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WO2025202929A1 - Procédés de production d'acides nucléiques - Google Patents

Procédés de production d'acides nucléiques

Info

Publication number
WO2025202929A1
WO2025202929A1 PCT/IB2025/053195 IB2025053195W WO2025202929A1 WO 2025202929 A1 WO2025202929 A1 WO 2025202929A1 IB 2025053195 W IB2025053195 W IB 2025053195W WO 2025202929 A1 WO2025202929 A1 WO 2025202929A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
composition
aspects
dna
recognition site
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/053195
Other languages
English (en)
Inventor
Wei Chen
Annie R CRUZ
Nicholas Jacob LUITJENS
Carrie Leanne SIMMS
Dillon Jarod SINANAN
Shuo Sui
Jun Sun
Karen Kiyoko TAKANE
Tian Tian
Christo Geevarghese VAIRAMON
Michael Casey VANDE VOORDE
Nichole Lea Wood
Yeou Hsiung Angel YU
Haitao Zhang
Xiaolu Zheng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Corp Belgium
Pfizer Corp SRL
Original Assignee
Pfizer Corp Belgium
Pfizer Corp SRL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pfizer Corp Belgium, Pfizer Corp SRL filed Critical Pfizer Corp Belgium
Publication of WO2025202929A1 publication Critical patent/WO2025202929A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase

Definitions

  • the present invention relates to an improved process for synthesis of nucleic acid molecules, in particular cell-free enzymatic synthesis of closed end DNA (ceDNA) and synthesis of RNA molecules via in vitro transcription (IVT).
  • ceDNA closed end DNA
  • IVT in vitro transcription
  • circular DNA molecules comprising: (a) an in vitro transcription (IVT) expression cassette; (b) a protelomerase (teIRL) sequence; and (c) a restriction endonuclease (RE) recognition site, wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 300 nucleotides or less.
  • the IVT expression cassette comprises a promoter, a gene of interest, and a poly-A tail.
  • the circular DNA molecules further comprise an origin of replication.
  • the circular DNA molecules further comprise an antibiotic resistance gene.
  • the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 200 nucleotides or less. In some embodiments, the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 30 nucleotides or less. In some embodiments, the circular DNA molecules comprises one or more ligated synthetic fragments.
  • dsRNA double-stranded RNA
  • IVT in vitro transcription
  • a restriction endonuclease (RE) recognition site wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 300 nucleotides or less; (b) treating the circular DNA molecule with a protelomerase to obtain a treated composition; (c) contacting the treated composition with an exonuclease to obtain a cleaned composition; (d) purifying the cleaned composition to obtain a first purified composition; (e) contacting the first purified composition with an endonuclease to obtain a digested composition; (f) purifying the digested composition to obtain a second purified composition; and (g) performing an in vitro transcription reaction to obtain an mRNA molecule, thereby reducing dsRNA formed in the IVT reaction.
  • RE restriction endonuclease
  • purifying the cleaned composition comprises a purification selected from the group consisting of proteinase K digestion, ultra-filtration/diafiltration (LIF/DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation.
  • purifying the digested composition comprises a purification selected from the group consisting of ultra- filtration/diafiltration (LIF/DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation.
  • the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 200 nucleotides or less.
  • the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 30 nucleotides or less.
  • the endonuclease comprises a type IIS restriction endonuclease.
  • the endonuclease is selected from the group consisting of Lgul, Eam1104l, and BspQI.
  • treating the circular DNA molecule with a protelomerase occurs in the absence of a surfactant.
  • treating the circular DNA molecule with a protelomerase occurs in the absence of Triton X-100.
  • dsRNA double-stranded RNA
  • IVT in vitro transcription
  • a restriction endonuclease (RE) recognition site wherein the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 300 nucleotides or less; (b) treating the circular DNA molecule with a protelomerase to obtain a treated composition; (c) contacting the treated composition with an exonuclease to obtain a cleaned composition; (d) purifying the cleaned composition to obtain a first purified composition; (e) contacting the first purified composition with an endonuclease to obtain a digested composition; (f) purifying the digested composition to obtain a second purified composition; (g) performing an in vitro transcription reaction to obtain an mRNA molecule, and (h) capturing the mRNA molecule using an oligo dT affinity ligand immobilized toa solid support, thereby reducing dsRNA formed in the IVT reaction.
  • RE restriction endonuclease
  • the solid support is selected from the group consisting of a chromatography resin and a magnetic bead.
  • treating the circular DNA molecule with a protelomerase occurs in the absence of a surfactant. In some embodiments, treating the circular DNA molecule with a protelomerase occurs in the absence of Triton X-100.
  • the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 200 nucleotides or less. In some embodiments, the teIRL sequence is downstream of the RE recognition site and separated from the RE recognition site by 30 nucleotides or less.
  • a level of dsRNA formed in the IVT reaction is reduced as compared to a level of dsRNA formed in a standard IVT reaction, wherein the standard IVT reaction is performed with a circular DNA molecule comprising a teIRL sequence downstream of an RE recognition site and separated from the RE recognition site by 301 nucleotides or more.
  • the level of dsRNA formed is measured by dot blot.
  • the level of dsRNA formed in the IVT reaction is reduced by at least two-fold.
  • the circular DNA molecule comprises one or more ligated synthetic fragments.
  • RNA molecules comprising: (a) obtaining a composition comprising: a. a circular double-stranded DNA (dsDNA) template, wherein the circular double-stranded DNA template comprises a teIRL sequence, b. a primer or primase, c. a deoxyribonucleotide triphosphate (dNTP), d. a pyrophosphate, and e.
