Enhanced DNA dendrimers and methods of use thereof

Abstract

Described herein are compositions comprising a DNA dendrimer and a cargo polynucleotide linked to a targeting moiety, in some embodiments, the cargo polynucleotide is directly linked to the DNA dendrimer. In some embodiments, the cargo polynucleotide is linked to the DNA dendrimer by an adaptor molecule. In some embodiments, the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding at least one molecule of interest, and has a topology selected from the group consisting of a complete circular polynucleotide, a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, and a linear polynucleotide with one open end and one closed end. In some embodiments, the cargo polynucleotide herein optionally comprises one or more of a DNA target sequence (DTS), a nuclear localization signal sequence (NLS), or a DNA dendrimer binding sequence.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 308,776, filed February 10, 2022, which is incorporated by reference in its entirety.

[0002] Embodiments of the present invention relate to DNA dendrimers and other molecules linked to or encoding targeting molecules, cargo molecules, adapter molecules, accessory molecules, or combinations thereof, and methods of their use and methods of making them. [Background technology]

[0003] In many research, diagnostic, and therapeutic applications, the target of action of a molecule, e.g., a reporter probe, a small molecule drug, a peptide, or a nucleic acid, is intracellular. For nucleic acids that require transcription and / or translation in a target cell, the nucleic acid must be safely transported to the cell nucleus. However, targeting a molecule to a selected cell type, achieving intracellular delivery to the cytosol, and then transporting to the desired intracellular compartment, e.g., the nucleus, is a challenging goal. Furthermore, therapeutic nucleic acid cargo must be safely protected from degradation and expressed in the nucleus in amounts that are neither too limited nor too abundant. Thus, there is a need for compositions that can reliably deliver molecules, such as therapeutic nucleic acids, to the nucleus of a target cell without degradation and have the ability to optimize expression. The present disclosure addresses this need and others. Summary of the Invention

[0004] Described herein is a composition comprising a DNA dendrimer linked to a targeting moiety and a cargo polynucleotide. In some embodiments, the cargo polynucleotide is directly linked to the DNA dendrimer. In some embodiments, the cargo polynucleotide is linked to the DNA dendrimer by an adaptor molecule. In some embodiments, the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding at least one molecule of interest, and has a topology selected from the group consisting of a complete circular polynucleotide, a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, and a linear polynucleotide with one open end and one closed end. In some embodiments, the cargo polynucleotide herein optionally comprises one or more of a DNA target sequence (DTS), a nuclear localization signal sequence (NLS), or both.

[0005] In some embodiments, the cargo polynucleotide comprises a DNA dendrimer binding sequence (DBS). In some embodiments, the DBS links the cargo polynucleotide to the DNA dendrimer. In some embodiments, the adapter molecule comprises a DBS and a cargo binding region. In some embodiments, the DBS links the adapter molecule to the DNA dendrimer. In some embodiments, the cargo binding region links the adapter molecule to the cargo polynucleotide. In some embodiments, the adapter molecule further comprises one or more of a purification region, a DTS, an NLS, a spacer, a cell penetrating peptide (CPP) sequence, a cleavage site, a flexible linker, or some combination thereof.

[0006] In some embodiments, any composition described herein may include an auxiliary molecule. In some embodiments, the auxiliary molecule can be associated with the DNA dendrimer. In some embodiments, the auxiliary molecule is linked to the DNA dendrimer. In some embodiments, the auxiliary molecule compacts the size of the cargo polynucleotide, the DNA dendrimer, or both.

[0007] Also described herein is a composition comprising a cargo polynucleotide, an auxiliary molecule, and a DNA dendrimer linked to a targeting moiety, wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding at least one molecule of interest, and has a topology selected from the group consisting of fully cyclic nucleotides, nicked cyclic nucleotides, linear nucleotides with closed 5' and 3' ends, linear nucleotides with open 5' and 3' ends, and linear nucleotides with one open end and one closed end. In some embodiments, the cargo polynucleotide is of fully cyclic topology and is not linked to a DNA dendrimer.

[0008] Also described herein is a plasmid comprising a plasmid backbone comprising at least two restriction sites, at least one promoter, and at least one coding sequence encoding at least one molecule of interest, which can form cargo polynucleotides with various topologies, in some embodiments, the various topologies are selected from the group consisting of fully cyclic nucleotides, nicked cyclic nucleotides, linear nucleotides with closed 5' and 3' ends, linear nucleotides with open 5' and 3' ends, and linear nucleotides with one open end and one closed end.

[0009] In some embodiments, a method of delivering a molecule of interest to the nucleus of a target cell is provided, the method comprising contacting the target cell with any of the compositions described herein, a targeting moiety binds to the target cell, allowing the composition to enter the target cell, and a cargo polynucleotide can enter the nucleus of the target cell. In some embodiments, a method of treating a disease is provided, the method comprising administering any of the compositions described herein to a subject to treat the disease, a targeting moiety binds to the target cell, allowing the composition to enter the target cell, and a cargo polynucleotide can enter the nucleus of the target cell. In some embodiments, a method of producing a cargo polynucleotide is provided, the method comprising adding at least one promoter and at least one coding sequence encoding at least one molecule of interest to a plasmid backbone to form any of the plasmids described herein, and then optionally contacting the plasmid with one or more restriction enzymes. [Brief description of the drawings]

[0010] [Figure 1] 1 shows a plasmid map and a diagram showing the individual topologies that a plasmid can have by contacting or not contacting the plasmid with certain restriction enzymes. [Diagram 2] The percentage (%) of GFP fluorescence for various polynucleotide topologies is shown. [Diagram 3] 1 shows a graph depicting the number of GFP positive cells per unit area for various polynucleotide topologies. [Figure 4] 1 shows the relative mean fluorescence of polynucleotides containing a DNA targeting sequence (DTS) compared to polynucleotides without a DTS in CHO-K1 cells. [Diagram 5] 1 shows the relative mean fluorescence of polynucleotides containing a DNA targeting sequence (DTS) compared to polynucleotides without a DTS in A427 cells. [Figure 6]1 shows the relative mean fluorescence of polynucleotides containing a DNA targeting sequence (DTS) compared to polynucleotides without a DTS in C2C12 cells. [Figure 7] 1 shows the relative integrated fluorescence of a polynucleotide containing a DNA targeting sequence (DTS) compared to a polynucleotide without a DTS in C2C12 cells. [Figure 8] 1 shows the relative peak fluorescence of a polynucleotide containing a DNA targeting sequence (DTS) compared to a polynucleotide without a DTS in C2C12 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although similar or equivalent methods and materials described herein can be used in the practice or testing, the preferred methods and materials are described herein. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the embodiments described herein will be apparent from the detailed description and claims of the present specification.

[0012] Recombinant engineering techniques and procedures, including nucleic acid and peptide synthesis, are generally performed according to conventional methods in the art and various general references provided throughout the specification (e.g., Sambrook et al, 2001, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., eds, 2005, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY; and Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC).

[0013] The terms "about" or "approximately" refer to a numerical value plus or minus 10% of the numerical value with which the term is used. Thus, about 50% means within the range of 45% to 55%.

[0014] Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural references.

[0015] As used herein, the terms "comprise," "have," "include," and their cognates mean "including but not limited to." Although various compositions, methods, and devices are described in terms of "comprising" (which should be interpreted to mean "including but not limited to") various components or steps, the compositions, methods, and devices may "consist essentially of" or "consist of" the various components or steps.

[0016] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or can be an immunoreactive portion of an intact immunoglobulin. Antibodies useful in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, and F(ab)2, as well as single chain antibodies (scFv), camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0017] As used herein, "complementary" refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., between two DNA molecules. If a nucleotide position in both molecules is occupied by a nucleotide that can normally base pair with each other, the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are substantially complementary to each other if at least about 50%, preferably at least 60%, and more preferably at least about 80% of the corresponding positions in each of the molecules are occupied by nucleotides that normally base pair with each other (e.g., A:T and G:C nucleotide pairs).

[0018] As used herein, "DNA-based carrier" refers to a delivery system that comprises deoxyribonucleic acid molecules.A non-limiting example of DNA-based carrier is DNA dendrimer.Other DNA-based carriers include double-stranded DNA, single-stranded DNA, and single-stranded hairpin DNA, or multimers thereof.

[0019] As used herein, "DNA dendrimer" or "dendrimer" refers to a matrix of polynucleotides exhibiting branches formed by the sequential or generational addition of branching layers to or from a core molecule, such as an initial monomer.

[0020] As used herein, an "initiator monomer" is a polynucleotide compound that serves to nucleate the formation of a dendrimer.

[0021] As used herein, "extension monomers" are polynucleotide compounds that can be attached to initiator monomers and / or to each other during construction of a dendrimer. The extension monomers form the layers of the dendrimer. The first layer of the dendrimer is the layer of extension monomers closest to the initiator monomer. The outer layer is the layer furthest from the initiator monomer and forms the surface of the dendrimer. Extension monomers are also referred to in the art as matrix monomers, matrix extension monomers, and matrix polynucleotide monomers.

[0022] As used herein, the terms "substitute," "substituted," "mutate," or "mutated" refer to changing, deleting, or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to create a variant of that sequence.

[0023] The term "polynucleotide" or "nucleic acid molecule" refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double- and single-stranded DNA and RNA are non-limiting examples of polynucleotides.

[0024] The term "polypeptide" or "protein" refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. In some embodiments, the term "peptide" may also be used.

[0025] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, including a substitution, insertion, or deletion.

[0026] Conventional notation for portraying polynucleotide sequences is used herein: the left-hand end of a single-stranded polynucleotide sequence is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction.

[0027] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence that controls or promotes the expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, while in other instances, this sequence may also include enhancer sequences and other regulatory elements that control or promote the expression of the gene product. The promoter / regulatory sequence may, for example, be one that causes the gene product to be expressed in an inducible manner. An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced only in the substantial presence of an inducer that corresponds to (e.g., binds to) the promoter.

[0028] As used herein, a "targeting moiety" refers to a molecule that binds to a molecule present on the cell surface of a target cell.

[0029] As used herein, "targeted DNA-based carrier" and "DNA-based carrier containing a targeting moiety" refer to a composition containing a DNA-based carrier and a targeting moiety. The targeting moiety can be linked to the DNA-based carrier directly or via a linker. Alternatively, the targeting moiety can be linked to another molecule, such as a cargo molecule or a second non-nucleic acid carrier in the composition. The DNA-based carrier is targeted by being present in the same composition with the targeted cargo or the targeted secondary carrier.

[0030] As used herein, the term "vector" refers to a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. It will be understood that a vector can be a naked nucleic acid or a nucleic acid complexed with a protein or lipid. Vectors include, but are not limited to, replicons (e.g., RNA replicons, bacteriophages) to which a fragment of DNA can be attached and replicated. Thus, vectors include, but are not limited to, RNA, autonomously self-replicating circular or linear DNA or RNA (i.e., "plasmids"), including both expression and non-expression plasmids. When a recombinant microorganism or cell culture is described as harboring an "expression vector," the expression vector includes both extrachromosomal circular and linear DNA as well as DNA integrated into the host chromosome(s). When a vector is maintained by a host cell, the vector may be stably replicated by the cell during mitosis as an autonomous structure or is integrated into the host's genome.

[0031] As used herein, a "promoter" refers to a synthetic or naturally derived molecule that can confer, activate, or enhance expression of a nucleic acid in a cell. A promoter can include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also include distal enhancer or repressor elements that can be located as far away as several thousand base pairs from the start site of transcription. Such promoter-enhancers can modify, for example, but not limited to, tissue specificity or transduction efficiency. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can regulate expression of genetic components constitutively, or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli, such as physiological stress, pathogens, metal ions, or inducers.

[0032] As used herein, the term "topology" refers to various structural arrangements of any vector, plasmid, or polynucleotide disclosed herein. For example, a polynucleotide may have a circular topology, such as a plasmid, in which the polynucleotide has no 5' or 3' ends. A polynucleotide disclosed herein may have a linear topology, in which the polynucleotide has 5' and 3' ends. A polynucleotide with a linear topology may have one or both ends of the polynucleotide covalently closed. In some embodiments, certain restriction enzymes, such as telomerase N (TelN), can covalently close DNA ends.

[0033] composition A. DNA-Based Carriers In some embodiments, the compositions disclosed herein include DNA-based carriers. DNA-based carriers include, but are not limited to, DNA dendrimers, double-stranded linear DNA, single-stranded linear DNA, and single-stranded hairpin DNA, formulated either as monomolecular structures or as conformations of several units crosslinked together (e.g., multimers). In some embodiments, the compositions include DNA dendrimers. DNA dendrimers are spherical particles (usually about 130 to about 150 nm in diameter, but can be designed to be other sizes) of mobile branches formed by DNA monomers hybridized to each other. Each DNA monomer is composed of two polynucleotide strands that share a central region of complementary sequences where the two strands hybridize to each other, leaving four single-stranded polynucleotide end portions. These end sequences are complementary between themselves, and therefore they can hybridize (within a layer) to the end sequences of other DNA monomers. Optionally, the DNA dendrimer includes DNA strands crosslinked by covalent bonds.

[0034] DNA dendrimers are commercially available. In addition, the structural design and construction of DNA dendrimers is generally known in the art. See, for example, U.S. Patent Nos. 5,175,270 and 6,274,723, each of which is incorporated herein by reference in its entirety. The initiating monomer constitutes approximately the center of the dendrimer, depending on the type of branching of the dendrimer. The three-dimensional assembly of the extension monomers around the initiating monomer constitutes the interior volume of the dendrimer. The outermost layer of the extension monomers forms the surface of the dendrimer. Thus, the assembly of the dendrimer results in a three-dimensional shape, usually (but not limited to) an approximately spherical shape comprising layers of extension monomers. The outer layer contains multiple binding sites.

