阅读说明：本技术 用于刺激视网膜细胞和治疗视力损失的方法 (Methods for stimulating retinal cells and treating vision loss ) 是由 R·R·伯奇 N·瓦格纳 J·A·格雷科 于 2018-05-08 设计创作，主要内容包括：蛋白质基视网膜下植入物为由于如年龄相关性黄斑变性(AMD)和视网膜色素变性(RP)等疾病而失明的患者提供了恢复视力的新方法。本发明涉及菌视紫红质基(BR基)视网膜植入物,该植入物可以在视网膜下位置植入以替代退化的感光细胞。(Protein-based subretinal implants provide a new method of restoring vision in patients who are blind due to diseases such as age-related macular degeneration (AMD) and Retinitis Pigmentosa (RP). The present invention relates to bacteriorhodopsin-based (BR-based) retinal implants that can be implanted in a subretinal location to replace degenerated photoreceptor cells.)

1. A method for treating a patient with vision loss due to retinal cell loss, comprising:

(a) implanting a bacteriorhodopsin-based retinal implant into the eye of a patient, and

(b) activating the implant with a light source,

wherein the implant stimulates retinal cells to provide the stimulation at a resolution of pixel size less than about 500 μm, or less than 350 μm, or less than 250 μm, or less than about 200 μm, or less than about 150 μm, or less than about 100 μm, or less than about 75 μm, or less than about 50 μm, or less than about 40 μm, or less than about 30 μm, or less than about 25 μm.

2. A method for treating a patient with vision loss due to retinal cell loss, comprising:

(a) implanting a bacteriorhodopsin-based retinal implant into the eye of a patient, and

(b) activating the implant with a light source,

wherein the implant stimulates retinal cells to provide stimulation at a resolution limited by: (i) the diffraction limit of the light source or (ii) the aperture and density of the ion-permeable substrate when the retinal implant further comprises the ion-permeable substrate.

3. A method for treating a patient with vision loss due to retinal cell loss, comprising:

(a) implanting a bacteriorhodopsin-based retinal implant into the eye of a patient, and

(b) activating the implant with a light source,

wherein the implant stimulates retinal cells to have about 4 pixels/mm²And a pixel size of about 500 μm, or about 8 pixels/mm²And a pixel diameter of about 350 μm, or about 16 pixels/mm²And a pixel diameter of about 250 μm, or about 25 pixels/mm²And a pixel diameter of about 200 μm, or about 44 pixels/mm²About 150 μmPixel diameter, or about 100 pixels/mm²And a pixel diameter of about 100 μm, or about 178 pixels/mm²And a pixel diameter of about 75 μm, or about 400 pixels/mm²And a pixel diameter of about 50 μm, or about 625 pixels/mm²And a pixel diameter of about 40 μm, or about 1111 pixels/mm²And a pixel diameter of about 30 μm, or about 1600 pixels/mm²And a resolution of about 25 μm pixel diameter provides the stimulus.

4. The method of any one of claims 1-3, wherein the microbial rhodopsin-based retinal implant comprises at least one substrate layer and a microbial rhodopsin film, wherein the microbial rhodopsin film comprises a plurality of individual layers of native microbial rhodopsin or a microbial rhodopsin mutant, wherein the individual layers of the native microbial rhodopsin or microbial rhodopsin mutant are alternated with cationic polymer layers, and wherein the microbial rhodopsin mutant is selected from the group consisting of chloride ion pump mutants, dipole mutants, light cycle mutants, gold binding mutants, ion pump mutants, Q state mutants, and combinations thereof.

5. The method of claim 4, wherein the light source is a light source emitting at a human visible wavelength of about 400 to about 700 nm.

6. The method of claim 4, wherein the intensity of the light source is less than about 100mW/cm²、50mW/cm²、40mW/cm²Or less than about 30mW/cm²Or less than about 20mW/cm²Or less than about 10mW/cm²Or less than about 5mW/cm²Or less than about 1mW/cm²。

7. The method of claim 5, wherein the light source is a pulsed light source.

8. The method of claim 5, wherein the light source is a continuous light source.

9. The method of any one of claims 1 to 3, wherein the implant is a biocompatible, ion-permeable retinal implant.

10. The method of claim 9, wherein the implant is made from a layer-by-layer assembly.

11. The method according to any one of claims 1 to 3, wherein the implant is implanted at a location selected from a subretinal location or a preretinal location.

12. The method of claim 11, wherein the implant is implanted in a subretinal location.

13. The method of claim 11, wherein the implant is implanted in a pre-retinal location.

14. The method of any one of claims 1 to 3, wherein the retinal cell is selected from the group consisting of a retinal ganglion cell, a retinal bipolar cell, a retinal photoreceptor cell, and a combination thereof.

15. The method of claim 14, wherein the retinal cell is selected from retinal ganglion cells.

16. The method of claim 14, wherein the retinal cell is selected from retinal bipolar cells.

17. The method of claim 14, wherein the retinal cell is selected from retinal photoreceptor cells.

18. The method of any one of claims 1 to 3, wherein the retinal implant comprises at least about 100 layers of native microbial rhodopsin or mutant microbial rhodopsin, or at least about 150 layers of native microbial rhodopsin or mutant microbial rhodopsin, or at least about 200 layers of native microbial rhodopsin or mutant microbial rhodopsin.

19. The method of any one of claims 1 to 3, wherein the retinal implant further provides a temporal resolution of at least about 30 milliseconds.

20. The method of any one of claims 1 to 3, wherein the retinal implant further provides a temporal resolution of at least about 20 milliseconds.

21. The method of any one of claims 1 to 3, wherein the retinal implant further provides a temporal resolution of at least about 10 milliseconds.

22. The method of any one of claims 1 to 3, wherein the retinal implant elicits an action potential of about 40 μ V to the cells.

23. The method of any one of claims 1 to 3, wherein the retinal implant elicits an action potential of about 30 μ V to the cells.

24. The method of any one of claims 1 to 3, wherein the retinal implant elicits an action potential of about 20 μ V to the cells.

25. The method of any one of claims 1 to 3, wherein the retinal implant provides a unidirectional proton gradient.

26. The method of claim 25, wherein the unidirectional proton gradient is directed at the retinal cell, wherein the retinal cell is selected from a retinal ganglion cell or a retinal bipolar cell.

