DNA origami encoding for gene expression and co-transfection

Abstract

The present invention relates to nucleic acid nanostructures comprising at least one scaffold strand and a plurality of staple strands, wherein the nanostructure, preferably the at least one scaffold strand, comprises at least one nucleic acid sequence encoding a gene. The present invention further relates to compositions comprising the nucleic acid nanostructures, and collections of nucleic acid sequences or collections of plasmids encoding the nucleic acid nanostructures. Furthermore, the present invention relates to nucleic acid nanostructures or compositions comprising nucleic acid nanostructures for use in medicine, preferably for use in methods for preventing, treating, and / or diagnosing diseases or disorders. The present invention also relates to methods for expressing genes from nucleic acid nanostructures, and to the use of the nanostructures or compositions for gene expression.

Description

[Technical Field]

[0001] The present invention relates to nucleic acid nanostructures comprising at least one scaffold strand and a plurality of staple strands, wherein the nanostructure, preferably the at least one scaffold strand, comprises at least one nucleic acid sequence encoding a gene. The present invention further relates to compositions comprising the nucleic acid nanostructures, and collections of nucleic acid sequences or collections of plasmids encoding the nucleic acid nanostructures. Furthermore, the present invention relates to nucleic acid nanostructures or compositions comprising nucleic acid nanostructures for use in medicine, preferably for use in methods for preventing, treating, and / or diagnosing diseases or disorders. The present invention also relates to methods for expressing genes from nucleic acid nanostructures, and to the use of the nanostructures or compositions for gene expression. [Background technology]

[0002] Delivering and expressing genes faces a series of obstacles, including how to package, target, and release the delivered nucleic acid. These obstacles become even more evident in the delivery of multi-component systems, such as those involving CRISPR-based technology and gene circuit construction, which require the delivery of precise and controllable amounts of several genes. Multiplexing to achieve genome or epigenome editing, transcriptional regulation, and / or gene circuit construction offers great potential for tailoring gene networks for therapeutic (re)programming, bioproduction, and basic research. However, despite rapid progress in these fields, simultaneous delivery and expression of multiple genes remains challenging, especially when attempting to deliver them in vivo.

[0003] Artificial structures formed from nucleic acids, such as DNA origami, have been discussed as having enormous potential for the field of biotechnology. DNA origami allows for the packaging of long single-stranded DNA into compact structures with unparalleled levels of structural programmability and homology, spatial addressability, and biocompatibility. Furthermore, multicomponent assemblies of different DNA origami structures can be achieved, with structures composed of 220 monomers and reaching sizes exceeding 1 GDa. However, while significant progress has been made in the use of DNA origami for applications such as drug delivery, sensing, and imaging, the development of DNA origami for gene therapy has been limited. To date, DNA origami has only been utilized as hybrids with either RNA or proteins for gene therapy research.

[0004] There remains a need for tools for efficiently expressing genes, such as mammalian genes. In particular, there remains a need for an assembly encoding two or more genes in a controlled stoichiometric ratio. Furthermore, there remains a need for simultaneous delivery and expression of one or more genes, for example, multiple genes. Furthermore, there remains a need for tools that can efficiently deliver and enable expression of genes in vivo. Summary of the Invention

[0005] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only those embodiments explicitly described. The description should be understood to support and encompass embodiments that combine two or more of the explicitly described embodiments, or that combine one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application, unless the context dictates otherwise.

[0006] In a first aspect, the present invention relates to a nucleic acid nanostructure comprising at least one scaffold strand and a plurality of staple strands, wherein the nanostructure, preferably the at least one scaffold strand, comprises at least one nucleic acid sequence encoding a gene.

[0007] In one embodiment, the nucleic acid nanostructure, preferably the at least one scaffold strand, comprises a first nucleic acid sequence encoding a gene and a second nucleic acid sequence encoding a gene, and optionally, the nucleic acid nanostructure, preferably the at least one scaffold strand, comprises multiple nucleic acid sequences encoding genes.

[0008] In one embodiment, the nucleic acid nanostructure comprises a first subunit and a second subunit, and preferably, the first subunit and the second subunit each comprise a nucleic acid sequence encoding a gene.

[0009] In one embodiment, the nanostructure comprises enhancer staple strands having a length in the range of about 60 to about 250 nucleobases, preferably about 80 to about 220 nucleobases, and more preferably about 90 to about 200 nucleobases; Optionally, the enhancer staple strand is configured to bind to the scaffold strand at the 5' end and / or the 3' end of the nucleic acid sequence encoding the gene.

[0010] In one embodiment, the nanostructure comprises enhancer staple strands having a length in the range of about 60 to about 250 nucleobases, preferably about 80 to about 220 nucleobases, and more preferably about 90 to about 200 nucleobases; Optionally, the enhancer staple strand comprises a nucleic acid sequence complementary to a nucleic acid sequence of the scaffold strand, the nucleic acid sequence of the scaffold strand being located at the 5' end and / or the 3' end of the nucleic acid sequence encoding the gene.

[0011] In one embodiment, the nanostructure, preferably the scaffold strand and / or at least one staple strand of the plurality of staple strands, comprises a nucleic acid sequence configured to form a loop structure, preferably an inverted terminal repeat nucleic acid sequence configured to form a hairpin. In one embodiment, the nanostructure, preferably the scaffold strand and / or at least one staple strand of the plurality of staple strands, comprises a loop structure, preferably a hairpin.

[0012] In one embodiment, the nucleic acid sequence configured to form the loop structure is configured so that the loop is formed at the 5' end and / or 3' end of the nucleic acid sequence encoding the gene.

[0013] In one embodiment, the nanostructure, preferably the scaffold strand and / or at least one staple strand of the plurality of staple strands, comprises a loop structure, preferably a hairpin, at the 5' end and / or 3' end of the nucleic acid sequence encoding the gene. In one embodiment, the nanostructure, preferably the at least one scaffold strand, comprises at least one nuclear targeting sequence, preferably a DNA nuclear targeting sequence, more preferably a simian virus 40 DNA nuclear targeting sequence, and optionally the nanostructure, preferably the at least one scaffold strand, comprises multiple nuclear targeting sequences.

[0014] In one embodiment, the nanostructure, preferably the at least one scaffold strand, comprises a promoter such as a CMV promoter, a terminator, a polyadenylation signal sequence, an intron, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element.

[0015] In one embodiment, the nanostructures have an aspect ratio ranging from about 1:1 to about 1000:1, preferably from 1.5:1 to about 20:1, and more preferably from about 2:1 to about 15:1.

[0016] In one embodiment, the scaffold strand comprises at least one nucleic acid sequence encoding the gene, a promoter, and a terminator, and optionally further comprises a nucleic acid sequence configured to form a loop structure, an intron, a DNA nuclear targeting sequence, a polyadenylation signal sequence, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element.

[0017] In one embodiment, the scaffold strand comprises at least one nucleic acid sequence encoding the gene, a promoter, and a terminator, and the scaffold strand optionally further comprises a loop structure, an intron, a DNA nuclear targeting sequence, a polyadenylation signal sequence, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element.

[0018] In one embodiment, the nucleic acid sequence encoding a gene is a nucleic acid sequence encoding a eukaryotic gene, preferably a nucleic acid sequence encoding a mammalian gene, more preferably a nucleic acid sequence encoding a human gene.

[0019] In a further aspect, the present invention relates to a composition, preferably a pharmaceutical composition, comprising a nucleic acid nanostructure as defined herein.

[0020] In a further aspect, the present invention relates to a collection of nucleic acid sequences or a collection of plasmids encoding the nucleic acid nanostructures defined herein.

[0021] In a further aspect, the present invention relates to a nucleic acid nanostructure as defined herein, or a composition as defined herein, for use in medicine, preferably for use in a method for preventing, treating and / or diagnosing a disease or disorder, preferably a genetic and / or immunological disease or disorder, optionally for use in gene therapy and / or immunotherapy.

[0022] In one embodiment, a nucleic acid nanostructure as defined herein, or a composition as defined herein, is for use in gene therapy and / or immunotherapy.

[0023] In a further aspect, the present invention provides a method of expressing a gene from a nucleic acid nanostructure, preferably a nucleic acid nanostructure as defined herein, comprising the steps of: i) providing a nucleic acid nanostructure, preferably a nucleic acid nanostructure as defined herein, comprising at least one nucleic acid sequence encoding a gene; ii) delivering the nucleic acid nanostructure provided in step i) to a cell, preferably wherein said delivering comprises transfecting or transforming said cell; iii) enabling the cell to express the gene; Optionally, said providing in step i) comprises providing a plasmid, preferably a phagemid, or a collection of plasmids, preferably a collection of phagemids, wherein said plasmid or collection of plasmids encodes said nucleic acid nanostructure; and preparing said nucleic acid nanostructure using said plasmid or collection of plasmids, preferably by using bacteriophages.

[0024] In a further aspect, the present invention relates to the use of a nanostructure as defined herein or a composition as defined herein for gene expression, preferably for in vitro gene expression.

[0025] In a further aspect, the present invention relates to a method for preventing, treating and / or diagnosing a disease or disorder, preferably a genetic and / or immunological disease or disorder, comprising administering a nanostructure as defined herein or a composition as defined herein to a patient in need thereof.

[0026] In one embodiment, the method for preventing, treating, and / or diagnosing a disease or disorder is a method of gene therapy and / or immunotherapy.

[0027] In one embodiment, the administering comprises administering an effective amount of a nanostructure as defined herein and / or a composition as defined herein to a patient in need thereof.

[0028] In a further aspect, the present invention relates to the use of a nucleic acid nanostructure as defined herein, or a composition as defined herein, for the manufacture of a medicament, e.g. a medicament for the prevention, treatment and / or diagnosis of a disease or disorder, preferably a genetic and / or immunological disease or disorder, optionally for gene therapy and / or immunotherapy.

[0029] In one embodiment, the nanostructures as defined herein, or the compositions as defined herein, are used for the manufacture of a medicament for gene therapy and / or immunotherapy. DETAILED DESCRIPTION OF THE INVENTION

[0030] An object of the present invention is to provide tools for efficiently expressing genes, such as eukaryotic genes, e.g., mammalian genes. In particular, an object of the present invention is to provide nanostructures encoding two or more genes in a controlled stoichiometric ratio. A further object of the present invention is to simultaneously deliver and express one or more genes, e.g., multiple genes. A further object of the present invention is to efficiently deliver and express genes in vitro, ex vivo, and in vivo.

[0031] The present inventors have successfully expressed genes from encoded DNA origami. In particular, the present inventors synthesized a library of custom ssDNA scaffolds for mammalian gene expression, for example, using bacteriophage-based production. The present inventors found that genes were readily expressed from nucleic acid nanostructures, regardless of the gene's location in the nanostructure or the nanostructure's geometry. The present inventors found that gene expression could be further enhanced by nucleic acid sequences configured to form loop structures, such as adeno-associated virus-inspired inverted terminal repeat (ITR) hairpin sequences, either upstream of the expression cassette or flanking either side of an expression cassette with a loop structure characterized on the staple, e.g., an ITR hairpin. Overall, the present inventors demonstrate highly efficient gene expression encoded within the nucleic acid nanostructures of the present invention, particularly the DNA origami structures of the present invention.

[0032] We have successfully delivered and expressed genes from encoded ssDNA scaffolds and customized DNA origami objects, as shown in Figure 1a. We found that our nucleic acid nanostructures easily deploy within the intracellular environment and efficiently express genes from the ssDNA scaffold strands, and that gene expression can be further optimized through targeted staple design. We also created and tested a library of ssDNA scaffolds optimized for increased gene expression, high-yield scaffold production and purity, and origami folding quality. Overall, we surprisingly found that the inclusion of at least one loop structure, preferably an ITR secondary DNA structure upstream of the expression cassette, and / or one or more nuclear targeting sequences, such as DTS sequences, e.g., three SV40 DTS sequences, enables highly efficient and robust gene expression without compromising scaffold production or origami folding quality. Finally, we demonstrate the efficient and controlled assembly of gene-encoded origami structures in stoichiometric ratios. These "plug-and-play" structural origamis have enabled the successful co-delivery and expression of numerous genes with unprecedented control.

