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WO2024115669A1 - Procede de production d'adn circulaire synthetique - Google Patents

Procede de production d'adn circulaire synthetique Download PDF

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Publication number
WO2024115669A1
WO2024115669A1 PCT/EP2023/083759 EP2023083759W WO2024115669A1 WO 2024115669 A1 WO2024115669 A1 WO 2024115669A1 EP 2023083759 W EP2023083759 W EP 2023083759W WO 2024115669 A1 WO2024115669 A1 WO 2024115669A1
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molecule
scdna
dna
recombinase
process according
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Jesper Tranholm GRØNLUND
Anders Bundgård SØRENSEN
Jens Vindahl KRINGELUM
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Evaxion Biotech AS
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Evaxion Biotech AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the present invention relates to the field of clinical DNA technology, including DNA vaccine technology.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • scDNA synthetic circular DNA
  • scDNA synthetic circular DNA
  • 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.
  • 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.
  • the new method overcomes several challenges associated with current cell-based methods for DNA production for clinical use, described above.
  • 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.
  • scDNA synthetic circular DNA
  • the invention in a 2 nd aspect, 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.
  • scDNA synthetic circular DNA
  • 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.
  • the invention in a 3 rd aspect, relates to a composition
  • 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.
  • 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.
  • 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.
  • a DNA molecule comprising loxP sites 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.
  • scDNA synthetic circular DNA
  • 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.
  • a DNA molecule comprising loxP sites 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.
  • scDNA synthetic circular DNA
  • top a synthetic circular DNA
  • bottom a linear by-product
  • 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.
  • scDNA synthetic circular DNA
  • pDNA E. coll-produced plasmid DNA
  • 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.
  • scDNA synthetic circular DNA
  • pDNA coll plasmid DNA
  • E coll plasmid DNA
  • E coll plasmid DNA
  • E coll plasmid DNA
  • E coll plasmid DNA
  • E electroporation device
  • 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.
  • synthetic circular DNA molecule 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.
  • PCR polymerase chain reaction
  • 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.
  • 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.
  • 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.
  • 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.
  • a polynucleotide such as a DNA molecule
  • thermal cycling as no temperature shifts are necessary for the amplification process using such a polymerase.
  • 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.
  • 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.
  • Temporal DNA molecule 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.
  • 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.
  • an "immune- stimulating sequence” defined below
  • 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.
  • universal buffer 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.
  • 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.
  • fed-batch enzymatic reaction 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).
  • 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.
  • “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.
  • immune-stimulating sequence 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.
  • therapeutic or prophylactic protein 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.
  • 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.
  • 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.
  • the amplification can be performed using different methods.
  • the amplification step comprises non-exponential amplification using an isothermal polymerase with strand displacement activity.
  • the isothermal polymerase also has 3'-5' exonuclease activity.
  • 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.
  • 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.
  • RCA Rolling Circle Amplification
  • an initial denaturation step is performed in the presence of random primers, after which the enzyme is added.
  • the enzyme recognizes primers bound to template DNA and starts polymerization/replication.
  • 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 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.
  • 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.
  • MDA Multiple Displacement Amplification
  • exponential amplification methods such as PCR
  • exponential isothermal methods such as exponential versions of RCA and loop-mediated isothermal amplification.
  • an advantage of using non-exponential amplification is that errors are not propagated, as they are with exponential amplification.
  • 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.
  • 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.
  • a recombinase selected from: Cre recombinase, Flippase recombinase, R recombinase, Lambda recombinase, HK101 recombinase, pSAM2 re
  • the circularization of the DNA product of the amplification step depends on the presence of suitable recombination sites in the template DNA.
  • 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.
  • the recombinase is Bxbl recombinase.
  • Bxbl recombinase is a recombinase belonging to the serine integrase subfamily of recombinases.
  • 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).
  • 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.
  • 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.
  • 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.
  • Lox72 has the following sequence: taccgTTCGTATA-ATGTATGC-TATACGAAcggta (SEQ ID NO: 11).
  • 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.
  • 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.
  • the relevant recombinase e.g. Cre recombinase
  • 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.
  • 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.
  • 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.
  • 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).
  • RDF Recombination Directionality Factor
  • 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.
  • 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.