  • dsDNA circular double-stranded DNA
  • dNTP deoxyribonucleotide triphosphate
  • dNTP deoxyribonucleotide triphosphate
  • a phi29 DNA polymerase (b) incubating the composition for a time period between about 20 and 30 hours at a temperature between about 30 and 45 °C to obtain an incubated composition; (c) treating the incubated composition with a protelomerase to obtain a treated composition; (d) contacting the treated composition with an exonuclease to obtain a cleaned composition; (e) purifying the cleaned composition to obtain a first purified composition; (f) contacting the first purified composition with a restriction endonuclease to obtain a digested composition; (g) purifying the digested composition to obtain a second purified composition; and (h) performing an in vitro transcription reaction, thereby producing the mRNA molecule.
  • steps (a) to (d) occur in the same reaction vessel.
  • the methods do not comprise a heat denaturation reaction prior to incubating the reaction.
  • the protelomerase is immobilized to a substrate.
  • the substrate is a N- hydroxy succinimide agarose bead.
  • purifying the cleaned composition comprises a purification selected from the group consisting of proteinase K digestion, ultra- filtration/diafiltration (UF/DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation.
  • purifying the digested composition comprises a purification selected from the group consisting of ultra- filtration/diafiltration (UF/DF), phenol-chloroform extraction, ammonium acetate+ethanol precipitation, and sodium acetate+ethanol precipitation.
  • treating the incubated composition with a protelomerase occurs in the absence of a surfactant.
  • the surfactant comprises Triton X-100.
  • methods of producing an mRNA molecule comprising: (a) obtaining a composition comprising: a. a circular double-stranded DNA (dsDNA) template, wherein the circular doublestranded DNA template comprises a teIRL sequence, b.
  • a primer or primase c. a deoxyribonucleotide triphosphate (dNTP), d. a pyrophosphate, and e. a phi29 DNA polymerase;
  • incubating the composition for a time period between about 20 and 30 hours at a temperature between about 30 and 45 °C to obtain an incubated composition;
  • treating the incubated composition with a protelomerase to obtain a treated composition;
  • contacting the treated composition with an exonuclease to obtain a cleaned composition;
  • purifying the digested composition to obtain a second purified composition;
  • performing an in vitro transcription reaction and (i) capturing the mRNA molecule using an oligo dT affinity ligand immobilized to a solid support, thereby producing the
  • the solid support is selected from the group consisting of a chromatography resin and a magnetic bead.
  • the phi29 DNA polymerase comprises a mutation resulting in increased thermostability and processivity.
  • the methods comprise repeating (a) and (b) one or more times.
  • the methods comprise repeating steps (a) - (h) using the second purified composition from step (g) as the circular double- stranded DNA template in step (a).
  • treating the incubated composition with a protelomerase occurs in the absence of a surfactant.
  • treating the incubated composition with a protelomerase occurs in the absence of Triton X-100.
  • the circular DNA molecule comprises one or more ligated synthetic fragments.
  • FIG. 1 is an exemplary diagram depicting closed end linear DNA production via multiple stage rolling cycle amplification and TelN protelomerase treatment.
  • the three stages include: 1) a first round of rolling circle amplification (RCA) of a circular DNA containing a TeIRL site to produce concatemeric DNA which is subjected to 2) multiple rounds of RCA to produce extended concatemeric DNA which is subjected to 3) TelN and exonuclease treatment to produce closed end DNA (ceDNA).
  • RCA rolling circle amplification
  • FIG. 2 is an exemplary diagram depicting a “one-pot” reaction to produce closed end linear DNA from a template.
  • Phi29 DNA polymerase, primer/primase, dNTPs, and circular DNA with a teIRL site is combined in a rolling circle amplification (RCA) reaction.
  • TelN protelomerase treatment results in ceDNA formation, followed by T5 exonuclease clean-up, and then EDTA/proteinase K enzyme inactivation and degradation.
  • the ceDNA is then purified, subjected to ultrafiltration/diafiltration, and then polished.
  • FIG. 3 is an exemplary agarose gel image analysis of closed end linear DNA synthesis.
  • Lane 1 is undigested plasmid DNA (pDNA);
  • lane 2 is plasmid DNA digested by Lgul;
  • lane 3 is rolling circle amplification (RCA) product using plasmid DNA as the template;
  • lane 4 is RCA product digested by Lgul;
  • lane 5 is closed end linear DNA formation by digesting RCA product with telN protelomerase;
  • lane 6 is clean-up of closed end linear DNA with T5 exonuclease; and
  • lane 7 is purified closed end linear DNA digested with Lgul.
  • FIG. 4 is an exemplary agarose gel image depicting pre-treatment of plasmid DNA with exonuclease for a rolling circle amplification (RCA) reaction.
  • RCA rolling circle amplification
  • FIG. 5 depicts an electropherogram of RNA made from ceDNA made with specific primers and a pDNA template with truncations in the poly(A) region, displaying an ideal peak shape. Overlayed with reference standard (lower line).
  • FIGS. 7A-C show exemplary plasmid configuration designs.
  • FIG. 7A depicts a 5’ TeIRL design with approximately 2000 base pairs between the restriction endonuclease recognition site (RS) and the TeIRL site.
  • FIG. 7B depicts a 3’ TeIRL design with approximately 30 base pairs between the RS and the TeIRL site.
