JPWO2019207563A5 - - Google Patents

Description

The present invention relates to particles of viral capsid units encapsulating nucleic acid origami structures.

Viruses are self-replicating molecular machines that package the genome and deliver it to the host. In many cases, the shell (or capsid) of spherical viruses self-assembles around the viral genome. This is the case for Simian Virus 40 (SV40), which has a well-characterized self-assembly system in vitro.

SV40 is a small, non-enveloped primate polyomavirus with a double-stranded (ds) circular DNA genome of 5.2 kb. DNA forms minichromosomes with a nucleosomal structure similar to the chromatin of the cell. The minichromosome is surrounded by a 50 nm diameter capsid composed of pentamers of the 72 basic capsid subunits, the major viral protein VP1, arranged in a T=7d icosahedral lattice. there is In addition to VP1, a single molecule of minor capsid protein VP2 or VP3 is tightly anchored to each pentamer. In the capsid, VP2 and VP3 are located internal to VP1. The triangulation number T may be different for different icosahedral viruses. For example, the icosahedral shape of herpesvirus has T=16, while adenovirus has T=25. Different triangulation numbers result in different numbers of capsid subunits.

SV40 self-assembles in vitro from its basic subunit, the VP1 capsid protein pentamer, and can encapsulate all kinds of nucleic acids. The size and shape of virus-like nanoparticles assembled in vitro depend on the type and length of the encapsulated nucleic acid. On short single-stranded (ss) RNAs (≤814 nucleotides), the SV40 pentamer is an icosahedral T=1 geometry, composed of 12 pentamers and a single RNA molecule. form 22 nm diameter capsids with If the RNA or single-stranded DNA is too long to fit into a T=1 particle, the pentamer is suggested to be of the 22 nm particle and the icosahedral T=3 or T=4 geometry. form a string of heterogeneous particles with diameters of 29-40 nm. On a single molecule of double-stranded DNA, SV40 always forms 50 nm particles composed of 72 pentamers with an icosahedral T=7 geometry. SV40 has also been shown to assemble in vitro around DNA from VP1 pentamers containing VP2 or VP3 molecules (Enomoto et al. Virology 2011 420:1-9).

The economics, ease of in vitro preparation of SV40-like nanoparticles, safety, and lack of immunogenicity make them highly promising for medical use as gene delivery vectors. However, problems exist that hinder their usefulness. One of these problems is the low compactness of nucleic acids, which occupy a large volume compared to the volume of the internal cavity of the capsid. The molecular mechanism of capsid assembly in vitro is based on the interrelationship between the stability of protein-protein interactions and the work required to confine the nucleic acid within the small cavity of the capsid. Flexibility and compactness of nucleic acids are important factors for effective encapsulation. There are strong indications that dsDNA, a rigid polymer and known for its long persistence, is a poor substrate for packaging. For example, hepatitis B virus (HBV) (Dhason et al. Virology 2012, 430(1):20-9) or cowpea chlorotic mottle virus (CCMV) (Mukherjee et al. Journal of the American Chemical Society 2006, 128(8):2538-9), distorted non-native structures are formed rather than distinct capsids of fixed size and shape when assembled on dsDNA. These structures are often seen as multiplets of capsids threaded through a single nucleic acid molecule, suggesting incomplete nucleic acid encapsulation. For SV40 assembled in vitro on dsDNA, small-angle X-ray scattering studies showed that the core of the particle had no electron density, DNA was excluded from the inner cavity of the capsid, and the capsid was virtually empty. (Saper et al. Nucleic Acids Research, 2013, 41(3):1569-1580). Unpackaged nucleic acid that is extruded from the capsid or non-native viral structure can hinder its utility as a gene delivery vector.

Nevertheless, control of the size and shape of self-assembled capsids is not straightforward using the more flexible ssRNA or ssDNA substrates. SV40 assembly on ssRNA results in multiplets of T=1 capsids or polymorphic capsids instead of native 50 nm T=7 virion-sized particles (Kler and Zlotnick (ACS Chem Biol. 2013, 8 (12 ):2753-61).In addition, cryo-EM 3D reconstructions show that RNA is partially extruded from the T=1 capsid (Kler and Zlotnick (ACS Chem Biol. 2013, 8(12): 2753-61) In CCMV, efficient assembly is limited by RNA size (Cadena-Nava et al. Journal of Virology 2012, 86(6):3318-26).

Another class of nanomaterials that can be encapsulated by viral capsid self-assembly are nanoparticles coated with anionic ligands, such as carboxyl or citrate acid groups (Chen et al. Journal of nanoscience and nanotechnology 2005, 5). 12): 2029-33; He et al. ACS nano 2013, 7(10): 8447-8454; Chen et al. Nano Letters 2006, 6(4): 611-5; 10):4275-82).

Encapsulation of nanoparticles is typically straightforward due to the pre-compact nature of these matrices, unlike nucleic acids. Studies of encapsulation of gold nanoparticles by Brome Mosaic Virus (BMV) have shown that template sizes and morphologies commensurate with the capsid cavity can enhance assembly efficiency (Daniel et al. ACS nano 2010, 4). 7):3853-60;Sun et al.Proceedings of the National Academy of Sciences of the United States of America 2007, 104(4):1354-9; of Materials chemistry 2008, 18(32): 3763-3774).

Surface charge density and other chemical and physical surface properties are also important for effective nanoparticle encapsulation (Daniel et al. ACS nano 2010, 4(7):3853-60; Kusters et al. .The Journal of Physical Chemistry B 2015, 119(5):1869-1880).

DNA origami is a technique of DNA self-folding that uses a set of short synthetic DNA oligonucleotides called staples that facilitate the folding of long single-stranded DNA (scaffolds) into predesigned nanometric structures. Staples are designed to complement different portions of the scaffold DNA, thereby bridging distant portions of the scaffold strands. This approach provides a platform for manipulating nanometric objects with 2D and 3D shapes with high precision and resolution. DNA origami technology has also been applied to RNA and double-stranded DNA scaffolds. DNA origami technology is used in a wide range of applications such as devices, biosensors, molecular machines, and nanorobots. The ability to load chemicals and nanoparticles into DNA origami structures is exploited for therapeutic applications. Such structures have been loaded with anticancer agents, photosensitizers, and gold particles for chemotherapy, photodynamic therapy, and photothermal therapy.

The highly compact forms of nucleic acids produced by DNA origami technology may allow complete encapsulation of large nucleic acid molecules by viral capsids. However, assembly of viral capsids around DNA origami can be difficult. The chemical and physical surface properties of DNA origami structures are distinct from those of gold nanoparticles that have so far served as substrates for assembly. Furthermore, an inherent property of using DNA origami as substrates for assembly is the inability of origami structures to readily form perfect spheres compared to gold nanoparticles. The discrepancy between the shape of the encapsulated nanoparticles and the native capsid would make the encapsulation results difficult to predict. As a result, this geometric limitation can adversely affect the efficiency of DNA origami encapsulation.

Various publications address the use of virus-like particles for delivery of nucleic acids. US Pat. No. 6,830,929 is directed to in vitro construction of SV40 virus and pseudovirus. Zlotnick A. (Biophysical Journal, 2013, 104, 1595-1604) is entitled "To Build a Virus on a Nucleic Acid Substrate". Handa H. (Virology, 2011, 420, 1-9), "In vitro reconstruction of SV40 particles that are composed of VP1/2/3 capsid proteins and nucleosomal DNA and direct effective "gene transfer". US Application Publication No. US2008/0131928 is directed to virus particle-like structures in physiological conditions and methods of forming the same. Other publications, including Mikkila et al. (Nano Lett. 2014, 14, 2196-2200) and Burns et al. reporting.

There remains a need in the art for efficient particles that can completely encapsulate large nucleic acid molecules and exhibit authentic viral structures that can be effectively used to transfer such nucleic acid molecules into cells. It is said that

The invention provides, in its embodiments, particles of at least one nucleic acid origami structure encapsulated by at least 12 capsid units, compositions comprising the same, and uses thereof.

