WO2024115669A1 - Process for production of synthetic circular dna - Google Patents

Abstract

The disclosure provides a novel process for producing a synthetic circular DNA (scDNA) molecule and a novel scDNA molecule which can be produced by the method. The disclosure further provides therapeutical methods using the scDNA molecule.

Description

PROCESS FOR PRODUCTION OF SYNTHETIC CIRCULAR DNA

FIELD OF THE INVENTION

The present invention relates to the field of clinical DNA technology, including DNA vaccine technology. In particular, the present invention relates to a novel method of producing a synthetic circular DNA (scDNA) molecule. The invention also relates to scDNA molecules, characterized by novel features, which can be produced using this method, as well as their use clinically.

BACKGROUND OF THE INVENTION

DNA can be used clinically for several purposes, including for DNA vaccination. This mode of vaccination, which has been investigated in detail since the early 1990'ies, is a technique where DNA is administered in a non-viral plasmid form to somatic cells of a mammal leading to expression of the genes comprised in the plasmid; in DNA vaccination the encoded material is immunogenic polypeptide(s), which upon production by the somatic cells will be able to induce an immune response. The first DNA vaccine for human use was approved in 2021.

Current production of DNA for use in clinical settings, including for DNA vaccination, is based on bacterial production of plasmid DNA. This presents several challenges for the production, including the need to keep the bacterial culture sterile for extended time periods. To help minimize the risk of contamination of bacterial cultures, production methods typically rely on use of expensive and highly specialized single-use consumables. These methods are also not easy to automate. They are therefore laborious as typically a great deal of manual handling is required. Once the bacteria have been harvested, they need to be lysed to release the plasmid DNA, which then must be further purified to remove all other bacterial components.

In addition to the technical issues, there are also biological issues. To amplify the plasmid DNA in a bacterial system, the DNA must contain an origin of replication, which is required for the initiation of replication of the plasmid DNA within the bacterium. However, if there is an escape, this can allow the plasmid to further spread to other, non-intended, bacteria. Furthermore, to prevent contamination and ensure the plasmid is not lost from the bacterial host during production, selection genes are used in the plasmid DNA. These typically confer resistance to an antibiotic that is supplemented into the growth media. The widespread use of antibiotic genes and antibiotics in large amounts is especially problematic as resistant species of infectious bacteria and microorganisms is on the rise, causing challenges to health care globally. Neither the replication origin nor the selection genes have any clinical relevancy for patients but are there purely to aid the production method.

Finally, not all DNA sequences can be reliably maintained in bacterial hosts. These can either be toxic to the cell in some intended or unintended way, resulting in low yield. Alternatively, changes can occur as part of the normal cellular processes in the bacterial host, such as through erroneous DNA repair mechanisms, insertion of transposable elements, etc.

Thus, there is a need for improved methods for production of DNA for use in clinical settings, to overcome the above-mentioned challenges.

OBJECT OF THE INVENTION

It is an object of the invention to provide a method for producing a synthetic circular DNA (scDNA) molecule useful in a DNA vaccine and/or for a different clinical use. It is further an object of the invention to provide scDNA molecules which have some improved features over other DNA vectors when it comes to their utility for clinical use.

SUMMARY OF THE INVENTION

It has been found by the present inventors that synthetic circular DNA (scDNA) molecules for clinical use can be generated by a new, purely synthetic process, which eliminates the need for cellular production steps. The process is fast, has a low error-rate, and results in pure scDNA molecules comprising a molecule of interest. The process can be fully automated, removing the need for large amounts of manual handling. As the method is entirely cell-free, there is no need for lysis, and purification is simplified as all components in the process are known. Cell-free production also ensures that all sequences of interest can be amplified, broadening the potential of DNA for clinical use, such as in DNA vaccines and immune- oncology. Furthermore, the present inventors have discovered that their process can be used to generate scDNA molecules which have some improved features for clinical use, such as scDNA molecules comprising a gene encoding a therapeutic molecule of interest but lacking an origin of replication and an antibiotic resistance gene, and scDNA molecules comprising an inactive recombination site, thereby avoiding accidental recombination events. Thus, the new method overcomes several challenges associated with current cell-based methods for DNA production for clinical use, described above.

So, in a first aspect the present invention relates to a process for producing a synthetic circular DNA (scDNA) molecule, the process comprising : an amplification step; wherein a template DNA molecule is amplified, and a circularization step; wherein the amplified DNA is circularized, wherein the circularization step comprises site-specific recombination.

In a 2^nd aspect, the invention relates to a synthetic circular DNA (scDNA) molecule which comprises at least one gene encoding a molecule of interest and which scDNA molecule does not comprise an origin of replication or an antibiotic resistance gene. A version of this aspect is a scDNA molecule obtainable by the process of the 1^st aspect of the invention as well as any embodiment thereof disclosed herein.

In a 3^rd aspect, the invention relates to a composition comprising a plurality of scDNA molecules, each of which is a scDNA molecule of the 2^nd aspect of the invention as well as any embodiments of thereof disclosed herein, wherein the majority of the scDNA molecules in the composition are in supercoiled monomeric form.

In 4^th and 5^th aspects of the invention is provided the scDNA molecule of the 2^nd aspect of the invention or the composition of the 3^rd aspect of the invention as well as any embodiments of these aspects disclosed herein for use as a medicament (4^th aspect) or for use in vaccination or gene therapy (5^th aspect).

A 6^th aspect of the invention relates to a method of treating a subject in need thereof, comprising administering to the subject an efficient amount of the scDNA molecule of the 2^nd aspect of the invention as well as any embodiment thereof disclosed herein or of the composition of the 3^rd aspect of the invention as well as any embodiment thereof disclosed herein.

LEGENDS TO THE FIGURES

Fig. 1 : Schematic drawing illustrating the principle of the circularization step according to the invention, using a DNA molecule comprising loxP sites as an example. On the left side is shown a linearized, amplified DNA molecule comprising loxP sites oriented in the same direction and flanking a payload. Recombination (indicated by the arrow) leads to the production of the two molecules shown on the right side: a synthetic circular DNA (scDNA) molecule (top) comprising a single loxP site and the payload, and a linear by-product (bottom) comprising a single loxP site.

Fig. 2: Schematic drawing illustrating the principle of the circularization step and the possibility for excluding undesired elements present in the template DNA molecule from the scDNA according to the invention, using a DNA molecule comprising loxP sites as an example. On the left side is shown a linearized, amplified DNA molecule comprising loxP sites oriented in the same direction and flanking a payload, as well as a selection marker and a replication origin not flanked by the loxP sites. Recombination (indicated by the arrow) leads to the production of the two molecules shown on the right side: a synthetic circular DNA (scDNA) molecule (top) comprising a single loxP site and the payload, and a linear by-product (bottom) comprising a single loxP site, the origin of replication, and the selection marker.