  • FIG. 7C depicts desirable on-target transcription resulting in transcription of the gene of interest (GOI) (top) and undesirable off-target transcription (bottom), which is a possible source of double-stranded RNA initiating from the ceDNA loop portion.
  • GOI gene of interest
  • bottom undesirable off-target transcription
  • FIG. 9 depicts generation 2 ceDNA yield by varying TelN additives with and without TelN removal (first column TelN, second column T5 for each sample).
  • IVTT In vitro transcription
  • RNA molecules e.g., mRNA, including modified mRNA molecules and/or self-amplifying RNA (saRNA), that are useful for producing clinical grade RNA, such as mRNA, of high purity and potency, consistently, reproducibly, and in compliance with current good manufacturing practices (cGMP).
  • the methods use a closed end DNA (ceDNA) template in an in vitro transcription (IVT) reaction to generate the RNA molecule and can be applied to a wide variety of constructs with varying 5’ UTRS, coding sequence lengths, 3’ UTRs, and 3’ ends.
  • the closed end linear DNA can then be purified by ultrafiltration/diafiltration (UF/DF) after proteinase K treatment and can be used as tempalte for in vitro transcription (IVT) to produce mRNA with similar IVT yield and main quality attributes as mRNA made from plasmid DNA.
  • UF/DF ultrafiltration/diafiltration
  • IVVT in vitro transcription
  • the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the inherent variation or standard deviation of error for the measurement or quantitation method being employed to determine the value.
  • the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the measurement or quantitation.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • essentially all is defined as “at least 95%”; if essentially all members of a group have a certain property, then at least 95% of members of the group have that property. In some instances, essentially all means equal to any one of, at least any one of, or between any two of 95, 96, 97, 98, 99, or 100 % of members of the group have that property.
  • compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
  • compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure.
  • the words “consisting of” (and any form of consisting of, such as “consist of’ and “consists of’) means including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.
  • an “isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and/or is not typically in the functional state and/or that is altered or removed from the natural state through human intervention.
  • a DNA naturally present in a living animal is not “isolated,” but a synthetic DNA, or a DNA partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the nucleic acid has been delivered.
  • the method for producing an RNA molecule includes (a) providing a sample having a linear ceDNA template, the ceDNA template includes an RNA polymerase promoter sequence operably linked to a sequence coding for a gene of interest and a 5' untranslated region (UTR) and/or a 3' UTR.
  • the ceDNA template includes an RNA polymerase promoter sequence operably linked to the respective RNA polymerase gene sequence, which is operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest.
  • the methods for producing an RNA molecule include (a) providing a sample having a linear ceDNA template, the ceDNA template includes an RNA-dependent RNA polymerase (RdRp) promoter sequence, located 5' to and operably linked to a subgenomic promoter, which is operably linked to a sequence coding for a gene of interest and a 5' untranslated region (UTR) and/or a 3' UTR.
  • RdRp RNA-dependent RNA polymerase
  • the methods for producing an RNA molecule disclosed herein include providing a sample having a ceDNA template, the ceDNA template having an RNA polymerase promoter sequence operably linked to a sequence coding for a gene of interest and a poly(A) tail sequence of 20-100 nucleotides.
  • the poly(A) tail can prevent degradation of the RNA molecule in a cell.
  • the plasmid ceDNA template includes a sequence coding for a poly(A) tail located 3' to the gene of interest.
  • poly(A) tail refers to a chain of adenine nucleotides.
  • the poly(A) tail includes 5-300 adenine nucleotides in length, e.g., at least, at most, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 adenine nucleotides in length, or any range or value derivable therein.
  • immediately downstream of the poly(A) tail coding sequence on the plasmid ceDNA template is a recognition site for a restriction endonuclease to linearize the plasmid. Linearization of the plasmid can mitigate transcriptional readthrough.
  • the plasmid ceDNA template is filtered into an appropriate solvent, e.g., a solvent selected from the group consisting of water, HEPES, Tris-CI and EDTA.
  • a solvent selected from the group consisting of water, HEPES, Tris-CI and EDTA.
  • the solvent includes 10 mM HEPES, 0.1 mM EDTA, and the like. Filtration occurs via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration/diafiltration.
  • In vitro transcription refers to a procedure that allows for DNA-directed synthesis of RNA molecules of any sequence, ranging in size from short oligonucleotides to several kilobases.
  • in vitro transcription involves engineering of a ceDNA template to include a bacteriophage promoter sequence (e.g., from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase.
  • the resulting RNA molecules are subsequently modified (e.g., by capping, splicing, the addition of a poly(A) tail, etc.).
  • the in vitro transcription reaction system includes the ceDNA template at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16,
  • the in vitro transcription reaction system includes the ceDNA template at a final concentration of 0.025 mg/mL. In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of 0.05 mg/mL. In some aspects, in vitro transcription reaction system includes the ceDNA template at a final concentration of 0.075 mg/mL. In some aspects, the in vitro transcription reaction system includes the ceDNA template at a final concentration of 0.1 mg/mL.
  • the in vitro transcription reaction system includes each nucleotide triphosphate (NTP) at a final concentration of, e.g., at least, at most, or about 0.4, 0.8, 1.0, 1.25, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein.
  • NTP nucleotide triphosphate
  • the in vitro transcription reaction system includes the nucleotide triphosphate GTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein.
  • the in vitro transcription reaction system includes the UTP or nucleotide triphosphate modified UTP at a final concentration of, e.g., at least, at most, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 mM, or any range or value derivable therein.
