Classifications

• • 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
• • 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
• • 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
  • A61K48/0075—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 delivery route, e.g. oral, subcutaneous
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• • 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
  • C12N5/0619—Neurons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • 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
• • 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
• • 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
  • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
  • C12N2529/10—Stimulation by light
• • 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
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the present disclosure relates to a protein comprising an amino acid sequence of an opsin, a nucleic acid molecule comprising a nucleic acid encoding the amino acid sequence of the protein, a nucleic acid construct comprising the nucleic acid molecule, a cell comprising the nucleic acid construct, and a medicine comprising the cell.
• Opsins are photoreceptor proteins that are ubiquitous in animals, and are classified into three types based on their light response characteristics. Vertebrate rhodopsin, a type of opsin, forms an active state when exposed to light (photostimulation), but does not return to its original dark state due to either light or heat reactions. On the other hand, many opsins other than animal rhodopsins, such as channelrhodopsin, can form an active state by light irradiation (photostimulation) and then return to a dark state by a thermal reaction.
• vertebrate rhodopsin also acquires photocycle and photoreversibility by introducing a single mutation at position 188, and from this, the residue at position 188 is activated.
• controlling the recovery from the dark state to the original dark state contributes to the diversification of the photoresponse properties of opsins.
• the present disclosure provides: (Item X1) A protein comprising an amino acid sequence of an opsin, the protein comprising an amino acid modification corresponding to position 188 when aligned with SEQ ID NO: 1 in the amino acid sequence of the opsin. (Item X2-1) A protein comprising an amino acid sequence of an opsin, the protein comprising an amino acid modification corresponding to G, T, S, or E at position 188 when aligned with SEQ ID NO: 1 in the amino acid sequence of an opsin. (Item X2-2) A protein comprising an amino acid sequence of an opsin, the protein comprising an amino acid modification corresponding to G at position 188 when aligned with SEQ ID NO: 1 in the amino acid sequence of the opsin.
• the amino acid sequence is 1) an amino acid sequence containing the above modification in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 34; 2) The sequence other than the modification site has at least about 80% identity with the sequence in 1), and the encoded protein has substantially the same biological activity as the protein obtained from the sequence in 1).
• the protein according to any one of the above items comprising: (Item X6) Furthermore, in the amino acid sequence of the opsins, the protein according to any one of the above items includes a modification of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1. (Item X7) Furthermore, in the amino acid sequence of the opsins, the protein according to any one of the above items includes a modification of the amino acid corresponding to E at position 122 when aligned with SEQ ID NO: 1.
• the protein according to any one of the above items comprising a modification of an amino acid corresponding to an amino acid belonging to (Item X9-2) Furthermore, in the amino acid sequences of the opsins, when aligned with SEQ ID NO: 1, there are amino acids that belong to part of the N-terminal domain (positions 1 to 11) and part of the extracellular third loop (positions 279 to 285). ) The protein according to any one of the above items, comprising a modification of an amino acid corresponding to an amino acid belonging to the group 2 to cysteine.
• the protein according to any one of the above items includes modification of amino acids corresponding to positions 2 and 282 when aligned with SEQ ID NO: 1.
• the protein according to any one of the above items, wherein the modification of the amino acids corresponding to positions 2 and 282 includes modification of the amino acids corresponding to positions 2 and 282 to cysteine.
• the opsin is a chimeric opsin.
• the opsin is a chimeric opsin in which the amino acid sequence that binds to G protein in the amino acid sequence of a certain opsin is replaced with the amino acid sequence that binds to G protein in another opsin.
• Proteins described in Section. (Item X10C)
• the opsins described in any one of the above items are chimeric opsins in which, among the amino acid sequences of a certain opsin, the amino acid sequence that binds to G protein is replaced with a functional sequence derived from another organism. protein.
• (Item X10D) The protein according to any one of the above items, wherein the opsin is a chimeric opsin of a homologous organism or a heterologous organism.
• (Item X10E) Protein according to any one of the above items, wherein said organism is selected from the group consisting of vertebrates, invertebrates and microorganisms.
• (Item X10F1) The protein according to any one of the above items, wherein the opsin is a chimeric opsin of a microbial opsin and a vertebrate opsin.
• the opsin is a chimeric opsin of a vertebrate opsin and an invertebrate opsin.
• the opsin is a chimeric opsin between microbial opsins.
• (Item X10F5) The protein according to any one of the above items, wherein the opsin is a chimeric opsin between vertebrate opsins.
• (Item X10F6) The protein according to any one of the above items, wherein the opsin is a chimeric opsin between invertebrate opsins.
• (Item X10) A protein according to any one of the above items, comprising the sequence SEQ ID NO: 1, 3, or 5.
• (Item X11) A nucleic acid molecule comprising a nucleic acid encoding the amino acid sequence of a protein according to any one of the above items.
• (Item X12) A nucleic acid construct comprising a nucleic acid molecule according to any one of the above items.
• (Item X13) A cell comprising a protein according to any one of the above items, a nucleic acid molecule according to any one of the above items, and/or a nucleic acid construct according to any one of the above items.
• (Item X14) A protein according to any one of the above items, a nucleic acid molecule according to any one of the above items, a nucleic acid construct according to any one of the above items and/or a protein according to any one of the above items.
• Medicines containing cells. A medicament according to any one of the above items for regenerating vision or for preventing or treating visual disorders or diseases.
• (Item 1) A composition comprising opsins that inactivate photoreceptor factors without releasing them.
• composition according to any one of the above items, wherein the opsins cause a transient change in cAMP concentration upon light stimulation.
• composition according to any one of the above items, wherein the transient cAMP concentration change is a decrease in cAMP concentration.
• the opsin comprises an amino acid sequence represented by any one of SEQ ID NOs: 1 to 34.
• the opsins include the G protein-coupled receptor rhodopsin.
• composition according to any one of the above items, wherein the G protein-coupled receptor rhodopsin is of mammalian origin (visual rhodopsin).
• the G protein-coupled receptor rhodopsin is a modified type.
• said modification comprises G188C when aligned with SEQ ID NO:1.
• the photoreceptor factor comprises retinal.
• composition according to any one of the above items, wherein the modification further comprises E122Q when aligned with SEQ ID NO:1.
• (Item Z1) A method for modifying or imparting visual function comprising using opsins that inactivate without releasing photoreceptor factors.
• Item Z2 A method of using opsins as an optical switch, which includes the step of using opsins that inactivate photoreceptive factors without releasing them.
• (Item Z3) A method comprising using opsins that inactivate photoreceptor factors without releasing them.
• (Item A1) A protein comprising an amino acid sequence of an opsin, wherein the protein is modified to be activated by light stimulation and then inactivated without releasing a photoreceptor factor; A protein whose opsins activate the Gs or Gq subfamily of G proteins.
• (Item A1a) The protein according to any one of the above items, wherein the opsins activate the Gs subfamily of G proteins.
• (Item A1b) The protein according to any one of the above items, wherein the opsins increase cAMP concentration by activating G protein.
• opsin is a chimeric opsin in which the intracellular second and third loops of the opsin are replaced with those of a G protein-coupled receptor for Gs or Gq activation. Proteins listed.
• the modification that inactivates the photoreceptor factor without releasing it after activation by light stimulation is achieved by modifying the amino acid corresponding to position 188 in the amino acid sequence when aligned with SEQ ID NO: 1. Protein according to any one of the above items. (Item A3) The protein according to any one of the above items, wherein the amino acid corresponding to position 188 includes G, T, S, or E.
• the protein according to any one of the above items comprising modification of the amino acid corresponding to position G188 when aligned with SEQ ID NO: 1 to cysteine.
• the amino acid sequence is 1) an amino acid sequence containing the above modification in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 34; 2) The sequence other than the modification site has at least about 80% identity with the sequence in 1), and the encoded protein has substantially the same biological activity as the protein obtained from the sequence in 1).
• the protein according to any one of the above items comprising: (Item A7) Furthermore, in the amino acid sequence of the opsins, the protein according to any one of the above items includes a modification of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1. (Item A8) The protein according to any one of the above items, wherein the amino acid corresponding to the 122nd position is E. (Item A9) The protein according to any one of the above items, wherein the amino acid corresponding to position G122 when aligned with SEQ ID NO: 1 in the amino acid sequence is modified to glutamine. (Item A10) Protein according to any one of the above items, wherein the photoreceptor factor comprises retinal.
• the protein according to any one of the above items comprising a modification of an amino acid corresponding to an amino acid belonging to the group A to cysteine.
• the protein according to any one of the above items includes modification of amino acids corresponding to positions 2 and 282 when aligned with SEQ ID NO: 1.
• the modification of the amino acids corresponding to positions 2 and 282 improves the thermal stability of the opsins.
• the protein according to any one of the above items, wherein the modification of the amino acids corresponding to positions 2 and 282 includes modification of the amino acids corresponding to positions 2 and 282 to cysteine.
• (Item A12a) A protein according to any one of the above items, comprising the sequence SEQ ID NO: 1, 3, or 5.
• (Item A12) A nucleic acid molecule comprising a nucleic acid encoding the amino acid sequence of a protein according to any one of the above items.
• (Item A13) A nucleic acid construct comprising a nucleic acid molecule according to any one of the above items.
• (Item A14) A cell comprising a protein according to any one of the above items, a nucleic acid molecule according to any one of the above items, and/or a nucleic acid construct according to any one of the above items.
• a medicament comprising a cell comprising a protein according to any one of the above items, a nucleic acid molecule according to any one of the above items, and/or a nucleic acid construct according to any one of the above items.
• the photocycle and photoreversibility of automatically returning to dark adaptation after light irradiation which could not be achieved with conventional techniques, can be achieved.
• the potential for application as an optogenetics tool was confirmed.
• FIG. 1 is a diagram showing the thermal recovery of the G188C mutant of bovine rhodopsin after irradiation with yellow light.
• FIG. 2 shows the photoresponse, retinal configuration, and G protein activation of the bovine rhodopsin G188C mutant.
• FIG. 3 is a diagram showing an increase in the speed of recovery of the photocycle characteristics of the bovine rhodopsin G188C mutant by introducing the E122Q mutation.
• FIG. 4 is a diagram showing light-induced suppression of intracellular cAMP levels by a bovine rhodopsin mutant.
• FIG. 5 shows photopigment formation of bovine rhodopsin G188C mutant after incubation with all-trans retinal.
• FIG. 1 is a diagram showing the thermal recovery of the G188C mutant of bovine rhodopsin after irradiation with yellow light.
• FIG. 2 shows the photoresponse, retinal configuration, and G protein activation
• FIG. 6 is a graph showing changes in cAMP concentration measured using chimeric opsin in which the intracellular second and third loops of the E122Q/G188C mutant of bovine rhodopsin were replaced with those of the mouse histamine H2 receptor.
• FIG. 7 is a diagram showing the acquisition of photocycle characteristics in the Xenopus tropicalis Open5m T188C mutant.
• FIG. 8 is a graph showing changes in cAMP concentration measured using the E122Q/G188C mutant of human rhodopsin.
• FIG. 9 is an alignment diagram of the amino acid sequences of opsins. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. Same as above. FIG.
• FIG. 10 is a graph showing changes in cAMP concentration measured using the G188C mutant of canine rhodopsin.
• FIG. 11 is a graph showing changes in cAMP concentration measured using the G188C mutant of medacalhodopsin.
• FIG. 12 is a graph showing changes in cAMP concentration measured using the G6A/G188C/N2C/D282C mutant and the V337A/G188C/N2C/D282C mutant of human rhodopsin.
• FIG. 10 is a graph showing changes in cAMP concentration measured using the G188C mutant of canine rhodopsin.
• FIG. 11 is a graph showing changes in cAMP concentration measured using the G188C mutant of medacalhodopsin.
• FIG. 12 is a graph showing changes in cAMP concentration measured using the G6A/G188C/N2C/D282C mutant and the V337A/G188C/N2C/D282
• FIG. 13 is a graph showing changes in cAMP concentration measured using human rhodopsin G188C/N2C/G3C/G280C mutant, G188C/N2C/G3C/S281C mutant, and G188C/G3C/N282C mutant.
• FIG. 14 is a graph showing the frequency of neural activity extracted from the extracellular potential of ganglion cells (RGCs) measured using a multi-electrode array (MEA).