According to some embodiments, the advantageous particles of the present invention are capable of efficient delivery of nucleic acids to target cells due to their ability to efficiently and completely encapsulate nucleic acids in the form of very compact origami structures. delivery. As exemplified herein, the encapsulated nucleic acid is completely encapsulated and protected within the shell made of the capsid unit and does not protrude or extrude from the particle. As demonstrated herein, advantageous particles of the invention exhibit an icosahedral viral structure that fully encapsulates nucleic acids and closely resembles viruses. Furthermore, without wishing to be bound by theory or mechanism, as demonstrated hereinbelow, advantageous particles that closely resemble virus structures are particles and nucleic acids that are recognized by cellular machinery. It is effective for gene delivery because it is transported and metabolized in cells as efficiently as the native virus, and the capsid may be opened by enzymatic reactions in the cell, after which the capsid is opened, followed by a specific structure is formed to facilitate nuclear uptake of the nucleic acid cargo. In contrast, particles that do not show high similarity to viruses are less likely to efficiently enter the nucleus. They may be phagocytosed by cells, but will eventually become degradable organelles. Thus, the particles of the invention exhibit a high potential for gene delivery activity.

It is disclosed herein that, according to some embodiments, DNA origami can be used as a new class of substrates for capsid assembly. To this end, as exemplified herein, SV40 capsid proteins and DNA origami structural molecules are packed into a near-spherical morphology and a suitable diameter that best matches the internal cavity of the native SV40 capsid. Used. As exemplified further herein below, particles with shells each containing capsid units made from one or more capsid proteins assemble around the core of a DNA origami structure, which can be highly efficiently can be encapsulated in In some exemplary embodiments, the cryo-EM structure of the resulting particles demonstrates that the outer shell forms a regular SV40 lattice with T=7d dodecahedral symmetry. . In some embodiments, within the core of the particle (i.e., inside the shell), structural features of the DNA origami such as parallel helices arranged in a "honeycomb" lattice, as demonstrated by the cryo-EM structure. can be identified, indicating that the DNA origami is encapsulated entirely within the capsid shell.

According to some exemplary embodiments, as demonstrated hereinbelow, the axis connecting two opposing honeycomb facets of the DNA origami structure is three Oriented slightly off the axis of rotational symmetry (eg, at a tilt angle of about −6.8° toward the axis of trifold symmetry), the DNA origami structure and capsid are not randomly oriented. SV40-like particles are therefore advantageously formed around DNA origami with high fidelity, efficiency and reproducibility, accurately reproducing the capsid and aligning the two lattices, the DNA origami and the SV40 capsid, against each other. Allowing precise positioning to form useful and active particles. In some embodiments, the DNA origami structure is randomly oriented with respect to the capsid.

According to some embodiments, particles are provided comprising a core and a shell, the core comprising at least one nucleic acid origami structure encapsulated by a shell, the shell comprising at least 12 capsid units. , the capsid unit comprises one or more capsid proteins.

According to some embodiments, the nucleic acid origami structure can be single-stranded DNA, double-stranded DNA, any form of single-stranded RNA, and any form of double-stranded DNA, modified nucleic acid, synthetic nucleic acid, or It can be any combination thereof, as well as variations thereof. In some embodiments, a nucleic acid origami structure may comprise multiple nucleic acid scaffolds or smaller origami structures, each optionally comprising two or more types of nucleic acids.

According to some embodiments, the shell of the particle can have a quasi-spherical shape. According to some embodiments, the shell of the particle may have an icosahedral shape. According to some embodiments, particles can be spherical or near-spherical.

According to some embodiments, the particles can have a diameter of about 10 to about 200 nanometers (nm). According to some embodiments, the particle diameter may be greater than about 20 nm.

According to some embodiments, a capsid unit may comprise at least 5 capsid proteins. According to some embodiments, each of the capsid units may comprise pentamers and/or hexamers of capsid proteins. In some embodiments, the capsid protein is a viral protein.

In some embodiments, the viral protein is from a virus selected from the viral family Polyomaviridae. In some embodiments, the viral protein is from human polyoma virus. In some embodiments, the viral protein is from a virus selected from the group consisting of SV40 virus, JC virus, or BK virus. In some embodiments, the capsid protein is from the SV40 virus.

According to some embodiments, the capsid protein may be selected from capsid protein VP1, capsid protein VP2, capsid protein VP3, or any combination thereof. In some embodiments, the capsid protein is VP1.

In some embodiments, an origami structure can be encapsulated by at least 72 capsid units. In some embodiments, the capsid unit comprises 5 VP1 molecules, 5 VP1 molecules and 1 VP2 molecule, 5 VP1 molecules and 1 VP3 molecule, and 5 VP1 molecules and 1 VP2 molecule or 1 VP3 molecules, or combinations of viral protein molecules selected from the group consisting of any combination thereof.

In some embodiments, the particles are for use in delivering nucleic acid molecules into target cells. In some embodiments, target cells are selected from in vitro cells or cells housed in a tissue or organism.

In some embodiments, compositions are provided that include a plurality of particles of the invention.

In some embodiments, use of particles comprising a core and a shell for delivery of nucleic acids to cells is provided, the core comprising at least one nucleic acid origami structure encapsulated by a shell, the shell comprising contains at least 12 capsid units, and a capsid unit contains one or more capsid proteins.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed description.

Electron micrographs of near-spherical DNA origami structures of various sizes. FIG. 1A—25 nm spheroidal origami structure constructed with 2873 nucleotide (nt) long single-stranded DNA, FIG. 1B—30 nm spheroidal origami constructed with 5386 nt long single-stranded DNA. Structures, FIG. 1C—35 nm spheroidal origami structure assembled with 7560 nt long single-stranded DNA, FIG. 1D—40 nm spheroidal origami structure composed of two hemispheres, one It is constructed with 7560 nt long single-stranded DNA and the second is constructed with 7249 nt single-stranded DNA. Negative stain transmission electron microscopy of particles. Figure 2A: Particle comprising VP1 assembled on a roughly spherical DNA origami structure with a diameter of 25 nm Figure 2B: Particle comprising VP1 assembled on a roughly spherical DNA origami structure with a diameter of 30 nm Figure 2C: Nearly spherical shape with a diameter of 35 nm FIG. 2D: Particles containing VP1 assembled on a nearly spherical DNA origami structure with a diameter of 40 nm. In all images assembly products are observed with floating origami structures and floating VP1 pentamers in the background. Eight cryo-EM 2D micrographs of SV40 VP1-constructed particles assembled on a 30 nm quasi-spherical DNA origami structure. Micrographs show 50 nm SV40-like particles composed of a shell and core exhibiting a characteristic viral spike (VP1 pentamer). In some of the cores, folded strands of the origami structure can be observed (seen as stripes). A model of wild-type SV40 (PDB entry 1SVA) superimposed on a 2D micrograph of SV40-like particles assembled on a 30 nm roughly spherical DNA origami structure, showing perfect alignment. 2D classification of cryo-EM micrographs grouping particles assembled on a 30 nm approximately spherical DNA origami structure into classes of similar particles, with each subclass representing hundreds of particles on average. Confocal micrographs of CV-1 cells incubated with SV40-like particles containing fluorescently labeled naked 30 nm origami structures or labeled 30 nm DNA origami structures. FIG. 4A: CV1 cells treated with fluorescently labeled naked (not packaged into viral capsid) origami structures (construction, control). FIG. 4B: CV1 cells incubated with viral capsids assembled in vitro around DNA origami structures, showing uptake of fluorescently labeled DNA (marked by arrows) into the cells. Cryo-EM and 3D reconstructions of T=7d icosahedral SV40-like particles assembled on 35 nm quasi-spherical DNA origami structures. FIG. 5A: Upper panel—Representative cryo-EM micrograph of SV40-like particles showing a homogeneous population of 50 nm particles. Scale bar represents 100 nm. The inset shows origami-filled (left) and empty (right) particles showing streaks (enlarged in middle panel). Bottom panel—average of 2D classes from a total of 48,778 particles, showing the presence of both empty particles and particles filled with DNA origami. FIG. 5B: 3D reconstruction of empty particles at 7.3 Å resolution (top panel—front view, middle panel—inside view) and the corresponding 2D class of 26,110 particles (bottom panel). FIG. 5C: 3D reconstruction of origami-filled particles at 25 Å resolution (top panel—front view, middle panel—interior view) and the corresponding 2D class of 19786 particles (bottom panel). Figure 5D: Demonstration of the icosahedral symmetry of the reconstructed grain. To demonstrate the icosahedral symmetry, the icosahedron is formed in the cryo-EM reconstructed origami-filled capsid by aligning its vertices with the center of the pentavalent pentamer in the shell. Manually attached to the surface. The 5-fold, 3-fold, and 2-fold axes of symmetry are marked as pentagons, triangles, and rhombuses on the facets of the icosahedron, respectively. FIG. 5E: Schematic of Caspar and Klug's calculation of the triangulation number T of the icosahedron. A pentagon indicates a pentavalent pentamer. T is obtained by the formula T=h**2+hk+k**2. For T = 7d, h = 2, and k = 1, h is represented by arrows 1 and 2 and K is represented by arrow 1″. Localization corresponds to T=7d. Internal view of cryo-EM 3D reconstruction of SV40-like particles assembled on a DNA origami, showing that the DNA origami is encapsulated as a whole. Figure 6A: Schematic of 35 nm DNA origami structure shown at different viewing angles; Figure 6B: 3D electron density map of 3D reconstruction of capsid filled with DNA origami. Views of the capsid filled with DNA origami at various viewing angles correspond to respective views of the DNA origami design in FIG. 6A. Pictograms showing three-dimensional representations of encapsulated 35 nm DNA origami with respect to capsid-aligned icosahedrons. The honeycomb facets of the DNA origami face the tilted icosahedral three-fold symmetry (Fig. 7B). When rotated, the axis connecting the two opposing honeycomb facets appears parallel to the origami helix marked by the dashed line and lies 6.8° off the icosahedron's three-fold symmetry axis. (Fig. 7A). FIG. 7C shows a rotated view of FIG. 7A. Fig. 4 is a pictogram showing an electrophoretic mobility shift assay (EMSA) analysis demonstrating the formation of stable VP1/DNA origami complexes. A 35 nm DNA origami was mixed with VP1 pentamers at a molar ratio of 400 pentamers per DNA origami structure. Assembly products were analyzed on a 0.7% agarose gel and stained with EtBr. M-size marker, 0-origami structure (no pentamer), 400-origami structure incubated with pentamer. Development of DNA origami under intracellular conditions (37° C. and 1 mM Mg+) demonstrated by the increased accessibility of DNA polymerases to enzymatic action.

