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WO2025019150A2 - Activation de canaux piézo1 et piézo2 pour la neuroprotection et la régénération axonale dans la rétine et le nerf optique - Google Patents

Activation de canaux piézo1 et piézo2 pour la neuroprotection et la régénération axonale dans la rétine et le nerf optique Download PDF

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Publication number
WO2025019150A2
WO2025019150A2 PCT/US2024/036414 US2024036414W WO2025019150A2 WO 2025019150 A2 WO2025019150 A2 WO 2025019150A2 US 2024036414 W US2024036414 W US 2024036414W WO 2025019150 A2 WO2025019150 A2 WO 2025019150A2
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piezo1
optic neuropathy
ion channel
subject
vector
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WO2025019150A3 (fr
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Wendy Liu
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • Sequence Listing is provided herewith as a Sequence Listing XML file, “STAN-2126WO” created on June 26, 2024 and having a size of 4,525 bytes.
  • the contents of the Sequence Listing XML file are incorporated by reference herein in their entirety.
  • Glaucoma is a leading cause of irreversible blindness worldwide characterized by the loss of retinal ganglion cells (RGCs).
  • RGCs retinal ganglion cells
  • IOP intraocular pressure
  • lowering IOP does not prevent blindness in up to a quarter of glaucoma patients.
  • optic neuropathies such as ischemic optic neuropathy, optic neuritis, hereditary optic neuropathies such as Leber’s Hereditary Optic Neuropathy and traumatic optic neuropathy.
  • Methods of treating an optic neuropathy or an optic nerve injury in a subject include administering an effective amount of an agonist of a Piezo mechanosensitive ion channel, a recombinant nucleic acid or an expression vector expressing a Piezo mechanosensitive ion channel, or a Piezo mechanosensitive ion channel to the subject.
  • pharmaceutical formulations comprising an agonist of a PiezoPiezo mechanosensitive ion channel, a recombinant nucleic acid or an expression vector expressing a Piezo mechanosensitive ion channel, or a Piezo mechanosensitive ion channel.
  • an agonist of a Piezo type mechanosensitive ion channel component 1 (PIEZO1) or an agonist of a Piezo type mechanosensitive ion channel component 2 (PIEZO2) is administered to a subject to treat an optic neuropathy or an optic nerve injury.
  • the agonist is 2-(5- ⁇ [(2,6- dich loropheny I) methy l]sulfanyl ⁇ - 1 ,3,4-thiadiazol-2-yl)pyrazine (Yodal ), 2-methyl-5-phenylfuran-3- carboxylic acid (Jedil ), or 2-methyl-5-thiophen-2-ylfuran-3-carboxylic acid (Jedi2).
  • the agonist of the Piezo mechanosensitive ion channel, the recombinant nucleic acid or expression vector expressing the Piezo mechanosensitive ion channel, or the Piezo mechanosensitive ion channel is administered to treat glaucoma, ischemic optic neuropathy, optic neuritis, or traumatic optic neuropathy.
  • a method of treating an optic neuropathy or an optic nerve injury in a subject comprising administering a therapeutically effective amount of an agonist of a Piezo mechanosensitive ion channel to the subject.
  • the agonist is an agonist of a Piezo type mechanosensitive ion channel component 1 (PIEZO1) or a Piezo type mechanosensitive ion channel component 2 (PIEZO2).
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • PIEZO2 Piezo type mechanosensitive ion channel component 2
  • the agonist of PIEZO1 is selected from the group consisting of 2-(5- ⁇ [(2,6-dich lorophenyl)methyl]sulfanyl ⁇ -1 ,3 ,4-th iadiazol-2-yl)pyrazine (Yodal ), 2-methyl-5- phenylfuran-3-carboxylic acid (Jedil ), and 2-methyl-5-thiophen-2-ylfuran-3-carboxylic acid (Jedi2).
  • the optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, or traumatic optic neuropathy.
  • the method increases survival of retinal ganglion cells compared to survival of retinal ganglion cells in the subject before treatment.
  • the method increases axon regeneration compared to axon regeneration in the subject before treatment.
  • the agonist is administered intravitreally.
  • the agonist is administered locally to the eye.
  • multiple cycles of treatment are administered to the subject.
  • the agonist is administered according to a weekly dosing regimen or intermittently.
  • the subject is an adult.
  • the subject is a mammalian subject.
  • the mammalian subject is a human subject.
  • composition comprising an agonist of a Piezo mechanosensitive ion channel for use in a method of treating an optic neuropathy or an optic nerve injury is provided.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • a method of treating an optic neuropathy or an optic nerve injury in a subject comprising administering a recombinant nucleic acid or vector comprising a coding sequence encoding a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ), wherein the PIEZO1 is expressed in vivo in the subject in a therapeutically effective amount sufficient to increase retinal ganglion cell survival or axon regeneration.
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • the recombinant nucleic acid is RNA or DNA.
  • the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the PIEZO1.
  • the vector comprises a promoter operably linked to the coding sequence encoding the PIEZO1.
  • the promoter is an ocular tissue-specific promoter, retina-specific promoter, or retinal ganglion cell -specific promoter.
  • the promoter is inducible.
  • the promoter is a constitutive promoter.
  • the coding sequence encoding the PIEZO1 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the PIEZO1 at the chromosomal locus.
  • the vector is a viral vector.
  • viral vectors include, without limitation, an adeno-associated virus vector, a lentivirus vector, or an adenovirus vector.
  • the recombinant nucleic acid or vector is administered locally to the eye.
  • the recombinant nucleic acid or vector is administered intravitreally.
  • the optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy or traumatic optic neuropathy.
  • a method of increasing axon regeneration and survival of retinal ganglion cells comprising introducing a recombinant nucleic acid or a vector comprising a coding sequence encoding a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ) into an eye of the subject, wherein the PIEZO1 is expressed in an effective amount sufficient to increase axon regeneration and survival of retinal ganglion cells.
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • the PIEZO1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence having at least about 80-100% sequence identity to the amino acid sequence of SEQ ID NO: 1 , including any percent identity within this range, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • composition comprising a recombinant nucleic acid or vector comprising a coding sequence encoding a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ) for use in a method of treating an optic neuropathy or an optic nerve injury is provided.
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • the optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • the recombinant nucleic acid is RNA or DNA.
  • the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the PIEZO1.
  • the vector is a viral vector such as, but not limited to, an adeno- associated virus vector, a lentivirus vector, or an adenovirus vector.
  • the composition is formulated for intravitreal administration or local administration to the eye.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • a method of treating an optic neuropathy or an optic nerve injury in a subject comprising genetically modifying the genome of the subject to increase expression of a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ).
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • the genome of the subject is genetically modified using a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas Cas-associated nuclease
  • meganuclease a meganuclease
  • ZFN zinc-finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR-mediated transcriptional activation (CRISPRa) is used to activate expression of PIEZO1 gene expression.
  • a recombinant nucleic acid or vector comprising a coding sequence encoding a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ) in the manufacture of a medicament or pharmaceutical composition for treating an optic neuropathy or an optic nerve injury in a subject in need thereof is provided.
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • a method of treating an optic neuropathy or an optic nerve injury in a subject comprising administering a therapeutically effective amount of a Piezo mechanosensitive ion channel to the subject.
  • the Piezo mechanosensitive ion channel is a Piezo type mechanosensitive ion channel component 1 (PIEZO1) or a Piezo type mechanosensitive ion channel component 2 (PIEZO2).
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • PIEZO2 Piezo type mechanosensitive ion channel component 2
  • the optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • the method increases survival of retinal ganglion cells compared to survival of retinal ganglion cells in the subject before treatment.
  • the method increases axon regeneration compared to axon regeneration in the subject before treatment.
  • the Piezo mechanosensitive ion channel is administered intravitreally.
  • the Piezo mechanosensitive ion channel is administered locally to the eye.
  • multiple cycles of treatment are administered to the subject.
  • the Piezo mechanosensitive ion channel is administered according to a weekly dosing regimen or intermittently.
  • the PIEZO1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence having at least about 80-100% sequence identity to the amino acid sequence of SEQ ID NO: 1 , including any percent identity within this range, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • composition comprising a Piezo mechanosensitive ion channel for use in a method of treating an optic neuropathy or an optic nerve injury is provided.
  • Piezo mechanosensitive ion channel in the manufacture of a medicament or pharmaceutical composition for treating an optic neuropathy or an optic nerve injury in a subject in need thereof is provided.
  • FIG. 1 Chemical formulas of Yoda 1 , romance 1 , and romance2.
  • FIG. 2 In an in vivo mouse model of optic nerve injury, activating Piezol channels with Yodal increases RGC survival by -50%.
  • FIG. 3 In an in vivo mouse model of optic nerve injury, activating Piezol channels with Yodal increases axon regeneration after optic nerve crush (ONC) over 2-fold at each distance from the injury site.
  • ONC represents a good and established model for mimicking glaucoma and other optic neuropathies more broadly.
  • FIG. 4 Elevated IOP leads to opening of mechanosensitive Piezo channels on RGCs and surrounding cell types (e.g. astrocytes and microglia), followed by increased calcium influx. Activation of downstream neuroprotective pathways leads to RGC survival and axon regeneration.
  • FIGS. 5A-5D Yodal rescues RGC cell count, axon count and electrical activity after glaucomatous injury.
  • FIG. 5A Glaucoma was induced in mice via injection of magnetic microbeads (MB). Mice were divided into 4 groups: Sham injection + vehicle, MB injection + vehicle, sham injection + Yodal and MB injection + Yoda 1. Injection of MB caused IOP elevation over 4 weeks.
  • FIG. 5B MB + Yodal eyes had significantly higher RGC counts than MB + vehicle at 4 weeks, and had similar cell counts than sham injected eyes.