Fig. 3 : Agarose gel pictures showing purified synthetic circular DNA (scDNA) produced by a process according to the present invention. The figure shows products obtained in two separate experiments (lanes 2 and 3, respectively). 2 (lane 3) or 3 (lane 2) DNA species are observable on the gel. Species number 1 (band number 1 in lanes 2 and 3) is the majority band and is supercoiled monomeric scDNA. The other two species (band number 2 in lane 2 and band number 3 in lanes 2 and 3) are likely supercoiled dimeric scDNA, such as supercoiled dimeric scDNA catenanes and/or concatemers, and/or non-supercoiled monomeric scDNA. A quantification of the percentage of the DNA found in each band is given in brackets (*Lane 2; * Lane 3).

Fig. 4: Agarose gel picture showing purified synthetic circular DNA (scDNA) produced by a process according to the present invention. The produced product (in lane 2) is ~95% supercoiled DNA and 5% concatemers, as indicated.

Fig. 5: Murine CCL19 (mCCL19) ELISA read-out as an indirect measure of the expression of the RBD antigen fragment from the SARS-CoV-2 spike protein in HEK293 cells transfected with either a synthetic circular DNA (scDNA) produced according to the invention and encoding RBD (and mCCL19) or an E. coll-produced plasmid DNA (pDNA) encoding the same. Three different batches of scDNA (R1-R3) were tested, and the DNA was diluted as indicated.

Fig. 6: ELISA read-out of RBD-specific IgG antibodies generated when vaccinating mice with either synthetic circular DNA (scDNA) produced according to the invention and encoding RBD or conventionally produced E. coll plasmid DNA (pDNA) encoding RBD or with a "mock" plasmid containing no antigen. Animals were either vaccinated with normal needle or an electroporation device (EP) to increase expression.

Fig. 7: Interferon-gamma (INF-y) ELISpot measuring the ability of synthetic circular DNA (scDNA) encoding RBD or conventionally produced E. coll plasmid DNA (pDNA) encoding the same or a "mock" plasmid containing no antigen to induce reactive T-cells upon vaccination of mice. The antigen fragment RBD that was used for re-stimulation was produced as three peptide pools across the RBD sequence, to assess T-cell responses across the protein (RBD pool 1-3). A positive control in the form of concavilin A (ConA) was used to ensure viability of the cells and their ability to produce INF-y upon stimulation.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

By "synthetic circular DNA molecule", as used herein, is meant a circular DNA molecule which has not been produced by conventional microbiological processes, i.e. which has not been produced in bacteria. Instead, it has been synthesized by an in vitro process, i.e. by an enzymatic process comprising separate enzymatic steps, including an amplification step such as polymerase chain reaction (PCR) or non-exponential amplification. This definition does not exclude the use of material in the process, such as use of a DNA plasmid as template DNA molecule, which has been obtained via a microbiological process.

By "non-exponential amplification" is meant a method of polynucleotide amplification which does not entail an exponential amplification phase where the number of polynucleotide molecules are doubled per each time unit passed. A typical example of a method having an exponential amplification phase is PCR. There are different non-exponential amplification methods, including some versions of Rolling Circle Amplification (RCA). The latter method will be described under Embodiments of the 1st aspect of the invention below.

By "isothermal polymerase" is meant a polymerase which amplifies a polynucleotide, such as a DNA molecule, independently of thermal cycling, as no temperature shifts are necessary for the amplification process using such a polymerase. In PCR-based methods, each amplification round is followed by an increase in temperature, which ensures that the newly formed double-stranded DNA molecules are denatured, leading to single-stranded molecules, which are ready for a new round of amplification. However, as is described in more detail under Embodiments of the 1st aspect of the invention below, a double-stranded DNA molecule may also be denatured enzymatically, e.g., by an isothermal polymerase having "strand displacement activity", meaning that the polymerase can detach a growing DNA-strand from its template strand, giving rise to a branching stretch of single-stranded DNA.

"Template DNA molecule", as used herein, should be taken to mean the starting material on which the amplification reaction is performed. The template DNA molecule can be, but is not limited to, a DNA plasmid. It may encode one or more "molecules of interest" (defined below) to be included in the final product, the synthetic circular DNA (scDNA). It may also contain "symmetric recombination sites" or "asymmetric recombination sites" (described later) flanking the one or more molecules of interest. Furthermore, it may include one or more other elements which should be included in the final product, such as an "immune- stimulating sequence" (defined below), and/or one or more elements which should not be included in the final product, such as an origin of replication and an antibiotic resistance gene, which elements can be necessary for the production of the template DNA molecule but not necessary or desirable in the final product.

By "universal buffer", as used herein, is meant a buffer which is suitable for the performance of at least two separate enzymatic reactions, which would normally or often be prepared to occur in separate buffers. When using a universal buffer for at least two separate enzymatic reactions, which have to occur sequentially, it is not necessary to exchange the buffer between at least these two steps, and this saves both time and reagents.

By "fed-batch enzymatic reaction", as used herein, is meant an enzymatic reaction wherein the substrates necessary for performing the enzymatic reaction are supplied not all at once, when starting the reaction, but gradually, in smaller doses, in the course of the reaction, such that the enzyme responsible for the enzymatic reaction is never saturated with substrate but functions on a sub-saturated level. The total amount of substrate given in a fed-batch enzymatic reaction may be smaller than or equal to the amount given in a reaction where everything is given at once. A fed-batch enzymatic reaction may increase the yield of an enzymatic reaction and decrease the error-rate. Regarding the latter, it has, e.g., been shown that an increase in dNTP concentration may affect DNA replication fidelity (Ganai and Johansson, Mol. Cell, 2016).

By "site-specific recombination" is meant a mechanism of recombining DNA in which a recombinase enzyme recognizes a certain short DNA sequence in a DNA molecule, termed a "recombination site", which divides the DNA molecule into two or more segments, and joins the recombination site with another corresponding (at least partially homologous) recombination site on another, or the same, DNA molecule. Then, the DNA strands are cleaved at both recombination sites, and the strands are re-joined, resulting in a rearrangement of the DNA segments, which may entail inversion or excision of a DNA segment. When two recombination sites flank a segment within a single DNA molecule, the orientation of the recombination sites relative to each other determine whether the DNA segment will be inverted or excised upon recombination. This concept, as well as the concept of "symmetric" and "asymmetric" recombination sites, will be described further below under Embodiments of the 1^st aspect of the invention.

By "supercoiling" is meant the process by which a DNA molecule becomes "supercoiled". In a relaxed DNA helix, there is a turn around the helical axis for app. every 10.4 base pairs. If the number of turns per base pair is increased compared to this, the DNA helix is said to be positively supercoiled; if the number of turns is decreased, the DNA helix is said to be negatively supercoiled.

A "molecule of interest", as used herein, can mean any molecule which can be expressed in a cell when encoded on an scDNA molecule according to the invention. It can be a peptide or protein but it can also be a microRNA (miRNA) or a different type of RNA. Preferably, the molecule of interest is a peptide or protein.