[0033] Advantageously, the nanostructures of the present invention utilize both the unique design possibilities of DNA origami structures as well as their ability to encode genetic information, and thus advantageously allow for the encoding and expression of large numbers of genes with controlled stoichiometry.

[0034] The present invention relates to nucleic acid nanostructures comprising at least one scaffold strand and a plurality of staple strands, wherein the nanostructure, preferably the at least one scaffold strand, comprises at least one nucleic acid sequence encoding a gene. Advantageously, the one or more genes can be efficiently expressed using the nanostructures of the present invention.

[0035] The term "nanostructure" as used herein refers to a nucleic acid nanostructure, preferably a DNA origami structure composed of one or more DNA origami subunits. In one embodiment, the nanostructure comprises or consists of a DNA origami structure. Nanostructures can be fabricated using readily available nucleic acid nanostructure technologies, such as DNA origami technology, which involves relatively less complex procedures for assembling nanostructures than standard nanofabrication techniques. In one embodiment, the nanostructures are fabricated at least in part using DNA origami technology. Due to the self-assembly of DNA origami structures and readily available software for designing the corresponding scaffolds and staple strands, this is a relatively less complex fabrication process compared to standard nanofabrication techniques. In one embodiment, the nanostructures of the present invention are DNA origami structures. In one embodiment, the nanostructures of the present invention have a maximum length of less than 1000 nm, e.g., in the range of about 10 nm to about 150 nm, preferably about 20 nm to about 100 nm.

[0036] In one embodiment, the nucleic acid nanostructures, e.g., DNA origami structures, of the present invention comprise at least one scaffold strand and a plurality of staple strands, e.g., single-stranded oligonucleotide staple strands. The term "staple strand," as used herein, refers to a single-stranded oligonucleotide molecule that is at least partially complementary to a scaffold strand. In one embodiment, the term "staple" refers to a staple strand among a plurality of staple strands. Generally, staple strands can be used, for example, to introduce coupling sites into DNA origami structures and / or DNA origami subunits. The term "plurality of staple strands," as used herein, refers to a plurality, e.g., at least three staple strands. For example, a plurality of staple strands can refer to at least 3, 4, 5, 6, 7, 8, 9, 10, or more staple strands. In one embodiment, a staple strand, particularly a staple strand among a plurality of staple strands, has a length of 20 to 100 nucleobases. In one embodiment, a staple strand of the plurality of staple strands has a length of 20 to 80 nucleobases and the enhancer staple strand(s) has a length of 90 to 250 nucleobases. In one embodiment, the enhancer staple strand(s) differ from a staple strand of the plurality of staple strands in that the enhancer staple strand(s) is / are at least 5, preferably at least 10 nucleobases longer than each of the staple strands of the plurality of staple strands.

[0037] The term "scaffold strand" refers to a single-stranded nucleic acid strand, e.g., a nucleic acid strand such as a single-stranded polynucleotide strand, preferably a DNA strand. In one embodiment, the scaffold strand constitutes and / or spans a major portion of the DNA origami structure and / or DNA origami subunit. In one embodiment, the scaffold strand has a length of 100 to 20,000 nucleobases, preferably 120 to 15,000 nucleobases, more preferably 260 to 11,000 nucleobases, e.g., 1,000 to 11,000 nucleobases. In one embodiment, the scaffold strand is a circular or linear scaffold strand. In one embodiment, the scaffold strand is a circular ssDNA scaffold strand. In one embodiment, the scaffold strand has a length of 260 to 20,000 nucleobases, the enhancer staple strand(s) have a length of 90 to 250 nucleobases, and each staple strand of the plurality of staple strands has a length of 20 to 80 nucleobases.

[0038] Nanostructures, such as DNA origami structures, can include at least one scaffold strand, i.e., a single-stranded polynucleotide scaffold DNA having a known sequence. The DNA origami structure can further include multiple single-stranded oligonucleotide staple strands, each of which can be at least partially complementary to at least one scaffold strand. Furthermore, each of the staple strands can be configured to bind to at least one scaffold strand, and the at least one scaffold strand can be folded and / or arranged to form a desired nanostructure. The term "strand" as used herein refers to a nucleic acid strand, e.g., a DNA and / or RNA strand, preferably a DNA strand. Three-dimensional nanostructures can be realized using DNA origami, i.e., by combining scaffold strands and staple strands to form the desired portions and overall device. Such designs can be implemented using software such as caDNAno. That is, nanostructures comprising multiple portions can be created from a single scaffold strand in some embodiments, while in other embodiments, portions of the nanostructure can be constructed using multiple scaffold strands.

[0039] In one embodiment, the shape of the nanostructure can be any shape, such as a brick, a rod, a triangle, a circle, a cube, a rectangle, a star, or any other shape. The nanostructures of the present invention can be of any length. In a preferred embodiment, the nanostructure comprises a maximum length, and in a particularly preferred embodiment, the maximum length is less than 1000 nm, preferably less than 500 nm, such as about 100 nm or less. In one embodiment, the terms "nanostructure," "nano object," and "nucleic acid nanostructure" are used interchangeably.

[0040] In one embodiment, each staple strand is configured to bind to at least one of the at least one scaffold strand at at least one, preferably two or more different locations. In one embodiment, the nucleic acid nanostructure of the present invention, e.g., DNA origami, comprises a scaffold strand and one or more staple strands. In one embodiment, the nucleic acid nanostructure of the present invention comprises 100 or fewer staple strands. In one embodiment, the nucleic acid nanostructure of the present invention comprises 10 or more DNA strands, preferably 15 or more DNA strands, e.g., at least one scaffold strand and at least 9 staple strands.

[0041] The terms "DNA origami structure," "DNA origami," and "DNA origami object," as used herein, refer to nanostructures that include DNA as a building material for creating nanoscale shapes. Preparing and / or providing a DNA origami involves folding, e.g., by self-assembly, one or more scaffold DNA strands into a defined shape using a plurality of rationally designed staple DNA strands. The scaffold strands are typically longer than the staple strands. The nucleic acid sequences of the staple strands are designed such that the staple strands hybridize to defined portions of the scaffold strands, and the hybridization results in a specific shape of the nanostructure.

[0042] In one embodiment, the term "nucleic acid," as used herein, relates to a nucleotide sequence, such as a ribonucleic acid or a deoxyribonucleic acid. In a preferred embodiment, the nucleic acid nanostructure is a DNA nanostructure. In a preferred embodiment, the nucleic acid nanostructure of the present invention is a DNA origami structure. In a preferred embodiment, the nucleic acid nanostructure comprises or consists of a DNA origami structure. In one embodiment, the nucleic acid nanostructure is provided in the form of a DNA origami structure. An advantage of nucleic acid nanostructures, such as DNA origami structures, is that nucleic acid nanostructures, such as DNA origami structures, can contain multiple genes, particularly with a desired stoichiometry, through their rational design. Advantageously, nucleic acid nanostructures, such as DNA origami structures, allow for precise control of the number of genes and their expression. Furthermore, the composition of the gene of interest and additional nucleic acid sequences can be precisely controlled. A further advantage of nucleic acid nanostructures, such as DNA origami structures, is that they can be stabilized against nucleases. In one embodiment, the nanostructures are configured to be stabilized against nucleases. A further advantage of DNA origami structures is that assembly, e.g., self-assembly, and purification of the structure(s) is more robust and simpler compared to non-DNA origami nucleic acid nanostructures, such as DNA tetrahedra or RNA assemblies. All embodiments described herein relating to "nucleic acid nanostructures" or "nucleic acid nanostructures of the invention" are meant to be understood to also relate to nanostructure(s) comprised by the compositions of the invention, nanostructures used in accordance with the invention, and nanostructure(s) provided in any method of the invention.

[0043] The term "nucleic acid sequence encoding a gene," as used herein, relates to a nucleic acid sequence encoding any gene of interest, for example, a gene involved in a pathological pathway, a gene suitable for vaccination, and / or a CRISPR-based gene. In one embodiment, the gene is selected from a prokaryotic gene, a viral gene, and a eukaryotic gene. In one embodiment, the gene is selected from a prokaryotic gene, such as a CRISPR-based gene, and a eukaryotic gene, such as a human gene. In a preferred embodiment, the gene is a eukaryotic gene, preferably a mammalian gene, for example, a human gene. An advantage of the nucleic acid nanostructures of the present invention is that they enable the expression of mammalian genes, for example, different mammalian genes with defined stoichiometry. A further advantage is that the nanostructures enable the expression of prokaryotic genes, for example, for CRISPR-based gene editing, and viral genes, for example, for DNA- or RNA-based vaccination. In a preferred embodiment, the nucleic acid sequence encoding a gene encodes a eukaryotic gene, preferably a mammalian gene, more preferably a human gene. In one embodiment, the nucleic acid nanostructure of the present invention, preferably at least one scaffold strand thereof, comprises a first nucleic acid sequence encoding a gene and a second nucleic acid sequence encoding a gene; optionally, the nucleic acid nanostructure, preferably at least one scaffold strand thereof, comprises multiple nucleic acid sequences encoding a gene. Advantageously, the nanostructure of the present invention, comprising a first nucleic acid sequence encoding a gene and a second nucleic acid sequence encoding a gene, e.g., comprising multiple nucleic acid sequences encoding genes, allows for efficient expression of a gene of interest with a defined stoichiometry. In one embodiment, the first nucleic acid sequence encoding a gene and the second nucleic acid sequence encoding a gene each encode a eukaryotic gene, preferably a mammalian gene, more preferably a human gene. Advantageously, the nanostructure of the present invention allows for the expression of numerous genes with controlled stoichiometry. In one embodiment, the first nucleic acid sequence encoding a gene and the second nucleic acid sequence encoding a gene may encode the same gene or different genes.In one embodiment, the nanostructures of the present invention comprise one or several copies of a gene of interest, e.g., one or more nucleic acid sequences encoding the gene, such as one or more first nucleic acid sequences encoding the gene and one or more second nucleic acid sequences encoding the gene.

[0044] In one embodiment, a nucleic acid nanostructure comprises a plurality of nucleic acid sequences encoding genes. In one embodiment, each nucleic acid sequence encoding a gene of the plurality of nucleic acid sequences encoding genes encodes a different gene. In one embodiment, each nucleic acid sequence encoding a gene of the plurality of nucleic acid sequences encoding genes encodes a gene that is different from the gene encoded by the other nucleic acid sequences encoding genes of the plurality of nucleic acid sequences encoding genes. Thus, a plurality of nucleic acid sequences encoding genes can comprise multiple encoded genes. In one embodiment, the nucleic acid sequence(s) encoding genes are positioned at any site on the nucleic acid nanostructure, preferably at any site on the scaffold strand.

[0045] In one embodiment, the nucleic acid nanostructure comprises a first subunit and a second subunit, and preferably, the first subunit and the second subunit each comprise a nucleic acid sequence encoding a gene. An advantage of a nucleic acid nanostructure comprising a first subunit and a second subunit, for example, a nucleic acid nanostructure comprising multiple subunits, is that each of the subunits can comprise one or more nucleic acid sequences encoding a gene, and thus the stoichiometry of the multiple nucleic acid sequences encoding the gene can be rationally designed. In one embodiment, the nucleic acid nanostructure comprises stacked first and second subunits, optionally multiple subunits. In one embodiment, the nucleic acid nanostructure comprises two or more stacked subunits comprising a first subunit and a second subunit. In one embodiment, the first and second subunits each comprise a nucleic acid sequence encoding a gene, and the nucleic acid sequence encoding the gene contained by the first subunit is the same as or different from the nucleic acid sequence encoding the gene contained by the second subunit. In one embodiment, the first and second subunits encode the same or different genes. In one embodiment, the first subunit comprises a first nucleic acid sequence encoding a gene, and the second subunit comprises a second nucleic acid sequence encoding a gene. In one embodiment, the first subunit and the second subunit, optionally each subunit of the plurality of subunits, each comprise a scaffold strand and multiple staple strands. In one embodiment, the nucleic acid nanostructure comprises a first subunit and a second subunit, each subunit comprising a scaffold strand comprising a nucleic acid sequence encoding a gene, each subunit comprising multiple staple strands, and optionally at least one subunit comprising an enhancer staple strand. The subunits of the nucleic acid nanostructure can be connected by any means, for example, shape complementarity, nucleic acid-mediated interactions such as nucleotide base stacking interactions, base pairing, and / or covalent bonds such as disulfide bridges. Advantageously, assembly of multiple DNA origami subunits comprising multiple genes can be achieved with a controlled stoichiometry ( FIG. 6 ) to enable simultaneous delivery of multiple components.This is particularly important in areas such as CRISPR-based technologies for gene / base editing or epigenetic regulation. Furthermore, the production of virus-like particles (VLPs), lentiviruses, and adeno-associated viruses, among others, additionally requires the delivery of multiple components in controlled ratios.

