CROSS REFERENCE TO RELATED APPLICATIONS

1001 ] This application claims priority to U.S. Provisional Application Nos. 62/812,017 filed February 28, 2019: 62/831 ,838 filed April 1 0, 2019; and 62/878,865 filed July 26, 2019, each of which is hereby incorporated in its entirety.

SEQUENCE LISTI NG

(001.11 The instant application contains a Sequence Listing which has been submitted

electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 3, 2020, is named 67000-1023_ WO__SL.txt and is 357,652 bytes in size.

FIELD OF THE INVENTION

[002] The present Invention is generally directed to using adeno-associated vims (AAV) vectors to deliver therapeutics to the eye, for example to the comeal endothelium. The present invention is also directed to compositions comprising the AAV vectors. Corneal dystrophies can be treated with the methods and compositions of the present invention.

BACKGROUND

Adeno-associated virus (A AV) is a small, replication-deficient parvovirus. AAV is about 20-24 nm long, with a densit of about 1.40-1.41 g/cc. AAV contains a single-stranded linear genomic DNA molecule approximately 4.7 kb in length. The single-stranded AAV genomic DNA can be either a plus strand, or a minus strand. AAV contains two open reading frames. Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). A A Vs contain a single iniron. Cis- acting sequences directing viral DNA replication (Rep), encapsidation/packaging and host cel! chromosome integration are contained within the ITRs. Three AAV promoters, p5, pl9, and p40 (named for their relative map locations) dri ve the expression of the two AAV internal open reading frames encoding rep an cap genes. The p5 and p 19 are the rep promoters. When coupled with the differential splicing of the single AAV intron, the two rep promoters 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 that are responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter, and encodes the three capsid proteins VP1 , VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single polyadenylationn site is located at map position 95 of the AAV genome. Muzyczka reviews the life cycle and genetics of AAV (Muzyczka, Current Topic in Microbiology and Immunology, 158:97-129 (1992)).

{02545219.1 } 1 [004] AAV infection is non-cytopathic in cultured cells. Natural infection of humans and other animals is silent and asymptomatic (does not cause disease). Because AAV infects many mammalian cells, there is the possibility of targeting many different tissues in vivo. In addition to dividing cells, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (i.e. extrachromosomal element). The AAV proviral genome is infective as cloned DNA in plasmids, which makes construction of recombinant genomes possible. Moreover, because the signals directing AAV replication, genome encapsidation, and integration are all contained with the ITRs of the AAV genome, some or all of the approximately 4.3 kb of the genome, encoding replication and structural capsid proteins (rep-cap) are contained within the ITRs of the AAV genome, and can be replaced with heterologous DNA, such as a gene cassette containing a promoter, a DNA of interest, and a polyadenylation signal. The rep and cap proteins may be provided in trans. AAV is a very stable and robust virus, and easily withstands conditions used to inactivate adenovirus (56°C to 65°C for several hours), therefore cold preservation of AAV less critical. And, AAV -infected cells are not resistant to super-infection. These unique properties of AAV make it useful as a vector for delivering foreign DNA to cells or subjects, for example, in gene therapy.

[005] Comeal dystrophy is a term for the heterogenous group of non-inflammatory bilateral diseases restricted to the cornea. They are grouped by the anatomical location within the cornea of the pathology. Most do not have any manifestations outside of the cornea and they result with comeal opacities and affect visual acuity (see

https://www.comealdystrophyfoundation.org/what-is-comeal- dystrophy).

[006] The cornea has three major regions that are affected by comeal dystrophies: comeal epithelium, stroma, endothelium. Anterior comeal dystrophies affect the comeal epithelium and its basement membrane and the superficial comeal stroma. Stromal comeal dystrophies affect the comeal stroma. Posterior comeal dystrophies affect Descemet membrane and the comeal endothelium. The most common posterior comeal dystrophy is Fuchs’ comeal endothelial dystrophy. [007] Recently, it has been found that certain pathological conditions or diseases are associated with mutations in the TCF4 gene, coding for transcription factor 4 protein (TCF4). Diseases associated with mutations in the TCF4 gene include Fuchs endothelial comeal dystrophy (FECD), posterior polymorphous comeal dystrophy (PPCD), primary sclerosing cholangitis (PSC), Pitt-Hopkins syndrome, distal 18q deletion, and schizophrenia.

[008] FECD is a condition that causes vision problems. It affects the cornea of the eye, in particular the endothelium. The cornea is located on the front surface of the eye, and comeal tissue contains five basic layers. The epithelium is the cornea’s outermost layer. The epithelium functions to block the passage of foreign material (e.g. dust, water, bacteria) into the eye and other layers of the cornea, and provides a smooth surface to absorb oxygen and cell nutrients from tears, distributing these nutrients to the rest of the cornea. The epithelial cells anchor and organize themselves on the basement membrane of the epithelium. Lying directly below the basement membrane of the epithelium is the Bowman’s layer, which is a transparent sheet of tissue composed of collagen fibers. Beneath Bowman’s layer is the stroma. The stroma comprises about 90% of the cornea’s thickness, and consists primarily of water and collagen. A thin, strong sheet of tissue, Descemet’s membrane is beneath the stroma. Descemet’s membrane is composed of collagen fibers, and is made by the endothelial cells that he beneath it. The endothelium is the layer below Descemet’s layer.

[009] The endothelium is the extremely thin innermost layer of the cornea and is vital to keeping the cornea clear. The comeal endothelium is a monolayer of amitotic cells that form a barrier between the comeal stroma and the aqueous humor. The comeal endothelial cells function by pumping fluid from the cornea to maintain the cornea at the correct thickness to preserve clarity. In some posterior comeal dystrophies, such as FECD, the comeal endothelium is diseased and cells die over the course of this progressive disease. As these cells die, the remaining cells expand to fill the space, and the layer loses the ability to properly function. This results in comeal edema and increased opacity, leading to a reduction in visual acuity. In advanced stages of the disease, blindness may ensue. Loss of vision due to FECD is the leading cause of comeal transplants in the USA.

[0010] Because the comeal endothelium is affected in these diseases, targeting them to deliver therapeutics could aid in stopping the progression of disease. One such methodology is adeno-associated viruses (AAVs), which can be packaged to deliver the therapeutic, and delivered via intracameral or intrastromal injection to come into contact with the cornea endothelium. Proteins or nucleotide sequences are commonly packaged into AAV vectors.

[0011] It has been suggested that genetic factors are associated with the occurrence of FECD. Genetic loci known to be associated with FECD include FCD1 to FCD4 , ZEB1/TCF8, SLC4A11, I.OXHDI . and COL8A2. One such genetic factor is trinucleotide repeat (TNR) expansions in the transcription factor 4 (I ^’ d A) gene. Most of the genetic predisposition for FECD is associated with a TNR in the third intron of the TCF4 gene. A repeat length of greater than 50 repeats is generally associated with a clinical diagnosis of FECD (Wieben et al, PLOS One, 7: 11, e49083 (2012)). Recently, it has been suggested that this TNR expansion causes aggregation of the affected TCF4 RNA, and sequestration of key RNA splicing factors (Mootha, et al, Invest. Ophthalmol. Vis. Sci., 55(l):33-42 (2014); Mootha, et al, Invest. Ophthalmol. Vis. Sci., 56(3):2003-l l (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 lead to global changes in gene expression, resulting in significant changes in cellular function, and cell death (Du et al, J. Biol. Chem., 290: 10, 5979-5990 (2015)).

[0012] Another genetic mutation that is associated with FECD occurs in the COL8A2 gene (Vedana et al., Clinical Opththalmology, 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 corneas from patients with mutations in COL8A2 have an irregular mosaic deposition of different amounts of COL8A1 and COL8A2, in a non- coordinated manner. Three point mutations of the COL8A2 lead to 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 related comeal disorder PPCD (which is characterized by changes in the Descemet’s membrane and endothelial layer of the cornea).

[0013] Although AAV vectors have been used to deliver gene editing therapeutics directly to the eye, this has generally only been shown for posterior portions of eye, such as the retina. Delivery of gene editing therapeutics to the anterior portions of the eye, such as the cornea, is far less well researched and documented. There remains a need to develop delivery techniques that can preferentially deliver therapeutics only to specific areas of the eye and to specific tissues or cells, particularly the anterior portions such as the cornea.

SUMMARY OF THE INVENTION

[0014] The present invention provides a method of delivering a therapeutic to the comeal endothelium, to treat diseases such as comeal dystrophies, for example, FECD. The methods of the invention utilize AAVs to deliver therapeutics directly to the eye, particularly the comeal endothelium. In certain embodiments, the AAVs are packaged with proteins, or nucleotides encoding the proteins, to be expressed in certain cells of the eyes. In other embodiments, the AAVs are packed with a CRISPR RNP complex (i.e. a complex with a Cas protein) to elicit directed gene editing in the eye, and in specific areas or cells of the eye. In some embodiments, the AAVs are packaged with a CRISPR gRNA complexed with a nucleotide sequence encoding a Cas protein. The present invention also provides compositions comprising the AAVs.