[0035] More specifically, DNA dendrimers can be prepared by a protocol having the following characteristics: (i) The starting material is a double-stranded duplex of DNA with 5' and 3' single-stranded overhangs or "binding arms" attached to the duplex trunk, referred to as "initiation monomers", a name that reflects their role in the construction of the dendrimer (e.g., four binding arms in total). The 5' and 3' binding arms of each initiation monomer are annealed to complementary binding arms of "extension monomers" of similar composition and morphology. (ii) A subset of the four binding arms of each extension monomer are complementary to the binding arms of the initiation monomer. The non-complementary binding arms of the extension monomer are inactive for annealing to the initiation monomer. Typically, four extension monomers can be annealed to the initiation monomer to give rise to a monolayer or monolayer dendrimer in solution. (iii) To add further layers of extension monomers to a dendrimer, typically similar but distinct extension monomers are added, each having a subset of four binding arms complementary to the binding arms on the dendrimer. This allows one-layer dendrimers to be converted into two-layer dendrimers, step-by-step, and so on until a dendrimer of the desired size is reached. Typically, three- or four-layer dendrimers are used.

[0036] After assembly, DNA dendrimers can be crosslinked to maintain and stabilize the structure of the dendrimer. Crosslinking of hybridized regions between monomers (i.e., intermonomer crosslinks), or between monomers and nucleic acids carrying detectable labels, and between stems (intramonomer crosslinks) can stabilize the structure of polynucleotide dendrimers. Similarly, any hybridized region of any DNA-based carrier can be crosslinked to stabilize the carrier. Individual units of the carrier can be crosslinked to multimolecular carriers. Such crosslinking chemistries are well known in the art. See, for example, Cimino et al., Annu. Rev. Biochem. 54:1151-1193 (1985), Shi et al., Biochemistry 25:5895-5902 (1986), and Cimino et al., Biochemistry 25:3013-3020 (1986). See also U.S. Pat. No. 4,196,281. Non-limiting examples of suitable cross-linking agents include psoralens (including, but not limited to, 8-methoxypsoralen and angelicin), mitomycin C, daunomycin, ethidium diazide, cisplatin, transplatin, carboplatin, 8-methoxypsoralen, mechlorethamine, oxaliplatin, and carbodiimide compounds, among others.

[0037] The polynucleotide chains used as monomers of dendrimers or other DNA-based carriers can be produced using standard techniques of nucleic acid synthesis. These techniques can be biological or chemical. The techniques and procedures are usually carried out according to conventional methods in the art and various general references (e.g., Sambrook et al., 2001, supra; Ausubel et al., eds., 2005, supra; and Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC).

[0038] In some embodiments, polynucleotides are chemically synthesized using methods known in the art. See, for example, Gait, 1985, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, England). In another embodiment, polynucleotides are enzymatically synthesized using polymerase chain reaction (PCR). One PCR method suitable for generating single-stranded polynucleotides is multi-cycle PCR using a single primer, which amplifies the single strand. As is well known in the art, nucleic acids can be purified by any suitable means before their use. For example, nucleic acids can be purified by reverse phase or ion exchange HPLC, size exclusion chromatography, or gel electrophoresis. Those skilled in the art will understand that the purification method depends in part on the size of the nucleic acid to be purified.

[0039] The outer layer of the DNA dendrimer may have at least two types of binding arms. These binding arms may be used to bind one or more types of moieties. In some embodiments, the DNA dendrimer of the composition may be bound or attached to a targeting moiety, cargo, auxiliary molecule, etc.

[0040] In some embodiments, the DNA-based carrier is associated with a secondary carrier that can function as a scaffold for the DNA-based carrier. Examples of such secondary carriers include, but are not limited to, liposomes, non-DNA dendrimers, polymeric carriers, microbubbles, paramagnetic and ferromagnetic particles, self-assembled polymers, polymersomes, filomicelles, albumin particles, lipoproteins, and the like. Self-assembled polymers are polymers formed by the self-assembly of unimolecular building blocks. These building blocks can be amphiphilic copolymers that contain hydrophilic components (e.g., but not limited to, polyethyleneimine or polyethylene glycol) and hydrophobic components (e.g., but not limited to, aliphatic polyesters) in a core-shell structure. These structures that are not maintained by direct conjugation or crosslinking of building blocks are known as micelles, and their morphology can vary, for example, from spherical micelles ("polymersomes") to elongated or thread-like rods ("filomicelles"). The size of these structures can also vary from nanometer to micrometer size ranges. Alternatively, these structures may be further chemically cross-linked to increase their stability once formed by self-assembly. Hydrophobic cargo may be embedded in the hydrophobic regions of the carrier, while hydrophilic cargo may be incorporated into the internal aqueous core or the external hydrophilic core.

[0041] In some embodiments, DNA-based carrier is linked to the surface of secondary carrier.In some embodiments, DNA-based carrier is not linked to secondary carrier, but is associated with secondary carrier by being present in the same composition.In some embodiments, cargo and targeting moiety can be linked to DNA-based carrier or scaffold carrier using the methods described herein and known in the art.

[0042] B. Targeting moiety In some embodiments, the DNA-based carrier is directed to a specific cell by linking a targeting moiety to the carrier, secondary carrier, and / or cargo. In some embodiments, the targeting moiety is linked to the DNA-based carrier. In some embodiments, the targeting moiety is linked to a secondary carrier. The targeting moiety can be an antibody, a naturally occurring ligand of a receptor or its functional derivative, a vitamin, a hormone, a small molecule mimic of a naturally occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an FN3 domain, an aptamer, a nucleic acid, a toxin, a component of a microorganism, or any other molecule that specifically binds to a cell surface molecule and induces endocytosis of the bound moiety.

[0043] Without being bound to any particular theory, the targeting moiety can specifically bind to a molecule on the cell surface of the target cell. The targeting moiety can bind to a cell surface molecule, and in some embodiments, the cell surface molecule can induce the endocytosis of the DNA-based carrier. Non-limiting examples of cell surface molecules that can be targeted include cell surface proteins, carbohydrates, and lipids. Cell surface molecules that can be targeted include molecules associated with classical endocytosis and molecules associated with non-classical endocytosis.

[0044] In some embodiments, the target cell surface molecule is a cell adhesion molecule (CAM). Cell adhesion molecules useful in the present invention include, but are not limited to, neurospecific adhesion molecules (e.g., NCAM) and systemic intercellular adhesion molecules. Systemic CAMs include intercellular adhesion molecules (e.g., ICAM-1, ICAM-2, ICAM-3), platelet / endothelial cell adhesion molecules (PECAM), activated leukocyte cell adhesion molecules (ALCAM), B-lymphocyte cell adhesion molecules (BL-CAM), vascular cell adhesion molecules (VCAM), mucosal vascular addressin cell adhesion molecules (MAdCAM), CD44, LFA-2 (CD2), LFA-3 (CD58), basigin (CD147), and the like. In some embodiments, the cell surface molecule is CD71.

[0045] In some embodiments, the targeting moiety is an antibody that specifically binds to a target cell surface molecule. In some embodiments, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a heavy chain antibody, and a biologically active fragment of an antibody, wherein the biologically active fragment is a Fab fragment, a F(ab')2 fragment, or a scFv fragment.

[0046] When the antibody used as a targeting moiety in the compositions and methods of the present invention is a polyclonal antibody (IgG), the antibody is generated by inoculating a suitable animal with the cell surface molecule to be targeted. The antibody produced in the inoculated animal that specifically binds to the cell surface molecule is then isolated from the body fluid obtained from the animal. The antibody can be generated in this manner in several non-human mammals, including but not limited to goats, sheep, horses, camels, rabbits, and donkeys. Methods for generating polyclonal antibodies are well known in the art and are described, for example, in Harlow, et al. (1988, Antibodies, A Laboratory Manual, Cold Spring Harbor, NY).

[0047] Monoclonal antibodies directed against the targeted full-length cell surface molecule or fragments thereof can be prepared using any of the well-known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and Tuszynski et al. (1988, Blood, 72:109-115). Human monoclonal antibodies can be prepared by the methods described in U.S. Patent Application Publication No. 2003 / 0224490. Monoclonal antibodies directed against an antigen are generated from mice immunized with the antigen using standard procedures cited herein. Nucleic acids encoding the monoclonal antibodies obtained using the procedures described herein can be cloned and sequenced using techniques available in the art, for example, as described in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and references cited therein.

[0048] To prepare a targeted DNA-based carrier, such as a DNA dendrimer, a targeting moiety can be linked to a DNA-based carrier, or to a secondary carrier or cargo that is linked to or associated with a composition that includes a DNA-based carrier. A single targeting moiety can be linked to a DNA-based carrier or secondary carrier. Alternatively, multiple (e.g., two or more) targeting moieties are linked to a DNA-based carrier, secondary carrier, or cargo. When multiple targeting moieties are linked to a carrier or cargo, the moieties can target the same cell surface molecule or can target different cell surface molecules. When targeting different cell surface molecules, these molecules can be associated with the same endocytosis pathway or different endocytosis pathways. The targeting moiety may not be associated with an endocytosis pathway. When targeting different cell surface molecules, the cell surface molecules can be present on the same cell type or on different cell types.

[0049] The linkage can be non-covalent or covalent. The targeting moiety can be linked to one or more of the polynucleotide strands that make up the DNA-based carrier. Alternatively, the targeting moiety is linked to a linker molecule, which is then linked to the DNA-based carrier. In some embodiments where the DNA-based carrier is a DNA dendrimer, the linker molecule is an oligonucleotide that contains a sequence that is substantially complementary to the sequence present in one of the binding arms on the surface of the DNA dendrimer. Thus, the targeting moiety is indirectly and non-covalently linked to the DNA dendrimer by hybridization to the binding arm of the oligonucleotide. This approach is also applicable to other DNA-based carriers. Optionally, the hybridized oligonucleotide is also crosslinked to the DNA dendrimer. Crosslinking chemistry is disclosed elsewhere herein. If not crosslinked, the hybridization between the DNA dendrimer and the oligonucleotide linked to the targeting moiety must last long enough under the conditions of use. Measurement of the binding free energy of nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Freier et al., 1986, Proc. Nat. Acad. Sci. USA 83: 9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109: 3783-3785; Chavali et al., 2005, Bioinformatics 21(20): 3918-3925).

[0050] In some embodiments, the linker is an IgG Fc-specific secondary antibody that is linked to a DNA-based carrier. In some embodiments, the antibody specifically binds to an IgG primary antibody, such as the Fc portion of a targeting moiety or an intervening antibody. The secondary antibody is preferably specific to the species that is the source of the primary antibody. For example, if the targeting moiety is a human IgG antibody, the secondary antibody is an anti-human IgG. In another aspect, the secondary antibody recognizes an epitope of the entire primary antibody, not just the Fc portion. Alternatively, the linker is an Fc gamma receptor that specifically binds to the Fc portion of an IgG antibody. In either case, the carrier can be easily linked to any targeting moiety that is an IgG antibody.

[0051] Non-covalent linkages include, but are not limited to, affinity binding pairs, such as biotin-streptavidin, and immunoaffinity, which have high enough affinity to maintain binding during use, and are well known in the art.The art is also replete with conjugation chemistries that are useful for covalently linking target moieties to DNA-based carriers, secondary carriers, or cargos, either directly or via linkers.Art-recognized covalent coupling techniques are disclosed, for example, in U.S. Patent Nos. 5,416,016, 6,335,435, 6,528,631, 6,861,514, and 6,919,439, which are incorporated herein by reference in their entirety.Other conjugation chemistries are disclosed in U.S. Patent Application Publication No. 20040249178, which is incorporated herein by reference in its entirety. Additional conjugation chemistries include p-hydroxybenzoic acid linkers (Chang-Po et al., 2002, Bioconjugate Chem. 13(3):525-529); native ligation (Stetsenko et al., 2000, J Org. Chem. 65:4900-4908); disulfide bridge conjugates (Oehlke et al., 2002, Eur J. Biochem. 269:4025-4032 and Rogers et al., 2004, Nuc Acids Res. 32(22) 6595-6604); maleimide linkers (Zhu et al., 1993, Antisense Res Dev. 3:265-275); thioester linkers (Ede et al., 1994, Bioconjugate Chem. 13(3):525-529); native ligation (Stetsenko et al., 2000, J Org. Chem. 65:4900-4908); disulfide bridge conjugates (Oehlke et al., 2002, Eur J. Biochem. 269:4025-4032 and Rogers et al., 2004, Nuc Acids Res. 32(22) 6595-6604); Chem. 5:373-378); Diels-Alder cycloaddition (Marchan et al., 2006. Nuc Acids Res. 34(3):e24, 2006 Feb. 14 Epub); U.S. Patent No. 6,656,730.For reviews of peptide-oligonucleotide conjugation chemistry, see also Tung et al., 2000, Bioconjugate Chem. 11:605-618; Zatsepin et al., 2005, Curr Pharm Des. 11(28):3639-3654; and Juliano, 2005, Curr Opin Mol. Ther. 7(2):132-136.

