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WO2024263946A2 - Procédés et compositions servant à la préparation d'acides nucléiques de vésicules extracellulaires qui préservent des informations de proximité spatiale et leurs applications - Google Patents

Procédés et compositions servant à la préparation d'acides nucléiques de vésicules extracellulaires qui préservent des informations de proximité spatiale et leurs applications Download PDF

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Publication number
WO2024263946A2
WO2024263946A2 PCT/US2024/035061 US2024035061W WO2024263946A2 WO 2024263946 A2 WO2024263946 A2 WO 2024263946A2 US 2024035061 W US2024035061 W US 2024035061W WO 2024263946 A2 WO2024263946 A2 WO 2024263946A2
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nucleic acid
extracellular vesicle
subject
proximity
cancer
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WO2024263946A3 (fr
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Anthony Schmitt
Kristin SIKKINK
Brian KILBURN
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Arima Genomics Inc
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Arima Genomics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • EVs Extracellular vesicles
  • L-EVs large oncosomes
  • LO large oncosomes
  • exosomes contain entire genomes with large fragments of dsDNA AMG-1022 and carry mutations of parental tumor cells. These data suggest diagnostic and prognostic value for EV DNA.
  • Liquid biopsies offer minimally invasive and practical clinical tools. Advancements in sequencing technologies have enabled the analysis of the genomic landscape of cancer using circulating cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Furthermore, liquid biopsies may overcome the limitation of tissue biopsies to capture the tumor heterogeneity and the dynamic evolution of cancer genomes. cfDNA has been used for non-invasive screening of chromosomal alterations in the circulation. However, CTCs are rare in most cancer types and cfDNA is fragmented ( ⁇ 160 bp).
  • cfDNA in early stage PCa and during treatment response might underrepresent the cancer genome, i.e. the levels of tumor DNA are lower, thus limiting the sensitivity of standard DNA analyses for detection of tumor-specific genomic alterations.
  • the prior art has only shown the extracellular vesicle DNA is high molecular weight and chromatinized (see, e.g. Vagner et al., cited above).
  • 3D chromatin structure could become partially or entirely destroyed during the process of being secreted or after being secreted while in EVs but before being harvested for measurement and analysis of spatial proximity relationships (e.g. crosslinking and proximity ligation).
  • spatial proximity relationships e.g. crosslinking and proximity ligation
  • Applicants have made the discovery that the DNA present in extracellular vesicles contains chromatin with 3D structure present. This observation supports the use of technologies that preserve and/or measure spatial proximity information of nucleic acids in EVs in numerous applications further discussed herein. Summary Provided in certain aspects are methods for preparing nucleic acid from an extracellular vesicle including the step of contacting an extracellular vesicle with one or more agents that preserve spatial-proximity relationships in the nucleic acid of the extracellular vesicle.
  • FIG.1 shows a schematic of the method used in an example of an embodiment of the invention.
  • FIGs.2A-2D show chr11 HiC interaction maps and a Pearson Correlation (PC) matrix of an example of an embodiment of the invention.
  • FIG.3 shows a first principal component eigenvector of an example of an embodiment of the invention.
  • FIG.4 depicts a zoomed in HiC heatmap showing the approx. locus coordinates: Chr8:103,500,000-110,000,000 an example of an embodiment of the invention.
  • FIGs.5A-5B show genome-wide HiC maps of an example of an embodiment of the invention.
  • FIGs.6A-6B show zoomed in HiC heatmaps of exemplary inter-chr translocation between chr3 and chr10 of an example of an embodiment of the invention.
  • FIGs.7A-7B show “genome scan plots” representing a virtual Capture-HiC analysis targeting the CACNB2 gene in an example of an embodiment of the invention.
  • FIGs.8A-8B show “genome scan plots” representing a virtual Capture-HiC analysis targeting the CAMTA1 gene in an example of an embodiment of the invention.
  • FIGs.9A-9B show “genome scan plots” representing a virtual Capture-HiC analysis targeting the PTEN gene in an example of an embodiment of the invention.