  • FIG. 5A Glaucoma was induced in mice via injection of magnetic microbeads (MB). Mice were divided into 4 groups: Sham injection + vehicle, MB injection + vehicle, sham injection + Yodal and MB injection + Yoda 1. Injection of MB caused IOP elevation over 4 weeks.
  • FIG. 5B MB + Yodal eyes had significantly higher RGC counts than MB + vehicle at 4 weeks
  • MB + Yodal eyes had significantly higher axon counts than MB + vehicle at 4 weeks, and had similar cell counts than sham injected eyes.
  • FIG. 5D MB + Yodal eyes had significantly higher P1-N2 amplitudes on pattern ERG than MB + vehicle at 4 weeks, and had similar amplitudes as sham injected eyes.
  • Methods of treating an optic neuropathy or an optic nerve injury in a subject are provided. Aspects of the methods include administering an effective amount of an agonist of a Piezo mechanosensitive ion channel, a recombinant nucleic acid or an expression vector expressing a Piezo mechanosensitive ion channel, or a Piezo mechanosensitive ion channel to the subject. Also provided are pharmaceutical formulations comprising an agonist of a Piezo mechanosensitive ion channel, a recombinant nucleic acid or an expression vector expressing a Piezo mechanosensitive ion channel, or a Piezo mechanosensitive ion channel.
  • the agonist of the Piezo mechanosensitive ion channel, the recombinant nucleic acid or expression vector expressing the Piezo mechanosensitive ion channel, or the Piezo mechanosensitive ion channel is administered to treat glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and/or symptom(s).
  • Those in need of treatment include those already inflicted (e.g., those with an optic neuropathy) as well as those in which prevention is desired (e.g., those with a genetic predisposition for developing optic neuropathy, those with increased susceptibility to optic neuropathy, those with an increased likelihood of optic neuropathy, those suspected of having optic neuropathy, etc.).
  • a therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration.
  • the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment.
  • the subject is suspected of having an increased likelihood of becoming inflicted.
  • a “therapeutically effective dose” or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy).
  • a therapeutically effective dose can be administered in one or more administrations.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; and farm animals such as sheep, goats, pigs, horses and cows.
  • isolated refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and/or in which the entity of interest is partially or substantially purified.
  • substantially purified generally refers to isolation of a substance (e.g., compound, molecule, agent) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample.
  • “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • “Pharmaceutically acceptable salt” includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts.
  • salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).
  • optical neuropathy refers to any condition or disease that causes damage to the optic nerve.
  • optic neuropathy include, without limitation, glaucoma; ischemic optic neuropathy, including anterior ischemic optic neuropathy, posterior ischemic optic neuropathy, and radiation optic neuropathy; optic neuritis, including single isolated optic neuritis (SION), relapsing isolated optic neuritis (RION), chronic relapsing inflammatory optic neuropathy (CRION), neuromyelitis optica (NMO) spectrum disorder, multiple sclerosis associated optic neuritis (MSON), and unclassified optic neuritis (UCON); traumatic optic neuropathy such as caused by trauma to the head or orbit; compressive optic neuropathy such as caused by lesions that compress the optic nerve, including tumors such as optic gliomas, meningiomas, hemangiomas, lymphangiomas, carcinoma, lymphoma, multiple myeloma, dermoid cysts, and inflammatory orbital pseudotumors, infectious lesions, and
  • Optic neuropathy can be caused by transient or permanent interruption of the blood supply to the optic nerve, traumatic injury that damages the optic nerve, inflammatory diseases, neurological disorders, infections (e.g., toxoplasmosis, herpes simplex), nutritional deficiency (e.g., deficiency of vitamins such as B1 , B9, B12, and copper), or drugs or toxins (e.g., certain antibiotics, arrhythmia medications (e.g., amiodarone), malaria medications, tuberculosis medications (e.g., ethambutol), anticancer medications, tobacco, nicotine, methanol, and ethylene glycol).
  • infections e.g., toxoplasmosis, herpes simplex
  • nutritional deficiency e.g., deficiency of vitamins such as B1 , B9, B12, and copper
  • drugs or toxins e.g., certain antibiotics, arrhythmia medications (e.g., amiodarone),
  • a agonist of a Piezo mechanosensitive ion channel such as 2-(5- ⁇ [(2,6-dichlorophenyl)methyl]sulfanyl ⁇ -1 ,3,4-thiadiazol-2-yl)pyrazine (Yodal ), 2-methyl-5-phenylfuran-3-carboxylic acid (Jedit ), or 2-methyl-5-thiophen-2-ylfuran-3-carboxylic acid (Jedi2), PIEZO1 , or a recombinant nucleic acid or vector comprising a coding sequence encoding PIEZO1 , or a genome modifying agent that modifies the genome of a subject to increase expression of PIEZO1 such as a Cas nuclease, meganuclease, zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN) is intended an amount that, when administered
  • a “positive therapeutic response” may include improving vision, preventing, delaying, or decreasing loss of vision, and/or alleviating eye pain.
  • a therapeutically effective dose or amount may increase survival of retinal ganglion cells and/or increase axon regeneration.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the type of condition and the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like.
  • An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.
  • protein refers to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and/or imino molecules. No particular size is implied by use of the terms “protein”, “peptide”, and “polypeptide”, and these terms are used interchangeably. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic).
  • synthetic oligopeptides, dimers, multimers e.g., tandem repeats, linearly-linked peptides), cyclized, branched molecules and the like, are included within the definition.
  • the terms also include molecules comprising one or more peptoids (e.g., N-substituted glycine residues) and other synthetic amino acids or peptides.
  • peptoids e.g., N-substituted glycine residues
  • other synthetic amino acids or peptides See, e.g., U.S. Patent Nos. 5,831 ,005; 5,877,278; and 5,977,301 ; Nguyen et al. (2000) Chem Biol. 7(7):463-473; and Simon et al. (1992) Proc. Natl. Acad. Sci.
  • Non-limiting lengths of peptides suitable for use in the present invention includes peptides of 3 to 5 residues in length, 6 to 10 residues in length (or any integer therebetween), 11 to 20 residues in length (or any integer therebetween), 21 to 75 residues in length (or any integer therebetween), 75 to 100 (or any integer therebetween), or polypeptides of greater than 100 residues in length.
  • polypeptides useful in this invention can have a maximum length suitable for the intended application.
  • the polypeptide is between about 3 and 100 residues in length.
  • one skilled in art can easily select the maximum length in view of the teachings herein.
  • peptides and polypeptides, as described herein, for example synthetic peptides may include additional molecules such as labels or other chemical moieties.
  • references to polypeptides or peptides also include derivatives of the amino acid sequences of the invention including one or more non-naturally occurring amino acids.
  • a first polypeptide or peptide is "derived from" a second polypeptide or peptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide encoding the second polypeptide or peptide, or (ii) displays sequence identity to the second polypeptide or peptide as described herein. Sequence (or percent) identity can be determined as described below.
  • derivatives exhibit at least about 50% percent identity, more preferably at least about 80%, and even more preferably between about 85% and 99% (or any value therebetween) to the sequence from which they were derived.
  • Such derivatives can include postexpression modifications of the polypeptide or peptide, for example, glycosylation, acetylation, phosphorylation, and the like.
  • Amino acid derivatives can also include modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the protein (or fragment thereof) maintains the desired activity (e.g., ability to increase survival of retinal ganglion cells and/or increase axon regeneration). These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification. Furthermore, modifications may be made that have one or more of the following effects: increasing survival of retinal ganglion cells and/or axon regeneration, increasing PIEZO1 biological activity, or facilitating purification, delivery, or cell processing. Proteins or biologically active fragments thereof can be made recombinantly, synthetically, or in tissue culture.
  • piezo type mechanosensitive ion channel component 1 or “PIEZO1 ” as used herein encompasses all forms of PIEZO1 and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain biological activity (e.g., ability to increase survival of retinal ganglion cells and/or increase axon regeneration).
  • a PIEZO1 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. A number of PIEZO1 nucleic acid and protein sequences are known. A representative sequence of a human PIEZO1 protein is presented in SEQ ID NO:1 . Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos.
  • NCBI National Center for Biotechnology Information
  • fragment is intended a molecule consisting of only a part of the intact full-length sequence and structure.
  • the fragment can include a C-terminal deletion an N- terminal deletion, and/or an internal deletion of the polypeptide.
  • Active fragments of a particular protein or polypeptide will generally include at least about 5-14 contiguous amino acid residues of the full length molecule, but may include at least about 15-25 contiguous amino acid residues of the full length molecule, and can include at least about 20-50 or more contiguous amino acid residues of the full length molecule, or any integer between 5 amino acids and the full length sequence, provided that the fragment in question retains biological activity (e.g., ability to increase survival of retinal ganglion cells and/or increase axon regeneration).
  • the term “derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.
  • variant refers to biologically active derivatives of the reference molecule that retain desired activity, such as ability to increase survival of retinal ganglion cells and/or increase axon regeneration, PIEZO1 biological activity, the ability to increase survival of retinal ganglion cells and/or increase axon regeneration for use in treating an optic neuropathy or an optic nerve injury, as described herein.
  • variants and analogs refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity, and which are “substantially homologous” to the reference molecule as defined below.
  • amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned.
  • the analogs will include the same number of amino acids but will include substitutions, as explained herein.
  • the term “mutein” further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and/or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like.
  • the term also includes molecules comprising one or more N-substituted glycine residues (a “peptoid”) and other synthetic amino acids or peptides.
  • the analog or mutein has at least the same biological activity as the native molecule.
  • Methods for making polypeptide analogs and muteins are known in the art and are described further below.
  • analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into four families: (1 ) acidic - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar - glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine.
  • Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5- 25, so long as the desired function of the molecule remains intact.
  • One of skill in the art may readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.
  • derivative is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, as long as the desired biological activity of the native polypeptide is retained.
  • Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.
  • Homology refers to the percent identity between two polynucleotide or two polypeptide molecules.
  • Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules.
  • substantially homologous also refers to sequences showing complete identity to the specified sequence.
  • identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl.
  • nucleotide sequence identity is available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra Nucleic Acid Hybridization, supra.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
  • transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f- mating are included.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
  • a "coding sequence” or a sequence which "encodes" a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
  • the boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • control elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • Encoded by refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.
  • Expression cassette or "expression construct” refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest.
  • An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well.
  • the expression cassette described herein may be contained within a plasmid construct.
  • the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • a signal which allows the plasmid construct to exist as single stranded DNA e.g., a M13 origin of replication
  • at least one multiple cloning site e.g., a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • Polynucleotide refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated.
  • Techniques for purifying polynucleotides of interest include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • transfection is used to refer to the uptake of foreign DNA by a cell.
  • a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981 ) Gene 13:197.
  • Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.
  • the term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.
  • a “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • target cells e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • vector construct e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • expression vector e transfer vector
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • Gene transfer refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells.
  • Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, adenoviruses, lentiviruses, alphaviruses, pox viruses, and vaccinia viruses.
  • a polynucleotide "derived from” a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence.
  • the derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.
  • a “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Gas") genes.
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system.
  • one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
  • Cas9 encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).
  • CRISPR type II clustered regularly interspaced short palindromic repeats
  • a Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA).
  • a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an exon or an intron of a gene), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence).
  • the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.
  • the Cas 9 protein naturally contains DNA endonuclease activity that depends on association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called a single guide RNA (“sgRNA”)).
  • sgRNA single guide RNA
  • the Cas9 associates with a DNA- targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence.
  • the Cas9 protein retains its natural enzymatic function, it will cleave target DNA to create a double-strand break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression.
  • CRISPR agent encompasses any agent (or nucleic acid encoding such an agent), comprising naturally occurring and/or synthetic sequences, that can be used in a Cas9-based system (e.g., a Cas9 or Cas9-like protein; any component of a DNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide; and the like).
  • a Cas9-based system e.g., a Cas9 or Cas9-like protein
  • any component of a DNA-targeting RNA e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.
  • a donor polynucleotide e.g., a donor polynucleotide; and the like.
  • a Cas9 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database.
  • NCBI National Center for Biotechnology Information
  • sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein, wherein the variant retains biological activity, such as Cas9 site-directed endonuclease activity. See also Fonfara et al. (2014) Nucleic Acids Res.
  • a gRNA will bind to a substantially complementary sequence and not to unrelated sequences.
  • a gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.
  • donor polynucleotide refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).
  • HDR homology directed repair
  • a "target site” or “target sequence” is the nucleic acid sequence recognized (i.e. , sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide.
  • gRNA guide RNA
  • the target site may be in an exon or an intron or a specific allele.
  • homology arm is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell.
  • the donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA.
  • the homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide.
  • the 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence” and "3' target sequence,” respectively.
  • the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • complementary refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to II, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine.
  • uracil when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated.
  • “Complementarity” may exist between two RNA strands, two DNA strands, or between a RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be “complementary” and able to form a duplex despite having less than perfect or less than 100% complementarity.
  • Two sequences are "perfectly complementary” or “100% complementary” if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region.
  • Two or more sequences are considered “perfectly complementary” or “100% complementary” even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other.
  • "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other.
  • a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an intron), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.
  • a “zinc-finger nuclease” or “ZFN” is an artificial DNA endonuclease generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain.
  • ZFNs can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences.
  • ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks.
  • ZFN agent encompasses a zinc finger nuclease and/or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.
  • a “transcription activator-like effector nuclease” or “TALEN” is an artificial DNA endonuclease generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain.
  • TALENS can be engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks.
  • TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks.
  • TALEN agent encompasses a TALEN and/or a polynucleotide comprising a nucleotide sequence encoding a TALEN.
  • administering a nucleic acid, such as a recombinant nucleic acid or vector encoding PIEZO1 , a CRISPR system or vector encoding a CRISPR system, a guide RNA, a donor polynucleotide (e.g., for HDR), a CRISPRa system, a vector encoding a ZFN, or a vector encoding a TALEN to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.
  • a nucleic acid such as a recombinant nucleic acid or vector encoding PIEZO1 , a CRISPR system or vector encoding a CRISPR system, a guide RNA, a donor polynucleotide (e.g., for HDR), a
  • Agents for treating an optic neuropathy or an optic nerve injury such as an agonist of PIEZO1 (e.g., 2-(5- ⁇ [(2,6-dichlorophenyl)methyl]sulfanyl ⁇ -1 ,3,4-thiadiazol-2-yl)pyrazine (Yodal ), 2-methyl-5- phenylfuran-3-carboxylic acid (Jedil ), or 2-methyl-5-thiophen-2-ylfuran-3-carboxylic acid (Jedi2)), PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1 can be formulated into pharmaceutical compositions optionally comprising one or more pharmaceutically acceptable excipients.
  • PIEZO1 e.g., 2-(5- ⁇ [(2,6-dichlorophenyl)methyl]sulfanyl ⁇ -1 ,3,4-thiadiazol-2-yl)pyra
  • excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
  • Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants.
  • a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient.
  • carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
  • the excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphat
  • a composition of the invention can also include an antimicrobial agent for preventing or deterring microbial growth.
  • antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
  • An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the agent, or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.
  • a surfactant can be present as an excipient.
  • exemplary surfactants include: polysorbates, such as “Tween 20” and “Tween 80,” and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.
  • Acids or bases can be present as an excipient in the composition.
  • acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.
  • Suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
  • the amount of the agent (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial).
  • a therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.
  • the amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred.
  • compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • solutions and suspensions are envisioned.
  • Additional preferred compositions include those for intravitreal or localized delivery such as by injection into the eye to treat an optic neuropathy or an optic nerve injury, increase survival of retinal ganglion cells, and/or increase axon regeneration.
  • compositions comprising the agent can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use.
  • the compositions comprising the agent are in unit dosage form, meaning an amount of a conjugate or composition of the invention appropriate for a single dose, in a premeasured or pre-packaged form.
  • compositions herein may optionally include one or more additional agents, such other drugs for treating an optic neuropathy or an optic nerve injury, or other medications.
  • compounded preparations may include at least one agent for treating an optic neuropathy or an optic nerve injury such as, but not limited to, corticosteroids such as prednisone, methylprednisolone, triamcinolone, and dexamethasone; pressure-lowering medications, including prostaglandin analogues, such as latanoprost, bimatoprost and travoprost; topical beta-adrenergic receptor antagonists, such as timolol, levobunolol, and betaxolol; alpha2-adrenergic agonists, such as brimonidine and apraclonidine; alpha agonists, such as epinephrine; miotic agents (parasympathomimetics), such as pilocarpine; acetylcholineste
  • At least one therapeutically effective cycle of treatment with a composition comprising an agent for treating an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • an agent for treating an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • a agonist of a Piezo mechanosensitive ion channel such as 2-(5- ⁇ [(2,6-dichlorophenyl)methyl]sulfanyl ⁇ -1 ,3,4- thiadiazol-2-yl)pyrazine (Yodal ), 2-methyl-5-phenylfuran-3-carboxylic acid (Jedil ), or 2-methyl-5- thiophen-2-ylfuran-3-carboxylic acid (Jedi2)
  • PIEZO1 or a recombinant nucleic acid or vector comprising a coding sequence encoding PIEZO1 , or a genome modifying agent that modifies the genome of a subject to increase expression of PIEZO1 such as a Cas nuclease, meganuclease, zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or CRISPRa system is intended an amount that,
  • a “positive therapeutic response” may include improving vision, preventing, delaying, or decreasing loss of vision, and/or alleviating eye pain.
  • a therapeutically effective dose or amount may increase survival of retinal ganglion cells and/or increase axon regeneration.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular type of agent employed to inhibit an optic neuropathy or an optic nerve injury, the mode of administration, and the like.
  • An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.
  • compositions comprising an agent for treating an optic neuropathy or an optic nerve injury such as an agonist of PIEZO1 (e.g., 2-(5- ⁇ [(2 ,6-dichlorophenyl) methy l]sulfanyl ⁇ - 1 ,3,4-thiadiazol-2-yl)pyrazine (Yodal ), 2-methyl-5- phenylfuran-3-carboxylic acid (Jedil ), or 2-methyl-5-thiophen-2-ylfuran-3-carboxylic acid (Jedi2)), a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1 , and/or one or more other therapeutic agents, such as one or more other drugs for treating an optic neuropathy or an optic nerve injury, or other medications.
  • an agent for treating an optic neuropathy or an optic nerve injury such as an agonist of PIEZO1 (e.g., 2-(5- ⁇ [
  • compounded preparations may include one or more additional agent for treating an optic neuropathy or an optic nerve injury such as, but not limited to, corticosteroids, including prednisone, methylprednisolone, triamcinolone, and dexamethasone; pressure-lowering medications, including prostaglandin analogues, such as latanoprost, bimatoprost and travoprost; topical beta-adrenergic receptor antagonists, such as timolol, levobunolol, and betaxolol; alpha2-adrenergic agonists, such as brimonidine and apraclonidine; alpha agonists, such as epinephrine; miotic agents (parasympathomimetics), such as pilocarpine; acetylcholinesterase inhibitors such as echothiophate; and carbonic anhydrase inhibitors, such as dorzolamide, brinzolamide, and aceta
  • compositions comprising an agent for treating an optic neuropathy or an optic nerve injury are typically, although not necessarily, administered intravitreally, topically, or locally. Additional modes of administration are also contemplated, such as orally, by infusion, or via injection (subcutaneously, intravenously, intramuscularly), and so forth.
