Ocular Delivery of Therapeutic Agents

Abstract

The present invention includes devices and methods of treatment for ocular diseases or disorders in an individual. Provided herein are devices and methods of delivering therapeutic agents (e.g., nucleic acid vectors) to targeted ocular cells (e.g., retinal cells), including therapeutic agents, and methods of administering therapeutic agents to an individual and methods of electrotransfer of therapeutic agents.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 163,350, filed March 19, 2021, U.S. Provisional Patent Application No. 63 / 167,296, filed March 29, 2021, U.S. Provisional Patent Application No. 63 / 293,297, filed December 23, 2021, U.S. Provisional Patent Application No. 63 / 167,463, filed March 29, 2021, U.S. Provisional Patent Application No. 63 / 316,699, filed March 4, 2022, and U.S. Provisional Patent Application No. 63 / 167,437, filed March 29, 2021, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing which has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on Mar. 21, 2022, is named 51503-066WO4_Sequence_Listing_3_21_22_ST25.txt and is 392,761 bytes in size.

[0003] FIELD OF THEINVENTION In general, the invention features ocular therapeutic agents and devices and methods for administering therapeutic agents to ocular cells. [Background technology]

[0004] background Visual impairment and blindness are major global health concerns, affecting millions of patients suffering from a wide variety of ocular pathologies. Retinal dystrophies are chronic and progressive disorders of visual function that arise due to genetic abnormalities in, for example, retinal cytoarchitecture (e.g., photoreceptors and / or retinal epithelial cells) and visual cycle pathways (e.g., the phototransduction and visual cycle pathways necessary to facilitate the conversion of light energy into perceptible neuronal signals). Visual impairment caused by retinal dystrophies ranges from poor peripheral or night vision to complete blindness, and severity usually increases with age. Due in part to complex biological mechanisms and limited access to the retina, safe and effective treatments for many retinal dystrophies remain lacking.

[0005] Recent developments in gene therapy have shown promise in the treatment of retinal dystrophies. However, current delivery methods often rely on the tropism of virion particles, such as adeno-associated virus (AAV) vectors. The success of such delivery methods depends on various factors, such as the location of the target tissue, the route of administration of the vector, and the host response. In addition, AAV vectors are limited by the size constraints of the therapeutic genes to be delivered, making such methods unsuitable for the delivery of many retinal genes. Thus, effective targeting of ocular cells remains a challenging endeavor, and improved approaches are needed for the effective delivery of therapeutic agents to retinal cells. Summary of the Invention

[0006] The present invention provides approaches for delivering therapeutic agents (e.g., nucleic acid vectors encoding therapeutic replacement proteins) to ocular cells (e.g., retinal cells). In some cases, the approaches described herein involve electrotransfer, a process in which the delivery of an electric field to ocular tissue (e.g., retina) using intraocular electrodes (e.g., placed in the vitreous or retina) facilitates the delivery of therapeutic agents (e.g., nucleic acid vectors, e.g., non-viral nucleic acid vectors, e.g., naked nucleic acid vectors, e.g., synthetic circular DNA vectors) to targeted ocular cells (e.g., retinal cells). Therapeutic agents, e.g., nucleic acid vectors for use in such methods, are also provided herein.

[0007] In one aspect, the invention provides a method for delivering a therapeutic agent (e.g., a nucleic acid vector, e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector) to a target retinal cell of an individual, the method comprising: (a) contacting an electrode with an interior region of the eye, wherein the extracellular space within the retina of the eye contains the therapeutic agent; and (b) while the electrode is in contact with the interior region of the eye, transmitting one or more (e.g., 4-12 or 6-10) pulses of electrical energy (e.g., current) through the electrode under conditions suitable for electrical introduction of the therapeutic agent to the target retinal cell. In some embodiments, the electrode is a monopolar electrode (e.g., a monopolar positive electrode disposed within the vitreous, or a monopolar negative electrode disposed within the retina, subretinal space, or a bleb created by subretinal injection of a therapeutic agent). In some embodiments, the electrode is a bipolar electrode (e.g., a bipolar electrode disposed such that the negative electrode is in contact with the retina, subretinal space, or a bleb created by subretinal injection of a therapeutic agent, and the positive electrode is within the vitreous). In other embodiments, the therapeutic agent is delivered to the extracellular space by subretinal injection (e.g., the therapeutic agent has already been administered subretinally and is in position for electrical introduction to the target retinal cells). In other embodiments, the therapeutic agent is delivered to the extracellular space by intravitreal injection. In some embodiments, delivery of the therapeutic agent to the extracellular space of the retina is also included as part of the above-mentioned method. In some embodiments, delivery of the therapeutic agent (e.g., a nucleic acid vector, e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector) is by subretinal injection. In other embodiments, delivery of the therapeutic agent is by intravitreal injection. In some embodiments where the therapeutic agent is a nucleic acid vector (e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector), the nucleic acid is expressed by the target cell (e.g., a retinal cell, e.g., a retinal pigment epithelium (RPE) cell and / or a photoreceptor cell). Thus, the delivery method described herein may possibly be a method of expressing a sequence of interest (e.g., a therapeutic sequence).

[0008] In some embodiments, the internal region of the eye in contact with the electrode includes the vitreous humor (e.g., the electrode is entirely within the vitreous humor). In some embodiments, the electrode is within 10 mm of the retina at the time of delivery of one or more pulses of electrical energy (e.g., within 10 mm, 5 mm, or 1 mm of the retina but not in direct contact with the retina). In some embodiments in which the electrode is in the vitreous humor, the electrode is a positive electrode and the voltage applied is a positive voltage (e.g., the electrode is in the vitreous humor, the electrode is a unipolar positive electrode, and the therapeutic agent is a nucleic acid vector (e.g., a DNA vector or an RNA vector), e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector.

[0009] In some embodiments, the electrodes are in direct contact with the retina (and / or subretinal bleb) during delivery of the one or more pulses of electrical energy. In some embodiments, the electrodes are 0.1 mm to 10 mm from the retina during delivery of the one or more pulses of electrical energy. In some embodiments, the electrodes are 0.5 mm to 10 mm from the retina during delivery of the one or more pulses of electrical energy.

[0010] In some embodiments, the internal eye region in contact with the electrode includes the retina. For example, the electrode may be entirely within the subretinal space or may be partially within the subretinal space (e.g., in contact with a subretinal bleb). In some embodiments in which the electrode is in contact with the retina, subretinal space, or subretinal bleb, the electrode is a negative electrode (e.g., cathodic), the voltage applied is a negative voltage (e.g., in which the electrode is in contact with the retina, subretinal space, or subretinal bleb, the electrode is a unipolar negative electrode (e.g., cathodic), and the therapeutic agent is a nucleic acid vector (e.g., any of the DNA or RNA vectors described herein), e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector.

[0011] In some embodiments, conditions suitable for electrical delivery of a therapeutic agent (e.g., a nucleic acid vector, e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector) to a target retinal cell include a voltage of 1 V / cm to 1,500 V / cm (1 V / cm to 10 V / cm (e.g., about 1 V / cm, about 2 V / cm, about 3 V / cm, about 4 V / cm, about 5 V / cm, about 6 V / cm, about 7 V / cm, about 8 V / cm, about 9 V / cm, or about 10 V / cm) at the target retinal cell, about 10 V / cm to about 100 V / cm (e.g., about 10 V / cm, about 20 V / cm, about 30 V / cm, about 40 V / cm, about 50 V / cm, or about 60 V / cm) at the target retinal cell. , about 60 V / cm, about 70 V / cm, about 80 V / cm, about 90 V / cm, or about 100 V / cm), about 100 V / cm to about 1,000 V / cm (e.g., about 200 V / cm, about 300 V / cm, about 400 V / cm, about 500 V / cm, about 600 V / cm, about 700 V / cm, about 800 V / cm, about 900 V / cm, or about 1,000 V / cm), or 1,000 V / cm to 1,500 V / cm (e.g., about 1,000 V / cm, about 1,100 V / cm, about 1,200 V / cm, about 1,300 V / cm, about 1,400 V / cm, or about 1,500 V / cm)). In some embodiments, the electric field strength at the target cell is between 50 V / cm and 300 V / cm, hi some embodiments, the electric field strength at the target cell is about 100 V / cm.

[0012] In some embodiments, conditions suitable for electrical delivery of a therapeutic agent (e.g., a nucleic acid vector, e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector) to a target retinal cell include a current of 10 μA to 1 A (e.g., 10 μA to 500 mA, 10 μA to 200 mA, 10 μA to 100 mA, 10 μA to 50 mA, or 10 μA to 25 mA; e.g., 50 μA to 500 mA, 100 μA to 200 mA, or 1 mA to 100 mA; e.g., 10 μA to 20 μA, 20 μA to 30 μA, 30 μA to 50 μA, 50 μA to 100 μA, 100 μA to 150 μA, 150 μA to 200 μA, 200 μA to 300 μA, 300 μA to 400 μA, 400 μA to 500 μA). A, 500 μA to 600 μA, 600 μA to 800 μA, 800 μA to 1 mA, 1 mA to 10 mA, 10 mA to 20 mA, 20 mA to 30 mA, 30 mA to 40 mA, 40 mA to 50 mA, 50 mA to 60 mA, 60 mA to 70 mA, 70 mA to 80 mA, 80 mA to 90 mA, 90 mA to 100 mA, 100 mA to 200 mA, 200 mA to 300 mA, 300 mA to 500 mA, or 500 mA to 1 A; for example, about 1 mA, about 5 mA about 10 mA, about 15 mA, about 20 mA, about 25 mA, about 30 mA, about 35 mA, about 40 mA, about 45 mA, about 50 mA, about 60 mA, about 70 mA, about 80 mA, about 90 mA, or about 100 mA).

[0013] In some embodiments, 1-3 pulses (e.g., 1 pulse, 2 pulses, or 3 pulses) of energy are delivered. In some embodiments, 4-12 pulses (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 pulses) of energy are delivered. In some embodiments, 1-12 pulses are administered. In some embodiments, 10-20 pulses (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pulses) are administered. In some embodiments, 8 pulses are administered.

[0014] In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 20 seconds. For example, the total number of pulses of electrical energy can be delivered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 seconds. The pulses of electrical energy can be, for example, a square waveform. The pulses of electrical energy can have an amplitude of 5V to 1,500V. For example, the pulses of electrical energy can have an amplitude of about 5V to 500V, about 500V to about 1,000V, or about 1,000V to about 1,500V. In some embodiments, the pulses of electrical energy have an amplitude of 5V to 500V. For example, the pulse of electrical energy can have an amplitude of about 5V, 10V, 15V, 20V, 25V, 30V, 40V, 50V, 100V, 125V, 150V, 175V, 200V, 225V, 250V, 275V, 300V, 325V, 350V, 375V, 400V, 425V, 450V, 475V, or 500V. In some embodiments, the pulse of electrical energy has an amplitude of about 5V to about 250V.

[0015] In some embodiments, the pulse of electrical energy has an amplitude of about 20V. In some embodiments, where the pulse of electrical energy has an amplitude of about 20V, the current is 5 mA to 50 mA (e.g., 10 mA to 40 mA, e.g., 5 mA to 10 mA, 10 mA to 15 mA, 15 mA to 20 mA, 20 mA to 30 mA, or 40 mA to 50 mA). In some embodiments, the pulse of electrical energy has an amplitude of about 40V. In some embodiments, where the pulse of electrical energy has an amplitude of about 40V, the current is 10 mA to 100 mA (e.g., 20 mA to 80 mA, or 30 mA to 70 mA, e.g., 10 mA to 20 mA, 20 mA to 30 mA, 30 mA to 40 mA, 40 mA to 50 mA, 50 mA to 60 mA, 60 mA to 70 mA, 70 mA to 80 mA, 80 mA to 90 mA, or 90 mA to 100 mA).

[0016] In some embodiments, the current resulting from the pulsed electric field is between 10 μA and 1 A (e.g., 10 μA to 500 mA, 10 μA to 200 mA, 10 μA to 100 mA, 10 μA to 50 mA, or 10 μA to 25 mA; e.g., 50 μA to 500 mA, 100 μA to 200 mA, or 1 mA to 100 mA; e.g., 10 μA to 20 μA, 20 μA to 30 μA, 30 μA to 50 μA, 50 μA to 100 μA, 100 μA to 150 μA, 150 μA to 200 μA, 200 μA to 300 μA, 300 μA to 400 μA, 400 μA to 500 μA, 500 μA to 600 μA, 600 μA to 800 μA, 8 00 μA to 1 mA, 1 mA to 10 mA, 10 mA to 20 mA, 20 mA to 30 mA, 30 mA to 40 mA, 40 mA to 50 mA, 50 mA to 60 mA, 60 mA to 70 mA, 70 mA to 80 mA, 80 mA to 90 mA, 90 mA to 100 mA, 100 mA to 200 mA, 200 mA to 300 mA, 300 mA to 500 mA, or 500 mA to 1 A; for example, about 1 mA, about 5 mA, about 10 mA, about 15 mA, about 20 mA, about 25 mA, about 30 mA, about 35 mA, about 40 mA, about 45 mA, about 50 mA, about 60 mA, about 70 mA, about 80 mA, about 90 mA, or about 100 mA).

[0017] In some embodiments, each of the pulses is about 0.01 ms to about 200 ms in duration, about 0.1 ms to about 200 ms in duration, or about 1 ms to about 200 ms in duration (e.g., 0.10 ms to about 200 ms in duration. For example, each of the pulses can be about 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 110 ms, 120 ms, 130 ms, 140 ms, 150 ms, 160 ms, 170 ms, 180 ms, 190 ms, or 200 ms in duration). In some embodiments, each of the pulses is about 20 ms in duration. In some embodiments, each of the pulses is about 50 ms in duration. In some embodiments, each of the pulses has a duration of about 0.01 ms to about 1 ms (e.g., 0.01 ms to 0.05 ms, 0.05 ms to 0.1 ms, 0.1 ms to 0.25 ms, 0.25 ms to 0.5 ms, 0.5 ms to 0.75 ms, or 0.75 ms to 1.0 ms; e.g., about 0.01 ms, about 0.05 ms, about 0.1 ms, about 0.2 ms, about 0.3 ms, about 0.4 ms, about 0.5 ms, about 0.6 ms, about 0.7 ms, about 0.8 ms, about 0.9 ms, or about 1.0 ms).

[0018] In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 20 seconds (e.g., within 6 to 12 seconds, e.g., within 1 to 3 seconds, within 3 to 6 seconds, within 6 to 10 seconds, within 10 to 15 seconds, or within 15 to 20 seconds, e.g., within 1 second, within 2 seconds, within 3 seconds, within 4 seconds, within 5 seconds, within 6 seconds, within 7 seconds, within 8 seconds, within 9 seconds, within 10 seconds, within 11 seconds, within 12 seconds, within 13 seconds, within 14 seconds, within 15 seconds, within 16 seconds, within 17 seconds, within 18 seconds, within 19 seconds, within 20 seconds).

[0019] In some embodiments, the pulse of energy is a square waveform. In some embodiments, the pulse of energy has an amplitude of 100V to 500V (e.g., 200V to 400V, e.g., 100V to 200V, 200V to 300V, 300V to 400V, or 400V to 500V, e.g., about 100V, about 120V, about 150V, about 200V, about 250V, about 300V, about 350V, about 400V, about 450V, or about 500V).

[0020] In some embodiments, the target retinal cells are retinal epithelial cells. In some embodiments, the target retinal cells are photoreceptors. In some embodiments, the target retinal cells are retinal epithelial cells and photoreceptors.

[0021] In some embodiments, the therapeutic agent is a nucleic acid vector, e.g., a DNA vector or an RNA vector. In some embodiments, the nucleic acid vector is a non-viral nucleic acid vector (e.g., a non-viral DNA vector or a non-viral RNA vector; e.g., a circular DNA vector or a circular RNA vector). In certain cases, the non-viral nucleic acid vector is a naked nucleic acid vector (e.g., a naked DNA vector (e.g., a naked circular DNA vector) or a naked RNA vector (e.g., a naked circular RNA vector). In some embodiments where the DNA vector is a circular DNA vector, the circular DNA vector lacks an origin of replication (e.g., a bacterial origin of replication), a drug resistance gene, and / or a recombination site.

[0022] In some aspects, the nucleic acid vector encodes a therapeutic replacement protein that replaces a protein that is endogenously expressed in healthy retinal cells, hi some aspects, the therapeutic replacement protein replaces a protein that is not endogenously expressed in target cells of an individual or that is non-functional in target cells of an individual.

[0023] In some embodiments, the therapeutic replacement protein is encoded by a coding sequence of greater than 4.5 kb.

[0024] In some embodiments, the therapeutic replacement protein is ABCA4 (e.g., human ABCA4 (e.g., ABCA4 having at least 95% sequence identity to SEQ ID NO:18, e.g., 100% sequence identity to SEQ ID NO:18)). In some embodiments, the method is a method of treating an ABCA4-associated retinal dystrophy (e.g., Stargardt's disease).

[0025] In some cases of any of the foregoing embodiments, the nucleic acid vector comprises a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96% sequence identity to SEQ ID NO:18, at least 97% sequence identity to SEQ ID NO:18, at least 98% sequence identity to SEQ ID NO:18, at least 99% sequence identity to SEQ ID NO:18, or 100% sequence identity to SEQ ID NO:18). In some cases, the nucleic acid vector is a naked nucleic acid vector (e.g., a naked DNA vector (e.g., a naked circular DNA vector (e.g., a plasmid DNA vector, a minicircle DNA vector, or a synthetic circular DNA vector lacking recombination sites (e.g., a supercoiled synthetic circular DNA vector)), a naked closed-end DNA vector, or a naked doggybone DNA vector), or a naked RNA vector (e.g., a naked circular RNA vector)) comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96% sequence identity to SEQ ID NO:18, at least 97% sequence identity to SEQ ID NO:18, at least 98% sequence identity to SEQ ID NO:18, at least 99% sequence identity to SEQ ID NO:18, or 100% sequence identity to SEQ ID NO:18). In some cases, the nucleic acid vector (e.g., a non-viral nucleic acid vector) comprises a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96% sequence identity to SEQ ID NO:18, at least 97% sequence identity to SEQ ID NO:18, at least 98% sequence identity to SEQ ID NO:18, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:18), wherein the nucleic acid vector is encapsulated in a nanoparticle, microparticle, liposome, or lipid nanoparticle. In some cases, such a nucleic acid vector comprises a CAG promoter.

[0026] In some cases, the nucleic acid vector comprises or consists of a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:19 (e.g., at least 96% sequence identity to SEQ ID NO:19, at least 97% sequence identity to SEQ ID NO:19, at least 98% sequence identity to SEQ ID NO:19, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:19). In some cases, the nucleic acid vector comprises or consists of the nucleic acid sequence of SEQ ID NO:19. In some cases, the nucleic acid vector is a naked nucleic acid vector (e.g., a naked DNA vector (e.g., a naked circular DNA vector (e.g., a plasmid DNA vector, a minicircle DNA vector, or a synthetic circular DNA vector lacking recombination sites (e.g., a supercoiled synthetic circular DNA vector)), a naked closed-end DNA vector, or a naked doggybone DNA vector), or a naked RNA vector (e.g., a naked circular RNA vector)) that comprises or consists of a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:19 (e.g., at least 96% sequence identity to SEQ ID NO:19, at least 97% sequence identity to SEQ ID NO:19, at least 98% sequence identity to SEQ ID NO:19, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:19). In some cases, the nucleic acid vector (e.g., a non-viral nucleic acid vector) comprises or consists of a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:19 (e.g., at least 96% sequence identity to SEQ ID NO:19, at least 97% sequence identity to SEQ ID NO:19, at least 98% sequence identity to SEQ ID NO:19, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:19), wherein the nucleic acid vector is encapsulated in a nanoparticle, microparticle, liposome, or lipid nanoparticle.

[0027] In some embodiments, the therapeutic replacement protein is encoded by a coding sequence of more than 4.5 kb. In some embodiments, the therapeutic replacement protein is MYO7A. In some embodiments, the method is a method of treating Usher syndrome type 1B in an individual.

[0028] In some embodiments, the therapeutic replacement protein is BEST1. In some embodiments, the method is a method of treating a bestrophinopathy associated with a BEST1 dominant mutation or a BEST1 recessive mutation.

[0029] In some embodiments, the therapeutic replacement protein is CFH. In some embodiments, the method is a method of treating age-related macular degeneration.

[0030] In another aspect, provided herein is a nucleic acid vector (or a pharmaceutical composition thereof) comprising a nucleic acid sequence driven by a CAG promoter encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96% sequence identity to SEQ ID NO:18, at least 97% sequence identity to SEQ ID NO:18, at least 98% sequence identity to SEQ ID NO:18, at least 99% sequence identity to SEQ ID NO:18, or 100% sequence identity to SEQ ID NO:18). In some cases, the nucleic acid vector is a naked nucleic acid vector (e.g., a naked DNA vector (e.g., a naked circular DNA vector (e.g., a plasmid DNA vector, a minicircle DNA vector, or a synthetic circular DNA vector lacking recombination sites (e.g., a supercoiled synthetic circular DNA vector)), a naked closed-end DNA vector, or a naked doggybone DNA vector), or a naked RNA vector (e.g., a naked circular RNA vector)) comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96% sequence identity to SEQ ID NO:18, at least 97% sequence identity to SEQ ID NO:18, at least 98% sequence identity to SEQ ID NO:18, at least 99% sequence identity to SEQ ID NO:18, or 100% sequence identity to SEQ ID NO:18). In some cases, the nucleic acid vector (e.g., a non-viral nucleic acid vector) comprises a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96% sequence identity to SEQ ID NO:18, at least 97% sequence identity to SEQ ID NO:18, at least 98% sequence identity to SEQ ID NO:18, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:18), wherein the nucleic acid vector is encapsulated in a nanoparticle, microparticle, liposome, or lipid nanoparticle.

[0031] In another aspect, the invention provides a nucleic acid vector (or a pharmaceutical composition thereof) comprising or consisting of a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:19 (e.g., at least 96% sequence identity to SEQ ID NO:19, at least 97% sequence identity to SEQ ID NO:19, at least 98% sequence identity to SEQ ID NO:19, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:19). In some cases, the nucleic acid vector comprises or consists of the nucleic acid sequence of SEQ ID NO:19. In some cases, the nucleic acid vector is a naked nucleic acid vector (e.g., a naked DNA vector (e.g., a naked circular DNA vector (e.g., a plasmid DNA vector, a minicircle DNA vector, or a synthetic circular DNA vector lacking recombination sites (e.g., a supercoiled synthetic circular DNA vector)), a naked closed-end DNA vector, or a naked doggybone DNA vector), or a naked RNA vector (e.g., a naked circular RNA vector)) that comprises or consists of a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:19 (e.g., at least 96% sequence identity to SEQ ID NO:19, at least 97% sequence identity to SEQ ID NO:19, at least 98% sequence identity to SEQ ID NO:19, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:19). In some cases, the nucleic acid vector (e.g., a non-viral nucleic acid vector) comprises or consists of a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:19 (e.g., at least 96% sequence identity to SEQ ID NO:19, at least 97% sequence identity to SEQ ID NO:19, at least 98% sequence identity to SEQ ID NO:19, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:19), wherein the nucleic acid vector is encapsulated in a nanoparticle, microparticle, liposome, or lipid nanoparticle.

[0032] In some embodiments, the therapeutic sequence or protein (eg, therapeutic replacement protein) is depicted in Table 1.

[0033] In another aspect, the present invention provides a method for delivering a non-viral (e.g., naked) synthetic circular DNA vector encoding a retinal protein (e.g., ABCA4, MYO7A or CEP290) to a target retinal cell of an individual (e.g., human), the method comprising: (a) contacting a monopolar needle electrode (e.g., a negative electrode, e.g., a cathode) with the individual's retina or subretinal bleb, where the extracellular space of the retina contains the synthetic circular DNA vector; and (b) applying 6-10 (e.g., 8) 20-40V pulses, each having a duration of 10-30 ms (e.g., about 20 ms) for 1 second to 30 seconds, e.g., about 8 seconds, to the electrode while the electrode is in contact with the retina or subretinal bleb. In some embodiments, the non-viral (e.g., naked) synthetic circular DNA vector is delivered to the extracellular space of the retina by subretinal injection. In some embodiments, delivery of a non-viral (e.g., naked) synthetic circular DNA vector to the extracellular space of the retina is also included as part of the aforementioned method. In some embodiments, the nucleic acid is expressed by a target cell (e.g., a retinal cell, e.g., an RPE cell and / or a photoreceptor cell). In some embodiments, the method treats or prevents an ocular disorder associated with a retinal protein expressed by the therapy.

[0034] In another aspect, the present invention provides a method of delivering a non-viral (e.g., naked) synthetic circular DNA vector encoding a retinal protein (e.g., ABCA4, MYO7A or CEP290) to a target retinal cell of an individual (e.g., human), the method comprising: (a) contacting a monopolar needle electrode (e.g., a monopolar positive needle electrode, e.g., an anode) with the vitreous humor of the individual such that the distal end of the electrode is within 1 mm of the retina, whereby the extracellular space of the retina contains the synthetic circular DNA vector; and (b) applying to the electrode 6-10 (e.g., 8) 20-40 V pulses, each having a duration of 10-30 ms (e.g., about 20 ms) for 1 second to 30 seconds, e.g., about 8 seconds, while the electrode is in contact with the vitreous humor within 1 mm of the retina. In some embodiments, the non-viral (e.g., naked) synthetic circular DNA vector is delivered to the extracellular space of the retina by subretinal injection. In some embodiments, delivery of non-viral (e.g., naked) synthetic circular DNA vectors to the extracellular space of the retina is also included as part of the aforementioned method. In some embodiments, the nucleic acid is expressed by the target cells (e.g., retinal cells, e.g., RPE cells and / or photoreceptor cells). In some embodiments, the method treats or prevents an ocular disorder associated with a therapeutically expressed retinal protein. The present invention also provides an approach for delivering or expressing a therapeutic agent (e.g., a nucleic acid vector encoding a therapeutic replacement protein) to an ocular cell (e.g., a retinal cell) by suprachoroidal administration. In some cases, the approaches described herein include electrical induction, a process in which the transmission of an electric field to an ocular tissue (e.g., the retina) facilitates the delivery of a therapeutic agent to a target ocular cell (e.g., a retinal cell).

[0035] In another aspect, the present invention provides a method for delivering a therapeutic agent (e.g., a nucleic acid vector, e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector) to a target retinal cell of an individual, the method comprising: (a) contacting an electrode with an internal region of the eye, wherein the extracellular space within the retina of the eye contains the therapeutic agent to be delivered by suprachoroidal injection; and (b) transmitting one or more pulses of electrical energy (e.g., electrical current) through the electrode while the electrode is in contact with the internal region of the eye, under conditions suitable for electrical introduction of the therapeutic agent to the target retinal cell. In some embodiments, the electrode is a monopolar electrode. In some embodiments, the electrode is a bipolar electrode.

[0036] In some embodiments, delivery of a therapeutic agent (e.g., a nucleic acid vector, e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector) to the extracellular space of the retina is also included as part of the aforementioned method. In some embodiments, delivery of the therapeutic agent is by suprachoroidal injection (e.g., bilateral suprachoroidal injection). In some embodiments, electrical induction is administered after delivery of the therapeutic agent. In some embodiments, electrical induction is administered before delivery of the therapeutic agent.

[0037] In some embodiments, the interior region of the eye in contact with the electrode includes the vitreous humor (e.g., the electrode is entirely within the vitreous humor). In some embodiments, the electrode is within 10 mm of the retina during delivery of the one or more pulses of electrical energy (e.g., within 10 mm of the retina, but not in direct contact with the retina). In some embodiments, the electrode is in direct contact with the retina during delivery of the one or more pulses of electrical energy. In some embodiments, the electrode is between 0.1 mm and 10 mm from the retina during delivery of the one or more pulses of electrical energy. In some embodiments, the electrode is between 0.5 mm and 10 mm from the retina during delivery of the one or more pulses of electrical energy.

[0038] In some embodiments, the interior region of the eye that is contacted by the electrode includes the retina, for example, the electrode may be completely within the subretinal space or may be partially within the subretinal space.

[0039] In some embodiments, conditions suitable for electrical delivery of a therapeutic agent (e.g., a nucleic acid vector, e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector) to a target retinal cell include a voltage of 1 V / cm to 1,500 V / cm (1 V / cm to 10 V / cm (e.g., about 1 V / cm, about 2 V / cm, about 3 V / cm, about 4 V / cm, about 5 V / cm, about 6 V / cm, about 7 V / cm, about 8 V / cm, about 9 V / cm, or about 10 V / cm) at the target retinal cell, about 10 V / cm to about 100 V / cm (e.g., about 10 V / cm, about 20 V / cm, about 30 V / cm, about 40 V / cm, about 50 V / cm, or about 60 V / cm) at the target retinal cell. , about 60 V / cm, about 70 V / cm, about 80 V / cm, about 90 V / cm, or about 100 V / cm), about 100 V / cm to about 1,000 V / cm (e.g., about 200 V / cm, about 300 V / cm, about 400 V / cm, about 500 V / cm, about 600 V / cm, about 700 V / cm, about 800 V / cm, about 900 V / cm, or about 1,000 V / cm), or 1,000 V / cm to 1,500 V / cm (e.g., about 1,000 V / cm, about 1,100 V / cm, about 1,200 V / cm, about 1,300 V / cm, about 1,400 V / cm, or about 1,500 V / cm)). In some embodiments, the electric field strength at the target cell is between 50 V / cm and 300 V / cm, hi some embodiments, the electric field strength at the target cell is about 100 V / cm.

[0040] In some embodiments, 1-3 pulses (e.g., 1 pulse, 2 pulses, or 3 pulses) of energy are delivered. In some embodiments, 4-12 pulses (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 pulses) of energy are delivered. In some embodiments, 1-12 pulses are administered. In some embodiments, 10-20 pulses (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pulses) are administered.

[0041] In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 20 seconds. For example, the total number of pulses of electrical energy can be delivered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 seconds. The pulses of electrical energy can be, for example, a square waveform. The pulses of electrical energy can have an amplitude of 5V to 1,500V. For example, the pulses of electrical energy can have an amplitude of about 5V to 500V, about 500V to about 1,000V, or about 1,000V to about 1,500V. In some embodiments, the pulses of electrical energy have an amplitude of 5V to 500V. For example, the pulse of electrical energy can have an amplitude of about 100V, 125V, 150V, 175V, 200V, 225V, 250V, 275V, 300V, 325V, 350V, 375V, 400V, 425V, 450V, 475V, or 500V. In some embodiments, the pulse of electrical energy has an amplitude of about 5V to about 250V.

[0042] In some embodiments, each of the pulses is about 0.01 ms to about 200 ms in duration, about 0.1 ms to about 200 ms in duration, or about 1 ms to about 200 ms in duration (e.g., 0.10 ms to about 200 ms in duration. For example, each of the pulses can be about 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 110 ms, 120 ms, 130 ms, 140 ms, 150 ms, 160 ms, 170 ms, 180 ms, 190 ms, or 200 ms in duration). In some embodiments, each of the pulses is about 20 ms in duration. In some embodiments, each of the pulses is about 50 ms in duration. In some embodiments, each of the pulses has a duration of about 0.01 ms to about 1 ms (e.g., 0.01 ms to 0.05 ms, 0.05 ms to 0.1 ms, 0.1 ms to 0.25 ms, 0.25 ms to 0.5 ms, 0.5 ms to 0.75 ms, or 0.75 ms to 1.0 ms; e.g., about 0.01 ms, about 0.05 ms, about 0.1 ms, about 0.2 ms, about 0.3 ms, about 0.4 ms, about 0.5 ms, about 0.6 ms, about 0.7 ms, about 0.8 ms, about 0.9 ms, or about 1.0 ms).

[0043] In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 20 seconds (e.g., within 6 to 12 seconds, e.g., within 1 to 3 seconds, within 3 to 6 seconds, within 6 to 10 seconds, within 10 to 15 seconds, or within 15 to 20 seconds, e.g., within 1 second, within 2 seconds, within 3 seconds, within 4 seconds, within 5 seconds, within 6 seconds, within 7 seconds, within 8 seconds, within 9 seconds, within 10 seconds, within 11 seconds, within 12 seconds, within 13 seconds, within 14 seconds, within 15 seconds, within 16 seconds, within 17 seconds, within 18 seconds, within 19 seconds, within 20 seconds).

[0044] In some embodiments, the pulse of energy is a square waveform. In some embodiments, the pulse of energy has an amplitude of 100V to 500V (e.g., 200V to 400V, e.g., 100V to 200V, 200V to 300V, 300V to 400V, or 400V to 500V, e.g., about 100V, about 120V, about 150V, about 200V, about 250V, about 300V, about 350V, about 400V, about 450V, or about 500V).

