DNA origami coding for gene expression and co-transfection

Abstract

The present invention relates to a nucleic acid nanostructure comprising at least one scaffold chain and a plurality of staple chains wherein the nanostructure, preferably the at least one scaffold chain, comprises at least one nucleic acid sequence encoding a gene. The invention further relates to a composition comprising the nucleic acid nanostructure, and to a set of nucleic acid sequences or a set of plasmids encoding a nucleic acid nanostructure. Furthermore, the present invention relates to a nucleic acid nanostructure or a composition comprising a nucleic acid nanostructure for use in a medicament, preferably in a method for preventing, treating and / or diagnosing a disease or condition. The invention also relates to a method for expressing a gene from a nucleic acid nanostructure, and to the use of a nanostructure or composition for gene expression.

Description

Technical Field

[0001] 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. The present invention further relates to a composition comprising a nucleic acid nanostructure and to a collection of nucleic acid sequences or a collection of plasmids encoding a nucleic acid nanostructure. In addition, the present invention relates to a nucleic acid nanostructure or a composition comprising a nucleic acid nanostructure for use in a medicament, preferably for use in a method of preventing, treating and / or diagnosing a disease or disorder. The present invention also relates to a method for expressing a gene from a nucleic acid nanostructure and to the use of a nanostructure or a composition for gene expression. Background Art

[0002] The delivery and expression of genes face a series of obstacles, including how to package, target and release the nucleic acids to be delivered. These obstacles become particularly prominent in the delivery of multi-component systems, in which several genes need to be delivered in precise and adjustable amounts, such as in the case of technologies based on CRISPR and the construction of genetic circuits. The achievement of genome or epigenome editing, transcriptional regulation and / or genetic circuit construction through multiplexing offers great potential for customizing gene networks for therapeutic programming (reprogramming), bioproduction and basic research. However, despite the rapid progress in these fields, the synchronous delivery and expression of multiple genes remain challenging, especially when seeking in vivo delivery.

[0003] Artificial structures formed by nucleic acids such as DNA origami have been demonstrated to have great potential in the field of biotechnology. DNA origami enables the packaging of long single-stranded DNA into compact structures with unparalleled levels of structural programmability, homology, spatial addressability and biocompatibility. In addition, multi-component assemblies of different DNA origami structures can be achieved, which have a structure composed of 220 monomers and a size exceeding 1 GDa. However, despite the significant progress made in the use of DNA origami in applications such as drug delivery, sensing and imaging, the development of DNA origami in gene therapy remains insufficient. To date, DNA origami has only been used in gene therapy research as a hybrid with RNA or protein.

[0004] There is still a need for tools for the efficient expression of genes, such as mammalian genes. In particular, there is still a need for assemblies encoding two or more genes in a controlled stoichiometry. In addition, there is still a need for the synchronous delivery and expression of one or more genes (e.g., multiple genes). In addition, there is still a need for tools for the efficient delivery of genes and for achieving in vivo gene expression. Summary of the Invention

[0005] The various elements of the present invention will be described below. These elements are listed by way of specific examples, but it should be understood that the elements can be combined in any manner and in any number to yield additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. This specification should be understood to support and cover the following embodiments: embodiments that combine two or more of the explicitly described embodiments; or embodiments that combine one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Additionally, any permutation and combination of all the described elements in this application should be considered to be disclosed by the description of this application, unless the context otherwise indicates.

[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; wherein optionally, the nucleic acid nanostructure, preferably the at least one scaffold strand, comprises a plurality of nucleic acid sequences encoding genes.

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

[0009] In one embodiment, the nanostructure comprises enhanced staple strands, the length of the enhanced staple strands ranging from about 60 to about 250 nucleic acid bases, preferably from about 80 to about 220 nucleic acid bases, more preferably from about 90 to about 200 nucleic acid bases;

[0010] wherein optionally, the enhanced staple strands are configured to bind to the scaffold strand at the 5' end and / or 3' end of the nucleic acid sequence of the gene encoding.

[0011] In one embodiment, the nanostructure comprises enhanced staple strands, the length of the enhanced staple strands ranging from about 60 to about 250 nucleic acid bases, preferably from about 80 to about 220 nucleic acid bases, more preferably from about 90 to about 200 nucleic acid bases;

[0012] wherein optionally, the enhanced staple strands comprise nucleic acid sequences complementary to the nucleic acid sequence of the scaffold strand, wherein the nucleic acid sequence of the scaffold strand is located at the 5' end and / or 3' end of the nucleic acid sequence of the gene encoding.

[0013] In one embodiment, the nanostructure, preferably at least one staple strand of the scaffold strand and / or 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 at least one staple strand of the scaffold strand and / or the plurality of staple strands, comprises a loop structure, preferably a hairpin.

[0014] In one embodiment, the nucleic acid sequence configured to form a loop structure is configured such that a loop is formed at the 5' end and / or 3' end of the nucleic acid sequence of the coding gene.

[0015] In one embodiment, the nanostructure, preferably at least one staple strand of the scaffold strand and / or 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 of the coding 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; wherein optionally, the nanostructure, preferably the at least one scaffold strand, comprises a plurality of nuclear targeting sequences.

[0016] 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.

[0017] In one embodiment, the aspect ratio of the nanostructure ranges 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.

[0018] In one embodiment, the scaffold strand comprises the nucleic acid sequence of the at least one coding gene, a promoter, and a terminator; 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.

[0019] In one embodiment, the scaffold strand comprises the nucleic acid sequence of the at least one coding gene, a promoter, and a terminator; wherein 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.

[0020] In one embodiment, the nucleic acid sequence of the encoding 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.

[0021] In another aspect, the present invention relates to a composition, preferably a pharmaceutical composition, comprising a nucleic acid nanostructure as defined herein.

[0022] In another aspect, the present invention relates to a collection of nucleic acid sequences encoding a nucleic acid nanostructure as defined herein or a collection of plasmids.

[0023] In another aspect, the present invention relates to a nucleic acid nanostructure as defined herein or a composition as defined herein, wherein the nucleic acid nanostructure or the composition is for use in a medicament; preferably for a method of preventing, treating, and / or diagnosing a disease or disorder, preferably a genetic and / or immune disease or disorder; optionally for gene therapy and / or immunotherapy.

[0024] In one embodiment, the nucleic acid nanostructure as defined herein or the composition as defined herein is for gene therapy and / or immunotherapy.

[0025] In another aspect, the present invention relates to a method for expressing a gene from a nucleic acid nanostructure, preferably a nucleic acid nanostructure as defined herein, the method comprising:

[0026] i) providing a nucleic acid nanostructure comprising a nucleic acid sequence encoding at least one encoding gene, preferably a nucleic acid nanostructure as defined herein;

[0027] ii) delivering the nucleic acid nanostructure provided in step i) to a cell; wherein preferably, the delivery comprises transfecting or transforming the cell;

[0028] iii) causing the cell to express the gene;

[0029] wherein optionally, the providing in step i) comprises: providing a plasmid, preferably providing a phagemid, or a collection of plasmids, preferably a collection of phagemids, wherein the plasmid or the collection of plasmids encodes the nucleic acid nanostructure; and using the plasmid or the collection of plasmids, preferably by using a phage to prepare the nucleic acid nanostructure.

[0030] In another 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.

[0031] In another aspect, the present invention relates to a method for preventing, treating, and / or diagnosing a disease or disorder, preferably a genetic and / or immune disease or disorder, the method comprising administering to a patient in need thereof a nanostructure as defined herein or a composition as defined herein.

[0032] In one embodiment, the method for preventing, treating, and / or diagnosing a disease or disorder is a method of gene therapy and / or immunotherapy.

[0033] In one embodiment, the administering comprises administering to a patient in need thereof an effective amount of a nanostructure as defined herein and / or a composition as defined herein.

[0034] In another aspect, the present invention relates to the use of a nanostructure as defined herein or a composition as defined herein for the manufacture of a medicament (e.g., for preventing, treating, and / or diagnosing a disease or disorder, preferably a genetic and / or immune disease or disorder), optionally for gene therapy and / or immunotherapy.

[0035] In one embodiment, a nanostructure as defined herein or a composition as defined herein is used for the manufacture of a medicament for gene therapy and / or immunotherapy. Detailed Description

[0036] An object of the present invention is to provide tools for efficient gene expression (such as eukaryotic genes, e.g., mammalian genes). Specifically, an object of the present invention is to provide nanostructures that encode two or more genes in a controlled stoichiometry. In addition, an object of the present invention is to synchronously deliver and express one or more genes, e.g., multiple genes. Moreover, an object of the present invention is to efficiently deliver and express genes in vitro, ex vivo, and in vivo.

[0037] The inventors have successfully expressed genes from encoded DNA origami. Specifically, the inventors synthesized a library of custom ssDNA scaffolds for mammalian gene expression (e.g., using phage-based production). The inventors found that genes are readily expressed from nucleic acid nanostructures regardless of the position of the gene within the nanostructure or the shape of the nanostructure. The inventors found that gene expression can even be further enhanced by nucleic acid sequences configured to form loop structures (e.g., adeno-associated virus-inspired inverted terminal repeat (ITR) hairpin sequences) that are located upstream of the expression cassette or flank the loop structure (e.g., ITR hairpin) on both sides of the expression cassette, and the loop structure is provided on the staple. Overall, the inventors demonstrated efficient expression of genes encoded in the nucleic acid nanostructures of the present invention, particularly the DNA origami structures of the present invention.

[0038] The inventors have successfully delivered and expressed genes from encoded ssDNA scaffolds and custom DNA origami objects, as Figure 1 shown in a. The inventors have found that the nucleic acid nanostructures of the present invention readily unfold in the intracellular environment and efficiently express genes from the ssDNA scaffold strand, and have found that gene expression can be further optimized by targeted staple design. The inventors have further produced and tested a library of ssDNA scaffolds that are optimized for improved gene expression, high-yield scaffold production and purity, and origami folding quality. Overall, the inventors have unexpectedly found that including at least one loop structure (such as an ITR secondary DNA structure, preferably located upstream of the expression cassette) and / or including one or more nuclear targeting sequences (such as DTS sequences, for example three SV40 DTS sequences) enables efficient and robust gene expression without compromising scaffold production or origami folding quality. Finally, the inventors have demonstrated the efficient and controlled assembly of gene-encoded origami structures in a stoichiometric ratio. These "plug-and-play" architected origamis have successfully achieved the co-delivery and expression of gene arrays with unprecedented control.

[0039] Advantageously, the nanostructures of the present invention utilize both the ability to encode genetic information and the unique design possibilities of DNA origami structures. Thus, advantageously, the nanostructures of the present invention are capable of encoding and expressing gene arrays in a controlled stoichiometry.

[0040] 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. Advantageously, one or more genes can be efficiently expressed using the nanostructures of the present invention.

