Compositions and methods

Abstract

Provided herein are single strands of DNA that allow for the expression of specific proteins or non-coding RNAs in a host cell or tissue. Also provided herein are vectors, lipid nanoparticles, DNA nanostructures, compositions, and vaccines comprising the single strand of DNA provided herein and uses thereof; and methods of making the same.

Description

[0001] Compositions and methods

[0002] Field of the invention

[0003] The present invention is in the field of therapeutic nucleic acids, more particularly in the field of single-stranded DNA (ssDNA) vaccines and gene therapies.

[0004] Background

[0005] Advances in therapeutic nucleic acid technologies have allowed the development of nucleic acid vaccines and gene therapies targeted to a range of genetic and acquired diseases. Established nucleic acid vaccines and gene therapies utilise mRNA or doublestranded DNA (dsDNA), which are typically delivered to a target cell in a vector such as a virus, lipid nanoparticle, or liposome. Translation of the mRNA, or transcription and translation of the dsDNA, produces a polypeptide which may (in the case of a vaccine) be processed and presented as an antigen to a subject's immune system, thereby generating adaptive immunity; or (in the case, for example, of cancer or a genetic disease) may provide a functional heterologous version of an absent or dysfunctional endogenous protein, thereby genetically complementing a pathogenic phenotype. mRNA and dsDNA have been used in vaccination strategies against, for example, COVID-19; and a clinical trial of a non-integrating dsDNA therapy for cystic fibrosis is ongoing.

[0006] Despite their promise, however, mRNA and dsDNA vaccines and gene therapies face significant technical challenges. For example, dsDNA vaccines and therapies are often associated with high cellular toxicity, immunogenicity due to interactions with cGAS and STING pathways, and a risk of genomic integrations, contributing a low safety profile. Because of the inherent instability of mRNA, vaccines and gene therapies based on mRNA must be refrigerated until the point of use, which is disadvantageous in countries which lack appropriate cold chains. Furthermore, mRNAs are rapidly degraded in the body following administration, leading to transient expression of antigens and / or therapeutic polypeptides in vivo. Transient expression from mRNA can require high levels of mRNA to be delivered a patient to induce the robust adaptive immune response required for vaccination; and, in a gene therapy context, requires frequent re-administration to obtain a therapeutic effect.

[0007] There therefore exists a need for improved therapeutic nucleic acids, in particular therapeutic nucleic acid vaccines and gene therapies. Brief description of the invention

[0008] The invention provides compositions and methods that allow for the use of ssDNA molecules in a wide range of in vivo purposes. For example, the single strand of DNA and methods and uses described herein are suitable for use as a vaccine, for example a vaccine against infection with a pathogen or a disease such as cancer. The single strand of DNA and methods and uses described herein are suitable for use in gene therapies, for example by delivering appropriate therapeutic proteins or peptides. The single strand of DNA and methods and uses described herein are also suitable for delivering non-coding RNA to a cell or a subject, for example for RNAi based therapies or CRISPR based gene editing or silencing.

[0009] The single strand of DNA and methods described herein are considered to be particularly useful in context where long lengths of DNA are required, for example in the context of encoding large genes, or tandem arrays of genes; and / or in contexts where large quantities of DNA are required, for example in a short time, such as at the onset of a pandemic. Due to the stability of the single stranded DNA relative to mRNA, the present invention is also considered to be particularly suitable for context in which a cold chain is not appropriate or difficult to achieve.

[0010] Detailed description of the invention

[0011] The invention provides a range of nucleic acids such as a single strand of DNA (ssDNA), methods, compositions and medical uses, as set out below.

[0012] By a single strand of DNA we mean a strand of DNA that is single stranded - i.e. a single strand of DNA that itself is single stranded. As set out elsewhere herein the single strand of DNA may comprise some short regions of self-complementarity but is otherwise not double-stranded.

[0013] The single strand of DNA of the invention may be referred to as a ssDNA, or as a ssDNA construct.

[0014] The invention provides a single strand of DNA that comprises a first target sequence operably linked to a first promoter sequence, wherein the first promoter sequence drives expression of the first target sequence to produce a first RNA molecule either: i) directly from the single strand of DNA; or ii) following replication of the single strand of DNA into the double strand form.

[0015] It will be appreciated that the concept of the invention is a single strand of DNA that ultimately is used as a template for transcription, to produce the first RNA molecule (which as described herein can be a protein coding RNA that can then be translated, or is a non-coding RNA). The skilled person will realise that the sequence of the single strand of DNA can directly be bound by RNA polymerase to instigate transcription (i.e. is the antisense strand); or, the single strand of DNA may itself not comprise the promoter sequence that is recognised by RNA polymerase, and the first target sequence may not itself be transcribed, and may instead require amplification to a double stranded form of the single strand of DNA, where the complementary new strand of DNA comprises the required sequences, i.e. the single strand of DNA maybe the sense or antisense strand with respect to promoter sequences, first target sequences and other sequences, and the disclosure herein should be taken as applying to either a sense or antisense stand. The skilled person will understand therefore that references to particular features, such as a polyA tail are intended to refer to the necessary sequence so as to result in an RNA molecule that comprises a polyA tail, whether the single strand of DNA is sense or antisense with respect to that feature.

[0016] Accordingly in one embodiment the single strand of DNA is an antisense strand with respect to the promoter sequence and the first target sequence. In other embodiments the single strand of DNA is the sense strand with respect to the promoter sequence and the first target sequence.

[0017] The single strand of DNA of the invention may be designed so as to allow expression of the first target sequence in any cell, for example in a eukaryotic cell or a prokaryotic cell, or in an in vitro transcription or in vitro transcription / translation system. Preferable the single strand of DNA of the invention is designed so as to allow expression in a eukaryotic cell, preferably a mammalian cell, preferably a human cell. Accordingly in preferred embodiments the single strand of DNA of the invention is for expression in a eukaryotic cell, for example in a mammalian cell, for example a human cell. The skilled person will recognise the components that are required to ensure expression in a particular context.

[0018] In some embodiments, the single strand of DNA is entirely single stranded and / or is substantially single stranded. By substantially single stranded we mean that the majority of the nucleic acids in the single strand of DNA are not hybridised to other nucleotides within the single strand of DNA. For example a substantially single stranded DNA of the invention may comprise multiple regions of self-complementarity, but wherein the overall single stranded DNA of the invention has a majority of singlestranded, unhybridized nucleotides.

[0019] In some embodiments, the single strand of DNA contains no regions of selfcomplementarity. In other embodiments the single strand of DNA contains no regions of self-complementarity of sufficient length so as to allow internal hybridisation or substantial internal hybridisation. For example, in some embodiments the single strand of DNA contains no regions of self-complementarity: a) that are longer than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 base pairs; and / or b) where the combined length of two regions of the single strand of DNA that make up a particular self-complementary region constitute less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 2% or less than 1% of the total length of the single strand of DNA.

[0020] In some embodiments less than 90%, 85%, 80%, 75%, 70%, 65%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% or less of the nucleotides of the single strand of DNA are involved in selfcomplementarity.

[0021] In some embodiments the single strand of DNA of the invention does not comprise a first region of more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 continuous nucleotides that are complementary to a second region of the ssDNA of more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 continuous nucleotides.

[0022] In some embodiments the first target sequence of the ssDNA has no region that is complementary to a second region of the ssDNA. For example in some embodiments the first target sequence of the ssDNA does not have a region of more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 continuous nucleotides that are complementary to a second region of the ssDNA of more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 continuous nucleotides. In some embodiments, the single-stranded DNA (ssDNA) construct comprises at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000 or more nucleotides that are singlestranded and are not part of a terminal hairpin or loop structure. Preferably, such nucleotides are internal to the sequence or flanked on only one side by a complementary region, such that they remain single-stranded under physiological conditions. These features distinguish the constructs of the present invention from closed linear DNA (clDNA) and doggybone DNA (dbDNA), which typically exhibit complete self-complementarity or are fully constrained by terminal loop structures, thereby lacking such free single-stranded segments. It should be clear that the ssDNA of the invention may also comprise one or more terminal hairpin loops, but where it does, it also comprises the region of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000 or more nucleotides that are single stranded.

[0023] The single-stranded DNA (ssDNA) constructs of the invention are structurally distinct from closed linear DNA (clDNA) and doggybone DNA (dbDNA), which comprise covalently closed, double-stranded DNA fragments joined by terminal loops and share a continuous phosphate backbone across both strands. The ssDNA constructs disclosed herein preferably comprise at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, or up to 100% of their nucleotide sequence in an unpaired single-stranded state under physiological conditions.

[0024] In some embodiments, the single strand of DNA is a single stranded DNA molecule (ssDNA). In some embodiments, the ssDNA comprises: (a) a polynucleotide sequence that comprises a hybridization motif that is capable of hybridizing to a complementary polynucleotide sequence;(b) one or more target sequences that encodes a peptide, a protein, a nucleic acid-based adjuvant or a non-coding RNA; and (c) at least one promoter sequence operable linked to the one or more target sequences, wherein upon hybridization of the complementary polynucleotide sequence to the hybridization motif of the polynucleotide sequence, a level of expression of the one or more target sequences increased, as compared to a level of expression of the one or more target sequences in the absence of the hybridization. In some embodiments, the complementary polynucleotide sequence is present on an oligonucleotide that is not covalently attached to the single stranded DNA molecule. In some cases, the complementary oligonucleotide is present in a region of the single stranded DNA molecule. In some embodiments, the one or more target sequences encode an antigenic protein fused to a peptide sequence that, upon introduction of the ssDNA into a host cell, enhances presentation on a surface of the host cell. As will be understood, the single strand of DNA may hybridise to a further nucleic acid strand, for example a further DNA strand and / or further RNA strand.

[0025] Although in some embodiments the single strand of DNA is intentionally designed so as to not comprise any or substantially any self-complementarity, in other embodiments the single strand of DNA is designed so as to have intentional regions of self-complementarity, since under some circumstances such internal hybridisation has advantages.

[0026] Accordingly, in some embodiments, at least one portion of the single strand of DNA may hybridise to a second portion of the same single strand of DNA. In each case, the single strand of DNA or portion of the single strand of DNA becomes double stranded.

[0027] In some embodiments the single strand of DNA of the invention is designed to have an intentional self complementary region to a second region of the ssDNA. For example in some embodiments the ssDNA of the invention comprises a first region of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or at least 1000 contiguous nucleotides that are complementary to a second region of more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 contiguous nucleotides.

[0028] In some embodiments the single strand of DNA of the invention has a first region of between 10-1000 contiguous nucleotides, optionally between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides that are complementary to a second region of between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150- 500, 200-450, 250-400, or 300-350 contiguous nucleotides.

[0029] In some embodiments it is considered to be useful if a first region of the first promoter hybridises to a second region of the ssDNA. For example in some embodiments the single strand of DNA of the invention comprises a first promoter that has a first region of between 10-1000 contiguous nucleotides, optionally between 20-950, 30-900, 40- 850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides that are complementary to a second region of between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides. In some embodiments it is considered to be useful if a first region of the first promoter hybridises to a region of the first target sequence of the ssDNA. For example in some embodiments the single strand of DNA of the invention comprises a first promoter that has a first region of between 10-1000 contiguous nucleotides, optionally between 20- 950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides that are complementary to a region of the first target sequence of between 20-950, 30-900, 40-850, 50-800, 60- 750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides.

[0030] In some embodiments it is a non-promoter region of the ssDNA that hybridises to a region of the first target sequence of the ssDNA. For example in some embodiments the single strand of DNA of the invention comprises a first region that is between 10- 1000 contiguous nucleotides, optionally between 20-950, 30-900, 40-850, 50-800, 60- 750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides that is complementary to a region of the first target sequence of between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100- 550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides.

[0031] In some embodiments the second region of the single strand of DNA of between 10- 1000, 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides, is a region of the first target sequence, the promoter sequence or any other region of the single strand of DNA.

[0032] Accordingly, in some embodiments, the single strand of DNA comprises at least one region that is double stranded, wherein said double stranded region is generated via hybridisation between at least a first and second region of the same single strand of DNA that are substantially complementary to each other. In some embodiments the single strand of DNA comprises at least one region that is double stranded, wherein said double stranded region is generated via hybridisation between at least a first and second region of the same single strand of DNA that are substantially complementary to each other and: a) wherein the hybridisation between a first and second region of the single stand of DNA enhances expression of the first RNA molecule, for example wherein the first region of the single strand of DNA is in the promoter and the second region of the single strand of DNA is distal to the promoter; or b) wherein the first and second region of the single strand of DNA that are substantially complementary to each other are located at the 5' and the 3' end of the single strand of DNA so that the single strand of DNA is circularised upon hybridisation between the first and second regions.

[0033] By enhances we mean enhances expression relative to an equivalent construct that does not have the double stranded region.

[0034] In some embodiments, the single strand of DNA of the invention comprises a double stranded region, but the double stranded region is formed via hybridisation between the single strand of DNA of the invention and a separate complementary oligonucleotide. The separate oligonucleotide is not part of the single strand of DNA. In this embodiment the single strand of DNA of the invention can be considered to be a complex that comprises the single strand of DNA of the invention and the corresponding complementary oligonucleotide, where the complementary oligonucleotide is hybridised to the corresponding region of the single strand of DNA. Accordingly the invention also provides a nucleic acid complex comprising a single strand of DNA of the invention as described herein, hybridised to a separate complementary oligonucleotide.

[0035] In some embodiments then the single strand of DNA comprises a double stranded region formed via hybridisation between a region of the single stranded DNA and a separate complementary oligonucleotide. In some embodiments the region of the single stranded DNA is a region of the first promoter. In some embodiments the region of the single stranded DNA is a region of the first target sequence. In some embodiments the region of the single stranded DNA is a region of neither the first target sequence or the first promoter.

[0036] In some embodiments the separate complementary oligonucleotide hybridises to only one contiguous region of the single strand of DNA of the invention. In some embodiments the separate complementary oligonucleotide is not considered to be a staple oligonucleotide.

[0037] In some embodiments the single strand of DNA of the invention comprises at least two double stranded regions that are each formed by hybridisation to separate oligonucleotides. In some embodiments the single strand of DNA of the invention comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more double stranded regions, where each double stranded region is formed by hybridisation with individual separate oligonucleotides, i.e. a single complementary oligonucleotides only binds to one defined region of the single strand of DNA of the invention.

[0038] Accordingly the invention also provides a nucleic acid complex that comprises a single strand of DNA of the invention as described herein, hybridised to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more separate complementary oligonucleotides, wherein each oligonucleotide hybridises to only one contiguous region of the single strand of DNA of the invention, i.e a first complementary oligonucleotide hybridises to a first region of the single strand of DNA of the invention; a second complementary oligonucleotide hybridises to a second region of the single strand of DNA of the invention; a third complementary oligonucleotide hybridises to a third region of the single strand of DNA of the invention and so on.

[0039] Preferences for the positions of the regions are set out above.

[0040] In preferred embodiments the complex of the invention does not comprise an oligonucleotide that binds to two or more contiguous regions of the single strand of DNA of the invention, i.e. in preferred embodiments the complex does not comprise one or more staple oligonucleotides.

[0041] In some embodiments the complementary oligonucleotide is at least 5 nucleotides in length, optionally at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 nucleotides in length.

[0042] The skilled person will be aware that a single stranded DNA molecule is expected to experience some non-specific interactions between different regions of the nucleic acid. These are distinct to the specific Watson-Crick base pairing described above in the context of complementarity and hybridisation.

[0043] The skilled person will be aware that certain internal hybridisation events can give rise to the formation of particular secondary structures. Secondary structures in the single strand of DNA (and / or the corresponding RNA product) can impart various properties on the nucleic acid.

[0044] Accordingly in some embodiments the single strand of DNA may comprise a secondary structure. Secondary structures are known to the person skilled in the art. In some embodiments, the single strand of DNA comprises a hairpin, for example a hairpin at the 5' and / or 3' end of the single strand of DNA. For example in some embodiments the ssDNA of the invention is linear with a hairpin structure at one or both ends. In other embodiments the ssDNA is circular and comprises one or more internal hairpin structures.

[0045] In some embodiments, the single strand of DNA comprises a secondary structure so as to: a) increase stability of the single stand of DNA; and / or b) to increase expression from the promoter.

[0046] Expression of the first target sequence is driven from the first promoter, located within the ssDNA. The promoter may be any suitable promoter. For example in preferred embodiments the ssDNA of the invention is for expression of the first target sequence in a eukaryotic cell and so will comprise a suitable promoter, appropriate for the host organism. For example where the first target sequence is for expression in a human cell, the first promoter is a promoter that is able to function in a human cell.

[0047] It may be desirable to direct or restrict expression of a target sequence to a particular cell type. Cell-specific expression of a target sequence may be achieved by selecting a suitable promoter. Accordingly, in some embodiments, the promoter is a promoter capable of driving transcription in a eukaryotic cell, for example in a mammalian cell, for example in a human cell, for example in a dendritic cell, T cell and / or B cell.

[0048] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter.

[0049] The skilled person will understand how to choose a suitable promoter depending on the intended use. For example the promoter may be selected from the group comprising or consisting of: a Cytomegalovirus (CMV) Immediate-Early promoter; Simian Virus 40 (SV40) promoter; CAT promoter; Muscle Creatine Kinase promoter (MCK); tissue or cell-specific promoter; Human Elongation Factor 1 Alpha (EFla) Promoter; Tetracycline-Inducible Promoter; a MiniPromoter; U6 promoter; Hl promoter, CD19 promoter; CD2 promoter; KRT5 promoter; FoxP3 promoter; and CAG promoter.

[0050] In some embodiments, the promoter sequence comprises a Cytomegalovirus (CMV) Immediate-Early promoter sequence, a Simian Virus 40 (SV40) promoter sequence, a CAT promoter sequence, a Muscle Creatine Kinase promoter (MCK), or a tissue or celltype specific promoter sequence. In some instances, the cell-type specific promoter sequence comprises a dendritic cell promoter sequence, a B cell promoter sequence or a T cell specific promoter sequence.

[0051] The promoter may be naturally occurring. The promoter may be engineered, and based on a naturally occurring promoter. The promoter may be a synthetic promoter and have no direct relationship to any naturally occurring promoter.

[0052] For example, in some embodiments, the promoter is selected from the group comprising or consisting of: Cytomegalovirus (CMV) Immediate-Early promoter; Simian Virus 40 (SV40) promoter; Muscle Creatine Kinase promoter (MCK); or a tissue or cell-type specific promoter, for example dendritic cell, B cell and / or T cell specific promoter. These promoters are considered to be particularly useful in the context of vaccines, for example for the delivery of antigens to dendritic cells, B cells and T cells.

[0053] In some embodiments, the promoter sequence comprises a Human Elongation Factor 1 Alpha (EFla) promoter sequence, a Tetracycline-Inducible promoter sequence, or a MiniPromoter sequence. In some embodiments, the promoter is selected from the group comprising or consisting of: Human Elongation Factor 1 Alpha (EFla) Promoter; Tetracycline-Inducible Promoter; CAT promoter; a MiniPromoter; or a tissue or celltype specific promoter. These promoters are considered to be particularly useful in the context of gene therapy. For example for delivering therapeutic proteins to target cells for example target mammalian cells.

[0054] In some embodiments, the promoter sequence comprises a U6 promoter sequence, an Hl promoter sequence, or a CMV promoter sequence. In some embodiments, the promoter is selected from the group comprising or consisting of: U6 promoter; Hl promoter; or a CMV promoter; or a tissue or cell-type specific promoter. These promoters are considered to be particularly useful for the expression of non-coding RNAs, i.e. where the first RIMA molecule is a non-coding RNA.

[0055] The promoter may be a strong promoter or a weak promoter. In some embodiments the promoter is a strong promoter, for example is selected from the group comprising or consisting of the CMV promoter, SP6 promoter or T7 promoter. In some embodiments, the promoter sequence comprises a CMV promoter sequence, a SP6 promoter sequence, or a T7 promoter sequence.