[0052] C. Cargo The compositions provided herein may also include a cargo. In some embodiments, the cargo is directly or indirectly bound to the DNA dendrimer. The cargo bound to the DNA dendrimer is associated with the DNA dendrimer via a linker or other type of molecule (adapter), etc. One, two, or more different cargoes may be delivered by the targeted DNA-based carrier. Cargoes that may be delivered by the compositions disclosed herein include, but are not limited to, various drugs, such as, but not limited to, therapeutic agents, imaging agents, monitoring agents, chemotherapeutic agents, anti-carcinogenic agents, anti-angiogenic agents, tumor suppressors, antibacterial agents, enzyme supplements, gene expression regulators, and expression constructs that include nucleic acids encoding therapeutic proteins or nucleic acids. In some embodiments, the cargo may be an exogenous or endogenous substance. Cargoes include any molecule that induces an effect in a cell, including any protein, nucleic acid, small molecule, carbohydrate, or lipid. Cargoes may be peptides, proteins (including enzymes, antibodies, and peptide hormones), cytoskeletal ligands, nucleic acids, small molecules, non-peptide hormones, etc. The nucleic acid and cargo polynucleotides that can be delivered by the method of the present invention include synthetic and natural nucleic acid substances, including DNA, RNA, transposon DNA, antisense nucleic acid, dsRNA, siRNA, transcription RNA, messenger RNA, ribosomal RNA, small nucleolar RNA, microRNA, ribozyme, plasmid, and expression construct. When the cargo is or comprises a nucleic acid, the nucleic acid can be a separate entity from the DNA-based carrier. In these embodiments, the DNA-based carrier is not a cargo itself.

[0053] In some embodiments, the cargo is a polynucleotide that comprises at least one promoter and at least one coding sequence that encodes at least one molecule of interest. For example, the cargo polynucleotide may comprise one promoter and two or more coding sequences that encode different molecules of interest, or one promoter for each coding sequence present. In some embodiments, the promoter or promoters are tissue-specific promoters that transcribe or translate at least one molecule of interest only when the composition that comprises the cargo polynucleotide is present in a target cell that is compatible with the tissue-specific promoter.

[0054] In some embodiments, the molecule of interest is a gene product, such as an enzyme, a protein, a receptor, etc. In some embodiments, the cargo polynucleotide encodes two or more molecules of interest. In some embodiments, at least one of the molecules of interest is a protein that is tagged with a detectable molecule, such as, but not limited to, a protein that can be detected by bioluminescence, fluorescence, radioactive signals, or some combination thereof. In some embodiments, such molecules of interest are useful for biodistribution studies and other quantitative and / or real-time assessments.

[0055] In some embodiments, the cargo polynucleotide may have one or more structures or topologies. For example, the cargo polynucleotide may exist as a fully circular double-stranded DNA or RNA with no free 5' or 3' ends. In some embodiments, the cargo polynucleotide is a plasmid. In some embodiments, the cargo polynucleotide may be a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, and a linear polynucleotide with one open end and one closed end. In some embodiments, the nicked circular polynucleotide is a fully circular double-stranded DNA or RNA with at least one break in one strand of the DNA or RNA. In some embodiments, the nicks may result from the action of a nicking restriction enzyme. In some embodiments, the linear polynucleotide has defined 5' and 3' ends, with either end or both optionally closed by covalent bonds.

[0056] Different topologies or structures can be used for different attachment to the DNA dendrimer.

[0057] Provided herein is a plasmid comprising a plasmid backbone comprising at least two restriction sites, at least one promoter, and at least one coding sequence encoding at least one molecule of interest, which can form cargo polynucleotides with various topologies, such as those described herein. For example, a gene cassette comprising a promoter sequence and a coding sequence encoding at least one molecule of interest can be inserted into the plasmid backbone. The resulting plasmid can have a completely circular topology, and if such a topology is desired, no further steps are required. However, if a different topology is desired, the plasmid can be contacted with one or more restriction enzymes to cleave one or both nucleotide strands of the plasmid and generate a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open end and one closed end. In some embodiments, the closed ends of the linear polynucleotide are formed using telomerase N (TelN).

[0058] In some embodiments, the cargo polynucleotide is transported to the nucleus of the cell. In some embodiments, the cargo polynucleotide may also include one or more of a DNA targeting sequence (DTS) or a nuclear localization signal (NLS). DTS and NLS are short amino acid sequences that target proteins and associated nucleic acids for import into the nucleus, respectively. For example, an NLS that may be encoded by a cargo polynucleotide has the amino acid sequence YPDEVKRKKKP (SEQ ID NO: 1) or SLLESPFDKPDEVKRKKKPPTSHQSDATAEDDSSSKKK (SEQ ID NO: 2). These are non-limiting examples of NLS sequences, and any NLS may be used. In some embodiments, at least one DTS, at least one NLS, or both may be present anywhere within or relative to the cargo polynucleotide. For example, the DTS, NLS, or both may be present upstream or downstream of at least one promoter of the cargo polynucleotide. In such molecules, the polypeptide is linked to the cargo polynucleotide.

[0059] In some embodiments, one or more DTS or NLS sequences may be attached or linked to a cargo polynucleotide rather than being present within the cargo polynucleotide. In some embodiments, one or more DTS or NLS sequences may be included in any adapter molecule described herein, where the adapter molecule is linked to a cargo polynucleotide. In some embodiments, one or more DTS or NLS sequences may be directly linked to a cargo polynucleotide without associating with a corresponding adapter molecule. In some embodiments, a DTS or NLS sequence may be linked to a cargo polynucleotide via a modified nucleotide. For example, a DTS or NLS sequence may be attached at a nick site to a nicked circular cargo polynucleotide, optionally with a modified nucleotide first inserted at the nick site.

[0060] In some embodiments, the cargo, including the cargo polynucleotides described herein, may be directly linked to the DNA dendrimer by any of the covalent or non-covalent interactions or bonds described herein, such as hydrogen bonds. Thus, in some embodiments, the cargo polynucleotide comprises a DNA dendrimer binding sequence (DBS) that links the cargo polynucleotide to the DNA dendrimer. In some embodiments, the DBS comprises a nucleic acid sequence that is complementary to the nucleic acid sequence of the DNA dendrimer. For example, the DBS comprises a nucleic acid sequence that is complementary to at least one binding arm of the DNA dendrimer. In some embodiments, the DBS binds to the DNA dendrimer by hydrogen bonds. In some embodiments, the DBS comprises a nucleic acid sequence of TAGAGGTAACAACTAGCGTACAA (SEQ ID NO: 3). In some embodiments, the DBS sequence further comprises a polythymine sequence at the 5' or 3' end of the sequence. For example, in some embodiments, the DBS comprises a nucleic acid sequence of TAGAGGTAACAACTAGCGTACAATTTTTTTTTT (SEQ ID NO: 4). In some embodiments, the DBS comprises the nucleic acid sequence of CCTCAGCTTGTACTCTAGTTGTTACCTCTAATGCTGGACCTCAGC (SEQ ID NO: 22). In some embodiments, the DBS comprises the nucleic acid sequence of CCTCAGCACCCTACAGAGTAACCTAGATTGATCAAACACCTCAGC (SEQ ID NO: 23). These are non-limiting examples of DBS molecules, and any DBS sequence that is complementary to the arms of a dendrimer may be used.

[0061] D. Adaptor molecules The cargo may be linked to the DNA dendrimer by an adapter molecule as described herein. In some embodiments, the adapter molecule comprises a DNA dendrimer binding sequence (DBS) that links the adapter molecule to the DNA dendrimer, and a cargo binding region that links to the cargo, e.g., a cargo polynucleotide as described herein.

[0062] In some embodiments, the DBS comprises a nucleic acid sequence that is complementary to the nucleic acid sequence of the DNA dendrimer. For example, the DBS comprises a nucleic acid sequence that is complementary to at least one binding arm of the DNA dendrimer. In some embodiments, the DBS binds to the DNA dendrimer by hydrogen bonding. In some embodiments, the DBS comprises any DBS sequence disclosed herein. In some embodiments, any DBS sequence that is complementary to the dendrimer arm can be used.

[0063] In some embodiments, the cargo binding region may be linked to the cargo polynucleotide by various linkers known in the art. For example, the cargo binding region may comprise a nucleic acid sequence that is complementary to at least a portion of the cargo polynucleotide and / or that is linked to the cargo by DNA ligation. In another example, the cargo binding region may be linked to the cargo polynucleotide by chemical coupling, including but not limited to, click chemistry or EDC crosslinking. In some embodiments, the cargo binding region comprises at least one nucleotide having an amine, azide, or other reactive group. In some embodiments, the cargo binding region comprises at least one cysteine ​​residue.

[0064] The adaptor molecule may also have other regions or features included. In some embodiments, such regions or features may be between the DBS and the cargo binding region, but may be present in any order within the adaptor molecule.

[0065] In some embodiments, the adapter molecule further comprises at least one purification region. In some embodiments, the purification region comprises at least one purification or affinity tag. The purification or affinity tag can be any type of chemical or amino acid tag known in the art, such as a polyhistidine tag, including but not limited to His6, His12, etc.

[0066] In some embodiments, the adapter molecule further comprises a DTS, an NLS, or both, as described herein. In some embodiments, the DTS and NLS regions are between the DBS and the cargo binding region. In some embodiments, the NLS has an amino acid sequence of YPDEVKRKKKP (SEQ ID NO: 1) or SLLESPFDKPDEVKRKKKPPTSHQSDATAEDDSSSKKK (SEQ ID NO: 2). In some embodiments, the DTS or NLS region further comprises at least one spacer that is either before or after the STS or NLS region. When two spacers are present, they are immediately before and after the DTS, NLS, or both. Spacers are generally known in the art and include a variety of flexible or semi-flexible amino acids or molecules. In some embodiments, the spacer or spacers include polyglycine, optionally with alanine and / or serine residues. In some embodiments, the spacer or spacers have an amino acid sequence of GGGG (SEQ ID NO: 5). In some embodiments, the spacer or spacers comprise polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic-glycolic acid copolymer (PGLA), or any combination thereof. In some embodiments, the spacer or spacers comprise a saturated or unsaturated hydrocarbon chain containing 3 to 6 carbons, which may be optionally substituted.

[0067] In some embodiments, the adapter molecule further comprises a cell penetrating peptide sequence (CPP). CPPs are a group of short peptides that have the ability to increase transduction through membranes and can be used to aid in the transport of molecules through cell membranes (for review, see Xu et al., 2019, J Control Release, 309:106-124). Various CPPs can be included in the adapter molecule to aid in the entry of the DNA dendrimer-based composition into the target cell or target nucleus. For example, in some embodiments, the CPP has an amino acid sequence of QPRRRPRRKKRG (SEQ ID NO: 6). In some embodiments, the CPP is present between the DBS and the cargo binding region.

[0068] In some embodiments, the cargo is separable from the DNA dendrimer. Thus, in some embodiments, the adapter molecule further comprises one or more cleavage sites. The cleavage site can be anywhere between the DBS and the cargo binding region. In some embodiments, the cleavage site is adjacent to the DBS or approximately adjacent to the DBS. For example, in some embodiments, the cleavage site is a valine-citrulline / p-aminobenzylcarbamate (Val-Cit) cleavable linker. Val-Cit linkers are known in the art and are used to release cargo from targeting moieties in drug conjugation. Other cleavable linkers known in the art may be included, such as self-cleaving ribozyme cleavage sites or tunable pH-sensitive linkers (see, for example, Choy et al., Bioconjugate Chem. (2016) 27(3):824-830).

[0069] In some embodiments, the adapter molecule further comprises at least one flexible linker. In some embodiments, the flexible linker is between the DNS and the cargo binding region. In some embodiments, the at least one flexible linker is or comprises polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic-glycolic acid copolymer (PGLA), (GG)n, (GGGGS)n (SEQ ID NO: 7), (GGGGA)n (SEQ ID NO: 27), or any combination thereof, where each n is independently 1 to 5.

[0070] The adaptor molecules described herein may have none, some, or all of any of the elements disclosed herein.For example, in addition to DBS and cargo binding regions, adaptor molecules may include one or more purification regions, DTS, NLS, CPP, spacer, cleavage site, flexible linker, or any combination thereof.Non-limiting examples of adaptor molecules are listed below. [Table 1]

[0071] In some embodiments, a composition is provided that comprises a targeting moiety and a cargo as disclosed herein, wherein the targeting moiety and the cargo are linked by an adapter molecule that comprises at least one DTS. In some embodiments, the targeting moiety / adapter molecule / cargo composition with DTS does not comprise a DNA dendrimer.

[0072] In some embodiments, the composition comprises a sequence shown in the table below: In some embodiments, the composition comprises from the 5' to the 3' end a sequence shown in the table below. [Table 2]

[0073] In some embodiments, the composition comprises from the 5' to 3' end the sequences of SEQ ID NO:4 and SEQ ID NO:28. In some embodiments, the composition comprises from the 5' to 3' end the sequences of SEQ ID NO:4, SEQ ID NO:29, and SEQ ID NO:1. In some embodiments, the composition comprises from the 5' to 3' end the sequences of SEQ ID NO:4, SEQ ID NO:29, CitV, SEQ ID NO:5, SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:28. In some embodiments, the composition comprises from the 5' to 3' end the sequences of SEQ ID NO:4, SEQ ID NO:29, CitV, SEQ ID NO:6, SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:28.

[0074] In some embodiments, the composition comprises, from the 5' to 3' end, the sequences of SEQ ID NO:4 and SEQ ID NO:28, wherein the sequences are conjugated to each other. In some embodiments, the composition comprises, from the 5' to 3' end, the sequences of SEQ ID NO:4, SEQ ID NO:29, and SEQ ID NO:1, wherein the sequences are conjugated to each other. In some embodiments, the composition comprises, from the 5' to 3' end, the sequences of SEQ ID NO:4, SEQ ID NO:29, CitV, SEQ ID NO:5, SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:28, wherein the sequences are conjugated to each other. In some embodiments, the composition comprises, from the 5' to 3' end, the sequences of SEQ ID NO:4, SEQ ID NO:29, CitV, SEQ ID NO:6, SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:28, wherein the sequences are conjugated to each other.

