or more nucleotide sequences are preferentially expressed in said cornea.

101. The method of claim 100, wherein the target gene is TCF4 or COL8A2.

102. 101. The method of claim 100, wherein at least one target sequence is selected from the group consisting of SEQ ID NOs: 1-1084.

103. 101. The method of claim 100, wherein at least one target sequence is specific to the TCF4 gene, said target sequence being selected from SEQ ID NOs: 1-190.

104. 101. The method of claim 100, wherein the target sequence is specific to the COL8A2 gene, and said target sequence is selected from SEQ ID NOs: 191-1084.

105. 101. The method of claim 100, wherein two guide RNAs are used.

106. 101. The method of claim 100, wherein at least one guide RNA comprises a crRNA sequence that is complementary to at least one target sequence selected from SEQ ID NOs: 1-1084.

107. 101. The method of Claim 100, wherein at least one guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 1089-1278.

108. The method of claim 100, wherein the TNR is located in intron 3 of the TCF4 gene.

109. 101. The method of claim 100, wherein at least one guide RNA comprises a guide sequence that is complementary to at least one target sequence of the TCF4 gene, said target sequence being selected from SEQ ID NOs: 1-190.

110. 110. The method of claim 109, wherein two guide RNAs are used, one guide RNA complementary to a target sequence 5' of the TNR and the other guide RNA complementary to a target sequence 3' of the TNR.

111. 101. The method of claim 100, wherein the point mutation is located in the COL8A2 gene and the target sequence is selected from SEQ ID NOs: 1064-1084.

112. The method of claim 100, wherein the AAV vector comprises a Cas protein.

113. 113. The method of claim 112, wherein the Cas protein is a Cas9 nuclease.

114. The method of claim 100, wherein the TNR is located in intron 3 of the TCF4 gene.

115. The method of claim 113, wherein the Cas9 nuclease cleaves the TNR.

116. The method of claim 100, wherein two gRNAs are used, each comprising a guide sequence, one guide sequence directing a nuclease to a first position on intron 3 of the TCF4 gene and another guide sequence directing a nuclease to a second position on intron 3 of the TCF4 gene.

117. 114. The method of claim 113, wherein the nuclease cleaves intron 3 of TCF4 and excises a fragment of nucleic acid between the first and second cleavage sites.

118. 118. The method of claim 117, wherein the excised fragment of nucleic acid comprises one or more TNRs.

119. 101. The method of claim 100, suitable for treating a disease or condition associated with TNR.

120. 101. The method of claim 100, suitable for treating a disease or condition associated with a TNR in intron 3 of the TCF4 gene.

121. 101. The method of claim 100, suitable for treating a disease or condition associated with a point mutation in the COL8A2 gene.

122. The method of claim 100, wherein the vector further comprises a repair template.

123. 123. The method of claim 122, wherein the repair template is selected from the group consisting of a DNA repair template, an mRNA repair template, an siRNA repair template, an miRNA repair template, and an antisense oligonucleotide repair template.

124. 101. The method of claim 100, wherein the vector further comprises a promoter.

125. The method of claim 124, wherein the promoter is optimized for use with an AAV5, AAV6, or AAV8 vector.

126. The method of claim 124, wherein the promoter is tissue-specific and active in the eye when operably linked to the AAV vector or a nucleotide sequence that is complementary to at least one mutant allele on a target gene.

127. The method of claim 126, wherein the tissue-specific promoter is active in the cornea.

128. The method of claim 127, wherein the tissue-specific promoter is active in the corneal endothelium.

129. 101. The method of claim 100, wherein the vector is administered to the subject via injection into the eye.

130. 101. The method of claim 100, wherein the vector is administered to the subject via injection into the anterior segment of the eye.

131. The method of claim 100, wherein the vector system is administered to the corneal stroma, the limbus, the epithelial surface of the cornea, or the endothelial membrane of the cornea.

132. 101. The method of claim 100, wherein the vector system is administered to the subject via intracameral (IC) injection.

133. 101. The method of claim 100, wherein the one or more nucleotide sequences are preferentially expressed in corneal endothelial cells compared to other cells of the eye following IC injection.

134. 101. The method of claim 100, suitable for treating an ophthalmic disease or condition.

135. 135. The method of claim 134, wherein the disease or condition of the eye is a disease or condition of the cornea.

136. 136. The method of claim 135, wherein the disease or condition of the cornea is a superficial corneal dystrophy, anterior corneal dystrophy, corneal stromal dystrophy, or posterior corneal dystrophy.