[0056] In some embodiments the promoter is selected from the group comprising or consissint of: Cytomegalovirus (CMV) Immediate-Early promoter; Simian Virus 40 (SV40) promoter; CAT promoter; Muscle Creatine Kinase promoter (MCK); Human Elongation Factor 1 Alpha (EFla) Promoter; Tetracycline-Inducible Promoter; a MiniPromoter; U6 promoter; Hl promoter, CD19 promoter; CD2 promoter; KRT5 promoter; CAT promoter; FoxP3 promoter; and CAG promoter, tissue specific promoter; cell type specific promoter; dendritic cell type specific promoter; B cell specific promoter; T cell specific promoter.

[0057] In some embodiments, the single strand of DNA further comprises a first enhancer sequence. Enhancers are well known to the skilled person and aid in increasing expression of the first target sequence from the promoter. Enhancers can be cell-type specific. For example in some embodiments the enhancer enhances expression of the first target sequence in a specific cell type. Enhancers can act as an enhancer when in the single strand of DNA itself, or can act as an enhancer once transcribed in to RNA. Enhancers can be located in the 5'UTR, 3'UTR or both 5' and 3' UTR. Accordingly in one embodiment the single strand of DNA or the first RNA molecule comprises at least one enhancer sequence is located in the 5'UTR, 3'UTR or in the 5'UTR and 3'UTR.

[0058] In some embodiments the single strand of DNA comprises a sequence that when transcribed into RNA is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments the single strand of DNA or the first RNA molecule comprises a WPRE. In some embodiments the WPRE is located in the 3'UTR of the RNA molecule.

[0059] The enhancer may be any enhancer. Examples of suitable enhancers include: a) viral enhancers, for example the 72-bp enhancer element from Simian Virus 40 (SV40), the enhancer from CMV, and / or the enhancer from Rous sarcoma virus; b) Ubiquitous / Constitutive Enhancers, for example the enhancer from the human elongation factor 1 alpha (EFla) enhancer; c) tissue / Cell-Type Specific Enhancers, for example the muscle creatine kinase (MCK) enhancer for muscle cells; d) Super-enhancers associated with genes defining cell identity; e) Synthetic / Designed Enhancers; and / or f) inducible enhancers, for example an inducible enhancer that response to hormones or small molecules.

[0060] In some embodiments, an enhancer sequence comprises a viral enhancer sequence, a ubiquitous / constitutive enhancer sequence, a tissue specific enhancer sequence, a celltype specific enhancer sequence, a super-enhancer sequence, a synthetic / designed enhancer sequence or an inducible enhancer sequence. For example the promoter region may also contain the CMV enhancer and / or CMV intron. Where the promoter is the EF-la promoter the promoter region may also comprise the EFl-a intron.

[0061] In some embodiments it is considered to be useful if the region of the ssDNA that is the enhancer hybridises to a second region of the ssDNA. For example in some embodiments the single strand of DNA of the invention comprises a region that is an enhancer and that has a region of between 10-1000 contiguous nucleotides, optionally between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides that are complementary to a second region of between 20-950, 30-900, 40-850, 50-800, 60- 750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides.

[0062] In some embodiments it is considered to be useful if the region of the ssDNA that is the enhancer hybridises to a region of the first target sequence of the ssDNA. For example in some embodiments the single strand of DNA of the invention comprises a region that is an enhancer and that has a region of between 10-1000 contiguous nucleotides, optionally between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80- 650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides that are complementary to a region of the first target sequence of between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150- 500, 200-450, 250-400, or 300-350 contiguous nucleotides.

[0063] In some preferred embodiments the single strand of DNA of the invention comprises a Kozak sequence around the start codon site.

[0064] In some embodiments the single strand of DNA or first RNA molecule comprises one enhancer. In other embodiments the single strand of DNA or first RNA molecule comprises at least two or more enhancers. For example in some embodiments the single strand of DNA or first RNA molecule comprises at least two enhancers of any of (a)-(f) above, for example a combination of the SV40 enhancer with a muscle-specific enhancer for muscle targeting.

[0065] The single strand of DNA of the invention has many uses, including therapeutic uses. To achieve some of these uses, in some embodiments the single strand of DNA has to be delivered to a particular cell. Once inside the cell the single strand of DNA is transported to the nucleus where in some embodiments it is replicated to a double strand form. The promoter then drives transcription, and where the first target sequence is a protein coding sequence, the transcript is translated along with other mRNAs in the cell. In some instances, the ssDNA when introduced into a host cell encodes one or more target sequences, optionally including a protein, a non-coding RNA, or a coding RNA.

[0066] To aid in this process, in some embodiments the single strand of DNA comprises a nuclear localisation signal. The nuclear localisation signal may in some preferred embodiments be a DNA Targeting Sequence (DTS). For example in some embodiments the DTS is the SV40 DTS, or the 81 base pairs of Sox2 regulatory region 2 (SRR2) or functional variant thereof.

[0067] The single stand of DNA of the invention may comprise more than one nuclear localisation signal, for example more than one DTS. In some embodiments the ssDNA of the invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nuclear localisation signals, for example in some embodiments the ssDNA of the invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more DTSs. The more than one DTS may be multiple copies of the same DTS, or may be copies of different DTS.

[0068] In other embodiments the single strand of DNA comprises inverted-terminal repeat like hairpin motifs.

[0069] It will be appreciated that disclosures herein can be combined, so that for example in some embodiments the single strand of DNA comprises substantially no internal complementarity but does comprise inverted-terminal repeat like hairpin motifs to aid in expression.

[0070] Similarly, the single strand of DNA may in some embodiments be linear, but comprises caps formed by hairpin loops at the 5' end and / or the 3' end.

[0071] In some embodiments the single strand of DNA is linear and does not comprise any hair pin loops.

[0072] In some embodiments the single strand of DNA is circular. There are various means for producing circular single stranded DNA. For example in some embodiments the single strand of DNA is circularised via complementary base pairing of the 5' end and the 3' end of the single strand of DNA. Accordingly in some embodiments the single strand of DNA has a 5' and 3' terminal sequence that are complementary to one another or substantially complementary to one another sufficient to allow hybridisation.

[0073] In the present examples, circular ssDNA (cssDNA) is produced through a scalable, fermentation-based process in Escherichia coli. During bacterial culture, the ssDNA is packaged into phage-like particles. After harvest, the particles are lysed and the cssDNA is purified. This method does not rely on complementary base pairing between the 5' and 3' ends for circularization. The cssDNA can be used directly in its circular form - usually retaining a small backbone — or optionally processed post-purification via site-specific cleavage through DNAzymes to yield a linear version without backbone and could be re-circularised as described in the paragraph.

[0074] Other means of producing circular ssDNA (cssDNA) are by enzymatic or chemical ligation methods, often involving short synthetic DNA splints or using a "one-pot" cyclization scheme. One approach involves using T4 DNA ligase with a complementary ssDNA splint to juxtapose reactive ends of precursor segments for ligation. Another method involves cyanogen bromide and a triple-helical splint complex.

[0075] In some embodiments the single strand of DNA is linear, and is capable of circularising when in a cellular environment, for example a eukaryotic cell, for example a mammalian cell, for example a human cell; and or is capable of circularising in an in vitro transcription translation system, for example in a mammalian in vitro transcription translation system.

[0076] In some embodiments the single strand of DNA is linear and does not circularise when in a cellular environment.

[0077] Since in some preferred embodiments the single strand of DNA is for expression in a cell, in some instances the single strand of DNA comprises modifications that aid in expression, or for example render the nucleic acid more stable in the cellular context, or that help to make the single strand of DNA less immunogenic or less toxic. For example in some embodiments the single strand of DNA is modified in such a way so as to enhance expression in a mammalian cell, for example a human cell.

[0078] In some embodiments the modification is to reduce immunogenicity, for example reduce immunogenicity in a mammalian cell. In some instances the modification is methylation. In some embodiments the ssDNA of the invention is less immunogenic, for example less immunogenic to a mammal such as a human or mouse relative to the immunogenicity of an equivalent dsDNA. Immunogenicity can be determined my monitoring the expression of cytokines such as IFN-y, IL-6, and / or TNF-a in response to administration. In some embodiments the ssDNA of the invention is less immunogenic, for example less immunogenic to a mammal such as a human or mouse relative to the immunogenicity of an equivalent dsDNA as determined by a reduced expression of one or more cytokines, for example one or more of IFN-y, IL-6, and / or TNF-a in response to administration of the ssDNA of the invention relative to the expression of the same one or more cytokines in response to administration of an equivalent dsDNA construct.

[0079] In some embodiments the ssDNA is not modified to make it less immunogenic, but is regardless less immunogenic that dsDNA.

[0080] In some embodiments the ssDNA of the invention is less toxic than the equivalent dsDNA construct. For example in some embodiments the single stranded DNA constructs of the invention demonstrate reduced cellular toxicity, for example reduced toxicity for example reduced liver toxicity relative to a corresponding dsDNA construct. In some embodiments the reduced toxicity is a reduced toxicity or reduced liver toxicity as determined by reduced expression levels of ALT (alanine transaminase) and / or AST (aspartate transaminase) relative to the expression levels of ALT and / or AST when administered an equivalent dsDNA construct. ALT and AST are enzymes found in the liver cells. Elevated levels of ALT and AST in the blood indicates liver damage or disease. In some embodiments the ssDNA of the invention results in reduced toxicity in mice, for example female BALB / c mice, as demonstrated by a reduced level of ALT and / or AST relative to the expression level of ALT and / or AST when administered an equivalent dsDNA construct at day 18 following administration of the construct.

[0081] In some embodiments the ssDNA of the invention is more stable, for example is stable at room temperature, for a longer period of time that an equivalent dsDNA and / or RNA. In some embodiments the modification is to make the single strand of DNA act as an adjuvant. For example non-methylated DNA is able to act as an adjuvant, so in some embodiments the modification is de-methylation.

[0082] The ssDNA may be found episomally in the cell, and may comprise sequences that aid in maintaining the ssDNA in the cell; or the ssDNA may comprise sequences that aid in integration into the cell genome.

[0083] In some embodiments the ssDNA is intended to be integrated into the host genome and comprises sequences that aid in genome integration. One means of integration makes use of the transposon system. Accordingly in some embodiments the ssDNA of the invention comprises two inverted repeats that are recognised by a transposase enzyme, for example inverted repeats from a transposon system. Exemplary transposon systems include Piggyback and Sleeping Beauty. In some embodiments then the ssDNA of the invention comprises two inverted repeats from the Piggyback transposon system. In some embodiments then the ssDNA of the invention comprises two inverted repeats from the Sleeping Beauty transposon system. In these instances, to allow for transposition, the relevant transposase is required. The transposase enzyme may be encoded with a target sequence in the ssDNA of the invention and which comprises the relevant inverted repeats. Alternative the transpose may be provided to the cell separately, for example via a plasmid or direct exposure of the cell to transposase protein.

[0084] The skilled person will appreciate how to arrange the two inverted repeats such that the intermediate sequence is the sequence that will be transposed into the genome.

[0085] In some other embodiments the ssDNA is not for genomic integration and does not comprise sequences for genomic integration.

[0086] The skilled person will appreciate that in some embodiments the single strand of DNA of the invention is substantially single stranded, but comprises small regions of complementary sequences that can hybridise to form short double stranded regions. Such single stranded DNA constructs of the invention are still considered to be substantially single stranded.

[0087] In some embodiments the target sequence is expressed in the cell episomally. In some embodiments the ssDNA comprises sequences that aid in episomal maintenance, such as an episomal maintenance element such as a scaffold / matrix attachment region (S / MAR). Scaffold matrix attachment regions are known and any S / MAR may be used with the ssDNA. In some embodiments the S / MAR is selected from the S / MAR from ApoB and / or IFN-beta. In some embodiments, the episomal maintenance element is not immunogenic and / or does not induce an immune response.

[0088] In some embodiments the single strand of DNA of the invention is not to be maintained episomally in the cell, and the single strand of DNA does not comprise an episomal maintenance element.

[0089] It is considered to be advantageous in some situations to enable the expression of multiple proteins or peptides, and / or multiple non-coding RNAs within the same cell or tissue. An advantage of the present invention is that it is possible to produce large amounts of single strand DNA, and large amounts of long read single strand DNA (see for example WO 2018054571). The ability to produce long strands of single strand DNA opens up the possibility of encoding multiple genes / non-coding RNAs on the same physical single strand DNA molecule; and / or encoding large proteins or large noncoding RNA for delivery into a subject for example for therapeutic purposes. Since the ssDNA of the invention does not need to be packaged into a capsid of a restricted size, there is considered to be no limit to the length / size of the ssDNA that can be made and delivered to a cell.

[0090] Accordingly in one embodiment the single strand of DNA of the invention additionally comprises a second target sequence, or a third, fourth, fifth, sixth, seventh, eighth, ninth or tenth or more target sequence. The single strand of DNA may comprise any number of target sequences. The target sequences may all be different, or at least two or more of the target sequences may be the same. For example in some embodiments: each target sequence is the same as the first target sequence; or the single strand of DNA comprises at least two target sequences that are different to each other. In some embodiments, the ssDNA encodes one or more target sequences. In some cases, the one or more target sequences are present in any order or orientation.

[0091] The target sequences may be present in the single strand of DNA in any order or orientation.

[0092] In some embodiments at least two of the target sequences are operably linked to different promoters. Preferably the multiple target sequences that present in the single strand of DNA are arranged in a tandem array. In some embodiments two or more of the target sequences present in the single strand of DNA are arranged in a tandem array. In preferred embodiments all of the target sequences present in the single strand of DNA are arranged in the same orientation so that transcription from a single promoter located at one end of the DNA (for example the 5' end) is able to drive transcription through all of the target sequences from a single promoter to produce a single transcript.

[0093] Accordingly in some embodiments all of the target sequences are present in the single strand of DNA in a tandem array. In some embodiments, the one or more target sequences are present in a ssDNA in a tandem array. In some embodiments all of the target sequences present in single strand of DNA in a tandem array are operably linked to the same promoter. In some embodiments, the one or more target sequences are present in a tandem array, and are operably linked to the same promoter sequence.

[0094] In preferred embodiments then where the single strand of DNA comprises multiple target sequences, the target sequences are transcribed into a single RNA molecule.

[0095] It will be appreciated that in these scenarios, to allow initiation of translation from each of the target sequences (in scenarios where the target sequence is a protein coding sequence), the RNA molecule may comprise an internal ribosome entry site (IRES) that is present 5' to each target sequence (each protein coding target sequence), so that each target sequence is translated, for example wherein the IRES is an IRES from encephalomyocarditis virus (EMCV) or poliovirus. Since the RNA molecule is transcribed from the single strand of DNA of the invention, the single strand of DNA of the invention also comprises sequences that when transcribed into RNA are capable of acting as an IRES.

[0096] In addition to IRES, where any one or more target sequences is a protein coding sequence that requires translation, the skilled person will understand that appropriate start and stop codon are also necessary.

[0097] In some instances, spacer sequences are present between the IRES and one or more target sequences.

[0098] As well as or instead of using one or more IRES, the skilled person may transcribe the target sequences as one long standard transcript, but which includes sequences such as the 2A peptide sequence (for example the P2A sequence, T2A sequence, E2A sequence and F2A sequence). 2A peptides are a class of 18-22 aa-long peptides, which can induce ribosomal skipping during translation of a protein in a biological cell. These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. 2A peptides can be introduced artificially to help generate polyproteins from a single ORF, by causing the ribosome to fail at making a peptide bond, and then resume translation, producing the second, separate protein. See for example Liu et al 2017 Nature 7 Article 2193; and Donnelly et al 2001 Journal of General Virology 82 Analysis of the aphthovirus 2A / 2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'.

[0099] Regardless of how many target sequences are present in the single strand of DNA of the invention, proper transcription requires a transcription terminator.

[0100] Of course, the single strand of DNA of the invention may comprise multiple promoter / target sequence units. For example may comprise a first promoter associated with a first set of target sequences (that may be one or more target sequences) target sequence and a second promoter associated with a second set of target sequences (that may be one or more target sequences). Multiple promoters can then be used to achieve differential effects. For example one promoter may be a constitutive promoter whilst a second promoter may be an inducible promoter. The promoters may interact via expressed inducers or repressors to build up complex genetic circuits using logic gates.

[0101] As set out elsewhere, the single strand of DNA of the invention may correspond to the sense or antisense strand of a particular sequence. For example in some instances, RNA polymerase is able to directly associate with the single strand of DNA of the invention and initiate transcription. In other cases RNA polymerase is not able to associate directly with the single strand of DNA of the invention, but is able to associate with the corresponding complementary strand which will be produced following replication of the single strand of DNA, for example replication in the nucleus of a eukaryotic cell. The skilled person will know how to design the various component parts depending on which strand is being employed.

[0102] As set out above, an advantage of the present invention is the ability to produce and package large amounts of nucleic acid sequence that can code for large proteins, large non-coding RNAs, multiple copies of the same protein / non-coding RIMA and / or multiple copies of different proteins / non-coding RNAs.

[0103] Accordingly in some embodiments the single strand of DNA is:

[0104] At least 500 nucleotides, or at least 1000 nucleotides or at least 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, 10000, 10250, 10500, 10750, 11000,

[0105] 11250, 11500, 11750, 12000, 12250, 12500, 12750, 13000, 13250, 13500, 13750,

[0106] 14000, 14250, 14500, 14750, 15000, 15250, 15500, 15750, 16000, 16250, 16500,

[0107] 16750, 17000, 17250, 17500, 17750, 18000, 18250, 18500, 18750, 19000, 19250,

[0108] 19500, 19750, or at least 20000 nucleotides in length; and / or

[0109] Between 1000 and 20000, 1250 and 19750, 1500 and 19500, 1750 and 19250, 2000 and 19000, 2250 and 18750, 2500 and 18500, 2750 and 18250, 3000 and 18000, 3250 and 17750, 3500 and 17500, 3750 and 17250, 4000 and 17000, 4250 and 16750, 4500 and 16500, 4750 and 16250, 5000 and 16000, 5250 and 15750, 5500 and 15500, 5750 and 15250, 6000 and 15000, 6250 and 14750, 6500 and 14500, 6750 and 14250, 7000 and 14000, 7250 and 13750, 7500 and 13500, 7750 and 13250, 8000 and 13000, 8250 and 12750, 8500 and 12500, 8750 and 12250, 9000 and 12000, 9250 and 11750, 9500 and 11500, 9750 and 11250, 10000 and 11000, 10250 and 10750 nucleotides in length.

[0110] In some preferred embodiments the single strand of DNA is: at least 5000 nucleotides in length. In other preferred embodiments the single strand of DNA is at least 7000 nucleotides in length. In other preferred embodiments the single strand of DNA is a least 9000 nucleotides in length or at least 10,000 nucleotides in length.

[0111] The single strand of DNA of the invention has therapeutic uses, since it can be used to deliver antigens or therapeutic proteins (for example) to a subject or a cell in need thereof. Accordingly in preferred embodiments the single strand of DNA is designed so as to allow expression of the first target sequence to produce a first RNA molecule in eukaryotes, for example in mammals, for example in humans. As set out above, promoters, terminators etc can be chosen by the skilled person to allow transcription and downstream translation (where required) in a eukaryote for example a mammal, for example a human.

[0112] In some embodiments the single strand of DNA is used to produce a protein or a peptide. As set out above the single strand of DNA may comprise multiple target sequences and some in some embodiments where the single strand of DNA does comprise multiple target sequences, at least one of the target sequences may be a sequence that encodes a protein or peptide.

[0113] The first RIMA molecule may be a transcript of a protein or peptide, i.e. the first RNA is capable of being translated into a protein or peptide, i.e. the first RNA is a protein coding RNA.

[0114] As set out above, the single strand of DNA may be sense or antisense with respect to any transcript that is intended to be produced. Accordingly in some embodiments the first target sequence is: a) an antisense strand that encodes a first protein or peptide and is able to be directly transcribed to produce the first RNA molecule; or b) a sense strand that is able to be transcribed to produce the first RNA molecule once the single strand of DNA is replicated to produce the antisense strand.

