WO2023187394A1 - Expression génique contrôlable - Google Patents

Expression génique contrôlable Download PDF

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Publication number
WO2023187394A1
WO2023187394A1 PCT/GB2023/050842 GB2023050842W WO2023187394A1 WO 2023187394 A1 WO2023187394 A1 WO 2023187394A1 GB 2023050842 W GB2023050842 W GB 2023050842W WO 2023187394 A1 WO2023187394 A1 WO 2023187394A1
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triplex
nucleic acid
stranded
section
double
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PCT/GB2023/050842
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English (en)
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Katherine DUNN
Alexander SPEAKMAN
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The University Court Of The University Of Edinburgh
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Priority claimed from GBGB2204729.4A external-priority patent/GB202204729D0/en
Priority claimed from GBGB2207592.3A external-priority patent/GB202207592D0/en
Application filed by The University Court Of The University Of Edinburgh filed Critical The University Court Of The University Of Edinburgh
Publication of WO2023187394A1 publication Critical patent/WO2023187394A1/fr

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs

Definitions

  • the present invention relates to methods and constructs for controlling gene expression, in particular by manipulating pH to direct the formation of DNA triplexes.
  • Optogenetics Background, Methodological Advances and Potential Applications for Cardiovascular Research and Medicine. Front Bioeng Biotechnol. 2020;7:466.). Again, regulatory proteins are required to detect light and control gene expression, and more complex genetic systems are necessary to achieve full control. Optogenetic systems are also difficult to scale-up to industrial levels due to light attenuation when working with larger volumes.
  • nucleic acid molecule for controllable gene expression comprising: a double-stranded section, comprising a sequence for expression; and a single-stranded overhanging section configured to bind to the double-stranded section to form a nucleic acid triplex.
  • sequence for expression may be operably linked to a regulatory element, optionally wherein the regulatory element comprises a promoter.
  • the formation of said triplex may be reversible.
  • the formation of said triplex may be pH dependent.
  • the sequence for expression may encode a protein or an RNA.
  • the formation of said triplex may control, reduce, slow, inhibit or prevent transcription of the sequence for expression.
  • the single-stranded section may reversibly bind to all or part of the double-stranded section or to any regulatory element/promoter and/or to a sequence region downstream of any regulatory element/promoter, optionally comprising all or part of the sequence for expression, to form the nucleic acid triplex.
  • the single-stranded overhanging section may comprise a triplex- forming domain, wherein the triplex-forming domain may be configured to reversibly bind to the double-stranded section to form the nucleic acid triple helix.
  • the single-stranded overhanging section may further comprise a single-stranded nucleic acid linker region disposed between the triplex-forming domain and the double-stranded section.
  • the length of the linker region may be selected to optimise the reversible binding of the triplex-forming domain.
  • the nucleic acid molecule may be obtainable by a method comprising asymmetric PCR.
  • the method may comprise: (a) the method set out in Figure 3; or
  • a method of producing a nucleic acid molecule for controllable gene expression comprising: obtaining first and second nucleic acid templates; preferentially amplifying, by asymmetric PCR, opposite strands of the first and second nucleic acid templates to produce first and second single-stranded nucleic acids; and hybridising the first single-stranded nucleic acids with the second single-stranded nucleic acids.
  • a nucleic acid molecule for controllable gene expression comprising: a double-stranded section comprising a first sequence region for expression; and a single-stranded overhanging section configured to reversibly bind to the double-stranded section to form a nucleic acid triplex, wherein the single-stranded section comprises: a triplex-forming domain, configured to reversibly bind to the double-stranded section to form the nucleic acid triple helix; and a linker region, disposed between said triplex-forming domain and the double-stranded section, wherein the nucleic acid molecule is produced by a method comprising steps of: determining a target region of the double-stranded section for binding to the triplex- forming domain; and selecting a length of the linker region to optimise the binding of the triplex-forming domain to the target region to form the nucleic acid triplex.
  • the method may further comprise determining a distance between the target region and the single-stranded section, and wherein the length of the linker region is selected based on said distance.
