Patent Application
20200010854
Corneal dystrophies are a group of disorders that are generally inherited, bilateral, symmetric, slowly progressive, and not predominantly related to environmental or systemic factors (1,2). Corneal dystrophies can affect any anatomic layer, cell type, or tissue of the cornea and result in loss of corneal clarity and reduction in vision (1,3). Corneal dystrophies as a group affect >4% of the US population, and corneal transplantation is definitive treatment for corneal dystrophies of sufficient severity to cause significant vision loss. Fuchs endothelial corneal dystrophy (FECD) is the most common corneal dystrophy affecting approximately 4% of the US population. Approximately 70% of FECD cases are caused by a microsatellite trinucleotide repeat expansion in the transcription factor 4 (TCF4) gene (4). Additional microsatellite expansion diseases have been described (5).
Thus there is a great need for novel and improved therapies for treating disorders affecting ocular and non-ocular tissues, like corneal dystrophies and microsatellite expansion diseases affecting the eye and other tissues and organs throughout the body.
Described herein are methods for treating disorders affecting ocular and non-ocular tissues, such as corneal dystrophies and microsatellite expansion diseases. The methods use a nuclease system, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) 9 (CRISPR-Cas9), to cut and/or repair genomic DNA. The CRISPR-Cas9-based gene editing can be used to inactivate or correct gene mutations causing corneal dystrophies and microsatellite expansion diseases, thereby providing a gene therapy approach for these groups of diseases.
One aspect of the invention relates to a method for treating a disorder affecting ocular tissue in a subject, the method comprising administering to the ocular area of the subject a therapeutically effective amount of a nuclease system comprising a genome targeted nuclease and a guide DNA comprising at least one targeted genomic sequence.
In certain embodiments, the nuclease can be provided as a protein, RNA, DNA, or an expression vector comprising a nucleic acid that encodes the nuclease.
In certain embodiments, the guide DNA can be provided as an RNA molecule (gRNA), DNA molecule, or an expression vector comprising a nucleic acid that encodes the gRNA.
In certain embodiments, the guide DNA may be provided as one, two, three, four, five, six, seven, eight, nine, or ten RNA molecules (gRNA), DNA molecules, or expression vectors comprising a nucleic acid that encodes the gRNA, or any combination thereof.
In certain embodiments, the nuclease system can be CRISPR-Cas9.
In certain embodiments, the nuclease system inactivates or excises gene mutations.
In certain embodiments, the system further comprises a DNA double-stranded break (DSB) repair system.
In certain embodiments, the DSB repair system comprises a repair template in combination with or without a Non-Homologous End-Joining (NHEJ) or Homology Directed Repair (HDR) targeted to the one or more CRISPR-Cas9 cleavage site, said site corrects or edits a genomic mutation.
In certain embodiments, the DSB repair system is provided by the host cell machinery.
In certain embodiments, the genome targeted nuclease can be Cas9.
In certain embodiments, the disorder can be a corneal dystrophy or microsatellite expansion disease.
In certain embodiments, the ocular area can be the cornea.
In certain embodiments, the guide DNA comprises at least one, two, three, four, five, six, seven, eight, nine, or ten targeted genomic sequences.
In certain embodiments, the target genomic sequences are selected from any one of the nucleotide sequences set forth in SEQ ID NOs: 1-172 and 174-342, or any combination thereof.
In certain embodiments, the nuclease system can be administered topically to the surface of the eye.
In certain embodiments, the nuclease system can be administered on or outside the cornea, sclera, to the intraocular, subconjunctival, sub-tenon, or retrobulbar space, or in or around the eyelids.
In certain embodiments, the nuclease system can be administered by implantation, injection, or virally.
Another aspect of the invention relates to a method for treating a disorder affecting non-ocular tissue in a subject, the method comprising administering to the non-ocular tissue of the subject a therapeutically effective amount of a nuclease system comprising a genome targeted nuclease and a guide DNA comprising at least one targeted genomic sequence.