  • FIGs.10A-10B show “genome scan plots” representing a virtual Capture-HiC analysis targeting the BCR gene in an example of an embodiment of the invention.
  • FIGs.11A-11B show “genome scan plots” representing a virtual Capture-HiC analysis in K562 cells in an example of an embodiment of the invention.
  • nucleic acid enrichment a method for nucleic acid enrichment, the method including: subjecting target nucleic acid from a nucleic acid sample to nucleic acid cleavage conditions in which nucleic acid fragments are generated; subjecting the target nucleic acid fragments to linking conditions in which proximity ligated nucleic acid molecules are generated; contacting the proximity ligated nucleic acid molecules with a composition comprising a plurality of oligonucleotide probes described herein under hybridization conditions in which hybridization AMG-1022 complexes comprising proximity ligated nucleic acid hybridized to oligonucleotide probes are generated; isolating the complexes; and analyzing nucleic acid in the complexes.
  • nucleic acid(s), nucleic acid molecule(s), nucleic acid fragment(s), target nucleic acid(s), nucleic acid template(s), template nucleic acid(s), nucleic acid target(s), target nucleic acid(s), polynucleotide(s), polynucleotide fragment(s), target polynucleotide(s), polynucleotide target(s), and the like may be used interchangeably throughout the disclosure.
  • RNA e.g., message RNA (mRNA), small interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA, transacting small interfering RNA (ta-siRNA), natural small interfering RNA (nat-siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), non-coding RNA (ncRNA), transfer-messenger RNA (tmRNA), precursor messenger RNA (pre-mRNA), small Cajal body-specific RNA (scaRNA), piwi- interacting RNA (piRNA), endoribonu
  • mRNA message RNA
  • siRNA small interfering RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • microRNA transacting small interfering RNA
  • ta-siRNA small interfering RNA
  • nat-siRNA natural small interfering
  • a nucleic acid may be, or may be from, a plasmid, phage, virus, bacterium, autonomously replicating sequence (ARS), mitochondria, centromere, artificial chromosome, chromosome, or other nucleic acid able to replicate or be replicated in vitro or in a host cell, a cell, a cell nucleus or cytoplasm of a cell in certain embodiments.
  • a template nucleic acid in some embodiments can be from a single chromosome (e.g., a nucleic acid sample may be from one chromosome of a sample obtained from a diploid organism).
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
  • nucleic acid is used interchangeably with locus, gene, cDNA, and mRNA encoded by a gene.
  • the term also may include, as equivalents, derivatives, variants and AMG-1022 analogs of RNA or DNA synthesized from nucleotide analogs, single-stranded ("sense” or “antisense,” “plus” strand or “minus” strand, “forward” reading frame or “reverse” reading frame) and double-stranded polynucleotides.
  • a nucleotide or base generally refers to the purine and pyrimidine molecular units of nucleic acid (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)).
  • a nucleotide or base generally refers to the purine and pyrimidine molecular units of nucleic acid (e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)).
  • a nucleic acid e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)
  • a nucleic acid e.g., adenine (A), thymine (T), guanine (G), and cytosine (C)
  • the base thymine is replaced with uracil (U).
  • Nucleic acid length or size may be expressed as a number of bases.
  • Target nucleic acids may be any nucleic acids of interest.
  • Nucleic acids may be polymers of any length composed of deoxyribonucleotides (i.e., DNA bases), ribonucleotides (i.e., RNA bases), or combinations thereof, e.g., 10 bases or longer, 20 bases or longer, 50 bases or longer, 100 bases or longer, 200 bases or longer, 300 bases or longer, 400 bases or longer, 500 bases or longer, 1000 bases or longer, 2000 bases or longer, 3000 bases or longer, 4000 bases or longer, 5000 bases or longer.
  • deoxyribonucleotides i.e., DNA bases
  • ribonucleotides i.e., RNA bases
  • 10 bases or longer 20 bases or longer, 50 bases or longer, 100 bases or longer, 200 bases or longer, 300 bases or longer, 400 bases or longer, 500 bases or longer, 1000 bases or longer, 2000 bases or longer, 3000 bases or longer, 4000 bases or longer, 5000 bases or longer.