27. The method of claim 26, wherein the implant is implanted at a location selected from a subretinal location or a preretinal location.

28. The method of claim 25, wherein the microbial rhodopsin-based retinal implant comprises microbial rhodopsin molecules such that they are uniformly oriented.

29. The method according to any one of claims 1 to 3, wherein the retinal implant is used at or below physiological temperature.

30. The method according to any one of claims 1 to 3, wherein the retinal implant is used at physiological pH.

31. The method of any one of claims 1 to 3, wherein the loss of vision is caused by loss of retinal photoreceptor cells.

32. The method of claim 4, wherein the base layer is an ion permeable layer and is on one or both sides of the implant.

33. The method of claim 32, wherein the substrate layer is selected from the group consisting of polyethylene terephthalate (PET), 2-hydroxyethyl methacrylate (HEMA), 2-phenylethyl methacrylate (PEM), polyethylene glycol (PEG), Methyl Methacrylate (MMA), Polydimethylsiloxane (PDMS) macromer, poly (vinyl pyrrolidone) (PVP), poly (p-xylylene) (also known as parylene), polyvinyl alcohol (PVA) hydrogel, and combinations thereof.

34. The method of claim 32, wherein the base layer is less than 100 μ ι η thick and contains a uniformly distributed array of pores having diameters less than about 100 μ ι η.

35. The method of any one of claims 1-3, wherein the microbial rhodopsin-based retinal implant is based on native microbial rhodopsin.

36. The method of any one of claims 1 to 3, wherein the microbial rhodopsin-based retinal implant is based on a microbial rhodopsin mutant.

37. The method of claim 36, wherein the mutant is a Q-state mutant.

38. The method of claim 37, wherein the mutant has been optimized to have a high dipole moment.

39. The method of claim 37, wherein the mutant has been optimized so as to form a high-efficiency Q state.

40. A method for stimulating retinal cells, comprising:

(a) contacting the retinal cells with a microbial rhodopsin-based retinal implant,

(b) the implant is activated with a light source and,

wherein

(i) Wherein the implant stimulates retinal cells to provide stimulation at a resolution of pixel size less than about 500 μm, or less than about 350 μm, or less than about 250 μm, or less than about 200 μm, or less than about 150 μm, or less than about 100 μm, or less than about 75 μm, or less than about 50 μm, or less than about 40 μm, or less than about 30 μm, or less than about 25 μm; or

(ii) Wherein the implant stimulates retinal cells to provide stimulation at a resolution limited by: (i) a diffraction limit of the light source or (ii) an aperture and density of the ion-permeable substrate when the retinal implant further comprises the ion-permeable substrate; or

(iii) Wherein the implant stimulates retinal cells to have about 4 pixels/mm²And a pixel size of about 500 μm, or about 8 pixels/mm²Has a pixel density of about 350 μm and a pixel diameter of about 350 μm, or hasAbout 16 pixels/mm²And a pixel diameter of about 250 μm, or about 25 pixels/mm²And a pixel diameter of about 200 μm, or about 44 pixels/mm²And a pixel diameter of about 150 μm, or about 100 pixels/mm²And a pixel diameter of about 100 μm, or about 178 pixels/mm²And a pixel diameter of about 75 μm, or about 400 pixels/mm²And a pixel diameter of about 50 μm, or about 625 pixels/mm²And a pixel diameter of about 40 μm, or about 1111 pixels/mm²And a pixel diameter of about 30 μm, or about 1600 pixels/mm²And a resolution of about 25 μm pixel diameter provides the stimulus.

41. A retinal implant for treating a patient with vision loss due to retinal cell loss, comprising a retinal implant that stimulates retinal cells to provide stimulation at a resolution of pixel size less than about 500 μ ι η, or less than about 350 μ ι η, or less than about 250 μ ι η, or less than about 200 μ ι η, or less than about 150 μ ι η, or less than about 100 μ ι η, or less than about 75 μ ι η, or less than about 50 μ ι η, or less than about 40 μ ι η, or less than about 30 μ ι η, or less than about 25 μ ι η.

42. A retinal implant for treating a patient with vision loss due to retinal cell loss, comprising a retinal implant that stimulates retinal cells to provide stimulation at a resolution limited by: (i) the diffraction limit of the light source or (ii) the aperture and density of the ion-permeable substrate when the retinal implant further comprises the ion-permeable substrate.

43. A retinal implant for treating a patient with vision loss due to retinal cell loss comprising stimulating retinal cells to have about 4 pixels/mm²And a pixel size of about 500 μm, or about 8 pixels/mm²Pixel density of (2) and pixels of about 350 μmDiameter, or about 16 pixels/mm²And a pixel diameter of about 250 μm, or about 25 pixels/mm²And a pixel diameter of about 200 μm, or about 44 pixels/mm²And a pixel diameter of about 150 μm, or about 100 pixels/mm²And a pixel diameter of about 100 μm, or about 178 pixels/mm²And a pixel diameter of about 75 μm, or about 400 pixels/mm²And a pixel diameter of about 50 μm, or about 625 pixels/mm²And a pixel diameter of about 40 μm, or about 1111 pixels/mm²And a pixel diameter of about 30 μm, or about 1600 pixels/mm²And a resolution of about 25 μm pixel diameter provides a stimulating retinal implant.

44. The retinal implant of any one of claims 41 to 43, wherein the microbial rhodopsin-based retinal implant comprises at least one substrate layer and a microbial rhodopsin film, wherein the microbial rhodopsin film comprises a plurality of individual layers of native microbial rhodopsin or a microbial rhodopsin mutant, wherein individual layers of the native microbial rhodopsin or microbial rhodopsin mutant are alternated with layers of a cationic polymer, and wherein the microbial rhodopsin mutant is selected from the group consisting of a chloride ion pump mutant, a dipole mutant, a light cycle mutant, a gold binding mutant, an ion pump mutant, a Q-state mutant, and combinations thereof.

45. The retinal implant of claim 44, wherein the light source is a light source emitting at a human visible wavelength of about 400 to about 700 nm.

46. The retinal implant of claim 44, wherein the intensity of the light source is less than about 100mW/cm²、50mW/cm²、40mW/cm²Or less than about 30mW/cm²Or less than about 20mW/cm²Or less than about 10mW/cm²Or less than about 5mW/cm²Or less than about1mW/cm²。

47. The retinal implant of claim 45, wherein the light source is a pulsed light source.

48. The retinal implant of claim 45, wherein the light source is a continuous light source.

49. The retinal implant according to any one of claims 41 to 43, wherein the implant is a biocompatible, ion-permeable retinal implant.