In certain embodiments, the nuclease can be provided as a protein, RNA, DNA, or an expression vector comprising a nucleic acid encoding the nuclease.
In certain embodiments, the guide DNA can be provided as an RNA molecule (gRNA), DNA molecule, or an expression vector comprising a nucleic acid that encodes the gRNA.
In certain embodiments, the nuclease system can be CRISPR-Cas9.
In certain embodiments, the nuclease system inactivates or excises gene mutations.
In certain embodiments, the method further comprises a DNA double-stranded break (DSB) repair system.
In certain embodiments, the DSB repair system comprises a repair template in combination with a Non-Homologous End-Joining (NHEJ) or Homology Directed Repair (HDR) targeted to the one or more CRISPR-Cas9 cleavage site, said site corrects or edits a genomic mutation.
In certain embodiments, the genome targeted nuclease can be Cas9.
In certain embodiments, the disorder can be microsatellite expansion disease.
In certain embodiments, the guide DNA comprises at least one, two, three, four, five, six, seven, eight, nine, or ten targeted genomic sequences.
In certain embodiments, the target genomic sequences are selected from any one of the nucleotide sequences set forth in SEQ ID NOs: 1-172 and 174-342, or any combination thereof.
In certain embodiments, the nuclease system is administered topically, intravascularly, intradermally, transdermally, parenterally, intravenously, intramuscularly, intranasally, subcutaneously, regionally, percutaneously, intratracheally, intraperitoneally, intraarterially, intravesically, intratumorally, inhalationly, perfusionly, lavagely, directly via injection, or orally via administration and formulation.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Described herein are methods for treating eye disorders, such as corneal dystrophies and microsatellite expansion diseases. The methods use a nuclease system, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) 9 (CRISPR-Cas9), to cut, nick, and/or repair genomic DNA.
As used herein, the term “eye disease” may encompass disorders of the eye including, but not limited to corneal dystrophies and microsatellite expansion diseases.
As used herein, the term “corneal dystrophy” or “corneal dystrophies” describes a group of disorders that are generally inherited, bilateral, symmetric, slowly progressive, and not predominantly related to environmental or systemic factors (1,2). Corneal dystrophies, include (but may not be limited to) the following: Epithelial Basement Membrane Dystrophy (aka Map-Dot-Fingerprint Dystrophy, Cogan Microcystic Epithelial Dystrophy, Anterior Basement Membrane Dystrophy); Epithelial Recurrent Erosion Dystrophies (aka Franceschetti Corneal Dystrophy, Dystrophia Smolandiensis, Dystrophia Helsinglandica); Subepithelial Mucinous Corneal Dystrophy; Meesmann Corneal Dystrophy (aka Juvenile Hereditary Epithelial Dystrophy, Stocker Holt Dystrophy); Lisch Epithelial Corneal Dystrophy (aka Band-Shaped and Whorled Microcystic Dystrophy); Gelatinous Drop-like Corneal Dystrophy (aka Subepithelial Amyloidosis, Primary Familial Amyloidosis (of Grayson)); Reis-Bucklers Corneal Dystrophy (aka Corneal Dystrophy of Bowman layer, type I (CDB I), Geographic Corneal Dystrophy (of Weidle), Atypical Granular Corneal Dystrophy, Granular Corneal Dystrophy, Type 3, Anterior Limiting Membrane Dystrophy, Type 1, Superficial Granular Corneal Dystrophy); Thiel-Behnke Corneal Dystrophy (aka Corneal Dystrophy of Bowman layer, Type II (CDB2), Honeycomb-Shaped Corneal Dystrophy, Anterior Limiting Membrane Dystrophy, Type II, Curly Fibers Corneal Dystrophy, Waardenburg-Jonkers Corneal Dystrophy); Lattice Corneal Dystrophy, Type 1 (Classic) (aka Biber-Haab-Dimmer