Methods of increasing the number of ocular cells

Abstract

Described are methods of increasing the number of ocular cells, such as corneal endothelial cells or retinal pigment epithelial cells, in a subject. The methods include applying ultrasonic energy to the target ocular area (e.g., endothelium) of the subject's eye, followed by contacting the target ocular area of the eye with a plurality of ocular cells, such as, corneal endothelial cells or retinal pigment epithelial cells. Also described is a system for ocular cell delivery in a subject with increased integration into the target ocular area.

Description

METHODS OF INCREASING THE NUMBER OF OCULAR CELLSRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application 63 / 636,649, filed April 19, 2024, to Emmetrope Ophthalmics LLC, titled “METHODS OF INCREASING THE NUMBER OF OCULAR CELLS,” the entirety of the disclosure of which is hereby incorporated by this reference.TECHNICAL FIELD

[0002] The subject matter disclosed herein is generally directed to methods of increasing the number of ocular cells in a subject comprising application of ultrasonic energy to a target ocular tissue in a subject comprising application of ultrasonic energy to the target ocular tissue of the subject followed by contacting the target ocular tissue with a plurality of ocular cells.BACKGROUND

[0003] Ultrasound energy is used to emulsify cataract during cataract surgery. The use of ultrasound, while removing the cataract, can cause a significant loss of corneal endothelial cells. In some cases, the loss of corneal endothelial cells can be about 10%, about 15% or about 20% of the corneal endothelial cells present prior to cataract surgery.

[0004] Delivery of cells for use in regeneration of cells within the eye is problematic in the art. Although cell therapy for ocular regeneration, such as cell injection for endothelial cell dysfunction and stem cell transplantation for retinal neuroprotection have been studied, these procedures are challenging technically, have varying rates of success, and are not yet commercially available for many types of ocular diseases or disorders, including diseases of the cornea (including but not limited to endothelial dystrophies), diseases of retinal ganglion cells and the optic nerve (including but not limited to glaucoma, ischemic optic neuropathies, other optic neuropathies), and diseases of retinal photoreceptors and retinal pigment epithelium (including but not limited to Leber’s congenital amaurosis, retinitis pigmentosa and age-related macular degeneration).

[0005] Unlike cell transplantation therapy in other organs, simply injecting therapeutic cells or even other therapeutic agents into the eye generally does not work as they do not remain localized and stick to or become incorporated into the patient’s tissue. For example, healthy corneal endothelial cells are inefficiently incorporated in a pre-clinical model of corneal endothelial dysfunction when those cells are injected into the anterior chamber of the eye (e.g.,Mimura et al., Invest. Ophthalmol. Vis. Sci. 2005, 46(10): 3637-44). Similarly, healthy retinal ganglion cells are not incorporated into the correct retinal layer by simple injection into a cavity of the eye. Most current cell therapy technology for the eye lack techniques for controlling the cells’ localization in vivo. A stem cell transplantation clinical trial for retinitis pigmentosa uses subretinal injection of hematopoietic stem cells with no mechanism for controlling the localization of the stem cells to the subretinal space and to keep them from floating or migrating away in the fluid of the eye. As another example, corneal endothelial cells injected into the anterior chamber of the eye will simply fall by gravity away from the cornea and not properly attach unless they are co-injected with a molecule that promotes cell adhesion (Kinoshita et al., N Engl J Med. 2018, 378(11): 995-1003).

[0006] There is a need for methods for increasing the number of ocular cells following a surgical procedure, such as corneal endothelial cells following cataract surgery.

[0007] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.SUMMARY

[0008] Described herein are methods of increasing the number of ocular cells (e.g., corneal endothelial cells or retinal pigment epithelial cells), in a subject comprising application of ultrasonic energy to a target ocular tissue in a subject comprising application of ultrasonic energy to the target ocular tissue of the subject followed by contacting the target ocular tissue with a plurality of ocular cells.

[0009] Also described herein are methods of increasing the number of corneal endothelial cells in a subject comprising application of ultrasonic energy to the corneal endothelium of the subject followed by contacting the endothelium with a plurality of corneal endothelial cells. Other aspects concern corneal endothelial cells for use in increasing the number of corneal endothelial cells in the corneal endothelium following application of ultrasonic energy to the corneal endothelium.

[0010] In some embodiments, the application of ultrasonic energy is associated with cataract surgery. In certain embodiments, the subject suffers from decreased corneal endothelial cells. The decreased corneal endothelial cells may result from a disease such as Fuch’s endothelial corneal dystrophy (FECD), posterior polymorphous corneal dystrophy (PPCD), phakic or pseudophakic bullous keratopathy (ABK / PBK), blunt trauma endothelial dysfunction, congenital hereditary endothelial dystrophy (CHED), iridocorneal endothelial(ICE) syndrome, refractory glaucoma, or any other condition that results in decreased corneal endothelial cells.

[0011] The contacting of the endothelium with a plurality of corneal endothelial cells may occur thirty seconds to seven days after the application of ultrasonic energy.

[0012] The ultrasonic energy is applied to the endothelium for a time and dose sufficient to induce integration of ocular cells.