[0015] In a particular aspect, the present invention provides a composition comprising:

a) a nucleotide sequence, or portion thereof, of an AAV vector; and b) 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 diseases or conditions in the cornea; a nucleic acid capable of down-regulating gene expression of at least one mutant allele on a target gene associated with diseases or conditions in the cornea; and/or at least one nucleotide sequence, or portion thereof, that codes for a protein to be expressed in the eye.

[0016] In a another aspect, the present invention provides a method of expressing a protein in an eye of a subject in need thereof comprising:

a) providing one or more adeno-associated (AAV) vectors comprising a nucleotide sequence that encodes said protein; and

b) administering the AAV vector to the eye.

[0017] In another aspect, the present invention provides a method for repairing a gene expressed in the cornea in a subject in need thereof, the method 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 diseases or conditions in the cornea; and

b) administering the delivery system to the cornea of the subject.

When the term“repairing” is used, it is also meant to include inducing repair.

[0018] In yet another aspect, the present invention provides a method of treating a disease or condition of the cornea caused by a mutant allele of a gene that comprises trinucleotide repeats (TNRs) and/or a point mutation in a subject in need thereof, said method comprising: a) excising at least a portion of the trinucleotide repeats (TNRs) within the gene,

comprising:

i) providing an AAV5, AAV6, or AAV8 vector which comprises one or more nucleotide sequences coding for one or more guide RNAs targeting a sequence within the TNRs, 5’ of the TNRs, 3’ of the TNRs, or combination thereof; and ii) administering the vector to the cornea; and/or

b) correcting the point mutation of the gene or gene product comprising:

i) providing an AAV5, AAV6, or AAV8 vector comprising one or more

nucleotide sequences coding one or more guide RNAs targeting a sequence in the gene associated with a point mutation in the gene product; and ii) administering the vector to the cornea;

wherein said one or more nucleotide sequences are preferentially expressed in the cornea after intracameral injection.

[0019] In another aspect, the present invention provides a method of treating a disease or condition of the cornea caused by a mutant allele of a gene that comprises trinucleotide repeats (TNRs) and/or a point mutation in a subject in need thereof, said method comprising:

a) excising at least a portion of the trinucleotide repeats (TNRs) within the gene, comprising:

i) providing an AAV5, AAV6, or AAV8 vector which comprises one or more nucleotide sequences coding for one or more guide RNAs targeting a sequence within the TNRs, 5’ of the TNRs, 3’ of the TNRs, or combination thereof; and

ii) administering the vector to the cornea; and/or b) Correcting the point mutation of the gene or gene product comprising: i) providing an AAV5, AAV6, or AAV8 vector comprising one or more nucleotide sequences coding one or more guide RNAs targeting a sequence in the gene associated with a point mutation in the gene product; and

ii) administering the vector to the cornea.

[0020] In another aspect, the present invention provides a method for down-regulating expression of a cornea gene in a subject in need thereof, the method comprising

administering to the subject a delivery system comprising:

a) a nucleotide sequence, or portion thereof, of an AAV vector;

b) a nucleic acid capable of down-regulating gene expression of at least one mutant allele on a target gene associated with diseases or conditions in the cornea; and c) administering the delivery system to the cornea.

[0021] In another aspect, the present invention provides a method of preferentially expressing a protein in endothelial cells of the cornea in a subject in need thereof, comprising:

a) providing one or more adeno-associated (AAV) vectors comprising a nucleotide sequence, or portion thereof, that encodes said protein; and b) administering the AAV vector to the cornea.

[0022] These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the compositions and methods as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1: Figure 1 is an illustration of the layers of the cornea (see

https://discoveryeye.org/treatment-comeal-scratches-and-a brasions/).

[0024] Figure 2: Figure 2 is an illustration of the structure of the mouse eye, and a depiction of intracameral and intravitreal injection into the eye. [0025] Figure 3: Figure 3 depicts the in vivo images of a mouse eye after intracameral delivery of AAV5-eGFP. Panels A-D show images from the OD eye (“OD” refers to Oculus Dexter which is latin for the right eye). Panels E-H show images from the OS eye (“OS” refers to Oculus Sinister which is latin for the left eye). Panel A provides a reference for panel B. Panel E provides a reference for panel F. Panels B & F show the image which demonstrates fluorescence in the cornea from the AAV5-eGFP. Panels C & G shows the fundus image and panels D & H show the image which demonstrates no fluorescence in the retina. Two dots of fluorescence are detected in the OS retina shown by arrows in panel H.

[0026] Figure 4: Figure 4 depicts the immunohistochemistry of the same eyes shown in Fig. 3. AAV5-eGFP was delivered by intracameral injection. The OS eye was separated into a cornea flat mount (panel A, magnified insert shown in panel B) and a retina flat mount (panel C, magnified insert shown in panel D). Staining shows eGFP localized to the cornea endothelium and a few cells staining in the retina. The OD eye was collected whole and processed for cross-sections shown in panel E. Staining shows eGFP localized to the cornea endothelium and not in the retina. Magnified inserts are shown in panels F-G. Panel F shows the cornea endothelium layer. Panel G shows the retina, where the exposure time had to be increased to capture a positive signal not seen in panel E. (green=endogenous eGFP, red=secondary staining using primary antibody to eGFP).

[0027] Figure 5: Figure 5 depicts the in vivo images of a mouse eye after intracameral delivery of AAV6-eGFP. Panel A provides a reference for panel B. Panel B shows the image which demonstrates fluorescence in the cornea from the AAV6-eGFP. Panel C shows the fundus image and panel D shows the image which demonstrates fluorescence in the retina.

[0028] Figure 6: Figure 6 depicts the immunohistochemistry of the same mouse eye shown in Fig. 5. Staining demonstrates that AAV6-eGFP is present in the comeal endothelium, stroma, and ciliary body (green=endogenous eGFP, red=secondary staining using primary antibody to eGFP, blue= DAPI stained nuclei). The white rectangle in panel A indicates the zoomed-in area shown in panel B. The left arrow in panel B indicates the positive comeal stroma layer. The right arrow in panel B indicates the positive comeal endothelium layer. [0029] Figure 7: Figure 7 depicts the in vivo images of a mouse eye after intracameral delivery of AAV8-eGFP. Panel A provides a reference for panel B. Panel B shows the image which demonstrates fluorescence in the cornea from the AAV8-eGFP. Panel C shows the fundus image and panel D shows the image which demonstrates fluorescence in the retina.

[0030] Figure 8: Figure 8 depicts the immunohistochemistry of the same mouse eye shown in Fig. 7. Staining demonstrates that AAV8-eGFP is present in the comeal endothelium, stroma, and ciliary body (green=endogenous eGFP, red=secondary staining using primary antibody to eGFP, blue= DAPI stained nuclei). The white rectangle in panel A indicates the zoomed-in area shown in panel B. The left arrow in panel B indicates the positive comeal stroma layer. The right arrow in panel B indicates the positive comeal endothelium layer.

[0031] Figure 9: Figure 9 depicts the ELISA results of eGFP protein levels from 4 mice (whole eyes) for each of the AAV serotypes, such as AAV5-eGFP, AAV6-eGFP, AAV8- eGFP, delivered by intracameral route. Two mice that received PBS+0.001% pluronic acid were included as controls for each of the AAV serotypes tested. Means with SEM are shown.

[0032] Figure 10: Figure 10 is a composite figure that depicts the in vivo fluorescence images and immunochemistry results of AAV2-eGFP, AAV5-eGFP, AAV6-eGFP, AAV8- eGFP, and AAV9-eGFP after IC delivery into the mouse eye.

DETAILED DESCRIPTION

[0033] 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.

[0034] Headings are used solely for organizational purposes, and are not intended to limit the invention in any way.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the inventions belong. All patents, patent applications, published applications and publications, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety for any purpose. 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.

[0036] Comeal dystrophy is a term for the heterogenous group of non-inflammatory bilateral diseases restricted to the cornea. They are grouped by the anatomical location of the pathology within the cornea. Most do not have any manifestations outside of the cornea and they result with comeal opacities and affect visual acuity (see

https://www.comealdystrophyfoundation.org/what-is-comeal- dystrophy).

[0037] Anterior comeal dystrophies affect the comeal epithelium and its basement membrane and the superficial comeal stroma. Stromal comeal dystrophies affect the comeal stroma. Posterior comeal dystrophies affect Descemet membrane and the comeal endothelium. The most common posterior comeal dystrophy is Fuchs’ comeal endothelial dystrophy.

[0038] The cornea has three major regions that are affected by comeal dystrophies: comeal epithelium, stroma and endothelium. AAV5 targets the comeal endothelium after IC delivery and could be utilized to deliver gene therapy for posterior comeal dystrophies. Both AAV6 and AAV8 can target the comeal stroma, endothelium, and ciliary body after IC delivery and could be utilized to deliver gene therapy for comeal stromal dystrophies and posterior comeal dystrophies. As some anterior comeal dystrophies affect both the epithelium and the superficial comeal stroma, AAV6 and AAV8 could deliver gene therapy to the stroma.