  • the concentration of NTPs in the reaction is 0.4mM, 0.8mM, 1mM, 1.25mM, 3.mM, 5mM, 6mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, or 10mM.
  • the IVT reaction system includes magnesium ion, for example, as a magnesium salt, such as any one of magnesium chloride and magnesium acetate.
  • the in vitro transcription reaction system includes the magnesium at a final concentration of, e.g., at least, at most, or about 12, 13, 14, 15, 16, 16.5, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92,
  • the Mg:NTP ratio can be maintained at a ratio of, e.g., at least, at most, or about, 0, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , or 2.2 mM Mg/mM NTP, or any range or value derivable therein.
  • one or more reaction components are added during in vitro transcription by occasional bolus feeds, semi-continuous feeds, or continuous feeds.
  • a continuous feed of at least 1 NTP can be delivered at flow rates of, e.g, at least, at most, or about 0, 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, or 4 mL/L/min.
  • a continuous feed of a cation such as magnesium can be delivered at concentrations of, e.g., at least, at most, or about 0, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 .0 mM/min, or any range or value derivable therein
  • the in vitro transcription (IVT) reaction system includes a buffer.
  • Exemplary buffers for the IVT reaction system may include Tris and/or HEPES.
  • the in vitro transcription reaction system includes the buffer at a pH of, e.g., at least, at most, or about 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1 , 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein.
  • the buffer is Tris-HCI, pH 8.0.
  • the in vitro transcription reaction system includes 40 mM Tris HCI, pH 8.0.
  • a pyrophosphatase is included in the in vitro transcription reaction system.
  • the pyrophosphatase may cleave the inorganic pyrophosphate generated following each nucleotide incorporation into two units of inorganic phosphate, which may reduce the likelihood of magnesium co- precipitating with pyrophosphate to form magnesium pyrophosphate.
  • Pyrophosphatase in certain aspects may be diluted in pyrophosphatase buffer and present in the reaction at concentrations of 0.01mll/uL, 0.02mll/uL, 0.05mll/uL, 0.08mll/uL, 0.1mll/uL, 0.2mll/uL, 0.8mll/uL, or 2mU/uL.
  • the in vitro transcription reaction system includes a polyamine.
  • Exemplary polyamines include spermine, putrescene, and spermidine.
  • 1 mM spermidine is included.
  • 2.0 mM spermidine is included.
  • 2.15 mM spermidine is included.
  • the in vitro transcription reaction system lacks a polyamine.
  • the in vitro transcription reaction system lacks spermidine.
  • the IVT reaction system includes a reducing reagent, such as, for example, DTT (dithiothreitol), e.g., at least, at most, or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 mM, or any range or value derivable therein.
  • the reducing agent is selected from the group consisting of dithiothreitol (DTT), dithioerythritol (DTE), Tris(2- carboxyethyl)phosphine (TCEP) and beta-mercaptoethanol.
  • the IVT reaction system includes 1 mM DTT. In some aspects, the IVT reaction system includes 5 mM DTT. In some aspects, the IVT reaction system includes 10 mM DTT. In some aspects, the IVT reaction system includes 20 mM DTT. In some aspects, the IVT reaction system lacks a reducing agent. In some aspects, the IVT reaction system does not contain DTT. In some aspects, the IVT reaction system does not contain added DTT beyond the protective amount of DTT present in the T7 RNA polymerase storage solution.
  • the in vitro transcription reaction proceeds, for example, at about 37°C for about 4 hours or about 240 minutes, e.g., at least, at most, or about 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, or 240 minutes, or any range or value derivable therein.
  • the in vitro transcription reaction proceeds, for example, at less than 50°C for less than 4 hours, such as for example, at least, at most, or about 50°C, 49°C, 48°C, 47°C, 46°C, 45°C, 44°C, 43°C, 42°C, 41 °C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31 °C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21 °C, or about 20°C, for at least, at most, or about 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,
  • the in vitro transcription reaction proceeds at about 37°C, at greater than 120 minutes and less than 360 minutes, preferably greater than 120 minutes and less than 300 minutes, more preferably greater than 120 minutes and less than 260 minutes. In some preferred aspects, the in vitro transcription reaction proceeds at about 37°C for about 150 minutes.
  • yields per in vitro transcription reaction may be at least 0.3 mg of RNA per mL starting volume of IVT reaction to about 20 mg of RNA per mL starting volume of IVT reaction.
  • the total yield per in vitro transcription reaction of RNA molecule produced having at least 90% of the intended full length transcript may be at least 2 mg RNA/mL, 3 mg RNA/mL, 4 mg RNA/mL, preferably at least 5 mg RNA/mL, 6 mg RNA/mL, 7 mg RNA/mL, 8 mg RNA/mL, 9 mg RNA/mL, 10 mg RNA/mL, 11 mg RNA/mL, 12 mg RNA/mL, 13 mg RNA/mL, 14 mg RNA/mL, 15 mg RNA/mL, 16 mg RNA/mL, 17 mg RNA/mL, 18 mg RNA/mL, 19 mg RNA/mL, or 20 mg RNA/mL starting volume of IVT reaction.
  • the total yield per in vitro transcription reaction of RNA molecule produced having at least 90% of the intended full length transcript is at least 17 mg RNA/mL starting volume of IVT reaction.
  • the methods of producing RNA molecules by contacting the ceDNA sample with the in vitro transcription reaction system described herein produces a first composition having an uncapped RNA molecule. In some aspects, at least 30% of the RNA molecules in the first composition includes uncapped RNA molecules.