[0013] In some embodiments, the corneal endothelial cells comprise magnetic nanoparticles having a mean diameter of no more than 200 nm affixed, bound, or attached to a surface of the corneal endothelial cells. The magnetic nanoparticle may be affixed to the surface of the corneal endothelial cells by means of an antibody. Some magnetic nanoparticles comprise iron in any ferromagnetic form. Some magnetic nanoparticles have a diameter of less than 100 nm.

[0014] In certain embodiments, the corneal endothelial cells comprising magnetic particles are positioned on the corneal endothelium using magnetic field(s). In some embodiments, the magnetic field is supplied by a plurality of electromagnets.

[0015] In some embodiments, the corneal endothelial cells adhere to the corneal endothelium using an adhesion promoter such as additional drug treatments given with the endothelial cells or given separately from the endothelial cells. In some embodiments, when the adhesion promoter is provided separately from the endothelial cells, the adhesion promoter is provided concurrently with the endothelial cells. In other embodiments, the adhesion promoter is provided after the endothelial cells have been delivered.

[0016] The subject is a human in some embodiments. The corneal endothelial cells are human in certain embodiments.

[0017] Some corneal endothelial cells are derived from stem cells or progenitor cells. Some corneal endothelial cells are derived from any cell line with tight junctions.

[0018] In some embodiments, the corneal endothelial cells are injected onto the endothelium. In certain embodiments, the corneal endothelial cells are injected onto a portion of eye and directed to the endothelium with one or more magnetic fields.

[0019] In some embodiments, the corneal endothelial cells are injected within thirty seconds to seven days after cataract surgery.

[0020] Also described herein are methods of increasing the number of retinal pigment epithelium (RPE) cells in a subject comprising application of ultrasonic energy to the retinal pigment epithelium of the subject followed by contacting the epithelium with a plurality of retinal pigment epithelium cells. Other aspects concern retinal pigment epithelium cells for usein increasing the number of retinal pigment epithelium cells in the retinal pigment epithelium following application of ultrasonic energy to the retinal pigment epithelium.

[0021] In some embodiments, the contacting of the epithelium with a plurality of retinal pigment epithelial cells may occur thirty seconds to seven days, one minute to three days, or two minutes to one day after the application of ultrasonic energy.

[0022] In some embodiments, the ultrasonic energy is applied to the epithelium for a time and dose sufficient to induce better integration of ocular cells.

[0023] In some embodiments, the retinal pigment epithelial cells comprise magnetic nanoparticles having a mean diameter of no more than 200 nm affixed, bound, or attached to a surface of the retinal pigment epithelial cells. The magnetic nanoparticle may be affixed to the surface of the retinal pigment epithelial cells by means of an antibody. Some magnetic nanoparticles comprise iron in any ferromagnetic form. Some magnetic nanoparticles have a diameter of less than 100 nm.

[0024] In certain embodiments, the retinal pigment epithelial cells comprising magnetic particles are positioned under the neural retina using magnetic field(s). In some embodiments, the magnetic field is supplied by a plurality of electromagnets.

[0025] In some embodiments, the retinal pigment epithelial cells adhere to the retinal pigment epithelium or sub-retinal space using an adhesion promoter such as additional drug treatments given with the retinal pigment epithelial cells or given separately from the retinal pigment epithelial cells. In some embodiments, when the adhesion promoter is provided separately from the epithelial cells, the adhesion promoter is provided concurrently with the epithelial cells. In other embodiments, the adhesion promoter is provided after the epithelial cells have been delivered.

[0026] The subject is a human in some embodiments. The retinal pigment epithelial cells are human in certain embodiments.

[0027] Some retinal pigment epithelial cells are derived from stem cells or progenitor cells. Some retinal pigment epithelial cells are derived from any cell line with tight junctions.

[0028] In some embodiments, the retinal pigment epithelial cells are injected onto the epithelium. In certain embodiments, the retinal pigment epithelial cells are injected onto a portion of eye and directed to the retinal pigment epithelium or sub-retinal space with one or more magnetic fields.DETAILED DESCRIPTION

[0029] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0030] The term “subject” refers to an animal, for example, a mammal, in particular a human. The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human.

[0031] The term “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable mater, composition, or vehicle, such as a liquid, diluent, excipient, or solvent involved in carrying or transporting the subject magnetic endothelial cells. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to a subject to whom it is administered.

[0032] The term “target tissue” refers to any specific tissue type or location, e.g., within an organ or tissue, to which it is desirable to deliver the therapeutic agent. For example, the therapeutic agent may be delivered to a target ocular tissue, or to a specific region of the eye, e.g., to the cornea, optic nerve, neural retina, etc.

[0033] The term “ocular cell” refers to corneal cells, retinal cells, lens cells, and iris cells. These cells are in pathway of incoming light.

[0034] The terms “ultrasonic energy”, “ultrasound”, “ultrasound energy”, and “therapeutic ultrasound” are used interchangeably herein to refer to sound with frequencies greater than 20 kilohertz. Ultrasonic devices operate with frequencies from 20 kHz up to several gigahertz. In example embodiments, the ultrasound is between 27 to 60 kHz, such as 40 kHz. Ultrasound used for therapeutic benefit often uses focused ultrasound waves, however, unfocused ultrasound waves may also be used. High-intensity focused ultrasound (HIFU), or MR-guided focused ultrasound surgery (MR-guided focused ultrasound ablation), is an incisionless therapeutic technique that uses non-ionizing ultrasonic waves to heat or ablate tissue. Ultrasound sources may be used for cataract treatment by phacoemulsification in which the internal lens of the eye is broken down into small pieces that may then be aspirated.