[0075] E. Auxiliary molecules The compositions herein may also include one or more auxiliary molecules. Auxiliary molecules as described herein may be used for a variety of tasks, including, but not limited to, protecting DNA dendrimers and cargo polynucleotides from nuclease degradation, enhancing transfection efficiency, aiding in delivery to the nucleus, and compacting the size of the DNA dendrimer, cargo polynucleotide, or both. The auxiliary molecules may be covalently or non-covalently linked to the DNA dendrimer in the same or similar manner as the targeting moiety and cargo. However, in some embodiments, the one or more auxiliary molecules are not linked to the DNA dendrimer and may be associated with the composition by other means.

[0076] Without wishing to be bound by theory, because many polynucleotide structures have negatively altered amine backbones, auxiliary molecules with sufficient positive charge can associate with the composition by charge attraction.Thus, in some embodiments, one or more auxiliary molecules associate with the composition by charge attraction.In some embodiments, one or more auxiliary molecules have a net positive charge high enough to allow one or more auxiliary molecules to interact with and optionally compact the negatively charged DNA dendrimer.In some embodiments, one or more auxiliary molecules have a net positive charge low enough to avoid aggregation or other toxic side effects on cells.

[0077] In addition, such auxiliary molecules can also be associated with the cargo polynucleotide and the DNA dendrimer simultaneously. Thus, in some embodiments, the compositions described herein include a cargo polynucleotide, an auxiliary molecule, and a DNA dendrimer linked to a targeting moiety, where the cargo polynucleotide is not covalently linked to the DNA dendrimer. The auxiliary molecule can associate the cargo polynucleotide with the DNA dendrimer in this manner, regardless of the topology of the cargo polynucleotide. However, when the cargo polynucleotide has a completely circular topology without any nicks, sites, or regions for direct covalent linkage to the DNA dendrimer or adapter molecule, one or more auxiliary molecules can associate the cargo polynucleotide and the DNA dendrimer with each other to form the composition.

[0078] The auxiliary molecules described herein can be used individually, as repeats, or in combination within one molecular entity or in a complex mixture of individual molecular entities. In some embodiments, the auxiliary molecules can be added to the composition before the association of the cargo, the DNA dendrimer, and optionally the adapter molecule. In some embodiments, the auxiliary molecules can be added to the composition after the association of the cargo, the DNA dendrimer, and optionally the adapter molecule.

[0079] In some embodiments, the auxiliary molecule is composed of a biocompatible peptide, a polymer, or both. In some embodiments, the auxiliary molecule can include a cell penetrating peptide (CPP) sequence, a nuclear localization signal (NLS) sequence, or both. In some embodiments, the CPP sequence is any CPP sequence disclosed herein. In some embodiments, the NLS sequence is any NLS sequence disclosed herein.

[0080] In some embodiments, one or more auxiliary molecules have the amino acid sequence of ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 12), WRRRGFGRRR (SEQ ID NO: 13), GRKKRRQRRRPQ (SEQ ID NO: 14), PKKKRKV (SEQ ID NO: 15), GLFHAIAHFIHGGWHGLIHGWYG (SEQ ID NO: 16), WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 17), HHHHHHHHH (SEQ ID NO: 18), (KK)n (where n=2-15), or any combination thereof. In some embodiments, the auxiliary molecule has the amino acid sequence of GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 19). In some embodiments, the auxiliary molecule has an amino acid sequence of HHHHHHHHHATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO:20). In some embodiments, the auxiliary molecule has an amino acid sequence of GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVGRKKRRQRRRPQWRRRGFGRRR (SEQ ID NO:21). In some embodiments, the auxiliary molecule has an amino acid sequence of KKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO:24). In some embodiments, the auxiliary molecule has an amino acid sequence of KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO:25). In some embodiments, the auxiliary molecule has an amino acid sequence of KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO:26).

[0081] In some embodiments, polyethylene glycol (PEG) can be added to any auxiliary molecule disclosed herein. The PEG molecule can be any PEG known in the art, including various molecular weights. For example, in some embodiments, the PEG is PEG400 or PEG2000. In some embodiments, the combination of the PEG molecule and the auxiliary molecule increases the circulation time of the auxiliary molecule, shields the positive charge, enhances the stability of the composition, or any combination thereof. In some embodiments, the PEG molecule is attached to the N-terminus of the auxiliary molecule.

[0082] In some embodiments, the auxiliary molecules are added to the composition at a specific concentration. In some embodiments, the auxiliary molecules are added to the composition to bring the ionic balance of the entire composition within an acceptable range of nitrogen / phosphate (N / P) ratios. The N / P ratio is the number of nitrogen groups in the auxiliary molecule relative to the number of phosphorus groups in any nucleic acid molecule in the composition. In some embodiments, the higher the N / P ratio, the more auxiliary molecules are added to the composition. In some embodiments, the N / P ratio is calculated using the following formula:

number

[0083] In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.5 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 1 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 2 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 3 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 4 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 5 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 6 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 7 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 8 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 9 to 10. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 9. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 8. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 7. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 6. In some embodiments, the adjunct molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 5. In some embodiments, the adjunct molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 4.In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 3. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 2. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 1. In some embodiments, the auxiliary molecule is added to the composition such that the composition has an ion balance of an N / P ratio of 0.25 to 5.

[0084] F. Qualification Modified nucleic acids may be used throughout the compositions described herein, including DNA dendrimers, cargo, adapter molecules, and auxiliary molecules. Non-limiting examples of such chemical modifications include, but are not limited to, independently, phosphate backbone modifications (e.g., phosphorothioate internucleotide linkages), nucleotide sugar modifications (e.g., 2'-O-methyl nucleotides, 2'-O-allyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxyribonucleotides), nucleotide base modifications (e.g., nucleotides containing "universal bases", 5-C-methyl nucleotides), and non-nucleotide modifications (e.g., abasic nucleotides, inverted deoxy abasic residues), or combinations of these modifications. In addition, oligonucleotides with morpholino backbone structures (U.S. Pat. No. 5,034,506) or polyamide backbone structures (Nielsen et al., 1991, Science 254:1497) may also be used. These and other chemical modifications can retain the in vivo biological activity of the nucleic acids while dramatically increasing the serum stability, potency, duration of effect, and / or specificity of these compounds. Nucleic acids containing modified internucleoside linkages can be synthesized using reagents and methods that are well known in the art. For example, methods for synthesizing nucleic acids containing phosphonate phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate, formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate, dimethylene-sulfide (-CH2-S-CH2), dimethylene-sulfoxide (-CH2-SO-CH2), dimethylene-sulfone (-CH2-SO2-CH2), 2'-O-alkyl, and 2'-deoxy-2'-fluoro phosphorothioate internucleoside linkages are well known in the art (see Uhlmann et al., 1990, Chem. Rev. 90:543-584; Schneider et al., 1990, Tetrahedron Lett. 31:335, and references cited therein).

[0085] The examples of oligonucleotide modifications described herein are not exhaustive, and it will be understood that the compositions include further modifications that serve to enhance the therapeutic or other properties of the oligonucleotide without significantly altering the basic sequence of the oligonucleotide. Similarly, protein cargoes may be modified as described elsewhere herein.

[0086] In addition, the linkages described herein can be non-covalent or covalent. Covalent linkages include linkages that are susceptible to cleavage upon internalization into a cell. Such linkages include pH-labile, photolabile, and radiation-labile linkages, and are well known in the art. The cargo can be linked to an oligonucleotide that includes a sequence that is substantially complementary to the linkage arm, substantially complementary to a sequence present in one of the linkage arms on the surface of the DNA dendrimer, or substantially complementary to a portion of the single-stranded sequence of any DNA-based carrier. The oligonucleotide can further include a nucleic acid cargo. The linkage arm of the DNA dendrimer, or a portion of any DNA-based carrier, can be designed to include a sequence that is complementary to a sequence of a known nucleic acid molecule (e.g., genomic DNA, cDNA, RNA, plasmid, etc.) to directly link (by hydrogen bonding) the nucleic acid cargo to the DNA dendrimer. The linkage arm, branch, or body of the DNA dendrimer, or a portion of any DNA-based carrier, can be designed to include a sequence that is a cargo (e.g., a DNA oligonucleotide).

[0087] Pharmaceutical Compositions and Kits In some embodiments, a pharmaceutical composition of the composition is provided. In some embodiments, the composition of the present invention further comprises a pharma- ceutically acceptable carrier. For therapeutic applications, the composition may be prepared as a pharmaceutical composition containing an effective amount of the cargo, composition, domain, or molecule as an active ingredient in a pharma- ceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with an active compound. A pharma- ceutically acceptable carrier is separate from the DNA dendrimer or other components as described herein. In some embodiments, the pharmaceutical composition does not have any additional pharma- ceutically acceptable carrier. Such vehicles may be liquids, such as water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The composition may contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickening agents, lubricants, and coloring agents. The concentration of the molecules disclosed herein in such pharmaceutical formulations may vary widely, i.e., less than about 0.5% by weight, usually at least about 1% to as much as 15 or 20%, and is selected primarily based on the required dose, fluid volume, viscosity, etc., according to the particular mode of administration selected. Suitable vehicles and formulations (including other human proteins, e.g., human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DBed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp691-1092, see especially pp. 958-989.

[0088] In some embodiments, any composition disclosed herein may include suitable formulation agents known in the art, such as stabilizers, buffers, excipients, etc., other than the auxiliary molecules or other components as described herein. For example, suitable formulation agents include, but are not limited to, purified water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, polymers such as polyethylene glycol, propylene glycol, PEG400, glycerin, DMA, ethanol, benzyl alcohol, citric acid / sodium citrate (pH 3), citric acid / sodium citrate (pH 5), tris(hydroxymethyl)aminomethane HCl (pH 7.0), 0.9% saline, 1.2% saline, silicone, wax, petroleum jelly, polyethylene glycol, propylene glycol, liposomes, sugars such as mannitol and lactose, and other substances depending on the particular type of formulation used.

[0089] The mode of administration for therapeutic applications of the compositions disclosed herein can be any suitable route that delivers an agent to a recipient, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or pulmonary administration; transmucosal (oral, intranasal, intravaginal, rectal) administration using tablet, capsule, solution, powder, gel, particle formulations; and containment in a syringe, implantable device, osmotic pump, cartridge, micropump; or other means recognized by the skilled artisan as being well known in the art. Site-specific administration can be achieved, for example, by intra-articular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intrasinus, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

[0090] The pharmaceutical composition may be supplied as a kit comprising a container containing the pharmaceutical composition as described herein. The pharmaceutical composition may be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted prior to injection. Such a kit may further comprise written information regarding the indications and usage of the pharmaceutical composition.

[0091] method In some embodiments, a method of delivering a molecule of interest to a target cell is provided. In some embodiments, the molecule of interest is delivered to the nucleus of the target cell. In some embodiments, the method includes contacting the target cell with any of the compositions or pharmaceutical compositions disclosed herein. Without being bound by any theory, the targeting moiety can bind to the target cell and facilitate the composition's entry into the target cell, where the cargo polynucleotide can be transported to the nucleus of the target cell. In some embodiments, the cargo polynucleotide is linked to an adapter molecule, such as any adapter molecule disclosed herein. In some embodiments, the cargo polynucleotide, the adapter molecule, or both, further comprises a DTS, an NLS, or both, to aid in transport to the nucleus of the target cell. In some embodiments, the adapter molecule comprises a CPP. In some embodiments, the cargo polynucleotide expresses the molecule of interest in the nucleus of the target cell. In some embodiments, the composition of the pharmaceutical composition can express two or more molecules of interest. In some embodiments, the promoter or promoters of the cargo polynucleotide are tissue-specific promoters. In some embodiments, the targeting moiety is a tissue-specific targeting moiety. In some embodiments, the composition or pharmaceutical composition comprises two or more tissue-specific targeting moieties. In some embodiments, the targeting moiety can be any type disclosed herein.

[0092] In some embodiments, a method of treating a disease is provided. In some embodiments, the method includes contacting a target cell with any of the compositions or pharmaceutical compositions disclosed herein. In some embodiments, the targeting moiety binds to the target cell and facilitates the composition entering the target cell. In some embodiments, the cargo polynucleotide can enter the nucleus of the target cell. In some embodiments, the cargo polynucleotide is linked to a DNA dendrimer. In some embodiments, the cargo polynucleotide is linked to an adapter molecule, such as any adapter molecule disclosed herein. In some embodiments, the cargo polynucleotide, the adapter molecule, or both, further comprises a DTS, an NLS, or both to aid in transport to the nucleus of the target cell. In some embodiments, the adapter molecule comprises a CPP. In some embodiments, the cargo polynucleotide expresses a molecule of interest in the nucleus of the target cell. In some embodiments, the pharmaceutical composition composition can express two or more molecules of interest. In some embodiments, the promoter or promoters of the cargo polynucleotide are tissue-specific promoters. In some embodiments, the targeting moiety is a tissue-specific targeting moiety. In some embodiments, the composition or pharmaceutical composition comprises two or more tissue-specific targeting moieties. In some embodiments, the targeting moieties can be of any type disclosed herein.

[0093] In some embodiments, the composition is administered to the subject by a route of administration as described herein, hi some embodiments, the composition is administered parenterally, for example, intravenously.

