Nucleic acid construct that encodes chimeric rhodopsin

Abstract

To provide prevention and suppression of the progression of retinal diseases, and enhancement of visual cognitive behavioral function and visual function.SOLUTION: Provided are: a nucleic acid comprising a nucleic acid sequence encoding a chimeric protein including at least part of an ion-transporting receptor rhodopsin and at least part of a G protein-coupled receptor rhodopsin, and a nucleic acid sequence encoding a signal sequence, and a nucleic acid comprising a nucleic acid sequence encoding a chimeric protein including at least part of an ion-channeling receptor rhodopsin and at least part of a G protein-coupled receptor rhodopsin; and a nucleic acid construct comprising these nucleic acids. Use of the nucleic acid or nucleic acid construct provides prevention and suppression of the progression of retinal diseases, and enhancement of visual cognitive behavioral function and visual function.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to the prevention and slowing of progression of retinal diseases, the improvement of visual-cognitive-behavioral functions, and the enhancement of visual function. [Background technology]

[0002] Rhodopsin is a light-sensitive receptor with a seven-transmembrane structure in the retina of humans and animals, and is also used in medical applications. Summary of the Invention [Means for solving the problem]

[0003] The present inventors have discovered that a chimeric protein of two types of rhodopsin, an ion transporter rhodopsin and a G protein-coupled receptor rhodopsin, has the effects of preventing and slowing the progression of retinal diseases, improving visual cognitive behavioral functions, and enhancing visual function.The present inventors have also discovered that a novel nucleic acid construct encoding a chimeric rhodopsin incorporating an endoplasmic reticulum transport signal has significantly high expression efficiency and is effective for clinical application.

[0004] Thus, the present disclosure provides: (Item X1) A nucleic acid comprising a nucleic acid sequence encoding a chimeric protein comprising at least a portion of an ion transport receptor rhodopsin and at least a portion of a G protein-coupled receptor rhodopsin, and a nucleic acid sequence encoding a signal sequence. (Item X2) The nucleic acid according to item X1, wherein the signal sequence is an endoplasmic reticulum export signal sequence. (Item X3) The nucleic acid according to item X1 or 2, wherein the nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO: 1 or 26. (Item X4) The nucleic acid according to any one of items X1 to X3, further comprising a nucleic acid sequence encoding a FLAG tag. (Item X5) The nucleic acid according to any one of items X1 to X4, wherein the nucleic acid comprises the nucleic acid sequence shown in SEQ ID NO:3. (Item X6) The nucleic acid according to any one of items X1 to X3, wherein the nucleic acid is the nucleic acid sequence shown in SEQ ID NO:26. (Item X7) A polypeptide consisting of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin, and a signal sequence. (Item X8) The polypeptide according to item X7, wherein the signal sequence is an endoplasmic reticulum export signal sequence. (Item X9) The polypeptide according to item X8 or 9, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 26. (Item X10) The nucleic acid according to item X1, comprising a nucleic acid sequence encoding the polypeptide according to any one of items X7 to X9. (Item X11) The nucleic acid of item X10, further comprising a nucleic acid sequence encoding a FLAG tag. (Item X12) The nucleic acid according to item X10 or 11, comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:4. (Item X13) A nucleic acid comprising a nucleic acid sequence encoding a chimeric protein comprising at least a portion of an ionotropic receptor rhodopsin and at least a portion of a G protein-coupled receptor rhodopsin. (Item X14) The nucleic acid according to item X13, wherein the nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO: 7. (Item X15) A polypeptide comprising a chimeric protein of a portion of an ionotropic receptor rhodopsin and a portion of a G protein-coupled receptor rhodopsin. (Item X16) The polypeptide according to item X15, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO: 8. (Item X17) The nucleic acid according to item X13, comprising a nucleic acid sequence encoding the polypeptide according to item X15 or 16. (Item X18) The nucleic acid according to item X17, comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:8. (Item X19) A nucleic acid construct comprising a nucleic acid according to any one of items X1 to X6 and 10 to 12 and / or a nucleic acid according to any one of items X13 to 14 and 17 to 18, and a nucleic acid operably linked to the nucleic acid that enables expression in a cell. (Item X20) The nucleic acid construct of item X19, further comprising a vector. (Item X21) The nucleic acid construct according to item X20, wherein the vector is a viral vector. (Item X22) The nucleic acid construct of item X20 or 21, wherein the vector is a retroviral vector, a lentiviral vector, or an adeno-associated viral (AAV) vector. (Item X23) The nucleic acid construct according to any one of items X20 to 22, wherein the vector is an AAV vector. (Item X24) The nucleic acid construct of item X23, wherein the AAV vector is AAV-DJ, AAV-2, or AAV-6. (Item X25) A composition for gene transfer, comprising the nucleic acid according to any one of items X1 to 6 and 10 to 12, the nucleic acid according to any one of items X13 to 14 and 17 to 18, or the nucleic acid construct according to any one of items X19 to 24. (Item X26) A cell comprising one or more of the nucleic acid according to any one of items X1 to X6 and 10 to 12, the polypeptide according to any one of items X7 to 9, the nucleic acid according to any one of items X13 to 14 and 17 to 18, the polypeptide according to items X15 to 16, or the nucleic acid construct according to any one of items X19 to 24. (Item X27) The cell according to item X26, wherein the cell is a retinal cell. (Item X28) A pharmaceutical composition comprising one or more of the nucleic acid according to any one of items X1 to 6 and 10 to 12, the polypeptide according to any one of items X7 to 9, the nucleic acid according to any one of items X13 to 14 and 17 to 18, the polypeptide according to items X15 to 16, the nucleic acid construct according to any one of items X19 to 24, the composition for gene transfer according to item X25, or the cell according to any one of items X26 to 27. (Item X29) The pharmaceutical composition according to item X28 for treating, preventing or inhibiting the progression of a retinal disease, disorder or condition. (Item X30) The pharmaceutical composition according to item X28 for improving visual cognitive behavioral function. (Item X31) The pharmaceutical composition according to item X28 for enhancing visual function. (Item X32) The pharmaceutical composition according to item X31 for enhancing object recognition function. (Item A1) 1. A method of treating, preventing, or inhibiting the progression of a retinal disease, disorder, or condition in a subject, comprising: A method comprising the step of administering to a subject an effective amount of one or more of the nucleic acid described in any one of items X1 to 6 and 10 to 12, the polypeptide described in any one of items X7 to 9, the nucleic acid described in any one of items X13 to 14 and 17 to 18, the polypeptide described in items X15 to 16, the nucleic acid construct described in any one of items X19 to 24, the composition for gene transfer described in item X25, or the cell described in any one of items X26 to 27. (Item A2) 1. A method for improving visual cognitive behavioral function in a subject, comprising: A method comprising the step of administering to a subject an effective amount of one or more of the nucleic acid described in any one of items X1 to 6 and 10 to 12, the polypeptide described in any one of items X7 to 9, the nucleic acid described in any one of items X13 to 14 and 17 to 18, the polypeptide described in items X15 to 16, the nucleic acid construct described in any one of items X19 to 24, the composition for gene transfer described in item X25, or the cell described in any one of items X26 to 27. (Item A3) 1. A method of enhancing visual function in a subject, comprising: A method comprising the step of administering to a subject an effective amount of one or more of the nucleic acid described in any one of items X1 to 6 and 10 to 12, the polypeptide described in any one of items X7 to 9, the nucleic acid described in any one of items X13 to 14 and 17 to 18, the polypeptide described in items X15 to 16, the nucleic acid construct described in any one of items X19 to 24, the composition for gene transfer described in item X25, or the cell described in any one of items X26 to 27. (Item A4) 1. A method for enhancing object recognition in a subject, comprising: A method comprising the step of administering to a subject an effective amount of one or more of the nucleic acid described in any one of items X1 to 6 and 10 to 12, the polypeptide described in any one of items X7 to 9, the nucleic acid described in any one of items X13 to 14 and 17 to 18, the polypeptide described in items X15 to 16, the nucleic acid construct described in any one of items X19 to 24, the composition for gene transfer described in item X25, or the cell described in any one of items X26 to 27. (Item B1) Use of one or more of the nucleic acid according to any one of items X1 to 6 and 10 to 12, the polypeptide according to any one of items X7 to 9, the nucleic acid according to any one of items X13 to 14 and 17 to 18, the polypeptide according to items X15 to 16, the nucleic acid construct according to any one of items X19 to 24, the composition for gene transfer according to item X25, or the cell according to any one of items X26 to 27 in the manufacture of a medicament for treating, preventing, or suppressing the progression of a retinal disease, disorder, or symptom. (Item B2) Use of one or more of the nucleic acid according to any one of items X1 to 6 and 10 to 12, the polypeptide according to any one of items X7 to 9, the nucleic acid according to any one of items X13 to 14 and 17 to 18, the polypeptide according to items X15 to 16, the nucleic acid construct according to any one of items X19 to 24, the composition for gene transfer according to item X25, or the cell according to any one of items X26 to 27 in the manufacture of a medicament for improving visual cognitive behavioral function. (Item B3) Use of one or more of the nucleic acid according to any one of items X1 to 6 and 10 to 12, the polypeptide according to any one of items X7 to 9, the nucleic acid according to any one of items X13 to 14 and 17 to 18, the polypeptide according to items X15 to 16, the nucleic acid construct according to any one of items X19 to 24, the composition for gene transfer according to item X25, or the cell according to any one of items X26 to 27 in the manufacture of a medicine for enhancing visual function. (Item B4) Use of one or more of the nucleic acid according to any one of items X1 to 6 and 10 to 12, the polypeptide according to any one of items X7 to 9, the nucleic acid according to any one of items X13 to 14 and 17 to 18, the polypeptide according to items X15 to 16, the nucleic acid construct according to any one of items X19 to 24, the composition for gene transfer according to item X25, or the cell according to any one of items X26 to 27 in the manufacture of a pharmaceutical for enhancing object recognition function. (Item B5) Use of the nucleic acid according to any one of Items X1 to X6 and 10 to 12, the nucleic acid according to any one of Items X13 to 14 and 17 to 18, the gene of the nucleic acid construct according to any one of Items X19 to 24, or the cell according to any one of Items X26 to 27 in the manufacture of a pharmaceutical for introducing a gene. (Item C1) A nucleic acid according to any one of items X1 to 6 and 10 to 12, a polypeptide according to any one of items X7 to 9, a nucleic acid according to any one of items X13 to 14 and 17 to 18, a polypeptide according to items X15 to 16, a nucleic acid construct according to any one of items X19 to 24, or a cell according to any one of items X26 to 27, for treating, preventing, or suppressing the progression of a retinal disease, disorder, or symptom. (Item C2) A nucleic acid according to any one of items X1 to 6 and 10 to 12, a polypeptide according to any one of items X7 to 9, a nucleic acid according to any one of items X13 to 14 and 17 to 18, a polypeptide according to items X15 to 16, a nucleic acid construct according to any one of items X19 to 24, or a cell according to any one of items X26 to 27, for improving visual cognitive behavioral function. (Item C3) A nucleic acid according to any one of items X1 to 6 and 10 to 12, a polypeptide according to any one of items X7 to 9, a nucleic acid according to any one of items X13 to 14 and 17 to 18, a polypeptide according to items X15 to 16, a nucleic acid construct according to any one of items X19 to 24, a composition for gene transfer according to item X25, or a cell according to any one of items X26 to 27, for enhancing visual function. (Item C4) A nucleic acid according to any one of items X1 to 6 and 10 to 12, a polypeptide according to any one of items X7 to 9, a nucleic acid according to any one of items X13 to 14 and 17 to 18, a polypeptide according to items X15 to 16, a nucleic acid construct according to any one of items X19 to 24, or a cell according to any one of items X26 to 27, for enhancing object recognition function. (Item C5) A nucleic acid according to any one of Items X1 to X6 and 10 to 12, a nucleic acid according to any one of Items X13 to 14 and 17 to 18, or a nucleic acid construct according to any one of Items X19 to 24, for introducing a gene. (Item 1) A nucleic acid comprising a nucleic acid sequence encoding a chimeric protein comprising at least a portion of an ion transport receptor rhodopsin and at least a portion of a G protein-coupled receptor rhodopsin, and a nucleic acid sequence encoding a signal sequence. (Item 2) 2. The nucleic acid according to item 1, wherein the signal sequence is an endoplasmic reticulum export signal sequence. (Item 3) 3. The nucleic acid according to item 1 or 2, wherein the nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO: 1. (Item 4) 4. The nucleic acid according to any one of items 1 to 3, further comprising a nucleic acid sequence encoding a FLAG tag. (Item 5) 5. The nucleic acid according to any one of items 1 to 4, wherein the nucleic acid comprises the nucleic acid sequence shown in SEQ ID NO:3. (Item 6) A polypeptide consisting of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin, and a signal sequence. (Item 7) 7. The polypeptide according to item 6, wherein the signal sequence is an endoplasmic reticulum export signal sequence. (Item 8) 9. The polypeptide according to item 7 or 8, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO:2. (Item 9) 9. The nucleic acid according to Item 1, comprising a nucleic acid sequence encoding the polypeptide according to any one of Items 6 to 8. (Item 10) 10. The nucleic acid of item 9, further comprising a nucleic acid sequence encoding a FLAG tag. (Item 11) 11. The nucleic acid according to item 9 or 10, comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:4. (Item 12) A nucleic acid comprising a nucleic acid sequence encoding a chimeric protein comprising at least a portion of an ionotropic receptor rhodopsin and at least a portion of a G protein-coupled receptor rhodopsin. (Item 13) 13. The nucleic acid according to item 12, wherein the nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO: 7. (Item 14) A polypeptide comprising a chimeric protein of a portion of an ionotropic receptor rhodopsin and a portion of a G protein-coupled receptor rhodopsin. (Item 15) Item 15. The polypeptide according to item 14, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO:8. (Item 16) 16. The nucleic acid according to Item 12, comprising a nucleic acid sequence encoding the polypeptide according to Item 14 or 15. (Item 17) 17. The nucleic acid according to item 16, comprising a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO:8. (Item 18) A nucleic acid construct comprising the nucleic acid according to any one of Items 1 to 5 and 9 to 11 and / or the nucleic acid according to any one of Items 12 to 13 and 16 to 17, and a nucleic acid operably linked to the nucleic acid that enables expression in a cell. (Item 19) 19. The nucleic acid construct of item 18, further comprising a vector. (Item 20) 20. The nucleic acid construct of item 19, wherein the vector is a viral vector. (Item 21) 21. The nucleic acid construct of item 19 or 20, wherein the vector is a retroviral vector, a lentiviral vector, or an adeno-associated viral (AAV) vector. (Item 22) 22. The nucleic acid construct according to any one of items 19 to 21, wherein the vector is an AAV vector. (Item 23) 23. The nucleic acid construct of item 22, wherein the AAV vector is AAV-DJ, AAV-2, or AAV-6. (Item 24) A composition for gene transfer, comprising the nucleic acid according to any one of Items 1 to 5 and 9 to 11, the nucleic acid according to any one of Items 12 to 13 and 16 to 17, or the nucleic acid construct according to any one of Items 18 to 23. (Item 25) A cell comprising one or more of the nucleic acid according to any one of Items 1 to 5 and 9 to 11, the polypeptide according to any one of Items 6 to 8, the nucleic acid according to any one of Items 12 to 13 and 16 to 17, the polypeptide according to Items 14 to 15, or the nucleic acid construct according to any one of Items 18 to 23. (Item 26) 26. The cell of item 25, wherein the cell is a retinal cell. (Item 27) A pharmaceutical composition comprising one or more of the nucleic acid according to any one of Items 1 to 5 and 9 to 11, the polypeptide according to any one of Items 6 to 8, the nucleic acid according to any one of Items 12 to 13 and 16 to 17, the polypeptide according to Items 14 to 15, the nucleic acid construct according to any one of Items 18 to 23, the composition for gene transfer according to Item 24, or the cell according to any one of Items 25 to 26. (Item 28) 28. The pharmaceutical composition according to item 27, for treating, preventing or inhibiting the progression of a retinal disease, disorder or condition. (Item 29) 28. The pharmaceutical composition according to item 27, for improving visual cognitive behavioral function. (Item 30) 28. The pharmaceutical composition according to item 27, for enhancing visual function. (Item 31) 31. The pharmaceutical composition according to item 30, for enhancing object recognition function.