  • the first composition includes at least, at most, or about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range or value derivable therein, uncapped RNA molecules.
  • RNA molecule produced by the methods described herein may be a non-coding and/or a coding RNA.
  • a non-coding RNA (ncRNA) molecule includes a functional RNA molecule that is not translated into a peptide or polypeptide.
  • Non-coding RNA molecules may include highly abundant and functionally important RNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as RNAs such as snoRNAs, microRNAs, siRNAs, snRNAs, guideRNAs, circularRNAs, exRNAs, and piRNAs and long ncRNAs.
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • RNAs such as snoRNAs, microRNAs, siRNAs, snRNAs, guideRNAs, circularRNAs, exRNAs, and piRNAs and long ncRNAs.
  • the RNA molecule is an mRNA molecule that includes a modified nucleotide (herein referred to as a “modified RNA molecule” or “modified mRNA molecule”).
  • the RNA molecule is a selfamplifying RNA molecule.
  • Coding RNA includes a functional RNA molecule that may be translated into a peptide or polypeptide.
  • the coding RNA molecule includes at least one open reading frame coding for at least one peptide or polypeptide.
  • the coding RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs).
  • the coding RNA molecule may be a messenger RNA (mRNA) molecule, viral RNA molecule or selfamplifying RNA molecule (saRNA, also referred to as a replicon).
  • mRNA messenger RNA
  • saRNA selfamplifying RNA molecule
  • the RNA molecule is an mRNA.
  • the RNA molecule is a saRNA.
  • Self-amplifying RNA refers to RNA with the ability to replicate itself.
  • Self-amplifying RNA molecules may be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest.
  • a self-amplifying RNA molecule is typically a positive-strand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNAs.
  • RNAs may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen.
  • the overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNAs and so the encoded gene of interest, e.g., a viral antigen, becomes a major polypeptide product of the cells.
  • the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. Preferably epitopes capable of eliciting either a helper T-cell response or a cytotoxic T-cell response or both.
  • the RNA molecule has a 3' poly(A) tail, that is, a stretch of consecutive adenosine residues, that may be attached to the 3' end of the RNA.
  • the poly(A) tail may increase the half-life of the RNA molecule.
  • the RNA molecule may further include a poly(A) polymerase recognition sequence (e.g., AALIAAA) near its 3' end.
  • the 3' poly(A) tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues.
  • the RNA molecule includes at least 20 consecutive adenosine residues and at most 40 consecutive adenosine residues. In some preferred aspects, the RNA molecule includes about 40 consecutive adenosine residues. In some aspects, the RNA molecule includes about 80 consecutive adenosine residues.
  • Poly(A) tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyper-polyadenylation may signal for RNA degradation. Exemplary designs include a poly(A) tails of about 40 As, about 80 As. In some aspects, the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly(A) tail sequence.
  • the RNA molecule produced by the in vitro transcription reaction described herein is purified, e.g., including filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration/diafiltration.
  • the methods of producing RNA molecules described herein further include capping uncapped RNA molecules by contacting the uncapped RNA molecules with a capping reaction system, which includes any one of guanylyltransferase, s-adenosyl-L-methionine (SAM), guanosine triphosphate (GTP), and 2'-O-methyltransferase, and any combination thereof, to produce a capped RNA molecule.
  • a capping reaction system which includes any one of guanylyltransferase, s-adenosyl-L-methionine (SAM), guanosine triphosphate (GTP), and 2'-O-methyltransferase, and any combination thereof, to produce a capped RNA molecule.
  • An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) that includes mRNA triphosphatase, guanylyltransferase and guanine-7- methytransferase, which catalyzes the construction of N7-monomethylated cap 0 structures).
  • VCE Vaccinia Virus Capping Enzyme
  • Cap 0 structure plays an important role in maintaining the stability and translational efficacy of the RNA molecule.
  • the 5' cap of the RNA molecule may be further modified by a 2'-O- Methyltransferase which results in the generation of a cap 1 structure (m7Gppp [m2 '-O] N), which may further increase translation efficacy.
  • the RNA molecule may be enzymatically capped at the 5' end using Vaccinia guanylyltransferase, guanosine triphosphate and S-adenosyl-L-methionine to yield cap 0 structure.
  • An inverted 7-methylguanosine cap is added via a 5' to 5' triphosphate bridge.
  • use of a 2'-O-methyltransferase with Vaccinia guanylyltransferase yields the cap 1 structure where in addition to the cap 0 structure, the 2'-OH group is methylated on the first transcribed nucleotide.
  • SAM S-adenosyl-L-methionine
  • SAM S-adenosyl-L-methionine
  • RNase inhibitor is not included in the enzymatic capping reaction.
  • the enzymatic capping reaction step is performed under constant mixing.
  • the RNA molecule is not co-transcriptionally capped.
  • the capping reaction system includes enzymatic 5' capping that is performed as follows.
  • the final 1X buffer conditions includes the following: at least, at most, or about 50 mM Tris HCI, pH 8, 5 mM KCI, 1 mM MgCI2, 0.5 mM GTP, 0.2 mM S-adenosyl-methionine and 1 mM dithiothreitol. In some aspects, the final 1X buffer does not include dithiothreitol.
  • the capping reaction occurs in the vessel in which the IVT reaction was performed.
  • the IVT reaction is diluted between 3-fold and 10-fold, e.g., at least, at most, or about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, before the capping reaction.
  • the IVT reaction is diluted with Tris pH 7.0 buffer.