[0094] In some embodiments, the cargo of the composition is a therapeutic agent, and the method is used to alleviate a disorder or disease or provide a preventive treatment for a disorder or disease. The method is carried out by administering any of the compositions or pharmaceutical compositions provided herein to an individual in need thereof. Without being bound by any theory, by avoiding or limiting lysosomal delivery and degradation of the cargo and successfully delivering the cargo to the cell nucleus, the compositions and pharmaceutical compositions described herein may allow for a reduced dose of the therapeutic agent compared to prior art delivery methods. Advantageously, the reduced dose also reduces the risk of potential side effects. In some embodiments, the therapeutic agent is a polypeptide or a small molecule drug. In other embodiments, the therapeutic agent comprises a nucleic acid. The therapeutic molecule may be any therapeutic molecule that can be encoded by a polynucleotide. Non-limiting examples of types of therapeutic molecules that can be encoded in the expression cassettes of the present invention include, but are not limited to, polypeptide enzymes, cytokines, hormones, antibodies, e.g., intrabodies or scFvs, suicide genes, e.g., HSV-TK, molecules that inhibit angiogenesis, molecules that increase angiogenesis, tumor suppressors, e.g., p53 and p21, pro-apoptotic molecules, e.g., TRAIL, transcription factors, receptors, ligands, immunogenic molecules, anti-proliferative molecules, agonists, antagonists, anti-inflammatory molecules, antibiotics, antidepressants, prodrugs, antihypertensives, antioxidants, etc. Therapeutic molecules that include nucleic acids can be nucleic acids that regulate the expression of genes in vivo. Such nucleic acids include antisense molecules, siRNAs, and ribozymes.

[0095] Thus, the methods herein provide novel therapeutic approaches for a wide range of diseases and conditions, including cancer or cancerous diseases, infectious diseases, eye diseases, cardiovascular diseases, nervous system diseases, prion diseases, inflammatory diseases, autoimmune diseases, metabolic diseases, genetic diseases, lung diseases, kidney diseases, liver diseases, mitochondrial diseases, endocrine diseases, reproductive related diseases and conditions, graft versus host disease, and any other indication that may respond to the level of a gene product expressed in a cell or individual. In some embodiments, the disease is a genetic disease or disorder, including, but not limited to, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, Huntington's disease, muscular atrophy, Machado-Joseph disease, acanthocytic chorea, spastic paraplegia, myotonic dystrophy, fragile X syndrome, fragile X ataxia syndrome, spinocerebellar ataxia, frontotemporal dementia, amyotrophic lateral sclerosis, myotonic dystrophy, cystic fibrosis, and Down's syndrome. In some embodiments, the disease has been shown to be treatable or potentially treatable by gene therapy, including, but not limited to, multiple myeloma, B-cell lymphoma, melanoma, Leber's congenital amaurosis, spinal muscular atrophy, lipoprotein lipase deficiency, metachromatic leukodystrophy, mantle cell lymphoma, large B-cell lymphoma, beta thalassemia, vascular endothelial growth factor peripheral arterial disease, cerebral adrenoleukodystrophy, squamous cell carcinoma of the head and neck, adenosine deaminase deficiency, and B-cell lymphoblastic leukemia.

[0096] The art is replete with exemplary molecules and associated diseases or disorders in which patients may benefit from expression or inhibition of expression of one or more molecules. For example, the assessment of altered expression of gene families in various human cancers has been explored (US Patent Application Publication No. 20060168670). In addition, tissue-specific expression levels for thousands of genes have been mapped by expression profiling (Alon et al., 1999, Proc. Natl. Acad. Sci. USA 96:6745-50; Iyer et al., 1999, Science 283:83-87; Khan et al., 1998, Cancer Res. 58:5009-13; Lee et al., 1999, Science 285:1390-93; Wang et al., 1999, Gene 229:101-08; and Whitney et al., 1999, Ann. Neurol. 46:42). Thus, one of skill in the art will be able to select molecules useful in the practice of the present invention without undue experimentation.

[0097] In some embodiments, a method for producing a cargo polynucleotide is provided, comprising adding at least one promoter and at least one coding sequence encoding at least one molecule of interest to a plasmid backbone to form a plasmid, and then optionally contacting the plasmid with one or more restriction enzymes. In some embodiments, the plasmid is contacted with one or more restriction enzymes to form various topologies, including nicked cyclic nucleotides, linear nucleotides with closed 5' and 3' ends, linear nucleotides with open 5' and 3' ends, or linear nucleotides with one open end and one closed end. In some embodiments, the cargo polynucleotide is then ligated to an adaptor molecule, which is then ligated to a DNA dendrimer to form a composition. In some embodiments, the plasmid is not contacted with one or more restriction enzymes to form a complete cyclic nucleotide topology. In some embodiments, the cargo polynucleotide is then contacted with a DNA dendrimer linked to a targeting moiety in the presence of a possible auxiliary molecule, such that the cargo polynucleotide and the DNA dendrimer associate with each other to form a composition. In some embodiments, the auxiliary molecule is any auxiliary molecule described herein. In some embodiments, the auxiliary molecule compacts the size of the cargo polynucleotide, the DNA dendrimer, or both.

[0098] In some embodiments, the plasmid containing at least one coding sequence is amplified by traditional methods in bacteria, such as E. coli. In some embodiments, the plasmid containing at least one coding sequence is amplified by rolling circle amplification or other non-bacterial methods. For example, amplification can be performed using enzymatic synthesis of covalently closed linear DNA as described in U.S. Pat. No. 11,149,302 and U.S. Patent Application Publication No. US2019 / 0185924, both of which are incorporated by reference in their entirety.

[0099] Embodiment Embodiments provided herein also include, but are not limited to, the following. 1. A targeting moiety; an adaptor molecule-cargo polynucleotide complex; Compositions comprising DNA dendrimers linked to or associated with.

[0100] 2. The composition of embodiment 1, wherein the DNA dendrimer is linked to the adapter molecule-cargo polynucleotide complex by electrostatic interactions, covalent bonds, non-covalent bonds, or hydrogen bonds with the adapter molecule.

[0101] 3. The composition of embodiment 1 or 2, wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding at least one molecule of interest, and wherein the cargo polynucleotide is a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open end and one closed end, wherein the one open end and the one closed end can be at either the 5' or 3' end of the polynucleotide.

[0102] 4. The composition of embodiment 2, wherein the cargo polynucleotide further comprises at least one DNA targeting sequence (DTS).

[0103] 5. The composition of embodiment 4, wherein the at least one DTS is upstream of the at least one promoter.

[0104] 6. The composition of embodiment 4, wherein the at least one DTS is downstream of the at least one promoter.

[0105] 7. The composition of any one of embodiments 4 to 6, wherein the cargo polynucleotide comprises two or more DTSs.

[0106] 8. The composition of embodiment 7, wherein the two or more DTSs are adjacent to each other or nearly adjacent to each other.

[0107] 9. The composition of embodiment 7, wherein the two or more DTSs are not adjacent to each other or not nearly adjacent to each other.

[0108] 10. The composition of any one of embodiments 1 to 5, wherein the cargo polynucleotide further comprises at least one nuclear localization signal sequence (NLS).

[0109] 11. The composition of embodiment 10, wherein the at least one NLS is present upstream of the at least one promoter.

[0110] 12. The composition of embodiment 10, wherein the at least one NLS is downstream of the at least one promoter.

[0111] 13. The composition of any one of embodiments 6 to 8, wherein the cargo polynucleotide comprises at least one DTS and at least one NLS.

[0112] 14. The composition of any one of embodiments 1 to 9, wherein the adapter molecule comprises a DNA dendrimer binding sequence (DBS) and a cargo binding region (CBR).

[0113] 15. The composition of embodiment 14, wherein the adaptor is linked to the DNA dendrimer via the DBS.

[0114] 16. The composition of embodiment 14 or 15, wherein the DBS comprises a nucleic acid sequence complementary to a nucleic acid sequence of the DNA dendrimer.

[0115] 17. The composition of any one of embodiments 14 to 16, wherein the DBS comprises the nucleic acid sequence TAGAGGTAACAACTAGCGTACAA (SEQ ID NO: 3), CCTCAGCTTGTACTCTAGTTGTTACCTCTAATGCTGGACCTCAGC (SEQ ID NO: 22), or CCTCAGCACCCTACAGAGTAACCTAGATTGATCAAACACCTCAGC (SEQ ID NO: 23).

[0116] 18. The composition of any one of embodiments 14 to 17, wherein the DBS further comprises a polythymine sequence at either the 5' or 3' end of the DBS.

[0117] 19. The composition of embodiment 18, wherein the DBS comprises the nucleic acid sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTTT (SEQ ID NO: 4).

[0118] 20. The composition according to any one of embodiments 14 to 19, wherein the cargo binding region (CBR) may be linked to the cargo polynucleotide by DNA ligation, i.e. linked to the polynucleotide by a covalent bond, e.g. a phosphodiester bond.

[0119] 21. The composition according to any one of embodiments 14 to 19, wherein the CBR is linked to the cargo polynucleotide by chemical coupling, i.e., by a covalent bond, for example a disulfide bond, to the polynucleotide.

[0120] 22. The composition of any one of embodiments 14 to 21, wherein the cargo binding region comprises at least one cysteine.

[0121] 23. The composition of any one of embodiments 14 to 22, wherein the adapter molecule further comprises a tag, such as, for example, a tag that can be used as an affinity tag for isolating / purifying a complex of the composition.

[0122] 24. The composition of embodiment 23, wherein the tag is located between the DBS and the CBR.

[0123] 25. The composition of embodiment 24, wherein the tag is a polyhistidine tag.

[0124] 26. The composition of any one of embodiments 14 to 25, wherein the adapter molecule further comprises at least one nuclear localization signal sequence (NLS).

[0125] 27. The composition of embodiment 26, wherein the at least one NLS is between the DBS and the cargo binding region or overlaps with the DBS and the cargo binding region.

[0126] 28. The composition of embodiment 26 or 27, wherein the at least one NLS comprises one or more NLS sequences disclosed herein.

[0127] 29. The composition of any one of embodiments 26 to 28, wherein the at least one NLS comprises the amino acid sequence YPDEVKRKKKP (SEQ ID NO: 1).

[0128] 30. The composition according to any one of embodiments 26 to 28, wherein the at least one NLS comprises the amino acid sequence SLLESPFDKPDEVKRKKKPPTSHQSDATAEDDSSSKKK (SEQ ID NO: 2).

[0129] 31. The composition of any one of embodiments 26 to 30, wherein the at least one NLS further comprises at least one spacer, which is present either before or after the at least one NLS.

[0130] 32. The composition of embodiment 31, wherein the at least one NLS comprises two spacers present before or after the at least one NLS, for example, one before the NLS and one after the NLS, or both spacers before or after the NLS.

[0131] 33. The composition of embodiment 31 or 32, wherein the spacer or spacers comprise polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic-glycolic acid copolymer (PGLA), or any combination thereof.

[0132] 34. The composition according to embodiment 31 or 32, wherein the spacer or spacers comprise a polyglycine sequence, optionally with alanine and / or serine residues.

[0133] 35. The composition of embodiment 34, wherein the spacer or spacers have the amino acid sequence GGGG (sequence number 5).

[0134] 36. The composition of any one of embodiments 14 to 35, wherein the adapter molecule further comprises at least one DNA targeting sequence (DTS).

[0135] 37. The composition of embodiment 36, wherein the at least one DTS is between the DBS and the CBR or overlaps the DBS and the CBR.

[0136] 38. The composition of any one of embodiments 14 to 37, wherein the adapter molecule further comprises a cell-penetrating peptide sequence (CPP).

[0137] 39. The composition of embodiment 38, wherein the CPP is between the DBS and the CBR or overlaps with the DBS and the CBR.

[0138] 40. The composition of embodiment 38 or 39, wherein the CPP comprises a CPP disclosed herein.

[0139] 41. The composition of any one of embodiments 38-40, wherein the CPP comprises the amino acid sequence QPRRRPRRKKRG (SEQ ID NO: 6).

[0140] 42. The composition of any one of embodiments 14 to 41, wherein the adapter molecule further comprises at least one cleavage site.

[0141] 43. The composition of embodiment 42, wherein the at least one cleavage site is between the DBS and the CBR or overlaps the DBS and the CBR.

[0142] 44. The composition of embodiment 42 or 43, wherein the cleavage site is adjacent to the DBS.

[0143] 45. The composition of any one of embodiments 42-44, wherein the cleavage site is a Val-Cit linker.

[0144] 46. ​​The composition of any one of embodiments 14 to 45, wherein the adapter molecule further comprises at least one flexible linker.

[0145] 47. The composition of embodiment 46, wherein the at least one flexible linker is between the DNS and the CBR.

[0146] 48. The composition of embodiment 46 or 47, wherein the at least one flexible linker is selected from the group consisting of polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic-glycolic acid copolymer (PGLA), (GG)n, (GGGGS)n, or (GGGGA)n, where each n is independently 1 to 5.

[0147] 49. The composition of any one of embodiments 1 to 47, wherein the composition further comprises an auxiliary molecule.

[0148] 50. The composition of embodiment 49, wherein the auxiliary molecule is linked to the DNA dendrimer.

[0149] 51. The composition of embodiment 49, wherein the auxiliary molecule is not linked to the DNA dendrimer.

[0150] 52. The composition of any one of embodiments 49 to 51, wherein the auxiliary molecule compacts the size of the cargo polynucleotide, the DNA dendrimer, or both.

[0151] 53. The composition of any one of embodiments 49 to 52, wherein the auxiliary molecule is capable of assisting delivery to the nucleus.

