Compositions and methods for the treatment of hereditary macular degeneration

Abstract

The present invention provides compositions and methods for treating and / or alleviating hereditary macular degeneration (IMD) disorders. [Solution] A gene therapy composition and method are provided for targeting ATP-binding cassette subfamily A member 4 (ABCA4) or a functional fragment thereof in a patient, thereby treating or alleviating hereditary macular degeneration, including Stargardt disease or other diseases including retinal degeneration.

Description

Technical Field

[0001] The present invention is partially related to therapies, such as methods, compositions, and products for treating and / or alleviating inherited macular degeneration (IMD).

[0002] Priority This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 017,442, filed Apr. 29, 2020, which is hereby incorporated by reference in its entirety.

[0003] Description of Electronically Submitted Text File This application contains an ASCII-formatted sequence listing submitted electronically herewith via EFS-Web. The ASCII copy created on Apr. 28, 2021, is named SAL-002PR_Sequence_Listing_ST25.txt and is 64,498 bytes in size. The sequence listing is hereby incorporated by reference in its entirety.

Background Art

[0004] Macular degeneration is a condition in which the cells of the macula, found in the center of the retina, the tissue in the back of the eye that senses light, are damaged. Usually, vision loss occurs gradually and typically affects both eyes at different rates. Inherited macular degeneration (IMD), also known as macular dystrophy (MD), refers to a group of genetic injuries that cause ophthalmoscopically visible abnormalities in the retina.

[0005] First described in 1909 by German ophthalmologist Karl Stargardt, Stargardt's disease (STGD) is the most common form of macular degeneration (IMD). It is typically a recessive genetic disorder of the retina. Other names for the disease include Stargardt macular dystrophy (SMD), juvenile macular degeneration, or macular fundus. STGD typically causes vision loss in childhood or adolescence, although occasionally the vision loss may not be noticed until late adulthood. STGD causes progressive damage, or degeneration, of the macula, a small area in the center of the retina responsible for vision straight ahead. The global incidence of STGD is estimated to be 1 in 8,000 to 10,000 people.

[0006] STGD is one of several genetic disorders that cause macular degeneration, characterized by progressive vision loss due to the loss of light-sensing photoreceptor cells in the retina. Loss of central vision dramatically reduces the ability to read, write, and walk safely in the surrounding environment, significantly reducing the person's quality of life. Recessive Stargardt disease (STGD1) is by far the most common form of Stargardt disease, caused by mutations in ATP-binding cassette subfamily A member 4 (ABCA4). The ABCA4 gene / protein is expressed in photoreceptor (PR) cells. STGD1 manifests as the accumulation of lipofuscin, a fluorescent mixture of partially digested proteins and lipids, in the lysosomal compartment of the retinal pigment epithelium (RPE), which leads to photoreceptor degeneration. The RPE plays a role in regulating the immune response through the expression of mRNA and proteins that associate with the complement portion of the immune system, a crucial component of innate immunity. Age-related macular degeneration (AMD) is a disease that shares significant similarities with STGD1 and is associated with RPE lipofuscin accumulation and complement dysregulation. Lenis et al., Proc Natl Acad Sci USA. 2017 Apr 11;114(15):3987-3992.

[0007] Another form of STGD is STGD4, a rare dominant deletion in the PROM1 gene. (Kniazeva et al. Am J Hum Genet. 1999;64:1394-1399.) STGD3, also known as Stargardt-like dystrophy, is another rare dominant form of STGD, caused by mutations in the elongation of the very long-chain fatty acid-like 4 gene (ELOVL4). (Agbaga et al. Invest Ophthalmol Vis Sci. 2014;55:3669-3680.)

[0008] Existing therapies for IMD include deuterated vitamin A, microcurrent stimulation (MCS), RPE transplantation, nutritional supplements, stem cell therapy, and complement system modulation. However, despite these efforts, there is currently no effective treatment for common IMD, particularly STGD. The commonly used adeno-associated virus (AAV) does not contain the capacity for genes with coding sequences exceeding 5kb, including ABCA4 (6.8kb), the gene responsible for STGD, among other retinal disorders, making the development of gene therapies for IMD difficult.

[0009] Therefore, there is a need for compositions and methods to efficiently prevent and treat IMDs such as Stargardt disease, as well as other macular dystrophy. [Overview of the Initiative]

[0010] In various embodiments, the present invention provides compositions and methods for treating and / or mitigating hereditary macular degeneration (IMD) disorders, which are a leading cause of blindness worldwide. Examples of IMD include Stargardt disease, as well as other macular dystrophi (MD) including Best disease, X-linked retinoschisis, pattern dystrophy, Sorsby retinal dystrophy, and autosomal dominant drusen. The compositions and methods of the present invention utilize gene transmodification constructs comprising transposon expression vectors that employ sequence-specific or locus-specific transposition (SLST) to correct gene deficiencies associated with these diseases. The described compositions and methods employ a non-viral mode of gene transduction; therefore, the drawbacks associated with the use of viral vectors are overcome.

[0011] In some embodiments, a composition is provided comprising a gene transfection construct comprising (a) a nucleic acid encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein or a functional fragment thereof, (b) a retina-specific promoter, and (c) a non-viral vector comprising one or more transposase recognition sites and one or more inverted terminal repeats (ITRs) or end sequences.

[0012] The gene therapy described herein can be carried out using a transposon-based vector system, either on the same vector as the gene to be introduced (cis), on a different vector (trans), or with the assistance of a transposase provided as RNA. The transposon-based vector system can operate under the control of a retina-specific promoter.

[0013] In the embodiment, for example, a transposase derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo sapiens, and / or an engineered version thereof, is used to insert the ABCA4 gene or a functional fragment thereof into the patient's genome.

[0014] In embodiments, the transposase is a Myotis lucifugus transposase (MLT, or MLT transposase) comprising the amino acid sequence of SEQ ID NO: 10, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity thereto, and one or more mutations selected from L573X, E574X, and S2X, where X is any amino acid or non-amino acid, and optionally X is A, G, or deletion. In embodiments, the mutations are L573del, E574del, and S2A.

[0015] In some embodiments, the MLT transposase comprises an amino acid sequence having mutations L573del, E574del, and S2A (SEQ ID NO: 10), in addition to one or more mutations that confer high activity (or high activity mutations). In some embodiments, the high activity mutation is one or more of the S8X, C13X, and N125X mutations, where X is optionally any amino acid or non-amino acid, and optionally X is P, R, or K. In some embodiments, the mutations are S8P, C13R, and N125K. In some embodiments, the MLT transposase has the S8P and C13R mutations. In some embodiments, the MLT transposase has the N125K mutation. In some embodiments, the MLT transposase has all three mutations: S8P, C13R, and N125K.

[0016] The compositions described may be delivered to host cells using lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise one or more molecules selected from neutral or structural lipids (e.g., DSPCs), cationic lipids (e.g., MC3s), cholesterol, PEG-conjugated lipids (CDM-PEGs), and targeted ligands (e.g., N-acetylgalactosamine (GalNAc)). In some embodiments, the LNPs comprise GalNAc or another ligand for uptake mediated by the asialoglycoprotein receptor (ASGPR) into cells having a mutated ABCA4 or other gene (e.g., ELOVL4, PROM1, BEST1, or PRPH2).

[0017] In some embodiments, methods are provided for preventing or reducing the rate of photoreceptor loss in a patient, which may be in vivo or ex vivo methods. Accordingly, in some embodiments, methods are provided that include administering a composition according to embodiments of the present disclosure to a patient in need. In some embodiments, an ex vivo method is provided for preventing or reducing the rate of photoreceptor loss in a patient, which includes (a) contacting cells obtained from a patient (self) or another individual (allogeneic) with the composition described, and (b) administering the cells to a patient in need.

[0018] In some embodiments, methods are provided for treating and / or alleviating a class of IMDs (also known as macular dystrophy (MD)), including STGD, Best disease, X-associated retinoschisis, pattern dystrophy, Sorsby retinal dystrophy, and autosomal dominant drusen.

[0019] In some embodiments, methods are provided for treating and / or alleviating IMD, which can be carried out in vivo or ex vivo. In some embodiments, the method comprises administering a composition according to embodiments of the present disclosure to a patient in need. In some embodiments, the method for treating and / or alleviating IMD comprises (a) contacting cells obtained from a patient or another individual with the composition of the present disclosure, and (b) administering the cells to a patient in need.

[0020] IMD may be STGD, and in some embodiments, STGD may be STGD type 1 (STGD1). In some embodiments, STGD may be STGD type 3 (STGD3) or STGD type 4 (STGD4) disease. IMD may be characterized by mutations in one or more of ABCA4, ELOVL4, PROM1, BEST1, and PRPH2. ABCA4 mutations may be autosomal recessive or dominant mutations. The methods according to this disclosure enable reduction, decrease, or mitigation of symptoms of IMD, such as Stargardt disease, including improvement in distance visual acuity and / or a decrease in the rate of photoreceptor loss, compared to the absence of treatment. In some embodiments, the methods result in an improvement of up to approximately 20 / 200 or more in best corrected visual acuity (BCVA).

[0021] The compositions and methods according to embodiments of this disclosure are substantially non-immunogenic, do not cause any uncontrollable side effects, and may, in some cases, be effectively delivered via a single dose. Prevention or reduction of photoreceptor loss may be robust and permanent. The compositions and methods described reduce or prevent lipofuscin accumulation in the retina (e.g., RPE and / or Bruch's membrane), reduce or prevent the formation of retinal pigment epithelium (RPE) fragments, and improve the patient's distance visual acuity.

[0022] In some aspects of this disclosure, isolated cells comprising compositions according to embodiments of this disclosure are provided.

[0023] In some embodiments, the method provides improved distance visual acuity and / or a reduced rate of photoreceptor loss compared to the absence of treatment. The method can also result in an improvement of up to approximately 20 / 200 or more in best corrected visual acuity (BCVA). In some embodiments, the method results in an improvement in the shape of the retina or fovea, as measured by fundus autofluorescence (FAF) or spectral domain optical coherence tomography (SD-OCT). Similarly, other imaging techniques may also be used.

[0024] The described method improves the patient's vision. In some embodiments, the method results in a reduction or prevention of one or more of wavy vision, blind spots, haze, loss of depth perception, sensitivity to glare, color vision impairment, and difficulty adapting to low light (delayed dark adaptation) in the patient.

[0025] In some embodiments, the method according to the present disclosure eliminates the need for steroid treatment. Additionally or alternatively, the method may eliminate the need for soraprazan, isotretinoin, dobesilate, 4-methylpyrazole, ALK-0019 (C20-deuterated vitamin A), fenretinide (a synthetic form of vitamin A), LBS-500, A1120, emixustat, fenofibrate, abicipar pegol, and other therapeutic agents. However, in some embodiments, the composition and method involve the use of one or more additional therapeutic agents selected from soraprazan, isotretinoin, dobesilate, 4-methylpyrazole, ALK-0019 (C20-deuterated vitamin A), fenretinide (a synthetic form of vitamin A), LBS-500, A1120, emixustat, fenofibrate, abicipar pegol, and other therapeutic agents.

[0026] Other aspects and certain embodiments of the invention will become apparent from the following detailed description.