  • DNase I can be added to degrade residual ceDNA template from the IVT reaction.
  • DNase I is added at a concentration between at least, at most, or about 1 ll/pg of DNA to 10 ll/pg of DNA, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 ll/pg of DNA, or any range or value derivable therein.
  • CaCh can be added as a co-factor for DNase I at a concentration between at least, at most, or about 0.1 mM to 4 mM, e.g., 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 mM, or any range or value derivable therein.
  • pyrophosphatase is added into the capping reaction. Pyrophosphatase assists with degrading pyrophosphate, which is the inhibitory by-product that is generated by the IVT reaction or by the capping reaction.
  • the capping reaction is conducted under 37 °C for 30 minutes to 2 hours, e.g., at least, at most, or about 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47,
  • the capping reaction is conducted at a temperature greater than 20°C and less than 50°C, e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50°C.
  • the step of capping the uncapped RNA molecules results in at least, at most, or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% capped RNA molecules of the total of RNA molecules (capped and uncapped), or any range or value derivable therein.
  • Purity may be determined as described herein, e.g., via reverse phase HPLC or Fragment analyzer or Bioanalyzer chip-based electrophoresis and measured by, e.g., peak area of full-length RNA molecule relative to total peak.
  • the RNA molecule produced by the methods described herein may be contacted with DNase I and CaCl2 to enzymatically digest ceDNA template following the in vitro transcription reaction.
  • the IVT reaction may include DNase I and CaCl2 additions as well as an additional treatment with EDTA and proteinase K.
  • the EDTA may quench any cationic metal species, including magnesium, and the proteinase K may digest proteins present in the IVT reaction, reducing their size.
  • the methods described herein do not include contacting the RNA molecule produced by the methods described herein with DNase I to enzymatically digest ceDNA template following the in vitro transcription reaction.
  • the linear ceDNA template is removed from the in vitro transcription reaction system, for example, the ceDNA template is separated from the RNA molecule via chromatography.
  • the RNA molecule binds to an affinity substrate while the ceDNA template flow through and is removed.
  • the poly(A) capture-based affinity purification is oligo(dT) purification.
  • a polythymidine ligand may be immobilized to a derivatized chromatography resin. The mechanism of purification may involve hybridization of the poly(A) tail of the RNA molecule to the oligonucleotide ligand, wherein the ceDNA template will not bind.
  • purified material is substantially free of one or more impurities or contaminants including the linear ceDNA template and/or reverse complement transcription products described herein and for instance is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% pure; more preferably, at least 98% pure, and more preferably still at least 99% pure.
  • the method for production of an RNA molecule may include additional purification steps after the in vitro transcription, e.g., an ion exchange chromatography step, a hydrophobic interaction chromatography (HIC) step, a ceramic hydroxyapatite (CHA) chromatography step, and/or an ultrafiltration/diafiltration (LIF/DF) step.
  • an ion exchange chromatography step e.g., a hydrophobic interaction chromatography (HIC) step, a ceramic hydroxyapatite (CHA) chromatography step, and/or an ultrafiltration/diafiltration (LIF/DF) step.
  • HIC hydrophobic interaction chromatography
  • CHA ceramic hydroxyapatite
  • LIF/DF ultrafiltration/diafiltration
  • the method of producing RNA molecules described herein may produce an RNA molecule that is at least 30% full-length transcript, or at least, at most, or about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% full-length transcript, or any range or value derivable therein. Purity may be determined as described herein, e.g., via reverse phase HPLC or Bioanalyzer chip-based electrophoresis and measure by, e.g., peak area of full-length RNA molecule relative to total peak.
  • the DNA template and resulting RNA molecule of the present invention include a gene of interest.
  • the gene of interest encodes a polypeptide of interest selected from, e.g., biologies, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties or those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery.
  • the sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g., GenBank.
  • the RNA molecule includes a coding region for an antigen preferably derived from a pathogen associated with infectious disease which are preferably selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Area no bacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia
  • the RNA molecules of the present disclosure encode a viral polypeptide or fragment thereof, including naturally occurring or engineered variants thereof, for prophylaxis against a virus in humans.
  • the viral polypeptide does not comprise a coronavirus polypeptide.
  • the viral polypeptide does not comprise a severe acute respiratory syndrome (SARS) virus polypeptide.
  • the viral polypeptide does not comprise a SARS-CoV-2 polypeptide.
  • the RNA molecules of the present disclosure do not encode a coronavirus polypeptide or fragment thereof, including naturally occurring or engineered variants thereof. In some aspects, the RNA molecules of the present disclosure do not encode a SARS virus polypeptide or fragment thereof, including naturally occurring or engineered variants thereof. In some aspects, the RNA molecules of the present disclosure do not encode a SARS-CoV-2 virus polypeptide or fragment thereof, including naturally occurring or engineered variants thereof.
  • the RNA molecules of the present disclosure are not used for prophylaxis against a coronavirus in humans. In some aspects, the RNA molecules of the present disclosure are not used for prophylaxis against a SARS virus in humans. In some aspects, the RNA molecules of the present disclosure are not used for prophylaxis against SARS-CoV-2 in humans.
  • insertion of a teIRL recognition sequence (56 bp; Deneke et al., The protelomerase of temperate Escherichia coli phage N15 has cleavingjoining activity, PNAS 97(14):7721-7726 (2000)) into a plasmid can enable the plasmid to perform rolling-circle-amplification (RCA) and lead to a closed-end DNA (ceDNA) construct.
