WO2024168210A1

WO2024168210A1 - Gene therapies for the front of the eye

Info

Publication number: WO2024168210A1
Application number: PCT/US2024/015101
Authority: WO
Inventors: Kenneth Mandell
Original assignee: Aldevron LLC
Current assignee: Aldevron LLC
Priority date: 2023-02-10
Filing date: 2024-02-09
Publication date: 2024-08-15
Anticipated expiration: 2025-08-10
Also published as: KR20250141827A; CN121002188A; EP4662321A1

Abstract

The present invention involves therapies for ocular diseases or disorders in individuals. Provided herein are methods of delivering nucleic acid vectors to ocular cells in the anterior segment of the eye (e.g., corneal endothelium, trabecular meshwork, etc.) involving methods of administering nucleic acid vectors to the individual and methods of electrotransfer of nucleic acid vectors to express therapeutic transgenes. Also provided are nucleic acid vectors for ocular disease, such as Fuchs' dystrophy.

Description

GENE THERAPIES FOR THE FRONT OF THE EYE

FIELD OF THE INVENTION

In general, the invention features nucleic acid vectors and methods of administering nucleic acid vectors to ocular cells in the front of the eye.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of, and claims priority to, US Provisional Application No. 63/444,740, filed February 10, 2023, which is hereby incorporated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

This application contains a Sequence Listing in XML format submitted electronically herewith via Patent Center. The contents of the XML copy, created on February 8, 2024, is named “IGT-012PC_135234-5012. xml” and is 11 ,089 bytes in size. The Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Many ocular disorders involving dysfunction of tissues in the front of the eye, such as corneal dysfunctions, have a high unmet medical need. For instance, Fuchs’ dystrophy is a disease of the corneal endothelium characterized by gradual loss of vision due its progressive degeneration. Symptoms include reduced corneal endothelial cell counts with polymegathism and pleomorphism, corneal guttata (drop-like deposits), corneal edema, decreased visual acuity and contrast sensitivity, glare, diurnal fluctuations in vision, and pain. Onset can occur early in life or later; it is estimated to affect 4% of the U.S. population above age 40. Fuchs’ dystrophy can be caused by autosomal dominant inherence of known mutations, which can cause symptoms through mechanisms such as oxidative stress, mitochondrial dysregulation, endoplasmic reticulum-associated mechanisms such as protein misfolding, apoptosis, epithelial- mesenchymal transition, RNA toxicity, and repeat-associated non-ATG transition. Currently, the only treatments available are tissue transplant-based therapies, such as penetrating keratoplasty and Descemet’s stripping endothelial keratoplasty/Descemet’s membrane endothelial keratoplasty. There is a clear unmet medical need for therapeutic options for front of the eye disorders. SUMMARY OF THE INVENTION

The invention provides therapeutic methods for front of the eye disorders (e.g., Fuchs’ dystrophy), methods of delivering nucleic acid vectors to front of the eye tissues and cells, and therapeutic compositions (circular DNA vectors) for treating Fuchs’ dystrophy.

In one aspect of the invention, provided are methods of expressing a transgene in an ocular cell of an anterior segment of an individual. In some embodiments, the method includes: (a) administering a circular DNA vector to the anterior segment (e.g., the anterior chamber or corneal stroma) of the eye, wherein the circular DNA vector lacks one or more components of a plasmid backbone and encodes the transgene; (b) positioning one or more electrodes (e.g., one, two, three, four, or more electrodes) in and/or around the eye; and (c) transmitting electrical energy through the one or more electrodes at conditions suitable for electrotransfer of the circular DNA vector into the ocular cell of the anterior segment, thereby expressing the transgene in the ocular cell of the anterior segment. In some embodiments, the circular DNA vector is expressed in the ocular cell of the anterior segment eight days following administration. In some embodiments, the transgene expressed by the circular DNA vector in the ocular cell of the anterior segment is more persistent than a transgene encoded by a plasmid DNA vector encoding the transgene. In some embodiments, the circular DNA vector in the ocular cell of the anterior segment is less immunogenic than a plasmid DNA vector encoding the transgene. In some embodiments, the one or more components of the plasmid backbone lacking in the circular DNA vector comprises a drug resistance gene and/or an origin of replication.

In some embodiments, the circular DNA vector is a nonviral circular DNA vector, e.g., a naked circular DNA vector. In some embodiments, the DNA vector is a synthetic circular DNA vector. In some embodiments, the DNA vector comprises an origin of replication and/or lacks a selectable marker. In some embodiments, the 3’ end of the transgene is linked to the 5’ end of a promoter of the transgene by a sequence comprising the bacterial origin of replication, wherein the sequence comprising the bacterial origin of replication is less than 50 bp or less than 100 bp in length. In embodiments, the sequence comprising the bacterial replication origin is less than 50 bp in length, and the circular DNA vector lacks a selectable marker. In various embodiments, the vector further lacks a recombination site. In some embodiments, the vector comprises a transposase scar. In some embodiments, the origin of replication is a ColE2-P9 origin of replication or functional variant thereof.

In embodiments, the sequence comprising the bacterial replication origin directly connects the 3’ end of the therapeutic sequence to the 5’ end of the therapeutic sequence. In embodiments, the circular DNA vector has about 200 base pairs (bp) or less, or about 150 bp or less, or about 100 bp or less, or about 75 bp or less, or about 50 bp or less of bacterially-derived sequences.

In embodiments, the replication origin is from a ColE2-related plasmid, and which is optionally ColE2-P9. In such embodiments, the replication origin is recognized by a ColE2-P9 replication protein. An exemplary ColE2-P9 replication protein comprises the amino acid sequence of SEQ ID NO: 1.

In embodiments, the replication origin is 40 bp or less in length. In some embodiments, the replication origin is 36 bp or less in length, or 34 bp or less in length, or 32 bp of less in length, or 30 bp or less in length, or 28 bp or less in length. For example, the replication origin may have the nucleotide sequence of SEQ ID NO: 2, or may be a functional variant or truncated variant thereof.

In embodiments, the replication origin is a truncated ColE2-P9 replication origin, where one strand of the replication origin comprises or consists of the nucleotide sequence of SEQ ID NO: 5.

In embodiments, the replication origin is a truncated ColE2-P9 replication origin, wherein one strand of the replication origin comprises or consists of the nucleotide sequence of SEQ ID NO: 6.

In embodiments, the replication origin is a truncated ColE2-P9 replication origin, wherein one strand of the replication origin comprises or consist of the nucleotide sequence of SEQ ID NO: 7.

In embodiments, the replication origin is a truncated ColE2-P9 replication origin, wherein one strand of the replication origin comprises or consists of the nucleotide sequence of SEQ ID NO: 8.

In embodiments, the replication origin is a truncated ColE2-P9 replication origin, wherein one strand of the replication origin comprises or consists of the nucleotide sequence of SEQ ID NO: 9.

In embodiments, the replication origin is a truncated ColE2-P9 replication origin, wherein one strand of the replication origin comprises or consists of the nucleotide sequence of SEQ ID NO: 10.

In embodiments, the replication origin is a truncated ColE2-P9 replication origin, wherein one strand of the replication origin comprises or consists of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In embodiments, the replication origin comprises or consists of the nucleic acid sequence of X1X2X3X4X5TGTTATCTGATAAGGCTTATCTGGTCTX6X7 (SEQ ID NO: 11), wherein each X is selected from A, T, C, or G. In some embodiments: X1 is A, T, or C; X2 is A, T, or C; X3 is A, T, or G; X4 is A, T, or C; X5 is A, T, or G; X6 is C; X7 is A.

In an aspect, the present disclosure provides an engineered bacterial cell for replicating the circular DNA vector. The engineered bacterial cell comprises: (a) the circular DNA vector of the present disclosure, and (b) a Rep gene encoding a bacterial replication protein that binds to the bacterial replication origin of the circular DNA vector, wherein the Rep gene replicates the circular DNA vector.

In some embodiments, the therapeutic sequence comprises a transposase overhang sequence, which may be (without limitation) TTAA. In such embodiments, the bacterial cell comprises a transposase protein, wherein the transposase protein hydrolyzes DNA adjacent to a transposase overhang sequence. In some embodiments, the transposase protein is encoded by a transposase gene expressed by the engineered bacterial cell, and may be integrated into the bacterial genome. In some embodiments, the engineered bacterial cell further comprises an insertion sequence excision enhancer (I EE), which can be encoded by a gene that is integrated into the bacterial genome. In some embodiments, the engineered bacterial cell further comprises a closed-ended linear DNA molecule comprising a plasmid backbone. The plasmid backbone may comprise a selectable marker (which may be an antibiotic resistance gene and/or a counterselection marker).

In various embodiments, the replication origin is the only bacterial sequence in the circular DNA vector. In embodiments, the engineered bacterial cell (e.g., in culture) comprises at least 10 copies of the circular DNA vector on average. In various embodiments, the circular DNA vector is monomeric. In some embodiments, the bacterial cell in culture comprises a mean copy number of the circular DNA vector per engineered bacterial cell of at least 10, or at least 15, or at least 20.

In another aspect, the disclosure provides an engineered bacterial cell for producing the circular DNA vector, the cell comprising: (a) a plasmid template, wherein the plasmid template comprises: (i) a first segment comprising a therapeutic sequence and a sequence comprising a bacterial replication origin, wherein the first segment is flanked by two transposase overhang sequences; and (ii) a second segment comprising a plasmid backbone, wherein the second segment is flanked by a left end (LE) repeat and a right end (RE) repeat, wherein the LE repeat and the RE repeat can be bound by the transposase protein; and (b) a Rep gene encoding a bacterial replication protein that binds to the bacterial replication origin. In various embodiments, the bacterial cell further comprises a transposase protein, wherein the transposase protein hydrolyzes DNA adjacent to a transposase overhang sequence. In some embodiments, the engineered bacterial cell further comprises (e.g., produces): (c) a circular DNA vector comprising the therapeutic sequence, the sequence comprising the bacterial replication origin, and one of the two transposase overhang sequences; and/or (d) a linear closed-ended DNA molecule comprising the plasmid backbone flanked by the LE repeat and the RE repeat.

In some embodiments, the transposase protein is encoded by a transposase gene expressed by the engineered bacterial cell, and which may be integrated into the bacterial genome. In some embodiments, the engineered bacterial cell further expresses an I EE, wherein the I EE may be encoded by a gene that is integrated into the bacterial genome.