[0035] The term “magnetic nanoparticle” refers to a particle on the nanometer scale, i.e., 1-500 nm, having magnetic properties.

[0036] The words “exemplary,” “example,” or various forms thereof are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” or as an “example” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Furthermore, examples are provided solely for purposes of clarity and understanding and are not meant to limit or restrict the disclosed subject matter or relevant portions of this disclosure in any manner. It is to be appreciated that a myriad of additional or alternate examples of varying scope could have been presented but have been omitted for purposes of brevity.

[0037] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within thescope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0038] All amounts, ratios, and percentages are by weight unless otherwise indicated. The articles “a”, “an”, and “the” each refer to one or more, unless otherwise indicated by the context of the specification. The disclosure of ranges includes the range itself and also anything subsumed therein, as well as endpoints. For example, disclosure of a range of 2.0 to 4.0 includes not only the range of 2.0 to 4.0, but also 2.1, 2.3, 3.4, 3.5, and 4.0 individually, as well as any other number subsumed in the range. Furthermore, disclosure of a range of, for example, 2.0 to 4.0 includes the subsets of, for example, 2.1 to 3.5, 2.3 to 3.4, 2.6 to 3.7, and 3.8 to 4.0, as well as any other subset subsumed in the range. As used herein “about,” “approximately,” and “substantially” mean within a percent difference of less than or equal to 20%, 10%, 5%, 3%, 2%, or 1%. Similarly, the disclosure of Markush groups includes the entire group and also any individual members and subgroups subsumed therein.

[0039] In one aspect, this application discloses methods of increasing the number of ocular cells in a subject comprising application of ultrasonic energy to a target ocular tissue in a subject comprising application of ultrasonic energy to the target ocular tissue of the subject followed by contacting the target ocular tissue with a plurality of ocular cells. In some embodiments, the ocular cells are corneal endothelial cells. In other embodiments, the ocular cells are retinal pigment epithelial cells. In some embodiments, the methods or ocular cells are administered to subjects for preventative or prophylactic purposes.

[0040] In another aspect, this application discloses ocular cells for use in increasing the number of ocular cells in a target ocular tissue of a subject following application of ultrasonic energy to the target ocular tissue. In some embodiments, the ocular cells are corneal endothelial cells. In other embodiments, the ocular cells are retinal pigment epithelial cells.

[0041] Reference is made to US Patent 9,078,932, titled “Magnetic cells for localizing delivery and tissue repair.” Reference is also made to Moysidis SN, Alvarez-Delfin K, Peschansky VJ, Salero E, Weisman AD, Bartakova A, Raffa GA, Merkhofer RM Jr, Kador KE, Kunzevitzky NJ, Goldberg JL. Magnetic field-guided cell delivery with nanoparticle- loaded human corneal endothelial cells. Nanomedicine. 2015 Apr;l l(3):499-509. Reference is also made to Xia X, Atkins M, Dalal R, Kuzmenko O, Chang KC, Sun CB, Benatti CA, Rak DJ, Nahmou M, Kunzevitzky NJ, Goldberg JL. Magnetic Human Corneal Endothelial Cell Transplant: Delivery, Retention, and Short-Term Efficacy. Invest Ophthalmol Vis Sci. 2019 Jun 3;60(7):2438-2448.

[0042] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.Corneal endothelial cells and methods of use

[0043] In one aspect, this application discloses a method of increasing the number of corneal endothelial cells in a subject comprising application of ultrasonic energy to the cornea, corneal endothelial cell layer, or inner surface of the cornea of the subject followed by contacting the cornea, corneal endothelial cell layer, or inner surface of the cornea with a plurality of corneal endothelial cells.

[0044] In another aspect, this application discloses corneal endothelial cells for use in increasing the number of corneal endothelial cells in the cornea, corneal endothelial cell layer, or inner surface of the cornea in a subject following application of ultrasonic energy to the cornea, corneal endothelial cell layer, or inner surface of the cornea.

[0045] In some embodiments, the application of ultrasonic energy may be associated with cataract surgery. In some embodiments, the application of ultrasonic energy may be delivered separately from cataract or other surgery. In some embodiments, the subject may suffer from diseased corneal endothelial cells. In some embodiments, the subject may suffer from decreased corneal endothelial cells. In further embodiments, the corneal endothelial cells may be injected within thirty seconds to seven days after cataract surgery. In some embodiments, the corneal endothelial cells may be injected within one minute to three days or two minutes to one hour after cataract surgery.

[0046] In some embodiments, the contacting of the inner surface of the cornea with a plurality of corneal endothelial cells may occur thirty seconds to seven days after the application of ultrasonic energy. In other embodiments, the contacting may occur one minute to three days after the application of ultrasonic energy and in further embodiments, the contacting may occur two minutes to one hour after the application of ultrasonic energy.