137. 137. The method of claim 136, wherein the disease or condition of the cornea is a posterior corneal dystrophy.

138. 138. The method of claim 137, wherein the posterior corneal dystrophies are selected from the group consisting of Fuchs endothelial corneal dystrophy (FECD; both early and late onset), posterior polymorphous dystrophies (PPCD; types 1, 2, and 3), congenital endothelial dystrophies (types 1 and 2), and X-linked endothelial corneal dystrophies.

139. 139. The method of claim 138, wherein the corneal dystrophy is FECD.

140. 1. A method for downregulating expression of a gene expressed in the cornea in a subject in need thereof, comprising: a) a nucleotide sequence of an AAV vector or a portion thereof; b) a nucleic acid capable of downregulating gene expression of at least one mutant allele on a target gene associated with said corneal disease or condition; c) administering the delivery system to the subject, the delivery system comprising:

141. 141. The method of claim 140, wherein the nucleic acid capable of downregulating gene expression is a CRISPR system nucleic acid sequence.

142. The method of claim 140, wherein the nucleic acid capable of downregulating gene expression is a CRISPR-Cas system.

143. The method of claim 140, wherein the CRISPR-Cas system is a CRISPR-Cas9 system.

144. The method of claim 140, wherein the nucleic acid capable of downregulating gene expression is selected from the group consisting of antisense RNA, antagomir RNA, siRNA, and shRNA.

145. 141. The method of claim 140, further comprising at least one nucleotide sequence that is complementary to at least one mutant allele on a target gene associated with the corneal disease or condition.

146. The method of claim 140, wherein the target gene is TCF4 or COL8A2.

147. 147. The method of claim 146, wherein at least one target sequence is selected from the group consisting of SEQ ID NOs: 1-1084.

148. 147. The method of claim 146, wherein at least one target sequence is specific to the TCF4 gene, said target sequence being selected from SEQ ID NOs: 1-190.

149. 147. The method of claim 146, wherein the target sequence is specific to the COL8A2 gene, and said target sequence is selected from SEQ ID NOs: 191-1084.

150. The method of claim 140, wherein the AAV vector serotype is selected from the group consisting of AAV5, AAV6, and AAV8.

151. The method of claim 140, wherein the AAV vector serotype is AAV5.

152. The method of claim 140, wherein the AAV vector serotype is AAV6.

153. The method of claim 140, wherein the AAV vector serotype is AAV8.

154. 141. The method of claim 140, wherein the delivery system further comprises a repair template.

155. 155. The method of claim 154, wherein the repair template is selected from the group consisting of a DNA repair template, an mRNA repair template, an siRNA repair template, an miRNA repair template, and an antisense oligonucleotide repair template.

156. 141. The method of claim 140, wherein the delivery system further comprises a promoter.

157. The method of claim 156, wherein the promoter is optimized for use with an AAV5, AAV6, or AAV8 vector.

158. The method of claim 156, wherein the promoter is tissue-specific and active in the eye when operably linked to the AAV vector or a nucleotide sequence that is complementary to at least one mutant allele on a target gene.

159. The method of claim 158, wherein the tissue-specific promoter is active in the cornea.

160. 160. The method of claim 159, wherein the tissue-specific promoter is active in the corneal endothelium.

161. 141. The method of claim 140, wherein the target gene is preferentially expressed in the anterior segment of the eye.

162. The method of claim 140, wherein the delivery system and / or the nucleotide sequence capable of downregulating at least one mutant allele on at least one target gene is preferentially expressed in the anterior region following IC injection.

163. 141. The method of claim 140, suitable for treating an eye disease or condition.

164. 164. The method of claim 163, wherein the disease or condition of the eye is a disease or condition of the cornea.

165. 165. The method of claim 164, wherein the disease or condition of the cornea is a superficial corneal dystrophy, anterior corneal dystrophy, corneal stromal dystrophy, or posterior corneal dystrophy.

166. 166. The method of claim 165, wherein the disease or condition of the cornea is a posterior corneal dystrophy.

167. 167. The method of claim 166, wherein the posterior corneal dystrophies are selected from the group consisting of Fuchs endothelial corneal dystrophy (FECD; both early and late onset), posterior polymorphous dystrophies (PPCD; types 1, 2, and 3), congenital endothelial dystrophies (types 1 and 2), and X-linked endothelial corneal dystrophies.

168. 168. The method of claim 167, wherein the corneal dystrophy is FECD.

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