Dystrophy); Lattice Corneal Dystrophy, Type 2 (aka Familial Amyloidosis (Finnish Type or Gelsolin Type), Meretoja Syndrome); Lattice Corneal Dystrophy, Type III; Lattice Corneal Dystrophy, Type IIIA; Lattice Corneal Dystrophy, Type I/IIIA; Lattice Corneal Dystrophy, Type IV; Polymorphic (Corneal) Amyloidosis; Granular Corneal Dystrophy, Type 1 (aka Corneal Dystrophy Groenouw Type I); Granular Corneal Dystrophy, Type 2 (aka Avellino Dystrophy, Combined Granular-Lattice Dystrophy); Macular Corneal Dystrophy (aka Groenouw Corneal Dystrophy Type II, Fehr Speckled Dystrophy); Schnyder Corneal Dystrophy (aka Schnyder Crystalline Corneal Dystrophy (SCCD), Schnyder Crystalline Dystrophy Sine Crystals, Hereditary Crystalline Stromal Dystrophy of Schnyder, Crystalline Stromal Dystrophy, Central Stromal Crystalline Corneal Dystrophy, Corneal Crystalline Dystrophy of Schnyder, Schnyder Corneal Crystalline Dystrophy); Congenital Stromal Corneal Dystrophy (aka Congenital Hereditary Stromal Dystrophy); Fleck Corneal Dystrophy (aka François-Neetens Speckled (Mouchetée) Corneal Dystrophy); Posterior Amorphous Corneal Dystrophy (aka Posterior Amorphous Stromal Dystrophy); Central Cloudy Dystrophy of Francois; Pre-Descemet Corneal Dystrophy; Fuchs Endothelial Corneal Dystrophy (aka Endoepithelial Corneal Dystrophy); Posterior Polymorphous Corneal Dystrophy (aka Posterior Polymorphous Dystrophy, Schlichting Dystrophy); Congenital Hereditary Endothelial Dystrophy (aka Maumenee Corneal Dystrophy); X-linked Endothelial Corneal Dystrophy.
All of the above disorders are caused by known or putative genetic mutations. Corneal dystrophies yet to be described will be caused by known or putative genetic mutations. Thus, all genetic corneal dystrophies can be amenable to the nuclease system, like CRISPR-Cas9, for gene therapy involving correction or inactivation of the mutant allele.
As used herein, “microsatellite sequences”, also called short tandem repeats, are short DNA sequences (usually 2-5 nucleotides) which are repeated, typically in the range of 5-50 times. These sequences are present throughout the human genome and can become mutated and/or increased in the number of repeats. Some microsatellite sequences, if they expand beyond a certain length, can result in microsatellite expansion diseases. All known or yet to be described microsatellite expansion diseases will be caused by expansions in known or putative genes. Thus, all microsatellite expansion diseases can be amenable to CRISPR-Cas9 gene therapy involving correction or inactivation of the mutant allele.
Microsatellite expansion diseases as used herein may encompasses diseases that affect ocular and non-ocular tissues, including (but may not be limited to) the following disorders: Blepharophimosis, ptosis and epicanthus inversus syndactyly; Cleidocranial dysplasia; Congenital central hypoventilation syndrome, Haddad syndrome DM (Myotonic dystrophy); FRAXA (Fragile X syndrome); FRAXE (Fragile XE mental retardation); FRDA (Friedreich's ataxia); Fuchs' Endothelial Corneal Dystrophy; FXTAS (Fragile X-associated tremor/ataxia syndrome); Hand-foot-genital syndrome; HD (Huntington's disease); Holoprosencephaly; Mental retardation with growth hormone deficiency; Mental retardation, epilepsy, West syndrome, Partington syndrome; Oculopharyngeal muscular dystrophy; SBMA (Spinal and bulbar muscular atrophy); SCA1 (Spinocerebellar ataxia Type 1); SCA12 (Spinocerebellar ataxia Type 12); SCA17 (Spinocerebellar ataxia Type 17); SCA2 (Spinocerebellar ataxia Type 2); SCA3 (Spinocerebellar ataxia Type 3 or Machado-Joseph disease); SCA6 (Spinocerebellar ataxia Type 6); SCAT (Spinocerebellar ataxia Type 7); SCA8 (Spinocerebellar ataxia Type 8); Synpolydactyly.