[0005] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0006] This disclosure demonstrates that gene expression of chimeric proteins is more efficient and sensitive, and functions more effectively. This disclosure allows for superior visual regeneration. This disclosure not only achieved increased expression levels and light sensitivity in MEA compared to conventional constructs, but also confirmed significant central light response regeneration in VEP. When applied to human visual regeneration gene therapy for retinal diseases, this is expected to restore vision in darker conditions and expand the visual field. Furthermore, it is possible to achieve the regeneration and improvement of light-dark discrimination and visual cognitive behavioral functions, the regeneration of object recognition functions, and the suppression of disease progression. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows the configurations of a nucleic acid construct encoding a chimeric rhodopsin (first nucleic acid construct) and a nucleic acid construct encoding a chimeric rhodopsin to which a signal sequence has been added (nucleic acid construct of the present disclosure). [Figure 2] FIG. 2 shows the results of a multi-electrode array test in mice injected with the first nucleic acid construct. [Figure 3] FIG. 3 shows the results of a multi-electrode array study in mice injected with the nucleic acid constructs of the present disclosure. [Figure 4] Figure 4 is a graph quantifying the results of Figures 2 and 3. Within the stimulation intensity range of 1x1014 to 16 photons / cm2 / s, the nucleic acid construct of the present disclosure had a significantly higher firing frequency. [Figure 5]Figure 5 is a graph quantifying the results of Figures 2 and 3. At a stimulation intensity of 1 x 1015 photons / cm2 / s, the nucleic acid construct of the present disclosure resulted in a significantly higher number of firing cells per unit area. [Figure 6] FIG. 6 is a graph evaluating the wavelength sensitivity of mice injected with nucleic acid constructs of the present disclosure. [Figure 7] FIG. 7 shows the results of a global assessment of visual evoked potentials in mice injected with a first nucleic acid construct and mice injected with a nucleic acid construct of the present disclosure. [Figure 8] Figure 8 shows the space designed for the experiment to evaluate the object recognition function. Tablet devices were placed on both sides of the space containing the mouse, and the brightness was set to 10 lux, with one side displaying a video of a mouse and the other displaying an empty mouse cage. [Figure 9] FIG. 9 is a diagram showing the results of an evaluation test of the object recognition function. [Figure 10] FIG. 10 shows the results of measuring the GPCR activity of proteins encoded by the nucleic acid constructs of the present disclosure using GloSensor™. [Figure 11] FIG. 11 shows the results of measuring the GPCR activity of a chimeric protein of ionotropic rhodopsin and G protein-coupled receptor rhodopsin using GloSensor™. [Figure 12] FIG. 12 shows the results of measuring the ion transport ability of a chimeric protein of ionotropic rhodopsin and G protein-coupled receptor rhodopsin by the patch clamp method. [Figure 13] FIG. 13 shows experimental data in which each gene was forced to be expressed in HEK293T cells using the lipofection method, and the change in cAMP concentration was measured with and without light stimulation. DETAILED DESCRIPTION OF THE INVENTION

[0008] The present disclosure will now be described, illustrating the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0009] (definition, etc.) The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0010] As used herein, "rhodopsin" refers to a protein containing a pigment called retinal, which is activated by exposure to light and transmits visual signals to the brain. Ion transport receptor rhodopsins, typically derived from microorganisms, retain retinal even when irradiated with light and can be repeatedly activated by absorbing light. However, unlike G protein-coupled receptor rhodopsins, typically derived from animals, they cannot activate G proteins. In contrast, the chimeric rhodopsin of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin provided by the present disclosure is believed to have enhanced functionality compared to conventional rhodopsins. In particular, when the ion transport receptor rhodopsin is preferably derived from a microorganism and can be repeatedly used, and when the G protein-coupled receptor rhodopsin is derived from an animal, preferably a mammal, it is possible to obtain high activity mediated by endogenous G proteins while maintaining the ability to repeatedly activate. Without wishing to be bound by theory, the chimeric proteins utilized in the present disclosure are expressed in mammals such as rodents and primates while retaining sufficient activity, as demonstrated in animal models, and therefore can achieve the effects of preventing and inhibiting the progression of retinal diseases, disorders, or symptoms, in particular preventing or inhibiting the progression of retinitis pigmentosa, or can bring about improvements in visual cognitive behavioral functions (e.g., improved light / dark judgment function, improved light aversion function, and / or danger avoidance function), or can have the effect of enhancing visual functions such as improved visual acuity.

[0011] As used herein, the term "ion transport receptor rhodopsin" refers to any rhodopsin that has the function of transporting ions, and examples thereof include ion pump receptor rhodopsin and ion channel receptor rhodopsin.

[0012] For ion transport receptor rhodopsins, the three-dimensional conformational compatibility with the G protein activation loop and membrane translocation efficiency are considered important. In particular, ion transport receptor rhodopsins derived from algae or microorganisms have good three-dimensional conformational compatibility with the G protein activation loop and membrane translocation efficiency. Among them, those from the genera Gloeobacter or Guillardia are preferred. In particular, Gloeobacter violaceus, a microorganism belonging to the genus Gloeobacter, and Guillardia theta, a microorganism belonging to the genus Guillardia, are preferred. Furthermore, rhodopsin from a microorganism belonging to the genus Gloeobacter (e.g., SEQ ID NO: 14) or a microorganism belonging to the genus Guillardia (e.g., SEQ ID NO: 16) is preferably used in combination with a mammalian G protein-coupled receptor rhodopsin, among animal-derived G protein-coupled receptor rhodopsins, preferably a G protein-coupled receptor rhodopsin from an artiodactyl such as cattle (e.g., SEQ ID NO: 12) or a primate such as a human (e.g., SEQ ID NO: 10).Algae such as those from the genera Gloeobacter and Guillardia are also preferred in that they have the important property of being well expressed in both Escherichia coli, a true bacterium, and human cells, a eukaryote.

[0013] As used herein, the term "ion pump receptor rhodopsin" refers to any pump-type rhodopsin that has the function of transporting ions. Upon sensing light, it functions by actively transporting ions such as hydrogen ions, chloride ions, or sodium ions.

[0014] As used herein, the term "ionotropic receptor rhodopsin" refers to any channel-type rhodopsin that has the function of transporting ions. Upon sensing light, it functions by allowing ions such as hydrogen ions, chloride ions, or sodium ions to flow into the cell.

[0015] As used herein, "G protein-coupled receptor rhodopsin" refers to rhodopsin, a type of receptor present on the cytoplasmic membrane of eukaryotic cells or on membranes within the cell. G protein-coupled receptors have seven alpha helices that span the cytoplasmic membrane, with the N-terminus present extracellularly and the C-terminus present intracellularly. They are said to have three extracellular loops (ECL1 / 2 / 3) and three intracellular loops (ICL1 / 2 / 3). Rhodopsin is composed of an apoprotein and the chromophore retinal. When retinal absorbs light, it isomerizes, causing a conformational change in the protein, which then drives the intracellular signaling pathway via the G protein.

[0016] As used herein, the term "retinal disease, disorder, or symptom" refers to any disease, disorder, or symptom related to the retina, including retinal degenerative diseases (retinitis pigmentosa, age-related macular degeneration, etc.), retinopathies (e.g., diabetic retinopathy, proliferative retinopathy, simple retinopathy, etc.), floaters, retinal breaks, retinal detachment (e.g., rhegmatogenous retinal detachment, non-rhegmatogenous retinal detachment, etc.), and the present invention can prevent, treat, or inhibit the progression of retinitis pigmentosa, age-related macular degeneration, myopic maculopathy, macular dystrophy, diabetic retinopathy, retinal detachment, etc. Examples of disorders or symptoms include disorders of visual acuity, contrast sensitivity, light-dark adaptation, color vision, etc., and symptoms related thereto.

[0017] As used herein, the term "visual cognitive behavioral function" refers to the function of visual information perceived by the visual organs (eyes, etc.) as a behavior of a target organism, such as the ability to judge light and dark, the ability to avoid light, or the ability to avoid danger. This function can be confirmed not only by confirming light sensitivity but also by actually verifying it using animal models, etc.

[0018] In this specification, the term "light and dark judgment function" refers to the ability or function to judge light and dark. The term "improvement" refers to any improvement in the light and dark judgment function, and includes, for example, not only becoming able to judge light and dark when one was previously unable to do so, but also improving to the point where one can finally distinguish between light and dark.

[0019] As used herein, the term "light avoidance function" refers to the ability or function to move away from a light source or avoid bright light. Improvement in this function refers to the recovery or enhancement of the ability to avoid bright light.

[0020] In this specification, the term "crisis avoidance function" refers to the function or ability to avoid a crisis based on visual function. Improvement of the crisis avoidance function includes not only the restoration of the crisis avoidance ability but also an increase in its level.

[0021] As used herein, "enhancement" or "enhancement" of "visual function" refers to the improvement, strengthening, or enhancement of any visual function (e.g., visual acuity, color vision, contrast sensitivity, light-dark adaptation, etc.).

[0022] As used herein, "improved visual acuity" refers to an improvement or recovery of visual acuity. For example, in humans, visual acuity can be measured using a visual acuity test using Landolt rings, as well as a Snellen chart or an E chart, and expressed as decimal or fractional visual acuity. These can also be expressed as logMAR visual acuity. In mice, visual acuity can be measured using visual stimuli in which the spatial frequency of a light-dark striped pattern is manipulated. Experimentally, visual evoked potentials can also be measured to determine visual acuity.

[0023] In this specification, "object recognition function" refers to the function or ability to recognize an object visually. The "object recognition function" requires not only the "ability to distinguish between light and dark" but also a certain level of "visual acuity." The improvement may be any improvement in the object recognition function, and may include, for example, the ability to recognize something that the object recognition function was previously unable to do, as well as an improvement to the point where the object recognition function can finally be recognized.

[0024] As used herein, the term "retinal degenerative disease" refers to any disease caused by degeneration of the retina, and examples thereof include retinitis pigmentosa and age-related macular degeneration.

[0025] As used herein, "retinitis pigmentosa" refers to a genetic disease characterized by retinal abnormalities, resulting in widespread degeneration of retinal photoreceptors and pigment epithelial cells. It presents with three symptoms: night blindness (difficulty seeing in the dark), constricted visual field (narrowed visual field), and decreased visual acuity. Degeneration of only rod photoreceptor cells is called rod dystrophy, while degeneration of both rod and cone photoreceptor cells is called rod-cone dystrophy. While research is underway on gene therapy, artificial retinas, retinal regeneration, and photoreceptor cell protection treatments, no established treatment has been established. Because the disease is bilaterally progressive and can often lead to social blindness as early as childhood, achieving progress control is highly significant.

[0026] In this specification, "retinitis pigmentosa" refers to not only autosomal recessive inheritance, but also autosomal dominant inheritance and X-linked recessive inheritance. The most common type is autosomal recessive inheritance, which accounts for about 35% of all cases, followed by autosomal dominant inheritance, which accounts for 10% of all cases, and the least common type is X-linked inheritance (X-linked recessive inheritance), which accounts for about 5% of all cases.

[0027] As used herein, "inhibition of progression" refers to the inhibition of the progression of a certain disease (e.g., retinitis pigmentosa), and inhibition encompasses not only a reduction in the rate of deterioration compared to the absence of treatment, but also maintenance or improvement of the disease level. The absence of disease onset corresponds to "prevention of onset." As used herein, "onset" refers to the onset of subjective symptoms of a disease after a state in which no subjective symptoms are present, and examples of subjective symptoms include night blindness, narrowed visual field, photophobia, decreased visual acuity, and color vision abnormalities.

[0028] As used herein, the terms "onset" and "immediately after" refer to within a certain period of time from the time when the patient first experiences symptoms, such as, but not limited to, within one year, within six months, or within three months.

[0029] As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term also encompasses those assembled into complex polypeptide chains. 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 (e.g., conjugation with a labeling moiety). This definition also encompasses, for example, polypeptides containing one or more analogs of an amino acid (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art. As used herein, "amino acid" refers collectively to an organic compound containing an amino group and a carboxyl group. When an antibody according to an embodiment of the present disclosure comprises a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Furthermore, any amino acid in the amino acid sequence may form a salt or solvate. Any amino acid in the amino acid sequence may be in the L- or D-form. In such cases, the protein according to the embodiment of the present disclosure can be said to contain the above-mentioned "specific amino acid sequence." Known chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, glycosylation, etc.). Natural or non-natural amino acids may be used as long as they satisfy the objectives of the present disclosure.

[0030] As used herein, the term "chimera" (protein, rhodopsin) refers to a state in which genetic information derived from different organisms is mixed in the same entity (in this case, protein, rhodopsin, etc.). A chimeric protein, for example, contains a mixture of gene sequences derived from two or more organisms. The sequence information contained in a chimeric protein may include sequences other than those derived from the organisms being mixed.

[0031] As used herein, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. This term also includes "oligonucleotide derivatives" or "polynucleotide derivatives." "Oligonucleotide derivatives" or "polynucleotide derivatives" refer to oligonucleotides or polynucleotides that contain derivatives of nucleotides or have unusual internucleotide bonds, and are used interchangeably. Specific examples of such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide has been converted to a phosphorothioate bond, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide has been converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which the ribose and phosphodiester bond in the oligonucleotide have been converted to a peptide nucleic acid bond, oligonucleotide derivatives in which the uracil in the oligonucleotide has been substituted with C-5 propynyl uracil, oligonucleotide derivatives in which the uracil in the oligonucleotide has been substituted with C-5 thiazole uracil, oligonucleotide derivatives in which the cytosine in the oligonucleotide has been substituted with C-5 propynyl cytosine, oligonucleotide derivatives in which the cytosine in the oligonucleotide has been substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in the DNA has been substituted with 2'-O-propyl ribose, and oligonucleotide derivatives in which the ribose in the oligonucleotide has been substituted with 2'-methoxyethoxy ribose. Unless otherwise specified, a particular base sequence is also intended to encompass its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Note that a nucleic acid sequence may be referred to as a base sequence, a nucleic acid sequence, a nucleotide sequence, or the like, all of which have the same meaning.Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Depending on the context, the term "nucleic acid" as used herein may be used interchangeably with gene, DNA such as cDNA, RNA such as mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotide" may be natural or non-natural. As used herein, nucleic acid may be DNA or RNA.

[0032] As used herein, the term "gene" refers to a factor that determines a genetic trait, and may refer to a "polynucleotide," an "oligonucleotide," and a "nucleic acid."

[0033] As used herein, "nucleic acid construct," "construct," or "gene construct" are used interchangeably and are nucleic acid molecules that comprise nucleic acids isolated from naturally occurring genes or combined and juxtaposed in a manner that does not occur in nature, and a vector.

[0034] As used herein, "homology" of genes refers to the degree of identity between two or more gene sequences. Generally, "homology" refers to a high degree of identity or similarity. "Identity" refers to the degree of correspondence between identical amino acid sequences, and "similarity" refers to the degree of correspondence between sequences, including identical amino acids as well as amino acids with similar properties. Therefore, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether two genes are homologous can be determined by direct sequence comparison or, in the case of nucleic acids, by hybridization under stringent conditions. When directly comparing two gene sequences, the genes are homologous if their DNA sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Thus, as used herein, a "homologue" or "homologous gene product" refers to a protein in another species, preferably a mammal, that performs the same biological function as a protein component of a complex as further described herein. Such a homologue may also be referred to as an "orthologous gene product." It is understood that such homologues, homologous gene products, orthologous gene products, etc., can also be used so long as they are consistent with the purposes of the present disclosure.

[0035] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes. Herein, comparisons of amino acid and nucleotide sequence similarity, identity, and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.2.28 (published April 2, 2013) (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). The identity values ​​used herein generally refer to the values ​​obtained when aligned using the above-mentioned BLAST under default conditions. However, if a higher value is obtained by changing the parameters, the highest value is used as the identity value. When identity is evaluated in multiple regions, the highest value among them is used as the identity value. Similarity is a numerical value that takes into account not only identity but also similar amino acids. When comparing amino acid sequences using BLAST, the algorithm Blastp can be used with default settings. Measurement results are quantified as positives or identities. The homology of amino acid sequences or nucleotide sequences can be determined using the BLAST algorithm by Karlin and Altschul. Programs called BLASTN and BLASTX have been developed based on this algorithm (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When analyzing nucleotide sequences using BLASTN based on BLAST, parameters are set, for example, to score = 100 and word length = 12. When analyzing amino acid sequences using BLASTX based on BLAST, parameters are set, for example, to score = 50 and word length = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are known (http: / / www.ncbi.nlm.nih.gov).

[0036] The nucleic acids or proteins used in the present disclosure may include sequences in which one or more amino acids or nucleotides have been substituted, deleted, and / or added to the target amino acid or base sequence. Here, "one or more" in the full-length amino acid sequence of the chimeric protein generally refers to 50 amino acids or less, preferably 30 amino acids or less, and more preferably 10 amino acids or less (e.g., 5 amino acids or less, 3 amino acids or less, or 1 amino acid or less). Furthermore, in the amino acid sequence of a domain, "one or more" generally refers to 6 amino acids or less, preferably 5 amino acids or less, and more preferably 4 amino acids or less (e.g., 3 amino acids or less, 2 amino acids or less, or 1 amino acid or less). To maintain the biological activity of the chimeric protein of the present disclosure, it is desirable that the mutated amino acid residue be mutated to another amino acid whose amino acid side chain properties are conserved. For example, the nature of the amino acid side chains may 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-containing side chains (S, T, Y), amino acids with sulfur-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), and amino acids with aromatic-containing side chains (H, F, Y, W) (the characters in parentheses represent the single-letter symbols of the amino acids). These are also referred to herein as "conservative substitutions." It is known that proteins having modified amino acid sequences by deletion, addition, and / or substitution of one or more amino acid residues with other amino acids can maintain their biological activity (Mark, D.F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666; Zoller, M.J. & Smith, M., Nucleic Acids Research (1982) 10, 6487-6500; Wang, A. et al., Science 224, 1431-1433; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).Therefore, in one embodiment of the present disclosure, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, or any of these values ​​or less. Chimeric proteins with deletions or other modifications can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library. For site-directed mutagenesis, for example, the KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used. From mutant antibodies with deletions or other modifications, antibodies with activity similar to that of the wild-type antibody can be selected by various characterization methods such as FACS analysis and ELISA.

[0037] As used herein, the term "signal sequence" refers to an amino acid sequence that, when functionally linked to a protein or peptide, promotes transport of the linked protein or peptide to a functional location. When the protein linked to a signal sequence is a membrane protein, an endoplasmic reticulum targeting signal peptide or an endoplasmic reticulum export signal peptide may be linked.

[0038] As used herein, an "endoplasmic reticulum targeting signal peptide" refers to an amino acid sequence consisting primarily of hydrophobic amino acids of approximately 5 to 10 amino acids that is added to the amino terminus of a protein to promote targeting to the endoplasmic reticulum. If deletion or mutation of a specific amino acid sequence in a protein known to target to the endoplasmic reticulum significantly reduces targeting to the endoplasmic reticulum, the specific amino acid sequence can be determined to be an endoplasmic reticulum targeting signal.