[0039] Table D1 shows comeal dystrophies and certain genes associated therewith

((Klintworth, 2009. Comeal dystrophies. Orphanet J. Rare Dis., 4, 7. doi: 10.1186/1750- 1172-4-7). Table Dl. Summary of the comeal dystrophies: modes of inheritance, gene loci, genes and the categories of the International Committee for the Classification of Comeal Dystrophies

(IC3D) categories.

*Category 1 : A well-defined comeal dystrophy in which the gene has been mapped and identified and specific mutations are known.

Category 2: A well-defined comeal dystrophy that has been mapped to 1 or more specific chromosomal loci, but the gene(s) remains to be identified.

Category 3: A well-defined comeal dystrophy in which the disorder has not yet been mapped to a chromosomal locus.

Category 4: A suspected new, or previously documented comeal dystrophy, although the evidence for it, being a distinct entity, is not yet convincing.

[0040] Table D2 is from Moore, C. B. T., Christie, K. A., Marshall, I, & Nesbit, M. A. (2018). Personalised genome editing - The future for comeal dystrophies. Prog Retin Eye Res, 65, 147-165. doi: 10.1016/j.preteyeres.2018.01.004.

Table D2. List of known comeal dystrophies, including associated inheritance pattern, gene locus and causative genes.

[0041] Delivering AAVs directly to the eye, for example by intracameral injection, can result in a viral targeting tropism to the cornea. Delivering AAV5 via intracameral injection results in a viral targeting tropism to the cornea endothelium, and not to other ocular structures. This targeted tropism could deliver the therapeutic to the affected structure, while sparing other ocular structures, decreasing the risk of off-target effects. Intracameral delivery of AAV6 or AAV8 also demonstrates targeting to the comeal endothelium. However, both AAV6 and AAV8 also display tropism to other corneal and anterior structures, as well as the retina, when delivering a gene using the ubiquitous CAG promoter.

[0042] AAV is a small virus consisting of two open reading frames, Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). When used for gene therapy, the Rep and Cap open reading frames are removed, and the desired gene, together with a promoter to drive transcription of the desired gene, is inserted between the ITRs.

[0043] CRISPR nucleotides (e.g. gRNA and/or nucleotides coding for Cas proteins) can be packaged between the ITRs, creating a viral vector for targeted delivery of therapeutics. In some embodiments, the CRISPR nucleotide gRNA is packaged with a Cas protein (e.g. Cas9 nuclease) to form a ribonucleoprotein (RNP) complex. However, the AAVs can also be packaged with nucleotides encoding other proteins. AAVs are preferred viral vectors because they can infect both dividing and non-dividing cells, and are associated with a lack of pathogenicity.

[0044] AAV vectors can thus be used to preferentially target certain layers of the cornea. AAV5, for example, specifically targets cornea endothelium. The specificity of AAV vectors reduces the risk for off-target effects of therapeutics that are delivered via the AAV vectors.

[0045] In certain embodiments, the AAV vectors can comprise one or more nucleotide sequences that are complementary to at least one target sequence on a target gene.

[0046] In some embodiments, the AAV vectors can comprise one or more nucleotide acid editing systems. Nucleotide editing systems include, but are not limited to a CRISPR system, an siRNA, an shRNA, an miRNA, an antisense RNA, or an antagomir RNA.

[0047] 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.

[0048] In certain embodiments, the AAV vectors can be used for targeted gene therapy in the cornea, by delivering CRISPR complexes targeting genes involved in comeal dystrophies, such as Fuchs endothelial comeal dystrophy (FECD). FECD is associated with trinucleotide repeat (TNR) expansions in the transcription factor 4 ( TCF 4) gene. Most of the genetic predisposition for FECD is associated with a TNR in the third intron of the TCF4 gene.

FECD is a condition that affects the cornea of the eye, in particular the endothelium. Comeal dystrophies are also associated with mutations in the COL8A gene. Mutations of the COL8A gene lead to a Gln455Lys, Gln455Val, or Leu450Trp mutation in the gene product.

[0049] By delivering CRISPR complexes (gRNA plus a Cas protein, or a nucleotide encoding a Cas protein) to the cornea endothelium, the TNRs, or a portion thereof, can be excised from the TCF4 gene in the comeal endothelium, without affecting the TCF4 gene in other parts of the eye.

[0050] In certain embodiments, CRISPR complexes are packaged into one or more AAV vectors. The CRISPR complexes may target either the TNRs of the TCF4 gene, or the mutant alleles of the COL8A2 gene.

[0051] In certain embodiments, the AAV vectors may be delivered by themselves. In other embodiments, the AAV vectors may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid for delivery.

Definitions

[0052] 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 forms as well, unless the context clearly indicates otherwise.

[0053] In this application, the use of“or” means“and/or” unless stated otherwise.

[0054] As used herein, the terms“comprises” and/or“comprising” specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Furthermore, to the extent that the terms “includes,”“having,”“has,”“with,”“composed ,”“comprised” or variants thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term“comprising.”

[0055] As used herein, ranges and amounts can be expressed as“about” a particular value or range. “About” is intended to also include the exact amount. Hence“about 5 percent” means “about 5 percent” and also“5 percent.”“About” means within typical experimental error for the application or purpose intended.

[0056] As used herein,“treatment” refers to any delivery, administration, or application of a therapeutic for a disease or condition. Treatment may include curing the disease, inhibiting the disease, slowing or stopping the development of the disease, ameliorating one or more symptoms of the disease, or preventing the recurrence of one or more symptoms of the disease.

[0057] As used herein,“FECD” refers to Fuchs endothelial comeal dystrophy. FECD includes patients who have the condition, as well as individuals who do not have symptoms, but have a genetic disposition to FECD.

[0058] As used herein,“AAV” refers to an adeno-associated virus. AAV is a non-enveloped vims that is icosahedral, is about 20 to 24 nm long with a density of about 1 40 1.41 g/cc, and contains a single stranded linear genomic DNA molecule approximately 4.7 kb in length.

The single stranded AAV genomic DNA can be either a plus strand, or a minus strand. In certain embodiments, the term“AAV” or“AAV vector” refers to an AAV that has been modified so that a therapeutic, 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.

[0059] As used herein,“AAV serotype” means a sub-division of AAV that is identifiable by serologic or DNA sequencing methods and can be distinguished by its antigenic character.

[0060] As used herein, a“vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior 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 an autonomously replicating plasmid or a virus. “Vector” may also include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds liposomes, lipid nanoparticles, non-lipid nanoparticles, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like. Preferably, the vector is an AAV vector.

[0061] As used herein,“RNA” refers to a molecule comprising one or more ribonucleotide residues. A“ribonucleotide” is a nucleotide with a hydroxyl group at the 2’ position of the 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 nucleotides or chemically synthesized nucleotides or deoxynucleotides.

[0062] As used herein“inhibitory RNA” means a nucleic acid molecule that contains a sequence that is 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), ribozymes, antagomirs, and antisense oligonucleotides.

[0063] As used herein,“shRNA” refers to an RNA molecule comprising an antisense region, a loop portion, and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem. Following post-transcriptional processing, the shRNA is converted to siRNA by a cleavage mediated by the enzyme Dicer, which is a member of the RNase III family.

[0064] As used herein,“siRNA” refers to any small RNA molecule capable of inhibiting or down-regulating gene expression by mediating RNA interference in a sequence specific manner. [0065] As used herein,“antisense RNA” or“antisense oligonucleotides” are short, synthetic pieces of nucleic acid whose sequence is complementary to the mRNA that codes for a protein. Antisense RNA binds to the mRNA and blocks transcription.

[0066] As used herein, an“antagomir” or“antagomir RNA” refers to small synthetic RNA that are complementary to a specific microRNA (miRNA) target, optionally with either mispairing at the cleavage site or one or more base modifications to inhibit cleavage.

[0067] As used herein,“micro RNA” or“miRNA” refers to a single-stranded RNA molecule of about 21-23 nucleotides in length, which regulates gene expression. miRNA molecules are partially complementary to one or more mRNA, and their main function is to down- regulate gene expression.

[0068] As used herein,“TNRs” refers to trinucleotide repeats (i.e. multiple repetitions of three base pairs). The term“TNR expansion” refers to a higher than normal number of TNRs. For example, about 50 or more TNRs in intron 3 of TCF4 would be considered a TNR expansion.

[0069] As used herein“CRISPR” means a bacterial adaptive immune system known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) sequences.

[0070] As used herein,“guide RNA” and“gRNA” are used interchangeably, and refer to RNA sequences that are directed to a target DNA sequence. The gRNA contains a CRISPR RNA (crRNA) and transactivating crRNA (trRNA or tracrRNA). The crRNA and the trRNA may be associated on a single RNA molecule, referred to as a single guide RNA (sgRNA). Alternatively, the crRNA and trRNA may be disassociated on separate RNA molecules, and form a dual guide RNA (dgRNA). In some embodiments, the gRNA is chemically modified, and comprises one or more modified nucleosides or nucleotides. Modification of nucleosides and nucleotides can include one or more of: i) alteration, e.g. replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone; ii) alteration, e.g. replacement, of a constituent of the ribose sugar, such as, for example, the 2’-hydroxyl on the ribose sugar; iii) complete replacement of the phosphate moiety with“dephospho” linkers; iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase; v) replacement or modification of the ribose-phosphate backbone; vi) modification of the 3’ end or 5’ end of the oligonucleotide, e.g. removal, modification, or replacement of a terminal phosphate group, or conjugation of a moiety, cap, or linker; and vii) modification or replacement of the sugar.