Brief Description of the Drawings

[0027] [Figure 1] Schematic diagram of a vector that can be used in transfection, transfer efficacy, and expression studies in retinal cell lines. [Figure 2] Shows the lipid nanoparticle structure used in some embodiments of the present disclosure. [Figure 3]This document shows GFP expression in 661W mouse photoreceptor cells 24 hours after transfection with a variable lipofection reagent, and either MLT transposase 1 (MLT with the N125K mutation) or MLT transposase 2 (MLT with the S8P / C13R mutation) of the present disclosure, compared to untransfected cells. The top row shows untransfected 661W mouse photoreceptor cells, cells transfected with transposons containing L3 (Lipofectamine 3000) and MLT1, and cells transfected with transposons containing L3 and MLT2. The middle row shows untransfected 661W mouse photoreceptor cells, cells transfected with transposons containing LTX (Lipofectamine LTX&PLUS) and MLT1, and cells transfected with transposons containing LTX and MLT2. The bottom row shows untransfected 661W mouse photoreceptor cells, cells transfected with transposons containing MAX (Lipofectamine Messenger MAX) and MLT1, and cells transfected with transposons containing MAX and MLT2. [Figure 4] This shows the stable incorporation of donor DNA (GFP) via translocation in mouse photoreceptor cell line 661W after four passages over 15 days. The rows show the results on days 3, 6, 9, 12, and 15, and the columns show the results for untransfected cells, cells transfected with donor DNA only, cells transfected with donor DNA and MLT1, and cells transfected with donor DNA and MLT2. [Figure 5] This bar graph shows the results of FACS analysis of the stable integration of donor DNA (GFP) via translocation in mouse photoreceptor cell line 661W on day 15. The percentage of GFP expression (%) is shown for untransfected cells, cells transfected with donor DNA only ("+GFP only"), cells transfected with donor DNA and MLT1 ("MLT1+GFP"), and cells transfected with donor DNA and MLT2 ("MLT2+GFP"). [Figure 6] The image shows GFP expression in ARPE-19 cells 24 hours after transfection. The top panel shows untransfected ARPE-19 cells, cells transfected with a transposon containing only L3, cells transfected with a transposon containing L3 and MLT1, and cells transfected with a transposon containing L3 and MLT2. The middle panel shows untransfected ARPE-19 cells, cells transfected with a transposon containing only LTX, cells transfected with a transposon containing LTX and MLT1, and cells transfected with a transposon containing LTX and MLT2. The bottom panel shows untransfected ARPE-19 cells, cells transfected with a transposon containing only MAX, cells transfected with a transposon containing MAX and MLT1, and cells transfected with a transposon containing MAX and MLT2. [Figure 7] This image shows higher-resolution images of the visible GFP expression of MLT transposase 1 and MLT transposase 2 24 hours after transfection. [Figure 8] This shows the stable integration of donor DNA (GFP) into the photoreceptor cell line ARPE19 using MLT transposase 2 (MLT2). The rows show the results at days 4, 8, 12, and 15, and the columns show the results for cells transfected with donor DNA alone, as well as cells transfected with both donor DNA and MLT2. [Figure 9] This bar graph shows the results of FACS analysis of the stable integration of donor DNA (GFP) by translocation in ARPE19 cell lines after four generations of cell division. The percentage of GFP expression (%) is shown for untransfected cells, cells transfected with donor DNA only ("+GFP only"), cells transfected with donor DNA and MLT1 ("MLT1+GFP"), and cells transfected with donor DNA and MLT2 ("MLT2+GFP"). [Figure 10] Images of the left eye (Figure 10A) and right eye (Figure 10B) of mouse 1-1L injected with PBS are shown. [Figure 11] Images of the right eye of mice 3-1L and 3-1R injected with DNA only (Figures 11A and 11C), and the left eye of mice 3-1L and 3-1R injected with donor DNA and MLT2 (Figures 11B and 11D) are shown. [Figure 12] Images of the right eye of mouse 4-1R injected with donor DNA (Figure 12A) and MLT2 (Figure 12B) are shown. [Figure 13] Figure 13A shows the right eye of a mouse 4-NP injected with donor DNA only, and Figure 13B shows the left eye injected with both donor DNA and MLT2. [Figure 14] Figure 14A shows the right eye of mouse 4-1L injected with donor DNA only, and Figure 14B shows the left eye injected with both donor DNA and MLT2. [Figure 15] Figure 15A shows the right eye of a mouse 5-BP injected with donor DNA only, and Figure 15B shows the left eye injected with both donor DNA and MLT2. [Figure 16] This disclosure presents experimental designs to evaluate the efficacy of translocation of 661W mouse photoreceptor cells and retinal epithelial (ARPE19) cells using DNA donors and RNA helpers, according to several embodiments of this disclosure. [Figure 17] The images show the left and right eyes of mice (top and bottom panels, respectively), taken 21 days after subretinal injection, with or without the MLT transposase used for transfection ("+MLT"). [Modes for carrying out the invention]

[0028] This invention is based in part on the discovery that nonviral, capsid-free gene therapy methods and compositions can be used to prevent or reduce the rate of photoreceptor loss in patients. The nonviral gene therapy methods according to this disclosure find use in retinal-directed gene therapy for hereditary macular degeneration (IMD). In some embodiments, the methods and compositions find use in retinal-directed gene therapy for Stargardt disease (STGD) caused by mutations in ATP-binding cassette subfamily A member 4 (ABCA4). The described methods and compositions utilize the transfer of ABCA4 or another gene or functional fragment thereof from a gene transconstruct into the host genome. The described methods and compositions reduce or prevent lipofuscin accumulation in the retina (e.g., RPE and / or Bruch membrane, and photoreceptors) and improve the patient's distance vision.

[0029] STGD is characterized by macular atrophy and peripheral plaques in the retinal pigment epithelium (RPE). The ABCA4 gene encodes a transmembrane protein (ABCA4 protein) found in rod and cone photoreceptors, specifically involved in the transport of vitamin A intermediates such as N-retinilysine-phosphatidylethanolamine (N-RPE) to the RPE. ABCA4 is responsible for the clearance of all-trans-retinal (reactive vitamin A aldehyde) from photoreceptor cells, and loss of ABCA4 function leads to the accumulation of bis-retinoids (such as N-RPE) on the outer segment membrane of photoreceptor cells, ultimately causing lipofuscin formation. This ultimately results in high levels of lipofuscin accumulation in the RPE (and thus increased retinal autofluorescence), as well as progressive RPE loss and photoreceptor cell loss.

[0030] Mutations in ABCA4 are associated with a wide range of phenotypes, including rod-cone dystrophy (in STGD disease, cones and rods die) and retinitis pigmentosa (degradation and loss of cells in the retina). See, for example, Song et al., JAMA, Ophthalmol. 2015;133(10):1198-1203. Similarly, mutations in other genes that cause MD also result in a variety of phenotypes that differ among patients.

[0031] As mentioned above, the use of adeno-associated virus (AAV) vectors for gene therapy involving ABCA4 is hindered by the size of ABCA4 (6.8kb), which exceeds the 4.5kb–5.0kb capacity of AAV. Therefore, equine lentivirus (EIAV) is used for gene delivery via subretinal injection. (Kong et al., Gene Ther. 2008;15(19):1311-1320.) Another approach to address the relatively large size of ABCA4 was to split the gene across two AAV vectors so that the two transgene fragments would combine within the host cell. (Dyka et al., Hum Gene Ther. 2019;Nov;30(11):1361-1370.)

[0032] The compositions and methods of the present disclosure provide nonviral delivery of transgenes that replace mutant copies of ABCA4 or other target genes(s). Accordingly, the compositions and methods of the present disclosure provide transgene constructs targeting ABCA4 or a functional fragment thereof for correcting pathogenic variants in a patient’s genome and thus preventing or reducing the rate of photoreceptor loss in the patient. Accordingly, in some aspects of the present disclosure, a composition is provided comprising a transgene construct including (a) a nucleic acid encoding the ABCA4 protein or a functional fragment thereof, (b) a retina-specific promoter, and (c) a nonviral vector comprising one or more transposase recognition sites and one or more inverted terminal repeats (ITRs) or end sequences.

[0033] In some embodiments, the ABCA4 protein is the human ABCA4 protein or a functional fragment thereof. In some embodiments, the gene encoding human ABCA4 is human ABCA4 (GenBank Acc.No.NM_000350). The nucleic acid encoding human ABCA4 may include a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 1, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto. In some embodiments, the nucleic acid encoding human ABCA4 includes the nucleotide sequence of SEQ ID NO: 2, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

[0034] In some embodiments, the nucleic acid encoding human ABCA4 comprises a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 1, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto. In some embodiments, the nucleic acid encoding human ABCA4 comprises a nucleotide sequence of SEQ ID NO: 2, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

[0035] Sequence ID 1 is as follows:

[0036] [ka]

[0037] In this embodiment, human ABCA4 is encoded by a variant including a codon-optimized version having at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto. Sequence ID 2 is as follows:

[0038] [ka]

[0039] [ka]

[0040] [ka]

[0041] In some embodiments, this disclosure relates to compositions and methods for gene delivery via bitransposons and transposase systems. Transposable factors are naturally occurring, universally found nonviral gene delivery vehicles. Transposon-based vectors have the ability to stably integrate the introduced gene construct into the cell genome and to express it for extended periods. In general, bitransposons and transposase systems function via a cleavage and paste mechanism, in which the transposon DNA containing the desired introduced gene(s) is integrated into chromosomal DNA by a transposase enzyme at a repetitive sequence site.

[0042] As will be understood in the art, a transposon often comprises a central open reading frame encoding a transgene and terminal repeat sequences at the 5' and 3' ends of the transposon. A translated transposase binds to the 5' and 3' sequences of the transposon and performs the transposition function.

[0043] In embodiments, the term transposon is used synonymously with transposable factors, referring to polynucleotides capable of inserting copies of themselves into other polynucleotides. The term transposon is well known to those skilled in the art and includes a category of transposons that can be distinguished based on sequence arrangement, e.g., short inverted repeats (ITRs) and / or directly repeated long terminal repeats (LTRs) at each end. In some embodiments, the transposons described herein can be described as piggyBac-like factors, e.g., transposon factors characterized by traceable excision that recognizes the TTAA sequence and restores the sequence of the insertion site to the original TTAA sequence after the transposon is removed.

[0044] In some embodiments, the nonviral vector is a transposon-mediated gene transfer system (e.g., a DNA plasmid transposon system) flanked by an ITR recognized by a transposase. In some embodiments, the ITR is flanked by a nucleic acid encoding the ABCA4 gene. The nonviral vector operates as a transposon-based vector system containing heterologous polynucleotides (also called transgenes) flanked by two ends recognized by a transposase. The transposon ends may be exact or inaccurate repeats and contain ITRs oriented opposite to each other. The transposase acts on the transposon ends, thereby "cleaving" the transposon (along with its ends) from the vector and "attaching" or incorporating the transposon into the host genome. In embodiments, the transposase is provided as a DNA expression vector, or as an expressible RNA or protein so that long-term expression of the transposase does not occur in transgenic cells.

[0045] In the embodiment, the gene transfer system is a nucleic acid (DNA) encoding a transposon and is referred to as "donor DNA." In the embodiment, the nucleic acid encoding the transposase is helper RNA (i.e., mRNA encoding the transposase), and the nucleic acid encoding the transposon is donor DNA (or DNA donor transposon). In the embodiment, the donor DNA is incorporated into a plasmid. In the embodiment, the donor DNA is a plasmid.

[0046] DNA donor transposons, which are mobile factors that utilize a "cleavage and paste" mechanism, include donor DNA adjacent to two end sequences in mammals, including humans (Homo sapiens) (e.g., Myotis lucifugus, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, and Pan troglodytes), or inverted end repeats (ITRs) in other organisms such as insects (e.g., Trichnoplusia ni) or amphibians (Xenopus species). Genomic DNA is excised by double-strand breaks at the host donor site, and the donor DNA is incorporated into this site. Dual systems using bioengineered transposons and transposases include (1) a source of active transposases that "cleave" at specific nucleotide sequences such as TTAA, and (2) DNA sequences adjacent to the recognition end sequence or ITR that are moved by the transposase. By moving DNA sequences, it becomes possible to insert intervening nucleic acids or transgenes into specific nucleotide sequences (i.e., TTAAs) without using DNA footprints.

[0047] In one embodiment, the transposase is a Myotis lucifugus transposase (MLT, or MLT transposase) comprising the amino acid sequence of SEQ ID NO: 10, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In another embodiment, the transposase is a Myotis lucifugus transposase (MLT, or MLT transposase) comprising the amino acid sequence of SEQ ID NO: 9, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity thereto, and S2X, where X is any amino acid or non-amino acid, and optionally X is A, G, or a deletion.

[0048] In embodiments, the transposase is a Myotis lucifugus transposase (MLT, or MLT transposase) comprising the amino acid sequence of SEQ ID NO: 9, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity thereto, and S2X, where X is any amino acid or non-amino acid, optionally X is A or G, the C-terminal deletion is selected from L573X and E574X, and X is non-amino acid. In embodiments, the mutations are L573del, E574del, and S2A.

[0049] In this embodiment, the MLT transposase has the amino acid sequence of SEQ ID NO: 10, which includes mutations L573del, E574del, and S2A: (Sequence ID 10) Or it contains an amino acid sequence that is identical to it by at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, at least approximately 98%, or at least approximately 99%.

[0050] In some embodiments, the MLT transposase containing the amino acid sequence of SEQ ID NO: 10 has the following nucleotide sequence: Alternatively, it is encoded by a nucleotide sequence having at least approximately 90% identity, or at least approximately 93%, or at least approximately 95%, or at least approximately 97%, or at least approximately 98%, or at least approximately 99% identity.

