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WO2024226813A1 - Procédés de traitement d'une maladie oculaire - Google Patents

Procédés de traitement d'une maladie oculaire Download PDF

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WO2024226813A1
WO2024226813A1 PCT/US2024/026291 US2024026291W WO2024226813A1 WO 2024226813 A1 WO2024226813 A1 WO 2024226813A1 US 2024026291 W US2024026291 W US 2024026291W WO 2024226813 A1 WO2024226813 A1 WO 2024226813A1
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gene
ipsc
editing agent
subject
comeal
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PCT/US2024/026291
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English (en)
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Allen O. EGHRARI
John D. Gottsch
Jinchong XU
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The Johns Hopkins University
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Publication of WO2024226813A1 publication Critical patent/WO2024226813A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure relates to the area of generating an engineered corneal endothelial cell from induced pluripotent stem cells and uses thereof.
  • it relates to engineering a comeal endothelial cell by using gene-editing agents to repair gene mutations in the cell.
  • Fuchs endothelial comeal dystrophy which affects 1 in 40 Americans, is characterized by progressive loss of comeal endothelial cells, thickening of Descemenf s membrane and deposition of extracellular matrix in the form of guttae. When the number of endothelial cells becomes critically low, the cornea swells and causes loss of vision. The clinical course of FECD usually spans 10-20 years, often requiring transplantation of endothelial cells to restore vision and comeal clarity'. Over the last several decades genetic studies have detected several genes, as well as areas of chromosomal loci associated with the disease. However, although keratoplasty 7 has been successful at visual rehabilitation, graft rejection and lack of suitable donor tissue for transplantation continue to be impediments to reduce worldwide comeal blindness.
  • iPSC induced pluripotent stem cell
  • the iPSC is obtained from blood of the subject. In some embodiments, the iPSC is a peripheral blood mononuclear cell (PBMC) originated iPSC.
  • PBMC peripheral blood mononuclear cell
  • the gene-editing agent comprises CRISPR/Cas9 components.
  • the gene-editing agent comprises a guide RNA (gRNA), wherein the gRNA is targeted to the target gene of the iPSC.
  • the target gene comprises a COL8A2 gene.
  • the target gene comprises a L450W mutation in the COL8A2 gene.
  • the gene-editing agent alters the L450W mutation in the COL8A2 gene.
  • the gene-editing agent further comprises a PAM sequence.
  • the PAM sequence comprises a TGG sequence.
  • the ocular disease comprises Fuchs endothelial comeal dystrophy (FECD), keratoconus, keratoconjunctivitis sicca (KCS). hepes virus infections, varicella-zoster virus infections, irido-comeal endothelial syndrome (ICE), pterygium, Stevens Johnson Syndrome (SJS), comeal ulcers, or bullous keratopathy.
  • FECD Fuchs endothelial comeal dystrophy
  • KCS keratoconjunctivitis sicca
  • hepes virus infections varicella-zoster virus infections
  • ICE irido-comeal endothelial syndrome
  • SJS Stevens Johnson Syndrome
  • comeal ulcers or bullous keratopathy.
  • the ocular disease is Fuchs dystrophy.
  • Also provided herein are methods of generating an engineered comeal endothelial cell comprising: (a) obtaining an induced pluripotent stem cell (iPSC) from a subject; (b) delivering a gene-editing agent into the iPSC, wherein the gene-editing agent alters a target gene of the iPSC; and (c) differentiating the altered iPSC into an engineered comeal endothelial cell.
  • iPSC induced pluripotent stem cell
  • the iPSC is obtained from blood of the subject. In some embodiments, the iPSC is a peripheral blood mononuclear cell (PBMC) originated iPSC.
  • PBMC peripheral blood mononuclear cell
  • the gene-editing agent comprises CRISPR/Cas9 components.
  • the gene-editing agent comprises a guide RNA (gRNA), wherein the gRNA is targeted to the target gene of the iPSC.
  • the target gene comprises a COL8A2 gene.
  • the target gene comprises a L450W mutation in the COL8A2 gene.