By "immune-stimulating sequence", as used herein, is meant one or more sequences which, in addition to the potential immune stimulation caused by the molecule of interest which may be encoded by the plasmid according to the invention, stimulates the immune system in some way. Examples of such immune-stimulating sequences are given below.

By "therapeutic or prophylactic protein" is meant any peptide or protein which has some therapeutic or prophylactic effect on a disease in a subject to which it is administered. It may for example be a vaccine antigen, which gives a prophylactic effect, but it may also be, e.g., an enzyme, which enzyme is absent or defect in a subject suffering from a genetic disease, and which is then supplied to this subject, yielding a therapeutic effect.

A "vaccine antigen" is a substance that can be recognized specifically by the immune system (e.g. when bound by antibodies or, alternatively, when fragments of the antigen bound to MHC molecules are being recognized by T cell receptors) and is capable of eliciting immunity, meaning that a host that has an established memory immunity against the antigen will mount a specific immune response against the antigen. Furthermore, it should be suitable for use in a vaccine and be able to provide some degree of protection against the disease it targets.

Embodiments of the 1st aspect of the invention

In the process described herein, a synthetic circular DNA (scDNA) molecule is produced by a process comprising : an amplification step; wherein a template DNA molecule is amplified, and a circularization step; wherein the amplified DNA is circularized, wherein the circularization step comprises site-specific recombination. In its most basic version, the process for production of an scDNA molecule according to the present invention consists of only two steps: an amplification step and a circularization step. The circularization is performed using site-specific recombination.

Amplification

The amplification can be performed using different methods.

In some embodiments, the amplification step comprises non-exponential amplification using an isothermal polymerase with strand displacement activity.

In preferred embodiments, the isothermal polymerase also has 3'-5' exonuclease activity.

In some embodiments, the polymerase is selected from the group consisting of isothermal polymerase from the bacteriophage Phi29 (Phi29 DNAP), isothermal polymerase from the bacteriophage B103, isothermal polymerase from the bacteriophage M2(Y), isothermal polymerase from the bacteriophage Nf, Large (Klenow) fragment of Polymerase I, Large fragment of Bsu DNA polymerase, and Bst DNA polymerase.

In preferred embodiments, the polymerase is Phi29 DNAP.

Phi29 DNAP can be used for Rolling Circle Amplification (RCA), which takes advantage of the strand displacement activity of the enzyme. In RCA, an initial denaturation step is performed in the presence of random primers, after which the enzyme is added. At this step, the enzyme recognizes primers bound to template DNA and starts polymerization/replication. When the enzyme encounters another primer binding site, from which replication has also started, its strand displacement activity allows it to displace the newly produced DNA strand and continue its polymerization/elongation. The displacement generates a new singlestranded DNA template for more random primers to anneal to and the process is repeated. The resulting amplified DNA is highly branched.

Non-exponential amplification (defined above) is a preferred amplification method for use in the process according to the present invention. Even more preferred is non-exponential versions of RCA, especially RCA using Phi29 DNAP. However, other amplification methods may be used, e.g., the non-exponential amplification method Multiple Displacement Amplification (MDA), exponential amplification methods, such as PCR, and exponential isothermal methods, such as exponential versions of RCA and loop-mediated isothermal amplification. An advantage of using an isothermal polymerase (defined above) is that no thermocycler is needed, as it is with PCR.

Furthermore, an advantage of using non-exponential amplification is that errors are not propagated, as they are with exponential amplification.

Circularization

The circularization is performed using site-specific recombination (defined above).

Site-specific recombination may be performed using a variety of recombinase enzymes, working on a variety of recombination sites.

In some embodiments, the site-specific recombination in the circularization step is performed using a recombinase selected from: Cre recombinase, Flippase recombinase, R recombinase, Lambda recombinase, HK101 recombinase, pSAM2 recombinase, Beta recombinase, CinH recombinase, ParA recombinase, y5 recombinase, Bcbl recombinase, Bxbl recombinase, PhiC31 recombinase, and TP901 recombinase.

The circularization of the DNA product of the amplification step depends on the presence of suitable recombination sites in the template DNA. For the circularization to occur correctly, two recombination sites must be present in the amplification product, flanking the DNA segment which is to be included in the scDNA molecule. This means that one recombination site is present upstream, and one recombination site is present downstream, of the DNA segment. This DNA segment will normally, but not always, contain one or more elements (also termed the "payload"), e.g., a gene encoding a molecule of interest. The orientation of the recombination sites relative to each other is also essential: The two recombination sites flanking the DNA segment must be oriented in the same direction. This will lead to excision of the DNA segment upon recombination. The excised DNA segment is in the form of a circular DNA molecule (Figure 1).

Suitable recombination sites are recombination sites that are compatible with the recombinase which is chosen for the circularization (such as any one of the recombinases listed above).

The recombination sites used in the present invention may be symmetric or asymmetric. This concept will be explained below. In some embodiments, the recombinase is Bxbl recombinase.

Bxbl recombinase is a recombinase belonging to the serine integrase subfamily of recombinases.

In some embodiments, the recombinase is Cre recombinase. Cre recombinase works on locus of X-over Pl loxP) recombination sites. The wildtype (wt) loxP recombination site is a 34 base-pair (bp) sequence comprising two 13 bp inverted repeats flanking an 8 bp asymmetric spacer region. The spacer region determines the orientation of the loxP site. As described above, the recombination sites must be oriented in the same direction for the circularization step of the process according to the invention to work. The wt loxP sequence is: ATAACTTCGTATA-ATGTATGC-TATACGAAGTTAT (SEQ ID NO: 1).

Several variants of the wt loxP site exist. As used herein, the term "loxP site" should be taken to include any one of such variants. Some examples of loxP sites are given in Table 1.

Table 1 : Examples of /oxP sites. Adapted from Missirlis et al. , BMC Genomics, 2006.

Nucleotides in small letters indicate mutations compared to the loxP wt sequence.

As can be seen from Table 1, some loxP sites contain mutations in the 8 bp spacer region compared to the loxP wt sequence (such as, e.g., Iox511')_l and others contain mutations in one of the two 13 bp flanking regions (such as Iox71 and Iox66'). The latter variants thus contain flanking regions which are not perfect inverted repeats of each other.

When recombination occurs between two loxP recombination sites, the two recombination sites are each cut, and then the DNA strands are rejoined in a manner such that one of the flanking regions is swapped (exchanged) between the two recombination sites. When the two recombination sites are identical (or "symmetric"), this exchange does not have any effect on the two recombination sites present in the one or more DNA molecules after the recombination event - these recombination sites will be identical to the ones present in the molecule(s) prior to recombination.