[0041] As used herein, the term "nanostructure" 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. The nanostructure can be fabricated using readily available nucleic acid nanostructure techniques (e.g., DNA origami techniques), which involve relatively less complex procedures for assembling nanostructures compared to standard nanofabrication techniques. In one embodiment, the nanostructure is at least partially fabricated using DNA origami techniques. Due to the self-assembly of DNA origami structures and the readily available software for designing the corresponding scaffold strands and staple strands, this is a relatively less complex manufacturing process compared to standard nanofabrication techniques. In one embodiment, the nanostructure of the present invention is a DNA origami structure. In one embodiment, the maximum length of the nanostructure of the present invention is less than 1000 nm, for example, in the range of about 10 nm to about 150 nm, preferably about 20 nm to about 100 nm.

[0042] In one embodiment, the nucleic acid nanostructure (e.g., DNA origami structure) of the present invention comprises at least one scaffold strand and multiple staple strands (e.g., single-stranded oligonucleotide staple strands). As used in the present invention, the term "staple strand" shall refer to a single-stranded oligonucleotide molecule that is at least partially complementary to the scaffold strand. In one embodiment, when referring to a "staple", it refers to one staple strand among multiple staple strands. Generally, staple strands can be used to introduce, for example, coupling sites into the DNA origami structure and / or DNA origami subunits. As used herein, the term "multiple staple strands" refers to multiple, for example, at least three staple strands. For example, multiple staple strands can refer to at least 3, 4, 5, 6, 7, 8, 9, 10 or more staple strands. In one embodiment, the length of the staple strand, particularly the staple strand among the multiple staple strands, is from 20 to 100 nucleic acid bases. In one embodiment, the length of the staple strand among the multiple staple strands is from 20 to 80 nucleic acid bases, and the length of the enhanced staple strand is from 90 to 250 nucleic acid bases. In one embodiment, the enhanced staple strand differs from the staple strand among the multiple staple strands in that it is at least 5 (preferably at least 10) nucleic acid bases longer than each staple strand among the multiple staple strands.

[0043] The term "scaffold strand" refers to a nucleic acid strand, preferably a DNA strand, such as a single-stranded nucleic acid strand, for example a single-stranded polynucleotide strand. In one embodiment, the scaffold strand composes and / or runs through a major part of a DNA origami structure and / or a DNA origami subunit. In one embodiment, the length of the scaffold strand is from 100 to 20,000 nucleic acid bases, preferably from 120 to 15,000 nucleic acid bases, more preferably from 260 to 11,000 nucleic acid bases, such as from 1,000 to 11,000 nucleic acid bases. 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 length of the scaffold strand is from 260 to 20,000 nucleic acid bases, the length of the enhanced staple strand is from 90 to 250 nucleic acid bases, and the length of the staple strands among the multiple staple strands is from 20 to 80 nucleic acid bases.

[0044] A nanostructure, such as a DNA origami structure, can comprise at least one scaffold strand, i.e., a single-stranded polynucleotide scaffold DNA with a known sequence. The DNA origami structure can further comprise multiple single-stranded oligonucleotide staple strands, wherein each staple strand can be at least partially complementary to at least one scaffold strand. Additionally, each staple strand can be configured to bind to the at least one scaffold strand, wherein the at least one scaffold strand can be folded and / or arranged such that a desired nanostructure can be formed. As used herein, the term "strand" refers to a nucleic acid strand, such as a DNA and / or RNA strand, preferably a DNA strand. DNA origami can be used, i.e., three-dimensional nanostructures can be achieved by combining scaffold strands and staple strands to form the required parts and whole devices. Such designs can be carried out, for example, using software such as caDNAno. That is, in some embodiments, a nanostructure comprising multiple parts can be composed of one scaffold strand, while in other embodiments, multiple scaffold strands can be utilized to construct the parts of the nanostructure.

[0045] In one embodiment, the shape of the nanostructure can be any shape, such as brick-shaped, rod-shaped, triangular, circular, cuboid (i.e., rectangular), star-shaped or any other shape. The nanostructure of the present invention can have 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", "nanoobject" and "nucleic acid nanostructure" can be used interchangeably.

[0046] In one embodiment, each staple strand is configured to bind to at least one, preferably two or more different positions of at least one of the at least one scaffold strand. In one embodiment, the nucleic acid nanostructure of the present invention, such as 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 staple strands. In one embodiment, the nucleic acid nanostructure of the present invention comprises ≥10 DNA strands, preferably ≥15 DNA strands, such as at least one scaffold strand and at least nine staple strands.

[0047] As used herein, the terms "DNA origami structure", "DNA origami", and "DNA origami object" refer to nanostructures that contain DNA as a building material to fabricate nanoscale shapes. Preparing and / or providing DNA origami involves using multiple rationally designed staple DNA strands to fold one or more scaffold DNA strands into a defined shape, e.g., by self-assembly. The scaffold strand is typically longer than the staple strand. The nucleic acid sequence of the staple strand is designed such that the staple strand hybridizes to a defined portion of the scaffold strand, and a specific shape of the nanostructure is obtained due to the hybridization.

[0048] In one embodiment, as used herein, the term "nucleic acid" refers to a nucleotide sequence, such as ribonucleic acid or 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. The advantage of nucleic acid nanostructures such as DNA origami structures is that, due to their rational design, nucleic acid nanostructures such as DNA origami structures can contain multiple genes, especially in the desired stoichiometry. Advantageously, through nucleic acid nanostructures such as DNA origami structures, the number of genes and their expression can be precisely controlled. In addition, the arrangement of genes and other nucleic acid sequences of interest can be precisely controlled. Another advantage of nucleic acid nanostructures such as DNA origami structures is that they can be stabilized against nucleases. In one embodiment, the nanostructure is configured to be stabilized against nucleases. Another advantage of DNA origami structures is that, compared to non-DNA origami nucleic acid nanostructures (such as DNA tetrahedra or RNA assemblies), the assembly (e.g., self-assembly) and purification of the structure are more robust and simple. All embodiments described herein regarding "a / the nucleic acid nanostructure" or "a / the nucleic acid nanostructure of the present invention" should be understood to equally refer to the nanostructures contained in the compositions of the present invention, the nanostructures used according to the present invention, and the nanostructures provided in any method of the present invention.

[0049] As used herein, the term "nucleic acid sequence encoding a gene" refers to a nucleic acid sequence encoding any gene of interest, such as 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 prokaryotic genes, viral genes, and eukaryotic genes. In one embodiment, the gene is selected from prokaryotic genes such as CRISPR-based genes and eukaryotic genes such as human genes. In a preferred embodiment, the gene is a eukaryotic gene, preferably a mammalian gene, such as a human gene. One advantage of the nucleic acid nanostructures of the present invention is that they are capable of expressing mammalian genes, such as different mammalian genes with defined stoichiometry. Another advantage is that the nanostructures are capable of expressing prokaryotic genes (e.g., for CRISPR-based gene editing) and viral genes (e.g., 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 the at least one scaffold strand, comprises a first nucleic acid sequence encoding a gene and a second nucleic acid sequence encoding a gene; wherein optionally, the nucleic acid nanostructure, preferably the at least one scaffold strand, comprises a plurality of nucleic acid sequences encoding genes. Advantageously, the nanostructures 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 a plurality of nucleic acid sequences encoding genes) are capable of efficiently expressing the gene of interest with 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. The nanostructures of the present invention advantageously allow the expression of gene arrays 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 the gene of interest, such as one or more nucleic acid sequences encoding genes, such as one or more first nucleic acid sequences encoding genes and one or more second nucleic acid sequences encoding genes.

[0050] In one embodiment, the nucleic acid nanostructure comprises a plurality of nucleic acid sequences encoding genes. In one embodiment, each nucleic acid sequence of the plurality of nucleic acid sequences encoding genes encodes a different gene. In one embodiment, the gene encoded by each nucleic acid sequence of the plurality of nucleic acid sequences encoding genes is different from the gene encoded by the nucleic acid sequence of other encoding genes in the plurality of nucleic acid sequences encoding genes. Thus, the plurality of nucleic acid sequences encoding genes can comprise a plurality of encoded genes. In one embodiment, the nucleic acid sequence of the encoding gene is located at any site of the nucleic acid nanostructure, preferably at any site of the scaffold strand.

[0051] In one embodiment, the nucleic acid nanostructure comprises a first subunit and a second subunit; wherein preferably, each of the first subunit and the second subunit comprises a nucleic acid sequence encoding a gene. The advantage of a nucleic acid nanostructure comprising a first subunit and a second subunit (e.g., a nucleic acid nanostructure comprising a plurality of subunits) is that each subunit can comprise one or more nucleic acid sequences encoding genes, and thus the stoichiometry of the plurality of nucleic acid sequences encoding genes can be rationally designed. In one embodiment, the nucleic acid nanostructure comprises a first subunit and a second subunit, optionally comprising a plurality of stacked subunits. In one embodiment, the nucleic acid nanostructure comprises two or more stacked subunits, and the two or more stacked subunits comprise a first subunit and a second subunit. In one embodiment, each of the first subunit and the second subunit comprises a nucleic acid sequence encoding a gene, wherein the nucleic acid sequence encoding a gene comprised by the first subunit is the same as or different from the nucleic acid sequence encoding a gene comprised by the second subunit. In one embodiment, the first subunit and the second subunit 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, each of the first subunit and the second subunit, optionally each subunit of the plurality of subunits, comprises a scaffold strand and a plurality of staple strands. In one embodiment, the nucleic acid nanostructure comprises a first subunit and a second subunit, wherein each subunit comprises a scaffold strand that comprises a nucleic acid sequence encoding a gene, and wherein each subunit comprises a plurality of staple strands, optionally wherein at least one subunit comprises enhanced staple strands. The subunits of the nucleic acid nanostructure can be connected in any manner, such as by shape complementarity, by nucleobase stacking interactions, by nucleic acid-mediated interactions such as base pairing and / or by covalent binding such as disulfide bridges. Advantageously, a controlled stoichiometric ratio ( Figure 6)Implement the assembly of multiple DNA origami subunits containing multiple genes, enabling the synchronous delivery of multiple components. This is particularly important for gene / base editing or epigenetic regulation in fields such as CRISPR-based technologies. In addition, the production of virus-like particles (VLPs), lentiviruses, adeno-associated viruses, etc. additionally requires the delivery of multiple components at a controlled ratio.