[0051] In some embodiments, the MLT transposase (for example, an MLT transposase having an amino acid sequence that is at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical thereto) contains one or more high-activity mutations that confer high activity to the MLT transposase. In embodiments, the high-activity mutation is one or more of the S8X, C13X, and N125X mutations compared to the amino acid sequence of SEQ ID NO: 10 or its functional equivalent, where X is optionally any amino acid or non-amino acid, and optionally X is P, R, or K. In embodiments, the mutations are S8P, C13R, and N125K. In some embodiments, the MLT transposase has the S8P and C13R mutations. In some embodiments, the MLT transposase has the N125K mutation. In some embodiments, the MLT transposase has all three mutations: S8P, C13R, and N125K.

[0052] In some embodiments, the MLT transposase corresponds to the nucleotide sequence (SEQ ID NO: 12) of an amino acid (SEQ ID NO: 13) having an N125K mutation, compared to the amino acid sequence of SEQ ID NO: 10.

[0053] [ka]

[0054] Alternatively, a nucleotide sequence having at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity with respect to it (codons corresponding to the N125K mutation are underlined and in bold).

[0055] [ka]

[0056] Alternatively, it may be coded by an amino acid sequence having at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity (amino acids corresponding to the N125K mutation are underlined and in bold).

[0057] In some embodiments, an MLT transposase encoded by the nucleotide sequence of SEQ ID NO: 12 and having the amino acid sequence of SEQ ID NO: 13 is referred to as MLT transposase 1 (or MLT1).

[0058] In some embodiments, the MLT transposase has a nucleotide sequence (SEQ ID NO: 14) corresponding to an amino acid (SEQ ID NO: 15) with S8P and C13R mutations, compared to the amino acid sequence of SEQ ID NO: 10 or its functional equivalent (SEQ ID NO: 14 and SEQ ID NO: 15 are as follows):

[0059] [ka]

[0060] Alternatively, a nucleotide sequence having at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity with respect to it (codons corresponding to the S8P and C13R mutations are underlined and in bold).

[0061] [ka]

[0062] Alternatively, it may be encoded by an amino acid sequence having at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity (amino acids corresponding to the S8P and C13R mutations are underlined and in bold).

[0063] In some embodiments, an MLT transposase encoded by the nucleotide sequence of SEQ ID NO: 14 and having the amino acid sequence of SEQ ID NO: 15 is referred to as MLT transposase 2 (or MLT2).

[0064] In some embodiments, the transposase is derived from the Tc1 / mariner transposon. See, for example, Plasterk et al. Trends in Genetics. 1999;15(8):326-32.

[0065] In some embodiments, the transposase is from the Sleeping Beauty transposon system (see, e.g., Cell. 1997;91:501-510) or the piggyBac transposon system (see, e.g., Trends Biotechnol. 2015 Sep;33(9):525-33.doi:10.1016 / j.tibtech.2015.06.009.Epub 2015 Jul 23).

[0066] In some embodiments, the transposase is of the LEAP-IN1 type or LEAP-IN transposon system (Biotechnol J.2018 Oct;13(10):e1700748.doi:10.1002 / biot.201700748.Epub 2018 Jun 11).

[0067] In some embodiments, the nonviral vector includes a LEAP-IN1 type LEAPIN transposase (ATUM, Newark, CA). The LEAPIN transposase system includes a transposase (e.g., transposase mRNA) and a vector containing one or more target genes (transposons), selection markers, regulatory factors, etc., adjacent to the congenital inverted terminal repeats (ITRs) and transposition recognition motifs (TTATs) of the transposon. Upon co-transfection of the vector DNA and transposase mRNA, the transiently expressed enzyme catalyzes the highly efficient and precise incorporation of a single copy of the transposon cassette (all sequences between ITRs) at one or more sites throughout the host cell genome. Hottentot et al. In Genotyping: Methods and Protocols. White SJ, Cantsilieris S, eds: 185-196. (New York, NY: Springer): 2017. pp. 185-196. LEAPIN transposases produce stable transgene constructs with various advantageous features, including the integration of single copies at multiple genomic loci, primarily in open chromatin segments. Because there is no payload limitation, multiple independent transcription units can be expressed from a single construct. The integrated transgenes maintain their structural and functional integrity, and maintaining transgene integrity ensures the desired chain ratio in all recombinant cells.

[0068] In some embodiments, ABCA4 is operably conjugated to a promoter that can influence the overall expression level and cell specificity of the transgene (e.g., ABCA4 or a functional fragment thereof).

[0069] In some embodiments, the promoter is a CAG promoter (cytomegalovirus (CMV) enhancer fused to a chicken β-actin promoter and a rabbit β-globin splice acceptor) (1732 bp), which is expressed both in vivo and in vitro at the RPE and visual receptor levels. In some embodiments, the CAG promoter has the following nucleotide sequence (SEQ ID NO: 16):

[0070] [ka]

[0071] or include variants having at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, or at least approximately 98% identity thereto.

[0072] In some embodiments, the promoter is optionally a CMV enhancer, a chicken beta-actin promoter, and a rabbit beta-globin splice acceptor site (CAG), which include the nucleic acid sequence of SEQ ID NO: 16 or a variant having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

[0073] In some embodiments, the promoter is tissue-specific, i.e., retina-specific. In embodiments where the transposase is a transposase that encodes a DNA sequence, such a DNA sequence is also operably ligated to the promoter. A variety of promoters can be used, including tissue-specific promoters, inducible promoters, constitutive promoters, and the like.

[0074] In some embodiments, the retina-specific promoter is a retinal pigment epithelium (RPE) promoter and may be RPE65 (retinal pigment epithelium-specific 65kDa protein gene), IRBP (photoreceptor-retinoid-binding protein), or VMD2 (vitiligo macular dystrophy 2) promoter.

[0075] The RPE65, IRBP, and VMD2 promoters are described, for example, in Aguirre. Invest Ophthalmol Vis Sci. 2017;58(12):5399-5411.doi:10.1167 / iovs.17-22978. An example of the RPE65 promoter, which may be used in some embodiments, is given below:

[0076] [ka]

[0077] Or a functional fragment of a variant having at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, or at least approximately 98% identity thereto.

[0078] The human photoreceptor-retinoid-binding protein (IRBP) promoter has been demonstrated to rescue photoreceptors from progressive degeneration. al-Ubaidi & Baehr. J. Cell Biol. 1992;119:1681-1687. An example of an IRBP promoter (1325 bp) that can be used in several embodiments (adapted from Bobola et al., J. Biol. Chem. 1995;270:1289-1294) is as follows:

[0079] [ka]

[0080] Or a functional fragment of a variant having at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, or at least approximately 98% identity thereto.

[0081] The human VMD2 promoter has been shown to specifically and exclusively target transgene expression in RPE cells in vivo after a single subretinal injection (in dogs). See Guziewicz et al., PloS One vol.8,10 e75666.15 Oct.2013, doi:10.1371 / journal.pone.0075666. An example of the VMD2 promoter sequence (624 bp), which is the upstream region of the BEST1 gene and may be used in some embodiments (see Esumi et al., J.Biol.Chem.2004;279(18):19064-19073), is as follows:

[0082] [ka]

[0083] Or a functional fragment of a variant having at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, or at least approximately 98% identity thereto.

[0084] In some embodiments, the retina-specific promoter is a photoreceptor promoter, optionally selected from β-phosphodiesterase (PDE), rhodopsin kinase (GRK1), CAR (cone arrestin), retinitis pigmentosa 1 (RP1), and L-opsin. The PDE and RP1 promoters, as well as the rhodopsin (Rho) promoter, have been shown to drive photoreceptor-specific expression in vitro. (Kan et al., Molecular Therapy, vol.15, Suppl.1, S258, May 01, 2007.) An example of a PDE promoter (200 bp) that may be used in some embodiments (e.g., described in Di Polo et al., Nucleic Acids Res. 1997;25(19):3863-3867) is as follows:

[0085] [ka]

[0086] Or a functional fragment of a variant having at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, or at least approximately 98% identity thereto.

[0087] The human rhodopsin kinase (GRK1) gene promoter has been shown to be active and specific to rod and cone photoreceptors, and due to its small size and demonstrated activity in cones, it is a promoter choice for somatic gene transfection and gene therapy targeting rods and cones. Khani et al., Investigative Ophthalmology & Visual Science September 2007; vol. 48: 3954-3961. Examples of GRK1 promoters (295 bp) that may be used in several embodiments (see Khani et al., 2007, McDougald et al., Mol Ther Methods Clin Dev. 2019; 13: 380-389. Published 2019 Mar 28) are as follows:

[0088] [ka]

[0089] Or a functional fragment of a variant having at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, or at least approximately 98% identity thereto.

[0090] The CAR promoter has also been shown to drive strong expression in the retina. Dyka et al., Adv Exp Med Biol. 2014;801:695-701. In some embodiments, the CAR promoter (2026bp) (see McDougald et al., Mol Ther Methods Clin Dev. 2019;13:380-389. Published 2019 Mar 28) is as follows:

[0091] [ka]

[0092] Or a functional fragment of a variant having at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, or at least approximately 98% identity thereto.

[0093] The human L-opsin promoter has been shown to induce high levels of GFP expression in mouse photoreceptors. Ye et al., Hum Gene Ther. 2016;27(1):72-82. In some embodiments, the L-opsin promoter (1726 bp) (see Lee et al., Vision Res. 2008 Feb;48(3):332-8) is as follows:

[0094] [ka]

[0095] Or a functional fragment of a variant having at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 93%, at least approximately 95%, at least approximately 97%, or at least approximately 98% identity thereto.

[0096] In this embodiment, the retina-specific promoter is an RPE promoter that includes the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a variant having at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

[0097] In embodiments, the retina-specific promoter is a photoreceptor promoter comprising a functional fragment of the nucleic acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a variant having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

[0098] In embodiments, the nonviral vector may contain at least one pair of inverted end repeats at the 5' and 3' ends of the transposon. In embodiments, the inverted end repeats are sequences located at one end of the vector that, when used in combination with complementary sequences located at the opposing ends of the vector, can form a hairpin structure. The pair of inverted end repeats are involved in the transposation activity of the transposon of the nonviral vector of the disclosure, specifically, in DNA addition or removal and excision of DNA of interest. In one embodiment, at least one pair of inverted end repeats appears to be the minimum sequence required for transposation activity in the plasmid. In another embodiment, the vector of the disclosure may contain at least two, three, or four pairs of inverted end repeats. As will be understood by those skilled in the art, the terminal sequences required to facilitate cloning may be as short as possible and therefore may contain as few inverted repeats as possible. Accordingly, in one embodiment, the vector of the disclosure may contain one or fewer pairs, two or fewer pairs, three or fewer pairs, or four or fewer pairs of inverted end repeats. In one embodiment, the vector of the disclosure may contain only one inverted end repeat.

[0099] In embodiments, the inverted end repeats of the present invention may form either perfect inverted end repeats (or synonymously referred to as “perfect inverted repeats”) or incomplete inverted end repeats (or synonymously referred to as “incomplete inverted repeats”). As used herein, the term “perfect inverted repeat” refers to two identical DNA sequences oriented in opposite directions. In contrast, the term “incomplete inverted repeat” refers to two DNA sequences that are similar to each other except that they contain some mismatches. These repeats (i.e., both perfect and incomplete inverted repeats) are transposase binding sites.

[0100] In some embodiments, the ITR of the nonviral vector is optionally that of a piggyBac-like transposon containing a TTAA repeat sequence, and / or the ITR is adjacent to ABCA4. The piggyBac-like transposon transposes through a "cleave and paste" mechanism, and the piggyBac-like transposon may contain a TTAA repeat sequence. The piggyBac transposon is a transposon system frequently used for genetic recombination and does not require DNA synthesis during the actual transposition event. The piggyBac factor can be cleaved from the donor chromosome by a transposase, and the passaged donor DNA can be rejoined with a DNA ligase. (Zhao et al. Translational lung cancer research, 2006;5(1):120-125.) The piggyBac transposon demonstrates precise excision, i.e., the ability to return the sequence to its pre-incorporation state. Yusa. piggyBac Transposon. Microbiol Spectr. 2015 Apr;3(2). In some embodiments, the gene transposition construct contains Super piggyBac® (SPB) transposase. See Barnett et al. Blood 2016;128(22):2167.