In any of the aforementioned embodiments for expressing a transgene in an ocular cell, step (a) may include administering the circular DNA vector intracamerally or into the corneal stroma. In some embodiments, step (b) includes positioning the one or more electrodes (e.g., one or more needle electrodes, e.g. monopolar needle electrodes) intracamerally, and step (c) comprises transmitting electrical energy through the one or more intracamerally positioned electrodes. In some embodiments, the intracameral position of the one or more electrodes is within 5 mm of the corneal endothelium.

In some of any of the aforementioned embodiments, the ocular cell of the anterior segment that expresses the circular DNA vector is corneal cell, trabecular meshwork cell, iris cell, lens cell, ciliary body cell, and/or Schlemm’s canal cell. In some embodiments, the ocular cell of the anterior segment is a corneal endothelial cell or a corneal stromal cell.

In some embodiments, the transgene encodes a protective factor that promotes corneal endothelial cell survival. In some embodiments, the protective factor modulates a nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway (e.g., an Nrf2 agonist), a Rho-kinase (ROCK) signaling pathway (e.g., a ROCK inhibitor), a transforming growth factor beta (TGF-B) signaling pathway (e.g., a TGF-B inhibitor), or a fibroblast growth factor 1 (FGF-1) signaling pathway. In some embodiments, the circular DNA vector silences, corrects, or replaces a mutated gene associated with Fuchs’ dystrophy, e.g., solute carrier family 4 member 11 (SLC4A11), TCF8, TCF, LOXHD1 , AGBL1 , DMPK, ZEB1 , or COL8A2.

In another aspect, the invention provides a method of expressing a therapeutic protein in an ocular cell of an anterior segment of an individual. In some embodiments, the method includes: (a) administering a nucleic acid vector to the anterior segment (e.g., the anterior chamber or corneal stroma) of the eye, wherein the nucleic acid vector encodes the therapeutic protein; (b) positioning one or more electrodes in and/or around the eye; and (c) transmitting electrical energy through the one or more electrodes at conditions suitable for electrotransfer of the nucleic acid vector into the ocular cell of the anterior segment, thereby expressing the therapeutic protein in the ocular cell of the anterior segment.

In some embodiments, the therapeutic protein is a protective factor that promotes corneal endothelial cell survival. In some embodiments, the protective factor modulates a Nrf2 signaling pathway, a ROCK signaling pathway, a TGF-B signaling pathway, or a FGF-1 signaling pathway. In some embodiments, the nucleic acid vector silences, corrects, or replaces a mutated gene associated with Fuchs’ dystrophy, e.g., SLC4A11 , TCF8, TCF, LOXHD1 , AGBL1 , DMPK, ZEB1 , or COL8A2.

In some embodiments, the individual has Fuchs’ dystrophy.

In another aspect, provided are methods of treating Fuchs’ dystrophy in an individual in need thereof, the method comprising: (a) administering a nucleic acid vector to the anterior segment (e.g., the anterior chamber or corneal stroma) of the eye in the individual, wherein the nucleic acid vector encodes a protective factor that promotes corneal endothelial cell survival; (b) positioning one or more electrodes in and/or around the eye; and (c) transmitting electrical energy through the one or more electrodes at conditions suitable for electrotransfer of the nucleic acid vector into the ocular cell of the anterior segment, thereby expressing the protective factor in the ocular cell of the anterior segment in an amount sufficient to treat Fuchs’ dystrophy.

In another aspect, provided are methods of treating Fuchs’ dystrophy in an individual in need thereof, the method comprising: (a) administering a nucleic acid vector to the anterior segment (e.g., the anterior chamber or corneal stroma) of the eye in the individual, wherein the nucleic acid vector silences, corrects, or replaces a mutated gene associated with Fuchs’ dystrophy; (b) positioning one or more electrodes in and/or around the eye; and (c) transmitting electrical energy through the one or more electrodes at conditions suitable for electrotransfer of the nucleic acid vector into the ocular cell of the anterior segment , thereby silencing, correcting, or replaces a mutated gene in an amount sufficient to treat Fuchs’ dystrophy.

In some embodiments, the nucleic acid vector lacks one or more components of a plasmid backbone. In some embodiments, the one or more components of the plasmid backbone lacking in the circular DNA vector comprises a drug resistance gene and/or an origin of replication. In some embodiments, the nucleic acid vector is a circular DNA vector, e.g., a nonviral circular DNA vector, e.g., a naked circular DNA vector. In some embodiments, the DNA vector is a synthetic circular DNA vector. In some embodiments, the DNA vector comprises an origin of replication and/or lacks a selectable marker. In some embodiments, the 3’ end of the transgene is linked to the 5’ end of a promoter of the transgene by a sequence comprising the bacterial origin of replication, wherein the sequence comprising the bacterial origin of replication is less than 100 bp in length. In some embodiments, the origin of replication is a ColE2-P9 origin of replication or functional variant thereof.

In some embodiments, the individual is a mammal, e.g., a human.

In another aspect, provided is a circular DNA vector (e.g., an isolated and/or engineered circular DNA vector) comprising: (a) a eukaryotic promoter; (b) a coding sequence, wherein the coding sequence: (i) encodes a protective factor that promotes corneal endothelial cell survival; or (ii) silences, corrects, or replaces a mutated gene associated with Fuchs’ dystrophy; and (c) a bacterial replication origin that is less than 50 bp in length, wherein the circular DNA vector lacks a selectable marker (e.g., a drug resistance gene). In some embodiments, the 3’ end of the coding sequence is linked to the 5’ end of a promoter by a sequence comprising the bacterial origin of replication, wherein the sequence comprising the bacterial origin of replication is less than 100 bp in length. In some embodiments, the origin of replication is a ColE2-P9 origin of replication or functional variant thereof (e.g., a truncated ColE2-P9 origin as described). In some embodiments, the protective factor modulates a Nrf2 signaling pathway, a ROCK signaling pathway, a TGF-B signaling pathway, or an FGF-1 signaling pathway. In some embodiments, the gene associated with Fuchs’ dystrophy is SLC4A11 , TCF8, TCF, LOXHD1 , AGBL1 , DMPK, ZEB1 , or COL8A2.

In another aspect, the invention provides a pharmaceutical composition comprising: (a) the circular DNA vector of any of the aforementioned embodiments of any of the preceding aspects; and (b) a suitable carrier for use in delivering the pharmaceutical composition to an individual.

In another aspect, provided are methods of delivering the circular DNA vector of any of the previous embodiments of any of the previous aspects to an ocular cell of the anterior segment of an individual (e.g., a corneal endothelial cell of an individual), the method comprising: (a) administering the circular DNA vector to the anterior segment of the eye (e.g., by intracameral injection or injection into the corneal stroma); (b) positioning one or more electrodes in and/or around the eye (e.g., one or more electrodes (e.g., needle electrodes) intracamerally); and (c) transmitting electrical energy (e.g., electrical pulses) through the one or more electrodes at conditions suitable for electrotransfer of the circular DNA vector into the ocular cell of the anterior segment, thereby delivering the circular DNA vector to the ocular cell of the anterior segment (e.g., corneal endothelial cell). In some embodiments, the circular DNA vector is expressed in the ocular cell of the anterior segment s (e.g., corneal endothelial cells) eight days following administration. In some embodiments, the transgene expressed by the circular DNA vector in the ocular cell of the anterior segment is more persistent than a transgene encoded by a plasmid DNA vector encoding the transgene. In some embodiments, the circular DNA vector in the ocular cell of the anterior segment is less immunogenic than a plasmid DNA vector encoding the transgene. In some embodiments, the one or more components of the plasmid backbone lacking in the circular DNA vector comprises a drug resistance gene and/or an origin of replication. In some embodiments, the individual is a mammal, e.g., a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A is a schematic drawing showing the relative positions of a DNA injection needle and an electrode needle in the anterior segment of the eye, as situated in Example 1. DNA was administered to the anterior chamber (intracamerally), and pulsed electric fields were transmitted through a single monopolar electrode in the anterior chamber (intracamerally).

FIG. 1 B is a fluorescent live image showing GFP expression in the rabbit eye on postoperative day 7 using a RetCam with gonioscopic lens.

FIG. 1C is a fluorescent image showing GFP expression in corneal endothelium (green). NaK ATPase is red, and DAPI is blue.

FIG. 2A is a schematic drawing showing the relative positions of a DNA injection needle and an electrode needle in the anterior segment of the eye, as situated in Example 2. DNA was administered to the corneal stroma, and pulsed electric fields were transmitted through a single monopolar electrode in the anterior chamber (intracamerally).

FIG. 2B is a fluorescent live image showing widespread GFP expression in the rabbit eye on postoperative day 7 using a RetCam with gonioscopic lens. FIG. 20 is a fluorescent image showing GFP expression in corneal endothelium (green). NaK ATPase is red, and DAPI is blue.

DETAILED DESCRIPTION

I. Definitions

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

As used herein, the terms “anterior segment” of the eye and “front of the eye” are used interchangeably to refer to the region of the eye containing the cornea, iris, ciliary body and lens as well as the spaces of the anterior and posterior chambers filled with aqueous humor.

As used herein, “electrotransfer” refers to movement of a molecule (e.g., a nucleic acid, e.g., a naked nucleic acid) across a membrane of a target cell (e.g., from outside to inside the target cell, e.g., a corneal endothelial cell) that is caused by transmission of an electric field (e.g., a pulsed electric field) to the microenvironment in which the cell resides (e.g., the anterior segment of the eye). Electrotransfer may occur as a result of electrophoresis, i.e., 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 current), based on a charge of the molecule. Electrophoresis can induce electrotransfer, for example, by moving a molecule (e.g., a nucleic acid, e.g., a naked nucleic acid) into proximity of a cell membrane to allow a biotransport process (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, electrotransfer may occur as a result of electroporation, i.e., generation of pores in the target cell caused by transmission of an electric field (e.g., a pulsed electric field), wherein the size, shape, and duration of the pores are suitable to accommodate movement of a molecule (e.g., a nucleic acid, e.g., a naked nucleic acid) from outside the target cell to inside the target cell. Thus, in some instances, electrotransfer occurs as a result of a combination of electrophoresis and electroporation.

The terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of a polynucleotide or an amino acid product or protein in a biological sample (e.g., retina). "Expression" generally refers to the process by which gene- encoded information is converted into the structures present and operating in the cell. Therefore, according to the invention, "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or post-translational modification of the protein. Fragments of the transcribed polynucleotide, the translated protein, or the post-translationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the protein, e.g., by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a protein, and also those that are transcribed into RNA but not translated into a protein (for example, transfer and ribosomal RNAs).

As used herein, “delivering,” “to deliver,” and grammatical variations thereof, refers to causing an agent (e.g., a therapeutic agent) to access a target cell. The agent can be delivered by administration of the agent to an individual having the target cell (e.g., systemically or locally administering the agent) such that the agent gains access to the organ or tissue in which the target cell resides. Additionally, or alternatively, the agent can be delivered by applying a stimulus to a tissue or organ harboring the agent, wherein the stimulus causes the agent to enter the target cell. Thus, in some instances, an agent is delivered to a target cell by transmitting an electric field into a tissue harboring the agent at conditions suitable for electrotransfer of the agent into a target cell within the tissue.

As used herein, "administering" refers to a method of giving a dosage of a therapeutic agent (e.g., a nucleic acid vector described herein) of the disclosure or a composition thereof to an individual. The compositions utilized in the methods described herein can be administered intraocularly, for example, intracamerally (i.e., into the aqueous humor), into the cornea (e.g., into the corneal stroma), intravitreally, subretinally, or periocularly. Additionally, or alternatively, the composition can be delivered intravenously, subcutaneously, intradermally, percutaneously, intramuscularly, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, peritoneally, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, orally, topically, transdermally, conjunctivally, subtenonly, intracamerally, subretinally, retrobulbarly, intracanalicularly, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. The compositions 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 being administered, and the severity of the condition, disease, or disorder being treated).

As used herein, the terms “vector” and “nucleic acid vector” are used interchangeably and refer to a nucleic acid molecule capable of delivering a therapeutic sequence to which is it linked into a target cell in which the therapeutic sequence can then be transcribed, replicated, processed, and/or expressed in the target cell. After a target cell or host cell processes the therapeutic sequence of the vector, the therapeutic sequence 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 into which additional DNA segments may be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector (e.g., adeno-associated viral (AAV) vector), wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “recombinant vectors” or “expression vectors”). Any of the nucleic acid vectors described herein may be referred to as “isolated nucleic acid vectors.”

As used herein, the term “circular DNA vector” refers to a DNA vector in a circular form. Such circular form is typically capable of being amplified into concatemers by rolling circle amplification. A linear double-stranded nucleic acid having conjoined strands at its termini (e.g., covalently conjugated backbones, e.g., by hairpin loops or other structures) is not a circular vector, as used herein. The term “circular DNA vector” is used interchangeably herein with the terms “covalently closed and circular DNA vector” and “C³DNA.” A skilled artisan will understand that such circular vectors include vectors that are covalently closed with supercoiling and complex DNA topology, as is described herein. In particular embodiments, a circular DNA vector is supercoiled (e.g., monomeric supercoiled). In other embodiments, a circular DNA vector is relaxed open circular (covalently closed without supercoiling). In certain instances, a circular DNA vector lacks a bacterial origin of replication. In some embodiments, a circular DNA vector comprises a bacterial origin of replication. In other instances, a circular DNA vector may lack one or more components of a plasmid backbone (e.g., a selectable marker) but may include an origin of replication (e.g., a ColE2-P9 origin of replication or truncated version thereof). 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 that corresponds to a portion of a first recombinase attachment site and a second sequence that corresponds to a portion of a second recombinase attachment site. One example of a hybrid recombination site is attR, which is a product of site-specific recombination and includes a first sequence that corresponds to a portion of attP and a second sequence that corresponds to a portion of attB. Alternatively, recombination sites can be generated from Cre/Lox recombination. Thus, a vector generated from Cre/Lox recombination (e.g., a vector including a LoxP site) includes a recombination site, as used herein. Other site-specific recombination events that generate recombination sites involve, e.g., lambda integrase, FLP recombinase, and Kw recombinase. Nucleic acid sequences that result from non-site-specific recombination events (e.g., ITR- mediated intermolecular recombination) are not recombination sites, as defined herein.

As used herein, the terms “individual” and “subject” are used interchangeably and include any mammal in need of treatment or prophylaxis, e.g., by a therapeutic circular DNA vector, or pharmaceutical composition thereof, described herein. In some embodiments, the individual or subject is a human. In other embodiments, the individual or subject is a non-human mammal (e.g., a non-human primate (e.g., a monkey), a mouse, a pig, a rabbit, a cat, or a dog). The individual or subject may be male or female.

As used herein, an “effective amount” or “effective dose” of a nucleic acid vector, or pharmaceutical composition thereof, refers to an amount sufficient to achieve a desired biological, pharmacological, or therapeutic effect, e.g., when administered to the individual according to a selected administration form, route, and/or schedule. As will be appreciated by those of ordinary skill in this art, the absolute amount of a particular composition that is effective can vary depending on such factors as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, etc. Those of ordinary skill in the art will further understand that an “effective amount” can be contacted with cells or administered to a subject in a single dose or through use of multiple doses. An effective amount of a composition to treat a disease may slow or stop disease progression or increase partial or complete response, relative to a reference population, e.g., an untreated or placebo population, or a population receiving the standard of care treatment.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, which can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and improved prognosis. In some embodiments, therapeutic circular DNA vectors of the invention are used to delay development of a disease or to slow the progression of a disease (e.g., reduction of corneal thickness or visual acuity).

As used herein, a “target cell” refers to a cell that expresses a therapeutic protein encoded by a therapeutic gene.

The terms “level of expression” or “expression level” are used interchangeably and generally refer to the amount of a polynucleotide or an amino acid product or protein in a biological sample (e.g., corneal endothelium). “Expression” generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, according to the invention, “expression” may refer to transcription into a polynucleotide, translation into a protein, or post- translational modification of the protein. Fragments of the transcribed polynucleotide, the translated protein, or the post-translationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post- translational processing of the protein, e.g., by proteolysis. “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a protein, and also those that are transcribed into RNA but not translated into a protein (for example, transfer and ribosomal RNAs).The terms “a” and “an” mean “one or more of.” For example, “a cell” is understood to represent one or more cells. As such, the terms “a” and “an,” “one or more of a (or an),” and “at least one of a (or an)” are used interchangeably herein.

As used herein, the term “about” refers to a value within ± 10% variability from the reference value, unless otherwise specified.

II. Methods

Provided are methods of administering nucleic acid vectors to the front of the eye using electrotransfer. Such methods include methods of expressing a transgene encoded by a nucleic acid vector and methods of treating disease (e.g., Fuchs’ dystrophy) by administering nucleic acid vectors that encode protective factors that promote corneal endothelial cell survival, or by administering nucleic acid vectors that silence, correct, or replace a mutated gene associated with Fuchs’ dystrophy. Fuchs’ Dystrophy

The methods provided herein are useful in treating diseases of the anterior segment of the eye, such as Fuchs’ dystrophy, which is characterized by gradual loss of vision due to progressive degeneration of the corneal endothelium. The present methods provide a treatment for Fuchs’ dystrophy by administering nucleic acid vectors that (a) encode protective factors that promote corneal endothelial cell survival or (b) silence, correct, or replace a mutated gene associated with Fuchs’ dystrophy, and transfecting corneal endothelial cells with these vectors by electrotransfer.

In some embodiments, methods of treating Fuchs’ dystrophy involve administering nucleic acid vectors that express protective factors known to promote corneal endothelial cell survival. Exemplary protective factors include modulators of Nrf2, ROCK, TGF-B, and FGF-1. In some embodiments, the protective factor modulates a nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway (e.g., an Nrf2 agonist), a Rho-kinase (ROCK) signaling pathway (e.g., a ROCK inhibitor), a transforming growth factor beta (TGF-B) signaling pathway (e.g., a TGF-B inhibitor), or a fibroblast growth factor 1 (FGF-1) signaling pathway. Thus, such protective factors can be cell-bound (e.g., intracellular or membrane- bound in the transfected cell) or secreted into the extracellular space (e.g., accessible to one or more additional cell types or extracellular components of the anterior segment of the eye).

In other embodiments, methods of treating Fuchs’ dystrophy involve administering nucleic acid vectors that silence (e.g., through inhibitory nucleic acid (e.g., shRNA)), correct (e.g., through gene editing, e.g., CRISPR), or replace (e.g., replacement with a functional version of the same or functionally similar or equivalent gene) a mutated gene associated with (i.e., known to cause or contribute to) Fuchs’ dystrophy. Genes associated with Fuchs’ dystrophy are known in the art and include, e.g., SLC4A11 , TCF8, TCF, LOXHD1 , AGBL1 , DMPK, ZEB1 , or COL8A2. Specific information on these mutations (e.g., nucleotide and amino acid changes) are identified and discussed in Liu et al., Eye Vis. 2021 :8(1):24, which is herein incorporated by reference in its entirety.

In some embodiments, the mutation in the gene is an autosomal dominant mutation.

Administration of Therapeutic Agents

Provided herein are methods of administering nucleic acid vectors (e.g., any of the nucleic acid vectors described herein), or pharmaceutical compositions thereof, to the front of the eye as a means to deliver a therapeutic agent into a target cell in the front of the eye of an individual (e.g., a human patient). In some instances, the nucleic acid vector is administered to the eye such that the nucleic acid vector enters the extracellular space of an anterior segment of the eye (e.g., the anterior chamber or the corneal stroma). Once the nucleic acid vector is in the anterior extracellular space upon administration, it can subsequently be electrotransferred into the target retinal cell upon transmission of electrical energy reaching into the anterior of the eye, e.g., though transmission of electrical energy from an electrode positioned in, on, or near the eye (e.g., within the anterior chamber).

In some embodiments, the nucleic acid vector is administered prior to transmitting an electrical field. For instance, a nucleic acid vector can be administered within 24 hours preceding transmission of an electric field (e.g., within 20 hours, within 18 hours, within 16 hours, within 14 hours, within 12 hours, within 10 hours, within 8 hours, within 6 hours, within 4 hours, within 3 hours, within 2 hours, within 90 minutes, within 60 minutes, within 45 minutes, within 30 minutes, within 20 minutes, within 15 minutes, within 10 minutes, within 5 minutes, within 4 minutes, within 3 minutes, within 2 minutes, within 1 minute, within 45 seconds, within 30 seconds, within 20 seconds, within 15 seconds, within 10 seconds, or within 5 seconds preceding transmission of an electric field). In some embodiments, the nucleic acid vector is administered as part of a method described herein.