According to some embodiments, advantageous particles are provided comprising at least one nucleic acid origami structure encapsulated by at least 12 capsid units, the capsid units comprising one or more capsid proteins. In some embodiments, the particle has a core and a shell, the shell being made up of capsid units and the core comprising a nucleic acid origami structure encapsulated within the shell. Compositions comprising a plurality of particles and their use for transferring/transfecting/delivering nucleic acids encapsulated in the particles to target cells are further provided.

Definitions To facilitate understanding of the present invention, some terms and phrases are defined below. These terms and phrases are intended to be descriptive so that the terms or phrases herein can be interpreted by a person of ordinary skill in the art in light of the teachings and guidance presented herein in combination with the knowledge of those of ordinary skill in the art. It should be understood that this is intended to be a non-limiting example.

As referred to herein, the terms "nucleic acid", "nucleic acid molecule", "oligonucleotide", "polynucleotide" and "nucleotide" may be used interchangeably herein. These terms refer to deoxyribonucleotides (DNA), ribonucleotides, in the form of discrete fragments, or as components of larger constructs, linear or branched, single-stranded, double-stranded, triple-stranded, or hybrids thereof. Of interest are polymers of (RNA) and their modified forms. The term also includes RNA/DNA hybrids. Polynucleotides can include DNA or RNA sense and antisense oligonucleotides or polynucleotide sequences. DNA or RNA molecules include, but are not limited to, complementary DNA (cDNA), genomic DNA, synthetic DNA, recombinant DNA, or hybrids thereof, or, for example, mRNA, shRNA, siRNA, miRNA, anti It can be an RNA molecule such as sense RNA. Each possibility is a separate embodiment. These terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent internucleoside linkages, as well as oligonucleotides having non-naturally occurring portions that function similarly to their respective naturally occurring portions. . In some embodiments, the nucleic acid is in the form of an origami structure.

Origami Structures As used herein, the terms "nucleic acid origami structure" and "origami structure" may be used interchangeably. These terms refer to nucleic acid molecules that are folded into a specific three-dimensional shape at the nanoscale. Origami structures are well known to those skilled in the art and are described in Rothemund et al. , 2006, Nature 440:297-302. Briefly, long strands of nucleic acid (scaffolds) are folded using short nucleic acid strands, also known as "staple" strands. The binding of these staple strands leads to cubic conformational changes in long strands, and many staple strands can be used to provide specific folding. Further heating and cooling cycles are repeated to optimize folding, binding and removal of staple strands.

Origami structures already folded into a specific shape can be purchased commercially. Companies that sell origami structures include, but are not limited to, tilibit nanosystems, and Eurofins Genomics.

In some embodiments, the origami structure comprises a nucleic acid strand. In some embodiments, the origami structure comprises single-stranded DNA, double-stranded DNA, any form of single- and double-stranded RNA (including mRNA, shRNA, siRNA, miRNA, antisense RNA, etc.). , modified nucleic acids, synthetic nucleic acids, any combination thereof, and variations thereof. Each possibility is a separate embodiment. In some embodiments, an origami structure comprises one nucleic acid molecule. In some embodiments, an origami structure comprises two or more nucleic acid molecules. In some embodiments, an origami structure can comprise a combination of two or more types of nucleic acid molecules. In some embodiments, the nucleic acid is compacted into an origami structure rather than linear from an origami structure. In some embodiments, an origami structure can include one or more nucleic acid scaffolds, and each scaffold can be composed of one or more types of nucleic acids. For example, a scaffold can comprise a DNA molecule linked to an RNA molecule.

In some embodiments, an origami structure can include one or more smaller origami structures, and each smaller origami structure can be composed of one or more types of nucleic acids. In some embodiments, staples consist of one or more types of nucleic acids. In some embodiments, the staple and scaffold each consist of a different type of nucleic acid.

The origami structure can take any shape desired for a particular application. In some embodiments, the shape of the origami structure is selected from the group consisting of spheres, cubes, pyramids, cones, springs, and spherical shapes. In some embodiments, the origami structure is configured to fill or be encapsulated by the shell. In some embodiments, the origami structure is spherical. In some embodiments, the origami structure is spherical in nature. In some embodiments, the origami structure is approximately spherical. In some embodiments, the origami structure is spherical and configured to fill the virus-like particles.

In some embodiments, nucleic acid origami structures are capable of unfolding intracellularly. In some embodiments, nucleic acid origami structures are capable of unfolding under the conditions of the intracellular environment. In some embodiments, nucleic acid origami structures are designed such that they can unfold under the conditions of the intracellular environment.

In some embodiments, nucleic acid origami structures are capable of being expressed in cells. In some embodiments, the nucleic acid template of the origami structure is capable of being expressed intracellularly. In some embodiments, a nucleic acid origami structure is capable of being expressed intracellularly after it has been deployed intracellularly.

In some embodiments, the nucleic acid origami structure is capable of unfolding upon release from the protein capsid shell after it has been introduced into the cell. In some embodiments, nucleic acid origami structures are capable of unfolding upon release from the protein capsid shell in the intracellular environment. In some embodiments, the nucleic acid origami structure is designed so that it can unfold when released from the protein capsid shell in the intracellular environment.

In some embodiments, the staples of the nucleic acid origami structure are allowed to disassemble within the cell such that the nucleic acid template of the origami structure is released into the cell. In some embodiments, disassembly of the staples of the nucleic acid origami structure is affected by intercellular conditions such as, but not limited to, temperature, salt concentration, magnesium concentration, pH, etc., or any combination thereof. In some embodiments, the staples of the nucleic acid origami structure are designed to disassemble at intracellular conditions of temperature and/or intracellular conditions of magnesium concentration to release the nucleic acid template of the origami structure.