[0052] In one embodiment, the nanostructure comprises enhanced staple strands, the length of the enhanced staple strands ranging from about 60 to about 250 nucleic acid bases, preferably about 80 to about 220 nucleic acid bases, more preferably about 90 to about 200 nucleic acid bases, such as about 154 bases. In one embodiment, the enhanced staple strands are configured to bind to the scaffold strand at the 5' end and / or 3' end of the nucleic acid sequence encoding the gene. The inventors have found that the enhanced staple strands stabilize the nanostructure and enhance gene expression from the nanostructure. In addition, the inventors unexpectedly found that efficient expression of genes, such as mammalian genes, can be achieved through nanostructures containing enhanced staple strands. In one embodiment, the term "enhanced staple strand" refers to a staple strand having a length of at least 60 nucleic acid bases, preferably at least 80 nucleic acid bases, more preferably at least 90 nucleic acid bases, such as a length ranging from about 90 to about 250 nucleic acid bases. In one embodiment, the terms "enhanced staple strand", "staple strand having a length of at least 60 nucleic acid bases", "continuous staple strand" and "stabilizing staple strand" are used interchangeably. In one embodiment, the enhanced staple strands bind to the scaffold strand, thereby forming one or more continuous double-helical domains with the scaffold, the domains containing at least 10 base pairs, preferably at least 15 base pairs, more preferably at least 20 base pairs, even more preferably at least 30 base pairs, even more preferably at least 80, 85 or 90 base pairs. In one embodiment, the enhanced staple strands provide a long region of dsDNA, thus facilitating the recognition and binding of polymerases and / or other accessory proteins required for gene expression.

[0053] The staple chains among the plurality of staple chains generally span from one region of the scaffold chain to other regions of the scaffold chain, thereby forming a folded pattern. The staple chains among the plurality of staple chains generally span multiple regions of the scaffold chain. In contrast, the enhanced staple chains perform fewer or no such typical spans, thereby maintaining continuity along one or two regions of the scaffold chain. In one embodiment, the enhanced staple chain binds to the scaffold chain along one or two regions of the scaffold chain. In one embodiment, the enhanced staple chain binds to the scaffold chain along one or two regions of consecutive nucleic acids in the nucleic acid sequence of the scaffold chain. In one embodiment, the enhanced staple chain comprises or consists of one or two portions, wherein if the enhanced staple chain comprises or consists of one portion, at least 90% of the nucleic acid bases of the one portion bind to the region of the scaffold chain; and wherein if the enhanced staple chain comprises or consists of two portions, at least 90% of the nucleic acid bases of the first portion of the two portions bind to the first region of the scaffold chain, and at least 90% of the nucleic acid bases of the second portion of the two portions bind to the second region of the scaffold chain. In one embodiment, the enhanced staple chain is arranged such that it is generally parallel to the longitudinal extension of the nanostructure as a whole or at least in part. In one embodiment, the "enhanced" in the term "enhanced staple chain" means that the enhanced staple chain binds to at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, even more preferably at least 80, 85 or 90 consecutive nucleic acids in the nucleic acid sequence of the scaffold chain; wherein preferably, the assembly of the nanostructure and gene expression therefrom are enhanced. In one embodiment, the scaffold chain comprises the nucleic acid sequence of the coding gene, and the enhanced staple chain binds to at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, even more preferably at least 80, 85 or 90 consecutive nucleic acids in the nucleic acid sequence of the scaffold chain at the 5'-end and / or 3'-end of the nucleic acid sequence of the coding gene. In one embodiment, the enhanced staple chain binds to at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, even more preferably at least 80, 85 or 90 consecutive nucleic acids in the nucleic acid sequence of the scaffold chain at the 5'-end of the nucleic acid sequence of the coding gene, and the enhanced staple chain binds to at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 30, even more preferably at least 80, 85 or 90 consecutive nucleic acids in the nucleic acid sequence of the scaffold chain at the 3'-end of the nucleic acid sequence of the coding gene.For example, the enhanced staple chain may have a first part (e.g., a first half) and a second part (e.g., a second half), and the first part (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, even more preferably at least 80, 85 or 90 consecutive nucleic acids in the nucleic acid sequence of the scaffold chain at the 5' end of the nucleic acid sequence of the encoded gene, and the second part (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, even more preferably at least 80, 85 or 90 consecutive nucleic acids in the nucleic acid sequence of the scaffold chain at the 3' end of the nucleic acid sequence of the encoded gene. The inventors unexpectedly found that the side-by-side arrangement of enhanced staple chains on both sides of the expression cassette results in a significant increase in transfection efficiency and gene expression. In one embodiment, the scaffold chain comprises the nucleic acid sequence of the encoded gene and a polyadenylation signal sequence, wherein optionally, the enhanced staple chain binds to the scaffold chain at the 5' end of the nucleic acid sequence of the encoded gene and at the 3' end of the polyadenylation signal sequence. In one embodiment, the enhanced staple chain is configured such that it binds to the scaffold chain to form a circular structure, such as a circular scaffold chain or a circular expression cassette.

[0054] In one embodiment, the nanostructure, preferably at least one staple strand of the scaffold strand and / or 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, when the nucleotide sequences of two regions of a nucleic acid strand, such as two regions of the same strand, are generally complementary when read in reverse and form a double helix ending with an unpaired loop through base pairing, a loop structure is formed. For example, an inverted terminal repeat nucleic acid sequence can be a single-stranded nucleotide sequence that is immediately followed downstream by its reverse complement. The intervening nucleotide sequence between the initial sequence and its reverse complement can be of any length, including zero. Advantageously, nanostructures comprising nucleic acid sequences configured to form loop structures (e.g., inverted terminal repeat nucleic acid sequences such as adeno-associated virus-inspired inverted terminal repeat (ITR) hairpin sequences) exhibit efficient gene expression. In one embodiment, the nanostructure (e.g., the scaffold strand and / or at least one staple strand 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 a loop structure is configured such that a loop is formed at the 5' end and / or 3' end of the nucleic acid sequence of the coding gene. In one embodiment, the scaffold strand comprises a nucleic acid sequence configured to form a loop structure, which is preferably upstream of the nucleic acid sequence of the coding gene and more preferably upstream of the expression cassette comprising the nucleic acid sequence of the coding gene. In one embodiment, a staple strand, such as a staple strand among a plurality of staple strands, comprises a nucleic acid sequence configured to form a loop structure, preferably a staple strand flanking both sides of the nucleic acid sequence of the coding gene and more preferably a staple strand flanking both sides of the expression cassette comprising the nucleic acid sequence of the coding gene. In one embodiment, the nucleic acid sequence configured to form a loop structure comprises or consists of a sequence as defined in any one 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, as used herein, the term "nucleic acid sequence encoding a loop structure" refers 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 is formed by the nucleic acid sequence configured to form a loop structure alone, or by the nucleic acid sequence configured to form a 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 nucleic acid sequence configured to form a loop structure and the second nucleic acid sequence configured to form a loop structure each form a loop structure and / or jointly form a loop structure. The inventors unexpectedly found that by including nucleic acid sequences encoding loop structures (such as adeno-associated virus-inspired inverted terminal repeat (ITR) hairpin sequences), gene expression efficiency can be enhanced, for example, when such sequences are located upstream of the expression cassette, or by setting loop structures on the staple strands to flank both sides of the expression cassette. In one embodiment, the nucleic acid sequence configured to form a loop structure is located upstream or downstream of the nucleic acid sequence encoding the 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 the gene, particularly upstream or downstream of the expression cassette. In one embodiment, the nucleic acid sequence configured to form a loop structure is located on one or both staple strands, and the one or both staple strands bind to the scaffold strand upstream or downstream of the nucleic acid sequence encoding the gene, particularly upstream or downstream of the expression cassette.

[0055] 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; wherein optionally, the nanostructure, preferably the at least one scaffold strand, comprises a plurality of nuclear targeting sequences. In one embodiment, the nuclear targeting sequence comprises or consists of the sequence as defined in SEQ ID NO.21. The DNA nuclear targeting sequence (DTS) is a consensus motif recognized by transcription factors and can be used to transport DNA from the cytosol to the nucleus through nuclear pores. For example, since the Simian virus 40 DNA nuclear targeting sequence (SV40 DTS) is recognized by various TFs, it can be used as a DTS. Advantageously, nanostructures containing one or more DTS sequences (such as three SV40 DTS sequences) enable efficient and robust gene expression. In one embodiment, the nucleic acid nanostructure comprises one, two, or three nuclear targeting sequences, preferably DNA nuclear targeting sequences. The inventors unexpectedly found that by including nuclear targeting sequences (e.g., DNA nuclear targeting sequences such as SV40 (Simian vacuolating virus 40) DTS) in the nanostructure, preferably the scaffold strand, transfection efficiency can be further improved. The inventors found that for both dividing cells and non-dividing (arrested) cells, the maximum effect can be obtained by including 1-3 repeats of nuclear targeting sequences (such as 1-3 repeats of SV40 sequences) Figure 5 ).

[0056] 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 the DNA downstream of the promoter (e.g., a bacterial or eukaryotic promoter, such as a mammalian promoter) into a single RNA transcript. A terminator, particularly a transcription terminator, is typically a segment of a nucleic acid sequence that marks the end of a gene or operon during transcription, such as a mammalian terminator, such as SV40, hGH, BGH, and rbGlob terminators. In one embodiment, the terminator is an SV40 terminator, an hGH terminator, a BGH terminator, or an rbGlob terminator. For example, a terminator mediates transcription termination by providing a signal in the newly synthesized transcript RNA that triggers the process of releasing the transcript RNA from the transcription complex. Polyadenylation is the addition of a poly(A) tail to an RNA transcript, typically messenger RNA (mRNA). The poly(A) tail enhances the nuclear export, translation, and stability of the mRNA. A polyadenylation signal sequence typically contains a consensus sequence and / or a terminator sequence for adding the poly(A) tail (polyadenylation). In one embodiment, the polyadenylation signal sequence contains the AAUAAA motif. In one embodiment, the polyadenylation signal sequence contains the sequence defined in or consisting of 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 contains the sequence defined in or consisting of SEQ ID NO.16. A woodchuck hepatitis virus post-transcriptional regulatory element can be a DNA sequence that forms a tertiary structure that enhances expression when the sequence is transcribed. In one embodiment, the woodchuck hepatitis virus post-transcriptional regulatory element contains the sequence defined in or consisting of SEQ ID NO.17.

[0057] In one embodiment, the nucleic acid nanostructure, preferably the at least one scaffold strand and / or at least one staple strand 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); and 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); optionally, wherein the at least one scaffold strand of the nucleic acid nanostructure is a ssDNA scaffold strand.

[0058] 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: an inverted terminal repeat nucleic acid sequence (such as an adeno-associated virus-inspired inverted terminal repeat hairpin sequence); 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); optionally, wherein 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: an adeno-associated virus-inspired inverted terminal repeat hairpin sequence; 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); optionally, wherein 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: 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); optionally, wherein at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0061] In one embodiment, at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand, wherein 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).

[0062] In one embodiment, the nucleic acid nanostructure is an ssDNA scaffold strand, wherein 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).