  • the gene-editing agent alters the L450W mutation in the COL8A2 gene.
  • the gene-editing agent further comprises a PAM sequence.
  • the PAM sequence comprises a TGG sequence.
  • FIG. 1 shows images of comeal endothelial cells grown from induced pluripotent stem cells from blood cells from a subject.
  • FIG. 2A shows genes expressed in comeal endothelium but not in stem cell, confirming the cells have differentiated from stem cells into comeal endothelium.
  • FIG. 2B shows genes expressed in stem cells but not in comeal endothelial cells.
  • FIG. 3 shows images of iPSC-derived comeal endothelial cells showing the same characteristics as comeal endothelial cells from the eye.
  • FIG. 4 show s images of comeal endothelial cells grown from the blood of a patient with the L450W mutation in COL8A2 demonstrating decreased cell density and fewer interactions between cells.
  • FIG. 5 shows an exemplary schematic of the L450W mutation in COL8A2 gene and the correction of the mutation in induced pluripotent stem cells using CRISPR.
  • FIG. 6 shows images of comeal endothelial cells from gene-corrected iPSCs looking similar in number and appearance when compared to normal comeal endothelium.
  • FIG. 7 shows gene expression analysis results showing that the presence of L450W genetic mutation in COL8A2 affects gene expression, and CRISPR correction of the L450W mutation restores gene expression levels to a profile similar to normal cells.
  • FIGs. 8A-8B show' image analysis show ing that qualities of normal borders are missing when cells have the L450W mutation in COL8A2, and after gene correction, the cells create healthy borders with tight junctions.
  • iPSC induced pluripotent stem cell
  • Also provided herein are methods of generating an engineered comeal endothelial cell the method including (a) obtaining an induced pluripotent stem cell (iPSC) from a subject; (b) delivering a gene-editing agent into the iPSC, wherein the gene-editing agent alters a target gene of the iPSC; and (c) differentiating the altered iPSC into an engineered comeal endothelial cell.
  • iPSC induced pluripotent stem cell
  • administration typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is. or is included in. the composition.
  • routes may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
  • administration may be ocular, oral, parenteral, topical, etc.
  • administration may be bronchial (e.g..
  • buccal, dermal which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.
  • enteral intra-arterial, intradermal, intragastric, intramedullary', intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic).
  • administration may involve only a single dose.
  • administration may involve application of a fixed number of doses.
  • administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing.
  • administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • a “cell” can refer to either a prokary otic or eukaryotic cell, optionally obtained from a subject or a commercially available source.
  • “delivering'’ or “gene delivery ” can refer to the introduction of an exogenous polynucleotide into a host cell, irrespective of the method used for the introduction.
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery' complexes) as well as techniques facilitating the delivery' of “naked” polynucleotides (e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • vector-mediated gene transfer e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery' complexes
  • techniques facilitating the delivery' of “nucleotides e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides.
  • the introduced polynucleotide may be stably or transiently maintained in the host cell.
  • Stable maintenance ty pically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • an extrachromosomal replicon e.g., a plasmid
  • a nuclear or mitochondrial chromosome e.g., a nuclear or mitochondrial chromosome.
  • a polynucleotide can be inserted into a host cell by a gene delivery molecule.
  • gene delivery molecules can include, but are not limited to, liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus.
  • adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles ty pically used in the art which have been described for expression in a variety' of eukary otic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • an engineered polypeptide refers to the aspect of having been manipulated by the hand of man.
  • a polypeptide is considered to be “engineered” when the polypeptide sequence is altered or manipulated.
  • an engineered polypeptide comprises a sequence that includes one or more amino acid mutations, deletions and/or insertions that have been introduced by the hand of man into a reference polypeptide sequence.
  • an engineered polypeptide includes a polypeptide that has been fused (i. e. , covalently linked) to one or more additional polypeptides by the hand of man, to form a fusion polypeptide that would not naturally occur in vivo.
  • a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols).
  • new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols.
  • derivatives and/or progeny of an engineered polypeptide or cell are typically still referred to as ‘‘engineered” even though the actual manipulation was performed on a prior entity.