[0047] In some embodiments, the ultrasonic energy may be applied to the cornea, corneal endothelial cell layer, or inner surface of the cornea for a time and dose sufficient to induce better integration of corneal endothelial cells. In some embodiments, the ultrasonic energy is delivered during cataract surgery. In other embodiments, the ultrasonic energy is delivered thirty seconds to seven days before or after cataract surgery. In some embodiments, theultrasonic energy may be delivered inside the eye. In other embodiments, the ultrasonic energy is delivered with a delivery probe on the outside of the eye.

[0048] In some embodiments, the corneal endothelial cells may comprise magnetic nanoparticles having a mean diameter of no more than 200 nm affixed, bound, or attached to a surface of the corneal endothelial cells. In further embodiments, the magnetic nanoparticle may be affixed to the surface of the corneal endothelial cells by means of an antibody. In some embodiments, the magnetic nanoparticle may comprise iron in any ferromagnetic form. In some embodiments, the magnetic nanoparticle may have a diameter of less than 100 nm. In further embodiments, endothelial cells comprising magnetic particles may be positioned on the corneal endothelium using magnetic field(s). In still further embodiments, the magnetic field(s) may be supplied by a plurality of electromagnets.

[0049] In some embodiments, the corneal endothelial cells may adhere to the corneal endothelium using an adhesion promoter. In some embodiments, the subject may be a human. In some embodiments, the corneal endothelial cells may be human cells. In some embodiments, the corneal endothelial cells may be derived from stem cells, progenitor cells, or any cell line with tight junctions.

[0050] In some embodiments, the corneal endothelial cells may be injected into the eye and be directed to the inner surface of the cornea by gravity. In other embodiments, the corneal endothelial cells may be injected into the eye and be directed to the inner surface of the cornea with one or more magnetic fields.Retinal pigment epithelial cells and methods of use

[0051] In another aspect, this application discloses a method of increasing the number of retinal pigment epithelial cells in a subject comprising application of ultrasonic energy to the neural retina or retinal pigment epithelial cell layer of the subject followed by contacting the neural retina or retinal pigment epithelial cell layer with a plurality of retinal pigment epithelial cells.

[0052] In another aspect, this application discloses retinal pigment epithelial cells for use in increasing the number of retinal pigment epithelial cells in the neural retina or retinal pigment epithelial cell layer in a subject following application of ultrasonic energy to the neural retina or retinal pigment epithelial cell layer .

[0053] In some embodiments, the application of ultrasonic energy may be associated with surgery. In some embodiments, the subject may suffer from diseased retinal pigment epithelialcells. In some embodiments, the subject may suffer from decreased retinal pigment epithelial cells.

[0054] In some embodiments, the contacting of the neural retina with a plurality of retinal pigment epithelial cells may occur thirty seconds to seven days after the application of ultrasonic energy. In other embodiments, the contacting may occur one minute to three days after the application of ultrasonic energy and in further embodiments, the contacting may occur two minutes to one hour after the application of ultrasonic energy.

[0055] In some embodiments, the ultrasonic energy may be applied to the neural retina or retinal pigment epithelial cell layer for a time and dose sufficient to induce better integration of retinal pigment epithelial cells . In some embodiments, the ultrasonic energy is delivered during surgery. In other embodiments, the ultrasonic energy is delivered thirty seconds to seven days before or after surgery. In some embodiments, the ultrasonic energy may be delivered inside the eye. In other embodiments, the ultrasonic energy is delivered with a delivery probe on the outside of the eye.

[0056] In some embodiments, the retinal pigment epithelial cells may comprise magnetic nanoparticles having a mean diameter of no more than 200 nm affixed, bound, or attached to a surface of the retinal pigment epithelial cells. In further embodiments, the magnetic nanoparticle may be affixed to the surface of the retinal pigment epithelial cells by means of an antibody. In some embodiments, the magnetic nanoparticle may comprise iron in any ferromagnetic form. In some embodiments, the magnetic nanoparticle may have a diameter of less than 100 nm. In further embodiments, retinal pigment epithelial cells comprising magnetic particles may be positioned on the retinal pigment epithelium using magnetic field(s). In still further embodiments, the magnetic field(s) may be supplied by a plurality of electromagnets.

[0057] In some embodiments, the retinal pigment epithelial cells may adhere to the retinal pigment epithelium using an adhesion promoter. In some embodiments, the subject may be a human. In some embodiments, the retinal pigment epithelial cells may be human cells. In some embodiments, the retinal pigment epithelial cells may be derived from stem cells, progenitor cells, or any cell line with tight junctions.

[0058] In some embodiments, the retinal pigment epithelial cells may be injected into the eye and be directed to the inner surface of the neural retina by gravity. In other embodiments, the retinal pigment epithelial cells may be injected into the eye and be directed to the inner surface of the neural retina with one or more magnetic fields.Magnetic cell delivery

[0059] The magnetic cells may be placed in a suitable pharmaceutical carrier. Suitable pharmaceutical carriers for ocular application are known in the art, for example those described in Remington: The Science and Practice of Pharmacy, 21stEd. (2005). In some aspects, at least 50% the volume of the pharmaceutically acceptable carrier is removed from the eye at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least an hour after injection of the composition comprising a magnetized therapeutic agent and a volume of pharmaceutically acceptable carrier. In certain implementations, the pharmaceutically acceptable carrier is removed from the eye no more than 3 hours, no more than 2 hours, or no more than 1 hour after injection of the composition comprising a magnetized therapeutic agent and a volume of pharmaceutically acceptable carrier. In other embodiments, such as corneal endothelial injections, the carrier is not removed.