[0115] Reference to the first target sequence being a protein or peptide encoding sequence is intended to capture both the sense and antisense embodiments that result in the production of said protein or peptide.

[0116] Where the first target sequence is a protein or peptide encoding sequence, the first target sequence comprises one or more sequences that when transcribed into the first RNA molecule form a 5'UTR, and / or 3'UTR and / or a polyA tail.

[0117] The 5'UTR and / or 3'UTR may comprise sequences that form RNA secondary structures that regulate gene expression in eukaryotes, for example wherein: a) the 5'UTR comprises: i) one or more stable hairpin and / or stem-loop structures to downregulate gene expression; and / or ii) an internal ribosome entry site (IRES), for example wherein the IRES is an IRES from encephalomyocarditis virus (EMCV) or poliovirus; and / or b) the 3'UTR comprises: i) one or more stem-loop structures and / or AU rich elements; and / or ii) one or more miRNA binding sites.

[0118] In some embodiments the first protein or peptide is an antigenic protein or peptide. In some embodiments the first protein or peptide comprises an antigenic protein or peptide, for example the protein or peptide itself may not be antigenic until processed by a cell to produce an antigenic fragment. In some embodiments, the one or more target sequences encode an antigenic protein. In some embodiments, the one or more target sequences further encodes a peptide sequence that is covalently linked to the antigenic protein.

[0119] Antigenic proteins or peptides are presented (at least in mammals such as humans) to the immune system via complex formation with proteins of the major histocompatibility proteins (MHC). Major histocompatibility complex (MHC) proteins are crucial components of the immune system that play a key role in recognizing foreign molecules and initiating immune responses. MHC proteins are glycoproteins found on the cell surface. They are categorized into two main classes: MHC Class I: Found on the surface of almost all nucleated cells. MHC Class 1 presents peptides from within the cell) to cytotoxic T cells (CD8+ T cells). This is essential for the immune system to recognize and destroy infected or cancerous cells; MHC Class II: Primarily expressed on antigen- presenting cells (APCs) such as dendritic cells, macrophages, and B cells. MHC Class II presents peptides from outside the cell to helper T cells (CD4+ T cells). This is crucial for the activation of T cells and the coordination of the immune response.

[0120] Different processing routes can direct a particular protein or peptide for presentation via MHC I or MHC II, for example ubiquitination tends to direct the presentation via MHC I, whereas targeting to the lysosome directs presentation via MHC II.

[0121] Other proteins that are not antigens, such as receptors, require localisation to the cell surface. In these instances too in some embodiments the single strand of DNA may comprise a signal peptide, a short peptide sequence that is fused to the target protein / antigen and that aids in direction of the protein to the endoplasmic reticulum and / or cell surface. Accordingly any of the single strand of DNA of the invention that encodes a target sequence that encodes a protein or peptide may also comprise a signal peptide sequence, arranged so as to be expressed as part of the target protein or peptide.

[0122] In some embodiments then the first protein or peptide that may be an antigenic protein or peptide or may comprise an antigen protein or peptide, or that may be a therapeutic protein or for example other cell surface protein or peptide comprises one or more sequences to enhance presentation of the antigen protein or peptide or fragments thereof, or enhance direction of the protein or peptide to the endoplasmic reticulum or cell surface, for example: a) presentation by the major histocompatibility proteins (MHC), for example MHC I and / or MHC II; and / or b) presentation by antigen presenting cells, for example dendritic cells, macrophages and / or B cells; and / or c) localisation to the cell surface.

[0123] The one or more sequences to enhance presentation of the first protein or peptide fragment or localisation on the cell surface may be: a) a ubiquitin sequence; b) an Endoplasmic Reticulum (ER) signal peptide, for example the signal peptide from tissue plasminogen activator (tPA); c) the Lysosome-Associated Membrane Protein 1 (LAMP-1); d) a chemokine ligand, for example XCL1; and / or e) the CD28 leader peptide.

[0124] The ubiquitin sequence and the ER signal peptide direct the protein or peptide for presentation via MHC I. Fusion of the antigen with ubiquitin can target it for degradation by the proteasome, enhancing the generation of peptide fragments for presentation via MHC class I molecules.

[0125] The LAMP-1 protein can be fused to the target antigenic protein or peptide to direct them to the lysosomal pathway, enhancing presentation via MHC class II molecules and stimulating helper T cell responses.

[0126] Fusion of antigens with chemokines like XCL1 can target the antigen to specific dendritic cell subsets, enhancing antigen presentation and immune response.

[0127] In some embodiments the first protein or peptide is a protein or peptide present in a pathogen, for example wherein the pathogen is a bacterial pathogen, viral pathogen, fungal pathogen or a protist pathogen. Such embodiments have clear uses in the context of vaccines to prevent or treat infection with a pathogen, as described herein. The protein or peptide present in a pathogen may be any protein or peptide present in a pathogen. Preferably the protein or peptide present in a pathogen is an antigen protein or peptide, or is a protein or peptide that comprises an antigen protein or peptide fragment, for example following intracellular processing.

[0128] In some embodiments the first protein or peptide is a disease-causing or disease- associated protein or peptide, such as a cancer antigen or a cancer neoantigen, or an autoimmune antigen or a neurological antigen, or is a protein or peptide that comprises a cancer antigen or cancer neoantigen, or comprises an autoimmune antigen or comprises a neurological antigen for example following intracellular processing. Such embodiments have clear uses in the context of vaccines to prevent or treat diseases, such as treat or prevent cancer or metastasis, treat or prevent autoimmune diseases or treat or prevent neurological diseases, as described herein. The protein or peptide present that is a cancer antigen or cancer neoantigen may be any protein or peptide that is a cancer antigen or cancer neoantigen.

[0129] In the context of proteins or peptides from a pathogen, the protein or peptide may be: a) a viral antigen, for example i) an influenza antigen, for example Influenza Hemagglutinin, for example from H5Nx strains; ii) a coronavirus antigen, for example from SARS-CoV-2, SARS-CoV, HCoV NL63, HCoV HKU1, or MERS-CoV; for example a spike protein, for example the spike protein from any one or more of from SARS-CoV-2, SARS-CoV, HCoV NL63, HCoV HKU1, or MERS-CoV; iii) a HIV antigen, for example HIV-1 ENV or HIV-1 GAG; or iv) a Hepatitis B antigen, for example Hepatitis B Surface Antigen (HBsAg); b) a bacterial antigen, for example i) an antigen from Mycobacterium tuberculosis, for example

[0130] Ag85 Complex, for example from any of Ag85A, Ag85B, Ag85C); ESAT- 6; CFP-10 ii) an antigen from Bacillus anthracis, for example Protective Antigen (PA); iii) an antigen from Clostridium tetani, for example tetanus toxin (TeNT) iv) an antigen from Escherichia coli, for example Colonization Factor Antigen I (CFA / I); v) Salmonella enterica, for example Flagellin (FliC), Invasin Proteins (e.g., SipA, SipC); vi) Streptococcus pneumoniae, for example Pneumolysin (Ply), PspA (Pneumococcal Surface Protein A); vii) Helicobacter pylori, for example Urease Subunits (UreA, UreB); viii) Listeria monocytogenes, for example Listeriolysin O (LLO); ix) Shigella spp., for example Ipa Proteins (e.g., IpaB, IpaC); x) Vibrio cholerae, for example Cholera Toxin (CT), for example the B subunit of cholera toxin (CTB); c) a fungal antigen, for example i) Candida albicans, for example Agglutinin-like sequence 3 (Als3), Secreted aspartyl proteinases (Sap), Heat shock protein 90 (Hsp90), Hyphal wall protein 1 (Hwpl), or Enolase (Enol); ii) Aspergillus fumigatus, for example Aspf3 (Allergen), Catalase, Gell (1,3-p-glucanosyltransferase), or Pmp20 (Putative Plasma Membrane Protein); iii) Cryptococcus neoformans, for example Glucosylceramide (GlcCer), Mannoproteins (MP98, MP88), Phospholipase Bl (Plbl), or Capsular polysaccharide (GXM); iv) Coccidioides posadasii / immitis, for example Antigen 2 / PRA (Prolinerich antigen), Chitinase, Urease, Fba (Fructose-bisphosphate aldolase), v) Histoplasma capsulatum, for example Histone 2B (H2B), Heat shock protein 60 (Hsp60), Catalase B (CatB), Yps3 (Yeast protein 3); vi) Paracoccidioides brasiliensis, for example Glycoprotein 43 (Gp43) peptide 10 (PIO), Paracoccin, Triosephosphate isomerase (Tpi), vii) Pneumocystis jirovecii, for example Major surface glycoprotein (Msg), Kexin, Monosaccharide transporter (Mst); and / or g) a protist antigen.

[0131] The single strand of DNA of the invention may comprise multiple target sequences each encoding different antigens from different strains of the same pathogen species, for example the single strand of DNA of the invention may allow for the expression or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different antigens from different influenza strains.

[0132] In the context of a first protein or peptide that is a disease-causing or disease- associated protein or peptide, such as a cancer antigen or a cancer neoantigen, or an autoimmune antigen or a neurological antigen, or is a protein or peptide that comprises a cancer antigen or cancer neoantigen, or comprises an autoimmune antigen or comprises a neurological antigen : the cancer antigen may be NY-ESO-1, MAGE-A3, HER2 / neu; or PSA; the Autoimmune Disease Antigen may be insulin, Myelin Basic Protein (MBP); and / or the Neurological Disease Antigen may be Amyloid Beta (A ) or Tau Protein. A mutant form of ubiquitin (e.g., G76V) can be used to prevent deubiquitination and stabilize the fusion protein.

[0133] In addition to an antigenic component, vaccine formulations also often comprise an adjuvant. Adjuvants are essential components of many vaccines, enhancing the immune response to the antigen, modulating the type of immunity generated, and allowing for lower doses of antigens to be used effectively. Some adjuvants are inorganic, such as alum adjuvants, such as aluminium hydroxide or aluminium phosphate. However some adjuvants are of a type that can be expressed by a cell, for example are protein-based or nucleic acid based. In some embodiments therefore it is possible for the single strand of DNA of the invention to encode one or more adjuvants as set out here. Accordingly in some embodiments one or more of the target sequences of the single strand of DNA of the invention encodes a protein or nucleic acid based adjuvant. In preferred embodiments the single strand of DNA encodes both one or more antigenic proteins or peptides, or proteins or peptides that comprise an antigenic portion, and also encode one or more protein or nucleic acid based adjuvants.

[0134] The adjuvant may be any protein or nucleic acid based adjuvant. Exemplary adjuvants that are considered to be suitable for use in the invention are: a) Bacterial protein adjuvants, for example FomA porin from Fusobacterium; MOMP from Shigella flexneri, Porin from Shigella dystenteriae; OmpU from Vibrio cholerae; PorB from Neisseria meningitidis; OmpC and OmpF from Samlonella thyphi, ESAT-6 from Mycobaceterium tuberculosis; rBCSP31 from Brucella abortus; DnaJ- AA146Ply from Steptococcus spneumonia; Endopeptidase O from Steptococcus pneumoinae; Cholera toxin from Vibrio cholerae; B-pentamers of LT-IIa and LT-IIb from E. coli; 0mpl6 from Brucella abortus; BLS from Brucella spp. GrpE from M. tuberculosis; RpfE from M. tuberculosis; Rv0652 from M. tuberculosis; HBHA from from M. tuberculosis; P97 protein from Mycoplasma hyopneumoniae; Flagellin from Salmonella species; Entolimod (CBLB502) optimised from Salmonella flagellin; b) One or more cytokines, for example wherein the cytokine is selected from the group comprising or consisting of Interleukin-12 (IL-12), Interleukin-2 (IL-2), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF); c) one or more cytokines, for example wherein the chemokine is CCL21 (Secondary Lymphoid Tissue Chemokine, SLC); d) one or more co-stimulatory molecules, for example wherein the costimulatory molecule is CD40 Ligand (CD40L), or B7-1 (CD80) and B7-2 (CD86); e) one or more Toll-Like Receptor (TLR) Ligands, for example wherein the Toll- Like Receptor (TLR) Ligand is CpG Oligodeoxynucleotides (CpG ODNs) f) Flt3 Ligand (Flt3L); and / or g) Heat Shock Proteins (HSPs) or any combinations thereof.

[0135] As set out above, the single strand of DNA of the invention is considered to be useful in the context of vaccination against infections or diseases such as cancer, autoimmune disease and neurological diseases.

[0136] However in the same or different embodiments, the first target sequence, or at least one of the target sequences encodes a non-antigenic protein or peptide. For instance in some embodiments the first target sequence or at least one of the target sequences encodes a therapeutic protein or peptide.

[0137] In some embodiments the therapeutic protein or peptide is capable of replacing a defective or deficient protein associated with a genetic disorder. It will be clear than that the single strand of DNA of the invention can be used in gene therapy.

[0138] The genetic disorder can be any genetic disorder where expression of a particular protein of non-coding RNA can exert a therapeutic effect. In some particular embodiments the genetic disorder is selected from the group comprising or consisting of: adenosine deaminase severe combined immunodeficiency (ADA-SCID), neuronal ceroid lipofuscinosis, aspartylglucosaminuria, Batten disease, beta-thalassemia, chronic granulomatous disease (CGD), cystic fibrosis, cystinosis, Duchenne muscular dystrophy, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Huntingdon's disease, Hurler disease, Leber's congenital amaurosis, Maroteau-Lamy disease, metachromatic leukodystrophy, Morquio disease type A, Morquio disease type B, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type Cl, Niemann-Pick disease type C2, Pompe disease, retinitis pigmentosa, Sandhoff disease, sickle cell disease, Spinal Muscular Atrophy, X-ALD, hemophilia A, hemophilia B, Hunter disease (MPS II), lysosomal storage disorder, Maroteaux-Lamy disease (MPS VI), Neuronal ceroid lipofuscinosis (NCL) including CLN1 disease, Sanfilippo disease type A (MPS IIIA), Sanfilippo disease type B (MPS IIIB), Sanfilippo disease type C (MPS IIIC), Sanfilippo disease type D (MPS HID), Schindler disease type I, Schindler disease type II, Sly disease (MPS VII), Tay-Sachs disease. In some embodiments the first protein or peptide is a therapeutic protein or peptide, but does not necessarily replace or complement a faulty gene or protein or peptide. For example the first protein or peptide may be collagen Villa, insulin, EPO, an antibody such as an anti-HER2 antibody or an anti-TNF antibody, factor VIII, GLP-1 analogs, Calcitonin, Buserelin, Desmopressin and / or Oxytocin.

[0139] The single strand of DNA of the invention may comprise multiple target sequences each encoding different therapeutic proteins or peptides, for example the single strand of DNA of the invention may allow for the expression or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different therapeutic proteins or peptides.

[0140] The single strand of DNA of the invention may comprise multiple target sequences each encoding different types and combinations or protein or peptide, for example may comprise a combination of antigens from the same or different strains of the same pathogen species, and may comprise one or more therapeutic proteins or peptides, for example the single strand of DNA of the invention may allow for the expression or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different antigens from different influenza strains.

[0141] As mentioned elsewhere herein, the first target sequence that is present in the single strand of DNA of the invention can in some instances be transcribed and translated into a protein. However in other instances the first target sequence may be a sequence that encodes a non-coding RNA.

[0142] Accordingly in some embodiments the first target sequence is a non-coding RNA, for example a functional RNA, for example selected from the group comprising or consisting of: a) a gRNA b) an RNA designed to have sequence complementarity to a host target RNA so as to cause RNAi when expressed in a host cell, for example an miRNA or a short hairpin RNA (shRNA); and / or c) a IncRNA.

[0143] As will be apparent to the skilled person, in some instances the first target sequence is designed so that when transcribed the RNA has a sequence that is complementary or substantially complementary to a host target RNA sequence (as mentioned elsewhere the skilled person will appreciate that the single strand of DNA may be designed so that the transcript produced from the single strand of DNA is itself complementary to the target; or, since the single strand of DNA may be sense or antisense, the single strand of DNA may designed so that only following replication to the double stranded form, is transcription initiated, so generating the RIMA with a sequence that is complementary to the target).

[0144] In some embodiments the single strand of DNA is designed so that the RNA transcript has sequence complementarity to a host target RNA that is RNA transcribed from an oncogene, for example wherein the oncogene is selected from the group comprising or consisting of KRAS, EGFR (Epidermal Growth Factor Receptor), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), DMD (Dystriophin).

[0145] In some instances the single strand of DNA comprises at least a first target sequence that encodes a protein or peptide, and at least a second target sequence that is not a protein or peptide coding sequence and wherein the RNA is a functional RNA. The protein or peptide may be any protein or peptide and the RNA may be any functional RNA.

[0146] Particular uses of this embodiment relate to the use of CRISPR gene editing or gene regulation. For example in some embodiments the protein or peptide is a Cas protein or peptide, or equivalent thereof, and the RNA is a gRNA. In some instances the gRNA is designed to have sequence complementarity to a viral genome or viral nucleic acid, for example wherein the virus is selected from the group comprising or consisting of HBV for example wherein the viral nucleic acid is cccDNA (Covalently Closed Circular DNA), or HIV Provirus.

[0147] As indicated above, the single strand of DNA of the invention can comprise multiple target sequences. Each of these target sequences can encode any number and any combination of proteins or peptides, and / or non-coding RNAs.

[0148] For example in some embodiments the single strand of DNA comprises a second target sequence, or a third, fourth, fifth, sixth, sevenths, eighth, ninth or tenth or more target sequences and wherein each target sequence encodes, or encodes following replication of the single strand of DNA , any one or more of: a) a protein or peptide that is an antigenic protein or peptide or comprises an antigenic protein or peptide; b) a protein or peptide that is a therapeutic protein or peptide; c) an adjuvant protein or peptide; and / or d) a non-coding RIMA, for example a functional RNA, for example selected from the group comprising or consisting of: i) a gRNA, ii) an RNA designed to have sequence complementarity to a host target RNA so as to cause RNAi when expressed in a host cell; and / or iii) a IncRNA.

[0149] In some embodiments the single strand of DNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sequences that each encodes, or encodes following replication of the single strand of DNA , an antigenic protein or peptide.

[0150] In some embodiments the single strand of DNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sequences that each encodes, or encodes following replication of the single strand of DNA any combination of: a) a therapeutic protein or peptide; b) a functional RNA, for example selected from the group comprising or consisting of: i) a gRNA, ii) an RNA designed to have sequence complementarity to a host target RNA so as to cause RNAi when expressed in a host cell; and / or iii) a IncRNA; and / or c) an antigenic protein or peptide.

[0151] The single strand of DNA of the invention may comprise multiple copies of the same target sequence; may comprise multiple target sequences where each target sequence is different; or may comprise some target sequences that are the same as each other, as well as comprising some unique target sequences.

[0152] In some embodiments at least two of the target sequences encode, or encodes following replication of the single strand of DNA, different antigenic proteins or peptides, for example at least 3, 4, 5, 6, 7, 8, 9 or at least 10 of the target sequences encode, or encode following replication of the single strand of DNA, different antigenic proteins or peptides.

[0153] In the same or different embodiments, at least two of the target sequence encode, or encode following replication of the single strand of DNA , the same antigenic protein or peptide, for example wherein at least 3, 4, 5, 6, 7, 8, 9 or at least 10 of the target sequences encode, or encode following replication of the single strand of DNA, the same antigenic proteins or peptides.

[0154] It will be appreciated that there are some instances where when the single strand of DNA encodes more than one antigenic protein or peptide, it may be beneficial to direct the antigenic protein or peptide towards different MHC presentation pathways. Accordingly in some embodiments at least two of the target sequences encode, or encode following replication of the single strand of DNA, an antigenic protein or peptide that each comprise one or more sequences to enhance presentation of the protein or peptide or fragments thereof, and wherein at least two of the antigenic proteins or peptides comprise different sequences to enhance presentation of the protein or peptide or fragments thereof, for example wherein the sequences to enhance presentation of the protein or peptide or fragments thereof are selected from the group comprising or consisting of: a) a ubiquitin sequence; b) an Endoplasmic Reticulum (ER) signal peptide, for example the signal peptide from tissue plasminogen activator (tPA); c) the Lysosome-Associated Membrane Protein 1 (LAMP-1); d) a chemokine ligand, for example XCL1; and / or e) the CD28 leader peptide.