[0152] 54. The composition of any one of embodiments 49 to 53, wherein the auxiliary molecule is capable of protecting the cargo polynucleotide, the DNA dendrimer, or both, from nuclease degradation.

[0153] 55. The composition of any one of embodiments 49 to 54, wherein the auxiliary molecule is capable of enhancing the transfection efficiency of the composition.

[0154] 56. The composition of any one of embodiments 49-55, wherein the auxiliary molecule comprises the amino acid sequence ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 12), WRRRGFGRRR (SEQ ID NO: 13), GRKKRRQRRRPQ (SEQ ID NO: 14), PKKKRKV (SEQ ID NO: 15), GLFHAIAHFIHGGWHGLIHGWYG (SEQ ID NO: 16), WEAALAALAEALAELAEHLAEALAEALEALAA (SEQ ID NO: 17), HHHHHHHHH (SEQ ID NO: 18), (KK)q, where q is 2 to 15.

[0155] 57. The composition of any one of embodiments 49 to 56, wherein the auxiliary molecule comprises the amino acid sequence GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 19).

[0156] 58. The composition of any one of embodiments 49-56, wherein the auxiliary molecule comprises the amino acid sequence HHHHHHHHHHATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 20).

[0157] 59. The composition of any one of embodiments 49-56, wherein the auxiliary molecule comprises the amino acid sequence GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVGRKKRRQRRRPQWRRRGFGRRR (SEQ ID NO: 21).

[0158] 60. The composition of any one of embodiments 49-56, wherein the auxiliary molecule comprises the amino acid sequence KKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 24).

[0159] 61. The composition of any one of embodiments 49-56, wherein the auxiliary molecule comprises the amino acid sequence KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 25).

[0160] 62. The composition of any one of embodiments 49-56, wherein the auxiliary molecule comprises the amino acid sequence of KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 26).

[0161] 63. The composition of any one of embodiments 1 to 62, wherein the targeting moiety is selected from the group consisting of an antibody, a naturally occurring ligand of a receptor or a functional derivative thereof, a vitamin, a hormone, a small molecule mimetic of a naturally occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, or any other molecule that specifically binds to a cell surface molecule and induces endocytosis of the bound moiety, or any combination thereof.

[0162] 64. A composition comprising a cargo polynucleotide, an auxiliary molecule, and a DNA dendrimer linked to a targeting moiety.

[0163] 65. The composition of embodiment 64, wherein the targeting moiety is selected from the group consisting of an antibody, a naturally occurring ligand of a receptor or a functional derivative thereof, a vitamin, a hormone, a small molecule mimetic of a naturally occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, or any other molecule that specifically binds to a cell surface molecule and induces endocytosis of the bound moiety, or any combination thereof.

[0164] 66. The cargo polynucleotide comprises at least one promoter, and at least one coding sequence encoding at least one molecule of interest, and optionally a DNA binding sequence (DBS); the cargo polynucleotide is a complete circular polynucleotide (e.g., a plasmid), a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open end and one closed end; The composition of embodiment 64 or 65, wherein said one open end and one closed end may be present at either the 5' or 3' end of said cargo polynucleotide.

[0165] 67. The composition of embodiment 66, wherein the cargo polynucleotide is a closed circular polynucleotide.

[0166] 68. The composition of embodiment 66, wherein the cargo polynucleotide is a nicked circular polynucleotide.

[0167] 69. The composition of any one of embodiments 64 to 68, wherein the cargo polynucleotide is not covalently linked to the DNA dendrimer.

[0168] 70. The composition of any one of embodiments 64 to 69, wherein the cargo polynucleotide further comprises at least one DNA targeting sequence (DTS).

[0169] 71. The composition of embodiment 70, wherein the DTS is present within the cargo polynucleotide.

[0170] 72. The composition of embodiment 70, wherein the DTS is linked to the cargo polynucleotide.

[0171] 73. The composition of embodiment 72, wherein the cargo polynucleotide is a nicked circular polynucleotide and the DTS is linked to the cargo polynucleotide at the point of the single-stranded DNA break (i.e., at the position of the nick).

[0172] 74. The composition of any one of embodiments 64 to 73, wherein the cargo polynucleotide further comprises at least one nuclear localization signal sequence (NLS).

[0173] 75. The composition of any one of embodiments 64 to 74, wherein the cargo polynucleotide comprises at least one DTS and at least one NLS.

[0174] 76. The composition of any one of embodiments 66 to 75, wherein the DBS comprises the nucleic acid sequence TAGAGGTAACAACTAGCGTACAA (SEQ ID NO: 3), CCTCAGCTTGTACTCTAGTTGTTACCTCTAATGCTGGACCTCAGC (SEQ ID NO: 22), or CCTCAGCACCCTACAGAGTAACCTAGATTGATCAAACACCTCAGC (SEQ ID NO: 23).

[0175] 77. The composition of embodiment 76, wherein the DBS further comprises a polythymine sequence at either the 5' or 3' end of the DBS.

[0176] 78. The composition described in embodiment 77, wherein the DBS comprises the nucleic acid sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTTT (sequence number 4).

[0177] 79. The composition of any one of embodiments 64-78, wherein the auxiliary molecule is linked to the DNA dendrimer.

[0178] 80. The composition of any one of embodiments 64-79, wherein the auxiliary molecule is not linked to the DNA dendrimer.

[0179] 81. The composition of any one of embodiments 64 to 80, wherein the auxiliary molecule compacts the size of the cargo polynucleotide, the DNA dendrimer, or both.

[0180] 82. The composition of any one of embodiments 64-81, wherein the auxiliary molecule is capable of assisting delivery to the nucleus.

[0181] 83. The composition of any one of embodiments 64-82, wherein the auxiliary molecule is capable of protecting the cargo polynucleotide, the DNA dendrimer, or both, from nuclease degradation.

[0182] 84. The composition of any one of embodiments 64 to 83, wherein the auxiliary molecule is capable of enhancing the transfection efficiency of the composition.

[0183] 85. The composition of any one of embodiments 64 to 84, wherein the presence of the auxiliary molecule promotes the association of the cargo polynucleotide and the DNA dendrimer with each other.

[0184] 86. The composition of any one of embodiments 64-85, wherein the auxiliary molecule has a positive charge.

[0185] 87. The composition of any one of embodiments 64-86, wherein the auxiliary molecule comprises the amino acid sequence of ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 12), WRRRGFGRRR (SEQ ID NO: 13), GRKKRRQRRRPQ (SEQ ID NO: 14), PKKKRKV (SEQ ID NO: 15), GLFHAIAHFIHGGWHGLIHGWYG (SEQ ID NO: 16), WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 17), HHHHHHHHHH (SEQ ID NO: 18), (KK)q, or any combination thereof, wherein q is 2 to 15.

[0186] 88. The composition of any one of embodiments 64 to 87, wherein the auxiliary molecule comprises the amino acid sequence GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 19).

[0187] 89. The composition of any one of embodiments 64-87, wherein the auxiliary molecule comprises the amino acid sequence HHHHHHHHHHATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 20).

[0188] 90. The composition of any one of embodiments 64-87, wherein the auxiliary molecule comprises the amino acid sequence GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVGRKKRRQRRRPQWRRRGFGRRR (SEQ ID NO: 21).

[0189] 91. The composition of any one of embodiments 64-87, wherein the auxiliary molecule comprises the amino acid sequence KKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 24).

[0190] 92. The composition of any one of embodiments 64-87, wherein the auxiliary molecule comprises the amino acid sequence KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 25).

[0191] 93. The composition of any one of embodiments 64-87, wherein the auxiliary molecule comprises the amino acid sequence of KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 26).

[0192] 94. The composition of any one of embodiments 64-93, wherein the auxiliary molecule further comprises a polyethylene glycol (PEG) molecule linked to the auxiliary molecule.

[0193] 95. The composition described in embodiment 94, wherein the polyethylene molecule is PEG2000.

[0194] 96. The composition of embodiment 94 or 95, wherein the PEG molecule is attached to the N-terminus of the auxiliary molecule.

[0195] 97. The composition of any one of the preceding embodiments, wherein the coding sequence of the cargo polynucleotide encodes an antibody, an enzyme, a protein, miRNA, siRNA, antisense RNA, or the like.

[0196] 98. The composition of any one of the preceding embodiments, wherein the nitrogen / phosphate (N / P) ratio of the composition is 0.5 to 10.

[0197] 99. The composition described in embodiment 98, wherein the N / P ratio of the composition is 2 to 5.

[0198] 100. A pharmaceutical composition comprising the composition according to any one of embodiments 1 to 99 and a pharma- ceutically acceptable carrier.

[0199] 101. A method for delivering a molecule of interest to the nucleus of a target cell, the method comprising contacting the target cell with a composition described in any one of embodiments 1 to 99 or the pharmaceutical composition described in embodiment 100, wherein the targeting moiety binds to the target cell.

[0200] 102. The method of embodiment 101, wherein the cargo polynucleotide expresses the molecule of interest in the nucleus of the target cell.

[0201] 103. The method of embodiment 101 or 102, wherein the cargo polynucleotide expresses two or more molecules of interest in the nucleus of the target cell.

[0202] 104. The method of any one of embodiments 101 to 103, wherein the cargo polynucleotide comprises a tissue-specific promoter.

[0203] 105. The method of any one of embodiments 101-104, wherein the targeting moiety is a tissue-specific targeting moiety.

[0204] 106. The method of any one of embodiments 101-105, wherein the composition or pharmaceutical composition comprises two or more targeting moieties.

[0205] 107. A method for treating a disease, the method comprising administering to a subject a composition described in any one of embodiments 1 to 99 or a pharmaceutical composition described in embodiment 100 to treat the disease, wherein the targeting moiety binds to a target cell.

[0206] 108. The method of embodiment 107, wherein the cargo polynucleotide expresses the molecule of interest in the nucleus of the target cell.

[0207] 109. The method of embodiment 107 or 108, wherein the cargo polynucleotide expresses two or more molecules of interest in the nucleus of the target cell.

[0208] 110. The method of any one of embodiments 107 to 109, wherein the cargo polynucleotide comprises a tissue-specific promoter.

[0209] 111. The method of any one of embodiments 107-110, wherein the targeting moiety is a tissue-specific targeting moiety. 112. The method of any one of embodiments 107-111, wherein the composition or pharmaceutical composition comprises two or more targeting moieties.

[0210] 113. A plasmid comprising a plasmid backbone comprising at least two restriction enzyme recognition sites, at least one promoter, at least one coding sequence encoding at least one molecule of interest, and optionally a DNA dendrimer binding sequence (DBS), wherein the plasmid is capable of forming cargo polynucleotides having various structures depending on whether the plasmid has one or more 5' and 3' ends, or none of them.

[0211] 114. The plasmid of embodiment 113, wherein the plasmid is a complete circular polynucleotide, a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, and a linear polynucleotide with one open end and one closed end.

[0212] 115. The plasmid of embodiment 113, wherein the nicked cyclic nucleotide, the linear nucleotide with closed 5' and 3' ends, the linear nucleotide with open 5' and 3' ends, and the linear nucleotide with one open end and one closed end are formed by cleaving the plasmid with one or more restriction enzymes.

[0213] 116. The plasmid described in any one of embodiments 113 to 115, wherein the plasmid further comprises at least one DNA targeting sequence (DTS).

[0214] 117. The plasmid of embodiment 116, wherein the at least one DTS is present in the plasmid backbone, i.e., between or overlapping the at least two restriction enzyme recognition sites.

[0215] 118. The plasmid of embodiment 116 or 117, wherein the at least one DTS is present upstream of the at least one promoter.

[0216] 119. The plasmid of embodiment 116 or 117, wherein the at least one DTS is present downstream of the at least one promoter.

[0217] 120. The plasmid of any one of embodiments 113 to 119, wherein the cargo polynucleotide further comprises at least one nuclear localization signal sequence (NLS).

[0218] 121. The plasmid of embodiment 120, wherein the at least one NLS is present upstream of the at least one promoter.

[0219] 122. The plasmid of embodiment 120, wherein the at least one NLS is present downstream of the at least one promoter.

[0220] 123. The plasmid of any one of embodiments 116 to 122, wherein the cargo polynucleotide comprises at least one DTS and at least one NLS.

[0221] 124. The composition of any one of embodiments 116 to 123, wherein the DBS comprises the nucleic acid sequence TAGAGGTAACAACTAGCGTACAA (SEQ ID NO: 3), CCTCAGCTTGTACTCTAGTTGTTACCTCTAATGCTGGACCTCAGC (SEQ ID NO: 22), or CCTCAGCACCCTACAGAGTAACCTAGATTGATCAAACACCTCAGC (SEQ ID NO: 23).

[0222] 125. The composition of embodiment 124, wherein the DBS further comprises a polythymine sequence at either the 5' or 3' end of the DBS.

[0223] 126. The composition described in embodiment 125, wherein the DBS comprises the nucleic acid sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTTT (sequence number 4).

[0224] 127. The plasmid of any one of embodiments 116 to 126, wherein the at least one promoter is a tissue-specific promoter.

[0225] 128. The plasmid according to any one of embodiments 116 to 126, wherein the plasmid comprises a polynucleotide sequence encoding two or more molecules of interest.

[0226] 129. A method for producing a composition according to any one of embodiments 1 to 63, comprising the steps of: contacting the plasmid according to any one of embodiments 113 to 129 with one or more restriction enzymes that cleave at said restriction enzyme recognition sites to form a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open end and one closed end; ligating the cargo polynucleotide to the adapter molecule to form an adapter molecule-cargo polynucleotide complex; and linking the adaptor molecule-cargo polynucleotide complex and a targeting moiety to a DNA dendrimer to form the composition.