  • a system for modulating or controlling gene expression comprising: a double-stranded nucleic acid comprising a first sequence region; and a single-stranded nucleic acid, configured to reversibly bind to the double-stranded nucleic acid section to form a nucleic acid triplex, wherein, in use, the presence of said triplex reduces or prevents transcription of the first sequence region.
  • the double-stranded nucleic acid may be a double-stranded DNA section, the first sequence region coding for a protein or an RNA; the single-stranded nucleic acid may be a single-stranded DNA section, configured to reversibly bind to the double-stranded DNA section to form an DNA triplex; and in use, the presence of said triplex may reduce or prevent transcription of the first sequence region.
  • the double-stranded and single-stranded DNA may be comprised within a single DNA molecule, wherein said single-stranded section is an overhanging section.
  • the reversible binding of the single-stranded nucleic acid section to the double-stranded nucleic acid section to form the nucleic acid triplex may be pH- dependent, and the system may further comprise means for controlling transcription of the first sequence region by manipulating a pH of the reaction solution.
  • the means for controlling transcription of the first sequence region by manipulating a pH of the reaction solution may comprise a device configured to apply an electrical input to the reaction solution.
  • a method of modulating or controlling gene expression comprising: (a) obtaining a reaction solution comprising: a double-stranded nucleic acid section comprising a first sequence region, said first sequence region coding for a protein or an RNA; and a single-stranded nucleic acid section, configured to reversibly bind to the double-stranded nucleic acid section to form a nucleic acid triplex, wherein the presence of said triplex reduces or prevents transcription of the first sequence region; and (b) modulating or controlling transcription of the first sequence region by manipulating a pH of the reaction solution.
  • a method of producing an mRNA vaccine comprising: (a) obtaining a reaction solution comprising: a double- stranded DNA section comprising a first sequence region, said first sequence region coding for an RNA transcript for use in an mRNA vaccine; and a single-stranded DNA section, configured to reversibly bind to the double-stranded DNA section to form a DNA triplex, wherein the presence of said triplex reduces or prevents transcription of the first sequence region; (b) controlling transcription of the first sequence region by manipulating a pH of the reaction solution; and (c) modifying the transcribed RNA transcript.
  • modifying the transcribed RNA transcript may comprise adding a 5’ m?G cap to the transcript.
  • step (c) may be performed simultaneously with step (b) and in the same reaction solution.
  • a method of in vitro protein or RNA expression comprising: (a) obtaining a reaction solution comprising: a double-stranded nucleic acid section comprising a first sequence region, said first sequence region coding for a protein or an RNA; and a single-stranded nucleic acid section, configured to reversibly bind to the double-stranded nucleic acid section to form a nucleic acid triplex, wherein the presence of said triplex reduces or prevents transcription of the first sequence region; and (b) controlling or modulating transcription of the first sequence region by manipulating a pH of the reaction solution.
  • manipulating the pH of the reaction solution may comprise applying an electrical input to the reaction solution.
  • manipulating the pH of the reaction solution may comprise selecting and/or varying one or more of a duration, current, voltage, or polarity of the electrical input.
  • manipulating the pH of the reaction solution may comprise applying first and second electrical inputs to the reaction solution, optionally asynchronously.
  • the first and second electrical inputs may be of opposite polarity.
  • the first and second electrical inputs may each increase the pH of the reaction solution. Said first and second electrical inputs may have the same polarity.
  • the first electrical input may increase the pH of the reaction solution and the second electrical input may decrease the pH of the reaction solution.
  • the first and second electrical inputs may have opposite polarity.
  • the first electrical input may decrease the pH of the reaction solution and the second electrical input may increase the pH of the reaction solution.
  • the first and second electrical inputs may have opposite polarity.
  • the first and second electrical inputs may each decrease the pH of the reaction solution.
  • the first and second electrical inputs may have the same polarity.
  • manipulating the pH of the reaction solution may comprise adding one or more of: an acidic compound; or a basic compound, to the reaction solution.
  • a triplex-forming oligonucleotide for modulating transcription and/or gene expression.
  • the transcription or gene expression may occur in a cell free system.