[0039] As used herein, an "endoplasmic reticulum export signal peptide" refers to amino acids that promote the transport of proteins from the endoplasmic reticulum to other organelles, such as the Golgi apparatus. Examples of such signal peptides include the ER2 sequence. When a specific amino acid sequence is added to a protein known to remain in the endoplasmic reticulum, if the protein is significantly transported from the endoplasmic reticulum compared to a protein without the specific amino acid sequence, the specific amino acid sequence can be determined to be an endoplasmic reticulum export signal peptide.

[0040] In one embodiment of the present disclosure, the amino acid sequence and nucleic acid sequence of the chimeric protein of the present disclosure may have 70% or more, 80% or more, or 90% or more identity or similarity to a reference sequence. As used herein, with respect to an amino acid sequence or a nucleic acid sequence, "70% or more" may mean, for example, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% or more, "80% or more" may mean, for example, 80, 85, 90, 95, 96, 97, 98, or 99% or more, and "90% or more" may mean, for example, 90, 95, 96, 97, 98, or 99% or more, or may be within a range of any two of these values. "Homology" may be calculated by calculating the percentage of homologous amino acids in two or more amino acid sequences according to methods known in the art. Before calculating the percentage, the amino acid sequences of the amino acid sequences to be compared are aligned, and gaps are introduced into some of the amino acid sequences if necessary to maximize the percentage of identical amino acids. Methods for alignment, percentage calculation, comparison, and related computer programs are well known in the art (e.g., BLAST, GENETYX, etc.). In the case of "identity," the percentage of identical amino acids is calculated, and in the case of "similarity," the percentage of similar amino acids is calculated. Similar amino acids include, but are not limited to, amino acids that can be conservatively substituted.

[0041] As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to conditions commonly used in the art. Such polynucleotides can be obtained by colony hybridization, plaque hybridization, Southern blot hybridization, or other methods using a polynucleotide selected from the polynucleotides disclosed herein as a probe. Specifically, the term refers to a polynucleotide that can be identified by hybridizing a filter onto which colony- or plaque-derived DNA has been immobilized in the presence of 0.7 to 1.0 M NaCl at 65°C, followed by washing the filter at 65°C using 0.1 to 2x SSC (saline-sodium citrate) solution (1x SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). Examples of "stringent conditions" that can be used include the following: (1) using low ionic strength and high temperature for washing (e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C); (2) using a denaturing agent such as formamide during hybridization (e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer, pH 6.5, and 750 mM sodium chloride and 75 mM sodium citrate at 42°C); or (3) overnight incubation at 37°C in a solution containing 20% ​​formamide, 5x SSC, 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA, followed by washing the filter with 1x SSC at approximately 37-50°C. The formamide concentration may be 50% or more, and the washing time may be 5, 15, 30, 60, or 120 minutes or more.Several factors, such as temperature and salt concentration, may affect the stringency of a hybridization reaction. For details, see Ausubel et al., *Current Protocols in Molecular Biology*, *Wiley Interscience Publishers*, (1995). Examples of "highly stringent conditions" include 0.0015 M sodium chloride, 0.0015 M sodium citrate, and 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. Hybridization can be performed according to methods described in experimental manuals such as *Molecular Cloning 2nd ed., *Current Protocols in Molecular Biology*, Supplement 1-38, and *DNA Cloning 1: Core Techniques, A Practical Approach*, Second Edition, *Oxford University Press* (1995). Sequences that hybridize under stringent conditions preferably exclude sequences containing only A or T sequences. Moderately stringent conditions can be readily determined by one of skill in the art based on, for example, the length of the DNA, and are set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vol. 3, Vol. 1, pp. 7.42-7.45, Cold Spring Harbor Laboratory Press, 2001, and include, for nitrocellulose filters, the use of a pre-wash solution of 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 2xSSC-6xSSC (or other similar hybridization solutions, such as Stark's solution in about 50% formamide at about 42°C) at about 40-50°C, and wash conditions of 0.5xSSC, 0.1% SDS at about 60°C.Thus, polypeptides as used in this disclosure also include polypeptides encoded by nucleic acid molecules that hybridize under high or moderate stringency conditions to nucleic acid molecules encoding the polypeptides specifically described in this disclosure.

[0042] As used herein, a "purified" substance or biological factor (e.g., nucleic acid, protein, etc.) refers to a substance or biological factor from which at least a portion of naturally associated factors has been removed. Therefore, typically, the purity of the biological factor in a purified biological factor is higher (i.e., concentrated) than in the state in which the biological factor normally exists. As used herein, the term "purified" means that preferably 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 of the same type of biological factor is present. The substance or biological factor used in the present disclosure is preferably a "purified" substance. As used herein, an "isolated" substance or biological factor (e.g., nucleic acid, protein, etc.) refers to a substance or biological factor from which naturally associated factors have been substantially removed. The term "isolated" as used herein does not necessarily refer to purity, as this may vary depending on the purpose, but where necessary, preferably means that 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 of the same type of biological factor is present. The substances used in this disclosure are preferably "isolated" substances or biological factors.

[0043] As used herein, a "corresponding" amino acid, nucleic acid, or portion refers to an amino acid or nucleotide in a polypeptide or polynucleotide molecule (e.g., rhodopsin) that has or is predicted to have the same function as a given amino acid, nucleotide, or portion in a polypeptide or polynucleotide to which it is compared. In particular, in an enzyme molecule, it refers to an amino acid that is located in a similar position in the active site and contributes similarly to catalytic activity, and in a composite molecule, it refers to a corresponding portion (e.g., heparan sulfate, etc.). For example, in the case of an antisense molecule, it may be a similar portion in an orthologue corresponding to a specific portion of the antisense molecule. A corresponding amino acid may be, for example, a specific amino acid that is cysteinylated, glutathionylated, forms an S-type disulfide bond, oxidized (e.g., oxidation of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, or the like. Alternatively, a corresponding amino acid may be an amino acid responsible for dimerization. Such a "corresponding" amino acid or nucleic acid may be a region or domain spanning a certain range. Therefore, in such cases, it is referred to herein as a "corresponding" region or domain. Such corresponding regions or domains are useful in the present disclosure when designing composite molecules.

[0044] As used herein, a "corresponding" gene (e.g., a polynucleotide sequence or molecule) refers to a gene (e.g., a polynucleotide sequence or molecule) in a given species that has or is predicted to have the same function as a given gene in a species used as a reference for comparison. When multiple genes with such a function exist, the term refers to genes that have the same evolutionary origin. Thus, a gene corresponding to a given gene may be its ortholog. Corresponding rhodopsins can be found for each human rhodopsin in other animals (particularly mammals). Such corresponding genes can be identified using techniques well known in the art. Therefore, for example, a corresponding gene in a given animal (e.g., a mouse), or a reference gene (e.g., rhodopsin) for the corresponding gene, can be found by searching a database containing the animal's sequences using a sequence such as SEQ ID NOs: 9-16 as a query sequence.

[0045] As used herein, the terms "portion," "fragment," or "fragment" refer to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). The length of a fragment can be varied appropriately depending on its purpose. For example, the lower limit of the length for a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and lengths represented by integers not specifically recited herein (e.g., 11) may also be suitable as lower limits. Furthermore, for polynucleotides, the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and lengths represented by integers not specifically recited herein (e.g., 11) may also be suitable as lower limits. As used herein, such fragments are understood to fall within the scope of the present disclosure, for example, if the full-length fragment functions as a marker or target molecule, as long as the fragment itself also functions as a marker or target molecule.

[0046] In accordance with the present disclosure, the term "activity" as used herein refers to the function of a molecule in the broadest sense. Activity generally includes, but is not limited to, the biological, biochemical, physical, or chemical function of a 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 a specific subcellular location. Where applicable, the term also relates to the function of a protein complex in the broadest sense. As used herein, "biological activity" includes, for example, activation of a light reaction.

[0047] As used herein, the term "functional equivalent" refers to any entity that has the same intended function as the original entity but a different structure. Therefore, functional equivalents of "rhodopsin" or its chimera are understood to encompass not only rhodopsin or its chimera itself, but also mutants or variants of rhodopsin or its chimera (e.g., amino acid sequence variants, etc.) that have the biological activity of rhodopsin or its chimera, as well as rhodopsin or its antibodies, or mutants or variants of rhodopsin or its chimera, upon acting (e.g., nucleic acids encoding rhodopsin or its chimera, or mutants or variants of rhodopsin or its chimera, and vectors, cells, etc. containing such nucleic acids). Functional equivalents of the present disclosure may include insertions, substitutions, and / or deletions of one or more amino acids in the amino acid sequence, or additions to one or both termini. As used herein, the phrase "insertion, substitution, and / or deletion of one or more amino acids in an amino acid sequence, or addition to one or both termini thereof" refers to modification by a well-known technical method such as site-directed mutagenesis, or by natural mutation, resulting in the substitution of a number of amino acids to the extent that would occur naturally. The modified amino acid sequence may be one in which, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted, or deleted, or added to one or both termini thereof. The modified amino acid sequence may preferably be an amino acid sequence in which the rhodopsin amino acid sequence has one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions.

[0048] As used herein, the terms "drug," "agent," or "factor" (all of which correspond to the English term "agent") are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, or electricity) that can achieve the intended purpose. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands), etc.), and composite molecules thereof.

[0049] For parenteral administration, the formulation may be contained in a unit-dose ampule, a multi-dose container, or a tube, and may also contain additives such as stabilizers, buffers, preservatives, isotonicity agents, etc. For parenteral administration, the formulation may be formulated into a powder that can be redissolved in an appropriate carrier (sterilized water, etc.) at the time of use.

[0050] Examples of parenteral administration include intravitreal administration, subconjunctival administration, intracameral administration, and ophthalmic administration, with intravitreal administration being preferred. The composition of the present disclosure can be administered to humans by the methods described above for treatment, prevention, progression suppression, and the like.

[0051] As used herein, "treatment" refers to preventing, preferably maintaining, more preferably alleviating, and even more preferably eliminating, the worsening of a certain disease or disorder (e.g., retinal degenerative disease) when that condition has developed, and includes the potential for symptom improvement or prevention of the patient's disease or one or more symptoms associated with the disease. Preliminary diagnosis followed by appropriate treatment is referred to as "companion treatment," and diagnostic agents used for this purpose are sometimes referred to as "companion diagnostic agents." Since the present disclosure targets genetic diseases, patients may be treated after genetic testing.

[0052] As used herein, the term "therapeutic agent" broadly refers to any drug capable of treating a target condition (e.g., retinal degenerative disease, etc.). In one embodiment of the present disclosure, the "therapeutic agent" may be a pharmaceutical composition containing an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition can be prepared, for example, by mixing the active ingredient with the carrier using any method known in the technical field of pharmaceuticals. Furthermore, the therapeutic agent may be in any form used as long as it is used for treatment, and may be the active ingredient alone or a mixture of the active ingredient with any other ingredient. Furthermore, the shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution).

[0053] As used herein, "prevention" refers to preventing a certain disease or disorder (e.g., retinal degenerative disease) from occurring before that state is reached. Diagnosis can be performed using the agent of the present disclosure, and, if necessary, the agent of the present disclosure can be used to prevent, for example, retinal degenerative disease, or preventative measures can be taken. As used herein, the term "prophylactic drug (agent)" broadly refers to any drug that can prevent a target state (e.g., a disease such as retinal degenerative disease).

[0054] As used herein, the term "kit" refers to a unit in which the components to be provided (e.g., nucleic acids, nucleic acid constructs, cells transfected with a nucleic acid of interest, test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.) are provided, typically separated into two or more compartments. This kit format is preferred when the objective is to provide a composition that, for reasons of stability, should not be provided in a mixed state, but is preferably mixed immediately before use. Such kits advantageously include instructions or manuals describing how to use the components to be provided (e.g., nucleic acids, nucleic acid constructs, cells transfected with a nucleic acid of interest, test agents, diagnostic agents, therapeutic agents), or how to handle the reagents. When the kit is used herein as a reagent kit, the kit typically includes instructions describing how to use the test agents, diagnostic agents, therapeutic agents, antibodies, etc.

[0055] As used herein, the term "active ingredient" refers to an ingredient contained in a composition, etc. of the present disclosure in an amount necessary to achieve the intended effect of treatment, prevention, or progression inhibition, and other ingredients may also be contained as long as the effect is not impaired to below the desired level. Furthermore, the medicament, composition, etc. of the present disclosure may be formulated. Furthermore, 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 formulation, etc.

[0056] As used herein, "instructions" (including package inserts and labels used by the U.S. FDA) are written instructions to a physician or other user on how to use the present disclosure. The instructions include instructions on how to use the detection method, diagnostic reagent, or administer a medicine or the like of the present disclosure. The instructions may also include instructions for oral or intraretinal administration (e.g., by injection) as the administration site. The instructions are prepared in accordance with a format specified by the regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and clearly state that they have been approved by the regulatory agency. The instructions are so-called package inserts or labels, and are typically provided in paper form, but are not limited thereto and may also be provided in the form of electronic media (e.g., a website provided on the Internet, email, etc.).

[0057] (Preferred embodiment) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0058] (New constructs of chimeric rhodopsins) The present disclosure provides a novel nucleic acid construct of a chimeric rhodopsin. The chimeric rhodopsin used in the present disclosure may be any type as long as it can achieve the objectives of the present disclosure. The chimeric rhodopsin used in the present disclosure is typically a chimeric protein comprising at least a portion of an ion transport receptor rhodopsin and at least a portion of a G protein-coupled receptor rhodopsin. To give a typical example, fusing a portion of an animal-derived G protein-coupled receptor rhodopsin with a portion of a reusable microbial ion transport receptor rhodopsin can maintain the repetitive activation function of the microbial ion transport receptor ionotropic receptor rhodopsin while achieving high activity mediated by endogenous G proteins via the G protein-coupled receptor. Further generating the nucleic acid construct of the present disclosure from this can further improve the therapeutic, ameliorative, preventative, and progression-suppressive effects against retinal diseases, disorders, and symptoms.

[0059] In one aspect, the present disclosure provides a nucleic acid construct encoding a chimeric rhodopsin comprising at least a portion of an ion transport receptor rhodopsin and at least a portion of a G protein-coupled receptor rhodopsin, and a signal sequence. In another aspect, the present disclosure provides a nucleic acid construct of a chimeric rhodopsin comprising at least a portion of an ionotropic receptor rhodopsin and at least a portion of a G protein-coupled receptor rhodopsin. The ionotropic receptor rhodopsin can be an algal rhodopsin. The alga may be Guillardia theta. In a preferred embodiment, the chimeric rhodopsin of the present disclosure has the amino acid sequence of the cytoplasmic second loop and / or the cytoplasmic third loop of the rhodopsin from Guillardia theta replaced with the amino acid sequence of the cytoplasmic second loop and / or the cytoplasmic third loop of a G protein-coupled receptor rhodopsin.

[0060] In one embodiment, the ion transport receptor rhodopsin used in the chimeric protein of the present disclosure can be an ion pump receptor rhodopsin or an ion channel receptor rhodopsin. In a preferred embodiment, the ion transport receptor rhodopsin is preferably derived from a microorganism, typically a cyanobacterium (blue-green bacteria), such as a rhodopsin derived from a microorganism belonging to eubacteria such as the genus Gloeobacter, or a eukaryote such as the genus Volvox, Chlamydomonas, or Guillardia. Examples of the genus Gloeobacter include Gloeobacter violaceus. Examples of the genus Volvox include Volvox carteri. Examples of the genus Chlamydomonas include Chlamydomonas reinhardtii, etc. Examples of the genus Guillardia include Guillardia theta, etc.

[0061] In one embodiment, the G protein-coupled receptor rhodopsin used in the chimeric protein of the present disclosure is typically derived from an animal, with rhodopsin derived from rodents, artiodactyls, perissodactyls, primates, carnivorans, etc. being preferred, with artiodactyls or primates being more preferred, and primate rhodopsin being even more preferred. Preferred G protein-coupled receptor rhodopsins include, for example, rhodopsins derived from cows, humans, mice, rats, cats, dogs, pigs, sheep, horses, etc. Of these, rhodopsins derived from cows or humans are particularly preferred.

[0062] In certain embodiments, the chimeric protein encoded by the nucleic acid construct or the like of the present disclosure is a chimeric protein comprising a portion of an ion transport receptor rhodopsin and a portion of a G protein-coupled receptor rhodopsin, and has a seven-transmembrane structure. In the present disclosure, the chimeric protein comprising a portion of an ion transport receptor rhodopsin and a portion of a G protein-coupled receptor rhodopsin is preferably designed to have both the function of repeatedly activating the ion transport receptor rhodopsin and the G protein activity of the G protein-coupled receptor rhodopsin at a high level. From this perspective, in order to maintain high levels of both activities and, in particular, to achieve high visual function regeneration, the chimeric protein encoded by the nucleic acid construct of the present disclosure preferably has the amino acid sequence of the cytoplasmic loop 2 and / or the cytoplasmic loop 3 of the amino acid sequence of the ion transport receptor rhodopsin replaced with the amino acid sequence of the cytoplasmic loop 2 and / or the cytoplasmic loop 3 of the G protein-coupled receptor rhodopsin. The "second loop on the cytoplasmic side" and "third loop on the cytoplasmic side" refer to the loops located second and third from the N-terminus, respectively, among the seven loops.