[0045] In some embodiments, the target retinal cells are retinal epithelial cells. In some embodiments, the target retinal cells are photoreceptors. In some embodiments, the target retinal cells are retinal epithelial cells and photoreceptors.

[0046] In some embodiments, the therapeutic agent is a nucleic acid vector, e.g., a DNA vector or an RNA vector. In some embodiments, the nucleic acid vector is a non-viral nucleic acid vector (e.g., a non-viral DNA vector or a non-viral RNA vector; e.g., a circular DNA vector or a circular RNA vector). In certain cases, the non-viral nucleic acid vector is a naked nucleic acid vector (e.g., a naked DNA vector (e.g., a naked circular DNA vector) or a naked RNA vector (e.g., a naked circular RNA vector). In some embodiments where the DNA vector is a circular DNA vector, the circular DNA vector lacks an origin of replication (e.g., a bacterial origin of replication), a drug resistance gene, and / or a recombination site.

[0047] In some aspects, the nucleic acid vector encodes a therapeutic replacement protein that replaces a protein that is endogenously expressed in healthy retinal cells, hi some aspects, the therapeutic replacement protein replaces a protein that is not endogenously expressed in target cells of an individual or that is non-functional in target cells of an individual.

[0048] In some embodiments, the therapeutic replacement protein is encoded by a coding sequence of more than 4.5 kb. In some embodiments, the therapeutic replacement protein is MYO7A. In some embodiments, the method is a method of treating Usher syndrome type 1B in an individual.

[0049] In some embodiments, the therapeutic replacement protein is BEST1. In some embodiments, the method is a method of treating a bestrophinopathy associated with a BEST1 dominant mutation or a BEST1 recessive mutation.

[0050] In some embodiments, the therapeutic replacement protein is CFH. In some embodiments, the method is a method of treating age-related macular degeneration.

[0051] In another aspect, the present invention provides a method for treating retinal dystrophy, comprising suprachoroidally injecting a circular DNA vector (e.g., a naked circular DNA vector) into the eye of an individual having a retinal dystrophy characterized by the absence of expression of a retinal protein. In some embodiments, the circular DNA vector comprises one or more therapeutic genes encoding therapeutic replacement proteins for replacing the retinal protein. In some embodiments, the circular DNA vector lacks a bacterial origin of replication and / or a drug resistance gene (e.g., the circular DNA vector lacks a bacterial origin of replication, a drug resistance gene, and a recombination site). In some embodiments, the method further comprises: (a) contacting an electrode with an internal region of the eye; and (b) transmitting one or more pulses of electrical energy (e.g., electrical current) through the electrode while the electrode is contacting the internal region of the eye, under conditions suitable for electrical introduction of the circular DNA vector into a target retinal cell. In some embodiments, the electrode is a monopolar electrode. In some embodiments, the electrode is a bipolar electrode.

[0052] In some embodiments, the interior region of the eye in contact with the electrode includes the vitreous humor (e.g., the electrode is entirely within the vitreous humor). In some embodiments, the electrode is between 0.1 mm and 10 mm from the retina at the time of delivery of the one or more pulses of electrical energy.

[0053] In some embodiments, the interior region of the eye that is contacted by the electrode includes the retina, for example, the electrode may be completely within the subretinal space or may be partially within the subretinal space.

[0054] In some embodiments, conditions suitable for electrical delivery of a therapeutic agent to a target retinal cell include a voltage of 1 V / cm to 1,500 V / cm (1 V / cm to 10 V / cm (e.g., about 1 V / cm, about 2 V / cm, about 3 V / cm, about 4 V / cm, about 5 V / cm, about 6 V / cm, about 7 V / cm, about 8 V / cm, about 9 V / cm, or about 10 V / cm) at the target retinal cell, about 10 V / cm to about 100 V / cm (e.g., about 10 V / cm, about 20 V / cm, about 30 V / cm, about 40 V / cm, about 50 V / cm, about 60 V / cm, about 70 V / cm, about 80 V / cm, In some embodiments, the electric field strength at the target cell is between 50V / cm and 300V / cm. In some embodiments, the electric field strength at the target cell is about 100 V / cm.

[0055] In some embodiments, 4-12 pulses (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 pulses) of energy are delivered, while in some embodiments, 10-20 pulses (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pulses) are administered.

[0056] In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 20 seconds. For example, the total number of pulses of electrical energy can be delivered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 seconds. The pulses of electrical energy can be, for example, a square waveform. The pulses of electrical energy can have an amplitude of 5V to 1,500V. For example, the pulses of electrical energy can have an amplitude of about 5V to 500V, about 500V to about 1,000V, or about 1,000V to about 1,500V. In some embodiments, the pulses of electrical energy have an amplitude of 5V to 500V. For example, the pulse of electrical energy can have an amplitude of about 100V, 125V, 150V, 175V, 200V, 225V, 250V, 275V, 300V, 325V, 350V, 375V, 400V, 425V, 450V, 475V, or 500V. In some embodiments, the pulse of electrical energy has an amplitude of about 5V to about 250V.

[0057] In some embodiments, each of the pulses is about 0.01 ms to about 200 ms in duration, about 0.1 ms to about 200 ms in duration, or about 1 ms to about 200 ms in duration (e.g., 0.10 ms to about 200 ms in duration. For example, each of the pulses can be about 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 110 ms, 120 ms, 130 ms, 140 ms, 150 ms, 160 ms, 170 ms, 180 ms, 190 ms, or 200 ms in duration). In some embodiments, each of the pulses is about 20 ms in duration. In some embodiments, each of the pulses is about 50 ms in duration.

[0058] In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 20 seconds (e.g., within 6 to 12 seconds, e.g., within 1 to 3 seconds, within 3 to 6 seconds, within 6 to 10 seconds, within 10 to 15 seconds, or within 15 to 20 seconds, e.g., within 1 second, within 2 seconds, within 3 seconds, within 4 seconds, within 5 seconds, within 6 seconds, within 7 seconds, within 8 seconds, within 9 seconds, within 10 seconds, within 11 seconds, within 12 seconds, within 13 seconds, within 14 seconds, within 15 seconds, within 16 seconds, within 17 seconds, within 18 seconds, within 19 seconds, within 20 seconds).

[0059] In some embodiments, the pulse of energy is a square waveform. In some embodiments, the pulse of energy has an amplitude of 100V to 500V (e.g., 200V to 400V, e.g., 100V to 200V, 200V to 300V, 300V to 400V, or 400V to 500V, e.g., about 100V, about 120V, about 150V, about 200V, about 250V, about 300V, about 350V, about 400V, about 450V, or about 500V).

[0060] In some embodiments, the target retinal cells are retinal epithelial cells. In some embodiments, the target retinal cells are photoreceptors. In some embodiments, the target retinal cells are retinal epithelial cells and photoreceptors.

[0061] In some embodiments, the therapeutic agent is a nucleic acid vector, e.g., a DNA vector or an RNA vector. In some embodiments, the nucleic acid vector is a non-viral nucleic acid vector (e.g., a non-viral DNA vector or a non-viral RNA vector; e.g., a circular DNA vector or a circular RNA vector). In certain cases, the non-viral nucleic acid vector is a naked nucleic acid vector (e.g., a naked DNA vector (e.g., a naked circular DNA vector) or a naked RNA vector (e.g., a naked circular RNA vector). In some embodiments where the DNA vector is a circular DNA vector, the circular DNA vector lacks an origin of replication (e.g., a bacterial origin of replication), a drug resistance gene, and / or a recombination site.

[0062] In some aspects, the nucleic acid vector encodes a therapeutic replacement protein that replaces a protein that is endogenously expressed in healthy retinal cells, hi some aspects, the therapeutic replacement protein replaces a protein that is not endogenously expressed in target cells of an individual or that is non-functional in target cells of an individual.

[0063] In some embodiments, the therapeutic replacement protein is encoded by a coding sequence of more than 4.5 kb. In some embodiments, the therapeutic replacement protein is MYO7A. In some embodiments, the method is a method of treating Usher syndrome type 1B in an individual.

[0064] In some embodiments, the therapeutic replacement protein is BEST1. In some embodiments, the method is a method of treating a bestrophinopathy associated with a BEST1 dominant mutation or a BEST1 recessive mutation.

[0065] In some embodiments, the therapeutic replacement protein is CFH. In some embodiments, the method is a method of treating age-related macular degeneration.

[0066] In some embodiments, the therapeutic replacement protein is shown in Table 1.

[0067] The present invention also provides devices and methods for delivering therapeutic agents (e.g., nucleic acid vectors) to target cells via electrical transfer. Such devices and methods generally employ the transmission of an electric field into a tissue by the device to facilitate the delivery of therapeutic agents to target cells within the tissue. The devices are designed to transmit an electric field whose shape matches the internal topography of the target tissue interface (e.g., a substantially flat, curved, or spherical topography), thereby increasing the number of target cells exposed to the effective electric field, which in turn improves the efficiency of electrical transfer of therapeutic agents. In certain uses of such devices, retinal cells can be transfected with high efficiency using nucleic acid vectors.

[0068] In one aspect, the device includes a sheath having a proximal end, a distal end, and a longitudinal axis therebetween. The device further includes an elongated conductor having a proximal portion and a distal portion within the sheath, the elongated conductor including a preformed shape memory material and retractable within the sheath from a proximal position to a distal position. In the proximal position, the distal portion of the elongated conductor is substantially straight. In the distal position, the distal portion of the elongate conductor extends beyond the distal end of the sheath, and the shape memory material of the distal portion of the elongate conductor is radially relaxed to form a preformed angle (e.g., about 10° to about 170°, e.g., about 20° to about 160°, e.g., about 30° to about 150°, e.g., about 45° to about 135°, e.g., about 60° to about 120°, e.g., about 70° to about 110°, e.g., about 80° to about 100°, e.g., about 85° to about 95°, e.g., about 10°, 20°, 30°, 45°, 50°, 55°, 60°, 65°) relative to the longitudinal axis of the sheath. , 70°, 71°, 72°, 73°, 74°, 75°, 76°, 77°, 78°, 79°, 80°, 81°, 82°, 83°, 84°, 85°, 86°, 87°, 88°, 89°, 90°, 91°, 92°, 93°, 94°, 95°, 96°, 97°, 98°, 99°, 100°, 101°, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°, 150°, 160°, or 170°). In some embodiments, the electrode is a substantially planar electrode. In some embodiments, the preformed angle is substantially a right angle. In some embodiments, the preformed angle is about 70° or about 110°.

[0069] In another aspect, the device includes a sheath having a proximal end, a distal end, and a longitudinal axis therebetween. The device further includes an elongated conductor having a proximal portion and a distal portion within the sheath, the elongated conductor including a preformed shape memory material and retractable within the sheath from a proximal position to a distal position. In the proximal position, the distal portion of the elongated conductor is substantially straight. In the distal position, the distal portion of the elongated conductor extends beyond the distal end of the sheath, and the shape memory material of the distal portion of the elongated conductor is radially relaxed to form an electrode approximately perpendicular to the longitudinal axis of the sheath. In some aspects, the electrode is a substantially flat electrode.

[0070] In another aspect, the device includes a sheath having a proximal end, a distal end, and a longitudinal axis therebetween. The device further includes an elongated conductor having a proximal portion and a distal portion within the sheath, the elongated conductor including a preformed shape memory material and retractable within the sheath from a proximal position to a distal position. In the proximal position, the distal portion of the elongated conductor is substantially straight. In the distal position, the distal portion of the elongated conductor extends beyond the distal end of the sheath, and the shape memory material of the distal portion of the elongated conductor is radially relaxed to form an electrode that is substantially perpendicular to the longitudinal axis of the sheath. In some embodiments, the electrode is a substantially flat electrode. In some embodiments, the substantially perpendicular angle is about 70° or about 110°.

[0071] In some embodiments, the device further comprises a handle having a proximal end and a distal end, and the sheath may be connected (e.g., secured) to the handle.

[0072] In some embodiments, the proximal end of the sheath is connected to (eg, disposed within) the handle.

[0073] In some aspects, the distal portion of the handle includes a hollow region between an inner surface of the handle and the elongate conductor therein, and the proximal end of the sheath is disposed within the hollow region in the handle.

[0074] In some embodiments, the proximal end of the sheath is disposed within the hollow region at least 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 55 mm, 60 mm, 65 mm, 70 mm, 80 mm, 85 mm, 90 mm, 95 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, 200 mm, or more.

[0075] In some embodiments, the handle is cylindrical.

[0076] In some embodiments, the handle further comprises a cap at the distal and / or proximal end of the handle.

[0077] In some aspects, the device further includes an actuator configured to slide the elongated conductor between the proximal and distal positions.

[0078] In some embodiments, the proximal end of the sheath and / or the elongated conductor are connected to an actuator. The actuator can be configured to slide the elongated conductor between a proximal position and a distal position. In some embodiments, the actuator is a slider. The slider has a proximal end and a distal end and is attached to the elongated conductor. The slider is configured to retract the elongated conductor from the distal position to the proximal position along the longitudinal axis of the sheath.

[0079] In some aspects, the slider includes a proximal position and a distal position. In the proximal position, the proximal end of the sheath can be disposed at or proximal to the distal end of the slider. In the distal position, the proximal end of the sheath can be disposed between the proximal end of the slider and the distal end of the slider.

[0080] In some embodiments, the slider is configured to stop upon sliding to a distal position and / or a proximal position.

[0081] In some aspects, the slider is disposed at a distal location and the distal portion of the elongate conductor extends beyond the distal end of the sheath, and the shape memory material of the distal portion of the elongate conductor can be radially relaxed to form a substantially planar electrode at a preformed angle relative to the longitudinal axis of the sheath.

[0082] In some aspects, the slider is disposed at a proximal position and the distal portion of the elongate conductor is substantially straight.

[0083] In some aspects, the slider includes a control member disposed externally of the handle. The control member and the slider may be integral. Alternatively, the control member and the slider may not be integral.

[0084] In another aspect, the device includes a handle having a proximal end and a distal end. The device further includes a sheath having a proximal end, a distal end, and a longitudinal axis therebetween. The sheath may be connected to (e.g., immobilized) the handle. The proximal end of the sheath may be connected to (e.g., disposed within) the handle. The device also includes an elongated conductor having a proximal portion and a distal portion within the sheath, the elongated conductor including a preformed shape memory material and retractable within the sheath from a proximal position to a distal position. In the proximal position, the distal portion of the elongated conductor is substantially straight. In the distal position, the distal portion of the elongate conductor extends beyond the distal end of the sheath, and the shape memory material of the distal portion of the elongate conductor is radially relaxed to form a preformed angle (e.g., about 10° to about 170°, e.g., about 20° to about 160°, e.g., about 30° to about 150°, e.g., about 45° to about 135°, e.g., about 60° to about 120°, e.g., about 70° to about 110°, e.g., about 80° to about 100°, e.g., about 85° to about 95°, e.g., about 10°, 20°, 30°, 45°, 50°, 55°, 60°, 65°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, 170°, 180°, 190°, 20°, 210°, 220°, 230°, 240°, 250°, 260°, 270°, 280°, 290°, 30°, 32°, 33°, 34°, 35°, 36°, 37°, 38°, 39°, 40°, 41°, 42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°, 50°, 55°, 50°, 55°, 56°, 57°, 58°, 59° 95°, 96°, 97°, 98°, 99°, 100°, 101°, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°, 150°, 160°, or 170°). The device also includes a slider having a proximal end and a distal end and attached to the elongated conductor. The slider is configured to retract the elongated conductor from a distal position to a proximal position along the longitudinal axis of the sheath. In some embodiments, the preformed angle is about 70° or about 110°.

[0085] In some aspects, the device further includes a sheath connected (e.g., fixed) to the slider. The elongated conductor may be present within the sheath connected to the slider. In some aspects, the sheath connected to the slider is nested with the sheath connected to the handle (e.g., fixed). The sheath connected to the slider may be configured to be surrounded by the sheath connected to the handle or a portion thereof. For example, the sheath connected to the slider may have a diameter smaller than the diameter of the sheath connected to the handle. Alternatively, the sheath connected to the slider may surround the sheath connected to the handle or a portion thereof. For example, the sheath connected to the slider may have a diameter larger than the diameter of the sheath connected to the handle. In some aspects, the sheath connected to the slider is connected to the elongated conductor, for example, at a proximal end of the elongated conductor.

[0086] In some embodiments, the distal end of the sheath comprises a needle (eg, a hypodermic needle).

[0087] In some embodiments, the device further comprises insulation, for example, between a proximal portion of the elongate conductor and the sheath.

[0088] In some embodiments, the sheath comprises an electrically conductive material.

[0089] The inner diameter of the sheath can be from about 0.01 mm to about 5 mm, for example, from about 0.1 mm to about 1 mm, for example, from about 0.2 mm to about 0.5 mm, for example, from about 0.2 mm to about 0.3 mm. For example, the inner diameter of the sheath can be 0.01 mm to about 5 mm, for example, about 0.01 mm to about 0.1 mm, for example, about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, for example, about 0.1 mm to about 1 mm, for example, about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm, for example, about 1 mm to about 5 mm, for example, about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm. In some embodiments, the sheath has an inner diameter of about 0.1 mm to about 1 mm. In some embodiments, the sheath has an inner diameter of about 0.2 mm to about 0.3 mm.

[0090] The outer diameter of the sheath, which is larger than the inner diameter, can be from about 0.01 mm to about 5 mm, for example, from about 0.1 mm to about 1 mm, for example, from about 0.2 mm to about 0.5 mm, for example, from about 0.2 mm to about 0.3 mm. For example, the inner diameter of the sheath can be from 0.01 mm to about 5 mm, for example, from about 0.01 mm to about 0.1 mm, for example, about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, for example, from about 0.1 mm to about 1 mm, for example, about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm, for example, from about 1 mm to about 5 mm, for example, about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0091] The thickness of the sheath can be about 0.01 mm to about 1 mm, e.g., about 0.01 mm to about 0.1 mm, e.g., about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, e.g., about 0.1 mm to about 1 mm, e.g., about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm. The thickness of the sheath may be substantially uniform throughout, or may have different thicknesses in different portions or regions of the sheath.

[0092] The diameter of the conductor can be about 50% to about 99% of the inner diameter of the sheath. For example, the diameter can be about 55% to about 95%, about 60% to about 90%, about 65% to about 85%, 70% to about 80%, or about 75%.

[0093] The elongated conductor may be substantially cylindrical (e.g., cylindrical wire). The cross section of the sheath may be substantially circular or elliptical. The diameter of the conductor may be about 0.01 mm to about 5 mm, for example, about 0.1 mm to about 1 mm, for example, about 0.2 mm to about 0.5 mm, for example, about 0.2 mm to about 0.3 mm. For example, the inner diameter of the sheath can be 0.01 mm to about 5 mm, for example, about 0.01 mm to about 0.1 mm, for example, about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, for example, about 0.1 mm to about 1 mm, for example, about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm, for example, about 1 mm to about 5 mm, for example, about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm. In some embodiments, the diameter of the conductor is about 0.2 mm. In some embodiments, the elongated conductor has a diameter of about 100 μm to about 200 μm. In some embodiments, the elongated conductor has a diameter of about 150 μm.

[0094] The diameter of the conductor may be substantially uniform throughout, or may have different thicknesses in different portions or regions of the conductor.

[0095] In some embodiments, a substantially flat electrode is about 2 mm to about 15 mm (e.g., about 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm) in one or more dimensions (e.g., both dimensions) perpendicular to the longitudinal axis.

[0096] In some aspects, the substantially planar electrode is substantially symmetric about the longitudinal plane.

[0097] In some embodiments, the substantially planar electrode is convex.

[0098] In some embodiments, the elongated conductor is a wire and the substantially planar electrode comprises a distal portion of the wire.

[0099] In some aspects, the distal portion of the wire includes a preformed angle (e.g., a preformed right angle) on the longitudinal plane, where the preformed angle (e.g., the preformed right angle) is between the substantially planar electrode and the proximal portion of the wire.

[0100] In some embodiments, the substantially flat electrode is a spiral. For example, the spiral can include about 1 to about 5 (e.g., 1, 2, 3, 4, or 5) turns about the longitudinal axis. In some embodiments, the spiral includes (e.g., consists of) 3 turns about the longitudinal axis. In some embodiments, the spiral includes (e.g., consists of) 2 turns about the longitudinal axis. For example, FIG. 3 shows a spiral having 2 turns about its longitudinal axis.

[0101] In some embodiments, the substantially flat electrode extends 1 mm or less (e.g., 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm or less) distal or proximal from the preformed angle (e.g., a preformed right angle). In some embodiments, the substantially flat electrode extends 1 mm or less (e.g., 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm or less) distal from the preformed angle (e.g., a preformed right angle). In some embodiments, the device does not include anything distal to the substantially flat electrode.

[0102] In some embodiments, the device is monopolar.

[0103] In some embodiments, the device is bipolar, where the device further comprises an auxiliary electrode in electrical communication with the substantially planar electrode. The auxiliary electrode may be part of the sheath or may be connected to the sheath.

[0104] In some embodiments, the proximal portion of the elongate conductor is connected to a voltage source and / or a waveform controller.

[0105] In another aspect, the invention features a method of delivering an agent (e.g., an agent of interest, e.g., a therapeutic agent) to a target cell of a patient using the device described herein. In some embodiments, the invention features a method of delivering an agent (e.g., an agent of interest (e.g., a therapeutic agent) or a sequence of interest (e.g., a therapeutic sequence)) to a target cell of a patient using the device described herein. The method includes inserting a sheath (or a sheath including a needle) through an external tissue surface (e.g., the sclera) of the subject, and sliding an elongated conductor to a distal position to form a substantially planar electrode. The method further includes placing the substantially planar electrode in electrical communication with a tissue interface separating the target cell from the substantially planar electrode. The method also includes transmitting one or more pulses of electrical energy through the substantially planar electrode under conditions suitable for electrical introduction of the agent (e.g., a therapeutic agent) to the target cell. In some embodiments where the therapeutic agent is a nucleic acid vector (e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector), the nucleic acid is expressed by the target cell (e.g., a retinal cell, e.g., an RPE cell and / or a photoreceptor cell). Thus, the delivery methods described herein can potentially be methods for expressing a sequence of interest (e.g., a therapeutic sequence).

[0106] In some embodiments, a substantially flat electrode is within about 10 mm (e.g., within 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, 0.5 mm, 0.45 mm, 0.40 mm, 0.35 mm, 0.30 mm, 0.25 mm, 0.20 mm, 0.15 mm, 0.10 mm, 0.05 mm, or less) of the tissue interface. A substantially planar electrode can be 0.05 mm to 5 mm (e.g., about 0.5 mm, 0.10 mm, 0.15 mm, 0.20 mm, 0.25 mm, 0.30 mm, 0.35 mm, 0.40 mm, 0.45 mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm) from the tissue interface upon delivery of one or more pulses. In some embodiments, a substantially planar electrode is about 1 mm from the tissue interface upon delivery of one or more pulses.

[0107] The target cells can be within about 5 mm (e.g., 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, 0.5 mm, 0.45 mm, 0.4 mm, 0.35 mm, 0.30 mm, 0.25 mm, 0.20 mm, 0.15 mm, 0.10 mm, or 0.05 mm) of the tissue interface. For example, the target cells can be about 0.01 mm to about 1 mm (e.g., about 0.01 mm to about 0.1 mm, e.g., about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, e.g., about 0.1 mm to about 1 mm, e.g., about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm) from the tissue interface.

[0108] In some embodiments, conditions suitable for electrical introduction of an agent (e.g., a therapeutic agent) into a target cell include a voltage of about 10 V / cm to about 1,500 V / cm (e.g., about 10 V / cm to about 100 V / cm, e.g., about 10 V / cm, 20 V / cm, 30 V / cm, 40 V / cm, 50 V / cm, 60 V / cm, 70 V / cm, 80 V / cm, 90 V / cm, or 100 V / cm, e.g., about 100 V / cm to about 1, The electric field strength may be about 1,000V / cm, e.g., about 200V / cm, 300V / cm, 400V / cm, 500V / cm, 600V / cm, 700V / cm, 800V / cm, 900V / cm, or 1,000V / cm, e.g., about 1,000V / cm to about 1,500V / cm, e.g., about 1,110V / cm, 1,200V / cm, 1,300V / cm, 1,400V / cm, or 1,500V / cm). In some embodiments, the electric field strength in the target cells is 50V / cm to 300V / cm. In some embodiments, the electric field strength in the target cells is about 100V / cm.

[0109] In some embodiments, 1 to 12 pulses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 pulses) of electrical energy are delivered. In some embodiments, 2 to 12 pulses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 pulses) of electrical energy are delivered. In some embodiments, 3 to 12 pulses (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 pulses) of electrical energy are delivered. In some embodiments, 4 to 12 pulses (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 pulses) of electrical energy are delivered.

[0110] In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 20 seconds. For example, the total number of pulses of electrical energy can be delivered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 seconds. The pulses of electrical energy can be, for example, a square waveform. In some embodiments, the pulses of electrical energy have an amplitude of 5V to 1,500V. For example, the pulses of electrical energy can have an amplitude of about 5V to 500V, about 500V to about 1,000V, or about 1,000V to about 1,500V. In some embodiments, the pulses of electrical energy have an amplitude of 5V to 500V. For example, the pulse of electrical energy can have an amplitude of about 100V, 125V, 150V, 175V, 200V, 225V, 250V, 275V, 300V, 325V, 350V, 375V, 400V, 425V, 450V, 475V, or 500V. In some embodiments, the pulse of electrical energy has an amplitude of about 5V to about 250V.

[0111] In some embodiments, conditions suitable for electrical introduction of an agent into a target cell include a voltage at the target cell of 5V to 100V (e.g., 10V to 80V, 15V to 70V, 20V to 60V, or 30V to 50V; e.g., about 10V, about 15V, about 20V, about 25V, about 30V, about 35V, about 40V, about 45V, about 50V, about 55V, about 60V, about 65V, or about 70V).

[0112] In some embodiments, each of the pulses is about 1 ms to about 200 ms, for example, about 1 ms to about 100 ms. For example, each of the pulses can be about 1 ms, 2 ms, 3 ms, 4 ms, 5 ms, 6 ms, 7 ms, 8 ms, 9 ms, 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 110 ms, 120 ms, 130 ms, 140 ms, 150 ms, 160 ms, 170 ms, 180 ms, 190 ms, or 200 ms. In some embodiments, each of the pulses is from about 50 ms.

[0113] In some embodiments, the agent (e.g., therapeutic agent) has been previously administered to the tissue. In other embodiments, the method further comprises administering the agent (e.g., therapeutic agent). The agent (e.g., therapeutic agent) may be administered simultaneously or sequentially with one or more pulses.

[0114] In any of the foregoing embodiments, the agent (e.g., therapeutic agent) can be a nucleic acid (e.g., a non-viral nucleic acid, such as a non-viral particle nucleic acid or a naked nucleic acid). The nucleic acid can be DNA or RNA (e.g., circular DNA or circular RNA).

[0115] In some embodiments, the target cell is a retinal cell. The retinal cell can be, for example, a retinal pigment epithelial (RPE) cell, a photoreceptor cell, or a ganglion cell.

[0116] In some aspects, the therapeutic agent is administered intravitreally, subretinally, or topically to the eye.

[0117] In some aspects, the therapeutic agent is administered to the suprachoroid. [Brief description of the drawings]