  • a method of co-transcriptional RNA modification comprising: (a) obtaining a reaction solution comprising: a double-stranded DNA section comprising a first sequence region, said first sequence region coding for an RNA transcript; and a single-stranded DNA section, configured to reversibly bind to the double-stranded DNA section to form a DNA triplex, wherein the presence of said triplex reduces or prevents transcription of the first sequence region; (b) controlling transcription of the first sequence region by manipulating a pH of the reaction solution; and (c) modifying the transcribed RNA transcript, wherein step (c) is performed simultaneously with step (b) and in the same reaction solution, optionally wherein modifying the transcribed RNA transcript comprises adding a 5’ m?G cap to the transcript.
  • FIGS 1 and 2 show schematically DNA constructs for controllable gene expression
  • Figure 3 shows schematically a method for producing a DNA construct for controllable gene expression
  • Figure 4 is a photograph of an electrophoresis gel, demonstrating the effectiveness of the method shown in Figure 2 for producing an exemplary construct for controllable gene expression
  • Figures 5 and 6 are graphs demonstrating triplex formation (as measured by fluorescence) as a function of pH;
  • Figure 7 is a graph demonstrating triplex formation (as measured by fluorescence) during electrolysis
  • Figure 8 is a graph demonstrating pH-dependent control of gene expression (as measured by fluorescence).
  • Figure 9 is a graph demonstrating differential pH-dependent control of gene expression (as measured by fluorescence) when using a triplex-forming construct and a non-triplex forming control;
  • Figure 10 is a graph showing the ratio of the fluorescence, shown in Figure 8, observed using the triplex-forming construct to that observed for the non-triplex-forming controls;
  • Figure 11 is a graph demonstrating iSpinach fluorescence kinetics of electrically activated transcription reactions, with both triplex forming constructs and non-triplex controls and two different lengths of electrolysis duration (26s and 107s);
  • Figure 12 is a graph demonstrating normalised triplex vs non-triplex transcription electrolysis activated reaction kinetics
  • Figures 13-24 are bar charts showing ratios between the amplitude of fluorescence observed from constructs expressing iSpinach during experiments at different pH values;
  • Figures 25A-28B are heat maps showing ratios between the amplitude of fluorescence observed from constructs expressing iSpinach during experiments at pH 8.89 and pH 7.62;
  • Figures 29A-30B are bar charts showing the observed fluorescence from constructs expressing iSpinach and subjected to electrolysis for Os, 26s, 54s, or 107s;
  • Figures 31-46 are graphs showing normalised fluorescence observed from constructs expressing iSpinach and subjected to electrolysis for Os, 26s, 54s, or 107s;
  • Figure 47A shows heat maps demonstrating the relative pH-sensitivity of expression in triplex-forming constructs and non-triplex-forming control constructs
  • Figure 47B shows corresponding heat maps according to a revised, reanalysis of the data
  • Figures 51-66 are graphs showing normalised fluorescence observed from constructs expressing iSpinach and subjected to chemical pH control;
  • Figure 67 shows heat maps demonstrating the relative pH-sensitivity of expression in triplex-forming constructs and non-triplex-forming control constructs, when subjected to chemical pH control;
  • Figures 68A and 68B are graphs comparing iSpinach spectra of samples with and without co-transcriptional capping by Vaccinia Capping Enzyme (VCE), and Figure 68C is a graph showing iSpinach fluorescence referenced against Ant-GTP fluorescence plotted over time;
  • Figure 69A is a graph showing the effect of triplex formation on the concentration of nucleotides (X), uncapped RNA (Y), and capped RNA (Z), according to a simplified model of co-transcriptional capping
  • Figure 69B is a graph showing the ratio of capped (Z) to total (Z+Y) in the presence (Modulated) and the absence (Control) of triplex formation.
  • the present disclosure provides products, systems, uses and methods which may be used to modulate in vitro and/or in vivo gene expression, RNA transcription and/or protein synthesis.
  • the products, systems, uses and methods may be used to control or modulate transcription in a cell-free medium.
  • a further advantage of the various disclosed constructs, methods, uses and systems is that the modulation of gene expression is tunable, allowing the user to exert a precise control.
  • the system may be tuned to react as desired to electrical controls.