[0046] In one embodiment, the nanostructure comprises an enhancer staple strand having a length ranging from about 60 to about 250 nucleobases, preferably from about 80 to about 220 nucleobases, more preferably from about 90 to about 200 nucleobases, e.g., about 154 bases. In one embodiment, the enhancer staple strand is configured to bind to the scaffold strand at the 5' and / or 3' end of the nucleic acid sequence encoding the gene. The inventors have found that the enhancer staple strand stabilizes the nanostructure and increases gene expression from the nanostructure. Furthermore, surprisingly, the inventors have found that efficient expression of genes, such as mammalian genes, can be achieved with nanostructures comprising enhancer staple strands. In one embodiment, the term "enhancer staple strand" refers to a staple strand having a length of at least 60 nucleobases, preferably at least 80 nucleobases, more preferably at least 90 nucleobases, e.g., a length ranging from about 90 to about 250 nucleobases. In one embodiment, the terms "enhancer staple strand," "staple strand having a length of at least 60 nucleobases," "contiguous staple strand," and "stabilizing staple strand" are used interchangeably. In one embodiment, an enhancer staple strand is attached to a scaffold strand such that one or more continuous double-helical domains are formed with the scaffold comprising at least 10 nucleobase pairs, preferably at least 15 nucleobase pairs, more preferably at least 20 nucleobase pairs, even more preferably at least 30 nucleobase pairs, and even more preferably at least 80, 85, or 90 nucleobase pairs. In one embodiment, the enhancer staple strand provides a long region of dsDNA to aid in the recognition and binding of polymerases and / or other helper proteins required for gene expression.

[0047] The staple strands of the plurality of staple strands typically cross from one region of the scaffold strand to another region of the scaffold strand, thereby creating a folding pattern. The staple strands of the plurality of staple strands typically cross multiple regions of the scaffold strand. In contrast, the enhancer staple strands perform fewer or no of these typical crossings, remaining continuous along one or two regions of the scaffold strand. In one embodiment, the enhancer staple strands bind to the scaffold strand along one or two regions of the scaffold strand. In one embodiment, the enhancer staple strands bind to the scaffold strand along one or two regions of the continuous nucleic acid of the nucleic acid sequence of the scaffold strand. In one embodiment, an enhancer staple strand comprises or consists of one or two portions, and when an enhancer staple strand comprises or consists of one portion, at least 90% of the nucleobases of the one portion are bound to a region of the scaffold strand, and when an enhancer staple strand comprises or consists of two portions, at least 90% of the nucleobases of a first portion of the two portions are bound to a first region of the scaffold strand, and at least 90% of the nucleobases of a second portion of the two portions are bound to a second region of the scaffold strand. In one embodiment, an enhancer staple strand is oriented such that it, or at least a portion of it, is substantially parallel to the longitudinal extension of the nanostructure. In one embodiment, the term "enhancer" in the phrase "enhancer staple strand" means that the enhancer staple strand binds to at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, and even more preferably at least 80, 85, or 90 consecutive nucleic acids in the nucleic acid sequence of the scaffold strand, preferably enhancing nanostructure assembly and gene expression therefrom.In one embodiment, the scaffold strand comprises a nucleic acid sequence encoding the gene, and the enhancer staple strand binds at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, and even more preferably at least 80, 85, or 90 contiguous nucleic acids of the nucleic acid sequence of the scaffold strand at the 5'-end and / or 3'-end of the nucleic acid sequence encoding the gene. In one embodiment, the enhancer staple strand binds at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, and even more preferably at least 80, 85, or 90 contiguous nucleic acids of the nucleic acid sequence of the scaffold strand at the 5'-end of the nucleic acid sequence encoding the gene, and the enhancer staple strand binds at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, and even more preferably at least 80, 85, or 90 contiguous nucleic acids of the nucleic acid sequence of the scaffold strand at the 3'-end of the nucleic acid sequence encoding the gene. For example, an enhancer staple strand can have a first portion, e.g., a first half, and a second portion, e.g., a second half, wherein the first portion, e.g., the first half, binds to at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, and even more preferably at least 80, 85, or 90 contiguous nucleic acids of the nucleic acid sequence of the scaffold strand at the 5' end of the nucleic acid sequence encoding the gene, and the second portion, e.g., the second half, binds to at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, and even more preferably at least 80, 85, or 90 contiguous nucleic acids of the nucleic acid sequence of the scaffold strand at the 3' end of the nucleic acid sequence encoding the gene. Surprisingly, the inventors have found that enhancer staple strands positioned adjacent to either side of an expression cassette result in a significant increase in gene transfer efficiency and gene expression.In one embodiment, a scaffold strand comprises a nucleic acid sequence encoding the gene of interest and a polyadenylation signal sequence, and optionally an enhancer staple strand is attached to the scaffold strand at the 5' end of the nucleic acid sequence encoding the gene of interest and the 3' end of the polyadenylation signal sequence. In one embodiment, the enhancer staple strand is configured to be attached to the scaffold strand so as to form a circular structure, e.g., a circular scaffold strand or a circular expression cassette.

[0048] In one embodiment, the nanostructure, preferably the scaffold strand and / or at least one staple strand of the plurality of staple strands, comprises a nucleic acid sequence configured to form a loop structure, preferably an inverted terminal repeat nucleic acid sequence configured to form a hairpin. For example, a loop structure is formed when two regions of a nucleic acid strand, e.g., two regions of the same strand, are typically complementary in nucleotide sequence when read in opposite directions and base pair to form a double helix that ends in an unpaired loop. For example, an inverted terminal repeat nucleic acid sequence can be a single-stranded sequence of nucleotides followed downstream by its reverse complement. The intervening sequence of nucleotides between the initial sequence and its reverse complement can be of any length, including zero. Advantageously, nanostructures comprising nucleic acid sequences configured to form a loop structure, e.g., inverted terminal repeat nucleic acid sequences such as adeno-associated virus-inspired inverted terminal repeat (ITR) hairpin sequences, exhibit highly effective gene expression. In one embodiment, a nanostructure, e.g., the scaffold strand and / or at least one staple strand of the plurality of staple strands of the nanostructure, comprises a nucleic acid sequence configured to form a loop structure, preferably an inverted terminal repeat nucleic acid sequence, such as an adeno-associated virus-inspired inverted terminal repeat hairpin sequence. In one embodiment, the nucleic acid sequence configured to form the loop structure is configured such that the loop is formed at the 5'-end and / or 3'-end of a nucleic acid sequence encoding the gene. In one embodiment, the scaffold strand comprises a nucleic acid sequence configured to form a loop structure, preferably upstream of a nucleic acid sequence encoding the gene, more preferably upstream of an expression cassette comprising the nucleic acid sequence encoding the gene. In one embodiment, a staple strand, e.g., a staple strand of a plurality of staple strands, comprises a nucleic acid sequence configured to form a loop structure, preferably a staple strand adjacent to either side of a nucleic acid sequence encoding the gene, more preferably a staple strand adjacent to either side of an expression cassette comprising the nucleic acid sequence encoding the gene. In one embodiment, the nucleic acid sequence configured to form a loop structure comprises or consists of a sequence set forth in any of SEQ ID NOs: 13-14.In one embodiment, the nanostructure comprises a nucleic acid sequence encoding a loop structure, preferably an inverted terminal repeat nucleic acid sequence encoding a hairpin. In one embodiment, the nanostructure comprises a loop structure, preferably a hairpin. In one embodiment, the term "nucleic acid sequence encoding a loop structure," as used herein, relates to a nucleic acid sequence configured to form a loop structure. In one embodiment, the nucleic acid sequence configured to form a loop structure is configured such that the loop structure is formed by the nucleic acid sequence configured to form the loop structure alone, or by the nucleic acid sequence configured to form the loop structure and an additional nucleic acid sequence, e.g., an additional nucleic acid sequence configured to form a loop structure. In one embodiment, the nucleic acid nanostructure comprises a first nucleic acid sequence configured to form a loop structure and a second nucleic acid sequence configured to form a loop structure. In one embodiment, the first and second nucleic acid sequences configured to form a loop structure each form a loop structure and / or jointly form a loop structure. Surprisingly, the inventors have found that enhanced gene expression efficiency can be achieved by including a nucleic acid sequence encoding a loop structure, such as an adeno-associated virus-inspired inverted terminal repeat (ITR) hairpin sequence, either upstream of the expression cassette or adjacent to either side of an expression cassette having a loop structure characterized on the staple strand. In one embodiment, the nucleic acid sequence configured to form the loop structure is located upstream or downstream of the nucleic acid sequence encoding a gene, particularly upstream or downstream of the expression cassette. In one embodiment, the loop structure is formed upstream or downstream of the nucleic acid sequence encoding a gene, particularly upstream or downstream of the expression cassette. In one embodiment, the nucleic acid sequence configured to form the loop structure is located on one or two staple strands that bind to the scaffold strand upstream or downstream of the nucleic acid sequence encoding a gene, particularly upstream or downstream of the expression cassette.

[0049] In one embodiment, the nanostructure, preferably the at least one scaffold strand, comprises at least one nuclear targeting sequence, preferably a DNA nuclear targeting sequence, more preferably a simian virus 40 DNA nuclear targeting sequence. Optionally, the nanostructure, preferably the at least one scaffold strand, comprises multiple nuclear targeting sequences. In one embodiment, the nuclear targeting sequence comprises or consists of the sequence set forth in SEQ ID NO: 21. A DNA nuclear targeting sequence (DTS) is a consensus motif recognized by transcription factors and can be used to transport DNA from the cytoplasm to the nucleus through nuclear pores. For example, the simian virus 40 DNA nuclear targeting sequence (SV40 DTS) can be used as a DTS because it is recognized by various TFs. Advantageously, nanostructures comprising one or more DTS sequences, such as three SV40 DTS sequences, enable highly efficient and robust gene expression. In one embodiment, the nucleic acid nanostructure comprises one, two, or three nuclear targeting sequences, preferably a DNA nuclear targeting sequence. Surprisingly, the inventors have found that gene transfer efficiency can be further increased by including a nuclear targeting sequence, e.g., a DNA nuclear targeting sequence such as the SV40 (simian vacuolating virus 40) DTS, within the nanostructure, preferably the scaffold strand. The inventors have found maximal efficacy for both dividing and non-dividing (arrested) cells with one to three nuclear targeting sequence repeats, such as one to three SV40 sequence repeats (Figure 5).

[0050] In one embodiment, the nanostructure, preferably the at least one scaffold strand, comprises a promoter such as a CMV promoter, a terminator, a polyadenylation signal sequence, an intron, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element. A promoter is typically a DNA sequence to which a protein binds to initiate transcription of a single RNA transcript from DNA downstream of the promoter, e.g., a bacterial or eukaryotic promoter such as a mammalian promoter. Terminators, particularly transcription terminators, are typically sections of nucleic acid sequences that indicate the end of a gene or operon being transcribed, e.g., mammalian terminators such as the SV40, hGH, BGH, and rbGlob terminators. In one embodiment, the terminator is the SV40 terminator, hGH terminator, BGH terminator, or rbGlob terminator. For example, terminators mediate transcription termination by providing a signal in newly synthesized transcribed RNA that triggers a process to release the transcribed RNA from the transcription complex. Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically a messenger RNA (mRNA). The poly(A) tail enhances the nuclear export, translation, and stability of the mRNA. A polyadenylation signal sequence typically comprises a consensus sequence for the addition of a poly(A) tail (polyadenylation) and / or a terminator sequence. In one embodiment, the polyadenylation signal sequence comprises an AAUAAA motif. In one embodiment, the polyadenylation signal sequence comprises or consists of the sequence set forth in SEQ ID NO: 19. A Kozak sequence can be a nucleic acid motif that functions as a protein translation initiation site in a transcript, e.g., a eukaryotic mRNA transcript. In one embodiment, the Kozak sequence comprises or consists of the sequence set forth in SEQ ID NO: 16. A woodchuck hepatitis virus post-transcriptional regulatory element can be a DNA sequence that, when transcribed, creates a tertiary structure that enhances expression. In one embodiment, the woodchuck hepatitis virus post-transcriptional regulatory element comprises or consists of the sequence set forth in SEQ ID NO:17.