50. The retinal implant of claim 49, wherein the implant is made in a layer-by-layer assembly.

51. The retinal implant according to any one of claims 41 to 43, wherein the implant is implanted in a location selected from a subretinal location or a pre-retinal location.

52. The retinal implant of claim 51, wherein the implant is implanted in a subretinal location.

53. The retinal implant of claim 51, wherein the implant is implanted in a pre-retinal location.

54. The retinal implant according to any one of claims 41 to 43, wherein the retinal cells are selected from the group consisting of retinal ganglion cells, retinal bipolar cells, retinal photoreceptor cells, and combinations thereof.

55. A retinal implant according to claim 54 wherein the retinal cells are selected from retinal ganglion cells.

56. The retinal implant according to claim 54, wherein the retinal cells are selected from retinal bipolar cells.

57. The retinal implant of claim 54, wherein the retinal cells are selected from retinal photoreceptor cells.

58. The retinal implant of any one of claims 41 to 43, wherein the retinal implant comprises at least about 100 layers of native microbial rhodopsin or mutant microbial rhodopsin, or at least about 150 layers of native microbial rhodopsin or mutant microbial rhodopsin, or at least about 200 layers of native microbial rhodopsin or mutant microbial rhodopsin.

59. The retinal implant according to any one of claims 41 to 43, wherein the retinal implant further provides a temporal resolution of at least about 30 milliseconds.

60. The retinal implant according to any one of claims 41 to 43, wherein the retinal implant further provides a temporal resolution of at least about 20 milliseconds.

61. The retinal implant according to any one of claims 41 to 43, wherein the retinal implant further provides a temporal resolution of at least about 10 milliseconds.

62. The retinal implant according to any one of claims 41 to 43, wherein the retinal implant elicits an action potential of about 40 μ V to the cells.

63. The retinal implant according to any one of claims 41 to 43, wherein the retinal implant elicits an action potential of about 30 μ V to the cells.

64. The retinal implant according to any one of claims 41 to 43, wherein the retinal implant elicits an action potential of about 20 μ V to the cells.

65. The retinal implant according to any one of claims 41 to 43, wherein the retinal implant provides a unidirectional proton gradient.

66. The retinal implant of claim 65, wherein the unidirectional proton gradient is directed to retinal cells, wherein the retinal cells are selected from retinal ganglion cells or retinal bipolar cells.

67. The retinal implant of claim 66, wherein the implant is implanted in a location selected from a subretinal location or an preretinal location.

68. The retinal implant of claim 65, wherein the microbial rhodopsin-based retinal implant comprises microbial rhodopsin molecules such that they are uniformly oriented.

69. The retinal implant according to any one of claims 41 to 43, wherein the retinal implant is used at or below physiological temperatures.

70. The retinal implant according to any one of claims 41 to 43, wherein the retinal implant is used at physiological pH values.

71. The retinal implant according to any one of claims 41 to 43, wherein the loss of vision is caused by a loss of retinal photoreceptor cells.

72. The retinal implant of claim 44, wherein the base layer is an ion permeable layer and is on one or both sides of the implant.

73. The retinal implant of claim 72, wherein the base layer is selected from the group consisting of polyethylene terephthalate (PET), 2-hydroxyethyl methacrylate (HEMA), 2-phenylethyl methacrylate (PEM), polyethylene glycol (PEG), Methyl Methacrylate (MMA), Polydimethylsiloxane (PDMS) macromer, poly (vinyl pyrrolidone) (PVP), poly (p-xylylene) (also known as parylene), polyvinyl alcohol (PVA) hydrogel, and combinations thereof.

74. The retinal implant of claim 72, wherein the base layer is less than 100 μm thick and contains a uniformly distributed array of pores less than about 100 μm in diameter.

75. The retinal implant according to any one of claims 41 to 43, wherein the microbial rhodopsin-based retinal implant is based on native microbial rhodopsin.

76. The retinal implant according to any one of claims 41 to 43, wherein the microbial rhodopsin-based retinal implant is based on a microbial rhodopsin mutant.

77. The retinal implant of claim 76, wherein the mutant is a Q-state mutant.

78. The retinal implant of claim 77, wherein the mutant has been optimized to have a high dipole moment.

79. The retinal implant of claim 77, wherein the mutant has been optimized so as to form a high-efficiency Q-state.

Technical Field

Protein-based subretinal implants provide a new method of restoring vision in patients who are blind due to age-related macular degeneration (AMD) and Retinitis Pigmentosa (RP). The present invention relates to bacteriorhodopsin-based (BR-based) retinal implants that can be implanted in a subretinal location to replace degenerated photoreceptor cells.

Background

The retina is a multi-layered tissue lining the concave inner surface of the back of the eye. The photoreceptor cells inside the retina are activated by light entering the eye and convert the light signals into electrochemical signals, which are transmitted to the retinal nerve cells. The retinal nerve cells accordingly transmit signals to the visual center of the brain via the optic nerve, thereby enabling the brain to perceive visual images. Photoreceptor cells are broadly classified into rods and cones, and these cells are named for their shape. However, the cone cells contain the photopigments necessary for color vision, and the rod cells contain the photopigment rhodopsin, which is highly sensitive to light, thus enabling vision under dim light conditions (e.g., night conditions). Rod cells are sensitive enough to be activated by a single photon, whereas cone cells require tens to hundreds of photons to be activated.

When activated by photons, rhodopsin (which is the photopigment of rod cells) undergoes a conformational change. Rhodopsin consists of a seven-transmembrane protein called opsin, which is covalently bound to a prosthetic group called retinal, which is a derivative of vitamin a. Unactivated retinal exists in the 11-cis form, however, light stimulation causes conformational changes to the all-trans form. Conformational changes in the retina cause covalent bonds to bind corresponding conformational changes in the opsin polypeptide, thereby triggering a second messenger cascade within the photoreceptor cell that results in the signal being transmitted to the appropriate retinal nerve cells. These signals are transmitted along the optic nerve to the visual center of the brain, which enables the brain to process visual inputs and perceive visual images.