[0063] In one embodiment, the chimeric protein encoded by the nucleic acid construct of the present disclosure advantageously has an amino acid sequence in which glutamic acid at position 132 of the amino acid sequence of SEQ ID NO: 14 (GR) is substituted with glutamine. Examples of glutamine-substituted amino acid sequences include, but are not limited to, the amino acid sequence of SEQ ID NO: 5.

[0064] Methods for obtaining nucleic acids such as DNA of the present disclosure include, but are not limited to, known methods such as obtaining cDNA by reverse transcription from mRNA (e.g., RT-PCR), preparing from genomic DNA, synthesizing by chemical synthesis, and isolating from a genomic DNA library or a cDNA library (see, for example, JP-A-11-29599).

[0065] Herein, a chimeric protein encoded by a nucleic acid construct of the present disclosure can be prepared, for example, by using a transformant into which an expression vector containing the nucleic acid construct of the present disclosure has been introduced. For example, first, the transformant is cultured under appropriate conditions to synthesize the chimeric protein encoded by the nucleic acid construct of the present disclosure. The synthesized protein can then be recovered from the transformant or culture medium to obtain the chimeric protein of the present disclosure.

[0066] More specifically, the nucleic acid construct of the present disclosure can be prepared by inserting the nucleic acid construct of the present disclosure into an appropriate expression vector. A "suitable vector" is any vector that can maintain replication or self-replicate in various prokaryotic and / or eukaryotic hosts, and can be selected appropriately depending on the intended use. For example, a high-copy vector can be selected when obtaining a large amount of nucleic acid, such as the nucleic acid construct of the present disclosure, and an expression vector can be selected when obtaining a polypeptide (chimeric protein). Specific examples include, but are not limited to, known vectors such as those described in Japanese Patent Application Laid-Open No. 11-29599.

[0067] Furthermore, expression vectors can be used not only to synthesize chimeric proteins but also in compositions of the present disclosure. That is, compositions of the present disclosure may contain, as an active ingredient, an expression vector incorporating the nucleic acid construct of the present disclosure. By directly introducing such an expression vector into a human, it can be used for the treatment, prevention, and suppression of progression of retinal diseases, disorders, or symptoms. In this case, a vector that can be introduced into human cells is used. Suitable examples of such vectors include adeno-associated virus vectors (AAV vectors) and lentivirus vectors.

[0068] The method for introducing the vector can be appropriately selected depending on the type of vector and host, etc. Specific examples include, but are not limited to, known methods such as the protoplast method and the competent method when bacteria are used as hosts (see, for example, JP 11-29599 A). Furthermore, when an expression vector is used as an active ingredient in the agent for regenerating visual function or the agent for preventing visual function decline of the present disclosure, the vector can be introduced, for example, by intraocular injection of the above-mentioned AAV vector.

[0069] The host into which the expression vector is introduced may be any host that is compatible with the expression vector and can be transformed, and specific examples thereof include, but are not limited to, known natural cells or artificially established cells such as bacteria, yeast, animal cells, and insect cells (see JP 11-29599 A), or animals such as humans and mice. The transformant can be cultured in an appropriate nutrient medium selected from known nutrient media depending on the type of transformant, and the temperature, pH of the nutrient medium, culture time, etc. can be adjusted appropriately so that the chimeric protein can be obtained easily in large quantities (see, for example, JP 11-29599 A).

[0070] Methods for isolating and purifying chimeric proteins are not particularly limited, and include known methods such as methods that utilize solubility, methods that utilize differences in molecular weight, and methods that utilize charge (see, for example, JP-A-11-29599).

[0071] In one embodiment, a nucleic acid construct or the like of the present disclosure may include any of the following: (A) a base sequence comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 26; (B) a polynucleotide comprising a nucleic acid sequence shown in (A) that contains one or more nucleotide substitutions, additions, deletions, or combinations thereof; (C) a polynucleotide comprising a nucleic acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence shown in (A) or (B), and encoding a polypeptide having biological activity; (D) a polynucleotide comprising a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleic acid sequences shown in (A) to (C) or its complementary sequence, and encoding a polypeptide having biological activity; (E) a polynucleotide that is an allelic variant of any one of the nucleic acid sequences (A) to (D), and encodes a polypeptide having biological activity; (F) a polynucleotide encoded by a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, or 27; (G) a polynucleotide encoding a polypeptide having biological activity, comprising the amino acid sequence of (F) containing one or more amino acid substitutions, additions, deletions, or a combination thereof; (H) a polynucleotide encoding a biologically active polypeptide having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in (F) or (G); or (I) A polynucleotide comprising a fragment of the nucleic acid sequence shown in (F) to (H); and the chimeric protein encoded by the polynucleotide has biological activity.

[0072] In specific embodiments, nucleic acid constructs etc. of the present disclosure may encode a polypeptide comprising the following amino acid sequence: (a) an amino acid sequence set forth in SEQ ID NO: 2, 4, or 27, or a fragment thereof; (b) a polypeptide having biological activity, comprising the amino acid sequence of (a) containing one or more amino acid substitutions, additions, deletions, or a combination thereof; (c) a polypeptide having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence shown in (a) or (b), and having biological activity; (d) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, or 27; (e) a polypeptide having biological activity encoded by a nucleic acid sequence containing one or more nucleotide substitutions, additions, deletions, or combinations thereof in the nucleic acid sequence shown in (d); (f) a polypeptide having biological activity encoded by a nucleic acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence shown in (d) or (e); (g) A polypeptide having biological activity, encoded by a nucleic acid sequence that hybridizes under stringent conditions with a polynucleotide comprising any one of the nucleic acid sequences set forth in (d) to (f) or its complementary sequence; (h) a biologically active polypeptide encoded by an allelic variant of the nucleic acid sequence of any one of (d) to (g); or (i) A chimeric protein of the present disclosure may comprise a polypeptide comprising a fragment of the amino acid sequence set forth in (a) through (h) and have biological activity. Alternatively, the chimeric protein of the present disclosure may comprise an amino acid sequence encoded by a nucleic acid set forth in any of the following: (aa) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, or 27, or a nucleotide sequence set forth in SEQ ID NO: 1, 3, or 26 (bb) a nucleic acid having a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, or 27 or a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 26 (cc) A nucleic acid having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2, 4, or 27, and having biological activity. (dd) a nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 2, 4, or 27, and having biological activity; or (aaa) the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 26, or a fragment thereof; (bbb) a nucleic acid having at least 70%, at least 80%, at least 90%, at least 95% identity to (aaa); (ccc) a base sequence with one or more nucleotide substitutions, additions, and / or deletions relative to (aaa) or (bbb); A nucleotide sequence that hybridizes under stringent conditions to any of (ddd) (aaa) to (ccc), and wherein the chimeric protein has biological activity.

[0073] In certain embodiments, the nucleic acid of the present disclosure may be a nucleic acid sequence that shares at least six or more triplets with the nucleic acid sequence set forth in SEQ ID NO: 1, 3, or 26. In another embodiment, the nucleic acid construct of the present disclosure is a nucleic acid sequence encoding the same amino acid as SEQ ID NO: 1, 3, or 26, selected from the group consisting of the nucleic acid sequence shown in SEQ ID NO: 1, 3, or 26, and the nucleic acid sequence shown in SEQ ID NO: 6, 9 to 13, 15, 16, 18 to 22, 27 to 29, 31 to 36, 39, 40, 43, 45, 48, 50, 51, 53 to 55, 58, 59, 61, 65 to 73, 75 to 84, 86, 88, 89, 93, 97, 98, 100, 101, 104, 106 to 108, 110, 112, 114, 115, 122, 123, 125, 128, 131, 133, 139, 143, 145, 146, 155, 157, 162, 165, 167, 169 to 171, 172, 173, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 2 4, 176, 179, 182, 183, 186-189, 193-198, 204, 205, 207, 209, 212, 215, 216, 218-220, 224, 225, 227, 228, 230, 231, 233-235, 238, 240, 242, 243, 246, 247, 249, 251, 253-255, 2 At least one of the triplets encoding amino acids 57 to 259, 261 to 264, 266 to 270, 272, 273, 275, 276, 279, 281 to 287, 289 to 291, 296 to 299, 302 to 305, 307 to 316, 318, 319, and 321 to 330 may contain a common nucleic acid sequence.

[0074] The nucleic acid sequence encoding the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin described above preferably has the following nucleic acid sequence: (A) a base sequence comprising the nucleotide sequence set forth in SEQ ID NO: 17 or 18; (B) a polynucleotide comprising a nucleic acid sequence shown in (A) that contains one or more nucleotide substitutions, additions, deletions, or combinations thereof; (C) a polynucleotide comprising a nucleic acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence shown in (A) or (B), and encoding a polypeptide having biological activity; (D) a polynucleotide comprising a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleic acid sequences shown in (A) to (C) or its complementary sequence, and encoding a polypeptide having biological activity; (E) a polynucleotide that is an allelic variant of any one of the nucleic acid sequences (A) to (D), and encodes a polypeptide having biological activity; (F) a polynucleotide encoded by a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 19 or 25; (G) a polynucleotide encoding a polypeptide having biological activity, comprising the amino acid sequence of (F) containing one or more amino acid substitutions, additions, deletions, or a combination thereof; (H) a polynucleotide encoding a biologically active polypeptide having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence shown in (F) or (G); or (I) A polynucleotide comprising a fragment of the nucleic acid sequence shown in (F) to (H).

[0075] In a specific embodiment, the nucleic acid sequence encoding the second loop on the cytoplasmic side of the above-mentioned G protein-coupled receptor rhodopsin is preferably a nucleic acid sequence that shares at least two triplets with the nucleic acid sequence shown in SEQ ID NO:17.

[0076] Alternatively, the second loop on the cytoplasmic side of the above-mentioned G protein-coupled receptor rhodopsin preferably has an amino acid sequence encoded by the nucleic acid described below.

[0077] (i) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 19 or 25; (ii) a nucleic acid having a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence complementary to a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 19 or 25; (iii) a nucleic acid having a nucleotide sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 19 or 25; (iv) a nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having 70% or more homology to the amino acid sequence set forth in SEQ ID NO: 19 or 25; Alternatively, the nucleic acid encoding the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin is preferably any one of the following:

[0078] In a specific embodiment, the nucleic acid sequence encoding the third loop on the cytoplasmic side of the above-mentioned G protein-coupled receptor rhodopsin is preferably a nucleic acid sequence that shares at least one triplet with the nucleic acid sequence shown in SEQ ID NO: 19 or 25.

[0079] (i) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 19 or 25; (ii) a nucleic acid having a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence complementary to a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 19 or 25; (iii) a nucleic acid having a nucleotide sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 19 or 25; (iv) a nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 19 or 25: (x) a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 19 or 25 or a fragment thereof; (y) a nucleic acid having at least 70%, at least 80%, at least 90%, at least 95% identity to (x); (z) a nucleic acid having one or more nucleotide substitutions, additions, and / or deletions relative to (x) or (y); (w) a nucleic acid that hybridizes under stringent conditions to any of (x) to (z); and the loop has biological activity.

[0080] The nucleic acid sequence encoding the third loop on the cytoplasmic side of the above-mentioned G protein-coupled receptor rhodopsin preferably has the following nucleic acid sequence: (A) a base sequence comprising the nucleotide sequence set forth in SEQ ID NO: 20 or 21; (B) a polynucleotide comprising a nucleic acid sequence shown in (A) that contains one or more nucleotide substitutions, additions, deletions, or combinations thereof; (C) a polynucleotide comprising a nucleic acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence shown in (A) or (B), and encoding a polypeptide having biological activity; (D) a polynucleotide comprising a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleic acid sequences shown in (A) to (C) or its complementary sequence, and encoding a polypeptide having biological activity; (E) a polynucleotide that is an allelic variant of any one of the nucleic acid sequences (A) to (D), and encodes a polypeptide having biological activity; (F) a polynucleotide encoded by a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22; (G) a polynucleotide encoding a polypeptide having biological activity, comprising the amino acid sequence of (F) containing one or more amino acid substitutions, additions, deletions, or a combination thereof; (H) a polynucleotide encoding a biologically active polypeptide having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in (F) or (G); or (I) A polynucleotide comprising a fragment of the nucleic acid sequence shown in (F) to (H).

[0081] The third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin preferably has an amino acid sequence encoded by any of the following nucleic acids: (l) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 22; (k) a nucleic acid having a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence complementary to a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 22; (m) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 22 in which one or more amino acids have been substituted, deleted, and / or added; (n) A nucleic acid consisting of a base sequence encoding an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% homology to the amino acid sequence set forth in SEQ ID NO: 22.

[0082] Alternatively, the nucleic acid encoding the third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin is preferably any one of the following: (l) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 22; (k) a nucleic acid having a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence complementary to a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 22; (m) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 22 in which one or more amino acids have been substituted, deleted, and / or added; (n) a nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% homology to the amino acid sequence set forth in SEQ ID NO: 22; (xx) a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 20 or a fragment thereof; (yy) a nucleic acid having at least 70%, at least 80%, at least 90%, at least 95% identity to (xx); (zz) a nucleic acid having one or more nucleotide substitutions, additions, and / or deletions relative to (xx) or (yy); or A nucleic acid that hybridizes under stringent conditions to any of (ww)(xx) to (zz); and the loop has biological activity.

[0083] The present disclosure also provides: (A) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, or 27, or a fragment thereof; (B) a nucleotide sequence set forth in SEQ ID NO: 1, 3, or 26, or a fragment thereof; (C) a nucleic acid having at least 70%, at least 80%, at least 90%, or at least 95% identity to (A) or (B); (D) A base sequence having one or more nucleotide substitutions, additions, and / or deletions relative to any of (A) to (C); (E) a nucleotide sequence that hybridizes to any one of (A) to (D) under stringent conditions; The present invention also provides a nucleic acid having one of the following sequences, wherein the protein encoded by the nucleic acid has biological activity.

[0084] In one aspect, the present disclosure provides a nucleic acid comprising a nucleic acid sequence encoding a chimeric protein of an ionotropic receptor rhodopsin and a G protein-coupled receptor rhodopsin. Examples of ionotropic receptor rhodopsins include, but are not limited to, rhodopsins derived from microorganisms belonging to eukaryotes such as the genera Volvox, Chlamydomonas, and Guillardia. In a preferred embodiment, the ionotropic receptor rhodopsin is Guillardia theta rhodopsin, as a member of the Guillardia genus, and the G protein-coupled receptor rhodopsin is bovine rhodopsin.

[0085] In one embodiment, the nucleic acid construct etc. of the present disclosure may include any of the following: (A) a base sequence comprising the nucleotide sequence set forth in SEQ ID NO: 7; (B) a polynucleotide comprising a nucleic acid sequence shown in (A) that contains one or more nucleotide substitutions, additions, deletions, or combinations thereof; (C) a polynucleotide comprising a nucleic acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence shown in (A) or (B), and encoding a polypeptide having biological activity; (D) a polynucleotide comprising a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleic acid sequences shown in (A) to (C) or its complementary sequence, and encoding a polypeptide having biological activity; (E) a polynucleotide that is an allelic variant of any one of the nucleic acid sequences (A) to (D), and encodes a polypeptide having biological activity; (F) a polynucleotide encoded by a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8; (G) a polynucleotide encoding a polypeptide having biological activity, comprising the amino acid sequence of (F) containing one or more amino acid substitutions, additions, deletions, or a combination thereof; (H) a polynucleotide encoding a biologically active polypeptide having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in (F) or (G); or (I) A polynucleotide comprising a fragment of the nucleic acid sequence shown in (F) to (H); and the chimeric protein encoded by the polynucleotide has biological activity.

[0086] In certain embodiments, the nucleic acid of the present disclosure may be a nucleic acid sequence that shares at least 14 triplets with the nucleic acid sequence set forth in SEQ ID NO:7. In another embodiment, the nucleic acid construct of the present disclosure is a nucleic acid sequence encoding the same amino acid as SEQ ID NO: 7, which is a nucleic acid sequence selected from the group consisting of the nucleic acid sequence shown in SEQ ID NO: 7 and 1, 2, 4 to 9, 11 to 17, 21, 22, 27 to 30, 33, 34, 36 to 41, 43, 45, 48, 49, 51, 54, 56 to 58, 60, 63, 65, 68, 70, 71 to 75, 77 to 78, 81, 83, 84, 86, 89, 90, 92, 93, 95, 97 to 99, 102, 103, 111, 113, 114, 123, 125, 130, 131 to 137, 139, 142, 143, 146, 148 to 153, 156, 160, 161, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 7, 168, 170, 171, 174-176, 180, 182, 183, 187, 188, 190, 191, 196, 197, 199, 200, 202, 204, 208, 212-214, 217, 219, 226, 229, 232, 236-238, 240, 242, 243, 247, 248, 251 , 252, 258, 263-265, 267, 269, 271, 272, 274, 276-280, 282-284, 289, 290, 291, 294, 297-299, 302, 304, 307, and 310 may contain a common nucleic acid sequence.