In some embodiments, the staples of the nucleic acid origami structure may consist or consist of one or more oligonucleotides joined by hydrazone chemical ligation. In some embodiments, the staple of the nucleic acid origami structure consists or may consist of at least two oligonucleotides joined by hydrazone chemical ligation. In some embodiments, a staple of a nucleic acid origami structure may consist or consist of two oligonucleotides joined by hydrazone chemical ligation. In some embodiments, the staples of the nucleic acid origami structure can consist or consist of one or more modified oligonucleotides that can be joined by hydrazone chemical ligation.

In some embodiments, the staples of the nucleic acid origami structure consist or may consist of one or more oligonucleotides joined by disulfide bonds. In some embodiments, the staple of the nucleic acid origami structure consists or may consist of at least two oligonucleotides joined by disulfide bonds. In some embodiments, the staple of the nucleic acid origami structure consists or may consist of two oligonucleotides joined by a disulfide bond. In some embodiments, the staples of the nucleic acid origami structure can consist of or consist of one or more modified oligonucleotides that can be joined by disulfide bonds.

In some embodiments, staples may comprise, consist of, or consist of oligonucleotides joined by hydrazone chemical ligation, oligonucleotides joined by disulfide bonds, or any combination thereof.

In some embodiments, the origami structural staples are degraded under the acidic conditions of cellular endosomes, allowing release of the origami structural nucleic acid template. In some embodiments, the origami-structured staples are designed to degrade under the acidic conditions of cellular endosomes, releasing the origami-structured nucleic acid template.

In some embodiments, the staples of the origami structure can be reduced intracellularly to release the nucleic acid template of the origami structure. In some embodiments, the staples of the origami structure can be reduced under intracellular conditions to release the nucleic acid template of the origami structure. In some embodiments, the staples of the origami structure are designed to be reduced under intracellular conditions to release the nucleic acid template of the origami structure.

In some embodiments, the diameter of the origami sphere (or near sphere) is 10-200, 10-180, 10-160, 10-140, 10-120, 10-100, 10-80, 10-60, 20-200, 20-180, 20-160, 20-140, 20-120, 20-100, 20-80, or 20-60 nanometers (nm). Each possibility represents a separate embodiment of the present invention. In some embodiments, the diameter of the origami sphere is 10-200 nm. In some embodiments, the diameter of the origami sphere is greater than 10, 20, 22, 25, or 30 nm. Each possibility represents a separate embodiment of the present invention. In some embodiments, the diameter of the origami sphere is greater than 22 nm. In some embodiments, the diameter of the origami sphere is greater than 25 nm.

In some embodiments, the distance from the center to the perimeter of the origami structure is 10-200, 10-180, 10-160, 10-140, 10-120, 10-100, 10-80, 10-60, 20-200, 20-180, 20-160, 20-140, 20-120, 20-100, 20-80, or 20-60 nanometers (nm). Each possibility represents a separate embodiment of the present invention. In some embodiments, the distance from the center to the perimeter of the origami structure is 10-200 nm. In some embodiments, the center-to-perimeter distance of the origami structure is greater than 10, 20, 22, 25, or 30 nm. Each possibility represents a separate embodiment of the present invention. In some embodiments, the center-to-perimeter distance of the origami structure is greater than 17 nm. In some embodiments, the center-to-perimeter distance of the origami structure is greater than 20 nm. In some embodiments, the center-to-perimeter distance of the origami structure is about 17 nm. In some embodiments, the center-to-perimeter distance of the origami structure is about 20 nm. In some embodiments, the center-to-perimeter distance of the origami structure is about 30 nm.

Capsid Units and Capsid Proteins As used herein, the terms "capsid unit" or "capsid subunit" refer to a structural unit composed of one or more capsid proteins; Refers to the protein shell. In some embodiments, the capsid unit is a capsomer. In some embodiments, a combination of one or more capsid units form the shell of the particles of the invention. In some embodiments, combinations of 12 or more capsid units form the shell of the particles of the invention. In some embodiments, the capsid protein is of natural sources. In some embodiments, the capsid protein is at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% the native viral capsid protein. % sequence homology. Each possibility is a separate embodiment. In some embodiments, the viral protein is from a virus selected from the viral family Polyomaviridae. In some embodiments, the capsid protein is of a virus selected from the group consisting of SV40 virus and human polyoma virus including, for example, JC virus or BK virus. In some embodiments, the Polyomaviridae virus is Merkel cell polyomavirus, Trichodysplasia spinulosapoliomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BK polyomavirus. from the group consisting of Omavirus, JC Polyomavirus, KI Polyomavirus, WU Polyomavirus, Human Polyomavirus 6, Human Polyomavirus 7, MW Polyomavirus, STL Polyomavirus, and Lyon IARC Polyomavirus can be selected. Each possibility is a separate embodiment. In some embodiments, the capsid protein is from the SV40 virus. In some embodiments, the capsid protein is a modified SV40 capsid protein. In some embodiments, the capsid protein is of artificial origin. In some embodiments, engineered capsid proteins may be modified or mutated forms of known viral capsid proteins. In some embodiments, engineered capsid proteins may comprise chimeric proteins derived from one or more known viral capsid proteins. In some embodiments, engineered capsid proteins may comprise chimeric proteins comprising one or more domains from one or more viral capsid proteins. In some embodiments, the engineered capsid protein may comprise non-viral regions or domains, eg, the engineered protein may comprise non-viral sequences instead of, or in addition to, viral sequences or domains. In some embodiments, an artificial capsid protein may contain one or more functional domains capable of mimicking a natural viral capsid protein. In some embodiments, the capsid protein may comprise any modified or mutated form of a known capsid protein. In some embodiments, the capsid protein can be any modified or mutated form of the viral capsid protein. In some embodiments, the capsid protein can be a modified or mutated form of the SV40 capsid protein. In some embodiments, the capsid protein may comprise any modified or mutant form of the SV40 capsid protein. In some embodiments, the capsid protein may be or comprise a genetically engineered form of a known viral capsid protein. In some embodiments, the capsid protein may comprise one or more domains of known viral capsid proteins from various sources. In some embodiments, a combination of one or more types of viral capsid proteins can form a capsid unit. In some embodiments, a combination of several capsid units can form the shell of the particle. In some exemplary embodiments, the shell of the particle comprises at least 12 capsid units. In some embodiments, each capsid unit may comprise at least one viral protein. In some embodiments, each capsid unit may comprise at least 5 viral proteins. In some embodiments, the capsid unit is a pentamer of capsid proteins. In some embodiments, the capsid unit forms a shell having a dodecahedral shape. In some embodiments, the particles of the invention have a dodecahedral shape. In some embodiments, the icosahedral shape has 12 vertices, 2-fold, 3-fold, and 5-fold axes of symmetry, and a characteristic number of structures defined by the icosahedral triangulation number. For structures with units, the icosahedral triangulation number can be calculated according to the system of Caspar and Klug (Physical principles in the construction of regular viruses. Cold Spring Harb Symp Quant Biol. 1962; 27: 1-24). can. The icosahedral shape can be determined based on the system of Caspar and Klug (Physical principles in the construction of regular viruses. Cold Spring Harb Symp Quant Biol. 1962; 27:1-24). In some embodiments, the dodecahedral geometry relates to capsid units arranged in the form of hollow quasi-spherical structures. In some embodiments, the dodecahedral capsid is as described in Wang and Zlotnick et al. Viruses 2018, 10:25, contain capsids with geometric defects in the dodecahedral lattice.

In some embodiments, the term "quasi-spherical" is intended to encompass hollow structures having an outer shell surface disposed about an inner core. In some embodiments, a quasi-spherical shape is a geometric shape such as a regular dodecahedron, a defective dodecahedron, an imperfect dodecahedron, a perfect sphere, a near sphere, a hemisphere, an ellipsoid, etc. can include

According to some embodiments, the particles have a dodecahedral shape. In some embodiments, the particles have a quasi-spherical shape. In some embodiments, particles can be spherical, hemispherical, or near-spherical.