  • nucleic acids are polymers composed of deoxyribonucleotides (i.e., DNA bases), ribonucleotides (i.e., RNA bases), or combinations thereof, e.g., 10 bases or less, 20 bases or less, 50 bases or less, 100 bases or less, 200 bases or less, 300 bases or less, 400 bases or less, 500 bases or less, 1000 bases or less, 2000 bases or less, 3000 bases or less, 4000 bases or less, or 5000 bases or less.
  • Nucleic acid may be single-stranded or double-stranded.
  • Single-stranded DNA ssDNA
  • Single-stranded DNA can be generated by denaturing double-stranded DNA by heating or by treatment with alkali, for example.
  • ssDNA is derived from double- stranded DNA (dsDNA).
  • Nucleic acid e.g., genomic DNA, nucleic acid targets, oligonucleotides, probes, primers
  • dsDNA double- stranded DNA
  • Nucleic acid e.g., genomic DNA, nucleic acid targets, oligonucleotides, probes, primers
  • a complementarity region being capable of hybridizing to another nucleic acid, or having a hybridization region.
  • hybridization generally refer to a nucleotide sequence that base-pairs by non-covalent bonds to a region of a nucleic acid.
  • adenine (A) forms a base pair with thymine (T), and guanine (G) pairs with cytosine (C) in DNA.
  • thymine (T) is replaced by uracil (U).
  • U uracil
  • A is complementary to T and G is complementary to C.
  • RNA A is complementary to U and vice versa.
  • a (in a DNA strand) is complementary to U (in an RNA strand).
  • complementary or “complementarity” or “capable of hybridizing” refer to a nucleotide sequence that is at least partially complementary.
  • nucleotide sequence may be partially AMG-1022 complementary to a target, in which not all nucleotides are complementary to every nucleotide in the target nucleic acid in all the corresponding positions.
  • the percent identity of two nucleotide sequences can be determined by aligning the sequences for optimal comparison purposes. When the total number of positions is different between the two nucleotide sequences, gaps may be introduced in the sequence of one or both sequences for optimal alignment.
  • % identity # of identical positions/total # of positions ⁇ 100.
  • % identity # of identical positions/total # of positions ⁇ 100.
  • extra or missing bases within a sequence are expressed as gaps in an alignment and may or may not be factored into a percent identity calculation.
  • a percent identity calculation may include a number of mismatches and gaps or may include a number of mismatches only.
  • hybridizing refers to binding of a first nucleic acid molecule to a second nucleic acid molecule under low, medium or high stringency conditions, or under nucleic acid synthesis conditions.
  • Hybridizing can include instances where a first nucleic acid molecule binds to a second nucleic acid molecule, where the first and second nucleic acid molecules are complementary.
  • “specifically hybridizes” refers to preferential hybridization under nucleic acid synthesis conditions of a primer, oligonucleotide, or probe, to a nucleic acid molecule having a sequence complementary to the primer, oligonucleotide, or probe compared to hybridization to a nucleic acid molecule not having a complementary sequence.
  • specific hybridization includes the hybridization of a primer, oligonucleotide, or probe to a target nucleic acid sequence that is complementary to the primer, oligonucleotide, or probe.
  • Primer, oligonucleotide, or probe sequences and length can affect hybridization to target nucleic acid sequences.
  • low, medium or high stringency conditions may be used to effect primer/target, oligonucleotide/target, or probe/target annealing.
  • stringent conditions refers to conditions for hybridization and washing. Methods for hybridization reaction temperature condition optimization are known, and can be found, e.g., in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are described in the aforementioned reference and either can be used.
  • Non-limiting examples of stringent hybridization conditions include, for example, hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45oC, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50oC.
  • Another example of stringent hybridization conditions includes hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45oC, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55oC.
  • a further AMG-1022 example of stringent hybridization conditions includes hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45oC, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60oC.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45oC, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65oC. More often, stringency conditions can include 0.5 M sodium phosphate, 7% SDS at 65oC, followed by one or more washes at 0.2X SSC, 1% SDS at 65oC. Stringent hybridization temperatures also can be altered (generally, lowered) with the addition of certain organic solvents, such as formamide for example.
