[go: up one dir, main page]

WO2025096912A1 - Procédés d'évaluation de contamination et d'intégrité d'arn - Google Patents

Procédés d'évaluation de contamination et d'intégrité d'arn Download PDF

Info

Publication number
WO2025096912A1
WO2025096912A1 PCT/US2024/054080 US2024054080W WO2025096912A1 WO 2025096912 A1 WO2025096912 A1 WO 2025096912A1 US 2024054080 W US2024054080 W US 2024054080W WO 2025096912 A1 WO2025096912 A1 WO 2025096912A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
molecular beacon
rna sequence
nucleic acid
binds
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/US2024/054080
Other languages
English (en)
Inventor
Craig T. MARTIN
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.)
University of Massachusetts Amherst
Original Assignee
University of Massachusetts Amherst
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 University of Massachusetts Amherst filed Critical University of Massachusetts Amherst
Publication of WO2025096912A1 publication Critical patent/WO2025096912A1/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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • RNA-based therapeutics There is a growing understanding of the role of ribonucleic acid (RNA) in various diseases, which has led to the promotion of RNA-based therapeutics.
  • RNA-based therapeutics there are significant challenges to developing RNA-based therapeutics including, but not limited to, distinguishing between purified RNA sequences and defective RNA sequences.
  • RNA manufacturing and/or isolation Some common problems in RNA manufacturing and/or isolation include the presence of undesirable RNA structures including, but not limited to 1) double stranded RNA (dsRNA) at RNA 3’ end, 2) truncated RNA (from RNA polymerase stopping, or terminating, early), 3) exonucleolytic degradation from the 5’ and 3’ ends of the RNA (shortening the RNA from its ends) and 4) endonucleolytic cleavage in the middle of the RNA.
  • dsRNA double stranded RNA
  • truncated RNA from RNA polymerase stopping, or terminating, early
  • exonucleolytic degradation from the 5’ and 3’ ends of the RNA shortening the RNA from its ends
  • endonucleolytic cleavage in the middle of the RNA Some common problems in RNA manufacturing and/or isolation include the presence of undesirable RNA structures including, but not limited to 1) double stranded RNA (dsRNA) at RNA
  • RNA-based therapeutics Given limitations of developing, manufacturing, and/or preparing RNA-based therapeutics, there is a need to address the aforementioned problems mentioned above by developing new methods to assess the quality and/or quantity of RNA samples of any length.
  • the compounds, compositions, and methods disclosed herein address these and other needs.
  • the present disclosure provides methods of detecting defective, unwanted, undesirable, and/or unexceptional ribonucleic acids (RNAs).
  • RNAs defective, unwanted, undesirable, and/or unexceptional ribonucleic acids
  • the present disclosure also provides methods of measuring quantity and/or quality of an RNA.
  • the present disclosure also provides methods validating a personalized therapeutic composition comprising an RNA.
  • a method of detecting a defective RNA in a sample comprising isolating or manufacturing an RNA sequence, contacting the RNA sequence with at least two molecular beacons, wherein the at least two molecular beacons comprise a first and a second nucleic acid, wherein the first nucleic acid is covalently linked to a first fluorescent molecule and a first quencher, and wherein the second nucleic acid is covalently linked to a second fluorescent molecule and a second quencher, and detecting decreased fluorescence emitted by a second molecular beacon relative to the fluorescence emitted by a first molecular beacon when the sample comprises the defective RNA.
  • RNA sequence in a sample, the method comprising isolating or manufacturing the RNA sequence, binding a first molecular beacon to an end of the RNA sequence and binding a second molecular beacon to an opposite end of the RNA sequence, wherein a full-length RNA sequence binds the first and second molecular beacon and a defective RNA sequence binds the first molecular beacon, detecting an RNA quantity from the first molecular beacon binding the RNA sequence, and detecting an RNA quality from a ratio of the second molecular beacon binding the RNA sequence relative to the first molecular beacon binding the RNA sequence.
  • a method of validating a personalized therapeutic composition comprising isolating or manufacturing an RNA sequence, binding a first, a second, and a third molecular beacon to the RNA sequence, wherein the first molecular beacon binds at an end of the RNA sequence, the second molecular beacon binds at an opposite end of the RNA sequence, and the third molecular beacon binds between the ends of the RNA sequence, detecting a full-length RNA sequence that binds the first, second and third molecular beacons, and incorporating the full-length RNA sequence into the personalized therapeutic composition.
  • the first fluorescent molecule and the first quencher are linked at opposite ends of the first nucleic acid. In some embodiments, the second fluorescent molecule and the second quencher are linked at opposite ends of the second nucleic acid. In some embodiments, the first and second fluorescent molecules are the same. In some embodiments, the first and second fluorescent molecules are different. In some embodiments, the first and second quenchers are the same. In some embodiments, the first and second quenchers are different.