[0118] [Figure 1] FIG. 1 is an anatomical cross-sectional view of the eye showing structures relevant to the methods described herein. [Diagram 2] 2A-2D are diagrams illustrating a method of delivering a therapeutic agent to a target retinal cell of an individual. The white lines represent the flow of current. FIG. 2A illustrates intravitreal pulsed electric field transmission in which a positive voltage is transmitted by a monopolar needle electrode in the vitreous humor near the target retinal cell. FIG. 2B illustrates subretinal pulsed electric field transmission in which a negative voltage is transmitted by a monopolar needle electrode in the subretinal space (e.g., in a bleb) at or near the target retinal cell. FIG. 2C illustrates intravitreal pulsed electric field transmission in which a positive voltage is transmitted by a monopolar planar electrode in the vitreous humor near the target retinal cell. FIG. 2D illustrates intravitreal pulsed electric field transmission in which a negative voltage is transmitted by a monopolar planar electrode in the vitreous humor near the target retinal cell. [Diagram 3]3A-3E are diagrams illustrating a method of suprachoroidal delivery of a therapeutic agent to a target retinal cell of an individual. The white lines represent the flow of current. FIG. 3A illustrates suprachoroidal injection of a pharmaceutical composition. The white arrows indicate the distribution path of the pharmaceutical composition upon injection throughout the suprachoroidal space toward the posterior region of the eye (i.e., toward the target retinal cell, e.g., toward the macula). FIG. 3B illustrates intravitreal pulsed electric field transmission in which a positive voltage is transmitted by a monopolar needle electrode in the vitreous humor near the target retinal cell. FIG. 3C illustrates intravitreal pulsed electric field transmission in which a negative voltage is transmitted by a monopolar needle electrode in the vitreous humor near the target retinal cell. FIG. 3D illustrates intravitreal pulsed electric field transmission in which a positive voltage is transmitted by a planar needle electrode in the vitreous humor near the target retinal cell. FIG. 3E illustrates intravitreal pulsed electric field transmission in which a negative voltage is transmitted by a planar needle electrode in the vitreous humor near the target retinal cell. [Figure 4] 4A and 4B are schematic diagrams showing a device described herein. Fig. 4A shows a cross-section of a device with a sheath and an elongated conductor in a retracted position such that the distal portion of the elongated conductor is substantially straight. The insulation between the elongated conductor and the sheath is also shown. Fig. 4B shows the device with the elongated conductor in a deployed position such that the distal portion of the elongated conductor is radially relaxed to form a substantially flat electrode perpendicular to the longitudinal axis of the sheath. [Diagram 5] 5 is a schematic diagram of a bipolar device with an elongated conductor in a deployed position such that a distal portion of the elongated conductor is radially relaxed to form a substantially planar electrode perpendicular to the longitudinal axis of the sheath. An auxiliary electrode is present on the device at the outer surface of the sheath, proximal to the distal end of the sheath. [Figure 6] 6 is a schematic diagram showing a substantially flat electrode in a deployed position. The elongated conductor is helical in shape, making approximately two turns about the longitudinal axis. Also shown is the insulation between the elongated conductor and the sheath. [Figure 7]7A-7C are a series of electrical simulation plots showing the voltage distribution (V / cm) across a cross section of an eye containing a needle electrode posterior to the vitreous humor. FIG. 7A shows a needle electrode offset by 0.25 mm from the vitreous humor-retina interface. FIG. 7B is an enlarged view of a portion of FIG. 7A showing details of the vitreous humor-retina interface. FIG. 7C shows a needle electrode offset by 0.95 mm from the vitreous humor-retina interface. [Figure 8] 8A-8C are a series of electrical simulation plots showing the voltage distribution (V / cm) across a cross section of an eye including a substantially flat electrode posterior to the vitreous humor. FIG. 8A shows a needle electrode offset from the vitreous humor-retina interface by 0.95 mm. FIG. 8B is an enlarged view of a portion of FIG. 8A showing details of the vitreous humor-retina interface. FIG. 8C shows a substantially flat electrode offset from the vitreous humor-retina interface by 0.25 mm. [Figure 9] 9A and 9B are a set of simulated plots showing the voltage (e.g., potential) across a cross section of an eye containing a 20 V electrode posterior to the vitreous humor (0.4 mm from the vitreous humor-retina interface). Figure 9A shows a needle electrode. Figure 9B shows a spiral (substantially flat) electrode. [Figure 10] 10 is a schematic diagram of a device having a handle and a slider, with the proximal end of the sheath disposed on the distal end of the handle. The elongated conductor is disposed along the longitudinal axis within the handle and attached to the slider. [Figure 11] FIG. 11 is a schematic diagram of a device having a handle and slider, where the proximal end of the sheath extends beyond the surface of the distal end of the handle into a hollow region of the handle. [Figure 12]12A-12C are schematic diagrams of a device having a handle and a slider. The handle is cylindrical and includes a cap at each of the distal and proximal ends. The slider further includes a control member that fits within the handle and moves the slider. FIG. 12A shows a device having a first sheath connected to an elongated conductor. The device further includes a second sheath connected to the slider. FIG. 12B shows an exploded view of the handle and slider. The slider may include an internal element connected to the handle. FIG. 12C shows a perspective view of FIG. 12B. [Figure 13] 13 is a set of schematic diagrams showing the dimensions of the cap located at the distal end of the slider, shown in inches. [Figure 14] 14 is a set of schematic diagrams showing the dimensions of the cap located at the proximal end of the slider, shown in inches. [Figure 15] 15 is a set of diagrams showing the dimensions of an exemplary handle, shown in inches. [Figure 16] Figure 16 is a set of schematic diagrams showing the dimensions of a sheath (18 gauge hypodermic needle), shown in inches. [Figure 17] FIG. 17 is a set of schematic diagrams showing the dimensions of the handle control members, shown in inches. [Figure 18] Figure 18 is a schematic diagram showing the dimensions of the insulator (polyimide tube), in inches. [Figure 19] Figure 19 is a set of schematic diagrams showing the dimensions of the slider, shown in inches. [Figure 20] Figure 20 is a set of schematic diagrams showing the dimensions of a sheath (23 gauge hypodermic needle), units are shown in inches. [Figure 21]Figures 21A and 21B are confocal scanning laser ophthalmoscopy (cSLO) images measuring GFP fluorescence in pig eyes after electrical introduction of GFP-expressing DNA. Figure 21A shows the fluorescence at baseline (before electrical introduction) from the nasal (left) or temporal (right) direction. Figure 21B shows the fluorescence 7 days (final endpoint) after electrical introduction from the nasal (left) or temporal (right) direction. [Figure 22] Figures 22A-22D are optical coherence tomography (OCT) images showing structural integrity in pig eyes after electrical introduction of GFP-expressing DNA and no detectable inflammation. Figures 22A and 22B are images from baseline (before electrical introduction). Figures 22C and 22D are images 7 days (final endpoint) after electrical introduction from either the nasal or temporal direction. [Figure 23] Figures 23A and 23B are photomicrographs showing the histology of an adult pig eye after electrotransfer of a synthetic circular DNA vector encoding GFP using a monopolar needle electrode placed in the subretinal bleb for electrotransfer. Figure 23A shows immunohistochemistry (IHC) where GFP expression (blue staining) is detected in both the photoreceptor (PR) and retinal pigment epithelium (RPE) layers. Cone opsin is stained yellow. Figure 23B shows H&E staining of the retina after electrotransfer, showing preservation of retinal cytoarchitecture. [Figure 24] Figures 24A and 24B are photomicrographs showing IHCs from adult pig eyes after electrotransfer of a synthetic circular DNA vector encoding GFP using a monopolar needle electrode, the distal end of which was placed within the vitreous within 1 mm of the retina. In Figure 24A, GFP is stained blue and RPE65 is stained yellow. In Figure 24B, GFP is stained blue and cone opsin is stained yellow. [Diagram 25] Figure 25 is a photomicrograph showing IHC of an adult pig eye after electrotransfer of a synthetic circular DNA vector encoding GFP using a bipolar needle electrode with the distal end (negative electrode) placed in the subretinal bleb and the positive electrode on the needle proximal to the negative electrode placed in the vitreous. GFP is stained blue and cone opsin is stained yellow. [Figure 26]Figures 26A and 26B are photomicrographs showing the histology of an adult pig eye after administration of a synthetic circular DNA vector encoding GFP without electrotransduction. Figure 26A shows IHC, where no significant GFP expression (blue staining) was observed. Cone opsin stains yellow. Figure 26B shows H&E staining of the retina. [Figure 27] Figures 27A and 27B are photomicrographs showing the histology of adult pig eyes after sham electrotransduction of the PBS control. Figure 27A shows IHC, and no GFP expression (blue staining) was observed. Figure 27B shows H&E staining of the retina after electrotransduction, showing preservation of retinal cytoarchitecture. [Figure 28] Figures 28A and 28B are photomicrographs showing IHC staining of an adult pig eye after electrotransfer of a synthetic circular DNA vector encoding GFP using a substantially flat electrode as shown in Figure 6. Figure 28A shows blue GFP and yellow RPE staining. Figure 28B shows blue GFP and yellow cone opsin staining. [Figure 29] Figures 29A-29E are a set of photomicrographs showing a time course of GFP expression in cultured induced RPE cells. Each figure has four panels; the top left panel of each figure shows GFP fluorescence in cells incubated with a synthetic circular DNA vector encoding GFP in the absence of electrical transfer; the top right panel of each figure shows GFP fluorescence in cells incubated with a synthetic circular DNA vector encoding GFP in the presence of electrical transfer; the bottom left panel of each figure is a brightfield image showing the morphology of induced retinal pigment epithelial (iRPE) cells incubated with a synthetic circular DNA vector encoding GFP in the absence of electrical transfer; the bottom right panel of each figure is a brightfield image showing the morphology of iRPE cells incubated with a synthetic circular DNA vector encoding GFP in the presence of electrical transfer. Figure 29A shows cells at day 4 of the time course, Figure 29B shows cells at day 21 of the time course, Figure 29C shows cells at day 32 of the time course, Figure 29D shows cells at day 40 of the time course, and Figure 29E shows cells at day 49 of the time course. [Diagram 30]Figure 30 is a bar graph showing mRNA expression of a synthetic circular DNA vector encoding the ABCA4 transgene (C3-ABCA4) electrotransferred into porcine eyes in vivo, as measured by qPCR. Endogenous (endo) porcine ABCA4 is shown for comparison. PBS was injected and sham electrotransferred using the same PEF conditions as the negative control. mRNA expression levels were quantified in the neural retina (NR) and RPE / choroid (RPE / Cho). [Diagram 31] Figure 31 is a bar graph showing mRNA expression of synthetic circular DNA vectors encoding GFP and MYO7A transgenes electrotransferred into two pig eyes in vivo, as measured by qPCR. For comparison, endogenous (endo) porcine MYO7A is shown in each eye. mRNA expression levels were quantified in the neural retina (NR) and RPE / choroid (RPE / Cho). [Diagram 32] Figures 32A and 32B are photomicrographs showing the histology of a pig retina 6 days after electrotransfer of an 8,656 bp synthetic circular DNA vector encoding human ABCA4 (C3-ABCA4). Figure 32A shows ABCA4 protein stained blue (indicated by solid arrow) and RPE65 stained brown (indicated by dashed arrow). Figure 32B shows ABCA4 protein stained blue and rhodopsin stained yellow. Arrows indicate double staining (green). [Diagram 33] 33 is a photomicrograph showing the histology of adult pig retina after electrotransfer of C3-ABCA4. ABCA4 protein stains blue (indicated by arrows). [Diagram 34] Figure 34 is a photomicrograph showing the histology of human retina (untreated). Endogenous ABCA4 protein stains blue (indicated by arrows). [Diagram 35]Figure 35 is a photograph of a Western blot showing ABCA4 protein expression in iRPE cells in vitro. Lane 1 is a negative control. Lanes 2-4 were loaded with samples from cells transfected with plasmid (lanes 2 and 3) or synthetic circular DNA vector (lanes 3 and 4). The transgene was the same between lanes 1 and 3 and between lanes 2 and 4, between the plasmid and the synthetic DNA vector. [Diagram 36] Figures 36A-36F are photomicrographs showing the fluorescence of iRPE cells after electroporation-mediated transfection of synthetic circular DNA encoding ABCA4 and a plasmid encoding ABCA4 in vitro. Figures 36A-36C show ZO-1 / GFP (Figure 36A), ABCAA4 (Figure 36B), and superimposed ZO-1 / GFP and ABCA4 (Figure 36C) after transfection with synthetic circular DNA encoding ABCA4. Figures 36D-36F show ZO-1 / GFP (Figure 36D), ABCAA4 (Figure 36E), and superimposed ZO-1 / GFP and ABCA4 (Figure 36F) after transfection with plasmid ABCA4. [Figure 37] Figure 37 is a photograph of a Western blot showing MYO7A protein expression in iRPE cells in vitro. Lane 1 was loaded with a sample from cells transfected with a plasmid encoding GFP. Lanes 2 and 3 were loaded with samples from cells transfected with a plasmid encoding MYO7A. Lane 4 was loaded with a sample from cells transfected with a synthetic circular DNA vector encoding the same MYO7A transgene as lane 3. [Figure 38]Figures 38A-38F are photomicrographs showing fluorescence of iRPE cells after electroporation-mediated transfection of synthetic circular DNA encoding MYO7A and a plasmid encoding MYO7A in vitro. Figures 38A-38C show ZO-1 / GFP (Figure 38A), MYO7A (Figure 38B), and superimposed ZO-1 / GFP and MYO7A (Figure 38C) after transfection with synthetic circular DNA encoding MYO7A. Figures 38D-38F show ZO-1 / GFP (Figure 38D), MYO7A (Figure 38E), and superimposed ZO-1 / GFP and MYO7A (Figure 38F) after transfection with plasmid MYO7A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0119] Detailed Description Provided herein are therapeutic agents (and pharmaceutical compositions thereof) and methods for their delivery to ocular cells, such as retinal cells. Therapeutic agents (e.g., nucleic acid vectors encoding therapeutic proteins) can be delivered to ocular cells (e.g., retinal cells) by injection of the therapeutic agent and / or delivery of electrical energy (e.g., electric current) to a target tissue (e.g., the retina). Thus, in some cases, the approaches described herein include electrotransduction, a process in which delivery of an electric field to an ocular tissue (e.g., the retina) facilitates delivery of a therapeutic agent (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site)) to a target ocular cell (e.g., a retinal cell, e.g., a photoreceptor and / or a retinal pigment epithelial cell). Additionally or alternatively, the methods of the present invention include administration of a therapeutic agent (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site)) to an individual. For example, in certain embodiments of the methods described herein, a therapeutic agent (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene and / or a recombination site)) is administered to an individual (e.g., by suprachoroidal administration) and then delivered to a target cell (e.g., a retinal cell) by electrotransduction.

[0120] The invention also features devices and methods for the electrical introduction of therapeutic agents into target cells, such as retinal cells (e.g., retinal pigment epithelial cells, photoreceptor cells, or ganglion cells). The device includes a sheath having a retractable elongated conductor that transmits electrical energy to the target cells via a substantially planar electrode. The device generates an electric field that is suited to the target tissue topography, increases the area of ​​cells exposed to the electric field, and can tolerate misalignment more than electrodes that lack a planar structure (e.g., conventional needle or wire electrodes). Second, some aspects of the device and its method of use advantageously require lower voltage settings than, for example, needle or straight wire electrodes. The device can provide improved transfection because the electrodes generate an electric field that covers a greater depth and a greater diameter of the target tissue compared to, for example, straight wire electrodes. Additionally, the electrodes cover a larger volume than other devices, such as wire electrodes. The device is also less sensitive to changes in position from the target tissue (e.g., retina) than wire electrodes. Additionally, by providing a rounded or spiral electrode, the device has an atraumatic interface with its target (eg, the retina) as opposed to a sharp feature that points at the target.

[0121] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and with reference to published texts which provide those skilled in the art with a general guide to many of the terms used in this application. In the event of a conflict between a definition set forth herein and a definition in a referenced publication, the definition provided herein shall control.

[0122] As used herein, the terms "superior choroid" and "superior choroidal space" are used interchangeably to refer to the space (or volume) and / or potential space (or potential volume) in the region of the eye between the sclera and the choroid, which joins anteriorly in the region of the scleral spur and posteriorly by the transscleral connection of the short posterior ciliary vessels to the choroid. The suprachoroidal space is primarily composed of dense layers of long pigmented projections from each of the two adjacent tissues; however, spaces can arise in this region as a result of accumulation of fluid or other substances in the suprachoroidal space and adjacent tissues. The suprachoroidal space can be expanded by accumulation of fluid due to some disease state of the eye or as a result of some trauma or surgical intervention. In some embodiments, fluid accumulation is intentionally created by injection of a pharmaceutical composition into the suprachoroidal space to create and / or further expand the suprachoroidal space.

[0123] As used herein, the term "microneedle" refers to a conduit body having a base, shaft, and tip suitable for insertion into the sclera and / or other ocular tissue, and has dimensions suitable for minimally invasive insertion and injection of formulations as described herein. The length of the microneedle (i.e., the length of the shaft of the microneedle and the bevel height of the microneedle) does not exceed 2 mm, and the diameter of the microneedle does not exceed 600 microns.

[0124] As used herein, "electrotransduction" refers to the movement of a molecule (e.g., a nucleic acid, e.g., a naked nucleic acid) across the membrane of a target cell (e.g., from the outside to the inside of a target cell, e.g., a retinal cell) caused by the delivery of an electric field (e.g., a pulsed electric field) to the microenvironment in which the cell resides (e.g., the retina). Electrotransduction can occur as a result of electrophoresis, i.e., the movement of a molecule (e.g., a nucleic acid, e.g., a naked nucleic acid) along an electric field (e.g., in the direction of the electric current) based on the charge of the molecule. Electrophoresis can induce electrotransduction by, for example, moving the molecule (e.g., a nucleic acid, e.g., a naked nucleic acid) close to the cell membrane to allow in vivo transport processes (e.g., endocytosis, including pinocytosis or phagocytosis) or passive transport (e.g., diffusion or lipid partitioning) to carry the molecule into the cell. Additionally or alternatively, electrical introduction can occur as a result of electroporation, i.e., the generation of pores in target cells caused by the transmission of an electric field (e.g., a pulsed electric field), where the size, shape, and duration of the pores are suitable for accommodating the movement of molecules (e.g., nucleic acids, e.g., naked nucleic acids) from the outside of the target cell to the inside of the target cell. Thus, in some cases, electrical introduction occurs as a result of a combination of electrophoresis and electroporation.

[0125] As used herein, the term "relax" and its grammatical derivatives refer to the change in shape of a structure from a constrained shape to an unconstrained shape driven by the unloading of elastic potential energy. Shape memory materials (e.g., shape memory alloys, e.g., NiTi) can relax to a preformed shape when structural constraints are removed. For example, a preformed shape memory wire housed in a rigid sheath is straight until it is unsheathed, at which point the shape memory material relaxes to its preformed shape.

[0126] As used herein, a "spiral" refers to a path of points in a plane that moves around a center point while either moving away from the center point or moving toward the center point.

[0127] As used herein, a "substantially flat electrode" refers to an electrode in which two of its vertical dimensions (e.g., Cartesian dimensions, e.g., depth and width) are each at least twice its third vertical dimension (e.g., length). In some embodiments, a substantially flat electrode refers to an electrode in which two of its vertical dimensions are each at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, or at least 100 times or more than its third vertical dimension.

[0128] As used herein, the term "circular DNA vector" refers to a DNA molecule in a circular form. Such circular forms can typically be amplified into concatemers by rolling circle amplification. A linear double-stranded nucleic acid with a strand attached to its end (e.g., a backbone covalently conjugated, e.g., by a hairpin loop or other structure) is not a circular vector as used herein. The term "circular DNA vector" is used interchangeably herein with the term "covalently closed circular DNA vector." One skilled in the art will understand that such circular vectors include vectors that are covalently closed in supercoiled and complex DNA topologies, as described herein. In some embodiments, the circular DNA vector is not supercoiled (e.g., open circular). In certain embodiments, the circular DNA vector is supercoiled. In certain cases, the circular DNA vector lacks a bacterial origin of replication.

[0129] As used herein, a "cell-free" method of making a circular DNA vector refers to a method that does not rely on any containment of DNA in a host cell, such as a bacterial (e.g., E. coli) host cell, to facilitate any step of the method, from providing a template DNA vector (e.g., a plasmid DNA vector) to making a circular DNA vector. For example, a cell-free method is carried out in one or more synthetic vessels (e.g., glass or plastic tubes, bioreactors, vessels, tanks, or other suitable vessels) in a suitable solution (e.g., a buffer solution) to which enzymes and other agents can be added to facilitate DNA amplification, modification, and isolation. A cell-free production method may use template DNA generated in a cell.

[0130] As used herein, the term "recombination site" refers to a nucleic acid sequence that is a product of site-specific recombination, which includes a first sequence corresponding to a portion of a first recombinase binding site and a second sequence corresponding to a portion of a second recombinase binding site. An example of a hybrid recombination site is attR, which is a product of site-specific recombination, which includes a first sequence corresponding to a portion of attP and a second sequence corresponding to a portion of attB. Alternatively, a recombination site can be generated from Cre / Lox recombination. Thus, a vector generated from Cre / Lox recombination (e.g., a vector containing a LoxP site) includes a recombination site as used herein. Other site-specific recombination events that generate recombination sites include, for example, lambda integrase, FLP recombinase, and Kw recombinase. Nucleic acid sequences resulting from non-site-specific recombination events (e.g., ITR-mediated intermolecular recombination) are not recombination sites as defined herein.

[0131] As used herein, the term "protein" refers to multiple amino acids linked together via peptide bonds (i.e., as a primary structure), including non-covalently linked multimeric (e.g., dimeric, trimeric, etc.) proteins (e.g., proteins having quaternary structure). Thus, the term "protein" encompasses peptides, natural proteins, recombinant proteins, and fragments thereof. In some embodiments, a protein has a primary structure under physiological conditions and no secondary, tertiary, or quaternary structure. In some embodiments, a protein has a primary and secondary structure under physiological conditions and no tertiary or quaternary structure. In certain embodiments, a protein has a primary, secondary, and tertiary structure under physiological conditions but no quaternary structure (e.g., a monomeric protein having one or more folded alpha helices and / or beta sheets). In some embodiments, any of the proteins described herein have a length of at least 25 amino acids (e.g., 50-1,000 amino acids).

[0132] The terms "therapeutic sequence," "therapeutic gene," and "heterologous gene" are used interchangeably to refer to an administered transgene (e.g., as part of a DNA vector or a self-replicating RNA molecule). A therapeutic gene can be a mammalian gene that encodes a protein that is endogenously expressed by the individual receiving the therapeutic gene or that replaces a non-functional mutant protein expressed by the individual.

[0133] As used herein, the term "mutation-associated disorder", "mutation associated with disorder", or protein or gene "associated" disorder (e.g., ABCA4-associated retinal dystrophy) refers to the correlation between the disorder and the mutation in a gene or protein. In some embodiments, the mutation-associated disorder is known or suspected to be caused in whole or in part, or directly or indirectly, by the mutation. For example, a subject with a mutation may be at risk of developing the disorder, and the risk may further depend on other factors, such as other (e.g., independent) mutations (e.g., in the same or different genes), or environmental factors.

[0134] The term "ABCA4" refers to any native ABCA4 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), as well as functionally equivalent or improved variants (e.g., natural or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof, unless otherwise specified. Functionally equivalent and improved variants can be determined based on known ABCA4 signaling. ABCA4 encompasses full-length unprocessed ABCA4, as well as any form of ABCA4 resulting from natural processing in cells. An exemplary human ABCA4 sequence is provided as National Center for Biotechnology Information (NCBI) Reference Sequence: NG_009073. In some cases, ABCA4 is encoded by a therapeutic gene having at least 95% sequence identity to SEQ ID NO:16 (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:16). In some cases, ABCA4 is encoded by a therapeutic gene having at least 95% sequence identity to SEQ ID NO:17 (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:17). In some cases, the ABCA4 protein has at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:18).

[0135] The term "MYO7A" refers to any naturally occurring MYO7A (also known as DFNB2, MYU7A, NSRD2, USH1B, DFNA11, or MYOVIIA) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), as well as functionally equivalent or improved variants (e.g., natural or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof, unless otherwise specified. Functionally equivalent variants and improved variants can be determined based on known MYO7A signaling. MYO7A encompasses full-length unprocessed MYO7A, as well as any form of MYO7A resulting from natural processing in cells. An exemplary human MYO7A sequence is provided as National Center for Biotechnology Information (NCBI) Gene ID:4647. In some cases, MYO7A is encoded by a therapeutic gene having at least 95% sequence identity to any one of SEQ ID NO:1 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1). In some cases, MYO7A encoded by a therapeutic gene has at least 95% sequence identity to any one of SEQ ID NO:2-9 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO:2-9).

[0136] The term "bestrophin 1 (BEST1)" refers to any naturally occurring BEST1 (also known as ARB, BMB, BEST, RP50, VMD2, TU15B, or Best1V1 delta2) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), as well as functionally equivalent or improved variants (e.g., natural or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof, unless otherwise specified. Functionally equivalent and improved variants are consistent with known BEST1 signaling (e.g., Ca in RPE cells). 2+ Signal transduction). BEST1 encompasses full-length unprocessed BEST1, as well as any form of BEST1 resulting from natural processing in cells. An exemplary human BEST1 sequence is provided as National Center for Biotechnology Information (NCBI) Gene ID:7439. In some cases, BEST1 is encoded by a therapeutic gene having at least 95% sequence identity to SEQ ID NO:10 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:10). In some cases, the BEST1 encoded by the therapeutic gene has at least 95% sequence identity to any one of SEQ ID NOs:11-13 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs:11-13).

[0137] The term "Complement Factor H (CFH)" refers to any naturally occurring CFH (also known as FH, HF, HF1, HF2, HUS, FHL1, AHUS1, AMBP1, ARMD4, ARMS1, or CFHL3) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats), as well as functionally equivalent or improved variants (e.g., natural or synthetic variants), such as mutants, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof, unless otherwise indicated. Functionally equivalent and improved variants are those that are involved in known CFH signaling (e.g., Ca in RPE cells). 2+ Signal transduction). CFH encompasses full-length unprocessed CFH as well as any form of CFH resulting from natural processing in cells. An exemplary human CFH sequence is provided as National Center for Biotechnology Information (NCBI) Gene ID:3075. In some cases, CFH is encoded by a therapeutic gene having at least 95% sequence identity to SEQ ID NO:14 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:14). In some cases, the CFH encoded by the therapeutic gene has at least 95% sequence identity to SEQ ID NO:15 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:15).

[0138] As used herein, a "variant" of a therapeutic gene, replicase, or a fragment thereof differs from a reference amino acid sequence, such as a naturally occurring amino acid sequence or a single amino acid sequence, by at least one amino acid residue. In this context, the difference in at least one amino acid residue may consist, for example, of a mutation, deletion, or insertion of an amino acid residue into another amino acid. A variant may be a homolog, isoform, or transcriptional variant of a therapeutic protein or a fragment thereof as defined herein, where the homolog, isoform, or transcriptional variant is characterized by a degree of identity or homology, respectively, as defined herein.

[0139] In some cases, the variant of the therapeutic gene or a fragment thereof contains at least one amino acid substitution (e.g., 1 to 100 amino acid substitutions, 1 to 50 amino acid substitutions, 1 to 20 amino acid substitutions, 1 to 10 amino acid substitutions, e.g., 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, 8 amino acid substitutions, 9 amino acid substitutions, or 10 amino acid substitutions). Substitutions in which amino acids from the same class are exchanged for one another are called conservative substitutions. In particular, these are amino acids with aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chain or amino acids whose side chains are capable of forming hydrogen bridges, e.g., side chains with hydroxyl functions. A conservative nature may, for example, substitute an amino acid having a polar side chain with another amino acid having a corresponding polar side chain, or, for example, substitute an amino acid characterized by a hydrophobic side chain with another amino acid having a corresponding hydrophobic side chain (e.g., threonine (serine) with serine (threonine) or isoleucine (leucine) with leucine (isoleucine)). In certain aspects, variants of a protein or fragment thereof may be encoded by a nucleic acid according to the invention, wherein at least one amino acid residue of the amino acid sequence comprises at least one conservative substitution compared to a reference sequence, such as a respective naturally occurring sequence.

[0140] In some cases, insertions, deletions, and / or non-conservative substitutions are also encompassed by the term variant, for example at positions that do not cause substantial alteration of the three-dimensional structure of the protein. Alterations in the three-dimensional structure due to one or more insertions or one or more deletions can be easily determined by those skilled in the art, for example using CD spectroscopy (circular dichroism spectroscopy).

[0141] To determine the percentage of identity of two sequences (e.g., nucleic acid sequences, e.g., DNA, RNA or amino acid sequences), the sequences can be aligned for subsequent comparison with each other. For this purpose, gaps can be inserted into the sequence of the first sequence, and the components at the corresponding positions of the second sequence can be compared. If a position in the first sequence is occupied by the same component as the corresponding position in the second sequence, the two sequences are identical at this position. The percentage of identity of two sequences is a function of the number of identical positions divided by the total number of positions. The percentage of identity of two sequences can be determined using a mathematical algorithm. A preferred but non-limiting example of a mathematical algorithm that can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm can be incorporated, for example, into the BLAST program. Sequences having a degree of identity to the sequences of the present invention can be identified by this program.

[0142] As used herein, the term "isolated" means artificially produced and not integrated into a natural host genome. For example, an isolated nucleic acid vector includes a nucleic acid vector encapsulated in a lipid envelope (e.g., a liposome) or a polymer matrix. In some embodiments, the term "isolated" refers to a DNA vector that is (i) amplified in vitro (e.g., in a cell-free environment), e.g., by rolling circle amplification or polymerase chain reaction (PCR); (ii) recombinantly produced by molecular cloning; (iii) purified, e.g., by restriction endonuclease cleavage and gel electrophoretic fractionation, or column chromatography; or (iv) synthesized, e.g., by chemical synthesis. An isolated nucleic acid vector is one that can be readily manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector for which the 5' and 3' restriction sites are known or for which the polymerase chain reaction (PCR) primer sequences are disclosed is considered to be isolated, while a nucleic acid sequence that exists in its natural state in its natural host is not considered to be isolated. An isolated nucleic acid vector may be substantially purified, but need not be.

[0143] As used herein, the term "naked" refers to a nucleic acid molecule (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site) that is not complexed (e.g., enclosed, conjugated, or non-covalently linked) with another agent. Thus, a nucleic acid in an envelope (e.g., a lipid envelope) or a matrix of covalently or non-covalently linked units (e.g., a synthetic polymer matrix or a peptide or protein matrix) is not a naked nucleic acid molecule as used herein. A naked nucleic acid molecule may be co-formulated (e.g., in solution) with an agent that is not complexed with the naked nucleic acid molecule, such as a buffer. In some cases of the present invention, the pharmaceutical composition comprises a naked circular DNA vector. One example of naked DNA is the covalently closed circular DNA (C3-DNA) described herein.

[0144] As used herein, a "vector" refers to a nucleic acid molecule capable of carrying a sequence of interest (e.g., a therapeutic gene, a therapeutic sequence, or a heterologous gene) that is linked to a target cell where the therapeutic gene can then be replicated, processed, and / or expressed in the target cell. After the target or host cell processes the sequence of interest (e.g., genome) of the vector, the sequence of interest (e.g., genome) is not considered a vector. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop containing a bacterial backbone to which additional DNA segments can be linked. Another type of vector is a phage vector. Another type of vector is a viral vector, where additional DNA segments can be linked to the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Additionally, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors" or "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0145] As used herein, "target cell" refers to the cell that expresses the regulatory protein encoded by therapeutic gene.In some embodiments, target cell is retinal cell.For example, in certain embodiments, target cell is RPE cell.In other embodiments, target cell is photoreceptor.In certain embodiments, RPE cell and photoreceptor are target cell.

[0146] As used herein, the term "individual" includes any mammal (e.g., a mammal with a retinal dystrophy) in need of the therapeutic or preventive methods described herein. In some embodiments, the individual is a human. In other embodiments, the individual is a non-human mammal (e.g., a non-human primate (e.g., monkey), mouse, pig, rabbit, cat, or dog). The subject may be male or female. In one embodiment, the individual has Usher syndrome type 1B. In some embodiments, the individual has a Best1 dominant mutation or a BEST1 recessive mutation, such as autosomal recessive bestrophinopathy, Best vitelliform macular dystrophy, BEST1 adult-onset vitelliform macular dystrophy, or bestrophinopathy associated with autosomal dominant vitreoretinochoroidopathy. In some embodiments, the individual has age-related macular degeneration.

[0147] As used herein, an "effective amount" or "effective dose" of a therapeutic agent (e.g., a nucleic acid vector) or a composition thereof refers to an amount sufficient to achieve a desired biological and / or pharmacological effect when administered to an individual, for example, according to a selected administration form, route and / or schedule. As will be understood by those skilled in the art, the absolute amount of a particular composition that is effective may vary depending on factors such as the desired biological or pharmacological endpoint, the agent delivered, the target tissue, etc. Those skilled in the art will further understand that an "effective amount" may be contacted with a cell or administered to a subject in a single dose or by the use of multiple doses. An effective amount of a composition for treating an eye disease may slow or stop disease progression (e.g., visual function), increase partial or complete response (e.g., visual function) compared to a reference population, such as an untreated or placebo population, or a population receiving standard care treatment.

[0148] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that seeks to alter the natural course of the individual being treated, which may be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing direct or indirect pathological consequences of disease, slowing the rate of disease progression, improving or alleviating the disease state, and improving prognosis. In some embodiments, the nucleic acid vectors (e.g., circular DNA vectors) of the present invention are used to delay the onset of disease or slow the progression of disease.

[0149] By "reduce or inhibit" is meant the ability to cause an overall decrease, preferably of 20% or more, more preferably of 50% or more, and most preferably of 75%, 85%, 90%, 95% or more.

[0150] The terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of polynucleotide or amino acid product or protein in a biological sample (e.g., retina). "Expression" generally refers to the process by which the information encoded in a gene is converted into a structure present and operating in a cell. Thus, according to the present invention, "expression" of a gene can refer to transcription into a polynucleotide, translation into a protein, or post-translational modification of a protein. Transcribed polynucleotides, translated proteins, or post-translationally modified protein fragments are also considered to be expressed, regardless of whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a protein, for example, by proteolysis. "Expressed genes" include those that are transcribed into polynucleotides as mRNA and then translated into proteins, as well as those that are transcribed into RNA but not translated into proteins (e.g., transfer and ribosomal RNA).

[0151] As used herein, "delivering", "deliver" and grammatical variations thereof refer to allowing an agent (e.g., a therapeutic agent) to access a target cell. The agent may be delivered by administration of the agent to an individual having the target cell (e.g., systemic or local administration of the agent) so that the agent gains access to the organ or tissue in which the target cell resides. Additionally or alternatively, the agent may be delivered by applying a stimulus to the tissue or organ carrying the agent, which causes the agent to enter the target cell. Thus, in some cases, the agent is delivered to the target cell by transmitting an electric field to the tissue carrying the agent under conditions suitable for electrical introduction of the agent to the target cell in the tissue.

[0152] As used herein, "administering" refers to a method of giving an individual a dose of a therapeutic agent of the invention (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene and / or a recombination site)) or a composition thereof (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising a nucleic acid vector). The compositions utilized in the methods described herein can be administered intraocularly, e.g., suprachoroidally. The compositions utilized in the methods described herein can be administered intraocularly, e.g., intravitreally, subretinal, or periocularly. Additionally or alternatively, the composition can be delivered intravenously, subcutaneously, intradermally, transdermally, intramuscularly, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, peritoneally, subconjunctivally, intravesicularly, mucosally, intracardially, intraumbilically, orally, topically, transdermally, conjunctivally, subtendinously, intracamerally, subretinally, retrobulbarly, intracanalicularly, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by local perfusion bath directly target cells, by catheter, by lavage, in cream, or in lipid composition. The composition utilized in the methods described herein can be administered systemically. The method of administration can vary depending on various factors (e.g., the compound or composition administered and the severity of the condition, disease or disorder being treated).

[0153] "Administered in combination with" refers to the administration of multiple therapeutic components as part of the same treatment regimen. The therapeutic agent of the present invention (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial replication origin, drug resistance gene and / or recombination site)) can be administered in combination with pulsed electric field therapy, e.g., as part of the same outpatient treatment or over multiple days. Additionally or alternatively, the nucleic acid vector of the present invention (e.g., a circular DNA vector) can be administered in combination with another therapeutic agent (e.g., as part of the same pharmaceutical composition or as separate pharmaceutical compositions, at the same time or at different times).

[0154] The term "a" means "one or more." For example, "a cell" is understood to represent one or more cells. Thus, the terms "a," "one or more," and "at least one of" are used interchangeably herein.

[0155] As used herein, the term "about" refers to values ​​within ±10% variation from a reference value, unless otherwise specified.

[0156] II. Therapeutic Agents and Compositions The present invention includes therapeutic agents for treating ocular diseases and disorders. Any therapeutic agent suitable for treating ocular diseases (e.g., retinal dystrophy) by delivery to ocular target cells (e.g., retinal cells) is contemplated as part of the present invention. Such therapeutic agents include nucleic acid vectors (e.g., non-viral DNA vectors, e.g., circular DNA vectors lacking bacterial replication origins, drug resistance genes and / or recombination sites), therapeutic proteins, small molecule drugs, and pharmaceutical compositions thereof. Exemplary nucleic acid vectors include circular DNA vectors (e.g., circular DNA vectors encoding therapeutic replacement proteins (e.g., proteins that replace proteins endogenously expressed in healthy cells), including ABCA4, MYO7A, BEST1 and CFH). Any of the nucleic acid vectors described herein can be part of a pharmaceutical composition in a pharmaceutically acceptable carrier.