In some embodiments, the capsid protein is a viral protein (VP). In some embodiments, the capsid protein may be selected from capsid protein VP1 (VP1), capsid protein VP2 (VP2), capsid protein VP3 (VP3), and combinations thereof. Each possibility is a separate embodiment. In some embodiments, the capsid protein is VP1.

In some embodiments, VP1, VP2, and VP3 refer to Polyomaviridae VP1, VP2, and VP3, respectively.

In some embodiments, VP1, VP2, and VP3 refer to JC VP1, JC VP2, and JC VP3, respectively.

In some embodiments, VP1, VP2, and VP3 refer to BK VP1, BK VP2, and BK VP3, respectively.

In some embodiments, VP1, VP2, and VP3 refer to SV40 VP1, SV40 VP2, and SV40 VP3, respectively.

In some embodiments, the capsid protein is a viral protein (VP), or modified form thereof. In some embodiments, the capsid protein is capsid protein VP1 (VP1) and/or modified forms thereof, capsid protein VP2 (VP2) and/or modified forms thereof, capsid protein VP3 (VP3) and/or modified forms thereof morphology, or any combination thereof. Each possibility is a separate embodiment. In some embodiments, modified forms of VP1, VP2, and VP3 refer to modified forms of Polyomaviridae VP1, VP2, and VP3, respectively. In some embodiments, variants of VP1, VP2, and VP3 refer to variants of JC VP1, JC VP2, JC VP3, respectively. In some embodiments, variants of VP1, VP2, and VP3 refer to variants of BK VP1, BK VP2, and BK VP3, respectively. In some embodiments, variants of VP1, VP2, and VP3 refer to variants of SV40 VP1, SV40 VP2, and SV40 VP3, respectively.

Methods for encapsulating nucleic acids into capsids are well known to those of skill in the art and are also provided herein below. Mukherjee et al, PLoS One, 2:e765 (2007) and S. Kler, et al. , JACS chemical biology 8, 2753-2761 (2013) can be consulted for further guidance.

Encapsulation As used herein, the term “encapsulated” with respect to an origami structure should be understood in its plain sense, i.e., the origami structure is completely surrounded by capsid units. there is In some embodiments, the origami structure is surrounded entirely by a capsid unit. In some embodiments, the origami structure is not exposed on the exterior portion of the capsid unit. In some embodiments, the capsid unit forms a shell that encapsulates a closed core in which the origami structure resides/houses. In some embodiments, an origami nucleic acid is more completely encapsulated than a less compact form of the same nucleic acid.

In some embodiments, one origami structure is encapsulated by at least 12 capsid units. In some embodiments, two or more origami structures are encapsulated by at least 12 capsid units.

In some embodiments, the origami structure is encapsulated by a shell of at least 12, 32, 42, 72, 92, 122, 132, 162, 212, 252, 272, 282, or 312 capsid units. Each possibility represents a separate embodiment of the present invention. In some embodiments, an origami structure can be encapsulated by at least 72 capsid units. In some embodiments, the origami structure is encapsulated by at least 72 capsid pentamers. In some embodiments, the capsid unit of the shell may be the same or different in the number or composition of capsid proteins that make up the capsid unit of a native viral capsid. In some embodiments, all of the capsid units or some of the capsid proteins are all VP1 or modified/mutated forms thereof. In some embodiments, the capsid proteins of all or some of the capsid units are VP1 and VP2 or modified/mutated forms thereof. In some embodiments, the capsid proteins of all or some of the capsid units are VP1 and VP3 or modified/mutated forms thereof. In some embodiments, the capsid proteins of all or some of the capsid units are VP1, VP2 and VP3 or modified/mutated forms thereof. In some embodiments, the capsid unit can be composed of at least five viral proteins. In some embodiments, the capsid unit may be composed of pentamers of viral proteins. In some embodiments, the capsid unit may be composed of pentamers of the VP1 protein. In some embodiments, the capsid unit may be composed of a pentamer of VP1 protein, optionally with one or more additional viral proteins such as VP2 and/or VP3. In some embodiments, the capsid unit comprises 5 VP1 molecules, 5 VP1 molecules and 1 VP2 molecule, 5 VP1 molecules and 1 VP3 molecule, 5 VP1 molecules and 1 VP2 molecule or 1 VP3 molecule , or any combination thereof. Each possibility is a separate embodiment. In some embodiments, the origami structure is encapsulated by at least 360 capsid proteins. In some embodiments, the origami structure comprises at least 72 VP1 pentamers, each pentamer consisting only of a VP1 molecule and variants thereof, each pentamer comprising one VP2 molecule and variants thereof. 72 VP1 pentamers, 72 VP1 pentamers each containing one VP3 molecule and modified forms thereof, or each pentamer containing one VP2 molecule or one VP3 molecule and modified forms thereof are encapsulated by any combination of at least 72 VP1 pentamers, including

In some embodiments, the ratio of nucleic acid origami structures to capsid proteins that are mixed to produce the particles of the invention is at least 1:100, 1:200, 1:300, 1:400 in molar concentrations. , 1:500, 1:600, 1:700, 1:750, 1:800, 1:900, 1:1000, or 1:2000. Each possibility represents a separate embodiment of the present invention. In some embodiments, the ratio of nucleic acid origami structure to capsid protein, on a molar basis, is at least 1:200. In some embodiments, the ratio of origami structure to capsid protein is, on a molar basis, 1:100 to 1:5000, 1:100 to 1:2000, 1:100 to 1:1000, 1:100 to 1: 750, 1:200-1:5000, 1:200-1:2000, 1:200-1:1000, or 1:200-1:750. Each possibility represents a separate embodiment of the present invention. In some embodiments, the ratio of origami structure to capsid protein mixed to produce the particles of the invention is 1:200 to 1:2000 in molarity.

In some embodiments, the ratio of origami structure to capsid unit in the particle is at least 1:12, 1:32, 1:42, 1:72, 1:92, 1:122, 1:132, 1: 162, 1:212, 1:252, 1:272, 1:282, or 1:312. Each possibility represents a separate embodiment of the present invention. In some embodiments, the ratio of nucleic acid origami structure to capsid structural units in the particles is at least 1:12, 1:32, 1:42, 1:72, 1:92, 1:122, 1:132, 1:162, 1:212, 1:252, 1:272, 1:282, or 1:312. Each possibility represents a separate embodiment of the present invention.

In some embodiments, particles comprising at least one origami structure encapsulated by a capsid unit are virus-like particles (VLPs). In some embodiments, the VLP can successfully transfer the encapsulated nucleic acid to the target cell so that the nucleic acid can be expressed in the target cell. In some embodiments, the VLP is capable of transferring an encapsulated nucleic acid origami structure into a target cell, such that the nucleic acid is optionally deployed within the target cell prior to intracellular deployment. It can and allows expression. In some embodiments the cell is in vitro. In some embodiments, the cells are housed in a tissue or organism. In some embodiments, the VLP is capable of transferring the encapsulated nucleic acid origami structure to an organism in vivo so that the nucleic acid can be expressed in vivo in target cells or tissues. In some embodiments, the VLPs are non-pathogenic. In some embodiments, the nucleic acid encapsulated in the particles is not of viral origin.

In some embodiments, the diameter of the particles is 20-500, 20-400, 20-300, 20-250, 20-200, 20-150, 20-100, 30-500, 30-400, 30- 300, 30-250, 30-200, 30-150, 30-100, 40-500, 40-400, 40-300, 40-250, 40-200, 40-150, 40-100, 50-500, 50-400, 50-300, 50-250, 50-200, 50-150, or 50-100 nm. Each possibility represents a separate embodiment of the present invention. In some embodiments, the particles are 20-300 nm in diameter. In some embodiments, the particle diameter is greater than 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 nm. Each possibility represents a separate embodiment of the present invention. In some embodiments, the particle diameter is that of a virus-like particle.