[0155] In some embodiments at least two of the target sequences encode, or encode following replication of the single strand of DNA, the same antigenic protein or peptide and each protein or peptide comprises one or more sequences to enhance presentation of the first protein or peptide or fragments thereof, but wherein the one or more sequences to enhance presentation of the first protein or peptide or fragments thereof present in the first target sequence are different to those of the second target sequence.

[0156] The single strand of DNA of the invention may take any physical form. For example in some embodiments the single strand of DNA may be linear or circular. As set out above by single strand of DNA we mean that that the DNA of the invention is single stranded, or substantially single stranded - i.e. a single strand of single stranded DNA. We do not include the meaning of a single strand of double-stranded DNA.

[0157] In some embodiments the single strand of DNA of the invention is not a closed linear DNA. In some embodiments the single strand of DNA of the invention is not doggy bone DNA (dbDNA). As set out above, the features of the single strand of DNA of the invention distinguish from closed linear DNA and dbDNA, for example in some embodiments the single strand of DNA of the invention comprises regions of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000 or more nucleotides that are single stranded and that are not part of one or more terminal hairpins or loop structures (it should be clearthat the ssDNA of the invention may also comprise one or more terminal hairpin loops, but where it does, it also comprises the region of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000 or more nucleotides that are single stranded); in the same or other embodiments the ssDNA constructs of the invention comprise at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, or up to 100% of their nucleotide sequence in an unpaired single-stranded state under physiological conditions.

[0158] In some embodiments the single strand of DNA of the invention does not comprise a telomerase target sequence or a protelomerase target sequence.

[0159] In some embodiments the single strand of DNA of the invention does not comprise a hairpin loop at the 5', 3' or 5' and 3' termini.

[0160] In some embodiments the ssDNA of the invention does not comprise a sequence that is or encodes a bacterial origin of replication.

[0161] In some embodiments the ssDNA of the invention does not comprise unmethylated CpG motifs.

[0162] In some embodiments the ssDNA of the invention does not comprise a bacterial selection marker.

[0163] In some embodiments the ssDNA of the invention does not comprise an antibiotic resistance gene, for example does not comprise an antibiotic resistance gene that is operably linked to elements which would allow expression of the antibiotic resistance gene in a mammalian cell, for example in a human cell.

[0164] The ssDNA of the invention is not a conjugative plasmid, and does not encode a conjugative plasmid and / or does not comprise a nic site.

[0165] The single strand of DNA of the invention may be made by any means. One particularly useful method of producing the single strand of DNA is as described in WO 2018 / 054571. Such a method is able to produce very large quantities of single stranded DNA in a very short time. Such an approach is critical for scalable production of vaccines, and in particular is critical in cases such as pandemics where large amounts of new vaccines are required in a very short time period. Current methods of producing alternative vaccines, such as those that utilise mRNA, are not able to achieve the scale and speed of production as are available for the production of vaccines comprising single strands of DNA. The combination of this method of production set out in WO 2018 / 054571, and methods of production set out herein, and vaccines comprising single strands of DNA is considered to be particularly powerful.

[0166] For example in some embodiments the single strand of DNA of the invention is produced by expressing ssDNA from a dsDNA template vector that comprises at least one, preferably at least two or more, copies of the single strand of DNA of the invention, wherein the one or each copy of the single strand of DNA is flanked by a self-cleaving DNA sequence, to produce a precursor template ssDNA. Accordingly the invention provides a dsDNA template vector that comprises at least one, preferably at least two or more, copies of the sequence corresponding to the single strand DNA of the invention, wherein the dsDNA vector comprises self-cleaving DNA sequences either side of each copy of the sequence corresponding to the single strand DNA of the invention.

[0167] In preferred embodiments expression from the dsDNA template vector occurs in a microbial cell culture. Accordingly the invention also provides a cell, for example a microbial cell, that comprises the dsDNA template vector. The microbial cell may be any microbial cell. Exemplary microbial cells may be selected from the group comprising or consisting of: a) a bacterial cell for example an E. coli cell, for example a K12-derived E. coli safety strain, for example DH5alpha, XL-lblue or JM109; or b) a yeast cell.

[0168] In preferred embodiments the dsDNA template vector is a phagemid, for example further comprising: a) a packaging sequence; b) component(s) ensuring propagation of the phagemid during cell division; and / or c) a selection marker, typically an antibiotic resistance gene.

[0169] Following expression of precursor template ssDNA from the dsDNA template, the method may further comprise isolating the single strand of DNA from the microbial cell or cell culture media. Following isolation, where the precursor template ssDNA comprises self-cleaving DNA sequences, the precursor template ssDNA is digested under reaction conditions where the self-cleaving DNA sequences become active to produce the single strand of DNA, for example wherein said conditions comprise the addition of Zn2 + .

[0170] The self-cleaving DNA sequences may be self-cleaving desoxyribozymes or DNAzymes, for example Zn 2+ -dependent DNAzymes, for example I-R3.

[0171] In some embodiments the dsDNA template vector comprises two, three, four or more, sequences that correspond to the single strand of DNA of the invention, for example wherein each of the sequences is separated with a self-cleaving DNA sequence as set out herein.

[0172] It will be clear to the skilled person that where the dsDNA template vector is a phagemid, additional components are required for appropriate assembly and release of the DNA containing phage-like particles. In view of this, in some embodiments, expression of the template precursor ssDNA from the dsDNA template vector occurs in the presence of a helper plasmid or a helper phage which comprises: a) genes encoding the proteins of a bacteriophage, e.g. M13 bacteriophage; b) component(s) ensuring propagation of the helper plasmid during cell division; and / or c) a selection marker, for example an antibiotic resistance gene.

[0173] In preferred embodiments, of producing the single strand of DNA of the invention, the microbial cell is a bacterial cell and the single strand of DNA is packaged into phagelike particles which are secreted from the bacterial cells, and wherein the phage-like particles are isolated from the cell culture and the phagemid ssDNA is purified from the phage-like particles.

[0174] It will be clear to the skilled person that where the single strand of DNA of the invention is made according to the method set out above, that utilises self-cleaving DNA sequences, that following cleavage of those sequences, the 5' and 3' end of the single strand of DNA will have fixed end sequences, i.e. sequences that correspond to the scars remaining from cleavage of the self-cleavage DNA sequence. Accordingly in some embodiments the single strand of DNA of the invention comprises 5' and 3' end sequences are the scars that remain following DNAzyme cleavage.

[0175] An advantage of the present system over the delivery of proteins using mRNA is that single stranded DNA is inherently more stable that mRNA. DNA contains the sugar deoxyribose, while RNA contains ribose. The absence of a 2’-hydroxyl group in DNA’s deoxyribose makes it chemically more stable than RNA. RNA also undergoes spontaneous hydrolytic cleavage approximately 100 times faster than DNA. This increased susceptibility in RNA is attributed to the intramolecular attack of the 2’- hydroxyl function on the neighbouring phosphate diester, leading to a 2’,3’-cyclic phosphate. DNA’s lack of a 2’-OH group makes it less prone to such cleavage, enhancing its stability over an organism's lifetime. Furthermore, the DNA duplex is more hydrated compared to the RNA duplex. This difference in hydration levels is speculated to influence their stability, with the more stable RNA duplex having the lowest hydration level. Although specific hydration numbers are not provided, the general trend indicates that lower hydration correlates with higher stability in single strand of DNA duplexes. High temperatures increase the rate of spontaneous and irreversible RIMA backbone hydrolysis, a phenomenon not typically observed for DNA.

[0176] In some embodiments the single strand of DNA of the invention is stable at a temperature of at least 18°C, or at least 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or 26°C, for example stable for at least 24 hours, 48 hours, 1 week, 2 weeks, 4 weeks, 2 months, 6 months, 1 year or more.

[0177] As set out above, the invention also provides a dsDNA template vector from which the single strand of DNA of the invention may be produced. In some embodiments the vector comprises multiple repeats of the sequence corresponding to the single strand of DNA of the invention, for example wherein the multiple repeats are arranged in a head-to-tail orientation, for example wherein the dsDNA vector comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more repeats.

[0178] In some instances the dsDNA vector of the invention is a phagemid. In some instances the phagemid further comprises: a) a packaging sequence; b) component(s) ensuring propagation of the phagemid during cell division; and / or c) a selection marker, typically an antibiotic resistance gene.

[0179] The phagemid may further comprise: a) genes encoding the proteins or parts of a protein of a bacteriophage, for example proteins or parts of a protein of the M13 bacteriophage; b) component(s) ensuring propagation of the helper plasmid during cell division; and / or c) a selection marker, for example an antibiotic resistance gene.

[0180] It will be appreciated that since there are clear therapeutic uses of the single strand of DNA of the invention, the single strand of DNA the invention must be administered to a subject in need thereof, for example administered to a mammal, for example to a human. Accordingly the invention provides various formulations of the single strand of DNA of the invention that are suitable for delivery of the single strand of DNA to a subject, or to a specific tissue or site in the subject. Preferences for features of the formulations are as defined elsewhere herein.

[0181] In some embodiments the formulation is a lipid nanoparticle formulation. The invention therefore provides a lipid nanoparticle (LNP) comprising one or more of the single strands of DNA of the invention. Preferences for features of the of the single strand of DNA are as set out elsewhere herein. In some embodiments of the LNP, the single strand of DNA has a length greater than lkb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, lOkb, or more.

[0182] In some embodiments the single strand of DNA is not contained within a viral vector, for example is not contained with an adenovirus, and AAV and / or a lentivirus.

[0183] In some embodiments the single strand of DNA is contained within a viral vector, for example is contained with an adenovirus, and AAV and / or a lentivirus.

[0184] In some embodiments the single strand of DNA is not complexed with any other nucleic acid strands. In some instances it may contain internal hybridisations, but in this embodiment the single strand of DNA of the invention is the only nucleic acid molecule present. In some embodiments the single strand of DNA of the invention is not a nucleic acid nanostructure or is not part of a nucleic acid nanostructure.

[0185] In other embodiments the single strand of DNA is complexed with additional nucleic acid molecules and in some instances is considered to be a nucleic acid nanostructure.

[0186] The invention also provides various nucleic acid based nanostructures that comprise one or more single strand of DNA of the invention. Nucleic acid nanostructures that are capable of expressing genes are described in W02024105115. For example the invention provides a nucleic acid nanostructure comprising at least one scaffold strand and a plurality of staple strands, wherein said nanostructure comprises at least one single strand of DNA according to the invention.

[0187] Preferences for features of the nucleic acid based nanostructure are as defined elsewhere herein.

[0188] In some embodiments said nucleic acid nanostructure comprises a first subunit and a second subunit; wherein, said first subunit and said second subunit each comprise a single strand of DNA according to the invention, for example wherein the nucleic acid nanostructure comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more subunits, for example where each subunit comprises a single strand of DNA of the invention.

[0189] The invention also provides a LNP comprising the nucleic acid nanostructure according to the invention. As will be appreciated various of the single strand DNA of the invention, LNPs, dsDNA template vectors and other components described herein may be provided as a composition. Accordingly the invention provides a composition comprising one or more single strands of DNA according to the invention, the LNP to the invention, and / or the nucleic acid nanostructure according to the invention.

[0190] In some embodiments the composition is formulated for administration to a subject, for example wherein the subject is a mammal, for example a human. In some embodiments the composition is a pharmaceutical composition. In some embodiments the composition or pharmaceutical composition further comprises one or more pharmaceutically acceptable carrier and / or excipients. In the same or different embodiments the composition or pharmaceutical composition further comprises an adjuvant.

[0191] In some embodiments the composition or pharmaceutical composition further comprises components required for Nanojet administration.

[0192] In some embodiments the composition or pharmaceutical composition is formulated for delivery of the one or more single strands of DNA according to the invention, the LNP according to the invention, and / or the nucleic acid nanostructure according the invention to an immune cell, for example to a dendritic cell or a macrophage.

[0193] In some embodiments the composition or pharmaceutical composition does not comprise a viral vector, for example does not comprise an adenovirus, and AAV and / or a lentivirus.

[0194] As described herein, the single strand of DNA provided herein may suitably be used as a vaccine, therapeutic, and / or prophylactic, in the treatment of or vaccination against one or more disease(s) disclosed herein. Accordingly, the LNP provided herein, nucleic acid nanostructure provided herein, and / or composition provided herein may suitably be used as a vaccine, therapeutic, and / or prophylactic, in the treatment of or vaccination against one or more disease(s) disclosed herein. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treatment of or vaccination against disease(s) are as set out elsewhere herein.

[0195] Accordingly, provided herein is a vaccine comprising one or more single strand of DNA provided herein, the LNP provided herein, the nucleic acid nanostructure provided herein, and / or the composition provided herein. Also provided herein is the single strand of DNA provided herein, the LNP provide herein, the nucleic acid nanostructure provided herein, the composition provided herein, and / or the vaccine provided herein for use in a method of treating or preventing an infection with a pathogen, for example a viral, bacterial, fungal or protist pathogen in a subject. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treatment of infection with a pathogen are as set out elsewhere herein.

[0196] The invention also provides a method of treating or preventing and infection with a pathogen, for example a viral, bacterial, fungal or protist pathogen in a subject said method comprising administering the single strand of DNA provided herein, the LNP provided herein, the nucleic acid nanostructure provided herein the composition provided herein, and / or the vaccine provided herein to the subject. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treatment of infection with a pathogen are as set out elsewhere herein.

[0197] The invention also provides the use of a single strand of DNA provided herein, the LNP provided herein, the nucleic acid nanostructure provided herein, the composition provided herein and / or the vaccine provided herein for the manufacture of a medicament for the treatment or preventing and infection with a pathogen, for example a viral, bacterial, fungal or protist pathogen in a subject. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treatment of infection with a pathogen are as set out elsewhere herein.

[0198] The invention also provides the single strand of DNA provided herein, the LNP provided herein, the single strand of DNA nanostructure provided herein, the composition provided herein and / or the vaccine provided herein for use in a method of treating or preventing a disease in a subject. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treating or preventing a disease are as set out elsewhere herein.

[0199] The invention also provides a method of treating or preventing a disease in a subject wherein said method comprises administering a single strand of DNA provided herein, the LNP provided herein, the single strand of DNA nanostructure provided herein, the composition according provided herein and / or the vaccine provided herein to a subject. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treating or preventing a disease are as set out elsewhere herein. The invention also provides the use of a single strand of DNA provided herein, the LNP provided herein, the single strand of DNA nanostructure provided herein, the composition provided herein and / or the vaccine provided herein for the manufacture of a medicament for the treatment or preventing and infection with a disease in a subject. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treating or preventing a disease are as set out elsewhere herein.

[0200] In some embodiments of the single strand of DNA, LNP, single strand of DNA nanostructure, composition or vaccine for use, method, or use, the first target sequence is: a) an antisense strand that encodes a first protein or peptide and is able to be directly transcribed to produce the first RNA molecule or b) a sense strand that is able to be transcribed to produce the first RNA molecule once the single strand of DNA is replicated to produce the antisense strand; and wherein the first protein or peptide is a therapeutic protein or peptide.

[0201] The invention also provides the single strand of DNA provided herein, the LNP provided herein, the nucleic acid nanostructure provided herein, the LNP provided herein, the composition provided herein and / or the vaccine provided herein for use in a method of treating or preventing cancer in a subject. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treating or preventing cancer are as set out elsewhere herein.

[0202] The invention also provides a method of treating or preventing cancer in a subject wherein said method comprises administering a single strand of DNA provided herein, the LNP provided herein, the single strand of DNA nanostructure provided herein, the LNP provided herein, the composition provided herein and / or the vaccine provided herein to a subject. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treating or preventing cancer are as set out elsewhere herein.

[0203] The invention also provides the use of a single strand of DNA provided herein, the LNP provided herein, the single strand of DNA nanostructure provided herein, the composition provided herein and / or the vaccine provided herein for the manufacture of a medicament for the treatment or prevention of cancer in a subject. Preferences for features of the ssDNA, such as the target sequences that are relevant in the context of treating or preventing cancer are as set out elsewhere herein. In some embodiments of the single strand of DNA, LNP, single strand of DNA nanostructure, composition or vaccine for use provided herein, method provided herein, or use provided herein, the first target sequence is: a) an antisense strand that encodes a first protein or peptide and is able to be directly transcribed to produce the first RIMA molecule or b) a sense strand that is able to be transcribed to produce the first RNA molecule once the single strand of DNA is replicated to produce the antisense strand; and wherein the first protein or peptide is an antigenic protein or peptide or comprises an antigenic protein or peptide, and wherein the antigenic protein or peptide is a cancer antigen or a cancer neoantigen.

[0204] Also provided herein is a method of producing a single strand of DNA provided herein, wherein said method comprises: a) providing a dsDNA template vector that is a phagemid and that comprises: i) at least one copy of the sequence of the single strand of DNA provided herein wherein the one or each copy of the single strand of DNA is flanked by a selfcleaving DNA sequence, to produce a precursor template ssDNA; ii) component(s) ensuring propagation of the phagemid during cell division; iii) a selection marker, typically an antibiotic resistance gene; and iv) a packaging sequence, v) and for example one or more phage genes, that encode for phage proteins (Genlll); b) introducing the dsDNA template vector into a microbial cell either as a double strand, or as a single stranded DNA packaged in a bacterial phage protein coat, for example an E. coli cell; c) culturing the microbial cell under conditions so as to produce a population of cells and allow the cells to produce phage-like particles comprising the at least one copy of the single strand of DNA provided herein that are released from the cells; and d) harvesting the phage-like particles from the culture media; and e) extracting the ssDNA from the phage and exposing said ssDNA to conditions so as to allow self-cleavage of the self-cleaving DNA sequence; f) isolating the single strand of DNA provided herein; and wherein the cell also expresses: i) genes encoding the proteins of a bacteriophage, for example genes encoding the proteins of M13 bacteriophage, for example wherein said genes are present on a helper plasmid; and ii) for example component(s) ensuring propagation of the helper plasmid, when present, during cell division.

[0205] Also provided herein is a method of producing a single strand of DNA provided herein, wherein said method comprises:

[0206] Step (a) culturing a microbial cell that comprises: i) at least one copy of the single strand of DNA provided herein present on a phagemid, wherein the one or each copy of the single strand of DNA is flanked by a self-cleaving DNA sequence, to produce a precursor template ssDNA, wherein the phagemid comprises a selection marker, for example an antibiotic resistance gene, and a packaging sequence; and ii) one or more phage genes, that encode for phage proteins (Genlll); and iii) genes encoding the proteins of a bacteriophage, for example genes encoding the proteins of M13 bacteriophage, for example wherein said genes are present on a helper plasmid under conditions so as to produce a population of cells and allow the cells to produce phage comprising the at least one copy of the single strand of DNA provided herein that are released from the cells.

[0207] The invention also provides a method of producing the single strand of DNA of the invention wherein said method comprises:

[0208] Step (a) culturing a microbial cell that comprises: i) at least one copy of the sequence of the single strand of DNA of the invention present on a phagemid, wherein the one or each copy of the single strand of DNA is flanked by a self-cleaving DNA sequence, to produce a precursor template ssDNA, wherein the phagemid comprises a packaging sequence; ii) one or more phage genes, that encode for phage proteins (Genlll); and iii) genes encoding the proteins of a bacteriophage, optionally genes encoding the proteins of M13 bacteriophage, optionally wherein said genes are present on a helper plasmid; and optionally comprises a selection marker, optionally an antibiotic resistance marker; wherein said culturing is under conditions so as to produce a population of cells and allow the cells to produce phage-like particles comprising the at least one copy of the single strand of DNA of the invention that are released from the cells; step (b) harvesting the phage-like particles from the culture media; step (c) extracting the ssDNA from the phage-like particles and exposing said ssDNA to conditions so as to allow self-cleavage of the self-cleaving DNA sequence; and step (d) isolating the single strand of DNA of the invention; and wherein steps (a), (b), (c) and (d) are completed within a 48 hour, 72 hour, or 96 hour, 108 hour, 120 hour, 132 hour, 144 hour, 156 hour, 168 hour, 180 hour period and produce: i) 50g, 75g, 100g, 125g, 150g, 175g, 200g of isolated single strand of DNA of the invention 1-18; and / or ii) 50mg / L, 75mg / L, lOOmg / L, 125mg / L, 150mg / L, 175mg / L, 200mg / L of isolated single strand of DNA of the invention.