  • a subject refers to an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms).
  • a subject is suffering from a relevant disease, disorder or condition.
  • a subject is susceptible to a disease, disorder, or condition.
  • a subject displays one or more symptoms or characteristics of a disease, disorder or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • treating means a reduction in the number, frequency, severity', or duration of one or more (e.g., two, three, four, five, or six) symptoms of a disease or disorder in a subject (e.g.. any of the subjects described herein), and/or results in a decrease in the development and/or worsening of one or more symptoms of a disease or disorder in a subject.
  • iPSC induced pluripotent stem cell
  • an “ocular disease” refers to a condition or disease that affects the human eye and visual system, wherein an ocular disease interferes with the ability' of the eye to function properly and/or negatively affects the visual clarity of the eye.
  • an ocular disease can include, but is not limited to, conjunctivitis, dry eye, macular degeneration, diabetic retinopathy, glaucoma, cataracts, keratoconus, or Fuchs dystrophy.
  • the ocular disease comprises Fuchs endothelial comeal dystrophy (FECD).
  • the ocular disease is Fuchs dystrophy.
  • Fuchs dystrophy fluid builds up in the cornea of the front of the eye, causing the cornea to swell and thicken.
  • Fuchs dystrophy can affect both eyes and cause vision to gradually worsen over years.
  • Symptoms of Fuchs dystrophy can include eye pains, discomfort in bright lights, fluctuating eyesight throughout the day, halos and/or glares from bright lights, and blurry vision combined with poor contrast in colors.
  • treatments for Fuchs dystrophy can include eyedrops, eye ointment, comeal transplant surgery, and/or endothelial keratoplasty 7 .
  • an “induced pluripotent stem cell (iPSC)’' is a ty pe of pluripotent stem cell derived from adult somatic cells that has been genetically engineered to an embryonic stem (ES) cell-like state through the forced expression of genes and factors important for maintaining the defining properties of ES cells.
  • Induced pluripotent stem cells can be derived from skin or blood cells that been reprogrammed back into an embry onic-like pluripotent state, wherein the iPSCs are enabled as an unlimited source of any type of human cell needed for therapeutic purposes.
  • an iPSC is obtained from blood of a subject.
  • the iPSC is a peripheral blood mononuclear cell (PBMC) originated iPSC.
  • PBMC peripheral blood mononuclear cell
  • a “gene-editing agent” can refer to an agent that can target and bind to a specific sequence in DNA.
  • a gene-editing agent comprises CRISPR/Cas9 components.
  • CRISPR refers to a technique of sequence specific genetic manipulation relying on the clustered regularly interspaced short palindromic repeats pathway, which unlike RNA interference regulates gene expression at a transcriptional level.
  • a “Cas effector'’ or “CRISPR-associated protein’' can refer to an enzyme or protein that uses CRISPR sequences as a guide to recognize and cleave specific nucleic acid strands that are complementary to the CRISPR sequence.
  • a gene-editing Cas effector can associate with a CRISPR RNA sequence to bind to, and alter DNA or RNA target sequences.
  • the gene-editing agent comprises a gene-editing Cas effector.
  • the gene-editing Cas effector comprises a Cas9 protein, a Cas 13b protein, or a Casl3d protein.
  • a gene-editing Cas effector can be a Cas9 endonuclease that makes a double-stranded break in a target DNA sequence.
  • a gene-editing Cas effector can be a Casl2a nuclease that also makes a doublestranded break in a target DNA sequence.
  • a gene-editing Cas effector can be a Cas 13 nuclease which targets RNA.
  • a gene-editing Cas effector comprises a Cas9 protein, a Casl3b protein, or a Casl3d protein.
  • the gene-editing Cas effector comprises a nuclease dead Cas9 (dCas9) protein.
  • the gene-editing Cas effector comprises a Cas 13b protein.
  • the gene-editing Cas effector comprises a Cas 13d protein.
  • the gene-editing agent further comprises a guide RNA (gRNA), wherein the gRNA is targeted to an individual gene of a cell.