  • the teIRL sequence was inserted upstream of 5’ to the Sbfl site in a self-amplified RNA (saRNA) construct or upstream of the T7 promoter in a Xcml site (20 bp) of a modRNA construct.
  • saRNA self-amplified RNA
  • Xcml site 20 bp
  • teIRL sequences were also inserted at 402bp downstream of the poly(A) region of a modRNA plasmid.
  • the VSV-G sequence was subcloned into the Pmel insertion site.
  • the reaction from Example 3 was treated with proteinase K to reach a value of 1.2 U per mL of reaction and then concentrated ammonium sulfate solution was added to a final concentration of 250mM ammonium sulfate.
  • the solution was loaded into a tangential filtration system with 300KDa Hydrosart membrane equilibrated with DF1 buffer as 250 mM Ammonium Sulfate, 10 mM Tris, 1mM EDTA, pH 7.0.
  • the solution was concentrated ⁇ 4X to achieve DNA concentration ⁇ 1.2 - 1.5 g/L.
  • concentrated solution was diafiltrated for 10 diafiltration volumes (DV) of DF1 buffer followed by 10 DV of DF2 buffer (10DV of 10mM Tris, pH7).
  • DV diafiltration volumes
  • a pDNA construct containing an Influenza antigen (p130) was pretreated with Exonuclease III as described in Example 6.
  • the pDNA lot used had known truncations in its polyA region.
  • the pretreated pDNA was amplified as described in Example 1 using either Tth primase or specific primers.
  • the resulting RCA DNA was converted to closed end DNA as described in Example 3 and purified as described in Example 4.
  • Results along with starting pDNA quality are displayed in Table 9. This data displays the ability of specific primers to mitigate poor polyA integrity from starting pDNA template compared with Tth primase which exacerbates the issue.
  • a pDNA construct containing self-amplifying RNA sequences and a VSV-G antigen (R048) was amplified as described in Example 1 using random primers. Temperature and reaction duration were held consistent at 30C and 18 hours, respectively. Dilution (ratio of TelN reaction volume to RCA reaction volume) and TelN protelomerase dose (units per ug of DNA input) were varied from 2.5X-8X and 1-2 hours, respectively.
  • the ceDNA was purified as described in Example 4. Results can be found in Table 11 demonstrating achieving an effective ceDNA yield with the variety of conditions.
  • reaction buffers for TelN digestion use Triton X-100 as a surfactant.
  • Reaction buffers for TelN digest were formulated with and without Triton X-100 for comparison.
  • a pDNA construct containing self-amplifying RNA sequences and VSV-G antigen (R048) was amplified as described in Example 1 using random primers, converted to close-end DNA as described in Example 3, and purified as described in Example 4. Results can be found in Table 13, demonstrating effective ceDNA yield using buffers with and without Triton X-100.
  • Table 14 dsRNA levels in mRNA produced from ceDNA constructs and p lasmid controls EXAMPLE 11 TELN IMMOBILIZATION
  • construct p292 was selected with the 3’ teIRL design for 2nd generation ceDNA production, considering this new configuration rendered low dsRNA impurity during IVT procedure (Table 16).
  • Table 16 mRNA quality attributes from 2 nd generation ceDNA as IVT template
  • Table 17 shows ceDNA produced with constructs p292, containing 3’ teIRL and poly 30L70 and p095 containing 3’ teIRL and a poly 80A where TelN enzyme is immobilized using NHS (N- hydroxy succinimide) agarose.
  • NHS N- hydroxy succinimide
  • Table 17 Immobilized TelN in 1 st generation and 2 nd generation ceDNA and mRNA quality
  • the experiment for p292 was a comparison between using 10 mg TelN protein vs 5 mg TelN protein with NHS immobilization of the TelN during incubation with the RCA concatemer during production of first generation ceDNA.
  • the experiment for p095 was a comparison between using specific primers vs primase in the first-generation RCA step before the (immobilized) TelN step.
  • the 1 :2.5 diluted RCA concatemer was incubated at 37°C for 6 hours with TelN immobilized using NHS beads on a rotating platform. The DNA material was then eluted from the column, leaving the TelN on the NHS beads. T5 exonuclease was added to the material post TelN and incubated at 37°C for 4 hours to remove open ended DNA. The resulting ceDNA was extracted from a phenol chloroform solution to remove protein and precipitated with NH4OAC and pure ethanol then resuspended in EB buffer. The 1 st generation ceDNA yield was > 0.5 mg ceDNA /ml RCA.
  • the first generation ceDNA was denatured and used as a template for 2nd generation RCA, TelN and T5 exonuclease. During the production of 2nd generation ceDNA, TelN was not removed prior to the T5 exonuclease step. After T5 exonuclease, purified ceDNA was extracted with phenol chloroform and precipitated with NH4OAc and pure ethanol and resuspended in EB buffer. The resulting 2nd generation ceDNA was then linearized and transcribed to produce mRNA.
  • a polymerase chain reaction using ⁇ 0.5 pM of specifically-designed forward & reverse primers, 0.02 ng/pl DNA template, 1X Q5 High-Fidelity 2X Master Mix (NEW ENGLAND BIOLABS), and water (qs) was performed (2min denature at 98°C, 20 cycles of [10sec at 98°C, 30sec at 69°C, 4min 30sec at 72°C], 5min at 72°C, then hold at 4°C) to amplify the gene fragment and remove the flanking regions.