[0209] The invention also provides a method of producing nucleic acid for use in a vaccine wherein the method produces: i) 50g, 75g, 100g, 125g, 150g, 175g, 200g of isolated single strands of DNA; and / or ii) 50mg / L, 75mg / L, lOOmg / L, 125mg / L, 150mg / L, 175mg / L, 200mg / L of isolated single strands of DNA; in a time period of 48 hours, 72 hours, or 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, 168 hours, or 180 hours, and wherein the method comprises any of the methods of producing the single strand of DNA of the invention as set out herein.

[0210] In some embodiments, the method further comprises step (b) harvesting the phagelike particles from the culture media. In some embodiments, the method further comprises step (c) extracting the ssDNA from the phage and exposing said ssDNA to conditions so as to allow self-cleavage of the self-cleaving DNA sequence. In some embodiments, the method further comprises step (d) isolating the single strand of DNA provided herein.

[0211] A particular advantage of the invention is that the methods provided herein may be used to generate a large quantity of a single strand of DNA or ssDNA in a short period of time, for example in a shorter period of time than existing methods. Accordingly, in some embodiments, steps (a); (a) and (b); (a), (b) and (c); or (a), (b), (c) and (d) are completed within a 48 hour, 72 hour, or 96 hour, 108 hour, 120 hour, 132 hour, 144 hour, 156 hour, 168 hour, 180 hour period.

[0212] In some embodiments: a) 50g, 75g, 100g, 125g, 150g, 175g, 200g of isolated single strand of DNA is produced; and / or b) 50mg / L, 75mg / L, lOOmg / L, 125mg / L, 150mg / L, 175mg / L, 200mg / L of isolated single strand of DNA is produced.

[0213] In some embodiments: a) 50g, 75g, 100g, 125g, 150g, 175g, 200g of isolated single strand of DNA is produced; and / or b) 50mg / L, 75mg / L, lOOmg / L, 125mg / L, 150mg / L, 175mg / L, 200mg / L of isolated single strand of DNA is produced in a 48 hour, 72 hour, or 96 hour, 108 hour, 120 hour, 132 hour, 144 hour, 156 hour, 168 hour, 180 hour period.

[0214] The invention also provides a method of producing a vaccine comprising one or more single strands of DNA of the invention wherein said method comprises producing a single strand of DNA of the invention as set out herein, and formulating the isolated single strand of DNA of step (f) as a vaccine.

[0215] The invention also provides a method of producing a LNP vaccine comprising one or more single strand of DNA provided herein, wherein said method comprises producing a single strand of DNA provided herein according to a method as provided herein, and combining the isolated single strand of DNA of step (f) with lipids.

[0216] Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, the invention provides: a) an antisense single strand of DNA that encodes three different antigens from three different strains of influenza; b) a linear single strand of DNA that encodes a antigen from a first bacterial species wherein said antigen is fused to ubiquitin and also encodes an antigen from a second bacterial species wherein said antigen from the second bacterial species is fused to a chemokine ligand; c) a LNP comprising a circular single strand of DNA of the invention, wherein the single strand of DNA of the invention comprises a first sequence that encodes a Cas protein at three or more sequences that encode different gRNAs, allowing for targeting of the Cas protein to at least three different loci in a host; d) a composition formulated for nanojet administration wherein the composition comprises a single strand of DNA of the invention, and wherein the first sequence encodes a viral antigen.

[0217] In this way the skilled person will appreciate how different features from different passages within this text can be combined together.

[0218] The invention is also illustrated in the following numbered paragraphs:

[0219] 1. A single strand of DNA that: comprises a first target sequence operably linked to a first promoter sequence, wherein the first promoter sequence drives expression of the first target sequence to produce a first RNA molecule either: i) directly from the single strand of DNA; or ii) following replication of the single strand of DNA into the double strand form.

[0220] 2. The single strand of DNA of paragraph 1 wherein the single strand of DNA is entirely single stranded and / or is substantially single stranded.

[0221] 3. The single strand of DNA of any of paragraphs 1 or 2 wherein the single strand of DNA contains no regions of self-complementarity, optionally no regions of selfcomplementarity of sufficient length so as to allow internal hybridisation, optionally no regions of self-complementarity: a) that are longer than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 base pairs; and / or b) where the combined length of two regions of the single strand of DNA that make up a particular self-complementary region constitute less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 2% or less than 1% of the total length of the single strand of DNA. 4. The single strand of DNA of any of paragraphs 1-3 wherein less than 90%, 85%, 80%, 75%, 70%, 65%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the nucleotides of the single strand of DNA are involved in self-complementarity.

[0222] 5. The single strand of DNA of any of paragraphs 1-4 wherein the single strand of DNA does not comprise a first region of more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 continuous nucleotides that are complementary to a second region of the ssDNA of more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 continuous nucleotides.

[0223] 6. The single strand of DNA of any of paragraphs 1-5 wherein the first target sequence of the ssDNA has no region that is complementary to a second region of the ssDNA.

[0224] 7. The single strand of DNA of any of paragraphs 1-5 wherein the first target sequence of the ssDNA does not have a region of more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 continuous nucleotides that are complementary to a second region of the ssDNA of more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 continuous nucleotides.

[0225] 8. The single strand of DNA of paragraph 1 and 4-7 wherein the single strand of DNA comprises at least one region that is double stranded, wherein said double stranded region is generated via hybridisation between at least a first and second region of the single strand of DNA that are substantially complementary to each other, optionally: a) wherein the hybridisation between a first and second region of the single stand of DNA enhances expression of the first RNA molecule, optionally wherein the first region of the single strand of DNA is in the promoter and the second region of the single strand of DNA is distal to the promoter; or b) wherein the first and second region of the single strand of DNA that are substantially complementary to each other are located at the 5' and the 3' end of the single strand of DNA so that the single strand of DNA is circularised upon hybridisation between the first and second regions; and / or c) wherein the first region is a region of the ssDNA that is not the target sequence and that is between 10-1000 contiguous nucleotides, optionally between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides and that is complementary to the second region that is a region of the first target sequence of between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80- 650, 90-600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides.

[0226] 9. The single strand of DNA of any one of paragraphs 1-8 wherein the single strand of DNA comprises a hairpin, optionally a hairpin at the 5' and / or 3' end of the single strand of DNA.

[0227] 10. The single strand of DNA of paragraph 1 or 4-9 wherein the single strand of DNA comprises a secondary structure so as to: a) increase stability of the single stand of DNA; and / or b) to increase expression from the promoter.

[0228] 11. The single strand of DNA of any of the preceding paragraphs wherein the promoter is a promoter capable of driving transcription in a eukaryotic cell, optionally in a mammalian cell, optionally in a human cell, optionally in a dendritic cell, T cell and / or B cell.

[0229] 12. The single strand of DNA of any of the preceding paragraphs wherein the promoter is a constitutive promoter.

[0230] 13. The single strand of DNA of any of the preceding paragraphs wherein the promoter is an inducible promoter.

[0231] 14. The single strand of DNA of any of the preceding paragraphs wherein the promoter is selected from the group comprising or consisting of: Cytomegalovirus (CMV) Immediate-Early promoter; Simian Virus 40 (SV40) promoter; CAT promoter; Muscle Creatine Kinase promoter (MCK); Human Elongation Factor 1 Alpha (EFla) Promoter; Tetracycline-Inducible Promoter; a MiniPromoter; U6 promoter; Hl promoter, CD19 promoter; CD2 promoter; KRT5 promoter; FoxP3 promoter; and CAG promoter.

[0232] 15. The single strand of DNA of any of the preceding paragraphs wherein the promoter is selected from the group comprising or consisting of: Cytomegalovirus (CMV) Immediate-Early promoter; Simian Virus 40 (SV40) promoter; Muscle Creatine Kinase promoter (MCK); or a tissue or cell-type specific promoter, optionally dendritic cell, B cell and / or T cell specific promoter, optionally where the T cell specific promoter is a CD2 promoter or FoxP3 promoter.

[0233] 16. The single strand of DNA of any of the preceding paragraphs wherein the promoter is selected from the group comprising or consisting of: Human Elongation Factor 1 Alpha (EFla) Promoter; Tetracycline-Inducible Promoter; a CAT promoter; a MiniPromoter; or a tissue or cell-type specific promoter.

[0234] 17. The single strand of DNA of any of the preceding paragraphs wherein the promoter is selected from the group comprising or consisting of: U6 promoter; Hl promoter; or a CMV promoter; or a tissue or cell-type specific promoter.

[0235] 18. The single strand of DNA of any of paragraphs 1-17 further comprising a first enhancer sequence, wherein the first enhancer sequence enhances expression of the first target sequence, optionally enhances expression of the first target sequence in a specific cell type, optionally wherein at least one enhancer sequence is located in the 5'UTR, 3'UTR or in the 5'UTR and 3'UTR; optionally wherein the enhancer is selected from the group comprising or consisting of: a) a viral enhancer, optionally the 72-bp enhancer element from Simian Virus 40 (SV40), the enhancer from CMV, and / or the enhancer from Rous sarcoma virus; b) a Ubiquitous / Constitutive Enhancers, optionally the enhancer from the human elongation factor 1 alpha (EFla) enhancer; c) a tissue / Cell-Type Specific Enhancers, optionally the muscle creatine kinase (MCK) enhancer for muscle cells; d) Super-enhancers associated with genes defining cell identity; e) Synthetic / Designed Enhancers f) inducible enhancers, optionally an inducible enhancer that response to hormones or small molecules; optionally wherein the single strand of DNA comprises more than one enhancer, optionally comprises combinations of at least two enhancers, optionally at least two enhancers of any of (a)-(f), optionally a combination of the SV40 enhancer with a muscle-specific enhancer for muscle targeting.

[0236] 19. The single strand of DNA according to any of the preceding paragraphs further comprising any one or more of: a) a nuclear localisation signal; and / or b) inverted-terminal repeat like hairpin motifs; and / or c) a DNA targeting sequence (DTS), optionally a DTS from SV40 or the 81 base pair DTS of Sox2 regulatory region 2 (SRR2) or functional variant thereof.

[0237] 20. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is linear, optionally wherein the single strand of DNA comprises caps formed by hairpin loops at the 5' end and / or the 3' end. 21. The single strand of DNA of any of paragraphs 1-19 wherein the single strand of DNA is circular, optionally wherein in the single strand of DNA is circularised via complementary base pairing of the 5' end and the 3' end.

[0238] 22. The single strand of DNA of any of paragraphs 1-20 wherein the single strand of DNA is linear, and capable of circularising when in a cellular environment, optionally a eukaryotic cell, optionally a mammalian cell, optionally a human cell; and or is capable of circularising in an in vitro transcription translation system, optionally in a mammalian in vitro transcription translation system.

[0239] 23. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA comprises any one or more modifications, optionally: a) a modification to reduce immunogenicity, optionally reduce immunogenicity in a mammalian cell, optionally wherein the modification is methylation; b) a modification to enhance expression, optionally enhance expression in a mammalian cell, optionally wherein the modification comprises or consists of any one or more of: c) a modification to make the single strand of DNA act as an adjuvant, optionally wherein the modification is non-methylation.

[0240] 24. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is less immunogenic than an equivalent dsDNA construct, optionally wherein administration of the single strand of DNA of any of the preceding claims, optionally to a human or to a mouse, results in reduced expression of one or more cytokines, optionally one or more of IFN-y, IL-6, and / or TNF-a, relative to the expression of the same one or more cytokines in response to administration of an equivalent dsDNA.

[0241] 25. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is less toxic than the equivalent dsDNA, optionally wherein the ssDNA of the invention results in reduced toxicity in humans or mice, for example female BALB / c mice, as demonstrated by a reduced level of ALT and / or AST relative to the expression level of ALT and / or AST when administered an equivalent dsDNA construct at day 18 following administration of the construct.

[0242] 26. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA additionally comprises a second target sequence, or a third, fourth, fifth, sixth, seventh, eighth, ninth or tenth or more target sequence.

[0243] 27. The single strand of DNA of paragraph 26 wherein each target sequence is the same as the first target sequence.

[0244] 28. The single strand of DNA of paragraph 26 wherein the single strand of DNA comprises at least two target sequences that are different to each other. 29. The single strand of DNA of any of paragraphs 26-28 wherein the target sequences present in the single strand of DNA are arranged in a tandem array / .

[0245] 30. The single strand of DNA of paragraph 29 wherein all of the target sequences present in the tandem array are operably linked to the same promoter.

[0246] 31. The single strand of DNA of any of paragraphs 26-30 wherein at least two of the target sequences are operably linked to different promoters.

[0247] 32. The single strand of DNA of any of paragraphs 26-31 wherein the target sequences are transcribed into a single RNA molecule.

[0248] 33. The single strand of DNA of paragraph 32 wherein the single RNA molecule comprises: a) an internal ribosome entry site (IRES) that is present 5' to each target sequence, so that each target sequence is translated, optionally wherein the IRES is an IRES from encephalomyocarditis virus (EMCV) or poliovirus; and / or b) a sequence that is translated into a 2A peptide sequence, optionally the P2A sequence, T2A sequence, E2A sequence and F2A sequence.

[0249] 34. The single strand of DNA of any of paragraphs 32 or 33 wherein each target sequence of the single RNA molecule comprises a start and stop codon.

[0250] 35. The single strand of DNA of any of paragraphs 32 or 33 wherein spacer sequences are present between the IRES and one or more target sequences.

[0251] 36. The single strand of DNA of any of the preceding paragraphs further comprising a transcription terminator.

[0252] 37. The single strand of DNA according to any of paragraphs 33-36 wherein the promoter is a strong promoter, optionally a CMV promoter, SP6 promoter or T7 promoter.

[0253] 38. The single strand of DNA according to any of the preceding paragraphs, wherein the single strand of DNA is:

[0254] At least 500 nucleotides, or at least 1000 nucleotides or at least 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, 10000, 10250, 10500, 10750, 11000,

[0255] 11250, 11500, 11750, 12000, 12250, 12500, 12750, 13000, 13250, 13500, 13750,

[0256] 14000, 14250, 14500, 14750, 15000, 15250, 15500, 15750, 16000, 16250, 16500,

[0257] 16750, 17000, 17250, 17500, 17750, 18000, 18250, 18500, 18750, 19000, 19250,

[0258] 19500, 19750, or at least 20000 and / or

[0259] Between 1000 and 20000, 1250 and 19750, 1500 and 19500, 1750 and 19250, 2000 and 19000, 2250 and 18750, 2500 and 18500, 2750 and 18250, 3000 and 18000, 3250 and 17750, 3500 and 17500, 3750 and 17250, 4000 and 17000, 4250 and 16750, 4500 and 16500, 4750 and 16250, 5000 and 16000, 5250 and 15750, 5500 and 15500, 5750 and 15250, 6000 and 15000, 6250 and 14750, 6500 and 14500, 6750 and 14250, 7000 and 14000, 7250 and 13750, 7500 and 13500, 7750 and 13250, 8000 and 13000, 8250 and 12750, 8500 and 12500, 8750 and 12250, 9000 and 12000, 9250 and 11750, 9500 and 11500, 9750 and 11250, 10000 and 11000, 10250 and 10750 nucleotides in length.

[0260] 39. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is designed so as to allow expression of the first target sequence to produce a first RNA molecule in eukaryotes, optionally in mammals, optionally in humans, optionally wherein: a) the first RNA molecule is a transcript that encodes a protein or peptide; and / or b) the first RNA is a protein coding RNA; or c) the first RNA is a non-coding RNA.

[0261] 40. The single strand of DNA according to any of paragraphs 1-39 wherein the first target sequence is: a) an antisense strand that encodes a first protein or peptide and is able to be directly transcribed to produce the first RNA molecule; or b) a sense strand that is able to be transcribed to produce the first RNA molecule once the single strand of DNA is replicated to produce the antisense strand.

[0262] 41. The single strand of DNA according to any of the preceding paragraphs wherein the first target sequence comprises one or more sequences that when transcribed into the first RNA molecule form a 5'UTR, a 3'UTR and / or a polyA tail.

[0263] 42. The single strand of DNA according to paragraph 41 wherein the 5'UTR and / or 3'UTR comprise sequences that form RNA secondary structures that regulate gene expression in eukaryotes, optionally wherein: a) the 5'UTR comprises: i) one or more stable hairpin and / or stem-loop structures to downregulate gene expression; and / or ii) an internal ribosome entry site (IRES), optionally wherein the IRES is an IRES from encephalomyocarditis virus (EMCV) or poliovirus; and / or b) the 3'UTR comprises: i) one or more stem-loop structures and / or AU rich elements; and / or ii) one or more miRNA binding sites.

[0264] 43. The single strand of DNA according to any one of paragraphs 40-42 wherein the first protein or peptide is an antigenic protein or peptide or comprises an antigenic protein or peptide.

[0265] 44. The single strand of DNA according to any of the preceding paragraphs wherein the first protein or peptide comprises one or more sequences to enhance presentation of the protein or peptide or fragments thereof, optionally: a) presentation by the major histocompatibility proteins (MHC), optionally MHC I and / or MHC II; and / or b) presentation by antigen presenting cells, optionally dendritic cells, macrophages and / or B cells; and / or c) localisation to the cell surface.

[0266] 45. The single strand of DNA according to paragraph 44 wherein the one or more sequences to enhance presentation of the first protein or peptide fragment is: a) a ubiquitin sequence; b) an Endoplasmic Reticulum (ER) signal peptide, optionally the signal peptide from tissue plasminogen activator (tPA); c) the Lysosome-Associated Membrane Protein 1 (LAMP-1); d) a chemokine ligand, optionally XCL1; and / or e) the CD28 leader peptide.

[0267] 46. The single strand of DNA according to any of paragraphs 40-45 wherein the first protein or peptide is a protein or peptide present in a pathogen, optionally wherein the pathogen is a bacterial pathogen, viral pathogen, fungal pathogen or a protist pathogen.

[0268] 47. The single strand of DNA according to any of paragraphs 40-46 wherein the first protein or peptide is a cancer antigen or a cancer neoantigen.