  • the method comprises two, three, four, or more molecular beacons.
  • the defective RNA comprises a double stranded RNA (dsRNA), a partially double stranded RNA (dsRNA), a truncated RNA, or a degraded RNA.
  • the first and second nucleic acid comprise a hairpin nucleic acid or a linear nucleic acid. In some embodiments, the hairpin nucleic acid transforms into the linear nucleic acid in the presence of a full-length RNA.
  • the first molecular beacon binds within a first or last 250 base pairs (bps) of the RNA sequence. In some embodiments, the second molecular beacon binds within a first or last 250 bps of the RNA sequence. In some embodiments, the second molecular beacon binds after a polyadenylate (poly A) tail of the RNA sequence.
  • poly A polyadenylate
  • the first molecular beacon emits a fluorescent signal when bound to the RNA sequence. In some embodiments, the second molecular beacon emits a fluorescent signal when bound to the RNA sequence.
  • the first molecular beacon comprises a first nucleic acid, a first fluorescent molecule, and a first quencher.
  • the second molecular beacon comprises a second nucleic acid, a second fluorescent molecule, and a second quencher.
  • the ratio comprises the fluorescent signal from the second molecular beacon relative to the fluorescent signal from the first molecular beacon.
  • the method detects the defective RNA.
  • the first molecular beacon measures a quantity of the RNA sequence.
  • the first and second molecular beacons measure a quality of the RNA sequence.
  • the third molecular beacon identifies a subject- specific RNA sequence.
  • the third molecular beacon comprises a third nucleic acid, a third fluorescent molecule, and a third quencher.
  • the third nucleic acid is complementary to the subject-specific RNA sequence.
  • the third molecular beacon comprises a hairpin nucleic acid or a linear nucleic acid.
  • the first, second, and third fluorescent molecules are the same. In some embodiments, the first, second, and third fluorescent molecules are different. In some embodiments, the first, second, and third quenchers are the same. In some embodiments, the first, second, and third quenchers are different.
  • the third molecular beacon emits a fluorescent signal when bound between the ends of the RNA sequence.
  • the personalized therapeutic composition comprises the full- length RNA sequence and a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, or a cream.
  • a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, or a cream.
  • the personalized therapeutic composition is administered to the subject.
  • the personalized therapeutic composition is administered with an additional therapeutic composition.
  • Figures 1A-1B show the structures of the molecular beacon alone ( Figure 1 A) and in the presence of a target nucleic acid sequence (Figure IB).
  • Figure 1A shows the unbound molecular beacon, fluorescent molecule F is localized sufficiently near a quencher molecule Q that the fluorophore shows no (or diminished) fluorescence intensity.
  • Figure IB shows the binding on the target RNA, the duplex holding F and Q in proximity is disrupted, such that the increase distance is sufficient to eliminate (or reduce) the quenching of F by Q, leading to an increase fluorescence intensity from F.
  • Figure 2 shows the differential beacon responses.
  • the illustration shows 3 classes of RNA.
  • Figure 2 (#1) shows the desired RNA.
  • Figure 2 (#2) shows the RNA containing a self- encoded extension at the 3’ end, resulting in regions of dsRNA.
  • Figure 2 (#3) shows the truncated RNAs. All three classes bind to a beacon targeting a sequence towards the 5’ end of the RNA. Class 1 binds a beacon targeting a sequence near the 3’ end of the RNA.
  • Figure 4 shows the swapping of beacon sequences with respect to position.
  • Two different classes of RNAs were prepared: the first is presented in Figures 3A-3D.
  • the second class exchanges the sequence originally at position 961 of the RNA with the sequence originally at position 961. In other words, the sequence originally at position 11 is moved to position 961 and the sequence originally at position 961 is moved to position 11.
  • Figure 5 shows an example application of real-time monitoring in a flow reactor.
  • Illustration of a representative flow reactor comprised of three identical, parallel reactor chambers combined into one outflow. The outflow is then sampled, with that small stream split into two identical streams.
  • To one stream beacon 1 is added and fluorescence intensity is measured.
  • To the second stream beacon 2 is added and fluorescence intensity is measured.
  • the concentration and quality of the product RNA inferred by fluorescence informs reactor parameters (e.g., flow rate) to provide real-time feedback control.
  • the metrics can feed back to valves that either direct the product to further processing, or in the case of metrics not meeting expectation, direct the product to waste or alternative collection.