• Figure 14A shows an untreated rd1 mouse (no light response is observed because it is blind).
• FIG. 14B shows that the human rhodopsin G188C/N2C/D282C mutant was introduced using a viral vector
• Figure 14C shows that the intracellular second and third loops of the human rhodopsin G188C/N2C/D282C mutant were transferred to the human histamine H2 receptor
• Figure 14D shows the case where the intracellular second and third loops of the human rhodopsin E122Q/G188C/N2C/D282C mutant were replaced with those of the human histamine H2 receptor.
• FIG. 15 is a graph showing changes in cAMP concentration measured using the N2C/D282C mutant and the N2C/D282C/G188C mutant of bovine rhodopsin.
• opsin refers to a protein that binds to retinal, a pigment that functions as a photoreceptor, or an analog thereof, and utilizes retinal or an analog thereof as a chromophore.
• Opsin varies depending on the species of organism and the type of photoreceptor cell, but examples include rod opsin and cone opsin (eg, blue opsin, green opsin, red opsin).
• Opsin as used herein includes, but is not limited to, melanopsin, encephalopsin, OPN5, RGR, and peropsin.
• animal opsin is a G protein-coupled receptor (GPCR) with a seven-transmembrane structure, and constitutes rhodopsin, which exhibits photoreceptive ability when the pigment retinal binds to it.
• GPCR G protein-coupled receptor
• Opsin transmits external light signals into cells by activating trimeric G proteins upon photoreception.
• Opsins can be broadly classified into microbial type opsins (Type I opsins) and animal type opsins (Type II opsins), and animal type opsins can be further classified into vertebrate visual opsins, vertebrate nonvisual opsins, and invertebrate opsins. Can be done.
• vertebrate non-visual opsins and invertebrate opsins are called bistable opsins, and like microbial opsins, they include opsins that use all-trans retinal as a chromophore.
• Microbial opsins, vertebrate nonvisual opsins, and some invertebrate opsins are opsins that do not release retinal from their constituent rhodopsins even when they receive light, and can be used as opsins of the present disclosure.
• Opsins that can be advantageously used in the present disclosure are opsins that do not release retinal upon reception of light, such as microbial opsins, vertebrate non-visual opsins, and some invertebrate opsins, Bistable opsins, including insect opsins, can also be included.
• Opsins used in the present disclosure may not strictly correspond to microbial-type opsins, vertebrate non-visual opsins, or invertebrate opsins, but may have functions equivalent to those advantageously used types of opsins. It can be advantageously used as long as it has functional equivalents.
• the specific amino acid sequences of opsins are shown in SEQ ID NOs: 1 to 34, and the 188th amino acid is shown in the alignment diagram in FIG.
• the 188th amino acid corresponds to G, which is the 188th amino acid of SEQ ID NO: 1 when aligned with SEQ ID NO: 1 using CLUSTRAL W (Version 2.0, released in 2007) (Fig. If the sequence number is different, the specific number may be different, such as G for SEQ ID NO: 2, G for SEQ ID NO: 3, G for SEQ ID NO: 4, and G for SEQ ID NO: 5.
• T in SEQ ID NO: 31 T in SEQ ID NO: 32, T in SEQ ID NO: 33, and T in SEQ ID NO: 34.
• SEQ ID NO: 34 T in SEQ ID NO: 34.
• the 122nd amino acid is the 122nd amino acid of SEQ ID NO: 1 when aligned using SEQ ID NO: 1 and CLUSTRAL W (Version 2.0, released in 2007).
• E it refers to the equivalent of E, specifically, E in SEQ ID NO: 2, E in SEQ ID NO: 3, E in SEQ ID NO: 4, E in SEQ ID NO: 5, L in SEQ ID NO: 6, I in SEQ ID NO: 7, and E in SEQ ID NO: 4.
• I for SEQ ID NO: 8 L for SEQ ID NO: 9, I for SEQ ID NO: 10, Q for SEQ ID NO: 11, M for SEQ ID NO: 12, L for SEQ ID NO: 13, I for SEQ ID NO: 14, I for SEQ ID NO: 15, and I for SEQ ID NO: 16.
• rhodopsin is a protein that contains a pigment called retinal inside, which is activated by light and transmits visual signals to the brain.
• rhodopsin not only means that it contains retinal but also may mean opsin (a protein portion), in which case it is interpreted interchangeably with opsin.
• the ion-transporting receptor rhodopsin which is typically derived from microorganisms, does not release retinal even when irradiated with light, so it can be repeatedly activated by absorbing light. Like the protein-coupled receptor rhodopsin, it cannot activate G proteins.
• the protein of the present disclosure may contain any opsins, and may also include chimeric opsins, as long as the object of the present disclosure can be achieved.
• Chimeric opsins can be produced by combining parts of two or more opsins known in the art. Each opsin to be combined may be derived from the same organism or from different organisms. Those skilled in the art can appropriately select appropriate opsins according to the intended use and/or function of the opsin to provide an appropriate chimeric opsin, and in this case, the chimeric opsin corresponding to amino acid 188 of SEQ ID NO: 1 Any modified amino acid, preferably modified to cysteine, can be used in the present disclosure.
• Opsins that can constitute chimeric opsins include, for example, rod opsin and cone opsin (e.g., blue opsin, green opsin, red opsin), melanopsin, encephalopsin, OPN5, RGR, and peropsin. Often, they may be microbial-type opsins (Type I opsins) or animal-type opsins (Type II opsins), and may be selected from vertebrate non-visual opsins, invertebrate opsins, and the like.
• chimeric opsins examples include chimeric opsins of G protein-coupled receptors and animal-type opsins, e.g., at least a portion of a mammalian-type opsin and a Gs- or Gq-activated G.
• Examples include chimeric proteins comprising at least a portion of protein-coupled receptors.
• Gs or Gq activity can be obtained by fusing a portion of a G protein-coupled receptor for Gs or Gq activation to a portion of a mammalian opsin.
• the amino acid sequences of the intracellular second loop and/or the intracellular third loop that couple with a G protein are linked to other G proteins. It is replaced with the amino acid sequence of the intracellular second loop and/or the intracellular third loop of the type receptor, or a functional sequence.
• the protein of the present disclosure can light-dependently activate Gi-type G proteins to lower intracellular cAMP levels, or light-dependently activate Gs-type G proteins to increase intracellular cAMP levels. , or can activate Gq-type G proteins to increase intracellular Ca 2+ levels.
• the chimeric proteins utilized in this embodiment of the present disclosure can be expressed with sufficient activity in mammals, such as rodents and primates, so that the retinal
• mammals such as rodents and primates
• visual cognitive behavioral functions e.g., improvement of light/dark judgment function, improvement of photopic aversion function, and/or (crisis avoidance function) is brought about, or visual functions such as visual acuity are enhanced.
• the G protein-coupled receptor rhodopsin used in the present disclosure includes human rhodopsin, bovine rhodopsin, and canine rhodopsin.
• retinal is a type of vitamin A1, also called retinaldehyde or retinene, and refers to a substance that constitutes rhodopsin, a visual substance contained in rod photoreceptor cells of the retina.
• Retinal which forms rhodopsin by combining with opsin, has a molecular shape of 11-cis-retinal, and upon exposure to light, it isomerizes to all-trans retinal, which releases its bond with opsin. All-trans-retinal then converts back to 11-cis-retinal in the visual cycle and binds to opsin.
• All-trans retinal is phototoxic and has been implicated in various retinal and other vision-related diseases, including age-related macular degeneration, Stargardt disease, fundus maculata, and recessive retinitis pigmentosa. Suggested.
• retinal analog refers to a compound that has properties substantially equivalent to retinal, and refers to a naturally occurring natural type, or a modification, addition, or addition that is different from the natural type. Including those with modifications such as substitution of functional groups.
• retinal analogs include natural analogs such as A2-retinal (3,4-dehydro retinal), A3-retinal (3-hydroxy retinal), A4-retinal (4-hydroxy retinal), and 9 Examples include artificial analogs such as -ethyl-retinal and 9-propyl-retinal.
• Akimori Wada Pharmaceutical Journal 141 (4), 557-577, 2021-04-01 can be referred to.
• visual impairment refers to any disease, disorder, or symptom related to vision such as the retina, including retinal degenerative diseases (retinitis pigmentosa, age-related macular degeneration, etc.) and retinopathy (e.g., diabetes). retinopathy, proliferative retinopathy, simple retinopathy, etc.), floaters, retinal tears, retinal detachment (e.g., rhegmatogenous retinal detachment, non-rhegmatogenous retinal detachment, etc.), and retinitis pigmentosa.
• retinal degenerative diseases retinitis pigmentosa, age-related macular degeneration, etc.
• retinopathy e.g., diabetes
• retinopathy proliferative retinopathy
• simple retinopathy floaters
• retinal tears e.g., rhegmatogenous retinal detachment, non-rhegmatogenous retinal detachment, etc.
• disorders or symptoms can also include disorders of visual acuity, contrast sensitivity, light/dark adaptation, color vision, and symptoms related thereto.
• protein As used herein, "protein,” “polypeptide,” “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. Amino acids may be natural or non-natural, or may be modified amino acids. The term can also encompass multiple polypeptide chains assembled into a complex. The term also encompasses naturally occurring or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling moiety).
• amino acid is a general term for organic compounds having an amino group and a carboxyl group.
• amino acid sequence may be chemically modified.
• any amino acid in the amino acid sequence may form a salt or a solvate.
• any amino acid in the amino acid sequence may be L-type or D-type.
• the protein according to the embodiment of the present disclosure includes the above-mentioned "specific amino acid sequence.”
• Chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modification (e.g., acetylation, myristoylation, etc.), C-terminal modification (e.g., amidation, glycosylphosphatidylinositol addition, etc.), or side chain modification. Modifications (eg, phosphorylation, glycosylation, etc.) are known. It may be natural or non-natural as long as it meets the objectives of this disclosure.
• chimera refers to a state in which multiple pieces of genetic information derived from the same or different organisms are mixed in the same entity (in this case, protein, etc.).
• a chimeric protein for example, contains a mixture of gene sequences derived from two or three or more organisms.
• the sequence information contained in the chimeric protein may include sequences other than the sequences derived from the organisms to be mixed.
• polynucleotide As used herein, "polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives.” The terms “oligonucleotide derivative” and “polynucleotide derivative” refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or have unusual linkages between nucleotides, and are used interchangeably.
• oligonucleotides include, for example, 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides.
• Examples include oligonucleotide derivatives substituted with '-methoxyethoxyribose.
• a particular base sequence may also include conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences thereof, as well as the explicitly indicated sequence. It is intended to include.
• a nucleic acid sequence is also referred to as a nucleotide sequence, a nucleic acid sequence, a nucleotide sequence, etc. in addition to a base sequence, but these terms have the same meaning.
• degenerate codon substitutions create sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and/or deoxyinosine residue.
• nucleic acid is also used interchangeably herein with gene, DNA such as cDNA, RNA such as mRNA, oligonucleotide, and polynucleotide.
• nucleotides may be natural or non-natural. Nucleic acids herein can be DNA or RNA.
• gene refers to a factor that defines genetic traits, and “gene” may refer to "polynucleotide,” “oligonucleotide,” and “nucleic acid.”
• nucleic acid construct As used herein, "nucleic acid construct,” “construct,” or “gene construct” are used interchangeably with nucleic acids isolated from naturally occurring genes or combined and juxtaposed in a non-naturally occurring manner. , a vector.
• homology of genes refers to the degree of identity between two or more gene sequences, and generally, having “homology” refers to a high degree of identity or similarity. say. "Identity” refers to the degree to which sequences of identical amino acids correspond, and “similarity” refers to the degree to which sequences correspond, including not only the same amino acids but also amino acids with similar properties. Therefore, the higher the homology between two genes, the higher the identity or similarity of their sequences. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, by hybridization under stringent conditions.
• the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% identical. , 95%, 96%, 97%, 98% or 99% identical, then the genes have homology.
• a "homolog” or “homologous gene product” refers to a protein in another species, preferably a mammal, that exerts the same biological function as the protein component of the complex described further herein. means.
• homologues also include “orthologous gene products” and "paralogous gene products,” and for the purposes of this disclosure, such homologs, homologous gene products, orthologous gene products, paralogous gene products, etc. It is understood that also can be used.