  • a lysis procedure may include a lysis step with EDTA/Proteinase K, a binding buffer step with high amounts of salts (e.g., guanidinium chloride (GuHCl), sodium acetate) and isopropanol, and binding DNA in this solution to silica-based column.
  • Nucleic acids can be present in and obtained from blood (e.g., from the blood of a human subject).
  • sources for EVs are blood, blood plasma, blood serum and urine.
  • EVs are obtained from a body fluid sample chosen from whole blood, blood plasma, blood serum, amniotic fluid, saliva, urine, pleural effusion, bronchial lavage, bronchial aspirates, breast milk, colostrum, tears, seminal fluid, peritoneal fluid, pleural effusion, and stool.
  • a body fluid sample chosen from whole blood, blood plasma, blood serum, amniotic fluid, saliva, urine, pleural effusion, bronchial lavage, bronchial aspirates, breast milk, colostrum, tears, seminal fluid, peritoneal fluid, pleural effusion, and stool.
  • the term “obtain” includes obtaining a sample of EVs or nucleic acids derived from EVs directly (e.g., collecting a sample, e.g., a test sample) or obtaining a sample from another who has collected a sample.
  • EVs may be a product of cellular secretion and/or nucleic
  • nucleic acids are derived from EVs from blood plasma or blood serum from a test subject. In some aspects, nucleic acids derived from EVs are degraded. In certain aspects, nucleic acids derived from EVs comprise cancer nucleic acid (e.g., cancer DNA). In certain aspects, nucleic acids are derived from EVs comprising tumor nucleic acid (e.g., tumor DNA). Nucleic acid derived from EVs can include different nucleic acid species, and therefore is referred to herein as "heterogeneous" in certain embodiments.
  • Nucleic acid may be provided for conducting methods described herein with or without processing of the sample(s) containing the nucleic acid.
  • nucleic acid is provided for conducting methods described herein after processing of the sample(s) containing AMG-1022 the nucleic acid.
  • a nucleic acid can be extracted, isolated, purified, partially purified or amplified from the sample(s).
  • isolated refers to nucleic acid removed from its original environment (e.g., the natural environment if it is naturally occurring, or a host cell if expressed exogenously), and thus is altered by human intervention (e.g., "by the hand of man") from its original environment.
  • isolated nucleic acid can refer to a nucleic acid removed from a subject (e.g., a human subject).
  • An isolated nucleic acid can be provided with fewer non-nucleic acid components (e.g., protein, lipid) than the amount of components present in a source sample.
  • a composition comprising isolated nucleic acid can be about 50% to greater than 99% free of non-nucleic acid components.
  • a composition comprising isolated nucleic acid can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of non-nucleic acid components.
  • purified can refer to a nucleic acid provided that contains fewer non-nucleic acid components (e.g., protein, lipid, carbohydrate) than the amount of non-nucleic acid components present prior to subjecting the nucleic acid to a purification procedure.
  • a composition comprising purified nucleic acid may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of other non-nucleic acid components.
  • purified can refer to a nucleic acid provided that contains fewer nucleic acid species than in the sample source from which the nucleic acid is derived.
  • a composition comprising purified nucleic acid may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free of other nucleic acid species.
  • small fragments of nucleic acid e.g., 30 to 500 bp fragments
  • nucleic acid is provided for conducting methods described herein without prior processing of the sample(s) containing the nucleic acid.
  • nucleic acid may be analyzed directly from a sample without prior extraction, purification, partial purification, and/or amplification.
  • cleavage conditions include contacting the sample nucleic acid with an endonuclease, such as a restriction endonuclease, and sometimes the restriction endonuclease cleaves the sample nucleic acid at ⁇ GATC and G ⁇ ANTC, where “ ⁇ ” represents the cut site.
  • sample nucleic acid is contacted with two or more restriction AMG-1022 enzyme types.
  • linking conditions comprise contacting the nucleic acid fragments with a ligase under conditions in which ends of fragments in proximity are joined.