[0227] 130. A method for producing a composition according to any one of embodiments 64 to 99, comprising the steps of: contacting the plasmid according to any one of embodiments 113-129, comprising a DNA dendrimer binding sequence (DBS), with one or more restriction enzymes that cleave at said restriction enzyme recognition sites to form a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open end and one closed end; linking said cargo polynucleotide and a targeting moiety to a DNA dendrimer to form said composition.

[0228] 131. A method for producing a composition according to any one of embodiments 64 to 99, comprising the steps of: The method of any one of embodiments 113 to 129, comprising contacting an uncleaved circular plasmid with a DNA dendrimer linked to a targeting moiety together with an auxiliary molecule capable of compressing the size of the cargo polynucleotide and the DNA dendrimer, such that the cargo polynucleotide and the DNA dendrimer associate with each other to form the composition.

[0229] 132. A kit comprising the composition according to any one of embodiments 1 to 99, the pharmaceutical composition according to embodiment 100, the plasmid according to any one of embodiments 113 to 129, or any combination thereof. EXAMPLES

[0230] The following examples are provided for illustrative purposes only, and the claims should in no way be construed as being limited to these examples, but rather as encompassing any variations that become evident as a result of the teachings provided herein. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield substantially similar results.

[0231] Example 1: Characterization of multiple topologies of polynucleotide cargo To determine how the topological structure of various polynucleotide cargoes can affect expression efficiency, gene cassettes containing luciferase and eGFP driven by the CMV promoter were inserted into a plasmid backbone containing multiple restriction enzyme recognition sites, including SalI, BbcCI, and Tel. To generate the various topologies, the resulting plasmids were cut with the following restriction enzymes, listed in Table 1. A description of the various topologies is shown in Figure 1. [Table 3]

[0232] Once formed, the various polynucleotide cargos were transfected into CHO-K1 cells using equal molar amounts of each topology variation. GFP expression was measured over time using cell imaging and quantitative fluorescence measurements. The percentage of GFP positive cells and overall fluorescence intensity were compared. The results are shown in Figures 2 and 3. The unmodified complete circular (i.e., plasmid) and linear topologies with two closed ends generally had the best overall fluorescence activity, but all topologies showed active fluorescence that is useful for altering the overall activity of a particular therapeutic agent as required.

[0233] Example 2: Localization in non-dividing cells In many cases, it is important that the cargo polynucleotide is delivered to the nucleus of the target cell for expression of the encoded molecule of interest. When cells divide, the nuclear membrane naturally breaks down, allowing direct access from the cytoplasm. However, when cells do not undergo mitosis, the nuclear membrane remains intact and is largely impermeable to plasmids. Candidate strategies are the inclusion of at least one copy of a DNA targeting sequence (DTS), a nuclear localization sequence (NLS), or both in the cargo polynucleotide, an adaptor molecule that can associate with the cargo polynucleotide, or both. In a healthy cytoplasm, transcription factors that contain an NLS can bind to the DTS and recruit the cargo for nuclear transport. Similarly, the NLS can directly recruit the necessary importins that read the cargo for nuclear transport. To test this general strategy, a polynucleotide plasmid expressing eGFP was loaded with a DTS and compared to a plasmid expressing eGFP without the DTS in three different cell lines: fast-growing CHO-K1, intermediate-growing A427, and relatively slowly dividing differentiated C2C12 myoblasts.

[0234] Overall, both the DTS-free plasmid (P1A) and the DTS plasmid (P1B) were able to express GFP in CHO-K1 (Figure 4) and A427 cell lines (Figure 5). This was expected since both cell lines undergo sufficient mitosis to allow easy access to the nucleus. However, in the C2C12 cell line, where lack of proliferation or slow growth rate limits access to an otherwise intact nucleus, the DTS-containing plasmid cargo showed significantly better expression compared to the control plasmid in relative mean fluorescence (Figure 6), relative integrated fluorescence (Figure 7), and relative peak fluorescence (Figure 8). For Figures 4-8, approximately 5,000 cells were plated per well, and imaging and quantification of fluorescence was performed 24 hours after the first transfection. Significance was determined using one-way analysis of variance and Tukey's multiple comparison test between groups; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0235] Example 3: Further characterization of multiple topologies of polynucleotide cargo Additional polynucleotide cargo plasmids were generated and tested.

[0236] Reporter constructs All reporter constructs contain bacterial selection sequences (antibiotic resistance, origin of replication (ORI)), SV40-DTS, codon-optimized firefly luciferase (Luc2_CO), codon-optimized enhanced green fluorescent protein (eGFP_CO), 2A self-cleaving peptide (P2A), and bovine growth hormone polyadenylation (bgh-polyA) signal. In addition, a TelN protelomerase sequence was used along with one of two DBS sequences (SEQ ID NO:22 or SEQ ID NO:23) that allow binding of the plasmid to the DNA dendrimer nanoparticles by complementary hybridization. Two nickase enzyme sites flanking the DBS enzyme were used to expose single-stranded DNA (ssDNA) sequences to selected binding arms of the synthetic scaffold.

[0237] Three reporter constructs were made. Reporter constructs 1 and 2 share the cytomegalovirus (CMV) enhancer and promoter, known for its robust and ubiquitous expression in mammalian cell lines. Both reporter constructs also share a Kozak sequence for translation initiation in eukaryotes. Reporter construct 1 has the DBS sequence of SEQ ID NO: 22, while reporter construct 2 has the DBS sequence of SEQ ID NO: 23. Reporter construct 3 has the DBS sequence of SEQ ID NO: 22, but has a different promoter: muscle-specific promoter mCK8e. All three reporter constructs have various cleavage sites for restriction enzymes, and two nickase sites were placed for plasmid manipulation. The region containing the selected enzyme cleavage sites and binding sequences is referred to as the "CB backbone element" region. An additional feature in this region is the TelN protelomerase recognition site. This sequence is a palindrome and, when recognized by the enzyme, forms two covalently closed ends (designated "TelR" and "TelL") at the cleavage site.

[0238] In addition, a construct of a molecule of interest was made in which the DNA insert coding construct is greater than 10K base pairs. This construct has many of the same sequences found in the reporter construct: bacterial selection moiety, SV40-DTS, mCK8e promoter, Kozak sequence, and TelN protelomerase site. An additional promoter, the T7 promoter, is incorporated in front of the coding sequence for enhanced expression in bacteria. A similar restriction enzyme site and two nickase sites are present near the DBS sequence (SEQ ID NO:22).

[0239] method Restriction enzymes used only in the reporter construct are designated with "†" and those used only in the 10K insert construct are designated with "‡". Nickases designated as "Nt" are used only in constructs with the DBS site of SEQ ID NO: 22. Nickases designated as "Nb" are used only in constructs with the DBS site of SEQ ID NO: 23. Any enzyme or nickase without a designation is universal and may be used in any of the constructs listed.

[0240] 1. Open nicked circular (OpC) The construct with the DBS site of SEQ ID NO:22 is incubated with Nt.BbvCI at a ratio of 1 μg of plasmid to 1 unit (U) of enzyme (10,000 U / mL, New England Biolabs, R0632L, lot number: 10141032). The construct with the DBS site of SEQ ID NO:23 is incubated with Nb.BbvCI at a ratio of 1 μg of plasmid to 1 U of enzyme (10,000 U / mL, New England Biolabs, R0631L, lot number: 10150965). The reaction is then incubated at 50° C. for 1 hour at 300 revolutions per minute (rpm).

[0241] The appropriate capture sequence for the DBS site is then added to the plasmid in 20 μmol excess. The reaction is then heated to 90° C. for 2 minutes to allow the nicked strand to separate from the plasmid. The reaction is then slowly cooled to room temperature over 3.5 hours to allow the capture sequence to bind to the nicked sequence, leaving the attachment site open.

[0242] Ion exchange chromatography (IEX) is then used to separate the nicked plasmid from the captured nicked sequence.The fractions containing the peak of interest are then combined, and the OpC plasmid is extracted using ethanol (EtOH) precipitation.The pellet is then dried and reconstituted in a buffer of choice.

[0243] 2. Nicked linear (LN) The construct is incubated with TelN protelomerase at a ratio of 1 μg of plasmid to 1 U of enzyme (20,000 U / mL, New England Biolabs, M0651B-HC2, Lot Number: 10151691). The reaction is incubated for 4 hours at 30° C. and 300 rpm. A buffer exchange into nuclease-free water is performed to remove Triton-X from the initial buffer.

[0244] The construct with DBS site of SEQ ID NO:22 linearized with TelN is incubated with Nt.BbvCI at a ratio of 1 μg of plasmid to 1 unit (U) of enzyme (10,000 U / mL, New England Biolabs, R0632L, lot number: 10141032). The construct with DBS site of SEQ ID NO:23 is incubated with Nb.BbvCI at a ratio of 1 μg of plasmid to 1 U of enzyme (10,000 U / mL, New England Biolabs, R0631L, lot number: 10150965). The reaction is then incubated at 50° C. for 1 hour at 300 rpm.

[0245] The appropriate capture sequence for the DBS site is then added to the plasmid in 20 μmol excess. The reaction is then heated to 90° C. for 2 minutes to allow the nicked strand to separate from the plasmid. The reaction is then slowly cooled to room temperature over 3.5 hours to allow the capture sequence to bind to the nicked sequence, leaving the attachment site open.

[0246] IEX is then used to separate the nicked plasmid from the captured nicked sequence.The fractions containing the peak of interest are then combined and the LN plasmid is extracted using EtOH precipitation.The pellet is then dried and reconstituted in the buffer of choice.

[0247] 3. 1 open end, 1 closed end (1co) The construct is incubated with TelN protelomerase at a ratio of 1 μg of plasmid to 1 U of enzyme (20,000 U / mL, New England Biolabs, M0651B-HC2, Lot Number: 10151691). The reaction is incubated for 4 hours at 30° C. and 300 rpm. A buffer exchange into nuclease-free water is performed to remove Triton-X from the initial buffer.

[0248] MfeI† (20,000U / mL, New England Biolabs, R3589L, Lot No: 10150327) or EagI‡ (100,000U / mL, New England Biolabs, R3505M, Lot No: 10157687) is incubated with the TelN-linearized construct at a ratio of 1 μg of plasmid to 1 U of enzyme. Reactions are incubated at 37°C for 1 hour at 300 rpm. EtOH precipitation is used to precipitate the linearized plasmid with one open end prior to the nickase reaction.

[0249] The construct with the DBS site of SEQ ID NO:22 is incubated with Nt.BbvCI at a ratio of 1 μg of plasmid to 1 unit (U) of enzyme (10,000 U / mL, New England Biolabs, R0632L, lot number: 10141032). The construct with the DBS site of SEQ ID NO:23 is incubated with Nb.BbvCI at a ratio of 1 μg of plasmid to 1 U of enzyme (10,000 U / mL, New England Biolabs, R0631L, lot number: 10150965). The reaction is then incubated at 50° C. for 1 hour at 300 rpm.

[0250] The appropriate capture sequence for the DBS site is then added to the plasmid in 20 μmol excess. The reaction is then heated to 90° C. for 2 minutes to allow the nicked strand to separate from the plasmid. The reaction is then slowly cooled to room temperature over 3.5 hours to allow the capture sequence to bind to the nicked sequence, leaving the attachment site open.

[0251] IEX is then used to separate the nicked plasmid from the captured nicked sequence.The fractions containing the peak of interest are then combined and the 1co plasmid is extracted using ethanol (EtOH) precipitation.The pellet is then dried and reconstituted in a buffer of choice.

[0252] 4. 2 open ends (2oe) MfeI† (20,000U / mL, New England Biolabs, R3589L, Lot No: 10150327) or EagI‡ (100,000U / mL, New England Biolabs, R3505M, Lot No: 10157687) is incubated with the construct at a ratio of 1 μg of plasmid to 1 U of enzyme. Reactions are incubated at 37°C for 1 hour at 300 rpm. EtOH precipitation is used to precipitate the linearized 2oe plasmid prior to reconstitution in the buffer of choice.

[0253] The construct with the DBS site of SEQ ID NO:22 is incubated with Nt.BbvCI at a ratio of 1 μg of plasmid to 1 unit (U) of enzyme (10,000 U / mL, New England Biolabs, R0632L, lot number: 10141032). The construct with the DBS site of SEQ ID NO:23 is incubated with Nb.BbvCI at a ratio of 1 μg of plasmid to 1 U of enzyme (10,000 U / mL, New England Biolabs, R0631L, lot number: 10150965). The reaction is then incubated at 50° C. for 1 hour at 300 revolutions per minute (rpm).

[0254] The appropriate capture sequence for the DBS site is then added to the plasmid in 20 μmol excess. The reaction is then heated to 90° C. for 2 minutes to allow the nicked strand to separate from the plasmid. The reaction is then slowly cooled to room temperature over 3.5 hours to allow the capture sequence to bind to the nicked sequence, leaving the attachment site open.

[0255] IEX is then used to separate the nicked plasmid from the captured nicked sequence.The fractions containing the peak of interest are then combined and the 1co plasmid is extracted using ethanol (EtOH) precipitation.The pellet is then dried and reconstituted in a buffer of choice.

[0256] Verification results Reporter construct 1 was used for all experiments unless otherwise noted.