[0101] In some embodiments, other non-viral gene transfer tools, such as the Sleeping Beauty transposon system, may be used. See, for example, Aronovich et al. Human Molecular Genetics, 2011;20(R1),R14-R20.

[0102] In some embodiments, the transposon system sequence can be codon-optimized to provide improved mRNA stability and protein expression in mammalian systems.

[0103] In various embodiments, the gene transfer construct may be any suitable gene transfer construct, such as a nucleic acid construct, plasmid, or vector. In various embodiments, the gene transfer construct is DNA. In some embodiments, the gene transfer construct is RNA. In some embodiments, the gene transfer may have both DNA and RNA sequences.

[0104] In embodiments, the nucleic acid comprises polymeric forms of nucleotides of any length, which are ribonucleotides, deoxyribonucleotides, or analogs or derivatives thereof. Embodiments provide double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA, and RNA-DNA hybrids. Embodiments provide transcription-activated polynucleotides, such as methylated or capped polynucleotides. In embodiments, the composition is mRNA or DNA.

[0105] In embodiments, the nonviral vector is a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to a control sequence, the control sequence providing expression of the polypeptide-encoding polynucleotide. In embodiments, the nonviral vector comprises a promoter sequence, as well as transcription and translation stop signal sequences. Such vectors include, among others, chromosomal and episomal vectors, e.g., vectors derived from bacterial plasmids, transposons, yeast episomes, insertion factors, yeast chromosomal elements, and combinations thereof. The construct may contain a control region that regulates and generates expression.

[0106] In some embodiments, the gene transconstruct may be codon-optimized. In the embodiments described, the nucleic acid encoding ABCA4, or a functional fragment thereof, functions as a transgene integrated into a host genome (e.g., the human genome) to provide a desired clinical outcome. Transgene codon optimization may be used to optimize the therapeutic potential of the transgene and its expression in the host organism. Codon optimization is performed to match the codon usage in the transgene with the abundance of transfer RNA (tRNA) for each codon in the host organism or cell. Codon optimization methods are known in the art and are described, for example, in WO2007 / 142954, which is incorporated herein by reference in its entirety. Optimization strategies include, for example, modification of the translation initiation region, alteration of mRNA structural factors, and the use of different codon biases.

[0107] The gene transfect construct includes several other regulatory factors selected to ensure stable expression of the construct. Thus, in some embodiments, the nonviral vector is a DNA plasmid that may contain one or more insulator sequences that prevent or mitigate activation or inactivation of the adjacent gene. In some embodiments, the one or more insulator sequences include an HS4 insulator (1.2-kb5′-HS4 chicken β-globin (cHS4) insulator factor) and a D4Z4 insulator (tandem macrosatellite repeats associated with facioscapulohumeral muscular dystrophy (FSHD)). In some embodiments, the sequences of the HS4 and D4Z4 insulators are as described in Rival-Gervier et al. Mol Ther. 2013 Aug;21(8):1536-50, which are incorporated herein by reference in their entirety. In some embodiments, the gene of the gene transfect construct is transposable in the presence of a transposase. In some embodiments, the nonviral vector according to embodiments of this disclosure includes a nucleic acid construct encoding a transposase. The transposase may be an RNA transposase plasmid. In some embodiments, the nonviral vector further comprises a nucleic acid construct encoding a DNA transposase plasmid. In some embodiments, the transposase is an in vitro transcribed mRNA transposase. The transposase is capable of excising a gene from a gene transposition construct and / or transferring the gene to a site-specific or locus-specific genomic region.

[0108] A composition comprising a gene transposition construct according to this disclosure may comprise one or more nonviral vectors. Furthermore, the transposase may be located on the same (cis) or different (trans) vector as the transposon containing the transgene. Thus, in some embodiments, the transposase and the transposon containing the transgene are in a cis configuration, such that they are contained within the same vector. In some embodiments, the transposase and the transposon containing the transgene are in a trans configuration, such that they are contained within different vectors. The vector is any nonviral vector according to this disclosure.

[0109] In some embodiments, the transposase is derived from and / or manipulated versions thereof of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo sapiens. In some embodiments, the transposase specifically recognizes ITR. The transposase may contain a DNA or RNA sequence encoding a Bombyx mori, Xenopus tropicalis, or Trichoplusia ni protein. See, for example, U.S. Patent No. 10,041,077, which is incorporated in its entirety herein by reference.

[0110] However, in some embodiments, transposases may be directly introduced into cells as proteins, for example, using cell-permeable peptides (e.g., described in Ramsey and Flynn. Pharmacol. Ther. 2915;154:78-86), small molecules containing salts and propane betaine (e.g., described in Astolfo et al. Cell 2015;161:674-690), or electroporation (e.g., described in Morgan and Day. Methods in Molecular Biology 1995;48:63-71).

[0111] In some embodiments, the transposon system may be implemented as described, for example, in U.S. Patent No. 10,435,696, which is incorporated herein by reference in its entirety.

[0112] In some embodiments, the described composition comprises a transgene (e.g., ABCA4 or a functional fragment thereof) and a transposase in a specific ratio. In some embodiments, a transgene-to-transposase ratio is selected to improve the efficiency of transposase activity. The transgene-to-transposase ratio may depend on the concentration of the transfected gene construct and other factors. In some embodiments, the ratio of nucleic acid encoding ABCA4 or a functional fragment thereof to nucleic acid construct encoding the transposase is about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5. In some embodiments, the ratio of nucleic acid encoding the ABCA4 protein to nucleic acid construct encoding the transposase is about 2:1. In some embodiments, a composition comprising a gene construct is provided. In the embodiment, the composition comprises (a) a nucleic acid encoding an ATP-binding cassette subfamily A member 4 (ABC) transporter (ABCA4) protein or a functional fragment thereof; (b) a CAG promoter; and (c) a nonviral vector comprising one or more transposase recognition sites and one or more inverted terminal repeats (ITRs) or end sequences, wherein the ABCA4 protein is a human ABCA4 protein or a functional fragment thereof comprising a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 1, or a variant having at least about 95% identity thereto.

[0113] In some embodiments, compositions comprising a gene transfection construct are provided. In embodiments, the composition comprises (a) a nucleic acid encoding an ATP-binding cassette subfamily A member 4 (ABC) transporter (ABCA4) protein or a functional fragment thereof; (b) a CAG promoter; and (c) a nonviral vector comprising one or more transposase recognition sites and one or more inverted terminal repeats (ITRs) or end sequences, wherein the ABCA4 protein is human ABCA4 or a functional fragment thereof, encoded by the nucleotide acid sequence of SEQ ID NO: 2 or a variant having at least about 95% identity thereto.

[0114] In some embodiments, a method is provided for treating and / or alleviating hereditary macular degeneration (IMD), comprising: (a) contacting cells obtained from a patient or another individual with the composition described in claim 62; (b) contacting the cells with a nucleic acid construct encoding a transposase and / or an engineered version thereof derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo sapiens, wherein the ratio of nucleic acid encoding the ABCA4 protein or a functional fragment thereof to the nucleic acid construct encoding the transposase is about 2:1; and (c) administering the cells to a patient in need.

[0115] In some embodiments, the non-viral vector is a conjugated polynucleotide sequence introduced into cells by various transfection methods, such as those using lipid particles. In some embodiments, the composition comprising the gene transconstruct comprises delivery particles. In some embodiments, the delivery particles comprise lipid-based particles (e.g., lipid nanoparticles (LNPs)), cationic lipids, or biodegradable polymers). Lipid nanoparticle (LNP) delivery of gene transconstructs offers certain advantages, including transient non-integrated expression to limit potential off-target events and immune responses, as well as efficient delivery with the ability to transport large cargo. LNPs have been used to deliver mRNA to the retina. See Patel et al., J Control Release. 2019 Jun 10;303:91-100. doi:10.1016 / j.jconrel.2019.04.015.Epub 2019 Apr 12. Furthermore, for example, U.S. Patent No. 10,195,291 describes the use of LNPs for the delivery of RNA interference (RNAi) therapeutic agents.

[0116] In some embodiments, the compositions according to embodiments of the present disclosure are in the form of LNPs. In some embodiments, the LNPs include 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol); phosphatidylcholine (PC); triolein (glyceryl trioleate); and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). It contains one or more lipids selected from 2K and 1,2-distearol-sn-glycerol-3-phosphocholine (DSPC).

[0117] In some embodiments, LNPs adapted from Patel et al., J Control Release 2019;303:91-100 are shown in Figure 2. As shown in Figure 2, LNPs may comprise one or more of the following: structural lipids (e.g., DSPC), PEG-conjugated lipids (CDM-PEG), cationic lipids (MC3), cholesterol, and targeted ligands (e.g., GalNAc).

[0118] In some embodiments, the composition may have lipids and polymers in various ratios, where the lipids may be selected from, for example, DOTAP, DC-Chol, PC, triolein, and DSPE-PEG, and the polymers may be, for example, PEI or polylactic acid-coglycolic acid (PLGA). In addition, any other lipids and polymers may be used. In some embodiments, the ratio of lipids to polymers is about 0.5:1, or about 1:1, or about 1:1.5, or about 1:2, or about 1:2.5, or about 1:3, or about 3:1, or about 2.5:1, or about 2:1, or about 1.5:1, or about 1:1, or about 1:0.5.

[0119] In some embodiments, the LNP includes cationic lipids, non-limiting examples of which include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), and 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA). ), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or Examples include its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen19-yl4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-')amino)ethyl)(2hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazandiyl)didodecane-2-ol (Tech G1), or mixtures thereof.

[0120] In some embodiments, the LNP comprises one or more molecules selected from polyethyleneimine (PEI), poly(lactic acid-coglycolic acid) (PLGA), and N-acetylgalactosamine (GalNAc), which are suitable for hepatic delivery. In some embodiments, the LNP comprises, for example, a hepatic-targeting compound described in U.S. Patent No. 5,985,826, which is incorporated herein in whole by reference. GalNAc is known to target the asialoglycoprotein receptor (ASGPR) expressed in mammalian hepatocytes. See Hu et al. Protein Pept Lett. 2014;21(10):1025-30.

[0121] In some cases, the gene transfection constructs of the present disclosure may be formulated or compounded with PEI, or derivatives thereof such as polyethyleneimine-polyethylene glycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethylene glycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.

[0122] In some embodiments, the LNP is a conjugated lipid, and non-limiting examples include, but are not limited to, polyethylene glycol (PEG)-lipids comprising PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be, for example, PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (C14), PEG-dipalmityloxypropyl (C16), or PEG-distearyloxypropyl (C18).

[0123] In some embodiments, the nanoparticles are particles having a diameter of less than about 1000 nm. In some embodiments, the nanoparticles of the Disclosure have a maximum dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In some embodiments, the nanoparticles of the Invention have a maximum dimension in the range of about 50 nm to about 150 nm, or about 70 nm to about 130 nm, or about 80 nm to about 120 nm, or about 90 nm to about 110 nm. In some embodiments, the nanoparticles of the Invention have a maximum dimension (e.g., diameter) of about 100 nm.

[0124] In some embodiments, the compositions according to the Disclosure may be delivered via in vivo recombination methods. In some embodiments, recombination according to the Disclosure may be carried out via ex vivo methods.

[0125] Accordingly, in some embodiments, methods are provided for preventing or reducing the rate of photoreceptor loss in a patient, comprising administering a composition according to any embodiment or combination of embodiments of the present disclosure to a patient in need thereof. The methods include delivering the composition via a preferred route, including administration by injection.

[0126] In some embodiments, the method and composition can provide permanent prevention or reduction of the rate of photoreceptor loss, thus reducing or eliminating the need for additional therapeutic agents. For example, in some embodiments, the method is carried out in the absence of steroid treatment. The method may be substantially non-immunogenic.

[0127] In some embodiments, the present invention provides an ex vivo gene therapy approach. Accordingly, in some embodiments, a method is provided for preventing or reducing the rate of photoreceptor loss in a patient, comprising (a) contacting cells obtained from a patient (autologous) or another individual (allogeneic) with a composition according to embodiments of the present disclosure, and (b) administering the cells to a patient in need.

[0128] In some embodiments, methods are provided for treating and / or alleviating hereditary macular degeneration (IMD), comprising administering a composition according to embodiments of the present disclosure to a patient in need thereof. In such in vivo methods, the composition is administered using any of the techniques described herein.