Any suitable means of anterior ocular administration known in the art or described herein may be used as part of the methods provided herein. Methods of delivering a nucleic acid vector to a target retinal cell include administering the nucleic acid vector to the eye by intraocular injection (e.g., intracameral injection) or intraocular implant. In some embodiments of any of the methods described herein, the administration of the nucleic acid vector is via an intraocular implant (e.g., a controlled release or depot implant). In other embodiments, the administration of the nucleic acid vector is not via an intraocular implant.

In some instances, administration of the nucleic acid vector is non-surgical. For example, in some embodiments, administration of the nucleic acid vector does not utilize general anesthesia and/or does not involve retrobulbar anesthesia (i.e. , retrobulbar block). Additionally, or alternatively, administration of the nucleic acid vector does not involve injection using a needle larger than 28 gauge.

Additionally, or alternatively, administration of the nucleic acid vector does not involve use of a guidance mechanism that is typically required for ocular drug delivery via shunt or cannula. In some instances, administration of the nucleic acid vector is by injection (e.g., microneedle injection) into an outer tissue of the eye, e.g., the sclera, cornea, corneal stroma, conjunctiva, subconjunctival space, or subretinal space. Alternatively, administration of the nucleic acid vector is by injection (e.g., microneedle injection) into a site proximal to the outer tissue, such as the trabecular meshwork, ciliary body, or aqueous humor.

In some instances, administration of the nucleic acid vector is by topical administration or eyedrop.

Any of the nucleic acid vectors, or pharmaceutical compositions thereof, described herein can be administered to a subject in a dosage from 1 pg to 10 mg of DNA (e.g., from 5 pg to 5.0 mg, from 10 pg to 2.0 mg, or from 100 pg to 1.0 mg of DNA, e.g., from 10 pg to 20 pg, from 20 pg to 30 pg, from 30 pg to 40 pg, from 40 pg to 50 pg, from 50 pg to 75 pg, from 75 pg to 100 pg, from 100 pg to 200 pg, from 200 pg to 300 pg, from 300 pg to 400 pg, from 400 pg to 500 pg, from 500 pg to 1.0 mg, from 1.0 mg to 5.0 mg, or from 5.0 mg to 10 mg of DNA, e.g., about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 600 pg, about 700 pg, about 750 pg, about 1.0 mg, about 2.0 mg, about 2.5 mg, about 5.0 mg, about 7.5 mg, or about 10 mg of DNA).

Transmission of Electric Fields

Methods of delivering nucleic acid vectors (e.g., circular DNA vectors) to the front of the eye include transmitting electrical energy into the tissue in which the target ocular cell resides. Such methods involve electrotransfer of the therapeutic agent from the extracellular space in the anterior segment of the eye (e.g., the anterior chamber or corneal stroma) into the target ocular cell (e.g., corneal endothelium). For example, in some instances in which an individual is being treated for a disease of the anterior segment of the eye (e.g., Fuchs’ dystrophy), the method involves transmitting electrical energy into the retina to cause electrotransfer of nucleic acid vector from the extracellular space in or near the cornea into a corneal endothelial cell.

In some aspects of the present invention, an electrode is positioned within the interior of the individual’s eye (e.g., within the anterior segment, e.g., in the anterior chamber or corneal stroma), and an electric field is transmitted through the electrode into a target ocular tissue at conditions suitable for electrotransfer of the nucleic acid vector into the target cell (e.g., corneal endothelium). An electric field transmitted into a target ocular tissue can promote transfer of a nucleic acid vector (e.g., circular DNA vector) into a target ocular cell. Such electrotransfer can occur through any one of several mechanisms (and combinations thereof), including electrophoresis, electrokinetically driven drug uptake, and/or electroporation. Transmission of electric fields involve conditions suitable for such mechanisms. Suitable means of generating electric fields for electrotransfer of nucleic acids in mammalian tissue 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.

Various means of generating and transmitting an electric field into a tissue are contemplated herein as part of the present methods. Devices and systems having electrodes suitable for transmitting electric fields in mammalian tissues are commercially available and can be useful in the methods disclosed herein. In some instances, the electric field is transmitted through an electrode comprising a needle (e.g., a needle positioned within the vitreous humor or in the subretinal space). Suitable needle electrodes include CLINIPORATOR® electrodes marketed by IGEA® and needle electrodes marketed by AMBU®. Other electrodes that may be adapted for use in the front of the eye are described in International Patent Publication No. WO 2022/198138, which is herein incorporated by reference in its entirety.

Electrodes (e.g., needle electrodes) for use in the present methods may be monopolar. In some embodiments involving electrotransfer using a monopolar electrode, a ground electrode is attached to the individual (e.g., attached to the skin of an individual) at a point other than the eye. In some embodiments, the ground electrode is a pad contacting the skin of the buttocks, leg, torso, neck (e.g., the posterior of the neck), or head (e.g., the posterior or temple of the head) of the individual. In some embodiments, the monopolar electrode transmits electrical energy upon becoming positively charged. In some embodiments, the monopolar electrode transmits electrical energy upon becoming negatively charged.

Alternatively, electrodes may be bipolar. In a bipolar embodiment, an auxiliary electrode may be in electrical communication with the primary electrode. The auxiliary electrode may be proximal to the primary electrode (i.e., closer to the operator), e.g., part of, or connected to, a sheath housing a primary wire electrode. In some embodiments involving electrotransfer using a bipolar electrode, electrical energy (e.g., current) is transmitted upon application of a positive voltage to the primary electrode and a negative voltage to the auxiliary electrode. In some embodiments involving electrotransfer using a bipolar electrode, electrical energy (e.g., current) is transmitted upon application of a negative voltage to the primary electrode and a positive voltage to the auxiliary electrode. It will be appreciated that a variety of suitable electrical parameters and algorithms thereof may be used. The voltage source may be configured to generate an electric field strength, e.g., at a target cell (e.g., a corneal endothelial cell), from about 10 V/cm to about 1 ,500 V/cm (e.g., from 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., from about 100 V/cm to about 1 ,000 V/cm, e.g., about 200 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., from 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 is be configured to generate an electric field strength, e.g., at a target cell, from about 10 V/cm to about 1 ,000 V/cm (e.g., from about 10 V/cm to 500 V/cm or from about 500 V/cm to about 1 ,000 V/cm). In some embodiments, the field strength is from 50 V/cm to 300 V/cm. In some embodiments, the field strength is about 100 V/cm at the target cell (e.g., the target retinal cell).

In some embodiments, the total number of pulses of electrical energy are delivered within 1-60 seconds (e.g., within 1-5 seconds, 5-10 seconds, 10-15 seconds, 15-20 seconds, 20-30 seconds, 30-40 seconds, 40-50 seconds, or 50-60 seconds). In some embodiments, the total number of pulses of electrical energy are delivered within 1-20 seconds. For example, the total number of pulses of electrical energy may be delivered within 1-5 seconds, 5-10 seconds, 10-15 seconds, or 15-20 seconds, e.g., 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 may be, e.g., square waveforms. The pulses of electrical energy may have an amplitude from 5 V to 500 V. For example, the pulses of electrical energy may have an amplitude of about 5 V, 10 V, 15 V, 20 V, 25 V, 30 V, 35V, 40 V, 45 V, 50 V, 60 V, 70 V, 80 V, 90 V, 100 V, 125 V, 150 V, 175 V, 200 V, 225V, 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 pulses of electrical energy have an amplitude of about 5-250 V (e.g., about 20 V). Any of the aforementioned voltages can be the tops of square-waveforms, peaks in sinusoidal waveforms, peaks in sawtooth waveforms, root mean square (RMS) voltages of sinusoidal waveforms, or RMS voltages of sawtooth waveforms.

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 transmitted during use. In some embodiments, about 4-12 pulses of electrical energy are transmitted during use.

In some embodiments, each of the pulses of electrical energy is from about 10 ms to about 200 ms. For example, each of the pulses of electrical energy may 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 of the pulses of electrical energy is from about 50 ms. In some embodiments, each of the pulses of electrical energy is less than 10 ms. For example, each of the pulses of electrical energy may be from about 10 ps to about 10 ms, e.g., from about 10 ps to about 100 ps, e.g., about 20 ps, 30 ps, 40 ps, 50 ps, 60 ps, 70 ps, 80 ps, 90 ps, or 100 ps, e.g., from about 100 ps to about 1 ms, e.g., about 200 ps, 300 ps, 400 ps, 500 ps, 600 ps, 700 ps, 800 ps, 900 ps, 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.

As demonstrated in Examples 1 and 2, the relative positioning of electrodes and injection can determine the area of transfection and transgene expression, e.g., by corneal endothelial cells.

In one instance of the invention, any of the nucleic acid vectors described herein can be injected into the corneal stroma, and an intracamerally positioned electrode can transmit energy in the aqueous humor. This configuration can result in widespread expression within (e.g., and specific to) the corneal endothelium.

Alternatively, any of the nucleic acid vectors described herein can be injected intracamerally, and an intracamerally positioned electrode can transmit energy in the aqueous humor. This configuration can result in localized expression within (e.g., and specific to) the corneal endothelium, at a region near the electrode. In some instances, the method includes repositioning the electrode one or more times and repeating the transmission of energy to transfect two or more regions of the corneal endothelium.

III. Compositions

The present invention provides therapeutic compositions (e.g., nucleic acid vectors and pharmaceutical compositions thereof) useful for treatment of Fuchs’ dystrophy. In some instances, the present invention provides covalently closed circular DNA (C³DNA) vectors (e.g., C³DNA vectors that lack one or more components of a plasmid backbone) useful for treatment of Fuchs’ dystrophy.