[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 promoter (such as the 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, for example one or more SV40 DTS sequences); optionally, wherein 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 promoter (such as the 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, for example one or more SV40 DTS sequences); optionally, wherein at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0065] 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 the 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, for example one or more SV40 DTS sequences); optionally, wherein at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0066] 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 the CMV promoter); a polyadenylation signal sequence; at least one nucleic acid sequence encoding a gene; and one or more SV40 DTS sequences; optionally, wherein at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0067] 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 the CMV promoter); a polyadenylation signal sequence; at least one nucleic acid sequence encoding a gene; and one or three SV40 DTS sequences; optionally, wherein at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0068] 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 the CMV promoter); a polyadenylation signal sequence; at least one nucleic acid sequence encoding a gene; and one or three SV40 DTS sequences; wherein at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0069] 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: the CMV promoter; a polyadenylation signal sequence; at least one nucleic acid sequence encoding a gene; and one or three SV40 DTS sequences; wherein at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand.

[0070] 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: the CMV promoter; a polyadenylation signal sequence; at least one nucleic acid sequence encoding a gene; and three SV40 DTS sequences; wherein at least one scaffold strand of the nucleic acid nanostructure is an ssDNA scaffold strand, and the shape of the nanostructure is triangular.

[0071] According to the present invention, the term "ssDNA" shall refer to single-stranded DNA. Thus, the term "ssDNA scaffold" shall refer to a single-stranded DNA scaffold, and the term "ssDNA scaffold strand" shall refer to a single-stranded DNA scaffold strand.

[0072] In one embodiment, the aspect ratio of the nanostructure ranges 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. In one embodiment, the aspect ratio is the ratio of the sizes of the nanostructure in different dimensions, such as the ratio of the longitudinal extension to the transverse extension. In one embodiment, the maximum longitudinal extension of the nanostructure along the longitudinal axis is greater than the maximum transverse extension along the transverse axis. For example, a nanostructure with an aspect ratio of 20:1 may have a longitudinal extension of 20 nm and a transverse extension of 1 nm.

[0073] In one embodiment, the scaffold strand comprises a nucleic acid sequence encoding the at least one gene, a promoter, and a terminator; 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 a nucleic acid sequence encoding the at least one gene, a promoter, and a terminator; 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.

[0074] 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 the pharmaceutical composition, comprises a pharmaceutically acceptable excipient. The composition of the present invention, preferably the pharmaceutical composition, should be formulated to be compatible with its intended route of administration. In a particularly preferred embodiment, examples of the route of administration of the drug and / or nanostructure of the present invention include: intravenous, oral, intranasal, intrathecal, intraarterial, intradermal, subcutaneous, transdermal (topical), intraventricular, intracerebral, intratumoral, transmucosal, rectal, vaginal, bronchial, parenteral administration, and any other clinically / medically acceptable method of administering drugs and / or compounds.

[0075] In a further aspect, the present invention relates to a collection of nucleic acid sequences encoding a nucleic acid nanostructure as defined herein or a collection of plasmids. In one embodiment, the collection of nucleic acid sequences comprises one or more nucleic acid sequences encoding a nucleic acid nanostructure as defined herein or consists of the same. In one embodiment, the collection of nucleic acid sequences comprises a scaffold strand and multiple staple strands. In one embodiment, the collection of plasmids comprises one or more plasmids encoding a nucleic acid nanostructure as defined herein or consists of the same. In one embodiment, the plasmid is a phagemid. In one embodiment, the collection of plasmids is a collection of phagemids.

[0076] In another aspect, the present invention relates to a nucleic acid nanostructure as defined herein or a composition as defined herein for use in a medicament. In one embodiment, a nucleic acid nanostructure as defined herein or a composition as defined herein is for use in a method of preventing, treating and / or diagnosing a disease or disorder, preferably a genetic and / or immune disease or disorder. 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. Advantageously, the nanostructures of the present invention are capable of highly efficient gene expression, such as mammalian genes, which is very useful for the prevention, treatment and diagnosis of genetic diseases or disorders and immune diseases or disorders. For example, the nanostructures of the present invention can be used in gene therapy and immunotherapy by administering the nanostructures to a patient in need and expressing a gene (e.g., a gene that the patient lacks and / or a gene involved in the pathology) from the nanostructures in the patient. In one embodiment, the gene therapy and / or immunotherapy comprises vaccination, preferably vaccination using or consisting of the nanostructure or composition.

[0077] In another aspect, the present invention relates to a method of expressing a gene from a nucleic acid nanostructure, preferably a nucleic acid nanostructure as defined herein, the method comprising:

[0078] i) providing a nucleic acid nanostructure comprising at least one nucleic acid sequence encoding a gene, preferably a nucleic acid nanostructure as defined herein;

[0079] ii) delivering the nucleic acid nanostructure provided in step i) to a cell; wherein preferably, the delivery comprises transfecting or transforming the cell;

[0080] iii) causing the cell to express the gene;

[0081] wherein optionally, the providing in step i) comprises: providing a plasmid, preferably providing a phagemid, or a collection of plasmids, preferably a collection of phagemids, wherein the plasmid or collection of plasmids encodes the nucleic acid nanostructure; and using the plasmid or collection of plasmids, preferably by using a phage, to prepare the nucleic acid nanostructure. In one embodiment, the collection of plasmids is a collection of plasmids as defined herein.

[0082] In one embodiment, the method of 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, optionally further comprising electroporation, liposome transfection, 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 hydroperforation. In one embodiment, the delivery in step ii) is carried out using: electroporation, liposome transfection, 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 hydroperforation. For example, chemically induced endocytosis can comprise peptide-targeted endocytosis, protein-targeted endocytosis, polysaccharide-targeted endocytosis, carbohydrate-targeted endocytosis, lipid-targeted endocytosis, and / or aptamer-targeted endocytosis.

[0083] In one embodiment, allowing the cell to express the gene in step iii) comprises culturing the cell in a cell culture at a temperature range of preferably about 25 °C to about 40 °C, preferably about 30 °C to about 38 °C, more preferably at about 37 °C, for example for about 1 hour to about 72 hours. In one embodiment, allowing the cell to express the gene in step iii) comprises providing suitable growth conditions for the cell. 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.

[0084] In one embodiment, in the context of the methods of the present invention, the term "providing a nucleic acid nanostructure" encompasses providing an assembled nucleic acid nanostructure and / or providing the building materials for the nucleic acid nanostructure, such as a scaffold strand and one or more staple strands. In one embodiment, the method of preparing a nanostructure comprises the steps of allowing the nanostructure to self-assemble and purifying the self-assembled nanostructure. For example, providing a nucleic acid nanostructure may comprise the steps of allowing self-assembly and subsequent purification. In one embodiment, allowing self-assembly comprises mixing at least one scaffold strand and one or more staple strands, optionally further comprising adjusting the ionic strength (e.g., by adding about 10 mM to about 20 mM MgCl2), and / or further comprising using a temperature profile that runs through a series of temperatures. In one embodiment, the purification comprises removing the remaining excess staple strands, for example, by precipitation (such as PEG precipitation), filtration, and / or liquid chromatography. In one embodiment, the step of self-assembly and / or allowing self-assembly comprises 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 a period of 1 minute to 45 minutes, preferably 10 minutes 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 with progressive cooling, preferably at a rate of 1°C per hour from about 58°C to about 50°C. Those skilled in the art will understand that the self-assembly protocol depends on the design of the nanostructure and / or the nucleic acid sequence, and the protocol can be adjusted according to known nanostructure preparation protocols.

[0085] In one embodiment, when referring to a method, the method is an in vivo, ex vivo, in vitro, or in situ method, such as an in vitro method. In one embodiment, when referring to a use, the use is an in vivo, ex vivo, in vitro, or in situ use, such as 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.

[0086] 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 for highly efficient expression of a molecule of interest (e.g., a protein of interest) in cell culture, such as for large-scale production of therapeutic proteins, such as mammalian enzymes or antibodies.

[0087] In another aspect, the present invention relates to a method for preventing, treating, and / or diagnosing a disease or disorder, preferably a genetic and / or immune disease or disorder, the method comprising administering to a patient in need thereof a nanostructure as defined herein or a composition as defined herein. 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 to a patient in need thereof an effective amount of a nanostructure as defined herein and / or a composition as defined herein. As used herein, the term "patient" can refer to a human or an animal. As used herein, the term "effective amount" refers to an amount sufficient to elicit a desired effect, such as an amount sufficient for in vivo imaging labeling.

[0088] In another aspect, the present invention relates to the use of a nanostructure as defined herein or a composition as defined herein for the manufacture of a medicament (e.g., for preventing, treating, and / or diagnosing a disease or disorder, preferably a genetic and / or immune disease or disorder), optionally for gene therapy and / or immunotherapy. For example, the nanostructure can comprise a gene that is lacking in the patient.

[0089] As used herein, the terms "of the present invention", "according to the present invention", "in accordance with the present invention", etc. are intended to refer to all aspects and embodiments of the present invention described and / or claimed herein.

[0090] As used herein, the term "comprising" shall be construed to cover both "including" and "consisting of", both of which meanings are specifically intended in accordance with embodiments of the present invention and are thus separately disclosed. As used herein, "and / or" shall be understood to expressly disclose each of the two specified features or components, with or without the other. For example, "A and / or B" shall be understood to expressly disclose (i) A, (ii) B, and (iii) each of A and B, as if each case were listed separately herein. In the context of the present invention, the terms "about" and "approximately" indicate a range of precision that a person skilled in the art will understand to still ensure the technical effect of the feature being discussed. The terms generally represent a deviation of ±20%, ±15%, ±10%, and for example ±5% from the indicated numerical value. As will be understood by a person of ordinary skill in the art, for a given numerical value of a technical effect, such specific deviations will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have greater such deviations than artificial or engineering technical effects. When an indefinite or definite article is used in reference to a singular noun, such as "a / an" or "the", this includes the plural form of the said noun, unless specifically stated otherwise. Description of the Drawings

[0091] The present invention will now be further described by reference to the following drawings.

[0092] All methods mentioned in the following description of the drawings are performed as detailed in the examples.

[0093] Figure 1Shows the folding and expression of genes from an origami structure, as well as the effects of gene position and origami aspect ratio on gene expression. a: Schematic of the overall workflow: (i) Generation of ssDNA from plasmid DNA via a phagemid; and then (ii) Folding into a 20-helix bundle (20HB) DNA origami object. (iii) Delivery of the object to cells and assessment of gene expression from the origami structure by positive fluorescence readout. The CMV promoter sequence is shown in blue, the gene encoding for enhanced green fluorescent protein (EGFP) is in green, and polyA is in purple. b: The upper and lower insets give the cylinder models and negative-staining transmission electron micrographs of 20HB, 12HB, and 32HB, respectively (scale bar 100 nm, inset 20 nm) (HB: helix bundle). The color coding shows the positions of the scaffold features, e.g., 20HB-ext shows the CMV (blue), EGFP (green), and polyA (purple) coding sequences presented along the outer helix, while 20HB-int presents the sequences encoding EGFP and polyA within the inner helix. c: Transfection efficiency in HEK293T cells (sc: scaffold; st: staple). d: Schematic explaining the internal cross-linking initiated by UV irradiation. In the UV-welded structures 20HB-ext-W and 20HB-int-W, EGFP expression is silenced. e: Transfection efficiency of 20HB(-ext), 32HB, and 12HB structures visible by electroporation in HEK293T cells. The data collected in c and e were quantified using flow cytometry and presented as the mean ± standard deviation (s.d.) of n = 3 independent biological experiments. Individual data points overlap. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison (*p ≤ 0.05, ns p > 0.05).