  • Figures 6A-6C show the independent quality metrics of RNA produced under varying added concentrations of NaCl. Transcription using a gapped promoter allows the use of increasing concentrations of added NaCl to decrease 3’ extensions during RNA synthesis. Equal amounts of total RNA generated under different conditions (and treated to remove salt and other small molecules) were transformed into HEK293T cells and cells were grown for 48 h. A) Since the mRNA encodes nanoluciferase, luminescence from the cells reports on the translatability of the transfected RNA (dsRNA down-regulates translation in the reporter cells). B-C) RT-qPCR analysis of different genes well-known to be up-regulated by contaminant dsRNA. B: interferon IFN-01. C: cellular pattern recognition receptor MDA5 (Melanoma Differentiation-Associated protein 5). I:C refers to commercial polyLC, which is a commercially available molecule widely used as a standard to mimic dsRNA impurities.
  • Figures 7A-7C show the electrophoretic characterizations of RNA pools.
  • Figures 7A- 7C show that the RNA was transcribed under the indicated concentrations of added NaCl was analyzed by polyacrylamide gel electrophoresis (PAGE) and by BioAnalyzer 2100 (Agilent).
  • Figure 7C shows the original data
  • Figure 7B shows a gel-like representation of the data in Figure 7B (as provided by the instrument).
  • the electropherograms (incorrectly) show the presence of truncated RNA, with more at 300 mM added NaCl and less at 400 mM added NaCl.
  • the results presented in Figures 8A-8B show that, instead, the signal at apparent shorter lengths is likely due to residual structure throughout the RNA, perhaps arising from differences in desalting. This is consistent with a similar agreement in the responses presented in Figures 6A-6C.
  • Figures 8A-8B show the expansion of data in Figures 3A-3D. Comparison of results from Figure 8 A show beacon position 11 and Figure 8B show beacon position 961 from Figure 3D. Note that the ratios for RNA synthesized at 300 mM added NaCl are very close to those for RNA synthesized at 400 mM added NaCl.
  • mRNA refers to messenger ribonucleic acid, or single stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is translated by a ribosome in the process of synthesizing a protein. mRNA is created during the process of transcription, where a gene is converted into a primary transcript mRNA (or pre-mRNA). The primary transcript is further processed through RNA splicing to only contain regions that will encode protein.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • blastn a tool that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website.
  • the “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).
  • a “variant,” “mutant,” or “fragment” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250).
  • a variant polynucleotide may show, for example, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides over a certain defined length relative to a reference polynucleotide.
  • truncated RNA refers to a type of damaged RNA molecule wherein 5’ and/or 3’ end of the RNA has become degraded over time or the synthesis of the RNA molecule is prematurely terminated leading to the loss of function(s).
  • cleaved RNA refers to another type of damaged RNA molecule wherein an endonuclease or exonuclease enzymes targets and degrades the RNA molecule, leading to the loss of function(s). It should be noted that the endonuclease enzyme begins degrading between the 5’ and 3’ ends of the RNA, while the exonuclease enzyme begins degrading at either the 5’end or the 3’end of the RNA.
  • downstream refers to a direction of transcription, the direction of transcription being from a promoter sequence to an RNA-encoding sequence.
  • the direction of transcription is 3’ to 5’.
  • the direction of transcription is 5’ to 3’.
  • Upstream means in a direction opposite the direction of transcription.
  • Upstream and downstream may be used in reference to either strand of a double-stranded DNA molecule even when relative to a sequence on one strand of a double-stranded DNA molecule.
  • interaction refers to an action that occurs as two or more objects have an effect on one another either with or without physical contact.
  • cell, proteins, and other macromolecules can have said effects on one another to impact biological functions, such as cell/tumor growth, cell death, and cell signaling pathways.
  • the molecular beacons provided herein contact and interact with RNA sequences to distinguish full-length RNAs from defective RNAs and/or RNA contamination.
  • administer refers to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra- arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir.
  • parenteral includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
  • detect refers to observing a presence or absence of an output signal released for the purpose of sensing of physical phenomenon. An event or change in environment is sensed and signal output released in the form of light, and the presence of the signal output is observed. Alternatively, an event or change in environment is not sensed and no signal output is released, and the presence of the signal output is not observed.
  • a “fluorophore” is a fluorescent molecule that can re-emit light upon light excitation.