[0051] In one embodiment, the nucleic acid nanostructure, preferably the at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises a nucleic acid sequence configured to form a loop structure, preferably an inverted terminal repeat nucleic acid sequence such as an adeno-associated virus-inspired inverted terminal repeat hairpin sequence, as well as a promoter such as a CMV promoter, a terminator, a polyadenylation signal sequence, an intron, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0052] In one embodiment, the nucleic acid nanostructure, preferably the at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises an inverted terminal repeat nucleic acid sequence, such as an adeno-associated virus-inspired inverted terminal repeat hairpin sequence, as well as a promoter such as a CMV promoter, a terminator, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0053] In one embodiment, the nucleic acid nanostructure, preferably the at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises an adeno-associated virus-inspired inverted terminal repeat hairpin sequence, as well as a promoter such as a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, a Kozak sequence, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0054] In one embodiment, the nucleic acid nanostructure, preferably the at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises an adeno-associated virus-inspired inverted terminal repeat hairpin sequence, as well as a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, a Kozak sequence, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and optionally, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0055] In one embodiment, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand, and the nucleic acid nanostructure comprises an adeno-associated virus-inspired inverted terminal repeat hairpin sequence, as well as a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, a Kozak sequence, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0056] In one embodiment, the nucleic acid nanostructure is an ssDNA scaffold strand, and the nucleic acid nanostructure comprises an adeno-associated virus-inspired inverted terminal repeat hairpin sequence, a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, a Kozak sequence, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0057] In one embodiment, the nucleic acid nanostructure, preferably the at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises a promoter such as a CMV promoter, a terminator, a polyadenylation signal sequence, an intron, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and one or more nuclear targeting sequences such as one or more DTS sequences, e.g., one or more SV40 DTS sequences, and optionally, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0058] In one embodiment, the nucleic acid nanostructure, preferably the at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises a promoter such as a CMV promoter, a terminator, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and one or more nuclear targeting sequences such as one or more DTS sequences, e.g., one or more SV40 DTS sequences, and optionally, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0059] In one embodiment, the nucleic acid nanostructure, preferably at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises a promoter such as a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, and one or more nuclear targeting sequences such as one or more DTS sequences, e.g., one or more SV40 DTS sequences, and optionally, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0060] In one embodiment, the nucleic acid nanostructure, preferably at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises a promoter such as a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, and one or more SV40 DTS sequences, and optionally, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0061] In one embodiment, the nucleic acid nanostructure, preferably at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises a promoter such as a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, and one or three SV40 DTS sequences, and optionally, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0062] In one embodiment, the nucleic acid nanostructure, preferably at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises a promoter such as a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, and one or three SV40 DTS sequences, and the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0063] In one embodiment, the nucleic acid nanostructure, preferably at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, and one or three SV40 DTS sequences, and the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0064] In one embodiment, the nucleic acid nanostructure, preferably at least one scaffold strand of the nanostructure and / or at least one staple strand of the plurality of staple strands, comprises a CMV promoter, a polyadenylation signal sequence, at least one nucleic acid sequence encoding a gene, and three SV40 DTS sequences, the at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand, and the shape of the nanostructure is triangular.

[0065] According to the present invention, the term "ssDNA" refers to single-stranded DNA. Thus, the term "ssDNA scaffold" refers to a single-stranded DNA scaffold, and the term "ssDNA scaffold strand" refers to a single-stranded DNA scaffold strand.

[0066] In one embodiment, the nanostructure has an aspect ratio ranging from about 1:1 to about 1000:1, preferably from 1.5:1 to about 20:1, and more preferably from about 2:1 to about 15:1. In one embodiment, the aspect ratio is the ratio of the size of the nanostructure in different dimensions, e.g., the ratio of longitudinal extension to lateral extension. In one embodiment, the nanostructure has a maximum longitudinal extension along the longitudinal axis that is greater than the maximum lateral extension along the lateral axis. For example, a nanostructure having an aspect ratio of 20:1 may have a longitudinal extension of 20 nm and a lateral extension of 1 nm.

[0067] In one embodiment, the scaffold strand comprises at least one nucleic acid sequence encoding the gene of interest, a promoter, and a terminator, and optionally further comprising a nucleic acid sequence configured to form a loop structure, an intron, a DNA nuclear targeting sequence, a polyadenylation signal sequence, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element. In one embodiment, the nanostructure, preferably the scaffold strand, comprises an expression cassette comprising at least one nucleic acid sequence encoding the gene of interest, a promoter, and a terminator, and optionally further comprising a nucleic acid sequence configured to form a loop structure, an intron, a DNA nuclear targeting sequence, a polyadenylation signal sequence, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element.

[0068] In a further aspect, the present invention relates to a composition, preferably a pharmaceutical composition, comprising a nucleic acid nanostructure as defined herein. In one embodiment, the composition, preferably a pharmaceutical composition, comprises a pharmaceutically acceptable excipient. The composition, preferably a pharmaceutical composition, of the present invention shall be formulated to be compatible with its intended route of administration. In particularly preferred embodiments, examples of routes of administration of the medicaments and / or nanostructures of the present invention include intravenous, oral, intranasal, intrathecal, intraarterial, intradermal, subcutaneous, transdermal (topical), intraventricular, intraparenchymal, intratumoral, transmucosal, rectal, vaginal, bronchial, parenteral administration, and any other clinically / medically accepted method for administering medicaments and / or compounds.

[0069] In a further aspect, the present invention relates to a collection of nucleic acid sequences or a collection of plasmids encoding the nucleic acid nanostructures defined herein. In one embodiment, the collection of nucleic acid sequences comprises or consists of one or more nucleic acid sequences encoding the nucleic acid nanostructures defined herein. In one embodiment, the collection of nucleic acid sequences comprises a scaffold strand and a plurality of staple strands. In one embodiment, the collection of plasmids comprises or consists of one or more plasmids encoding the nucleic acid nanostructures defined herein. In one embodiment, the plasmids are phagemids. In one embodiment, the collection of plasmids is a collection of phagemids.

[0070] In a further aspect, the present invention relates to a nucleic acid nanostructure as defined herein or a composition as defined herein for use in medicine. In one embodiment, the nucleic acid nanostructure as defined herein or the composition as defined herein is for use in a method for preventing, treating, and / or diagnosing a disease or disorder, preferably a genetic and / or immunological disease or disorder. In one embodiment, the nucleic acid nanostructure as defined herein or the composition as defined herein is for use in gene therapy and / or immunotherapy. Advantageously, the nanostructures of the present invention allow for the efficient expression of genes, such as mammalian genes, which are highly useful in the prevention, treatment, and diagnosis of genetic and immunological diseases or disorders. For example, the nanostructures of the present invention can be used in gene therapy and immunotherapy by administering the nanostructure to a patient in need thereof and expressing a gene from the nanostructure in the patient, e.g., a gene defective in the patient and / or a gene involved in a pathology. In one embodiment, the gene therapy and / or immunotherapy comprises or consists of vaccination, preferably vaccination using the nanostructure or composition.

[0071] In a further aspect, the present invention provides a method of expressing a gene from a nucleic acid nanostructure, preferably a nucleic acid nanostructure as defined herein, comprising the steps of: i) providing a nucleic acid nanostructure, preferably a nucleic acid nanostructure as defined herein, comprising at least one nucleic acid sequence encoding a gene; ii) delivering the nucleic acid nanostructure provided in step i) to a cell, preferably wherein said delivering comprises transfecting or transforming said cell; iii) enabling the cell to express the gene; Optionally, said providing in step i) comprises providing a plasmid, preferably a phagemid, or a collection of plasmids, preferably a collection of phagemids, wherein said plasmid or collection of plasmids encodes said nucleic acid nanostructure, and preparing said nucleic acid nanostructure using said plasmid or collection of plasmids, preferably by using bacteriophage, hi one embodiment, said collection of plasmids is a collection of plasmids as defined herein.

[0072] In one embodiment, the method for expressing the gene is an in vitro or ex vivo method. In one embodiment, delivering the nanostructure to the cell comprises contacting the nanostructure with the cell, and optionally further comprises electroporation, lipofection, endocytosis, such as chemically induced endocytosis and / or receptor-mediated endocytosis, phagocytosis, membrane fusion, heat shock, calcium phosphate, liposomes, nanoparticles, gene gun, microinjection, sonoporation, photoporation, magnetofection, and / or hydroporation. In one embodiment, the delivering in step ii) is performed using electroporation, lipofection, endocytosis, such as chemically induced endocytosis and / or receptor-mediated endocytosis, phagocytosis, membrane fusion, heat shock, calcium phosphate, liposomes, nanoparticles, gene gun, microinjection, sonoporation, photoporation, magnetofection, and / or hydroporation. For example, chemically induced endocytosis can include peptide-targeted endocytosis, protein-targeted endocytosis, polysaccharide-targeted endocytosis, carbohydrate-targeted endocytosis, lipid-targeted endocytosis, and / or aptamer-targeted endocytosis.

[0073] In one embodiment, allowing the cell(s) to express the gene in step iii) comprises culturing the cell(s) in a cell culture, preferably at a temperature ranging from about 25°C to about 40°C, preferably from about 30°C to about 38°C, more preferably about 37°C, for example, for about 1 hour to about 72 hours. In one embodiment, allowing the cell(s) to express the gene in step iii) comprises providing suitable growth conditions for the cells. In one embodiment, the cell is a eukaryotic or prokaryotic cell, such as a mammalian cell, a fungal cell, a yeast cell, or a bacterial cell. In one embodiment, the cell is a mammalian cell.

[0074] In one embodiment, in the context of the methods of the invention, the term "providing a nucleic acid nanostructure" includes providing an assembled nucleic acid nanostructure and / or providing building materials for a nucleic acid nanostructure, e.g., a scaffold strand and one or more staple strands. In one embodiment, a method of preparing a nanostructure includes allowing self-assembly of the nanostructure and purifying the self-assembled nanostructure. For example, providing a nucleic acid nanostructure can include allowing self-assembly and subsequent purification. In one embodiment, allowing self-assembly includes mixing at least one scaffold strand with one or more staple strands, optionally further including adjusting the ionic strength by adding, e.g., about 10 mM to about 20 mM MgCl2, and / or using a temperature protocol that implements a temperature series. In one embodiment, purifying includes removing remaining excess staple strands by, e.g., precipitation, such as PEG precipitation, filtration, and / or liquid chromatography. In one embodiment, the self-assembly and / or allowing self-assembly includes a denaturation step and a cooling step. In one embodiment, the denaturation step is carried out at a temperature of 50°C to 80°C, preferably 60°C to 70°C, for example, about 65°C, for 1 minute to 45 minutes, preferably 10 to 20 minutes, for example, about 15 minutes. In one embodiment, the cooling step is carried out at a temperature of 0°C to 70°C, preferably 20°C to 60°C, for example, about 50°C to 58°C. In a preferred embodiment, the cooling step is carried out as a gradual cooling, preferably from about 58°C to about 50°C, with a decrease of 1°C per hour. Those skilled in the art will understand that the protocol for self-assembly will depend on the nanostructure design and / or nucleic acid sequence, and that the protocol can be adjusted according to known protocols for preparing nanostructures.

[0075] In one embodiment, when a method is mentioned, the method is an in vivo, ex vivo, in vitro, or in situ method, e.g., an in vitro method. In one embodiment, when a use is mentioned, the use is an in vivo, ex vivo, in vitro, or in situ use, e.g., an in vitro use. In one embodiment, the nanostructures of the present invention are for in vivo or in vitro use, preferably in vivo use.

[0076] In a further aspect, the present invention relates to the use of a nanostructure as defined herein or a composition as defined herein for gene expression, preferably for in vitro gene expression. For example, the nanostructures of the present invention can be used to efficiently express a molecule of interest, e.g., a protein of interest, in cell culture, for example, for large-scale production of therapeutic proteins such as mammalian enzymes or antibodies.