[0071] As used herein, the“guide sequence” refers to an about 20 base-pair sequence within the crRNA or trRNA that is complementary to a target sequence. The guide sequence directs the gRNA to a target sequence for cleavage by a nuclease.

[0072] As used herein,“target sequence” refers to a sequence of nucleic acids, within the genomic DNA of the subject, to which a gRNA directs a nuclease for cleavage of the target sequence. For example, a Cas protein may be directed by a gRNA to a target sequence, where the gRNA hybridizes with the target sequence, and the nuclease cleaves the target sequence. Target sequences include both the positive and negative strands 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 may specifically bind under conditions sufficient for binding to occur.

[0073] As used herein, the term“CRISPR complex” refers to a combination of a gRNA and an endonucleotide encoding for a Cas protein (gRNA: Cas endonucleotide), or a combination of a gRNA and a Cas protein (gRNA: Cas protein). As used herein, a“ribonucleoprotein” (RNP) refers to a gRNA: Cas protein complex. The CRISPR complexes of the present invention may be directed to and cleave a target sequence either within the TNRs, or flanking the TNRs (5’ or 3’) of the TCF4 gene. The CRISPR complexes may also be directed to cleave a target sequence in the COL8A gene. As used herein, a“protospacer adjacent motif’ or“PAM” refers to a nucleotide sequence that must be adjacent to a target nucleotide sequence. The required PAM depends on the specific CRISPR system used. For example, in the CRISPR/Cas system derived from Streptococcus pyogenes, the target DNA must immediately precede a 5’-NGG PAM (where“N” is any nucleobase followed by two guanine nucleobases) for optimal cutting. Although Streptococcus pyogenes Cas9 also recognizes the 5’-NAG PAM, it appears to cut less efficiently at these PAM sites. Other Cas9 orthologs (e.g. derived from Staphylococcus aureus) require different PAM sequences.

[0074] As used herein,“indels” means insertion/deletion mutations that consist of a number of nucleotides that are either inserted or deleted at the site of double-stranded breaks (DSBs) in the nucleic acid of the DNA.

[0075] As used herein,“excision fragment” or“excision fragments” refers to deletions of a consecutive number of nucleotides (such as TNRs) that may occur when two or more gRNA are used together with a Cas mRNA or Cas protein.

[0076] As used herein,“promoter” means a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate specific transcription of a polynucleotide sequence. Preferred are promoters that are operable for AAV vectors, preferably AAV5, AAV6, and/or AAV8, and tissue specific promoters, preferably specific for the eye, more preferably specific for the cornea, and most preferably specific for the endothelium of the cornea. AAV promoters include, for example, an 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,

EF1 alpha, GRK, MT, MMTV, TY, RU486, RHO, RHOK, CBA, chimeric CMV-CBA, MLP, RSV, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, functional fragments thereof, etc. In AAV packaged with heterologous DNA, a promoter normally associated with heterologous nucleic acid can be used, or a promoter normally associated with the AAV vector, or a promoter not normally associated with either, can be used.

[0077] As used herein,“constitutive promoter” is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies 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, Rous sarcoma virus (RSV) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, functional fragments thereof, or combinations thereof.

[0078] As used herein,“inducible promoter” is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. 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. the comeal edothelium.

[0079] As used herein, a“tissue specific promoter” is a nucleotide sequence which, when operably linked with a polynucleotide which 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.

[0080] As used herein, a“promoter/regulatory sequence” means a nucleic acid sequence which is 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 an enhancer sequence and other regulatory elements which are required for expression of the gene product.

[0081] As used herein,“under transcriptional control” or“operably linked” means that the promoter is in the correct location and orientation in relation to a polynucleotide to control initiation of transcription by RNA polymerase and expression of the polynucleotide. These include promoters, a 3’ UTR, or a 5’ UTR. The promoter may be recognized by RNA polymerase III (Pol III), such as, but limited to, U6 and HI Pol III promoters. The Pol III promoters may be, for example, mouse or human.

[0082] As used herein,“gene editing” or“nucleic acid editing” refers to modification of the nucleic acid sequence of a target gene. [0083] As used herein,“nucleic acid editing system” or“gene editing system” refers to a method that can be used for performing gene editing or nucleic acid editing. Nucleic acid editing systems and gene editing systems include CRISPR systems, and interfering RNAs.

[0084] As used herein,“delivery system” refers to materials used to deliver nucleic acids to target cells. Such materials may include viral vectors such as AAV vectors and

pharmaceutically acceptable ingredients.

[0085] As used herein,“modulation” or“modification” includes decreasing or inhibiting expression or function, of for example, a gene or protein, as well as increasing expression or function, of for example, a gene or protein. As used herein,“modulation” or“modification” also includes complete restoration of gene function, which includes replacing mutated part(s) of a gene or replacing the mutant gene with a wild-type version.

[0086] As used herein,“down-regulating” or“down-regulation” means a reduction in expression or transcription of a target nucleotide sequence. Down-regulation may be partial or temporary reduction in the expression or transcription of a target nucleotide sequence. Down-regulation may be a complete elimination of the expression or transcription of a target nucleotide sequence.

[0087] As used herein,“knockdown” refers to a partial or temporary reduction in expression or transcription of a target nucleotide sequence. This may be accomplished by administering a complementary nucleotide sequence that binds to the target sequence. Knockdown can be elicited by antisense oligonucleotides, siRNA, and the like.

[0088] As used herein,“knockout” refers to complete elimination of the expression or transcription of a target nucleotide sequence. Knockout may be elicited, for example, by use of a CRISPR system to cleave the target nucleotide sequence out of the target gene.

[0089] As used herein, non-homologous end joining (NHEJ) is a DNA repair mechanism which is a re-ligation of break ends after cleavage of a target nucleotide sequence. [0090] As used herein,“homologous repair/homology directed repair (HR/HDR)” refers to DNA repair which is a process of homologous recombination where a DNA template is used to provide the homology necessary for precise repair of a double-strand break. The repair may consist of insertions of desired sequences, or modification of the target sequence.

[0091] As used herein,“repair template” refers to the DNA template used in HR/HDR.

[0092] As used herein,“subject” means a living organism. Preferably, a subject is a mammal, such as a human, non-human primate, rodent, or companion animal such as a dog, cat, cow, pig, etc.

Modulation of gene expression

[0093] Gene expression can be modulated by administering to a subject in need thereof a composition comprising a nucleotide editing system.

[0094] In one embodiment, modulating expression of a target gene comprises administering to the subject a composition, wherein the composition comprises a nucleic acid editing system comprising at least one nucleotide sequence that is complementary to at least one allele on a target gene associated with comeal dystrophies. In certain embodiments, the at least one nucleotide sequence that is complementary to at least one allele on a target gene is selected from an siRNA, an shRNA, an miRNA, an antisense RNA, or an antagomir RNA.

Administration of the composition

[0095] In certain embodiments, the composition is administered by itself.

[0096] In preferred embodiments, the composition comprises an adeno-associated virus (AAV) vector, or a nucleotide sequence or portion thereof encoding an AAV vector.

AAV

[0097] Adeno-associated vims (AAV) is a small, replication-deficient parvovims. AAV is about 20-24 nm long, with a density of about 1 40 1.41 g/cc. AAV contains a single-stranded linear genomic DNA molecule approximately 4.7 kb in length. The single-stranded AAV genomic DNA can be either a plus strand, or a minus strand. AAV contains two open reading frames, Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). AAVs contain a single intron. Cis-acting sequences directing viral DNA replication (Rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters, p5, pi 9, and p40 (named for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The p5 and pi 9 are the rep promoters. When coupled with the differential splicing of the single AAV intron, the two rep promoters 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 that are responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter, and encodes the three capsid proteins VP1, VP2, and VP3.

Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single polyadenylation site is located at map position 95 of the AAV genome. Muzyczka reviews the life cycle and genetics of AAV (Muzyczka, Current Topics in Microbiology and Immunology , 158:97-129 (1992)).

[0098] AAV infection is non-cytopathic in cultured cells. Natural infection of humans and other animals is silent and asymptomatic (does not cause disease). Because AAV infects many mammalian cells, there is the possibility of targeting many different tissues in vivo. In addition to dividing cells, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (i.e. extrachromosomal element). The AAV proviral genome is infective as cloned DNA in plasmids, which makes construction of recombinant genomes possible. Moreover, because the signals directing AAV replication, genome encapsidation, and integration are all contained with the ITRs of the AAV genome, some or all of the approximately 4.3 kb of the genome, encoding replication and structural capsid proteins (rep-cap) are contained within the ITRs of the AAV genome, and can be replaced with heterologous DNA, such as a gene cassette containing a promoter, a DNA of interest, and a polyadenylation signal. The rep and cap proteins may be provided in trans.