  • the chemicals are sometimes used alone as a tracer in fluids, as a dye for staining certain structures, as an enzyme substrate, or as a probe/indicator. More commonly they are covalently bonded to a macromolecule to serve as a marker for bioactive reagents (i.e.: antibodies, peptides, nucleic acids, etc.)
  • Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods such as fluorescent imaging and spectroscopy. It should be understood that throughout this disclosure fluorophore and fluorescent molecule are used interchangeably .
  • a ’’quencher refers to a molecule or compound capable of either exhibiting a dipoledipole interaction or exhibiting electron transfer processes with the fluorophore, and re- emitting the energy in another form of thermal energy such as, for example heat energy or light energy.
  • Quantity refers to a measurable finite or total number of a composition, nucleic acid, or compound.
  • quality refers to a measurable characterization, such as for example purity, structural integrity, and/or function(s), of a composition, nucleic acid, or compound.
  • “Pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
  • the term When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
  • “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations.
  • a carrier for use in a composition will depend upon the intended route of administration for the composition.
  • the preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005.
  • physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICSTM (BASF; Florham Park, NJ).
  • buffers such as phosphate buffer
  • compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.
  • Molecular beacons are oligonucleotide dual-labeled probes that quench fluorescence for a stem-loop/hairpin structure in the native state and fluoresce upon hybridization with a target nucleic acid sequence.
  • a typical molecular beacon probe is about 25 nucleotides long with the middle 15 nucleotides being complementary to the target DNA or RNA, and typically do not base pair with one another. The remaining approximately 10 nucleotides at the 5’ and 3’ ends are complementary to each other and form the hairpin structure.
  • Typical molecular beacons can also be divided into 4 segments: (1) a loop, having about 15 bases pairs, complementary to the target DNA or RNA; (2) a stem, formed by the binding of the 5’ end to the 3’ end of the probe (roughly about 5 to 7 base pairs); (3) a 5’ fluorophore covalently attached to the probe; and (4) a 3’ quencher covalently attached to the probe.
  • it may have a (5) a 3’ fluorophore covalently attached to the probe; and (6) a 5’ quencher covalently attached to the probe.
  • the molecular beacons disclosed herein can be shorter relative to typical molecular beacons used in the art.
  • the present disclosure provides molecular beacons that are about 15-20 nucleotides in length. Similar to typical molecular beacons, the present disclosure also provides molecular beacons with a loop (about 10 - 15 nucleotide residues), a stem (about 5 nucleotide residues), a 5’ fluorophore, and a 3’ quencher.
  • the molecular beacons disclosed herein can comprise a fluorescent molecule/fluorophore known in the art and can comprise a quencher known in the art.
  • Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy, in the form of light, at a second wavelength.
  • the fluorophore is derived from the fluorescein/rhodamine family of fluorophores including, but not limited to Fluorescein (6- FAM); 6-FAM (NHS Ester); Fluorescein deoxy thymine(dT); SUN; Hexachlorofluorescein (HEX); 6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein (JOE); MAX (NHS Ester); tetrachlorofluorescein (TET); 5 -Carboxy fluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); TAMRA (NHS Ester); Texas Red 615; 5-ROX (carboxy-X-r
  • quenchers are molecules capable of absorbing the electromagnetic energy of the electronic excited states of fluorophores.
  • quenchers to be attached to the 3’ end of the molecular beacon includes black hole quenchers (BHQ) (such as, for example BHQ-1, BHQ-2, BHQ-3, 3’-BHQ-2 CPG, BHQ phosphoramidites, BHQ-1- dT, BHQ-2-dT, 3 ’-BHQ-1 CPG, 3 ’-BHQ-3 CPG, and derivatives thereof), Iowa Black quenchers (such as, for example Iowa Black FQ and Iowa Black RQ), blackberry quenchers (such as, for example BBQ-650-CE Phosphoramidite, BBQ-650-dT-CE Phosphoramidite, BBQ-650 CPG, 3’-BBQ-650 CPG, 3’-BBG-650 CPG II, 3’-BBQ-650 CPG III, and derivative thereof), eclipse quenchers (such as, for example eclipse quencher phosphoramidite, MGB
  • BHQ black
  • any fluorophore and any quencher disclosed herein can be paired together to form a molecular beacon needed to perform the desired effect.
  • the present disclosure provides methods of detecting a defective, unwanted, undesirable, and/or unexceptional ribonucleic acids (RNAs).
  • RNAs ribonucleic acids
  • the present disclosure also provides methods of measuring quantity and/or quality of an RNA, even in the presence of large excess of free nucleosides or nucleotides. It should be noted that the present disclosure differs from conventional methods that rely on measuring absorption (including, but not limited to NanoDrop), but cannot distinguish RNA from precursor molecules, such as DNA or substrate ribonucleoside triphosphates (NTP’s).
  • NTP substrate ribonucleoside triphosphates
  • the present disclosure also provides methods validating a personalized therapeutic composition comprising an RNA.