[0269] 48. The single strand of DNA according to any of paragraphs 40-47 wherein the first protein or peptide is: a) a viral antigen, optionally i) an influenza antigen, optionally Influenza Hemagglutinin, optionally from H5Nx strains; ii) a coronavirus antigen, optionally from SARS-CoV-2, SARS-CoV, HCoV NL63, HCoV HKU1, or MERS-CoV; optionally a spike protein, optionally the spike protein from any one or more of from SARS-CoV-2, SARS-CoV, HCoV NL63, HCoV HKU1, or MERS-CoV; iii) a HIV antigen, optionally HIV-1 ENV or HIV-1 GAG; or iv) a Hepatitis B antigen, optionally Hepatitis B Surface Antigen (HBsAg); b) a cancer antigen, optionally wherein the cancer antigen is NY-ESO-1, MAGE-A3, HER2 / neu; or PSA; c) an Autoimmune Disease Antigen, optionally insulin, Myelin Basic Protein (MBP); d) a Neurological Disease Antigens, optionally Amyloid Beta (A ) or Tau Protein; e) a bacterial antigen, optionally i) an antigen from Mycobacterium tuberculosis, optionally Ag85 Complex, optionally from any ofAg85A, Ag85B, Ag85C); ESAT-6; CFP-10 ii) an antigen from Bacillus anthracis, optionally Protective Antigen (PA); iii) an antigen from Clostridium tetani, optionally tetanus toxin (TeNT) iv) an antigen from Escherichia coli, optionally Colonization Factor Antigen I (CFA / I); v) Salmonella enterica, optionally Flagellin (FliC), Invasin Proteins (e.g., SipA, SipC); vi) Streptococcus pneumoniae, optionally Pneumolysin (Ply), PspA (Pneumococcal Surface Protein A); vii) Helicobacter pylori, optionally Urease Subunits (UreA, UreB); viii) Listeria monocytogenes, optionally Listeriolysin O (LLO); ix) Shigella spp., optionally Ipa Proteins (e.g., IpaB, IpaC); x) Vibrio cholerae, optionally Cholera Toxin (CT), optionally the B subunit of cholera toxin (CTB); f) a fungal antigen, optionally i) Candida albicans, optionally Agglutinin-like sequence 3 (Als3), Secreted aspartyl proteinases (Sap), Heat shock protein 90 (Hsp90), Hyphal wall protein 1 (Hwpl), or Enolase (Enol); ii) Aspergillus fumigatus, optionally Aspf3 (Allergen), Catalase, Gell (1,3-p-glucanosyltransferase), or Pmp20 (Putative Plasma Membrane Protein); iii) Cryptococcus neoformans, optionally Glucosylceramide (GlcCer), Mannoproteins (MP98, MP88), Phospholipase Bl (Plbl), or Capsular polysaccharide (GXM); iv) Coccidioides posadasii / immitis, optionally Antigen 2 / PRA (Proline-rich antigen), Chitinase, Urease, Fba (Fructose-bisphosphate aldolase), v) Histoplasma capsulatum, optionally Histone 2B (H2B), Heat shock protein 60 (Hsp60), Catalase B (CatB), Yps3 (Yeast protein 3); vi) Paracoccidioides brasiliensis, optionally Glycoprotein 43 (Gp43) peptide 10 (PIO), Paracoccin, Triosephosphate isomerase (Tpi), vii) Pneumocystis jirovecii, optionally Major surface glycoprotein (Msg), Kexin, Monosaccharide transporter (Mst); and / or g) a protist antigen.

[0270] 49. The single strand of DNA according to any of paragraphs 1-48 wherein the single strand of DNA comprises at least one adjuvant sequence that encodes an adjuvant protein, optionally wherein the adjuvant protein is selected from the group comprising or consisting of: a) Bacterial protein adjuvants, optionally FomA porin from Fusobacterium; MOMP from Shigella flexneri, Porin from Shigella dysenteriae; OmpU from Vibrio cholerae; PorB from Neisseria meningitidis; OmpC and OmpF from Salmonella typhi, ESAT-6 from Mycobaceterium tuberculosis; rBCSP31 from Brucella abortus; DnaJ-AA146Ply from Streptococcus pneumoniae; Endopeptidase O from Streptococcus pneumoniae; Cholera toxin from Vibrio cholerae; B- pentamers of LT-IIa and LT-IIb from Escherichia coll; 0mpl6 from Brucella abortus; BLS from Brucella spp.; GrpE from M. tuberculosis; RpfE from M. tuberculosis; Rv0652 from M. tuberculosis; HBHA from M. tuberculosis; P97 protein from Mycoplasma hyopneumoniae; Flagellin from Salmonella species; Entolimod (CBLB502) optimised from Salmonella flagellin; b) One or more cytokines, optionally wherein the cytokine is selected from the group comprising or consisting of Interleukin-12 (IL-12), Interleukin-2 (IL- 2), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF); c) one or more cytokines, optionally wherein the chemokine is CCL21 (Secondary Lymphoid Tissue Chemokine, SLC); d) one or more co-stimulatory molecules, optionally wherein the costimulatory molecule is CD40 Ligand (CD40L), or B7-1 (CD80) and B7-2 (CD86); e) one or more Toll-Like Receptor (TLR) Ligands, optionally wherein the Toll-Like Receptor (TLR) Ligand is CpG Oligodeoxynucleotides (CpG ODNs) f) Flt3 Ligand (Flt3L); and / or g) Heat Shock Proteins (HSPs) or any combinations thereof.

[0271] 50. The single strand of DNA according to any one of paragraphs 40-49 wherein the first protein or peptide is a therapeutic protein or peptide.

[0272] 51. The single strand of DNA of paragraph 50 wherein the therapeutic protein or peptide is capable of replacing a defective or deficient protein associated with a genetic disorder.

[0273] 52. The single strand of DNA of paragraph 51 wherein the genetic disorder is selected from the group comprising or consisting of: adenosine deaminase severe combined immunodeficiency (ADA-SCID), neuronal ceroid lipofuscinosis, aspartylglucosaminuria, Batten disease, betathalassemia, chronic granulomatous disease (CGD), cystic fibrosis, cystinosis, Duchenne muscular dystrophy, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Huntingdon's disease, Hurler disease, Leber's congenital amaurosis, Maroteau-Lamy disease, metachromatic leukodystrophy, Morquio disease type A, Morquio disease type B, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type Cl, Niemann-Pick disease type C2, Pompe disease, retinitis pigmentosa, Sandhoff disease, sickle cell disease, Spinal Muscular Atrophy, X-ALD, hemophilia A, hemophilia B, Hunter disease (MPS II), lysosomal storage disorder, Maroteaux-Lamy disease (MPS VI), Neuronal ceroid lipofuscinosis (NCL) including CLN1 disease, Sanfilippo disease type A (MPS IIIA), Sanfilippo disease type B (MPS IIIB), Sanfilippo disease type C (MPS IIIC), Sanfilippo disease type D (MPS HID), Schindler disease type I, Schindler disease type II, Sly disease (MPS VII), Tay-Sachs disease.

[0274] 53. The single strand of DNA according to any of paragraphs 1-39 wherein the first target sequence is a non-coding RNA, optionally a functional RNA, optionally selected from the group comprising or consisting of: a) a gRNA; b) an RNA designed to have sequence complementarity to a host target RNA so as to cause RNAi when expressed in a host cell, optionally a miRNA or a short hairpin RNA (shRNA); and / or c) a IncRNA.

[0275] 54. The single strand of DNA of paragraph 53 wherein the RNA is designed to have sequence complementarity to a host target RNA that is RNA transcribed from an oncogene, optionally wherein the oncogene is selected from the group comprising or consisting of KRAS, EGFR (Epidermal Growth Factor Receptor), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), DMD (Dystriophin). 55. The single strand of DNA according to any of paragraphs 1-54 wherein the single strand of DNA comprises at least a first target sequence that encodes a protein or peptide, and at least a second target sequence that is not a protein or peptide coding sequence and wherein the RNA is a functional RNA.

[0276] 56. The single strand of DNA according to paragraph 55 wherein the protein or peptide is a Cas protein, and wherein the RNA is a corresponding gRNA.

[0277] 57. The single strand of DNA of paragraph 56, wherein the gRNA is designed to have sequence complementarity to a viral genome or viral nucleic acid, optionally wherein the virus is selected from the group comprising or consisting of HBV optionally wherein the viral nucleic acid is cccDNA (Covalently Closed Circular DNA), or HIV Provirus.

[0278] 58. The single strand of DNA according to any of the preceding paragraphs wherein the single strand of DNA comprises a second target sequence, or a third, fourth, fifth, sixth, sevenths, eighth, ninth or tenth or more target sequence, and wherein each target sequence encodes, or encodes following replication of the single strand of DNA, any one or more of: a) a protein or peptide that is an antigenic protein or peptide or comprises an antigenic protein or peptide; b) a protein or peptide that is a therapeutic protein or peptide; c) an adjuvant protein or peptide; and / or d) a non-coding RNA, optionally a functional RNA, optionally selected from the group comprising or consisting of: i) a gRNA; ii) an RNA designed to have sequence complementarity to a host target RNA so as to cause RNAi when expressed in a host cell; and / or iii) a IncRNA.

[0279] 59. The single strand of DNA according to paragraph 58 wherein the single strand of DNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sequences that each encodes, or encodes following replication of the single strand of DNA, an antigenic protein or peptide.

[0280] 59a. The single strand of DNA according to paragraph 58 wherein the single strand of DNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sequences that each encodes, or encodes following replication of the single strand of DNA a therapeutic protein or peptide.

[0281] 59b. The single strand of DNA according to paragraph 58 wherein the single strand of DNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sequences that each encodes, or encodes following replication of the single strand of DNA a non-coding RNA, for example a functional RNA, for example selected from the group comprising or consisting of: i) a gRNA, ii) an RIMA designed to have sequence complementarity to a host target RNA so as to cause RNAi when expressed in a host cell; and / or iii) a IncRNA. 59c. The single strand of DNA according to paragraph 58 wherein the single strand of DNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sequences that each encodes, or encodes following replication of the single strand of DNA any combination of: a) a therapeutic protein or peptide; or b) a functional RNA, for example selected from the group comprising or consisting of: i) a gRNA, ii) an RNA designed to have sequence complementarity to a host target RNA so as to cause RNAi when expressed in a host cell; and / or iii) a IncRNA; and / or c) an antigenic protein or peptide.

[0282] 60. The single strand of DNA according to paragraph 58 or 59 wherein at least two of the target sequences encode, or encodes following replication of the single strand of DNA, different antigenic proteins or peptides, optionally at least 3, 4, 5, 6, 7, 8, 9 or at least 10 of the target sequences encode, or encode following replication of the single strand of DNA, different antigenic proteins or peptides.

[0283] 61. The single strand of DNA according to paragraph 58-60 wherein at least two of the target sequences encode, or encode following replication of the single strand of DNA, the same antigenic protein or peptide, optionally wherein at least 3, 4, 5, 6, 7, 8, 9 or at least 10 of the target sequences encode, or encode following replication of the single strand of DNA, the same antigenic proteins or peptides.

[0284] 62. The single strand of DNA according to any of paragraphs 58-61 wherein at least two of the target sequences encode, or encode following replication of the single strand of DNA, an antigenic protein or peptide that each comprise one or more sequences to enhance presentation of the protein or peptide or fragments thereof, and wherein at least two of the antigenic proteins or peptides comprise different sequences to enhance presentation of the protein or peptide or fragments thereof, optionally wherein the sequences to enhance presentation of the protein or peptide or fragments thereof are selected from the group comprising or consisting of: a) a ubiquitin sequence; b) an Endoplasmic Reticulum (ER) signal peptide, optionally the signal peptide from tissue plasminogen activator (tPA); c) the Lysosome-Associated Membrane Protein 1 (LAMP-1); d) a chemokine ligand, optionally XCL1; and / or e) the CD28 leader peptide.

[0285] 63. The single strand of DNA according to any of paragraphs 58-62 wherein at least two of the target sequences encode, or encode following replication of the single strand of DNA, the same antigenic protein or peptide and each protein or peptide comprises one or more sequences to enhance presentation of the first protein or peptide or fragments thereof, but wherein the one or more sequences to enhance presentation of the first protein or peptide or fragments thereof present in the first target sequence are different to those of the second target sequence.

[0286] 64. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is not a closed linear DNA.

[0287] 65. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is not doggybone DNA (dbDNA).

[0288] 66. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is not closed linear DNA.

[0289] 67. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA comprises at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000 or more nucleotides that are single-stranded and are not part of a terminal hairpin or loop structure.

[0290] 68. The single strand of DNA of any of the preceding paragraphs wherein the ssDNA of any of the preceding paragraphs comprises one or more terminal hair pin loops and also comprises a further region of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000 or more nucleotides that are single stranded.

[0291] 69. The single strand of DNA of any of the preceding paragraphs comprising at least 5%, 10%, 15%, 20%, 25%, 50%, 75%, or up to 100% of their nucleotide sequence in an unpaired single-stranded state under physiological conditions.

[0292] 70. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA does not comprise a telomerase target sequence or a protelomerase target sequence.

[0293] 71. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA does not comprise a hairpin loop at the 5', 3', or 5' and 3' termini.

[0294] 72. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA does not comprise a sequence that is or encodes a bacterial origin of replication.

[0295] 73. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA does not comprise unmethylated CpG motifs.

[0296] 74. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA does not comprise a bacterial selection marker.

[0297] 75. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA does not comprise an antibiotic resistance gene, for example does not comprise an antibiotic resistance gene that is operably linked to elements which would allow expression of the antibiotic resistance gene in a mammalian cell, for example in a human cell. 76. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is not a conjugative plasmid, and does not encode a conjugative plasmid and / or does not comprise a nic site.

[0298] 76a. The single strand of DNA according to any of the preceding paragraphs wherein the single strand of DNA does not comprise any phage-derived sequences.

[0299] 76b. The single strand of DNA according to any of the preceding paragraphs wherein the single strand of DNA does not comprise a phage packaging signal, optionally does not comprise an ml3 phage packaging signal.

[0300] 76c. The single strand of DNA according to any of the preceding paragraphs wherein : a) the single strand of DNA comprises a sequence that when transcribed into RNA is a WPRE; b) the first RNA molecule comprises a WPRE optionally where the WPRE is located in the 3'UTR of the first RNA molecule.

[0301] 77. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA comprises one or more sequences that aid in maintaining the ssDNA in the cell, for example maintaining the ssDNA episomally in the cell.

[0302] 78. The single strand of DNA of paragraph 77 wherein the sequence that aids in maintaining the ssDNA in the cell is an episomal maintenance element such as a scaffold / matrix attachment region (S / MAR).

[0303] 79. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is for integration into a host cell genome.

[0304] 80. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA comprises two inverted repeats from a transposon system.

[0305] 81. The single strand of DNA of paragraph 80 where the transposon system is the Piggyback transposon system or the Sleeping Beauty transposon system.

[0306] 82. The single strand of DNA of any of the preceding paragraphs wherein the ssDNA comprises two inverted repeats from the Piggyback transposon and / or two inverted repeats from the Sleeping Beauty transposon.

[0307] 83. The single stand of DNA of any of the preceding paragraphs wherein the target sequence, or at least one of the target sequences encodes a protein or peptide that is a transposase.

[0308] 84. The single strand of DNA of any of the preceding paragraphs where the single strand of DNA comprises two inverted repeats from a transposon and wherein the target sequence or one the at least one target sequences encodes a protein or peptide that is a transposase that recognises the two inverted repeats.

[0309] 85. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is able to be maintained in a cell, optionally a mammalian cell optionally a human cell. 86. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA comprises an episomal maintenance element.

[0310] 87. The single strand of DNA according to paragraph 86 wherein the episomal maintenance element is a scaffold / matrix attachment region (S / MAR).

[0311] 89. The single strand of DNA according to any of the preceding paragraphs wherein the ssDNA is not for genomic integration and does not comprise sequences for genomic integration.

[0312] 90. The single strand of DNA according to any of the preceding paragraphs wherein the single strand of DNA is not to be maintained episomally in the cell, and the single strand of DNA does not comprise an episomal maintenance element.

[0313] 91. The single strand of DNA of any of paragraphs 1-90 wherein the single strand of DNA was produced by expressing ssDNA from a dsDNA template vector that comprises at least one copy of the single strand of DNA of any of paragraphs 1-90, wherein the one or each copy of the single strand of DNA is flanked by a self-cleaving DNA sequence, to produce a precursor template ssDNA, wherein said expression is in a microbial cell culture, optionally: a) a bacterial cell culture, optionally an E. coli cell, optionally a K12- derived E. coli safety strain, optionally DH5alpha (DH5a), XL-lblue or JM109; or b) a yeast cell culture.

[0314] 92. The single strand of DNA of paragraph 91 wherein the dsDNA template vector is a phagemid, optionally further comprising: a) a packaging sequence; b) component(s) ensuring propagation of the phagemid during cell division; and / or c) a selection marker, typically an antibiotic resistance gene.

[0315] 93. The single strand of DNA of paragraph 91 or 92 wherein following expression of the precursor template ssDNA from the dsDNA template vector, the ssDNA is isolated from the microbial cell or cell culture media.

[0316] 94. The single strand of DNA of any of paragraphs 91-93 wherein the precursor template ssDNA is digested under reaction conditions where the self-cleaving DNA sequences become active to produce the single strand of DNA , optionally wherein said conditions comprise the addition of Zn2+.

[0317] 95. The single strand of DNA of any of paragraphs 91-94 wherein the self-cleaving DNA sequences are self-cleaving deoxyribozymes or DNAzymes, optionally Zn2+- dependent DNAzymes, optionally I-R3. 96. The single strand of DNA of any of paragraphs 91-95 wherein the dsDNA template vector comprises two, three, four or more, single strand of DNA as defined in any of paragraphs 1-90.

[0318] 97. The single strand of DNA of any of paragraphs 91-96 wherein expression of ssDNA from the dsDNA template vector occurs in the presence of a helper plasmid or a helper phage which comprises: a) genes encoding the proteins of a bacteriophage, e.g., M13 bacteriophage; b) component(s) ensuring propagation of the helper plasmid during cell division; and / or c) a selection marker, optionally an antibiotic resistance gene.

[0319] 98. The single strand of DNA of any of paragraphs 91-97 wherein the microbial cell is a bacterial cell and the single strand of DNA is packaged into phage-like particles which are secreted from the bacterial cells, and wherein the phage-like particles are isolated from the cell culture and the phagemid ssDNA is purified from the phage-like particles.

[0320] 99. The single strand of DNA of any of paragraphs 1-98 wherein the single strand of DNA further comprises fixed 5' and 3' end sequences, optionally wherein the fixed 5' and 3' end sequences are the scars that remain following DNAzyme cleavage.

[0321] 100. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is stable at a temperature of at least 18°C, or at least 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or 26°C, optionally stable for at least 24 hours, 48 hours, 1 week, 2 weeks, 4 weeks, 2 months, 6 months, 1 year or more.

[0322] 101. A dsDNA vector comprising a sequence corresponding to the single strand of DNA of any of paragraphs 1-100.

[0323] 102. The dsDNA vector of paragraph 101 wherein the vector comprises multiple repeats of the sequence corresponding to the single strand of DNA of any of paragraphs 1-90, optionally wherein the multiple repeats are arranged in a head-to-tail orientation, optionally wherein the dsDNA vector comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more repeats.

[0324] 103. The dsDNA vector of paragraph 101 wherein the vector is a phagemid, and optionally further comprises: a) a packaging sequence; b) component(s) ensuring propagation of the phagemid during cell division; and / or c) a selection marker, typically an antibiotic resistance gene.

[0325] 104. The dsDNA vector of paragraph 103 wherein the phagemid further comprises: a) genes encoding the proteins or parts of a protein of a bacteriophage, optionally proteins or parts of a protein of the M13 bacteriophage; b) component(s) ensuring propagation of the helper plasmid during cell division; and / or c) a selection marker, optionally an antibiotic resistance gene.

[0326] 105. A lipid nanoparticle (LNP) comprising one or more of the single strand of DNA of any one of paragraphs 1-100.

[0327] 106. The LNP of paragraph 105 wherein the single strand of DNA has a length greater than lkb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, lOkb, or more.

[0328] 107. The single strand of DNA according to any of paragraphs 1-100 wherein the single strand of DNA is not contained within a viral vector, optionally is not contained with an adenovirus, and AAV and / or a lentivirus.

[0329] 108. A nucleic acid nanostructure comprising at least one scaffold strand and a plurality of staple strands, wherein said nanostructure comprises at least one single strand of DNA according to any of paragraphs 1-100.

[0330] 109. The nucleic acid nanostructure according to paragraph 108 wherein said nucleic acid nanostructure comprises a first subunit and a second subunit; wherein, said first subunit and said second subunit each comprise a single strand of DNA according to any of paragraphs 1-58, optionally wherein the nucleic acid nanostructure comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more subunits, optionally where each subunit comprises a single strand of DNA according to any of paragraphs 1-100.

[0331] 109a. A nucleic acid complex comprising a single strand of DNA of any of the preceding claims and at least one separate oligonucleotide, wherein the at least one separate oligonucleotide is hybridised to one contiguous region of the single strand of DNA of any of the preceding claims.

[0332] 109b. The nucleic acid complex according to paragraph 109a comprising a single strand of DNA of any of the preceding claims and at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separate oligonucleotides, wherein each of the separate oligonucleotides is hybridised separately to different contiguous regions of the single strand of DNA of any of the preceding claims.

[0333] 110. A LNP comprising the nucleic acid nanostructure according to any of paragraphs 108-109 or the nucleic acid complex of paragraphs 109a or 109b.