However, if the two recombination sites are not identical (i.e., they are "asymmetric"), recombination may lead to a change in the sequences of both recombination sites. Though, this is not always the case. If, for example, one recombination site is loxP wt and the other is Iox71, recombination will lead to one Iox71 site and one loxP wt site, i.e. the same recombination sites that were present in the starting material. On the other hand, if, for example, one recombination site is Iox71 and the other is Iox66, recombination between these sites will lead to two new recombination sites: one comprising the mutated left flanking region of Iox71 as well as the mutated right flanking region of !ox66, and one loxP wt site. In this case, the recombination site having both mutated left and right flanking regions (termed 10x72) will not be recognizable by the Cre enzyme, and thus it will be inactive. Lox72 has the following sequence: taccgTTCGTATA-ATGTATGC-TATACGAAcggta (SEQ ID NO: 11). Similarly, recombination between non-identical attP and attB sites in the integrase subfamilies of recombinases leads to the formation of non-identical attL and attR sites, and vice versa. Whilst none of these sites need to be mutated, they are still considered asymmetrical.

In embodiments of the present invention, the template DNA molecule is designed in such a way that the two recombination sites surrounding the gene of interest are asymmetrical, and so that after recombination, one of these recombination sites, i.e. the one that is left in the scDNA molecule, is not recognizable by the relevant recombinase, e.g. Cre recombinase, and thus is inactive, whereas the other recombination site, i.e. the one that is left in the linear by-product, is active. Ensuring that the recombination site left in the scDNA molecule is inactive has the advantage that no accidental recombination can occur inside a cell during clinical use of the plasmid. Furthermore, as long as a recombination reaction is reversible, which is the case when active recombination sites are left in both the scDNA molecule and the linear by-product, the reaction will occur both ways, and thus non-recombined template DNA molecules and recombined scDNA molecules and linear by-products will be present in an equilibrium. However, when the reaction becomes irreversible by ensuring that the recombination site left in the scDNA molecule is inactive, the equilibrium is disturbed, and the reaction becomes much more efficient in the direction where the scDNA molecule is generated from the template DNA molecule. Thus, the process becomes much more productive, and a higher yield of scDNA can be achieved from the same amount of template DNA molecule (the efficiency of the reaction may even increase from about 20-30% to about 100%). It should be noted that for some recombinases, additional factors may be required for the reaction to be reversible. For the integrase subfamilies of recombinases, for example, the reversibility of the reaction is dependent on the presence or absence of a Recombination Directionality Factor (RDF) protein (Lewis and Hatfull, Nucleic acids research, 2001; Merrick et al., ACS Synth. Biol., 2018). Thus, the absence of this protein alone will ensure that an integrase-mediated recombination reaction is irreversible.

In some embodiments, the template DNA molecule comprises two symmetric recombination sites, which recombination sites are oriented in the same direction, flanking a gene encoding a molecule of interest.

In some embodiments, the template DNA molecule comprises two asymmetric recombination sites, which recombination sites are oriented in the same direction, flanking a gene encoding a molecule of interest. In further embodiments, the recombination results in an scDNA molecule comprising an inactive recombination site and the gene encoding the molecule of interest. However, in other embodiments, the recombination results in an scDNA molecule comprising an active recombination site and the gene encoding the molecule of interest.

In some embodiments, the recombination sites may comprise half-sites of a restriction enzyme recognition site in their spacer regions, so that when recombination occurs between two recombination sites, each having different halves of the restriction enzyme recognition site, it leads to the assembly of a complete restriction enzyme recognition site in the linear by-product and no restriction enzyme recognition site in the scDNA molecule. This may enable cleavage of the linear by-product after the recombination event, aiding in its degradation.

The restriction enzyme recognition site may be a Pmel recognition site.

It has been found by the present inventors that the process for producing an scDNA molecule according to the present invention has some advantages relating to the DNA product which can be produced by the process. Due to the synthetic nature of the production process, there is no need for an origin of replication or antibiotic resistance gene (also called a selection marker), as the DNA is not replicated and selected for in bacteria. Therefore, it is possible to produce a simpler and "cleaner" DNA construct for clinical use, which does not contain these elements that are unnecessary for the clinical use. Furthermore, the present production method does not depend on the use of antibiotics, and there is no risk of escape of variants comprising antibiotic resistance genes, which may be spread to other bacteria.

In some cases, the template DNA molecule according to the present invention is produced by conventional microbiological processes and may possess both an origin of replication and an antibiotic resistance gene. In this case, it is possible to ensure that these elements are removed during the circularization step by designing the template DNA molecule such that the two recombination sites flank only the elements that are to be included in the scDNA molecule - such as a gene of interest, so that when recombination occurs, the gene of interest will end up in the scDNA molecule, and the origin of replication and the antibiotic resistance gene will end up in the linear by-product (Figure 2).

In some embodiments, the template DNA molecule comprises an origin of replication and/or an antibiotic resistance gene, and these features are removed as part of the circularization and are retained in the linear by-product. Linearization

In addition to the steps of amplification and circularization, the process according to the present invention may include a linearization step.

Thus, in some embodiments, the process further comprises a linearization step; wherein the amplified DNA is linearized, comprising cleavage of the amplified DNA by an endonuclease, which linearization step is performed after the amplification step and before the circularization step.

In some embodiments, the endonuclease is Xhol.

Other endonucleases (also called restriction enzymes) may be useful for linearizing the amplification product. The person skilled in the art will know how to select a suitable endonuclease for the cleavage of a specific DNA sequence, based on principles such as avoidance of off-target cleavage.

Digestion

In some embodiments, the process further comprises a digestion step; wherein non-circularized and circularized nicked amplified DNA is removed, which digestion step is performed after the circularization step.

The presence of linear DNA in the reaction after the circularization step may interfere with downstream processes. Nicked circular DNA cannot be supercoiled and is therefore undesirable. Thus, these two potential side-products should preferably be removed, e.g. by digestion.

Digestion may be performed using an exonuclease. The exonuclease may digest linear and/or nicked circular DNA. Preferably, the exonuclease will digest both linear and nicked circular DNA.

In preferred embodiments, the exonuclease is T5 exonuclease.

In other embodiments, the exonuclease is Exonuclease V. However, this exonuclease does not digest nicked circular DNA. In other embodiments, the exonuclease is Exonuclease III, VIII or T7. However, these exonucleases do not digest all single stranded DNA under standard reaction conditions, and Exonuclease VIII additionally does not digest nicked circular DNA.

Supercoilinq

It has surprisingly been discovered by the present inventors that the end-product of the process according to the present invention is supercoiled, even in embodiments of the method where a supercoiling step is not performed. This is surprising because in a cell DNA is supercoiled by an enzymatic process using gyrases. In the present method, which is fully synthetic, the DNA supercoils even when it has not been in the presence of gyrases. Negative supercoiling is normally a prerequisite for introduction of a circular DNA molecule into humans. It may help prevent damage caused by mechanical stress during injection. Thus, the present inventors have demonstrated that they can achieve a supercoiled product using one step less than what would have been expected to be required (no supercoiling step, e.g. using gyrases, is required). This is highly advantageous, as it saves both time and resources.