[0077] In a further aspect, the present invention relates to a method for preventing, treating, and / or diagnosing a disease or disorder, preferably a genetic and / or immunological disease or disorder, comprising administering a nanostructure as defined herein or a composition as defined herein to a patient in need thereof. In one embodiment, the method for preventing, treating, and / or diagnosing a disease or disorder is a method of gene therapy and / or immunotherapy. In one embodiment, the administering comprises administering an effective amount of a nanostructure as defined herein and / or a composition as defined herein to a patient in need thereof. The term "patient", as used herein, may relate to a human or an animal. The term "effective amount", as used herein, relates to an amount sufficient to induce a desired effect, e.g., sufficient labeling for in vivo imaging.

[0078] In a further aspect, the invention relates to the use of a nucleic acid nanostructure as defined herein, or a composition as defined herein, for the manufacture of a medicament, e.g., a medicament for preventing, treating, and / or diagnosing a disease or disorder, preferably a genetic and / or immunological disease or disorder, optionally for gene therapy and / or immunotherapy. For example, the nanostructure may comprise a gene that is deficient in a patient.

[0079] The terms "of the invention," "in accordance with the invention," "according to the invention," and the like, when used herein, are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0080] As used herein, the term "comprising" should be interpreted as encompassing both "including" and "consisting of," and both meanings are specifically intended and therefore individually disclosed embodiments according to the present invention. As used herein, "and / or" should be understood as a specific disclosure of each of two specific features or components, with or without the other. For example, "A and / or B" should be considered a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were described individually herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical value of ±20%, ±15%, ±10%, and, for example, ±5%. As will be understood by a person skilled in the art, the specific deviation from the numerical value of a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a greater deviation than an artificial or engineered technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g., "a", "an" or "the", this includes a plural of that noun unless otherwise stated.

[0081] The invention will now be further described with reference to the following drawings.

[0082] All methods referred to in the following figure legends were performed as detailed in the Examples. [Brief explanation of the drawings]

[0083] [Figure 1]Gene folding and expression from origami structures, as well as the effects of gene position and origami aspect ratio on gene expression, are shown. a) Schematic of the overall workflow: ssDNA is produced from plasmid DNA via phagemid (i) and then folded into 20-helix bundle (20HB) DNA origami objects (ii). The objects were delivered to cells, and gene expression from the origami structures was assessed by detecting a positive fluorescent readout (iii). The CMV promoter sequence is shown in blue, the gene encoding enhanced green fluorescent protein (EGFP) is shown in green, and polyA is shown in purple. b) Cylinder models and negative-stained transmission electron micrographs of 20HB, 12HB, and 32HB are shown in the top and bottom panels, respectively (scale bar 100 nm, inset 20 nm). (HB: helix bundle). Coloring indicates the location of scaffold features. For example, 20HB-ext indicates the CMV (blue), EGFP (green), and poly(A) (purple) coding sequences along the outer helix, while 20HB-int indicates the EGFP and poly(A) coding sequences within the inner helix. (c) Gene transfer efficiency in HEK293T cells (sc: scaffold; st: staple). (d) Schematic illustrating internal crosslinking via UV irradiation. UV-welded constructs 20HB-ext-W and 20HB-int-W silenced EGFP expression. (e) Gene transfer efficiency in HEK293T cells by electroporation observed for 20HB(-ext), 32HB, and 12HB constructs. Data collected in (c) and (e) were quantified using flow cytometry and presented as the mean ± standard deviation (sd) for n = 3 biologically independent experiments. Individual data points are overlaid. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons (*p≦0.05, ns p>0.05). [Figure 2]Optimization of gene expression through alternative staple designs and scaffold orientations is shown. Schematics of the scaffold routing and deployed scaffolds for the 20HB-LPv2 and 20HB-Circ designs, a and b, respectively. 20HB-LPv2 incorporates two consecutive 154-mer staples (enhancer staples, pink), while the 20HB-Circ design routes the 200-mer staple (pink) to serve as a splint for bringing together the 5' and 3' ends of the CMV. c, TEM micrographs of 20HB-LPv2 and 20HB-Circ. Scale bar 100 nm. d, Delivery into HEK293T cells via electroporation revealed statistically significantly increased gene transfer efficiency for samples 20HB-LP, 20HB-LPv2, and 20HB-Circ compared to standard 20HB (20HB-LP containing two consecutive 93-mer staples (enhancer staples)). e, Previously used scaffolds encoded the "coding strand," with the expression cassette present in the 5' to 3' orientation ("sc_EGFP1," top panel). We designed and produced a scaffold encoding the reverse complement of the expression cassette, thus the "template strand" ("sc_EGFP2," bottom panel). f, HEK293T cells transduced with either the sc_EGFP2 scaffold + staple mixture or the scaffold alone showed significantly higher transfection efficiency than that of the sc_EGFP1 counterpart. No significant difference in transfection efficiency was observed from the 20HB construct. g, EGFP mean fluorescence intensity (MFI) for the 20HB, 20HB-LPv2, and 20HB-Circ constructs folded using either the coding strand or the template strand as a scaffold. The 20HB and 20HB-Circ constructs did not show significant differences in MFI, but a significant difference was observed for the 20HB-LPv2 construct. Data collected in d, f, and g were quantified using flow cytometry and are presented as mean ± sd for n = 3 biologically independent experiments, with individual data points overlaid.Statistical analysis in d was performed using one-way ANOVA with Tukey's multiple comparisons, while statistical analysis in f and g was performed using Student's t-test (*p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001, ns p>0.05). Advantageously, efficient gene expression is achieved by nanostructures comprising a nucleic acid sequence encoding a gene in the scaffold strand and a nucleic acid sequence encoding a gene in a plurality of staple strands. [Figure 3] Figure 1 shows the enhancement of gene expression via alternative scaffold sequences. a) sc_EGFP1 represents the initial scaffold design, while sc_EGFP3 / 4 / 5 / 6 represent scaffold designs containing additional sequence features, such as ITRs (light pink) or ITR-binding domains (ITR*), Kozak sequences (black), and WPREs (dark pink). b and c) Comparison of gene transfer efficiency as determined by EGFP+ cells (b) and the mean fluorescence intensity of EGFP+ cells (c). Data collected in a and b were quantified using flow cytometry and presented as mean ± SD for n = 3 biologically independent experiments, with individual data points overlaid. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns p > 0.05). [Figure 4]Enhancement of gene expression through scaffold sequence design is shown. a) The 20HB design of the sc_EGFP5 scaffold contains an external single-stranded loop to allow the ITR sequence to self-anneal and form a hairpin structure. The 20HB design of the sc_EGFP6 scaffold contains two external loops to expose the ITR-binding domain, allowing the ITR hairpin staple to anneal. b, c) Gene transfer efficiency and MFI observed for the 20HB scaffolds folded into sc_EGFP5 and sc_EGFP6. Data collected in b and c were quantified using flow cytometry and are presented as the mean ± SD for n = 3 biologically independent experiments, with individual data points overlaid. Statistical analysis for b and c was performed using one-way ANOVA with Tukey's multiple comparisons (*p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, ns p > 0.05). d) Representative epifluorescence microscopy images showing EGFP expression from cells transfected with DNA origami objects folded with sc_EGFP1, sc_EGFP5, and sc_EGFP6. The images in the bottom row have been intentionally enhanced for contrast to reveal EGFP-positive cells with low EGFP intensity in the sc_EGFP1 sample. Images represent one of three biologically independent experiments, and similar results were observed each time. Scale bar: 100 μm. [Figure 5]Summary and optimization of structural transfection is shown. a) Comparison of transfection efficiency (%) and EGFP MFI (AU) across all investigated constructs, grouped by scaffold. sc_EGFP1 20HB-ext was used as an internal control in all experiments, and EGFP MFI is expressed as fold change compared to this sample. Three clusters are highlighted: 1, structures with high folding quality but low overall gene expression (gray); 2, structures with low folding quality and moderate expression levels (orange); and 3, structures with moderate to high folding quality and high expression levels (yellow). b) Representative epifluorescence microscopy images showing EGFP expression by successfully transfected HEK293T cells after optimization of electroporation settings. For each condition, eGFP expression (green), cells (phase contrast), and overlays are shown. Images represent one of two biologically independent experiments, and similar results were observed each time. Scale bar 100 μm. [Figure 6] Plasmid designs with various numbers of SV40 DTS sequences included (0x, 1x, 3x, and 6x SV40 repeats) for the production of custom scaffolds are shown. [Figure 7] Figure 1 shows the production and characterization of custom scaffolds and corresponding DNA origami structures. a) Agarose gel showing all custom scaffolds produced and the corresponding purified DNA origami structures. b) Representative negative-stain TEM image showing the 20HB DNA origami structures of each of the custom scaffolds produced. Scale bar 100 nm. [Figure 8](c) Flow cytometry histogram plot showing cell cycle populations of actively dividing and chemically arrested HEK293T cells. (d) Quantification of mCherry+ cells (%) in dividing and chemically arrested HEK293T populations 24 hours after electroporation with 20HB-mCh. [Figure 9]Figure 1 shows the effect of including SV40 DTS sequences in DNA origami structures on gene expression in dividing and arrested HEK293T cells after electroporation. a) Gene transfer efficiency (bar graph) and mean fluorescence intensity (square symbols) 24 hours after electroporation in normally dividing HEK293T cells with DNA origami structures containing one or several DTSs compared to control structures without DTS sequences. b) Gene transfer efficiency (bar graph) and mean fluorescence intensity (square symbols) 24 hours after electroporation in arrested HEK293T cells with DNA origami structures containing one or several DTSs compared to control structures without DTS sequences. Data were quantified using flow cytometry and presented as mean ± SD for n = 3 biologically independent experiments. One-way ANOVA was performed to test for statistically significant differences in gene expression compared to the control. For normally dividing cells (a), inclusion of one copy of the SV40 DTS resulted in a statistically significant increase in gene transfer efficiency (*p≦0.05) and mean fluorescence intensity (**p≦0.01) compared to controls without the DTS sequence. For arrested cells (b), inclusion of three copies of the SV40 DTS resulted in a statistically significant increase in gene transfer efficiency (**p≦0.01) and mean fluorescence intensity (****p≦0.0001) compared to controls without the DTS sequence. Inclusion of one copy did not result in a statistically significant increase in gene transfer efficiency for arrested cells, but did result in a statistically significant increase in mean gene expression (****p≦0.0001). [Figure 10]We demonstrate that delivery of multimeric origami assemblies enables co-delivery of genes at defined ratios. a) Cylindrical model of DNA origami objects for programmed assembly via shape-complementary protrusions and recesses. Schematics show the unique interaction patterns for building higher-order assemblies of dimers (i), trimers (i and ii), and tetramers (i, ii, iii, and iv). b) Representative comparative tomographic slices through dimer, trimer, and tetramer structures taken from a sample with mixed assembly products. Scale bar 100 nm. c) Schematic showing passivated overhangs to inhibit assembly and assembly assisted via complementary 5-nt sticky ends. d) Co-transfection (mCherry+ / EGFP+) efficiency in HEK293T cells after delivery of mCherry and EGFP as individual monomers (passivated) or as dimers connected via 5-nt or 8-nt sticky ends. e, Co-transfection (mCherry+ / EGFP+) efficiency in HEK293T cells with assembled multimeric DNA origami structures containing mCherry- and EGFP-encoded monomers at mCherry:EGFP ratios of 1:1, 1:2, and 1:3. EGFP MFI (AU) is shown on the right y-axis. Data collected in d and e were quantified using flow cytometry and presented as mean ± SD for n = 3 biologically independent experiments, with individual data points overlaid. Statistical analysis for d and e was performed using one-way ANOVA with Tukey's multiple comparisons (*p ≤ 0.05, **p ≤ 0.01, ns p > 0.05). f, Top: Schematic design of mCherry and EGFP monomer blocks for no assembly (passivation) or assembly into dimeric, trimeric, or tetrameric structures at mCherry:EGFP ratios of 1:1, 1:2, and 1:3, from left to right. Bottom: Representative epifluorescence microscopy images show expression of mCherry (red), EGFP (green), or co-expression (yellow) by successfully transfected HEK293T cells. Cell nuclei are shown in blue. Scale bar 100 μm. Images represent one of three biologically independent experiments, and similar results were observed each time. [Figure 11]DNA origami triangles for gene expression. a) Scaffold design with three SV40 DTS sequences for the production of custom scaffolds. The mCherry-encoding phagemid has 8064 bases. b) Representative negative-stain TEM image showing the mCherry-encoding DNA origami structure (triangular structure) on the produced custom scaffold. Scale bar: 50 nm. c) Representative epifluorescence microscopy image showing mCherry expression from HEK293T cells transfected with DNA origami objects 48 hours after electroporation of the mCherry-encoding origami triangles. Scale bar: 50 μm.