  • constructs may be designed to have a transition midpoint (where the triplex is most stable) at a desired pH. This allows, for example, for gene expression to be precisely controlled by manipulating pH within a range specifically chosen for compatibility with a given reaction mixture or product.
  • the constructs can also be designed to adjust the sensitivity of gene expression to changes in pH, again allowing for tunable and precise control.
  • Figures 1 and 2 show schematically DNA constructs 1 for modulating (for example controlling) gene expression (lengths shown in Fig. 2 are not necessarily accurate).
  • the DNA construct 1 is a chimeric, single-stranded/double-stranded linear DNA molecule comprising a double-stranded section 2 and a (long), single-stranded 5’ overhang 3.
  • the double-stranded section 2 comprises a promoter 4, a gene 5 for expression, and a terminator sequence 6.
  • the single-stranded overhang 3 comprises a triplex-forming domain 7, and a linker region 8 disposed between the triplex-forming domain 7 and the double-stranded section 2.
  • the triplex-forming domain 7 is configured to bind to (or interact with) the double-stranded section 2 to form a DNA triplex capable of reducing or preventing transcription of the gene 5.
  • the presence or formation of the triplex blocks or slows the progress of transcription machinery, e.g. RNA polymerase, or inhibits its binding to the DNA sequence.
  • the triplex-forming domain 7 is configured to bind in the major-groove of the double- stranded section 2 via Hoogsteen or reverse Hoogsteen base-pairing.
  • the sequence of the triplex-forming domain 7 can, therefore, be designed to target a specific region of the double-stranded section 2 in order to optimise modulation (e.g. suppression) of the transcription of the gene 5 when the triplex is present.
  • the triplex-forming domain 7 binds to the double-stranded section 2 at a triplex- forming domain binding region or “target region” 9 downstream of the promoter 4.
  • the target region 9 may be selected from the promoter 4, a region downstream of the promoter 4, the gene for expression 5, or overlapping portions thereof.
  • the length of the linker region 8 may also be selected to optimise the binding of the triplex-forming domain 7 to the desired target region 9.
  • the length of the linker region may vary between different constructs.
  • the length of the linker region 8 may be selected from between a minimum length required for the triplex-forming domain 7 to extend to the target region 9 (e.g. downstream of the promoter 4), and a maximum length above which binding of the triplex-forming domain 7 to the target region 9 becomes less thermodynamically favourable.
  • the skilled person could also prepare and test various different constructs (e.g. having different linker lengths) to determine an optimum linker length for a specific application.
  • the length of the linker region 8 may be selected according to the following equation:
  • X may be at least: 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.
  • the formation of the triplex is reversible, and highly pH-dependent.
  • the lower the pH the greater the stability of the triplex structure.
  • the triplex structure is only stable at low pH, for example at pH 7.5 or less.
  • the pH of the transition midpoint may, however, be higher or lower.
  • the triplex-forming domain 7 is configured to bind to the double-stranded section 2 to form a parallel triplex. Construct production via asymmetrical PCR
  • Fig. 3 shows schematically an exemplary method for producing DNA constructs for controlled or modulated gene expression (such as those shown in Fig. 1 or 2 and described above). Due to the complex sequences required and the limitations of known chemical oligonucleotide synthesis techniques, a method of producing the constructs via asymmetrical PCR has been developed.
  • a first DNA template in the form of a plasmid comprises a promoter, a target region, a gene for controlled expression, and a terminator sequence.
  • a second DNA plasmid template comprises the promoter, target region, gene and terminator, plus an additional “tail” region (which will form the single-stranded overhang). Both templates are amplified using forward and reverse primer pairs.
  • a reaction solution comprising the triplex-forming construct 1 is contained in a first, isolated reaction compartment.
  • a second reaction compartment contains a “sacrificial” solution, electrically coupled to the reaction solution via a salt bridge.
  • An electrode is inserted into each reaction mixture and connected to a controllable power supply. The pH of the reaction solution may then be increased by electrolysing with the cathode in the reaction mixture, and or decreased by the reverse polarity.
  • the pH can also be adjusted to a desired level by varying the length of time the electrical input is applied for, or by varying the current or voltage used.