  • a method of detecting or assessing a defective RNA in a sample comprising isolating or manufacturing an RNA sequence, contacting the RNA sequence with at least two molecular beacons, wherein the at least two molecular beacons comprise a first and a second nucleic acid, wherein the first nucleic acid is covalently linked to a first fluorescent molecule and a first quencher, and wherein the second nucleic acid is covalently linked to a second fluorescent molecule and a second quencher, and detecting decreased fluorescence emitted by a second molecular beacon relative to the fluorescence emitted by a first molecular beacon when the sample comprises the defective RNA.
  • a method of measuring quantity and quality of an RNA sequence in a sample comprising isolating or manufacturing the RNA sequence, binding a first molecular beacon to an end of the RNA sequence and binding a second molecular beacon to an opposite end of the RNA sequence, wherein a full-length RNA sequence binds the first and second molecular beacon and a defective RNA sequence binds the first molecular, detecting an RNA quantity from the first molecular beacon binding the RNA sequence, and detecting an RNA quality from a ratio of the second molecular beacon binding the RNA sequence relative to the first molecular beacon binding the RNA sequence.
  • a sample having a high ratio has higher quality RNA than a sample having a low ratio.
  • a method of validating a personalized therapeutic composition comprising isolating or manufacturing an RNA sequence, binding a first, a second, and a third molecular beacon to the RNA sequence, wherein the first molecular beacon binds at an end of the RNA sequence, the second molecular beacon binds at an opposite end of the RNA sequence, and the third molecular beacon binds between the ends of the RNA sequence, detecting a full-length RNA sequence that binds the first, second and third molecular beacons, and incorporating the full-length RNA sequence into the personalized therapeutic composition.
  • first molecular beacon and the second molecular beacon of any preceding aspect bind at opposite ends of the RNA sequence.
  • first molecular beacon binds at or near the 5’ end of the RNA sequence
  • second molecular beacon binds at or near the 3 ’ end of the RNA sequence.
  • reverse of the preceding example is also true.
  • one or more molecular beacons is a quencher- free (QF) molecular beacon comprising a nucleic acid sequence covalently linked to a fluorescent molecule (fluorophore), wherein the nucleic acid sequence comprises a region of repeated nucleotides near the 5’ end or the 3’ end.
  • the region of repeated nucleotides serve as an alternative quencher.
  • the region of repeated nucleotides includes, but is not limited to guanosines repeats (G) n , and CAG repeats (CAG) n .
  • RNA sequence can be isolated from a tissue or cell source, or can be manufactured by manually assembling adenine, guanine, cytosine, and uracil nucleotides into an RNA sequence.
  • the first fluorescent molecule is linked to the 5’ end of the first nucleic acid and the first quencher is linked to the 3’ end of the first nucleic acid.
  • the second fluorescent molecule is linked to the 5’ end of the second nucleic acid and the second quencher is linked to the 3 ’ end of the second nucleic acid.
  • the first fluorescent molecule is linked to the 3’ end of the first nucleic acid and the first quencher is linked to the 5’ end of the first nucleic acid.
  • the second fluorescent molecule is linked to the 3’ end of the second nucleic acid and the second quencher is linked to the 5 ’ end of the second nucleic acid.
  • the first and second fluorescent molecules are the same. In some embodiments, the first and second fluorescent molecules are different. In some embodiments, the first and second quenchers are the same. In some embodiments, the first and second quenchers are different.
  • the method comprises two, three, four, or more molecular beacons.
  • the defective RNA comprises a double stranded RNA (dsRNA), a truncated RNA, or a cleaved RNA.
  • the defective RNA comprises partially dsRNA, wherein a region of the RNA is single stranded, however a smaller region, usually at the ends, is double stranded (See Figure 2, item 2).
  • the first and second nucleic acid comprise a hairpin nucleic acid or a linear nucleic acid.
  • the hairpin nucleic acid transforms into the linear nucleic acid in the presence of a full-length RNA.
  • a first molecular beacon targets and binds at or near a 5’ region of the RNA sequence. In some embodiments, the first molecular beacon binds with the first 250bps of the RNA sequence. In some embodiments, the first molecular beacon binds with the first 100bps of the RNA sequence. In some embodiments, the first molecular beacon binds within the first 50 bps of the RNA sequence. In some embodiments, the first molecular beacon binds with the first 25 base pairs (bps) of the RNA sequence. In some embodiments, the first molecular beacon binds with the first 10 base pairs (bps) of the RNA sequence. In some embodiments, the first molecular beacon binds with the first 5 base pairs (bps) of the RNA sequence. In some embodiments, the first molecular beacon binds within the first 5, 6, 7, 8, 9,