[0087] In specific embodiments, nucleic acid constructs etc. of the present disclosure may encode a polypeptide comprising the following amino acid sequence: (a) the amino acid sequence set forth in SEQ ID NO: 8 or a fragment thereof; (b) a polypeptide having biological activity, comprising the amino acid sequence of (a) containing one or more amino acid substitutions, additions, deletions, or a combination thereof; (c) a polypeptide having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence shown in (a) or (b), and having biological activity; (d) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8; (e) a polypeptide having biological activity encoded by a nucleic acid sequence containing one or more nucleotide substitutions, additions, deletions, or combinations thereof in the nucleic acid sequence shown in (d); (f) a polypeptide having biological activity encoded by a nucleic acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the nucleic acid sequence shown in (d) or (e); (g) A polypeptide having biological activity, encoded by a nucleic acid sequence that hybridizes under stringent conditions with a polynucleotide comprising any one of the nucleic acid sequences set forth in (d) to (f) or its complementary sequence; (h) a biologically active polypeptide encoded by an allelic variant of the nucleic acid sequence of any one of (d) to (g); or (i) A chimeric protein of the present disclosure may comprise a polypeptide comprising a fragment of the amino acid sequence set forth in (a) through (h) and have biological activity. Alternatively, the chimeric protein of the present disclosure may comprise an amino acid sequence encoded by a nucleic acid set forth in any of the following: (aa) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 8 or a nucleotide sequence set forth in SEQ ID NO: 7 (bb) a nucleic acid having a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 8 or a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 7 (cc) A nucleic acid having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 8, and having biological activity. (dd) a nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 8 and having biological activity; or (aaa) the nucleotide sequence set forth in SEQ ID NO: 7 or a fragment thereof; (bbb) a nucleic acid having at least 70%, at least 80%, at least 90%, at least 95% identity to (aaa); (ccc) a base sequence with one or more nucleotide substitutions, additions, and / or deletions relative to (aaa) or (bbb); A nucleotide sequence that hybridizes under stringent conditions to any of (ddd) (aaa) to (ccc), and wherein the chimeric protein has biological activity.

[0088] The present disclosure also provides: (A) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 8 or a fragment thereof; (B) a nucleotide sequence set forth in SEQ ID NO: 7 or a fragment thereof; (C) a nucleic acid having at least 70%, at least 80%, at least 90%, or at least 95% identity to (A) or (B); (D) A base sequence having one or more nucleotide substitutions, additions, and / or deletions relative to any of (A) to (C); (E) a nucleotide sequence that hybridizes to any one of (A) to (D) under stringent conditions; The present invention also provides a nucleic acid having one of the following sequences, wherein the protein encoded by the nucleic acid has biological activity.

[0089] As used herein, representative examples of "biological activity" include the function of a G protein-coupled receptor (e.g., membrane translocation efficiency) possessed by the loop, as well as the ability to prevent and inhibit the progression of retinal diseases (e.g., retinitis pigmentosa), visual cognitive behavioral functions (e.g., improved light-dark discrimination, improved photophobia and / or danger avoidance), and enhanced visual acuity. In the case of a loop, biological activity includes, but is not limited to, functions such as conformational compatibility and membrane translocation efficiency. Alternatively, the function of a loop may be evaluated based on the function of the entire protein (here, rhodopsin) into which it is incorporated.

[0090] In the present disclosure, the chimeric proteins of the present disclosure and nucleic acids encoding them have been found to have uses in preventing or inhibiting the progression of retinal diseases, disorders or symptoms, improving visual cognitive behavioral functions (e.g., improved light / dark judgment, improved light aversion and / or danger avoidance) or object recognition functions, and for providing visual function enhancement effects such as improved visual acuity.

[0091] Retinal degenerative diseases, including retinitis pigmentosa and atrophic age-related macular degeneration, are among the eye diseases for which there is currently no cure, but this disclosure may provide a fundamental treatment for these diseases. It is said that there are a total of over 130 million patients worldwide, and in Japan, retinitis pigmentosa is the third leading cause of acquired blindness, and age-related macular degeneration is the fourth leading cause. Given the large number of patients and the severity of visual impairment, there has been a great desire for the development of a treatment, and this disclosure may provide a solution.

[0092] Like the central nervous system, photoreceptors, the primary visual neurons, cannot regenerate once lost. However, in retinitis pigmentosa and atrophic age-related macular degeneration, bipolar cells and retinal ganglion cells, which correspond to second- and third-order visual neurons, are preserved, which is thought to be one of the reasons why this disclosure is effective. This disclosure is a minimally invasive, safe, and long-term gene transfer therapy using optogenetics, which promises to regenerate vision. Unlike conventional methods that introduce light-activated ion channels, this method utilizes a more physiological phototransduction pathway that utilizes endogenous G protein signaling cascades and channels, enabling efficient and safe visual regeneration. While conventional light-activated ion channel transfer methods have been limited to regeneration in patients with already advanced retinal degeneration, this method does not require the retinal metabolic regeneration system known as the Visual Cycle, which is required for normal phototransduction, and therefore may also have the potential to inhibit the progression of retinal degeneration. This demonstrates its applicability not only to patients with advanced retinal degeneration, but also to preventing progression in patients in the early stages of the disease.

[0093] In one aspect, the present disclosure provides a nucleic acid comprising a nucleic acid sequence encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin, and a nucleic acid sequence encoding a signal sequence. In one embodiment, the signal sequence is an endoplasmic reticulum transport signal sequence or an endoplasmic reticulum export signal sequence. In a specific embodiment, the signal sequence is an endoplasmic reticulum export signal sequence. In a more specific embodiment, the endoplasmic reticulum export signal is an ER2 signal.

[0094] It has been reported that ER export signals have a positive effect on membrane transport of proteins (FEBS Lett. 2001 Mar 30;493(2-3):129-33.). Although evaluation of ER2 alone has not been performed, it has been reported that the peak current increases by approximately 1.7 times. On the other hand, Nature. 2010 Jan 7;463(7277):98-102 found no current enhancement effect due to the addition of a signal sequence.

[0095] In one embodiment, the nucleic acid of the disclosure comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 1 or 26. In some embodiments, the nucleic acid of the disclosure comprises or consists of a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 1 or 26.

[0096] In one embodiment, the nucleic acid of the present disclosure may further comprise a nucleic acid sequence encoding a FLAG tag. In one embodiment, the nucleic acid of the present disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments, the nucleic acid of the present disclosure comprises a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 3.

[0097] In one aspect, the present disclosure provides a polypeptide comprising a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin, and a signal sequence. In some embodiments, the polypeptide of the present disclosure consists of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin, and a signal sequence. In another embodiment, the signal sequence is an endoplasmic reticulum transport signal sequence or an endoplasmic reticulum export signal sequence. In certain embodiments, the signal sequence is an endoplasmic reticulum export signal sequence.

[0098] In one embodiment, a polynucleotide of the disclosure comprises or consists of a sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 or 27. In certain embodiments, a polynucleotide of the disclosure comprises or consists of a polynucleotide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 2 or 27.

[0099] In one embodiment, the disclosure comprises or consists of a polypeptide encoded by a polynucleotide of the disclosure. In some embodiments, the polypeptide of the disclosure comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2 or 27. In certain embodiments, the polypeptide encoded by a polynucleotide of the disclosure comprises or consists of a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or 27.

[0100] In one embodiment, the present disclosure comprises or consists of a nucleotide sequence of the present disclosure. In some embodiments, the nucleic acid of the present disclosure comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 4. In certain embodiments, the nucleic acid of the present disclosure comprises or consists of a nucleic acid having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0101] In one aspect, the present disclosure provides a nucleic acid comprising a nucleic acid sequence encoding a chimeric protein of an ionotropic receptor rhodopsin and a G protein-coupled receptor rhodopsin. In one embodiment, the nucleic acid of the present disclosure may comprise a nucleic acid sequence encoding a signal sequence. In certain embodiments, the signal sequence is an endoplasmic reticulum transport signal sequence or an endoplasmic reticulum export signal sequence. In certain embodiments, the signal sequence is an endoplasmic reticulum export signal sequence.

[0102] In one embodiment, the nucleic acid of the disclosure comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleic acid of the disclosure comprises or consists of a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 7.

[0103] In one embodiment, the nucleic acid of the present disclosure can include a nucleic acid sequence encoding any FLAG tag.

[0104] In one aspect, the present disclosure provides a polypeptide comprising a chimeric protein of an ionotropic receptor rhodopsin and a G protein-coupled receptor rhodopsin, and a signal sequence. In some embodiments, the polypeptide of the present disclosure consists of a chimeric protein of an ionotropic receptor rhodopsin and a G protein-coupled receptor rhodopsin, and a signal sequence. In another embodiment, the signal sequence is an endoplasmic reticulum transport signal sequence or an endoplasmic reticulum export signal sequence. In certain embodiments, the signal sequence is an endoplasmic reticulum export signal sequence.

[0105] In one embodiment, a polypeptide of the disclosure comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, a polypeptide of the disclosure comprises or consists of a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

[0106] In one embodiment, the present disclosure comprises or consists of a nucleic acid encoding a polypeptide of the present disclosure. In some embodiments, the nucleic acid of the present disclosure comprises or consists of a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the nucleic acid of the present disclosure comprises or consists of a nucleic acid encoding a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8.

[0107] In one aspect, the present disclosure provides a nucleic acid construct, comprising the nucleic acid of the present disclosure and a nucleic acid that allows expression in a cell, operably linked to the nucleic acid.In one embodiment, the nucleic acid construct of the present disclosure further comprises a vector.In some embodiments, the vector is selected from the group consisting of a viral vector, a plasmid vector, a cosmid vector, an artificial chromosome vector, and a fosmid vector.In certain embodiments, the vector is a viral vector.In another embodiment, the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector (AAV), a retroviral vector, and a lentiviral vector.In certain embodiments, the viral vector is an adeno-associated viral vector (AAV).

[0108] In one embodiment, the AAV is AAV-DJ, AAV-2, or AAV-6. In a preferred embodiment, the AAV may be AAV-DJ or AAV-6. The infection efficiency of bipolar cells is higher with AAV-DJ and AAV-6 than with AAV-2.

[0109] In one aspect, the present disclosure relates to a composition for gene transfer comprising a nucleic acid or nucleic acid construct of the present disclosure. In one embodiment, the composition for gene transfer of the present disclosure is administered by injection. In another embodiment, the composition for gene transfer of the present disclosure is administered intravitreally. In certain embodiments, the composition for gene transfer of the present disclosure may be provided together with a preservative solution, and in some embodiments, the preservative solution may be a buffer solution. In other embodiments, the composition for gene transfer of the present disclosure may be provided in a container. In certain embodiments, the container storing the composition for gene transfer of the present disclosure may be a syringe.

[0110] In another aspect, the present disclosure provides cells comprising the nucleic acids, polypeptides, or nucleic acid constructs of the present disclosure. In some embodiments, the cells of the present disclosure may be retinal cells. In another embodiment, the cells of the present disclosure may be provided as a cell preparation. The cell preparation includes cells and a cell preservation solution. In some embodiments, the cell preservation solution may be a culture medium or a buffer solution. In other embodiments, the cells of the present disclosure may be provided in a container. In certain embodiments, the container containing the cells of the present disclosure may be a syringe.

[0111] In another aspect, the present disclosure provides a pharmaceutical composition comprising the nucleic acid, polypeptide, nucleic acid construct, gene transfer composition, or cell of the present disclosure. In one embodiment, the pharmaceutical composition of the present disclosure may be a pharmaceutical composition for treating, preventing, or inhibiting the progression of a retinal disease, disorder, or symptom. In one embodiment, the pharmaceutical composition of the present disclosure may be a pharmaceutical composition for improving visual cognitive behavioral function. In one embodiment, the pharmaceutical composition of the present disclosure may be a pharmaceutical composition for enhancing visual function. In one embodiment, the pharmaceutical composition of the present disclosure may be a pharmaceutical composition for improving object recognition function. In the present disclosure, the prevention or inhibition of the progression of a retinal disease, disorder, or symptom, typified by the inhibition of the progression of retinal degeneration pigmentopathy, has been confirmed by demonstration in the experiments shown in Examples 1 to 10.

[0112] (Improvement of visual cognitive behavioral functions) Functions such as improvements in visual cognitive behavioral functions (e.g., improvement in light / dark judgment function, improvement in light aversion function, and / or danger avoidance function) have been verified using experimental models in the present disclosure, and are considered to have significant effects. The effects of visual cognitive behavioral functions (e.g., improvement in light / dark judgment function, improvement in light aversion function, and / or danger avoidance function) were demonstrated by the results of a light / dark box choice test (LDT) as demonstrated in Example 5. Visual cognitive behavioral functions are functions that can be confirmed not only by confirming the light sensitivity of the visual organs, but also by verifying whether they are actually manifested as behavior in animal models, etc. The verification in experiments such as Example 5 can be considered one of the achievements of the present disclosure. Improvements in visual cognitive behavioral functions include improvements, strengthening, or enhancement of visual acuity, contrast sensitivity, light / dark adaptation, color vision, etc.

[0113] (Visual enhancement and vision improvement) The function of improving vision has been verified using an experimental model in this disclosure, and it can be said that it has a remarkable effect. The enhancement of visual function such as improving vision has been confirmed by the experiment of visual evoked potentials (VEP) as exemplified in Example 4.

[0114] (Improved object recognition function) Functions such as improvement of object recognition function have been verified using an experimental model in the present disclosure, and it can be said that they produce significant effects. Functions such as improvement of object recognition function have been confirmed by demonstrating them through experiments using the object recognition test (ORT), as exemplified in Example 6. Visual evoked potential (VEP) experiments showed that the input of light stimuli reached the central nervous system (brain), and LDT showed that this was output as an aversion response, but it was not clear whether vision had recovered to a level where objects could be recognized. The results shown in Example 6 confirming recovery of vision to a level where objects could be recognized are of great clinical significance.

[0115] In one aspect, the present disclosure provides a method for treating, preventing, or inhibiting the progression of an ocular disease, disorder, or symptom in a subject, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid, polypeptide, nucleic acid construct, gene transfer composition, cell, or pharmaceutical composition of the present disclosure.

[0116] In one embodiment, the disease, disorder, or symptom is a retinal degenerative disease, and examples of retinal degenerative diseases include retinitis pigmentosa and age-related macular degeneration, and more preferably retinitis pigmentosa.

[0117] In a preferred embodiment, the retinitis pigmentosa covered by the present disclosure is autosomal dominant, preferably RHO autosomal dominant.

[0118] In a preferred embodiment, the present disclosure is used to prevent or slow the progression of retinitis pigmentosa.

[0119] In a preferred embodiment, the present disclosure is administered to a subject before or shortly after the onset of a disease, disorder, or symptom, for example, but not limited to, within one year, preferably within six months, three months, or one month of the onset (e.g., the appearance of subjective symptoms).

[0120] In one specific embodiment, the composition or vector of the present disclosure is administered once. Single administration of the present disclosure has been shown to be effective and is thought to result in good patient compliance.

[0121] In one particular embodiment, the amount of vector used of the present disclosure is 0.1 x 10 11 ~10×10 11 vg / eye unit dose, for example, the lower limit is 0.01 × 10 11 vg / eye, 0.02×10 11 vg / eye, 0.03×10 11 vg / eye, 0.04×10 11 vg / eye, 0.05×10 11 vg / eye, 0.06×1011 vg / eye, 0.07×10 11 vg / eye, 0.08×10 11 vg / eye, 0.09×10 11 vg / eye, 0.1×10 11 vg / eye, 0.2×10 11 vg / eye, 0.3×10 11 vg / eye, 0.4×10 11 vg / eye, 0.5×10 11 vg / eye, etc., with an upper limit of 2 × 10 11 vg / eye, 3×10 11 vg / eye, 4×10 11 vg / eye, 5×10 11 vg / eye, 6×10 11 vg / eye, 7×10 11 vg / eye, 8×10 11 vg / eye, 9×10 11 vg / eye, 10×10 11 vg / eye, 15×10 11 vg / eye, 20×10 11 vg / eye, 30×10 11 vg / eye, 40×10 11 vg / eye, 50×10 11 vg / eye, etc.

[0122] In another aspect, the present disclosure provides a method for improving visual cognitive behavioral function, the method comprising administering to a subject a therapeutically effective amount of a nucleic acid, polypeptide, nucleic acid construct, gene transfer composition, cell, or pharmaceutical composition of the present disclosure.

[0123] In yet another aspect, the present disclosure provides a method for enhancing visual function, the method comprising administering to a subject a therapeutically effective amount of a nucleic acid, polypeptide, nucleic acid construct, gene transfer composition, cell, or pharmaceutical composition of the present disclosure.

[0124] In certain aspects, the present disclosure provides a method for improving object recognition function, the method comprising administering to a subject a therapeutically effective amount of a nucleic acid, polypeptide, nucleic acid construct, gene transfer composition, cell, or pharmaceutical composition of the present disclosure.

[0125] In one aspect, the present disclosure provides use of a nucleic acid, polypeptide, nucleic acid construct, gene transfer composition, cell, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating, preventing, or inhibiting the progression of an ocular disease, disorder, or condition in a subject.

[0126] In another aspect, the present disclosure provides use of a nucleic acid, a polypeptide, a nucleic acid construct, a composition for gene transfer, a cell, or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for improving visual cognitive behavioral function.

[0127] In yet another aspect, the present disclosure provides use of a nucleic acid, polypeptide, nucleic acid construct, gene transfer composition, cell, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for enhancing visual function.

[0128] In certain aspects, the present disclosure provides use of a nucleic acid, polypeptide, nucleic acid construct, gene transfer composition, cell, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for improving object recognition function. (General technology)

[0129] The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art and are described in, for example, Current Protocols in Molecular Biology (http: / / onlinelibrary.wiley.com / book / 10.1002 / 0471142727) and Molecular Cloning: A Laboratory Manual (Fourth Edition) (http: / / www.molecularcloning.com), the relevant portions of which (possibly in their entirety) are incorporated herein by reference.

[0130] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when "within a range" of "two values" is specified, the range includes the two values ​​themselves.

[0131] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0132] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0133] Examples are described below. Where necessary, the handling of animals used in the following examples complied with the standards established by Keio University and other institutions, as well as other relevant ethical standards and guidelines, and was carried out in accordance with the Declaration of Helsinki. While the reagents used were specifically those listed in the examples, equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science Inc., etc.) can also be used.