  • compositions comprising an agent for treating an optic neuropathy or an optic nerve injury may be administered by injection into the eye.
  • the particular preparation and appropriate method of administration can be chosen to target the agent to the optic nerve or retinal ganglion cells. Local treatment may avoid some side effects of systemic therapy.
  • the pharmaceutical preparation can be in the form of a liquid solution or suspension immediately prior to administration, but may also take another form such as a syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, or the like.
  • the pharmaceutical compositions comprising an agent for treating an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • an agent for treating an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art.
  • the pharmaceutical compositions comprising the agent for treating an optic neuropathy or an optic nerve injury (e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1 ) and/or other drugs for treating an optic neuropathy or an optic nerve injury, and/or other agents are in a sustained-release formulation, or a formulation that is administered using a sustained-release device.
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • other drugs for treating an optic neuropathy or an optic nerve injury are in a sustained-release formulation, or a formulation that is administered using a sustained-release device.
  • Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained-release effect with a non-sustained-release pharmaceutical composition.
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • the actual dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered. Therapeutically effective amounts can be determined by those skilled in the art, and will be adjusted to the particular requirements of each particular case.
  • multiple therapeutically effective doses of a composition comprising an agent for treating an optic neuropathy or an optic nerve injury (e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1 ) will be administered according to a daily dosing regimen or intermittently.
  • a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth.
  • a composition comprising the agent for treating an optic neuropathy or an optic nerve injury (e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1 ) will be administered once-weekly, twice-weekly or thrice-weekly for an extended period of time, such as for 1 , 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, and so forth.
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression
  • “twice- weekly” or “two times per week” is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses.
  • “thrice weekly” or “three times per week” is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present invention, this type of dosing is referred to as “intermittent” therapy.
  • a subject can receive intermittent therapy (i.e., once-weekly, twice-weekly or thrice-weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved.
  • intermittent therapy i.e., once-weekly, twice-weekly or thrice-weekly administration of a therapeutically effective dose
  • the agents can be administered by any acceptable route of administration as noted herein below.
  • the amount administered will depend on the potency of the agent for treating an optic neuropathy or an optic nerve injury (e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1 ) and/or other agents administered, the severity of the optic neuropathy or optic nerve injury, the magnitude of the effect desired, and the route of administration.
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • other agents administered e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of
  • the agent (again, preferably provided as part of a pharmaceutical preparation) can be administered alone or in combination with one or more other therapeutic agents, such as other agents for treating an optic neuropathy or an optic nerve injury, or other medications used to treat a particular condition or disease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth.
  • the specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods.
  • Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof.
  • Preferred compositions are those requiring dosing no more than once a day.
  • the agent for treating an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • the agent for treating an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • the agent for treating an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • concurrent therapy is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy.
  • concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising the agent for treating an optic neuropathy or an optic nerve injury (e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1 ) and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating an optic neuropathy or an optic nerve injury, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen.
  • a pharmaceutical composition comprising the agent for treating an optic neuropathy or an optic nerve injury (e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1 ) and a dose of a pharmaceutical composition comprising at least one other agent, such as another
  • the agent for treating an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • an optic neuropathy or an optic nerve injury e.g., an agonist of PIEZO1 , PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 , or a genome editing agent that increases expression of PIEZO1
  • one or more other therapeutic agents can be administered in at least one therapeutic dose.
  • Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.
  • Toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 5 Q (the dose lethal to 50% of the population) or the LD wo (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • Recombinant nucleic acids and vectors comprising coding sequences encoding PIEZO1 or a biologically active fragment or variant thereof that can increase survival of retinal ganglion cells and/or increase axon regeneration are provided.
  • an RNA or DNA comprising a coding sequence encoding PIEZO1 is provided.
  • a recombinant nucleic acid or a vector comprising a coding sequence encoding PIEZO1 is administered to a subject, wherein the PIEZO1 is expressed in vivo in the subject in an effective amount sufficient to increase survival of retinal ganglion cells and/or increase axon regeneration.
  • nucleic acid e.g., DNA or RNA
  • a nucleic acid such as a recombinant nucleic acid comprising a coding sequence encoding PIEZO1 or a recombinant expression vector comprising a coding sequence encoding PIEZO1 into a host cell
  • any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell.
  • Suitable methods include, e.g., nucleic acid delivery by encapsulation in lipid nanoparticles (LNPs), viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)- mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle- mediated nucleic acid delivery, and the like.
  • LNPs lipid nanoparticles
  • PEI polyethyleneimine
  • DEAE-dextran mediated transfection DEAE-dextran mediated transfection
  • liposome-mediated transfection particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle- mediated nucleic acid delivery, and the like.
  • Introducing a recombinant nucleic acid or expression vector into a cell or cells can occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing the recombinant nu
  • a PIEZO1 polypeptide-encoding nucleic acid can be provided as RNA, such as a messenger RNA (mRNA), wherein translation of the mRNA results in production of the PIEZO1 polypeptide in the subject.
  • RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the PIEZO1 polypeptide).
  • the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., encapsulated in LNPs, microinjection, electroporation, transfection, etc.).
  • Nucleic acids may be provided to the cells using well-developed transfection techniques; see, e.g., Angel and Yanik (2010) PLoS One 5(7): e11756, and the commercially available TransMessenger® reagents from Qiagen, StemfectTM RNA Transfection Kit from Stemgent, and TranslT®-mRNA Transfection Kit from Minis Bio LLC. See also Beumer et al. (2008) Proc. Natl. Acad. Sci. USA 105(50):19821 -19826.
  • nucleic acids are introduced into cells by encapsulation in LNPs.
  • LNP transfection techniques see, e.g., Wang et al. (2023) J. Mater. Chem.
  • LNPs for nucleic acid delivery are commercially available, for example, from LipExoGen Biotech (Baltimore, MD), Avanti Polar Lipids, Inc. (Alabaster, AL), Lonza Biologies (Hayward, CA), and Exelead, Inc. (Indianapolis, IN).
  • a vector may be provided directly to a target host cell, for example, by contacting the host cell with the vector (e.g., a recombinant expression vector comprising a coding sequence encoding a PIEZO1 polypeptide) such that the vector is taken up by the cells.
  • the vector e.g., a recombinant expression vector comprising a coding sequence encoding a PIEZO1 polypeptide
  • Methods of transfecting cells are well known in the art, and include, without limitation, electroporation, calcium chloride transfection, microinjection, and lipofection.
  • cells can be contacted with viral particles comprising viral expression vectors.
  • Nucleic acids encoding PIEZO1 can be inserted into an expression vector to create an expression cassette capable of producing the PIEZO1 in a suitable host cell.
  • Expression cassettes typically include control elements operably linked to a coding sequence, which allow for the expression of a gene in vivo in the subject species.
  • control elements operably linked to a coding sequence, which allow for the expression of a gene in vivo in the subject species.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.
  • Promoters can be used to drive expression by an RNA polymerase (e.g., pol I, pol II, pol III).
  • Suitable promoters can be derived from viruses (i.e., viral promoters) or an organism, including prokaryotic or eukaryotic organisms.
  • Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et aL, Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et aL, Nucleic Acids Res. 2003 Sep. 1 ; 31 (17)), and human H1 promoter (H1 ), and the like.
  • LTR mouse mammary tumor virus long terminal repeat
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • the promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/“ON” state) or an inducible promoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF” is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein).
  • a promoter is a spatially restricted promoter (e.g., tissuespecific promoter or cell type-specific promoter controlled by a transcriptional control element, enhancer, etc.).
  • a promoter is a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process).
  • inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art.
  • inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal -regulated promoters (e
  • the promoter is a spatially restricted promoter (i.e., cell type-specific promoter, tissue-specific promoter, organ-specific, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells.
  • Spatially restricted promoters may be regulated by enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell).
  • the promoter is a tissue-specific promoter.
  • the promoter is a cell type-specific promoter.
  • the transcriptional control element (e.g., the promoter) is functional in a targeted tissue, targeted cell type, or targeted cell population.
  • the transcriptional control element can be functional in ocular tissue, a neuron, a retinal cell, or a retinal ganglion cell.
  • the promoter is a reversible promoter.
  • Suitable reversible promoters including reversible inducible promoters are known in the art.
  • Such reversible promoters may be isolated and derived from any of a variety of organisms. Modification of reversible promoters derived from a first organism for use in a second (different) organism is well known in the art.
  • Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters
  • a suitable promoter can include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline- reg dated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Then, 5:491 - 496; Wang et al., 1997, Gene Then, 4:432-441 ; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. BiotechnoL, 16:757-761 ).
  • hypoxia response elements e.g., Gal4 response elements, lac repressor response element
  • small molecule control systems such as tetracycline- reg dated systems and the RU-486 system
  • spatially restricted promoters include, but are not limited to, neuron-specific promoters, photoreceptor-specific promoters, retinal ganglion cell-specific promoters, etc.
  • the promoter is a neuron-specific promoter.
  • neuronspecific promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Pat. No. 6,649,811 , U.S. Pat. No.
  • NSE neuron-specific enolase
  • AADC aromatic amino acid decarboxylase
  • a neurofilament promoter see, e.g., GenBank HUMNFL, L04147
  • a synapsin promoter see, e.g., GenBank HUMSYNIB, M55301
  • a thy-1 promoter see, e.g., Chen et al. (1987) Cell 51 :7-19; and Llewellyn et al. (2010) Nat. Med.
  • a serotonin receptor promoter see, e.g., GenBank S62283; a tyrosine hydroxylase promoter (TH) (see, e.g., NucL Acids. Res. 15:2363-2384 (1987) and Neuron 6:583-594 (1991 )); a GnRH promoter (see, e.g., Radovick et aL, Proc. Natl. Acad. Sci.