  • Methodology for preparing proximity ligated nucleic acid is known in the art, and non-limiting examples of such methodology are referred to as Hi-C, 3C, 4C, ChiA-PET and variants thereof (e.g., capture Hi-C), as described additionally herein.
  • oligonucleotide probes include a capture agent, and the complexes are isolated by contacting the complexes with a solid phase comprising a capture agent counterpart that specifically binds to the capture agent under binding conditions.
  • a solid phase sometimes is a plurality of beads, such as magnetic or SEPHAROSE (TM) beads for example.
  • a capture agent is selected from biotin, avidin and streptavidin
  • the capture agent counterpart is a molecule that specifically binds to the capture agent and independently is selected from biotin, avidin and streptavidin.
  • Hybridization complexes can be isolated by contacting the complexes with a solid phase that includes a capture agent counterpart under conditions in which the capture agent counterpart of the solid phase specifically binds to a capture agent associated with the oligonucleotide probes of the hybridization complexes, and separating the complexes bound to the solid phase from complexes not bound to the solid phase. Any suitable method for carrying out proximity ligation may be used.
  • a Hi- C method typically includes the following steps: (1) digesting the DNA of a chromatin sample with a restriction enzyme (or more broadly, fragmenting), where a non-limiting example of a chromatin sample is chromatin obtained from solubilized and decompacted FFPE (formalin- fixed paraffin embedded) tissue; (2) labelling the digested ends by filling in the 5’-overhangs, such as with biotinylated nucleotides; and (3) ligating the spatially proximal fragmented ends, thus preserving spatial-proximity information of the nucleic acids.
  • a restriction enzyme or more broadly, fragmenting
  • a HiC method may include: purifying and enriching biotin–labelled ligation junction fragments, preparing a library from the enriched fragments and sequencing the library.
  • Another example of a proximity ligation method may include the following steps: (1) digestion of a chromatin sample with a restriction enzyme (or fragmentation), where a non-limiting example of a chromatin sample is chromatin obtained from solubilized and decompacted FFPE (formalin-fixed paraffin embedded) tissue; (2) blunting the digested or fragmented ends or omission of the blunting procedure; and (3) ligating the spatially proximal ends, thus preserving spatial-proximal contiguity information.
  • FFPE fragment-fixed paraffin embedded
  • further steps can include: size selection and affinity purification to purify and enrich ligated fragments, which represent ligation junction fragments, preparing a library from the enriched fragments and sequencing the library.
  • proximity ligated nucleic acid molecules are generated in situ (i.e., within a nucleus).
  • Capture HiC a further step is included where ligation AMG-1022 products containing certain nucleic acid sequences are enriched using one or more capture probes (see e.g., International Patent Application Publication No. WO 2014/168575).
  • a capture probe generally includes a short sequence of nucleotides or oligonucleotide (e.g., 10-500 bases in length) capable of hybridizing to another nucleotide sequence.
  • a capture probe includes a label, e.g., a label for selectively purifying specific nucleic acid sequences of interest. Labels are discussed herein and can include, for example, a biotin or digoxigenin label.
  • Processes that include preparing hybridization complexes comprising proximity ligated nucleic acid hybridized to oligonucleotide probes and then isolating the complexes can enrich the relative abundance of polynucleotides in sample nucleic acid complementary to the oligonucleotide probe polynucleotides, which are referred to as “target polynucleotides” herein.
  • target polynucleotides As oligonucleotide probes described herein include polynucleotides complementary to oncogene introns, exons, promoters, and external regions, such probes are useful for enriching oncogene polynucleotides in a sample.
  • a subset of hybridization complexes containing proximity ligated nucleic acid hybridized to probes described herein typically is enriched for oncogene target polynucleotides.
  • Target polynucleotides e.g., oncogene oligonucleotides
  • oncogene oligonucleotides generally are enriched in a subset of hybridization complexes containing proximity ligated nucleic acid hybridized, or that was hybridized, to the probes, relative to all proximity ligated nucleic acid prepared from a nucleic acid sample.
  • the abundance (e.g., percentage) of target polynucleotides generally is greater in the subset of hybridization complexes containing proximity ligated nucleic acid hybridized, or that was hybridized, to the probes, relative to the abundance of target polynucleotides (e.g., percentage) in all proximity ligated nucleic acid prepared from a nucleic acid sample.