[0157] Nucleic Acid Vectors The nucleic acid vectors of the present invention include non-viral nucleic acid vectors (e.g., non-viral DNA vectors or non-viral RNA vectors, such as circular DNA vectors and circular RNA vectors). In certain cases, the nucleic acid vector (e.g., non-viral nucleic acid vector) is a naked nucleic acid vector (e.g., naked DNA (e.g., naked circular DNA (e.g., synthetic circular DNA) or naked linear DNA (e.g., closed-end DNA or doggy-gibone DNA)) or naked RNA (e.g., naked circular RNA).

[0158] Some embodiments of the present invention include circular DNA vectors. In some cases, the circular DNA vectors useful for carrying the therapeutic genes (e.g., therapeutic replacement genes) described herein can be plasmid DNA vectors. In certain cases of the present invention, the circular DNA vectors differ from conventional plasmid DNA vectors in that they lack plasmid backbone elements (e.g., bacterial elements such as (i) a bacterial origin of replication and / or (ii) a drug resistance gene). In some embodiments, the circular DNA vectors encoding any of the therapeutic genes (e.g., therapeutic replacement genes) described herein lack recombination sites (e.g., synthetic circular DNA vectors made using cell-free processes). In alternative embodiments, the circular DNA vectors described herein include recombination sites (e.g., minicircle DNA vectors).

[0159] The circular DNA vector of the present invention can persist in cells (e.g., in quiescent cells, such as postmitotic cells) as episomes. The vectors provided herein can lack bacterial plasmid DNA components, such as immunogenic components (e.g., immunogenic bacterial signatures (e.g., CpG motifs)), or components that are additionally or alternatively associated with reduced persistence (e.g., CpG islands). For example, in some embodiments, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or essentially all) of the DNA lacks one or more elements of bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial signatures (e.g., CpG motifs)) or components additionally or otherwise associated with reduced persistence (e.g., CpG islands). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or essentially all) of the DNA lacks CpG methylation. In some embodiments, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or essentially all) of the DNA lacks a bacterial methylation signature, such as Dam methylation and Dcm methylation.For example, in some embodiments, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or essentially all) of the GATC sequences are unmethylated (e.g., by Dam methylase). Additionally or alternatively, the vector comprises DNA in which at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or essentially all) of the CCAGG and / or CCTGG sequences are unmethylated (e.g., by Dcm methylase).

[0160] In some embodiments of any of the foregoing vectors, the DNA vector is persistent in vivo (e.g., circularity and non-bacterial (i.e., by in vitro (e.g., cell-free) synthesis) associated with long-term transcription or expression of a therapeutic gene in the DNA vector). In some embodiments, the persistence of the circular DNA vector is 5%-50% greater, 50%-100% greater, 1-5-fold, or 5-10-fold (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more) greater than a reference vector (e.g., a circular vector produced in bacteria or having one or more bacterial signatures not present in the vectors of the invention). In some embodiments, the circular DNA vector of the present invention lasts for 1 week to 4 weeks, 1 month to 4 months, 4 months to 1 year, 1 year to 5 years, 5 years to 20 years, or 20 years to 50 years (e.g., at least 1 week, at least 2 weeks, at least 1 month, at least 4 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years, or at least 50 years).

[0161] The circular DNA vector of the present invention may include a promoter operably linked to the 5' side of a therapeutic gene (e.g., a therapeutic replacement gene). A promoter is operably linked to a therapeutic gene (e.g., a therapeutic replacement gene) if the promoter is capable of transcription of the therapeutic gene (e.g., a therapeutic replacement gene). Promoters that can be used as part of the circular DNA vector include constitutive promoters, inducible promoters, native promoters, and tissue-specific promoters. Examples of constitutive promoters include cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with a RSV enhancer), SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1-alpha promoter. In certain embodiments of the present invention, the circular DNA vector includes a CMV promoter. In some embodiments, the circular DNA vector includes a CAG promoter.

[0162] Alternatively, the circular DNA vector of the present invention comprises an inducible promoter. Inducible promoters allow the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions, such as acute phase, specific differentiation state of cells, or only in replicating cells. Examples of inducible promoters regulated by exogenously supplied promoters include zinc-inducible sheep metallothionine (MT) promoter, T7 polymerase promoter system, ecdysone insect promoter, tetracycline repression system, tetracycline induction system, RU486 induction system, and rapamycin induction system. Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, specific differentiation state of cells, or only in replicating cells. Inducible promoters and induction systems are available from a variety of commercial sources.

[0163] The circular DNA vector of the present invention may also contain a polyadenylation sequence 3' to the sequence encoding the self-replicating RNA molecule. Useful polyadenylation sequences include extended polyadenylation sequences of more than 20 nt (e.g., more than 25 nt, more than 30 nt, more than 35 nt, more than 40 nt, more than 50 nt, more than 60 nt, more than 70 nt, or more than 80 nt, e.g., 20-100 nt, 30-100 nt, 40-100 nt, 50-100 nt, 60-100 nt, 70-100 nt, 80-100 nt, 100-200 nt, 200-300 nt, or 300-400 nt, or more).

[0164] Circular DNA vectors, which lack bacterial elements such as DNA replication origins and / or drug resistance genes, can persist in an individual longer than conventional DNA vectors (eg, plasmids) and longer than naked RNA.

[0165] Circular DNA vectors can have a variety of sizes and shapes. Circular DNA vectors carrying therapeutic genes (e.g., therapeutic replacement genes) of the present invention can be 2.5 kb to 20 kb in length (e.g., 5 kb to 19 kb, 6 kb to 18 kb, 7 kb to 16 kb, 8 kb to 14 kb or 9 kb to 12 kb, e.g., 5 kb to 6 kb, 6 kb to 7 kb, 7 kb to 8 kb, 8 kb to 9 kb, 9 kb to 10 kb, 10 kb to 11 kb, 11 kb to 12 kb, 12 kb to 13 kb, 13 kb to 14 kb). , 14kb to 15kb, 15kb to 16kb, 16kb to 18kb, or 18kb to 20kb in length, e.g., about 3kb, about 4kb, about 5kb, about 6kb, about 7kb, about 8kb, about 9kb, about 10kb, about 10.5kb, about 11kb, about 11.5kb, about 12kb, about 12.5kb, about 13kb, about 14kb, about 15kb, about 16kb, about 17kb, about 18kb, about 19kb, or about 20kb in length).

[0166] Circular DNA vectors useful as part of the present invention can be readily synthesized by various means known in the art and described herein. For example, circular DNA vectors lacking plasmid backbone elements (e.g., bacterial elements such as (i) bacterial origin of replication and / or (ii) drug resistance genes) can be made using in vitro (cell-free) methods that can provide purer compositions compared to bacterial-based methods. Such in vitro synthesis methods can include the use of phage polymerases such as Phi29 polymerase as a replication tool, for example using rolling circle amplification. Certain methods of in vitro synthesis of circular DNA vectors are further described in WO 2019 / 178500, which is incorporated herein by reference.

[0167] In some cases, the nucleic acid vector is a non-viral nucleic acid vector (e.g., the nucleic acid vector is not encapsulated in a viral capsid). Additionally or alternatively, in some embodiments, the nucleic acid vector is not encapsulated in an envelope (e.g., a lipid envelope) or matrix (e.g., a polymer matrix) and is not physically associated (e.g., covalently or non-covalently bound) with a solid structure (e.g., a particulate structure) before and after administration to an individual. In some embodiments, the nucleic acid vector is not bound to any adjacent nucleic acid vector such that in a solution of nucleic acid vectors, each nucleic acid vector is free to diffuse independently of adjacent nucleic acid vectors. In some embodiments, the nucleic acid vector is associated with another agent in the liquid solution, such as a charge-changing molecule or a stabilizing molecule.

[0168] The nucleic acid vector can be a naked nucleic acid vector, i.e., a nucleic acid vector that is not complexed (e.g., encapsulated, conjugated, or non-covalently linked) with another agent. The naked nucleic acid molecule can be co-formulated (e.g., in solution) with an agent that is not complexed with the naked nucleic acid molecule, such as a buffer and / or an agent that is Generally Recognized As Safe (GRAS) by the U.S. Food and Drug Administration.

[0169] Therapeutic Genes The nucleic acid vectors described herein include therapeutic genes, such as therapeutic genes or therapeutic sequences that code for therapeutic replacement proteins. The therapeutic replacement proteins can replace proteins endogenously expressed in healthy cells, such as healthy retinal cells, or non-functional mutant proteins expressed by the individual being treated. Thus, it will be understood that the nucleic acid vectors of the present invention that code for therapeutic replacement proteins can be administered as gene replacement therapy and / or gene augmentation therapy.

[0170] The therapeutic genes of the present invention include ocular genes (e.g., genes encoding proteins expressed in ocular tissues such as the retina). In some embodiments, the therapeutic sequence (e.g., therapeutic gene) is selected from the group consisting of MYO7A, BEST1, CFH, CEP290, USH2A, ADGRV1, CDH23, CRB1, PCDH15, RPGR, ABCA4, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, C3, IFT172, COL11A1, TUBGCP6, KIAA1549, CACNA1F, SNRNF200, PRPF8, VCAN, USH2A, HMCN1, RPE65, NR2E3, NRL, RHO, RP1, RP2, or OFD1. In some embodiments, the therapeutic sequence (e.g., therapeutic gene) is an autosomal dominant gene. In some embodiments, the therapeutic sequence (e.g., therapeutic gene) is an autosomal recessive gene. In some embodiments, the therapeutic sequence (eg, therapeutic gene) is an X-linked gene.

[0171] In some embodiments, the therapeutic protein encoded by the nucleic acid vector is a retinal pigment epithelium specific protein, adrenoceptor alpha 2A, amyloid beta (A4) precursor protein, complement component 3, complement component 5, complement factor D (adipsin), thrombospondin receptor, complement component 5 receptor 1, HIF1A, nerve growth factor receptor, STAT3, VEGFA, PDGFR, VEGFR1 / 2, plasminogen, tyrosine kinase, mTOR, factor III, cadherin, chemokine receptor (3 / 4), integrin A5, placenta growth factor, protein tyrosine phosphatase, S1PR1, vRaf, TGF beta, HtrA serine peptidase 1, TNF receptor 10A, NOTCH4, insulin-like growth factor binding protein 7, Ras responsive element binding protein 1, constitutive factor H, constitutive factor B, complement component 3, complement component 2, complement factor I, hepatic lipase, cofactor 1, complement component 2, complement component ... lestriyl ester transfer protein, translocase 40 of the mitochondrial outer membrane, mitochondrial superoxide dismutase 2, tenascin XB, type X collagen alpha 1, myelin basic protein, type VIII collagen alpha 1, bestrophin 1, carbohydrate (N-acetylglucosamine 6-0) sulfotransferase 6, retinitis pigmentosa GTPase, guanylate cyclase system (2D, A1A), calcium channel (A2, LA1F), peripherin 2, cadherin 1, choroideremia (Rab escort protein 1), guanylate cyclase 2D, peripherin 2, mitochondrial-encoded ATP synthase, mitochondrial-encoded cytochrome, mitochondrial-encoded NADH dehydrogenase, mitofusin 2, optic neuropathy 1, 3' repair exonuclease 1, 3' repair exonuclease 1, DICER1, HIF-PHD, Hey 1, dominant negative CCR3, anti-eotaxin mAb, Dcr1, Sema3E, VEGF trap, PDGF trap Nitrin1R, aA crystallin, aB crystallin, Hey 2, Sirtuins such as SIRT1, DR4-Fc, DR5-Fc, PD1R, RhoJ, sFLT-1, IGFR I-Fc, IGFBP7, PEDF, NPPB, CD59, PLEKHA1, RPE65, and / or PDE.

[0172] Nucleic acid vectors carrying these therapeutic sequences (e.g., therapeutic genes) are useful for treating ocular diseases or disorders, such as retinal dystrophy associated with the transgene carried by the nucleic acid vector (e.g., ABCA4-associated retinal dystrophy, MYO7A-associated retinal dystrophy, or BEST1-associated retinal dystrophy), including Usher syndrome (e.g., Usher syndrome type 1B), retinitis pigmentosa (RP), diabetic eye disease (e.g., diabetic retinopathy or diabetic macular edema), dry eye, cataracts, retinal vein occlusion (e.g., central retinal vein occlusion or branch retinal vein occlusion), retinal artery occlusion, macular edema (e.g., macular edema following retinal vein occlusion), macular degeneration (e.g., age-related macular degeneration (AMD)), wet macular degeneration (e.g., , wet AMD), dry macular degeneration (e.g., dry AMD), or neovascular AMD), geographic atrophy, refractive and accommodative disorders, keratoconus, amblyopia, glaucoma, Stargardt's disease, endophthalmitis, conjunctivitis, uveitis (e.g., posterior uveitis), retinal detachment, corneal ulcer, dacryocystitis, Duane's constriction syndrome, optic neuritis, choroidal neovascularization, choroidal ischemia, or hypertensive retinopathy. Nucleic acid vectors carrying these therapeutic genes are useful for treating symptoms of ocular diseases or disorders, such as any of the above diseases or disorders, or ocular symptoms of more widespread disorders, such as hypotension, hypertension, infection, sarcoid or sickle cell disease. In some aspects, the therapeutic genes are useful for treating acute diseases. In other aspects, the therapeutic genes are useful for treating chronic diseases.

[0173] Other therapeutic sequences or genes (e.g., therapeutic genes encoding therapeutic proteins) useful within the nucleic acid vectors described herein include genes encoding retinal proteins other than any one or more of the proteins listed herein.

[0174] The therapeutic sequence or gene of any of the nucleic acid vectors described herein (e.g., a therapeutic gene encoding a therapeutic replacement protein) may encode a functionally equivalent fragment of any of the proteins described herein, or a variant thereof. A fragment of a protein or variant thereof encoded by a nucleic acid vector according to the present invention may comprise an amino acid sequence having at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity) with a reference amino acid sequence (e.g., the amino acid sequence of the respective naturally occurring full-length protein or variant thereof). In some embodiments, the therapeutic gene is selected from Table 1.

[0175] In any of the polycistronic nucleic acid vectors described herein, cleavage sites can be designed between protein-coding regions. For example, a furin-P2A site can separate any of the protein-coding genes described herein. Ribozymes can also be incorporated into the RNA molecule to cleave sites downstream of protein-coding genes. In some embodiments, T2A, E2A, F2A, or any other suitable self-cleavage site (e.g., a viral cleavage site) can separate any of the protein-coding genes described herein.

[0176] In some embodiments, the therapeutic sequence (e.g., therapeutic gene) is greater than 4.5 Kb in length (e.g., one or more therapeutic genes, together or each alone, are greater than 4.5 Kb in length, e.g., 4.5 Kb to 25 Kb, 4.6 Kb to 24 Kb, 4.7 Kb to 23 Kb, 4.8 Kb to 22 Kb, 4.9 Kb to 21 Kb, 5.0 Kb to 20 Kb, 5.5 Kb to 18 Kb, 6.0 Kb to 17 Kb, 6.5 Kb to 16 Kb, 7.0 Kb to 15 Kb, 7.5 Kb to 14 Kb, 8.0 Kb to 13 Kb, 8.5 Kb to 12.5 Kb, 9.0 Kb to 1 2.0Kb, 9.5Kb to 11.5Kb, or 10.0Kb to 11.0Kb, for example, 4.5Kb to 8Kb, 8Kb to 10Kb, 10Kb to 15Kb, 15Kb to 20Kb, or more, for example, 4.5Kb to 5.0Kb, 5.0Kb to 5.5Kb, 5.5Kb to 6.0Kb, 6.0Kb to 6.5Kb, 6.5Kb to 7.0Kb, 7.0Kb to 7.5Kb, 7.5Kb to 8.0Kb, 8.0Kb to 8.5Kb, 8.5Kb to 9.0Kb, 9.0Kb to 9.5Kb, 9.5Kb to 10Kb, 10Kb~10.5Kb, 10.5Kb~11Kb, 11Kb~11.5Kb, 11.5Kb~12Kb, 12Kb~12.5Kb, 12.5Kb~13Kb, 13Kb~13.5Kb, 13.5Kb~14Kb, 14Kb~14.5Kb, 14.5Kb ~15Kb, 15Kb~15.5Kb, 15.5Kb~16Kb, 16Kb~16.5Kb, 16.5Kb~17Kb, 17Kb~17.5Kb, 17.5Kb~18Kb, 18Kb~18.5Kb, 18.5Kb~19Kb, 19Kb~19.5Kb, 19.5Kb to 20Kb, 20Kb to 21Kb, 21Kb to 22Kb, 22Kb to 23Kb, 23Kb to 24Kb, 24Kb to 25Kb, or more, e.g., about 4.5Kb, about 5.0Kb, about 5.5Kb, about 6.0Kb, about 6.5Kb, about 7.0Kb, about 7.5Kb, about 8.0Kb, about 8.5Kb, about 9.0Kb, about 9.5Kb, about 10Kb, about 11Kb, about 12Kb, about 13Kb, about 14Kb, about 15Kb, about 16Kb, about 17Kb, about 18Kb, about 19Kb, about 20Kb, or more). In some embodiments, the therapeutic gene is greater than 2.5 Kb (eg, between 2.5 Kb and 10 Kb, between 2.5 Kb and 8 Kb, or between 2.5 Kb and 6 Kb).

[0177] In some embodiments, the therapeutic sequence (eg, therapeutic gene) is greater than 8 Kb (eg, between 8 Kb and 15 Kb, between 8 Kb and 12 Kb, between 8 Kb and 10 Kb, or between 8 Kb and 9 Kb).

[0178] In some cases, the nucleic acid vector has a nucleic acid sequence driven by a CAG promoter that encodes an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96% sequence identity to SEQ ID NO:18, at least 97% sequence identity to SEQ ID NO:18, at least 98% sequence identity to SEQ ID NO:18, at least 99% sequence identity to SEQ ID NO:18, or 100% sequence identity to SEQ ID NO:18). In some cases, the nucleic acid vector is a naked nucleic acid vector (e.g., a naked DNA vector (e.g., a naked circular DNA vector (e.g., a plasmid DNA vector, a minicircle DNA vector, or a synthetic circular DNA vector lacking recombination sites (e.g., a supercoiled synthetic circular DNA vector)), a naked closed-end DNA vector, or a naked doggybone DNA vector), or a naked RNA vector (e.g., a naked circular RNA vector)) comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96% sequence identity to SEQ ID NO:18, at least 97% sequence identity to SEQ ID NO:18, at least 98% sequence identity to SEQ ID NO:18, at least 99% sequence identity to SEQ ID NO:18, or 100% sequence identity to SEQ ID NO:18). In some cases, the nucleic acid vector (e.g., a non-viral nucleic acid vector) comprises a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:18 (e.g., at least 96% sequence identity to SEQ ID NO:18, at least 97% sequence identity to SEQ ID NO:18, at least 98% sequence identity to SEQ ID NO:18, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:18), wherein the nucleic acid vector is encapsulated in a nanoparticle, microparticle, liposome, or lipid nanoparticle.

[0179] Additionally or alternatively, the nucleic acid vector comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:19 (e.g., at least 96% sequence identity to SEQ ID NO:19, at least 97% sequence identity to SEQ ID NO:19, at least 98% sequence identity to SEQ ID NO:19, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:19). In some cases, the nucleic acid vector comprises or consists of the nucleic acid sequence of SEQ ID NO:19. In some cases, the nucleic acid vector is a naked nucleic acid vector (e.g., a naked DNA vector (e.g., a naked circular DNA vector (e.g., a plasmid DNA vector, a minicircle DNA vector, or a synthetic circular DNA vector lacking recombination sites (e.g., a supercoiled synthetic circular DNA vector)), a naked closed-end DNA vector, or a naked doggybone DNA vector), or a naked RNA vector (e.g., a naked circular RNA vector)) that comprises or consists of a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:19 (e.g., at least 96% sequence identity to SEQ ID NO:19, at least 97% sequence identity to SEQ ID NO:19, at least 98% sequence identity to SEQ ID NO:19, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:19). In some cases, the nucleic acid vector (e.g., a non-viral nucleic acid vector) comprises or consists of a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:19 (e.g., at least 96% sequence identity to SEQ ID NO:19, at least 97% sequence identity to SEQ ID NO:19, at least 98% sequence identity to SEQ ID NO:19, at least 99% sequence identity to SEQ ID NO:19, or 100% sequence identity to SEQ ID NO:19), wherein the nucleic acid vector is encapsulated in a nanoparticle, microparticle, liposome, or lipid nanoparticle.

[0180] Any of the aforementioned therapeutic sequences or therapeutic genes (e.g., therapeutic nucleic acid vectors, e.g., non-viral nucleic acid vectors, e.g., naked nucleic acid vectors, e.g., synthetic circular DNA vectors) can be used to treat a disease or disorder (e.g., in an individual in need thereof, e.g., an individual having a disease or disorder or at risk of developing a disease or disorder). Thus, provided herein is the use of any of the aforementioned therapeutic sequences or therapeutic genes (e.g., therapeutic nucleic acid vectors, e.g., non-viral nucleic acid vectors, e.g., naked nucleic acid vectors, e.g., synthetic circular DNA vectors) for treating or preventing any associated disorder by any of the therapeutic methods and uses described herein (e.g., Section III). Further provided herein is any of the aforementioned therapeutic sequences or therapeutic genes (e.g., therapeutic nucleic acid vectors, e.g., non-viral nucleic acid vectors, e.g., naked nucleic acid vectors, e.g., synthetic circular DNA vectors) for using to treat or prevent any associated disorder by any of the therapeutic methods and uses described herein (e.g., Section III).

[0181] Pharmaceutical Compositions The invention also provides methods that include administration of a pharmaceutical composition having a therapeutic agent (e.g., any of the nucleic acid vectors described herein (e.g., a circular DNA vector)) in a pharma- ceutically acceptable carrier. For example, in some cases, the pharmaceutical composition administered in conjunction with the methods described herein includes a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site) that encodes a therapeutic replacement protein that replaces a protein endogenously expressed in healthy retinal cells, and a pharma- ceutically acceptable carrier.

[0182] In some cases, the pharmaceutical composition comprises a non-viral nucleic acid vector (e.g., the pharmaceutical composition is substantially free of viral capsids). Additionally or alternatively, the pharmaceutical composition may comprise a nucleic acid vector that is not encapsulated in an envelope (e.g., a lipid envelope) or matrix (e.g., a polymer matrix) and is not physically associated (e.g., covalently or non-covalently bound) with a solid structure (e.g., a particulate structure) before and after administration to an individual. In some aspects of the pharmaceutical composition, the nucleic acid vector is not bound to any adjacent nucleic acid vector such that in a solution of the nucleic acid vectors, each nucleic acid vector is free to diffuse independently of the adjacent nucleic acid vectors. In some aspects of the pharmaceutical composition, the nucleic acid vector is associated with another agent in the liquid solution, such as a charge-modifying molecule or a stabilizing molecule.

[0183] The pharmaceutical composition may include the nucleic acid vector in a naked form, i.e., the nucleic acid vector is not complexed (e.g., encapsulated, conjugated, or non-covalently linked) with another agent. In such pharmaceutical compositions, the naked nucleic acid molecule may be co-formulated (e.g., in solution) with an agent that is not complexed with the naked nucleic acid molecule, such as a buffer and / or an agent that is generally recognized as safe (GRAS) by the U.S. Food and Drug Administration.

[0184] In some cases of the present invention, the pharmaceutical composition comprises a naked circular DNA vector.

[0185] Pharmaceutically acceptable carriers may include excipients and / or stabilizers that are non-toxic to individuals at the dosage and concentration used.In some embodiments, the pharma-ceutically acceptable carrier is a pH buffered aqueous solution.Examples of pharma-ceutically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween, polyethylene glycol (PEG) and Pluronics.

[0186] A pharmaceutical composition having a therapeutic agent (e.g., a nucleic acid vector, such as a circular DNA vector) of the present invention may include a pharma- ceutically acceptable carrier. When the composition is provided in liquid form, the carrier may be water (e.g., pyrogen-free water), isotonic saline, or an aqueous buffer solution, such as a phosphate or citrate buffer. The injection of the pharmaceutical composition may be performed in water or a buffer solution, such as an aqueous buffer solution containing a sodium salt (e.g., at least 50 mM sodium salt), a calcium salt (e.g., at least 0.01 mM calcium salt), or a potassium salt (e.g., at least 3 mM potassium salt). According to certain embodiments, the sodium, calcium, or potassium salts may be present in the form of their halides, such as chlorides, iodides, or bromides, their hydroxides, carbonates, bicarbonates, or sulfates, and the like. Without being limited thereto, examples of sodium salts include NaCl, NaI, NaBr, Na2CO2, NaHCO2, and Na2SO4. Examples of potassium salts include, for example, KCl, KI, KBr, K2CO2, KHCO2, and K2SO4. Examples of calcium salts include, for example, CaCl2, CaI2, CaBr2, CaCO2, CaSO4, and Ca(OH)2. In addition, organic anions of the above cations may be included in the buffer. According to a particular embodiment, the buffer suitable for injection purposes as defined above may contain a salt selected from sodium chloride (NaCl), calcium chloride (CaCl2) or potassium chloride (KCl), and further anions may be present. CaCl2 may also be replaced by another salt, such as KCl. In some embodiments, the salts in the injection buffer are present in a concentration of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl), and at least 0.01 mM calcium chloride (CaCl2). The injection buffer may be hypertonic, isotonic, or hypotonic relative to a particular reference medium, i.e., the buffer may have a higher, the same, or a lower salt content relative to a particular reference medium, preferably such concentrations of the aforementioned salts may be used that do not cause cell damage due to osmosis or other concentration effects.The reference medium may be a liquid, such as blood, lymph, cytosolic fluid, other body fluids, or a common buffer solution. Such common buffer solutions or liquids are known to those skilled in the art. Lactated Ringer's solution is particularly preferred as the liquid base.

[0187] One or more compatible solid or liquid fillers, diluents, or encapsulating compounds may be suitable for administration to humans. The components of the pharmaceutical composition according to the invention can be mixed with the nucleic acid vector according to the invention as defined herein in such a way that there is no interaction that would substantially reduce the pharmaceutical effectiveness of the (pharmaceutical) composition according to the invention under typical conditions of use. Pharmaceutically acceptable carriers, fillers and diluents can have a sufficiently high purity and a sufficiently low toxicity to make them suitable for administration to the individual to be treated. Some examples of compounds that can be used as pharma- ceutically acceptable carriers, fillers or components thereof are sugars such as lactose, glucose, trehalose and sucrose; starches such as corn starch or potato starch; dextrose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oils derived from cocoa butter; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; or alginic acid.

[0188] The choice of a pharma- ceutically acceptable carrier can be determined according to the way in which the pharmaceutical composition is to be administered.

[0189] Suitable unit dose forms for injection include sterile solutions of water, saline, and mixtures thereof. The pH of such solutions can be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid, and collagen matrices. Suitable pharma- ceutically acceptable carriers for topical application include those suitable for use in lotions, creams, gels, etc. When the pharmaceutical composition is administered orally, tablets, capsules, etc. are the preferred unit dose forms.

[0190] Additional additives that may be included in the pharmaceutical composition are emulsifying agents, such as Tweens; wetting agents, such as sodium lauryl sulfate; coloring agents; pharmaceutical carriers; stabilizers; antioxidants; and preservatives.

[0191] The pharmaceutical composition according to the present invention can be provided in liquid or dry (e.g., lyophilized) form. In a particular embodiment, the nucleic acid vector of the pharmaceutical composition is provided in lyophilized form. The lyophilized composition comprising the nucleic acid vector of the present invention can be reconstituted before administration in a suitable buffer, such as lactate Ringer's solution, Ringer's solution or phosphate buffer, preferably based on an aqueous carrier.

[0192] In certain embodiments of the invention, any of the nucleic acid vectors of the invention can be complexed with one or more cationic or polycationic compounds, such as cationic or polycationic polymers, cationic or polycationic peptides or proteins, such as protamine, cationic or polycationic polysaccharides, and / or cationic or polycationic lipids.

[0193] According to certain embodiments, the nucleic acid vectors of the invention may be complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles. Thus, in one embodiment, the compositions of the invention comprise liposomes, lipoplexes, and / or lipid nanoparticles that contain a therapeutic agent (e.g., a nucleic acid vector, e.g., a circular DNA vector).

[0194] Lipid-based formulations can be effective delivery systems for nucleic acid vectors due to their biocompatibility and ease of large-scale production. Cationic lipids have been widely explored as synthetic materials for delivering nucleic acids. After mixing together, the nucleic acid is condensed by the cationic lipid to form lipid / nucleic acid complexes known as lipoplexes. These lipid complexes can protect the genetic material from the action of nucleases and deliver the genetic material into cells by interacting with the negatively charged cell membrane. Lipoplexes can be prepared by directly mixing positively charged lipids with negatively charged nucleic acids at physiological pH.

[0195] Conventional liposomes contain a lipid bilayer, which may be composed of cationic, anionic or neutral phospholipids and cholesterol, which surrounds an aqueous core. Both the lipid bilayer and the aqueous space can incorporate hydrophobic or hydrophilic compounds, respectively. The characteristics and behavior of liposomes in vivo can be modified by adding hydrophilic polymer coatings, such as polyethylene glycol (PEG), to the liposome surface to impart steric stabilization. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides and carbohydrates) to their surface or to the ends of the attached PEG chains.

[0196] Liposomes are colloidal lipid-based and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment. They can exist as spherical vesicles and can range in size from 20 nm to several microns. Cationic lipid-based liposomes can complex with negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer the biocompatibility, low toxicity, and potential for large-scale production required for in vivo clinical use. Liposomes can fuse with the plasma membrane for uptake; once inside the cell, the liposomes are processed via the endocytic pathway and the genetic material is then released from the endosome / carrier into the cytoplasm.

[0197] Cationic liposomes can serve as a delivery system for DNA and / or RNA. Cationic lipids such as MAP (1,2-dioleoyl-3-trimethylammonium propane) and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate) can form complexes or lipoplexes with negatively charged nucleic acids to form nanoparticles through electrostatic interactions, providing high in vitro transfection efficiency. In addition, neutral lipid-based nanoliposomes for nucleic acid vector delivery are available, for example, neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomes.

[0198] Thus, in one aspect of the invention, the nucleic acid vector of the invention is complexed with cationic lipids and / or neutral lipids, thereby forming liposomes, lipid nanoparticles, lipoplexes or neutral lipid-based nanoliposomes.

[0199] In a particular embodiment, the pharmaceutical composition according to the invention comprises a nucleic acid vector of the invention formulated with a cationic or polycationic compound and / or a polymeric carrier. Thus, in a further embodiment of the invention, the nucleic acid vector as defined herein can be optionally formulated with a ratio of about 5:1 (w / w) to about 0.25:1 (w / w), such as about 5:1 (w / w) to about 0.5:1 (w / w), such as about 4:1 (w / w) to about 1:1 (w / w) or about 3:1 (w / w) to about 1:1 (w / w), such as about 3:1 (w / w) to about 2:1 (w / w) of the nucleic acid vector to the cationic or polycationic compound and / or the polymeric carrier. or optionally with a nitrogen / phosphate (N / P) ratio between the nucleic acid vector and the cationic or polycationic compound and / or polymeric carrier in the range of about 0.1 to 10, e.g., in the range of about 0.3 to 4 or 0.3 to 1, e.g., in the range of about 0.5 to 1 or 0.7 to 1, e.g., in the range of about 0.3 to 0.9 or 0.5 to 0.9. For example, the N / P ratio between the nucleic acid vector and one or more polycations is in the range of about 0.1 to 10, including the ranges of about 0.3 to 4, about 0.5 to 2, about 0.7 to 2, and about 0.7 to 1.5.

[0200] The nucleic acid vectors described herein can also be associated with vehicles, transfection agents or complexing agents to increase transfection efficiency and / or expression of the regulated gene according to the present invention.