[0084] Reference will now be made to the following examples, which are presented to illustrate but not to limit the invention. [Example]

[0085] Example 1: Materials and Methods Scaffold production. Our customized scaffold design and cloning methods are detailed in Examples 2 and 6. Briefly, gene fragments from EGFP-containing plasmids (Addgene plasmids #13031 and #105530, with and without ITR sequences, respectively) were assembled with a fragment for bacterial resistance from a phage origin of replication (Addgene plasmid #126854) using either Golden Gate or digestion-ligation cloning. Plasmids were verified using restriction digestion and DNA sequencing (Eurofins genomics, Ebersberg, Germany). The exact primer sequences and methods can be found in Examples 2 and 6, and the sequences of the custom scaffolds can be found in SEQ ID NOS: 1-11. Exemplary sequences of staple strands can be found in SEQ ID NOS: 76-1507.

[0086] Production of ssDNA custom scaffolds was carried out as previously described 1、2Briefly, chemically competent DH5α E. coli cells were cotransformed with the plasmid of interest and a helper plasmid (Addgene plasmid #120346). Single colonies were picked and grown in a 5 mL preculture (2xYT, 30 μg / mL kanamycin, 30 μg / mL carbenicillin) for approximately 10 hours before being transferred to 750 mL of 2xYT (30 μg / mL kanamycin, 30 μg / mL carbenicillin, 5 mM MgCl2) in an Ultra Yield flask (Thomson). Cells were then grown overnight at 37°C in a shaking incubator. Bacteria were pelleted by centrifugation (45 min, 4500 g), and the supernatant was collected. Phagemid particles were precipitated from the supernatant by adding polyethylene glycol 8000 (PEG-8000, final concentration 3% w / w) and NaCl (final concentration 0.5 M), incubated with stirring at room temperature for 1 h, and then collected by centrifugation (45 min, 4500 g, 4 °C). The pellet was resuspended in 4 mL of 1x TE buffer (10 mM Tris, 1 mM EDTA, pH 8) and centrifuged again (15 min, 16000 g, 4 °C) to remove residual bacterial components. The ssDNA scaffold was then extracted via phagemid lysis and purified via ethanol precipitation.

[0087] Design, folding, and purification of DNA origami. All origami objects were folded in a standardized "folding buffer" containing 5 mM Tris base, 1 mM EDTA, and 5 mM NaCl, pH 8 (FoBx), plus x mM MgCl. All reactions were subjected to a thermal annealing ramp in a Tetrad (Bio-Rad) thermal cycling device. The exact folding conditions for each structure are shown in Tables 2 and 3. Staple strands were purchased from Integrated DNA Technologies, as exemplified in Example 6, and used with standard desalting unless otherwise specified. Origami objects were purified by either PEG precipitation or gel purification, as previously described. 3、4 .

[0088] Assembly of multicomponent DNA origami structures. To assemble origami subunits into dimer, trimer, and tetramer samples, monomers were mixed in molar ratios in 1x FoB5 buffer and incubated at 37°C for 48 hours. Passivated samples were treated identically.

[0089] UV welding. UV-weldable samples were designed with additional thymine bases located at all potential staple cross-sections and UV-crosslinked as previously described [5] with UV light (310 nm, 2 h) using an Asahi Spectra xenon light source (300 W, MAX-303) equipped with a high-transmission bandpass filter centered at 310 nm (XAQA310, Asahi Spectra). Samples were in FoB10 buffer during UV cross-linking.

[0090] PAGE purification of Ultramer. Long staple oligomers (93mer, 154mer, and 200mer) were purchased from IDT as Ultramers and purified in-house via denaturing urea polyacrylamide gel electrophoresis (Urea-PAGE). Bands corresponding to the correct MW were excised, crushed, and then 1x TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.00) was added. Pure Ultramers were recovered via EtOH precipitation, redissolved in MilliQ HO, and stored at 4°C.

[0091] Gel electrophoresis. For characterization of PCR products and plasmids, we used 1% agarose gels containing 0.5x TBE buffer (22.25 mM Tris base, 22.25 mM boric acid, 0.5 mM EDTA). Gel electrophoresis was performed in the same buffer at a voltage of 110 V for 1 h. To characterize assembled origami and scaffolds, we used 2% agarose gels containing 0.5x TBE buffer and 5.5 mM MgCl. Gel electrophoresis was performed in the same buffer at a voltage of 90 V for 1–2 h, and the gels were placed in a water bath for cooling. All gels were imaged using a Typhoon FLA9500 laser scanner (GE Healthcare) with a pixel size of 50 μm / pixel.

[0092] Negative staining TEM. Samples were incubated for 30–60 seconds on glow-discharged copper TEM grids (FCF400-CU, Electron Microscopy Sciences). Grids were then stained for 30 seconds (2% aqueous uranyl formate, 25 mM NaOH). Imaging was performed at magnifications of 21,000–42,000×. Data were acquired using an FEI Tecnai T12 microscope (120 kV, Tietz TEMCAM-F416 camera) with SerialEM software. Images were processed using ImageJ. 5 TEM micrographs were high-pass filtered to remove long-range staining gradients, and contrast was auto-leveled using Adobe Photoshop CS5.

[0093] Tilt series were performed from -50° to +50°, and photomicrographs were acquired at 2° increments. Tomograms were then generated using filtered backprojection and processed in Etomo (IMOD) to obtain tomograms. 6 The Gaussian filter used cutoffs of 0.25-0.5 and a falloff of 0.035.

[0094] Cell culture. HEK293T cells (DSMZ) were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, catalog number 31966047) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, catalog number F9665). Cells were grown in a humidified incubator at 37°C with 5% CO2.

[0095] Cell cycle arrest. HEK293T cells were arrested 24 hours prior to electroporation using arrest medium (DMEM supplemented with 10% FBS and 5 ng / μL aphidicolin (Sigma-Aldrich, catalog number A0781) dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich). Cells were maintained in arrest medium for the entire duration of the experiment.

[0096] Electroporation. Electroporation experiments were performed according to the manufacturer's protocol (Neon™ transfection protocol, ThermoFisher). Briefly, HEK293T cells were washed with phosphate-buffered saline (PBS) and harvested using TryplE. Cells were pelleted by centrifugation (5 min, 300 g), resuspended in PBS, and counted. Cells were centrifuged again (5 min, 300 g) and then 5 × 10 6 The cells were resuspended in Buffer R (Neon™ Transfection System) at a concentration of 1000 cells / mL. Mixtures for each condition were prepared so that each electroporation event contained 0.5 μg of total DNA, and the volume was supplemented to a total of 1 μL with 1× FOB5 buffer (folding buffer, 1 mM Tris, 1 mM EDTA, 5 mM NaCl, 5 mM MgCl2), which was then mixed with 9 μL of cell suspension. Electroporation was performed using a 10 μL gene transfer tip with a pulse voltage of 1150 V and two pulses with a width of 20 ms. After electroporation, the cells were immediately transferred to a 48-well plate previously prepared with poly-L-lysine coating and 240 μL of complete DMEM growth or arrest medium.

[0097] After 48 hours, samples were imaged using an EVOS™ M7000 Imaging System, and gene transfer efficiency was quantified via flow cytometry. For arrest experiments, cells were analyzed 24 hours after electroporation to avoid extensive cell death. Briefly, samples were acquired using an Attune Nxt Flow Cytometer and software (Thermo Fisher). For analysis, a total of 20,000 single-cell events were recorded, gated on side-scatter area versus height. EGFP was excited with a 488 nm laser, and emission was measured with a 530 / 30 nm bandpass filter. mCherry was excited with a 561 nm laser, and emission was measured with a 620 / 15 nm bandpass filter. Untreated cells and cells electroporated with buffer alone served as negative controls. Cells electroporated with the corresponding EGFP plasmid served as positive controls. Cell cycle arrest was confirmed by flow cytometric cell cycle analysis. Cells were stained with FxCycle™ Far Red Stain (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's protocol. The dye was excited with a 638 nm laser and emission was measured with a 670 / 14 nm bandpass filter. Data were analyzed after acquisition using FlowJo software (v10.7.1).

[0098] Statistics and reproducibility. Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc. v9). Data are presented as mean ± standard deviation, with individual data points representing biological replicates. Specific analyses performed are detailed in the corresponding figure legends. For all tests, differences were considered significant at p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

[0099] Example 2: Further Materials and Methods Scaffold cloning. Plasmids encoding custom scaffolds were created via standard cloning techniques. All plasmids except sc_EGFP3 were created via Golden Gate assembly using either Esp3I (NEB catalog no. R0734) or BsaI-HF® v2 (NEB catalog no. R3733) with T4 DNA ligase (NEB catalog no. M0202). For each plasmid, the appropriate clean site was introduced by PCR, and assembly was performed according to the manufacturer's protocol.

[0100] The exception, sc_EGFP3, was assembled via digestion and ligation; compatible enzyme cleavage sites were added to the fragments of interest by PCR. The fragments of interest were digested with the enzymes EcoNI and PacI (NEB catalog numbers R0521 and R0547, respectively), and then ligated using NEB T4 DNA ligase as described above.

[0101] For all constructs, touchdown PCR was performed using the primers (see Table 1). In all cases, PCR products were verified by agarose gel electrophoresis (AGE), bands were excised, and fragments were extracted according to the manufacturer's protocol (Qiagen QIAquick Gel Extraction Kit). [Table 1-1] [Table 1-2]

[0102] DNA origami folding. Nucleic acid nanostructures, such as CS3_EGFP3, CS3_EGFP1, CS3_EGFP4, CS3_EGFP4A, CS3_EGFP4B, CS3EGFP5, were successfully produced and folded using the conditions defined in Tables 2 and 3. [Table 2] [Table 3]

[0103] Example 3: Structural integrity analysis. The structural integrity of the nucleic acid nanostructures of the present invention was analyzed. For example, the structural integrity of 20 HB after electroporation was analyzed. The structural integrity of 20 HB was maintained when diluted with RPMI 1640 medium. Furthermore, 20 HB was stable both in electroporation buffer (EB, Buffer R in the kit) and after electroporation using the Neon™ Gene Transfer System. The nucleic acid nanostructures of the present invention exhibit advantageous structural integrity.

[0104] Example 4: Results Genes are easily expressed from DNA origami regardless of the location of the gene or the shape of the origami. Our initial investigation was to determine the fundamental parameters of origami design for mammalian cell expression. To do this, we created a customized circular ssDNA scaffold encoding enhanced green fluorescent protein (EGFP) in the 5' to 3' direction (coding strand), as shown in Figure 1a. Thus, cells successfully expressing EGFP from nucleic acid nanostructures can be monitored via fluorescence detection. We used two observable quantities in this study: the percentage of cells exhibiting green fluorescence (termed transfection efficiency) and the fluorescence intensity per cell, which we use as a proxy for expression efficiency. We used electroporation as a method for directly delivering origami to cells to avoid issues such as cellular uptake and endosomal escape, which can confound data interpretation and instead focus on parameters that directly affect expression. We used electroporation via the Neon™ transfection system, which left the DNA origami structure intact (and prevented its aggregation).

[0105] The custom EGFP scaffold (sc_EGFP1) was expressed with high yield and purity via phagemid production, and the scaffold folded efficiently into the designed target object (Figure 1b). To address whether the spatial location of the gene within the DNA origami object affects expression, we designed two 20-helix bundle (20HB) variants in which the EGFP gene was positioned either on the outside (20HB-ext) or inside (20HB-int) of the multilayered DNA origami (Figure 1b, first two panels). Gene expression occurred from both 20HB variants in human embryonic kidney 293T (HEK293T) cells after electroporation (Figure 1c). We found no statistically significant differences in either gene transfer or expression efficiency from the two objects.