• Amino acids may be referred to herein by either their commonly known three-letter symbol or the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly recognized one-letter codes.
• comparisons of similarity, identity, and homology of amino acid and base sequences are calculated using default parameters using BLAST, a sequence analysis tool.
• the identity search can be performed using, for example, NCBI's BLAST 2.2.28 (published on April 2, 2013) (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993).
• the identity value in this specification usually refers to the value obtained when alignment is performed using the above-mentioned BLAST under default conditions.
• identity is taken as the identity value. If identity is evaluated in multiple areas, the highest value among them is taken as the identity value. Similarity is a numerical value that takes into account similar amino acids in addition to identity.
• Blastp can be used as an algorithm with default settings. The measurement results are quantified as Positives or Identities. Homology of amino acid sequences and base sequences can be determined by the algorithm BLAST by Karlin and Altschul. Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215:403-410, 1990).
• BLAST and Gapped When using BLAST programs, use the default parameters for each program. Specific techniques for these analysis methods are publicly known (http://www.ncbi.nlm.nih.gov.).
• the nucleic acid or protein used in the present disclosure may include a target amino acid or base sequence in which one or more amino acids or nucleotides are substituted, deleted, and/or added.
• “one or more” usually means within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (for example, within 5 amino acids, within 3 amino acids, 1 amino acid).
• "one or more” usually means 6 amino acids or less, preferably 5 amino acids or less, and more preferably 4 amino acids or less (for example, 3 amino acids or less, 2 amino acids or less, 1 amino acid or less). amino acids).
• amino acid residue to be mutated be mutated to another amino acid in which the properties of the amino acid side chain are conserved.
• the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), aromatic-containing side chains
• amino acids (H, F, Y, W) having the following (each in parentheses represents a one-letter code for an amino acid).
• “several” may be, for example, 10, 8, 6, 5, 4, 3, or 2, or may be less than or equal to any of these values.
• Chimeric proteins with deletions etc. can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library.
• site-directed mutagenesis method for example, KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used. It is possible to select antibodies that have the same activity as the wild type from mutant antibodies in which deletions or the like have been introduced by performing various characterizations such as FACS analysis and ELISA.
• the amino acid sequence and nucleic acid sequence of the modified protein and/or chimeric protein of the present disclosure have an identity or similarity of 70% or more, 80% or more, or 90% or more to a reference sequence. It may have.
• 70% or more with respect to an amino acid sequence or a base sequence may be, for example, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more, etc. % or more" may be, for example, 80, 85, 90, 95, 96, 97, 98, 99% or more.
• “90% or more” may be, for example, 90, 95, 96, 97, 98, 99%.
• “Homology” may be calculated as the ratio of the number of homologous amino acids in two or more amino acid sequences according to a method known in the art. Before calculating the ratio, the amino acid sequences of the group of amino acid sequences to be compared are aligned, and gaps are introduced into part of the amino acid sequences if necessary to maximize the ratio of identical amino acids. Methods for alignment, ratio calculation, comparison, and related computer programs are conventionally well known in the art (eg, BLAST, GENETYX, etc.). In the case of "identity”, the proportion of identical amino acids is calculated, and in the case of “similarity”, the proportion of similar amino acids is calculated. Similar amino acids include, but are not limited to, amino acids that allow conservative substitutions.
• a polynucleotide that hybridizes under stringent conditions refers to well-known conditions commonly used in the field.
• Such a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like using a polynucleotide selected from the polynucleotides of the present disclosure as a probe. Specifically, hybridization is performed at 65°C in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA is immobilized, and then 0.1 to 2 times the concentration of NaCl is used.
• a polynucleotide that can be identified by washing a filter at 65°C using SSC (saline-sodium citrate) solution (the composition of a 1x SSC solution is 150mM sodium chloride and 15mM sodium citrate).
• SSC saline-sodium citrate
• the following conditions can be adopted as "stringent conditions”.
• low ionic strength and high temperature for cleaning e.g., 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C
• a denaturing agent such as formamide during hybridization
• sequences that hybridize under stringent conditions preferably include sequences containing only the A sequence or only the T sequence.
• Moderate stringency conditions can be readily determined by those skilled in the art based on, for example, the length of the DNA and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, No. 3, Vol. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001 and for nitrocellulose filters, a pre-wash solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); Other similar hybridizations such as Stark's solution in about 50% formamide, 2 ⁇ SSC-6 ⁇ SSC (or Stark's solution in about 50% formamide at about 42°C) at about 40-50°C.
• polypeptides as used in this disclosure include those encoded by nucleic acid molecules that hybridize under highly or moderately stringent conditions to nucleic acid molecules encoding the polypeptides specifically described in this disclosure. Also included are polypeptides.
• a "purified" substance or biological factor refers to a substance or biological factor from which at least a portion of the factors naturally associated with the substance or biological factor has been removed. . Therefore, the purity of the biological agent in a purified biological agent is typically higher (ie, more concentrated) than the state in which the biological agent normally exists.
• the term "purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight. It means that the same type of biological agent is present.
• the substances or biological agents used in this disclosure are preferably "purified" substances.
• an "isolated" substance or biological agent is one that has been substantially removed from the factors that naturally accompany it. say something
• the term “isolated” as used herein does not necessarily have to be expressed in purity, as it varies depending on its purpose, but where necessary, preferably at least 75% by weight, more preferably means that at least 85%, even more preferably at least 95%, and most preferably at least 98% by weight of the same type of biological agent is present.
• the substances used in this disclosure are preferably "isolated" substances or biological agents.
• a "corresponding" amino acid or nucleic acid or moiety refers to a given amino acid or nucleotide or moiety in a polypeptide or polynucleotide molecule (e.g., rhodopsin) that is the basis for comparison.
• a polypeptide or polynucleotide molecule e.g., rhodopsin
• amino acids or nucleotides that have or are predicted to have similar effects, and especially in enzyme molecules refer to amino acids that are present in similar positions in the active site and make similar contributions to catalytic activity.
• a complex molecule refers to the corresponding part (for example, heparan sulfate, etc.).
• an antisense molecule in an antisense molecule, it can be a similar portion in an ortholog that corresponds to a particular portion of the antisense molecule.
• Corresponding amino acids can be identified, for example, by cysteination, glutathionylation, SS bond formation, oxidation (e.g., oxidation of methionine side chains), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. amino acids.
• the corresponding amino acid may be an amino acid responsible for dimerization.
• Such "corresponding" amino acids or nucleic acids may be a range of regions or domains. In such cases, therefore, the regions or domains are referred to herein as "corresponding" regions or domains. Such corresponding regions or domains are useful in designing complex molecules in this disclosure.
• a "corresponding" gene in this case, it may be a polynucleotide sequence or molecule encoding an opsin, etc.
• a gene that has or is predicted to have in this case, it may be a polynucleotide sequence or molecule that encodes an opsin, etc.
• a gene corresponding to a given gene may be an ortholog of that gene.
• corresponding genes can be identified using techniques well known in the art.
• a corresponding gene in a certain animal e.g., a mouse
• a reference gene e.g., opsin, etc.
• a corresponding gene in a certain animal can be found in a database containing the animal's sequences using a specific sequence as a query sequence. It can be found by searching.
• fragment refers to a polypeptide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). or polynucleotide.
• the length of the fragment can be changed as appropriate depending on the purpose; for example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (e.g., 11, etc.) are also suitable as lower limits. obtain.
• examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides, which are specifically listed here.
• a length expressed as an integer may also be suitable as a lower limit.
• such fragments are included within the scope of the present disclosure, for example, if the full-length version functions as a marker or target molecule, as long as the fragment itself also functions as a marker or target molecule. is understood.
• the term "activity" herein refers to the function of a molecule in the broadest sense.
• Activity generally includes, but is not intended to be limiting, a biological, biochemical, physical, or chemical function of the molecule.
• Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of other molecules. stability, and the ability to localize to specific subcellular locations.
• the term also relates to the function of protein complexes in the broadest sense.
• biological activity includes activation of photoreactions and the like.
• a functional equivalent refers to any object that has the same intended function but a different structure with respect to the original entity.
• a functional equivalent of an "opsin” or a chimera thereof is not an opsin or a chimera thereof per se, but a variant or variant (e.g., an amino acid sequence variant, etc.) of an opsin or a chimera thereof;
• a chimera has a biological effect, and at the time of action, it can change into an opsin or its antibody itself or a mutant or variant of this opsin or a chimera thereof (e.g., an opsin or a chimera thereof or a variant thereof).
• the invention includes nucleic acids encoding opsins or chimeric mutants or variants thereof, and vectors, cells, etc. containing the nucleic acids.
• Functional equivalents of the present disclosure may include insertions, substitutions and/or deletions of one or more amino acids, or additions to one or both ends of the amino acid sequence.
• insertion, substitution, and/or deletion of one or more amino acids, or addition to one or both ends of an amino acid sequence refers to the well-known method such as site-directed mutagenesis. It means that a modification has been made by a technical method or by natural mutation, such as substitution of a plurality of amino acids to the extent that they can occur naturally.
• the modified amino acid sequence may include insertions, substitutions, or deletions of, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, particularly preferably 1 to 2 amino acids. It can be deleted or added to one or both ends.
• the modified amino acid sequence is preferably one in which the amino acid sequence has one or more (preferably one or several or one, two, three, or four) conservative substitutions in the amino acid sequence of opsin. It's okay.
• drug drug
• agent agent
• factor factor
• any term can be used as long as it can achieve the intended purpose. It may also be a substance or other element (eg, light, radioactivity, heat, energy such as electricity).
• Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, DNA such as cDNA and genomic DNA, and RNA such as mRNA), Saccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, information transmitters, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)) , complex molecules thereof, but are not limited to them.
• proteins proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, DNA such as cDNA and genomic DNA, and RNA such as mRNA), Saccharides, oligosacchari
• oral administration it may be formulated into various forms such as tablets, granules, fine granules, powders, and capsules, and binders, encapsulating agents, excipients, and lubricants commonly used in formulations may be used. Additives such as thickeners, disintegrants, and wetting agents may also be included.
• preparations for oral administration may be formulated as liquids such as oral solutions, suspensions, emulsions, syrups, etc., or as dry formulations that are redissolved at the time of use. It's okay.
• the formulation may be formulated in unit-dose ampoules or multi-dose containers or tubes, and may also contain additives such as stabilizers, buffers, preservatives, and tonicity agents. It may be included.
• the preparation for parenteral administration may be formulated into a powder that can be redissolved in an appropriate carrier (sterile water, etc.) at the time of use.
• parenteral administration examples include intraocular administration such as intravitreal administration, subretinal administration, subchoroidal administration, and intracameral administration, and extraocular administration such as subconjunctival administration, subtenon's capsule administration, and ophthalmic administration. Intravitreal administration is preferred.
• the composition of the present disclosure can be used for treatment, prevention, inhibition of disease progression, etc. by administering it to humans in the manner described above.
• Treatment refers to preventing the worsening of a disease or disorder (e.g., vesicular transport disorder or apoptosis) when such a condition occurs, preferably, It refers to maintaining the status quo, more preferably alleviating, and even more preferably eliminating, and includes being able to exhibit a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease.
• Performing a diagnosis in advance and providing appropriate treatment is called “companion treatment,” and the diagnostic agent used for this purpose is sometimes called a “companion diagnostic agent.” Since the present disclosure targets genetic diseases, genes may be tested in advance to treat patients.
• a “therapeutic drug” in a broad sense refers to any drug that can treat a target condition (eg, retinal degenerative disease, etc.).
• a “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers.
• the pharmaceutical composition can be produced, for example, by mixing the active ingredient and the carrier described above, and by any method known in the technical field of pharmaceutical science.
• the therapeutic agent is not limited in its usage form as long as it is used for treatment, and may be an active ingredient alone or a mixture of an active ingredient and any other ingredient.
• the shape of the carrier is not particularly limited, and may be, for example, solid or liquid (eg, buffer).
• prevention refers to preventing a certain disease or disorder (for example, retinal degenerative disease) from developing into such a condition before it occurs. Diagnosis can be performed using the drug of the present disclosure, and if necessary, the drug of the present disclosure can be used to prevent, for example, retinal degenerative diseases, or to take preventive measures.