The degree of supercoiling of the produced circular DNA molecules can be assessed by running the DNA on an agarose gel, where supercoiled monomeric DNA will be separated from non-supercoiled DNA and multimeric supercoiled DNA, such as catenanes and concatemers. The fraction of the DNA which is in supercoiled monomeric form can then be quantified by gel analysis software. Figure 3 shows an example of such a quantification of supercoiling of circular DNA product obtained in two separate experiments by the process according to the present invention (an embodiment of the process not comprising a supercoiling step). As can be seen, a high fraction of the circular DNA was found to be in supercoiled monomeric form: 80% and 92% of the DNA, respectively. This underscores that a supercoiling step is not necessary when using the production process according to the present invention.

Although a large fraction of the DNA product is already in supercoiled monomeric form in the absence of a supercoiling step, a supercoiling step may be performed to increase this fraction further or to increase the level of negative supercoiling.

In some embodiments, the process further comprises a supercoiling step; wherein negative supercoiling is introduced into the circularized amplified DNA, which supercoiling step is performed after the circularization step and, if the process includes a digestion step according to embodiments of the first aspect, either before or after the digestion step. In some embodiments, the negative supercoiling is introduced into the circularized amplified DNA using a gyrase.

In some embodiments, the gyrase is Escherichia coli GyrA₂B₂.

In other embodiments, the gyrase is a gyrase derived from any one of: Staphylococcus aureus, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Acinetobacter baumannii, and Clostridium difficile.

Additional conditions of the process

In some embodiments, two or more steps of the process are carried out in the same universal buffer.

The concept of a universal buffer is explained under Definitions above. It is the use of a single buffer for at least two consecutive enzymatic reactions, preferably all enzymatic reactions, in the process according to the present invention. When using a universal buffer, the number of buffer changes is limited, resource spending is minimized, and the procedure is simplified.

In some embodiments, at least one of the enzymatic reactions occurs as a fed-batch enzymatic reaction.

The concept of a fed-batch enzymatic reaction is explained under Definitions above. It covers the stepwise addition of substrate to an enzymatic reaction, instead of adding the whole amount at once when the reaction begins. When an enzymatic reaction occurs as a fed-batch enzymatic reaction, it may increase its efficiency and/or decrease its error-rate.

Elements of the scDNA molecule

The scDNA molecule according to the present invention may comprise a gene encoding a molecule of interest.

As described under Definitions above, the molecule of interest may, e.g., be a vaccine antigen. This could be an antigen for a vaccine against a microorganism, such as a virus, a bacterium or a parasite. It could also be an antigen for a cancer vaccine, such as an antigen comprising one or more patient-specific neo-epitopes. The gene encoding the molecule of interest will preferably be operably linked to a eukaryotic promoter sequence, such as the nucleotide sequence of a strong eukaryotic promoter. The compositions and methods herein may involve the use of any particular eukaryotic promoter, and a wide variety are known, e.g., a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoter. The promoter can be heterologous with respect to the host cell. The promoter used may be a constitutive promoter. The promoter used may include an enhancer region and an intron region to improve expression levels, such as is the case when using a CMV promoter. In some embodiments, the gene encoding the molecule of interest comprises a signal sequence for secretion.

In some embodiments, the scDNA molecule further comprises at least one immune- stimulating sequence.

Depending on the purpose of the scDNA molecule, i.e. its specific clinical use, it may in some cases be an advantage if the molecule contains one or more immune-stimulating sequence(s) (ISS). This may for example be an advantage if the scDNA is to be used as a DNA vaccine.

The aim of using ISS in a DNA vaccine is to enhance T-cell responses towards the encoded antigen, in particular Thl cell responses, which are elicited by agonists of the toll-like receptors TLR3, TLR7-TLR8, and TLR9 and/or cytosolic RNA receptors such as, but not limited to, RIG-1, MDA5 and LGP2 (Desmet et al., Nat. Rev. Imm., 2012). One possibility of employing ISS is to mimic a bacterial infection activating TLR9 by stimulating with nonmethylated CG-rich motifs (so-called CpG motifs) of six bases with the general sequence NNCGNN (which have a 20-fold higher frequency in bacterial DNA than in mammalian DNA). Thus, such CpG motifs could be incorporated directly in the scDNA backbone. Since CpG sequences exert an effect irrespectively of their position in a longer DNA molecule, their position could in principle be anywhere in the scDNA molecule, as long as the presence of the CpG motif does not interfere with the molecule's ability to express the coding regions of the vaccine antigen. When CpG sequences are present in the molecule backbone (which thereby becomes "self-adjuvating"), any number of possible NNGCNN or NNCGNN sequences can according to the invention be present, either as identical sequences or in the form of nonidentical sequences of the CpG motif, or in the form of palindromic sequences that can form stem-loop structures. For instance, the following CpG motifs are of interest: AACGAC and GTCGTT, but also CTCGTT, and GCTGTT.

Another possibility is to mimic an RNA viral infection to activate TLR3 by encoding a dsRNA in the scDNA molecule backbone, which will be transcribed into RNA after vaccination - in this case the DNA vaccine hence encodes the immunological adjuvant. This approach can include DNA sequences that encode hairpin RIMA with lengths of up to 100 base pairs, where the sequence is unspecific.

Purification

After the production of the scDNA molecule according to the method described above, the molecule may be purified.

The person skilled in the art will know how to select a method suitable for purifying scDNA molecules, such as a standard method for purifying DNA plasmids.

Embodiments of the 2^nd aspect of the invention

The 2^nd aspect of the invention provides a synthetic circular DNA (scDNA) molecule. The scDNA molecule is e.g. obtainable by the process described in embodiments of the 1^st aspect of the invention and the embodiments described under Embodiments of the 1^st aspect of the invention above are hence also relevant embodiments of the 2^nd aspect of the invention.

Generally, the 2^nd aspect of the invention provides a synthetic circular DNA (scDNA) molecule which comprises at least one gene encoding a molecule of interest and which scDNA molecule does not comprise an origin of replication or an antibiotic resistance gene.

As described above under Embodiments of the 1^st aspect of the invention, an advantage of the process according to the present invention is that a plasmid can be generated which does not contain elements which are unnecessary for its clinical use, such as origins of replication and antibiotic resistance genes.

In some embodiments of the 2^nd aspect of the invention, the scDNA molecule has been produced by the process according to certain embodiments of the 1^st aspect of the invention.

In some embodiments of the 2^nd aspect of the invention, the scDNA molecule has not been produced by a process comprising a supercoiling step.

In some embodiments of the 2^nd aspect of the invention, the scDNA molecule (further) comprises a recombination site.

In some embodiments, the recombination site is inactive. In some embodiments, the recombination site is a loxP site.