  • expression of the gene 5 may be increased or decreased, or pulsed on and off with tight control.
  • the combination of the triplex-forming construct 1 and electrical manipulation of pH described above allows for precise control of transcription.
  • the pH of the reaction solution may be controlled chemically, by the controlled addition of one or more acids or bases.
  • the term “gene for expression” is used to describe a sequence for transcription into a desired product.
  • the gene may be transcribed into an mRNA for translation into a desired protein.
  • the desired product may also be the transcribed RNA itself, for example a non-coding RNA, or an mRNA for use in an mRNA vaccine as described below.
  • the constructs and methods described herein therefore have a number of useful applications.
  • the constructs and methods described herein may be used as part of a cell-free, transcription/translation system (e.g. for the production of therapeutic proteins).
  • Many therapeutic proteins such as monoclonal antibodies or cytokines are both expensive and difficult to produce by known methods, particularly when developing and manufacturing a modified version of the protein that is suitable for packaging into a pill or tablet format.
  • the present methods allow for clinically relevant proteins to be produced more easily, in the small amounts which are often required for treatment, and without the modifications needed for long term storage.
  • the expertise needed to control gene expression is dramatically reduced.
  • the input could precisely turn expression on and off in order to produce an exact dosage amount of protein.
  • This method may also provide a significant cost reduction in the production of therapeutic proteins, as due to the nature of in vitro transcription/translation systems a universal expression solution can be bulk produced, with the only difference between proteins being the sequence of the DNA template provided. This would have the potential to improve drug administration and reduce costs in the treatment of many life threatening and long-term diseases such as cancer and autoimmune diseases.
  • RNA transcripts must be modified with a 5’ m7G cap to allow it to be accepted and read by mammalian cells.
  • the most efficient and well established method of doing this is via an enzymatic capping reaction, which is done as a separate reaction to the initial transcription.
  • RNA transcription is a fast process, which means that transcription will outpace downstream reactions.
  • the industrial RNA transcription mixture is purified, then capped, then purified again (Rosa SS, Prazeres DMF, Azevedo AM, Marques MPC.
  • mRNA vaccines manufacturing Challenges and bottlenecks. Vaccine.
  • the present methods allow for transcription and modification to be performed (e.g. at a 1 :1 ratio), allowing a single reaction mixture to produce modified mRNA and eliminating both purification and separate capping modification steps from the manufacturing process. This greatly streamlines large-scale manufacturing, reducing both time and costs for the emerging market of mRNA vaccine production.
  • a triplex-forming domain may be provided in the form of a triplex-forming oligonucleotide which is a separate DNA molecule to the double-stranded DNA section encoding the protein or RNA.
  • the triplex-forming domain may bind in the minor groove of the double-stranded section to form the triplex.
  • EDGE Electrically Directed Gene Expression
  • EDGE Electrically Directed Gene Expression
  • Triple helices, or triplexes are DNA nanostructures that are capable of the inhibition of gene expression, and can be designed to require specific pH conditions in order to form Electrolysis can be induced by applying a voltage to a cell- free transcription solution, this alters the pH, leading to a reversible conformational change in the triplex (Fig. 7). When the triplex is closed, transcription is inhibited (Fig.
  • T7D An exemplary construct “T7D” was designed, produced, and analysed,
  • the T7D construct comprises a 40-nucleotide triplex-forming domain targeting a region downstream of a T7 promoter, and 54-nucleotide linker region.
  • the sequence of the top-strand of the T7D construct corresponds to SEQ ID NO: 3, and the bottom strand (which includes the single-stranded overhanging region) corresponds to SEQ ID NO: 5).
  • the T7D construct also encodes for the fluorescent RNA aptamer, iSpinach.
  • the T7D construct was produced according to the method shown in Fig. 3.
  • Fig. 4 panels A-C demonstrate that this method is effective for producing the construct (shown by effective hybridisation into products of the correct size).
  • the sizes observed for the T7D top strand are shown in lane 2, the bottom strand is shown in lane 4, and the hybridised T7D construct is shown in lane 6.