Nucleic Acid Vectors

Provided herein are nucleic acid vectors that include any of the transgenes or coding sequences described herein. The nucleic acid vectors can be produced according to methods for production of plasmid DNA vectors, nanoplasmid vectors (as described in, e.g., WO 2008/153733 and WO 2014/035457), minicircle DNA vectors (as described in, e.g., U.S. Patent Nos. 8,828,726 and 9,233,174), mini-intronic plasmids (described in, e.g., Lu et al., Mol. Ther. 2013, 21:954 and U.S. Patent No. 9,347,073), synthetic circular DNA vectors as described herein and in WO 2019/178500, closed-ended DNA vectors (as described, e.g., in U.S. 2020/0283794 and U.S. 2021/0071197), doggybone DNA vectors (as described, e.g., in U.S. 2015/0329902 and U.S. Patent No. 9,499,847), or ministring DNA vectors (as described, e.g., in U.S. Patent Nos. 9,290,778 and USRE48908E1). In particular embodiments, any of the nucleic acid vectors described herein comprise a therapeutic sequence.

In some instances, the nucleic acid vectors are C³DNA vectors that persist intracellularly (e.g., in dividing or in quiescent cells, such as post-mitotic cells) as episomes, e.g., in a manner similar to AAV vectors. In any of the embodiments described herein, a circular DNA vector maybe a non-integrating vector. C³DNA vectors provided herein can be naked DNA vectors, devoid of components inherent to viral vectors (e.g., viral proteins) and substantial components of bacterial plasmid DNA, such as immunogenic components (e.g., immunogenic bacterial signatures (such as CpG islands or CpG motifs) or components additionally, or otherwise associated with reduced persistence (e.g., CpG islands or CpG motifs). Circular DNA vectors feature one or more therapeutic sequences and may lack plasmid backbone elements, such as (i) a bacterial origin of replication and/or (ii) a drug resistance gene and/or (iii) a recombination site. Synthetic circular DNA vectors lacking an origin or replication can be synthesized through various means known in the art and described herein. Synthesis methods may involve use of phage polymerase, such as Phi29 polymerase, as a replication tool using, e.g., rolling circle amplification. Particular methods of cell-free synthesis of synthetic circular DNA vectors are further described in, for example, WO 2019/178500, which is hereby incorporated by reference.

In other embodiments, therapeutic circular DNA vectors described herein can be nonsynthetic vectors (e.g., containing bacterial backbone sequences, such as origin of replication and/or recombination sites).

Such nucleic acid vectors described herein (e.g., circular DNA vector lacking a plasmid backbone element) can be in vivo (e.g., bacterially)-produced , and may lack a selectable marker (e.g., drug resistance gene) and optionally a recombination site or transposase scar, e.g., by using engineered bacterial cells to produce circular DNA vectors from a parental plasmid. Such bacterially produced circular DNA (e.g., C³DNA) vectors lacking a selectable marker can include any of the features described in International Patent Application No. PCT/US2022/082078, which is herein incorporated by reference in its entirety. Bacterial cells (e.g., E. coli) can be engineered to contain a Rep gene encoding a bacterial replication protein, which is optionally integrated into the bacterial genome. The engineered cells can be transfected with a parental plasmid having a vector sequence and a backbone sequence. The vector sequence includes an on sequence (e.g., a ColE2-P9 replication origin) corresponding to the Rep gene and does not include a selectable marker. The backbone sequence includes a selectable marker and does not include the ori sequence included in the vector sequence. The parental plasmid may also have restriction enzyme recognition sequences, or site-specific recombination, or transposase recognition sequences flanking the vector sequence arranged so that the plasmid backbone sequence can be separated from the vector sequence inside the cell by restriction digestion, site-specific recombination, or transposase action. In the case of restriction digestion, the circular DNA vector is then formed by self-ligation of the vector sequence. In the case of site-specific recombination or transposase action, the circular DNA vector is formed as recombination or transposase action is completed. Expression of the rep protein after separation of the vector sequence and formation of the circular DNA vector can maintain the circular DNA vector at a high copy number, despite the circular DNA vector lacking a selectable marker. In contrast, maintenance of the plasmid backbone sequence in the engineered bacterial cell after separation can be avoided by changing the culture conditions to remove selective pressure for the selectable marker. Culturing of a population of bacterial cells with a high copy number of circular DNA vector under conditions in which the parental plasmid is not maintained can efficiently produce a high yield of highly pure C³DNA vector with a backbone of less than 100 bp (and an origin of less than 50 bp). Such methods are described in WO 2023/122625 and U.S. 63/509,458 (filed June 21 , 2023), which are hereby incorporated by reference in their entireties.

One benefit of using a transposase-based system is the ability to further reduce the backbone size within the C³DNA DNA vector. For instance, use of a site-specific recombinase results in a recombination site (e.g., an attachment site) within the vector, near or adjacent to the replication origin. In contrast, use of a transposase allows the replication origin to directly connect the 5’ end of the therapeutic sequence to 3’ end of the therapeutic sequence without intervening sequences. In some instances, use of a transposase allows for a “scarless” backbone by positioning the resulting sequence of the transposition (the transposase overhang) within the therapeutic sequence without modifying the function of the therapeutic sequence. As an example, piggybac transposase produces a four-bp transposase overhang of TTAA. By positioning the plasmid backbone within the sequence of interest at a TTAA site, one can design the system such that, upon transposase-mediated excision of the plasmid backbone from the sequence of interest, the original sequence of interest is restored, leaving only the original TTAA sequence as the transposase scar. This leaves the backbone within the C³DNA DNA vector free of a transposase scar. Thus, the plasmid backbone sequences in the vector can consist entirely of replication origin.

Additionally, or alternatively, the transposase scar may be positioned within the vector backbone (e.g., within the sequence containing the replication origin). For instance, if the parental plasmid contains inverted repeats (left-end) and (right-end) flanking the backbone, and or transposase overhang sequences flanking the therapeutic sequence, the transposase scar will be positioned between the 3’ and 5’ ends of the sequence of interest (e.g., next to the origin of replication).

In some embodiments, the engineered bacterial cells for producing the C³DNA DNA vector of this disclosure include a Rep gene encoding a bacterial replication protein directing replication from ColE2-P9 origin, and which may be integrated into the bacterial genome. Alternatively, the Rep gene is included on an extrachromosomal DNA molecule such as, for example, a plasmid or a bacterial artificial chromosome (“BAC”). The engineered bacterial cells further comprise a parental plasmid comprising a vector sequence and a backbone sequence. The vector sequence includes a replication origin (ori) sequence corresponding to the Rep gene and does not include a selectable marker. The backbone sequence includes a gene encoding a selectable marker and does not include the ori sequence included in the vector sequence. The parental plasmid also has enzyme recognition sequences (e.g., restriction enzyme recognition sequences, site-specific recombination sequences, or transposase recognition sequences) flanking the vector sequence arranged so that the plasmid backbone sequence can be separated from the vector sequence inside the cell by restriction digestion, transposition, or sitespecific recombination.

In some embodiments, a short origin of replication is used in the C³DNA DNA vector to minimize bacterial sequences, such as a ColE2-P9 replication origin, or a functional variant thereof. In such embodiments, the Rep gene encodes a ColE2-P9 replication protein. In some exemplary embodiments, the Rep gene encodes a ColE2-P9 replication protein that has the amino acid sequence set forth in SEQ ID NO: 1 (or a functional variant thereof, for example, having at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity thereto). Other suitable replication proteins include replication proteins encoded by naturally-occurring plasmids, including, for example, those that are related to ColE2-P9, such as ColE3-CA38. In some exemplary embodiments, the ori (e.g., one strand) comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 2. In some embodiments, the ori sequence is a functional fragment of the ColE2-P9 ori sequence that has the DNA sequence (on one strand) set forth in SEQ ID NO: 2. The 40 base pair functional fragment set forth in SEQ ID NO: 2 is capable of supporting vector replication in a cell expressing the ColE2-P9 replication protein. In some embodiments, the ori is ColE2-P9 origin and is no more than about 40 nucleotides in length, or no more than 38 nucleotides in length, no more than 37 nucleotides in length, or no more than 36 nucleotides in length, or no more than 34 nucleotides in length, or no more than 30 nucleotides in length. In various embodiments, the ColE2-P9 origin is from 20 to 40 nucleotides in length, or from 30 to 40 nucleotides in length, or from 34 to 40 nucleotides in length, thereby minimizing bacterial-derived sequences in the C³DNA vector. In some embodiments, the ori sequence is a naturally occurring ori sequence.

In some instances, the ori sequence is a functional variant of a naturally occurring ori, such as, for example, an ori sequence that has been modified to be shorter than a corresponding naturally occurring ori sequence, while still retaining the ability to support replication initiation. Such functional variants of the ColE2-P9 replication origin include SEQ ID NOs: 3-11. Such sequences are shown herein as a single strand for convenience, although it is recognized that the origin will be present in the vector as double-stranded DNA. In some embodiments, the functional variant has 1 , 2, 3, 4, or 5 nucleotide substitutions with respect to an origin sequence of SEQ ID NOS: 3-11.

In some instances, C³DNA vectors provided herein are naked DNA vectors and are devoid of components inherent to viral vectors (e.g., viral proteins) and substantial components 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). For example, in some embodiments, the C³DNA vector contains 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 99%, 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 99%, or essentially all) of the DNA lacks CpG methylation. In some embodiments, the C³DNA DNA vector contains 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 99%, or essentially all) of the DNA lacks bacterial methylation signatures, such as Dam methylation and Dem methylation. For examples, in some embodiments, the C³DNA DNA vector contains 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 99%, or essentially all) of the GATC sequences are unmethylated (e.g., by Dam methylase). Additionally, or alternatively, the C³DNA DNA vector contains 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 99%, or essentially all) of the CCAGG sequences and/or CCTGG sequences are unmethylated (e.g., by Dem methylase).

In some embodiments, the C³DNA vector lacking one or more components of a plasmid backbone is persistent in vivo (e.g., the C³DNA vector lacking one or more components of a plasmid backbone exhibits improved expression persistence (e.g., intra-cellular persistence and/or trans-generational persistence) and/or therapeutic persistence relative to a reference vector, e.g., a circular DNA vector having a plasmid backbone).

In some embodiments, expression of a C³DNA vector lacking one or more components of a plasmid backbone persists for at least two weeks, at least three weeks, at least four weeks, at least six weeks, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year, or longer after administration.