In some embodiments, the loxP site is an inactive iox72 site with the sequence of SEQ ID NO: 11.

In some embodiments, the molecule of interest is a therapeutic or prophylactic protein.

In some embodiments, the molecule of interest is a vaccine antigen.

Embodiments of the 3^rd aspect of the invention

The 3^rd aspect of the invention provides a composition comprising a plurality of the scDNA molecule according to embodiments of the 2^nd aspect of the invention, wherein the majority of the scDNA molecules in the composition are in supercoiled monomeric form.

In some embodiments, at least 70% of the scDNA molecules are in supercoiled monomeric form, such as at least 71%, such as at least 72%, such as at least 73%, such as at least

74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least

78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least

82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least

86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least

90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least

94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least

98%, such as at least 99%, and such as 100%.

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier and/or diluent and/or excipient.

The choice of carriers, diluents and excipients will generally follow the state-of-the-art as disclosed in, e.g., "DNA vaccines - methods and protocols" (Sousa, 2021).

Embodiments of the 4^th-6^th aspects of the invention

The 4^th aspect of the invention provides the scDNA molecule or composition according to embodiments of the 2^nd and 3^rd aspects of the invention for use as a medicament. The 5^th aspect of the invention provides the scDNA molecule or composition according to embodiments of the 2^nd and 3^rd aspects of the invention for use in vaccination, such as prophylactic vaccination or therapeutic vaccination; however, any therapy that relies on delivery of DNA plasmids can take advantage of the scDNA molecules of the present invention, which hence finds practical use in gene therapy settings alongside the utility in DNA vaccination.

The exact choice of disease targeted is determined by the skilled artisan, but for gene therapy, inborn errors of metabolism as well as diseases caused by mutated genes or rare allelic variations are targets for administration of the presently disclosed DNA molecules and compositions comprising these. Likewise, DNA vaccination strategies can target infectious diseases (typically as prophylactic treatment) and cancers (typically as therapy) by effecting expression of protective antigens that can trigger a specific adaptive immune response against the pathological cells.

The related 6^th aspect of the invention provides a method of treating a subject in need thereof, comprising administering to the subject an efficient amount of the scDNA molecule or of the composition according to embodiments of the 2^nd and 3^rd aspects of the invention.

The embodiments described above for the scDNA molecule according to the 2^nd aspect of the invention (described under Embodiments of the 1^st aspect of the invention and Embodiments of the 2^nd aspect of the invention above) are also applicable to the scDNA molecule for the use according to the 4^th and 5^th aspects of the invention, to the composition according to the 3^rd aspect of the invention, and to the method of treatment according to the 6^th aspect of the invention.

Route of administration and formulation

The scDNA according to the present invention may be administered to a subject by one or more of several different routes, and it may be formulated in various ways.

Routes of administration include, but are not limited to, intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraoccular and oral, as well as topical, transdermal, by inhalation or suppository, or to mucosal tissue such as by lavage to vaginal, rectal, urethral, buccal and sublingual tissue. In other words, the route of administration can be selected from any one of parenteral routes, such as via the intramuscular route, the intradermal route, transdermal route, the subcutaneous route, the intravenous route, the intra-arterial route, the intrathecal route, the intramedullary route, the intraventricular route, the intraperitoneal route, the intranasal route, the vaginal route, the intraocular route, or the pulmonary route; it can be administered via the oral route, the sublingual route, the buccal route, or the anal route; or it can be administered topically.

Typical routes of administration include intramuscular, intradermal and subcutaneous injection. The scDNA may be administered by means including, but not limited to, traditional syringes, needleless injection devices, such as PharmaJet® devices, "microprojectile bombardment gene guns", or other physical methods such as electroporation, "hydrodynamic method" or ultrasound. The scDNA can be delivered by any method that can be used to deliver DNA as long as the DNA is expressed and the molecule of interest is produced in the cell.

In some embodiments, scDNA disclosed herein is delivered via or in combination with known transfection reagents such as cationic liposomes, fluorocarbon emulsion, cochleate, tubules, gold particles, biodegradable microspheres, or cationic polymers. Cochleate delivery vehicles are stable phospholipid calcium precipitants consisting of phosphatidyl serine, cholesterol, and calcium; this nontoxic and noninflammatory transfection reagent can be present in a digestive system. Biodegradable microspheres comprise polymers such as poly(lactide-co- glycolide), a polyester that can be used in producing microcapsules of DNA for transfection. Lipid-based microtubes often consist of a lipid of spirally wound two layers packed with their edges joined to each other. When a tubule is used, the nucleic acid can be arranged in the central hollow part thereof for delivery and controlled release into the body of a subject.

An scDNA molecule, such as a DNA vaccine, can also be delivered to mucosal surfaces via microspheres. Bioadhesive microspheres can be prepared using different techniques and can be tailored to adhere to any mucosal tissue including those found in eye, nasal cavity, urinary tract, colon and gastrointestinal tract, offering the possibilities of localized as well as systemic controlled release of, e.g., vaccines. Application of bioadhesive microspheres to specific mucosal tissues can also be used for localized vaccine action. In some embodiments, an alternative approach for mucosal vaccine delivery is the direct administration to mucosal surfaces of an scDNA molecule which encodes the gene for a specific protein antigen.

The scDNA molecules disclosed are formulated according to the mode of administration to be used. Typically, the scDNA molecules are injectable compositions, they are sterile, and/or pyrogen free and/or particulate free. In preferred embodiments, an isotonic formulation is used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In preferred embodiments, isotonic solutions such as phosphate buffered saline are used. An alternative solution is Tyrode's buffer. In some embodiments, stabilizers include gelatine and albumin. In some embodiments, a stabilizing agent that allows the formulation to be stable at room or ambient temperature for extended periods of time, such as LGS or other poly-cations or poly-anions, is added to the formulation.

The pharmaceutically acceptable carrier or diluent in the pharmaceutical composition disclosed herein is preferably in the form of a buffered solution. Parenteral vehicles include sodium chloride solution, Ringer's dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and antimicrobials include antioxidants, chelating agents, inert gases and the like. Preferred preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B.

In preferred embodiments, the buffered solution is phosphate buffered saline (PBS), and in preferred embodiments the PBS has the composition 137 mM NaCI, 2.7 mM KCI, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4. The concentration of the PBS (or alternatives) is typically about 35% v/v, but depending on the water content of suspended scDNA molecules, the concentration may vary considerably - since the buffer is physiologically acceptable, it can constitute any percentage of the aqueous phase of the composition.

Additional carrier substances may be included and can contain proteins, sugars, etc. Such carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline.