  • a second molecular beacon targets and binds at or near a 3’ region of the RNA sequence. In some embodiments, the second molecular beacon binds within the last 250 bps of the RNA sequence. In some embodiments, the second molecular beacon binds within the last 100 bps of the RNA sequence. In some embodiments, the second molecular beacon binds within the last 50 bps of the RNA sequence.
  • the second molecular beacon binds within the last 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,
  • RNA sequence 242, 243, 244, 245, 246, 247, 248, 249, 250, or more bps of the RNA sequence.
  • the present disclosure also allows for one of skill in the art to detect very long dsRNA elements, and can place the molecular beacon 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,
  • someone practiced in the art may prefer to detect only very long dsRNA elements and so may choose to place the beacon 100, 150, 200 or more bases from the 3’ end.
  • the 3’ end of RNA is the last region to be synthesized.
  • the poly(A) tail is encoded in the precursor DNA sequence.
  • a poly adenylated tail (poly (A) tail) of adenosine monophosphates is added to the 3’ end of the RNA.
  • the addition of the poly(A) tail occurs by polyadenylation.
  • the second molecular beacon binds after a polyadenylate (poly A) tail of the RNA sequence.
  • one of skill in the art can include 5 or more heterogeneous nucleotides following the poly(A) tail, thus providing the molecular beacon a binding site to bind very close to the 3’ end.
  • characterization would typically precede the enzymatic addition of a poly(A) tail.
  • the second molecular beacon does not bind the RNA sequence in a defective RNA.
  • the first molecular beacon emits a fluorescent signal when bound to the RNA sequence.
  • the second molecular beacon emits a fluorescent signal when bound to the RNA sequence.
  • the second molecular beacon does not emit the fluorescent signal in the presence of the defective RNA.
  • Spectroscopy relates to the production, measurement, and interpretation of spectra arising from the interaction of electromagnetic energy with matter.
  • spectroscopic methods including, but not limited to fluorescence spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, atomic spectroscopy, infrared (IR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy, that ultimately allow for the detection, monitoring, quantification, and analyses of biological molecules, such as DNA, RNA, and/or proteins.
  • UV-Vis ultraviolet-visible
  • IR infrared
  • NMR nuclear magnetic resonance
  • the present disclosure provides methods of using spectroscopy, such as, for example fluorescence spectroscopy, in combination with the molecular beacons to distinguish between full-length RNA and defective/contaminating RNA.
  • the method detects the fluorescent signal using a spectroscopy technique, or derivatives thereof. In some embodiments, the method measures quantity and quality of the RNA sequence. In some embodiments, the ratio comprises the fluorescent signal from the second molecular beacon relative to the fluorescent signal from the first molecular beacon.
  • a research grade spectrometer is replaced by a limited wavelength light source, such as a light emitting diode (LED) with appropriate filter, for excitation of the fluorophore, and a simple sensor, combined with an appropriate filter, to detect fluorescent light.
  • a limited wavelength light source such as a light emitting diode (LED) with appropriate filter, for excitation of the fluorophore
  • a simple sensor combined with an appropriate filter, to detect fluorescent light.
  • the defective RNA sequence does not bind the second molecular beacon.
  • the first molecular beacon comprises a first nucleic acid, a first fluorescent molecule, and a first quencher.
  • the second molecular beacon comprises a second nucleic acid, a second fluorescent molecule, and a second quencher.
  • the third molecular beacon comprises a third nucleic acid, a third fluorescent molecule, and a third quencher.
  • the first nucleic acid is complementary to the 5 ’end of the RNA sequence.
  • the second nucleic acid is complementary to the 3’ end of the RNA sequence.
  • the personalized therapeutic composition comprises the full- length RNA sequence and a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, or a cream.
  • a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, or a cream.
  • the personalized therapeutic composition is administered to the subject.
  • the personalized therapeutic composition is administered with an additional therapeutic composition including, but not limited to an inhibitor, an antibody, an antibiotic, an antiviral, an anti-inflammatory compound, an anesthetic, a sedative, or combinations thereof.
  • the method of any preceding aspect can be performed “off-line”, wherein the molecular beacon of any preceding aspect is manually delivered to a target RNA sequence for assessing the quantity, quality, and/or accuracy of the target RNA sequence.
  • the method of any preceding aspect can also be performed “in-line”, wherein the molecular beacon of any preceding aspect is incorporated into an automated process or instrument to allow for more efficient and automatic assessments of quantity, quality, and/or accuracy of the target RNA sequence.
  • Example 1 A method to assess RNA contamination and integrity both off-line and on-line.