[0060] In particular embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% volume of the pharmaceutically acceptable carrier is removed from the eye about 5-30 minutes after injection of the composition comprising a magnetized therapeutic agent and a volume of pharmaceutically acceptable carrier. For example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% volume of the pharmaceutically acceptable carrier is removed 10-30 seconds after injection, 30 seconds- 1 minute after injection, 1-2 minutes after injection, 2-5 minutes after injection, 5-10 minutes after injection, 5-15 minutes after injection, 5-20 minutes after injection, 5-25 minutes after injection, 5-30 minutes after injection, 10-15 minutes after injection, 10-20 minutes after injection, 10-25 minutes after injection, 10-30 minutes after injection, 15-20 minutes after injection, 15-25 minutes after injection, 15-30 minutes after injection, 20-25 minutes after injection, 20-30 minutes after injection, or 25-30 minutes after injection. Thus, in some implementations, between 50-99% volume (for example, 50-60%, 50- 70%, 50-80%, 50-90%, 50-95%, 50-99%, 60-70%, 60-80%, 60-90%, 60-95%, 60-99%, 70- 80%, 70-90%, 70-95%, 70-99%, 80-90%, 80-95%, 80-99%, 90-95%, 90-99%, 95-99%, or any other range between 50-99%) is removed or evacuated after injection of the composition comprising a magnetized therapeutic agent and a volume of pharmaceutically acceptable carrier. In another aspect, no more than 99% volume of the pharmaceutically acceptable carrieris evacuated after injection of the composition comprising magnetized cells and a volume of pharmaceutically acceptable carrier.

[0061] In some embodiments, the surgically related bleb is removed 25-30 seconds or SO- 35 seconds after injection. In other embodiments, the surgically related bleb is not removed.

[0062] Fixed / rare earth magnets, electromagnets, or superconducting magnets may be used to direct the magnetic cells to the desired location within the eyes. The fixed / rare earth magnets, electromagnets, or superconducting magnets may comprise sufficient field density uniformity and magnetic field gradient to direct the cells and hold them in place. Specifics of magnetic field strength may vary by need, such that stronger fields / gradients being used when the magnet is required to act at greater distances, across larger areas or surfaces of damaged cells, and weaker fields / gradients may be used when the magnet can be localized closer to the target tissues. The magnetic cells may be directed to the target tissue and with a first magnetic field of a first strength, with the magnetic field then (or thereabout) being modulated to further refine their movement and shape the tissue. The magnetic field may be modulated to be a second magnetic field of a second strength less than the first strength to merely hold the cells in a desired location until a desired cell adhesion is achieved, rather than using the stronger first field to move the cells.

[0063] The magnetic field may also be modulated by activating different zones, portions, or areas of the magnet or magnetic body to encourage movement of migrations of the cells to particular areas. Magnetic cell migration may be directed in real time while observing the placement of cells, such as when cells include a visual marker and may be observed visually during placement. Cell placement may also be observed or monitored indirectly or in any other suitable way. In some instances, multiple layers or strata of cells may be placed with a delay between layering so as to provide time for adhesion of a first layer, and then a subsequent second (or any number or 1 + n) layer of cells may be added. In some instances, a first layer and subsequent layer of cells may comprise a same or similar footprint, while in other instances a footprint of subsequent layers of cells may vary and may include different cell types.

[0064] The magnetic body may be disposed within the patient rather than having the magnet being externally applied, such as being affixed in a patch to the surface of the eye external to the eyelid centered over the cornea (so as to drive, push, or pull the magnetic cells, such as magnetic human corneal endothelial cells that are floating in the anterior chamber towards the corneal endothelium). The magnetic body will be disposed within the patient and proximate the area being treated, such as within the eye socket and behind the eye of the patient (so as to attract or pull the magnetic cells, such as magnetic human retinal pigment epithelialcells injected in the subretinal space to a desired location on or underneath the neural retina in the eye).

[0065] In certain implementations, the magnetic force is applied by a magnet positioned behind the eye of the patient. In other implementations, the magnetic force is applied by a magnet coupled to the patient on the rear of the sclera, behind the neural retina. The magnet may be coupled to the patient through stitches or any other acceptable manner of coupling. In some aspects, the magnet is positioned behind the eye of the patient in a reversible manner so that the magnet is removable once magnetic force is no longer required.

[0066] In some embodiments, the magnet is left in position for a desirable length of time, during which time the cells may begin to adhere, bond, attach, or grow into place and / or the therapeutic agent may be delivered to and interact with the appropriate tissue. For example, the magnet may be left in place for 3 minutes to 7 days, and in some instances more typically 5 minutes to 1 day. The magnetic field would help direct the magnetic therapeutic agent to the desired site in the subretinal space. In a particular embodiment wherein, the therapeutic agent is a magnetic nanoparticle-bound cell, the magnetic field directs the cells to the specific site where the nanoparticle-bound cells affix to the endothelial cornea surface of the subject, after which time natural cell adhesion would take place, the magnetic field may be removed, and the magnet could be removed. In certain implementations, the application of the magnetic force to the eye is stopped once the appropriate volume of the pharmaceutically acceptable carrier has been removed.