[0334] 111. A composition comprising one or more single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, and / or the nucleic acid nanostructure according to any of paragraphs 108-109. 112. The composition according to paragraph 111 wherein the composition is formulated for administration to a subject, optionally wherein the subject is a mammal, optionally a human.

[0335] 113. The composition according to any of paragraphs 111 or 112 further comprising a pharmaceutically acceptable carrier and / or excipients.

[0336] 114. The composition according to any of paragraphs 111-113 further comprising an adjuvant.

[0337] 115. The composition according to any of paragraphs 111-114 further comprising components required for Nanojet administration.

[0338] 116. The composition according to any of paragraphs 111-115 wherein the composition is formulated for delivery of the one or more single strand of DNA according to any of paragraphs 1-100, the LNP of any of paragraphs 105 or 106, and / or the nucleic acid nanostructure according to any of paragraphs 108-109 to an immune cell, optionally to a dendritic cell or a macrophage.

[0339] 117. The composition according to any of paragraphs 111-116 wherein the composition does not comprise a viral vector, optionally does not comprise an adenovirus, and AAV and / or a lentivirus.

[0340] 118. A vaccine comprising one or more single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, the nucleic acid nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 80, and / or the composition according to any of paragraphs 111-117.

[0341] 119. The single strand of DNA according to any of paragraphs 1-100, the LNP of any of paragraphs 105 or 106, or the nucleic acid complex of paragraphs 109a or 109b, the nucleic acid nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 80, the composition according to any of paragraphs 111-117 and / or the vaccine according to paragraph 118 for use in a method of treating or preventing an infection with a pathogen, optionally a viral, bacterial, fungal or protist pathogen in a subject.

[0342] 120. A method of treating or preventing and infection with a pathogen, optionally a viral, bacterial, fungal or protist pathogen in a subject said method comprising administering the single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, the nucleic acid nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 110, the composition according to any of paragraphs 111-117 and / or the vaccine according to paragraph 118 to the subject.

[0343] 121. Use of a single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, the nucleic acid nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 110, the composition according to any of paragraphs 111-117 and / or the vaccine according to paragraph 118 for the manufacture of a medicament for the treatment or preventing and infection with a pathogen, optionally a viral, bacterial, fungal or protist pathogen in a subject.

[0344] 122. The single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, the single strand of DNA nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 110, the composition according to any of paragraphs 111-117 and / or the vaccine according to paragraph 118 for use in a method of treating or preventing a disease in a subject.

[0345] 123. A method of treating or preventing a disease in a subject wherein said method comprises administering a single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, the single strand of DNA nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 110, the composition according to any of paragraphs 111-117 and / or the vaccine according to paragraph 118 to a subject.

[0346] 124. Use of a single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, the single strand of DNA nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 110, the composition according to any of paragraphs 111- 117 and / or the vaccine according to paragraph 118 for the manufacture of a medicament for the treatment or preventing and infection with a disease in a subject.

[0347] 125. The single strand of DNA, nucleic acid complex, LNP, single strand of DNA nanostructure, composition or vaccine according to paragraph 122, method according to paragraph 123 or use according to paragraphs 124 wherein the first target sequence is: a) an antisense strand that encodes a first protein or peptide and is able to be directly transcribed to produce the first RNA molecule or b) a sense strand that is able to be transcribed to produce the first RNA molecule once the single strand of DNA is replicated to produce the antisense strand; and wherein the first protein or peptide is a therapeutic protein or peptide.

[0348] 126. The single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, the nucleic acid nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 110, the composition according to any of paragraphs 111-117 and / or the vaccine according to paragraph 118 for use in a method of treating or preventing cancer in a subject.

[0349] 127. A method of treating or preventing cancer in a subject wherein said method comprises administering a single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, the single strand of DNA nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 110, the composition according to any of paragraphs 111-117 and / or the vaccine according to paragraph 118 to a subject.

[0350] 128. Use of a single strand of DNA according to any of paragraphs 1-100, or the nucleic acid complex of paragraphs 109a or 109b, the LNP of any of paragraphs 105 or 106, the single strand of DNA nanostructure according to any of paragraphs 108-109, a LNP according to paragraph 110, the composition according to any of paragraphs 111- 117 and / or the vaccine according to paragraph 118 for the manufacture of a medicament for the treatment or prevention of cancer in a subject.

[0351] 129. The single strand of DNA, nucleic acid complex, LNP, single strand of DNA nanostructure, composition or vaccine according to paragraph 126, method according to paragraphs 127 or use according to paragraphs 128 wherein the first target sequence is: a) an antisense strand that encodes a first protein or peptide and is able to be directly transcribed to produce the first RNA molecule; or b) a sense strand that is able to be transcribed to produce the first RNA molecule once the single strand of DNA is replicated to produce the antisense strand; and wherein the first protein or peptide is an antigenic protein or peptide or comprises an antigenic protein or peptide, and wherein the antigenic protein or peptide is a cancer antigen or a cancer neoantigen.

[0352] 130. A method of producing a single strand of DNA of any of paragraphs 1-100, wherein said method comprises: a) providing a dsDNA template vector that is a phagemid and that comprises: i) at least one copy of the single strand of DNA of any of paragraphs 1-100 wherein the one or each copy of the single strand of DNA is flanked by a self-cleaving DNA sequence, to produce a precursor template ssDNA; ii) component(s) ensuring propagation of the phagemid during cell division; iii) a selection marker, typically an antibiotic resistance gene; and iv) a packaging sequence; and optionally v) one or more phage genes, that encode for phage proteins (Genlll); b) introducing the dsDNA template vector into a microbial cell either as a double strand, or as a single stranded DNA packaged in a bacterial phage protein coat, optionally an E. coli cell; c) culturing the microbial cell under conditions so as to produce a population of cells and allow the cells to produce phage-like particles comprising the at least one copy of the single strand of DNA of any of paragraphs 1-100 that are released from the cells; and d) harvesting the phage-like particles from the culture media; and e) extracting the ssDNA from the phage-like particles and exposing said ssDNA to conditions so as to allow self-cleavage of the self-cleaving DNA sequence; f) isolating the single strand of DNA of any of paragraphs 1-100; and wherein the cell also expresses: i) genes encoding the proteins of a bacteriophage, optionally genes encoding the proteins of M13 bacteriophage, optionally wherein said genes are present on a helper plasmid; and ii) optionally component(s) ensuring propagation of the helper plasmid, when present, during cell division.

[0353] 131. A method of producing a single strand of DNA of any of paragraphs 1-100, wherein said method comprises:

[0354] Step (a) culturing a microbial cell that comprises: i) at least one copy of the single strand of DNA of any of paragraphs 1-100 present on a phagemid, wherein the one or each copy of the single strand of DNA is flanked by a self-cleaving DNA sequence, to produce a precursor template ssDNA, wherein the phagemid comprises a selection marker, optionally an antibiotic resistance gene, and a packaging sequence; and ii) one or more phage genes, that encode for phage proteins (Genlll); and iii) genes encoding the proteins of a bacteriophage, optionally genes encoding the proteins of M13 bacteriophage, optionally wherein said genes are present on a helper plasmid under conditions so as to produce a population of cells and allow the cells to produce phage comprising the at least one copy of the single strand of DNA of any of paragraphs 1-100 that are released from the cells.

[0355] 132. The method of paragraph 131 further comprising step (b) harvesting the phagelike particles from the culture media.

[0356] 133. The method of paragraph 132 further comprising step (c) extracting the ssDNA from the phage and exposing said ssDNA to conditions so as to allow self-cleavage of the self-cleaving DNA sequence.

[0357] 134. The method of paragraph 133 further comprising step (d) isolating the single strand of DNA of any of paragraphs 1-100.

[0358] 135. The method of any of paragraphs 131-134 wherein steps (a); (a) and (b); (a), (b) and (c); or (a), (b), (c) and (d) are completed within a 48 hour, 72 hour, or 96 hour, 108 hour, 120 hour, 132 hour, 144 hour, 156 hour, 168 hour, 180 hour period.

[0359] 136. The method of any of paragraphs 131-135 wherein : a) 50g, 75g, 100g, 125g, 150g, 175g, 200g of isolated single strand of DNA is produced; and / or b) 50mg / L, 75mg / L, lOOmg / L, 125mg / L, 150mg / L, 175mg / L, 200mg / L of isolated single strand of DNA is produced.

[0360] 137. The method of any of paragraphs 131-136 wherein : a) 50g, 75g, 100g, 125g, 150g, 175g, 200g of isolated single strand of DNA is produced; and / or b) 50mg / L, 75mg / L, lOOmg / L, 125mg / L, 150mg / L, 175mg / L, 200mg / L of isolated single strand of DNA is produced in a 48 hour, 72 hour, or 96 hour, 108 hour, 120 hour, 132 hour, 144 hour, 156 hour, 168 hour, 180 hour period.

[0361] 138. A method of producing a vaccine comprising one or more single strand of DNA of any of paragraphs 1-100 wherein said method comprises producing a single strand of DNA of any of paragraphs 1-100 according to paragraph 130, and formulating the isolated single strand of DNA of step (f) as a vaccine.

[0362] 39. A method of producing a LNP vaccine comprising one or more single strand of DNA of any of paragraphs 1-100 wherein said method comprises producing a single strand of DNA of any of paragraphs 1-100 according to paragraph 130, and combining the isolated single strand of DNA of step (f) with lipids.

[0363] Figure legends Figure 1 - Schematic representation of exemplary constructs. Topology describes the topology of the ssDNA used within this application. + / - -strand refers to the sensestrand (+) or the antisense-strand (-). Sequences are given in 5' to 3' order on the resulting ssDNA. Boxes are not representative of length.

[0364] Figure 2 - GFP & Luciferase co-expression. HEK293T cells were transfected using lipofectamine reagent with lOOng of ssDNA Construct 5 and 7 (encoding GFP and Luciferase, separated by a P2A sequence).

[0365] Figure 3 - Expression of GFP with ssDNA vectors containing sense (+) and antisense (-) expression cassettes (ssDNA Construct 1 and 4)

[0366] Figure 4 - Expression of HA from circular ssDNA constructs 2 and 3 in HEK293T cells.

[0367] Figure 5 - GFP expression with LNP-formulated ssDNA Construct 1

[0368] Figure 6 - (A) linear or circular GFP encoding ssDNA are capable of expressing GFP in cells (Construct 7). (B)Expression of GFP from circular ssDNA constructs 7 (no DTS) and 10 (3xDTS) in HEK cells.

[0369] Figure 7 - GAS-STING activation by ssDNA and dsDNA in THP-1 cells.

[0370] Figure 8 - Overview of ssDNA constructs.

[0371] Examples

[0372] Constructs used in the Examples are shown in Figure 1 and 8. These are non-limiting Exemplary embodiments of the ssDNA of the invention and the skilled person will know that the various features and elements of these constructs can be replaced with equivalent versions of the element; and that various elements may be combined to produce new ssDNA constructs.

[0373] Design of ssDNA plasmid vectors optimized for expression and immunogenicity

[0374] Various factors such as regulatory elements, nuclear localisation signals for nuclear delivery and transcription, linear and circular ssDNA, sense and missense expression will be tested to optimise the single strand of DNA of the invention. Scalable production methods specifically for the production of the single strand of DNA of the invention will be developed, with upstream and downstream processing optimised.

[0375] In vitro evaluation for target sequence production

[0376] The ability of various iterations of single strands of DNA of the invention will be tested for the ability to transcribe the target sequence, and produce protein or peptides, or non-coding RNAs. The ability of antigenic proteins or peptides produced in this way will be tested for the ability to stimulate immune cells.

[0377] In vivo testing in mice and rabbits

[0378] Promising candidates will be rapidly advanced to in vivo testing in mice and rabbits to assess immunogenicity, safety, and protection against viral challenge.

[0379] Scalable production of single strands of DNA suitable for therapeutic purposes In parallel with the above, a scalable GMP manufacturing process will be developed for clinical trial material production.

[0380] Protection against pathogen infection

[0381] The efficacy of the single strand of DNA of the invention in providing protection to various pathogens will be demonstrated by the use of established animal challenge models will build confidence in the platform.

[0382] Delivery methods

[0383] Various delivery methods will be investigated to determine the optimal delivery method for a given context, including the testing of LNP encapsulation and Jet Injection.

[0384] Case studies

[0385] 1 - Development of an ssDNA vaccine against a newly identified influenza strain with high pandemic potential (avian flu H5Nx), from antigen selection to preclinical immunogenicity and challenge studies in animal models.

[0386] 2 - Adaptation of the platform to target a novel coronavirus, demonstrating the speed and flexibility of the approach in responding to unexpected outbreaks.

[0387] 3 - Comparison of ssDNA vaccines to conventional plasmid DNA and mRNA vaccines in terms of immunogenicity, manufacturing timelines, and stability. Example 1 - Expression of multiple target sequences from a single ssDNA

[0388] See Figure 2

[0389] Constructs used: Construct 5, Construct 7

[0390] HEK293T cells were transfected using lipofectamine reagent with lOOng of ssDNA Construct 5 (CMV promoter) and Construct 7 (EFla promoter). Both constructs encode GFP and Luciferase, separated by a P2A sequence. 24 hours post transfection, GFP expression was analyzed via flow cytometry while luciferase activity was quantified by addition of substrate and quantification of chemiluminescence on a plate reader.

[0391] Figure 2 shows the results from transfection with Construct 5 and Construct 7, both generate single transcripts which are translated into two separate proteins due to ribosome skipping at the P2A sequence to generate active GFP and active luciferase.

[0392] In summary, using ssDNA it is possible to express more than one gene from the same expression cassette, producing two functional proteins from a single promoter.

[0393] Example 2 - Expression from ssDNA occurs from a sense or antisense template

[0394] See Figure 3

[0395] Constructs used:_Construct 1 and Construct 4

[0396] Jurkat cells were electroporated with ssDNA constructs Construct 1 (GFP expression cassette in sense (+) orientation) and Construct 4 (construct backbone identical to Construct 1, but expression cassette in antisense (-) orientation) using a nucleofector device (Lonza). GFP expression was analyzed via flow cytometry after 24 hours.

[0397] Both + and - strand expression cassettes mediate similar levels of GFP expression, showing that delivery of ssDNA in either sense or antisense orientations results in expression of the target sequence in cells.

[0398] Example 3 - Expression of antigenic proteins with MHC targeting peptides allows cell surface expression of the antigen

[0399] See Figure 4

[0400] Constructs used: Construct 2 and Construct 3

[0401] This data demonstrates the ability of ssDNA carrying antigen-encoding sequences to enter cells, and express the functional antigen with subsequent targeting to the cell surface with MHC targeting peptides. HEK293T cells were transfected with circular ssDNA Constructs 2 and 3 encoding hemagglutinin (HA), using Lipofectamine 2000 at two concentrations. HA expression was assessed 48 hours post-transfection by flow cytometry using two anti-HA monoclonal antibodies (antibody 1 and antibody 2) followed by FITC-labelled secondary staining. Expression efficiencies ranged from 35-60% depending on the antibody used, with FI6 showing stronger detection. Controls included plasmid-transfected cells and secondary-antibody-only staining to confirm specificity.

[0402] The data is presented in Figure 4. In summary, antigen (HA) expression was confirmed in up to 35-40% of cells with antibody 1 detection (vs. 70% for plasmid) and up to 60% of cells with antibody 2 detection (vs. >80% for plasmid). The expression is specific, with no unspecific binding was observed.

[0403] This experiment demonstrates the expression of antigenic proteins (HA) from ssDNA constructs and the subsequent detection of the antigens by antigen-specific antibodies. The addition of the MHC-enhancing features (signal peptide) maintains a functionally active antigen. HA is a surface glycoprotein of influenza viruses and a key antigenic target in vaccine development, demonstrating the utility of ssDNA constructs for vaccination against influenza. The efficacy of up to 60% HA+ cells from circular ssDNA affirms the viability of ssDNA for antigen delivery in vaccine applications.

[0404] There is no reason to suppose that the delivery of antigens is restricted to HA - as demonstrated by the data herein it is possible to express a range of target sequences from ssDNA inside cells for presentation on the cell surface. The data demonstrates the ability of the present invention to deliver sequences encoding antigens to cells, for example for the purpose of vaccination.

[0405] Example 4 - Inclusion of a DNA nuclear targeting (DTS) sequence enhances expression

[0406] See Figure 6b

[0407] Constructs used: Construct 7 and Construct 10

[0408] HEK293T cells were transfected using Lipofectamine reagent with ssDNA Constructs 7 and 10. Both constructs encode GFP and luciferase separated by a P2A self-cleaving peptide, under the control of an EFla promoter. Construct 10 additionally contains three tandem repeats of the SV40 DNA Targeting Sequence (DTS), designed to enhance nuclear import of the ssDNA template. GFP expression was analyzed via flow cytometry 48 hours post-transfection.

[0409] As shown in Figure 6b, inclusion of the DTS elements in Construct 10 led to increased GFP expression compared to the DTS-negative control (Construct 7). This indicates that nuclear targeting sequences such as SV40 DTS can enhance the intracellular delivery and / or nuclear access of ssDNA vectors, leading to higher transgene expression.

[0410] Example 5 - LNP Delivery

[0411] See Figure 5

[0412] Constructs used: Construct 1 ssDNA Construct 1 was formulated into LNPs using the LipidLaunch LNP-102 exploration (Cayman Chemical) at an N:P ratio of 8. HEK cells were transfected with LNP-formulated ssDNA and after 24 hours, GFP expression was assessed via flow cytometry.

[0413] Figure 5 shows that_LNPs are a suitable means for delivering ssDNA to cells.

[0414] Example 6 - Linear and circular ssDNA is capable of expressing proteins in mammalian cells

[0415] See Figure 6A

[0416] Construct used: Construct 7

[0417] In brief, HEK293 cells were transfected with 100 ng of either circular or linearized Construct 7 using Lipofectamine. eGFP expression levels were subsequently analyzed through flow cytometry 48 hours post-transfection.

[0418] Example 7- LNP Delivery

[0419] The primary goal was to evaluate the immunogenicity and safety of ssDNA-LNP constructs relative to dsDNA and mRNA-LNPs. A secondary aim was to assess in vivo expression from ssDNA cargo.

[0420] The innate immune response was evaluated by measuring cytokines including IFN-y, IL-6, TNF-a, and others post-administration. Liver toxicity was assessed using ALT and AST levels on day 18.

[0421] Luciferase expression was monitored through in-life and ex vivo imaging of the liver.

[0422] Example 8 - Therapeutic Protein Expression

[0423] Construct tested - Construct 6 encoding collagen

[0424] Circular ssDNA construct 6, encoding a therapeutic collagen variant, has been successfully synthesized and ssDNA has been purified. Mammalian cells (e.g., HEK293T or primary fibroblasts) have been transfected with construct 6 using Lipofectamine- based delivery. Collagen expression will be quantified by ELISA from culture supernatants collected 48-72 hours post-transfection.

[0425] Conclusion:

[0426] This experiment aims to demonstrate that ssDNA constructs can encode functional therapeutic proteins relevant for gene therapy. Collagen, being a structural extracellular matrix protein, is a representative example of a large, secreted, and clinically relevant therapeutic target.

[0427] Example 9 - ssDNA does not activate the cGAS-STING pathway

[0428] See Figure 7

[0429] To evaluate potential innate immune activation, circular ssDNA and two doublestranded DNA controls were transfected into THP-1 human monocyte cells using Lipofectamine 2000. Cells were exposed to increasing concentrations of each construct (4, 40, 400, and 4000 ng / mL). After 4 hours of incubation, cells were lysed, and the production of 2',3'-cGAMP — an indicator of cGAS-STING activation— was measured using a competitive ELISA kit.

[0430] As shown in Figure 7, poly(dG:dC) induced robust production of cGAMP, confirming activation of the cGAS-STING pathway. In contrast, no significant cGAMP signal was detected following transfection with the ssDNA at any dose tested. dsDNA produced intermediate levels of cGAMP. These results confirm that ssDNA, when delivered under identical conditions, does not activate the cGAS-STING pathway in THP-1 cells. ssDNA constructs may have lower immunostimulatory potential than dsDNA, supporting their suitability for therapeutic use.