[0201] In some cases, the pharmaceutical composition includes a nucleic acid vector complexed with one or more polycations (e.g., protamine or oligofectamine), for example as a particle (e.g., nanoparticle or microparticle). Further cationic or polycationic compounds that can be used as transfection agents, complexing agents or particles (e.g. nanoparticles or microparticles) include cationic polysaccharides, such as chitosan, polybrene, cationic polymers, such as polyethyleneimine (PEI), cationic lipids, such as DOTMA: [1-(2,3-dioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPE, LEAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristoxypropyldimethylhydroxyethylammonium bromide, MAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(α-trimethylammonioa), cetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]trimethylammonium, oligofectamine, or cationic or polycationic polymers, e.g. modified polyamino acids such as β-amino acid polymers or inverse polyamides, modified polyethylenes such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified amidoamines such as pAMAM (poly(amidoamine)), polybeta amino esters (PBAE) or modified PBAEs (e.g. polymers described in U.S. Pat. No. 8,557,231 ;PEGylated PBAEs, such as those described in U.S. Patent Application No. 2018 / 0112038; any suitable polymer disclosed in Green et al., Acc. Chem. Res. 2008, 41(6):749-759, such as diamine-terminated 1,4 butanediol diacrylate-co-5-amino-1-pentanol polymer; any suitable modified PBAEs described in WO 2020 / 077159 or WO 2019 / 070727; or PBAEs disclosed in Shen et al., Sci. Adv. 2020, 6:eaba1606. 457), dendrimers such as polypropylamine dendrimers or pAMAM-based dendrimers, polyimine(s) such as PEI: poly(ethyleneimine), poly(propyleneimine), polyallylamine, cyclodextrin-based polymers, dextran-based polymers, sugar backbone-based polymers such as chitosan, silane backbone-based polymers such as PMOXA-PDMS copolymers, block polymers consisting of a combination of one or more cationic blocks (e.g. selected from the cationic polymers listed above) with one or more hydrophilic or hydrophobic blocks (e.g. polyethylene glycol);

[0202] In some cases, the pharmaceutical composition comprises a nucleic acid vector encapsulated in a nanoparticle or microparticle, e.g., a biodegradable nanoparticle or microparticle (e.g., a cationic biodegradable polymer nanoparticle or microparticle, such as a PBAE or a modified PBAE (e.g., a polymer of formula (I) in WO 2019 / 070727, or PBAE 457 disclosed in Shen et al., Sci. Adv. 2020, 6:eaba1606), or a PEG-PBAE (or modified PBAE) copolymer) or a pH-sensitive nanoparticle or microparticle (e.g., a nanoparticle (ECO) having a polymer of formula (I) in U.S. Pat. No. 10,792,374).

[0203] According to a particular embodiment, the pharmaceutical composition of the present invention comprises a therapeutic agent, for example a nucleic acid vector (e.g., a circular DNA vector) encapsulated in or bound to a polymeric carrier. The polymeric carrier used according to the present invention may be a polymeric carrier formed by disulfide-bridged cationic components. The disulfide-bridged cationic components may be the same as each other or different. The polymeric carrier may also comprise additional components. It is also particularly preferred that the polymeric carrier used according to the present invention comprises a mixture of cationic peptides, proteins or polymers crosslinked by disulfide bonds as described herein and optionally additional components as defined herein. In this context, the disclosure of WO 2012 / 013326 is incorporated herein by reference. In this context, the cationic components forming the basis of the polymeric carrier by disulfide bridges are typically selected from any suitable cationic or polycationic peptides, proteins or polymers suitable for this purpose, in particular any cationic or polycationic peptides, proteins or polymers capable of complexing additional nucleic acids contained in the nucleic acid vector or composition as defined herein, thereby preferably condensing the nucleic acid vector. The cationic or polycationic peptides, proteins or polymers can be linear molecules; however, branched cationic or polycationic peptides, proteins or polymers can also be used.

[0204] All disulfide bridged cationic or polycationic proteins, peptides or polymers of the polymeric carrier that may be used to complex the nucleic acid vectors according to the invention included as part of the pharmaceutical composition of the present invention may contain at least one SH moiety (e.g. at least one cysteine ​​residue or any further chemical group exhibiting an SH moiety) capable of forming a disulfide bond upon condensation with at least one further cationic or polycationic protein, peptide or polymer as the cationic component of the polymeric carrier referred to herein.

[0205] Such polymeric carriers used to complex the nucleic acid vectors of the present invention may be formed by disulfide-bridged cationic (or polycationic) components. In particular, such cationic or polycationic peptides or proteins or polymers of the polymeric carriers containing or further modified to contain at least one SH moiety may be selected from proteins, peptides and polymers as complexing agents.

[0206] In other embodiments, the pharmaceutical compositions according to the present invention may be administered naked, without association with any additional vehicles, transfection agents, or complexing agents.

[0207] Any of the aforementioned pharmaceutical compositions (e.g., a pharmaceutical composition comprising any of the aforementioned therapeutic sequences or therapeutic genes (e.g., a therapeutic nucleic acid vector, e.g., a non-viral nucleic acid vector, e.g., a naked nucleic acid vector, e.g., a synthetic circular DNA vector)) can be used to treat a disease or disorder (e.g., in an individual in need thereof, e.g., an individual having a disease or disorder or at risk of developing a disease or disorder). Thus, provided herein is the use of any of the aforementioned pharmaceutical compositions for treating or preventing any associated disorder according to any of the therapeutic methods and uses described herein (e.g., Section III). Further provided herein is any of the aforementioned pharmaceutical compositions for use in treating or preventing any associated disorder according to any of the therapeutic methods and uses described herein (e.g., Section III).

[0208] III. Treatment Methods and Uses Provided herein are methods of delivering a therapeutic agent (e.g., a nucleic acid vector encoding a therapeutic replacement protein) to ocular cells of an individual (e.g., a human patient). Such approaches may include (a) electrical introduction to facilitate delivery of the therapeutic agent to target cells in the individual, (b) administration of the therapeutic agent to the individual, or both (a) and (b). Such methods include administration of any of the therapeutic agents or pharmaceutical compositions described herein, such as a nucleic acid vector or a pharmaceutical composition thereof (e.g., a pharmaceutical composition comprising a naked nucleic acid vector). Also provided herein are methods of treating an ocular disease or disorder in an individual by a) electrical introduction to facilitate delivery of the therapeutic agent to target cells in the individual, (b) administration of the therapeutic agent to the individual, or both (a) and (b). Particular ocular diseases that can be treated using such compositions and methods include ABCA4-associated retinal dystrophies (e.g., Stargardt disease), MYO7A-associated retinal dystrophies (e.g., Usher syndrome type 1B), bestrophinopathy associated with BEST1 dominant or BEST1 recessive mutations (e.g., autosomal recessive bestrophinopathy, Best vitelliform macular dystrophy, BEST1 adult-onset vitelliform macular dystrophy, or autosomal dominant vitreoretinochoroidopathy), and age-related macular degeneration.

[0209] Eye Diseases and Individuals The therapeutic agents and pharmaceutical compositions described herein can be used to treat various ocular diseases or disorders. In some cases, the ocular disease or disorder is a retinal disease or disorder, such as a retinal dystrophy (e.g., a retinal dystrophy characterized by a reduced level of functional expression (e.g., lack of functional expression) of a retinal protein in an individual compared to a reference (e.g., a healthy level of functional expression). In some embodiments, the ocular disease or disorder (e.g., a retinal disease or disorder) is a single gene disorder. In some embodiments, the ocular disease or disorder (e.g., a retinal disease or disorder) is a recessive genetic disorder. In some embodiments, the individual has or is expected to develop an ocular disease or disorder (e.g., a retinal disease or disorder) caused by a heterozygous mutation. In other embodiments, the individual has or is expected to develop an ocular disease or disorder (e.g., a retinal disease or disorder) caused by a homozygous mutation.

[0210] In some embodiments (e.g., in embodiments where the individual is being treated for an ABCA4-associated retinal dystrophy, such as Stargardt's disease or cone-rod dystrophy), the retinal protein is ABCA4. In such embodiments, the individual may be an adult, teenager, or child with retinal degeneration due to an ABCA4 mutation (e.g., a biallelic ABCA4 mutation). In some cases, the individual has macular degeneration due to a recessive biallelic ABCA4 mutation. The individual may have retinal degeneration of any severity due to a biallelic ABCA4 mutation.

[0211] In some embodiments (eg, those in which the individual is being treated for Usher Syndrome Type 1B), the retinal protein is MYO7A.

[0212] In some embodiments (e.g., those in which an individual is being treated for a bestrophinopathy associated with a BEST1 dominant mutation or a BEST1 recessive mutation, e.g., autosomal recessive bestrophinopathy, Best vitelliform macular dystrophy, autosomal dominant vitreoretinochoroidopathy, BEST1 adult-onset vitelliform macular dystrophy, autosomal dominant microcornea, cone-rod dystrophy, early onset cataract posterior polar uveoma syndrome, or retinitis pigmentosa), the retinal protein is BEST1.

[0213] In some embodiments (eg, those in which the individual is being treated for age-related macular degeneration), the retinal protein is CFH.

[0214] In some embodiments, the ocular disease or disorder is Usher syndrome (e.g., Usher syndrome type 1B), autosomal recessive Bestrophinopathy, autosomal dominant Best vitelliform macular dystrophy, macular degeneration (e.g., age-related macular degeneration (AMD), wet macular degeneration (e.g., wet AMD), dry macular degeneration (e.g., dry AMD) or neovascular AMD), geographic atrophy, retinitis pigmentosa (RP), diabetic eye disorder (e.g., diabetic retinopathy or diabetic macular edema), or ophthalmic disorder (e.g., diabetic retinopathy or diabetic macular edema). retinal vein occlusion (e.g., central retinal vein occlusion or branch retinal vein occlusion), retinal artery occlusion, macular edema (e.g., macular edema following retinal vein occlusion), refractive and accommodative disorders, keratoconus, amblyopia, glaucoma, Stargardt's disease, endophthalmitis, conjunctivitis, uveitis (e.g., posterior uveitis), retinal detachment, corneal ulcer, dacryocystitis, Duane's constriction syndrome, optic neuritis, choroidal neovascularization, choroidal ischemia, or hypertensive retinopathy.

[0215] In some embodiments, the ocular disease or disorder is a retinal dystrophy (e.g., a Mendelian retinal dystrophy). In some embodiments, the retinal dystrophy is selected from the group consisting of Leber's congenital amaurosis (LCA), Stargardt's disease, pseudoxanthoma elasticum, cone-rod dystrophy, exudative vitreoretinopathy, Joubert syndrome, congenital stationary night blindness type 1C (CSNB-1C), age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.

[0216] In some cases, the methods provided herein are useful for treating symptoms of such ocular diseases or disorders, such as any of the above diseases or disorders, or ocular symptoms of more widespread disorders, such as hypotension, hypertension, infection, sarcoid or sickle cell disease. In some aspects, the disease is an acute ocular disease. In other aspects, the disease is a chronic ocular disease.

[0217] In some embodiments, the individual being treated is a human patient. In some embodiments, the individual is a pediatric human patient, e.g., a person who is 21 years of age or younger at the time of diagnosis or treatment. In some embodiments, the pediatric human patient is 16 years of age or younger at the time of treatment. In other embodiments, the individual is 22 to 40 years of age at the time of treatment. In other embodiments, the individual is 41 to 60 years of age at the time of treatment. In other embodiments, the individual is 61 years of age or older at the time of treatment. In some cases, the individual is male. In other cases, the individual is female.

[0218] Administration of therapeutic drugs Provided herein are methods of administering a therapeutic agent (e.g., a nucleic acid vector (e.g., any of the nucleic acid vectors described herein)) or pharmaceutical composition thereof to the eye as a means of delivering the therapeutic agent to a target retinal cell of an individual (e.g., a human patient). For reference, an anatomical diagram of the eye is shown in FIG. 1. In some cases, the nucleic acid vector is administered to the eye such that the nucleic acid vector enters the extracellular space of the posterior region of the eye (e.g., the retina (e.g., the macula)). Once the nucleic acid vector enters the posterior extracellular space upon administration (e.g., as a naked formulation, encapsulated in a nanoparticle or microparticle (e.g., lipid nanoparticle or microparticle), or released from a nanoparticle or microparticle), the nucleic acid vector can then be electrotransduced into the target retinal cell upon transmission of electrical energy that reaches the posterior region of the eye (e.g., the retina (e.g., the macula)), for example, via transmission of electrical energy from an electrode placed in the vitreous or subretinal space. In some cases, the nucleic acid vector is administered to the eye such that the nucleic acid vector enters the extracellular space of the posterior region of the eye (e.g., the posterior suprachoroid or posterior choroid). Once the nucleic acid vector enters the posterior extracellular space upon administration, the nucleic acid vector can then be electrotransduced into the target retinal cells upon transmission of electrical energy that reaches the posterior portion of the eye (e.g., the posterior suprachoroidal or posterior choroidal), for example, via transmission of electrical energy from an electrode placed in the vitreous or subretinal space.

[0219] In some embodiments, the nucleic acid vector is administered prior to the methods described herein (e.g., prior to the method of transmitting an electric field to retinal tissue). For example, the nucleic acid vector can be administered within 24 hours prior to the transmission of the electric field (e.g., within 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or 5 seconds prior to the transmission of the electric field). In some embodiments, the nucleic acid vector is administered after the methods described herein (e.g., after the method of transmitting an electric field to retinal tissue), for example, when the nucleic acid vector is released from the nanoparticle or microparticle over time. For example, the nucleic acid vector can be administered within 24 hours after delivery of the electric field (e.g., within 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, or 5 seconds after delivery of the electric field). In some embodiments, the nucleic acid vector is administered as part of a method described herein.

[0220] Any suitable means of ocular administration known in the art or described herein can be used as part of the methods provided herein. The method of delivering therapeutic agents to target retinal cells includes administering a nucleic acid vector to the eye by intraocular injection (e.g., injection into the back of the eye or the front of the eye, e.g., suprachoroidal injection, intravitreal injection, subretinal injection, periocular injection, sub-Tenon injection, posterior juxtascleral injection, anterior chamber injection, subconjunctival injection, or retrobulbar injection) or intraocular implant. In some aspects of any of the methods described herein, administration of the nucleic acid vector is via intraocular implant (e.g., controlled release or depot implant, intravitreal implant, subconjunctival implant, or episcleral implant). In other aspects, administration of the nucleic acid vector is not via intraocular implant (e.g., controlled release or depot implant, intravitreal implant, subconjunctival implant, or episcleral implant). In some aspects of any of the methods described herein, administration of the nucleic acid vector is via iontophoresis (e.g., the method includes administering the nucleic acid vector to the intraocular space by iontophoresis as described herein, followed by delivery to the retina by transmitting an electrical current through electrodes in contact with the interior region of the eye). In other aspects, administration of the nucleic acid vector does not include iontophoresis.

[0221] In some cases, administration of nucleic acid vector is non-surgical.For example, in some embodiments, administration of nucleic acid vector does not utilize general anesthesia and / or does not include retrobulbar anesthesia (i.e., retrobulbar block).Additionally or alternatively, administration of nucleic acid vector does not include injection using a needle larger than 28 gauge.Additionally or alternatively, administration of nucleic acid vector does not include the use of a guidance mechanism that is typically required for ocular drug delivery via shunt or cannula.

[0222] In some cases, the nucleic acid vector is administered by injection (e.g., microneedle injection) into an external tissue of the eye, such as the choroidal space, sclera, cornea, corneal stroma, conjunctiva, subconjunctival space, or subretinal space. Alternatively, the nucleic acid vector is administered by injection (e.g., microneedle injection) into a site proximal to an external tissue, such as the trabecular meshwork, ciliary body, aqueous humor, or vitreous humor.

[0223] The microneedle for injecting the nucleic acid vector into the eye includes a hollow microneedle, which may include an elongated housing for holding the proximal end of the microneedle. The microneedle may further include a means for directing the formulation therethrough. For example, the means may be a flexible or rigid conduit in fluid communication with the base or proximal end of the microneedle. The means may also include a pump or other device for creating a pressure gradient to induce fluid flow through the device. The conduit may be operatively connected to a source of the formulation. The source may be any suitable container. In one embodiment, the source may be in the form of a conventional syringe. The source may be a disposable unit dose container. In one embodiment, the microneedle has an effective length of about 50 μm to about 2000 μm. In another specific embodiment, the microneedle has an effective length of about 150 μm to about 1500 μm, about 300 μm to about 1250 μm, about 500 μm to about 1250 μm, about 500 μm to about 1500 μm, about 600 μm to about 1000 μm, or about 700 μm to about 1000 μm. In one embodiment, the effective length of the microneedle is about 600 μm, about 700 μm, about 800 μm, or about 1000 μm. In various embodiments, the proximal portion of the microneedle has a maximum width or cross-sectional dimension of about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 50 μm to about 500 μm, about 100 μm to about 400 μm, about 200 μm to about 600 μm, or about 100 μm to about 250 μm, and an opening diameter of about 5 μm to about 400 μm. In certain embodiments, the proximal portion of the microneedle has a maximum width or cross-sectional dimension of about 600 μm. In various embodiments, the microneedle has a bevel height of 50 μm to 500 μm, 100 μm to 500 μm, 100 μm to 400 μm, 200 μm to 400 μm, or 300 μm to 500 μm.

[0224] The microneedles may have an aspect ratio (width:length) of about 1:1.5 to about 1:10. In one embodiment, the aspect ratio of the microneedles is about 1:3 to about 1:5. In another embodiment, the aspect ratio of the microneedles is about 1:4 to about 1:10.

[0225] In certain embodiments, the microneedle may be designed such that the tip portion of the microneedle is substantially the only portion of the microneedle that is inserted into the ocular tissue (i.e., the tip portion is greater than 75% of the total length of the microneedle, greater than 85% of the total length of the microneedle, or greater than about 95% of the total length of the microneedle). In other certain embodiments, the microneedle may be designed such that the tip portion is the only portion of the microneedle that is inserted into the ocular tissue, generally having a length of less than about 75% of the total length of the microneedle, less than about 50% of the total length of the microneedle, or less than about 25% of the total length of the microneedle. For example, in one embodiment, the microneedle has a total effective length of 500 μm to 1500 μm, where the tip portion has a length of less than about 400 μm, less than about 300 μm, or less than about 200 μm.

[0226] In one embodiment, the height of the bevel is 100 μm to about 500 μm. In another embodiment, the height of the bevel is 500 μm or less, 450 μm or less, 400 μm or less, or 350 μm or less. In another embodiment, the height of the bevel is 200 μm to 500 μm, 100 μm to 700 μm, or 200 μm to about 700 μm. In yet another embodiment, the height of the bevel is 500 μm to 900 μm, 500 μm to 800 μm, or 500 μm to 700 μm. In this manner, the configuration of the bevel can be sufficiently sharp so that the distal end punctures the target tissue and penetrates into the vitreous without (i) substantially elastically deforming the target tissue, or (ii) damaging the internal structures of the eye, such as the lens or retina.

[0227] The microneedles useful in the method can be made of various biocompatible materials, including metals, glass, semiconductor materials, ceramics, or polymers. Examples of suitable metals include pharmaceutical grade stainless steel, gold, titanium, nickel, iron, gold, tin, chromium, copper, platinum, and their alloys. Suitable polymers can be biodegradable or non-biodegradable. Examples of suitable biocompatible biodegradable polymers include polylactide, polyglycolide, polylactide-co-glycolide (PLGA), polyanhydrides, polyorthoesters, polyetheresters, polycaprolactone, polyesteramides, poly(butyric acid), poly(valeric acid), polyurethanes, and copolymers and blends thereof. Representative non-biodegradable polymers include various thermoplastics or other polymeric structural materials known in the manufacture of medical devices. Examples include nylon, polyester, polycarbonate, polyacrylate, ethylene-vinyl acetate and other acyl-substituted cellulose acetate polymers, non-degradable polyurethanes, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulfonated polyolefins, polyethylene oxide, blends and copolymers thereof. Biodegradable microneedles can provide a high level of safety compared to non-biodegradable microneedles, such that they are essentially harmless even if they are unnoticed and dislodged into the eye tissue.

[0228] In certain instances, administration of the nucleic acid vector is by suprachoroidal injection, which can be achieved in a minimally invasive, non-surgical manner. For example, suprachoroidal injection can provide nucleic acid delivery over a larger tissue area and to less accessible target tissues in a single administration compared to other types of administration (e.g., subretinal injection). Without wishing to be bound by theory, upon entering the suprachoroidal space, the pharmaceutical composition can flow circumferentially toward the retina-choroidal tissues, macula, and optic nerve of the posterior segment of the eye. In addition, a portion of the injected pharmaceutical composition can remain in the suprachoroidal space as a depot, or can remain in the tissues covering the suprachoroidal space, such as the sclera, near the microneedle insertion site, and then function as an additional depot of the pharmaceutical composition that can diffuse into the suprachoroidal space and other adjacent posterior tissues.

[0229] Choroidal injection can be performed using any suitable method known in the art or described herein.For example, in some cases, the nucleic acid vector is administered suprachoroidally via a microneedle (e.g., a hollow microneedle).In some cases, the nucleic acid vector is administered suprachoroidally via a microneedle array.Exemplary microneedles suitable for use in suprachoroidal administration of the nucleic acid vectors described herein are described, for example, in U.S. Patent Application No. 2017 / 0273827, which is incorporated herein by reference.

[0230] Choroidal injection can be performed using methods known in the art. For example, the microneedle tip can be placed in the eye so that the formulation flows into the choroidal space and the posterior ocular tissues surrounding the choroidal space. In one embodiment, the microneedle is inserted in the sclera of the eye. In one embodiment, the flow of the drug into the choroidal space is achieved without contacting the underlying tissues, such as choroidal and retinal tissues, with the microneedle. In some embodiments, one or more microneedles are inserted vertically or at an angle of 80° to 100° into the eye, for example, into the sclera, to reach the choroidal space with a short penetration distance. Exemplary methods suitable for use in choroidal administration of the nucleic acid vectors described herein are described, for example, in International Publication No. WO 2014 / 074823, which is incorporated herein by reference.

[0231] In some embodiments, the device includes an array of two or more microneedles. For example, the device may include an array of 2-1000 (e.g., 2-100) microneedles. In one embodiment, the device includes 1-50 microneedles. The array of microneedles may include a mixture of different microneedles. For example, the array may include microneedles having various lengths, base diameters, tip shapes, spacing between microneedles, drug coatings, etc. In embodiments where the microneedle device includes an array of two or more microneedles, the angle at which a single microneedle extends from the base may be independent of the angle at which another microneedle in the array extends from the base.

[0232] In some cases, the present methods of delivering a therapeutic agent (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene and / or a recombination site)) include intravitreal administration of the therapeutic agent. Intravitreal administration can be performed using any suitable method known in the art or described herein. For example, intravitreal injection methods including the InVitria Injection Assistant (FCI Ophthalmics, Pembroke, MA), the Rapid Access Vitreal Injection (RAVI) Guide (Katalyst Surgical, Chesterfield, MO), the Doi-Umeatsu Intravitreal Injection Guide (Duckworth&Kent Ltd., England), the Malosa Intravitreal Injection Guide (Beaver-Visitec International, Waltham, MA), or an automatic injection guide are contemplated herein.

[0233] The present invention includes methods in which a nucleic acid vector is administered suprachoroidally via a device (e.g., a microinjector device) that includes a cannula and / or a microneedle (e.g., any of the microneedles described above). Exemplary devices suitable for use in suprachoroidal administration of the nucleic acid vectors described herein are described, for example, in U.S. Pat. No. 10,722,396, U.S. Design Patent No. 750223S1, and Hancock et al., Expert Opinion on Drug Delivery 2021, DOI: 10.1080 / 17425247.2021.1867532, each of which is incorporated herein by reference.

[0234] In some cases, suprachoroidal injections are performed, for example, within the pars plana, 1-5 mm from the limbus. Microneedles for use in such injections can be designed to have a length that substantially matches the scleral thickness at the pars plana (e.g., 400 μm to 600 μm, e.g., about 500 μm).

[0235] In some embodiments of any of the methods described herein that include a suprachoroidal injection, the suprachoroidal injection is a bilateral suprachoroidal injection (e.g., split into two injections). In other embodiments, the suprachoroidal injection is a unilateral suprachoroidal injection (e.g., a single injection).

[0236] In some cases, the method of delivering a therapeutic agent to targeted retinal cells comprises systemically administering (eg, intravenously or orally) a nucleic acid vector.

[0237] Any suitable dose of the nucleic acid vector may be administered. For example, in an embodiment involving subretinal administration of a naked nucleic acid vector, each eye may be injected with one or more blebs, each having a volume of 20 to 500 μL (e.g., 50 to 250 μL; e.g., 50 to 100 μL, 100 to 150 μL, 150 to 200 μL, or 200 to 250 μL; e.g., about 50 μL, about 75 μL, about 100 μL, about 150 μL, or about 200 μL), e.g., one bleb, two blebs, three blebs, four blebs, or more per eye. In embodiments involving subretinal administration of a naked nucleic acid vector, the injection volume (e.g., pharmaceutical composition) may range from 0.5 mg / mL to 5 mg / mL (e.g., 1.0 mg / mL to 2.5 mg / mL; e.g., 0.5 mg / mL to 1.0 mg / mL, 1.0 mg / mL to 1.5 mg / mL, 1.5 mg / mL to 2.0 mg / mL, 2.0 mg / mL to 2.5 mg / mL, 2.5 mg / mL to 3.0 mg / mL, 3.0 mg / mL to 4.0 mg / mL, or for example, about 0.5 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, about 2.0 mg / mL, about 2.5 mg / mL, about 3.0 mg / mL, about 4.0 mg / mL, or about 5.0 mg / mL. In certain cases (e.g., when a naked nucleic acid vector is administered subretinally), the injection volume (e.g., pharmaceutical composition) contains the nucleic acid vector at a concentration of 1.5 mg / mL.In some embodiments involving subretinal administration, the naked nucleic acid vector may be administered in a concentration of 20 μg to 2.0 mg (e.g., 100 μg to 1.0 mg, or 200 μg to 500 μg; e.g., 20 μg to 50 μg, 50 μg to 100 μg, 100 μg to 150 μg, 150 μg to 200 μg, 200 μg to 250 μg, 250 μg to 300 μg, 300 μg to 350 μg, 350 μg to 400 μg, 400 μg to 500 μg, 500 μg to 750 μg, 750 μg to 1.0 mg, 1.0 mg to 1.5 mg, or 1.5 mg to 2.0 mg; e.g., about 20 μg, about 25 μg, about 30

[0043] The present invention is directed to a method for administering the present invention to an eye in an amount of about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, or about 2.0 mg. In some embodiments, including subretinal administration, the naked nucleic acid vector is 8 ~10 15 vector copies (e.g., DNA vector molecules, e.g., circular DNA vector molecules) (e.g., 10 8 ~10 9 pieces, 10 9 ~10 10 pieces, 10 10 ~10 11 pieces, 10 11 ~10 12 pieces, 10 12 ~10 13 pieces, 10 13 ~10 14 Pieces or 10 14 ~10 15 copies of the vector; for example, about 1x10 11 Vector copies, approximately 5x10 11 Vector copies, approximately 1x10 12 Vector copies, approximately 5x10 12 Vector copies, approximately 1x10 13 Vector copies, approximately 2.5x1013 vector copies, or approximately 5x10 13 In a specific embodiment, the naked nucleic acid vector is administered to each eye in an amount of about 2.5×10 13 In another embodiment, the naked nucleic acid vector is administered subretinally at a total dose of about 5×10 vector copies per eye (e.g., two 75 μL blebs per eye). 12 In another embodiment, the naked nucleic acid vector is administered subretinally at a total dose of about 5×10 vector copies per eye (e.g., two 75 μL blebs per eye). 11 Administer subretinally for a total dose of 10 vector copies per eye (e.g., two 75 µL blebs per eye).

[0238] Electric field transmission A method for delivering a therapeutic agent (e.g., a nucleic acid vector) to an eye includes transmitting electrical energy to a tissue in which a target ocular cell is present. Such a method includes electrical introduction of a therapeutic agent from the extracellular space at the back of the eye (e.g., the choroidal space, the choroid, the retina, or the vitreous body) to a target ocular cell (e.g., a retinal cell). For example, in some cases where an individual is being treated for a retinal disease or disorder, the method includes transmitting electrical energy to the retina to cause electrical introduction of a therapeutic agent (e.g., a nucleic acid vector) from the extracellular space of the retina to one or more retinal cell types (e.g., photoreceptor and / or retinal pigment epithelial cells).

[0239] In some aspects of the invention, an electrode is placed inside the eye of an individual, and an electric field is delivered through the electrode to a target ocular tissue (e.g., the retina) under conditions suitable for electrical delivery of a therapeutic agent (e.g., a nucleic acid vector) to a target cell (e.g., a target retinal cell). The electric field (e.g., a pulsed electric field (PEF)) delivered to the target ocular tissue can promote the transfer of a nucleic acid vector (e.g., a circular DNA vector) to the target ocular cell. Such electrical delivery can occur by any one of several mechanisms (and combinations thereof), including electrophoresis, electrokinetically driven drug uptake, and / or electroporation. The delivery of the electric field includes conditions suitable for such mechanisms. Suitable means for generating an electric field for electrical delivery of nucleic acids in mammalian tissues are known in the art, and any suitable means known in the art or described herein can be adapted for use as part of the present invention.

[0240] Various means of generating and transmitting electric fields into tissue are contemplated herein as part of the method. Devices and systems with electrodes suitable for transmitting electric fields in mammalian tissue are commercially available and can be useful in the methods disclosed herein. In some cases, the electric field is transmitted through an electrode that includes a needle (e.g., a needle placed in the vitreous humor or subretinal space). Suitable needle electrodes include the CLINIPORATOR® electrode marketed by IGEA® and the needle electrode marketed by AMBU®. The electrode (e.g., needle electrode) can be made of any suitable conductive material, such as metal or metal alloy, e.g., platinum, stainless steel, nickel, titanium, and combinations thereof, e.g., platinum / iridium alloy or nitinol.

[0241] In some embodiments, the electrode used as part of the methods described herein is a substantially flat electrode, such as any of the substantially flat electrodes described in U.S. Patent Application Nos. 63 / 163,350, 63 / 167,296, and 63 / 293,297, the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, the electrode used as part of the methods described herein is a substantially flat electrode as described herein (e.g., see the Device section below). Such a substantially flat electrode is composed of a shape memory material (e.g., a shape memory alloy) that allows the structure of the elongated conductor (e.g., a wire electrode) to relax into a preformed substantially flat electrode when uncovered. The substantially flat electrode is approximately perpendicular to the longitudinal axis of the sheath and / or proximal portion of the wire (e.g., the area that does not include the substantially flat electrode). Those skilled in the art will understand that in some embodiments, a substantially flat electrode may not be completely flat. For example, in some embodiments, two of its vertical dimensions (e.g., Cartesian dimensions such as depth and width) are each at least twice as long as its third vertical dimension (e.g., length). In some embodiments, a substantially flat electrode refers to an electrode in which two of its vertical dimensions are each at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least twenty times, at least thirty times, at least forty times, at least fifty times, at least sixty times, at least seventy times, at least eighty times, or at least one hundred times, or more, of its third vertical dimension. Thus, in some cases, the longitudinal dimension of a substantially flat electrode is less than 10% of the radial dimension of the substantially flat electrode (e.g., at its outermost radial point). In some cases, the longitudinal dimension of a substantially flat electrode is less than 5% of its radial dimension (e.g., at its outermost radial point).

[0242] In certain embodiments, the spatial configuration of the electrode is manufactured to optimize its conductive properties and / or apply a desired electric field to a target region of the cell. Thus, if the eye includes a curvature, the shape of the electrode may also include a curvature (e.g., a convex shape) that matches or approximates the shape of the eye or a portion thereof (e.g., the retina), for example.

[0243] In some examples, the elongated conductor is a wire and the substantially flat electrode is a distal portion of the wire. In some cases, the distal tip of the wire (or a point along the wire within 5 mm (e.g., within 4 mm, within 3 mm, within 2 mm, within 1 mm, within 0.5 mm, or within 0.1 mm) of the distal tip of the wire) is at the outermost radial point of the substantially flat electrode. The distal portion of the wire may include a preformed right angle (or a substantially right angle, e.g., about 70°, 75°, 80°, 85°, 86°, 87°, 88°, 89°, 90°, 91°, 92°, 93°, 94°, 95°, 100°, 105°, or 110°) on the longitudinal plane, where the preformed right angle is between the substantially flat electrode and the proximal portion of the wire.

[0244] In some embodiments, the substantially flat electrode extends 1 mm or less (e.g., 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm or less) distal or proximal from the preformed right angle. In some embodiments, the substantially flat electrode extends 1 mm or less (e.g., 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm or less) distal from the preformed right angle. In some embodiments, the device includes nothing distal to the substantially flat electrode (e.g., the substantially flat electrode is free to contact the tissue surface).

[0245] In some embodiments, a substantially flat electrode is about 2 mm to about 15 mm (e.g., about 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm) in one or more dimensions (e.g., both dimensions) perpendicular to the longitudinal axis.