• preventive drug (agent) in a broad sense refers to any drug that can prevent a desired condition (eg, vesicular transport disorder, apoptosis, etc.).
• kit refers to parts that should be provided, usually divided into two or more compartments (e.g., nucleic acids, nucleic acid constructs, cells transfected with the target nucleic acid, test reagents, diagnostic reagents, therapeutic A unit in which drugs, antibodies, labels, instructions, etc.) are provided.
• a kit may be used when a combination of a diagnostic agent and a therapeutic agent is provided.
• kits when the purpose is to provide a composition that should not be provided mixed for stability etc., such as certain unstable pharmaceuticals, but is preferably mixed immediately before use.
• This kit format is preferred.
• kits preferably include the provided parts (e.g., nucleic acids, nucleic acid constructs, cells transfected with the nucleic acids of interest, how to use test, diagnostic, or therapeutic agents, or reagents). It is advantageous to be provided with instructions or instructions describing how the , instructions on how to use therapeutic drugs, antibodies, etc.
• active ingredient refers to an ingredient that is contained in the amount necessary for the composition of the present disclosure to achieve the desired therapeutic, preventive, or progression-suppressing effect, and the effect is less than the desired level. Other ingredients may also be included as long as they do not impair the quality.
• the medicament, composition, etc. of the present disclosure may be formulated.
• the route of administration of the medicament, composition, etc. of the present disclosure may be either oral or parenteral, and can be appropriately determined depending on the form of the preparation and the like.
• instructions describe instructions for a physician or other user on how to use the present disclosure.
• This instruction sheet includes words instructing the detection method of the present disclosure, how to use the diagnostic agent, or administering the medicine.
• the instructions may include language instructing oral administration or retinal administration (for example, by injection) as the administration site.
• This instruction is prepared in accordance with the format prescribed by the regulatory authority of the country where this disclosure will be made (for example, the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and is approved by that regulatory authority. It will be clearly stated that it was received. Instructions are so-called package inserts or labels, and are usually provided in paper media, but are not limited to this, and include, for example, electronic media (e.g., homepages provided on the Internet, electronic It may also be provided in a format such as email).
• the present disclosure provides a protein comprising an amino acid sequence of an opsin, the protein comprising an amino acid modification corresponding to position 188 when aligned with SEQ ID NO: 1 in the amino acid sequence of the opsin. provided.
• Opsin is a light-sensitive G protein-coupled receptor and is ubiquitous in animals. All opsins have common structural elements, including seven transmembrane domains, which bind the light-absorbing chromophore retinal to the opsin's Lys296 (based on the bovine rhodopsin numbering system) via a Schiff base bond. Opsins function in both visual and non-visual photoreception and are classified into several groups based on their amino acid sequences (Shichida and Matsuyama, 2009; Koyanagi and Terakita, 2014).
• Bovine rhodopsin is the most studied opsin (Yau and Hardie, 2009) and functions as a visual photoreceptor protein in the retina, binding 11-cis retinal in the dark.
• the meta II intermediate of rhodopsin is generated and couples with G protein.
• Meta II is a metastable active state and is spontaneously converted to Meta III (Heck et al., 2003).
• meta II is irradiated with light, the formation of meta III is induced instead of the original dark state (Bartl et al., 2001; Ritter et al., 2008).
• bistable opsins because they have two stable states, a dark state and an active state, which can be mutually converted by light.
• opsin which is the only one with photocyclic properties, has a cysteine residue at position 188, and this is known to be the basis of the photocyclic reaction of opsin (Sato et al., 2018 ).
• Open5L1 binds to all-trans retinal rather than 11-cis retinal in the dark, forming an active state.
• the Cys188-retinal adduct is dissociated by thermal rotation of the C11-C12 single bond in retinal, and the original dark state is regenerated.
• the ability of Opn5L1 to activate G proteins is controlled by a combination of photoisomerization and thermal isomerization of retinal, making it the first animal opsin whose activity is controlled by a photocycle reaction.
• the amino acid at position 188 when aligned with SEQ ID NO: 1 in the amino acid sequence of the opsin of the present disclosure may include an amino acid corresponding to G, T, S, or E.
• the modified opsins of the present disclosure can have these amino acids modified.
• the amino acid corresponding to position G188 when aligned with SEQ ID NO: 1 may be modified to cysteine.
• the amino acids corresponding to G, T, S, or E can be mentioned as described above, but any of these can be modified to cysteine. It is possible to obtain photocycle characteristics and photoreversibility. Therefore, in one embodiment of the present disclosure, the proteins of the present disclosure can be activated by light stimulation and then inactivated without releasing the photoreceptor factor.
• any opsin can be used as the opsin used in the present disclosure, such as G protein-coupled receptor rhodopsin, rod opsin, cone opsin (blue opsin, green opsin, Examples that can be used include, but are not limited to, red opsin, melanopsin, encephalopsin, OPN5 panopsin, RGR and peropsin.
• the opsins used in this disclosure include vertebrate, invertebrate Examples include, but are not limited to, those derived from animals or microorganisms, or chimeras derived from these organisms.
• Vertebrates include mammals, birds, reptiles, amphibians, fish (teleostea, cartilaginous fishes), or Invertebrates may include, but are not limited to, molluscs, arthropods, or cnidarians.
• Microorganisms include, but are not limited to, eugnaths. Can include, but are not limited to, bacteria, archaea, or fungi.
• the opsins of the present disclosure can be of mammalian origin.
• Rhodopsins can include, but are not limited to, those derived from mammals, such as rhodopsins derived from rodents, artiodactyls, perissodactyls, primates, carnivores, etc. Rhodopsin of primates may be advantageous. Opsins that may also be used include, for example, opsins of bovine, human, murine, rat, cat, canine, porcine, ovine, equine, etc. origin. Of these, those of bovine or human origin are particularly preferred.
• specific amino acid sequences of the opsins of the present disclosure include the sequences of SEQ ID NOs: 1 to 34 listed in Table 1, but are not limited to these sequences.
• nucleic acid sequences of the opsins of the present disclosure include the sequences of SEQ ID NOs: 35 to 68 listed in Table 2, but are not limited to these sequences.
• the proteins of the present disclosure are chimeric proteins comprising a portion of the G protein-coupled receptor rhodopsin and a portion of other G protein-coupled receptors, metabotropic glutamate receptors, adrenergic receptors. It has a seven-transmembrane structure.
• the amino acid sequence encoding the protein of the present disclosure is 1) an amino acid sequence containing the above modification in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 34; 2)
• the sequence other than the modification site has at least about 80% identity with the sequence in 1), and the encoded protein has substantially the same biological activity as the protein obtained from the sequence in 1).
• the amino acid sequence encoding the protein of the present disclosure has a sequence other than the modification site that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about
• the amino acid sequence can be 98%, at least about 99%, or 100% identical and the encoded protein has substantially the same biological activity as the protein obtained from sequence 1). .
• the protein of the present disclosure can include a modification of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1 in the amino acid sequence of opsins, and when aligned with SEQ ID NO: 1, Examples of the amino acid corresponding to position 122 include the amino acid corresponding to E at position 122.
• the modified opsins of the present disclosure can have these amino acids modified.
• the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1 may be modified to glutamine.
• the amino acid corresponding to position 122 of opsins used in the present disclosure the amino acid corresponding to E can be mentioned as described above, but any of these can be modified to glutamine to inhibit the collapse of meta II. can accelerate photocycle reactions.
• the opsins of the present disclosure include amino acids that belong to a part of the N-terminal domain (positions 1 to 11) when aligned with SEQ ID NO: 1, and the extracellular third loop. may include modifications of amino acids corresponding to a portion of the amino acids (positions 278 to 285).
• amino acids that belong to a part of the N-terminal domain (positions 1 to 11) and amino acids that belong to a part of the extracellular third loop (positions 278 to 285) The corresponding amino acid can be modified to cysteine.
• amino acids that correspond to part of the N-terminal domain positions 1 to 11
• amino acids that belong to part of the extracellular third loop positions 278 to 285
• amino acids corresponding to positions 2 and 282 when aligned with SEQ ID NO: 1 can also be used, and preferably these amino acids can be modified to cysteine. By performing such modifications, the thermal stability of the opsins of the present disclosure can be increased.
• the opsins of the present disclosure include the amino acid at position 1, amino acid at position 2, amino acid at position 3, amino acid at position 4, 5 Any one, two, three, four, or five of the following: amino acid at position 6, amino acid at position 7, amino acid at position 8, amino acid at position 9, amino acid at position 10, and amino acid at position 11. , 6, 7, 8, 9, 10, or 11 amino acids and positions 278, 279, and 280 of the extracellular third loop when aligned with SEQ ID NO: 1. , corresponds to any 1, 2, 3, 4, 5, 6, 7, or 8 amino acids of the amino acids at positions 281, 282, 283, 284, and 285 Modifications can be made with amino acids, preferably one or more such amino acids can be modified with cysteine.
• the modified amino acid sequence preferably has one or more amino acids (preferably one or several, or one, two, three, four) in the amino acid sequence of opsin. , 5, 6, 7, 8, 9, 10, or 11) conservative substitutions.
• Methods for obtaining nucleic acids such as DNA of the present disclosure include, but are not particularly limited to, methods for obtaining cDNA by reverse transcription from mRNA (for example, RT-PCR method), methods for preparing cDNA from genomic DNA, and methods for obtaining cDNA by chemical synthesis. Examples of known methods include methods for isolation from genomic DNA libraries and cDNA libraries (see, for example, Japanese Patent Application Laid-Open No. 11-29599).
• the chimeric protein can be prepared, for example, by using a transformant into which an expression vector containing a nucleic acid such as DNA encoding the chimeric protein described above has been introduced. For example, first, this transformant is cultured under appropriate conditions, and a chimeric protein encoded by this DNA or other nucleic acid is synthesized. The chimeric protein of the present disclosure can then be obtained by recovering the synthesized protein from the transformant or the culture solution.
• an "appropriate vector” may be one that can maintain replication or self-propagation in various prokaryotic and/or eukaryotic hosts, and can be appropriately selected depending on the purpose of use. For example, if you want to obtain a large amount of nucleic acid such as DNA, you can select a high copy vector, and if you want to obtain a polypeptide (chimeric protein), you can select an expression vector. Specific examples thereof are not particularly limited, and include, for example, the known vectors described in JP-A No. 11-29599.
• expression vectors can be used not only for synthesizing chimeric proteins, but also for the compositions of the present disclosure. That is, the composition of the present disclosure may contain as an active ingredient an expression vector into which the above-described nucleic acid construct of the present disclosure has been integrated. By directly introducing such an expression vector into humans, it can be used to treat, prevent, and inhibit the progression of retinal diseases, disorders, or symptoms.
• the vector used in this case is one that can be introduced into human cells. Suitable examples of such vectors include adeno-associated virus vectors (AAV vectors) and lentivirus vectors.
• the method for introducing the vector can be selected as appropriate depending on the type of vector and host. Specific examples thereof include, but are not particularly limited to, known methods such as the protoplast method and the competent method (for example, see Japanese Patent Application Laid-Open No. 11-29599) when bacteria are used as hosts. Furthermore, when the expression vector is used as an active ingredient of the visual function regenerating agent or the visual function decline prevention agent of the present disclosure, it can be introduced, for example, by intraocularly injecting the above-mentioned AAV vector or the like.
• the host into which the expression vector is introduced may be any host that is compatible with the expression vector and can be transformed. Specific examples include, but are not limited to, known natural cells such as bacteria, yeast, animal cells, and insect cells. Alternatively, artificially established cells (see Japanese Patent Application Laid-open No. 11-29599), or animals such as humans and mice can be mentioned.
• the transformant is cultured by selecting an appropriate nutrient medium from known nutrient media according to the type of transformant, and adjusting the temperature, pH of the nutrient medium, culture time, etc. so that the chimeric protein can be easily obtained in large quantities. This can be done by adjusting as appropriate (for example, see Japanese Patent Application Laid-Open No. 11-29599).
• the opsins of the present disclosure are provided as a nucleic acid molecule or a nucleic acid construct containing the nucleic acid molecule, and are provided as a medicine for realizing gene therapy.
• a nucleic acid molecule encoding an opsin or a nucleic acid construct containing the nucleic acid molecule that can be used in this embodiment is a protein comprising an amino acid sequence of an opsin, and in the amino acid sequence of the opsin, the amino acid sequence is SEQ ID NO: 1.