Various diseases and conditions that destroy retinal photoreceptor cells can result in partial or complete loss of vision. Two major diseases of the retina are age-related macular degeneration (AMD) and Retinitis Pigmentosa (RP). As a major cause of vision loss and blindness in the elderly, AMD causes degeneration of rod and cone photoreceptors located inside the macula at the center of the retina. In addition, AMD can affect central vision and thus cause difficulties in reading, driving, and other tasks requiring high contrast vision.

Another disease, RP, is a genetic disease in which rod degeneration occurs, leading to loss of vision and blindness. Loss of rod cells impairs visual ability in dim light and gradually reduces peripheral vision until the patient appears with tunnel vision and eventual blindness.

Many artificial retinal prototypes have been studied to date for the treatment of such retinal diseases and disorders, but each prototype has significant disadvantages. One of the most promising designs is the pre-retinal implant biochemical eye system (ArgusII), which was designed by researchers at the university of California, south and sold by Second Sight, Inc. The implant uses an external camera mounted on a pair of glasses connected to a microelectrode array by a connection cable. The electrode array provides electrical stimulation directly to the ganglion cells. In clinical trials, subjects were able to sense light with an 8 x 8 electrode array and were able to detect motion and recognize simple shapes. However, this design has the significant disadvantage that it requires external hardware, such as glasses and surgically implanted external devices. Furthermore, implants provide insufficient low resolution to restore functional vision, and the surgical complexity required for implantation limits the accessibility of the retinal surgeon population.

What is needed is a surgically less traumatic high resolution retinal implant that can at least partially restore vision quality in patients with vision loss due to photoreceptor cell loss as a result of retinal disease or injury.

In view of the foregoing, it is apparent that there is a continuing need to develop safe and effective methods of treating vision loss.

Disclosure of Invention

The protein-based subretinal implants of the present invention provide a novel method of restoring vision in patients who are blind due to AMD and RP. The ability of this ion-mediated protein-based implant to stimulate bipolar cells and ganglion cells of degenerated retina was demonstrated using ex vivo extracellular recordings of transgenic rats using P23H.

The present invention provides an implant that can operate at high resolution and can be activated by light conditions similar to indoor ambient lighting, such as a high pixel resolution approach for stimulating retinal cells and treating vision loss.

Drawings

Figure 1 shows the application of a protein-based retinal implant. Retinal degenerative diseases (including AMD and RP) are characterized by the loss of photoreceptor cells within the retina. Individuals affected by these diseases lose vision and eventually blindness. Bacteriorhodopsin-based (BR-based) retinal implants replace degenerated photoreceptor cells and are placed under the retina. Upon absorption of light, the implant creates an ion gradient that stimulates the rest of the retinal tissue. See Wangner, N.L.et al.InBionanotechnology: Biological Self-Assembly and its Applications; rehm, ed.Caister Academic Press, Norfolk, UK 2013, 205-; greco, j.a.et al.int.j.highspeed electron.syst.2017,26,1740012-1-17400120-20; and Chen, z.; big, R.R.trends Biotech.1993,11, 292-300.

Fig. 2 shows a schematic view of a retinal implant, and a diagram showing the arrangement of alternating layers of BR and polycation. See He, J. -A.et al.1998,14, 1674-. Protein-based retinal implants are based on electrostatic interactions between ion-permeable membranes, polycations and BR. The resulting multilayer film produces a unidirectional ionic gradient for stimulating degenerated retinal tissue.

Fig. 3 shows the absorption spectra of P23H rat green cone pigment and photoadaptive bacteriorhodopsin [ br (la) ], compared to the output from the three LEDs used in the experiment. Note that the red LED combines with the br (la) absorption spectrum with reasonable efficiency, but poorly with the rat green cone. Since rats have no red cones and P23H rat retinas degenerate, we can selectively activate the implant with a red LED.

Fig. 4A and 4B show the activation efficiency of P23H rat retinas in response to photoactivation of ion-mediated retinal implants. Fig. 4A demonstrates that retinal implants with high Optical Density (OD) when oriented to pump protons toward the retina (traces with closed squares and triangles, upper trace in fig. 4A and lower trace in fig. 4B) are capable of stimulating retinal tissue with high efficiency. If a lower OD implant is used (traces with open squares and triangles, lower trace in fig. 4A and upper trace in fig. 4B), the efficiency decreases significantly. When the implant orientation is reversed to pump protons away from the retina, as shown in fig. 4B, less to no activity is detected. The square and triangular data points refer to measured action potentials having positive and negative amplitudes, respectively. All data used a 30 μ V threshold for analysis.

Fig. 5A, 5B, 5C, 5D, 5E and 5F show the latency distribution of measured RGCs in P23H rats after photoactivation of the retinal implant. Fig. 5A, 5C and 5E correspond to the case where the implant is oriented to pump protons toward neural tissue ([ H)⁺]Increased). Fig. 5B, 5D and 5F are for comparison, where the implant is placed in the opposite orientation ([ H ])⁺]Decrease). The y-axis is adjusted so that it is the average number of observations per 100 scans. The intensity of the incident light (630nm, 1ms pulse) increased from top to bottom (5A/5B: 20.5 mW/cm)²；5C/5D：29.5mW/cm²；5E/5F：49.89mW/cm²)。

Fig. 6A and 6B show schematic diagrams of a Multiple Electrode Array (MEA) for extracellular registration experiments quantifying the spatial resolution of protein-based retinal implants (fig. 6A). Selective activation of electrode 5 in row E of an 8 x 8MEA is also shown (fig. 6B). The light induced action potential is limited to the RGC measured by the electrodes 5 inside the array, while all other signals are due to spontaneous activity. The incident beam has a diameter of 200 μm and is centered on the electrode 5. The y-axis is the normalized amplitude of the radiation beam and the normalized time range spanning 110.2 s.

Fig. 7A and 7B show a comparison of the absorption spectra of P23H rat green cones (fig. 7A) and light-adapted BR (BR; fig. 7B) with the output from a projector using either green or red interference filters employed. The coupling efficiency of the light source to the spectrum is shown on the right side of the figure. The red interference filter can be seen to couple with the bR absorption spectrum, but much less efficiently with the rat green cone pigment.

Fig. 8A, 8B and 8C show targeted irradiation of P23H rat retinas centered on a single electrode inside the MEA array. Fig. 8A, 8B and 8C correspond to selective activation of electrodes E3, E4 and E5, respectively. The upper graph in each plate illustrates the percentage of signal from each electrode after irradiation and each graph is normalized to the maximum value. The lower graph in each plate shows the position and relative beam spot size.