[0246] In some embodiments, the substantially flat electrode is substantially symmetrical about the longitudinal plane. In some embodiments, the substantially flat electrode is a spiral. For example, the spiral can include 1 to 5 (e.g., 1, 1.5, 2, 2.5, 3, 2.5, 4, 4.5, 5, 5.5, 6, 7, 8, or more) turns about the longitudinal axis. In some cases, the spiral has 2 to 5 turns about the longitudinal axis. In some cases, the spiral has 2 to 3 turns about the longitudinal axis. In certain embodiments, the spiral has 2 turns about the longitudinal axis. In some embodiments, the spiral has 3 turns about the longitudinal axis. Other suitable shapes include, for example, loop, concentric loop, paddle, mesh, grid, or umbrella shapes.

[0247] The substantially flat electrode can be made entirely or partially from a shape memory material (e.g., a shape memory alloy, e.g., NiTi) that can recover its original shape in the presence of a predetermined stimulus. For example, the shape memory material can relax to a preformed shape when a structural constraint is removed. As described herein, a preformed shape memory wire (e.g., a substantially flat electrode) housed in a rigid sheath is straight until it is desheathed, at which point the shape memory material relaxes to its preformed shape, such as a spiral. Shape memory materials are known in the art. In some embodiments, the shape memory material includes an alloy such as NiTi, CuAlNi, or CuZnAl. The shape memory material can be iron-based. In some embodiments, the shape memory material is NiTi. NiTi is an alloy of nickel and titanium (nitinol). The nitinol can include, for example, about 40% to about 70% nickel (eg, about 40%, 45%, 50%, 55%, 60%, 65%, or 70% nickel).

[0248] Electrodes for use in the methods (e.g., substantially planar or non-planar electrodes (e.g., substantially axial wire electrodes)) can be monopolar. In some embodiments involving electrical induction using monopolar electrodes, a ground electrode is attached to the individual at a point other than the eye (e.g., attached to the skin of the individual). In some embodiments, the ground electrode is a pad that contacts the skin of the individual's buttocks, leg, torso, neck (e.g., back of the neck), or head (e.g., back of the head). In some embodiments, the monopolar electrode transmits electrical energy when positively charged. In some embodiments, the monopolar electrode transmits electrical energy when negatively charged.

[0249] Alternatively, the electrode may be bipolar (e.g., a substantially planar or non-planar electrode may be bipolar (e.g., a substantially axial wire electrode may be bipolar)). In bipolar aspects, the auxiliary electrode may be in electrical communication with the primary electrode (e.g., a substantially planar or non-planar electrode (e.g., a substantially axial wire electrode)). The auxiliary electrode may be proximal (i.e., closer to the operator) to the primary electrode, e.g., part of or connected to the sheath that houses the primary wire electrode. In some aspects involving electrical introduction using bipolar electrodes, electrical energy (e.g., current) is transferred upon application of a positive voltage to the primary electrode and a negative voltage to the auxiliary electrode. In some aspects involving electrical introduction using bipolar electrodes, electrical energy (e.g., current) is transferred upon application of a negative voltage to the primary electrode and a positive voltage to the auxiliary electrode.

[0250] In some cases, the method of the present invention includes contacting an electrode (e.g., a substantially flat electrode or a substantially non-flat electrode (e.g., a substantially axial wire electrode)) with an internal region of the eye such that the electrical energy transmitted from the electrode is sufficient to cause electrical conduction at the target tissue (e.g., the retina, e.g., the macula). Thus, the method of the present invention may include placing the electrode in electrical communication with the target tissue (e.g., the retina, e.g., the macula). In certain cases, the internal region of the eye that contacts the electrode includes the vitreous humor. For example, the electrode may be wholly or partially disposed within the vitreous humor during transmission of the electric field. When the electrode is disposed within the vitreous humor (e.g., completely within the vitreous humor), the electrode may be disposed in electrical communication with the interface between the vitreous humor and the retina (e.g., the interface at the macula).

[0251] In any of the foregoing embodiments, proximity of the electrode (e.g., the tip of a substantially flat or needle electrode) to a target cell results in a voltage (e.g., potential) at the target cell that is within 20% of the amplitude of the waveform of the applied energy (e.g., within 19%, within 18%, within 17%, within 16%, within 15%, within 14%, within 13%, within 12%, within 11%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, or within 1% of the amplitude of the waveform of the applied energy).

[0252] It will be understood that various suitable electrical parameters and algorithms thereof may be used. The voltage source may be, for example, about 10 V / cm to about 1,500 V / cm (e.g., about 10 V / cm to about 100 V / cm, e.g., about 10 V / cm, 20 V / cm, 30 V / cm, 40 V / cm, 50 V / cm, 60 V / cm, 70 V / cm, 80 V / cm, 90 V / cm, or 100 V / cm, e.g., about 100 V / cm to about 1,000 V / cm, e.g., about 200 V / cm) at the target cell. , 300V / cm, 400V / cm, 500V / cm, 600V / cm, 700V / cm, 800V / cm, 900V / cm, or 1,000V / cm, e.g., about 1,000V / cm to about 1,500V / cm, e.g., about 1,110V / cm, 1,200V / cm, 1,300V / cm, 1,400V / cm, or 1,500V / cm). In some embodiments, the voltage source can be configured to generate, for example, an electric field strength at the target cell of about 10V / cm to about 1,000V / cm (e.g., about 10V / cm to 500V / cm or about 500V / cm to about 1,000V / cm). In some embodiments, the electric field strength is 50V / cm to 300V / cm. In some embodiments, the electric field strength is about 100 V / cm at the target cell.

[0253] In some embodiments, the voltage (e.g., potential) at the target cell is 5V to 100V (e.g., 10V to 80V, 15V to 70V, 20V to 60V, or 30V to 50V; e.g., about 10V, about 15V, about 20V, about 25V, about 30V, about 35V, about 40V, about 45V, about 50V, about 55V, about 60V, about 65V, about 70V). In some embodiments, the voltage (e.g., potential) at the target cell is 20V to 60V. In some embodiments, the voltage (e.g., potential) at the target cell is 30V to 50V, e.g., about 35V to 45V. In any of the foregoing embodiments, close proximity of the electrode to the target cell results in a voltage (e.g., potential) at the target cell that is within 20% of the amplitude of the waveform of the applied energy (e.g., within 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the amplitude of the waveform of the applied energy). For example, a 40V amplitude pulse from a monopolar intravitreal electrode placed near the retina may result in a voltage (e.g., potential) of 35V at the target retinal cell. It will be understood that the waveform amplitude required to achieve a given voltage at the target cell depends on the electrode configuration (e.g., monopolar vs. bipolar), electrode shape, the distance between the electrode and the target cell, and the material properties (e.g., conductivity) of the tissues (e.g., vitreous and retina).

[0254] In some embodiments, the current resulting from the pulsed electric field is between 10 μA and 1 A (e.g., 10 μA to 500 mA, 10 μA to 200 mA, 10 μA to 100 mA, 10 μA to 50 mA, or 10 μA to 25 mA; e.g., 50 μA to 500 mA, 100 μA to 200 mA, or 1 mA to 100 mA; e.g., 10 μA to 20 μA, 20 μA to 30 μA, 30 μA to 50 μA, 50 μA to 100 μA, 100 μA to 150 μA, 150 μA to 200 μA, 200 μA to 300 μA, 300 μA to 400 μA, 400 μA to 500 μA, 500 μA to 600 μA, 600 μA to 800 μA, 8 00 μA to 1 mA, 1 mA to 10 mA, 10 mA to 20 mA, 20 mA to 30 mA, 30 mA to 40 mA, 40 mA to 50 mA, 50 mA to 60 mA, 60 mA to 70 mA, 70 mA to 80 mA, 80 mA to 90 mA, 90 mA to 100 mA, 100 mA to 200 mA, 200 mA to 300 mA, 300 mA to 500 mA, or 500 mA to 1 A; for example, about 1 mA, about 5 mA, about 10 mA, about 15 mA, about 20 mA, about 25 mA, about 30 mA, about 35 mA, about 40 mA, about 45 mA, about 50 mA, about 60 mA, about 70 mA, about 80 mA, about 90 mA, or about 100 mA).

[0255] In some embodiments, the electrode is positioned within about 10 mm (e.g., within 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, or 0.5 mm, 0.45 mm, 0.40 mm, 0.35 mm, 0.30 mm, 0.25 mm, 0.20 mm, 0.15 mm, or 0.10 mm) of the retinal interface. The electrode can be 0.1 to about 0.5 mm (e.g., about 0.15 mm, 0.2 mm, 0.25 mm, 0.30 mm, 0.35 mm, 0.40 mm, 0.45 mm, or 0.5 mm) or about 0.5 mm to 5 mm (e.g., about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm) from the retinal interface during delivery of the one or more pulses. In some embodiments, the electrode (e.g., a substantially planar electrode) is within about 1 mm of the retinal interface during delivery of the one or more pulses.

[0256] The target cell (e.g., a target retinal cell, which may be a retinal cell of the macula) may be within about 5 mm (e.g., 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, or 0.5 mm) of the retinal interface (e.g., at the macula). For example, the target cell may be within about 0.01 mm to about 1 mm (e.g., about 0.01 mm to about 0.1 mm, e.g., about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, e.g., about 0.1 mm to about 1 mm, e.g., about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm) of the retinal interface.

[0257] It will be understood that various suitable electrical parameters and algorithms thereof may be used. The voltage source may be, for example, about 10 V / cm to about 1,500 V / cm (e.g., about 10 V / cm to about 100 V / cm, e.g., about 10 V / cm, 20 V / cm, 30 V / cm, 40 V / cm, 50 V / cm, 60 V / cm, 70 V / cm, 80 V / cm, 90 V / cm, or 100 V / cm, e.g., about 100 V / cm to about 1,000 V / cm, e.g., about 20 The voltage source may be configured to generate an electric field strength of 0 V / cm, 300 V / cm, 400 V / cm, 500 V / cm, 600 V / cm, 700 V / cm, 800 V / cm, 900 V / cm, or 1,000 V / cm, e.g., about 1,000 V / cm to about 1,500 V / cm, e.g., about 1,110 V / cm, 1,200 V / cm, 1,300 V / cm, 1,400 V / cm, or 1,500 V / cm. In some embodiments, the voltage source may be configured to generate an electric field strength of about 10 V / cm to about 1,000 V / cm (e.g., about 10 V / cm to 500 V / cm or about 500 V / cm to about 1,000 V / cm), for example, in a target cell (e.g., a retinal cell). In some embodiments, the electric field strength is between 50 V / cm and 300 V / cm. In some embodiments, the electric field strength is about 100 V / cm at the target cell (eg, the target retinal cell).

[0258] In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 60 seconds (e.g., within 1 to 5 seconds, 5 to 10 seconds, 10 to 15 seconds, 15 to 20 seconds, 20 to 30 seconds, 30 to 40 seconds, 40 to 50 seconds, or 50 to 60 seconds). In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 20 seconds. For example, the total number of pulses of electrical energy can be delivered within 1 to 5 seconds, 5 to 10 seconds, 10 to 15 seconds, or 15 to 20 seconds, e.g., within 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, 18 seconds, 19 seconds, or 20 seconds. The pulses of electrical energy can be, for example, a square waveform. The pulses of electrical energy can have an amplitude of 5V to 500V. For example, the pulse of electrical energy can have an amplitude of about 5V, 10V, 15V, 20V, 25V, 30V, 35V, 40V, 45V, 50V, 60V, 70V, 80V, 90V, 100V, 125V, 150V, 175V, 200V, 225V, 250V, 275V, 300V, 325V, 350V, 375V, 400V, 425V, 450V, 475V, or 500V. In some embodiments, the pulse of electrical energy has an amplitude of about 5-250V (e.g., about 20V). Any of the foregoing voltages can be the apex of a square waveform, the peak of a sinusoidal waveform, the peak of a sawtooth waveform, the root mean square (RMS) voltage of a sinusoidal waveform, or the RMS voltage of a sawtooth waveform.

[0259] In some embodiments, about 1-12 pulses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 pulses) of electrical energy are delivered during use. In some embodiments, about 4-12 pulses of electrical energy are delivered during use.

[0260] In some embodiments, each pulse of electrical energy is about 10 ms to about 200 ms. For example, each pulse of electrical energy can be about 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 110 ms, 120 ms, 130 ms, 140 ms, 150 ms, 160 ms, 170 ms, 180 ms, 190 ms, or 200 ms. In some embodiments, each pulse of electrical energy is from about 50 ms. In some embodiments, each pulse of electrical energy is less than 10 ms. For example, each pulse of electrical energy can be from about 10 μs to about 10 ms, e.g., from about 10 μs to about 100 μs, e.g., about 20 μs, 30 μs, 40 μs, 50 μs, 60 μs, 70 μs, 80 μs, 90 μs, or 100 μs, e.g., from about 100 μs to about 1 ms, e.g., about 200 μs, 300 μs, 400 μs, 500 μs, 600 μs, 700 μs, 800 μs, 900 μs, or 1 ms, e.g., from about 1 ms to about 10 ms, e.g., about 2 ms, 3 ms, 4 ms, 5 ms, 6 ms, 7 ms, 8 ms, 9 ms, or 10 ms.

[0261] An electric field suitable for electrical introduction can be delivered to the target ocular cells at or near the time of administration of a therapeutic agent (e.g., a nucleic acid vector (e.g., a circular DNA vector)) or a pharmaceutical composition thereof (e.g., as part of the same procedure). For example, the invention includes methods in which the electric field is delivered within one hour after administration of a therapeutic agent (e.g., a nucleic acid vector (e.g., a circular DNA vector)) or a pharmaceutical composition thereof (e.g., within 55 minutes, within 50 minutes, within 45 minutes, within 40 minutes, within 35 minutes, within 30 minutes, within 25 minutes, within 20 minutes, within 15 minutes, within 10 minutes, within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, within 90 seconds, within 60 seconds, within 45 seconds, within 30 seconds, within 20 seconds, within 15 seconds, within 10 seconds, within 9 seconds, within 8 seconds, within 7 seconds, within 6 seconds, within 5 seconds, within 4 seconds, within 3 seconds, within 2 seconds, or within 1 second after administration of a nucleic acid vector (e.g., a circular DNA vector) or a pharmaceutical composition thereof) (e.g., simultaneously with administration of a nucleic acid vector (e.g., a circular DNA vector) or a pharmaceutical composition thereof, or after administration but within any of the aforementioned time periods). In some embodiments, the electric field is delivered within 24 hours after administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof (e.g., within 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 8 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, or 2 minutes after administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof). In some embodiments, the electric field is delivered within 7 days after administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof (e.g., within 6 days, 5 days, 4 days, 3 days, or 2 days after administration of the nucleic acid vector (e.g., circular DNA vector) or pharmaceutical composition thereof).

[0262] An electric field suitable for electrical introduction can be delivered at or near the site of administration (e.g., injection) of a therapeutic agent (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site)) or a pharmaceutical composition thereof. For example, in some embodiments, a therapeutic agent (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site)) or a pharmaceutical composition thereof is administered intravitreally, and an electrode is positioned at or near the intravitreal administration site (e.g., within 10 mm, within 8 mm, within 6 mm, within 5 mm, within 4 mm, within 3 mm, within 2 mm, or within 1 mm of the intravitreal administration site) for delivery of the electric field. In other embodiments, the therapeutic agent is administered (e.g., injected) subretinally, and an electrode is placed at or near the subretinal administration site (e.g., within 10 mm, 8 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, or 1 mm of the subretinal administration site) for transmission of the electric field. In other embodiments, the therapeutic agent (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, drug resistance gene, and / or recombination site)) or a pharmaceutical composition thereof is administered suprachoroidally, and an electrode is placed at or near the suprachoroidal administration site (e.g., within 10 mm, 8 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, or 1 mm of the suprachoroidal administration site) for transmission of the electric field.

[0263] In some cases, the therapeutic agent (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site)) or a pharmaceutical composition thereof is administered to a location that is exposed to an electric field (or will be exposed to an electric field upon subsequent electric field transmission). In some embodiments, the therapeutic agent (e.g., a nucleic acid vector (e.g., a circular DNA vector ...) that is 1% or more of the maximum tissue voltage (e.g., at least 5% of the maximum tissue voltage, at least 10% of the maximum tissue voltage, at least 20% of the maximum tissue voltage, at least 30% of the maximum tissue voltage, at least 40% of the maximum tissue voltage, at least 50% of the maximum tissue voltage, at least 60% of the maximum tissue voltage, at least 70% of the maximum tissue voltage, at least 80% of the maximum tissue voltage, or at least 10% of the maximum tissue voltage). The electrical potential is delivered to a location that is exposed (or will be exposed to) a voltage that is at least 90% of the maximum tissue voltage, e.g., 1% to 10% of the maximum tissue voltage, 10% to 20% of the maximum tissue voltage, 20% to 30% of the maximum tissue voltage, 30% to 40% of the maximum tissue voltage, 40% to 50% of the maximum tissue voltage, 50% to 60% of the maximum tissue voltage, 60% to 70% of the maximum tissue voltage, 70% to 80% of the maximum tissue voltage, 80% to 90% of the maximum tissue voltage, 90% to 95% of the maximum tissue voltage, or 95% to 100% of the maximum tissue voltage.

[0264] Alternatively, the administration site may be in a region of tissue remote from the electric field. For example, administration of a therapeutic agent (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene and / or a recombination site)) or a pharmaceutical composition thereof may be systemic (e.g., intravenous), but the electric field is delivered intraocularly (e.g., in the vitreous humor or subretinal space).

[0265] In any of the methods described herein involving electrotransduction (e.g., by PEF), a paralytic agent may be administered according to standard procedures which may help reduce the risk and / or severity of muscle contraction upon delivery of electrical energy.

[0266] Treatment characterization The level or concentration of ocular proteins (e.g., retinal proteins) expressed from the nucleic acid vectors described herein may be expression levels, presence, absence, truncation, or alteration of the administered vector. It may be advantageous to correlate the level with assays of one or more clinical phenotypes or human disease biomarkers. The assays may be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof, and exosomes may be isolated using immunohistochemical methods such as enzyme-linked immunosorbent assay (ELISA) methods. The therapeutic genes delivered by the nucleic acid vectors described herein may be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis). The quantified polynucleotides may be analyzed to determine whether the polynucleotides may be of appropriate size and to ensure that degradation of the polynucleotides has not occurred. Degradation of polynucleotides may be confirmed by methods such as, but not limited to, HPLC-based purification methods, such as, but not limited to, agarose gel electrophoresis, strong anion exchange HPLC, weak anion exchange HPLC, reversed-phase HPLC (RP-HPLC), hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).

[0267] The effectiveness of treatment can be monitored, evaluated, and / or quantified using any suitable method known in the art or provided herein. For example, an individual treated for a retinal disease or disorder can be monitored periodically to evaluate the progression of retinal degeneration, for example, by testing visual acuity and visual field using standard tests. Additionally or alternatively, optical coherence tomography (OCT) (e.g., spectral domain OCT (SD-OCT)) can be performed to evaluate changes in retinal structure. In some cases, an individual treated by the methods described herein shows improvement or no further degradation of retinal structure as assessed by imaging endpoints such as fundus autofluorescence (FAF) and / or SD-OCT.

[0268] The safety and tolerability of treatment can be monitored, evaluated, and / or quantified using any suitable method known in the art or provided herein.For example, the individual treated for retinal disease or disorder can be monitored periodically to evaluate retinal damage, such as cataract formation, intraocular inflammation, or RPE depigmentation.In some embodiments, the individual treated according to the methods described herein does not show cataract formation, does not show intraocular inflammation up to 2 months after treatment (or shows intraocular inflammation less than grade 2 up to 2 months after treatment), and / or shows minimal retinal / RPE damage (e.g., RPE depigmentation).

[0269] In some cases, an individual is treated with a nucleic acid vector and electrical transfer according to any of the embodiments described herein only once in their lifetime (e.g., treatment of the disease or disorder continues for several years (e.g., 3-5 years, 5-10 years, 10-15 years, or at least 15 years). Alternatively, an individual may be treated exactly twice in their lifetime. Additionally or alternatively, an individual may be treated once every 2-3 years, once every 3-5 years, or once every 5-10 years.

[0270] IV. Equipment The devices described herein include a sheath having a proximal end, a distal end, and a longitudinal axis therebetween. The device includes an elongated conductor having a proximal portion and a distal portion within the sheath. In some embodiments (e.g., embodiments including planar electrodes), the elongated conductor is comprised of a preformed shape memory material and is retractable within the sheath from a proximal position (FIG. 4A) where the conductor is in a retracted position to a distal position (FIG. 4B) where the elongated conductor is deployed. In the proximal position, the distal portion of the elongated conductor is substantially straight. In the distal position, the distal portion of the elongated conductor extends beyond the distal end of the sheath, and the shape memory material of the distal portion of the elongated conductor is radially relaxed. When radially relaxed, the elongated conductor forms a substantially planar electrode that is approximately perpendicular to the longitudinal axis of the sheath.

[0271] Also featured is a device including a sheath having a proximal end, a distal end and a longitudinal axis therebetween. The device further includes an elongated conductor having a proximal portion and a distal portion within the sheath, the elongated conductor including a preformed shape memory material and retractable within the sheath from a proximal position to a distal position. In the proximal position, the distal portion of the elongated conductor is substantially straight. In the distal position, the distal portion of the elongate conductor extends beyond the distal end of the sheath, and the shape memory material of the distal portion of the elongate conductor is radially relaxed to form a preformed angle (e.g., about 10° to about 170°, e.g., about 20° to about 160°, e.g., about 30° to about 150°, e.g., about 45° to about 135°, e.g., about 60° to about 120°, e.g., about 70° to about 110°, e.g., about 80° to about 100°, e.g., about 85° to about 95°, e.g., about 10°, 20°, 30°, 45°, 50°, 55°, 60°, 65°) relative to the longitudinal axis of the sheath. , 70°, 71°, 72°, 73°, 74°, 75°, 76°, 77°, 78°, 79°, 80°, 81°, 82°, 83°, 84°, 85°, 86°, 87°, 88°, 89°, 90°, 91°, 92°, 93°, 94°, 95°, 96°, 97°, 98°, 99°, 100°, 101°, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°, 150°, 160°, or 170°). In some aspects, the electrode is a substantially planar electrode, hi some aspects, the preformed angle is substantially a right angle.

[0272] Components of such devices described herein are shown, for example, in Figures 13-20. Although these figures show various dimensions and parameters of each component, one of ordinary skill in the art will understand that these dimensions and parameters are exemplary and may be varied within the scope of the invention.

[0273] sheath The device includes a sheath in which the elongated conductors are deployed. The sheath is hollow and may include any suitable size or shape that allows the conductors to be deployed and retracted therein. The sheath may be substantially straight or curved. The sheath may be rigid or flexible, for example, to provide easy manipulation to reach the target area. The sheath has a substantial rigidity that allows the elongated conductors to remain constrained therein, for example, when in a retracted position.

[0274] The sheath may have a length of about 1 mm to about 100 cm, for example, about 1 cm to about 75 cm, about 2 cm to about 50 cm, about 5 cm to about 40 cm, about 10 cm to about 35 cm, or about 15 cm to about 20 cm. For example, the sheath may have a length of about 0.1 mm to about 1 mm, for example, about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm, for example, about 1 mm to about 10 mm, for example, about 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, for example, about 10 mm to about 100 mm, for example, about 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 9 mm, 10 mm, 12 mm, 14 mm, 16 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm, 31 mm, 32 mm, 33 mm, 34 mm, 35 mm, 36 mm, 37 mm, 38 mm, 39 mm, 40 mm, 41 mm, 42 mm, 43 mm, 44 mm, 45 mm, 46 mm, 47 mm, 48 mm, 49 mm, 50 mm, 51 mm, 52 mm, 53 mm, 54 mm, 55 mm, 56 mm, 57 mm, 58 mm, 59 mm, 60 mm The length may be about 1 cm to about 10 cm, for example, about 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm, for example, about 10 cm to about 100 cm, for example, about 15 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm, 50 cm, 55 cm, 60 cm, 65 cm, 70 cm, 75 cm, 80 cm, 85 cm, 90 cm, 95 cm, or 100 cm.

[0275] The sheath may be a substantially hollow tube or other suitable shape, including an inner diameter and an outer diameter that depend on the thickness of the sheath. The cross section of the sheath may be substantially circular or elliptical. The cross section of the sheath may be polygonal (e.g., triangular or square, etc.). In some embodiments, the outer cross section is a first shape (e.g., a circle, an ellipse, or a polygon, e.g., a triangle or square) and the inner cross section is a second shape (e.g., a circle, an ellipse, or a polygon, e.g., a triangle or square). The inner diameter of the sheath may be about 0.01 mm to about 5 mm, e.g., about 0.1 mm to about 1 mm, e.g., about 0.2 mm to about 0.5 mm, e.g., about 0.2 mm to about 0.3 mm. For example, the inner diameter of the sheath can be from 0.01 mm to about 5 mm, for example, from about 0.01 mm to about 0.1 mm, for example, about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, for example, from about 0.1 mm to about 1 mm, for example, about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm, for example, from about 1 mm to about 5 mm, for example, about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0276] The outer diameter of the sheath, which is larger than the inner diameter, can be from about 0.01 mm to about 5 mm, for example, from about 0.1 mm to about 1 mm, for example, from about 0.2 mm to about 0.5 mm, for example, from about 0.2 mm to about 0.3 mm. For example, the outer diameter of the sheath can be from 0.01 mm to about 5 mm, for example, from about 0.01 mm to about 0.1 mm, for example, about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, for example, from about 0.1 mm to about 1 mm, for example, about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm, for example, from about 1 mm to about 5 mm, for example, about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0277] The thickness of the sheath can be about 0.01 mm to about 1 mm, e.g., about 0.01 mm to about 0.1 mm, e.g., about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, e.g., about 0.1 mm to about 1 mm, e.g., about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm. The thickness of the sheath may be substantially uniform throughout, or may have different thicknesses in different portions or regions of the sheath.

[0278] The sheath may be constructed of a conductive material such as a metal or metal alloy. Suitable sheath materials include, for example, stainless steel, titanium, PEEK (e.g., machined, molded, or extruded) or polymers such as polyimide, composites such as woven polymers with epoxy, or ceramics. In some embodiments, the sheath is made of stainless steel. In some embodiments, the sheath is constructed of Nitinol. In some embodiments, the sheath is constructed of stainless steel and includes a polymer tip, for example to facilitate retraction of the electrode wire.

[0279] The distal end of the sheath is configured to contact the eye such that the electrodes can access an area in suitable proximity (e.g., electrical communication) with the desired target cells (e.g., in the vitreous humor near the surface of the retina). Thus, the distal end of the sheath can include a sharp feature, such as a pointed tip for puncturing the eye. The tip can be beveled (e.g., standard bevel, short bevel, or true short bevel). The distal end of the sheath can include a needle (e.g., a hypodermic needle). The needle can be of any suitable gauge or thickness, e.g., if desired, that allows an elongated conductor to pass therethrough and / or that matches the thickness of the sheath. The gauge of the needle can be, for example, about 7 to about 33 (e.g., about 10 to 30, e.g., 12 to 28, e.g., 15 to 28, e.g., 20 to 28, e.g., 20 to 25, e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33. In some embodiments, the needle is 19 gauge. In some embodiments, the needle is 23 gauge. In some embodiments, the needle is 25 gauge. In some embodiments, the needle is 30 gauge.

[0280] In some embodiments, the device includes a second sheath. The second sheath can be configured to be surrounded by the first sheath or a portion thereof. For example, the second sheath can have a diameter smaller than that of the first sheath. In some embodiments, the second sheath is connected to the elongated conductor, for example, at the proximal end of the elongated conductor. In some embodiments, the second sheath is connected to an actuator (e.g., a slider) as described herein.

[0281] In some embodiments, the device (e.g., a device with a planar electrode or a device with a non-planar needle electrode) includes a sheath connected to the handle and a sheath (e.g., a second sheath) connected to the slider (FIG. 13C). The elongated conductor may be present in the sheath connected to the slider. In some embodiments, the sheath connected to the slider is nested with the sheath connected to the handle. The sheath connected to the slider may be configured to be surrounded by the sheath connected to the handle or a portion thereof. For example, the sheath connected to the slider may have a diameter smaller than the diameter of the sheath connected to the handle. Alternatively, the sheath connected to the slider may surround the sheath connected to the handle or a portion thereof. For example, the sheath connected to the slider may have a diameter larger than the diameter of the sheath connected to the handle. In some embodiments, the sheath connected to the slider is connected to the elongated conductor, for example, at the proximal end of the elongated conductor.

[0282] The inner diameter of the second sheath can be from about 0.01 mm to about 5 mm, for example, from about 0.1 mm to about 1 mm, for example, from about 0.2 mm to about 0.5 mm, for example, from about 0.2 mm to about 0.3 mm. For example, the inner diameter of the second sheath can be from 0.01 mm to about 5 mm, for example, from about 0.01 mm to about 0.1 mm, for example, about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, for example, from about 0.1 mm to about 1 mm, for example, about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm, for example, from about 1 mm to about 5 mm, for example, about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0283] The outer diameter of the second sheath, which is larger than the inner diameter, can be about 0.01 mm to about 5 mm, for example, about 0.1 mm to about 1 mm, for example, about 0.2 mm to about 0.5 mm, for example, about 0.2 mm to about 0.3 mm. For example, the outer diameter of the second sheath can be from 0.01 mm to about 5 mm, for example, from about 0.01 mm to about 0.1 mm, for example, about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, for example, from about 0.1 mm to about 1 mm, for example, about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm, for example, from about 1 mm to about 5 mm, for example, about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0284] The thickness of the second sheath can be about 0.01 mm to about 1 mm, e.g., about 0.01 mm to about 0.1 mm, e.g., about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, e.g., about 0.1 mm to about 1 mm, e.g., about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm. The thickness of the second sheath can be substantially uniform throughout, or can have different thicknesses in different portions or regions of the second sheath.

[0285] The second sheath may be or include a needle (e.g., a hypodermic needle). The needle may be of any suitable gauge or thickness that allows, for example, the first sheath and / or the elongated conductor to pass therethrough and / or to match the thickness of the sheath, if desired. The gauge of the needle can be, for example, about 7 to about 33 (e.g., about 10 to 30, e.g., 12 to 28, e.g., 15 to 28, e.g., 20 to 28, e.g., 20 to 25, e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33. In some embodiments, the needle is 19 gauge. In some embodiments, the needle is 23 gauge. In some embodiments, the needle is 25 gauge. In some embodiments, the needle is 30 gauge.

[0286] An embodiment having two sheaths (e.g., a device having planar electrodes or a device having non-planar electrodes) may be particularly advantageous, for example, to prevent buckling of the elongated conductor within the first sheath. For example, if the elongated conductor is substantially straight and within the sheath, the contact force between the conductor and the sheath is greater than the force that would buckle the elongated conductor when pushed (FIG. 10). Thus, the elongated conductor may buckle and the distal end of the elongated conductor, including the substantially flat electrode, cannot be properly deployed through the sheath. A second sheath may allow for more efficient deployment of the substantially flat electrode without buckling the elongated conductor. In certain embodiments, buckling may be prevented by connecting the second sheath directly to the elongated conductor and / or the slider.

[0287] In another embodiment, buckling may also be prevented by extending or disposing the distal ends of the first sheath and / or second sheath within the handle (FIGS. 10, 11, and 12A).

[0288] In some embodiments, the sheath (e.g., the first sheath and / or the second sheath) includes a coating on the inside and / or outside of the sheath. The coating may be used, for example, to reduce friction between a sliding portion, such as an elongated conductor within the sheath and / or the second sheath (if used), and the first sheath.

[0289] Long and thin conductor The elongated conductor may be disposed within the sheath and deployed from within the sheath. The conductor may have a length of about 1 mm to about 100 cm, for example, about 1 cm to about 75 cm, about 2 cm to about 50 cm, about 5 cm to about 40 cm, about 10 cm to about 35 cm, or about 15 cm to about 20 cm. For example, the conductor may have a length of about 1 mm to about 10 mm, such as about 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, for example, about 10 mm to about 100 mm, for example, about 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, or 100 mm, for example, about 1 cm to about 10 cm, for example, about 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm, for example, about 10 cm to about 100 cm, for example, about 15 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, 45 cm, 50 cm, 55 cm, 60 cm, 65 cm, 70 cm, 75 cm, 80 cm, 85 cm, 90 cm, 95 cm, or 100 cm.