  • beacons were invented in the 1990’ s and are widely used to probe for specific nucleic acid sequences.
  • the basic principle is that the beacon normally forms a hairpin structure ( Figure 1 A).
  • a fluorescence molecule is covalently attached to the 5’ end of the DNA and quencher is attached to the 3’ end (or vice versa).
  • the quencher when in close proximity to the fluorophore, “quenches” the fluorescence excited state of the fluorescent molecule, such that beacon is non-fluorescent or “dark” when the adjacent base pairs in the stem form a duplex.
  • beacons are designed so that the sequence (or a part of the sequence) in the loop is complementary to the target sequence being assayed.
  • the stem is disrupted, and the fluorescence molecule and the quencher are physically separated; the fluorophore now exhibits its intrinsic fluorescence (Figure IB).
  • the present disclosure can distinguish the desired full-length RNA (1) from products 2 and 3 ( Figure 2).
  • the invention can also be implemented in-line in a microfluidics environment. The latter allows incorporation of this as a real-time monitor of both quantity and quality of the mRNA being produced.
  • Molecular beacons target unique sequences at the very beginning and the very end of the RNA.
  • the beacon at the 5 ’ end will bind to all RNA and so will report on total RNA quantity.
  • Full length/correct length RNA will bind both beacons ( Figure 3D, top strands), while partially double stranded RNA ( Figure 3D, middle strands) will not bind the beacon.
  • Truncated RNA ( Figure 3D, bottom strands) will also not bind the beacon.
  • the ratio of fluorescence intensity from the 3 ’ beacon relative to the 5’ beacon reports on the fraction of correct length I full-length RNA - the quality of the RNA.
  • Results from a probe at position 63 confirm the results at position 11.
  • the probe at position 782 is about 80% of the way along the RNA.
  • the two measurements (preliminary) at OmM and 300 mM NaCl are close in intensity, suggesting that the trend seen at 960 reflects double stranded RNA, rather than (randomly) truncated RNA.
  • the present disclosure demonstrates a valuable off-line assay.
  • An in-line approach is also demonstrated ( Figure 5).
  • the diagram shows a flow reactor but with the addition of a small sampling stream just beyond the (three in parallel) reactor.
  • a very small output stream is extracted in real time, flowing to a point that splits the stream into two streams.
  • a 5’- specific beacon is then mixed into one stream and a 3 ’-specific beacon is mixed into the other stream.
  • fluorescence intensity from the 5’ beacon would report on total RNA, while the ratio of intensities from the 3’ compared to the 5’ beacons reports on quality.
  • Each of these could then give feedback to control flow rates to optimize both yield and quality. These values also serve as quality control feedback. If the ratio drops below a threshold or the quantity drops below a threshold, valves redirect the flow away from the production stream until such time as a correction is applied.
  • each oncology patient has a different mutation in their cancer cell DNA.
  • the device described previously can be used to manufacture a large number of different sequences in parallel, one for each patient.
  • One concern in the parallelization is certifying that the final drug substance or drug product is the patient-specific sequence (at the moment, upstream processes, such as template DNA production is done prior to mRNA manufacturing, so there is potential for mix-ups or cross-contamination prior to single-path mRNA manufacturing).
  • a molecular beacon specific to the patient-specific mutation is generated at low scale (the supplier already has massive parallel DNA synthesis) and delivered to the platform.
  • a third channel (split from the dual channel path above) contains a patient- specific beacon to confirm, in real time, that the mRNA matches the patient mutation. Since this is near or at the final stage in manufacturing, it provides assurance that the desired product is delivered to the patient.
  • SEQ ID NO: 1 Sequence targeted by molecular beacon (first 11 nucleotides).
  • SEQ ID NO: 2 Sequence targeted by molecular beacon (first 63 nucleotides). AAGATTTCGTTGGGG
  • SEQ ID NO: 3 Sequence targeted by molecular beacon (first 782 nucleotides). GCTGTCCAATTTCTA
  • SEQ ID NO: 4 Sequence targeted by molecular beacon (first 961 nucleotides).
  • SEQ ID NO: 7 Example sequence with targets of molecular beacons underlined (while the DNA sequence is listed here, the corresponding RNA sequence is also disclosed).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés d'évaluation de la contamination (pureté) et de l'intégrité de l'ARN. La présente invention concerne des procédés de détection d'acides ribonucléiques (ARN) défectueux, indésirables, non-souhaités et/ou non exceptionnels. La présente divulgation concerne également des procédés de mesure de la quantité et/ou de la qualité d'un ARN. La présente invention concerne également des procédés de validation d'une composition thérapeutique personnalisée comprenant un ARN.
PCT/US2024/054080 2023-11-03 2024-11-01 Procédés d'évaluation de contamination et d'intégrité d'arn Pending WO2025096912A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363596017P 2023-11-03 2023-11-03
US63/596,017 2023-11-03