Detailed Description

The present invention relates to a method for treating a patient with vision loss due to retinal cell loss, comprising:

(a) implanting a bacteriorhodopsin-based retinal implant into the eye of a patient, and

(b) the implant is activated with a light source and,

wherein the implant stimulates retinal cells to provide stimulation at a resolution of less than about 500 μm, or less than about 350 μm, or less than about 250 μm, or less than about 200 μm, or less than about 150 μm, or less than about 100 μm, or less than about 75 μm, or less than about 50 μm, or less than about 40 μm, or less than about 30 μm, or less than about 25 μm in pixel size.

In another aspect, the invention relates to a method for treating a patient with vision loss due to retinal cell loss, comprising:

(a) implanting a bacteriorhodopsin-based retinal implant into the eye of a patient, and

(b) the implant is activated with a light source and,

wherein the implant stimulates retinal cells to provide stimulation at a resolution limited by: (i) the diffraction limit of the light source; or (ii) the pore size and density of the ion-permeable substrate (scaffold) when the retinal implant further comprises the ion-permeable substrate (scaffold).

In another aspect, the invention relates to a method of treating a patient with vision loss due to retinal cell loss, comprising:

(a) implanting a bacteriorhodopsin-based retinal implant into the eye of a patient, and

(b) the implant is activated with a light source and,

wherein the implant stimulates retinal cells to a concentration of about 4 pixels/mm²And a pixel size of about 500 μm, or about 8 pixels/mm²And a pixel density of about 350 μm in pixel diameter, or about 16 pixels/mm²And a pixel density of about 250 μm in pixel diameter, or about 25 pixels/mm²And a pixel diameter of about 200 μm, or about 44 pixels/mm²And a pixel diameter of about 150 μm, or about 100 pixels/mm²And a pixel diameter of about 100 μm, or about 178 pixels/mm²And a pixel diameter of about 75 μm, or about 400 pixels/mm²And a pixel diameter of about 50 μm, or about 625 pixels/mm²Has a pixel density of about 40 μm and a pixel diameter of about 1111 pixels/mm, or has a pixel diameter of about 1111 pixels/mm²And a pixel diameter of about 30 μm, or about 1600 pixels/mm²And a resolution of about 25 μm pixel diameter provides the stimulus.

In another aspect, the invention relates to a method wherein the microbial rhodopsin-based retinal implant comprises at least one matrix layer (scaffold) and a microbial rhodopsin membrane, wherein the microbial rhodopsin membrane comprises a plurality of individual layers of native microbial rhodopsin or a mutant of microbial rhodopsin, wherein the individual layers of native microbial rhodopsin or mutant of microbial rhodopsin alternate with a cationic polymeric layer, and wherein the microbial rhodopsin mutant is selected from the group consisting of a chloride-ion pump mutant, a dipole mutant, a light cycle mutant, a gold-binding mutant, an ion pump mutant, a Q-state mutant, and combinations thereof.

In another aspect, the invention relates to a method wherein the light source is a light source emitting at a human visible wavelength, i.e., about 400 to about 700 nm.

In another aspect, the present invention relates to a method wherein the intensity (power density) of the light source is less than about 100mW/cm²、50mW/cm²、40mW/cm²Or less than about 30mW/cm²Or less than about 20mW/cm²Or less than about 10mW/cm²Or less than about 5mW/cm²Or less than about 1mW/cm²。

In another aspect, the invention relates to a method wherein the light source is a pulsed light source.

In another aspect, the invention relates to a method wherein the light source is a continuous light source.

In another aspect, the invention relates to a method wherein the implant is a biocompatible, ion-permeable retinal implant.

In another aspect, the invention relates to a method wherein the implant is made from a layer-by-layer assembly.

In another aspect, the invention relates to a method wherein the implant is implanted in a location selected from a subretinal location or a preretinal location.

In another aspect, the invention relates to a method wherein the implant is implanted in a subretinal location.

In another aspect, the present invention is directed to a method 11 wherein an implant is implanted in a pre-retinal location.

In another aspect, the invention relates to a method wherein the retinal cell is selected from the group consisting of a retinal ganglion cell, a retinal bipolar cell, a retinal photoreceptor cell, and combinations thereof.

In another aspect, the invention relates to a method wherein the retinal cells are selected from retinal ganglion cells.

In another aspect, the invention relates to a method wherein the retinal cells are selected from retinal bipolar cells.

In another aspect, the invention relates to a method wherein the retinal cells are selected from retinal photoreceptor cells.

In another aspect, the invention relates to a method wherein the retinal implant comprises at least about 100 layers of native microbial rhodopsin or mutant microbial rhodopsin, or at least 1 about 50 layers of native microbial rhodopsin or mutant microbial rhodopsin, or at least about 200 layers of native microbial rhodopsin or mutant microbial rhodopsin.

In another aspect, the invention relates to a method wherein the retinal implant further provides a temporal resolution of at least about 30 milliseconds.

In another aspect, the invention relates to a method wherein the retinal implant further provides a temporal resolution of at least about 20 milliseconds.

In another aspect, the invention relates to a method wherein the retinal implant further provides a temporal resolution of at least about 10 milliseconds.

In another aspect, the invention relates to a method wherein the retinal implant elicits (provides) an action potential of about 40 μ V to the cells.

In another aspect, the invention relates to a method wherein the retinal implant elicits (provides) an action potential of about 30 μ V to the cells.

In another aspect, the invention relates to a method wherein the retinal implant elicits (provides) an action potential of about 20 μ V to the cells.

In another aspect, the invention relates to a method wherein the retinal implant provides a unidirectional proton gradient.

In another aspect, the invention relates to a method wherein the unidirectional proton gradient is directed to a retinal cell, wherein the retinal cell is selected from a retinal ganglion cell or a retinal bipolar cell.

In another aspect, the invention relates to a method wherein the implant is implanted in a location selected from a subretinal location or a preretinal location.

In another aspect, the invention relates to a method wherein the microbial rhodopsin-based retinal implant comprises microbial rhodopsin molecules such that they are consistently (equally) oriented.

In another aspect, the invention relates to a method wherein the retinal implant is used at or below physiologic temperatures (37℃.).