[0106] The aspect ratio of DNA origami has previously been reported to affect cellular uptake. 7、8 To elucidate whether aspect ratio influences expression, we designed a 12-helix bundle (12HB) ​​approximately 114 nm long, a 20HB-ext approximately 69 nm long, and a 32HB approximately 42 nm long, with the EGFP gene and its recognition sequence displayed in all cases on the outside of the bundle (Figure 1b). These objects have aspect ratios of approximately 15, 5, and 2 for 12HB, 20HB, and 32HB, respectively. EGFP and related genes are displayed as a long continuous region with minimal scaffold crossings in 12HB, while 32HB displays them within the shortest continuous region. When delivered to HEK293T cells, we found no statistically significant difference in gene transfer and expression efficiency from the 20HB and 32HB samples (Figure 1e). A slight decrease in gene transfer efficiency was observed from the 12HB sample compared to the 20HB and 32HB samples (p ≤ 0.05). However, cell density after electroporation was also low for the 12HB object.

[0107] Therefore, both gene transfer and expression efficiency were independent of how the gene of interest was packaged among our panel of tested DNA origami. This observation suggested that DNA origami unfolding occurred prior to gene expression. We tested this hypothesis with non-unfoldable EGFP-encoding constructs. To this end, we included extra thymidine residues in the staple strands of 20HB-ext and 20HB-int to enable internal crosslinking via UV spot welding. 9 The object was then internally stabilized by hundreds of UV-induced cyclobutane pyrimidine dimer bonds between the staple strands and the crossroads, which topologically prevented strand dissociation (Fig. 1d). When we delivered the UV-point-welded 20HB variant into HEK293T cells, we found a near-complete suppression of EGFP signal (Fig. 1c). Although exposure to UV irradiation may also have an inhibitory effect on gene expression from the plasmid, 10、11 The remarkable and nearly complete inhibition of gene expression from covalently cross-linked DNA origami supports our hypothesis that DNA origami must first be unfolded for gene expression.

[0108] Targeted engineered modifications in the promoter region enhance gene expression. We observed that electroporating a premixed, non-annealed cocktail of ssDNA scaffold and staple strands, which do not form structured objects, resulted in slightly higher gene transfer efficiency compared to administering the ssDNA scaffold alone (Figures 1c and 1e). We hypothesized that partial association between the staple and scaffold strands results in a double-stranded DNA region around the promoter region that enhances gene expression. Therefore, we tested whether simply increasing the average staple length of the DNA origami object would result in enhanced expression. This did not result in enhanced expression, suggesting the need for more targeted design. We redesigned the 20HB object to incorporate long, continuous staple segments (enhancer staples) without crossovers in the promoter region, resulting in structures with consecutive 93-mer and 154-mer staples (enhancer staples) flanking the expression region, at the 5' start region of the CMV promoter, and at the 3' end of the polyA sequence (20HB-LP and 20HB-LPv2, respectively). Figure 2a shows a schematic of the design and staple positioning of 20HB-LPv2. 12 Inspired by this, we also prepared a design in which a 200-mer staple acts as a splint between the 5' start of CMV and the 3' end of polyA, forming a partially double-stranded circular structure when unfolded (20HB-Circ) (Figure 2b). All designs readily folded into defined 20HB structures, as seen by direct TEM imaging (Figure 2c). Delivery of these structures into cells resulted in up to 50% enhancement in gene expression efficiency for the structures 20HB-LP, 20HB-LPv2, and 20HB-Circ when compared to the standard 20HB staple route (Figure 2d).

[0109] Next, we determined whether the orientation of the target gene on the scaffold affects gene expression. Because the scaffold is ssDNA, delivery of the coding strand requires the synthesis of a complementary sequence (template strand) prior to transcription. We created a "template strand" scaffold with a reverse-complementary gene sequence (sc_EGFP2, Figure 2e). Delivering these scaffolds with or without staples demonstrated a significant increase in gene transfection efficiency for the template strand compared to the coding strand (Figure 2f). However, when folded into 20HB DNA origami objects, the difference in gene transfection efficiency disappeared (Figure 2f, right). Thus, the overall gene transfection efficiency of 20HB does not depend on whether it has either the coding strand or the template strand as the scaffold. However, we observed a slight trend toward increased mean fluorescence intensity (MFI) of EGFP in EGFP-positive cells for all objects with template strand scaffolds relative to those using the coding strand as the scaffold (Figure 2g).

[0110] The inclusion of scaffold sequence features enhances gene expression. To further enhance gene expression, we included additional features in the scaffold sequence based on the ssDNA AAV2 expression cassette (Fig. 3a): upstream of EGFP, we added a Kozak sequence that functions as a protein translation site. 13 The chimeric intron and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were placed downstream of EGFP (before the polyA). The WPRE is thought to improve mRNA stability and protein yield. 14 In addition, we included inverted terminal repeats (ITRs) flanking the expression cassette. ITRs are palindromic sequences that form T-shaped hairpins and are used by adeno-associated viruses as origins of replication for their ssDNA genomes, among other functions. 15、16We attempted to produce a synthetic scaffold ssDNA containing all of these features (sc_EGFP3, Figure 3a). The scaffold was produced with low yield and quality, which we attributed to the repetitive ITR structure. To improve the yield and quality of the scaffold, we produced a series of additional scaffolds (sc_EGFP4 and sc_EGFP5, respectively) containing only a single ITR downstream or upstream of the expression cassette. Furthermore, we created a scaffold (sc_EGFP6) that contains partial sequences of both ITRs, but in which the ITR hairpin is provided by complementary staple oligonucleotides during DNA origami folding. For all of these scaffold variants, we produced 20HBs with standard staple designs.

[0111] We observed a trend toward increased transfection efficiency and increased gene expression in all cases relative to the scaffold-only control (Figure 3b). While 20HB samples folded from the "enhanced" scaffolds showed a similar range of transfection efficiency to that observed with 20HB folded from the sc_EGFP1 scaffold, there was a significant increase in MFI in positive cells for 20HB folded from the "enhanced" scaffolds sc_EGFP3 / 4 / 5 / 6 relative to the original sc_EGFP1 (Figure 3b), indicating that additional features in the scaffolds enhanced intracellular gene expression.

[0112] In our designs discussed so far, the ITR sequences were masked within the double-helical DNA domain of the object so that they were only available when the object denatured in the cell. We hypothesized that gene expression from 20HB could be further improved by positioning the ITR sequence motifs so that they could assemble into their hairpin secondary structure already during object folding (Figure 4a, design 20HB-ex). Furthermore, we included a continuous 154-mer staple (enhancer staple) in the 5' region of the promoter of design 20HB-exLP, encoded by both the sc_EGFP5 and 6 scaffolds. Indeed, delivery of these designs demonstrated up to 2-fold gene transfer enhancement (Figure 4b) and up to 6- and 9-fold increases in expression efficiency, as measured by MFI, respectively, compared to the original 20HB design using the sc_EGFP1 scaffold (Figure 4c, d).

[0113] We plotted the achieved transfection efficiency against the fold change in EGFP MFI (AU) for all designs, relative to sc_EGFP1 20HB-ext as an internal control (Figure 5a). The samples are separated into three clusters. Cluster 1 contains objects constructed from the sc_EGFP1 / 2 scaffold, characterized by high quality folding and high transfection efficiency but low overall gene expression. Cluster 2 contains objects based on the sc_EGFP3 / 4 / 5 / 6 scaffold, which had low quality folding and low transfection efficiency but enhanced gene expression. Cluster 3 then contained objects with improved folding quality, as well as improved transfection efficiency and improved gene expression, as in the case of 20HB-exLP for the sc_EGFP5 and 6 scaffolds. Finally, we further optimized transfection efficiency by titrating the amount of material administered and by varying the electroporation conditions, resulting in even higher transfection efficiency (approximately 80%) and MFI (Figure 5b).

[0114] Encoding active nuclear import into DNA origami We designed and investigated DNA origami objects that inherently encode instructions for active nuclear import in mammalian cells, using an mCherry gene expression cassette to enable a facile fluorescent readout for assessing successful nuclear access (Figure 6). We designed a DNA origami scaffold to encode mCherry expression and a DNA nuclear targeting sequence (DTS) within the origami structure. A custom ssDNA scaffold was designed to contain a CMV promoter, mCherry reporter gene, and poly(A) signal encoded in the 5' to 3' direction (coding strand). We also used a 72-bp simian virus 40 (SV40) DTS. 17 We chose to use a scaffold with either 0x, 1x, 3x, or 6x SV40 repeats incorporated into the scaffold design (Figure 6). We designed a 20-helix bundle (20HB) and oriented the scaffold so that the gene features and DTS sequences were on the outer helices of the object.

[0115] Four DNA origami structures corresponding to each custom scaffold were folded and purified. 20HB-mCh and 20HB-1xSV40 folded with high yields and no clear leading bands and no major structural impurities, whereas 20HB-3xSV40 and 20HB-6xSV40 showed some structural impurities (Figure 7). The 20HB-3xSV40 structure showed low levels of higher-order bands. Higher-order impurities were even more prominent in the 20HB-6xSV40 structure, so this structure was further gel-purified. We attribute the folding difficulty to an increased number of repetitive DTS sequences within the scaffold, which hinder folding.

[0116] To avoid passive nuclear import during mitosis, we were interested in both dividing cells and cells arrested in the G1 / S phase of the cell cycle. We tested whether cell cycle arrest would result in the expected decrease in gene expression, since passive nuclear transport should be inhibited. To this end, we transduced both dividing and arrested cells with 20HB-mCh via electroporation. Cells were qualitatively analyzed by fluorescence microscopy and quantified by flow cytometry. We observed a statistically significant decrease in the percentage of mCherry+ cells in the arrested cell population after electroporation (Figure 8). We then tested the 20HB variants, 20HB-mCh, 20HB-1xSV40, 20HB-3xSV40, and 20HB-6xSV40, in both dividing and chemically arrested HEK293T cells. Cells were quantitatively evaluated via flow cytometry for the percentage (%) of mCherry+ cells and MFI (AU), indicating gene expression levels, and values ​​were compared with those of the control 20HB-mCh (Figure 9). In dividing cells, we observed a slight increase in both the percentage of mCherry+ cells and MFI for both 20HB-1xSV40 and 20HB-3xSV40. Inclusion of the SV40 DTS sequence had a greater effect in arrested cells, with the percentage of mCherry+ cells increasing for 20HB-1xSV40 (approximately 1.4-fold) and even more for 20HB-3xSV40 (approximately 1.8-fold) when compared with 20HB-mCh. MFI also showed a similar trend, with a 3-fold increase for 20HB-1xSV40 and a 4.5-fold increase for 20HB-3xSV40 in chemically arrested cells. Interestingly, 20HB-6xSV40 consistently showed a low percentage of mCherry+ cells and a low MFI of mCherry expression, an observation we attribute to poor folding quality of the sample.

[0117] Multiplexed gene assemblies for co-transfection. We designed DNA origami objects encoding either mCherry or EGFP expression, enabling assembly and delivery at 1:1, 1:2, and 1:3 stoichiometric ratios of mCherry to EGFP (Fig. 10a, b, using scaffolds sc_mCherry5 and sc_EGFP5). Individual gene blocks were assembled using shape-complementary docking sites flanked by sequence-complementary sticky ends that were either 5 or 8 base pairs long (referred to as 5nt or 8nt sticky ends, respectively). 18 The mCherry and EGFP monomers were programmed to interact with each other via the α- and β-terminal domains. We also created control objects with inactivated docking sites passivated by five thymidine-long single-stranded overhangs (Fig. 10c). Either dimeric, trimeric, or tetrameric gene assemblies formed as designed, as viewed by negative-stain TEM tomography and AGE (Fig. 10b). The co-transfection efficiency of a 1:1 stoichiometric mixture of unlinked mCherry and EGFP monomers was approximately 5.4 + / - 1.4%. In contrast, we observed a co-transfection efficiency of approximately 17.5 + / - 2.9% when using pre-assembled dimeric objects containing both mCherry and EGFP as expressible genes (Fig. 10d, f). This nearly four-fold increase in co-transfection relative to gene delivery via separate objects indicates that the delivery and expression of both components are now linked and no longer random.