[0129] In some embodiments, the in vivo and ex vivo methods described herein can treat and slow the progression of various MDs, which are a heterogeneous group of diseases characterized by bilateral, symmetrical central vision loss. Examples of MDs include Stargardt disease, Best disease, X-linked retinoschisis, pattern dystrophy, Sorsby dystrophy, and autosomal dominant drusen. Best disease is an autosomal dominant condition associated with the disease-causing variant in Best1; X-linked retinoschisis (XLRS) is the most common form of juvenile-onset retinal degeneration in adolescent males; pattern dystrophy (PD) is a group of disorders characterized by a variable distribution of pigment deposits at the RPE level; Sorsby dystrophy (SFD) is a rare macular dystrophy that often results in bilateral central vision loss in the 50s; and autosomal dominant drusen (ADD) is an autosomal dominant condition characterized by drusen-like deposits in the macula, which may have a radial or honeycomb-like appearance. See Rahman et al., Br J Ophthalmol. 2020;104(4):451-460.

[0130] In some embodiments, an ex vivo method is provided for treating and / or alleviating IMD, comprising (a) contacting cells obtained from a patient or another individual with a composition according to embodiments of the present disclosure, and (b) administering the cells to a patient in need. In some embodiments, IMD is STGD. In some embodiments, STGD is STGD type 1 (STGD1). In some embodiments, STGD disease may be STGD type 3 (STGD3) or STGD type 4 (STGD4) disease.

[0131] In some embodiments, the IMD is characterized by a mutation in one or more of the following: ABCA4, ELOVL4, PROM1, BEST1, and PRPH2. In some embodiments, the ABCA4 mutation is an autosomal recessive mutation.

[0132] Mutations in ELOVL4 (extension of very long-chain fatty acids in protein 4) have been shown to cause STGD3, characterized by retinal degeneration. See Agbaga et al., PNAS September 2, 2008;105(35)12843-12848, and Zhang et al., Nat Genet. 2001;Jan;27(1):89-93. The clinical profile of STGD3 is very similar to that of STGD1.

[0133] PROM1 (prominin 1 gene) encodes pentaspan transmembrane glycoprotein, a protein localized to membrane protrusions. Yang et al., J Clin Invest. 2008;118(8):2908-2916. Mutations in the PROM1 gene have been shown to result in retinitis pigmentosa and Stargardt disease, and this gene is expressed from at least five surrogate promoters in a tissue-dependent manner. See, for example, Lonnroth et al., Int J Oncol. 2014;45(6):2208-2220.

[0134] The BEST1 gene provides instructions for the production of a protein called bethroffin-1, which appears to play a crucial role in normal vision. Mutations in the BEST1 gene cause retinal detachment and degeneration of photoreceptor cells (PRs) due to primary channelopathy in adjacent RPE cells. See Guziewicz et al., PNAS March 20, 2018 115(12)E2839-E2848, and Petrukhin et al., Nature Genetics 1998;vol.19:241-247. Disease-causing variants in BEST1 are associated with Best disease (BD), the second most common MD, affecting approximately 1 in 10,000 people. Rahman et al., Br J Ophthalmol. 2020 Apr;104(4):451-460. The BEST1 sequence variant is also responsible for at least four other phenotypes, including adult yolkoid MD, autosomal dominant vitreous choriopathopathy, autosomal recessive bethrophinopathy, and retinitis pigmentosa. Id.

[0135] The PRPH2 (peripherin-2) gene encodes a PR-specific tetraspanin protein called retinal degeneration slow (RDS), and mutations in PRPH2 have been shown to cause morphologies of retinitis pigmentosa and macular degeneration. (Conley & Naash. Cold Spring Harb Perspect Med. 2014 Aug 28;4(11):a017376.) Mutations in PRPH2 have been identified in patients with Stargardt macular degeneration.

[0136] Pathogenic mutations in one or more of ABCA4, ELOVL4, PROM1, BEST1, and PRPH2 can be corrected using the described methods for treating and / or alleviating the associated macular dystrophy condition.

[0137] One of the advantages of ex vivo gene therapy is the ability to "sample" transduced cells before administration to the patient. This enhances efficacy and allows for safety checks before introducing cells(s) into the patient. For example, the transduction efficiency and / or clonality of the incorporated cells can be evaluated before the injection of the product. This disclosure provides compositions and methods that can be effectively used for ex vivo genetic recombination.

[0138] In some embodiments, any of the in vivo and ex vivo methods described herein improve a patient's distance vision. In some embodiments, the methods are substantially non-immunogenic.

[0139] In some embodiments, the method requires a single dose, which simplifies the delivery of the composition and improves the overall patient experience. Many patients affected by various IMD disorders are children, and delivering permanent, substantially non-immunogenic treatments according to some embodiments of the present disclosure as a single dose simplifies the therapy delivery process and reduces the burden on the patient.

[0140] As mentioned above, lipofuscin accumulation in the rPE is associated with the development of STGD, age-related macular degeneration, and other retinal diseases. Lipofuscin clumps, which are yellowish substances that form spots, accumulate in and around the macula, impairing central vision. The main component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E), but lipofuscin also contains other bis-retinoids. A2E is a fluorescent substance that accumulates in the lysosomes of the RPE of the eye with age or in some retinal disorders such as STGD. RPE lipofuscin contains A2E and an additional fluorophore-A2E double-bonded isomer, iso-A2E. Photochemical studies of A2E and iso-A2E have shown that they exist in a photo-equilibrium state of 44(A2E):1(iso-A2E). See Parish et al., Proc Natl Acad Sci USA. 1998;95(25):14609-13. A2E has been shown to cause the accumulation of lipofuscin-like fragments in the retinal pleoplasm (RPE). Mihai & Washington. Cell Death & Disease 5, e1348 (2014). A2E may be the cause of RPE fragments found in the human eye, which include lipofuscin-like bodies, late lysosomes, abnormal glycogen and lipid deposits, and inclusions exhibiting heterogeneous electron density. Id. Therefore, A2E drives retinal aging and associated degeneration. Vitamin A aldehyde (retinaldehyde), a chemical precursor of A2E, also plays a role in the degenerative process. Id.

[0141] Therefore, lipofuscin accumulation in the retina, e.g., the RPE and / or the Bruch membrane beneath it, can be treated or mitigated by reducing the levels of one or more of retinaldehyde, A2E, and iso-A2E. In some embodiments, the method reduces or prevents the formation of RPE fragments. In some embodiments, vitamin A dimer accumulation in the RPE and Bruch membrane (BM) can be treated or mitigated by reducing the levels of one or more of retinaldehyde, A2E, and iso-A2E.

[0142] Accordingly, in some embodiments, the method provides reducing one or more of retinaldehyde, A2E, and iso-A2E compared to the level of one or more of retinaldehyde, N-retinylidene-N-retinylethanolamine (A2E), and iso-A2E without administration of the composition. In some embodiments, the level of one or more of retinaldehyde, A2E, and iso-A2E is reduced (compared to the level of one or more of retinaldehyde, A2E, and iso-A2E without administration of the composition) by at least more than about 40%. In some embodiments, the method provides a reduction of more than about 40%, or more than about 50%, or more than about 60%, or more than about 70%, or more than about 80%, or more than about 90%.

[0143] In some embodiments, nucleic acid constructs encoding transposases are administered to the patient. The transposases may be derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo sapiens, and / or modified versions thereof.

[0144] In some embodiments, ex vivo methods for preventing or reducing photoreceptor loss in patients include contacting cells with nucleic acid constructs encoding transposases and / or manipulated versions thereof derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo sapiens, respectively.

[0145] In some embodiments, methods for preventing or reducing the rate of photoreceptor loss in patients are carried out in the absence of steroid therapy. The effectiveness of AAV-based gene therapy has been improved by reducing the immune response using steroids such as glucocorticoid steroids (e.g., prednisone). However, steroid therapy is not without side effects. The compositions and methods of this disclosure may be substantially non-immunogenic and therefore eliminate the need for steroid therapy.

[0146] In some embodiments, however, the method is carried out in combination with steroid treatment.

[0147] In some embodiments, the method may be used to administer the composition described herein in combination with one or more additional therapeutic agents. Non-limiting examples of additional therapeutic agents include one or more anti-vascular endothelial growth factor (VEGF) therapeutic agents, including aflibercept (Eylea), ranibizumab (Lucentis), and bevacizumab (Avastin). Additional therapeutic agents may include deuterated vitamin A and / or other vitamins, or nutritional supplements (e.g., beta-carotene, lutein, and zeaxanthin).

[0148] Administration may be intravitreous or intraretinal. In some embodiments, administration is to RPE cells and / or photoreceptors. Compositions for nonviral gene therapy according to this disclosure may be administered via various delivery routes, including administration by injection. In some embodiments, the injection is intravitreous or intraretinal. In some embodiments, the injection is subvitreous or subretinal. In some embodiments, the injection is sub-RPE.

[0149] In some embodiments, in vitro or ex vivo methods for treating and / or alleviating IMD provide improved distance visual acuity and / or a reduced rate of photoreceptor loss compared to the absence of treatment. In some embodiments, the methods result in an improvement of up to approximately 20 / 200 or more in best corrected visual acuity (BCVA).

[0150] In some embodiments, methods for treating and / or alleviating IMD result in improvement of the retinal or foveal shape, as measured by fundus autofluorescence (FAF) or spectral domain optical coherence tomography (SD-OCT). FAF is a non-invasive retinal imaging diagnostic technique used to provide a lipofuscin density map in the retinal pigment epithelium. See Madeline et al., Int J Retin Vitr 2,12(2016), Sepah et al., Saudi J Ophthalmol. 2014;28(2):111-116, and Sparrow et al., Investigative Ophthalmology & Visual Science September 2010;vol.51:4351-4357.

[0151] SD-OCT is an interferometric technique that provides depth-analyzed tissue structure information encoded by the magnitude and delay of backscattered light through spectral analysis of interference fringe patterns. (Yaqoob et al., Biotechniques, vol.39, No.6S; published Online: 30 May 2018.) Other imaging techniques, including scanning laser ophthalmoscopic imaging (SLO), fluorescence lifetime imaging (FLIO), and two-photon microscopy (TPM), can also be used. Images (one-eyed or bi-eyed) acquired using a suitable technique can be analyzed to evaluate patient parameters, including fluorescence intensity. For example, FAF, characterized by a general increase in autofluorescence intensity, is an indicator of Stargardt disease in its early stages. (Burke et al., Invest Ophthalmol Vis Sci. 2014; 55: 2841-2852.)

[0152] In some embodiments, the method results in the reduction or prevention of one or more of the following in a patient: metamorphopsia, blind spot, blurred vision, loss of depth perception, glare sensitivity, color vision impairment, and difficulty adapting to dim lighting (delayed scotropia).

[0153] In some embodiments, the method may be used to administer the composition described herein in combination with one or more additional therapeutic agents. Non-limiting examples of additional therapeutic agents include one or more of soraprazan, isotretinoin, dovesilate, 4-methylpyrazole, ALK-0019 (C20 deuterated vitamin A), fenretinide (a synthetic form of vitamin A), LBS-500, A1120, emixustat, fenofibrate, and abasin capta dopegol. In some embodiments, the method eliminates the need for additional therapeutic agents, which may be any of the above therapeutic agents.

[0154] In some embodiments, the method eliminates the need for steroid treatment.

[0155] In some embodiments, the compositions according to this disclosure include pharmaceutically acceptable carriers, excipients, or diluents.

[0156] Methods for formulating suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005, and a Series of Textbooks and Monographs (Dekker, NY) in the Drugs and the Pharmaceutical Sciences series. For example, suitable pharmaceutical compositions for injectable use may include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders, for the immediate preparation of sterile injection solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile, and the fluid must be easily drawn up by syringe. It must be stable under manufacturing and storage conditions and stored in a manner that protects against microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugar, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride in the composition. The inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition can result in sustained absorption of the injectable composition.

[0157] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent containing one or a combination of the components listed above, and then, if necessary, sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components different from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying, which yield powders of the active component and any desired additional components from a pre-sterilized filtered solution.