  • TBR10 triplex-forming test construct
  • SDR10_BHQ2 top strand
  • C10 non-triplex control
  • Non-triplex controls (C10) differ from TBR10 only by the end 10 nucleotides in the ssDNA tail, which would otherwise be designed to form a triplex.
  • fluorophore labelled oligos was produced with fluorophore labelled oligos and used to investigate T7D dynamics. pH dependent triplex formation was tested in different pH level Britton Robinson Buffers and AceOTB buffers (/n vitro transcription solution).
  • AceOTB is a previously optimised RNA aptamer transcription buffer (OTB) (Millacura et al.
  • TXO Transcription-Only genetic circuits as a novel cell- free approach for Synthetic Biology bioRxiv 826230), modified to be acetate based. pH-dependent changes in relative fluorescence were observed for TBR10 and the shortened T7D construct (Figs. 5 and 6), indicating pH-dependent triplex-formation. The lower the pH, the greater the formation of the DNA triplex, and the lower the relative fluorescence observed. The differences observed between the constructs demonstrates the effect of differing triplex lengths, linker lengths, and base composition. No change in relative fluorescence was observed for the non-triplex- forming controls (Fig. 5).
  • AceOTB promotes much stronger triplex binding than Britton Robinson Buffer. Without wishing to be bound by theory, it may be that the higher magnesium concentrations in AceOTB may act serve to stabilise the DNA triplex.
  • a voltage source unit was used to electrolyse a sample of 1 M sodium sulphate containing TBR10 triplex forming, Cy5/BHQ labelled oligonucleotides, while being scanned by a fluorescence spectrophotometer.
  • the setup used a second, sacrificial cuvette connected by a salt bridge, and a graphite electrode in each cuvette.
  • pH was first increased by electrolysing with the cathode in the cuvette containing TBR10 (forward polarity: pH increasing), then decreased by switching the polarity and electrolysing again (reverse polarity: pH decreasing). This resulting change in pH is reflected by the change in fluorescence produced by the labelled oligonucleotides as it destabilises then reforms a triplex on demand.
  • T7D constructs were tested in various pH levels of AceOTB.
  • the product is iSpinach, a fluorescent RNA aptamer, allowing fluorescence to be used to quantify the amount of transcription occurring. These were measured as kinetics runs over time. As shown in Fig. 8, T7D showed pH-dependent transcription, with decreased levels of transcription at lower pH.
  • Fig. 9 compares the fluorescence observed for the iSpinach-expressing T7D construct to that of a comparable no-tail control. The ratio of the fluorescence for two constructs is shown in Fig. 10. These results confirm that the triplex-forming construct exhibits a different dependence on pH to the non-triplex-forming control.
  • Fig. 11 iSpinach fluorescence kinetics of electrically activated transcription reactions, with both triplex forming EDGE constructs and non-triplex controls and 2 different lengths of electrolysis duration (26s and 107s). Longer electrolysis durations induce a greater shift in pH, producing a greater increase in the rate of transcription of iSpinach due to the innate pH sensitivity of transcription reactions. These different shifts also induce different levels of triplex destabilisation, reducing triplex inhibition in EDGE constructs to different extents. Triplex forming EDGE constructs demonstrate lower leakage of transcription prior to electrolysis than non-triplex controls, due to the low pH inducing strong triplex formation. After electrolysis, triplex mediated inhibition remains present throughout the rest of the reaction as T7D triplexes are still relatively stable at these higher pH levels.
  • Fig. 12 Normalised triplex vs non-triplex transcription electrolysis activated reaction kinetics. Non-triplex transcription occurs more quickly prior to electrolysis due to the lack of triplex, causing the ratio to consistently drop.
  • the pH increases, the triplex is lifted, slowing the rate at which the ratio drops as EDGE construct transcription rate increases. As the reaction progresses, the pH drops, strengthening EDGE triplex formation and allowing triplex inhibition to return to previous levels, and causing the ratio to drop more quickly once again.
  • each construct codes for iSpinach.
  • a corresponding non-triplex-forming control construct was also produced for each of the
  • Each control is provided with the same triplex-forming domain binding region as its corresponding triplex-forming construct.
  • Table 1 List of constructs (Set 1).