  • the recombination results in an scDNA molecule comprising an inactive recombination site and the gene encoding the molecule of interest.
  • the recombination results in an scDNA molecule comprising an active recombination site and the gene encoding the molecule of interest.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the process according to the present invention may include a linearization step.
  • 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.
  • the endonuclease is Xhol.
  • endonucleases also called restriction enzymes
  • 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.
  • 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.
  • Digestion may be performed using an exonuclease.
  • the exonuclease may digest linear and/or nicked circular DNA.
  • the exonuclease will digest both linear and nicked circular DNA.
  • the exonuclease is T5 exonuclease.
  • 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.
  • 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.
  • a supercoiling step is not performed.
  • 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.
  • 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).
  • 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.
  • a supercoiling step may be performed to increase this fraction further or to increase the level of negative supercoiling.
  • 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.
  • the negative supercoiling is introduced into the circularized amplified DNA using a gyrase.
  • the gyrase is Escherichia coli GyrA 2 B 2 .
  • the gyrase is a gyrase derived from any one of: Staphylococcus aureus, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Acinetobacter baumannii, and Clostridium difficile.
  • two or more steps of the process are carried out in the same universal buffer.
  • At least one of the enzymatic reactions occurs as a fed-batch enzymatic reaction.
  • the scDNA molecule according to the present invention may comprise a gene encoding a molecule of interest.
  • 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.
  • 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.
  • the gene encoding the molecule of interest comprises a signal sequence for secretion.
  • the scDNA molecule further comprises at least one immune- stimulating sequence.
  • the scDNA molecule 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.
  • ISS immune-stimulating sequence
  • 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).
  • 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.
  • 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.
  • the following CpG motifs are of interest: AACGAC and GTCGTT, but also CTCGTT, and GCTGTT.
  • 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.
  • the molecule After the production of the scDNA molecule according to the method described above, the molecule may be purified.
  • 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.
  • 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.
  • scDNA synthetic circular DNA
  • 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.
  • the scDNA molecule has been produced by the process according to certain embodiments of the 1 st aspect of the invention.
  • the scDNA molecule has not been produced by a process comprising a supercoiling step.
  • the scDNA molecule (further) comprises a recombination site.
  • the recombination site is inactive. In some embodiments, the recombination site is a loxP site.
  • the loxP site is an inactive iox72 site with the sequence of SEQ ID NO: 11.
  • the molecule of interest is a therapeutic or prophylactic protein.
  • the molecule of interest is a vaccine antigen.
  • 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.
  • 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
  • 94% such as at least 95%, such as at least 96%, such as at least 97%, such as at least
  • the composition further comprises a pharmaceutically acceptable carrier and/or diluent and/or excipient.
  • 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.
  • 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 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the scDNA molecules are injectable compositions, they are sterile, and/or pyrogen free and/or particulate free.
  • an isotonic formulation is used.
  • additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose.
  • isotonic solutions such as phosphate buffered saline are used.
  • An alternative solution is Tyrode's buffer.
  • stabilizers include gelatine and albumin.
  • 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.
  • 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 Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4.
  • concentration of the PBS 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.
  • 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.
  • Figure 3 shows representative examples of scDNA obtained in a process according to the present invention.
  • 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).
  • the total amplification was app. 340-fold (7.5 pg to 2.57 mg).
  • the inventors have obtained DNA which after purification consisted of at least 80% negatively supercoiled monomeric scDNA ( Figure 3).
  • 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.
  • 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.
  • 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.
  • 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.
  • a 6-well plate was coated with Poly-D-lysine and seeded with lxlO 5 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.
  • plasmid DNA 4 ug was used for transfection, and 2 ug was used for the scDNA (due to its approximately 50% smaller molecular size).
  • the media were exchanged with serum-free HEK293 media for all wells, followed by supernatant and cell lysate harvesting on day 5.
  • mCCL19 murine CCL19
  • HRP horseradish peroxidase
  • TMB 3,3',5,5'-Tetramethylbenzidine
  • mice 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.
  • 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 H 2 SO 4 . Data was read using a standard plate-reader.
  • 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).
  • 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
  • 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
  • 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.
  • 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.

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Abstract

L'invention concerne un nouveau procédé de production d'une molécule d'ADN circulaire synthétique (ADNcs) et une nouvelle molécule d'ADNcs pouvant être produite selon le procédé. L'invention concerne en outre des procédés thérapeutiques utilisant la molécule d'ADNcs.
PCT/EP2023/083759 2022-11-30 2023-11-30 Procede de production d'adn circulaire synthetique Ceased WO2024115669A1 (fr)

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