• Opsins include a nucleic acid molecule encoding a protein containing a modification of the amino acid corresponding to position 188 when aligned, or a nucleic acid construct containing the nucleic acid molecule, and examples of opsins include those described elsewhere in this specification. can be used.
• the nucleic acid molecule encoding opsin includes, but is not limited to, nucleic acid molecules encoding melanopsin, encephalopsin, OPN5, RGR, and peropsin.
• the nucleic acid molecules used in this disclosure may be those encoding microbial-type opsins, animal-type opsins, and the like.
• the animal-type opsin may also be a nucleic acid molecule encoding a vertebrate visual opsin, a vertebrate non-visual opsin, an invertebrate opsin, and the like.
• Nucleic acid molecules that may be used in this disclosure may be those that encode bistable opsins, such as vertebrate non-visual opsins and invertebrate opsins.
• Opsin-encoding nucleic acid molecules used in the present disclosure include opsins of the microbial type, vertebrate nonvisual opsins, and invertebrate opsins, even if they do not strictly apply to these advantageously used types of opsins. Any nucleic acid molecule can be advantageously used as long as it encodes a functional equivalent having the same function.
• the nucleic acid molecule used in the present disclosure may be, for example, a nucleic acid molecule encoding a G protein-coupled receptor rhodopsin or a chimeric opsin of a G protein-coupled receptor rhodopsin.
• a nucleic acid molecule encoding the G protein-coupled receptor rhodopsin that can be used here is derived from an animal, preferably a mammal, the encoded protein retains the function of repeatedly activating and It is possible to obtain high activity through the sexual G protein.
• Methods for isolating and purifying chimeric proteins are not particularly limited, and known methods such as methods using solubility, methods using differences in molecular weight, methods using charge (for example, Japanese Patent Application Laid-Open No. 11-29599) (see official bulletin).
• the present disclosure provides a protein comprising an amino acid sequence of an opsin, the protein comprising a modification of the amino acid corresponding to position 188 when aligned with SEQ ID NO: 1 in the amino acid sequence of the opsin, A composition, compound, medicament, or method for regenerating vision, or for treating, preventing, or suppressing the progression of visual impairment or disease using a nucleic acid molecule encoding the same or a nucleic acid construct containing the nucleic acid molecule.
• I will provide a.
• the present disclosure provides a method for regenerating vision in a subject, or for treating, preventing, or suppressing the progression of visual impairment or disease in a subject, the method comprising administering a protein of the present disclosure to the subject.
• method can be provided.
• the present disclosure also provides the opsins of the present disclosure, or codes thereof, for producing a composition or medicament for regenerating vision or for treating, preventing, or suppressing the progression of visual impairment or disease in a subject.
• Use of the nucleic acid molecule can be provided.
• the opsins of the present disclosure may have the activity of not releasing retinal when receiving light.
• the diseases, disorders, or symptoms that can be targeted by the opsins of the present disclosure, the nucleic acid molecules encoding the same, or the nucleic acid constructs containing the nucleic acid molecules include vesicular transport disorders, diabetes, diabetic retinopathy, myopia. , macular degeneration (e.g. age-related macular degeneration), glaucoma, cataract, viral infection, corneal dystrophy, retinoblastoma, Alzheimer's disease, Parkinson's disease, lifestyle-related diseases, stroke, hearing loss, arrhythmia, heart failure, motor paralysis, autonomic disease Examples include, but are not limited to, neurological disorders, depression, anxiety, urinary and defecation disorders, and rehabilitation.
• the opsins of the present disclosure or a nucleic acid molecule encoding the same, or a nucleic acid construct comprising such a nucleic acid molecule can be a medicament directed against a disease, disorder, or condition in a human, or a non-human It can also be used as a medicine for treating diseases, disorders or symptoms in animals.
• the opsins of the present disclosure, proteins containing them, nucleic acid molecules encoding the opsins, and/or nucleic acid constructs containing the nucleic acid molecules can be provided as cells containing them. , these cells can also be used as medicine.
• a protein comprising an amino acid sequence of an opsin according to the present disclosure, which comprises a modification of the amino acid corresponding to position 188 when aligned with SEQ ID NO: 1 in the amino acid sequence of the opsin, It can also be used in optogenetics, where it can be used to control only specific neural activities in a target animal.
• Optogenetics is a technology that transforms target cells into a form that can be controlled by light by introducing genes encoding light-driven ion channels, pumps, enzymes, etc. to express light-driven proteins. This allows reversible, immediate, and bioorthogonal manipulation/control and analysis of specific cells (groups) on a millisecond timescale while the animal remains alive.
• the opsins of the present disclosure or the nucleic acid molecules encoding them can also be provided as a kit in combination with a companion diagnostic agent.
• a composition containing the active ingredient of the medicament of the present disclosure may be administered to a subject in the event of a disease or disorder such as a retina-related disease, or in the event that such a disease or disorder is expected to occur. By doing so, the therapeutic effect can be achieved. Therefore, the composition of the present disclosure can be used in combination with a companion diagnostic agent for pre-diagnosing vision-related disorders, diseases, or disorders, and after diagnosing or testing the genetic condition of the subject or the genes possessed by the subject. Compositions of the present disclosure can be administered only to subjects for whom it is expected to be effective.
• the composition or medicine of the present disclosure can also be provided as a nucleic acid medicine.
• polynucleotides may be introduced into the genome of cells to restore or modify genes and/or gene expression.
• therapeutic methods that introduce normal genes into human cells using vectors that can be introduced into human cells, such as various viral vectors, or other delivery systems, can be used.
• the method of introducing the vector can be appropriately selected depending on the type of vector and host, etc.
• the expression vector as an active ingredient of the composition of the present disclosure, for example, by intraocularly injecting an AAV vector etc. can be introduced.
• gene therapy is the insertion of a nucleic acid sequence (e.g., a transgene as defined herein) into the cells and/or tissues of an individual to treat a disease.
• a transgene can be a functional mutant allele that replaces or supplements a defective allele.
• Gene therapy involves methods that are inhibitory in nature, i.e., inhibit or reduce the expression, activity, or function of endogenous genes or proteins, such as unwanted or abnormal (e.g., pathogenic) genes or proteins. , or the insertion of a transgene that reduces Such a transgene may be exogenous. Exogenous molecules or sequences are understood to be molecules or sequences that are not normally present in the cells, tissues and/or individuals to be treated. Both acquired and congenital diseases are amenable to gene therapy.
• a "gene therapy vector” is any vector capable of delivering a polynucleotide encoding a therapeutic protein (eg, opsins, etc.) to a host, such as a patient.
• gene therapy vectors are targeted to particular host cells or organs, eg, for local delivery, eg, tissue-specific delivery.
• local delivery requires a protein encoded by an mRNA (e.g. a therapeutic protein) to be translated and expressed primarily in and/or by an organ, e.g. the liver, thereby requiring a depot, e.g. Forms a hepatic depot for production (and secretion).
• the gene therapy vector is configured to deliver the therapeutic protein polynucleotide to the patient's eye.
• gene therapy vectors deliver polynucleotides encoding therapeutic proteins to other tissues in a patient.
• the gene therapy vector delivers a polynucleotide encoding a therapeutic protein to the patient's optic nerve.
• the gene therapy vector is a viral vector, including, for example, a virus, a viral capsid, a viral genome, and the like.
• the gene therapy vector is a naked polynucleotide, eg, an episome.
• gene therapy vectors include polynucleotide complexes.
• Exemplary, non-limiting polynucleotide conjugates for use as gene therapy vectors include lipoplexes, polymersomes, polypexes, dendrimers, inorganic nanoparticles (e.g., polynucleotide-coated gold, silica, iron oxide, phosphorus, etc.). calcium acid, etc.).
• the gene therapy vectors described herein include a combination of viral vectors, naked polynucleotides, and polynucleotide complexes.
• the gene therapy vector is a viral vector, including a retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus.
• the gene therapy vector is an adeno-associated virus (AAV), such as serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. , or selected engineered or natural variants thereof.
• the polynucleotide also contains an adeno-associated virus (AAV) nucleic acid sequence.
• the gene therapy vector is a chimeric adeno-associated virus containing genetic elements from two or more serotypes.
• an AAV vector (referred to as AAV1/2 or AAV RC1/2) having a rep gene derived from AAV1 and a cap gene derived from AAV2 can be used to inject the polynucleotide of the therapeutic protein of the present disclosure into cells or cells of a patient in need thereof. may be used as a gene therapy vector for delivery to.
• the gene therapy vectors include AAV1/2, AAV1/3, AAV1/4, AAV1/5, AAV1/6, AAV1/7, AAV1/8, AAV1/9, AAV1/10, AAV1/11, AAV2/1, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2/10, AAV2/11, AAV3/1, AAV3/2, AAV3/ 4, AAV3/5, AAV3/6, AAV3/7, AAV3/8, AAV3/9, AAV3/10, AAV3/10, AAV4/1, AAV4/2, AAV4/3, AAV4/5, AAV4/6, AAV4/7, AAV4/8, AAV4/9, AAV4/10, AAV4/11, AAV5/1, AAV5/2, AAV5/3, AAV5/4, AAV5/6, AAV5/7, AAV5/8, AAV5/ 9, AAV5/10, AAV5/11, AAV6/1, AAV6/2, A
• Gao et al. “Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy”, PNAS 99(18): 11854-11859, Sep. 3, 2002, AAV vectors and chimeric virions , and their construction and use, are incorporated herein by reference.
• the gene therapy vector is a viral vector that has been pseudotyped (eg, engineered) to target specific cells (eg, retinal cells).
• target specific cells eg, retinal cells.
• Many of the advances in targeted gene therapy using viral vectors involve non-recombinant (non-genetic) or recombinant (genetic) modifications of the viral vector, such that the natural tropism of the viral vector is can be summarized as resulting in the pseudotyping, expansion, and/or retargeting of. (reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012:1-15).
• Non-genetic approaches typically utilize adapters that recognize both wild-type (unmodified) viral surface proteins and target cells.
• Soluble pseudoreceptors for wild-type virus
• polymers such as polyethylene glycol
• antibodies or parts thereof have been used as virus-binding domains of adapters, while natural peptides or vitamin ligands, as well as antibodies and their Some have been used for the cell binding domain of the adapters described above.
• retargeting of a viral vector to a target cell can be accomplished by binding the vector:adapter complex to a protein expressed on the surface of the target cell, such as a cell surface protein.
• Such approaches include AAV (Bartlett et al. (1999) Nat. Biotechnol. 74: 2777-2785), adenovirus (Hemminki et al. (2001) Cancer Res.
• a common approach is the recombinant genetic modification of the viral capsid protein and hence the recombinant surface of the viral capsid.
• the viral capsid is modified with a heterologous "scaffold" and then ligated to adapters.
• the adapter binds to the scaffold and target cells.
• scaffold For example (1) Antibody Fc-binding molecules (e.g.
• Fc receptors protein A, etc.
• (strepto)avidin that binds to biotinylated adapters
• Proteins that form isometric peptide bonds such as SpyCatcher that binds to SpyTagged adapters: protein binding pairs, etc. et al. (2008) Biochemical and Biophysical Research Communications 366: 769-774; Henning et al. (2002) Human Gene Therapy 13:1427-1439; Banerjee et al. (2011) Bioorganic and Medicinal Chemistry Letters 21:4985-4988 ), AAV (Gigout et al.
• the targeting ligand is inserted or attached directly into the viral capsid, ie, the protein viral capsid is modified to express the heterologous ligand.
• the ligand redirects, eg, binds, a receptor or marker that is preferentially or exclusively expressed on the target cell.
• Poxvirus (Guse et al. (2011) Expert Opinion on Biological Therapy 11:595-608; Galmiche et al. (1997) Journal of General Virology 78:3019-302 7; Paul et al. (2007) Viral Immunology 20:664 -671), paramyxovirus (Nakamura and Russell (2004) Expert Opinion on Biological Therapy 4:1685-1692; Hammond et al.
• the gene therapy vectors described herein include naked polynucleotides.
• a polynucleotide encoding a therapeutic polypeptide may be injected intravenously, directly into an organ to form a depot, eg, near the eye.