  • the fluorescence signal from ISpinach were measured as a function of time. Using automation (laboratory robot) and high-throughput methods (plate, plate-reader etc), data was captured from three replicate 96-well plates in the same run, where each plate had 48 wells that contained constructs.
  • an alternative measure of expression is computed by averaging the last five data points. The error is taken to be the population standard deviation of the five data points in question. If these quantities could not be calculated, the kinetic data in question would be omitted from analysis.
  • the ‘amplitude’ is the parameter from the fit or the alternative measure defined above.
  • Ratio H (High) Amplitude for pH 8.89 / Amplitude for pH 7.62
  • Ratio M (Medium) Amplitude for pH 7.62 / Amplitude for pH 6.92
  • Ratio L (Low) Amplitude for pH 6.92 / Amplitude for pH 6.1
  • Ratio L is of limited utility as expression levels are generally low at pH 6.1 .
  • Each ratio has an associated error, computed in the normal way as understood by the skilled person, by summing in quadrature the fractional errors of the contributing amplitudes.
  • Figs. 13-28B are bar charts showing Ratio H, M or L.
  • Crosses indicate instances where, for the pH window tested, the triplex- forming construct is more pH-sensitive than the corresponding control construct. Dots indicate instances where the (non-triplex-forming) control is more sensitive.
  • Figs. 25A- 28B are heat maps showing ratio H for different combinations of parameters. Crosses indicate regions where the triplex constructs show significant pH sensitivity in a region wherein the control constructs do not. Arrows show a visual approximation of the ratio values for the regions marked by crosses on the accompanying scale.
  • the results show dependence of expression on sequences flanking the promoter, intrinsic pH-dependence of transcription and the design of the triplex (percentage of TAT triplets, length of triplex-forming domain, length of linker region). Different constructs are sensitive to different pH windows. Without wishing to be bound by theory, if the triplex is too short, it is unlikely to be stable at any condition explored in these experiments. If the triplex-forming domain is very long, it may be more stable, but the chance of undesired secondary structures also increases. Changing the length of the triplex-forming domain also changes the number of bases between the promoter and the coding region, which can affect expression as the polymerase activity is affected by promoter flanking sequences.
  • the linker is too short, the triplex-forming domain is unlikely to be able to reach its target because the single-stranded tail would need to be occupy an extended conformation (which would occur with lower probability). If the linker is longer, the single-stranded tail has a larger volume of space to explore and the entropy penalty for triplex binding is larger, reducing the probability of triplex formation.
  • Changing the proportion of triplets that are TAT changes the pKa of the triplex, hence altering the pH at which the triplex is most stable.
  • Changing the bases also changes the thermodynamics of triplex/duplex formation, in addition to changing the flanking sequence of the promoter region.
  • the advantages of having the triplex in place are as follows:
  • the triplex-forming unit gives more choice over the conditions under which transcription will be switched on and off, expanding the possibility space.
  • the triplex-forming unit could be deployed to control expression with polymerase/promoter systems that have a lesser degree of pH-sensitivity than those used here. Further, with the triplexes we can decide exactly how sensitive the construct will be to pH.
  • Figs. 51 -66 show relative levels of transcription, as reported by the fluorescence signal from iSpinach, for each of the different constructs when subjected to chemical pH control.
  • the relative pH sensitivities of the constructs and non-triplex forming controls are shown by way of heatmaps in Fig. 67.
  • Samples were electrolysed for a set period of time: Os, 26s, 54s or 107s (using a different set of samples for each condition). Initially the reactions were at pH6.1 According to prior tests (not shown), a pulse of 26s was expected to take the pH to 7.25, a pulse of 54s was expected to take the pH to 7.5 and a pulse of 107s was expected to take the pH to 7.75. The electrolysing pulse was applied 40 minutes after the experiment started, for the stated duration. A customised system was used to apply a constant current of 130 ⁇ A, which normally equates to a voltage of around 3V.
  • the amplitude from the analysis is a measure of how much expression is occurring. For each construct, the analysis procedure yields one amplitude (and associated error) for the experiment in which there was no electrolysis, one amplitude for the experiment in which there was a 26s electrolysing pulse, one for the 54s pulse and one for the 107s pulse.