[0099] Several AAV serotypes have been identified, differing in their tropism (type of cell that they infect). Serotype AAVl shows tropism to the following tissues: CNS; heart; retinal pigment epithelium (RPE); and skeletal muscle. Serotype AAV2 shows tropism to the following tissues: CNS; kidney; photoreceptor cells; and RPE. Serotype AAV3 shows tropism mainly to the heart and liver. Serotype AAV4 shows tropism to the following tissues: CNS; lung; and RPE. Serotype AAV5 shows tropism to the following tissues: CNS; lung; photoreceptor cells; and RPE. Serotype AAV6 shows tropism to the following tissues: lung; and skeletal muscle. Serotype AAV7 shows tropism to the following tissues: liver; and skeletal muscle. Serotype AAV8 shows tropism to the following tissues: CNS; heart; liver; pancreas; photoreceptor cells; RPE; and skeletal muscle. Serotype AAV9 shows tropism for the following tissues: CNS; heart; liver; lung; and skeletal muscle. The tropism of AAV viruses may be related to the variability of the amino acid sequences of the capsid protein, which may bind to different functional receptors present on different types of cells.

[00100] Depending on the promoter included in the heterologous DNA cassette, it may be possible to target specific tissues in the eye. Modifying the capsid proteins may also enable specific infectivity of certain tissues or cells. In one embodiment, an AAV containing an Anc80 or Anc80L65 capsid protein is used for delivery of therapeutics directly to specific tissues in the eye. In some embodiments, the AAV viral particle comprises an AAV1,

AAV2, AAV3, AAV4, AAV5, AAV6 (e.g., a wild-type AAV6 capsid, or a variant AAV6 capsid such as ShHIO, as described in U.S. PG Pub. 2012/0164106), AAV7, AAV8,

AAVrh8, AAVrh8R, AAV9 (e.g., a wild-type AAV9 capsid, or a modified AAV9 capsid as described in U.S. PG Pub. 2013/0323226), AAV10, AAVrhlO, AAV11, AAV 12, a tyrosine capsid mutant, a heparin binding capsid mutant, an AAV2R471 A capsid, an AAVAAV2/2- 7m8 capsid, an AAV DJ capsid (e.g., an AAV-DJ/8 capsid, an AAV-DJ/9 capsid, or any other of the capsids described in U.S. PG Pub. 2012/0066783), AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1/AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2/HBoVl 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 comprising an amino acid substitution at one or more of positions R484, R487, K527, K532, R585 or R588, numbering based on VP1 of AAV2. In further embodiments, a AAV particle comprises capsid proteins of an AAV serotype from Classes A-F. In some embodiments, the rAAV viral particle comprises an AAV serotype 2 capsid. In further embodiments, the AAV serotype 2 capsid comprises AAV2 capsid protein comprising a R471A amino acid substitution, numbering relative to AAV2 VP1. In some embodiments, the vector comprises AAV1, AAV2, AAV 3, AAV4, AAV5, AAV6, AAV 7, AAV 8, AAVrh8, AAVrh8R, AAV9, AAV 10, AAVrhlO, AAV11, AAV 12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype inverted terminal repeats (ITRs). In some embodiments, the vector comprises AAV serotype 2 ITRs. In some embodiments, the AAV viral particle comprises one or more ITRs and capsid derived from the same AAV serotype. In other embodiments, the AAV viral particle comprises one or more ITRs derived from a different AAV serotype than the capsid of the rAAV viral particles. In some embodiments, the rAAV viral particle comprises an AAV2 capsid, and wherein the vector comprises AAV2 ITRs. In further embodiments, the AAV2 capsid comprises AAV2 capsid protein comprising a R471A amino acid substitution, numbering relative to AAV2 VP1 (see US Patent Publication

2017/0304465).

[00101] It has recently been shown that including a human rhodopsin kinase (hGRKl) promoter in an AAV5 vector results 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)).

[00102] It has also recently been shown that AAV virions with altered capsid proteins may impart greater tissue specific infectivity. For example, AAV6 with a variant capsid protein shows increased infectivity of retinal cells, compared to wild-type AAV capsid protein (US 8,663,624). A variant capsid protein comprising a peptide insertion between two adjacent amino acids corresponding to amino acids 570 ad 611 of VP1 of AAV2, or the corresponding position in a capsid protein of another AAV serotype, confers increased infectivity of retinal cells, compared to wild-type AAV (US 9,193,956).

Expression of protein in a cornea

[00103] To express specific proteins in a cornea, AAV vectors packaged with either an endonucleotide encoding the desired protein, or AAV vectors packaged with the desired protein may 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). [00104] In certain embodiments, the AAV vectors may be delivered without being enclosed in any particle or lipid vessels. In other embodiments, the AAV vectors may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid for delivery.

[00105] In some embodiments, the compositions and/or the AAV vectors can be delivered directly to the eye. The composition and/or AAV vector may be administered to the anterior chamber of the eye, the posterior chamber of the eye, the cornea, or the vitreous chamber of the eye. In preferred embodiments, the AAV vectors are administered directly to the aqueous humor of the anterior chamber, which is in contact with the cornea. More than one AAV vector such as a dual AAV vector system may be used for the purpose of modulating gene expression as defined in the present invention.

[00106] As explained above, there are several AAV serotypes, each exhibiting tropism for certain types of tissue. Although the AAV serotype used is not particularly limited, the AAV5, AAV6, and AAV8 serotypes are preferred AAV vectors for targeting comeal and anterior tissues in the eye.

[00107] To test the viral tropism of different AAV serotypes in the present invention, several serotypes were packaged with a nucleotide sequence encoding green fluorescent protein (GFP or eGFP). These AAV-eGFP complexes were delivered intracamerally into the eye. The fluorescence of the GFP could be measured in vivo, showing the localization of the AAV-GFP. The localization of the GFP could also be assessed by performing

immunohistochemistry on sections of the eye. The viral tropism of AAV5, as indicated by immunohistochemical staining, was localized to the comeal endothelium. The viral tropism of AAV6 was localized to the cornea endothelium, stroma and endothelium, and ciliary body, with some targeting to retinal cells. The viral tropism of AAV8 was localized to cornea endothelium and stroma, and ciliary body, with some targeting to retinal cells. The viral tropism of AAV2 and AAV9 was localized to both the posterior and anterior segments of the eye after IC administration, with greater expression in the posterior segment than the anterior segment.

[00108] The results show that AAV5, AAV6, and AAV8 show selective tropism for comeal tissues. When selective targeting to the cornea endothelium is desired, use of AAV5 is preferred. Use of other AAV serotypes (e.g. AAV2), which are less tissue selective, may lead to unwanted off-target effects.

[00109] In certain embodiments, the AAV vectors may be delivered by themselves. In other embodiments, the AAV vectors may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid for delivery.

[00110] In some embodiments, the AAV vectors can be delivered directly to the eye. The AAV vector may be administered to the anterior chamber of the eye, the posterior chamber of the eye, the cornea, or the vitreous chamber of the eye. In certain embodiments, the AAV vectors can be administered to the comeal stroma, comeal limbus, onto the epithelial surface of the cornea, or onto the endothelial membrane of the cornea. In preferred embodiments, the AAV vectors are administered directly to the aqueous humor of the anterior chamber which is in direct contact with the comeal endothelium.

Gene targeting using CRISPR complexes

[00111] In certain embodiments, a CRISPR complex is used to modify a specific nucleotide sequence of the DNA of a gene. The specific nucleotide sequence of the DNA of the gene is the“target sequence.”

[00112] A CRISPR complex is a combination of a gRNA and an endonucleotide encoding for a Cas protein (gRNA:Cas endonucleotide), or a combination of a gRNA and a Cas protein (gRNA:Cas protein).

[00113] The gRNA comprises RNA sequences that are directed to a target DNA sequence. The gRNA contains a CRISPR RNA (crRNA) and transactivating crRNA (trRNA or tracrRNA). The crRNA and the trRNA may be associated on a single RNA molecule, referred to as a single guide RNA (sgRNA). Alternatively, the crRNA and trRNA may be disassociated on separate RNA molecules, and form a dual guide RNA (dgRNA). The gRNA can be targeted to either the positive or negative strand of the DNA.

[00114] The gRNA guides the Cas component (i.e. endonucleotide encoding a Cas protein, or a 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 a guide sequence of a gRNA and its corresponding target sequence is at least about 50% or greater. For example, the degree of complementarity or identity may be about 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97% 98%, 99%, or 100%.