• Additional well-known methods for enhanced delivery of naked polynucleotides include, but are not limited to, electroporation, ultrasound perforation, use of a gene gun to eject polynucleotide-coated gold particles, magnetic particles, and hydrodynamic delivery.
• the gene therapy vectors described herein are polynucleotide complexes, such as, but not limited to, nanoparticles (e.g., polynucleotide self-assembled nanoparticles, polymer-based self-assembled nanoparticles, inorganic nanoparticles, lipid nanoparticles, semiconducting/metallic nanoparticles), gels and hydrogels, polynucleotide complexes containing cations and anions, microparticles, and any combination thereof.
• nanoparticles e.g., polynucleotide self-assembled nanoparticles, polymer-based self-assembled nanoparticles, inorganic nanoparticles, lipid nanoparticles, semiconducting/metallic nanoparticles
• gels and hydrogels polynucleotide complexes containing cations and anions, microparticles, and any combination thereof.
• polynucleotides disclosed herein can be formulated as self-assembled nanoparticles.
• polynucleotides may be used to create nanoparticles that can be used in delivery systems for polynucleotides (e.g., International Patent No. (See Application Publication No. 2012125987).
• polynucleotide self-assembled nanoparticles can include a polynucleotide core and a polymer shell as disclosed herein.
• the polymer shell can be any of the polymers described herein and known in the art.
• a polymer shell can be used to protect the polynucleotides within the core.
• these self-assembled nanoparticles can be microsponges formed of long polymeric polynucleotide hairpins that fold into crystalline "wrinkled” sheets. is formed and then self-assembled into microsponges. These microsponges are densely packed, sponge-like microparticles that can act as efficient carriers to deliver cargo to cells. Microsponges can be from 1 ⁇ m to 300 nm in diameter. Microsponges may be complexed with other agents known in the art to form larger microsponges. As a non-limiting example, microsponges may be complexed with agents, such as polycationic polyethylenimine (PEI), to form an outer layer and facilitate uptake by cells.
• PEI polycationic polyethylenimine
• This complex can form particles with a diameter of 250 nm that can remain stable at high temperatures (150 °C) (Grabow and Jaegar, Nature Materials 2012, 11:269-269; (incorporated into the specification). Furthermore, these microsponges may be able to exert an extraordinary degree of protection from degradation by ribonucleases.
• polymer-based self-assembled nanoparticles such as, but not limited to, microsponges, may be fully programmable nanoparticles. Nanoparticle geometry, size, and stoichiometry can be precisely controlled to create optimal nanoparticles for delivering cargo, such as, but not limited to, polynucleotides.
• polynucleotides can be formulated into inorganic nanoparticles (US Pat. No. 8,257,745, incorporated herein by reference in its entirety).
• Inorganic nanoparticles can include, but are not limited to, water-swellable clay materials.
• inorganic nanoparticles can include synthetic smectite clays made from simple silicates (e.g., U.S. Pat. No. 5,585,108 and U.S. Pat. 8,257,745).
• the polynucleotide is formulated in water-dispersible nanoparticles comprising semiconducting or metallic materials (U.S. Patent Application Publication No. 20120228565, incorporated herein by reference in its entirety). or may be formed of magnetic nanoparticles (US Patent Application Publication Nos. 20120265001 and 20120283503, incorporated herein by reference in their entirety). Water-dispersible nanoparticles may be hydrophobic nanoparticles or hydrophilic nanoparticles.
• the polynucleotides disclosed herein can be encapsulated in any hydrogel known in the art that is capable of forming a gel when injected into a subject.
• Hydrogels are networks of hydrophilic polymer chains and may be found as colloidal gels in which water is the dispersion medium. Hydrogels can include highly absorbent (can contain greater than 99% water) natural or synthetic polymers. Hydrogels also have a flexibility very similar to natural tissue due to their significantly higher water content.
• the hydrogels described herein can be used to encapsulate biocompatible, biodegradable, and/or porous lipid nanoparticles.
• the hydrogel may be an aptamer-functionalized hydrogel.
• Aptamer-functionalized hydrogels may be programmed to release one or more polynucleotides using polynucleotide hybridization (Battig et al., J. Am. Chem. Society. 2012 134: 12410-12413; incorporated herein by reference in its entirety).
• polynucleotides may be encapsulated within lipid nanoparticles, which in turn may be encapsulated within a hydrogel.
• polynucleotides may be encapsulated within a fibrin gel, fibrin hydrogel, or fibrin glue.
• the polynucleotide may be formulated into lipid nanoparticles or rapidly excretable lipid nanoparticles before being encapsulated within a fibrin gel, fibrin hydrogel, or fibrin glue.
• the polynucleotide may be formulated as a lipoplex before being encapsulated in a fibrin gel, hydrogel, or fibrin glue.
• Fibrin gels, hydrogels, and adhesives include two components: a fibrinogen solution and a calcium-rich thrombin solution (e.g., Spicer and Mikos, Journal of Controlled Release 2010.
• the concentrations of the components of the fibrin gel, hydrogel, and/or adhesive can be altered to change the properties of the gel, hydrogel, and/or adhesive, the mesh size of the network, and/or the degradation characteristics. , such as, but not limited to, altering the release properties of fibrin gels, hydrogels, and/or adhesives (see, for example, Spicer and Mikos, each of which is incorporated herein by reference in its entirety).
• This feature may be advantageous when used to deliver the polynucleotides disclosed herein. (See, e.g., Kidd et al. Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128, each of which is incorporated herein by reference in its entirety. ).
• polynucleotides disclosed herein may include cations or anions.
• the formulation includes metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ and combinations thereof.
• the formulation can include a polymer and a polynucleotide complexed with a metal cation (e.g., U.S. Pat. No. 6,265,389 and U.S. Pat. 6,555,525).
• polynucleotides may be formulated into nanoparticles and/or microparticles. These nanoparticles and/or microparticles can be shaped into any size shape and chemistry. As an example, nanoparticles and/or microparticles can be made using PRINT® technology by LIQUIDA TECHNOLOGIES.RTM (Morrisville, N.C.) (International Patent Application No. (See Publication No. 2007024323).
• polynucleotides can be formulated in nanojackets and nanoliposomes by Keystone Nano (State College, Pa.).
• Nanojackets are made from compounds naturally found in the body, including calcium, phosphates, or compounds that also contain small amounts of silicates. Nanojackets can range in size from 5 to 50 nm and can be used to deliver hydrophilic and hydrophobic compounds such as, but not limited to, polynucleotides, primary constructs, and/or polynucleotides.
• Nanoliposomes are made from lipids, including, but not limited to, naturally occurring lipids in the body.
• Nanoliposomes can range in size from 60-80 nm and can be used to deliver hydrophilic and hydrophobic compounds such as, but not limited to, polynucleotides, primary constructs, and/or polynucleotides.
• the polynucleotides disclosed herein are formulated in nanoliposomes, such as, but not limited to, ceramide nanoliposomes.
• the polynucleotide e.g., DNA
• the promoter is a tissue-specific promoter that drives gene expression in specific tissues.
• the tissue-specific promoter is a liver-specific enhancer/promoter derived from Serpina 1 and/or the TTR promoter.
• the promoter is a CMV promoter.
• the promoter is the ubiquitin C promoter.
• the polynucleotide also includes a "locus targeting nucleic acid sequence.” Sequences targeting the genetic locus allow for the integration of a polynucleotide encoding a therapeutic protein into the genome of a recipient host cell.
• the locus targeting sequence includes flanking homologous arms to enable homologous recombination.
• the locus targeting sequence includes a guide RNA sequence and a type II Cas enzyme (ie, CRISPR-Cas9 technology) to drive integration.
• the locus targeting sequence includes a guide zinc finger nuclease (ZFN) recognition sequence to drive integration.
• the locus targeting sequence includes a transcription activator-like effector nuclease (TALEN) recognition sequence to drive integration.
• the locus targeting sequence comprises a single residue pair nucleotide code used by a BuD-derived nuclease to drive integration.
• cell therapy using cells containing the composition of the present disclosure includes a treatment method in which retinal cells containing the composition of the present disclosure are transplanted.
• cells comprising the compositions of the present disclosure may be administered in conjunction with additional agents in addition to the cells.
• additional agents may be those commonly used in ophthalmological treatment, such as steroids, antibiotics, antibacterial substances, NSAIDs.
• additional agents may be included in the cell medicine of the present disclosure as a medicament, or may be provided in a separately administered form. In the case of a separately provided or administered form, it may be provided as a kit or a combination drug. When used as a kit or combination drug, an attached document describing how to use the drug may be included.
• the present disclosure provides a method for treating a disease, disorder or condition as described above before or immediately after the onset of the disease, disorder or condition, for example within one year, preferably within six months, three months after the onset (e.g. the onset of subjective symptoms). It is preferable that the drug is administered to a subject within a month, or within a month, but the administration is not limited thereto.
• the proteins, nucleic acid molecules, nucleic acid constructs, and/or cells of the present disclosure, or medicaments comprising them are administered once during the treatment period.
• the medicine and the like of the present disclosure are effective when administered once, and patient compliance is considered to be good.
• the amount of vector used when administering the medicament, etc. of the present disclosure to the eye, can be a unit dose of about 0.01 x 10 11 to about 100 x 10 11 vg/eye.
• the lower limit is about 0.01 ⁇ 10 11 vg/eye, about 0.02 ⁇ 10 11 vg/eye, about 0.03 ⁇ 10 11 vg/eye, about 0.04 ⁇ 10 11 vg/eye, Approximately 0.05 ⁇ 10 11 vg/eye, approximately 0.06 ⁇ 10 11 vg/eye, approximately 0.07 ⁇ 10 11 vg/eye, approximately 0.08 ⁇ 10 11 vg/eye, approximately 0.09 ⁇ 10 11 vg/eye, approximately 0.1 ⁇ 10 11 vg/eye, approximately 0.2 ⁇ 10 11 vg/eye, approximately 0.3 ⁇ 10 11 vg/eye, approximately 0.4 ⁇ 10 11 vg/eye, approximately 0.5 ⁇ 10 11 vg/eye, etc., with upper limits of about 2 ⁇ 10 11 vg/eye, about 3 ⁇ 10 11 vg
• the amount of the vector used is about 0.1 ⁇ 10 11 to about
• a unit dose of 1000 ⁇ 10 11 vg/kg can be given, for example, a lower limit of about 0.1 ⁇ 10 11 vg/kg, about 0.2 ⁇ 10 11 vg/kg, about 0.3 ⁇ 10 11 vg/kg, approximately 0.4 ⁇ 10 11 vg/kg, approximately 0.5 ⁇ 10 11 vg/kg, approximately 0.6 ⁇ 10 11 vg/kg, approximately 0.7 ⁇ 10 11 vg/kg, approximately 0 .8 ⁇ 10 11 vg/kg, about 0.9 ⁇ 10 11 vg/kg, about 1.0 ⁇ 10 11 vg/kg, etc., with an upper limit of about 20 ⁇ 10 11 vg/kg, about 30 ⁇ 10 11 vg/kg, about 40 x 10 11 vg/kg, about 50
• the opsins of the present disclosure proteins, nucleic acid molecules, nucleic acid constructs, and/or cells containing the same, or medicaments containing the same are used for preventing or suppressing the progression of visual impairment. can be used in combination.
• the same active ingredient can be used for the combined use, or different active ingredients can be used in combination.
• This plasmid was transfected into HEK293T cells using the calcium phosphate method. After culturing for 2 days, the transfected cells were collected by centrifugation, suspended in Buffer A (50 mM HEPES, 140 mM NaCl, 3 mM MgCl2, pH 6.5), and 11-cis or all-trans retinal was added. The photopigments were reconstituted. These were solubilized in Buffer A containing 1% dodecyl maltoside (DDM) and adsorbed onto a Rho1D4 (anti-bovine rhodopsin monoclonal antibody) affinity column to purify the dye.
• Buffer A 50 mM HEPES, 140 mM NaCl, 3 mM MgCl2, pH 6.5
• 11-cis or all-trans retinal was added.
• the photopigments were reconstituted. These were solubilized in Buffer A containing 1% dodecyl maltoside (DDM
• UV-visible absorption spectra were recorded using a UV-visible spectrophotometer (UV-2450, UV-2400, Shimadzu Corporation).