  • gRNA guide RNA
  • guide RNA or “gRNA” is a specific type of gRNA that combines tracrRNA (transactivating RNA), which binds to Cas9 to activate the complex to create the necessary strand breaks, and crRNA (CRISPR RNA). comprising complimentary nucleotides to the tracrRNA, into a single RNA construct. Exemplary methods of employing the CRISPR technique are described in WO 2017/091630, which is incorporated by reference in its entirety.
  • the guide RNA can recognize a target RNA. for example, by hybridizing to the target RNA.
  • the guide RNA comprises a sequence that is complementary to the target RNA.
  • the gRNA can include one or more modified nucleotides.
  • the gRNA has a length that is about 10 nt (e.g., about 20 nt, about 30 nt. about 40 nt, about 50 nt. about 60 nt, about 70 nt, about 80 nt, about 90 nt, about 100 nt, about 120 nt, about 140 nt, about 160 nt, about 180 nt. about 200 nt. about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, about 800 nt, about 900 nt, about 1000 nt, or about 2000 nt).
  • the gene-editing agent comprises a guide RNA (gRNA), wherein the gRNA is targeted to the target gene of the iPSC.
  • the target gene comprises a COL8A2 gene.
  • the target gene comprises a L450W mutation in the COL8A2 gene.
  • the gene-editing agent alters the L450W mutation in the COL8A2 gene.
  • the gene-editing agent alters the L450W mutation in the COL8A2 gene to a wild-type version of the COL8A2 gene.
  • the target gene comprises a LOXHD1 gene. In some embodiments, the target gene comprises a c,1639C>T (p.Arg547Cys) mutation in the LOXHD1 gene. In some embodiments, the gene-editing agent alters the c. 1639C>T (p.Arg547Cys) mutation in the LOXHD1 gene. In some embodiments, the gene-editing agent alters the c. I639C>T (p.Arg547Cys) mutation in the LOXHDlgene to a wild-type version of the LOXHD1 gene.
  • the target gene comprises a miR-184 gene. In some embodiments, the target gene comprises a +57C>T mutation in the miR-184 gene. In some embodiments, the gene-editing agent alters the +57C>T mutation in the miR-184 gene. In some embodiments, the gene-editing agent alters the +57C>T mutation in the miR-184 gene to a wild-type version of the miR-184 gene. In some embodiments, a gRNA is targeted to an miR- 184 gene. In some embodiments, a gRNA comprises or consists of the sequence shown in SEQ ID NO: 1.
  • the gene-editing agent further comprises a PAM sequence.
  • PAM photospacer adj acent motif' or “PAM” refers to a sequence that activates the nuclease domain of Cas9.
  • a PAM-presenting oligonucleotide can include an antisense synthetic oligonucleotide composed of alternating 2’0Me RNA and DNA bases and/or other variations of a PAM presenting oligonucleotide that can optimize the CRISPR/Cas9 system and generate specific cleavage of RNA targets without cross reactivity between non-target RNA or against genomic DNA.
  • the PAM sequence comprises a TGG sequence.
  • CRISPR-Cas systems for genome engineering are described, for example, in Jinek et al., Science 337:816-821 (2012); Cho et al., Nature Biotechnology 7 31:230-232 (2013); Cong et al., Science 339:819-823 (2013); Jinek et al., eLife 2:e00471 (2013); Mali et al.. Science 339:823-826 (2013); Qi et al., Cell 152: 1173-1183 (2013); Fu et al., Nature Biotechnology 31 :822-826 (2013); Fu et al.. Nature Biotechnology 31 :822-826 (2013); Hsu et al..
  • iPSC induced pluripotent stem cell
  • the “cornea” refers to the outermost, transparent layer of the eye, and composed of five layers that include the epithelium, Bowman's membrane, stroma, Descemets membrane, and endothelium.
  • the comeal endothelium (CE) is a monolayer of hexagonal cells which is critical in maintaining comeal clarity by mediating hydration through barrier and pump functions.