  • the un-normalised amplitudes are plotted for all constructs in Figs. 29A, B (triplex- forming construct) and 30A, B (non-triplex-forming controls). For each construct, four bars represent (from left-to-right): Os pulse; 26s pulse; 54s pulse; and 107s pulse.
  • the control constructs require shorter electrolysis pulses for activation than their triplex-based counterparts.
  • This is shown schematically in the heatmaps in Fig. 47A, where black indicates that the control is activated by shorter electrolysis pulses than the triplex, grey indicates the control and triplex behave similarly, white indicates that the triplex is activated by shorter pulses than the control, and cross-hatching indicates missing data (due to quality of fit results or ambiguity in the data, which can arise due to factors such as anomalously high values for the non- electrolysed case).
  • the data was classified by visual inspection.
  • the heatmaps reveal that it is extremely rare for the triplex construct to be activated more easily than the control. In many cases, the triplex and control construct respond similarly.
  • FIGs. 48 and 49 A specific example is shown in Figs. 48 and 49, using data gathered from Construct Number: 3 (Set 1 , 90_TFR40_42). Following a 26s pulse, an increase in fluorescence was observed for the non-triplex-forming control. In contrast, fluorescence observed for the triplex-forming construct remained low (comparable to that of non-electrolysed controls). Under these conditions, the presence of the triplex therefore inhibited gene expression. Following a 56s pulse however, fluorescence observed for the triplex- forming construct did increase. Unlike the 26s pulse, the 56s pulse was therefore sufficient to activate gene expression in the triplex-forming construct. This demonstrates the ability of the constructs and methods described herein to provide controllable gene expression, in particular through the use of electrical inputs.
  • Co-transcriptional capping co-transcriptional capping of iSpinach with fluorescent Ant-GTP
  • VCE Vaccinia Capping Enzyme
  • Integrated Buffer is a previously optimised buffer for in vitro co-transcriptional enzymatic mRNA capping using T7 RNA polymerase and VCE developed and tested by Nwokeoji, Chou, and Nwokeoji (Low Resource Integrated Platform for Production and Analysis of Capped mRNA. ACS Synth. Biol. 2023, 12, 329-339). Samples had either ultrapure water or VCE added, and were repeatedly scanned for emission spectra (350nm to 600nm) at excitation wavelengths of either 330nm or 465nm to excite Ant-GTP and iSpinach respectively.
  • IB Buffer 1X 50mM Tris, 10mM MgCl 2 , 1 mM DTT, 1.8mM spermidine, adjusted to pH 8 via 2M HCI;
  • Co-transcriptional capping may be modelled as a process that consists of two irreversible sub-processes i.e. X ⁇ Y ⁇ Z where X represents nucleotides, Y represents uncapped RNA and Z represents capped RNA.
  • the first process has a rate k 1 and the second process has a rate k 2 .
  • timescales may be used.
  • X falls off exponentially from an initial value taken as 1000 arbitrary units.
  • Y builds up and then starts to decay, while Z increases monotonically but slowly.
  • the second calculation is for a scenario in which one of our triplex-based constructs is employed, and at the point indicated by the arrow the triplex is closed, reducing k 1 to 0.0001 units from that point on.
  • This is the ‘modulated’ case (thick lines in Fig. 69A).
  • the concentration of Y starts to drop off after the triplex is closed.
  • SEQ ID NO: 1 SDR10_BHQ2

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Abstract

L'invention concerne une molécule d'acide nucléique pour l'expression génique contrôlable qui comprend : une section double brin, comportant une séquence pour l'expression ; et une section en porte-à-faux simple brin conçue pour se lier à la section double brin pour former un triplex d'acides nucléiques.
PCT/GB2023/050842 2022-03-31 2023-03-30 Expression génique contrôlable WO2023187394A1 (fr)

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GBGB2204729.4A GB202204729D0 (en) 2022-03-31 2022-03-31 Electrically directed gene expression (edge)
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GBGB2207592.3A GB202207592D0 (en) 2022-05-24 2022-05-24 Controllable gene expression
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