In another aspect, the invention relates to a method wherein the retinal implant is used at physiological pH values (about 7.0 to about 7.2).

In another aspect, the invention relates to a method wherein the loss of vision is caused by loss of retinal photoreceptor cells.

In another aspect, the invention relates to a method wherein the substrate layer is an ion permeable layer and is on one or both sides of the implant.

In another aspect, the invention relates to a method wherein the substrate layer is selected from the group consisting of polyethylene terephthalate (PET), 2-hydroxyethyl methacrylate (HEMA), 2-phenylethyl methacrylate (PEM), polyethylene glycol (PEG), Methyl Methacrylate (MMA), Polydimethylsiloxane (PDMS) macromer, poly (vinyl pyrrolidone) (PVP), poly (p-xylylene) (also known as parylene), polyvinyl alcohol (PVA) hydrogel, and combinations thereof.

In another aspect, the invention relates to a method wherein the base layer (scaffold) has a thickness of less than 100 μm and contains a uniformly distributed array of pores having diameters of less than about 100 μm.

In another aspect, the invention relates to a method wherein the microbial rhodopsin-based retinal implant is based on native microbial rhodopsin.

In another aspect, the invention relates to a method wherein the microbial rhodopsin-based retinal implant is based on a microbial rhodopsin mutant.

In another aspect, the invention relates to a method wherein the mutant is a Q-state mutant.

In another aspect, the invention relates to a method wherein the mutant has been optimized to have a high dipole moment.

In another aspect, the invention relates to a method wherein the mutant has been optimized so as to form a highly efficient Q state.

In another aspect, the invention relates to a method for stimulating retinal cells, comprising:

(a) contacting retinal cells with a microbial rhodopsin-based retinal implant, and

(b) the implant is activated with a light source and,

wherein

(i) The implant stimulates the retinal cells to provide stimulation at a resolution of pixel size less than about 500 μm, or less than about 350 μm, or less than about 250 μm, or less than about 200 μm, or less than about 150 μm, or less than about 100 μm, or less than about 75 μm, or less than about 50 μm, or less than about 40 μm, or less than about 30 μm, or less than about 25 μm; or

(ii) Wherein the implant stimulates retinal cells to provide stimulation at a resolution limited by: (i) the diffraction limit of the light source; or (ii) when the retinal implant further comprises an ion-permeable substrate (scaffold), the pore size and density of the ion-permeable substrate (scaffold); or

(iii) Wherein the implant stimulates retinal cells to have a size of about 4 pixels/mm²And a pixel size of about 500 μm, or about 8 pixels/mm²And a pixel density of about 350 μm in pixel diameter, or about 16 pixels/mm²And a pixel density of about 250 μm in pixel diameter, or about 25 pixels/mm²And a pixel density of about 200 μm in pixel diameter, or about 44 pixels/mm²And a pixel density of about 150 μm, or about 100 pixels/mm²And a pixel diameter of about 100 μm, or about 178 pixels/mm²And a pixel density of about 75 μm in pixel diameter, or about 400 pixels/mm²And a pixel diameter of about 50 μm, or about 625 pixels/mm²Pixel density ofAnd a pixel diameter of about 40 μm, or about 1111 pixels/mm²And a pixel density of about 30 μm in pixel diameter, or about 1600 pixels/mm²And a resolution of about 25 μm pixel diameter provides the stimulus.

In another aspect, the invention relates to a retinal implant for treating a patient with vision loss due to retinal cell loss, comprising a retinal implant that stimulates retinal cells to provide stimulation at a resolution having a pixel size of less than about 500 μm, or less than about 350 μm, 250 μm, or less than about 200 μm, or less than about 150 μm, or less than about 100 μm, or less than about 75 μm, or less than about 50 μm, or less than about 40 μm, or less than about 30 μm, or less than about 25 μm.

In another aspect, the present invention relates to a retinal implant for treating a patient with vision loss due to retinal cell loss, comprising a retinal implant that stimulates retinal cells to provide stimulation at a resolution limited by: (i) the diffraction limit of the light source; or (ii) the pore size and density of the ion-permeable substrate (scaffold) when the retinal implant further comprises the ion-permeable substrate (scaffold).

In another aspect, the invention relates to a retinal implant for treating a patient with vision loss due to retinal cell loss, comprising stimulating retinal cells to have about 4 pixels/mm²And a pixel size of about 500 μm, or about 8 pixels/mm²And a pixel density of about 350 μm in pixel diameter, or about 16 pixels/mm²And a pixel density of about 250 μm in pixel diameter, or about 25 pixels/mm²And a pixel density of about 200 μm in pixel diameter, or about 44 pixels/mm²And a pixel density of about 150 μm, or about 100 pixels/mm²And a pixel diameter of about 100 μm, or about 178 pixels/mm²And a pixel density of about 75 μm in pixel diameter, or about 400 pixels/mm²And a pixel diameter of about 50 μm, or about 625 pixels/mm²And a pixel density of about 40 μm in pixel diameter, or about 1111 pixels/mm²And a pixel diameter of about 30 μm, or about 1600 pixels/mm²And a resolution of about 25 μm pixel diameter provides a stimulating retinal implant.

In another aspect, the present invention relates to a retinal implant, wherein the microbial rhodopsin-based retinal implant comprises at least one matrix layer (scaffold) and a microbial rhodopsin membrane, wherein the microbial rhodopsin membrane comprises a plurality of individual layers of native microbial rhodopsin or a mutant of microbial rhodopsin, wherein the individual layers of native microbial rhodopsin or mutant of microbial rhodopsin alternate with a layer of a cationic polymer, and wherein the mutant of microbial rhodopsin is selected from the group consisting of chloride ion pump mutants, dipole mutants, light cycle mutants, gold binding mutants, ion pump mutants, Q state mutants, and combinations thereof.

In another aspect, the present invention relates to a retinal implant wherein the light source is a light source emitting at a human visible wavelength, i.e., about 400 to about 700 nm.

In another aspect, the present invention relates to a retinal implant wherein the intensity (power density) of the light source is less than about 100mW/cm²、50mW/cm²、40mW/cm²Or less than about 30mW/cm²Or less than about 20mW/cm²Or less than about 10mW/cm²Or less than about 5mW/cm²Or less than about 1mW/cm²。

In another aspect, the invention relates to a retinal implant in which the light source is a pulsed light source.

In another aspect, the invention relates to a retinal implant in which the light source is a continuous light source.