[0094] Figure 2Optimization of gene expression through alternative staple designs and scaffold orientations is shown. a and b: Schematic diagrams of the scaffold paths and the unfolded scaffolds for the 20HB-LPv2 and 20HB-Circ designs. The 20HB-LPv2 incorporates two consecutive 154-mer staples (enhanced staples; pink), and the 20HB-Circ design has been routed such that a 200-mer staple (pink) serves as a splint to join the 5'-start and 3'-ends of CMV. c: TEM micrographs of 20HB-LPv2 and 20HB-Circ, scale bar 100 nm. d: Delivery of samples into HEK293T cells by electroporation shows statistically significant improved transfection efficiencies of 20HB-LP, 20HB-LPv2, and 20HB-Circ compared to the standard 20HB (20HB-LP containing two consecutive 93-mer staples (enhanced staples)). e: The scaffolds used thus far encode the 'coding strand', where the expression cassette is presented in the 5'-to-3' direction ("sc_EGFP1", upper inset). Scaffolds encoding the reverse complementary sequence of the expression cassette, i.e., the 'template strand' ("sc_EGFP2", lower inset), were designed and produced. f: HEK293T cells transfected with a mixture of sc_EGFP2 scaffold + staples or with the scaffold alone show significantly higher transfection efficiencies than the sc_EGFP1 counterparts. No significant differences in transfection efficiency were observed for the 20HB constructs. g: Mean fluorescence intensity (MFI) of EGFP for 20HB, 20HB-LPv2, and 20HB-Circ constructs folded using the coding strand or the template strand as the scaffold. The 20HB and 20HB-Circ constructs did not show significant differences in MFI, while significant differences were observed for the 20HB-LPv2 construct. The data collected in d, f, and g were quantified using flow cytometry and are presented as the mean ± s.d. of n = 3 independent biological experiments, with individual data points overlapping. The statistical analysis in d was performed using one-way ANOVA and Tukey's multiple comparisons, while the 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 can be achieved whether the nanostructure contains the nucleic acid sequence encoding the gene in the scaffold strand or in multiple staple strands.

[0095] Figure 3Enhanced gene expression via alternative scaffold sequences is shown. a: Scaffold designs where sc_EGFP1 represents the initial scaffold design, and sc_EGFP3 / 4 / 5 / 6 include additional sequence features such as ITR (light pink) or ITR binding domain (ITR*), kozak sequence (black), and WPRE (dark pink). b and c: Comparison of transfection efficiency as determined by EGFP+ cells (b) and mean fluorescence intensity of EGFP+ cells (c). Data collected in a and b were quantified using flow cytometry and presented as mean ± s.d. of n = 3 independent biological experiments, with individual data points overlapping. 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).

[0096] Figure 4 Enhanced gene expression via scaffold sequence design is shown. a: The 20HB design of the sc_EGFP5 construct includes an external single-stranded loop to enable the ITR sequence to self-anneal and form a hairpin structure. The 20HB design of the sc_EGFP6 scaffold includes two external loops to expose the ITR binding domain, allowing the ITR hairpin staples to anneal. b, c: Transfection efficiency and MFI visible for the 20HB constructs folded using sc_EGFP5 and sc_EGFP6. Data collected in b and c were quantified using flow cytometry and presented as mean ± s.d. of n = 3 independent biological experiments, with individual data points overlapping. Statistical analysis of 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 show EGFP expression in cells transfected with DNA origami objects folded using sc_EGFP5 and sc_EGFP6 compared to sc_EGFP1. Images in the bottom row have been deliberately contrast-enhanced to show EGFP-positive cells with weaker EGFP intensity in the sc_EGFP1 sample. The images represent one experiment out of n = 3 independent biological experiments; similar results were observed each time. Scale bar 100 μm.

[0097] Figure 5Shows structural transfection summary and optimization. a: Comparison of transfection efficiency (%) of all studied constructs grouped by scaffold with EGFP MFI (arbitrary units). sc_EGFP1 20HB-ext was used as an internal control in all experiments, and EGFP MFI is expressed as fold change relative to this sample. Three clusters are highlighted: 1, constructs with high folding quality but low overall gene expression (grey); 2, constructs with low folding quality and moderate expression levels (orange); 3, constructs with moderate to high folding quality and high expression levels (yellow). b: Representative epifluorescence microscopy images of HEK293T cells expressing EGFP after successful transfection following optimized electroporation settings. eGFP expression (green), cells (phase contrast), and merged images are given for each condition. The images represent one experiment out of n = 2 independent biological experiments; similar results were observed each time. Scale bar 100 μm.

[0098] Figure 6 Shows plasmid designs containing different numbers of SV40 DTS sequences (0x, 1x, 3x, and 6x SV40 repeats) for generating custom scaffolds.

[0099] Figure 7 Shows the generation and characterization of custom scaffolds and corresponding DNA origami structures. a: Agarose gel shows all generated custom scaffolds and corresponding purified DNA origami structures. b: Representative negative stain TEM images show 20HB DNA origami structures generated from each custom scaffold. Scale bar 100 nm.

[0100] Figure 8 Shows that cell cycle arrest reduces gene delivery efficiency. Representative transmission and corresponding fluorescence images of dividing a and arrested b HEK293T cells 24 hours after electroporation with 20HB-mCh without any SV40 sequences. mCherry signal is shown in red, nuclei are in blue, scale bar 100 μm. c: Flow cytometry histograms show the cell cycle populations of actively dividing and chemically arrested HEK293T cells. (d): Quantification of mCherry+ cells (%) in populations of dividing and chemically arrested HEK293T cells 24 hours after electroporation with 20HB-mCh.

[0101] Figure 9Shows the effect of including the SV40 DTS sequence in a DNA origami structure on gene expression in split and arrested HEK293T cells after electroporation. a: In normally dividing HEK293T cells, transfection efficiency (bar graph) and mean fluorescence intensity (square symbols) 24 hours after electroporation with a DNA origami structure containing one or more DTS compared to a control structure without the DTS sequence. b: In arrested HEK293T cells, transfection efficiency (bar graph) and mean fluorescence intensity (square symbols) 24 hours after electroporation with a DNA origami structure containing one or more DTS compared to a control structure without the DTS sequence. Data were quantified using flow cytometry and are presented as the mean ± s.d. of n = 3 independent biological 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 transfection efficiency (*p ≤ 0.05) and mean fluorescence intensity (**p ≤ 0.01) compared to the control without the DTS sequence. For arrested cells (b), inclusion of three copies of the SV40 DTS resulted in a statistically significant increase in transfection efficiency (**p ≤ 0.01) and mean fluorescence intensity (****p ≤ 0.0001) compared to the control without the DTS sequence. For arrested cells, inclusion of one copy did not result in a statistically significant increase in transfection efficiency but did result in a statistically significant increase in mean gene expression (****p ≤ 0.0001).

[0102] Figure 10Delivery of the polyhedral origami assemblies enables co-delivery of genes at defined ratios. a: Cylindrical model of a DNA origami object programmed for assembly by shape-complementary protrusions and grooves. Schematic shows 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 tomographic slices comparison of dimeric, trimeric, and tetrameric structures, scale bar 100 nm, taken from samples containing the mixed assembly products. c: Schematic shows the passivated protruding ends that inhibit assembly, and assembly assisted by complementary 5-nt sticky ends. d: Co-transfection (mCherry+ / EGFP+) efficiency after delivery of mCherry and EGFP as individual monomers (passivated) or dimers linked by 5-nt or 8-nt sticky ends in HEK293T cells. e: Co-transfection (mCherry+ / EGFP+) efficiency in HEK293T cells by assembly of polymeric DNA origami constructs containing mCherry- and EGFP-encoding monomers with mCherry:EGFP ratios of 1:1, 1:2, and 1:3. The right y-axis gives the EGFP MFI (arbitrary units). Data collected in d and e were quantified using flow cytometry and are presented as mean ± s.d. of n = 3 independent biological experiments, with individual data points overlapping. Statistical analysis of d and e was performed using one-way ANOVA and Tukey's multiple comparisons (*p ≤ 0.05, **p ≤ 0.01, ns p > 0.05). f: Top: Schematic design of mCherry and EGFP monomer modules for non-assembled (passivated) or assembled 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 micrographs showing expression and co-expression (yellow) of mCherry (red), EGFP (green) in successfully transfected HEK293T cells. Nuclei are shown in blue, scale bar 100 μm. The images represent one experiment out of n = 3 independent biological experiments; similar results were observed each time.

[0103] Figure 11 DNA origami triangles for gene expression are shown. a: Scaffold design containing three SV40 DTS sequences for generating a custom scaffold, where the phagemid encoding mCherry has 8064 bases. b: Representative negative-staining TEM image of the mCherry-encoding DNA origami structure (triangular structure) of the generated custom scaffold. Scale bar: 50 nm. c: Representative epifluorescence micrograph showing mCherry expression from HEK293T cells transfected with the DNA origami object 48 h after electroporation of the mCherry-encoding origami triangle. Scale bar: 50 μm.

[0104] The following refers to examples, which are for illustrative purposes only and do not limit the present invention.

[0105] Example

[0106] Example 1: Materials and Methods

[0107] Generated by the scaffold.

[0108] For details of the design and cloning methods of the customized scaffolds of the present invention, see Examples 2 and 6. Briefly, gene fragments of plasmids containing EGFP (plasmids #13031 and #105530 from Addgene, which contain and do not contain the ITR sequence respectively) were assembled with fragments of bacteriophage replication origin bacterial resistance (plasmid #126854 from Addgene) using Golden Gate or digestion-ligation cloning. The plasmids were verified using restriction digestion and DNA sequencing (Eurofins genomics, Ebersberg Germany). Specific primer sequences and methods can be found in Examples 2 and 6, and the sequences of the customized scaffolds can be found in SEQ ID NO 1-11 .

[0109] The sequence of the exemplary staple chain can be found in SEQ ID NO 76-1507.

[0110] The generation of ssDNA customized scaffolds was carried out as previously described. 1,2Briefly, the plasmid of interest and the helper plasmid (plasmid #120346 from Addgene) were co-transformed into chemically competent DH5α Escherichia coli cells. Single colonies were picked and grown in 5 mL of pre-culture medium (2×YT, 30 μg / mL kanamycin, 30 μg / mL carbenicillin) for about 10 hours, and then transferred to 750 mL of 2×YT (30 μg / mL kanamycin, 30 μg / mL carbenicillin, 5 mM MgCl2) in an Ultra Yield flask (Thomson). The cells were then grown overnight in an orbital incubator at 37°C. The bacterial pellet was obtained by centrifugation (45 minutes, 4500 g), and the supernatant was collected. Polyethylene glycol 8000 (PEG-8000, final concentration 3% w / w) and NaCl (final concentration 0.5 M) were added to the supernatant to precipitate the phagemid particles, and the mixture was incubated with stirring at room temperature for 1 hour, and then collected by centrifugation (45 minutes, 4500 g, 4°C). The pellet was resuspended in 4 mL of 1×TE buffer (10 mM Tris, 1 mM EDTA, pH 8), and centrifuged again (15 minutes, 16000 g, 4°C) to remove residual bacterial components. Then, the ssDNA scaffold was extracted by phagemid lysis and purified by ethanol precipitation.