[00115] In some embodiments, the gRNA is chemically modified, and comprises one or more modified nucleosides or nucleotides. Modification of nucleosides and nucleotides can include one or more of: i) alteration, e.g. replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone (e.g. phosphorothioate or boranosphosphate linkages); ii) alteration, e.g. replacement, of a constituent of the ribose sugar, such as, for example, 2’-O-methyl and/or 2’-fluoro and/or 4-thio modifications; iii) complete replacement of the phosphate moiety with“dephospho” linkers; iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase; v) replacement or modification of the ribose-phosphate backbone; vi) modification of the 3’ end or 5’ end of the oligonucleotide, e.g. removal, modification, or replacement of a 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-azauridine, 5-hydroxyuridine, 5-aminouridine 5-methyluridine, 2- thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5- aminopseduridine, pseudoisocytidine 5-methylcytidine N-4-methyctidine, 2-thiocytidine, 5- azacytidine 5-hydroxycytidine, 5-aminocytidine, N-4-methylpseudoisocytidine, 2- thiopseudoisocytidine, 5-hydroxypseudoisocytidine, 5-aminopseudisocytidine, 5- methylpseudoisocytidie, 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.

[00116] In some embodiments the Cas component comprises Type-I, Type-II, or Type-Ill components. In certain embodiments, the Cas component is a nuclease. In some

embodiments the Cas nuclease is Cas9 or Cpfl. Preferably the Cas nuclease is Cas9. In some embodiments, the gene-editing molecule is a Cas protein (e.g, Cpfl, CasX , CasY, C2C2, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al , Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), Casio, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cul966, or homologs or modified versions thereof). In some embodiments, the Cas protein is a Cas9 protein (e.g., wild-type Cas9, a Cas9 nickase, a dead Cas9 (dCas9), or a split Cas9). In some embodiments, the Cas9 protein is a Streptococcus pyogenes Cas9 protein or Staphylococcus aureus Cas9 protein.

[00117] Once guided to the target sequence, the Cas nuclease cleaves the target sequence. This leads to double-stranded breaks in the DNA, or single-strand breaks if a nickase enzyme is used. Double-stranded breaks in the DNA can be repaired via non-homologous end joining (NHEJ), which is re-ligation of the break ends. NHEJ can produce indel mutations.

Alternatively, the DNA may be repaired via homologous repair (HR) or homology-directed repair (HDR). HR and HDR generate precise, defined modifications at the target locus in the presence of an exogenously introduced repair template. In certain embodiments, the repair template contains a nucleotide sequence encoding a desirable mutation on a target gene, and the nucleotide sequence is inserted at the target locus of the gene.

[00118] Some of the sequences disclosed herein include the following lists (see WO

2017/185054). SEQ ID NOs: 1-93 are target sequences 5’ of the TNRs in intron 3 of the TCF4 gene. SEQ ID NOs: 94-190 are target sequences 3’ of the TNRs in intron 3 of the TCF4 gene. SEQ ID NOs: 191-1063 are target sequences for the wild type COL8A2 gene. SEQ ID NOs: 1064-1069 are target sequences for the COL8A2 Gln455Lys mutation. SEQ ID NOs: 1070-1075 are target sequences for the COL8A2 Gln455al mutation. SEQ ID NOs: 1076-1084 are target sequences for the COL8A2 Leu450Trp mutation.

[00119] Table A shows the sequences for SEQ ID NOs: 1085-1088 (see Exemplary sequences from WO 2017/185054). [00120] The guide RNA and Cas components (i.e. the CRISPR complexes) are packaged into AAV vectors for delivery to a subject. In certain embodiments, the AAV vectors may be delivered by themselves. In other embodiments, the AAV vectors may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid for delivery.

[00121] In some embodiments, the AAV vectors can be delivered directly to the eye. The AAV vector may be administered to the anterior chamber of the eye, the posterior chamber of the eye, the cornea, or the vitreous chamber of the eye. In certain embodiments, the AAV vectors can be administered to the comeal stroma, comeal limbus, onto the epithelial surface of the cornea, or onto the endothelial membrane of the cornea. In preferred embodiments, the AAV vectors are administered directly to the aqueous humor of the anterior chamber which is in direct contact with the comeal endothelium.

TCF4 gene targeting

[00122] The TCF4 gene is located on chromosome 18. The cytogenic location is 18q21.2 (the long arm of chromosome 18 at position 21.2). The molecular location is on base pairs 55,222,311 to 55,635,993 on chromosome 18 (Homo sapiens Annotation Release 109, GRCh38.pl2 (NCBI)).

[00123] The target sequence may be within or flanking the TNRs in the TCF4 gene. A Cas nuclease is guided to the target sequence. In some embodiments, the Cas nuclease may be guided to a target sequence within the TNRs of the TCF4 gene. In other embodiments, the Cas nuclease may be guided to a target sequence flanking the TNR. For example, the Cas nuclease may be directed to a target sequence 5’ of the TNRs. Or the Cas nuclease may be directed to a target sequence 3’ of the TNRs. In some embodiments, the Cas protein may be directed by two or more gRNAs to two target sequences flanking the TNRs. In some embodiments, the Cas nuclease may be directed by two or more gRNAs to two target sequences, wherein one is within the TNRs of the TCF4 gene, and the other flanks the TNRs of the TCF4 gene. Target sequences for the TCF4 gene are chosen from SEQ ID NOs: 1- 190. SEQ ID NOs: 1-93 are target sequences 5’ of the TNRs in intron 3 of the TCF4 gene. SEQ ID NOs: 94-190 are target sequences 3’ of the TNRs in intron 3 of the TCF4 gene.

Guide sequences for the TCF4 gene are chosen from SEQ ID NOs: 1089-1278. (see

Sequence Listing) [00124] The one or more gRNA comprise a guide sequence that is complementary to a target sequence in the TCF4 gene, or the reverse complement of a target sequence in 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 a guide sequence of a gRNA and its corresponding target sequence is at least about 50% or greater. For example, the degree of complementarity or identity may be about 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97% 98%, 99%, or 100%.

[00125] In some embodiments, one gRNA is used. In other embodiments, a combination of two or more gRNA are used. In certain embodiments, a gRNA targeting a sequence 5’ of the TNRs is used in combination with a gRNA that targets a sequence 3’ to the TNRs, in order to excise the TNRs of the TCF4 gene. In some embodiments, a gRNA complementary to a target sequence chosen from SEQ ID NOs: 1-93 is used together with a gRNA

complementary to a target sequence chosen from SEQ ID NOs: 94-190. Table 1 shows target sequences and corresponding guide sequences (from Ex. 1 of WO 2017/185054). Table 2 shows combinations of guide sequences (From Ex. 1 of WO 2017/185054). (see Exemplary sequences from WO 2017/185054)

[00126] In embodiments wherein the CRISPR complex includes an endonucleotide encoding the 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, Rous sarcoma virus (RSV) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, 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. the comeal edothelium. [00127] In some embodiments, the nucleotide sequence encoding the gRNA may be operably linked to at least one transcriptional or translational control sequences. These include promoters, a 3’ UTR, or a 5’ UTR. The promoter may be recognized by RNA polymerase III (Pol III), such as, but limited to, U6 and HI Pol III promoters. The Pol III promoters may be, for example, mouse or human.

[00128] In certain embodiments, one or more gRNA are packaged in AAV vectors, in combination with either an endonucleotide sequence encoding a Cas protein, or a Cas protein (e.g. Cas9) (i.e. CRISPR complexes). The AAV serotype used is not particularly limited. Preferably, the AAV vectors are of the AAV5, AAV6, or AAV8 serotype.

[00129] The AAV-CRISPR complexes can be delivered directly into the eye via intracameral or intrastromal injection. In certain embodiments, the AAV vectors may be delivered by themselves. In other embodiments, the AAV vectors may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid for delivery.

[00130] In some embodiments, the AAV vectors can be delivered directly to the eye. The AAV vector may be administered to the anterior chamber of the eye, the posterior chamber of the eye, the cornea, or to the vitreous chamber of the eye. In certain embodiments, the AAV vectors can be administered to the comeal stroma, comeal limbus, onto the epithelial surface of the cornea, or onto the endothelial membrane of the cornea. In preferred embodiments, the AAV vectors are administered directly to the aqueous humor of the anterior chamber which is in direct contact with the comeal endothelium.

COL8A2 gene targeting

[00131] Mutations in the COL8A2 gene, and thus the mutations in the gene products, can also be treated with the methods and compositions described herein. This can be done by developing CRISPR complexes that target specific sequences in the COL8A2 gene that lead to the mutations.

[00132] In some embodiments, a CRISPR complex can be used to excise a target mutant nucleotide sequence on the COL8A2 gene, and excise a nucleotide sequence of the DNA encoding a mutated gene product. The DNA may then be repaired with the process of NHEJ, leading to the generation of indels and the loss of the mutant allele. In other embodiments, use of the CRISPR complexes can be done together with either an exogenous template for HR/HDR, or using the endogenous normal allele as a template for HR/HDR, resulting in correction of the nucleic acid mutation that leads to the amino acid mutation in the alpha 2 subunit of COL8. Mutations that can be corrected include: the Gln455Lys mutation, caused by the C.13640A nucleotide change; the Gln455Val mutation caused by the c.1363- 1364CA>GT nucleotide changes; or the Leu450Trp mutation caused by the c.1349T>G nucleotide change.