Publications (1)

Publication Number Publication Date
WO2025096912A1 true WO2025096912A1 (fr) 2025-05-08

Family

ID=95581505

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/054080 Pending WO2025096912A1 (fr) 2023-11-03 2024-11-01 Procédés d'évaluation de contamination et d'intégrité d'arn

Country Status (1)

Country Link
WO (1) WO2025096912A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180318409A1 (en) * 2015-10-22 2018-11-08 Modernatx, Inc. Cancer vaccines
US20190338357A1 (en) * 2018-05-03 2019-11-07 Becton, Dickinson And Company High throughput multiomics sample analysis
US20210180106A1 (en) * 2016-02-12 2021-06-17 Curevac Ag Method for analyzing rna

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180318409A1 (en) * 2015-10-22 2018-11-08 Modernatx, Inc. Cancer vaccines
US20210180106A1 (en) * 2016-02-12 2021-06-17 Curevac Ag Method for analyzing rna
US20190338357A1 (en) * 2018-05-03 2019-11-07 Becton, Dickinson And Company High throughput multiomics sample analysis

Similar Documents

Publication Publication Date Title
US20230357826A1 (en) Competitive probes for engineering signal generation
EP2551356B1 (fr) Méthode permettant de détecter une séquence de bases cible à l'aide d'une amorce compétitive
JP2021118697A (ja) デジタル検体分析
US20240035081A1 (en) Labeled nucleotide compositions and methods for nucleic acid sequencing
CN1348096A (zh) 一种均相特异性检测核酸的探针及应用方法
CN106834456B (zh) 一种采用荧光标记方法标记的y-str复合扩增检测试剂盒及其使用方法
JP2021531024A (ja) 分子内動力学的プローブ
EP1436426A4 (fr) Procedes destines a detecter des haplotypes genetiques par interaction avec des sondes
EP2297344B1 (fr) Procédé de marquage double pour mesurer la prolifération cellulaire
CN106086192A (zh) 他克莫司个性化用药相关基因的分型检测试剂盒
CN108998536A (zh) 一种人类微卫星不稳定性状态msi检测试剂盒及其检测方法
CN109097478A (zh) 一种人类微卫星不稳定性状态msi检测试剂盒及其检测方法
EP2804957B1 (fr) Procédé de détection d'adn génomique résiduaire et kit associé
KR102278402B1 (ko) 홍역 바이러스 및 백신주의 검출용 프라이머 및 프로브와 이를 이용한 검출 방법
KR20090052322A (ko) 물질의 검출 등에 유용한 핵산쇄와 그 방법
KR102080823B1 (ko) 형광염기유사체를 포함하는 분할 g-쿼드로플렉스를 이용한 표적 dna 검출방법
WO2025096912A1 (fr) Procédés d'évaluation de contamination et d'intégrité d'arn
Tang et al. A single quantum dot-based biosensor for DNA point mutation assay
US8367814B2 (en) Assay for BCR/ABL gene rearrangement
DE69724375T2 (de) Verfahren zur Messung der Polynukleotidkonzentration
CN1238456A (zh) 用于核酸检测的荧光定量pcr方法及其试剂盒
CN112481374B (zh) Hla-b*1502基因的检测方法、检测试剂盒及其应用
Bao et al. A leakage-resistant catalytic hairpin assembly for ultrasensitive detection of miRNA-146a in systemic lupus erythematosus
WO2023234648A1 (fr) Méthode personnalisée de traitement du cancer pour des patients atteints d'un cancer à l'aide d'une pcr numérique
US20180037941A1 (en) Method for detecting genetic mutation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24886965

Country of ref document: EP

Kind code of ref document: A1