In some embodiments, the particles of the invention allow encapsulation of longer nucleic acid molecules than could be encapsulated if the molecules were less compact. In some embodiments, particles of the invention are more stable than particles generated from less compact nucleic acid molecule templates. In some embodiments, the particles disclosed herein form particle populations that are more uniform in size and shape than particle populations generated from less compact nucleic acid molecule templates.

In some embodiments, particles are provided comprising at least one origami structure encapsulated by at least 12 capsid units.

In some embodiments, particles of the invention have a different triangulation number (dodecahedral shape) than particles generated from less compact nucleic acid molecule templates. In some embodiments, particles of the invention have an icosahedral shape that more closely resembles naturally occurring viruses than particles generated from less compact nucleic acid molecule templates. In some embodiments, the particles of the present invention have T=1, T=3, T=4, T=7, T=9, T=12, T=13, T=16, T=21, T =25, T=27, T=28, or T=31. Each possibility represents a separate embodiment of the present invention.

In some exemplary embodiments, particles of the present invention have a shape of T=7. In some embodiments, the particles of the present invention exhibit T=7d dodecahedral symmetry. In some exemplary embodiments, a T=7d icosahedral particle capsid has 12 pentavalent pentamers (eg, a VP1 pentamer surrounded by 5 other pentamers) and 60 It is composed of one hexavalent pentamer (eg, a VP1 pentamer surrounded by six other pentamers).

In some embodiments, the icosahedral surface lattice of the assembled capsid can include 5-fold, 3-fold, and 2-fold axes of symmetry.

In some embodiments, the DNA origami structures are not randomly positioned with respect to capsid shape. In some embodiments, the interior (core) of the particles is uniform from particle to particle, ie, the population of particles is substantially homogeneous. In some embodiments, the DNA origami structures are randomly positioned with respect to the capsid shape.

In some embodiments, the encapsulation efficiency of the DNA origami structure within the capsid shell is greater than about 1%, greater than about 2%, greater than about 3%, greater than about 5%, greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 99%. Each possibility is a separate embodiment.

In some embodiments, the encapsulation efficiency of the DNA origami structure within the capsid shell is at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, At least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. Each possibility is a separate embodiment.

In some embodiments, methods are provided for delivering/infecting/transfecting target cells with nucleic acids encapsulated in particles of the invention. In some embodiments, the particles of the invention are for use in delivering/infecting/transfecting target cells with nucleic acids encapsulated in the particles. In some embodiments, the particles of the invention are for use in delivering nucleic acid molecules to cells. In some embodiments, the particles of the invention are for use in delivering nucleic acid molecules into cells. In some embodiments, the particles of the invention are for use in delivering nucleic acid molecules into the cytoplasm of cells. In some embodiments, the particles of the invention are for use in delivering nucleic acid molecules into the nucleus of cells. In some embodiments, the particles of the invention are superior at delivering nucleic acid molecules into cells compared to VLPs (virus-like particles) containing less compact nucleic acid molecules. In some embodiments, the particles of the invention are superior at delivering nucleic acid molecules into cells compared to conventional viral vectors. The terms "better delivery" and "better delivery" are used interchangeably and include, but are not limited to, ease of delivery, accuracy of delivery, efficiency and ease of generation of particles, target Efficiency of expression of nucleic acids in cells, efficiency of target gene modification or inactivation in cells, increased amount of nucleic acid molecules entering the cell nucleus, increased amount of nucleic acid molecules entering the cell cytoplasm, delivery of larger nucleic acid molecules to cells ability to deliver multiple nucleic acid molecules to the same cell; ability to deliver any type of nucleic acid molecule to a cell regardless of their sequence; ability to deliver multiple types of nucleic acid molecules to the same cell; Any advantageous delivery can be referred to, including the ability to deliver the nucleic acid molecule to the same cell, the ability to deliver nucleic acid of viral origin, increased protein production from the nucleic acid molecule within the cell, and the like. In some embodiments, advantageous delivery refers to lack of immunogenicity of the particles of the invention. In some embodiments, the particles of the invention are for use in the delivery/infection/transfection of nucleic acid molecules to a desired target site. A target site can include any target site, such as, but not limited to, a cell, tissue, organ, microorganism, and the like. The target site can be an in vivo or in vitro target site.

In some embodiments the cell is in vitro. In some embodiments the cell is in vivo. In some embodiments, the cells are in vitro or in vivo. In some embodiments, the cells are human cells. In some embodiments the cells are mammalian cells.

In some embodiments, compositions are provided that include one or more particles of the invention. In some embodiments, the composition comprises a plurality of particles of the invention. In some embodiments the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, a composition may comprise a plurality of particles that may be the same or different in terms of size, composition, and/or identity of the particles and/or nucleic acids encapsulated in the capsid units of the particles.

In this application, unless otherwise stated, adjectives such as "substantially" and "about" that modify the state or relationship of the characteristic(s) of an embodiment of the present invention shall be used only if the state or characteristic is permissible. It is understood to mean defined within the scope, which is acceptable for operation for the applications for which the operation of the embodiments is intended. Unless otherwise stated, the word "or" in the specification and claims is to be considered an inclusive "or" rather than an exclusive or, and refers to at least one or any of the items it couples. indicates a combination of

In the description and claims of this application, each of the verbs "comprise," "include," and "have," as well as their cognates, refers to the object or plural objects of the verb. is used to indicate that is not necessarily the complete list of components, elements, or parts of the subject or subjects of the verb.

Other terms used herein are meant to be defined by their well-known meanings in the art.

In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are explained fully in the literature. See, eg, "Molecular Cloning: A Laboratory Manual," Sambrook et al. , (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.; M. , ed. (1994); Ausubel et al. , "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al. , "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 659, and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. Am. E. , ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique" by Freshney, Wiley-Liss, N.J. Y. (1994), Third Edition; "Current Protocols in Immunology", Volumes I-III Coligan J.; E. , ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); ion-A Laboratory Course Manual "CSHL Press (1996) See, these are all incorporated by reference. Other general references are provided throughout this document.

As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm±100 nm.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Please note that Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, and reference to "a polypeptide thereof" includes one or more polypeptides and their equivalents known to those of ordinary skill in the art. Contains references to objects, etc. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement serves as an antecedent basis for the use of such exclusive terms, such as "single", "only", etc., in connection with the recitation of claim elements, or for the use of "negative" limitations. intended to be

In those cases where traditional wording similar to "at least one of A, B, and C, etc." (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, A only, B only, C only, A and B together, A and C together, B and C together, and/or systems having A, B, and C together.). Virtually any disjunctive word and/or phrase presenting two or more alternative terms may be used to refer to one of the terms, any of the terms, or It will be further understood by those skilled in the art that it should be understood to contemplate the possibility of including both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

It is understood that specific features of the invention that are for clarity described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention which are for brevity described in the context of a single embodiment can also be provided separately or in any suitable subcombination. All combinations of embodiments related to this invention are specifically encompassed by this invention and are disclosed herein as if each combination were individually and expressly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically encompassed by the present invention, as if any such subcombination were individually and expressly disclosed herein; Disclosed herein.

Additional objects, advantages and novel features of the present invention will become apparent to those skilled in the art upon consideration of the following non-limiting examples. Additionally, each of the various embodiments and aspects of the present invention as delineated above and claimed in the claims section below finds experimental support in the following examples.

Various embodiments and aspects of the present invention, as delineated above and claimed in the claims section below, find experimental support in the following examples.

The following examples serve to illustrate certain embodiments and aspects of the invention and should not be construed as limiting its scope. Those skilled in the art will understand that many variations can be made in the specific embodiments disclosed herein while still achieving the same or similar results.

Materials and Methods Particle Generation (Generation of Virus-Like Particles (VLPs))
Self-assembly of particles in vitro requires the use of purified capsid proteins and nucleic acid origami structures. In the examples below, empty VP1-VLPs were generated as a source of VP1 pentamers for assembly. SV40 VP1 was produced in SF9 cells infected with a baculovirus expression vector (VP1 UniProt entry-P03087).