  • comeal endothelial dystrophies and surgical trauma can be major factors that contribute to loss of comeal endothelial cells (CECs) and a decrease in CE cell density.
  • Fuchs endothelial comeal dystrophy FECD is the leading cause of comeal transplantation.
  • iPSCs can offer autologous cell sources for replacement cell therapy, to replace or regenerate tissues by autologous transplantation.
  • an iPSC can be obtained from a biological sample from a subject.
  • the iPSC is obtained from blood of the subject.
  • the iPSC is a peripheral blood mononuclear cell (PBMC) originated iPSC.
  • PBMC peripheral blood mononuclear cell
  • US20220025325 provides methods of producing comeal endothelial cells (CECs) comprises isolating peripheral blood mononuclear cells (PBMCs) from a biological sample, reprograming the PBMCs to induce pluripotency expressing the pluripotent markers NANOG, OCT4, SOX2, SSEA4, TRA-1-60 and differentiation of the pluripotent stem cells including induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) to produce comeal endothelial cells (CECs).
  • PBMCs peripheral blood mononuclear cells
  • iPSCs induced pluripotent stem cells
  • hESCs human embryonic stem cells
  • the subject has been identified as having Fuchs dystrophy.
  • Cryopreserved PBMCs were reprogramed using a Sendai vims delivery system kit. according to the manufacturer's instmctions (Cytotune 2.0; Life Technologies, Carlsbad, CA, USA).
  • the PBMC vial was removed from liquid nitrogen and thawed at 37°C in a water bath.
  • the PBMCs were washed with medium (StemSpan; STEMCELL Technologies, Inc.) and cultured in StemSpan medium supplemented with 100 ng/rnL Feline McDonough Sarcoma-like tyrosine kinase 3 ligand (FLT-3L), 100 ng/mL stem cell factor, 20 ng/mL interleukin-3 (IL-3), and 20 ng/mL interleukin-6 (IL-6), termed complete medium hereafter, in a humidified incubator at 37°C supplemented with 5% CO2 for 4 days.
  • medium StemSpan; STEMCELL Technologies, Inc.
  • StemSpan medium supplemented with 100 ng/rnL Feline McDonough Sarcoma-like tyrosine kinase 3 ligand (FLT-3L), 100 ng/mL stem cell factor, 20 ng/mL interleukin-3 (IL-3), and 20 ng/mL interleukin-6 (IL-6), termed complete medium hereafter, in
  • MOI multiplicity of infection
  • the infected cells were centrifuged at 1000g for 30 minutes at room temperature.
  • the cells were resuspended in 1 mL complete medium, transferred to a single well of a 12-well plate, and cultured for 3 days in complete medium.
  • Embryonic stem cell-like putative iPSC colonies were selected and cultured on a Matrigel-coated (Coming) plate in mTeSRl medium (FIG. 1).
  • a specific L450W mutation was identified in COL8A2 (NP 001281276.1).
  • the CRISPR/Cas9 gene editing is used to make gene correction in human L450W-COL8A2 iPS cells (FIGs. 4-6). Briefly, the gene sequence is retrieved through HomoloGene (NCBI) and UCSC.
  • NCBI HomoloGene
  • UCSC HomoloGene
  • an evidence-based gRNA designer tool and database DeeoHF http://www.deephf.com/index/
  • DeeoHF http://www.deephf.com/index/
  • the gRNA is then ligated into px- gRNA-mCherry vector.
  • the gRNA cutting efficiency is evaluated by co-transfection of pCas9-GFP and px-gRNA-mCherry in 293T cells.
  • a T7 Endonuclease I digestion method is applied to screen the highest indel % efficiency following the standard protocol of NEB T7 Endonuclease I kit (NEB #E3321).
  • a 120bp of single-stranded oligo donor (ssODN) is designed for single-base substitutions by homologous recombination.
  • the ssODN is the reverse sequence of 50bp of Left arm plus 20bp of gRNA and 50bp of Right arm.
  • a new restriction enzy me is introduced in the ssODN with no change of the original protein sequence.