  • the promoter is retinal cell-specific promoter or a retinal ganglion cellspecific promoter.
  • retinal cell-specific promoters and retinal ganglion cell-specific promoters include, but are not limited to, RPE (vitelliform macular dystrophy, VMD2) cell-specific promoter, cone opsin promoter (COP), rod opsin promoter (ROP), PAX6 promoter, y-synuclein promoter (SNCG), human synapsin 1 gene promoter (hSyn), and a promoter containing 6.5 kb of the murine Thy-1.2 gene for expression in retinal ganglion cells.
  • RPE vitrelliform macular dystrophy, VMD2
  • COP cone opsin promoter
  • ROP rod opsin promoter
  • PAX6 promoter y-synuclein promoter
  • SNCG y-synuclein promoter
  • hSyn human synapsin 1 gene promoter
  • cell subtype-specific expression of PIEZO1 is achieved by using a recombination system, e.g., Cre-Lox recombination, Flp-FRT recombination, etc.
  • transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon.
  • a sequence for optimization of initiation of translation located 5' to the coding sequence, is also present.
  • transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et aL, supra, as well as a bovine growth hormone terminator sequence.
  • Enhancer elements may also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et aL, EMPO J. (1985) 4:761 , the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et aL, Proc. NatL Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et aL, Cell (1985) 41 :521 , such as elements included in the CMV intron A sequence.
  • LTR long terminal repeat
  • UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same.
  • Such sequences may include UTRs comprising an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • IRES permits the translation of one or more open reading frames from a vector.
  • the IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et aL, Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et aL, Biochem. Biophys. Res. Comm.
  • IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picornaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol.
  • EMCV encephalomyocarditis virus
  • IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et aL, Mol. Cell Endocrinol. (2003) 212:51 -61 ), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol.
  • IRES insulin-like growth factor 2
  • Clontech Mountain View, CA
  • Invivogen San Diego, GA
  • Addgene Cambridge, MA
  • GeneCopoeia Rockville, MD. See also IRESite: The database of experimentally verified IRES structures (iresite.org).
  • An IRES sequence may be included in a vector, for example, to express multiple protein products in combination.
  • a polynucleotide encoding a viral T2A peptide can be used to allow production of multiple protein products from a single vector.
  • 2A linker peptides are inserted between the coding sequences in the multicistronic construct.
  • the 2A peptide which is self-cleaving, allows coexpressed proteins from the multicistronic construct to be produced at equimolar levels.
  • 2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot- and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1 . See, e.g., Kim et al.
  • cells containing a construct encoding PIEZO1 are identified in vitro or in vivo by including a selection marker expression cassette in the construct.
  • Selection markers confer an identifiable change to the cell permitting positive selection of cells having the construct.
  • fluorescent or bioluminescent markers e.g., green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluoresecent protein, blue fluorescent protein, mCherry, mOrange, mPlum, Venus, YPet, phycoerythrin, or luciferase
  • cell surface markers e.g., GFP, dsRed, GUS, lacZ, CAT
  • drug selection markers such as genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, or histidinol may be used to identify cells.
  • enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
  • tk herpes simplex virus thymidine kinase
  • CAT chloramphenicol acetyltransferase
  • Any selectable marker may be used as long as it is capable of being expressed in the cell to allow identification of cells containing the construct. Further examples of selectable markers are well known to one of skill in the art.
  • the selection marker expression cassette encodes two or more selection markers. Selection markers may be used in combination, for example, a cell surface marker may be used with a fluorescent marker, or a drug resistance gene may be used with a suicide gene.
  • the selection marker expression cassette is multicistronic to allow expression of multiple selection markers in combination.
  • the multicistronic vector may include an IRES or viral 2A peptide to allow expression of more than one selection marker from a single vector.
  • a suicide marker is included as a negative selection marker to facilitate negative selection of cells. Suicide genes can be used to selectively kill cells by inducing apoptosis or converting a nontoxic drug to a toxic compound in genetically modified cells.
  • Examples include suicide genes encoding thymidine kinases, cytosine deaminases, intracellular antibodies, telomerases, caspases, and DNases.
  • a suicide gene is used in combination with one or more other selection markers, such as those described above for use in positive selection of cells.
  • a suicide gene may be used in cells containing constructs expressing PIEZO1 , for example, to improve their safety by allowing their destruction at will. See, e.g., Jones et al. (2014) Front. Pharmocol. 5:254, Mitsui et al. (2017) Mol. Ther. Methods Clin. Dev. 5:51 -58, Greco et al. (2015) Front. Pharmacol. 6:95; herein incorporated by reference.
  • the constructs encoding PIEZO1 can be administered to a subject using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. Genes can be delivered either directly to a subject or, alternatively, delivered ex vivo, to cells derived from the subject and the cells reimplanted in the subject.
  • Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et aL, Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et aL, H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191 ; WO 94/28938; WO 95/1 1984 and WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et aL, Hum Gene Ther 9:81 86, 1998, Flannery et aL, P
  • AAV adeno-associated virus
  • a retroviral vector e.g., a lentivirus, a y-retrovirus such as murine leukemia virus and feline leukemia virus, an avian retrovirus such as spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor
  • retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1 :5-14; Scarpa et al. (1991 ) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci.
  • Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al (2002) Science 295:868-872; Durand et al. (201 1) Viruses 3(2):132-159; herein incorporated by reference).
  • retroviral vectors are “defective”, i.e., unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line.
  • the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line.
  • Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells).
  • the appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles.
  • Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art (see, e.g., Kafri et al. (2004) Methods Mol Biol. 246:367-390, herein incorporated by reference).
  • adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et aL, J. Virol. (1993) 67:5911 -5921 ; Mittereder et aL, Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1 :51 -58; Berkner, K. L.
  • AAV vector systems have been developed for gene delivery.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941 ; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et aL, Molec. Cell. Biol.
  • Another vector system useful for delivering nucleic acids encoding PIEZO1 is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference).
  • Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the PIEZO1 include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus.
  • vaccinia virus recombinants expressing the PIEZO1 can be constructed as follows. The DNA encoding the particular PIEZO1 coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome.
  • the resulting TK-recombinant can be selected by culturing the cells in the presence of 5- bromodeoxyuridine and picking viral plaques resistant thereto.
  • avipoxviruses such as the fowlpox and canarypox viruses
  • canarypox viruses can also be used to deliver the genes.
  • Recombinant avipox viruses expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species.
  • the use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with, respect to the production of vaccinia viruses.
  • Molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et aL, J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
  • Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol. 70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072; as well as Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec.
  • chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; herein incorporated by reference in their entireties.
  • a vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest (for example, a PIEZO1 expression cassette) in a host cell.
  • cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery.
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
  • an amplification system can be used that will lead to high level expression following introduction into host cells.
  • a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene.
  • T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction.
  • the polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase.
  • International Publication No. WO 94/26911 Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Common. (1994) 200:1201 -1206; Gao and Huang, Nuc. Acids Res. (1993) 21 :2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:21 14-2120; and U.S. Pat. No. 5,135,855.
  • anelloviral vectors can be used to deliver genes.
  • An anellovector based on a virus of the Betatorquevirus genus, has been developed (see, e.g., Prince et al. (2024) (biorxiv.org/content/10.1101/2024.03.27.586964v1 ).
  • the vector comprises a self-amplifying transcomplementation of a universal recombinant anellovector (SATURN) system, which relies on a selfreplicating plasmid to provide viral proteins in trans that drive replication and capsid-dependent packaging of vector genomes.
  • SATURN universal recombinant anellovector
  • the SATURN system uses Cre-lox-based recombination to generate single unit-sized circular genomes inside a MOLT-4 production cell line. Capsid protein-dependent particles that encapsidate single stranded DNA vector genomes can be produced using the SATURN system.
  • the synthetic expression cassette of interest can also be delivered without a viral vector.
  • the synthetic expression cassette can be packaged as DNA or RNA in liposomes prior to delivery to the subject or to cells derived therefrom.
  • Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.
  • the ratio of condensed DNA to lipid preparation can vary but will generally be around 1 :1 (mg DNA:micromoles lipid), or more of lipid.
  • Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred.
  • Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081 ); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192), in functional form.
  • Cationic liposomes are readily available.
  • N[1 -2,3-dioleyloxy)propyl]-N,N,N- triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416).
  • Other commercially available lipids include (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).
  • Other cationic liposomes can be prepared from readily available materials using techniques well known in the art.
  • anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials.
  • Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
  • the liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).
  • MLVs multilammelar vesicles
  • SUVs small unilamellar vesicles
  • LUVs large unilamellar vesicles
  • the various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et aL, in Methods of Immunology (1983), Vol. 101 , pp. 512-527; Szoka et aL, Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et aL, Biochim. Biophys.
  • DNA and/or peptide(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et aL, Biochem. Biophys. Acta (1975) 394:483-491 . See, also, U.S. Pat. Nos. 4,663,161 and 4,871 ,488.
  • the expression cassette of interest may also be encapsulated, adsorbed to, or associated with, particulate carriers.
  • particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co- glycolides), known as PLG. See, e.g., Jeffery et aL, Pharm. Res. (1993) 10:362-368; McGee J. P., et aL, J MicroencapsuL 14(2):197-210, 1997; O'Hagan D. T., et aL, Vaccine 11 (2):149-54, 1993.
  • particulate systems and polymers can be used for the in vivo or ex vivo delivery of the nucleic acid of interest.
  • polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest.
  • DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods. See, e.g., Feigner, P.
  • Peptoids Zaerman, R. N., et al., U.S. Pat. No. 5,831 ,005, issued Nov. 3, 1998, herein incorporated by reference
  • Peptoids may also be used for delivery of a construct of the present invention.
  • biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering synthetic expression cassettes encoding PIEZO1.
  • the particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun.”