  • Target nucleic acid sometimes is modified as part of a nucleic acid analysis process.
  • a target nucleic acid can be modified to include an identifier (e.g., a tag, an indexing tag), a capture sequence, a label, an adapter, a restriction enzyme site, a promoter, an enhancer, an origin of replication, a stem loop, a complimentary sequence (e.g., a primer binding site, an annealing site), a suitable integration site (e.g., a transposon, a viral integration site), a modified nucleotide, a unique molecular identifier (UMI), the like or combinations thereof.
  • a nucleic acid or isolated nucleic acid comprises one or more adapters (e.g., sequencing adapters, also known as sequencing adapter oligonucleotides).
  • Sequencing adapters may comprise sequences complementary to flow-cell anchors, and sometimes are utilized to immobilize a nucleic acid to a solid support, such as the inside surface of a flow cell, for example. Adapters and other polynucleotide components described typically are not associated with the nucleic acid in vivo and thereby do not naturally occur with the nucleic acid.
  • analyzing the nucleic acid in the complexes includes sequencing the proximity ligated nucleic acid of the isolated complexes. Target nucleic acid sometimes is modified as part of a sequencing process.
  • non-naturally occurring AMG-1022 oligonucleotides that facilitate sequencing are joined to proximity ligated nucleic acid in the oligonucleotide probe-isolated proximity ligated nucleic acid, thereby forming adapter-modified nucleic acid.
  • Adapter-modified nucleic acid is optionally amplified by an amplification process known in the art, and the adapter-modified nucleic acid (or amplified adapter-modified nucleic acid) is then subjected to sequencing conditions to identify the polynucleotide sequence of proximity ligated nucleic acid. Additional aspects of nucleic acid analytical methodology are described herein.
  • nucleic acid from a sample can identify one or more structural variants in the nucleic acid relative to nucleic acid from a reference genome or from another sample in certain instances. Various types of structural variants that can be identified are described herein.
  • Samples Provided herein are methods and compositions for processing and/or analyzing nucleic acid. Nucleic acid utilized in methods and compositions described herein may be isolated from a sample obtained from a subject (e.g., a test subject). A subject can be any living or non-living organism, including but not limited to a human and a non-human animal.
  • Any human or non- human animal can be selected, and may include, for example, mammal, reptile, avian, amphibian, fish, ungulate, ruminant, bovine (e.g., cattle), equine (e.g., horse), caprine and ovine (e.g., sheep, goat), swine (e.g., pig), camelid (e.g., camel, llama, alpaca), monkey, ape (e.g., gorilla, chimpanzee), ursid (e.g., bear), poultry, dog, cat, mouse, rat, fish, dolphin, whale and shark.
  • a subject is a human.
  • a subject may be a male or female.
  • a subject may be any age (e.g., an embryo, a fetus, an infant, a child, an adult).
  • a subject may be a cancer patient, a patient suspected of having cancer, a patient in remission, a patient with a family history of cancer, and/or a subject obtaining a cancer screen.
  • a subject is an adult patient.
  • a subject is a pediatric patient.
  • a nucleic acid sample may be isolated or obtained from any type of suitable biological specimen or sample (e.g., a test sample).
  • a nucleic acid sample may be isolated or obtained from a single cell, a plurality of cells (e.g., cultured cells), cell culture media, conditioned media, a tissue, an organ, or an organism.
  • a nucleic acid sample is isolated or obtained from a cell(s), tissue, organ, and/or the like of an animal (e.g., an animal subject). In some instances, a nucleic acid sample may be obtained as part of a diagnostic analysis.
  • a sample or test sample may be any specimen that is isolated or obtained from a subject or part thereof (e.g., a human subject, a cancer patient, a tumor).