  • Human L450W-COL8A2 iPS cells (2 x io 6 cells/line) are then dissociated and transfected with pCas9-GFP, px-mCherry-gRNA and matched ssODN (targeting L450W mutation) using P3 Primary Cell buffer and CB150 program on a 4D-Nucleofector (Lonza, Walkersville).
  • transfected cells are then plated onto vitronectin-coated plate with mTeSRl medium in the presence of 10 pM ROCK inhibitor Y27632. Twenty-four hours after nucleofection, single GFP and mCherry double-positive cells are sorted into 96-well followed by picking up individual clones and performing PCR and restrictive enzyme screens 7 days after plating. The correct sequence-verified homozygous iPS clone (L450W corrected to right sequence) is saved for the evaluation of final mutation landscapes of base-edited by whole-genome sequencing (WGS).
  • WGS whole-genome sequencing
  • iPSCs were seeded on 35-mm Matrigel-coated plates (Coming) in 1: 12 dilution (80% confluent plate is split into 12 plates) on day 0 using cell dissociation buffer (Life Technologies). The iPSCs were grown for 4 days in medium (mTeSRl; STEMCELL Technologies, Inc.) (FIGs. 2A-2B and 3). On day 4.
  • medium mTeSRl; STEMCELL Technologies, Inc.
  • mTeSRl media was replaced with dual Smad inhibitors media containing 500 ng/mL human recombinant Noggin (R&D Systems, Minneapolis, MN, USA) and 10 pM SB431542 (MilliporeSigma) in a basal media of 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% nonessential amino acids (Life Technologies), 1 mM 1-glutamine (STEMCELL Technologies, Inc.), 0.1 mM 0- mercaptoethanol (MilliporeSigma), and 8 ng/mL bFGF (MilliporeSigma).
  • dual Smad inhibitors media w as replaced by cornea medium containing 0.1 * B27 supplement (Life Technologies), 10 ng/mL recombinant human platelet derived growth factor-BB (PDGF-BB; PeproTech, Rocky Hill, NJ, USA), and 10 ng/mL recombinant human Dickkopf related protein-2 (DKK-2; R&D Systems) in a basal media of 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% nonessential amino acids (Life Technologies), 1 mM 1-glutamine (STEMCELL Technologies, Inc.), 0.1 mM 0- mercaptoethanol (MilliporeSigma), and 8 ng/mL bFGF (MilliporeSigma).
  • the differentiating CECs were transferred to new Matrigel-coated plates (35 mm) and were grown in cornea medium for 13 additional days (FIGs. 7 and 8A-8B).

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  • Medicinal Chemistry (AREA)
  • Ophthalmology & Optometry (AREA)
  • Veterinary Medicine (AREA)
  • Mycology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention propose des procédés de traitement d'une maladie oculaire chez un sujet en ayant besoin, le procédé comprenant : (a) l'obtention d'une cellule souche pluripotente induite (iPSC) à partir du sujet ; (b) l'administration d'un agent d'édition de gène dans l'iPSC, l'agent d'édition de gène modifiant un gène cible de l'iPSC ; (c) la différenciation de l'iPSC modifiée en une cellule endothéliale cornéenne ; et (d) l'administration de la cellule endothéliale cornéenne au sujet, ce qui permet de traiter la maladie oculaire chez le sujet.
PCT/US2024/026291 2023-04-25 2024-04-25 Procédés de traitement d'une maladie oculaire WO2024226813A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200255859A1 (en) * 2017-07-31 2020-08-13 Reflection Biotechnologies Limited Cellular models of and therapies for ocular diseases
WO2022235586A1 (fr) * 2021-05-03 2022-11-10 Astellas Institute For Regenerative Medicine Procédés de génération de cellules endothéliales cornéennes matures

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200255859A1 (en) * 2017-07-31 2020-08-13 Reflection Biotechnologies Limited Cellular models of and therapies for ocular diseases
WO2022235586A1 (fr) * 2021-05-03 2022-11-10 Astellas Institute For Regenerative Medicine Procédés de génération de cellules endothéliales cornéennes matures

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