[0111] DNA origami design, folding, and purification.

[0112] All origami objects were folded in a standardized 'folding buffer' which contained, in addition to 5 mM Tris base, 1 mM EDTA and 5 mM NaCl (pH 8), x mM MgCl2 (FoBx). All reaction mixtures were subjected to a thermal annealing ramp program in a Tetrad (Bio-Rad) thermal cycler. The exact folding conditions for each construct are shown in Tables 2 and 3. As exemplified in Example 6, staple strands were purchased from Integrated DNA Technologies and standard desalting was used unless otherwise stated. Origami objects were purified by PEG precipitation or gel purification as previously described. 3,4

[0113] Assembly of multi-component DNA origami structures. To assemble origami subunits into dimeric, trimeric and tetrameric samples, the monomers were mixed at a molar ratio in 1×FoB5 buffer and incubated at 37°C for 48 hours. Passivated samples were treated in the same manner.

[0114] UV welding.

[0115] The UV-weldable samples were additionally designed with thymine bases at all potential staple crossover positions and were UV crosslinked by UV light (310 nm, 2 h) using an Asahi Spectra xenon light source (300 W, MAX-303) with a high-transmission bandpass filter centered at approximately 310 nm (XAQA310, Asahi Spectra Co., Ltd. (Asahi Spectra)) as previously described [5]. The samples were placed in FoB10 buffer during UV crosslinking.

[0116] PAGE purification of superpolymers.

[0117] Long staple oligomers (93-mer, 154-mer, and 200-mer) were purchased as supermolecules from IDT (IDT) and were internally purified by denaturing urea polyacrylamide gel electrophoresis (Urea-PAGE). Bands corresponding to the correct MW were excised and crushed, and then 1× TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.00) was added. The pure supermolecules were recovered by EtOH precipitation, redissolved in MilliQ H2O, and stored at 4 °C.

[0118] Gel electrophoresis.

[0119] For the characterization of PCR products and plasmids, 1% agarose gels containing 0.5× TBE buffer (22.25 mM tris base, 22.25 mM boric acid, 0.5 mM EDTA) were used. Gel electrophoresis was carried out at 110 V for 1 h in the same buffer solution. To characterize the assembled origami and scaffolds, the inventors used 2% agarose gels containing 0.5× TBE buffer and 5.5 mM MgCl2. Gel electrophoresis was carried out at 90 V for 1 h - 2 h in the same buffer solution, and the gels were cooled in a water bath. All gels were imaged using a Typhoon FLA 9500 laser scanner (GE Healthcare) with a pixel size of 50 μm / pixel.

[0120] Negative staining TEM.

[0121] The samples were incubated on glow-discharged copper TEM grids (FCF400-CU, Electron Microscopy Sciences) for 30 seconds to 60 seconds. Then, the grids were stained for 30 seconds (2% uranyl formate aqueous solution, 25 mM NaOH). Imaging was performed at magnifications of 21,000 - 42,000 times. Data were acquired using SerialEM software and a FEI Tecnai T12 microscope (120 kV, Tietz TEMCAM-F416 camera). Images were processed using ImageJ. 5 TEM micrographs were high-pass filtered to remove long-range staining gradients and the contrast was automatically adjusted using Adobe Photoshop CS5.

[0122] Tilt series were taken from -50° to +50° and micrographs were obtained at 2° increments, and then tomograms were generated using filtered back-projection and processed by Etomo (IMOD) to obtain the tomograms. 6 The Gaussian filter used a cut-off value between 0.25 and 0.5 and a decay of 0.035.

[0123] Cell culture.

[0124] 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 and 5% CO2.

[0125] Cell cycle arrest.

[0126] HEK293T cells were arrested 24 hours before electroporation using an 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 kept in the arrest medium throughout the experiment.

[0127] Electroporation.

[0128] The electroporation experiments were performed according to the manufacturer's protocol (Neon TMTransfection protocols were performed by Thermo Fisher. Briefly, HEK293T cells were washed with phosphate-buffered saline (PBS) and collected using TryplE. Cells were pelleted by centrifugation (5 minutes, 300 g), resuspended in PBS and counted. Cells were centrifuged again (5 minutes, 300 g) and then resuspended at a concentration of 5×10 6 cells / mL in Buffer R (Neon TM Transfection System). Mixtures were prepared for each condition such that each electroporation event contained 0.5 μg total DNA and was supplemented to a total volume of 1 μL with 1× FOB5 buffer (folding buffer, 1 mM Tris, 1 mM EDTA, 5 mM NaCl, 5 mM MgCl2), which was mixed with 9 μL of cell suspension. Electroporation was performed in a 10 μL transfection tip, applying two pulses at a pulse voltage of 1150 V and a width of 20 milliseconds. After electroporation, cells were immediately transferred to a 48-well plate pre-coated with poly-L-lysine and containing 240 μL of complete DMEM growth medium or arrest medium.

[0129] After 48 hours, samples were imaged using an EVOS TM M7000 imaging system and transfection efficiency was quantified by flow cytometry. For the 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). A total of 20,000 single-cell events (gated on side scatter area versus height) were recorded for analysis. EGFP was excited with a 488 nm laser and emission was measured through a 530 / 30 nm bandpass filter. mCherry was excited with a 561 nm laser and emission was measured through a 620 / 15 nm bandpass filter. Untreated cells and cells electroporated with buffer only were used as negative controls. Cells electroporated with the corresponding EGFP plasmid were used as positive controls. Cell cycle analysis was performed by flow cytometry to confirm cell cycle arrest. According to the manufacturer's protocol, cells were stained using FxCycle TM Far Red stain (Invitrogen, Thermo Fisher Scientific). The dye was excited with a 638 nm laser and emission was measured through a 670 / 14 nm bandpass filter. Post-acquisition data analysis was performed using FlowJo software (v10.7.1).

[0130] Statistics and reproducibility.

[0131] Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., v9). Data are presented as mean ± standard deviation, and individual data points representing biological replicates are shown. Specific analyses were performed as detailed in the corresponding figure legends. For all tests, p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****) were considered statistically significant.

[0132] Example 2: Additional Materials and Methods

[0133] Scaffold cloning.

[0134] Plasmids encoding custom scaffolds were generated by standard cloning techniques. All plasmids except sc_EGFP3 were generated by Golden gate assembly using Esp3I (New England Biolabs (NEB), catalog number R0734) or v2 (NEB, catalog number R3733) and T4 DNA ligase (NEB, catalog number M0202). For each plasmid, appropriate cleavage sites were introduced by PCR and assembly was performed according to the manufacturer's protocol.

[0135] The exception, sc_EGFP3, was assembled by digestion ligation and compatible enzyme cleavage sites were added to the fragment of interest by PCR. The fragment of interest was digested using the enzymes EcoNI and PacI (NEB, catalog numbers R0521 and R0547 respectively) and then ligated using NEB T4 DNA ligase as described above.

[0136] Touchdown PCR was performed on all constructs using primers (see Table 1). In all cases, PCR products were confirmed by agarose gel electrophoresis (AGE), bands were excised, and fragments were extracted according to the manufacturer's protocol (QIAquick Gel Extraction Kit from Qiagen).

[0137] Table 1 | Primer sequences for plasmid construction for custom scaffold generation.

[0138]

[0139]

[0140] DNA origami folding

[0141] Nucleic acid nanostructures, such as CS3_EGFP3, CS3_EGFP1, CS3_EGFP4, CS3_EGFP4A, CS3_EGFP4B, and CS3EGFP5, were successfully generated. The nucleic acid nanostructures were folded using the conditions defined in Tables 2 and 3.

[0142] Table 2 | Summary of the folding conditions for each structure and scaffold used.

[0143]

[0144] *For a 20 μL folding reaction, make up to a total volume of 20 μL with ddH2O if necessary.

[0145] The staple concentration used here was 500 μM.

[0146] Table 3 | Folding protocol for the folding reaction.

[0147]

[0148] Example 3: Structural integrity analysis.

[0149] Structural integrity analysis was performed on the nucleic acid nanostructures of the present invention. For example, the integrity of the 20HB structure after electroporation was analyzed. The structural integrity of 20HB was maintained when diluted in RPMI 1640 medium. In addition, 20HB remained stable both in the electroporation buffer (EB, buffer R in the kit) and after electroporation using the Neon TM transfection system. The nucleic acid nanostructures of the present invention showed favorable structural integrity.

[0150] Example 4: Results

[0151] Genes are easily expressed from DNA origami without being affected by gene position or origami shape.

[0152] The inventors' research first determined the basic parameters of the origami design that mammalian cells would express. To this end, the inventors produced custom circular ssDNA scaffolds that encoded enhanced green fluorescent protein (EGFP) in the 5' to 3' direction (coding strand), as Figure 1as shown in a). Therefore, cells that successfully express EGFP from the nucleic acid nanostructure can be monitored by fluorescence detection. In this study, the inventors used two observables: the fraction of cells showing green fluorescence (referred to as transfection efficiency), and the fluorescence intensity per cell, which the inventors used as a representative of expression efficiency. To circumvent issues that may interfere with data interpretation, such as cellular uptake and endosomal escape, the inventors used electroporation to directly deliver the origami to the cells and thus focused on the parameters directly affecting expression. The inventors used electroporation performed by the Neon TM transfection system, which does not damage the DNA origami structure (and prevents its aggregation).

[0153] A custom EGFP scaffold (sc_EGFP1) was expressed in high yield and purity by phagemid production, and the scaffold efficiently folded into the designed target object ( Figure 1 b). To investigate whether the spatial position of the gene in the DNA origami object affects expression, the inventors designed two 20-helix bundle (20HB) variants, in which the EGFP gene was located either on the outer side (20HB-ext) or the inner side (20HB-int) of the multi-layer DNA origami ( Figure 1 the first two small panels in b). After electroporation, gene expression occurred in both 20HB variants in human embryonic kidney 293T (HEK293T) cells ( Figure 1 c). The inventors found that there were no statistically significant differences in the transfection efficiency or expression efficiency of these two objects.