[0158] Therapeutic compounds may be prepared with carriers that will protect them from rapid release from the body, such as controlled-release formulations including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides (e.g., poly[1,3-bis(carboxyphenoxy)propane-co-sebacic acid](PCPP-SA) matrix, fatty acid dimer-sebacic acid (FAD-SA) copolymer, poly(lactide-co-glycolide)), polyglycolic acid, collagen, polyorthoesters, polyethylene glycol-coated liposomes, and polylactic acid may be used. Such formulations can be prepared using standard techniques or are commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared, for example, according to methods known to those skilled in the art, as described in U.S. Patent No. 4,522,811. For example, when administration to the surgical site is desirable, semi-solid, gelled, soft gel, or other formulations (including controlled-release formulations) may be used. Methods for preparing such formulations are known in the art and may include the use of biodegradable, biocompatible polymers. See, for example, Sawyer et al., Yale J Biol Med. 2006;79(3-4):141-152.

[0159] In embodiments, a method is provided for transforming cells using the gene transfection construct described herein in the presence of a transposase to produce stably transfected cells resulting from the stable integration of the gene of interest into the cell. In embodiments, the stable integration involves the introduction of polynucleotides into the cell's chromosomes or minichromosomes, thus becoming a relatively permanent part of the cell genome.

[0160] In embodiments, the present invention relates to determining whether a target gene, for example ABCA4, has been introduced into the host genome. In one embodiment, the method may include carrying out a polymerase chain reaction using a primer adjacent to the target gene, determining the size of the resulting amplified polymerase chain reaction product, and comparing the size of the resulting product with a reference size. If the size of the resulting product matches the expected size, the target gene has been successfully introduced.

[0161] In the embodiments, a host cell comprising a composition described herein (for example, a composition comprising, but not limited to, a gene transfection construct and / or a transposase) is provided. In the embodiments, the host cell is a prokaryotic or eukaryotic cell, for example, a mammalian cell.

[0162] In embodiments, a transgenic organism is provided which may include cells transformed by the method of the present disclosure. For example, the organism may be a mammal or an insect. If the organism is a mammal, examples of such organisms include, but are not limited to, mice, rats, monkeys, dogs, rabbits, etc. If the organism is an insect, examples of such organisms include, but are not limited to, fruit flies, mosquitoes, tobacco budworm larvae, etc.

[0163] The composition may be contained within a container, kit, pack, or dispenser, along with instructions for administration.

[0164] Also provided herein are kits comprising i) any of the aforementioned gene transfection constructs of the present invention and / or any of the aforementioned cells of the present invention, and ii) a container. In certain embodiments, the kit further includes instructions for using the kit. In certain embodiments, any of the aforementioned kits may further include a recombinant DNA construct comprising a nucleic acid sequence encoding a transposase.

[0165] The present invention can be further described by the following non-limiting embodiments.

[0166] definition As used herein, “a,” “an,” or “the” may mean one or more.

[0167] Furthermore, when used in relation to a referenced numerical value, the term "approximately" means adding or subtracting up to 10% of that referenced numerical value. For example, the phrase "approximately 50" encompasses a range from 45 to 55.

[0168] When used in connection with medical applications, an "effective dose" is the amount that is effective in producing a measurable treatment, prevention, or reduction in the rate of onset of the disease in question.

[0169] Where used herein, all compositional percentages refer to the total weight of the composition unless otherwise specified. Where used herein, the word “include” and its variations are intended to be non-limiting so that the enumeration of items in the list does not exclude other similar items that may also be useful in the compositions and methods of the Art. Similarly, the terms “can” and “may,” and their variations, are intended to be non-limiting so that an enumeration of which embodiments can or may include certain elements or features does not exclude other embodiments of the Art that do not include those elements or features.

[0170] The open term “comprising,” as a synonym for including, containing, or having, is used herein to describe and claim an invention, the present invention, or an embodiment of the present invention, and may instead be described using alternative terms such as “consisting of” or “essentially consisting of.”

[0171] As used herein, the words “preferred” and “preferred” refer to embodiments of the technology that provide a particular benefit under certain circumstances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the enumeration of one or more preferred embodiments does not mean that other embodiments are unhelpful and is not intended to exclude other embodiments from the scope of the Art.

[0172] The amount of the compositions described herein required to achieve a therapeutic effect may be determined empirically by conventional procedures for a particular purpose. Generally, to administer a therapeutic agent for a therapeutic purpose, the therapeutic agent is administered in a pharmacologically effective dose. "Pharmacologically effective dose," "pharmacologically effective dose," "therapeutic effective dose," or "effective dose" refers to an amount sufficient to produce a desired physiological effect or to achieve a desired result, particularly for the treatment of a disorder or disease. As used herein, an effective dose may include, for example, an amount sufficient to delay the onset of symptoms of a disorder or disease, to alter the course of symptoms of a disorder or disease (e.g., to slow the progression of symptoms of a disease), to reduce or eliminate one or more symptoms or signs of a disorder or disease, and to reverse the symptoms of a disorder or disease. Therapeutic benefits also include stopping or slowing the progression of an underlying disease or disorder, whether or not improvement is achieved.

[0173] Effective dose, toxicity, and therapeutic efficacy are, for example, LD50. 50 (Lethal dose for approximately 50% of the population), and ED 50The effective dose (the therapeutic dose in approximately 50% of the population) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage may vary depending on the dosage form used and the route of administration utilized. The dose-to-toxicity ratio is the therapeutic index, which is the LD50. 50 / ED 50 It can be expressed as a ratio. In some embodiments, compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can be initially estimated from an in vitro assay, including, for example, a cell culture assay. The dose can also be determined in a cell culture or in a suitable animal model using IC. 50 To achieve a circulating plasma concentration range including the specified components, formulations may be used in animal models. The levels of the described compositions in plasma may be measured, for example, by high-performance liquid chromatography. The effect of any particular dose may be monitored by a suitable bioassay. The dose is determined by a physician and may be adjusted, if necessary, to suit the observed effect of the treatment.

[0174] As used herein, “method of treatment” is equally applicable to the use of a composition for treating a disease or disorder described herein, and / or the use of a composition for use in the manufacture of a drug for treating a disease or disorder described herein. [Examples]

[0175] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention. In addition, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the technical scope of the present invention.

[0176] Example 1 - Design of a transposon expression vector The non-viral transposon expression vectors schematically shown in Figures 1A-1I are designed and cloned for in vitro, in vivo, and ex vivo studies of transfection, translocation efficacy, and expression in retinal cell lines.

[0177] Figure 1A shows a phosphoglycerate kinase (PGK)-GFP transposon construct containing a PGK promoter, used to determine the transposon (Tn):transposase (Ts) ratio and transposition efficacy by GFP fluorescence-activated cell sorting (FACS). Figures 1B and 1C show transposon constructs used to evaluate the efficacy of the retinal pigment epithelial promoter (RPEP) (Figure 1B) and the photoreceptor promoter (PRP) (Figure 1C) when they selectively maximize GFP expression (as determined by FACS) and copy number (as determined using droplet digital PCR (ddPCR) or quantitative PCR (qPCR) techniques).

[0178] Figure 1D shows a BEST-RPEP construct that can be used to assess ABCA4 expression by flow cytometry and ABCA4 copy number (e.g., using ddPCR or qPCR). Figure 1E similarly shows a BEST-PRP construct that can be used to assess ABCA4 expression by flow cytometry and ABCA4 copy number (e.g., using ddPCR or qPCR).

[0179] The transposon constructs shown in Figures 1F, 1G, 1H, and 1I will be used in the human iPSC and transgenic abca4- / - mouse studies discussed below. The constructs in Figures 1F and 1H contain the BEST-RPEP promoter, and the constructs in Figures 1G and 1I contain the BEST-PRP promoter.

[0180] Example 2 - Determination of the effect of different transposon (Tn):transposase (Ts) ratios The effect of different transposon (Tn):transposase (Ts) ratios is evaluated by stable green fluorescent protein (GFP) expression (>14 days) in retinal and non-retinal cell lines. The study includes establishing cultures of human retinal adherent cell lines (ARPE-19, RPE-1) and mouse photoreceptor cell line (661W). HEK293 (ABCA4 negative) and HeLa (ABCA4 positive) cell cultures are used as controls. In this example, the transposon vector shown in Figure 1A may be used. LEAPIN transposase technology (ATUM, Newark, CA) may also be used.

[0181] Different conditions for electroporation of established cell lines can be studied using transposon vectors expressing GFP driven by a constitutive promoter, for example, vectors designed as shown in Figure 1A. Cells can be transfected with gene transfect constructs having two, three, or more than three different Tn:Ts ratios. Conditions resulting in a culture containing a relatively large number of GFP-positive cells can be maintained in the culture by passage for 14 days. In these studies, 14 days is expected to be a sufficient period to cause transient loss of GFP expression. The transfected cultures are analyzed after 14 days by flow cytometry to determine the percentage of cells retaining GFP expression as a measure of stable expression. Cultures with more than 40% GFP expression can be analyzed by ddPCR or qPCR to determine the copy number.

[0182] Example 3 - Selection of RPE-specific promoter and photoreceptor promoter In this study, promoters are evaluated and selected based on their ability to induce specific and high levels of GFP expression in retinal cell lines derived from retinal pigment epithelium (RPE) or photoreceptors. In this example, transposon vectors shown in Figures 1B and 1C may be used. RPE (VMD2, IRBP, RPE65), photoreceptors [PDE, rhodopsin kinase (Rk or GRK1), CAR (cone arrestin), RP1, L-opsin], and nonspecific promoters (PGK, CAG, CMV) are cloned into transposon vectors to drive GFP expression. Using certain conditions (which can be identified as described in Example 2), the resulting constructs are transfected into two human RPE cell lines (ARPE-19, RPE-1), a mouse-derived photoreceptor cell line (661W), and two control cell lines (HEK293, HeLa). Relative expression levels are determined qualitatively (visually by eye or by flow cytometry), and promoters that are strongly expressed in RPE or photoreceptor cell lines and relatively low in control cells are considered retina-specific for the purpose of this assay.

[0183] In this study, ARPE-19, RPE-1, and 661W transfections using promoters considered to be RPE-specific and photoreceptor-specific were subcultured for approximately 14 days and analyzed by flow cytometry afterward. Different levels of GFP expression were considered as a measure of the relative strength of these promoters in the studied cell lines.

[0184] Example 4 - Demonstration of stable expression of human ABCA4 driven by a retina-specific promoter in retinal and non-retinal cell lines. HEK293 cells will be used to identify endogenous ABCA4-positive and negative controls. Transfected HEK293 cells have been shown to exhibit similar transport function to photoreceptor cells (Sabirzhanova et al., J Biol Chem 2015;290:19743-55, Quazi et al., Nat Commun 2012;3:925), and RT-PCR does not show endogenous ABCA4 expression in untransfected HEK293 (protein atlas), therefore HEK293 cells will be used. See Bauwens et al., Genet Med 2019;21:1761-71. In addition, HeLa cells express endogenous ABCA4 (protein atlas). To confirm that HEK293 cells can be used as a negative control and HeLa cells can be used as a positive control, cells will be labeled with an antibody against human ABCA4 using standard methods. Labeled cells are quantified by flow cytometry and visualized using immunocytochemistry techniques. In addition, the mRNA level of endogenous ABCA4 is quantified by ddPCR or RTqPCR.

[0185] In this study, RPE-specific promoters and photoreceptor promoters selected as described in Example 3 may be used. The selected promoters are cloned into transposon vectors, such as the transposon vectors shown in Figures 1D and 1E, to drive the expression of both human and mouse ABCA4. The transposon constructs are transfected using transfection conditions to human retinal adherent cell lines (ARPE-19, RPE-1) and photoreceptor cell line (661W), for example, as determined as described in Example 2. HEK293 (ABCA4-negative) and HeLa (ABCA4-positive) cells are used as untransfected controls. Cells are cultured for approximately 14 days by passage. After this period, the cultured cells are labeled with an anti-ABCA4 antibody, and the percentage of cells expressing ABCA4 is quantified by flow cytometry. The percentage of fluorescent cells analyzed by flow cytometry is used to monitor transfection efficiency.

[0186] In addition, the presence of the ABCA4 transcript is quantified by ddPCR or RT-qPCR using known methods.

[0187] Example 5 - Generation of transposon (Tn) and transposase (Ts) constructs for study in STGD patient iPSCs, transgenic abca4- / - mice, and large animal models. The objective of this study is to identify lead transposon (Tn) and transposase (Ts) constructs for in vivo, in vitro, and ex vivo testing in individual patient pluripotent stem cells (iPSCs), transgenic abca4- / - mice, and large animal models (e.g., abcd4 mutant Labrador Retrievers). Vector constructs shown in Figures 1F, 1G, 1H, and 1I may be used. Constructs may include luciferase (pLuc) or GFP genes, as well as photoreceptor and RPE-specific promoters.