[0134] Example 1: Vector Preparation DNA encoding the chimeric protein (GR / BvRh) was constructed as follows. The sequence corresponding to amino acids 137-145 from the N-terminus of the cytoplasmic second loop of Gloeobacter violaceus rhodopsin (GR) (SEQ ID NO: 14) was replaced with the sequence corresponding to amino acids 137-145 of bovine rhodopsin (BvRh) (SEQ ID NO: 12). The sequence corresponding to amino acids 198-206 from the N-terminus of the cytoplasmic third loop of GR was replaced with the sequence corresponding to amino acids 225-252 of bovine rhodopsin. Furthermore, the 132nd amino acid, glutamic acid, of GR was replaced with glutamine. DNA encoding this chimeric protein was inserted into the pCDNA3.1 vector. Alternatively, a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 23 was generated and inserted into the HindIII / XbaI site of the pCDNA3.1 vector as the DNA encoding the chimeric protein. The nucleotide sequence set forth in SEQ ID NO:23 was generated as follows: the sequence corresponding to amino acids 137-145 from the N-terminus, which corresponds to the cytoplasmic loop 2 of Gloeobacter violaceus rhodopsin (GR) (SEQ ID NO:14), was replaced with the nucleotide sequence set forth in SEQ ID NO:18, which corresponds to the loop 2 of bovine rhodopsin (BvRh) (SEQ ID NO:12) (encoding the amino acid sequence set forth in SEQ ID NO:19), and the sequence corresponding to amino acids 198-206 from the N-terminus, which corresponds to the cytoplasmic loop 3 of GR, was replaced with the nucleotide sequence set forth in SEQ ID NO:20, which corresponds to the loop 3 of bovine rhodopsin (encoding the amino acid sequence set forth in SEQ ID NO:22). Furthermore, the nucleotide sequence set forth in SEQ ID NO:3 was generated by changing some bases without changing the encoded amino acids.Specifically, 6, 9-13, 15, 16, 18-22, 27-29, 31-36, 39, 40, 43, 45, 48, 50, 51, 53-55, 58, 59, 61, 65-73, 75-84, 86, 88, 89, 93, 97, 98, 100, 101, 104, 106-108, 110, 112, 114, 115, 122, 123, 125, 128, 131, 133, 139, 143, 145, 146, 155, 157, 162, 165, 167, 169-171, 174, 176, 179, 182, 183, 186-189, 193-198, 204, The nucleic acids encoding the amino acids at positions 205, 207, 209, 212, 215, 216, 218-220, 224, 225, 227, 228, 230, 231, 233-235, 238, 240, 242, 243, 246, 247, 249, 251, 253-255, 257-259, 261-264, 266-270, 272, 273, 275, 276, 279, 281-287, 289-291, 296-299, 302-305, 307-316, 318, 319, and 321-330 were mutated without changing the encoded amino acids. Mutants were created using the quick-change method. The sequence portion used in bovine rhodopsin is completely identical to the amino acid sequence of human rhodopsin, so there is no problem in calling it human rhodopsin.

[0135] The EGFP or GR / BvRh gene was subcloned into the AAV2 shuttle plasmid to generate the viral expression constructs AAV2-CAGGS-EGFP-WPRE-pA (vector for EGFP expression) and AAV2-CAGGS-GR / BvRh-WPRE-pA (vector for chimeric protein expression). Viral vectors were packaged by transfecting HEK293 cells with three types of plasmids: the vector plasmid, the AAV vector plasmid, and the adenovirus helper plasmid. The viral vectors were purified using the cesium chloride method. In the vectors, "ITR" stands for "Inverted Terminal Repeat." "CAGGS" refers to the CAG promoter region. "WPRE" stands for "woodchuck hepatitis virus post-transcriptional regulatory element." "pA" refers to the peptide tag. "EGFP" stands for "enhanced green fluorescent protein."

[0136] The structures of a nucleic acid construct encoding a chimeric rhodopsin (hereinafter referred to as a first nucleic acid construct) and a nucleic acid construct encoding a chimeric rhodopsin to which a signal sequence has been added (hereinafter referred to as a nucleic acid construct of the present disclosure) are shown in Figure 1. ER2 shown in Figure 1 is a type of endoplasmic reticulum export signal.

[0137] Example 2: Multiple Electrode Array (MEA) Testing Using Nucleic Acid Constructs of the Present Disclosure The effect of the nucleic acid construct of the present disclosure on photoresponse was measured as described below.

[0138] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations were purchased from CLEA Japan, Inc. (multi-electrode array) Here, a multi-electrode array is a technique in which neurons are placed on an element with many electrodes, the electrical response of the neurons is recorded from outside the cells, and the waveform of the electrical response is analyzed to investigate the type of activated cells, the timing and magnitude of that activity, etc.

[0139] (Vector administration) Blind rd1 mice aged 10 weeks or older were injected with 1.0 × 10 AAV DJ-CAGGS- Chimeric rhodopsin (GR / BvRh)-WPRE-pA vector (first nucleic acid construct) or AAV DJ-CAGGS- Chimeric rhodopsin-sm (GR / BvRh-sm)-WPRE-pA (the nucleic acid construct of the present disclosure) to which a signal sequence had been added. 9 1 μl was administered by intravitreal injection at a concentration of vg / μl.

[0140] (measurement) The light responses of the mice were measured after 4 weeks post-injection, when gene expression peaked. Using a multielectrode array (MEA) experiment, the light responses of retinal ganglion cells were measured ex vivo using white LED light stimulation at various intensities.

[0141] (result) 1×10 17 photons / cm 2 / s, 1 × 10 16 photons / cm 2 / s, 1 × 10 15 photons / cm 2 / s, 1 × 10 14 photons / cm 2 / s and 1 × 10 13 photons / cm 2The results of performing a multi-electrode array with a light intensity of 1 × 10 / s are shown. The upper part of Figures 2 and 3 shows a raster plot of retinal ganglion cell firing, and in each graph, the vertical axis shows a histogram of firing frequency per second. The horizontal axis of the graph shows time (seconds). The lower part of each graph shows the chimeric protein construct introduced and the light intensity. 13 photons / cm 2 The light intensity of / s corresponds to the light intensity of a street with street lights or a hallway at home at night, and is 1 × 10 14 photons / cm 2 The light intensity of / s corresponds to the light intensity in a home, and is 1 × 10 15 photons / cm 2 / s corresponds to the light intensity in a shop, and is 1 × 10 16 photons / cm 2 / s corresponds to the outdoor light intensity on a cloudy day, and is 1×10 17 photons / cm 2 The light intensity of 1x10 / s corresponds to the light intensity outdoors on a sunny day. 14 photons / cm 2 / s stimulation (Figure 2), whereas the nucleic acid construct of the present disclosure showed a response at a light intensity of 1x10 13 photons / cm 2 When the first nucleic acid construct was expressed in a retinitis pigmentosa model mouse, responses were obtained up to a light intensity of 1 × 10 15 photons / cm 2 / s, electrical signals were emitted at a frequency higher than that of the negative control, whereas when the nucleic acid construct of the present disclosure was expressed, the frequency was 1 × 10 13 photons / cm 2 Up to a light intensity of 1 × 10 / s, ganglion cells fired at a higher frequency than the negative control. Furthermore, at each light intensity, the retinal ganglion cells transfected with the nucleic acid construct of the present disclosure tended to fire at a higher frequency than the retinal ganglion cells transfected with the first nucleic acid construct, particularly at a light intensity of 1 × 10 15 photons / cm 2At a light intensity of 1×10 / s, the nucleic acid construct of the present disclosure was found to have a firing frequency approximately three times higher than that of the first nucleic acid construct. 13 photons / cm 2 At a light intensity of 1 / s, the first nucleic acid construct showed almost no firing, whereas the nucleic acid construct of the present disclosure resulted in firing of retinal ganglion cells. Thus, it was demonstrated that expression of the chimeric protein of the nucleic acid construct of the present disclosure unexpectedly resulted in significantly superior light sensitivity compared to expression of the chimeric protein of the first nucleic acid construct. Additionally, 1x10 14~16 photons / cm 2 In the stimulation intensity range of 1 × 10 / s, the nucleic acid construct of the present disclosure produced significantly higher firing rates (Figure 4). 15 photons / cm 2 The results show that the multi-electrode array per unit area at 1x10 / s was performed. 15 photons / cm 2 The number of firing cells per unit area was also significantly higher at a stimulation intensity of 2.6 mm / s (Fig. 5). 2 The figure shows the number of retinal ganglion cells that fired per The retinal ganglion cells transfected with the first nucleic acid construct were 1 × 10 15 photons / cm 2 In the first nucleic acid construct, only about 2.7 cells fired in response to light with an intensity of 1 / s, whereas about 33 cells fired in response to light with the nucleic acid construct of the present disclosure. Thus, it was demonstrated that the nucleic acid construct of the present disclosure provides unexpectedly superior light sensitivity, more than 12 times that of the first nucleic acid construct.

[0142] (Example 3: Wavelength Sensitivity Evaluation) Seven weeks after injection of the nucleic acid construct of the present disclosure, the relative luminosity of 11-week-old male rd1 mice was measured for each wavelength. Light stimulation was performed using wavelength-specific LEDs, and the peak firing rate (spikes / sec) of 25 cells that responded was measured for each wavelength. The highest response value among all wavelengths was set to 1, and a ratio was calculated, and the average was measured. 1x10 14 photons / cm 2 Measurements were performed at a light stimulus intensity of 1 / s. The results showed that mice injected with the nucleic acid construct of the present disclosure exhibited the expected wavelength sensitivity (Figure 6). Because the amount of protein expression is proportional to the number of firing cells, it was thought that the expression level of the chimeric rhodopsin protein also increased to the same extent as the number of firing cells. It was thought that sensitivity would also increase as the amount of protein expression increased.

[0143] Example 4: Evaluation of visual evoked potentials The effect of the nucleic acid construct of the present disclosure on visual evoked potentials (VEPs) was measured as described below.

[0144] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations were purchased from CLEA Japan, Inc. (Method for assessing visual evoked potentials) Electrical signals emitted from the retina are transmitted to the primary visual cortex (V1) of the brain, activating nerve cells in this area. To confirm the visual regeneration effect at the central level, an experiment was also conducted in which electrodes were implanted in the brain to record nerve activity extracellularly. Specifically, the produced vector was injected intravitreally into a retinitis pigmentosa model mouse (rd1) mouse, and after anesthesia, a white LED placed 3 cm in front of the eye was used to project 0.1 cds / m 2Evoked potentials to a flash stimulus of 0.1 cds / m (this light intensity corresponds roughly to the light intensity of a street with streetlights or a home corridor at night) were measured using the PuREC acquisition system (manufactured by Mayo Co., Ltd.). 2 The flash stimulation was measured.

[0145] (Vector administration) The first nucleic acid construct or the nucleic acid construct of the present disclosure was injected at 1.0 × 10 cells / ml into blind rd1 mice aged 10 weeks or older. 9 The control group received the same amount of the AAV DJ-CAGGS-EGFP-WPRE-pA vector.

[0146] (measurement) VEPs were measured 4 weeks after injection, when gene expression peaked. One week before measurement, mice were sedated with a triple anesthesia (midazolam, medetomidine, and butorphanol tartrate administered at 4 mg / kg, 0.75 mg / kg, and 5 mg / kg body weight, respectively), and a measurement electrode was implanted in the skull near the visual cortex (1.5 mm anterior and 1.5 mm lateral to the lambdoid suture). After sedation with the triple anesthesia again, a 0.1 cds / m was emitted from a white LED placed 3 cm in front of the eyes. 2 Evoked potentials to flash stimuli were measured using the PuREC acquisition system (Mayo, Inazawa, Japan).

[0147] (result) In Figure 7, the vertical axis shows the amplitude (µV) of visual evoked potentials obtained from the visual cortex in response to light stimulation. The chimeric protein constructs introduced are shown at the bottom of the graph. A significant increase in amplitude was observed in mice treated with the nucleic acid construct of the present disclosure (56.4 ± 14.0 µV) compared to the control (17.86 ± 3.37 µV) and mice treated with the first nucleic acid construct (22.13 ± 8.38 µV). Treatment with the improved construct also showed a significant visual regeneration effect at the central level (Figure 7). Therefore, it was demonstrated that expression of the chimeric protein of the nucleic acid construct of the present disclosure results in significantly better light sensitivity than expression of the chimeric protein of the first nucleic acid construct.

[0148] (Example 5: Evaluation of light and dark recognition function) The effect of the nucleic acid construct of the present disclosure on light-dark recognition function was measured, as described below.

[0149] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations were purchased from CLEA Japan, Inc.

[0150] (Vector administration) The first nucleic acid construct or the nucleic acid construct of the present disclosure was injected at 1.0 × 10 cells / ml into blind rd1 mice aged 10 weeks or older. 9 The control group received the same amount of AAV DJ-CAGGS-EGFP-WPRE-pA vector.

[0151] (measurement) After 4 weeks post-injection, when gene expression peaked, the mice were tested in a light-dark box. A light-dark transition test (LDT) is performed to evaluate light-dark recognition function. Mice are placed in a light-dark box (an acrylic case measuring 415mm wide, 300mm high, and 250mm deep, divided into two sections by a partition; one section is lighted at 20 lux, and the other is a dark room, connected by a 5x5mm window), and their behavior is videotaped for 10 minutes. The ratio of time spent in the light and dark areas is measured and compared.

[0152] (result) Healthy mice avoid bright light and therefore spend less time in bright light, whereas blind mice (controls) have a roughly half-and-half ratio of 0.5. Furthermore, mice treated with the nucleic acid construct of the present disclosure show a significantly shorter time in bright light compared to mice treated with the first nucleic acid construct.

[0153] Example 6: Evaluation of object recognition function The effect of the nucleic acid construct of the present disclosure on object recognition function was measured as described below.

[0154] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations were purchased from CLEA Japan, Inc. (Method for evaluating object recognition function) To evaluate object recognition function, the constructed vector was injected intravitreally into retinitis pigmentosa model mice (rd1), and differences in behavior with and without video playback were observed. Tablet devices were placed on both sides of the space containing the mouse, and at 10 lux brightness, a video of a mouse was played on one side and an empty mouse cage on the other. The time spent in the area playing the mouse video and the area playing the video of an empty mouse cage were measured. The measurement period was 15 minutes, starting immediately after the central partition was removed.

[0155] (Vector administration) The nucleic acid construct of the present disclosure (AAV (2 / 6 / DJ)-CAGGS-chimeric rhodopsin (GR / BvRh)-WPRE-pA vector) was injected at 1.0 × 10 9 A 1µl dose at a concentration of 100µg / µl was administered intravitreally. A blindness control group received the same amount of the AAV DJ-CAGGS-EGFP-WPRE-pA vector. A comparison group was also administered a less sensitive microbial rhodopsin, AAV DJ-C1V1. Tablet devices were placed on both sides of the space containing the mice, and at 10 lux brightness, one displayed a video of a mouse, while the other displayed an empty mouse cage. The experimental space is shown in Figure 8.

[0156] (measurement) The time spent in the area showing the mouse video and the area showing the empty mouse cage video was measured for 15 minutes starting immediately after the central partition was removed.

[0157] (result) The ratio of time spent on the object video side (time spent in the area where the mouse video was played / measurement time) in the blinded control group (EGFP) was 0.495±0.019, while it was significantly higher at 0.555±0.06 in the DJ-type AAV chimera injection and 0.538±0.015 in the 6-type AAV chimera injection (Figure 9). In the experimental results shown in Figure 9, the vertical axis shows the object video side stay time ratio (time spent in the area where the mouse video is played / measurement time). An object video side stay time ratio of 0.5 indicates no mouse movement, and values ​​farther away from 0.5 are interpreted as indicating that the mouse has an object recognition function directly linked to visual ability. In the blinded control group (EGFP), the ratio of time spent in the object video side was 0.495±0.019, while in the DJ AAV chimera injection group it was 0.555±0.06, and in the 6 AAV chimera injection group it was 0.538±0.015, which were significantly higher. Furthermore, in the AAV2-incorporated vector (2-Chimera), the time spent in the cage where the video was played tended to decrease. This result may be due to the fact that the vector changes the appearance from normal, causing the mice to mistake it for a predator or other repelling object. The above results demonstrate that expression of the construct of the present disclosure restores vision to a level where objects can be recognized.

[0158] (Consideration) In DJ and type 6 mice, it is believed that vision was restored to a level where objects could be recognized. In type 2 mice, it is possible that the expression level in the target bipolar cells was low, preventing them from seeing objects, or that visual regeneration occurred, but the expression pattern was different, resulting in different perceptions and aversion to the mouse video.