In some embodiments, the C³DNA vector lacking one or more components of a plasmid backbone persists for at least about six months or at least one year, or at least 18 months, or years in targeted ocular cells. In some embodiments, the expression level of the C³DNA vector lacking one or more components of a plasmid backbone does not decrease by more than 90%, or by more than 50%, or by more than 25%, or by more than 10% in the 1 week or more, e.g., 2 weeks, 3 weeks, 5 weeks, 7 weeks, 9 weeks or more, 13 weeks or more, 18 weeks or more following transfection from levels observed within the first 1 , 2, or 3 days. In some embodiments, administration of the nucleic acid vector of this disclosure (e.g., to retinal cells) is no more than 4 times per year, or no more than 2 times per year, or no more than once per year, or even less frequently (e.g., once every two years).

In embodiments, the C³DNA vector is monomeric. In some embodiments, the C³DNA vector is supercoiled, e.g., following treatment with a topoisomerase (e.g., gyrase). In some embodiments, the C³DNA vector is a monomeric, supercoiled circular DNA molecule. In some embodiments, the C³DNA vector is nicked. In some embodiments, the C³DNA vector is open circular (relaxed open circular). In some embodiments, the C³DNA vector is double-stranded circular.

Therapeutic C³DNA DNA vectors described herein contain a therapeutic sequence, which may include one or more protein-coding domains and/or one or more non-protein coding domains. Therapeutic sequences can include any of the expression constructs disclosed herein.

In embodiments of C³DNA DNA vectors involving a non-protein coding therapeutic sequence, the therapeutic sequence lacks a protein-coding domain (e.g., a therapeutic proteincoding domain). For instance, in some embodiments, a therapeutic sequence includes a non- protein-coding therapeutic nucleic acid, such as a short hairpin RNA (shRNA)-encoding sequence or an immune activating therapeutic nucleic acid (e.g., a TLR agonist).

In some embodiments involving C³DNA DNA vectors, the therapeutic sequence is from 0.1 Kb to 100 Kb in length (e.g., the therapeutic gene sequence is from 0.2 Kb to 90 Kb, from 0.5 Kb to 80 Kb, from 1 .0 Kb to 70 Kb, from 1.5 Kb to 60 Kb, from 2.0 Kb to 50 Kb, from 2.5 Kb to 45 Kb, from 3.0 Kb to 40 Kb, from 3.5 Kb to 35 Kb, from 4.0 Kb to 30 Kb, from 4.5 Kb to 25 Kb, from 4.6 Kb to 24 Kb, from 4.7 Kb to 23 Kb, from 4.8 Kb to 22 Kb, from 4.9 Kb to 21 Kb, from 5.0 Kb to 20 Kb, from 5.5 Kb to 18 Kb, from 6.0 Kb to 17 Kb, from 6.5 Kb to 16 Kb, from 7.0 Kb to 15 Kb, from 7.5 Kb to 14 Kb, from 8.0 Kb to 13 Kb, from 8.5 Kb to 12.5 Kb, from 9.0 Kb to 12.0 Kb, from 9.5 Kb to 11.5 Kb, or from 10.0 Kb to 11.0 Kb in length, e.g., from 0.1 Kb to 0.5 Kb, from 0.5 Kb to 1.0 Kb, from 1.0 Kb to 2.5 Kb, from 2.5 Kb to 4.5 Kb, from 4.5 Kb to 8 Kb, from 8 Kb to 10 Kb, from 10 Kb to 15 Kb, from 15 Kb to 20 Kb in length, or greater, e.g., from 0.1 Kb to 0.25 Kb, from 0.25 Kb to 0.5 Kb, from 0.5 Kb to 1.0 Kb, from 1.0 Kb to 1.5 Kb, from 1.5 Kb to 2.0 Kb, from

2.0 Kb to 2.5 Kb, from 2.5 Kb to 3.0 Kb, from 3.0 Kb to 3.5 Kb, from 3.5 Kb to 4.0 Kb, from 4.0

Kb to 4.5 Kb, from 4.5 Kb to 5.0 Kb, from 5.0 Kb to 5.5 Kb, from 5.5 Kb to 6.0 Kb, from 6.0 Kb to 6.5 Kb, from 6.5 Kb to 7.0 Kb, from 7.0 Kb to 7.5 Kb, from 7.5 Kb to 8.0 Kb, from 8.0 Kb to 8.5

Kb, from 8.5 Kb to 9.0 Kb, from 9.0 Kb to 9.5 Kb, from 9.5 Kb to 10 Kb, from 10 Kb to 10.5 Kb, from 10.5 Kb to 11 Kb, from 11 Kb to 11.5 Kb, from 11.5 Kb to 12 Kb, from 12 Kb to 12.5 Kb, from 12.5 Kb to 13 Kb, from 13 Kb to 13.5 Kb, from 13.5 Kb to 14 Kb, from 14 Kb to 14.5 Kb, from 14.5 Kb to 15 Kb, from 15 Kb to 15.5 Kb, from 15.5 Kb to 16 Kb, from 16 Kb to 16.5 Kb, from 16.5 Kb to 17 Kb, from 17 Kb to 17.5 Kb, from 17.5 Kb to 18 Kb, from 18 Kb to 18.5 Kb, from 18.5 Kb to 19 Kb, from 19 Kb to 19.5 Kb, from 19.5 Kb to 20 Kb, from 20 Kb to 21 Kb, from 21 Kb to 22 Kb, from 22 Kb to 23 Kb, from 23 Kb to 24 Kb, from 24 Kb to 25 Kb in length, or greater, e.g., about 4.5 Kb, about 5.0 Kb, about 5.5 Kb, about 6.0 Kb, about 6.5 Kb, about 7.0 Kb, about 7.5 Kb, about 8.0 Kb, about 8.5 Kb, about 9.0 Kb, about 9.5 Kb, about 10 Kb, about 11 Kb, about 12 Kb, about 13 Kb, about 14 Kb, about 15 Kb, about 16 Kb, about 17 Kb, about 18 Kb, about 19 Kb, about 20 Kb in length, or greater). In some embodiments, the therapeutic sequence is at least 10 Kb (e.g., from 10 Kb to 15 Kb, from 15 Kb to 20 Kb, or from 20 Kb to 30 Kb; e.g., from 10 Kb to 13 Kb, from 10 Kb to 12 Kb, or from 10 Kb to 11 Kb; e.g., from 10-11 Kb, from 11-12 Kb, from 12-13 Kb, from 13-14 Kb, or from 14-15 Kb). In some embodiments, the therapeutic sequence is at least 1 ,100 bp in length (e.g., from 1,100 bp to 10,000 bp, from 1 ,100 bp to 8,000 bp, or from 1 ,100 bp to 5,000 bp in length). In some embodiments, the therapeutic sequence is at least 2,500 bp in length (e.g., from 2,500 bp to 15,000 bp, from 2,500 bp to 10,000 bp, or from 2,500 bp to 5,000 bp in length; e.g., from 2,500 bp to 5,000 bp, from 5,000 bp to 7,500 bp, from 7,500 bp to 10,000 bp, from 10,000 bp to 12,500 bp, or from 12,500 bp to 15,000 bp). In some embodiments, the therapeutic sequence is at least 8,000 bp, at least 9,000 bp, at least 10,000 bp, at least 11 ,000 bp, at least 12,000 bp at least 13,000 bp, at least 14,000 bp, at least 15,000 bp, at least 16,000 bp (e.g., 11 ,000 bp to 16,000 bp, 12,000 bp to 16,000 bp, 13,000 bp to 16,000 bp, 14,000 bp to 16,000 bp, or 15,000 bp to 16,000 bp). In particular embodiments, the therapeutic sequence is sufficiently large to encode a protein and is not an oligonucleotide therapy (e.g., not an antisense, siRNA, shRNA therapy, etc.).

In some embodiments, a nucleic acid vector includes a reporter sequence in addition to a therapeutic protein-encoding domain or a therapeutic non-protein encoding domain. Such reporter genes can be useful in verifying therapeutic gene sequence expression, for example, in specific cells and tissues. Reporter sequences that may be provided in a transgene include, without limitation, DNA sequences encoding p-lactamase, p-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. When associated with regulatory elements which drive their expression, the reporter sequences provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for [3-galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer. In some embodiments, the therapeutic sequence lacks a reporter sequence.

In some instances, the nucleic acid vector is a non-viral DNA vector (e.g., the DNA vector is not encapsulated within 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 a matrix (e.g., a polymer matrix) and is not physically associated with (e.g., covalently or non- covalently bound to) a solid structure (e.g., a particulate structure) prior to and upon administration to the individual. In some embodiments, the nucleic acid vector is untethered to any adjacent nucleic acid vectors 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 liquid solution, such as a charge-altering molecule or a stabilizing molecule.

The nucleic acid vector may be a naked DNA vector, i.e., not complexed with another agent (e.g., encapsulated within, conjugated to, or non-covalently bound to another agent). Naked DNA vectors may be co-formulated (e.g., in solution) with agents that are not complexed with the naked DNA vector, such as buffering agents and/or agents that are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration.

Pharmaceutical Compositions

The invention also provides methods involving administration of pharmaceutical compositions having a therapeutic agent (e.g., any of the nucleic acid vectors (e.g., circular DNA vectors) described herein) in a pharmaceutically acceptable carrier. In some instances, the pharmaceutical composition contains a non-viral nucleic acid vector (e.g., the pharmaceutical composition is substantially devoid of viral capsid). Additionally, or alternatively, the pharmaceutical composition may contain a nucleic acid vector that is not encapsulated in an envelope (e.g., a lipid envelope) or a matrix (e.g., a polymer matrix) and is not physically associated with (e.g., covalently or non-covalently bound to) a solid structure (e.g., a particulate structure) prior to and upon administration to the individual. In some embodiments of the pharmaceutical composition, the nucleic acid vector is untethered to any adjacent nucleic acid vectors 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 of the pharmaceutical composition, the nucleic acid vector is associated with another agent in liquid solution, such as a charge-altering molecule or a stabilizing molecule.

The pharmaceutical composition may contain the nucleic acid vector in naked form, i.e., the nucleic acid vector is not complexed with another agent (e.g., encapsulated within, conjugated to, or non-covalently bound to another agent). In such pharmaceutical compositions, naked nucleic acid molecules may be co-formulated (e.g., in solution) with agents that are not complexed with the naked nucleic acid molecule, such as buffering agents and/or agents that are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration.

In some instances of the present invention, a pharmaceutical composition includes a naked circular DNA vector.

Pharmaceutically acceptable carriers may include excipients and/or stabilizers that are nontoxic to the individual at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; 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 dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as tween, polyethylene glycol (PEG), and pluronics.