[0067] The fixed / rare earth magnets, electromagnets, or superconducting magnets may comprise sufficient field density uniformity and magnetic field gradient to direct the cells and hold them in place. In some aspects, upon being positioned in the appropriate location behind the eye, the magnet is capable of being turned on and off as magnetic force is required. Specifics of magnetic field strength may vary by need, such that stronger fields / gradients being used when the magnet is required to act at greater distances, across larger areas or surfaces of damaged cells, and weaker fields / gradients may be used when the magnet can be localized closer to the implanted particles and / or target tissues. The magnetic therapeutic agent may be directed to the target tissue and with a first magnetic field of a first strength, with the magnetic field then (or thereabout) being modulated to further refine their movement and shape the tissue. The magnetic field may be modulated to be a second magnetic field of a second strength less than the first strength to merely hold the magnetic therapeutic agent in a desired location until a desired adhesion or therapeutic delivery is achieved, rather than using the stronger first field to move the therapeutic agent.

[0068] The magnetic cells are magnetized with magnetic nanoparticles. The magnetic nanoparticles have a mean diameter of no larger than about 500 nm, typically no larger than 200 nm, though preferably no more than 100 nm. Particles that can be used include nanospheres, conjugates, micelles, colloids, aggregates, and complexes comprising ferromagnetic, paramagnetic, or superparamagnetic material, such as iron, nickel, cobalt, and alloys thereof, as suitable for in vivo use. For example, the magnetic nanoparticles may comprise iron in any ferromagnetic form, with or without an inert surface coating, with its surface chemically modified to allow the binding of an antibody, or antibody fragment, or protein, or sugar fragment that binds to cells. Persons of skill in the art will appreciate that compounds having excessive toxicity when used according to the method are to be avoided. It is expected that in many applications the particles will be endocytosed and excreted over time, and that small amounts of otherwise toxic particles may accordingly not present a problem when used in the method.

[0069] In general, the magnetic nanoparticles will have a diameter of between 5 and 500 nm, more particularly between 20 and 400 nm, most particularly between 40 and 100 nm. Difficulties in using nanoparticles over micron-scale particles include particle aggregation, particle tracking and observation, and the ability to mobilize particles by external magnetic fields, all of which are considerably easier when using micron-scale or larger particles. For this reason, previous efforts likely focused on micron-scale particles and ignored the possible advantages of using nanoparticles over micron-scale particles. Advantages include the ability to bind to cell surfaces without stimulating endocytosis; the ability to be shed from cell surfaces, or, when internalized, excreted from cells; and the ability to be excreted from the eye or the body when shed from cells.

[0070] Magnetic nanoparticles in various forms are already in use clinically and in research applications without any demonstrated toxicity. For example, superparamagnetic particles containing microcrystalline iron oxide nanoparticles (MION) of diameters of <50 nm have been used as MRI contrast agents. These particles have demonstrated neurologic non-toxicity and axonal transport of ferrous-based agents (Neuwell et al., Neurosurgery. 1994, 34: 777- 784). Published studies supporting the use of MRI contrast agent Ferridex (Advanced Magnetics and Berlex Laboratories) have found no deleterious effects. Furthermore, magnetically directed drug delivery, using tagged pharmaceuticals in the form of magnetic microspheres and magnetic polymer carriers, has shown success in delivering anti -neoplastic drugs and radio-isotypes to magnetically targeted areas in vivo (Schutt et al., Hybridoma. 1997, 16: 109-117; Lubbe et al., J Surg Res. 2001, 95: 200-206). In particular embodiments, themagnetic nanoparticles are those described in Miltenyi et al., Cytometry. 1990, 11 :231-238. For example, the magnetic nanoparticles are dextran coated and have diameters of 30 ± 20 nm (as determined by electron microscopy) or 65 ± 20 nm (as determined by dynamic light scattering). In particular embodiments, the magnetic nanoparticles are CliniMACS® from Miltenyi Biotec, which have a mean diameter of 50 nm.

[0071] In some aspects, the magnetic particles are coated. Coatings to be affixed to the magnetic particles include non-specific binding agents such as inert metals like gold or dextrans or polymers; and / or specific binding agents such as antibodies that are specific to cell-surface antigens. For example, antibodies directed against SSEA-1 bind to many types of stem cells, and nanoparticles coated with anti-SSEA-1 antibodies can be used to convert stem cells into magnetic stem cells. Similarly, many cells express specific surface receptors, and antibodies against these specific receptors conjugated to magnetic nanoparticles bind to these cells such as corneal endothelial cells and create magnetic corneal endothelial cells. Coatings are affixed to the particles by standard methods used broadly in the field (for example, see Schroder et al., J Immunol Methods . 1986, 93: 45-53; Douglas et al., Crit Rev Ther Drug Carrier Syst. 1987, 3: 233-261; Sestier et al., Electrophoresis. 1998, 19: 1220-1226; Perrin et al., J Immunol Methods. 1999, 224: 77-87; McCloskey et al., Cytometry. 2000, 40: 307-315; Tibbe et al., Cytometry. 2001, 43: 31-37).