As used herein, the term “eye”, “eye area” or “ocular area” of the subject encompasses the cornea, conjunctiva, sclera, fovea, macula, optic nerve, retina, lens, iris, pupil, to the intraocular, subconjunctival, sub-tenon, or retrobulbar space, or in or around the eyelids, and other anatomical features of the eye.
As used herein the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) 9 nuclease are an extremely versatile and accurate approach to cut and/or repair genomic DNA (6). CRISPR-Cas9-based gene editing can be used to inactivate or correct gene mutations causing corneal dystrophies and microsatellite expansion diseases, thereby providing a gene therapy approach for these groups of diseases. The naturally occurring CRISPR system from S. pyogenes has been modified to utilize a single guide RNA (gRNA) consisting of a 20 nucleotide (nt) target sequence and an additional structural RNA portion which binds the Cas9 double strand nuclease (6,7). The CRISPR-Cas9 system from S. pyogenes has the potential to cut at any 20 nt sequence adjacent to a 5′-NGG-3′ protospacer-adjacent motif (PAM), or alternate PAM sequences and bioinformatics provides tools to map target sites (8, 10). DNA cut by Cas9 is repaired by endogenous cellular mechanisms, including non-homologous end-joining (NHEJ), which produces insertion deletion mutations that can inactivate the original mutant allele. Thus, CRISPR-Cas9 can correct disease causing genetic mutations by cutting DNA in close enough proximity to a protein coding mutation to inactivate it through frameshifting. Alternatively, CRISPR-Cas9 can correct disease causing genetic mutations, either coding or non-coding, by cutting DNA on both sides of a mutation to excise it, or nicking on different strands flanking the mutation or repeat, if the distance is under 200 bp or so, or through the use of a repair template and homology directed repair (HDR) targeted to one or more CRISPR-Cas9 cleavage sites. Thus, specific mutant sequences can be gene edited and repaired.
CRISPR-Cas9 applied to corneal cells can correct the genetic defect causing corneal dystrophies and thus be used to treat these disorders. The CRISPR-Cas9 treatment could be administered topically to the surface of the eye, via implant, or via injection. The implant or injection could be administered to the cornea, sclera, to the intraocular, subconjunctival, sub-tenon, or retrobulbar space, or in or around the eyelids. CRISPR-Cas9 can also be applied outside the cornea or eye to treat other microsatellite expansion diseases in addition to Fuchs endothelial corneal dystrophy. CRISPR-Cas9 approaches to treat corneal dystrophies and microsatellite expansion diseases could employ single or multiple guide RNAs to inactivate or excise gene mutations, or using a repair template to correct gene mutations. In other embodiments, the CRISPR-Cas9 treatment may be applied to non-ocular tissue to correct the genetic defect causing microsatellite expansion diseases.
In certain embodiments, the routes of CRISPR-Cas9 treatment administration can vary with the location and nature of the cells or tissues to be contacted, and include, e.g., intravascular, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional, percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, direct injection, and oral administration and formulation, or any of the following routes of administration. The term “systemic administration” refers to administration in a manner that results in the introduction of the composition into the subject's circulatory system or otherwise permits its spread throughout the body. “Regional” administration refers to administration into a specific, and somewhat more limited, anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ. “Local administration” refers to administration of a composition or drug into a limited, or circumscribed, anatomic space, such as intratumoral injection into a tumor mass, subcutaneous injections, intradermal or intramuscular injections. Those of skill in the art will understand that local administration or regional administration may also result in entry of a composition into the circulatory system i.e., rendering it systemic to one degree or another. For example, the term “intravascular” is understood to refer to delivery into the vasculature of a patient, meaning into, within, or in a vessel or vessels of the patient, whether for systemic, regional, and/or local administration. In certain embodiments, the administration can be into a vessel considered to be a vein (intravenous), while in others administration can be into a vessel considered to be an artery. Veins include, but are not limited to, the internal jugular vein, a peripheral vein, a coronary vein, a hepatic vein, the portal vein, great saphenous vein, the pulmonary vein, superior vena cava, inferior vena cava, a gastric vein, a splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and/or femoral vein. Arteries include, but are not limited to, coronary artery, pulmonary artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and/or ciliary artery. It is contemplated that delivery may be through or to an arteriole or capillary.