• UV-2450 UV-2400
• Shimadzu Corporation For detailed analysis of the thermal response of the pigments, samples were kept at 0°C, 20°C, or 37°C using a cell holder with a temperature-controlled circulating water bath. The sample was exposed to yellow light from a 1 kW tungsten halogen lamp (Master HILUX-HR; RIKEN) through a Y-52 cutoff filter (Toshiba) or through a UV D-36 glass filter (AGC Techno Glass). irradiated with either ultraviolet light.
• a time-resolved CCD spectrophotometer (C10000 system, Hamamatsu Photonics) was used (Sakai et al.) to monitor the G188C mutant sample in the dark and under irradiation ( Spectra were acquired at different time points after 170 ⁇ s (yellow light from a xenon flash lamp through a Y-52 cut-off filter). The temperature of the sample was maintained at 37°C using a temperature controller (pqod, QUANTUM Northwest). The change in absorbance at ⁇ max was plotted as a function of time and fitted with a monoexponential function to determine the time constant for recovery to the original dark state.
• Retinal isomer analysis Retinal isomer analysis
• LC-10ATvp high performance liquid chromatography
• Shimadzu silica column
• silica column YMC-Pack SIL, particle size 3 ⁇ m, 150 ⁇ 6.0 mm, YMC
• Gi ⁇ was prepared by mixing rat Gi ⁇ 1 expressed in E. coli BL21 strain (Lee et al., 1994) and Gt ⁇ purified from bovine retina (Tachibanaki et al., 1997). All assay procedures were performed at 0°C.
• the assay mixture consisted of 10 nM dye, 600 nM G protein, 50 mM HEPES (pH 7.0), 140 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.01% DDM, 1 ⁇ M [35S]GTP ⁇ S, and 2 ⁇ M GDP.
• Bovine rhodopsin wild type and G188C mutant purified after reconstitution with 11-cis retinal were mixed with G protein solution and kept in the dark or irradiated with yellow light (>500 nm) for 1 min followed by UV light. 1 minute, or re-irradiation with yellow light for 1 minute.
• a [35S]GTP ⁇ S solution was added to the mixture of rhodopsin and G protein to initiate the GDP/GTP ⁇ S exchange reaction. After incubation for the selected time in the dark, an aliquot (20 ⁇ l) of the sample was removed into 200 ⁇ l of stop solution (20 mM Tris/Cl [pH 7.4], 100 mM NaCl, 25 mM MgCl2, 1 ⁇ M GTP ⁇ S, 2 ⁇ M GDP) and immediately transferred to nitro. [35S]GTP ⁇ S bound to G protein was trapped by filtration through a cellulose membrane. The amount of bound [35S]GTP ⁇ S was quantified by measuring membranes in a liquid scintillation counter (Tri-Carb 2910 TR; PerkinElmer).
• HEK293T cells The amount of cAMP in HEK293T cells was measured using GloSensor cAMP assay (Promega) according to the manufacturer's instructions and a previous report (Bailes and Lucas, 2013).
• GloSensor cAMP assay Promega
• HEK293T cells were seeded in 96-well plates at a density of 20,000 cells/well in low serum medium (D-MEM/F12 with 0.25% FBS). After culturing for 24 hours, the cells were transfected with 50 ng of rhodopsin plasmid and 50 ng of Glosensor 22F plasmid per well by polyethyleneimine transfection method.
• the equilibration medium was changed to containing a 2% dilution of GloSensor cAMP reagent stock solution in CO2-independent medium (Thermo Fisher Scientific), 10% FBS, 5 ⁇ M retinal.
• luminescence from the cells was measured using a microplate reader (SpectraMax L, Molecular Devices).
• SpectraMax L Molecular Devices
• cells were first treated with 2 ⁇ M forskolin to increase cAMP-dependent luminescence to a plateau level, followed by yellow luminescence from a 1 kW tungsten-halogen lamp through a Y-52 cutoff filter. Stimulation was applied with light for 30 seconds.
• Example 1 Acquisition of photocycle characteristics of bovine rhodopsin G188C mutant
• Figure 1 shows the thermostability of wild type ( Figure 1A), G188C ( Figure 1B), and G188C/N2C/D282C ( Figure 1C) mutants of bovine rhodopsin purified after incubation with 11-cis retinal.
• Absorption spectra were recorded after incubation for 0, 5, 10, 15, 20 minutes in the dark at 37°C (curves 1-5, respectively).
• FIG. 1D shows a schematic representation of retinal conformational changes in wild-type bovine rhodopsin. Dark state, Meta II, and Meta III contain 11-cis-15-anti retinal, all-trans-15-anti retinal, and all-trans-15-syn retinal, respectively.
• Figures 1E and F show the absorption spectra of the N2C/D282C ( Figure 1E) and G188C/N2C/D282C ( Figure 1F) variants of bovine rhodopsin purified after incubation with 11-cis retinal. Spectra were recorded at 20° C. in the dark (curve 1) and at 0, 5, 15, 30, 60, 120 minutes after yellow light irradiation (curves 2-7, respectively).
• the inset shows the difference spectra (curves 1 to 5, respectively) obtained by subtracting the spectra immediately after irradiation (curves 2 to E and F) from the spectra measured after irradiation (curves 3 to 7 in E and F).
• FIG. 1G shows the absorption spectrum of the G188C/N2C/D282C mutant measured at 37°C. Spectra were recorded in the dark (curve 1) and at 0.1, 10, 50, 100, and 1000 seconds after yellow light irradiation (curves 2 to 6, respectively).
• FIG. 1H shows the retinal isomer composition of the G188C/N2C/D282C mutant. Chromophores were extracted from samples taken before light irradiation and 0, 5, and 60 minutes after yellow light irradiation at 20°C, and retinal composition was analyzed by high performance liquid chromatography (HPLC).
• the G188C mutant was found to have significantly lower thermostability than the wild type. That is, the G188C mutant gradually decayed during incubation in the dark at 37°C (Fig. 1B), whereas the wild type was extremely stable under the same conditions (Fig. 1A). Therefore, we improved the thermal stability of the G188C mutant and analyzed the detailed molecular characteristics of the mutant. According to previous reports (Xie et al., 2003; Standfuss et al., 2007), two cysteine residues (N2C/D282C) were introduced into the mutant and thermal attenuation when incubated in the dark at 37°C. The rate was measured.
• Example 2 Acquisition of photoreversible properties of bovine rhodopsin G188C mutant
• Figure 2 shows the photoreaction, retinal arrangement, and G protein activation of the bovine rhodopsin G188C mutant.
• Figures 2A and B show the absorption spectra of purified bovine rhodopsin wild type ( Figure 2A) or G188C mutant ( Figure 2B) after incubation with 11-cis retinal at 0°C.
• Spectra were recorded in the dark (curve 1), after yellow light (>500 nm) irradiation (curve 2), after subsequent ultraviolet light (360 nm) irradiation (curve 3), and after yellow light re-irradiation (curve 4).
• the inset shows the spectral changes of the wild type (Fig. 2A) or the G188C mutant (Fig. 2B) upon yellow light irradiation (curve 1), followed by UV irradiation (curve 2), and yellow light re-irradiation (curve 3).
• the difference spectrum was calculated from the spectra shown in Figures 2A and 2B.
• FIGS. 2C and D show the isomeric composition of retinal of the wild type ( Figure 2C) and the G188C mutant ( Figure 2D). Chromophores were extracted from the samples before light irradiation, after yellow light irradiation, after subsequent UV irradiation, and after yellow light re-irradiation at 0° C., and the retinal composition was analyzed by high performance liquid chromatography (HPLC).
• FIG. 2E shows the Gi type with wild type G protein activation ability. Activation ability was measured in the dark (closed circle) and after yellow light irradiation (open circle).
• FIG. 2F shows the Gi type of G protein activation ability of the G188C mutant.
• the spectra are shown in the dark (curve 1), after yellow light (>500 nm) irradiation (curve 2), after subsequent UV (360 nm) irradiation (curve 3), after yellow light re-irradiation (curve 4), and after UV re-irradiation (curve 4).
• Curve 5 was recorded.
• the inset shows the spectral changes caused by yellow light irradiation (curve 1), followed by UV irradiation (curve 2), yellow light reirradiation (curve 3), and UV reirradiation (curve 4).
• the difference spectrum was calculated based on the spectrum shown in FIG. 2G.
• the wild type and G188C mutant were cooled to 0°C to prevent the thermal reaction of Meta II, and the spectral changes caused by yellow light and subsequent UV irradiation were measured. Irradiation of the wild type with yellow light resulted in the production of Meta II, and subsequent UV irradiation shifted the spectrum into the visible region, shifting ⁇ max ( ⁇ 470 nm) from that of the original dark state to the blue (Fig. 2A). Previous studies have revealed that this state is equivalent to meta-III (Fig. 1D).
• FIG. 3 shows an increase in the speed of recovery of the photocycle characteristics of the bovine rhodopsin G188C mutant due to the introduction of the E122Q mutation.
• FIG. 3A shows the absorption spectrum of the E122Q/G188C/N2C/D282C variant measured at 0°C. Spectra were recorded in the dark (curve 1) and at 0, 5, and 60 minutes after yellow light (>500 nm) irradiation (curves 2 to 4, respectively).
• the inset shows the difference spectra (curves 1 to 3, respectively) obtained by subtracting the spectrum before irradiation (curve 1 in FIG. 3A) from the spectrum measured after irradiation (curves 2 to 4 in FIG. 3A).
• FIG. 3B shows the retinal isomer composition of the E122Q/G188C/N2C/D282C mutant. Chromophores were extracted from samples before light irradiation and 0, 5, and 60 minutes after yellow light irradiation, and retinal composition was analyzed by high performance liquid chromatography (HPLC).
• FIG. 3C shows the absorbance spectrum of the E122Q/G188C/N2C/D282C mutant measured at 37°C.
• FIG. 3D is a graph showing a comparison of the heat recovery processes of G188C/N2C/D282C and E122Q/G188C/N2C/D282C.
• the differential absorbance at ⁇ max obtained by subtracting the spectrum before irradiation from the spectrum measured after irradiation, shown in FIGS.
• the photocycle rate of the E122Q/G188C/N2C/D282C mutant at 37°C was about 12 times faster than that of the G188C/N2C/D282C mutant (Fig. 3D).
• the lifetime of Meta II with a single mutation we succeeded in speeding up the photocycle reaction of the G188C mutant.
• FIG. 4 is a diagram showing light-induced suppression of intracellular cAMP levels by a bovine rhodopsin mutant.
• HEK293T transfected with N2C/D282C- (Fig. 4A, B), G188C/N2C/D282C- (Fig. 4C, D), E122Q/G188C/N2C/D282C- (Fig. 4E, F), and mock (Fig. 4G).
• cAMP levels in cells were measured at room temperature using a GloSensor cAMP assay.
• the light-dependent Gi activation ability was comparable between the wild type and the G188C mutant at 0°C, and no significant thermal recovery to the original dark state was observed in the G188C mutant. Therefore, the amount of intracellular cAMP in cultured cells was measured using a cAMP biosensor (GloSensor), and changes in luminescence of the biosensor triggered by bovine rhodopsin were compared and examined.
• the increase in cAMP levels induced by forskolin addition was attenuated by yellow light irradiation in N2C/D282C bovine rhodopsin-transfected cells (Fig. 4A, B), but not in mock-transfected cells (Fig.
• FIG. 5 shows photopigment formation of bovine rhodopsin G188C mutant after incubation with all-trans retinal.
• the absorption spectra of the wild type (FIG. 5A) or G188C mutant (FIG. 5B) purified after adding all-trans retinal to a suspension of rhodopsin-expressing cell membranes at 0° C. are shown.
• FIG. 5C shows the retinal isomer composition of the G188C mutant purified after addition of all-trans retinal.
• Chromophores were extracted from the samples after yellow light irradiation, subsequent ultraviolet irradiation, and yellow light re-irradiation, and retinal composition was analyzed by high performance liquid chromatography (HPLC). Photopigment regeneration is shown when 11-cis retinal (FIGS. 5D, E) or all-trans retinal (FIGS. 5F, G) is added to purified apoprotein of N2C/D282C.
• the spectra in Figure 5D were taken before addition (curve 1) and at 0, 3, 6, 15, 30, 60, 120 minutes after addition of 1.1 ⁇ M 11-cis retinal (curves 2-8).