Empty VLPs formed in SF9 cells were extracted by performing cell lysis and purified on a CsCl gradient. VLPs disassembled in vitro into basic VP1 pentameric capsid units. The pentamers were first lysed against 20 mM Tris-Cl, pH 8.9, 50 mM NaCl 2 mM DTT (dissociation buffer) containing 5 mM EDTA and then against dissociation buffer containing 2 mM EDTA. It was obtained by dissociating the VLPs by dialysis at °C. All dialysis steps were performed in the presence of complete protease inhibitors (Roche). The dialysate was centrifuged at 20,000 g for 30 min at 4° C. to precipitate aggregated proteins.

For particle assembly, nucleic acid origami structures were added to VP1 pentamers that were degraded with 2× assembly buffer (250 mM NaCl, 100 mM MOPS pH 7.2). A capsid spontaneously assembled around the nucleic acid. For the assembly reaction, the nucleic acid templates were: 2873 nt long single-stranded DNA structured into a 25 nm near-spherical origami structure, 5386 nt long ΦX174Virion DNA structured into a 30 nm near-spherical origami structure, two 7560 nt long single-stranded DNA based on the M13mp18 bacteriophage derivative, structured into a 35 nm near-spherical DNA origami structure composed of hemispheres and a 40 nm near-spherical DNA origami structure; is a 7249-nucleotide-long scaffold DNA strand based on M13mp18 bacteriophage derivatives, the second is based on a 7560-nucleotide-long scaffold DNA strand based on M13mp18 bacteriophage (all origami structures are from tilibit, Garching, Germany). commercially available from nanosystems).

Origami Structures As detailed above, origami structures were purchased from tilibit nanosystems, Garching, Germany. Similarly, a fluorescently labeled 30 nm near-sphere DNA origami composed of 5386 nt ΦX174Virion DNA containing 25 Cy5-modified staples was purchased.

Negative stain transmission electron microscopy (TEM)
A 3 μl drop of sample was applied to a glow discharge grid (carbon support film on 300 mesh Cu grid, Ted Pella, Ltd.). After 10-20 seconds, excess liquid was blotted with filter paper. Grids were incubated with 2% uranyl acetate stain for 30 seconds, blotted and dried in air. Samples were investigated by a FEI Tecnai 12G ² TWIN TEM operating at 120 kV and images were recorded by a 4K×4K FEI Eagle CCD camera.

Cryo-EM
Sample preparation For single particle analysis, 3 μl of VLP samples were applied to holey carbon grids (Quantifoil R1.2/1.3, Micro Tools GmbH, Germany) after 30 seconds of glow discharge treatment. Grids were blotted and vitrified by rapid immersion in liquid ethane at −182° C. using a Vitrobot (FEI, Eindhoven).

Data collection for single particle analysis:
Samples were imaged under low dose conditions with a FEI Tecnai F30 Polara microscope (FEI, Eindhoven) operating at 300 kV. Data sets were collected automatically using SerialEM with a K2 Summit direct electron detector camera mounted behind a 2.3 Å calibrated pixel size energy filter (Gatan Quantum GIF). The energy filter was set to remove electrons >±10 eV from the zero-loss peak energy. The K2 Summit camera was operated in counting mode with a dose rate of 10 electrons/pixel/sec on the camera. Each movie was dose split into 50 image frames and the total electron dose was 80e/Å^2.

Single Particle Reconstruction Dose-separated image stacks were aligned using MotionCorr2 and their defocus values estimated by Gctf. Aligned frame sums were used for further processing and the rest of the processing was done in RELION 2.1. 48778 particles were auto-selected and subjected to 2D classification using RELION2.1 with 100 classes. An initial 3D reference was prepared from 3200 empty capsid particles of different selected 2D class averages. 3D classification and refinement of empty capsids from 26,110 particles imposing dodecahedral symmetry (I1) was performed. The resulting empty capsid map was then used as an initial reference model for the 3D classification and refinement of the origami-filled capsid (19786 particles). Reconstruction of the 3D cryo-EM map of the origami-filled capsid was confirmed using a de-novo 3D initial model generated in RELION 3.0.

The correct handedness of the empty capsid was assessed by the quality of the fit to the SV40 crystal structure (PDB ID 1SVA) by measuring the correlation coefficient using the UCSF chimera protocol 'Fit in Map' and the correlation coefficient was 0.952 (as opposed to 0.907 on the contralateral side).

Confocal Microscopy Assay VLPs were isolated at capsid protein pentamer/DNA molar ratios of 200, 400, 750 (fluorescently labeled 30 nm DNA origami structures - 3.75 nM, 1.9 nM, 0.95 nM; VP1 pentamer - 750 nM). 25 μL of VLPs were then mixed with 300 μL of serum-free medium and layered over CV1 cells in 24 multiwell plates for 1 hour. 1 ml of serum-containing medium was then added to the wells. Intracellular uptake of fluorescent DNA was visualized by confocal microscopy after 24 hours.

Example 1: Assembly of SV40 VP1 in DNA origami Origami structural DNA in near-sphere morphology was used for particle preparation. Origami DNA structures of indicated diameters of 25 nm, 30 nm, 35 nm, and 40 nm (shown in the electron micrographs of FIGS. 1A-D) were purchased from tilibit nanosystems, Garching, Germany. Additionally, a 30 nm DNA origami structure with 25 staples modified with Cy5 fluorescent tags was used. All DNA origami structures were assembled into particles (VLPs) and used in the experiments described here.

A capsid unit made up of pentamers of the SV40 capsid protein VP1 was used to encapsulate the origami structure. VLPs were prepared using a pentamer/origami structure ratio of 400:1 (pentamer - 4 μM; origami structure - 10 nm) for negative staining TEM, cryo-EM and agarose gel analysis.

Example 2 Formation of Virus-Like Particles VLP formation was confirmed using negative staining transmission electron microscopy (TEM). VLPs were negatively stained with 2% uranyl acetate and imaged. Assembly of VP1 on 25 nm DNA origami structures leads to the formation of 36-38 nm particles (as shown in Figure 2A). Assembly of VP1 around a 30 nm diameter DNA origami structure leads to the formation of 50 nm particles (as shown in Figure 2B).

Assembly of VP1 around a 35 nm diameter near-spherical DNA origami structure leads to the formation of 50 nm particles (as shown in Figure 2C).

Assembly of VP1 around a near-spherical DNA origami structure with a diameter of 40 nm leads to the formation of 56 nm particles (as shown in Fig. 2D).

Particles obtained by assembly of VP1 around 25 nm and 40 nm DNA origami structures represent entirely new forms of SV40 particles with unnatural capsid sizes, suggesting T=3 or 4 and T=12 geometries, respectively. ing. These results suggest that T, the triangulation number characteristic of the icosahedral capsid shape, can be adjusted to accommodate the size of the origami structure. The results thus demonstrate a lot of flexibility in accommodating different DNA origami sizes, suggesting flexibility in bond angles between adjacent pentamers.

Example 3: Cryo-EM of SV40-like particles assembled on 30 nm quasi-spherical DNA origami structures
Cryo-EM 2D micrographs of eight typical particles formed by VP1 assembly on a 30 nm near-spherical DNA origami were performed. The resulting particles are substantially identical to SV40 in size and shape. The particles are composed of a shell (VP1 pentamer) and a core exhibiting characteristic viral spikes around the circumference. As shown in FIG. 3A, in some of the cores the folded strands of the origami structure are visible (seen as stripes). Demonstrating similarity to the native virus, a 3D model of wild-type SV40 (PDB entry 1SVA) shows perfect alignment when superimposed on synthetic particles (Fig. 3B). 2D classification of cryo-EM micrographs classifies particles into classes of similar particles, with each subclass representing hundreds of particles on average. As shown in FIG. 3C, all subtypes are very similar to each other and characteristic of viruses. 2D class averaging results in a disordered 'cloud' of density in the core of the particle, suggesting that the DNA origami structure is randomly oriented relative to the shape of the capsid. This suggests that the 30 nm DNA origami structure is smaller than the SV40 capsid cavity, leaving some degree of freedom for the origami structure within the capsid (below, the details of the DNA origami structure are clearly observed, the larger Class averages of particles encapsulating 35 nm origami structures, as well as can be seen by comparison with Fig. 7).