[0431] Equivalents The foregoing embodiments, instances, and examples are applicable to any of the aspects of the present disclosure and should be construed as such.

[0432] While the present disclosure has been described in terms of various aspects, embodiments, and examples, it is understood that variations, improvements, and equivalents will occur to the person skilled in the art. Such variations, improvements, and equivalents are contemplated by the present disclosure and fall within the scope of the matter disclosed and claimed herein.

Claims

Claims1. A single strand of DNA that: comprises at least a first target sequence operably linked to a first promoter sequence, wherein the first promoter sequence drives expression of the first target sequence to produce a first RIMA molecule either: i) directly from the single strand of DNA; or ii) following replication of the single strand of DNA into the double strand form where the single strand of DNA is single-stranded or substantially single stranded and wherein the first target sequence is for expression from the first promoter sequence in a mammalian cell or tissue.

2. The single strand of DNA of claim 1, wherein the single strand of DNA comprises at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, or 1000 or more nucleotides that are single-stranded and not part of a hairpin loop.

3. The single strand of DNA of any of claims 1 or 2 wherein the single strand of DNA does not comprise a first region of more than 500 continuous nucleotides that are complementary to a second region of the ssDNA of more than 500 continuous nucleotides.

4. The single strand of DNA of any of the preceding claims wherein the single strand of DNA comprises a first region that is between 10-1000 contiguous nucleotides, optionally between 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90- 600, 100-550, 150-500, 200-450, 250-400, or 300-350 contiguous nucleotides that is complementary to a second region of the single strand of DNA of between 10-1000, 20-950, 30-900, 40-850, 50-800, 60-750, 70-700, 80-650, 90-600, 100-550, 150- 500, 200-450, 250-400, or 300-350 contiguous nucleotides.

5. The single strand of DNA of claim 4 wherein the second region is a region of the first target sequence.

6. The single strand of DNA of claim 4 wherein the second region is a region of the promoter sequence.

7. The single stand of DNA of claim 4 wherein the first region is part of the first promoter.

8. The single strand of DNA of any of the preceding claims wherein the single strand of DNA is for integration into a host cell genome and comprises elements that allow genomic integration, optionally wherein the elements that allow genomic integration comprises two inverted repeats from a transposon system.

9. The single strand of DNA of any of the preceding claims wherein the single strand of DNA comprises two inverted repeats from a transposon system, optionally wherein the transposon system is the Piggyback transposon system or the Sleeping Beauty transposon system.

10. The single strand of DNA of any of claims 1-7 wherein the single strand of DNA is for episomal maintenance.

11. The single strand of DNA of any claims 1-7 and 10 wherein the single strand of DNA comprises one or more sequences that aid in maintaining the ssDNA episomally in the cell.

12. The single strand of DNA of claim 11 wherein the sequence that aids in maintaining the ssDNA in the cell is an episomal maintenance element such as a scaffold / matrix attachment region (S / MAR), optionally an S / MAR from ApoB and / or IFN-beta.

13. The single strand of DNA of any of the preceding claims wherein the single strand of DNA comprises at least one region that is double stranded, wherein said double stranded region is generated via hybridisation between at least a first and second region of the single strand of DNA that are substantially complementary to each other, and wherein the hybridisation between a first and second region of the single stand of DNA enhances expression of the first RNA molecule.

14. The single strand of DNA of any of the preceding claims wherein the single strand of DNA comprises at least one double-stranded region formed via hybridisation between a region of the single stranded DNA and at least one separate complementary oligonucleotide.

15. The single strand of DNA of claim 14 wherein the region of the single stranded DNA is a region of the first promoter.

16. The single strand of DNA of claim 14 wherein the region of the single stranded DNA is a region of the first target sequence.

17. The single strand of DNA of claim 14 wherein the region of the single stranded DNA is a region of neither the first target sequence or the first promoter.

18. The single strand of DNA of any of claims 14-17 wherein the complementary oligonucleotide is at least 5 nucleotides in length, optionally at least 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 nucleotides in length.

19. The single strand of DNA of any of claims 14-18 wherein the single strand of DNA comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more double stranded regions, where each double stranded region is formed by hybridisation with individual separate oligonucleotides.

20. The single strand of DNA of any of claims 1-10 further comprising: a) a cell-type specific promoter; and / or b) a first enhancer sequence, wherein the first enhancer sequence enhances expression of the first target sequence in a specific cell type.

21. The single strand of DNA of any of the preceding claims wherein the at least first target sequence encodes a first protein or peptide or following replication of the single strand of DNA into the double strand form encodes a first protein or peptide.

22. The single strand of DNA of any of the claims 1-20 wherein the at least first target sequence encodes a first non-coding RNA or following replication of the single strand of DNA into the double strand form encodes a first non-coding RNA.

23. The single strand of DNA of any of the preceding claims wherein the single strand of DNA additionally comprises a second target sequence, or a third, fourth, fifth, sixth, seventh, eighth, ninth or tenth or more target sequence.

24. The single strand of DNA of claim 23 wherein the second target sequence, or third, fourth, fifth, sixth, seventh, eighth, ninth or tenth or more target sequences present in the single strand of DNA are arranged in a tandem array and are operably linked to the same promoter so that the target sequences are transcribed into a single RNA molecule, and wherein the single RNA molecule comprises one or more 2A peptide sequences and / or one or more internal ribosome entry site (IRES) that is present 5' to each target sequence, so that each target sequence is translated as separate proteins or peptides.

25. The single stand of DNA of any of the preceding claims wherein the single strand of DNA comprises at least one target sequence that encodes a transposase.

26. The single strand of DNA according to any of the preceding paragraphs further comprising one or more DNA targeting sequences (DTS), optionally a DTS from SV40 or the 81 base pair DTS of Sox2 regulatory region 2 (SRR2) or functional variant thereof, optionally wherein the single stand of DNA comprises 1, 2 or 3 DTS.

27. The single strand of DNA according to any of the preceding claims wherein the first target sequence encodes a protein or peptide, or when in double stranded form encodes a protein or peptide, and further comprises one or more sequences to enhance localisation of the protein or peptide to the cell surface.

28. The single strand of DNA according to claim 27 wherein the one or more sequences to enhance localisation of the protein or peptide to the cell surface is: a) an Endoplasmic Reticulum (ER) signal peptide, optionally the signal peptide from tissue plasminogen activator (tPA) or CD28; b) a ubiquitin sequence; c) the Lysosome-Associated Membrane Protein 1 (LAMP-1); and / or d) a chemokine ligand, optionally XCL1.

29. The single strand of DNA of any of the preceding paragraphs wherein the single strand of DNA is linear, optionally wherein the single strand of DNA comprises caps formed by hairpin loops at the 5' end and / or the 3' end.

30. The single strand of DNA of any of claims 1-28 wherein the single strand of DNA is circular.

31. The single strand of DNA of any of the preceding claims wherein the first target sequence is an antisense strand that is able to be directly transcribed to produce the first RIMA molecule; or is a sense strand that is able to be transcribed to produce the first RNA molecule only once the single strand of DNA is replicated to produce the antisense strand.

32. The single strand of DNA according to any of the preceding claims wherein the at least first target sequence encodes (or when in double stranded form encodes) a first protein or peptide that is an antigenic protein or peptide or comprises an antigenic protein or peptide, and wherein the antigenic protein or peptide is selected from the group comprising or consisting of: a) a viral antigen; b) a cancer antigen; c) an Autoimmune Disease Antigen; d) a Neurological Disease Antigens; e) a bacterial antigen; f) a fungal antigen; and / or g) a protist antigen.

33. The single strand of DNA according to claim 32 wherein the first protein or peptide that is an antigenic protein or peptide or comprises an antigen protein or peptide comprises one or more sequences to enhance presentation of the antigen protein or peptide or fragments thereof on MHC I and / or MHC II.

34. The single strand of DNA according to claim 33 wherein the one or more sequences to enhance presentation of the first protein or peptide fragment is: a) a ubiquitin sequence; b) an Endoplasmic Reticulum (ER) signal peptide, optionally the signal peptide from tissue plasminogen activator (tPA); c) the Lysosome-Associated Membrane Protein 1 (LAMP-1); d) a chemokine ligand, optionally XCL1; and / or e) the CD28 leader peptide.

35. The single strand of DNA according to any of the preceding claims wherein at least one target sequence comprises a sequence that encodes (or when in double stranded form encodes) an adjuvant protein or peptide.

36. The single strand of DNA according to any of the preceding claims wherein at least one target sequence encodes (or when in double stranded form encodes) a protein or peptide, and wherein the protein or peptide is a therapeutic protein or peptide capable of replacing a defective or deficient protein associated with a genetic disorder.

37. The single strand of DNA according to any of the preceding claims wherein at least one target sequence encodes (or when in double stranded form encodes) a noncoding RNA selected from the group comprising or consisting of: a) a gRNA; b) an RNA designed to have sequence complementarity to a host target RNA so as to cause RNAi when expressed in a host cell, optionally a miRNA or a short hairpin RNA (shRNA); and / or c) a IncRNA; optionally wherein the RNA is designed to have sequence complementarity to a host target RNA that is RNA transcribed from an oncogene, optionally wherein the oncogene is selected from the group comprising or consisting of KRAS, EGFR (Epidermal Growth Factor Receptor), CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), DMD (Dystriophin).

38. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA comprises at least a first target sequence that encodes (or when in double stranded form encodes) a protein or peptide, and at least a second target sequence that encodes (or when in double stranded form encodes) a gRNA and wherein the protein or peptide is a Cas protein; optionally wherein the gRNA is designed to have sequence complementarity to a viral genome or viral nucleic acid, optionally wherein the virus is selected from the group comprising or consisting of HBV and wherein the viral nucleic acid is cccDNA (Covalently Closed Circular DNA), or HIV Provirus.

39. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sequences that each encodes, or encodes following replication of the single strand of DNA, an antigenic protein or peptide; and wherein at least two of the target sequences encode, or encodes following replication of the single strand of DNA, different antigenic proteins or peptides, optionally at least 3, 4, 5, 6, 7, 8, 9 or at least 10 of the target sequencesencode, or encode following replication of the single strand of DNA, different antigenic proteins or peptides.

40. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA is not a closed linear DNA.

41. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA is not doggybone DNA (dbDNA).

42. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise a telomerase target sequence or a protelomerase target sequence.

43. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise a hairpin loop at the 5', 3', or 5' and 3' termini.

44. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise a hairpin loop at the 5' and 3' termini.

45. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise a sequence that is or encodes a bacterial origin of replication.

46. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise unmethylated CpG motifs.

47. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise a bacterial selection marker.

48. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise an antibiotic resistance gene.

49. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise an antibiotic resistance gene that is operably linked to elements which would allow expression of the antibiotic resistance gene in a mammalian cell, for example in a human cell.

50. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA is not a conjugative plasmid, and does not encode a conjugative plasmid and / or does not comprise a nic site.

51. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise any phage-derived sequences.

52. The single strand of DNA according to any of the preceding claims wherein the single strand of DNA does not comprise a phage packaging signal, optionally does not comprise an ml3 phage packaging signal.

53. The single strand of DNA according to any of the preceding claims wherein: a) the single strand of DNA comprises a sequence that when transcribed into RNA is a WPRE; b) the first RNA molecule comprises a WPRE optionally where the WPRE is located in the 3'UTR of the first RNA molecule.

54. The single strand of DNA according to any of the preceding claims, wherein the single strand of DNA is:At least 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, 10000, 10250, 10500,10750, 11000, 11250, 11500, 11750, 12000, 12250, 12500, 12750, 13000, 13250,13500, 13750, 14000, 14250, 14500, 14750, 15000, 15250, 15500, 15750, 16000,16250, 16500, 16750, 17000, 17250, 17500, 17750, 18000, 18250, 18500, 18750,19000, 19250, 19500, 19750, or at least 20000 nucleotides in length.

55. A nucleic acid complex comprising a single strand of DNA of any of the preceding claims and at least one separate oligonucleotide, wherein the at least one separate oligonucleotide is hybridised to one contiguous region of the single strand of DNA of any of the preceding claims.

56. The nucleic acid complex according to claim 55 comprising a single strand of DNA of any of the preceding claims and at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more separate oligonucleotides, wherein each of the separate oligonucleotides is hybridised separately to different contiguous regions of the single strand of DNA of any of the preceding claims.

57. A lipid nanoparticle (LNP) comprising one or more of the single strands of DNA of any one of claims 1-54 or one or more of the nucleic acid complexes of claims 55 or 56.

58. A vaccine comprising one or more single strands of DNA according to any of claims 1-54 or nucleic acid complex of claims 55 or 56, or LNP of claim 57.

59. A cell comprising the single strand of DNA according to any of claims 1-54. or nucleic acid complex of claims 55 or 56, or LNP of claim 57.

60. The single strand of DNA according to any of claims 1-54, or nucleic acid complex of claims 55 or 56, or LNP of claim 57 or vaccine of claim 58 or cell of claim 59 for use in : a) a method of treating or preventing an infection with a pathogen, optionally a viral, bacterial, fungal or protist pathogen in a subject; b) a method of treating or preventing a disease in a subject. c) a method of treating or preventing cancer in a subject, optionally wherein the single strand of DNA, nucleic acid complex, LNP or vaccine is formulated for Nanojet delivery.

61. A method of producing the single strand of DNA of any of claims 1-54, wherein said method comprises:Step (a) culturing a microbial cell that comprises: i) at least one copy of the single strand of DNA of any of claims 1- 42 present on a phagemid, wherein the one or each copy of the single strand of DNA is flanked by a self-cleaving DNA sequence, to produce a precursor template ssDNA, wherein the phagemid comprises a packaging sequence; ii) one or more phage genes, that encode for phage proteins (Genlll); and iii) genes encoding the proteins of a bacteriophage, optionally genes encoding the proteins of M13 bacteriophage, optionally wherein said genes are present on a helper plasmid; and optionally comprises a selection marker, optionally an antibiotic resistance marker; wherein said culturing is under conditions so as to produce a population of cells and allow the cells to produce phage-like particles comprising the at least onecopy of the single strand of DNA of any of claims 1-42 that are released from the cells; step (b) harvesting the phage-like particles from the culture media; step (c) extracting the ssDNA from the phage-like particles and exposing said ssDNA to conditions so as to allow self-cleavage of the self-cleaving DNA sequence; and step (d) isolating the single strand of DNA of any of claims 1-42; and wherein steps (a), (b), (c) and (d) are completed within a 48 hour, 72 hour, or 96 hour, 108 hour, 120 hour, 132 hour, 144 hour, 156 hour, 168 hour, 180 hour period and produce: i) 50g, 75g, 100g, 125g, 150g, 175g, 200g of isolated single strand of DNA of any of claims 1-18; and / or ii) 50mg / L, 75mg / L, lOOmg / L, 125mg / L, 150mg / L, 175mg / L, 200mg / L of isolated single strand of DNA of any one of claims 1-18.

62. A method of producing nucleic acid for use in a vaccine wherein the method produces: i) 50g, 75g, 100g, 125g, 150g, 175g, 200g of isolated single strands of DNA; and / or ii) 50mg / L, 75mg / L, lOOmg / L, 125mg / L, 150mg / L, 175mg / L, 200mg / L of isolated single strands of DNA; in a time period of 48 hours, 72 hours, or 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, 168 hours, or 180 hours, and wherein the method comprises the method of claim 56.

63. An ex vivo method of expressing a protein or RNA in a cell, said method comprising transfecting a cell, optionally a mammalian cell, optionally a human cell, with one or more single strands of DNA of any of claims 1-54, the complex of claims 55 or 56, or the LNP of claim 57, optionally where the transfection is via electroporation, lipofection or via delivery of the ssDNA formulated in LNPs.

64. A single stranded DNA molecule (ssDNA) that comprises:(a) a self-hybridizing domain that comprises at least two regions, wherein the at least two regions comprises a first region of the self-hybridizing domain that is capable of hybridizing to a second region of the self-hybridizing domain;(b) one or more target sequences that encodes a peptide, a protein, a nucleic acid-based adjuvant or a non-coding RIMA; and(c) at least one promoter sequence operable linked to the one or more target sequences.

65. The ssDNA of claim 64, wherein the at least one promoter sequence drives expression of the one or more target sequences directly from the ssDNA.

66. The ssDNA of claims 54 or 65, wherein the one or more target sequences encode an antigenic protein fused to a peptide sequence that, upon introduction of the ssDNA into a host cell, enhances presentation on a surface of the host cell.

67. The ssDNA of claim 66, wherein the peptide sequence enhances presentation by a major histocompatibility protein (MHC) I protein, a MHC II protein, or both.

68. The ssDNA of claim 66, wherein the peptide sequence enhances presentation by one or more antigen presenting cells, optionally dendritic cells, macrophages and / or B cells.

69. The ssDNA of claims 66-68, wherein the antigenic protein comprises a viral antigen, a cancer antigen, an autoimmune disease antigen, a neurological disease antigen, a bacterial antigen, a fungal antigen or a protist antigen.

70. The ssDNA of any one of claims 64-69, wherein the at least one promoter sequence comprises a Cytomegalovirus (CMV) Immediate-Early promoter sequence, a Simian Virus 40 (SV40) promoter sequence, a CAT promoter sequence, a Muscle Creatine Kinase promoter (MCK), a Human Elongation Factor 1 Alpha (EFla) promoter sequence, a Tetracycline-Inducible promoter sequence, a MiniPromoter sequence, a U6 promoter sequence, an Hl promoter sequence, a SP6 promoter sequence, T7 promoter sequence, a tissue specific promoter sequence or a cell-type specific promoter sequence.

71. The ssDNA of any one of claims 64-70, wherein the ssDNA further comprises an enhancer sequence that is operably linked to the at least one promoter sequence resulting in increased expression of the one or more target sequences, as compared toan expression of the one or more target sequences in a comparable ssDNA that lacks the enhancer sequence.

72. The ssDNA of claim 71, wherein the enhancer sequence comprises a viral enhancer sequence, a ubiquitous / constitutive enhancer sequence, a tissue specific enhancer sequence, a cell-type specific enhancer sequence, a super-enhancer sequence, a synthetic / designed enhancer sequence or an inducible enhancer sequence.

73. The ssDNA of any one of claims 64-72, wherein the ssDNA further comprises an intron sequence that is positioned between the at least one promoter and an open reading frame encoding the one or more target sequences.

74. The ssDNA of claim 73, wherein the intron sequence comprises a CMV intron A sequence, an AAV intron sequence, or an elongation factor la first intron (EF-la) intron sequence.

75. The ssDNA of any one of claims 64-74 for use in treatment of a disease or condition.

76. The ssDNA of claim 75, wherein the ssDNA when administered to a subject in need thereof results in a reduction in one or more symptoms caused by the disease or condition.

77. The ssDNA of claim 75 or 76 wherein the ssDNA when administered to a subject in need thereof results in reduced immunogenicity, as compared to a level of immunogenicity in the subject when an mRNA or plasmid expressing the one or more target sequences is administered to the subject.

78. A single stranded DNA molecule (ssDNA) that comprises:(a) a polynucleotide sequence that comprises a hybridization motif that is capable of hybridizing to a complementary polynucleotide sequence;(b) one or more target sequences that encodes a peptide, a protein, a nucleic acid-based adjuvant or a non-coding RNA; and(c) at least one promoter sequence operable linked to the one or more target sequences, wherein upon hybridization of the complementary polynucleotide sequence to the hybridization motif of the polynucleotide sequence, a level of expression of the one or more target sequences increased, as compared to a level of expression of the one or more target sequences in the absence of the hybridization.

79. The ssDNA of claim 78, wherein the complementary polynucleotide sequence is present on an oligonucleotide that is not covalently attached to the single stranded DNA molecule.

80. The ssDNA of claim 78, wherein the complementary oligonucleotide is present in a region of the single stranded DNA molecule.

81. The ssDNA of claim 78, wherein the one or more target sequences encode an antigenic protein fused to a peptide sequence that, upon introduction of the ssDNA into a host cell, enhances presentation on a surface of the host cell.

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