[0257] 1. Confirmation of OpC Plasmid Restriction fragment length polymorphism (RFLP) analysis was used to assess the efficacy of double nickase on a 15% TBE-urea denaturing polyacrylamide gel. Before modification of the plasmid, it showed no gel band, but after nickase, the plasmid showed a 38 bp band, consistent with the expected cleavage band size. For RFLP, the plasmid was opened using TelN and then cut further upstream from the nick site with an additional restriction enzyme. This resulted in two smaller fragments (47 and 58 bp) from the nicked top strand and one longer fragment (209 bp) from the bottom strand. In addition, the 38 bp nickase band is present because this analysis was performed before purification by IEX. These bands were consistent with the expected size from the cleavage.

[0258] To observe whether the modified plasmid was still functional, HepG2 cells were transfected with reporter construct 1 of OpC topology using Lipofectamine2000 (catalog number: 11668019, ThermoFisher Scientific, Waltham, MA). Cells were seeded at approximately 2.5 × 104 cells / well one day before treatment. After 48 hours, the fluorescence of the cells due to eGFP expression was imaged (excitation / emission: 488 / 509 nm). The fluorescent signal was quantified using the sum of integrated fluorescence intensity, which allows individual pixels to be represented without bias. The results showed that reporter construct 1 showed a strong distribution of fluorescent signals.

[0259] 2. Confirmation of nicked linear (LN) plasmid For RFLP of the LN plasmid, only an additional restriction enzyme upstream of the nickase site was required because the plasmid had already been treated with TelN. As expected, in addition to the 38 bp band due to the nickase, bands of 47, 58, and 209 bp are present.

[0260] 3. Identification of one open end, one closed end (1co) plasmid RFLP performed on the 1co plasmid showed that before addition of nickase, a 132 bp band representing the removed closed end and a 30 bp band representing the confirmatory cleavage were present as expected. After addition of nickase, RFLPs of 132, 48, 46, and 38 bp bands were produced, all as expected, representing removal of the closed end, a smaller fragment of the nicked strand, and an excised portion of the DNA.

[0261] Example 4: Characterization of auxiliary molecules for complex formation and protection of polynucleotide cargo Nucleases are enzymes that cleave the phosphodiester bonds between nucleotides of nucleic acids. They are often found in DNA repair mechanisms, such as proofreading, Okazaki fragment processing, mismatch repair, base excision repair, nucleotide excision repair, and double-strand break repair. In nucleic acid-based gene delivery methods, nucleases can be potent inhibitors of activity by destroying the payload before it can be transcribed (in the case of DNA) or translated (in the case of ribonucleic acid (RNA)).

[0262] An additional factor to take into account for in vivo delivery is the size of nanocarriers with payload versus nanocarriers without payload. Pore size varies from cell type to cell type, and only substances of the same size are allowed to be taken up. DNA dendrimer nanocarriers have a size of about 60 nm. Depending on the topology, the payload can dramatically increase the particle size. Linearized pDNA previously measured was about 750 nm long after hybridization to DNA dendrimers. To allow delivery to cells with smaller pore sizes, the particles must be reduced or compressed.

[0263] In addition to size, particle size and protection can enhance the durability of the scaffold and cargo. Auxiliary molecules can compact the nanoparticles and protect them from degradative enzymes. These auxiliary molecules can be peptide-based, polymer-based, or hybrids of both to obtain the desired properties.

[0264] Several peptide-based and polymer-based auxiliary molecules were tested, as listed in Table 1 below. Peptide-based auxiliary molecules have a combination of positively charged and neutral residues, which not only allow nucleic acid complexation but also provide protection against nucleases. Polymer-based auxiliary molecules are composed of poly-L-lysine (PLL) of various sizes, which have a high complexation success rate but may show a reduced ability to protect the nucleic acid cargo from degradation by nucleases. In addition, PEG2000 was added to the N-terminus of Exc 1 (SEQ ID NO: 19) to form a hybrid auxiliary molecule (not shown in Table 1). [Table 4]

[0265] The amount of auxiliary molecule used for complex formation and protection of the plasmid construct is determined by the N / P ratio, a formula disclosed herein. The higher the N / P ratio, the more auxiliary molecule is present in the final composition. Once the N / P ratio is determined, the auxiliary molecule and plasmid solutions can be mixed. The solution is mixed with 10x phosphate buffered saline (PBS), which provides salts to aid in complex formation, and nuclease free water (NFW) to fill up the total volume. The auxiliary molecule can be used at a DNA concentration of about 0.05 μg / μL to about 0.5 μg / μL. For example, to achieve an N / P ratio of 3 with a final DNA concentration of 0.5 μg / μL using 300 ug from a plasmid solution with a concentration of 1.0 μg / μL and 1300 μg from an auxiliary molecule solution at 5.0 μg / μL, mix 300 μL of plasmid solution, 260 μL of auxiliary molecule solution, 6 μL of 10xPBS, and 34 μL of NFW to a total volume of 600 μL. The solution is then mixed at 300 rpm for 30 minutes at room temperature.

[0266] To confirm whether the auxiliary molecules were successful in complexing with the plasmid nucleic acid, the final product was run on a 1.1% agarose gel to observe whether free plasmid was still present after the reaction. The results showed that Exc 1 showed partial complexation at N / P ratios of 1 or 2, but complete complexation at N / P ratios of 5 and 8. Exc 2 showed no complexation at N / P ratio of 1 and partial complexation at N / P ratio of 5, while Exc 3 showed no complexation at N / P ratios of 1 or 5. The polymer-based auxiliary molecules function at lower N / P ratios due to their uniformly positive sequences. PLL P1, PLL P2, and PLL P3 all showed complete complexation at N / P ratios as low as 0.5. The hybrid PEG-Exc 1 auxiliary molecule showed complexation at N / P ratios similar to that of Exc 1 alone.

[0267] Nuclease protection was measured using a DNase I assay. Samples were subjected to 5U of DNase I (M030S, New England Biolabs) for 30 minutes at 37°C. This amount was determined by determining the amount of DNase I required to completely degrade 0.5ug of plasmid, which is the maximum amount of DNA that can be processed by the cleanup kit. Samples were then purified using a commercial kit from New England Biolabs (T1030L). After cleanup, samples were run on a 1.1% agarose gel to see if the complex, the plasmid, or both remained. Experiments were performed with Exc 1 complexed with plasmid DNA at N / P ratios of 5 and 8. Plasmid complexed with the transfection agent TurboFect™ (R0531, ThermoFisher Scientific) was also included as a control, as it has been demonstrated to have the ability to complex as well. All three solutions formed complexes with the DNA plasmid, but TurboFect™ was unable to protect the plasmid from degradation. Exc 1 showed little or no degradation at both N / P ratios. Additional experiments with PPL auxiliary molecules show that they exhibit weaker DNase protection. The PEG-Exc 1 hybrid protected comparably to Exc 1, indicating that PEG does not affect the stability of the auxiliary molecule.

[0268] To determine whether the PEG-Exc 1 hybrid auxiliary molecule causes any cytotoxicity, a CellTiter-Glo® luminescent cell viability assay (G7570, Promega) was performed on treated mouse cells (C2C12, CRL-1772, ATCC). Cells were incubated with the auxiliary molecule encapsulation conjugation (nanocarrier, plasmid, targeting moiety, and auxiliary molecule). Dose was based on plasmid mass (0.2 μg and 1.0 μg per well). Samples were tested in quintuplicate. At 0.2 μg per well, there was no statistical decrease in viability with either PEG-Exc 1 or TurboFect™ compared to control cells. At 1.0 μg per well, both PEG-Exc 1 and TurboFect™ affected viability compared to control cells.

[0269] Additional auxiliary molecules can be designed. The number of H1 sequences can be altered to compact the nucleic acid, and their addition can reduce the N / P ratio and overall stabilizer content. Different CPPs, e.g., arginine-8, can be added to vary transfection in both in vitro and in vivo models. Similarly, alternative NLS sequences can be explored to enhance nuclear delivery. Furthermore, targeting peptide sequences can be added to the base stabilizer during synthesis or chemically added via functional groups (e.g., click chemistry, amide, crosslinking). Finally, certain regions of the auxiliary molecule can be made neutral or positive for separation of charge regions for complexation, and new functional groups can be added to the termini or placed throughout the chain. For example, these can include binding sites for small molecules, antibodies, and peptide sequences.

Claims

1. 1. A composition comprising a DNA dendrimer linked to or associated with a targeting moiety and an adapter molecule-cargo polynucleotide complex, the adapter molecule of the adapter molecule-cargo polynucleotide complex comprises a DNA dendrimer binding sequence (DBS) and a cargo binding region (CBR); the cargo polynucleotide of the adapter molecule-cargo polynucleotide complex comprises at least one DNA targeting sequence (DTS), at least one nuclear localization signal sequence (NLS), at least one promoter, and a cargo sequence; the cargo sequence is an miRNA, an siRNA, an antisense RNA, or encodes an antibody, an enzyme, or a protein; the composition has a nitrogen / phosphate (N / P) ratio of 0.5 to 10; The composition.

2. The composition of claim 1 , wherein the adapter molecule is linked to the DNA dendrimer via the DBS, and the DBS comprises a nucleic acid sequence complementary to a nucleic acid sequence of the DNA dendrimer.

3. The DBS is TAGAGGTAACAACTAGCGTACAA (SEQ ID NO: 3), TAGAGGTAACAACTAGCGTACAATTTTTTTTTTT (SEQ ID NO: 4), CCTCAGCTTGTACTCTAGTTGTTACCTCTAATGCTGGACCTCAGC (SEQ ID NO: 22), or CCTCAGCACCCTACAGAGTAACCTAGATTGATCAAACACCTCAGC (SEQ ID NO: 23) The composition of claim 2, comprising the nucleic acid sequence:

4. The composition of claim 1 , wherein the CBR is linked to the cargo polynucleotide by DNA ligation or chemical coupling.

5. The composition of claim 1 , wherein the adapter molecule further comprises a tag, the tag being located between the DBS and the CBR.

6. 2. The composition of claim 1, wherein the adapter molecule further comprises at least one nuclear localization signal sequence (NLS), wherein the at least one NLS is located between the DBS and the CBR or overlaps the DBS and the CBR.

7. The at least one NLS is YPDEVKRKKKKP (SEQ ID NO: 1) or SLLESPFDKPDEVKRKKKPPTSHQSDATAEDDSSSKKK (SEQ ID NO: 2) The composition of claim 6, comprising the amino acid sequence:

8. 7. The composition of claim 6, wherein the at least one NLS further comprises at least one spacer present either before or after the at least one NLS, and the at least one spacer comprises polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic-glycolic acid copolymer (PGLA), a polyglycine sequence with alanine and / or serine residues, or any combination thereof.

9. 2. The composition of claim 1, wherein the adapter molecule further comprises at least one DNA targeting sequence (DTS) and a cell-penetrating peptide sequence (CPP), wherein the at least one DTS is located between the DBS and the CBR or overlaps with the DBS and the CBR, and the CPP is located between the DBS and the CBR or overlaps with the DBS and the CBR.

10. 2. The composition of claim 1, wherein the adapter molecule further comprises at least one cleavage site, wherein the at least one cleavage site is between the DBS and the CBR or overlaps the DBS and the CBR.

11. 2. The composition of claim 1, wherein the adapter molecule further comprises at least one flexible linker, wherein the at least one flexible linker is between the DNS and the CBR, and wherein the at least one flexible linker is selected from the group consisting of polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), polylactic-glycolic acid copolymer (PGLA), (GG)n, (GGGGS)n (SEQ ID NO: 7), or (GGGGA)n (SEQ ID NO: 27), wherein each n is independently 1 to 5.

12. the composition further comprises an adjunct molecule; The auxiliary molecule is ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 12), WRRRGFGRRR (SEQ ID NO: 13), GRKKRRQRRRPQ (SEQ ID NO: 14), PKKKRKV (SEQ ID NO: 15), GLFHAIAHFIHGGWHGLIHGWYG (SEQ ID NO: 16), WEAALAEAEAEALAEHLAEALAEALEALAA (SEQ ID NO: 17), HHHHHHHHHH (SEQ ID NO: 18), (KK)q, where q is 2 to 15; GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVATPKK STKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 19), HHHHHHHHHHATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 20), GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVGRKKRRQRRRPQWRRRGFGRRR (SEQ ID NO: 21), KKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 24), KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 25), or KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 26) The composition of claim 1 , comprising an amino acid sequence selected from:

13. The composition of claim 12 , wherein the auxiliary molecule is linked to the DNA dendrimer.

14. The composition of claim 12 , wherein the auxiliary molecule is not linked to the DNA dendrimer.

15. 2. The composition of claim 1, wherein the targeting moiety is selected from an antibody, a naturally occurring ligand of a receptor or a functional derivative thereof, a vitamin, a hormone, a small molecule mimetic of a naturally occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, any other molecule that specifically binds to a cell surface molecule and triggers endocytosis of the targeting moiety, or any combination thereof.

16. 2. The composition of claim 1, wherein the cargo polynucleotide is selected from a circular polynucleotide (e.g., a plasmid), a nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' ends, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open end and one closed end, wherein the one open end can be at either the 5' or the 3' end.

17. 17. The composition of claim 16, wherein the plasmid comprises a plasmid backbone comprising at least two restriction enzyme recognition sites, at least one promoter, at least one coding sequence encoding at least one molecule of interest, and optionally a DNA dendrimer binding sequence (DBS), and wherein the plasmid is capable of forming cargo polynucleotides with various structures depending on whether the plasmid has one or more 5' ends and 3' ends, or none of them.

18. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

19. 10. A method for delivering a molecule of interest to the nucleus of a target cell, the method comprising contacting the target cell with the composition of claim 1, wherein the targeting moiety binds to the target cell.

20. 10. A method of treating a disease, the method comprising administering to a subject the composition of claim 1 to treat the disease, wherein the targeting moiety binds to a target cell.

Images