  • specimens include fluid or tissue from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, or the like), umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), biopsy sample (e.g., from pre-implantation embryo; cancer biopsy), celocentesis sample, cells (blood cells, placental cells, embryo or fetal cells, fetal nucleated AMG-1022 cells or fetal cellular remnants, normal cells, abnormal cells (e.g., cancer cells)) or parts thereof (e.g., mitochondrial, nucleus, extracts, or the like), washings of female reproductive tract, urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk
  • a biological sample is a cervical swab from a subject.
  • a fluid or tissue sample from which nucleic acid is extracted may be acellular (e.g., cell-free).
  • a fluid or tissue sample may contain cellular elements or cellular remnants.
  • cancer cells may be included in the sample.
  • a sample can be a liquid sample.
  • a liquid sample can comprise extracellular nucleic acid (e.g., circulating cell-free DNA).
  • liquid samples include, but are not limited to, blood or a blood product (e.g., serum, plasma, or the like), urine, cerebrospinal fluid, saliva, sputum, biopsy sample (e.g., liquid biopsy for the detection of cancer), a liquid sample described above, the like or combinations thereof.
  • a sample is a liquid biopsy, which generally refers to an assessment of a liquid sample from a subject for the presence, absence, progression or remission of a disease (e.g., cancer).
  • a liquid biopsy can be used in conjunction with, or as an alternative to, a solid biopsy (e.g., tumor biopsy).
  • extracellular nucleic acid is analyzed in a liquid biopsy.
  • a biological sample may be blood, plasma or serum.
  • blood encompasses whole blood, blood product or any fraction of blood, such as serum, plasma, buffy coat, or the like as conventionally defined. Blood or fractions thereof often comprise nucleosomes. Nucleosomes comprise nucleic acids and are sometimes cell-free or intracellular. Blood also comprises buffy coats. Buffy coats are sometimes isolated by utilizing a ficoll gradient. Buffy coats can comprise white blood cells (e.g., leukocytes, T-cells, B-cells, platelets, and the like). Blood plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants.
  • Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. Fluid or tissue samples often are collected in accordance with standard protocols hospitals or clinics generally follow. For blood, an appropriate amount of peripheral blood (e.g., between 3 to 40 milliliters, between 5 to 50 milliliters) often is collected and can be stored according to standard procedures prior to or after preparation.
  • An analysis of nucleic acid found in a subject’s blood may be performed using, e.g., whole blood, serum, or plasma.
  • An analysis of tumor or cancer DNA found in a patient’s blood for example, may be performed using, e.g., whole blood, serum, or plasma.
  • a subject e.g., patient; cancer patient
  • a subject’s blood e.g., patient’s blood; cancer patient’s blood
  • a tube containing EDTA or a specialized commercial product such as Cell-Free DNA BCT (Streck, Omaha, NE) or Vacutainer SST (Becton Dickinson, Franklin Lakes, N.J.) to prevent blood clotting, and plasma can then be obtained from whole blood through AMG-1022 centrifugation. Serum may be obtained with or without centrifugation-following blood clotting.
  • a sample may be a tumor nucleic acid sample (i.e., a nucleic acid sample isolated from a tumor).
  • a sample generally refers to neoplastic cell growth and proliferation, whether malignant or benign, and may include pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” generally refer to the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
  • a sample is a blood sample, or a urine sample.
  • a sample comprises formalin-fixed, paraffin-embedded (FFPE) tissue.
  • FFPE formalin-fixed, paraffin-embedded
  • a sample comprises frozen tissue.
  • a sample comprises EVs derived from peripheral blood.
  • a sample comprises EVs derived from blood obtained from bone marrow.
  • a sample comprises EVs obtained from urine. In some embodiments, a sample comprises nucleic acids derived from EVs. In some embodiments, a sample comprises EVs originating from a tumor cell. In some embodiments, a sample comprises an EV or nucleic acid originating from a solid tumor. In some embodiments, a sample comprises an EV or nucleic acid originating from a blood tumor.
  • amplified or “amplification” or “amplification conditions” generally refer to subjecting a target nucleic acid in a sample to a process that linearly or exponentially generates amplicon nucleic acids having the same or substantially the same nucleotide sequence as the target nucleic acid, or part thereof.
  • the term “amplified” or “amplification” or “amplification conditions” refers to a method that comprises a polymerase chain reaction (PCR).