[0154] It has been previously reported that the aspect ratio of DNA origami affects cellular uptake 7,8 . To clarify whether the aspect ratio affects expression, the inventors designed a twelve-helix bundle (12HB) approximately 114 nm in length, a 20HB-ext approximately 69 nm in length, and a 32HB approximately 42 nm in length, in which the EGFP gene and recognition sequences were presented on the outer side of the bundle in all cases ( Figure 1 b). The aspect ratios of these objects were approximately 15 for 12HB, approximately 5 for 20HB, and approximately 2 for 32HB. In 12HB, EGFP and related genes were presented in a long continuous region with the fewest scaffold crossovers, while in 32HB they were presented in the shortest continuous region. When delivered to HEK293T cells, the inventors found no statistically significant differences in transfection and expression efficiency between the 20HB and 32HB samples ( Figure 1 e). The transfection efficiency of the 12HB sample was slightly decreased (p ≤ 0.05) relative to 20HB and 32HB. However, the cell density after electroporation of the 12HB object was also lower.

[0155] Thus, for both transfection and expression efficiency, how the gene of interest is packaged in the test DNA origami sets of the present invention is not important. This observation indicates that DNA origami unfolds prior to gene expression. The inventors tested this hypothesis with EGFP-encoding objects that cannot unfold. To this end, the inventors included additional thymidine residues in the staple strands of 20HB-ext and 20HB-int to enable internal cross-linking by UV spot welding. 9 The object was then internally stabilized by cyclobutane pyrimidine dimer bonds between and at the crossings of hundreds of UV-induced staple strands, which topologically prevents strand dissociation ( Figure 1 d). When the inventors delivered the UV spot-welded 20HB variants to HEK293T cells, the inventors found that the EGFP signal was almost completely inhibited ( Figure 1 c). Although exposure to UV radiation may also have an inhibitory effect on gene expression from plasmids 10,11 , gene expression from covalently cross-linked DNA origami was significantly and almost completely inhibited, which supports the inventors' hypothesis that DNA origami must unfold prior to gene expression.

[0156] Targeted design modification of the promoter region enhances gene expression.

[0157] The inventors observed that electroporation of a pre-mixed but non-annealed mixture of ssDNA scaffolds and staple strands (which did not form structured objects) produced a slightly higher transfection efficiency compared to the administration of ssDNA scaffolds alone ( Figure 1 c, 1e). The inventors hypothesized that partial binding occurred between the staple strands and the scaffold strands, forming double-stranded DNA regions around the promoter region to enhance gene expression. Therefore, the inventors tested whether simply increasing the average staple length in the DNA origami object would enhance expression and found that it did not, indicating that a more targeted design is needed. The inventors redesigned the 20HB object to incorporate long continuous staple segments (enhanced staples) without crossings in the promoter region, resulting in structures with continuous 93-mer and 154-mer staples (enhanced staples) that flank the expression region and are at the 5' start region of the CMV promoter and the 3' end of the polyA sequence (20HB-LP and 20HB-LPv2, respectively). Figure 2 a shows the schematic design and staple positioning of 20HB-LPv2. Inspired by a partial double-stranded hepatitis B genome 12 , the inventors also prepared a design in which 200-mer staples act as a splint between the 5' start of CMV and the 3' end of polyA to form a partially double-stranded circular structure upon unfolding (20HB-Circ) ( Figure 2b). All designs can be easily folded into the defined 20HB, as visible by direct TEM imaging ( Figure 2 c). Delivery of these objects to cells resulted in up to 50% enhanced gene expression efficiency for objects 20HB-LP, 20HB-LPv2, and 20HB-Circ compared to the standard 20HB staple path ( Figure 2 d).

[0158] Next, the inventors determined whether the orientation of the target gene on the scaffold affects gene expression. Since the scaffold is ssDNA, delivery of the coding strand requires synthesis of a complementary sequence (template strand) prior to transcription. The inventors produced a'scaffold template strand' (sc_EGFP2) containing the reverse complementary gene sequence ( Figure 2 e). Delivery of these stapled and non-stapled scaffolds demonstrated a significant increase in transfection efficiency of the template strand compared to the coding strand ( Figure 2 f). However, when folded into 20HB DNA origami objects, the difference in transfection efficiency disappeared ( Figure 2 f, right). Thus, the overall transfection efficiency of 20HB does not depend on whether the scaffold uses the coding strand or the template strand. However, the inventors observed that in all objects using the template strand scaffold, there was a slight increasing trend in the mean fluorescence intensity (MFI) of EGFP in EGFP-positive cells compared to those using the coding strand as the scaffold ( Figure 2 g).

[0159] Including scaffold sequence features enhances gene expression.

[0160] To further enhance gene expression, the inventors included additional features in the scaffold sequence based on the ssDNA AAV2 expression cassette ( Figure 3 a). The inventors placed the kozak sequence, which serves as a protein translation site, upstream of EGFP ( 13 ; and placed a chimeric intron and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) downstream of EGFP (before polyA). The WPRE is thought to improve mRNA stability and protein production. 14 Additionally, the inventors included inverted terminal repeats (ITRs) flanking the expression cassette. The ITRs are palindromic sequences that form T-shaped hairpins and are used by adeno-associated virus as the origin of replication for its ssDNA genome and have other functions. 15,16 The inventors attempted to produce a synthetic scaffold ssDNA (sc_EGFP3) containing all these features ( Figure 3a). The scaffolds produced had low production yields and poor quality, which the inventors attributed to the repetitive ITR structure. To improve scaffold yield and quality, the inventors produced another series of scaffolds that included only a single ITR either downstream or upstream of the expression cassette (sc_EGFP4 and sc_EGFP5, respectively). Additionally, the inventors produced scaffolds that included partial sequences of two ITRs, but where the ITR hairpins would be provided by complementary staple oligonucleotides during DNA origami folding (sc_EGFP6). 20HBs with a standard staple design were generated for all of these scaffold variants.

[0161] The inventors observed that in all cases, compared to the original sc_EGFP1 scaffold, the scaffold-only control groups showed a trend of increased transfection efficiency and enhanced gene expression ( Figure 3 b). Compared to the transfection efficiency ranges observed for the 20HBs folded from the sc_EGFP1 scaffold, the 20HB samples folded from the "enhanced" scaffolds showed similar transfection efficiency ranges, but the MFI in positive cells was significantly increased for the 20HBs folded from the "enhanced" scaffolds sc_EGFP3 / 4 / 5 / 6 compared to the original sc_EGFP1 ( Figure 3 b), meaning that additional features in the scaffolds enhanced intracellular gene expression.

[0162] In the inventors' designs discussed so far, the ITR sequences were hidden within the double-helical DNA domain of the object and would only become available after the object denatured intracellularly. The inventors hypothesized that by positioning the ITR sequence motifs to be able to assemble into their hairpin secondary structures during object folding ( Figure 4 a, design 20HB-ex), gene expression of the 20HB could be further improved. Additionally, the inventors included a continuous 154-mer staple (enhanced staple) at the 5' region of the promoter of the 20HB-exLP design, which was encoded by both the sc_EGFP5 and sc_EGFP6 scaffolds. Indeed, as measured by MFI, delivery of these designs showed up to 2-fold enhanced transfection compared to the original 20HB design using the sc_EGFP1 scaffold ( Figure 4 b) and up to 6-fold and 9-fold increased expression efficiency, respectively ( Figure 4 c, d).

[0163] The inventors plotted the fold change in transfection efficiency relative to EGFP MFI (arbitrary units) achieved for all designs, using sc_EGFP1 20HB-ext as an internal control. Figure 5a). The samples were divided into three clusters. Cluster 1 included objects constructed with the sc_EGFP1 / 2 scaffold, which had high-quality folding and high transfection efficiency but low overall gene expression. Cluster 2 included objects based on the sc_EGFP3 / 4 / 5 / 6 scaffold, which had low folding quality and low transfection efficiency but enhanced gene expression. Then, the folding quality of the objects in Cluster 3 was improved, as in the case of 20HB-exLP of sc_EGFP5 and sc_EGFP6 scaffolds, and both transfection efficiency and gene expression were improved. Finally, the inventors further optimized the transfection efficiency by titrating the amount of material administered and changing the electroporation conditions, thus achieving an even higher transfection efficiency (about 80%) and MFI( Figure 5 b).

[0164] Encoding active nuclear import into DNA origami

[0165] The inventors designed and studied DNA origami objects that are naturally encoded with active nuclear import instructions in mammalian cells, using the mCherry gene expression cassette to achieve a simple fluorescence readout to evaluate successful nuclear entry( Figure 6 ). The inventors designed DNA origami scaffolds to encode mCherry expression and a DNA nuclear targeting sequence (DTS) within the origami structure. The custom ssDNA scaffolds were designed to include a CMV promoter, mCherry reporter gene, and polyA signal encoded in the 5' to 3' direction (coding strand). The inventors chose to use the 72bp simian virus 40 (SV40) DTS 17 , and incorporated 0×, 1×, 3×, or 6× SV40 repeats in the scaffold design( Figure 6 ). The inventors designed a 20-helix bundle (20HB), and oriented the scaffold such that the gene features and DTS sequences were located on the outer helices of the object.

[0166] Four DNA origami structures corresponding to each custom scaffold were folded and purified. 20HB-mCh and 20HB-1×SV40 were folded in high yield, with a clear main band and no major structural impurities, while 20HB-3×SV40 and 20HB-6×SV40 showed some structural impurities( Figure 7 ). The 20HB-3×SV40 structure showed the presence of low levels of higher-order bands. Higher-order impurities were even more prominent in the 20HB-6×SV40 object, and thus this object was further gel-purified. The inventors attributed the folding difficulty to the increased number of repeated DTS sequences in the scaffold, which hindered folding.

[0167] The inventors focused on both mitotic cells and cells arrested in the G1 / S phase of the cell cycle to avoid passive nuclear uptake during mitosis. The inventors tested whether cell cycle arrest would result in the expected decrease in gene expression, as this should inhibit the occurrence of passive nuclear transport. For this purpose, the inventors transfected both dividing and arrested cells with 20HB-mCh by electroporation. The cells were qualitatively analyzed by fluorescence microscopy and quantified by flow cytometry. The inventors observed a statistically significant decrease in the percentage of mCherry+ cells after electroporation in the arrested cell population ( Figure 8 ). Next, the 20HB variants 20HB-mCh, 20HB-1×SV40, 20HB-3×SV40, and 20HB-6×SV40 were tested in both dividing and chemically arrested HEK293T cells. The mCherry+ cell proportion (%) and MFI (arbitrary units) of the cells were quantitatively evaluated by flow cytometry to represent the gene expression level, and the values were compared with the control 20HB-mCh ( Figure 9 ). In dividing cells, the inventors observed a slight increase in both the percentage of mCherry+ cells and the MFI for both 20HB-1×SV40 and 20HB-3×SV40. Inclusion of the SV40 DTS sequence was more effective in arrested cells, where the percentage of mCherry+ cells for 20HB-1×SV40 increased (by about 1.4-fold) compared to 20HB-mCh, and 20HB-3×SV40 increased even more (by about 1.8-fold). The MFI showed a similar trend, with a 3-fold increase for 20HB-1×SV40 and a 4.5-fold increase for 20HB-3×SV40 in chemically arrested cells. Notably, 20HB-6×SV40 consistently showed a lower proportion of mCherry+ cells and MFI of mCherry expression. The inventors attributed this observation to the lower folding quality of the samples.