In another aspect, the present invention relates to a retinal implant, wherein the implant is a biocompatible, ion-permeable retinal implant.

In another aspect, the present invention relates to a retinal implant, wherein the implant is made from a layer-by-layer assembly.

In another aspect, the invention relates to a retinal implant, wherein the implant is implanted in a location selected from a subretinal location or a preretinal location.

In another aspect, the invention relates to a retinal implant, wherein the implant is implanted in a subretinal location.

In another aspect, the invention relates to a retinal implant, wherein the implant is implanted in a pre-retinal location.

In another aspect, the invention relates to a retinal implant, wherein the retinal cells are selected from the group consisting of retinal ganglion cells, retinal bipolar cells, retinal photoreceptor cells, and combinations thereof.

In another aspect, the invention relates to a retinal implant, wherein the retinal cells are selected from retinal ganglion cells.

In another aspect, the invention relates to a retinal implant wherein the retinal cells are selected from retinal bipolar cells.

In another aspect, the invention relates to a retinal implant, wherein the retinal cells are selected from retinal photoreceptor cells.

In another aspect, the invention relates to a retinal implant, wherein the retinal implant comprises at least about 100 layers of native microbial rhodopsin or mutant microbial rhodopsin, or at least about 150 layers of native microbial rhodopsin or mutant microbial rhodopsin, or at least about 200 layers of native microbial rhodopsin or mutant microbial rhodopsin.

In another aspect, the present invention relates to a retinal implant, wherein the retinal implant further provides a temporal resolution of at least about 30 milliseconds.

In another aspect, the present invention relates to a retinal implant, wherein the retinal implant further provides a temporal resolution of at least about 20 milliseconds.

In another aspect, the present invention relates to a retinal implant, wherein the retinal implant also provides a temporal resolution of at least about 10 milliseconds.

In another aspect, the present invention relates to a retinal implant, wherein the retinal implant elicits (provides) an action potential of about 40 μ V to cells.

In another aspect, the present invention relates to a retinal implant, wherein the retinal implant elicits (provides) an action potential of about 30 μ V to cells.

In another aspect, the invention relates to a retinal implant, wherein the retinal implant elicits (provides) an action potential of about 20 μ V to cells.

In another aspect, the present invention relates to a retinal implant, wherein the retinal implant provides a unidirectional proton gradient.

In another aspect, the invention relates to a retinal implant in which a unidirectional proton gradient is directed to retinal cells, wherein the retinal cells are selected from retinal ganglion cells or retinal bipolar cells.

In another aspect, the invention relates to a retinal implant, wherein the implant is implanted in a location selected from a subretinal location or a preretinal location.

In another aspect, the invention relates to a retinal implant, wherein the microbial rhodopsin-based retinal implant comprises microbial rhodopsin molecules such that they are consistently (equally) oriented.

In another aspect, the invention relates to a retinal implant, wherein the retinal implant is used at or below physiological temperatures (37 ℃).

In another aspect, the present invention relates to a retinal implant, wherein the retinal implant is used at physiological pH values (about 7.0 to about 7.2).

In another aspect, the invention relates to a retinal implant in which vision loss is caused by loss of retinal photoreceptor cells.

In another aspect, the invention relates to a retinal implant in which the basal layer is an ion-permeable layer and is on one or both sides of the implant.

In another aspect, the present invention relates to a retinal implant wherein the substrate layer is selected from the group consisting of polyethylene terephthalate (PET), 2-hydroxyethyl methacrylate (HEMA), 2-phenylethyl methacrylate (PEM), polyethylene glycol (PEG), Methyl Methacrylate (MMA), Polydimethylsiloxane (PDMS) macromer, poly (vinyl pyrrolidone) (PVP), poly (p-xylylene) (also known as parylene), polyvinyl alcohol (PVA) hydrogel, and combinations thereof.

In another aspect, the invention relates to a retinal implant in which the basal layer (scaffold) is less than 100 μm thick and contains an array of uniformly distributed pores less than about 100 μm in diameter.

In another aspect, the invention relates to a retinal implant, wherein the microbial rhodopsin-based retinal implant is based on natural microbial rhodopsin.

In another aspect, the invention relates to a retinal implant, wherein the microbial rhodopsin-based retinal implant is based on a microbial rhodopsin mutant.

In another aspect, the invention relates to a retinal implant, wherein the mutant is a Q-state mutant.

In another aspect, the invention relates to a retinal implant in which the mutant has been optimized to have a high dipole moment.

In another aspect, the invention relates to a retinal implant in which the mutant has been optimized to form a highly efficient Q state.

These and other aspects of the invention will become apparent based on the disclosure herein.

Bacteriorhodopsin-based retinal implants:

the retinal implant of the present invention (fig. 1) uses a light activated protein (bacteriorhodopsin (BR)) as a photoactive medium to absorb incident light and induce proton dynamics to activate other neural circuits of the degenerated retina to stimulate visual response. See, berge, r.r.; nolenberger, m.ranaghan, m.j.; sandberg, d.j.; wagner, nicolel.protein-Based Artificial Retinas, U.S. patent 8,563,026; and Chen, z.; big, R.R., Protein Based arthritis trends Biotech.1993,11,292- "300. Bacteriorhodopsin is a very excellent candidate for this use, since the proton pumping function of proteins works with the same high quantum efficiency (65%) as the natural visual pigment rhodopsin. The protein also functions at high temperatures (>80 ℃), high light intensities, and high salt environments. The structure of the retinal implant allows the optical density of the protein to absorb a sufficient amount of incident light to create an ion gradient for retinal stimulation. This performance characteristic is facilitated by a layer-by-layer (LBL) electrostatic adsorption process for forming highly uniform thin films. The result is a membrane that can produce a unidirectional proton gradient. An automated LBL system was developed for this bottom-up fabrication method to form multi-layered implants. The high optical density of retinal implants circumvents the expression level problems inherent with gene therapy and optogenetic methods that attempt to express similar light sensitive proteins for light capture in the degenerated retina. Ex vivo extracellular recording experiments using P23H transgenic rats were used to demonstrate the ability of these ion-mediated protein-based repair procedures to stimulate bipolar cells and ganglion cells of degenerating the retina.