  • the product of the first PCR was used in a second round of PCR using explicitly-designed primers to add a poly A tail, a BspQI restriction site for IVT linearization, and Bsal restriction sites, along with four GC-rich nucleotide ligation site sequence on both the 5’ and 3’ ends.
  • This round of PCR required longer oligonucleotides, which have only partial binding to the amplicon, while the nonbinding sequences promote addition of the complement to the 5’ and 3’ ends of the amplicon.
  • the forward primer included sequences for the addition of a 5’ Bsal restriction site (GGTCTCN) followed by a four nucleotide GC-rich sequence compatible with T7 ligase to promote hybridization of GC-rich sticky ends.
  • the Ultramer reverse primer (>1OOnt; INTEGRATED DNA TECHNOLOGIES) partially binds to the 3’IITR and included non-binding complementary sequences for the addition of a poly A tail, BspQI restriction site (GCTCTTC), a four nucleotide GC-rich sequence, followed by a sequence complementary to a 3’ Bsal restriction site.
  • Fragment design was such that digestion with Bsal restriction endonuclease cleaved the double strand on both ends, leaving GC-rich complementary overhangs (sticky ends) on the 5’ ends of the sense and anti-sense strands.
  • Addition of T7 ligase hybridizes the complementary GC-rich sticky ends together resulting in a self-ligated minicircle containing a TeIRL sequence, 5’ UTR, GOI, 3’ UTR, poly A tail and a BspQI restriction site.
  • An exemplary minicircle using this single self-ligating fragment design was used in an IVT reaction and compared to IVT reactions using plasmid templates, as shown in Table 18.
  • Table 18 Data for comparison of ceDNA mRNA prepared from minicircle template in RCA versus other DNA template for IVT :
  • This method can also be performed with a multiple fragment design for ligation.
  • two or more synthetic fragments are designed so that the double stranded fragments hybridize GC-rich overhangs at the 5’ ends of each strand.
  • all fragments must contain on both 5’ ends of the sense and anti-sense strands: a) GC-rich four nucleotide sequences specifically complementary to the opposite strand of another fragment and b) Bsal restriction sites outside of the GC-rich nucleotide sequences (with 1 spacer nucleotide).
  • Fragments may then contain: a) >25 bp flanking sequences on both ends (outside all other sequences), b) TeIRL sequence, c) T7 promoter sequence, d) 5’UTR, d) GOI, f) the 3’UTR, and e) any other sequences needed.
  • One of the fragments will be a duplex Ultramer (INTEGRATED DNA TECHNOLOGIES) (complementary strands of olioonucleotides annealed together) that contains a poly A tail, GC-rich four nucleotide sequences and Bsal restriction site sequences on both ends. The fragments may be amplified via PGR.
  • Bsal digestion can include 0.1 pg/pl DNA template, 1X Cutsmart Buffer (NEW ENGLAND BIOLABS), 5 U/pg Bsal- HF v2 (NEW ENGLAND BIOLABS) and water (qs) and can be run at 37°C for > 1 hour, static.
  • the GC-rich nucleotide sequences are designed to promote hybridization of the 5’ overhang of the sense strand of one fragment to only the 5’ overhang of the anti-sense strand of a second fragment that is complementary. Each 5’ overhang sense strand must be explicitly complementary to only one 5’ overhang anti-sense strand.
  • the 5’ sticky end of the sense strand hybridizes with the complementary 5’ sticky end of the anti-sense strand, and then T7 DNA ligase forms phosphodiester bonds linking the hybridized nucleotides in the strands together to form a single fragment self-ligated minicircle or circular DNA from multiple ligated fragments.
  • This minicircle can contain: a) the TeIRL site, b) T7 promoter, c) 5’ UTR, d) GOI, e) 3’ UTR, f) restriction site for IVT linearization, g) the poly A tail, h) ligation site(s) - 5’ end sense strand GC-rich four nucleotide sticky ends joined with their complementary sequences from the 5’end anti-sense strand, and j) any other sequences needed. Ligation can occur at 4°C-37°C depending on fragment size(s) (bp) for > 2 hours, static.
  • Ligation components can include variable amounts of DNA (-2-12 ng/pl or ⁇ 1 pM), fragments in a 1 :1 molar ratio, 0.2 U/pl Bsal-HF v2 (NEW ENGLAND BIOLABS), 25 U/pl T7 DNA ligase, 0.2X-1X StickTogether DNA ligase buffer (NEW ENGLAND BIOLABS), variable amounts (2-15%) of 1 ,2- propanediol, and water (qs).
  • T5 exonuclease is added after ligation to remove non-circular (linear or nicked) double stranded DNA leaving only circular DNA components.
  • T5 exonuclease can be added at a final concentration of 0.1 U/pl. Digest at 37°C for 1-2 hours.
  • Purification of DNA can occur after PCR steps, after digestion, and/or after T5 exonuclease treatment to remove non-nucleic acid components (such as protein) (FIG. 10).
  • FIG. 11 An exemplary schematic for the minicircle process using a single self-ligating fragment design is depicted in FIG. 11.

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Abstract

L'invention concerne des procédés de production de molécules d'ARN dans une réaction de transcription in vitro. L'invention concerne également des procédés de production d'une molécule d'ARNm à partir d'un modèle d'ADN à extrémité fermée à l'aide d'un système de réaction in vitro.
PCT/IB2025/053195 2024-03-29 2025-03-26 Procédés de production d'acides nucléiques Pending WO2025202929A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
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WO2010086626A1 (fr) * 2009-01-30 2010-08-05 Touchlight Genetics Limited Production d'adn linéaire fermé
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