A pharmaceutical composition having a therapeutic agent of the invention (e.g., a nucleic acid vector, such as a circular DNA vector) may contain a pharmaceutically acceptable carrier. If the composition is provided in liquid form, the carrier may be water (e.g., pyrogen-free water), isotonic saline, or a buffered aqueous solution, e.g., a phosphate buffered solution or a citrate buffered solution. Injection of the pharmaceutical composition may be carried out in water or a buffer, such as an aqueous buffer, e.g., containing a sodium salt (e.g., at least 50 mM of a sodium salt), a calcium salt (e.g., at least 0.01 mM of a calcium salt), or a potassium salt (e.g., at least 3 mM of a potassium salt). According to a particular embodiment, the sodium, calcium, or potassium salt may occur in the form of their halogenides, e.g., chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Without being limited thereto, examples of sodium salts include NaCI, Nal, NaBr, Na2CO2, NaHCO2, and Na2SO4. Examples of potassium salts include, e.g., KOI, KI, KBr, K2CO2, KHCO2, and K2SO4. Examples of calcium salts include, e.g., CaCI2, Cal2, CaBr2, CaCO2, CaSO4, and Ca(OH)2. Additionally, organic anions of the aforementioned cations may be contained in the buffer. According to a particular embodiment, the buffer suitable for injection purposes as defined above, may contain salts selected from sodium chloride (NaCI), calcium chloride (CaCI2) or potassium chloride (KCI), wherein further anions may be present. CaCI2 can also be replaced by another salt, such as KCI. In some embodiments, salts in the injection buffer are present in a concentration of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium chloride (KCI), and at least 0.01 mM calcium chloride (CaCI2). The injection buffer may be hypertonic, isotonic, or hypotonic with reference to the specific reference medium, i.e., the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects. Reference media are can be liquids such as blood, lymph, cytosolic liquids, other body liquids, or common buffers. Such common buffers or liquids are known to a skilled person. Ringer- Lactate solution is particularly preferred as a liquid basis.

One or more compatible solid or liquid fillers, diluents, or encapsulating compounds may be suitable for administration to a person. The constituents of the pharmaceutical composition according to the invention are capable of being mixed with the nucleic acid vector according to the invention as defined herein, in such a manner that no interaction occurs, which would substantially reduce the pharmaceutical effectiveness of the (pharmaceutical) composition according to the invention under typical use conditions. Pharmaceutically acceptable carriers, fillers and diluents can have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to an individual being treated. Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers, or constituents 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 groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; or alginic acid.

The choice of a pharmaceutically acceptable carrier can be determined, according to the manner in which the pharmaceutical composition is administered.

Suitable unit dose forms for injection include sterile solutions of water, physiological saline, and mixtures thereof. The pH of such solutions may be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid, and collagen matrices.

The pharmaceutical composition according to the present invention may be provided in liquid or in dry (e.g., lyophilized) form. In a particular embodiment, the nucleic acid vector of the pharmaceutical composition is provided in lyophilized form. Lyophilized compositions including nucleic acid vector of the invention may be reconstituted in a suitable buffer, advantageously based on an aqueous carrier, prior to administration, e.g., Ringer-Lactate solution, Ringer solution, or a phosphate buffer solution.

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, e.g., cationic or polycationic polymers, cationic or polycationic peptides or proteins, e.g., protamine, cationic or polycationic polysaccharides, and/or cationic or polycationic lipids.

According to a particular embodiment, the nucleic acid vector of the invention may be complexed with lipids to form one or more liposomes, lipoplexes, or lipid nanoparticles. Therefore, in one embodiment, the inventive composition comprises liposomes, lipoplexes, and/or lipid nanoparticles comprising a therapeutic agent (e.g., a nucleic acid vector, e.g., a circular DNA vector).

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

Conventional liposomes include of a lipid bilayer that can be composed of cationic, anionic, or neutral phospholipids and cholesterol, which encloses an aqueous core. Both the lipid bilayer and the aqueous space can incorporate hydrophobic or hydrophilic compounds, respectively. Liposome characteristics and behavior in-vivo can be modified by addition of a hydrophilic polymer coating, e.g., polyethylene glycol (PEG), to the liposome surface to confer steric stabilization. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains.

Liposomes are colloidal lipid-based and surfactant-based delivery systems composed of a phospholipid bilayer surrounding an aqueous compartment. They may present as spherical vesicles and can range in size from 20 nm to a few microns. Cationic lipid-based liposomes are able to complex with negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications. 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.

Cationic liposomes can serve as delivery systems RNAs. Cationic lipids, such as MAP, (1 ,2- dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethyl-ammonium methyl sulfate) can form complexes or lipoplexes with negatively charged nucleic acids to form nanoparticles by electrostatic interaction, providing high in vitro transfection efficiency. Furthermore, neutral lipid-based nanoliposomes for nucleic acid vector delivery as e.g., neutral 1 ,2-dioleoyl-sn-glycero- 3-phosphatidylcholine (DOPC)-based nanoliposomes are available.

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

In a particular embodiment, a pharmaceutical composition according to the invention comprises the nucleic acid vector of the invention that is formulated together with a cationic or polycationic compound and/or with a polymeric carrier. Accordingly, in a further embodiment of the invention, the nucleic acid vector as defined herein is associated with or complexed with a cationic or polycationic compound or a polymeric carrier, optionally in a weight ratio selected from a range of about 5:1 (w/w) to about 0.25:1 (w/w), e.g., from about 5:1 (w/w) to about 0.5:1 (w/w), e.g., from about 4:1 (w/w) to about 1 : 1 (w/w) or of about 3: 1 (w/w) to about 1 : 1 (w/w), e.g., from about 3:1 (w/w) to about 2:1 (w/w) of nucleic acid vector to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a nitrogen/phosphate (N/P) ratio of nucleic acid vector to cationic or polycationic compound and/or polymeric carrier in the range of about 0.1-10, e.g., in a range of about 0.3-4 or 0.3-1, e.g., in a range of about 0.5-1 or 0.7-1 , e.g., in a range of about 0.3-0.9 or 0.5-0.9. For example, the N/P ratio of the nucleic acid vector to the one or more polycations is in the range of about 0.1 to 10, including a range of about 0.3 to 4, of about 0.5 to 2, of about 0.7 to 2 and of about 0.7 to 1.5.

The nucleic acid vectors described herein can also be associated with a vehicle, transfection or complexation agent for increasing the transfection efficiency and/or the expression of the modulatory gene according to the invention.

In some instances, the pharmaceutical composition contains a nucleic acid vector complexed with one or more polycations (e.g., protamine or oligofectamine). Further cationic or polycationic compounds that can be used as transfection or complexation agent may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N- trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPE, LEAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, MAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: 0,0- ditetradecanoyl-N-(a-trimethylammonioacetyl)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 polyaminoacids, such as [3-aminoacid-polymers or reversed polyamides, etc., modified polyethylenes, such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc., modified amidoamines such as pAMAM (poly(amidoamine)), etc., modified polybetaaminoester (PBAE), such as diamine end modified 1 ,4 butanediol diacrylate- co-5-amino-1-pentanol polymers, etc., dendrimers, such as polypropylamine dendrimers or pAMAM based dendrimers, etc., polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based polymers, chitosan, etc., silan backbone based polymers, such as PMOXA-PDMS copolymers, etc., block polymers consisting of a combination of one or more cationic blocks (e.g. selected from a cationic polymer as mentioned above) and of one or more hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.

According to a particular embodiment, the pharmaceutical composition of the invention includes the therapeutic agent, e.g., nucleic acid vector (e.g., circular DNA vector) encapsulated within or attached to a polymeric carrier. A polymeric carrier used according to the invention might be a polymeric carrier formed by disulfide-crosslinked cationic components. The disulfide- crosslinked cationic components may be the same or different from each other. The polymeric carrier can also contain further components. It is also particularly preferred that the polymeric carrier used according to the present invention comprises mixtures of cationic peptides, proteins or polymers and optionally further components as defined herein, which are crosslinked by disulfide bonds as described herein. In this context, the disclosure of WO 2012/013326 is incorporated herewith by reference. In this context, the cationic components that form basis for the polymeric carrier by disulfide-crosslinkage are typically selected from any suitable cationic or polycationic peptide, protein or polymer suitable for this purpose, particular any cationic or polycationic peptide, protein or polymer capable of complexing the nucleic acid vector as defined herein or a further nucleic acid comprised in the composition, and thereby preferably condensing the nucleic acid vector. The cationic or polycationic peptide, protein or polymer, may be a linear molecule; however, branched cationic or polycationic peptides, proteins or polymers may also be used.

Every disulfide-crosslinking cationic or polycationic protein, peptide or polymer of the polymeric carrier, which may be used to complex the nucleic acid vector according to the invention included as part of the pharmaceutical composition of the 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 linkage upon condensation with at least one further cationic or polycationic protein, peptide or polymer as cationic component of the polymeric carrier as mentioned herein.

Such polymeric carriers used to complex the nucleic acid vectors of the present invention may be formed by disulfide-crosslinked cationic (or polycationic) components. In particular, such cationic or polycationic peptides or proteins or polymers of the polymeric carrier, which comprise or are additionally modified to comprise at least one SH moiety, can be selected from proteins, peptides, and polymers as a complexation agent.

In other embodiments, the pharmaceutical composition according to the invention may be administered naked without being associated with any further vehicle, transfection, or complexation agent.

IV. Kits and Articles of Manufacture

In another aspect of the invention, an article of manufacture or a kit containing materials useful for the treatments described above is provided. 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, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a therapeutic agent of the invention (e.g., nucleic acid vector (e.g., a non-viral DNA vector, e.g., a circular DNA vector that lacks a bacterial original of replication, a drug resistance gene, and/or a recombination site)) or a pharmaceutical composition comprising the therapeutic agent of the invention. The label or package insert indicates that the composition is used for treating the disease or disorder of choice. The article of manufacture may further comprise a package insert indicating that the compositions can be used to treat a particular condition (e.g., Fuchs’ dystrophy). Alternatively, or additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable carrier, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, dextrose solution, or any of the pharmaceutically 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.