  • a gun powder discharge from a "gene gun” For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371 ,015; and 5,478,744.
  • needle-less injection systems can be used (Davis, H. L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).
  • compositions for delivery to a vertebrate subject are formulated into compositions for delivery to a vertebrate subject. These compositions may either be prophylactic or therapeutic.
  • the compositions will comprise a "therapeutically effective amount" of the nucleic acid of interest such that an amount of the PIEZO1 protein (or a biologically active fragment thereof) can be produced in vivo in the individual to which it is administered that brings about a positive therapeutic response with respect to treatment of the individual for an optic neuropathy or an optic nerve injury and/or an amount that increases survival of retinal ganglion cells and/or axon regeneration.
  • the exact amount necessary will vary depending on the subject being treated; the age and general condition of the subject to be treated; the degree of protection desired; the type of condition and severity of the condition being treated; the particular PIEZO1 protein produced and its mode of administration, among other factors.
  • An appropriate effective amount can be readily determined by one of skill in the art. Thus, a "therapeutically effective amount" will fall in a relatively broad range that can be determined through routine trials.
  • compositions will generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, surfactants and the like, may be present in such vehicles. Certain facilitators of nucleic acid uptake and/or expression can also be included in the compositions or coadministered.
  • pharmaceutically acceptable excipients or vehicles such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, surfactants and the like, may be present in such vehicles.
  • Certain facilitators of nucleic acid uptake and/or expression can also be included in the compositions or coadministered.
  • compositions can be administered directly to the subject (e.g., as described above) or, alternatively, delivered ex vivo, to cells derived from the subject, using methods such as those described above.
  • methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and can include, e.g., dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • the genome of a cell in the eye of a subject may be genetically modified to express or increase expression of PIEZO1.
  • Various gene editing approaches can be used for this purpose, including, without limitation, the use of genome editing systems comprising clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Gas) nucleases, meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Gas CRISPR-associated nucleases
  • ZFNs zinc-finger nucleases
  • TALENs transcription activator-like effector nucleases
  • DSB double-strand break
  • HDR homology-directed repair
  • the donor polynucleotide comprises a nucleotide sequence encoding PIEZO1 , which is flanked by a pair of homology arms responsible for targeting the donor polynucleotide to a genomic locus (e.g., intron or exon) where the coding sequence encoding the PIEZO1 is integrated into the genome.
  • the donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence.
  • the homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence encoding the PIEZO1 within the donor polynucleotide.
  • the 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence,” respectively.
  • the homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus.
  • a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence encoding the PIEZO1 is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e. , sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • the corresponding homologous nucleotide sequences in the genomic target sequence flank a specific site for cleavage and/or a specific site for introducing the nucleotide sequence encoding the PIEZO1.
  • the distance between the specific cleavage site and the homologous nucleotide sequences can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides).
  • the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.
  • a homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc.
  • the 5' and 3' homology arms are substantially equal in length to one another, e.g.
  • the 5' and 3' homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.
  • RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by altering its guide RNA sequence.
  • a target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site.
  • the gRNA can be designed with a sequence complementary to a sequence of the genomic target locus to target the nuclease-gRNA complex to a target site.
  • the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease.
  • CRISPR clustered regularly interspersed short palindromic repeats
  • Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases.
  • Cas proteins include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Casi o, Cas10d, CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx
  • a type II CRISPR system Cas9 endonuclease is used.
  • Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks
  • the Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced.
  • Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database.
  • NCBI National Center for Biotechnology Information
  • sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol.
  • the CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA.
  • the bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break.
  • gRNA guide RNA
  • Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.
  • PAM 5' protospacer-adjacent motif
  • the genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM).
  • the target site comprises 20-30 base pairs in addition to a 3 base pair PAM.
  • the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen.
  • Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.
  • the intron sequence of the TOR gene targeted by a gRNA comprises a mutation that creates a PAM within the intron, wherein the PAM promotes binding of the Cas9-gRNA complex to the intron.
  • the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 nucleotides in length.
  • the guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.
  • Cas12a is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cas12a does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cas12a for targeting than Cas9. Cas12a is capable of cleaving either DNA or RNA.
  • the PAM sites recognized by Cas12a have the sequences 5'-YTN-3' (where "Y” is a pyrimidine and “N” is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9.
  • Cas12a cleavage of DNA produces doublestranded breaks with sticky-ends having a 4 or 5 nucleotide overhang.
  • Cas12a see, e.g., Ledford et al. (2015) Nature. 526 (7571 ) :17-17, Zetsche et al. (2015) Cell. 163 (3):759- 771 , Murovec et al. (2017) Plant BiotechnoL J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; herein incorporated by reference.
  • C2c1 is another class II CRISPR/Cas system RNA-guided nuclease that may be used.
  • C2c1 similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites.
  • a description of C2c1 see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.
  • RNA- guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (Fokl- dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fokl.
  • dCas9 inactive Cas9
  • Fokl- dCas9 Fokl endonuclease
  • dCas9 portion confers guide RNA-dependent targeting on Fokl.
  • CRISPR-mediated transcriptional activation is used to activate expression of PIEZO1 gene expression.
  • CRISPRa is performed with a complex of a catalytically inactive Cas9 (dCas9) fused to a transcriptional activator domain and a guide RNA that targets the PIEZO1 gene.
  • An engineered nuclease-deactivated Cas9 (dCas9) is used to allow sequence-specific targeting without cleavage.
  • Nuclease-deactivated forms of Cas9 may be engineered by mutating catalytic residues at the active site of Cas9 to destroy nuclease activity.
  • nuclease deficient Cas9 protein from any species may be used as long as the engineered dCas9 retains gRNA-mediated sequence-specific targeting.
  • the nuclease activity of Cas9 from Streptococcus pyogenes can be deactivated by introducing two mutations (D10A and H841A) in the RuvC1 and HNH nuclease domains.
  • Other engineered dCas9 proteins may be produced by similarly mutating the corresponding residues in other bacterial Cas9 isoforms.
  • engineered nuclease-deactivated forms of Cas9 see, e.g., Qi et al. (2013) Cell 152:1173-1183, Dominguez et al. (2016) Nat. Rev. Mol. Cell. Biol. 17(1):5-15; herein incorporated by reference in their entireties.
  • the dCas9 protein can be designed to target the PIEZO1 gene by altering its guide RNA sequence.
  • a target-specific single guide RNA comprises a nucleotide sequence that is complementary to a target site, and thereby mediates binding of the dCas9-sgRNA complex by hybridization at the target site. Fusing the dCAS9 to a transcriptional activator allows specific activation of expression of the PIEZO1 gene.
  • the tetraloops and stemloops of the gRNA can be modified to contain binding sites (protein binding RNA aptamers) for trans-activating molecules. Any suitable CRISPRa system may be used to increase expression of the PIEZO1 gene.
  • dCas9 can be fused to VP64, a synthetic transcriptional activator.
  • VP64 consists of four tandem repeats of herpes simplex virus (HSV) protein 16, which is fused to the C-terminus of the Cas9 protein.
  • the gRNA is modified to include MS2 aptamers which recruit the MS2 complex consisting of the transactivation domains of NF-KB (p65) and heat shock transcription factor 1 (HSF1 ), which activate gene expression.
  • HRISPRa systems see, e.g., Bendixen et al. (2023) Mol Ther. 31 (7):1920-1937, Kampmann (2016) ACS Chem Biol. 13(2):406-416, Becirovic (2022) Cell Mol Life Sci. 79(2):130, and Konermann et al. (2015) Nature 517(7536):583-588; herein incorporated by reference in their entireties.
  • RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector such as a plasmid or viral vector). Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell, organoid, or organism.
  • RNA e.g., messenger RNA
  • DNA expression vector such as a plasmid or viral vector
  • a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a human cell or a non-human mammalian cell, such as a non-human primate cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.
  • a nucleic acid encoding the gRNA and/or RNA-guided nuclease is introduced into cells, the gRNA and/or RNA-guided nuclease can be transiently, conditionally, or constitutively expressed in the cell.
  • Recombinant nucleic acids encoding the gRNA, RNA-guided nuclease, and/or donor polynucleotide can be introduced into a cell using any suitable transfection technique such as, but not limited to electroporation, nucleofection, or lipofection.
  • a ribonucleoprotein complex of the gRNA and the RNA-guided nuclease may be introduced into a cell by microinjection into the cytoplasm or nucleus.
  • the CRISPR system is introduced into cells with a viral vector that encodes the RNA-guided nuclease and guide RNA (gRNA).
  • gRNA RNA-guided nuclease and guide RNA
  • Viral delivery of CRISPR components has been demonstrated using lentiviral, retroviral, adenovirus, and adeno-associated virus (AAV) vectors.
  • AAV adeno-associated virus
  • a gRNA and a messenger RNA encoding the RNA-guided nuclease can be introduced into cells, wherein the RNA-guided nuclease is produced by translation of the mRNA in the cytoplasm.
  • the gRNA and RNA-guided nuclease then form a complex in the cytoplasm and enter the nucleus.
  • RNA transfection of cells can be performed using electroporation, cationic-lipid- mediated transfection, or using liposomes or lipid nanoparticles (LNPs) encapsulating the gRNA and mRNA. See, e.g., Billingsley et al.
  • Donor polynucleotides and gRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-2311 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987).
  • Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et al., Meth. Enzymol. (1979) 68:109.
  • gRNA-donor polynucleotide cassettes can be produced by standard oligonucleotide synthesis techniques and subsequently ligated into vectors.
  • Zinc-finger nucleases are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain.
  • ZFNs can be engineered to target desired DNA sequences, which enables zinc-finger nucleases to cleave unique target sequences.
  • ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks.
  • ZFN agent encompasses a zinc finger nuclease and/or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.
  • Transcription activator-like effector nucleases are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain.