[0168] Multi-gene assemblies for co-transfection.

[0169] The inventors designed DNA origami objects encoding mCherry or EGFP expression to achieve the assembly and delivery of 1:1, 1:2, and 1:3 mCherry to EGFP stoichiometric ratios ( Figure 10 a, b, using the scaffolds sc_mCherry5 and sc_EGFP5). Each gene module was programmed to interact through shape-complementary docking sites 18 , which were arranged with sequence-complementary sticky ends five or eight base pairs long (referred to as 5nt or 8nt sticky ends, respectively). The inventors also prepared control objects with inactivated docking sites, which were passivated with single-stranded overhangs five thymidines long.Figure 10 c). As seen by the inventors through negative staining TEM tomography and AGE, the genetic assemblies of dimers, trimers or tetramers are formed as designed ( Figure 10 b). The co-transfection efficiency of non-linked mCherry and EGFP monomers mixed in a 1:1 stoichiometry was approximately 5.4 + / - 1.4%. In contrast, when using pre-assembled dimer objects that included both mCherry and EGFP as expressible genes, the inventors observed a co-transfection efficiency of approximately 17.5 + / - 2.9% ( Figure 10 d, f). The nearly four-fold increase in co-transfection compared to delivering genes as separate objects indicates that the delivery and expression of the two components are now linked to each other and no longer occur randomly.

[0170] Finally, the inventors delivered the polyhedral origami objects in the form of dimers, trimers and tetramers, where the ratio of mCherry:EGFP was 1:1, 1:2 and 1:3. The molar concentration of the polyhedral origami objects was kept consistent in each sample and thus the overall transfection efficiency remained comparable ( Figure 10 e, black bars). However, the expression level of EGFP was directly proportional to the number of monomers present within the object ( Figure 10 e (green bars)). Direct imaging of the cells using dual-channel fluorescence microscopy was consistent with the observations by flow cytometry ( Figure 10 f). Thus, the inventors successfully achieved gene delivery and expression in designed stoichiometric ratios by "clicking" genes together to form higher-order DNA origami assemblies.

[0171] Example 5: Discussion

[0172] Here, the inventors have studied gene expression from encoded DNA origami structures. The inventors presented scaffold and structural design features that allow for efficient gene expression. The nanostructures of the present invention are efficient 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, such as techniques that can even further optimize transfection efficiency and gene expression. Instead of or in addition to electroporation, chemical moieties, aptamers, peptides or antibodies can be included on the origami surface for targeted delivery and gene expression. The nanostructures of the present invention are of great value for therapeutic applications, such as scaffolds that include sequences encoding the expression of therapeutic proteins or gene editing techniques such as CRISPR-Cas for therapeutic gene delivery and vaccines. In addition, the present invention provides a valuable tool for exploring the intracellular or in vivo fate of DNA nanotechnology - hitherto, this research has been difficult because tracking molecules mainly attach to the staples rather than the scaffold.

[0173] Example 6: Exemplary Sequences

[0174] SEQ ID NO 1-11: Scaffold sequences

[0175] Sequences of scaffolds: 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).

[0176] SEQ ID NO 12-21: Specific features

[0177] CMV promoter / enhancer sequence (SEQ ID NO 12); 5' ITR sequence (SEQ ID NO 13); 3' ITR sequence (SEQ ID NO 14); chimeric intron sequence (SEQ ID NO 15); Kozac sequence (SEQ ID NO 16); WPRE sequence (SEQ ID NO 17); EGFP sequence (SEQ ID NO 18); bGH polyA sequence (SEQ ID NO 19); mCherry sequence (SEQ ID NO 20); and SV40 sequence (SEQ ID NO 21).

[0178] SEQ ID NO 22-75: Primer sequences (see also Table 1).

[0179] SEQ ID NO 76-1507 : Exemplary staple chain sequences

[0180] SEQ ID NO 76-177 : Staple sequence of sc_EGFP1; 20HB-ext.

[0181] SEQ ID NO 178-279 : Staple sequence of sc_EGFP1; 20HB-ext-W.

[0182] SEQ ID NO 280-381 : Staple sequence of sc_EGFP1; 20HB-int.

[0183] SEQ ID NO 382-483 : Staple sequence of sc_EGFP1; 20HB-int-W.

[0184] SEQ ID NO 484-584 : Staple sequence of sc_EGFP1; 32HB.

[0185] SEQ ID NO 585-692 : Staple sequence of sc_EGFP1; 12HB.

[0186] SEQ ID NO 693-766 : Staple sequence of sc_EGFP1; 20HB-LS.

[0187] SEQ ID NO 767-863 : Staple sequence of sc_EGFP1; 20HB-LP.

[0188] SEQ ID NO 864-957 : Staple sequence of sc_EGFP1; 20HB-LPv2.

[0189] SEQ ID NO 958-1055 : Staple sequence of sc_EGFP1; 20HB-Circ.

[0190] SEQ ID NO 1056-1157 : Staple sequence of sc_EGFP2; 20HB.

[0191] SEQ ID NO 1158-1279 : Staple sequence of sc_EGFP3; 20HB.

[0192] SEQ ID NO 1280-1392 : Staple sequence of sc_EGFP4; 20HB.

[0193] SEQ ID NO 1393-1507 : Staple sequence of sc_EGFP5; 20HB-exLP.

[0194] References

[0195] 1. Engelhardt, F. A. S. et al. Custom-Size, Functional, and Durable DNA Origami with Design-Specific Scaffolds. ACS Nano 13, 5015 - 5027 (2019).

[0196] 2. Praetorius, F. et al. Biotechnological mass production of DNA origami. Nature 552, 84 - 87 (2017).

[0197] 3. Stahl, E., Martin, T. G., Praetorius, F., and Dietz, H. Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions. Angewandte Chemie 126, 12949 - 12954 (2014).

[0198] 4. Wagenbauer, K. F. et al. How We Make DNA Origami. ChemBioChem 18, 1873 - 1885 (2017).

[0199] 5. Schindelin, J. et al. Fiji: an open - source platform for biological - image analysis. Nat Methods 9, 676 - 682 (2012).

[0200] 6. Kremer, J. R., Mastronarde, D. N., and McIntosh, J. R. Computer Visualization of Three - Dimensional Image Data Using IMOD. Journal of Structural Biology 116, 71 - 76 (1996).

[0201] 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).

[0202] 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).

[0203] 9. Gerling, T., Kube, M., Kick, B. and Dietz, H. Sequence-programmable covalent bonding of designed DNA assemblies. Science Advances (2018).

[0204] 10. Mitchell, D. L., Vaughan, J. E. and 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).

[0205] 11. Jiang, Y., Ke, C., Mieczkowski, P. A. and Marszalek, P. E. Detecting Ultraviolet Damage in Single DNA Molecules by Atomic Force Microscopy. Biophys J 93, 1758-1767 (2007).

[0206] 12. Wei, L. and Ploss, A. Hepatitis B virus cccDNA is formed through distinct repair processes of each strand. Nat Commun 12, 1591 (2021).

[0207] 13. Acevedo, J. M., Hoermann, B., Schlimbach, T. and Teleman, A. A. Changes in global translation elongation or initiation rates shape the proteome via the Kozak sequence. SciRep 8, 4018 (2018).

[0208] 14. Brun, S., Faucon-Biguet, N. and Mallet, J. Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy. Molecular Therapy 7, 782 - 789 (2003).

[0209] 15. Ping, H., Liu, X., Zhu, D., Li, T. and Zhang, C. Construction and Gene Expression Analysis of a Single-Stranded DNA Minivector Based on an Inverted Terminal Repeat of Adeno-Associated Virus. MolBiotechnol 57, 382 - 390 (2015).

[0210] 16. Cao, L., During, M., and Xiao, W. Replication competent helper functions for recombinant AAV vector generation. Gene Ther 9, 1199 - 1206 (2002).

[0211] 17. Dean, D. A., Dean, B. S., Muller, S., and Smith, L. C. Sequence Requirements for Plasmid Nuclear Import. Experimental Cell Research 253, 713 - 722 (1999).

[0212] 18. Gerling, T., Wagenbauer, K. F., Neuner, A. M., and Dietz, H. Dynamic DNA devices and assemblies formed by shape - complementary, non–base pairing 3D components. Science 347, 1446 - 1452 (2015).

[0213] The features of the invention disclosed in this specification, the claims, and / or the drawings can be used as materials for implementing the invention in various forms of the invention either individually or in any combination thereof.

Claims

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

2. The nucleic acid nanostructure according to 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; wherein optionally, the nucleic acid nanostructure, preferably the at least one scaffold strand, comprises multiple nucleic acid sequences encoding genes.

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

4. The nucleic acid nanostructure according to any one of the preceding claims, wherein the nanostructure comprises enhanced staple strands, the length of the enhanced staple strands ranging from about 60 to about 250 nucleic acid bases, preferably from about 80 to about 220 nucleic acid bases, more preferably from about 90 to about 200 nucleic acid bases; wherein optionally, the enhanced staple strands are configured to bind to the scaffold strand at the 5' end and / or 3' end of the nucleic acid sequence of the gene encoding.

5. The nucleic acid nanostructure according to any one of the preceding claims, wherein the nanostructure, preferably the scaffold strand and / or at least one of the multiple 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 according to claim 5, wherein the nucleic acid sequence configured to form a loop structure is configured to form a loop at the 5' end and / or 3' end of the nucleic acid sequence of the gene encoding.

7. The nucleic acid nanostructure according to 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; wherein optionally, the nanostructure, preferably the at least one scaffold strand, comprises multiple nuclear targeting sequences.

8. The nucleic acid nanostructure according to 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. The nucleic acid nanostructure according to any one of the preceding claims, wherein the aspect ratio of the nanostructure ranges 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. The nucleic acid nanostructure according to any one of the preceding claims, wherein the scaffold strand comprises the nucleic acid sequence of the at least one encoding gene, a promoter, and a terminator; 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.

11. A composition, preferably a pharmaceutical composition, comprising the 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 nanostructure as defined in any one of claims 1 to 10.

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

14. A method of expressing a gene from a nucleic acid nanostructure, preferably the nucleic acid nanostructure according to any one of claims 1 to 10, the method comprising i) providing a nucleic acid nanostructure comprising a nucleic acid sequence of at least one encoding gene, preferably the 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; wherein preferably, the delivery comprises transfecting or transforming the cell; iii) expressing the gene in the cell; wherein optionally, the providing in step i) comprises: providing a plasmid, preferably providing a phagemid, or a collection of plasmids, preferably providing a collection of phagemids, wherein the plasmid or collection of plasmids encodes the nucleic acid nanostructure; and using the plasmid or collection of plasmids, preferably preparing the nucleic acid nanostructure by using a phage.

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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