The CRISPR-Cas system may be used facilitate targeted genome editing in eukaryotic cells, including mammalian cells, such as human cells. To facilitate genome editing, the cell to be modified is co-transfected with an expression vector encoding Cas9 or the Cas9 protein, DNA, or RNA itself, along with a guide-RNA molecule itself, or an expression vector comprising a nucleic acid molecule encoding the guide-RNA molecule. For example, in certain embodiments, the introduction of Cas9 can be done by transfecting in Cas9 as a protein, RNA, DNA, or expression vector comprising a nucleic acid that encodes Cas9. In certain embodiments, the guide DNA can itself be administered directly as an RNA molecule (gRNA), DNA molecule, or as expression vector comprising a nucleic acid that encodes the gRNA.
While many different CRISPR-Cas systems could be modified to facilitate targeted genome modification, the most commonly used CRISPR-Cas system in targeted genome modification is the CRISPR-Cas9 system from S. pyogenes. The CRISPR-Cas9 system requires only a single protein, Cas9, to catalyze double-stranded DNA breaks at sites targeted by a guide-RNA molecule.
Multiple guide RNA sequences can be encoded in a single CRISPR array to facilitate the simultaneous editing of multiple sites within a cell's genome. For example, a pair of guide RNAs can target proximally located sequences to facilitate the deletion of the intervening sequence. In some embodiments, Cas9 is encoded by a codon-optimized sequence. Plasmids encoding Cas9, including codon-optimized plasmids and plasmids encoding engineered Cas9 nickase are publicly available from Addgene (http://www.addgene.org/CRISPR/).
Additional information on the application of CRISPR-Cas systems to targeted genome engineering can be found in Jinek et al., Science 337:816-821 (2012); Cho et al., Nature Biotechnology 31:230-232 (2013); Cong et al., Science 339:819-823 (2013); Jinek et al., eLife 2:e00471 (2013); Mali et al., Science 339:823-826 (2013); Qi et al., Cell 152:1173-1183 (2013); Fu et al., Nature Biotechnology 31:822-826 (2013); Fu et al., Nature Biotechnology 31:822-826 (2013); Hsu et al., Nature Biotechnology 31:827-832 (2013); Mali et al., Nature Biotechnology 31:833-838 (2013); Pattanayak et al., Nature Biotechnology 31:839-843 (2013) and WO/2013/142578, each of which is hereby incorporated by reference in its entirety.
In some embodiments of the methods provided herein, the target nucleic acid sequence is modified using a CRISPR/Cas system. In some embodiments, the CRISPR/Cas system is a CRISPR-Cas9 system. In some embodiments, the subject is administered a nucleic acid encoding Cas9 and a nucleic acid encoding a guide-RNA that is specific to a target nucleic acid sequence in the eye.
In some embodiments, the guide-RNA comprises a target-specific guide sequence (e.g., a sequence that is complementary to a sequence of the target DNA sequence) and a guide-RNA scaffold sequence. In some embodiments, the target-specific guide sequence is a nucleic acid sequence selected from any one of SEQ ID NOs: 1-172 and 174-342, or any combination thereof. The target-specific guide sequence may comprise two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty nucleic acid sequences selected from the nucleotide sequences set forth in SEQ ID NOs: 1-172 and 174-342.