[0290] The elongated conductor may be substantially cylindrical (e.g., a cylindrical wire). The cross section of the conductor may be substantially circular or elliptical. The cross section of the conductor may be polygonal, e.g., triangular, square, etc. The diameter of the conductor may be about 0.01 mm to about 5 mm, e.g., about 0.1 mm to about 1 mm, e.g., about 0.2 mm to about 0.5 mm, e.g., about 0.2 mm to about 0.3 mm. For example, the diameter of the sheath can be 0.01 mm to about 5 mm, for example, about 0.01 mm to about 0.1 mm, for example, about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, for example, about 0.1 mm to about 1 mm, for example, about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm, for example, about 1 mm to about 5 mm, for example, about 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm. In some embodiments, the diameter of the conductor is about 0.2 mm. The diameter of the conductor may be substantially uniform throughout or may have different diameters in different portions or regions of the conductor.

[0291] In some embodiments, the device includes multiple elongated conductors bundled together, for example within a sheath, hi one embodiment, the device includes two elongated conductors, each conductor having a cross section that is substantially semicircular, or half of an ellipse.

[0292] The diameter of the conductor may be about 50% to about 99% of the inner diameter of the sheath. For example, the diameter may be about 55% to about 95%, about 60% to about 90%, about 65% to about 85%, 70% to about 80%, or about 75%. The diameter of the conductor may be, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the inner diameter of the sheath.

[0293] The conductor may be composed of any suitable conductive material known in the art, such as a metal or metal alloy. In some cases, the conductor is composed of the same material as the sheath. In other cases, the conductor is a different material than the sheath. Suitable conductive materials useful for the conductor include, for example, platinum, platinum / iridium alloy, stainless steel, nickel, and titanium. In some embodiments, the conductor is made of an alloy of nickel and titanium alloy (e.g., Nitinol). Nitinol may contain, for example, about 40% to about 70% nickel (e.g., about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, or about 65% to about 70%, e.g., about 40%, 45%, 50%, 55%, 60%, 65%, or 70% nickel).

[0294] Substantially flat electrodes In some embodiments, the elongated conductor or a portion thereof (e.g., a distal portion) comprises a substantially flat electrode. The substantially flat electrode is comprised of a shape memory material (e.g., a shape memory alloy). The shape memory material allows the structure of the elongated conductor to relax to a preformed shape when a restraint (e.g., a structural element) is removed. For example, a preformed shape memory wire housed within a rigid sheath is restrained until it is desheathed, at which point the shape memory material relaxes to its preformed shape (e.g., a substantially flat electrode) as shown in Figures 4-6. In some embodiments, an actuator is used to deploy the substantially flat electrode (see, e.g., Figures 10 and 11).

[0295] In the devices described herein, the preformed shape can be a substantially flat electrode that is approximately perpendicular to the longitudinal axis of the sheath and / or the proximal portion of the elongated conductor (e.g., the region that does not include the substantially flat electrode). One skilled in the art will appreciate that in some embodiments, a substantially flat electrode may not be completely flat. For example, in some embodiments, two of its vertical dimensions (e.g., Cartesian dimensions such as depth and width) are each at least twice as large as its third vertical dimension (e.g., length). In some embodiments, a substantially flat electrode refers to an electrode in which two of its vertical dimensions are each at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, or at least 100 times or more larger than its third vertical dimension. Thus, in some cases, the longitudinal dimension of a substantially flat electrode is less than 10% of its radial dimension (e.g., its outermost radial point). In some cases, the longitudinal dimension of a substantially flat electrode is less than 5% of its radial dimension (e.g., its outermost radial point).

[0296] In certain embodiments, the spatial configuration of the electrode is manufactured to optimize its conductive properties and / or apply a desired electric field to a target region of the cell. Thus, if the eye includes a curvature, the shape of the electrode may also include a curvature (e.g., a convex shape) that matches or approximates the shape of the eye or a portion thereof (e.g., the retina), for example.

[0297] In some instances, the elongated conductor is a wire and the substantially flat electrode is a distal portion of the wire. In some instances, the distal tip of the wire (or a point along the wire within 5 mm (e.g., within 4 mm, within 3 mm, within 2 mm, within 1 mm, within 0.5 mm, or within 0.1 mm) of the distal tip of the wire) is at the outermost radial point of the substantially flat electrode. The distal portion of the wire can include a preformed right angle (or a substantially right angle, e.g., about 70°, 75°, 80°, 85°, 86°, 87°, 88°, 89°, 90°, 91°, 92°, 93°, 94°, 95°, 100°, 105°, or 110°) on the longitudinal plane; or a preformed angle of about 45° to about 135° (e.g., about 45°, about 50°, about 55°, about 60°, about 65°, about 115°, about 120°, about 125°, about 130°, or about 135°) on the longitudinal plane, where the preformed angle (e.g., the preformed right angle) is between the substantially flat electrode and the proximal portion of the wire.

[0298] In some embodiments, the shape memory material of the distal portion of the elongate conductor is radially relaxed to a preformed angle (e.g., about 10° to about 170°, e.g., about 20° to about 160°, e.g., about 30° to about 150°, e.g., about 45° to about 135°, e.g., about 60° to about 120°, e.g., about 70° to about 110°, e.g., about 80° to about 100°, e.g., about 85° to about 95°, e.g., about 10°, 20°, 30°, 45°, 50°, 55°, 60°, 65°, 70°, 71°, 72°, In some embodiments, the electrodes are arranged at an angle of about 73°, 74°, 75°, 76°, 77°, 78°, 79°, 80°, 81°, 82°, 83°, 84°, 85°, 86°, 87°, 88°, 89°, 90°, 91°, 92°, 93°, 94°, 95°, 96°, 97°, 98°, 99°, 100°, 101°, 102°, 103°, 104°, 105°, 106°, 107°, 108°, 109°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°, 150°, 160°, or 170°). In some embodiments, the electrodes are substantially flat electrodes.

[0299] In some embodiments, the substantially flat electrode extends 1 mm or less (e.g., 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm or less) distal or proximal from the preformed angle (e.g., a preformed right angle). In some embodiments, the substantially flat electrode extends 1 mm or less (e.g., 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm or less) distal from the preformed angle (e.g., a preformed right angle). In some embodiments, the device includes nothing distal to the substantially flat electrode (e.g., the substantially flat electrode is free to contact the tissue surface).

[0300] In some embodiments, a substantially flat electrode is about 2 mm to about 15 mm (e.g., about 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm) in one or more dimensions (e.g., both dimensions), e.g., perpendicular to the longitudinal axis or at a preformed angle to the longitudinal axis.

[0301] In some embodiments, the substantially flat electrode is substantially symmetrical about the longitudinal plane. In some embodiments, the substantially flat electrode is a spiral (FIG. 6). For example, the spiral can include 1-5 (e.g., 1, 1.5, 2, 2.5, 3, 2.5, 4, 4.5, 5, 5.5, 6, 7, 8, or more) turns about the longitudinal axis. In some cases, the spiral has 2-5 turns about the longitudinal axis. In some cases, the spiral has 2-3 turns about the longitudinal axis. In certain embodiments, the spiral has 2 turns about the longitudinal axis. In some embodiments, the spiral has 3 turns about the longitudinal axis. Other suitable shapes include, for example, loop, concentric loop, paddle, mesh, grid, or umbrella shapes. In some embodiments, the spiral consists of 3 turns about the longitudinal axis. In some embodiments, the spiral consists of 2 turns about the longitudinal axis (FIG. 6).

[0302] The substantially flat electrode can be made entirely or partially from a shape memory material (e.g., a shape memory alloy, e.g., NiTi) that can recover its original shape in the presence of a predetermined stimulus. For example, the shape memory material can relax to a preformed shape when a structural constraint is removed. As described herein, a preformed shape memory wire (e.g., a substantially flat electrode) housed in a rigid sheath is straight until it is desheathed, at which point the shape memory material relaxes to its preformed shape, such as a spiral. Shape memory materials are known in the art. In some embodiments, the shape memory material includes an alloy such as NiTi, CuAlNi, or CuZnAl. The shape memory material can be iron-based. In some embodiments, the shape memory material is NiTi. NiTi is an alloy of nickel and titanium (nitinol). The nitinol can include, for example, about 40% to about 70% nickel (eg, about 40%, 45%, 50%, 55%, 60%, 65%, or 70% nickel).

[0303] Insulation The device may include an insulator disposed between the elongated conductor and the sheath. The insulator may be disposed between a proximal portion of the elongated conductor and the sheath. The insulator prevents electrical contact between the sheath and the elongated conductor. The insulator may be made of any suitable material, such as glass, porcelain, or a polymeric (e.g., composite polymer) material. In some embodiments, the insulator is comprised of polyimide or polyetheretherketone (PEEK). In some embodiments, the insulator is comprised of polyvinylidene fluoride (PVDF), low density polyethylene (LDPE), a blend of polyolefin and ethylene acrylic acid copolymer, high density polyethylene (HDPE), fluorinated ethylene propylene (FEP), polyvinyl chloride (PVC), Parylene C, or a combination thereof. The insulating material may be deposited on the electrode surface or may be created via, for example, heat shrink tubing. The insulator can have a thickness of about 1 μm to about 100 μm, for example, about 5 μm to about 90 μm, about 10 μm to about 80, about 10 μm to about 50 μm, or about 20 μm to about 30 μm, for example, about 25 μm. For example, the insulator may have a thickness of about 1 μm to about 10 μm, e.g., about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm, e.g., about 10 μm to about 100 μm, e.g., about 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, or 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, or 100 μm.

[0304] In some embodiments, the device further comprises an adhesive, glue, or epoxy disposed between the elongated conductor and the insulator.

[0305] handle In some aspects, the described devices include a handle. In certain aspects, a proximal portion of the device includes a handle, e.g., for easy manipulation. The handle may be disposed on the sheath. The handle may be disposed on a proximal portion of the elongated conductor, for example. In some aspects, the device includes a handle for manipulating the sheath and a handle on a proximal portion of the elongated conductor, e.g., for manipulating the conductor within the sheath.

[0306] The handle may have a proximal end and a distal end (FIGS. 10, 11, and 15). In some embodiments, the proximal end of the sheath is connected to the handle (e.g., connected to and disposed within the handle). In some embodiments, the distal portion of the handle includes a hollow region between the inner surface of the handle and the elongated conductor therein. The proximal end of the sheath may extend into at least the hollow region within the handle. In some embodiments, the proximal end of the sheath extends at least 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 35 mm, 40 mm, 45 mm, 50 mm, 55 mm, 60 mm, 65 mm, 70 mm, 80 mm, 85 mm, 90 mm, 95 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, 200 mm, or more into the hollow region in the handle (FIG. 11).

[0307] In some embodiments, the handle is cylindrical (FIGS. 12A-12C). In some embodiments, the handle further comprises a cap at the distal and / or proximal end of the handle (FIGS. 12B, 13, and 14). For example, the handle may comprise a cap at each of the distal and proximal ends, e.g., to close a hollow portion of the handle.

[0308] In some embodiments, the handle can have a length of about 3 inches to about 10 inches, e.g., about 3 inches to about 9 inches, e.g., about 3 inches, 4 inches, 5 inches, 6 inches, 7 inches, 8 inches, 9 inches, or 10 inches. In some embodiments, the handle length is about 5 inches to about 6 inches, e.g., about 5.5 inches, e.g., about 5.425 inches (FIG. 15).

[0309] In some embodiments, the cap that fits within the distal and / or proximal ends of the handle has a length of about 0.1 inches to about 1.0 inches, e.g., about 0.1 inches, 0.2 inches, 0.3 inches, 0.4 inches, 0.5 inches, 0.6 inches, 0.7 inches, 0.8 inches, 0.9 inches, or 1.0 inches. In some embodiments, the length of the cap is about 0.2 inches to about 0.3 inches, e.g., about 0.28 inches (FIGS. 13 and 14).

[0310] Actuator The devices described herein may further include an actuator (e.g., a slider). The actuator (e.g., a slider) may be configured to slide the elongated conductor between a proximal position and a distal position, such as between its relaxed position and a covered position. The actuator may be a manual actuator. Alternatively, the actuator may be an electronically controlled actuator. In some aspects, the actuator is a piezoelectric actuator.

[0311] In some aspects, the actuator is operably coupled to the elongated conductor, hi some aspects, the actuator is present on the handle of the device.

[0312] In some embodiments, the actuator is a slider. The slider has a proximal end and a distal end and is attached (e.g., directly or indirectly) to the elongated conductor (see, e.g., FIGS. 10-12 and 27). The slider can be configured to retract the elongated conductor from a distal position to a proximal position along the longitudinal axis of the sheath. In some embodiments, the slider includes a proximal position and a distal position. In the proximal position, the proximal end of the sheath is disposed within the distal end of the slider or extends at least to the distal end of the slider. In the distal position, the proximal end of the sheath is disposed between the proximal and distal ends of the slider or extends between the proximal and distal ends of the slider. In some embodiments, the slider is hollow and the elongated conductor is disposed within the slider or extends entirely through the slider.

[0313] In some embodiments, the slider is configured to stop when it reaches a distal position and / or a proximal position. In some embodiments, the slider is disposed at a distal position and the distal portion of the elongate conductor extends beyond the distal end of the sheath. The shape memory material of the distal portion of the elongate conductor can be radially relaxed to form a substantially flat electrode at a preformed angle relative to the longitudinal axis of the sheath.

[0314] In some aspects, the slider is disposed at a proximal location and the distal portion of the elongated conductor is substantially straight. In some aspects, the slider further includes a control member disposed on the exterior of the handle. The control member may include a protrusion, knob, or other mechanism for easy control or ergonomic design of the slider. The control member and slider may be integral. Alternatively, the control member and slider may not be integral, e.g., may be separate parts.

[0315] In some embodiments, the length of the slider is from about 0.5 inches to about 5.0 inches, such as from about 0.5 inches to about 3.5 inches, such as from about 1.0 inches to about 2.5 inches, such as about 2.0 inches, such as about 1.925 inches (FIG. 19).

[0316] In some embodiments, the length of the control member is from about 0.1 inches to about 2.0 inches, e.g., about 0.2 inches, 0.3 inches, 0.4 inches, 0.5 inches, 0.6 inches, 0.7 inches, 0.8 inches, 0.9 inches, 1.0 inches, 1.1 inches, 1.2 inches, 1.3 inches, 1.4 inches, 1.5 inches, 1.6 inches, 1.7 inches, 1.8 inches, 1.9 inches, or 2.0 inches, e.g., from about 0.5 inches to about 1.0 inches, e.g., about 0.8 inches (FIG. 17).

[0317] Further elements The devices described herein may be monopolar. Alternatively, the devices may be bipolar. In bipolar aspects, the devices further include an auxiliary electrode in electrical communication with the substantially planar electrode. The auxiliary electrode may be part of the sheath or may be connected to the sheath.

[0318] The device may further include a voltage source. The device may further include a waveform controller. In some embodiments, the proximal portion of the elongated conductor is connected to a voltage source and / or a waveform controller.

[0319] The device may be configured for use with an endoscope or bronchoscope, for example, the device may be disposed at the distal end of a bronchoscope endoscope and deployed, for example, upon insertion into a subject.

[0320] V. How to Use the Device The invention features a method of using any of the devices described herein. In some cases, the invention provides a method of using the devices described herein to deliver a therapeutic agent to a target cell of an individual. The method includes inserting a sheath or needle through an external tissue surface of the individual and sliding an elongated conductor to a distal position to radially relax the preformed shape memory material, thereby forming a substantially planar electrode in the tissue. The method may include actuating a slider (e.g., to a distal position) to deploy the substantially planar electrode from its covered position. The method further includes placing the substantially planar electrode in electrical communication with a tissue interface separating the target cell from the substantially planar electrode. The method also includes transmitting one or more pulses of electrical energy (e.g., with a voltage source) through the substantially planar electrode under conditions suitable for electrical introduction of the therapeutic agent into the target cell.

[0321] In some embodiments, the substantially planar electrode is positioned within about 10 mm (e.g., within 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, or 0.5 mm, 0.45 mm, 0.40 mm, 0.35 mm, 0.30 mm, 0.25 mm, 0.20 mm, 0.15 mm, or 0.10 mm) of the tissue interface. A substantially planar electrode can be 0.1 to about 0.5 mm (e.g., about 0.15 mm, 0.2 mm, 0.25 mm, 0.30 mm, 0.35 mm, 0.40 mm, 0.45 mm, or 0.5 mm) or about 0.5 mm to 5 mm (e.g., about 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm) from the tissue interface during delivery of one or more pulses. In some embodiments, close proximity of the electrode to the target cell results in a voltage (e.g., potential) at the target cell that is within 20% of the amplitude of the waveform of the applied energy (e.g., within 19%, within 18%, within 17%, within 16%, within 15%, within 14%, within 13%, within 12%, within 11%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, or within 1% of the amplitude of the waveform of the applied energy). In some embodiments, the substantially planar electrode is within about 1 mm of the tissue interface during delivery of one or more pulses. In any of the foregoing embodiments, proximity of the electrode to the target cell results in a voltage (e.g., potential) at the target cell that is within 20% of the amplitude of the waveform of the applied energy (e.g., within 19%, within 18%, within 17%, within 16%, within 15%, within 14%, within 13%, within 12%, within 11%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, or within 1% of the amplitude of the waveform of the applied energy).

[0322] The target cells can be within about 5 mm (e.g., 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, or 0.5 mm) of the tissue interface. For example, the target cells can be within about 0.01 mm to about 1 mm (e.g., about 0.01 mm to about 0.1 mm, e.g., about 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, or 0.1 mm, e.g., about 0.1 mm to about 1 mm, e.g., about 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm) of the tissue interface.

[0323] It will be understood that various suitable electrical parameters and algorithms thereof may be used. The voltage source may be, for example, about 10 V / cm to about 1,500 V / cm (e.g., about 10 V / cm to about 100 V / cm, e.g., about 10 V / cm, 20 V / cm, 30 V / cm, 40 V / cm, 50 V / cm, 60 V / cm, 70 V / cm, 80 V / cm, 90 V / cm, or 100 V / cm, e.g., about 100 V / cm to about 1,000 V / cm, e.g., about 200 V / cm) at the target cell. , 300V / cm, 400V / cm, 500V / cm, 600V / cm, 700V / cm, 800V / cm, 900V / cm, or 1,000V / cm, e.g., about 1,000V / cm to about 1,500V / cm, e.g., about 1,110V / cm, 1,200V / cm, 1,300V / cm, 1,400V / cm, or 1,500V / cm). In some embodiments, the voltage source can be configured to generate, for example, an electric field strength at the target cell of about 10V / cm to about 1,000V / cm (e.g., about 10V / cm to 500V / cm or about 500V / cm to about 1,000V / cm). In some embodiments, the electric field strength is 50V / cm to 300V / cm. In some embodiments, the electric field strength is about 100 V / cm at the target cell.

[0324] In some embodiments, the voltage (e.g., potential) at the target cell is 5V to 100V (e.g., 10V to 80V, 15V to 70V, 20V to 60V, or 30V to 50V; e.g., about 10V, about 15V, about 20V, about 25V, about 30V, about 35V, about 40V, about 45V, about 50V, about 55V, about 60V, about 65V, or about 70V). In some embodiments, the voltage (e.g., potential) at the target cell is 20V to 60V. In some embodiments, the voltage (e.g., potential) at the target cell is 30V to 50V, e.g., about 35V to 45V. In any of the foregoing embodiments, close proximity of the electrode to the target cell results in a voltage (e.g., potential) at the target cell that is within 20% of the amplitude of the waveform of the applied energy (e.g., within 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the amplitude of the waveform of the applied energy). For example, a 40V amplitude pulse from a monopolar intravitreal electrode placed near the retina may result in a voltage (e.g., potential) of 35V at the target retinal cell. It will be understood that the waveform amplitude required to achieve a given voltage at the target cell depends on the electrode configuration (e.g., monopolar vs. bipolar), electrode shape, the distance between the electrode and the target cell, and the material properties (e.g., conductivity) of the tissues (e.g., vitreous and retina).

[0325] In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 60 seconds (e.g., within 1 to 5 seconds, 5 to 10 seconds, 10 to 15 seconds, 15 to 20 seconds, 20 to 30 seconds, 30 to 40 seconds, 40 to 50 seconds, or 50 to 60 seconds). In some embodiments, the total number of pulses of electrical energy is delivered within 1 to 20 seconds. For example, the total number of pulses of electrical energy can be delivered within 1 to 5 seconds, 5 to 10 seconds, 10 to 15 seconds, or 15 to 20 seconds, e.g., within 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 11 seconds, 12 seconds, 13 seconds, 14 seconds, 15 seconds, 16 seconds, 17 seconds, 18 seconds, 19 seconds, or 20 seconds. The pulses of electrical energy can be, for example, a square waveform. The pulses of electrical energy can have an amplitude of 5V to 500V. For example, the pulse of electrical energy can have an amplitude of about 5 V, 10 V, 15 V, 20 V, 25 V, 30 V, 35 V, 40 V, 45 V, 50 V, 60 V, 70 V, 80 V, 90 V, 100 V, 125 V, 150 V, 175 V, 200 V, 225 V, 250 V, 275 V, 300 V, 325 V, 350 V, 375 V, 400 V, 425 V, 450 V, 475 V, or 500 V. In some embodiments, the pulse of electrical energy has an amplitude of about 5 to 250 V (e.g., about 20 V).

[0326] In some embodiments, about 1-12 pulses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 pulses) of electrical energy are delivered during use. In some embodiments, about 4-12 pulses of electrical energy are delivered during use.

[0327] In some embodiments, each pulse of electrical energy is about 10 ms to about 200 ms. For example, each pulse of electrical energy can be about 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 110 ms, 120 ms, 130 ms, 140 ms, 150 ms, 160 ms, 170 ms, 180 ms, 190 ms, or 200 ms. In some embodiments, each pulse of electrical energy is from about 50 ms. In some embodiments, each pulse of electrical energy is less than 10 ms. For example, each pulse of electrical energy can be from about 10 μs to about 10 ms, e.g., from about 10 μs to about 100 μs, e.g., about 20 μs, 30 μs, 40 μs, 50 μs, 60 μs, 70 μs, 80 μs, 90 μs, or 100 μs, e.g., from about 100 μs to about 1 ms, e.g., about 200 μs, 300 μs, 400 μs, 500 μs, 600 μs, 700 μs, 800 μs, 900 μs, or 1 ms, e.g., from about 1 ms to about 10 ms, e.g., about 2 ms, 3 ms, 4 ms, 5 ms, 6 ms, 7 ms, 8 ms, 9 ms, or 10 ms.

[0328] The device may be used in combination with delivery of a therapeutic agent. For example, in some embodiments, the therapeutic agent has been previously administered to the tissue. In other embodiments, the method further comprises administering the therapeutic agent simultaneously with the delivery of the pulse of electrical energy. For example, in some embodiments, the therapeutic agent is administered simultaneously with the pulse of electrical energy. In some embodiments, the therapeutic agent is administered simultaneously with the pulse of electrical energy. In some embodiments, the therapeutic agent is administered prior to the pulse of electrical energy. In any of the above embodiments, the device may be configured to deliver the therapeutic agent (e.g., via a channel on or in the sheath, e.g., via a channel between the sheath and the insulator).

[0329] The therapeutic agent can be a nucleic acid (e.g., a non-viral nucleic acid (e.g., a naked nucleic acid vector), e.g., a non-viral DNA vector (e.g., a naked DNA vector)). The nucleic acid can be DNA or RNA (e.g., a circular DNA (e.g., naked circular DNA) or circular RNA (e.g., naked circular RNA)). The nucleic acid can be a vector, e.g., a vector containing a transgene. The vector can be, e.g., a non-viral vector (e.g., a naked non-viral vector, e.g., a naked non-viral DNA vector).

[0330] In some embodiments, the target cell is a cell within the eye, such as a retinal cell. The retinal cell can be, for example, a retinal pigment epithelium (RPE) cell, a photoreceptor cell, or a ganglion cell. The therapeutic agent can be, for example, administered intravitreally, subretinally, suprachoroidally, or locally to the eye. The compositions utilized in the methods described herein can be administered locally (e.g., on or in the eye) or systemically (e.g., intravenously). The method of administration can vary depending on various factors (e.g., the compound or composition administered and the severity of the condition, disease, or disorder being treated). In certain embodiments, the therapeutic agent is delivered via an intravitreal route. In certain embodiments, the therapeutic agent is delivered via a suprachoroidal route. In some embodiments, the device targets the intravitreal cavity of the eye.

[0331] In some aspects, the device may be used in any of the methods described herein.

[0332] VI. KITS AND PRODUCTS In another aspect of the present invention, an article of manufacture or kit is provided that includes materials useful for the above treatment. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition, alone or in combination with another composition, that is effective for treating, preventing, and / or diagnosing a condition, and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a therapeutic agent of the present invention (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site)) or a pharmaceutical composition that includes a therapeutic agent of the present invention. The label or package insert indicates that the composition is used to treat a selected disease or disorder. The article of manufacture may further include a package insert indicating that the composition can be used to treat a particular condition (e.g., Usher syndrome type 1B, autosomal recessive Bestrophinopathy, autosomal dominant Best vitelliform macular dystrophy, or macular degeneration (e.g., age-related macular degeneration (AMD)). Alternatively, or additionally, the article of manufacture may further include a second container comprising a pharma- ceutically acceptable carrier, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, dextrose solution, or any of the pharma- ceutically acceptable carriers disclosed above. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0333] In certain instances of the invention, kits are provided that include one or more elements of (i) any one or more of the above materials (e.g., any of the aforementioned therapeutic agents of the invention and / or one or more pharma- ceutically acceptable carriers) and (ii) an energy delivery device (e.g., a device including electrodes for transmitting an electric field to a tissue (e.g., the retina), such as any suitable device or system described above). In some embodiments, kits are provided herein that include a therapeutic agent of the invention (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site)) and electrodes. In some embodiments, kits are provided herein that include a pharmaceutical composition including a therapeutic agent of the invention (e.g., a nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector lacking a bacterial origin of replication, a drug resistance gene, and / or a recombination site)) and electrodes. EXAMPLES

[0334] The following are non-limiting examples of the methods and compositions described above. The following examples also provide non-limiting ways to implement and use the devices described above. Those skilled in the art will recognize that variations of the following examples are also encompassed by the description herein.

[0335] Example 1. Computational modeling of substantially planar electrodes Electric field distribution simulations were performed to compare the effect of the electric field delivered by a substantially planar electrode on retinal tissue to the effect of the electric field delivered by a needle electrode on retinal tissue, with each electrode design positioned for electrical introduction of a therapeutic agent into the retina (i.e., contacting the vitreous humor anterior to the retina). Figures 7A-7C show needle electrodes and Figures 8A-8C show substantially planar electrodes. Each electrode is monopolar.

[0336] Figures 7A-7C show a cross section of an eye containing a needle electrode posterior to the vitreous humor (shown on the graph as the lower segment of the circle in Figure 7A). When a voltage is applied, the needle electrode generates an elliptical electric field along the axis of its sheath. When the distal end of the needle electrode is positioned 0.25 mm from the vitreous humor-retina interface (Figures 7A and 7B), the volume of retinal tissue experiencing an electric field strength of >50 V / cm is 0.5 mm. 3 The volume of retinal tissue exposed to electric field strengths of >100 V / cm was 0.15 mm 3 The volume of retinal tissue exposed to electric field strengths of >150 V / cm was 0.075 mm 3 As shown in Figure 7C, when the distal tip of the needle electrode was positioned further away from the vitreous-retina interface (0.95 mm from the vitreous-retina interface), the volume of retinal tissue subjected to electric field strengths >50 V / cm was 0.4 mm 3 and none of the retinal tissue experienced a field strength of >100 V / cm. Thus, shifting the needle electrode 0.7 mm anteriorly resulted in a 100% reduction in the retinal volume experiencing a field strength of at least 100 V / cm.

[0337] Figures 8A-8C show that the electric field strength experienced by the retina during delivery by a substantially flat electrode is substantially less sensitive to electrode position. In this simulation, moving the substantially flat electrode 0.7 mm anteriorly reduced the retinal volume experiencing electric field strengths of at least 100 V / cm by 8%. When the distal end of the substantially flat electrode was positioned 0.25 mm from the vitreous-retina interface (Figure 8C), the volume of retinal tissue experiencing electric field strengths >50 V / cm was reduced by 1.87 mm. 3 The volume of retinal tissue exposed to electric field strengths of >100 V / cm was 1.11 mm 3 The volume of retinal tissue exposed to electric field strengths of >150 V / cm was 0.77 mm 3 As shown in Figure 8B, when the distal end of the substantially flat electrode was positioned further away from the vitreous-retina interface (0.95 mm from the vitreous-retina interface), the volume of retinal tissue subjected to electric field strengths >50 V / cm was 2.27 mm 3 The volume of retinal tissue exposed to electric field strengths of >100 V / cm was 1.02 mm3 none of the retinal tissues were subjected to electric field strengths >150 V / cm.

[0338] Thus, in addition to improved tolerance to changes in electrode position relative to the retina, the substantially planar electrode design provides access to a larger volume of the retina by the delivered electric field as compared to a needle electrode design.

[0339] Figures 9A and 9B show that when voltage is applied using a spiral electrode (Figure 9B) compared to a needle electrode (Figure 9A), the retinal potential matches the electrode voltage more closely. When the distal end of a needle electrode with a potential of 20 V was placed 0.4 mm from the vitreous-retinal interface (Figure 9A), the potential at the anterior of the retina was 10.8 V and the potential at the posterior of the retina (choroid-retinal interface) was 9.24 V. In comparison, when a spiral electrode with a potential of 20 V was placed 0.4 mm from the vitreous-retinal interface (Figure 9B), the potential at the anterior of the retina was 19.0 V and the potential at the posterior of the retina (choroid-retinal interface) was 17.9 V. This model demonstrates how a planar electrode design can improve the consistency of energy delivery and electrical induction across a volume of target tissue.

[0340] Example 2. Delivery of nucleic acid vectors to the retina using a device with substantially flat electrodes The bipolar electrode device shown in FIG. 5 is used to deliver a nucleic acid vector to a population of retinal pigment epithelial cells of an individual after diagnosis of a patient with an inherited retinal disorder characterized by a mutation in a gene encoding a retinal protein. The patient is prescribed a pharmaceutical composition including a non-viral DNA vector encoding a retinal protein, and a pharmaceutical composition including, for example, 20-150 (e.g., 50-150) microliters is administered to the patient's eye by subretinal or intravitreal injection. As part of the same procedure, a device having an elongated conductor retracted within a sheath is inserted into the vitreous humor of the eye containing the non-viral DNA vector. Using an actuator, the operator slides the elongated conductor distally relative to the sheath until the sheath is in its distal position, thereby forming a substantially flat electrode within the vitreous humor. Using a surgical microscope as a visual guide, the operator positions the substantially flat electrode in a substantially coplanar orientation relative to the target area of ​​the retina, offset by approximately 0.5 mm from the vitreous humor-retina interface. The operator delivers eight 50 V, 20 ms pulses through the electrode over a period of eight seconds at one pulse per second. Alternatively, the operator may choose to deliver eight 20V, 20ms pulses. The operator retracts the substantially planar electrode proximally into the sheath and removes the device from the patient's eye. The treatment is terminated and the patient is monitored over the following weeks and months for improvement in expression of the genes delivered by the treatment.

[0341] Example 3: Electrotransfer of a synthetic circular DNA vector encoding GFP in pig retina A supercoiled synthetic covalently closed loop (C) vector encoding GFP and lacking a bacterial replication origin, drug resistance genes and recombination sites. 3 )DNA vector (C 3 -GFP) was produced using Phi29 polymerase-mediated rolling circle amplification in a cell-free process according to the methods taught in WO 2019 / 178500.

[0342] 225ug of vector was administered by single bilateral subretinal injection into two subretinal blebs (75μL each) in each eye of Gottingen minipigs on day 1 of the study. Briefly, animals were anesthetized and placed in lateral recumbency. Topical proparacaine was applied to the eye. The conjunctival fornix was flushed with a 1:50 dilution of betadine solution / saline and the lid margin was wiped with undiluted 5% betadine solution. The eye was draped and a wire speculum was placed. A caliper was used to mark a spot 3.0mm posterior to the limbus on the superotemporal and superonasal sclera. A bipolar cautery was used to cauterize the sclera below the marked spot followed by topical application of undiluted 5% betadine solution. A microvitreoretinal blade with a 25 g valved cannula was inserted through the conjunctiva and sclera into each marked spot and advanced into the vitreous humor while the globe position was fixed using scleral fixation forceps. The trocar was positioned to face the posterior axis of the globe and then retracted, leaving the scleral port in place. A 31 g needle was then inserted tangentially through the limbus into the anterior chamber to remove 75 μL of aqueous humor. A surgical direct contact lens was placed on the cornea with sterile coupling gel. An intraocular illumination probe was inserted through one of the scleral ports to facilitate direct visualization of the posterior segment with a microscope. A subretinal injection cannula was inserted through the second port and advanced to the center of the vitreous. The small diameter injection cannula was then advanced until it contacted the retinal surface. The dosing solution was then slowly delivered to guide and fill the subretinal bleb. After visualizing adequate bleb formation, the injection was continued to deliver the entire dose volume (75 μL / bleb) into the subretinal space. Two injection blebs were administered into the nasal and temporal regions. Once the injection dose was delivered, the injection cannula and intraocular illumination probe were removed from the scleral port and the contact lens was removed from the cornea. Once the PEF was delivered, the scleral port was removed.