In particular instances of the invention, provided is a kit that includes (i) any one or more of the materials described above (e.g., any of the aforementioned therapeutic agents of the invention and/or one or more pharmaceutically acceptable carriers) and (ii) one or more elements of an energy delivery device (e.g., a device including an electrode for transmitting an electric field to a tissue (e.g., retina), such as any suitable devices or systems described above). In some embodiments, provided herein is a kit that includes 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)) and an electrode. In some embodiments, provided herein is a kit that includes a pharmaceutical composition comprising 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)) and an electrode.

EXAMPLES

Example 1 : GFP expression in the corneal endothelium by electrotransfer of C³DNA

Dutch Belted rabbits were anesthetized, and their eyes were prepared in a sterile fashion. Approximately 100 microliters of C3DNA containing the reporter gene GFP was injected into the anterior chamber of the rabbit eye. Pulsed electric field-mediated electrotransfer was conducted immediately following the C³DNA injection. A needle electrode was placed within the anterior chamber in close proximity to posterior surface of the cornea. FIG. 1 A shows the relative positions of the injection needle and electrode.

Eight -50V pulses of 5 ms duration were applied. Expression of GFP in the cornea was visualized in live animals on postoperative day 7 using a RetCam with gonioscopic lens and fluorescence filter (FIG. 1B). Rabbits were then sacrificed, and they eyes were enucleated and processed for histologic analysis. Localization of GFP within the corneal endothelium was confirmed by immunofluorescence microscopy using anti-GFP antibodies (FIG. 1C).

Example 2: GFP expression in the corneal stroma by electrotransfer of C³DNA Dutch Belted rabbits were anesthetized, and their eyes were prepared in a sterile fashion. Approximately 50 microliters of C³DNA containing the reporter gene GFP was injected the corneal stroma of the rabbit eye. Delivery of fluid intro the corneal stroma was evidenced by whitening of the cornea. Pulsed electric field-mediated electrotransfer was performed immediately following the C³DNA injection. The COMET electrode was placed within the anterior chamber in close proximity to posterior surface of the cornea. FIG. 2A shows the relative positions of the injection needle and electrode.

Eight -20V pulses of 20 ms duration were applied. Expression of GFP in the cornea was visualized in live animals on postoperative day 7 using a RetCam with gonioscopic lens and fluorescence filter. Surprisingly, widespread GFP expression was observed across a large portion of the corneal surface (FIG. 2B). Rabbits were then sacrificed, and they eyes were enucleated and processed for histologic analysis. Localization of GFP within the corneal stroma was confirmed by immunofluorescence microscopy using anti-GFP antibodies (FIG. 2C).

SEQUENCES

OTHER EMBODIMENTS

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

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

What is claimed is:

Claims

1 . A method of expressing a transgene in an ocular cell of an anterior segment of the eye in an individual, the method comprising:

(a) administering a circular DNA vector to the anterior segment of the eye, wherein the circular DNA vector lacks one or more components of a plasmid backbone and encodes the transgene;

(b) positioning one or more electrodes in and/or around the eye; and

(c) transmitting electrical energy through the one or more electrodes at conditions suitable for electrotransfer of the circular DNA vector into the ocular cell of the anterior segment, thereby expressing the transgene in the ocular cell of the anterior segment.

2. The method of claim 1 , wherein the circular DNA vector is expressed in the ocular cell of the anterior segment eight days following administration.

3. The method of claim 1 or 2, wherein the transgene expressed by the circular DNA vector in the ocular cell of the anterior segment is more persistent than a transgene encoded by a plasmid DNA vector encoding the transgene.

4. The method of any one of claims 1-3, wherein the circular DNA vector in the ocular cell of the anterior segment is less immunogenic than a plasmid DNA vector encoding the transgene.

5. The method of any one of claims 1-4, wherein the one or more components of the plasmid backbone lacking in the circular DNA vector comprises a drug resistance gene and/or an origin of replication.

6. The method of any one of claims 1-5, wherein the circular DNA vector is a nonviral circular DNA vector.

7. The method of claim 6, wherein the nonviral circular DNA vector is a naked circular DNA vector.

8. The method of claim 6 or 7, wherein the DNA vector is a synthetic circular DNA vector.

9. The method of any one of claims 1-7, wherein the DNA vector comprises an origin of replication.

10. The method of any one of claims 1-9, wherein the DNA vector lacks a selectable marker.

11. The method of claim 9, wherein the origin of replication is a ColE2-P9 origin of replication or functional variant thereof.

12. The method of any one of claims 1-11 , wherein step (a) comprises administering the circular DNA vector intracamerally or into the corneal stroma.

13. The method of any one of claims 1-12, wherein step (b) comprises positioning the one or more electrodes intracamerally and step (c) comprises transmitting electrical energy through the one or more intracamerally positioned electrodes.

14. The method of claim 12 or 13 wherein the intracameral position of the one or more electrodes is within 5 mm of the corneal endothelium.

15. The method of any one of claims 1-14, wherein at least one of the one or more electrodes is a needle electrode.

16. The method of any one of claims 1-5, wherein the ocular cell of the anterior segment that expresses the circular DNA vector is corneal cell, trabecular meshwork cell, iris cell, lens cell, ciliary body cell, and/or Schlemm’s canal cell.

17. The method of claim 16, wherein the corneal cell is a corneal endothelial cell or a corneal stromal cell.

18. The method of any one of claims 1-17, wherein the transgene encodes a protective factor that promotes corneal endothelial cell survival.

19. The method of claim 18, wherein the protective factor modulates a Nrf2 signaling pathway, a ROCK signaling pathway, a TGF-B signaling pathway, or a FGF-1 signaling pathway.

20. The method of any one of claims 1-19, wherein the circular DNA vector silences, corrects, or replaces a mutated gene associated with Fuchs’ dystrophy.

21. The method of claim 19, wherein the gene associated with Fuchs’ dystrophy is SLC4A11, TCF8, TCF, LOXHD1 , AGBL1, DMPK, ZEB1 , or COL8A2.

22. A method of expressing a therapeutic protein in an ocular cell of an anterior segment of an individual, the method comprising:

(a) administering a nucleic acid vector to the anterior segment of the eye, wherein the nucleic acid vector encodes the therapeutic protein;

(b) positioning one or more electrodes in and/or around the eye; and

(c) transmitting electrical energy through the one or more electrodes at conditions suitable for electrotransfer of the nucleic acid vector into the ocular cell of the anterior segment, thereby expressing the therapeutic protein in the ocular cell of the anterior segment.

23. The method of claim 22, wherein the therapeutic protein is a protective factor that promotes corneal endothelial cell survival.

24. The method of claim 23, wherein the protective factor modulates a Nrf2 signaling pathway, a ROCK signaling pathway, a TGF-B signaling pathway, or a FGF-1 signaling pathway.

25. The method of any one of claims 22-24, wherein the nucleic acid vector silences, corrects, or replaces a mutated gene associated with Fuchs’ dystrophy.

26. The method of claim 25, wherein the gene associated with Fuchs’ dystrophy is SLC4A11 , TCF8, TCF, LOXHD1 , AGBL1, DMPK, ZEB1 , or COL8A2.

27. The method of claim 25 or 26, wherein the individual has Fuchs’ dystrophy.

28. A method of treating Fuchs’ dystrophy in an individual in need thereof, the method comprising:

(a) administering a nucleic acid vector to the anterior segment of the eye in the individual, wherein the nucleic acid vector encodes a protective factor that promotes corneal endothelial cell survival;

(b) positioning one or more electrodes in and/or around the eye; and

(c) transmitting electrical energy through the one or more electrodes at conditions suitable for electrotransfer of the nucleic acid vector into the ocular cell of the anterior segment , thereby expressing the protective factor in the ocular cell of the anterior segment in an amount sufficient to treat Fuchs’ dystrophy.

29. A method of treating Fuchs’ dystrophy in an individual in need thereof, the method comprising:

(a) administering a nucleic acid vector to the anterior segment of the eye in the individual, wherein the nucleic acid vector silences, corrects, or replaces a mutated gene associated with Fuch’s dystrophy;

(b) positioning one or more electrodes in and/or around the eye; and

(c) transmitting electrical energy through the one or more electrodes at conditions suitable for electrotransfer of the nucleic acid vector into the ocular cell of the anterior segment , thereby silencing, correcting, or replacing the mutated gene in an amount sufficient to treat Fuchs’ dystrophy.

30. The method of claim 28 or 29, wherein the nucleic acid vector lacks one or more components of a plasmid backbone.

31. The method of any one of claims 28-30, wherein the nucleic acid vector comprises an origin of replication.

32. The method of any one of claims 28-31 , wherein the DNA vector lacks a selectable marker.

33. The method of claim 31 , wherein the origin of replication is a ColE2-P9 origin of replication or functional variant thereof.

34. A circular DNA vector comprising:

(a) a eukaryotic promoter;

(b) a coding sequence, wherein the coding sequence:

(i) encodes a protective factor that promotes corneal endothelial cell survival; or

(ii) silences, corrects, or replaces a mutated gene associated with Fuchs’ dystrophy; and

(c) a bacterial replication origin that is less than 50 bp in length, wherein the circular DNA vector lacks a selectable marker.

35. The circular DNA vector of claim 34, wherein the protective factor modulates a Nrf2 signaling pathway, a ROCK signaling pathway, a TGF-B signaling pathway, or a FGF-1 signaling pathway.

36. The circular DNA vector of claim 34, wherein the gene associated with Fuchs’ dystrophy is SLC4A11 , TCF8, TCF, LOXHD1 , AGBL1 , DMPK, ZEB1 , or COL8A2.

37. The circular DNA vector of any one of claims 34-36, wherein the 3’ end of the coding sequence is linked to the 5’ end of the promoter by a sequence comprising the bacterial origin of replication, wherein the sequence comprising the bacterial origin of replication is less than 100 bp in length.

38. A pharmaceutical composition comprising:

(a) the circular DNA vector of any one of claims 34-37 and

(b) a suitable carrier for use in delivering the pharmaceutical composition to an individual.

39. A method of delivering the circular DNA vector of any one of claims 34-38 to an ocular cell of the anterior segment of an individual, the method comprising:

(a) administering the circular DNA vector to the anterior segment of the eye;

(b) positioning one or more electrodes in and/or around the eye; and

(c) transmitting electrical energy through the one or more electrodes at conditions suitable for electrotransfer of the circular DNA vector into the ocular cell of the anterior segment, thereby delivering the circular DNA vector to the ocular cell of the anterior segment.