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
The present description is further illustrated by the following examples, which should not be construed as limiting in any way.
CRISPR-Cas9 guide RNAs (gRNAs) targeting known mutations causing corneal dystrophies were identified (Table 1a-1c). Human genomic sequences corresponding to gRNA IDs in Table 1 are listed in Table 2.
Mutations in transforming growth factor beta-induced (TGFBI) gene are known to cause several forms of corneal dystrophies including Reis-Bücklers corneal dystrophy, Thiel-Behnke corneal dystrophy, Lattice corneal dystrophy, Granular corneal dystrophy, type 1, and Granular corneal dystrophy, type 2 (1). Missense mutations at two hotspots, R124 and R555, account for nearly 50% of the TGFBI-related corneal dystrophies (9).
In order to demonstrate the feasibility of CRISPR-based treatments for corneal dystrophies, two Cas9 targeting sites were identified that overlap with the genomic sequence encoding both R124 and R555 of TGFBI: 5′-TCAGCTGTACACGGACCGCACGG-3′ (SEQ ID NO: 145), and 5′-AGAGAACGGAGCAGACTCTTGGG-3′(SEQ ID NO: 171), located in exons 4 and 12, respectively. Specific amino acid substitutions at these residues result in clinically distinct corneal dystrophies: R124C—Lattice corneal dystrophy, type I; R124H—Granular corneal dystrophy, type 2; R555W—Granular corneal dystrophy, type 1; and R555Q—Reis-Bücklers corneal dystrophy.
The two target sites were cloned in pH1v1 (Addgene 60244) as described (8), and HEK293 cells were co-transfected with Cas9 and guide RNA (gRNA) constructs. Forty-eight or sixty hours post transfection, genomic DNA was harvested and the sequence surrounding the target cut sites were amplified according to the primers listed in the Appendix A (see below). The PCR products were then purified and quantified before performing the T7 Endo I assay.
Briefly, 200 ng of PCR product was denatured and then slowly re-annealed to allow for the formation of heteroduplexes, T7 Endonuclease I was added to the PCR products and incubated at 37° C. for 25/30 minutes to cleave heteroduplexes. The reaction was stopped by putting PCR products on ice, purified and finally run on a 6% TBE PAGE gel to resolve the products. The gel was stained with SYBR-Gold/Diamond Nucleic Acid dye from Promega, visualized, and quantified using ImageJ. Non-homologous end joining (NHEJ) frequencies were calculated using the binomial-derived equation:
where the values of “a” and “b” are equal to the integrated area of the cleaved fragments after background subtraction and “c” is equal to the integrated area of the un-cleaved PCR product after background subtraction.
The results (
CRISPR-Cas9 approaches to treat corneal dystrophies and microsatellite expansion diseases could employ single or multiple guide RNAs to inactivate or excise gene mutations, or using a repair template and homology directed repair to correct a gene mutation. In the case of the TCF4 microsatellite expansion causing FECD, one or more gRNAs targeting a region on one side of a microsatellite expansion or regions on both sides of a microsatellite expansion could be used. Table 3 shows IDs and corresponding human genomic sequences for gRNA target sequences upstream of the TCF4 microsatellite expansion causing FECD. Table 4 shows IDs and corresponding human genomic sequences for gRNA target sequences downstream of the same TCF4 microsatellite expansion. These gRNAs or others in the TCF4 gene could be used in any combination to correct the microsatellite expansion causing FECD. A similar approach using one or more gRNAs targeting a region on one side of a microsatellite expansion or regions on both sides of a microsatellite expansion could be used for other microsatellite expansion diseases, including but not limited to those listed in Table 5.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention may become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. Such equivalents are intended to be encompassed by the following claims.
This application claims priority to U.S. Provisional Application No. 62/188,013, filed Jul. 2, 2015, the contents of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/040962 | 7/5/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62188013 | Jul 2015 | US |