Example 4 Delivery of Nucleic Acid Origami into Cells Fluorescent particles prepared with Cy5-tagged 30 nm origami DNA showed significant infection rates when added to CV1 cells. The results are shown in Figures 4A-B, where cells provided with naked origami DNA did not show any fluorescence after 24 hours (Figure 4A). However, as seen 24 hours post-infection, fluorescent particles assembled on Cy5-tagged origami DNA were found intracellularly, indeed one of the destinations of the intracellular trafficking pathway of wild-type SV40. It seems to accumulate in the Golgi, which is Fluorescent particles are marked with black arrows (Fig. 4B).

The results therefore demonstrate effective recognition and intracellular internalization of DNA-encapsulated particles.

Example 5: Cryo-EM and 3D reconstruction of T=7d dodecahedral SV40-like particles assembled on 35 nm quasi-spherical DNA origami structures Cryo-EM of SV40 VP1 assembled on 35 nm spherical DNA origami structures Micrographs show the presence of 50 nm virus-like particles resembling wild-type SV40 in size, globular shape, and characteristic viral pentamer (Fig. 5A, top panel). The mixture of particles was apparently composed of two populations: empty virus-like particles (VLPs) and virus-like particles filled with origami. Representative micrographs of empty and filled particles are shown enlarged (Fig. 5A, inset, middle panel), with DNA clearly distinguished as stripes within the core of the particle on the left, indicating the origami structure. Represents folded strands. A total of 48778 individual particles were automatically selected from 1666 micrographs to reconstruct the 3D model of the particles (RELION 2.1). The particles were subjected to 2D classification, confirming the presence of both empty and DNA-filled capsid class averages. (Fig. 5A, bottom panel).

A reconstruction of the 3D model of the empty particle was calculated from 26110 particles producing T=7d icosahedral capsids at a resolution of 7.3 Å (Fig. 5B, icosahedral triangulation Caspar and See below for Klug's calculation method).

Due to the asymmetry of the DNA origami, the 3D reconstruction of the origami-filled particles was performed without imposing icosahedral symmetry. The 3D reconstruction of 19786 particles containing an asymmetric DNA origami was calculated using the empty capsid structure as the initial model and is shown at ~25 A resolution in Figure 5C. The lower resolution of the structure compared to that of the empty particles is probably due to the lack of imposed symmetry. Despite the lack of symmetry averaging, the protein shell of the reconstructed origami-filled particle is clearly icosahedral (Fig. 5C upper panel, icosahedral triangulation number See below for calculations). Inside the shell, the parallel helices of the DNA origami lattice are clearly visible (Fig. 5C, middle panel). The reconstruction of the 3D model of the origami-filled capsid was confirmed using a de-novo 3D initial model.

As shown, the particles were positively twelve at T=7d, according to Caspar and Klug's theory of viral tiling (Physical principles in the construction of regular viruses. Cold Spring Harb Symp Quant Biol. 1962; 27:1-24). Clearly shows the hedron symmetry. First, the dodecahedral capsid has 5-fold, 3-fold, and 2-fold axes of rotation (see Figure 5D). To demonstrate the icosahedral symmetry, the icosahedron is formed in the cryo-EM reconstructed origami-filled capsid by aligning its vertices with the center of the pentavalent pentamer in the shell. Manually attached to the surface. The 5-fold, 3-fold, and 2-fold axes of symmetry are marked with pentagons, triangles, and rhombuses, respectively.

The SV40 T=7d icosahedral capsid then contains 12 pentavalent pentamers (VP1 pentamer surrounded by 5 other pentamers) and 60 hexavalent pentamers. It is composed of a body (VP1 pentamer surrounded by 6 other pentamers). As shown in FIG. 5E, pentavalent pentamers are indicated by pentagons. Black arrows indicate Caspar and Klug calculation of the icosahedron triangulation number (T). T is given by the formula T=h**2+hk+k**2, where for every pair of integers h and k are relative to the nearest located pentavalent pentamer on the icosahedral lattice. Define the position of the pentavalent pentamer. For T = 7d, h = 2, and k = 1, h is represented by arrows 1 and 2 and K is represented by arrow 1″. The localization of the mer clearly corresponds to T=7d (see FIG. 5E for explanation).

Overall, therefore, the results clearly show that the origami structure is encapsulated within the protein shell of the particle, which forms a regular SV40 lattice of T=7d icosahedral symmetry.

Example 6: Internal view of cryo-EM 3D reconstruction of SV40-like particles assembled on a 35 nm DNA origami, showing that the DNA origami is encapsulated as a whole.
A schematic diagram of the 35 nm DNA origami structure is shown at different viewing angles in FIG. 6A. A corresponding view of the 3D electron density map of the 3D reconstruction of the capsid filled with DNA origami is shown alongside different views of the schematic design of the DNA origami (Fig. 6B). The honeycomb lattice organization of the DNA origami is clearly visible (Fig. 6B top right), the 45 degree inward tilt of the honeycomb lattice (Fig. 6B top left), and perpendicular to the longitudinal axis connecting the two honeycomb planes (Fig. 6B bottom right). ) and horizontal (FIG. 6B bottom left). Each rotated view of the reconstructed particle accurately depicts the properties predicted by each view of the schematic design of the DNA origami.

Overall, therefore, all the structural features of the origami structure, the parallel helices arranged in a 'honeycomb' lattice, are clearly visible within the capsid in all possible projections, suggesting that the DNA origami resides within the protein shell of the particle. Indicates complete encapsulation.

Furthermore, the results shown in FIGS. 7A-C demonstrate that the 35 nm DNA origami structures are not randomly positioned relative to the shape of the capsid, as evidenced by the cross-sectional view shown, where the capsid was removed from the front. and because the capsid lattice is rotated, either the 3-fold (Fig. 7B), 2-fold (Fig. 7A), or 5-fold (Fig. 7C) symmetry axis of the regular icosahedron is drawn. In each case, the viewer appears to face different facets of the DNA origami. For example, the honeycomb facets can be displayed along an axis that is slightly off (6.8°) from the icosahedron 3-fold axis (Figs. 7A and 7B).

Example 7 Detection of Capsid Protein-Nucleic Acid Origami Structural Complexes Electrophoretic mobility shift assays (EMSA) provide evidence of protein-nucleic acid complex formation and observe pentamer-DNA origami interactions. use for

In the EMSA experiments, 35 nm DNA origami structures were mixed with VP1 pentamers at a molar ratio of 400 pentamers per DNA origami structure. Assembly products were analyzed on an agarose gel to confirm migration. The results are shown in FIG. Free DNA origami migrated with a dsDNA size marker of 4 kb. Upon incubation with VP1 protein, the free DNA origami band disappeared and was replaced by a slow-migrating, distinct band. The appearance of a new distinct band of higher molecular weight suggests the formation of a stable and distinct VP1/DNA origami complex.

Example 8 Unfolding of DNA Origami Under Intracellular Conditions DNA origami folding is reversible under intracellular conditions, as demonstrated by the increased accessibility to enzymatic action of DNA polymerases (FIG. 9). ). At 15° C. and 20 mM Mg+, the nucleic acid origami structure is stable and inaccessible to the enzymatic action of DNA polymerase (lanes 1, 2 and 4). The structure is unstable under intracellular conditions at 37° C. and 1 mM Mg+. Being an unstable structure, it is amenable to amplification by polymerases (lanes 3 and 5).

Example 9 Use of VP2 and VP3 in VLP Formation VP2 and VP3 are secondary capsid proteins that may enhance transnucleotide efficiency when combined with VP1 when forming VLPs. To generate sufficient quantities of VP2/3 for large-scale VLP preparations, they are expressed in SF9 cells together with VP1. The VP1+VP2 and VP1+VP3 pentamers are extracted and purified in a manner similar to the preparation of the VP1 pentamer. The VP1 + VP2 pentamers and VP1 + VP3 pentamers are mixed in specific ratios determined by standard testing to generate a DNA origami containing VLPs. The VLPs are then used to infect mammalian cells to observe increased efficiency of nucleotide transfer.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alterations, modifications and variations that fall within the spirit and broad scope of the appended claims.