[0343] Group-specific methods of pulsed electric field (PEF) conditions and results are described below.

[0344] Subretinal PEF using a monopolar needle electrode Within 5 min of injection, a monopolar needle electrode (negative pole, 0.2–2 mm long, diameter large enough to fit a 25-gauge trocar) was placed within the subretinal bleb (as shown in Figure 2B) and eight 20 ms electrical pulses were delivered at 20 V for 8 s. The average current measured under these conditions was 13.7 mA. Optical coherence tomography (OCT) and confocal scanning laser tomography (cSLO) were performed pretreatment and on day 7. On day 7, animals were euthanized and eyes were dissected to collect the retina and retinal pigment epithelium (RPE) as well as the choroid for staining.

[0345] Confocal scanning laser ophthalmoscopy (cSLO) images at day 7 showed widespread and uniform GFP fluorescence within the subretinal bleb (Figure 21B) compared to baseline (Figure 21A). Optical coherence tomography (OCT) images suggested the transfection was safe; no structural changes or inflammation were detected (Figures 22A-22D). H&E staining was consistent in showing no structural changes (Figure 23B). Histological images (IHC) showed GFP expression in both photoreceptor (PR) and retinal pigment epithelial (RPE) cells (Figure 23A).

[0346] Intravitreal PEF using a monopolar needle electrode A monopolar needle electrode (positive electrode; 0.2–2 mm long, diameter large enough to fit a 25-gauge trocar) was placed intravitreally such that the distal tip of the electrode was within 1 mm of the retina (as shown in Figure 2A). Eight 20 ms electrical pulses were delivered at 40 V for 8 s. The average current measured under these conditions was 26.7 mA. On day 6, animals were euthanized and eyes were dissected to retrieve the retina and retinal pigment epithelium (RPE) as well as the choroid for staining. GFP expression was observed in the RPE layer (Figures 24A and 24B). A similar PEF technique with the negative electrode placed intravitreously resulted in negative GFP staining of the RPE layer (data not shown).

[0347] Subretinal PEF using bipolar needle electrodes A bipolar needle electrode with a negative pole at the distal end and a positive pole on the proximal needle was positioned so that the negative pole was in the subretinal bleb and the positive pole was in the vitreous. Eight 20 ms electrical pulses were delivered at 40 V for 8 seconds. On day 6, animals were euthanized and eyes were dissected to retrieve the retina and retinal pigment epithelium (RPE) as well as the choroid for staining. GFP expression was observed in the RPE layer (Figure 25).

[0348] Intravitreal PEF using a monopolar planar electrode A monopolar spiral electrode, as shown in Figure 6, was placed in the vitreous humor within 1 mm of the target retinal tissue, and a dispersive patch was placed on the animal's abdomen. Electrical energy of +40 V (as shown in Figure 2C) or -40 V (as shown in Figure 2D) was delivered from the monopolar electrode in eight pulses, each pulse having a duration of 20 ms. The average current measured under these conditions was 32.1 mA. On day 7, the animals were euthanized and the eyes were dissected to collect the retina and retinal pigment epithelium (RPE) as well as the choroid for staining.

[0349] Minimal retinal degeneration was observed in both conditions. In eyes receiving a +40V pulse (positive intravitreal electrode), GFP staining was observed in retinal photoreceptor cells (FIGS. 28A and 28B). In eyes receiving a -40V pulse (negative intravitreal electrode), GFP staining was negative (data not shown).

[0350] Control As a control, the eye was treated with C without electrical induction by a pulsed electric field. 3 C-GFP was injected subretinal (FIGS. 26A and 26B), and PBS was injected subretinal with electrical induction by a pulsed electric field (FIGS. 27A and 27B). 3 In eyes injected with -GFP, no significant GFP labeling in the RPE was observed (FIG. 26A). In eyes treated with PBS, no nonspecific labeling was observed (FIG. 27A).

[0351] Example 4: Sustained expression of synthetic circular DNA vectors in iRPE cells Synthetic covalent ring closure (C 3To assess the persistence of expression of the DNA vector, induced retinal pigment epithelial (iRPE) cells were generated according to published methods, transfected with and without a pulsed electric field on day 1, and GFP expression was monitored over time. 3 The DNA vectors were as described in Example 1. iRPE cells were seeded in 6.5 mm transwell plates and 20 ug of synthetic C 3 DNA vectors were added in a total volume of 120 uL per transwell (upper chamber). A bipolar plate electrode assembly was placed above and below the cell membrane of each well with a distance of 4 mm between the electrode bars, and two pulses of 300-450 V with pulse durations of 5 or 20 s, respectively, were applied. Images were taken on days 4 (Figure 29A), 21 (Figure 29B), 32 (Figure 29C), 40 (Figure 29D), and 49 (Figure 29E). GFP expression was observed in cells transfected by electrotransduction at all time points, with no signs of degradation.

[0352] Example 5: Expression of human ABCA4 mRNA in pig retina by in vivo electrotransfer A synthetic covalently closed (C) vector encoding full-length human ABCA4 driven by the CAG promoter and lacking a bacterial replication origin, drug resistance genes, and recombination sites was 3 )DNA vector (C 3 The naked C. cerevisiae-ABCA4; 8656 bp; SEQ ID NO: 19) was produced using Phi29 polymerase-mediated rolling circle amplification in a cell-free process according to the methods generally taught in WO 2019 / 178500. 3 -ABCA4 was administered to pigs by subretinal injection (225ug / eye) and subretinal PEF was administered using a monopolar needle electrode (as shown in Figure 2B). 20V of electrical energy was delivered from the monopolar electrode (negative electrode) in eight pulses with each pulse having a duration of 20ms. Eyes were harvested and the neural retina (NR) layer and RPE / choroid layers were isolated. RNA was isolated from tissues and mRNA levels of the ABCA4 transgene and endogenous porcine ABCA4 were quantified by qPCR using standard methods.

[0353] As shown in Figure 30, ABCA4 transgene mRNA expression was detected in both the NR layer, which contains photoreceptors, and the RPE / choroid layer. In general, higher ABCA4 transgene mRNA expression was detected in the RPE / choroid layer compared to the neural retina layer. This study was carried out to confirm the efficacy of PEF-mediated electrical induction of C 3 1 shows that administration of -ABCA4 resulted in ABCA4 transgene expression in vivo.

[0354] Example 6: Expression of human MYO7A mRNA in pig retina by in vivo electrotransfer A synthetic C encoding full-length human MYO7A lacking the bacterial replication origin, drug resistance genes and recombination sites 3 DNA vector (C 3 Naked C. cerevisiae (-MYO7A) was produced using Phi29 polymerase-mediated rolling circle amplification in a cell-free process according to the methods generally taught in WO 2019 / 178500. 3 -MYO7A was administered by subretinal injection (225ug DNA per eye; 2.53 x 10 13 After injecting the vector copy number 1 (0.01), subretinal PEF was administered using a monopolar needle electrode (as shown in Figure 2B). 20 V of electrical energy was delivered from the monopolar electrode (negative electrode) in eight pulses with each pulse having a duration of 20 ms. Eyes were harvested and the neural retina (NR) layer and RPE / choroid layers were isolated. RNA was isolated from the tissues and mRNA levels of the ABCA4 transgene and endogenous porcine ABCA4 were quantified by qPCR using standard methods.

[0355] As shown in FIG. 31, MYO7A transgene mRNA expression was detected in both the NR layer, which contains photoreceptors, and the RPE / choroid layer. In general, higher MYO7A transgene mRNA expression was detected in the RPE / choroid layer compared to the neural retina layer. This study demonstrated that PEF-mediated electrotransfer enhances C-cell proliferation and cell death in the retina. 3 -MYO7A administration resulted in ABCA4 transgene expression in vivo.

[0356] Example 7: Expression of human ABCA4 protein in pig retina by in vivo electrotransfer A synthetic covalently closed (C) promoter-driven human ABCA4 lacking a bacterial replication origin, drug resistance genes, and recombination sites was 3 )DNA vector (C 3 C-ABCA4; 8656 bp; SEQ ID NO: 19) was generated using Phi29 polymerase-mediated rolling circle amplification in a cell-free process according to the methods generally taught in WO 2019 / 178500. 3 -ABCA4 was formulated in solution at a concentration of 1.5 mg / mL. Naked C-ABCA4 was administered by injecting two blebs of 75 μL each into the subretinal space of Göttingen minipigs. 3 - ABCA4 was administered (225ug DNA per eye; 2.53 x 10 per eye) 13 After injection, a monopolar needle electrode was placed into each subretinal bleb and eight 20 ms electrical pulses were delivered at 20 V. On day 6, animals were euthanized and eyes were dissected to collect the retina and RPE as well as the choroid for staining.

[0357] Widespread human ABCA4 protein expression was observed in the photoreceptor layer adjacent to the RPE (Figure 32A). Furthermore, human ABCA4 protein was expressed in the photoreceptor outer segments (Figure 32B, showing colocalization with rhodopsin). These data support the notion that human ABCA4 protein expression in pigs is a novel mechanism for the development of C. 3 This shows that in vivo electrotransduction of -ABCA4 resulted in widespread expression of human ABCA4 protein in the desired cell type (PR) and in the desired subcellular location (outer segment) within those cells. To confirm clinical feasibility, Figures 32 and 33 show identical localization of the human ABCA4 transgene in pigs (Figure 33) to human endogenous ABCA4 in the human eye (Figure 34).

[0358] Example 8: C in iRPE cells 3 Expression comparison of -ABCA4 and plasmid ABCA4 Induced retinal pigment epithelium (iRPE cells) were generated according to published methods and transfected in vitro with ABCA4 encoded by a plasmid or synthetic circular DNA vector generated by Phi29 polymerase-mediated rolling circle amplification in a cell-free process. Briefly, iRPE cells were seeded in laminin-coated 6-well plates and cultured for 48 h until 100% confluence. Cells were harvested with TrypLE, counted, and >2.5 × 10 per 24-well. 5 The cells were replated with 1 μg / well of ABCA4. DNA vector was added at 1 ug / well and cells were electroporated using the Neon transfection system at 1100V, 20 ms. Cells were incubated for 48 hours before antibody staining. Protein expression analysis revealed that the synthetic circular DNA vector expressed higher amounts of ABCA4 protein compared to the plasmid (Figure 35). Representative fluorescent images showing ABCA4 expression by the synthetic circular DNA vector are shown in Figures 36A-36C compared to expression by the plasmid vector shown in Figures 36D-36F.

[0359] Example 9: C in iRPE cells 3 Expression comparison of -MYO7A and plasmid MYO7A iRPE cells were generated according to published methods and transfected in vitro with MYO7A encoded by a plasmid or synthetic circular DNA vector generated by Phi29 polymerase-mediated rolling circle amplification in a cell-free process. Briefly, iRPE cells were seeded into laminin-coated 6-well plates and cultured for 48 hours until 100% confluence. Cells were harvested with TrypLE, counted, and >2.5 × 10 per 24-well. 5The cells were replated with 1 μg / well of DNA vector. The DNA vector was added at 1 ug / well and the cells were electroporated using the Neon transfection system at 1100V, 20 ms. The cells were incubated for 48 hours before antibody staining. Protein expression analysis (Figure 37) revealed that the synthetic circular DNA vector (lane 4) expressed higher amounts of MYO7A protein compared to the plasmid encoding the same MYO7A transgene (lane 3). Representative fluorescent images showing MYO7A expression by the synthetic circular DNA vector are shown in Figures 38A-38C compared to expression by the plasmid vector shown in Figures 38D-38F.

[0360] Example 10: Treatment of Stargardt's disease with subretinal DNA injection and subretinal PEF administration The patient is an adult with a biallelic ABCA4 mutation that causes retinal degeneration due to Stargardt disease. 3 - ABCA4 is provided in a naked form in an aqueous pharmaceutical composition and loaded into a subretinal delivery device. 150 μL of the pharmaceutical composition is administered subretinal to each eye of the patient (225 μg of DNA per eye; 2.53 × 10 13 Vector copy).

[0361] The patient is prepared for pulsed electric field (PEF) treatment. Within 30 minutes after subretinal injection of the circular DNA vector, an energy delivery device with an elongated wire electrode within a sheath is inserted into the vitreous humor of each eye as shown in Figure 2A. Using a surgical microscope as a visual guide, the exposed electrode is positioned completely within the vitreous humor approximately 0.5 mm from the retina centered on the macula. Eight pulses of a 40 volt waveform, each pulse having a duration of 20 milliseconds, are delivered by the electrode. The electrodes are removed from each eye and the treatment is complete.

[0362] After treatment, patients are monitored weekly to assess the progression of retinal degeneration. For this purpose, visual acuity and visual field are monitored using standard tests. OCT is performed to assess changes in retinal structure.

[0363] Example 11: Treatment of Usher syndrome type 1B with subretinal DNA injection and intravitreal PEF administration Patients are adults with an allelic MYO7A mutation causing retinal degeneration due to Usher syndrome type 1B.

[0364] A covalently closed circular DNA vector encoding MYO7A is generated using a cell-free method by phi-29-mediated rolling circle amplification adapted from the method described in WO 2021 / 055760. The circular DNA vector is provided in naked form in an aqueous buffered pharmaceutical composition and loaded into a subretinal delivery device.

[0365] 100 μL of the pharmaceutical composition is administered subretinal to each eye of the patient.

[0366] The patient is prepared for PEF treatment. Within 30 minutes after subretinal injection of the circular DNA vector, an energy delivery device with an elongated wire electrode within a sheath is inserted into the vitreous humor of each eye as shown in Figure 2A. Using a surgical microscope as a visual guide, the exposed electrode is positioned completely within the vitreous humor approximately 1 mm from the retina centered on the macula. Eight pulses of a 40 volt waveform, each pulse having a duration of 20 ms, are delivered by the electrode. The electrodes are removed from each eye and the procedure is complete.

[0367] After treatment, patients are monitored weekly to assess the progression of retinal degeneration. For this purpose, visual acuity and visual field are monitored using standard tests. OCT is performed to assess changes in retinal structure.

[0368] Example 12: Treatment of Usher syndrome type 1B with suprachoroidal DNA and PEF administration Patients are adults with retinal degeneration due to mutations in the MYO7A allele that causes retinal degeneration due to Usher syndrome type 1B.

[0369] A covalently closed circular DNA vector encoding MYO7A is synthesized using a cell-free method by phi-29-mediated rolling circle amplification adapted from the method described in WO 2021 / 055760. The circular DNA vector is provided in naked form in an aqueous buffered pharmaceutical composition and loaded into a delivery device having a microneedle configured for suprachoroidal administration, such as the device described in WO 2014 / 074823.

[0370] As shown in Figure 3A, 100 μL of the pharmaceutical composition is administered suprachoroidally to each eye of the patient. The circular DNA vector migrates through the suprachoroidal space toward the back of the eye, where it occupies the extracellular space surrounding the retina (the retina and / or the suprachoroidal space adjacent to the retina).

[0371] The patient is prepared for pulsed electric field treatment. Within 30 minutes after suprachoroidal injection of the circular DNA vector, an energy delivery device with an elongated wire electrode within a sheath is inserted into the vitreous humor of each eye as shown in Figures 3B-3E. Using a surgical microscope as a visual guide, the exposed electrode is positioned completely within the vitreous humor approximately 1 mm from the retina centered on the macula. Eight pulses of a 40 volt waveform, each pulse having a duration of 20 ms, are delivered by the electrode. The electrodes are removed from each eye and the procedure is complete.

[0372] After treatment, patients are monitored weekly to assess the progression of retinal degeneration. For this purpose, visual acuity and visual field are monitored using standard tests. OCT is performed to assess changes in retinal structure.

[0373] (Table 1) Sequence TIFF2024512520000001.tif244160TIFF2024512520000002.tif251160TIFF2024512520000003.tif251160TIFF2024512520000004.tif251160TIFF2024512520000005.tif251160TIFF2024512520000006.tif251160TIFF2024512520000007.tif251160TIFF2024512520000008.tif251160TIFF2024512520000009.tif251160TIFF2024512520000010.tif251160TIFF2024512520000011.tif251160TIFF2024512520000012.tif251160TIFF2024512520000013.tif251160TIFF2024512520000014.tif251160TIFF2024512520000015.tif251160TIFF2024512520000016.tif251160TIFF2024512520000017.tif251160TIFF2024512520000018.tif251160TIFF2024512520000019.tif251160TIFF2024512520000020.tif251160TIFF2024512520000021.tif251160TIFF2024512520000022.tif251160TIFF2024512520000023.tif251160TIFF2024512520000024.tif251160TIFF2024512520000025.tif251160TIFF2024512520000026.tif251160TIFF2024512520000027.tif251160TIFF2024512520000028.tif251160TIFF2024512520000029.tif251160TIFF2024512520000030.tif251160TIFF2024512520000031.tif251160TIFF2024512520000032.tif251160TIFF2024512520000033.tif251160TIFF2024512520000034.tif251160TIFF2024512520000035.tif251160TIFF2024512520000036.tif215160.

[0374] Other Aspects All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0375] While the invention has been described in relation to particular embodiments thereof, it will be understood that the invention is capable of further modifications, and this application is intended to cover any variation, use, or adaptation of the invention generally in accordance with the principles of the invention and which comes within known or customary practice within the art to which the invention pertains and which may be applied to the essential features described above, including such departures from the present disclosure as fall within the scope of the claims.

[0376] Other aspects are within the scope of the claims.

Claims

1. 1. An electrode for use in a method of delivering a therapeutic agent to targeted retinal cells of an individual, the method comprising: (a) contacting an electrode with an interior region of the eye, wherein an extracellular space within the retina of the eye contains a therapeutic agent; and (b) delivering one or more pulses of electrical energy through the electrode under conditions suitable for electrical delivery of the therapeutic agent to the targeted retinal cells while the electrode is in contact with the interior region of the eye; wherein, optionally, the method further comprises the step of delivering a therapeutic agent to the extracellular space of the retina; and, optionally, delivery of the therapeutic agent is by subretinal or intravitreal injection.

2. the interior area of ​​the eye in contact with the electrode comprises vitreous humor; and Optionally, the electrode is within 10 mm of the retina during delivery of the one or more pulses of electrical energy; or Optionally, the interior region of the eye contacted by the electrode includes the retina; and Optionally, the interior region of the eye contacted by the electrode includes the subretinal space.

2. An electrode for use according to claim 1.

3. the conditions suitable for electrical delivery of a therapeutic agent to a target retinal cell comprise an electric field strength at said target retinal cell of between 10 V / cm and 1,500 V / cm; and / or Optionally, 1 to 12 pulses of electrical energy are delivered; and / or Optionally, the total number of pulses of electrical energy is delivered within 1-20 seconds; and / or Optionally, the pulses of electrical energy are square waveforms; and / or Optionally, the pulse of electrical energy has an amplitude of between 5V and 250V; and / or Optionally, each of the pulses of electrical energy is between 10 and 200 milliseconds in duration. Electrode for use according to claim 1 or 2.

4. 4. The electrode for use according to any one of claims 1 to 3, wherein the target retinal cells are retinal epithelial cells or photoreceptors.

5. The therapeutic agent is a nucleic acid vector; and Optionally, the nucleic acid vector is a non-viral nucleic acid vector; and Optionally, the non-viral nucleic acid vector is a circular DNA vector; and / or Optionally, the nucleic acid vector is a non-viral nucleic acid vector encoding a therapeutic replacement protein that replaces a protein endogenously expressed in healthy retinal cells; and Optionally, the therapeutic replacement protein replaces a protein that is not endogenously expressed in the individual's target cells or that is non-functional in the individual's target cells; and / or Optionally, the therapeutic replacement protein is encoded by a coding sequence of greater than 4.5 kb; and / or Optionally, the therapeutic replacement protein is MYO7A; and Optionally, the method is a method of treating Usher syndrome type 1B in an individual. An electrode for use according to any one of claims 1 to 4.

6. The nucleic acid vector is a non-viral nucleic acid vector encoding a therapeutic replacement protein that replaces a protein endogenously expressed in healthy retinal cells; and Optionally, the therapeutic replacement protein replaces a protein that is not endogenously expressed in the individual's target cells or that is non-functional in the individual's target cells; and the therapeutic replacement protein is BEST1; and Optionally, the method is a method of treating a bestrophinopathy associated with a BEST1 dominant mutation or a BEST1 recessive mutation; or Optionally, the therapeutic replacement protein is CFH; and Optionally, the method is a method of treating age-related macular degeneration; or the therapeutic replacement protein is ABCA4; and Optionally, the method is a method of treating an ABCA4-associated retinal dystrophy; and Optionally, the ABCA4-associated retinal dystrophy is Stargardt's disease.

6. An electrode for use according to claim 5.

7. 1. An electrode for use in a method of delivering a therapeutic agent to targeted retinal cells of an individual, the method comprising: (a) contacting an electrode with an interior region of the eye, wherein the extracellular space within the retina of the eye contains a therapeutic agent to be delivered by suprachoroidal injection; and (b) delivering one or more pulses of electrical energy through the electrode under conditions suitable for electrical delivery of the therapeutic agent to the targeted retinal cells while the electrode is in contact with the interior region of the eye; wherein, optionally, the interior region of the eye in contact with the electrode comprises vitreous humor; and Optionally, the electrode is within 10 mm of the retina during delivery of the one or more pulses of electrical energy; and / or Optionally, the interior region of the eye in contact with the electrode includes the retina; Optionally, the interior region of the eye contacted by the electrode includes the subretinal space.

8. the conditions suitable for electrical delivery of a therapeutic agent to a target retinal cell comprise an electric field strength at said target retinal cell of between 10 V / cm and 1,500 V / cm; and / or Optionally, 1 to 12 pulses of electrical energy are delivered; and / or Optionally, the total number of pulses of electrical energy is delivered within 1-20 seconds; and / or Optionally, the pulses of electrical energy are square waveforms; and / or Optionally, the pulse of electrical energy has an amplitude of between 5V and 250V; and / or Optionally, each pulse of electrical energy is of duration between 10 and 200 milliseconds; and / or Optionally, the target retinal cells are retinal epithelial cells; and / or Optionally, the target retinal cells are photoreceptors; and / or Optionally, the therapeutic agent is a nucleic acid vector; and / or Optionally, the nucleic acid vector is a non-viral nucleic acid vector; and / or Optionally, the non-viral nucleic acid vector is a circular DNA vector; and / or Optionally, the non-viral nucleic acid vector encodes a therapeutic replacement protein that replaces a protein endogenously expressed in healthy retinal cells; and Optionally, the therapeutic replacement protein replaces a protein that is not endogenously expressed in the individual's target cells or that is non-functional in the individual's target cells; and / or Optionally, the therapeutic replacement protein is encoded by a coding sequence of more than 4.5 kb; and / or Optionally, the therapeutic replacement protein is MYO7A; and Optionally, the method is a method of treating Usher syndrome type 1B in an individual; or Optionally, the therapeutic replacement protein is BEST1; and Optionally, the method is a method of treating a bestrophinopathy associated with a BEST1 dominant mutation or a BEST1 recessive mutation; or Optionally, the therapeutic replacement protein is CFH; and Optionally, the method is a method for treating age-related macular degeneration.

8. An electrode for use according to claim 7.

9. (a) a sheath having a proximal end, a distal end, and a longitudinal axis therebetween; (b) an elongate conductor having a proximal portion and a distal portion within the sheath, the elongate conductor comprising a preformed shape memory material and retractable within the sheath from a proximal position to a distal position; (i) in a proximal position, a distal portion of the elongated conductor is substantially straight; and (ii) in a distal position, a distal portion of the elongate conductor extends beyond the distal end of the sheath, and the shape memory material of the distal portion of the elongate conductor relaxes radially to form a substantially planar electrode disposed at a preformed angle relative to the longitudinal axis of the sheath; or optionally, the shape memory material of the distal portion of the elongate conductor relaxes radially to form a substantially planar electrode perpendicular to the longitudinal axis of the sheath; or optionally, the shape memory material of the distal portion of the elongate conductor relaxes radially to form a substantially planar electrode substantially perpendicular to the longitudinal axis of the sheath; A long thin conductor Including, and optionally, the preformed angle is substantially a right angle; and optionally, the preformed angle is about 70° or about 110°; Or optionally, the preformed angle is from about 45° to about 135°; and optionally further comprising a handle having a proximal end and a distal end, the sheath being connected to the handle; and optionally, a proximal end of the sheath connected to a handle; and optionally, the handle is cylindrical; Device.

10. further comprising an actuator; and optionally, a proximal end of the sheath and / or the elongate conductor is connected to an actuator, the actuator configured to slide the elongate conductor between a proximal position and a distal position; and optionally, the actuator is a slider; and optionally, the slider has a proximal end and a distal end and is attached to the elongated conductor, the slider configured to retract the elongated conductor from a distal position to a proximal position along the longitudinal axis of the sheath; and optionally, the slider includes a proximal position and a distal position; (i) in the proximal position, a proximal end of the sheath is disposed at or proximal to a distal end of the slider; and (ii) in the distal position, a proximal end of the sheath is disposed between a proximal end of the slider and a distal end of the slider; and optionally, the slider is configured to stop upon sliding to a distal position and / or a proximal position.

10. The apparatus of claim 9.

11. The actuator according to claim 1, wherein the actuator is a slider; and Optionally, the slider is positioned at a distal location such that a distal portion of the elongate conductor extends beyond the distal end of the sheath and the shape memory material of the distal portion of the elongate conductor is radially relaxed to form a substantially planar electrode at a preformed angle relative to the longitudinal axis of the sheath; or Optionally, the slider is disposed at a proximal position and the distal portion of the elongate conductor is substantially straight; and / or Optionally, the slider includes a control member disposed externally of the handle; and Optionally, the control member and the slider may or may not be integral; 11. The apparatus of claim 10.

12. (a) a handle having a proximal end and a distal end; (b) a sheath connected to the handle, the sheath having a proximal end, a distal end, and a longitudinal axis therebetween; (c) an elongate conductor having a proximal portion and a distal portion within the sheath, the elongate conductor comprising a preformed shape memory material and retractable within the sheath from a proximal position to a distal position; (i) in a proximal position, a distal portion of the elongated conductor is substantially straight; and (ii) in a distal position, a distal portion of the elongate conductor extends beyond the distal end of the sheath, and the shape memory material of the distal portion of the elongate conductor is radially relaxed to form a substantially planar electrode disposed at a preformed angle relative to the longitudinal axis of the sheath; an elongated conductor; (d) a slider having a proximal end and a distal end and attached to the elongated conductor, the slider configured to retract the elongated conductor from a distal position to a proximal position along a longitudinal axis of the sheath; Including, and optionally, the preformed angle is about 70° or about 110°; and optionally, the preformed angle is from about 45° to about 135°. Device.

13. a sheath connected to the slider, the elongated conductor being within the sheath connected to the slider; and optionally, a sheath connected to the slider nested with a sheath connected to the handle; and optionally, a sheath connected to the slider is connected to the elongated conductor.

13. Apparatus according to any one of claims 9 to 12.

14. the distal end of the sheath includes a needle; and / or further comprising insulation between the proximal portion of the elongated conductor and the sheath; 14. Apparatus according to any one of claims 9 to 13.

15. the sheath comprises a conductive material; and / or Optionally, the sheath has an inner diameter of about 0.01 mm to about 1 mm; and Optionally, the sheath has an inner diameter of about 0.2 mm to about 0.3 mm; and / or Optionally, the elongated conductor has a diameter that is about 50% to about 99% of the inner diameter of the sheath.

15. Apparatus according to any one of claims 9 to 14.

16. the elongated conductor has a diameter of about 100 μm to about 200 μm; and / or Optionally, the elongated conductor has a diameter of about 0.2 mm; or Optionally, the elongated conductor has a diameter of about 150 μm.

16. The device according to any one of claims 9 to 15.

17. the substantially planar electrode has one or more dimensions perpendicular to the longitudinal axis that are between 2 mm and 15 mm; and optionally, the substantially planar electrode is between 2 and 15 mm in both dimensions perpendicular to the longitudinal axis; and / or Optionally, the substantially planar electrode is substantially symmetrical about a longitudinal plane; and / or Optionally, the substantially planar electrode is convex.

17. Apparatus according to any one of claims 9 to 16.

18. the elongated conductor is a wire and the substantially planar electrode comprises a distal portion of the wire; and optionally, a distal portion of the wire includes a preformed angle on the longitudinal plane, the preformed angle being between the substantially planar electrode and a proximal portion of the wire; and / or Optionally, the substantially planar electrode is a spiral; and optionally, the helix comprises 1 to 5 turns about the longitudinal axis; and optionally, the helix comprises three turns about the longitudinal axis; and / or Optionally, the substantially planar electrode extends no more than 1 mm distal or proximal to the preformed right angle; and optionally, the substantially planar electrode extends distally to the preformed right angle by 1 mm or less; and / or Optionally, the device includes nothing distal to the substantially planar electrode; and / or wherein the device is unipolar; or wherein the device is bipolar and further comprises an auxiliary electrode in electrical communication with the substantially planar electrode; and optionally an auxiliary electrode is part of or connected to the sheath; and / or Optionally, the device includes an elongated conductor having a proximal portion, and optionally, the proximal portion of the elongated conductor is connected to a voltage source and / or a waveform controller.

18. Apparatus according to any one of claims 9 to 17.

19. 19. An electrode for use in a method of delivering an agent to a target cell in a patient using a device according to any one of claims 9 to 18, said method comprising: (i) inserting a sheath through an external tissue surface of a patient; (ii) sliding the elongated conductor to a distal position to form a substantially planar electrode; (iii) placing the substantially planar electrode in electrical communication with a tissue interface separating the target cells from the substantially planar electrode; and (iv) delivering one or more pulses of electrical energy through the substantially planar electrode under conditions suitable for electrical delivery of the agent to the target cells; and optionally, the sheath comprises a needle, and step (i) comprises inserting the needle through an external tissue surface of the patient; and / or Optionally, the substantially planar electrode is within 10 mm of the tissue interface; and optionally, the substantially planar electrode is between 0.5 mm and 5 mm from the tissue interface during delivery of the one or more pulses; And optionally, the substantially planar electrode is about 1 mm from the tissue interface during delivery of the one or more pulses.

20. The target cells are within 5 mm of the tissue interface. and optionally, the target cells are between 0.01 mm and 1 mm from the tissue interface; and / or Optionally, the conditions suitable for electrical delivery of the agent to the target cell comprise an electric field strength at the target cell of between 10 V / cm and 1,500 V / cm; and optionally, the electric field strength at the target cell is between 50V / cm and 300V / cm; and optionally, the electric field strength at the target cell is about 100 V / cm.

20. An electrode for use according to claim 19.

21. 4 to 12 pulses of electrical energy are delivered; and optionally, the total number of pulses of electrical energy is delivered within 1 to 20 seconds; and optionally, the pulses of electrical energy are square waveforms; and / or Optionally, the pulse of electrical energy has an amplitude between 5V and 250V; and optionally, the pulse of electrical energy has an amplitude of about 250V.

21. An electrode for use according to claim 19 or 20.

22. The conditions suitable for electrical transfer of the agent into the target cell comprise a voltage at the target cell of between 5V and 100V; and optionally, the conditions suitable for electrical introduction of the agent into a target cell comprise a voltage at said target cell of between 10V and 80V; and optionally, the pulse of electrical energy has an amplitude of about 40V; and / or Optionally, each of the pulses is between 10 and 200 ms; and optionally, each of the pulses is about 50 ms.

22. An electrode for use according to any one of claims 19 to 21.

23. the agent has been previously administered to the tissue; Or optionally, the method further comprises administering an agent; and optionally, the agent is administered simultaneously or sequentially in one or more pulses; and / or Optionally, the agent is a nucleic acid; and optionally, the nucleic acid is a non-viral nucleic acid; and optionally, the non-viral nucleic acid is DNA or RNA; and / or Optionally, the agent is a therapeutic agent; and / or Optionally, the target cell is a retinal cell; and optionally, the retinal cell is a retinal pigment epithelium (RPE) cell or a photoreceptor cell or a ganglion cell.

23. An electrode for use according to any one of claims 19 to 22.

24. The therapeutic agent is administered intravitreally, subretinal, or topically; and optionally, the therapeutic agent is administered suprachoroidally; Electrode for use according to any one of claims 19 to 23.

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