EXAMPLE 1

Protocol for producing supercoiled scDNA

Given below is a specific protocol for a process of producing scDNA according to an embodiment of the present invention. Following this protocol, the inventors have repeatedly achieved a high yield of scDNA molecules comprising a high fraction of supercoiled monomeric scDNA molecules. As described under the subsection Supercoiling above, Figure 3 shows representative examples of scDNA obtained in a process according to the present invention. In the experiment reflected in Lane 3, a starting amount of 15 pg of template DNA molecule in the form of a plasmid resulted in a final scDNA yield of 2.57 mg. 50% of the template plasmid was backbone (the DNA segment which is not included in the scDNA molecule; app. 7.5 pg). Thus, the total amplification was app. 340-fold (7.5 pg to 2.57 mg). Furthermore, following this protocol, which does not comprise a supercoiling step, the inventors have obtained DNA which after purification consisted of at least 80% negatively supercoiled monomeric scDNA (Figure 3).

Example of protocol for production of scDNA:

500ng template plasmid is mixed with substrate (5mM dNTPs and 50pM primers) and 100U of Phi29 isothermal DNA polymerase in a suitable buffer in a total volume of ImL. The primers used are exonuclease resistant to minimize primer degradation by the exonuclease part of the isothermal DNA polymerase. 1.25U of Pyrophosphatase is also included in the reaction to remove pyrophosphate generated as a by-product of the DNA synthesis by the isothermal DNA polymerase. The reaction is incubated 24 hours at 30°C, 700rpm and with a heated lid at 105°C. After the amplification, the reaction is terminated by heat inactivation of the Phi29 isothermal DNA polymerase at 70°C for 20 minutes, 700rpm and with a heated lid at 105°C.

The amplified products are processed into linear monomers using 1500U of Xhol endonuclease in an appropriate buffer in a total volume of 1.03mL. The reaction is incubated for 4 hours at 37°C, 700rpm and no heated lid to increase mixing by convection. After the digestion, the reaction is terminated by heat inactivation of the endonuclease at 80°C for 15 minutes, 700rpm and no heated lid to increase mixing by convection.

The linear monomers are processed into a circular product and linear by-product(s) using 3000U of Cre recombinase in an appropriate buffer. The reaction is incubated 6 hours at 37°C, 700rpm with no heated lid to increase mixing by convection. After the recombination, the reaction is terminated by heat inactivation of the recombinase at 70°C for 15 minutes with no heated lid to increase mixing by convection.

The linear by-products are removed using 250U T5 exonuclease that specifically degrades linear single- and double-stranded DNA, as well as nicked circular DNA, in an appropriate buffer in a total volume of 1.4mL. The reaction is incubated for 2 hours at 37°C, 700rpm with no heated lid to increase mixing by convection. After the amplification, the reaction is terminated by purification of the circular DNA product. EXAMPLE 2

Pre-clinical testing of an scDNA encoding a vaccine antigen

To test the feasibility of using the process according to the invention to produce scDNA for vaccination purposes, an scDNA encoding the antigen RBD from the SARS-CoV-2 coronavirus spike protein was produced according to the protocol described in Example 1 and tested in vitro and in vivo as described below. As can be observed in Figure 4, lane 2, the material produced was highly pure, with ~95% supercoiled monomeric scDNA and only 5% concatemers. The quantification was performed by densiometric analysis using built-in software for an Ibright instrument. This level of pure supercoiled DNA is above the level currently used as a requirement for use in a clinical setting of 80%, supporting its usage in such a setting. A similar yield of 2.47 mg was obtained as that presented in Example 1.

In vitro expression of the vaccine antigen

The expression levels of the RBD antigen encoded by the scDNA were tested by ELISA in a human cell line (HEK293) and compared to the expression levels from an E. coli plasmid.

On day 1, a 6-well plate was coated with Poly-D-lysine and seeded with lxlO⁵ HEK293 cells/well, and on day 2, the cells were transfected using 10 uL Lipofectamine in 250 uL Opti- Mem medium with 8 ul P3000 pr well. For E. co//-produced plasmid DNA, 4 ug was used for transfection, and 2 ug was used for the scDNA (due to its approximately 50% smaller molecular size). On day 3, the media were exchanged with serum-free HEK293 media for all wells, followed by supernatant and cell lysate harvesting on day 5. An ELISA on the supernatant was performed using a commercially available murine CCL19 (mCCL19) capture antibody, since the tested construct encoded CCL19 in conjunction with the RBD antigen, and thus CCL19 was used as a surrogate measure for RBD antigen expression. The mCCL19 antibody was coated on standard ELISA plates. A standard curve was prepared covering 110,000 to 0 pg/mL of recombinantly produced CCL19 in PBS+1% BSA. All reagents were diluted in PBS+1% BSA, and for detection, a biotinylated detection antibody was used that recognizes a different epitope on mCCL19. Development was performed using streptavidin- fused horseradish peroxidase (HRP) and 3,3',5,5'-Tetramethylbenzidine (TMB).

The results are shown in Figure 5. The scDNA performed at the same level as the E. coli plasmid DNA, with the expression reaching the same OD=~3.0 at no dilution of the DNA prior to analysis. Subsequent dilutions of 1 : 10, 1 :50, and 1 : 100 resulted in the same performance for all tested replicates (Rl-3) of scDNA. In vivo antibody generation and T-cell activation

It was then tested whether vaccination with the RBD antigen-encoding scDNA could induce RBD-specific immune responses in animals.

A total of 36 C57BL/6 mice were used for the study, with 6 mice in each of 6 study groups. Mice were either vaccinated with a standard needle or an electroporation (EP) device intramuscularly in the left or right back leg. Mice were immunized on day 0 and day 28, with tailvein blood samples drawn on day 14 and day 27. Orbital blood and spleens were collected upon termination on day 42. The resulting blood samples were used to prepare sera for an ELISA assay, while spleens were homogenised to produce single-cell suspensions for use in an ELISpot assay.

Briefly, ELISA was performed using sera diluted from 1 :200 to 1 :409,600. The ELISA plates were coated with recombinant RBD, and RBD-specific IgG was detected using an HRP- conjugated polyclonal rabbit anti-mouse IgG detection antibody. The ELISA was developed using the TMB SLOW HRP substrate, and the reaction was stopped using 0.16M H₂SO₄. Data was read using a standard plate-reader.

For the ELISPot assay, 5x10® cells/mL splenocytes were seeded per well followed by overnight stimulation with 10 ug of RBD1 (a mixture of 15mer peptides derived from a segment corresponding to amino acid (aa) position 319-385 of RBD), RBD2 (the same but for aa position 391-457) or RBD3 (the same but for aa position 463-529). A capture antibody against mouse INF-y was added, followed by spot development with a secondary antibody (BD Mouse IFN-y ELISPOT pair, cat #551818) and BD Streptavidin-HRP+AEC (#557630 and #55195). The spots were detected using an ELISpot reader. A positive control in the form of 10 mg/mL concavilin A was used to measure the INF-y production potential of the used splenocytes, while unstimulated cells served as a negative control.