[00133] Target sequences for 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 in the amino acid sequence of the COL8 gene product, and may be considered the“wild type” COL8A2 gene sequence. Target sequences can be selected between Chrl:36097532-36100270 (hg38). Target sequences for the COL8A2 gene are selected from SEQ ID NOs: 191-1063. Target sequences for 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.

[00134] Target sequences to the mutant alleles can also be developed, based on the differences in the nucleotide sequences for the mutant alleles. Table 4 shows target sequences specific for the Glnl55Lys mutation, caused by the C.13640A nucleotide change (SEQ ID NOs: 1064-1069). Table 5 shows target sequences specific for the Gln455Val mutation, caused by the c 1363-1364CA>GT nucleotide changes (SEQ ID NOs: 1070-1075). Table 6 shows target sequences specific for Leu450Trp mutation, caused by the c 1349T>G nucleotide change (SEQ ID NOs: 1076-1084). The mutant alleles could be targeted using gRNA comprising guide sequences complementary to the target sequences, or comprising guide sequences complementary to the reverse complement of the target sequences.

[00135] In certain embodiments, one or more gRNA are packaged in AAV vectors, in combination with either an endonucleotide sequence encoding a Cas protein, or a Cas protein (e.g. Cas9) (i.e. CRISPR complexes). The AAV serotype used is not particularly limited. Preferably, the AAV vectors are of the AAV5, AAV6, or AAV8 serotype. [00136] The AAV-CRISPR complexes can be delivered directly into the eye via intracameral or intrastromal injection. In certain embodiments, the AAV vectors may be delivered by themselves. In other embodiments, the AAV vectors may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid for delivery.

[00137] In some embodiments, the AAV vectors can be delivered directly to the eye. The AAV vector may be administered to the anterior chamber of the eye, the posterior chamber of the eye, or the cornea. In certain embodiments, the AAV vectors can be administered to the comeal stroma, comeal limbus, onto the epithelial surface of the cornea, or onto the endothelial membrane of the cornea. In preferred embodiments, the AAV vectors are administered directly to the aqueous humor of the anterior chamber which is in direct contact with the comeal endothelium.

Exemplary embodiments

[00138] In certain embodiments, the mutant allele is encoded by a target sequence on the target gene.

[00139] In some embodiments, at least one nucleotide sequence that is complementary to at least one mutant allele on a target gene hybridizes to a target sequence on the target gene in a cell in the subject.

[00140] In certain embodiments the target gene is TCF4 or COL8A2.

[00141] In some embodiments, at least one target sequence is selected from the group consisting of SEQ ID NOs: 1-1084.

[00142] 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.

[00143] In some embodiments, the target sequence is specific to the COL8A2 gene and the target sequence is selected from SEQ ID NOs: 191-1084 [00144] In some embodiments, the nucleic acid editing system is a CRISPR system, an siRNA, an shRNA, an miRNA, an antisense RNA, or an antagomir RNA.

[00145] In some embodiments, the nucleic acid editing system is a CRISPR system.

[00146] In some embodiments, the nucleic acid editing system is a CRISPR-Cas system.

[00147] 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).

[00148] In some embodiments, the Cas gene encodes a Cas protein.

[00149] In some embodiments, the Cas protein encoded by the Cas gene is a Cas nuclease. [00150] In some embodiments, the Cas nuclease is Cas9.

[00151] In some embodiments, the guide RNA comprises a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA or trRNA).

[00152] In some embodiments, the guide RNA is a single guide RNA (sgRNA), and both the crRNA and the tracrRNA are combined on one guide RNA molecule.

[00153] In some embodiments, the guide RNA is a double guide RNA (dgRNA), and the crRNA and the tracrRNA are on separate RNA molecules, used at the same time, but not combined.

[00154] In some embodiments, the CRISPR-Cas system is a CRISPR-Cas9 system.

[00155] In some embodiments, the crRNA and tracrRNA form a complex with the nucleotide sequence encoding Cas9 nuclease. [00156] In some embodiments, the nucleotide sequence that is complementary to at least one mutant allele is a gRNA.

[00157] 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.

[00158] In some embodiments, at least one guide RNA comprises a guide sequence selected from the group consisting of SEQ ID NOs: 1089-1278.

[00159] In some embodiments, the delivery system, vector, gene editing system, or composition further comprises a repair template.

[00160] 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.

[00161] In some embodiments, the AAV vector serotype is selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO.

[00162] In some embodiments, the AAV vector serotype is AAV5, AAV6, or AAV8.

[00163] In some embodiments, the AAV vector serotype is AAV5.

[00164] In some embodiments, the AAV vector serotype is AAV6.

[00165] In some embodiments, the AAV vector serotype is AAV8.

[00166] In some embodiments, the delivery system, vector, nucleotide or gene editing system, or composition further comprises a promoter.

[00167] In some embodiments, the promoter is optimized for use with an AAV5, AAV6 or AAV8 vector. [00168] In some embodiments, the promoter is tissue specific, and when operably linked with the AAV vector or the nucleotide that is a sequence that is complementary to at least one mutant allele on a target gene is active in the eye.

[00169] In some embodiments, the tissue specific promoter is active in the cornea or other anterior ocular tissues.

[00170] In some embodiments, the tissue specific promoter is active in the endothelium of the cornea.

[00171] In some embodiments, the target gene is preferentially expressed in the anterior portion of the eye. Preferably, the target gene is preferentially expressed in the cornea, and most preferably, preferentially expressed in the endothelium of the cornea.

[00172] In some embodiments, the delivery system, vector, nucleotide or gene editing system, or composition is preferentially expressed in the anterior portion of the eye after IC injection. Preferably, the delivery system, vector, nucleotide or gene editing system, or composition is preferentially expressed in the cornea, and most preferably, preferentially expressed in the endothelium of the cornea, after IC injection.

[00173] In some embodiments, the delivery system, vector, nucleotide or gene editing system, or composition is suitable for treating a disease or condition in the eye.

[00174] In some embodiments, the disease or condition in the eye is a disease or condition of the cornea.

[00175] In some embodiments, the disease or condition of the cornea is a superficial comeal dystrophy, anterior comeal dystrophy, comeal stromal dystrophy, or posterior cornea dystrophy.

[00176] In some embodiments, the disease or condition of the cornea is a posterior comeal dystrophy. [00177] In some embodiments, the posterior comeal dystrophy is Fuchs endothelial comeal dystrophy (FECD; both early and late onset), posterior polymorphous dystrophy (PPCD; types 1, 2, and 3), congenital endothelial dystrophy (types 1 and 2), and X-linked endothelial comeal dystrophy.

[00178] In some embodiments, the comeal dystrophy is FECD.

EXAMPLES

Example 1. Methods of preparing and administering AAV vectors

AAV vectors

[00179] Wildtype AAV2, AAV5, AAV6, AAV8, and AAV9 vectors were produced by methods known in the art. Each AAV encoded for eGFP under the ubiquitous CAG promoter. Each AAV was supplied at lel3vg/mL in a PBS + 0.001% pluronic acid formulation.

Intracameral (IC) injections

[00180] Adult male C57BL/6J mice (10-11 weeks old) were purchased from Jackson Laboratories. All animal procedures and handling were conducted according to the ARVO Statement for the use of Animals and the Regeneron Pharmaceuticals IACUC reviewed protocol. Mice were anesthetized with ketamine/xylazine mixture by intraperitoneal injection. The eyes were rinsed with sterile saline followed by a drop of tropicamide (to dilate the pupil) with a drop of proparacaine (to numb the cornea). Using a Drummond Scientific Nanoject II microinjection device fitted with a pulled glass needle (sandpaper beveled), AAV solution was filled into the needle and used to inject 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. Bubbles were pushed into cornea followed by 1.5 pL of AAV solution, containing 1.5el0 vg. The needle was held still after the injection for 30 sec and then pulled out in a quick smooth motion. Both OD (right eye) and OS (left eye) of each animal were injected. Control animals received injections of PBS+0.001% pluronic acid instead of AAV solution. Genteal ointment was applied to each eye to prevent comeal drying and abrasion while the mouse was placed on its ventral side (to prevent leakage and pooling) to recover from anesthesia. Intravitreal injections

[00181] Adult male C57BL/6J mice (10-11 weeks old) were purchased from Jackson Laboratories. All animal procedures and handling were conducted according to the ARVO Statement for the use of Animals and the Regeneron Pharmaceuticals IACUC reviewed protocol. Mice were anesthetized with ketamine/xylazine mixture by intraperitoneal injection. The eyes were rinsed with sterile saline followed by a drop of tropicamide (to dilate the pupil) with a drop of proparacaine (to numb the cornea). Using a Drummond Scientific Nanoject II microinjection device fitted with a pulled glass needle (sandpaper beveled), AAV solution was filled into the needle and used to inject AAV solution into the vitreous humor of the vitreous chamber. The glass needle was injected through the sclera at the limbus of the eye into the vitreous chamber. 1.5 pL of AAV solution, containing 1.5el0 vg, was injected into the vitreous chamber using the microinjection device. The needle was pulled out in a quick smooth motion. Both OD (right eye) and OS (left eye) of each animal were injected. Control animals received injections of PBS+0.001% pluronic acid instead of AAV solution. Genteal ointment was applied to each eye to prevent comeal drying and abrasion while the mouse was placed on its ventral side (to prevent leakage and pooling) to recover from anesthesia.