[0159] (Example 7: Method for producing a chimeric protein (GtACR2 / BvRh) of ionotropic rhodopsin and G protein-coupled receptor rhodopsin) A chimeric protein (GtACR2 / BvRh) between ionotropic rhodopsin and G protein-coupled receptor rhodopsin was prepared using the same method as in Example 1. The sequence corresponding to the amino acids in the cytoplasmic loop 2 of Guillardia theta (GT) (SEQ ID NO: 15) was replaced with the amino acid sequence corresponding to bovine rhodopsin (BvRh) (SEQ ID NO: 12), and the sequence corresponding to the amino acids in the cytoplasmic loop 3 of GT was replaced with the amino acid sequence corresponding to bovine rhodopsin. DNA encoding this chimeric protein was inserted into the pCDNA3.1 vector. Alternatively, a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 6 was generated and inserted into the HindIII / XbaI site of the pCDNA3.1 vector as the DNA encoding the chimeric protein. The nucleic acid sequence set forth in SEQ ID NO: 7 was prepared by adding specific mutations to the prepared nucleic acid sequence. Specifically, 1, 2, 4-9, 11-17, 21, 22, 27-30, 33, 34, 36-41, 43, 45, 48, 49, 51, 54, 56-58, 60, 63, 65, 68, 70, 71-75, 77-78, 81, 83, 84, 86, 89, 90, 92, 93, 95, 97-99, 102, 103, 111, 113, 114, 123, 125, 130, 131-137, 139, 142, 143, 146, 148-153, 156, 160, 161, 165, 167, 168, 170, 171, 174-176, 180, 182 The bases encoding the amino acids at positions 183, 187, 188, 190, 191, 196, 197, 199, 200, 202, 204, 208, 212-214, 217, 219, 226, 229, 232, 236-238, 240, 242, 243, 247, 248, 251, 252, 258, 263-265, 267, 269, 271, 272, 274, 276-280, 282-284, 289, 290, 291, 294, 297-299, 302, 304, 307, and 310 were changed without changing the amino acids encoded.

[0160] Example 8: Measurement of GPCR activity of nucleic acid constructs of the present disclosure GPCR activity was measured by observing the fluorescence of GloSensor™ (Promega), which is used as an indicator of intracellular cAMP concentration.

[0161] (method) (material) ND7 / 23 cells were cultured and transfected with the GR / BvRh-double-EQ-linker-Venus-ER2 vector, which contained a Venus inserted nucleic acid construct of the present disclosure, and the pGloSensor™ (Promega) vector. The control group was transfected with the same amount of pcDNA3.1 (empty vector) and pGloSensor (Promega) vector. The transfected cells were cultured and washed with PBS, then detached using trypsin and EDTA and collected in a centrifuge tube. The cells were pelleted by centrifugation and suspended in fresh DMEM medium. Based on the cell concentration, the cells were replated at a concentration suitable for fluorescence microscopy observation. (Measurement of G protein-coupled receptor (GPCR) activity) An experimental system was constructed to analyze intracellular signal transduction induced by light stimulation. The details of the signal transduction pathway are as follows: photoreceptors express rhodopsin, a type of G protein-coupled receptor (GPCR), on their cell membranes, and rhodopsin binds to retinal. When light hits photoreceptors, the structure of retinal changes, activating rhodopsin. Activated rhodopsin activates G proteins (Gt in the retina) distributed near the cell membrane, and this Gt activates cGMP phosphodiesterase. cGMP phosphodiesterase is an enzyme that breaks down cGMP within the cell, so its activation reduces the intracellular cGMP concentration. Photoreceptor cells have cGMP-dependent ion channels on their cell membranes. When the intracellular cGMP concentration decreases, the ion permeability of these cGMP-dependent ion channels changes, causing a change in the membrane potential of the photoreceptor cells, resulting in the generation of an electrical signal. In this way, photoreceptor cells convert light signals into electrical signals. Here, since it is difficult to measure the cGMP concentration in the pathway mediated by the Gt-type G protein, in the present disclosure, activation of the G protein is measured by measuring the change in intracellular cAMP concentration caused by the pathway mediated by the Gi-type G protein, which is in the same G protein family. It is well known to those skilled in the art that Gt-type G proteins and Gi-type G proteins are cross-reactive (e.g., Xiang Li et al., "Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin," PNAS, December 6, 2005, Vol. 102, No. 49, pp. 17816-17821, p. 17817, left column, fourth paragraph, states, "Vertebrate rhodopsin is a G protein transducin that couples to an α subunit belonging to the Gi subfamily (15), thus raising the possibility that mammalian rhodopsin may couple to other Gi / o family members."). Therefore, measuring changes in intracellular cAMP concentration mediated by Gi-type G proteins has been commonly used to measure Gt activation in the retina. It should be noted that there are multiple types of G proteins, and the G protein present in photoreceptor cells is known to be Gt (Gαt). Gt-type G proteins are present only in some cells, such as photoreceptors, while Gs-, Gi-, and Gq-type G proteins are present in general neurons. Among them, Gs-type G proteins activate adenylate cyclase, increasing the intracellular cAMP concentration, while Gi-type G proteins inhibit adenylate cyclase, decreasing the intracellular cAMP concentration. In this experiment, we measured the change in intracellular cAMP concentration mediated by Gi-type G proteins to analyze the intracellular signaling pathway in response to light stimulation. The specific experimental method is as follows. Lipofectamine (R)The GR / BvRh-double-EQ-linker-Venus-ER2 vector and control vector were expressed in HEK293T cells using a 2000 nm microscope according to the manufacturer's instructions. Experiments to transfect the vectors into HEK293T cells were also carried out in parallel. These cultured cells were then incubated at 525 nm for 10 min. 16 photons / cm 2 Light stimulation was applied for 1 minute at a light intensity of 1 / s, and intracellular cAMP concentration was measured using a cAMP Gi kit (Cisbio) according to the manufacturer's instructions.

[0162] (measurement) The transfected cells were transferred to a CO2-independent culture medium containing retinal (10% FBS, 2% GloSensor™ stock solution). Changes in intracellular cAMP concentration were measured by recording changes in GloSensor™ fluorescence intensity. Measurements were performed using a plate reader equipped with a light irradiator according to the standard GloSensor™ assay protocol. Forskolin (final concentration 3.5 μM), which activates adenylyl cyclase, was administered to elevate intracellular cAMP concentration. After confirming that GloSensor™ intensity had reached a plateau, the cells were irradiated with 510 nm light (approximately 0.27 mW) for 2 minutes, approximately 35 minutes after forskolin administration. Furthermore, 464 nm light (approximately 2.8 mW) was irradiated for 2 minutes, approximately 50 minutes after forskolin administration. This experiment was performed twice, and the change in brightness of the GloSensor™ in each experiment was plotted as a graph (FIG. 10).

[0163] (result) In the control group, no difference in luminance was observed between light irradiation and no light irradiation (Figure 10A). On the other hand, in the group administered with the GR / BvRh-double-EQ-linker-Venus-ER2 vector, a decrease in GloSensor™ luminance was observed after 464 nm light irradiation compared to the group without light irradiation (Figure 10B). GloSensor™ luminance is known to correspond to a decrease in intracellular cAMP concentration, indicating that light stimulation reduced intracellular cAMP concentration in the group administered with the GR / BvRh-double-EQ-linker-Venus-ER2 vector. Therefore, it can be seen that the nucleic acid construct GR / BvRh-double-EQ-linker-Venus-ER2 of the present disclosure, in which Venus is inserted, has GPCR activity and can treat, prevent, or inhibit the progression of retinal diseases, disorders, or symptoms, improve visual cognitive behavioral function, and enhance visual function. Figure 13 shows experimental data obtained by forcibly expressing various genes in HEK293T cells using lipofection and measuring the change in cAMP concentration with and without light stimulation. The vertical axis shows the ΔHTRF ratio, and the horizontal axis shows the results for each gene. HTRF (Homogeneous Time-Resolved Fluorescence) is the ratio of exogenous reference cAMP to endogenous cAMP (HTRF ratio), measured using a fluorescent antibody against cAMP. As endogenous cAMP increases, the HTRF ratio decreases, and is inversely proportional to the cAMP concentration. The ΔHTRF ratio is the difference between the HTRF ratios with and without light irradiation; a larger ratio indicates a greater decrease in cAMP due to light stimulation, i.e., activation of G protein (Gi). In Figure 13, an experiment was conducted using 11 individuals, and the results were 4.6±11.5 for the negative control, 31.1±21.4 for the chimeric protein of the first nucleic acid construct, and 156.9±24.2 for the chimeric protein of the nucleic acid construct of the present disclosure. Thus, it was demonstrated that expression of the nucleic acid construct of the present disclosure results in significantly greater light sensitivity than expression of the chimeric protein of the first nucleic acid construct.

[0164] Example 9: Measurement of GPCR activity of a nucleic acid construct encoding a chimeric protein of an ionotropic rhodopsin and a G protein-coupled receptor rhodopsin GPCR activity was measured by observing the fluorescence of GloSensor™ (Promega), which is used as an indicator of intracellular cAMP concentration.

[0165] (method) (material) ND7 / 23 cells were cultured and transfected with the GtACR2tr / BvRh-double vector and the pGloSensor™ (Promega) vector. The control group was transfected with the same amount of pcDNA3.1 (empty vector) and pGloSensor (Promega) vector. The transfected cells were cultured and washed with PBS. Afterwards, the cells were detached using trypsin and EDTA and collected in a centrifuge tube. The cells were pelleted by centrifugation and suspended in fresh DMEM medium. Based on the cell concentration, the cells were replated at a concentration suitable for fluorescence microscopy.

[0166] (measurement) The transfected cells were transferred to a CO2-independent culture medium containing retinal (10% FBS, 2% GloSensor™ stock solution). Changes in intracellular cAMP concentration were measured by recording changes in GloSensor™ fluorescence intensity. Measurements were performed using a plate reader equipped with a light irradiator according to the standard GloSensor™ assay protocol. Forskolin (final concentration 3.5 μM), which activates adenylyl cyclase, was administered to elevate intracellular cAMP concentration. After confirming that GloSensor™ intensity had reached a plateau, the cells were irradiated with 510 nm light (approximately 0.27 mW) for 2 minutes, approximately 35 minutes after forskolin administration. Furthermore, 464 nm light (approximately 2.8 mW) was irradiated for 2 minutes, approximately 50 minutes after forskolin administration. This experiment was performed twice, and the change in brightness of the GloSensor™ in each experiment was plotted as a graph (FIG. 11).

[0167] (result) In the control group, no difference in brightness was observed between light irradiation and no light irradiation (Figure 11A). On the other hand, in the group administered with the GtACR2tr / BvRh-double vector, a decrease in GloSensor™ brightness was observed after 464 nm light irradiation compared to the group without light irradiation (Figure 11B). GloSensor™ brightness is known to correspond to a decrease in intracellular cAMP concentration, and this indicates that light stimulation reduced intracellular cAMP concentration in the group administered with the GtACR2tr / BvRh-double vector. Therefore, it can be seen that the GtACR2tr / BvRh-double nucleic acid construct of the present disclosure, in which Venus is inserted, has GPCR activity and can treat, prevent, or inhibit the progression of retinal diseases, disorders, or symptoms, improve visual cognitive behavioral function, and enhance visual function.

[0168] Example 10: Measurement of ion transport ability of nucleic acid construct encoding chimeric protein of ionotropic rhodopsin and G protein-coupled receptor rhodopsin Ion transport activity was measured by the patch clamp technique.

[0169] (material) ND7 / 23 cells were cultured and transfected with the GtACR2tr / BvRh-double vector, which encodes a chimeric protein consisting of an ion channel rhodopsin and a G protein-coupled receptor rhodopsin. Control cells were transfected with the same amount of the GtACR1 vector, which encodes the wild-type anion channel rhodopsin from Guillardia theta. The transfected cells were cultured in a medium containing retinal.

[0170] (measurement) Whole-cell patch clamp recordings were performed using a patch clamp apparatus, glass microelectrodes, and standard extracellular and intracellular solutions. Light exposure was performed using a microscope-mounted light exposure device, with 500 nm or 480 nm light for approximately 400 ms. The photocurrent response was recorded. The membrane potential was clamped between -80 mV and 20 mV (in 20 mV increments).

[0171] (result) In the control group, light irradiation induced a photocurrent (Figure 12). This photocurrent decayed over approximately 2000 ms after the end of light irradiation. In the GtACR2tr / BvRh-double vector-transfected group, light irradiation induced a large photocurrent. This photocurrent decayed over approximately 1000 ms. These results further demonstrated that GtACR2tr / BvRh-double has greater ion transport capacity and faster kinetics than the control group.

[0172] Because the resting membrane potential of photoreceptors is between -30mV and -50mV, GtACR2tr / BvRh-double is able to hyperpolarize the membrane potential through light stimulation, potentially leading to the treatment, prevention, or inhibition of progression of retinal diseases, disorders, or symptoms, improvement of visual cognitive behavioral functions, and enhancement of visual function.

[0173] Example 11: Preparation of a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence A nucleic acid construct is prepared comprising a nucleic acid sequence encoding a chimeric protein in which an endoplasmic reticulum export signal different from the endoplasmic reticulum export signal inserted in Example 2 is inserted into the nucleic acid sequence encoding the chimeric protein prepared in Example 1.

[0174] Example 12: Multielectrode array (MEA) test using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The effect of the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 on the light response was measured as follows.

[0175] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0176] (Vector administration) The AAV DJ-CAGGS-chimeric rhodopsin (GR / BvRh)-WPRE-pA vector (first nucleic acid construct) or the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 was injected at 1.0 × 10 9 1 μl at a concentration of vg / μl is administered by intravitreal injection.

[0177] (measurement) The light response of the mice will be measured from 4 weeks after injection, when gene expression reaches its peak. Using a multielectrode array (MEA) test, the light response of retinal ganglion cells will be measured ex vivo using white LED light stimulation at different intensities.

[0178] (result) 1x10 for the first nucleic acid construct 14photons / cm 2 Although a response can only be obtained at a light intensity of up to 1x10 / s stimulation, an improved response can be obtained with the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11. 14~16 photons / cm 2 In the stimulation intensity range of 1x10 / s, the firing frequency was significantly higher in the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11. 15 photons / cm 2 The number of firing cells per unit area was also significantly higher at a stimulus intensity of 1 / s.

[0179] Example 13: Evaluation of wavelength sensitivity using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The wavelength sensitivity of a nucleic acid construct containing a nucleic acid sequence encoding the signal sequence of Example 11 is evaluated. Seven weeks after injection of the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11, the relative luminosity of 11-week-old male rd1 mice was measured for each wavelength. Light stimulation was performed using a wavelength-specific LED, and the peak firing rate (spikes / sec) of 25 cells that responded was measured for each wavelength. The highest response value among all wavelengths was set to 1, and a ratio was calculated, and the average was measured. 1x10 14 photons / cm 2 Measurements are performed at a light stimulus intensity of 1 / s. The measurement results show that mice injected with the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 exhibit wavelength sensitivity as expected.

[0180] Example 14: Evaluation of visual evoked potentials using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The effect of the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 on visual evoked potential (VEP) was measured as follows.

[0181] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0182] (Vector administration) The first nucleic acid construct or the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 was injected at 1.0 × 10 9 The control group received the same amount of AAV DJ-CAGGS-EGFP-WPRE-pA vector.

[0183] (measurement) VEPs were measured 4 weeks after injection, when gene expression peaked. One week before the measurements, mice were anesthetized with a triple-dose mixture (midazolam, medetomidine, and butorphanol tartrate, 4 mg / kg, 0.75 mg / kg, respectively). The animals were sedated with a triple-anesthesia mixture, and a measuring electrode was implanted in the skull near the visual cortex (1.5 mm anterior and 1.5 mm lateral to the lambdoid suture). After the animals were sedated again with a triple-anesthesia mixture, a white LED (0.1 cds / m) was placed 3 cm in front of the eyes. 2 The evoked potentials to flash stimuli were measured using the PuREC acquisition system (Mayo, Inazawa, Japan).

[0184] (result) A significant increase in amplitude was observed in mice treated with the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 compared to the control and first nucleic acid construct-treated mice. Treatment with the improved construct also showed a significant visual regenerative effect at the central level.

[0185] Example 15: Evaluation of light / dark recognition function using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The effect of the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 on light-dark recognition function was measured as follows.

[0186] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0187] (Vector administration) The first nucleic acid construct or the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 was injected at 1.0 × 10 9 The control group received the same amount of AAV DJ-CAGGS-EGFP-WPRE-pA vector.

[0188] (measurement) After 4 weeks post-injection, when gene expression peaked, the mice were tested in a light-dark box. A light-dark transition test (LDT) is performed to evaluate light-dark recognition function. Mice are placed in a light-dark box (an acrylic case measuring 415mm wide, 300mm high, and 250mm deep, divided into two sections by a partition; one section is lighted at 20 lux, and the other is a dark room, connected by a 5x5mm window), and their behavior is videotaped for 10 minutes. The ratio of time spent in the light and dark areas is measured and compared.

[0189] (result) Healthy mice avoid bright light and therefore spend less time in bright light, whereas blind mice (controls) have a roughly half-and-half ratio of 0.5. Furthermore, mice treated by injection with a nucleic acid construct containing a nucleic acid sequence encoding the signal sequence of Example 11 show a significantly shorter time spent in bright light than mice treated by injection with the first nucleic acid construct.

[0190] Example 16: Evaluation of object recognition function using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The effect of the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 on the object recognition function was measured as follows.

[0191] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0192] (Vector administration) The nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11 was injected at 1.0 × 10 9 The mice were administered 1 μl of the vector intravitreally at a concentration of 100 μg / μl. The blindness control group received the same amount of the AAV DJ-CAGGS-EGFP-WPRE-pA vector. Tablets were placed on both sides of the mouse cage, and at 10 lux brightness, one side displayed a video of the mouse, while the other displayed an empty mouse cage. The experimental space is shown in Figure 8.

[0193] (measurement) The time spent in the area where the mouse video was playing and the area where the empty mouse cage video was playing was measured for 15 minutes starting immediately after the central partition was removed.

[0194] (result) The ratio of time spent on the object video side (time spent in the area where the mouse video is played / measurement time) in the blinded control group (EGFP) was approximately 0.5, whereas it was significantly higher in the nucleic acid construct containing the nucleic acid sequence encoding the signal sequence of Example 11.