The results of the antibody ELISA are shown in Figure 6, and the results of the T-cell activation ELISpot are shown in Figure 7. Vaccination with the RBD-encoding scDNA induced a similar level of RBD-specific IgG antibodies as vaccination with the RBD-encoding E. coli plasmid DNA. Furthermore, vaccination with the RBD-encoding scDNA induced a similar activation of RBD-specific T-cells as vaccination with the RBD-encoding E. coli plasmid DNA.

These data support the feasibility of using the process according to the invention to produce scDNA for vaccination purposes. LIST OF REFERENCES

Desmet, C. J. and Ishii, K. J. (2012). Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination. Nat. Rev. Immunol.; 12(7) :479-91.

Ganai, R. A. and Johansson, E. (2016). DNA replication - a matter of fidelity. Mol. Cell; 62(5) :745-55.

Lewis, J., and Hatfull, G. (2001). Control of directionality in integrase-mediated recombination: Examination of recombination directionality factors (RDFs) including Xis and Cox proteins. Nucleic acids research; 29:2205-16.

Merrick, C. A., Zhao, J., and Rosser, S. J. (2018). Serine integrases: Advancing synthetic biology. ACS Synth. Biol.; 7(2) :299-310.

Missirlis, P. I., Smailus, D. E., and Holt, R. A. (2006). A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination. BMC Genomics; 7:73.

Sousa, A. (ed.). (2021). DNA vaccines - methods and protocols. ISBN : 978-1-0716-0872-2 (DOI: 10.1007/978-1-0716-0872-2).

Claims

1. A process for producing a synthetic circular DNA (scDNA) molecule, the process comprising : an amplification step; wherein a template DNA molecule is amplified, and a circularization step; wherein the amplified DNA is circularized, wherein the circularization step comprises site-specific recombination.

2. The process according to claim 1, wherein the amplification step comprises nonexponential amplification using an isothermal polymerase with strand displacement activity.

3. The process according to claim 2, wherein the polymerase is selected from: Isothermal polymerase from the bacteriophage Phi29 (Phi29 DNAP), isothermal polymerase from the bacteriophage B103, isothermal polymerase from the bacteriophage M2(Y), isothermal polymerase from the bacteriophage Nf, Large (Klenow) fragment of Polymerase I, Large fragment of Bsu DNA polymerase, and Bst DNA polymerase.

4. The process according to claim 3, wherein the polymerase is Phi29 DNAP.

5. The process according to any one of the preceding claims, wherein the site-specific recombination in the circularization step is performed using a recombinase selected from: Cre recombinase, Flippase recombinase, R recombinase, Lambda recombinase, HK101 recombinase, pSAM2 recombinase, Beta recombinase, CinH recombinase, ParA recombinase, y5 recombinase, Bcbl recombinase, Bxbl recombinase, PhiC31 recombinase, and TP901 recombinase.

6. The process according to claim 5, wherein the recombinase is Cre recombinase.

7. The process according to claim 5, wherein the recombinase is Bxbl recombinase.

8. The process according to any one of the preceding claims, wherein the process further comprises a linearization step; wherein the amplified DNA is linearized, comprising cleavage of the amplified DNA by an endonuclease, which linearization step is performed after the amplification step and before the circularization step.

9. The process according to claim 8, wherein the endonuclease is Xhol.

10. The process according to any one of the preceding claims, wherein the process further comprises a digestion step; wherein non-circularized and circularized nicked amplified DNA is removed, which digestion step is performed after the circularization step.

11. The process according to any one of the preceding claims, wherein the process further comprises a supercoiling step; wherein negative supercoiling is introduced into the circularized amplified DNA, which supercoiling step is performed after the circularization step and, if the process includes a digestion step as described in claim 10, either before or after the digestion step.

12. The process according to claim 11, wherein the supercoiling is introduced into the circularized amplified DNA using a gyrase.

13. The process according to any one of the preceding claims, wherein two or more steps of the process are carried out in the same universal buffer.

14. The process according to any one of the preceding claims, wherein at least one of the enzymatic reactions occurs as a fed-batch enzymatic reaction.

15. The process according to any one of the preceding claims, wherein the template DNA molecule comprises two symmetric recombination sites, which recombination sites are oriented in the same direction, flanking a gene encoding a molecule of interest.

16. The process according to any one of claims 1-14, wherein the template DNA molecule comprises two asymmetric recombination sites, which recombination sites are oriented in the same direction, flanking a gene encoding a molecule of interest.

17. The process according to claim 16, wherein the recombination results in an scDNA molecule comprising an inactive recombination site and the gene encoding the molecule of interest.

18. The process according to any one of the preceding claims, wherein the template DNA molecule comprises an origin of replication and/or an antibiotic resistance gene, and wherein these features are removed as part of the circularization and are retained in the linear byproduct.

19. The process according to any one of claims 15-18, wherein the gene encoding the molecule of interest comprises a signal sequence for secretion.

20. The process according to any one of claims 15-19, wherein the scDNA molecule further comprises at least one immune-stimulating sequence.

21. A synthetic circular DNA (scDNA) molecule obtainable by the process according to any one of the preceding claims.

22. A synthetic circular DNA (scDNA) molecule which comprises at least one gene encoding a molecule of interest and which scDNA molecule does not comprise an origin of replication or an antibiotic resistance gene.

23. The scDNA molecule according to claim 21 or 22, which has been produced by the process according to any one of claims 1-10.

24. The scDNA molecule according to claim 23, wherein the scDNA molecule has not been produced by a process comprising a supercoiling step.

25. The scDNA molecule according to any one of claims 21-24, which comprises a recombination site.

26. The scDNA molecule according to claim 25, wherein the recombination site is inactive.

27. The scDNA molecule according to claim 25 or 26, wherein the recombination site is a loxP site.

28. The scDNA molecule according to claim 27, wherein the loxP site is an inactive Iox72 site with the sequence of SEQ ID NO: 11.

29. The scDNA molecule according to any one of claims 22-28, wherein the molecule of interest is a therapeutic or prophylactic protein.

30. The scDNA molecule according to any one of claims 22-29, wherein the molecule of interest is a vaccine antigen.

31. A composition comprising a plurality of scDNA molecules according to any one of claims 21-30, wherein the majority of the scDNA molecules in the composition are in supercoiled monomeric form.

32. The composition according to claim 31, wherein at least 70% of the scDNA molecules are in supercoiled monomeric form, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, and such as 100% of the scDNA molecules.

33. The composition according to claim 31 or 32, further comprising a pharmaceutically acceptable carrier and/or diluent.

34. The scDNA molecule according to any one of claims 21-30 or the composition according to any one of claims 31-33 for use as a medicament.

35. The scDNA molecule according to any one of claims 21-30 or the composition according to any one of claims 31-33 for use in vaccination or gene therapy.

36. A method of treating a subject in need thereof, comprising administering to the subject an efficient amount of the scDNA molecule according to any one of claims 21-30 or of the composition according to any one of claims 31-33.