[0118] Finally, we delivered multimeric origami objects in the form of dimers, trimers, and tetramers at mCherry:EGFP ratios of 1:1, 1:2, and 1:3. The molar concentrations of the multimeric origami objects were conserved across samples, and thus, the overall co-transfection efficiency remained comparable (Figure 10e, black bars). However, the EGFP expression level was directly proportional to the number of monomers present within the objects (Figure 10e, green bars). Direct imaging of cells using a two-channel fluorescence microscope was consistent with the observations made by flow cytometry (Figure 10f). Thus, we successfully delivered and expressed genes at designed stoichiometric ratios simply by "clicking" the genes together in higher-order DNA origami assemblies.

[0119] Example 5: Discussion Herein, we investigated gene expression from encoded DNA origami structures. We present scaffold and structural design features that enable highly efficient gene expression. The nanostructures of the present invention are highly effective tools for therapeutic gene delivery applications. In addition to electroporation, as described above, the nanostructures of the present invention can be delivered using techniques other than electroporation, for example, techniques that allow for even further optimization of gene transfer efficiency and gene expression. Instead of or in addition to electroporation, the inclusion of chemical moieties, aptamers, peptides, or antibodies on the origami surface can be used for targeted delivery and gene expression. The nanostructures of the present invention are invaluable for therapeutic applications, including, for example, designing scaffolds containing sequences that code for the expression of therapeutic proteins, or gene editing techniques such as CRISPR-Cas to enable therapeutic gene delivery and vaccines. Furthermore, the present invention provides a valuable tool for investigating the intracellular or in vivo fate of DNA nanotechnology, which has proven difficult to date due to the attachment of tracking molecules primarily on the staples rather than the scaffold.

[0120] Example 6: Exemplary Sequences SEQ ID NOs: 1 to 11: Scaffold sequences Scaffold sequences: sc_EGFP1 (SEQ ID NO: 1), sc_EGFP2 (SEQ ID NO: 2), sc_EGFP3 (SEQ ID NO: 3), sc_EGFP4 (SEQ ID NO: 4), sc_EGFP5 (SEQ ID NO: 5), sc_EGFP6 (SEQ ID NO: 6), sc_mCherry5 (SEQ ID NO: 7), sc_mCherry (SEQ ID NO: 8), sc_mCherry_1xSV40 (SEQ ID NO: 9), sc_mCherry_3xSV40 (SEQ ID NO: 10), and sc_mCherry_6xSV40 (SEQ ID NO: 11).

[0121] SEQ ID NOs: 12-21: Specific features Sequences of CMV promoter / enhancer (SEQ ID NO: 12), 5'ITR (SEQ ID NO: 13), 3'ITR (SEQ ID NO: 14), chimeric intron (SEQ ID NO: 15), Kozak (SEQ ID NO: 16), WPRE (SEQ ID NO: 17), EGFP (SEQ ID NO: 18), bGH polyA (SEQ ID NO: 19), mCherry (SEQ ID NO: 20), and SV40 (SEQ ID NO: 21).

[0122] SEQ ID NOs: 22 to 75: primer sequences (see also Table 1). SEQ ID NOs: 76-1507: Exemplary staple strand sequences. SEQ ID NOs: 76 to 177: staple sequences of sc_EGFP1, 20HB-ext. SEQ ID NOs: 178 to 279: sc_EGFP1, staple sequences of 20HB-ext-W. SEQ ID NOs: 280 to 381: staple sequences of sc_EGFP1, 20HB-int. SEQ ID NOs: 382 to 483: staple sequences of sc_EGFP1, 20HB-int-W. SEQ ID NOs: 484 to 584: staple sequences of sc_EGFP1, 32HB. SEQ ID NOs: 585 to 692: sc_EGFP1, 12HB staple sequences. SEQ ID NOs: 693 to 766: sc_EGFP1, 20HB-LS staple sequences. SEQ ID NOs: 767 to 863: sc_EGFP1, staple sequences of 20HB-LP. SEQ ID NOs: 864 to 957: staple sequences of sc_EGFP1 and 20HB-LPv2. SEQ ID NOs: 958 to 1055: staple sequences of sc_EGFP1 and 20HB-Circ. SEQ ID NOs: 1056 to 1157: staple sequences of sc_EGFP2, 20HB. SEQ ID NOs: 1158 to 1279: staple sequences of sc_EGFP3, 20HB. SEQ ID NOs: 1280 to 1392: staple sequences of sc_EGFP4, 20HB. SEQ ID NOs: 1393 to 1507: staple sequences of sc_EGFP5 and 20HB-exLP.

[0123] References 1. Engelhardt, F. A. S. et al. Custom-Size, Functional, and Durable DNA Origami with Design-Specific Scaffolds. ACS Nano 13, 5015 - 5027 (2019). 2. Praetorius, F. et al. Biotechnological mass production of DNA origami. Nature 552, 84 - 87 (2017). 3. Stahl, E., Martin, T. G., Praetorius, F. & Dietz, H. Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions. Angewandte Chemie 126, 12949 - 12954 (2014). 4. Wagenbauer, K. F. et al. How We Make DNA Origami. ChemBioChem 18, 1873 - 1885 (2017). 5. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676 - 682 (2012). 6. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer Visualization of Three-Dimensional Image Data Using IMOD. Journal of Structural Biology 116, 71 - 76 (1996). 7. Bastings, M. M. C. et al. Modulation of the Cellular Uptake of DNA Origami through Control over Mass and Shape. Nano Lett. 18, 3557 - 3564 (2018). 8.Wang,P.et al.Visualization of the Cellular Uptake and Trafficking of DNA Origami Nanostructures in Cancer Cells.J.Am.Chem.Soc.140,2478-2484(2018). 9.Gerling,T.,Kube,M.,Kick,B.&Dietz,H.Sequence-programmable covalent bonding of designed DNA assemblies.Science Advances(2018). 10.Mitchell,D.L.,Vaughan,J.E.&Nairn,R.S.Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and(6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA.Plasmid 21,21-30(1989). 11.Jiang,Y.,Ke,C.,Mieczkowski,P.A.&Marszalek,P.E.Detecting Ultraviolet Damage in Single DNA Molecules by Atomic Force Microscopy.Biophys J 93,1758-1767(2007). 12.Wei,L.&Ploss,A.Hepatitis B virus cccDNA is formed through distinct repair processes of each strand.Nat Commun 12,1591(2021). 13.Acevedo,J.M.,Hoermann,B.,Schlimbach,T.&Teleman,A.A.Changes in global translation elongation or initiation rates shape the proteome via the Kozak sequence.Sci Rep 8,4018(2018). 14.Brun,S.,Faucon-Biguet,N.&Mallet,J.Optimization of transgene expression at the posttranscriptional level in neural cells:implications for gene therapy.Molecular Therapy 7,782-789(2003). 15.Ping,H.,Liu,X.,Zhu,D.,Li,T.&Zhang,C.Construction and Gene Expression Analysis of a Single-Stranded DNA Minivector Based on an Inverted Terminal Repeat of Adeno-Associated Virus.Mol Biotechnol 57,382-390(2015). 16.Cao,L.,During,M.&Xiao,W.Replication competent helper functions for recombinant AAV vector generation.Gene Ther 9,1199-1206(2002). 17.Dean,D.A.,Dean,B.S.,Muller,S.&Smith,L.C.Sequence Requirements for Plasmid Nuclear Import.Experimental Cell Research 253,713-722(1999). 18. Gerling, T., Wagenbauer, KF, Neuner, AM & Dietz, H. Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components. Science 347, 1446-1452 (2015).

[0124] The features of the invention disclosed in the specification, the claims and / or the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims

1. A nucleic acid nanostructure comprising at least one scaffold strand and a plurality of staple strands, wherein the nanostructure, preferably the at least one scaffold strand, comprises at least one nucleic acid sequence encoding a gene.

2. 2. The nucleic acid nanostructure of claim 1, wherein the nucleic acid nanostructure, preferably the at least one scaffold strand, comprises a first nucleic acid sequence encoding a gene and a second nucleic acid sequence encoding a gene, and optionally, the nucleic acid nanostructure, preferably the at least one scaffold strand, comprises multiple nucleic acid sequences encoding genes.

3. The nucleic acid nanostructure of claim 1 or 2, wherein the nucleic acid nanostructure comprises a first subunit and a second subunit, and preferably the first subunit and the second subunit each comprise a nucleic acid sequence encoding a gene.

4. the nanostructures comprise enhancer staple strands having a length ranging from about 60 to about 250 nucleobases, preferably from about 80 to about 220 nucleobases, more preferably from about 90 to about 200 nucleobases; Optionally, the enhancer staple strands are configured to bind to the scaffold strands at the 5' and / or 3' ends of a nucleic acid sequence encoding the gene.

5. 10. The nucleic acid nanostructure of any one of the preceding claims, wherein the nanostructure, preferably the scaffold strand and / or at least one staple strand of the plurality of staple strands, comprises a nucleic acid sequence configured to form a loop structure, preferably an inverted terminal repeat nucleic acid sequence configured to form a hairpin.

6. The nucleic acid nanostructure of claim 5, wherein the nucleic acid sequence configured to form the loop structure is configured so that the loop is formed at the 5' end and / or 3' end of the nucleic acid sequence encoding the gene.

7. 10. The nucleic acid nanostructure of any one of the preceding claims, wherein the nanostructure, preferably the at least one scaffold strand, comprises at least one nuclear targeting sequence, preferably a DNA nuclear targeting sequence, more preferably a simian virus 40 DNA nuclear targeting sequence, and optionally the nanostructure, preferably the at least one scaffold strand, comprises multiple nuclear targeting sequences.

8. 10. The nucleic acid nanostructure of any one of the preceding claims, wherein the nanostructure, preferably the at least one scaffold strand, comprises a promoter such as a CMV promoter, a terminator, a polyadenylation signal sequence, an intron, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element.

9. 10. The nucleic acid nanostructure of any one of the preceding claims, wherein the nanostructure has an aspect ratio ranging from about 1:1 to about 1000:1, preferably from 1.5:1 to about 20:1, more preferably from about 2:1 to about 15:

1.

10. 10. The nucleic acid nanostructure of any one of the preceding claims, wherein the scaffold strand comprises at least one nucleic acid sequence encoding the gene, a promoter, and a terminator, and optionally further comprises a nucleic acid sequence configured to form a loop structure, an intron, a DNA nuclear targeting sequence, a polyadenylation signal sequence, a Kozak sequence, and / or a woodchuck hepatitis virus post-transcriptional regulatory element.

11. A composition, preferably a pharmaceutical composition, comprising a nucleic acid nanostructure according to any one of the preceding claims.

12. A collection of nucleic acid sequences or a collection of plasmids encoding the nucleic acid nanostructures of any one of claims 1 to 10.

13. A nucleic acid nanostructure according to any one of claims 1 to 10 or a composition according to claim 11 for use in medicine, preferably for use in a method for preventing, treating and / or diagnosing a disease or disorder, preferably a genetic and / or immunological disease or disorder, optionally for use in gene therapy and / or immunotherapy.

14. 11. A method for expressing a gene from a nucleic acid nanostructure, preferably a nucleic acid nanostructure according to any one of claims 1 to 10, comprising: i) providing a nucleic acid nanostructure comprising at least one nucleic acid sequence encoding a gene, preferably a nucleic acid nanostructure according to any one of claims 1 to 10; ii) delivering the nucleic acid nanostructure provided in step i) to a cell, preferably wherein said delivering comprises transfecting or transforming said cell; iii) enabling the cell to express the gene; Optionally, the method wherein said providing in step i) comprises providing a plasmid, preferably a phagemid, or a collection of plasmids, preferably a collection of phagemids, wherein said plasmid or collection of plasmids encodes said nucleic acid nanostructure; and preparing said nucleic acid nanostructure using said plasmid or collection of plasmids, preferably by using bacteriophages.

15. Use of the nanostructure according to any one of claims 1 to 10 or the composition according to claim 11 for gene expression, preferably for in vitro gene expression.

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