[0188] This study will demonstrate transmissibility by conducting in vivo studies in Abca4- / - transgenic mice or other animals using intraretinal delivery of transduced cells. Therefore, intraretinal injection of the construct (using the mouse Abca4a gene) into Abca4a- / - mice will be performed to demonstrate phenotypic correction. To demonstrate the safety, tolerability, and efficacy of the appropriate construct and administration procedure, a similar experiment will be designed in naturally occurring Abca4- / - Labrador Retriever dogs (see Makelainen et al., PLoS Genet 2019;15:e1007873). Biodistribution, dose-response, pharmacokinetic, pharmacodynamic, safety, and pathological studies will be conducted in Abca4- / - Labrador Retriever dogs (or other canid models) or non-human primates (cynomolgus monkeys, macaca fascicularis) in a GLP environment to reverse retinal pathology.

[0189] Example 6 – Use of MLT transposase for transposition of 661W mouse photoreceptor cells The objective of this study was to determine lipofection conditions for transposition of 661W photoreceptor cells using green fluorescent protein (GFP) driven by a CAG-GFP donor construct, using the MLT transposase (RNA helper) of this disclosure.

[0190] 661W cells were transfected with a donor transposon DNA (CAG-GFP):MLT transposase 1 and MLT transposase 2 mRNA (donor DNA:helper RNA) ratio of 10ug:5ug. Conditions that yielded a relatively large number of GFP-positive cells were maintained in the culture by passage for 7–14 days. 14 days was expected to be a sufficient period to cause transient loss of GFP expression. Cells were imaged at different time points after transfection to monitor expression and determine the conditions for eliminating GFP expression within 14 days. Optimal transfected cultures were imaged and analyzed by flow cytometry to determine the percentage of cells retaining GFP expression. Cultures with more than 40% GFP expression were analyzed by qPCR to determine the copy number.

[0191] In this study, the following reagents were used: donor DNA (>1 ug / ul, 300 ul, 1x TE buffer, endotoxin-free, sterile), helper RNA MLT transposase 1 (>500 ng / ul, 100 ul, nuclease-free water, sterile), and helper RNA MLT transposase 2 (>500 ng / ul, 100 ul, nuclease-free water, sterile). The reagents used in this study are shown in Table 1.

[0192] [Table 1]

[0193] result Figure 3 shows GFP expression in 661W mouse photoreceptor cells 24 hours after transfection using varying lipofection reagents and either MLT transposase 1 or MLT1 (containing the amino acid sequence of SEQ ID NO: 13) or MLT transposase 2 or MLT2 (containing the amino acid sequence of SEQ ID NO: 15) of the present disclosure, compared to untransfected cells.

[0194] Figure 4 shows the stable integration of donor DNA (GFP) via translocation in mouse photoreceptor cell line 661W after four passages over 15 days.

[0195] Figure 5 shows the results of FACS analysis of the stable integration of donor DNA (GFP) via translocation in mouse photoreceptor cell line 661W on day 15.

[0196] As shown in Figure 3, all untransfected cells showed no GFP expression. Using MLT transposase 1 for transfection resulted in GFP expression present in 661W cells after 24 hours. The same was observed with MLT transposase 2 (Figure 3). MAX+CAG-GFP expressed little GFP with either MLT transposase 1 or MLT transposase 2 transfection. L3+CAG-GFP expressed a small amount of GFP 24 hours after transfection. LTX+CAG-GFP expressed a moderate amount of GFP 24 hours after transfection. With LTX, 40-50% of cells expressed GFP 24 hours after transfection.

[0197] GFP expression persisted in transfected cells only under conditions where helper RNA (MLT transposase 1 or MLT transposase 2) was co-expressed with GFP donor DNA for an extended period (Figure 4). After four passages of cells over a 15-day period, the donor-only DNA lost its expression, while donor DNA containing either MLT transposase 1 or MLT transposase 2 (GFP) continued to express GFP.

[0198] FACS analysis was performed on day 15 for all four conditions (Figure 5). The FACS data suggest that MLT transposase 1 exhibits higher GFP expression compared to cells co-transfected with GFP donor DNA along with MLT transposase 2. Both MLT transposase 1 and MLT transposase 2 showed significantly higher GFP expression compared to conditions with donor DNA alone or untransfected cells.

[0199] In summary, these data indicate that, in lipofectamine, LTX (lipofectamine with PLUS reagent) is an effective reagent for transfecting 661W cells containing CAG-GFP and either MLT transposase 1 or MLT transposase 2. Both MLT transposase 1 and MLT transposase 2 exhibited similar GFP expression 24 hours after transfection, thus resulting in stable integration of donor DNA by transfection. In the 661W cell type, MLT transposase 1 showed more effective transfection compared to MLT transposase 2.

[0200] Example 7 - Transfection of ARPE-19 human retinal pigment epithelial cells using MLT transposase The objective of this study was to evaluate the effects of two different helper RNA transposases (MLT transposase 1 and MLT transposase 2) versus helper RNA transposases (Ts) on stable green fluorescent protein (GFP) expression in retinal cell lines using a CAG-GFP donor construct.

[0201] ARPE-19 cells were transfected with a ratio of 10 μg:5 μg donor transposon DNA (CAG-GFP):MLT transposase 1 and MLT transposase 2 mRNA (donor DNA:helper RNA). Conditions resulting in a culture containing a relatively large number of GFP-positive cells were maintained in the culture by passage for 7–14 days. 14 days was expected to be a sufficient period to cause transient loss of GFP expression. Cells were imaged at different time points after transfection to monitor expression and determine the conditions for eliminating GFP expression within 14 days. Optimal transfected cultures were imaged and analyzed by flow cytometry to determine the percentage of cells retaining GFP expression.

[0202] In this study, the following reagents were used: donor DNA (>1 ug / ul, 300 ul, 1x TE buffer, endotoxin-free, sterile), helper RNA MLT transposase 1 (>500 ng / ul, 100 ul, nuclease-free water, sterile), and helper RNA MLT transposase 2 (>500 ng / ul, 100 ul, nuclease-free water, sterile). The reagents used in this study are shown in Table 2.

[0203] [Table 2]

[0204] Figure 6 shows GFP expression in ARPE-19 cells 24 hours after transfection. In this experiment, ARPE-19 cells were seeded in 24-well plates. After 24 hours, the cells were transfected with three different transfection systems: L3 (Lipofectamine 3000, ThermoFisher catalog number L3000-001), LTX (Lipofectamine LTX&PLUS, ThermoFisher catalog number A12621), and MAX (Lipofectamine Messenger MAX, ThermoFisher catalog number LMRNA001). Subsequently, 24 hours after transfection, the cells were imaged for GFP.

[0205] Figure 7 shows higher-resolution images of the visible GFP expression of MLT transposase 1 and MLT transposase 2 24 hours after transfection.

[0206] Figure 8 shows the stable integration of donor DNA (GFP) in the photoreceptor cell line ARPE19 using MLT transposase 2.

[0207] Figure 9 shows that FACS analysis reveals stable GFP expression from the ARPE19 cell line after four generations of cell division.

[0208] As shown in the results of this study, all untransfected cells showed no GFP expression, as can be seen in Figure 6. Only L3 and CAG-GFP expressed the presence of GFP 24 hours after transfection. Only LTX and CAG-GFP expressed the greatest amount of GFP 24 hours after transfection. MAX and CAG-GFP also showed moderate GFP expression 24 hours after transfection. When MLT transposase 1 was added to the lipofection reagent and CAG-GFP, GFP expression was still present in the cells after 24 hours, but not as much as with the lipofection reagent and CAG-GFP alone. The same was true for MLT transposase 2 (see Figure 6). When comparing lipofection reagent + DNA containing both MLT transposase 1 (left column) and MLT transposase 2 (right column) side by side, MLT transposase 1 and MLT transposase 2 were similar in GFP expression efficiency, as can be seen in Figure 7.

[0209] It was found that donor DNA and GFP were stably incorporated into the ARPE19 cell line only when co-overexpressed with either MLT transposase 1 or MLT transposase 2 as a helper. To confirm that the visible signal was not transient, GFP expression was investigated over 15 days and four passages. The donor-only condition lost GFP expression after the second passage (see Figure 8).

[0210] Flow cytometry analysis revealed that MLT transposase 2 was significantly more effective in the stable transfer of donor (GFP) compared to other conditions, such as untransfected or donor only. MLT transposase 1 also appeared to be effective in the stable incorporation of GFP (Figure 9).

[0211] Lipofectamine & PLUS was an efficient lipofection reagent when using CAG-GFP alone, as well as when using both CAG-GFP and either MLT transposase 1 or MLT transposase 2. Both MLT transposase 1 and MLT transposase 2 exhibited similar GFP expression rates in these ARPE-19 cells. These data indicate that both MLT transposase 1 and MLT transposase 2 are efficient in the stable transfer of donor DNA into the genome. However, MLT transposase 2 is more effective than MLT transposase 1 in the stable integration of donor DNA in ARPE19 cell lines.

[0212] Example 8 - Mouse in vivo subretinal LNP dose pharmacodynamics using donor DNA (CAG-GFP) / MLT transposase The objective of this study was to analyze GFP expression levels in the mouse retina after subretinal injection of two doses (high and low) of lipid nanoparticle (LNP) formulations containing nucleic acids encoding donor DNA (CAG-GFP) and helper RNA (MLT transposase 2 or MLT2).

[0213] In this study, GFP expression in the mouse retina was measured after subretinal injection of two doses of lipid nanoparticle formulations containing donor DNA (CAG-GFP) and helper RNA (MLT transposase 2 or MLT2) in a 2:1 ratio. The "high" dose was 500 ng / uL (333 ng donor DNA / 166 ng helper RNA), and the "low" dose was 250 ng / uL (166 ng donor DNA / 83 ng helper RNA).

[0214] Retinal GFP expression in the photoreceptor layer and RPE cell layer was measured by immunohistochemistry (IHC).

[0215] Donor DNA (CAG-GFP) and MLT transposase 2 (MLT with S8P / C13R mutation), encapsulated together in lipid nanoparticles, were injected into the left eye. Only the donor DNA encapsulated in lipid nanoparticles was injected into the right eye. The goal was to demonstrate that MLT transposase 2 can transfect ARPE-19 cells into the retina without causing cell damage.

[0216] In this study, DNA encoding CAG-GFP (VB200819-1024gzm) and RNA encoding MLT transposase 2 (VB200926-1055qkq) were used. The LNP preparations contained cationic lipids, cholesterol, phospholipids, and PEG lipids. Table 2 contains information about the mice used in this experiment.

[0217] [Table 3]

[0218] result Images of mouse eyes were captured using the Phoenix MICRON IV® Retinal Imaging Microscope, a fundus imaging microscope.

[0219] Figures 10A and 10B show images of the left eye (Figure 10A) and right eye (Figure 10B) of mouse 1-1L injected with PBS.

[0220] Figures 11A, 11B, 11C, and 11D show images of the right eye of mice 3-1L and 3-1R injected with DNA only (Figures 11A and 11C), and the left eye of mice 3-1L and 3-1R injected with donor DNA and MLT2 (Figures 11B and 11D).

[0221] Figures 12A and 12B show images of the right eye of mouse 4-1R injected with donor DNA (Figure 12A) and MLT2 (Figure 12B).

[0222] Figures 13A and 13B show images of the right eye of a mouse 4-NP injected with donor DNA only (Figure 13A), and the left eye injected with both donor DNA and MLT2 (Figure 13B).

[0223] Figures 14A and 14B show images of the right eye of mouse 4-1L injected with donor DNA only (Figure 14A), and the left eye injected with both donor DNA and MLT2 (Figure 14B).

[0224] Figures 15A and 15B show images of the right eye of a mouse 5-BP injected with donor DNA only (Figure 15A), and the left eye injected with both donor DNA and MLT2 (Figure 15B).

[0225] Figure 16 shows the general setup for this study, and also indicates that the images were taken 21 days after subretinal injection. Figure 17 shows images of the left and right eyes (top and bottom panels, respectively) of mice taken 21 days after subretinal injection, with or without the MLT transposase used for transfection ("+MLT"). In Figure 17, the right eye is the control (donor DNA only) and the left eye is the treated eye (donor DNA + MLT2 transposase).