[0072] The magnetic nanoparticles can be endocytosed by a cell or affixed to the surface of the cells by any effective means known to those of skill in the art. For example, the magnetic particle may be affixed to the cell by means of an antibody, e.g., an antibody specific for a surface antigen present on the cell. Surface coatings comprising, for example, anti -LI, anti- trkB, anti-integrin, and cholera toxin subunit B may be placed on the magnetic particles for the purpose of attaching them to retinal ganglion cells. The magnetic particle may also be affixed to a cell using a specific ligand for which a receptor is present on the cell. For example, the magnetic particles can be functionalized for attachment to retinal ganglion cells using brain- derived neurotrophic factor (BDNF). Coated magnetic nanoparticles can be affixed to the outer surface of cells by co-incubation in a general media that affords adequate cell survival during the co-incubation period. In example embodiments, ex vivo cells are cultured with coated magnetic nanoparticles. The media is not generally found to be germane to the affixing process. In general, a balanced salt solution at a physiologic pH around 7.4 will suffice; supplements to the media that enhance cell survival during the process are the topic of other published work specific to the cell types being used and are not germane to this invention. Co-incubation time and temperature may depend on the specific cell type being converted into a magnetic cell; forexample, binding to retinal ganglion cells using magnetic nanoparticles coated with an anti- trkB antibody occurs maximally after 4 hours at 37°C but may also be performed at 4°C overnight. Excess magnetic nanoparticles not bound to cells can be washed away either by spinning the cells down in a centrifuge at a speed that pellets the cells but not the unbound magnetic nanoparticles, or by eluting the magnetic nanoparticle-bound cells away from the unbound cells using a magnetic field, or both.

[0073] Other means of attaching the magnetic particles to a therapeutic agent include nonspecific chemical modifications such as carboxy or amide groups, or coatings of sugars or dextrans, or coatings of polymers such as amino acid polymers like poly-lysine, or coatings of otherwise inert coatings that bind to a therapeutic agent.

[0074] The cells may be suspended in any pharmaceutically / physiologically acceptable medium or solution, such as for example, isotonic saline solution, culture medium, transport or storage medium suitable for in vivo delivery to a subject. Additional excipients and carriers may be added as are found suitable by those of skill in the art. Suitable solutions and delivery vehicles are described in Remington: The Science and Practice of Pharmacy, 21stEd. (2005). For some applications, 103-l 06cells will be delivered by injection in a volume of 3-300 pL, but more typically around 104cells in a volume of 10-200 pL.

[0075] In some embodiments, the cells are injected into a portion of the eye and directed to the target ocular tissue with one or more magnetic fields. The corneal endothelial cells may be injected into the anterior chamber in some methods of the disclosure.

[0076] In yet other embodiments, the method further comprises administration of a third, fourth, fifth, or sixth magnetized therapeutic agent following administration of the first and second magnetized therapeutic agent and evacuation of the respective pharmaceutically acceptable carriers. In such implementations, the application of the magnetic force to the eye is stopped once 50-99% volume of the volume of the pharmaceutically acceptable carrier in the composition of the last administered magnetized therapeutic agent is removed from the eye.

Claims

CLAIMSWhat is claimed is:

1. A method for increasing the number of ocular cells in a subject, comprising:(a) applying ultrasonic energy to a target ocular tissue in the subject; and(b) contacting the target ocular tissue with a plurality of ocular cells after the ultrasonic energy is applied.

2. A method for increasing the number of corneal endothelial cells in a subject, comprising:(a) applying ultrasonic energy to the cornea, corneal endothelial cell layer, or inner surface of the cornea of the subject; and(b) contacting the cornea, corneal endothelial cell layer, or inner surface of the cornea with a plurality of corneal endothelial cells after the ultrasonic energy is applied.

3. A method for increasing the number of retinal pigment epithelial (RPE) cells in a subject, comprising:(a) applying ultrasonic energy to the neural retina or retinal pigment epithelial cell layer of the subject; and(b) contacting the neural retina or retinal pigment epithelial cell layer with a plurality of retinal pigment epithelial cells after the ultrasonic energy is applied.

4. A method for enhancing ocular cell integration, comprising:(a) applying ultrasonic energy to a target ocular tissue;(b) administering ocular cells comprising magnetic nanoparticles to the eye; and(c) directing the magnetic nanoparticle-laden cells to the target ocular tissue using a magnetic field.

5. The method of claim 1, wherein the target ocular tissue comprises a cornea, a corneal endothelial cell layer, or an inner surface of the cornea of the subject and the ocular cells comprise corneal endothelial cells.

6. The method or ocular cells of any one of claims 1-4, wherein the application of ultrasonic energy is associated with cataract surgery.

7. The method of claim 5, wherein the ocular cells are injected within thirty seconds to seven days after cataract surgery.

8. The method of any one of claims 5-7, wherein the subject suffers from decreased corneal endothelial cells.

9. The method of any one of claims 5-7, wherein the ocular cells are injected into an eye of the subject and directed to the inner surface of the cornea by gravity.