[0195] Example 17: Preparation of a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence A nucleic acid construct is prepared comprising a nucleic acid sequence encoding a chimeric protein in which an endoplasmic reticulum targeting signal is inserted into the nucleic acid sequence encoding the chimeric protein prepared in Example 1.

[0196] Example 18: Multielectrode array (MEA) test using a nucleic acid construct containing a nucleic acid sequence encoding an endoplasmic reticulum targeting signal The effect of the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 on the light response was measured as described below.

[0197] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0198] (Vector administration) The AAV DJ-CAGGS-chimeric rhodopsin (GR / BvRh)-WPRE-pA vector (first nucleic acid construct) or the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 19 was injected at a dose of 1.0 × 10 9 1 μl at a concentration of vg / μl is administered by intravitreal injection.

[0199] (measurement) The light response of the mice will be measured from 4 weeks after injection, when gene expression reaches its peak. Using a multielectrode array (MEA) test, the light response of retinal ganglion cells will be measured ex vivo using white LED light stimulation at different intensities.

[0200] (result) 1x10 for the first nucleic acid construct 14 photons / cm 2Although a response can be obtained only at light intensities up to 1x10 / s stimulation, an improved response can be obtained with the nucleic acid construct of Example 17 containing a nucleic acid sequence encoding an endoplasmic reticulum targeting signal. 14~16 photons / cm 2 In the stimulation intensity range of 1x10 / s, the firing frequency was significantly higher for the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17. 15 photons / cm 2 The number of firing cells per unit area was also significantly higher at a stimulus intensity of 1 / s.

[0201] Example 19: Evaluation of wavelength sensitivity using a nucleic acid construct containing a nucleic acid sequence encoding an endoplasmic reticulum targeting signal The wavelength sensitivity of the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 is evaluated. Seven weeks after injection of the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17, the relative luminosity of 11-week-old male rd1 mice was measured for each wavelength. Light stimulation was performed using wavelength-specific LEDs, and the peak firing rate (spikes / sec) of 25 cells that responded was measured for each wavelength. The highest response value among all wavelengths was set to 1, and a ratio was calculated, and the average was measured. 1x10 14 photons / cm 2 Measurements were performed at a light stimulation intensity of 1 / s. The results showed that mice injected with the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 exhibited the expected wavelength sensitivity.

[0202] Example 20: Evaluation of visual evoked potentials using a nucleic acid construct containing a nucleic acid sequence encoding an endoplasmic reticulum targeting signal The effect of the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 on visual evoked potential (VEP) was measured as follows.

[0203] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0204] (Vector administration) The first nucleic acid construct or the nucleic acid construct comprising the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 was injected at 1.0 × 10 9 The control group received the same amount of AAV DJ-CAGGS-EGFP-WPRE-pA vector.

[0205] (measurement) VEPs were measured 4 weeks after injection, when gene expression peaked. One week before the measurements, mice were anesthetized with a triple-dose mixture (midazolam, medetomidine, and butorphanol tartrate, 4 mg / kg, 0.75 mg / kg, respectively). The animals were sedated with a triple-anesthesia mixture, and a measuring electrode was implanted in the skull near the visual cortex (1.5 mm anterior and 1.5 mm lateral to the lambdoid suture). After the animals were sedated again with a triple-anesthesia mixture, a white LED (0.1 cds / m) was placed 3 cm in front of the eyes. 2 The evoked potentials to flash stimuli were measured using the PuREC acquisition system (Mayo, Inazawa, Japan).

[0206] (result) A significant increase in amplitude was observed in mice treated with the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 compared to the control and first nucleic acid construct-treated mice. Treatment with the improved construct also showed a significant visual regeneration effect at the central level.

[0207] Example 21: Evaluation of light / dark recognition function using a nucleic acid construct containing a nucleic acid sequence encoding an endoplasmic reticulum targeting signal The effect of the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 on light-dark recognition function was measured as follows.

[0208] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0209] (Vector administration) The first nucleic acid construct or the nucleic acid construct comprising the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 was injected at 1.0 × 10 9 The control group received the same amount of AAV DJ-CAGGS-EGFP-WPRE-pA vector.

[0210] (measurement) Four weeks after injection, when gene expression reaches its peak, a light-dark transition test (LDT) is performed to evaluate light and dark recognition function. Mice are placed in a light-dark box (an acrylic case measuring 415mm wide, 300mm high, and 250mm deep, divided into two sections by a partition; one section is illuminated at 20 lux, the other is a dark room, and is connected by a 5x5mm window), and their behavior is videotaped for 10 minutes. The ratio of time spent in the light and dark areas is measured and compared.

[0211] (result) Healthy mice avoid bright light and therefore spend less time in bright light, whereas blind mice (controls) have a roughly half-and-half ratio of 0.5. Furthermore, mice treated with the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 show a significantly shorter time spent in bright light compared to mice treated with the first nucleic acid construct.

[0212] Example 22: Evaluation of object recognition function using a nucleic acid construct containing a nucleic acid sequence encoding an endoplasmic reticulum targeting signal The effect of the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 on the object recognition function was measured as described below.

[0213] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0214] (Vector administration) The nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17 was injected at 1.0 × 10 9 The mice were administered 1 μl of the vector intravitreally at a concentration of 100 μg / μl. The blindness control group received the same amount of the AAV DJ-CAGGS-EGFP-WPRE-pA vector. Tablets were placed on both sides of the mouse cage, and at 10 lux brightness, one side displayed a video of the mouse, while the other displayed an empty mouse cage. The experimental space is shown in Figure 8.

[0215] (measurement) The time spent in the area where the mouse video was playing and the area where the empty mouse cage video was playing was measured for 15 minutes starting immediately after the central partition was removed.

[0216] (result) The ratio of time spent on the object video side (time spent in the area where the mouse video is played / measurement time) in the blinded control group (EGFP) was approximately 0.5, whereas it was significantly higher in the nucleic acid construct containing the nucleic acid sequence encoding the endoplasmic reticulum targeting signal of Example 17.

[0217] Example 23: Comparative Example of GPCR Activity Measurement A nucleotide sequence encoding the amino acid indicated in the discarded sequence number is prepared, which is different from the nucleotide sequence indicated in SEQ ID NO: 7. The nucleic acid construct containing the nucleotide sequence indicated in SEQ ID NO: 7 and the nucleic acid construct containing the nucleotide sequence prepared in this example are transfected into ND7 / 23 cells.

[0218] When the intracellular cAMP concentration was measured using GloSensor™, it was found that the cAMP concentration was lower in cells transfected with a nucleic acid construct containing the nucleotide sequence shown in SEQ ID NO: 7 than in cells transfected with the nucleic acid construct prepared in this example. This indicates that the chimeric rhodopsin encoded by the nucleotide sequence shown in SEQ ID NO: 7 has stronger GPCR activity than the chimeric rhodopsin encoded by the nucleotide sequence prepared in this example.

[0219] Example 24: Comparative Example of Ion Transport Ability A nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 8, which is different from the nucleotide sequence set forth in SEQ ID NO: 7, is prepared. The nucleic acid construct containing the nucleotide sequence set forth in SEQ ID NO: 7 and the nucleic acid construct containing the nucleotide sequence prepared in this example are transfected into ND7 / 23 cells.

[0220] When the ion transport capacity of the chimeric rhodopsins encoded by each base sequence was measured using the patch clamp method, it was found that cells transfected with a nucleic acid construct containing the base sequence shown in sequence number 7 had greater ion transport capacity than cells transfected with the nucleic acid construct prepared in this example.

[0221] Example 25: Vector culture in adherent and suspension culture systems HEK293T cells or (adherent) HEK293 cells are cultured as an adherent culture system. HEK293 cells or CHO cells are cultured as a suspension culture system. After culture, the following plasmids are mixed and transfected into the cells (PEI: Polyethylenimine, and if necessary, the calcium phosphate method or DEAE-dextran method is used). pAAV-RC (rep and cap genes) pHelper pAAV-GOI (a gene of interest) Several days after transfection, cells are harvested and lysed using detergent to obtain the drug substance. The final product is then purified by affinity chromatography, ultracentrifugation, and filter purification. Purification can be performed according to the method described in Nathalie C and Joshua C., Methods & Clinical Development (2016) 3, 16002.

[0222] Example 26: Multielectrode array (MEA) test using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The effect of the base sequence shown in SEQ ID NO: 26 (encoding the amino acid sequence shown in SEQ ID NO: 27) on photoresponse was measured as described below.

[0223] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0224] (Vector administration) Blind rd1 mice aged 10 weeks or older were injected with 1.0 × 10 mAb of the AAV 6-CAGGS-chimeric rhodopsin (GR / BvRh)-WPRE-pA vector (first nucleic acid construct) or a nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26. 8 1 μl at a concentration of vg / μl is administered by intravitreal injection.

[0225] (measurement) The light response of the mice will be measured from 4 weeks after injection, when gene expression reaches its peak. Using a multielectrode array (MEA) test, the light response of retinal ganglion cells will be measured ex vivo using white LED light stimulation at different intensities.

[0226] (result) 1x10 for the first nucleic acid construct 14 photons / cm 2 Although responses can only be obtained at light intensities up to 1x10 / s stimulation, improved responses can be obtained with the nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26. 14~16 photons / cm 2 In the stimulation intensity range of 1x10 / s, the firing frequency was significantly higher in the nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26. 15 photons / cm 2 The number of firing cells per unit area was also significantly higher at a stimulus intensity of 1 / s.

[0227] Example 27: Evaluation of wavelength sensitivity using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The wavelength sensitivity of a nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26 is evaluated. Seven weeks after injection of a nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26, the relative luminosity of 11-week-old male rd1 mice at each wavelength was measured. Light stimulation was performed using a wavelength-specific LED, and the peak firing rate (spikes / sec) of 25 cells that responded was measured at each wavelength. The highest response value among all wavelengths was set to 1, and a ratio was calculated, and the average was measured. 1x1014 photons / cm 2 Measurements are performed at a light stimulus intensity of 1 / s. The results show that mice injected with a nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26 exhibit wavelength sensitivity as expected.

[0228] Example 28: Evaluation of visual evoked potentials using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The effect of a nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26 on visual evoked potential (VEP) was measured as described below.

[0229] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0230] (Vector administration) The first nucleic acid construct or the nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 26 was injected at 1.0 × 10 8 The control group received the same amount of the AAV 6-CAGGS-EGFP-WPRE-pA vector.

[0231] (measurement) VEPs are measured 4 weeks after injection, when gene expression reaches its peak. One week before measurement, mice are sedated with a triple-anesthesia (midazolam, medetomidine, and butorphanol tartrate administered at 4 mg / kg, 0.75 mg / kg, and 5 mg / kg body weight, respectively), and a measurement electrode is implanted in the skull near the visual cortex (1.5 mm anterior and 1.5 mm lateral to the lambdoid suture). After the mice are sedated again with the triple-anesthesia, a 0.1 cds / m field is emitted from a white LED placed 3 cm in front of the eyes. 2The evoked potentials to flash stimuli were measured using the PuREC acquisition system (Mayo, Inazawa, Japan).

[0232] (result) A significant increase in amplitude was observed in mice treated with the nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26 compared to control and first nucleic acid construct-treated mice. Treatment with the improved construct also showed a significant visual regenerative effect at the central level.

[0233] Example 29: Evaluation of light / dark recognition function using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The effect of a nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26 on light-dark recognition function was measured as described below.

[0234] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0235] (Vector administration) The first nucleic acid construct or the nucleic acid construct comprising the nucleic acid sequence set forth in SEQ ID NO: 26 was injected at 1.0 × 10 8 The control group received the same amount of the AAV 6-CAGGS-EGFP-WPRE-pA vector.

[0236] (measurement) A light-dark transition test (LDT) is performed 4 weeks after injection, when gene expression reaches its peak, to evaluate light and dark recognition function. Mice are placed in a light-dark box (an acrylic case measuring 415mm wide, 300mm high, and 250mm deep, divided into two sections by a partition; one section is illuminated at 10 lux, the other is a dark room, and is connected by a 5x5mm window), and their behavior is videotaped for 10 minutes. The ratio of time spent in the light and dark areas is measured and compared.

[0237] (result) Healthy mice avoid bright light and therefore spend less time in bright light, whereas blind mice (controls) have a roughly half-and-half ratio of 0.5. Furthermore, mice treated with a nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26 show a significantly shorter time in bright light than mice treated with the first nucleic acid construct.

[0238] Example 30: Evaluation of object recognition function using a nucleic acid construct containing a nucleic acid sequence encoding a signal sequence The effect of a nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26 on object recognition function was measured as described below.

[0239] (material and method) (animal) The rd1 mouse (Pde6b rd1 / rd1 C3H / HeJ Jcl mice carrying the above mutations are purchased from CLEA Japan, Inc.

[0240] (Vector administration) The nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26 was injected at 1.0 × 10 8The mice were administered 1 μl of the vector intravitreally at a concentration of 100 μg / μl. The blindness control group received the same amount of the AAV 6-CAGGS-EGFP-WPRE-pA vector. Tablets were placed on both sides of the mouse cage, and at 10 lux brightness, one side displayed a video of the mouse, while the other displayed an empty mouse cage. The experimental space is shown in Figure 8.

[0241] (measurement) The time spent in the area where the mouse video was playing and the area where the empty mouse cage video was playing was measured for 15 minutes starting immediately after the central partition was removed.

[0242] (result) The ratio of time spent on the object video side (time spent in the area where the mouse video is played / measurement time) in the blinded control group (EGFP) was approximately 0.5, whereas it was significantly higher in the nucleic acid construct containing the nucleic acid sequence set forth in SEQ ID NO: 26.

[0243] (Note) As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted solely by the scope of the claims. It is understood that the patents, patent applications, and literature cited in this specification are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2019-167553 (filed September 13, 2019) filed with the Japan Patent Office, and the contents of that application are incorporated by reference into this specification in their entirety as if set forth herein. [Industrial Applicability]

[0244] Novel nucleic acid constructs of chimeric rhodopsin are provided for preventing and inhibiting the progression of retinal diseases, improving visual cognitive behavioral functions (e.g., improving light-dark discrimination, improving light aversion and / or danger avoidance), object recognition, and enhancing visual acuity. Technologies based on such technologies are also provided that can be utilized in industries (such as pharmaceuticals). [Sequence List Free Text]

[0245] SEQ ID NO: 1: An example of a nucleic acid sequence consisting of a chimeric rhodopsin (GR / BvRh) and an endoplasmic reticulum export signal SEQ ID NO: 2: An example of an amino acid sequence consisting of chimeric rhodopsin (GR / BvRh) and an endoplasmic reticulum export signal SEQ ID NO: 3: An example of a nucleic acid sequence consisting of chimeric rhodopsin (GR / BvRh), an endoplasmic reticulum export signal, and a FLAG tag SEQ ID NO: 4: An example of an amino acid sequence consisting of chimeric rhodopsin (GR / BvRh), an endoplasmic reticulum export signal, and a FLAG tag SEQ ID NO: 5: An example of the amino acid sequence of chimeric rhodopsin (GR / BvRh) SEQ ID NO: 6: An example of the nucleic acid sequence of chimeric rhodopsin (GtACR2 / BvRh) SEQ ID NO: 7: An example of a nucleic acid sequence of chimeric rhodopsin (GtACR2 / BvRh) SEQ ID NO: 8: An example of the amino acid sequence of chimeric rhodopsin (GtACR2 / BvRh) SEQ ID NO: 9: Nucleic acid sequence of human rhodopsin (huRh) SEQ ID NO: 10: Amino acid sequence of human rhodopsin (huRh) SEQ ID NO: 11: Nucleic acid sequence of bovine rhodopsin (BvRh) SEQ ID NO: 12: Amino acid sequence of bovine rhodopsin (BvRh) SEQ ID NO: 13: Nucleic acid sequence of Gloeobacter violaceus Rhodopsin (GR) SEQ ID NO: 14: Amino acid sequence of Gloeobacter violaceus Rhodopsin (GR) SEQ ID NO: 15: Nucleic acid sequence of Guillardia theta anion channelrhodopsin2 (GtACR2) SEQ ID NO: 16: Amino acid sequence of Guillardia theta anion channelrhodopsin2 (GtACR2) SEQ ID NO: 17: An example of the nucleic acid sequence of the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin SEQ ID NO: 18: An example of the nucleic acid sequence of the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin SEQ ID NO: 19: An example of the amino acid sequence of the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin (corresponding to SEQ ID NO: 18) SEQ ID NO: 20: An example of the nucleic acid sequence of the third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin SEQ ID NO: 21: An example of the nucleic acid sequence of the third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin SEQ ID NO: 22: An example of the amino acid sequence of the third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin SEQ ID NO: 23: Example of a nucleic acid sequence of a chimeric rhodopsin (GR / BvRh) (corresponding to SEQ ID NO: 8). The start codon corresponds to nucleotides 43-45, and the stop codon corresponds to nucleotides 994-996. SEQ ID NO: 24: An example of the nucleic acid sequence of the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin SEQ ID NO: 25: An example of the amino acid sequence of the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin (corresponding to SEQ ID NO: 24) SEQ ID NO: 26: An example of a nucleic acid sequence consisting of a chimeric rhodopsin (GR / BvRh) and an endoplasmic reticulum export signal SEQ ID NO: 27: An example of an amino acid sequence consisting of chimeric rhodopsin (GR / BvRh) and an endoplasmic reticulum export signal

Claims

[Claim 1] A nucleic acid comprising a nucleic acid sequence encoding a chimeric protein comprising at least a portion of an ion transport receptor rhodopsin and at least a portion of a G protein-coupled receptor rhodopsin, and a nucleic acid sequence encoding a signal sequence.

Images