[0226] Figures 10A, 10B, 11A-11D, 12A, 12B, 13A, 13B, 14A, 14B, 15A, 15B, and 17 show images of the eyes of mice treated with a high dose of 500 ng / uL.

[0227] The results of this study demonstrate that subretinal injection of MLT transposase 2 encapsulated with donor DNA (CAG-GFP in this example) does not adversely affect the eyes of mice. As shown in Figures 14A and 14B, both eyes showed no visible damage and exhibited GFP expression 7 days after subretinal injection. Some variability in surgical efficiency was observed between animals and between the left and right eyes of the same animal.

[0228] In this study, we determined that the MLT transposase dose that leads to successful gene transfer from donor DNA is 500 ng / uL (333 ng of DNA / 166 ng of RNA).

[0229] In conclusion, this study demonstrates positive expression of the transgene (green fluorescent protein (GFP), used as the transgene example) upon injection of LNP under the retina of the eye. Transgene expression continued until day 21 (see Figure 17), demonstrating the feasibility of this approach for therapeutic use.

[0230] Equivalents While the present invention has been described in relation to its specific embodiments, further modifications are possible, and this application generally covers any variations, uses, or adaptations of the present invention in accordance with the principles of the invention, and should be understood to include deviations from the disclosure that are known or included in practice in the art to which the invention belongs, and that may be applied to the essential features described above, as well as deviations from the disclosure that are included in the scope of the appended claims.

[0231] Those skilled in the art will be able to recognize or confirm numerous equivalents of the specific embodiments described herein by means of simple, routine experiments. Such equivalents are intended to be included within the scope of the following claims.

[0232] Built-in by reference All patents and publications referenced herein are incorporated herein by reference in their entirety.

[0233] The publications discussed herein have been disclosed independently prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention is not entitled to precede such publications by prior art.

[0234] Where used herein, all headings are solely for the structural purposes of this specification and are not intended to limit disclosure in any way. The content of any individual section may be equally applicable to all sections.

Claims

1. A composition comprising a gene transfer construct, (a) A nucleic acid encoding an ATP-binding cassette subfamily A member 4 (ABC) transporter (ABCA4) protein, or a functional fragment thereof, (b) Retinal-specific promoter and (c) The composition comprising a nonviral vector having one or more transposase recognition sites and one or more inverted terminal repeats (ITRs) or end sequences.

2. The composition according to claim 1, wherein the gene transfer construct comprises DNA or RNA.

3. The composition according to claim 1 or 2, wherein the gene transfer construct is codon-optimized.

4. The composition according to any one of claims 1 to 3, wherein the ABCA4 protein is human ABCA4 protein or a functional fragment thereof.

5. The composition according to claim 4, wherein the nucleic acid encoding the human ABCA4 protein or a functional fragment thereof comprises a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 1, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

6. The composition according to claim 4, wherein the nucleic acid encoding the human ABCA4 protein or a functional fragment thereof comprises the nucleotide sequence of SEQ ID NO: 2, or a variant having at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

7. The composition according to any one of claims 1 to 6, wherein the retina-specific promoter is a human promoter.

8. The composition according to any one of claims 1 to 7, wherein the retina-specific promoter is optionally selected from a retina pigment epithelium-specific 65 kDa protein (RPE65) promoter, a photoreceptor-interretinoid-binding protein (IRBP) promoter, and a vitiligo macular dystrophy 2 (VMD2) promoter, or optionally selected from a photoreceptor promoter, PDE, rhodopsin kinase (GRK1), CAR (cone arrestin), RP1, and L-opsin, or a functional fragment of a variant having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

9. The composition according to any one of claims 1 to 8, wherein the promoter is a CMV enhancer, a chicken beta-actin promoter, and a rabbit beta-globin splice acceptor site (CAG), which optionally comprises a functional fragment of the nucleic acid sequence of Sequence ID No. 16, or a variant having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

10. The composition according to claim 8, wherein the RPE promoter comprises a functional fragment of a variant having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

11. The composition according to claim 8, wherein the photoreceptor promoter comprises a functional fragment of a variant having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, or at least about 98% identity thereto.

12. The composition according to any one of claims 1 to 11, wherein the nonviral vector is a DNA plasmid.

13. The composition according to claim 12, wherein the DNA plasmid comprises one or more insulator sequences that prevent or mitigate the activation or inactivation of nearby genes.

14. The ITR or the end sequence is optionally a piggyBac-like transposon containing a TTAA repeat sequence, and / or The composition according to any one of claims 1 to 13, wherein the ITR or the end sequence is adjacent to the nucleic acid encoding the ABCA4 protein.

15. The composition according to any one of claims 1 to 14, wherein the nonviral vector further comprises a transposase, optionally, a nucleic acid construct encoding an RNA transposase plasmid.

16. The composition according to any one of claims 1 to 14, further comprising a nucleic acid construct encoding a DNA transposase plasmid or an in vitro transcription mRNA transposase.

17. The composition according to claim 15 or 16, wherein the transposase is capable of excising and / or transposing the gene from the gene-transformed construct.

18. The transposase is Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus. discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo The composition according to claim 17, wherein the transposase is derived from and / or an engineered version thereof, and / or the transposase specifically recognizes the ITR or the end sequence.

19. The composition according to any one of claims 1 to 18, wherein the gene is transposable in the presence of a transposase.

20. The composition according to any one of claims 1 to 19, wherein the composition is in the form of lipid nanoparticles (LNPs).

21. The composition according to claim 20, comprising one or more lipids selected from 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2K), and 1,2-distearol-sn-glycerol-3-phosphocholine (DSPC).

22. The composition according to claim 20 or 21, comprising one or more molecules selected from polyethyleneimine (PEI), poly(lactic acid-coglycolic acid) (PLGA), and N-acetylgalactosamine (Gal-Nac).

23. Isolated cells comprising the composition according to any one of claims 1 to 22.

24. A method for preventing or reducing the rate of photoreceptor loss in a patient, comprising administering a composition according to any one of claims 1 to 22 to a patient in need thereof.

25. A method for preventing or reducing the rate of photoreceptor loss in a patient, (a) Contacting cells obtained from a patient or another individual with the composition according to any one of claims 1 to 22, (b) The method comprising administering the cells to a patient in need thereof.

26. The method according to claim 24 or 25, wherein the method improves the patient's distance visual acuity.

27. The method according to claim 24 or 25, wherein the method provides for selectively reducing one or more of retinaldehyde, A2E, and iso-A2E by more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% compared to the level of one or more of retinaldehyde, N-retinylidene-N-retinylethanolamine (A2E), and iso-A2E that is not administered.

28. The method according to claim 24 or 25, wherein the method optionally reduces or prevents lipofuscin accumulation in the RPE and / or Bruch membrane in the retina.

29. The method according to any one of claims 24 to 28, wherein the method is carried out in the absence of steroid treatment.

30. The method according to any one of claims 24 to 29, wherein the method is substantially non-immunogenic.

31. The method according to any one of claims 24 to 30, wherein the prevention of photoreceptor loss or the reduction of its rate is permanent.

32. The method according to any one of claims 24 to 31, wherein the method requires a single dose.

33. The method according to any one of claims 24 to 32, wherein the method reduces or prevents the formation of retinal pigment epithelium (RPE) fragments.

34. The method according to any one of claims 24 to 33, further comprising optionally administering a nucleic acid construct encoding a transposase derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo sapiens, and / or an engineered version thereof.

35. The method according to any one of claims 24 to 33, further comprising optionally contacting the cells with a nucleic acid construct encoding a transposase derived from Bombyx mori, Xenopus tropicalis, or Trichoplusia ni, and / or an engineered version thereof.

36. The method according to any one of claims 24 to 35, wherein the administration is performed intravitreously, intraretinally, subvitreously, or subretinally.

37. The method according to any one of claims 24 to 36, wherein the administration is to RPE cells and / or photoreceptors.

38. The method according to any one of claims 24 to 37, wherein the administration is by injection.

39. The method according to any one of claims 34 to 38, wherein the ratio of the nucleic acid encoding the ABCA4 protein or a functional fragment thereof to the nucleic acid construct encoding the transposase is about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:

5.

40. The method according to any one of claims 34 to 39, wherein the ratio of the nucleic acid encoding the ABCA4 protein or a functional fragment thereof to the nucleic acid construct encoding the transposase is about 2:

1.

41. A method for treating and / or alleviating hereditary macular degeneration (IMD), comprising administering a composition according to any one of claims 1 to 22 to a patient in need thereof.

42. A method for treating and / or alleviating hereditary macular degeneration (IMD), (a) Contacting cells obtained from a patient or another individual with the composition according to any one of claims 1 to 22, (b) The method comprising administering the cells to a patient in need thereof.

43. The method according to claim 41 or 42, wherein the IMD is STGD, and the STGD disease is optionally STGD type 1 (STGD1).

44. The method according to any one of claims 41 to 43, wherein the IMD is characterized by one or more mutations in one or more of ABCA4, ELOVL4, PROM1, BEST1, and PRPH2, and the ABCA4 mutation is optionally an autosomal recessive mutation.

45. The method according to any one of claims 41 to 44, wherein the method provides improved distance visual acuity and / or a reduction in the rate of photoreceptor loss compared to the absence of treatment.

46. The method according to any one of claims 41 to 45, wherein the method results in an improvement of up to approximately 20 / 200 in best corrected visual acuity (BCVA).

47. The method according to any one of claims 41 to 45, wherein the method results in an improvement in the shape of the retina or fovea as measured by fundus autofluorescence (FAF) or spectral domain optical coherence tomography (SD-OCT).

48. The method according to any one of claims 41 to 47, wherein the method results in a reduction or prevention of one or more of the following in the patient: wavy vision, blind spot, blurred vision, loss of depth perception, sensitivity to glare, color vision impairment, and difficulty adapting to dim lighting (delayed adaptation to darkness).

49. The method according to any one of claims 41 to 48, wherein the method eliminates the need for steroid treatment.

50. The method according to any one of claims 41 to 49, wherein the method improves the patient's distance visual acuity.

51. The method according to any one of claims 41 to 50, wherein the method is substantially non-immunogenic.

52. The method according to any one of claims 41 to 51, wherein the treatment and / or relief is permanent.

53. The method according to any one of claims 41 to 52, wherein the method requires a single dose.

54. The method according to any one of claims 41 to 53, wherein the method reduces or prevents the formation of retinal pigment epithelium (RPE) fragments.

55. The method according to any one of claims 41 to 54, further comprising optionally administering a nucleic acid construct encoding a transposase derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo sapiens, and / or an engineered version thereof.

56. The method according to any one of claims 41 to 55, wherein the administration is performed intravitreously or intraretinally.

57. The method according to any one of claims 41 to 56, wherein the administration is to RPE cells and / or photoreceptors.

58. The method according to any one of claims 41 to 57, wherein the administration is by injection.

59. The cells are optionally Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus. aegyptiacus, Phyllostomus discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo The method according to any one of claims 42 to 54, further comprising contacting with a nucleic acid construct encoding a transposase derived from sapiens and / or an engineered version thereof.

60. The method according to any one of claims 55 to 59, wherein the ratio of the nucleic acid encoding the ABCA4 protein or a functional fragment thereof to the nucleic acid construct encoding the transposase is about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:

5.

61. The method according to any one of claims 55 to 60, wherein the ratio of the nucleic acid encoding ABCA4 or a functional fragment thereof to the nucleic acid construct encoding the transposase is about 2:

1.

62. A composition comprising a gene transfer construct, (a) A nucleic acid encoding an ATP-binding cassette subfamily A member 4 (ABC) transporter (ABCA4) protein, or a functional fragment thereof, (b) CAG promoter and (c) A nonviral vector comprising one or more transposase recognition sites and one or more inverted terminal repeats (ITRs) or end sequences, The composition wherein the ABCA4 protein is human ABCA4 or a functional fragment thereof encoded by the nucleotide sequence of SEQ ID NO: 2, or a variant having at least about 95% identity thereto.

63. A method for treating and / or alleviating hereditary macular degeneration (IMD), (a) Contacting cells obtained from a patient or another individual with the composition described in claim 62, The cells are of Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus. discolor, Myotis myotis, Myotis lucifugus, Pteropus vampyrus, Pipistrellus kuhlii, Pan troglodytes, Molossus molossus, or Homo Contacting a nucleic acid construct encoding a transposase derived from sapiens and / or an engineered version thereof, wherein the ratio of ABCA4 or its functional fragment to the transposase is approximately 2:1, (c) The method comprising administering the cells to a patient in need thereof.

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