Example 2. Assessment of specificity of protein targeting to different tissues in the eve.

In Vivo imaging

[00182] In vivo imaging was performed at baseline prior to injections and at timepoints post injections using the Heidelberg Spectralis HRA+OCT (Heidelberg Engineering, Inc, Germany). Mice were anesthetized and a drop of tropicamide was applied to each eye to dilate the pupil, followed by a drop of proparacaine to numb the cornea. At each time point, infrared images and fluorescence images to detect AAV-eGFP fluorescence were taken of the posterior retinal fundus (+25 diopter small animal imaging lens) and the anterior cornea (anterior segment module). The FA modality on the Heidelberg Spectralis HRA+OCT was used to detect fluorescence of eGFP protein resulting from the AAV-eGFP injections.

Immunohistochemistrv

[00183] Mice that received AAV-eGFP injections, such as AAV5-eGFP, AAV6-eGFP, and AAV8-eGFP, by intraocular injection were euthanized for enucleation of their eyes. Control mice that received PBS+0.001% pluronic acid intraocular injections were euthanized for enucleation of their eyes. Each eye was enucleated and fixed in 4% PFA overnight at 4°C. The eyes were washed in PBS followed by incubation in 30% sucrose at 4°C for a minimum of 3 days. Eyes were then embedded in OCT embedding compound and a subset of the samples were sent for cross-sectioning by Histoserv Inc (Maryland). In order to amplify regions of AAV-eGFP localization, a primary antibody for eGFP was incubated on the slides containing cross-sectioned mouse eye tissues at 4°C overnight. The secondary antibody was conjugated to Alexa-Fluor 594 (red) to differentiate from the green endogenous eGFP unamplified signal. DAPI (blue) was added to the slides to label nuclei and aid in the identification of cellular types and regions.

[00184] The slides were imaged using the Keyence microscope (Keyence Corporation of America, Illinois, USA). Regions of green and/or red fluorescence were assessed for both anatomical ocular regions and cellular localization. eGFP protein measurement

[00185] Four mice (whole eyes) for each of the AAV serotypes, such as AAV5-eGFP, AAV6-eGFP, AAV8-eGFP, delivered by intraocular injections were euthanized for enucleation of their eyes. Two mice that received PBS+0.001% pluronic acid were included as controls for each of the AAV serotypes tested and were euthanized for enucleation of their eyes. Each eye was kept separate and processed as an individual sample. The eyes were immersed in IX cell extraction buffer PTR (provided in the ELISA kit) and were homogenized using a tissuelyzer with stainless steel beads. The samples were centrifuged and the protein containing lysate was collected. Total protein measurements were measured using the BCA kit (Pierce BCA Protein Assay kit, ThermoFisher). Samples were assayed in triplicates for eGFP protein expression using the GFP SimpleStep ELISA kit (Abeam). eGFP expression per eye was calculated as ng/pg of total protein isolated from the eye.

[00186] Transduction efficiency and tropism varied depending on the AAV serotype used. Using Heidelberg Spectralis in vivo imaging, regions of AAV transduction after IC administration were determined. AAV2, AAV6, AAV8, and AAV9were 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 targets only anterior ocular tissues. The data also indicate that that AAV5, AAV6, and AAV8 have a strong tropism for anterior regions after IC injections. Additionally, IC injections are also capable of delivering AAVs to the posterior tissues, as shown by the strong tropism of AAV2 and AAV9 to the posterior regions after IC injections.

Example 3. Correction of a gene mutation in the endothelial cells of the cornea

[00187] Corrections of target gene mutations such as mutations in TCF4 or COL8A2 in the endothelial cells of the cornea are done by administering a composition comprising a nucleic acid editing system comprising a CRISPR/Cas complex.

[00188] The CRISPR/Cas complex comprises a guide sequence that is complementary to a portion of the target gene containing the mutation and is directed to the target DNA sequence, and an endonucleotide encoding for a Cas nuclease.

[00189] The CRISPR/Cas complex is guided to the target sequence, and the Cas nuclease cleaves the target sequence. A gene insertion mutation is corrected by cleaving the target sequence, and repairing the break in the DNA. A gene mutation that is a change in a nucleotide is corrected by cleaving the mutated sequence nucleotide sequence, and repairing the DNA with a repair template comprising the nucleotide sequence of the wild-type gene.

[00190] The CRISPR/Cas complex is preferably packaged in an AAV vector, such as AAV5, AAV6 or AAV8. AAV vectors are produced by methods known in the art. Each AAV encodes for a target sequence under the ubiquitous CAG promoter. Each AAV is supplied at lel3g/mL in a PBS + 0.001% pluronic acid formulation.

[00191] The AAV vector packaged with the CRISPR/Cas complex is administered directly to the anterior chamber of the eye via intracameral injection. Mice carrying such mutations are anesthetized with ketamine/xylazine mixture by intraperitoneal injection. The eyes are rinsed with sterile saline followed by a drop of tropicamide (to dilate the pupil) with a drop of proparacaine (to numb the cornea). Using a Drummond Scientific Nanoject II microinjection device fitted with a pulled glass needle (sandpaper beveled), AAV solution is filled into the needle and used to inject 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 is allowed to leak out. Bubbles are pushed into cornea followed by 1.5 pL of AAV solution, containing 1.5el0 vg. The needle is held still after the injection for 30 sec and then pulled out in a quick smooth motion. Both OD (right eye) and OS (left eye) of each animal are injected. Control animals receive injections of PBS+0.001% pluronic acid instead of AAV solution. Genteal ointment is applied to each eye to prevent comeal drying and abrasion while the mouse is placed on its ventral side (to prevent leakage and pooling) to recover from anesthesia.

[00192] Corrections of gene expression is confirmed by dissecting corneas (as well as isolating endothelial cells from said corneas) from the eyes of treated and control mice, and doing DNA and/or RNA nucleic acid sequencing.

Example 4 - Downregulation of gene expression in the endothelial cells of the cornea.

[00193] Gene expression is downregulated by administering a composition comprising at least one inhibitory nucleotide sequence that is complementary to at least one allele on a target gene, selected from an siRNA, an shRNA, an miRNA, an antisense RNA, or an antagomir RNA. The target gene is any cornea mutated gene such as TCF4 or COL8A2. The inhibitory RNA is present in the composition by itself, or as part of a CRISPR/Cas complex.

[00194] The inhibitory RNA is packaged in an AAV vector similarly to Example 3. The inhibitory RNA is preferably packaged in an AAV vector, such as AAV5, AAV6 or AAV8. AAV vectors are produced by methods known in the art. Each AAV encodes for a target sequence under the ubiquitous CAG promoter. Each AAV is supplied at lel3g/mL in a PBS + 0.001% pluronic acid formulation.

[00195] Similarly to Example 3, the AAV vector packaged with the inhibitory RNA is administered directly to the anterior chamber of the eye via intracameral injection. Mice are anesthetized with ketamine/xylazine mixture by intraperitoneal injection. The eyes are rinsed with sterile saline followed by a drop of tropicamide (to dilate the pupil) with a drop of proparacaine (to numb the cornea). Using a Drummond Scientific Nanoject II microinjection device fitted with a pulled glass needle (sandpaper beveled), AAV solution is filled into the needle and used to inject 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 is allowed to leak out. Bubbles are pushed into the cornea followed by 1.5 pL of AAV solution, containing 1.5el0 vg. The needle is held still after the injection for 30 sec and then pulled out in a quick smooth motion. Both OD (right eye) and OS (left eye) of each animal are injected. Control animals receive injections of PBS+0.001% pluronic acid instead of AAV solution. Genteal ointment is applied to each eye to prevent comeal drying and abrasion while the mouse is placed on its ventral side (to prevent leakage and pooling) to recover from anesthesia.

[00196] Downregulation of gene expression is confirmed by dissecting corneas from the eyes of treated and control mice, and measuring the amount of the protein encoded by the gene in the samples via Western blot. Successful downregulation of gene expression results in reduced levels of the encoded protein in comeal tissue from treated mice versus control mice.

[00197] The present invention has been described in detail, including the preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and/or improvements on this invention that fall within the scope and spirit of the invention.

Exemplary sequences from WO 2017/1 5054

Table A. SEQ ID NOs: 1085-1088

{02556636.1} 46

Attorney Docket No : 67000-1023 WO

Table 1. TCF4 target sequences and corresponding guide sequences

Table 2. Combinations of TCF4 guide sequences

Table 3. Target sequences for wild-type COL8A2 gene

{02544745.1} 95

{02544753.1} 99

{02544766.1} 102 Table 4. Target sequences for COL8A2 with Gln455Lys mutation

Table 5. Target sequences for COL8A2 with Gln455Val mutation

Table 6. Target sequences for COL8A2 with Leu450Trp mutation