10. The method of claims 9 or 10 wherein the subject suffers from decreased retinal pigment epithelial cells.

11. The method of any one of claims 5-10, wherein the ultrasonic energy is applied to the cornea, the corneal endothelial cell layer, or the inner surface of the cornea for a time and dose sufficient to induce better integration of ocular cells.

12. The method of any one of claims 9-11, wherein the ultrasonic energy is applied to the neural retina or the retinal pigment epithelial cell layer for a time and dose sufficient to induce better integration of ocular cells.

13. The method or ocular cells of any one of claims 1-4 or 11-12 wherein the subject is treated for preventative purposes.

14. The method of any one of claims 1-4, wherein the contacting occurs thirty seconds to seven days after the application of ultrasonic energy, one minute to three days, or two minutes to one day.

15. The method of claims of anyone of claims 1-14, wherein the ocular cells comprise magnetic nanoparticles having a mean diameter of no more than 200 nm affixed, bound, or attached to a surface of the ocular cells.

16. The method of claim 16, wherein the magnetic nanoparticles are affixed to the surface of the target ocular tissue by means of an antibody.

17. The method of claim 15 or 16, wherein the magnetic nanoparticles comprise iron in any ferromagnetic form.

18. The method of any one of claims 15-17, wherein the magnetic nanoparticles have a diameter of less than 100 nm.

19. The method of any one of claims 14-18, wherein the ocular cells comprising magnetic particles are directed to the target ocular tissue using a magnetic field.

20. The method of claim 19, wherein the magnetic field is supplied by a plurality of electromagnets.

21. The method of any one of claims 1 or 4, wherein the ocular cells adhere to the target ocular tissue using an adhesion promoter.

22. The method of any one of claims 1-4, wherein the subject is a human.

23. The method of claims lor 4, wherein the ocular cells are human.

24. The method of any one of claims 1 or 4, wherein the ocular cells are derived from stem cells or progenitor cells.

25. The method of any one of claims 1-4, wherein the ocular cells are derived from any cell line with tight junctions.

26. The method of any one of claims 1 or 4, wherein the ocular cells are injected into an eye of the subject and directed to the target ocular tissue with one or more magnetic fields.

27. Use of ocular cells for increasing the number of ocular cells in a target ocular tissue of a subject, wherein said use comprises:(a) application of ultrasonic energy to the target ocular tissue; and(b) subsequent contacting of the target ocular tissue with said ocular cells.

28. Use of comeal endothelial cells for increasing the number of corneal endothelial cells in a cornea, corneal endothelial cell layer, or inner surface of the cornea of a subject, wherein the use comprises:(a) application of ultrasonic energy to the cornea, corneal endothelial cell layer, or inner surface of the cornea; and(b) contacting the same with said corneal endothelial cells.

29. Use of retinal pigment epithelial cells for increasing the number of retinal pigment epithelial cells in the neural retina or retinal pigment epithelial cell layer of a subject, wherein the use comprises:(a) application of ultrasonic energy to the neural retina or retinal pigment epithelial cell layer; and(b) contacting the same with said retinal pigment epithelial cells.

30. Use of magnetically modified ocular cells for increasing the number of ocular cells in a target ocular tissue of a subject, wherein the magnetically modified ocular cells comprise magnetic nanoparticles affixed to the surface of the cells, and wherein the use comprises: directing said magnetically modified ocular cells to the target ocular tissue using a magnetic field following application of ultrasonic energy to the target ocular tissue.

31. The use according to any one of claims 27-30, wherein the ocular cells are administered within 30 seconds to 7 days after the application of ultrasonic energy.

32. The use according to any one of claims 27-30, wherein the ocular cells are administered within 1 minute to 3 days, or 2 minutes to 1 hour, after the application of ultrasonic energy.

33. The use according to any one of claims 28 or 30, wherein the ultrasonic energy is applied during or after cataract surgery.

34. The use according to any one of claims 29 or 30, wherein the ultrasonic energy is applied during or after a surgical procedure on the eye.

35. The use according to claim 30, wherein the magnetic nanoparticles comprise iron and have a mean diameter of no more than 200 nm.

36. The use according to claim 30 or 35, wherein the magnetic nanoparticles are affixed to the cells via an antibody.

37. The use according to claim 30 or any claim depending thereon, wherein the magnetic field is supplied by fixed magnets, electromagnets, or superconducting magnets positioned external to or behind the eye.

38. The use according to any one of claims 27-37, wherein the ocular cells are selected from stem cells, progenitor cells, or differentiated cells with tight junctions.

39. The use according to claims 27 or 30, wherein the target ocular tissue is selected from the group consisting of: corneal endothelium, neural retina, and retinal pigment epithelial layer.

40. The use according to any one of claims 27-39, wherein the cells are suspended in a pharmaceutically acceptable carrier and at least 50% of the carrier is removed after injection.

41. The use according to claim 40, wherein the removal of the carrier occurs between 5- 30 minutes post-injection.

42. A system for ocular cell delivery comprising:(a) a source of ultrasonic energy configured to apply energy to a target ocular tissue;(b) a magnetic field generator configured to direct magnetically labeled ocular cells to the target ocular tissue; and(c) a pharmaceutical composition comprising ocular cells labeled with magnetic nanoparticles.