Molecular packing of BR proteins within the layers of the retinal implant structure results in high pixel density, which results in spatial resolution limited only by diffusion rate and lateral diffusion of protons through the ion-permeable scaffold. Importantly, the retinal implant is powered by incident light, which is an advantage over other electronic type implants that require external cables, electrodes, or power sources. Thus, the retinal implants of the present invention are less traumatic during surgery, which greatly reduces the time required for implantation and the chance of infection due to mechanical fixation. The implant will also allow the patient to scan the view with natural eye movement and provide normal visual perception without signal transmission delay.

Construction and design of multilayer films: the BR-based retinal implant of the present invention (fig. 2) was composed of a multilayer BR film formed by sequential LBL electrostatic adsorption using a custom LBL impregnator. LBL deposition of BR Using commercially available ion-permeable microfibers (trade name: BR)

) As a scaffold in retinal implants.

Has been successfully applied to the eye and can be used to directly bind proteinsIs attached and oriented on a surface. To utilize the ability of BR to generate a sufficient pH gradient to stimulate the retina, the protein is present at an optimal density

The surface regions are uniformly oriented and contain enough BR layer to adequately absorb the incident light while also forming a pH gradient. Furthermore, we have found it advantageous to provide the brackets with a suitable dimensional spacing, as described below.

Stability and biocompatibility of retinal implants: the long-term stability of BR to thermal and photochemical degradation is well documented in the literature. Proteins can withstand temperatures above 80 ℃, which far exceed normal human body temperatures. Furthermore, the cyclicity of the protein (i.e., the number of photochemical cycles that the protein can circulate before the sample degrades by 1/e) is approximately 106. This value is much greater than most synthetic photochromic materials. This stability is achieved by protein separation within the two-dimensional lattice of trimers (purple membrane) surrounded by a natural lipid membrane. The layer-by-layer (LBL) assembled retinal implants are held together by highly efficient electrostatic interactions that bind BR to polycations.

Overview of the mechanism of action: microbial rhodopsins are used as potential light emitting tools for the treatment of inherited retinal diseases. The luminescence method uses light-activated proteins expressed in AAV towards bipolar cells and ganglionic cells, including rhodopsin channel protein-2 (ChR-2) and bacteriorhodopsin (hR), instead of degenerating the photosensitivity of photoreceptor cells. These therapies take advantage of the fact that the neural network remains substantially intact for both RP and AMD, despite the loss of photoreceptor cells. Theoretical models have been used to demonstrate altered Na surrounding retinal cells⁺、K⁺And Cl^-Ion concentrations of ions can trigger nerve impulses, and light activated ion channels and agents such as ChR-2 (Na) can be used⁺/K⁺Channels) and HR (Cl)^-Pump) depolarizes and hyperpolarizes these nerve cells. Retinal Ganglion Cells (RGCs) contain epithelial Na embedded in the nerve cell membrane⁺Proton gated analogs of channels. Therefore, proton pumps (e.g., BR) have also been considered as optogenetic tools for restoring vision in patients with retinal degeneration. Lilley et al measured the threshold for proton induced retinal stimulation (-pH 6.5) and repeatedly demonstrated that a rapid millisecond pulse of extracellular acidification elicits a normal electrical activity pattern of RGCs. See Lilley, s.; LeTissier, p.; robbins, J., The Discovery and Characterization of a Proton-Gated Sodium Current in Ratretin Ganglion cells, J.Neurosci.2004,24, 1013-1022; lilley, s.; robbins, J., Characterisation of a Novel Proton-Gated Sodium Current cells J.Physiol.2001,531, 83P-84P. The optical cycle duration of BR of about 10ms and the ability to modulate local acidity make BR another viable candidate for ion-mediated stimulation of the retina. The present invention takes advantage of this principle by creating a stable biocompatible implantable prosthesis that functions with the same efficiency as natural pigments in photoreceptor cells.

The BR-based retinal implant of the present invention employs in part the same principles as ion-mediated optogenetic therapy, however this implant has some key advantages of a prosthesis that overcomes the inherent disadvantages of gene therapy approaches. First, RPE65 gene mutations, which are the subject of mainly optogenetic therapy, affect only about 6000 people worldwide, whereas the present invention has the potential to treat the 150 million RP population worldwide, which is affected by various gene mutations. In addition, optogenetic tools such as ChR-2 have λ at the blue edge of the visible spectrum (470 nm)_maxAnd BR has a width λ at 570nm_maxλ of the_maxSpanning a substantial central portion of the visible spectrum. This feature provides greater sensitivity without relying on shorter wavelengths adjacent to the UV region of the spectrum. Finally, the expression levels of non-native ChR-2 and HR proteins can be limited and unstable, resulting in the need for a dangerously high intensity light source to activate targeted nerve cells. Multilayer protein-based retinal prostheses circumvent this problem by using hundreds of layers of BR, which requires lower light intensity to create sufficient ion gradients.

Pixel modulation using Q-state mutants: in addition to the definition of proton pumps of light-activated proteinsIn addition to photochemical transport mechanisms, BR can also enter branched photo-cycles via sequential two-photon processes. The Q state contains 9-cis retinal color bodies (as opposed to all-trans color bodies in the BR resting state) which are hydrolyzed by proteins and produce an isolated blue-shifted photoproduct that is stable at ambient temperatures for about several years. See, berge, r.r.; gillespie, n.b.; izaguirre, e.w.; kusnetzow, a.; lawrence, a.f.; singh, d.; song, q.w.; schmidt, e.; stuart, j.a.; seetharaman, S.et al, Biomolecular Electronics: Protein-Based Association processors and Volumetric memories.J.Phys.chem.B 1999,103, 10746-10766. The Q state prevents the protein from undergoing light-induced light cycling, which provides a means to convert active pixels in protein-based retinal implants to inactive pixels. This feature may be advantageous for adjusting the pixel density of the implant before or after surgical implantation and for nullifying specific areas to prevent interference with the photoreceptor cells. The natural wild-type protein does not efficiently obtain this photoproduct, and various mutants of BR have been identified that provide greater availability and stability of the Q state. Large, r.r.; rangiajan, r.; McCleary, k.n. Bacteriorhodopsin protein variants and methods for long-term data storage (Bacteriorhodopsin protein variants and methods of use for long term datastore) us patent 8883719. The retinal implants described in the present invention can be made identically using either the wild-type form of the protein or using Q-state mutants, and the functions and stimulation methods described in the present invention can be modulated based on this unique photochemistry.