  • TALENS can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks.
  • target DNA in the cell e.g., the cell's genome
  • TALEN agent encompasses a TALEN and/or a polynucleotide comprising a nucleotide sequence encoding a TALEN.
  • the PIEZO1 protein (or a biologically active fragment thereof) can be prepared in any suitable manner (e.g., recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g. native, fusions, labeled, lipidated, amidated, acetylated, PEGylated, etc.).
  • the PIEZO1 protein may include naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing proteins are well understood in the art. Proteins are preferably prepared in substantially pure form (i.e. substantially free from other host cell or non-host cell proteins).
  • PIEZO1 nucleic acid and protein sequences may be derived from any source. A number of PIEZO1 nucleic acid and protein sequences are known. A representative human PIEZO1 sequence is presented in SEQ ID NOU and additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NM 001142864, NG_042229, HQ215520, KJ902019, NM_001077200, NM_001357349, NM 001037298, XM_038666802, XM_038666801 , XM_021093657, XM_021093656,
  • a PIEZO1 protein is generated using recombinant techniques.
  • Oligonucleotide probes can be devised based on the known sequences and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques and, e.g., restriction enzymes employed to truncate the gene at desired portions of the full-length sequence.
  • sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA.
  • sequences encoding proteins can also be produced synthetically, for example, based on the known sequences.
  • the nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired.
  • the complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981 ) Nature 292:756: Nambair et al. (1984) Science 223:1299: Jay et al. (1984) J. Biol. Chem. 259:6311 ; Stemmer et al. (1995) Gene 164:49-53.
  • Recombinant techniques are readily used to clone sequences encoding proteins that can then be mutagenized in vitro by the replacement of the appropriate base pair(s) to result in the codon for the desired amino acid.
  • a change can include as little as one base pair, effecting a change in a single amino acid, or can encompass several base pair changes.
  • the mutations can be effected using a mismatched primer that hybridizes to the parent nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex.
  • the primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located.
  • Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe.
  • the technique is also applicable for generating multiple point mutations. See, e.g., Dalbie- McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409.
  • coding sequences Once coding sequences have been isolated and/or synthesized, they can be cloned into any suitable vector or replicon for expression. (See, also, Examples). As will be apparent from the teachings herein, a wide variety of vectors encoding modified proteins can be generated by creating expression constructs which operably link, in various combinations, polynucleotides encoding proteins having deletions or mutations therein.
  • cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage A (E coli), pBR322 (E coll), pACYC177 (E.
  • Insect cell expression systems such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit).
  • Plant expression systems can also be used to produce the PIEZO1 protein.
  • PIEZO1 protein can also be produced.
  • virus-based vectors to transfect plant cells with heterologous genes.
  • a description of such systems see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221 ; andhackland et al., Arch. Virol. (1994) 139:1 -22.
  • Viral systems such as a vaccinia-based infection/transfection system, as described in Tomei et al., J. Virol. (1993) 7:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74:1103-11 13, will also find use with the present invention.
  • cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the DNA of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery.
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).
  • the PIEZO1 gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control' 1 elements), so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence. With the present invention, both the naturally occurring signal peptides or heterologous sequences can be used. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431 ,739; 4,425,437; 4,338,397. Such sequences include, but are not limited to, the TPA leader, as well as the honeybee mellitin signal sequence.
  • regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell.
  • Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.
  • the expression vector is then used to transform an appropriate host cell.
  • mammalian cell lines include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells ⁇ e.g., Hep G2), Vero293 cells, as well as others.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells ⁇ e.g., Hep G293 cells
  • E. coli E. coli
  • Bacillus subtilis Bacillus subtilis
  • Streptococcus spp. will find use with the present expression constructs.
  • Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.
  • Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
  • the fusion proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed.
  • the selection of the appropriate growth conditions is within the skill of the art.
  • the transformed cells secrete the PIEZO1 protein product into the surrounding media.
  • Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example using a tissue plasminogen activator (TPA) leader sequence, an interferon (yor a) signal sequence or other signal peptide sequences from known secretory proteins.
  • TPA tissue plasminogen activator
  • yor a interferon
  • the secreted PIEZO1 protein product can then be isolated by various techniques described herein, for example, using standard purification techniques such as but not limited to, hydroxyapatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.
  • standard purification techniques such as but not limited to, hydroxyapatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.
  • the transformed cells are disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the recombinant peptides or polypeptides substantially intact.
  • Intracellular proteins can also be obtained by removing components from the cell wall or membrane, e.g., by the use of detergents or organic solvents, such that leakage of the polypeptides occurs. Such methods are known to those of skill in the art and are described in, e.g., Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001 ).
  • methods of disrupting cells for use with the present invention include but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freezethaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS.
  • the particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, culture conditions and any pre-treatment used.
  • cellular debris is removed, generally by centrifugation, and the intracellularly produced peptides or polypeptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion-exchange chromatography, sizeexclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.
  • one method for obtaining the intracellular peptides or polypeptides of the present invention involves affinity purification, such as by immunoaffinity chromatography using antibodies (e.g., previously generated antibodies), or by lectin affinity chromatography.
  • Particularly preferred lectin resins are those that recognize mannose moieties such as but not limited to resins derived from Galanthus nivalis agglutinin (GNA), Lens culinaris agglutinin (LCA or lentil lectin), Pisum sativum agglutinin (PSA or pea lectin), Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinum agglutinin (ALIA).
  • GAA Galanthus nivalis agglutinin
  • LCA Lens culinaris agglutinin
  • PSA Pisum sativum agglutinin
  • NPA Narcissus pseudonarcissus agglutinin
  • ALOA All
  • the PIEZO1 protein can be conveniently synthesized chemically, for example by any of several techniques that are known to those skilled in the peptide art. See, e.g., Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W. C. Chan and Peter D. White eds., Oxford University Press, 1 st edition, 2000) ; N.
  • these methods employ the sequential addition of one or more amino acids to a growing peptide chain.
  • a suitable protecting group either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group.
  • the protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage.
  • the protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth.
  • any remaining protecting groups and any solid support, if solid phase synthesis techniques are used are removed sequentially or concurrently, to render the final peptide or polypeptide.
  • any remaining protecting groups and any solid support, if solid phase synthesis techniques are used are removed sequentially or concurrently, to render the final peptide or polypeptide.
  • Typical protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o- bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl and the like.
  • Typical solid supports are cross-linked polymeric supports. These can include divinylbenzene cross-linked-styrene-based polymers, for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene- benzhydrylaminopolystyrene copolymers.
  • divinylbenzene cross-linked-styrene-based polymers for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene- benzhydrylaminopolystyrene copolymers.
  • the PIEZO1 protein can also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985) 82:5131 -5135; U.S. Patent No. 4,631 ,21 1.
  • kits for treating a patient with an agonist of PIEZO1 e.g., 2-(5- ⁇ [(2,6- dichlorophenyl)methyl]sulfanyl ⁇ -1 ,3,4-thiadiazol-2-yl)pyrazine (Yodal ), 2-methyl-5-phenylfuran-3- carboxylic acid (Jedil ), or 2-methyl-5-thiophen-2-ylfuran-3-carboxylic acid (Jedi2)
  • PIEZO1 or a recombinant nucleic acid or an expression vector expressing PIEZO1 or a genome editing agent that increases expression of PIEZO1 such as a CRISPR system or other genome modifying agent (e.g., ZNFs, TALENS), as described herein.
  • a CRISPR system or other genome modifying agent e.g., ZNFs, TALENS
  • Kits may include unit doses of the formulations comprising the agonist of PIEZO1 , PIEZO1 , recombinant nucleic acid or expression vector expressing PIEZO1 , or genome editing agent suitable for use in the treatment methods described herein, e.g., in injectable doses, topical eye drops, or tablets.
  • Transfection agents may also be included in the kit such as lipid nanoparticles (LNPs), calcium phosphate, polyethyleneimine (PEI), DEAE-dextran, liposomes, and the like.
  • kits suitable for intravitreal administration are of particular interest, and in such embodiments the kit may further include a syringe or other device to accomplish such administration, which syringe or device may be pre-filled with a composition comprising the agonist of PIEZO1 , PIEZO1 , recombinant nucleic acid or expression vector expressing PIEZO1 , or genome editing agent.
  • the instructions can be printed on a label affixed to the container or can be a package insert that accompanies the container.
  • Kits may comprise one or more containers of the compositions described herein. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution.
  • the kit may also provide a delivery device pre-filled with a solution comprising a unit dose of the agonist of PIEZO1 , PIEZO1 , recombinant nucleic acid or expression vector expressing PIEZO1 , or genome editing agent, and a pharmaceutically acceptable excipient; and instructions to administer a unit dose according to a desired regimen or exemplary regimen dependent upon a patient’s age, weight, gender, and the like.
  • the subject kits may further include (in certain embodiments) instructions for practicing the subject methods.
  • These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
  • Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, Blu-ray, flash drive, and the like, on which the information has been recorded.
  • Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site. Examples of Non-Limiting Aspects of the Disclosure
  • a method of treating an optic neuropathy or an optic nerve injury in a subject comprising administering a therapeutically effective amount of an agonist of a Piezo mechanosensitive ion channel to the subject.
  • agonist is an agonist of a Piezo type mechanosensitive ion channel component 1 (PIEZO1) or a Piezo type mechanosensitive ion channel component 2 (PIEZO2).
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • PIEZO2 Piezo type mechanosensitive ion channel component 2
  • PIEZO1 is selected from the group consisting of 2-(5- ⁇ [(2,6-dichlorophenyl)methyl]sulfanyl ⁇ -1 ,3,4-thiadiazol-2-yl)pyrazine (Yodal ), 2- methyl-5-phenylfuran-3-carboxylic acid (Jedil ), and 2-methyl-5-thiophen-2-ylfuran-3-carboxylic acid (Jedi2).
  • optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • a composition comprising an agonist of a Piezo mechanosensitive ion channel for use in a method of treating an optic neuropathy or an optic nerve injury.
  • composition of aspect 14, wherein the agonist is an agonist of a Piezo type mechanosensitive ion channel component 1 (PIEZO1) or a Piezo type mechanosensitive ion channel component 2 (PIEZO2).
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • PIEZO2 Piezo type mechanosensitive ion channel component 2
  • composition of aspect 15, wherein the agonist of PIEZO1 is selected from the group consisting of 2-(5- ⁇ [(2,6-dichlorophenyl)methyl]sulfanyl ⁇ -1 ,3,4-th iadiazol-2-yl)pyrazine (Yodal ), 2-methyl-5-phenylfuran-3-carboxylic acid (Jedil ), and 2-methyl-5-thiophen-2-ylfuran-3- carboxylic acid (Jedi2).
  • a method of treating an optic neuropathy or an optic nerve injury in a subject comprising administering a recombinant nucleic acid or vector comprising a coding sequence encoding a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ), wherein the PIEZO1 is expressed in vivo in the subject in a therapeutically effective amount sufficient to increase retinal ganglion cell survival or axon regeneration.
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the PIEZO1 .
  • mRNA messenger RNA
  • the promoter is an ocular tissue-specific promoter, retina-specific promoter, or retinal ganglion cell -specific promoter.
  • optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • PIEZO1 comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1 .
  • a method of increasing axon regeneration and survival of retinal ganglion cells comprising introducing a recombinant nucleic acid or a vector comprising a coding sequence encoding a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ) into an eye of the subject, wherein the PIEZO1 is expressed in an effective amount sufficient to increase axon regeneration and survival of retinal ganglion cells.
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the PIEZO1 .
  • mRNA messenger RNA
  • the vector comprises a promoter operably linked to the coding sequence encoding the PIEZO1 .
  • the promoter is an ocular tissue-specific promoter, retina-specific promoter, or retinal ganglion cell -specific promoter.
  • the viral vector is an adeno-associated virus vector, a lentivirus vector, or an adenovirus vector.
  • PIEZO1 comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:
  • a composition comprising a recombinant nucleic acid or vector comprising a coding sequence encoding a Piezo type mechanosensitive ion channel component 1 (PIEZO1) for use in a method of treating an optic neuropathy or an optic nerve injury.
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • composition of aspect 46, wherein the optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • composition of aspect 47, wherein the recombinant nucleic acid is RNA or DNA.
  • composition of aspect 48 wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the PIEZO1 .
  • mRNA messenger RNA
  • the vector is a viral vector.
  • composition of aspect 50 wherein the viral vector is an adeno-associated virus vector, a lentivirus vector, or an adenovirus vector.
  • composition of any one of aspects 46-51 wherein the composition is formulated for intravitreal administration or local administration to the eye.
  • composition of any one of aspects 46-52, wherein the PIEZO1 comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a method of treating an optic neuropathy or an optic nerve injury in a subject comprising genetically modifying the genome of the subject to increase expression of a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ).
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas clustered regularly interspaced short palindromic repeats
  • ZFN zinc-finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR-mediated transcriptional activation (CRISPRa) is used to activate expression of PIEZO1 gene expression.
  • agonist of a Piezo mechanosensitive ion channel in the manufacture of a medicament or pharmaceutical composition for treating an optic neuropathy or an optic nerve injury in a subject in need thereof.
  • agonist is an agonist of a Piezo type mechanosensitive ion channel component 1 (PIEZO1) or a Piezo type mechanosensitive ion channel component 2 (PIEZO2).
  • PIEZO1 is selected from the group consisting of 2-(5- ⁇ [(2,6-dichlorophenyl)methyl]sulfanyl ⁇ -1 ,3,4-thiadiazol-2-yl)pyrazine (Yodal ), 2- methyl-5-phenylfuran-3-carboxylic acid (Jedil ), and 2-methyl-5-thiophen-2-ylfuran-3-carboxylic acid (Jedi2).
  • optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • a recombinant nucleic acid or vector comprising a coding sequence encoding a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ) in the manufacture of a medicament or pharmaceutical composition for treating an optic neuropathy or an optic nerve injury in a subject in need thereof.
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • PIEZO1 comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1 .
  • RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of the PIEZO1 .
  • mRNA messenger RNA
  • aspect 67 The use of aspect 62, wherein the vector comprises a promoter operably linked to the coding sequence encoding the PIEZO1 .
  • the promoter is an ocular tissue-specific promoter, retina-specific promoter, or retinal ganglion cell -specific promoter.
  • PIEZO1 is integrated into a chromosomal locus, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the PIEZO1 at the chromosomal locus.
  • the viral vector is an adeno-associated virus vector, a lentivirus vector, or an adenovirus vector.
  • a method of treating an optic neuropathy or an optic nerve injury in a subject comprising administering a therapeutically effective amount of a Piezo mechanosensitive ion channel to the subject.
  • Piezo mechanosensitive ion channel is a Piezo type mechanosensitive ion channel component 1 (PIEZO1) or a Piezo type mechanosensitive ion channel component 2 (PIEZO2).
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • PIEZO2 Piezo type mechanosensitive ion channel component 2
  • optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • PIEZO1 comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1 .
  • a composition comprising a Piezo mechanosensitive ion channel for use in a method of treating an optic neuropathy or an optic nerve injury.
  • Piezo mechanosensitive ion channel is a Piezo type mechanosensitive ion channel component 1 (PIEZO1 ) or a Piezo type mechanosensitive ion channel component 2 (PIEZO2).
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • PIEZO2 Piezo type mechanosensitive ion channel component 2
  • composition of aspect 87 or 88, wherein the optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • composition of 87-89, wherein the PIEZO1 comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1 .
  • PIEZO1 comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1 .
  • Piezo mechanosensitive ion channel is a Piezo type mechanosensitive ion channel component 1 (PIEZO1) or a Piezo type mechanosensitive ion channel component 2 (PIEZO2).
  • PIEZO1 Piezo type mechanosensitive ion channel component 1
  • PIEZO2 Piezo type mechanosensitive ion channel component 2
  • optic neuropathy is glaucoma, ischemic optic neuropathy, optic neuritis, hereditary optic neuropathy, or traumatic optic neuropathy.
  • PIEZO1 comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NOU .
  • Piezol is a mechanically activated cation channel expressed in many cell types, including in the eye and in retinal ganglion cells (RGCs).
  • RRCs retinal ganglion cells
  • Piezol may be a novel therapeutic target for glaucoma (Liu et al. (2023) Sci Rep 13(1 ):15871 ).
  • Yoda 1 is a synthetic small molecule discovered in a chemical screen of -3.25 million compounds, and was found to act as an agonist for both human and mouse Pieozl (Syeda et al, 2015). Yodal activates both mPiezol and hPiezol with an apparent EC50 of about 17 pM and 27 pM, respectively. It is a highly hydrophobic compound with a maximal water solubility of about 30 pM.
  • romance 1 and romance2 were later discovered also as chemical activators of Piezol , with an EC50 of about 200 pM and 158 pM, respectively. They are more water-soluble than Yodal (up to 2 mM). Yodal and Brussels 112 are currently not yet FDA approved for human use, but have been used in many in vitro and in vivo animal studies to activate Piezol .
  • Piezol and Piezo2 are candidate molecules for transducing IOP in the eye. 6 In animal models, Piezol is mostly expressed in non-neuronal cells, and regulates cardiovascular mechanotransduction, red blood cell volume and epithelial homeostasis. On the other hand, the Piezo2 channel is expressed in subsets of somatosensory neurons and Merkel cells, and is essential for light touch, proprioception and respiratory mechanotransduction.
  • Piezol and 2 are expressed in the optic nerve head of the mouse.
  • smFISH singlemolecule fluorescence in situ hybridization
  • Receptor Potential Vanilloid 4 Modulates Calcium Flux, Spiking Rate, and Apoptosis of Mouse Retinal Ganglion Cells. J Neurosci. 201 1 ;31 (19):7089-7101 .
  • Yodal treatment preserves RGC function using pattern electroretinogram (PERG), which measures RGC activity in response to contrast modulation of patterned visual stimuli.
  • PERG responses were robust in the uninjured sham group, which were significantly reduced 4 weeks after MB injection (FIG. 5D).
  • Yodal treatment preserved PERG responses to levels similar to those in uninjured sham retinas (FIG. 5D).

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Abstract

L'invention concerne des méthodes de traitement d'une neuropathie optique ou d'une lésion nerveuse optique chez un sujet. Des aspects des méthodes comprennent l'administration d'une quantité efficace d'un agoniste d'un canal ionique mécanosensible Piézo, d'un acide nucléique recombinant ou d'un vecteur d'expression exprimant un canal ionique mécanosensible Piézo, ou d'un canal ionique mécanosensible Piézo au sujet. L'invention concerne également des formulations pharmaceutiques comprenant un agoniste d'un canal ionique mécanosensible Piézo, un acide nucléique recombinant ou un vecteur d'expression exprimant un canal ionique mécanosensible Piézo, ou un canal ionique mécanosensible Piézo. Dans certains modes de réalisation, l'agoniste du canal ionique mécanosensible Piézo, l'acide nucléique recombinant ou le vecteur d'expression exprimant le canal ionique mécanosensible Piézo, ou le canal ionique mécanosensible Piézo sont administrés pour traiter le glaucome, la neuropathie optique ischémique, la névrite optique ou la neuropathie optique traumatique.
PCT/US2024/036414 2023-07-18 2024-07-01 Activation de canaux piézo1 et piézo2 pour la neuroprotection et la régénération axonale dans la rétine et le nerf optique Pending WO2025019150A2 (fr)

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