• Figure 5E shows the spectrum immediately after the addition of 11-cis retinal (curve 2 in Figure 5D) and the spectrum measured 3, 6, 15, 30, 60, and 120 minutes after the addition of 11-cis retinal (curves 3 to 8 in Figure 5D). Difference spectra were calculated by subtracting (curves 1 to 6, respectively).
• Figure 5F shows spectra measured before addition of 1.1 ⁇ M all-trans retinal (curve 1) and 0, 0.5, 1, 2, 6, 12, 16 hours after addition (curves 2-8).
• the difference spectrum in Figure 5G shows the spectrum immediately after the addition of all-trans retinal (curve 2 in Figure 5F) and the spectrum measured 0.5, 1, 2, 6, 12, and 16 hours after the addition of all-trans retinal (Fig.
• Figure 5I shows the spectrum immediately after the addition of 11-cis retinal (curve 2 in Figure 5H) to the spectrum measured 3, 6, 15, 30, 60, and 120 minutes after the addition of 11-cis retinal (curves 3 to 8 in Figure 5H). ) are shown (curves 1 to 6, respectively) calculated by subtracting the difference spectra.
• Figure 5J shows spectra measured before addition of 1.1 ⁇ M all-trans retinal (curve 1) and at 0, 0.5, 1, 2, 6, 12, 16 hours after addition (curves 2-8).
• the difference spectrum in Figure 5K shows the spectrum immediately after the addition of all-trans retinal (curve 2 in Figure 5J) and the spectrum measured 0.5, 1, 2, 6, 12, and 16 hours after the addition of all-trans retinal (Fig.
• FIG. 5L is a graph in which the regeneration process of photopigments of N2C/D282C and G188C/N2C/D282C by addition of all-trans retinal shown in FIGS. 5F and 5J was monitored by changes in absorbance at 500 nm.
• the wild type and G188C mutant were purified.
• the absorption spectrum of the wild type had almost no peaks in the visible light region and near ultraviolet light region (FIG. 5A).
• the absorption spectrum of the G188C variant has a peak in the visible region (curve 1 in Figure 5B), which may be due to the predominant incorporation of 11-cis and 9-cis retinal rather than all-trans retinal. ( Figure 5C).
• Example 6 Chimeric opsin
• intracellular second and third loops (amino acid numbers 140 to 152 and 225 to 251 of Gene ID: 509933, respectively)
• mouse histamine H2 receptor (amino acid numbers 121-134 and 203-232 of Gene ID: 15466, respectively)
• chimeric opsin was expressed in human-derived cultured cells HEK293.
• T188C mutant Since Xenopus aegypti Opn5m has threonine at position 188, a T188C mutant was created. This T188C mutant bound only all-trans, and its absorption spectrum showed an absorption maximum at 470 nm. When this received visible light, the absorption at 470 nm decreased once at both 20 degrees (FIG. 7a) and 37 degrees (FIGS. 7b and 7c), and then recovered over time. Furthermore, during this process, retinal changed from the all-trans type to the 11-cis or 13-cis type upon visible light reception, and returned to the all-trans type over time (FIG. 7d). These results indicate that the Aedes aegypti Open5mT188C mutant spontaneously returns to its original state after photoreception, and it can be said that it has acquired photocyclic properties.
• Example 8 Human rhodopsin
• the G188C mutant and the E122Q/G188C mutant were expressed in human-derived cultured cells HEK293 in the same manner as in the example for bovine rhodopsin.
• a modified luciferase Promega, GloSensor
• the cultured cells were irradiated with yellow light and the luminescence derived from the modified luciferase was compared.
• the intracellular second and third loops (amino acid numbers 140 to 152 and 225 to 251 of SEQ ID NO: 1, respectively) are connected to the human histamine H2 receptor.
• Chimeric opsins substituted with those of Gene ID: 3274 (amino acid numbers 121 to 134 and 204 to 233, respectively) were expressed in human-derived cultured cells HEK293.
• a modified luciferase Promega, GloSensor was coexpressed as a probe for cAMP, so that the luminescence would increase when the cAMP concentration was high.
• the cultured cells were irradiated with yellow light and the luminescence derived from the modified luciferase was compared.
• Fig. 8D human rhodopsin N2C/N282C in which the second and third intracellular loops were replaced
• Fig. 8E luminescence increased transiently upon light irradiation, and immediately observed to decrease. This result indicates that by introducing the G188C mutation, it is possible to modify the intracellular cAMP concentration so that it can be increased by light for a short period of time and then returned to its original level.
• Example 9 Canine rhodopsin
• canine Canis familiaris
• rhodopsin N2C/D282C the G188C variant was expressed in human-derived cultured cells HEK293 in the same manner as in the example with bovine rhodopsin.
• a modified luciferase Promega, GloSensor
• the cultured cells were irradiated with yellow light and the luminescence derived from the modified luciferase was compared.
• canine rhodopsin N2C/D282C FIG.
• a modified luciferase (Promega, GloSensor) was coexpressed as a probe for cAMP, so that the luminescence would increase when the cAMP concentration was high.
• the cultured cells were irradiated with yellow light and the luminescence derived from the modified luciferase was compared.
• the one with the G188C mutation (Fig. 10D) showed a transient increase in luminescence upon light irradiation, and observed to decrease. This result indicates that by introducing the G188C mutation, it is possible to modify the intracellular cAMP concentration so that it can be increased by light for a short period of time and then returned to its original level.
• Example 10 Medacharhodopsin
• the G188C mutant was expressed in human-derived cultured cells HEK293 in the same manner as in the example for bovine rhodopsin.
• a modified luciferase Promega, GloSensor was coexpressed as a probe for cAMP, so that the luminescence would increase when the cAMP concentration was high.
• the cultured cells were irradiated with yellow light and the luminescence derived from the modified luciferase was compared.
• Example 11 Additional mutations other than positions 188 and 122
• conservative amino acid substitutions were made in the N-terminal domain (positions 1 to 34) and the C-terminal domain (positions 308 onwards).
• the G6A mutation or the V337A mutation was introduced into the human rhodopsin G188C/N2C/D282C mutant and expressed in human-derived cultured cells HEK293.
• a modified luciferase Promega, GloSensor was coexpressed as a probe for cAMP, so that the luminescence would increase when the cAMP concentration was high.
• the cultured cells were irradiated with yellow light and the luminescence derived from the modified luciferase was compared. Similar to the human rhodopsin G188C/N2C/D282C mutant (Fig. 12A), the human rhodopsin G6A/G188C/N2C/D282C mutant (Fig. 12B) and the human rhodopsin V337A/G188C/N2C/D282C mutant (Fig. 12C). We observed that the luminescence decreased temporarily upon irradiation with light, and then recovered immediately. This result shows that these three mutants cause a short-term decrease in intracellular cAMP concentration with light.
• the intracellular cAMP concentration can be reduced by light. It is shown that he retains the ability to reduce time.
• Example 12 Introduction of cysteine mutations other than positions 2 and 282
• SEQ ID NO: 1 amino acids belonging to part of the N-terminal domain (positions 1 to 11) and part of the extracellular third loop ( Cysteine was modified in the amino acids corresponding to amino acids belonging to positions 278 to 285).
• the N2C/G3C/G280C mutation, N2C/G3C/S281C mutation, or G3C/N282C mutation was introduced into the human rhodopsin G188C mutant and expressed in human-derived cultured cells HEK293.
• a modified luciferase (Promega, GloSensor) was coexpressed as a probe for cAMP, so that the luminescence would increase when the cAMP concentration was high.
• the cultured cells were irradiated with yellow light and the luminescence derived from the modified luciferase was compared. Similar to the human rhodopsin G188C/N2C/D282C mutant (Fig. 13A), the human rhodopsin G188C/N2C/G3C/G280C mutant (Fig. 13B), the human rhodopsin G188C/N2C/G3C/S281C mutant (Fig.
• Example 13 Restoration of light-induced activity from the retina by G188C mutant
• a viral vector (rAAV-DJ) containing the G188C mutant coding sequence under the control of the CMV promoter was injected into the vitreous of 10-week-old rd1 mice (retinitis pigmentosa blindness model mouse).
• rAAV-DJ a viral vector containing the G188C mutant coding sequence under the control of the CMV promoter was injected into the vitreous of 10-week-old rd1 mice (retinitis pigmentosa blindness model mouse).
• AAV-DJ vector for more efficient and widespread gene transfer, and take AAV-2 as a benchmark that has already been applied clinically. Retinas were obtained 1 month later.
• Expression of the reporter gene (EGFP) appears throughout the retina and in both the ganglion cell layer (GCL) and the internal nuclear layer (INL).
• FIG. Figure 14A shows untreated rd1 mice.
• Figure 14B shows that the human rhodopsin G188C/N2C/D282C mutant was introduced using a viral vector, and
• Figure 14C shows that the intracellular second and third loops of the human rhodopsin G188C/N2C/D282C mutant were transferred to the human histamine H2 receptor.
• Figure 14D shows the case where the intracellular second and third loops of the human rhodopsin E122Q/G188C/N2C/D282C mutant were replaced with those of the human histamine H2 receptor. These mutants are the same as those described in Example 8 and elsewhere.
• Example 14 Evaluation of visual evoked potential
• VEPs visual evoked potentials
• Example 15 Evaluation of brightness recognition function
• LDT light-dark box shift test
• Example 16 Optogenetics ⁇ neural control>
• the intervention of cAMP is essential for the induction of branching and elongation of nerve axons, and as in the G188C mutant and Example 8, the intracellular second and third loops are replaced with those of the human histamine H2 receptor.
• the branching and elongation of axons in G188C mutant-introduced hippocampal neurons that were irradiated with blue light for 30 minutes was suppressed compared to those that were not irradiated.
• hippocampal neurons introduced with the G188C mutant chimera opsin that were irradiated with blue light for 30 minutes branching and elongation of axons were promoted compared to those that were not irradiated.
• Example 17 Calcium ion-transformable chimeric opsin
• a chimeric opsin in which the intracellular second and third loops were replaced with those of the human ⁇ 1A adrenergic receptor was expressed in human-derived cultured cells HEK293 for the G188C mutant. Ta. At this time, they co-expressed aequorin derived from the Aequora jellyfish as a probe for calcium ions, so that the luminescence would increase when the concentration of calcium ions was high. The cultured cells were irradiated with yellow light and the luminescence derived from aequorin was compared.
• SEQ ID NO: 1 Amino acid sequence of Homo sapiens rhodopsin NP_001372054.1
• SEQ ID NO: 2 Amino acid sequence of Mus musculus rhodopsin NP_663358.1
• SEQ ID NO: 3 Amino acid sequence of Canis familiaris rhodopsin CAA50502.1
• SEQ ID NO: 4 Amino acid sequence of Gallus gallus rhodopsin NP_001384426.1
• SEQ ID NO: 6 Amino acid sequence of Homo sapiens blue cone opsin NP_001372054.1
• SEQ ID NO: 7 Amino acid sequence of Homo sapiens red cone opsin NP_064445.2
• SEQ ID NO: 8 Homo Amino acid sequence of sapiens green cone opsin

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Genetics & Genomics (AREA)
• Zoology (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biophysics (AREA)
• Gastroenterology & Hepatology (AREA)
• Toxicology (AREA)
• Biomedical Technology (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Public Health (AREA)
• Cell Biology (AREA)
• Biotechnology (AREA)
• Immunology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Epidemiology (AREA)
• Endocrinology (AREA)
• Ophthalmology & Optometry (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Wood Science & Technology (AREA)
• Neurology (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Neurosurgery (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (1)

Family

ID=90099743

Family Applications (1)

Country Status (5)

Cited By (1)

Citations (12)

• 2023
  • 2023-08-30 JP JP2024544545A patent/JPWO2024048688A1/ja active Pending
  • 2023-08-30 EP EP23860450.8A patent/EP4582544A1/en active Pending
  • 2023-08-30 CN CN202380062525.5A patent/CN120225677A/zh active Pending
  • 2023-08-30 WO PCT/JP2023/031644 patent/WO2024048688A1/ja not_active Ceased
  • 2023-08-30 KR KR1020257010345A patent/KR20250061742A/ko active Pending

Patent Citations (13)

Non-Patent Citations (57)

Also Published As

Similar Documents

Legal Events