  • Detecting a structural variant (SV) described herein using amplification may include use of a primer(s) designed to hybridize to a region upstream (e.g., 5’) of one or more SV breakpoints, and/or hybridize to a region downstream (e.g., 3’) of one or more SV breakpoints, hybridize to a region AMG-1022 adjacent to one or more SV breakpoints, and/or hybridize to a region spanning one or more SV breakpoints.
  • a nucleic acid analysis comprises fluorescence in situ hybridization (FISH).
  • a sequencing process generates short sequencing reads or “short reads.”
  • the nominal, average, mean or absolute length of short reads sometimes is about 10 continuous nucleotides to about 250 or more contiguous nucleotides.
  • the nominal, average, mean or absolute length of short reads sometimes is about 50 continuous nucleotides to about 150 or more contiguous nucleotides.
  • a nucleic acid analysis comprises a method that preserves spatial-proximal relationships and/or spatial-proximal contiguity information (see e.g., International PCT Application Publication No. WO2019/104034; International PCT Application Publication No. WO2020/106776; International PCT Application Publication No.
  • Spatial- proximal contiguity information can be preserved by proximity ligation, by solid substrate- mediated proximity capture (SSPC), by compartmentalization with or without a solid substrate or by use of a Tn5 tetramer.
  • Methods that preserve spatial-proximal contiguity information may be based on proximity ligation or may be based on a different principle where spatial proximity is inferred.
  • Methods based on proximity ligation may include, for example, 3C, 4C, 5C, Hi-C, TCC, GCC, TLA, PLAC-seq, HiChIP, ChIA-PET, Capture-C, Capture-HiC, single-cell HiC, sciHiC, AMG-1022 single-cell 3C, single-cell methyl-3C, DNAase HiC, Micro-C, Tiled-C, and Low-C.
  • Methods where spatial proximity is inferred based on a principle other than proximity ligation may include, for example, SPRITE, scSPRITE, Genome Architecture Mapping (GAM), ChIA-Drop, imaging- based approaches using labeled probes and visualization of probe-bound DNA (e.g.
  • Nucleic acid molecules that preserve spatial-proximity information can be fragmented and sequenced using short-read sequencing methods (e.g., Illumina, nucleic acid fragments of lengths approximately 500 bp) or intact molecules that preserve spatial-proximity information can be sequenced using long-read sequencing (e.g., Oxford Nanopore, PacBio, or others, nucleic acid fragments of lengths approximately 5kbp or greater). Nucleic acid molecules that preserve spatial-proximity information can be fragmented and sequenced using synthetic long-read sequencing methods (e.g. Illumina Complete Long Reads, Universal Sequencing Technology TELL-seq, and MGI stLFR).
  • short-read sequencing methods e.g., Illumina, nucleic acid fragments of lengths approximately 500 bp
  • long-read sequencing e.g., Oxford Nanopore, PacBio, or others, nucleic acid fragments of lengths approximately 5kbp or greater.
  • Nucleic acid molecules that preserve spatial-proximity information can be fragmented
  • a sample can be a fixed sample that is embedded in a material such as paraffin (wax).
  • a sample can be a formalin fixed sample.
  • a sample is formalin-fixed paraffin-embedded (FFPE) sample.
  • FFPE formalin-fixed paraffin-embedded
  • a formalin-fixed paraffin-embedded sample can be a tissue sample or a cell culture sample.
  • a tissue sample has been excised from a patient and can be diseased or damaged.
  • a tissue sample is not known to be diseased or damaged.
  • a formalin-fixed paraffin-embedded sample can be a formalin-fixed paraffin-embedded section, block, scroll or slide.
  • methods that preserve spatial-proximal contiguity information comprise methods that generate proximity ligated nucleic acid molecules (e.g., using proximity ligation).
  • a proximity ligation method is one in which natively occurring spatially proximal nucleic acid molecules are captured by ligation to generate ligated products.
  • Proximity ligation methods generally capture spatial-proximity information in the form of ligation products, whereby a ligation junction is formed between two natively spatially proximal nucleic acids.
  • reagents that generate proximity ligated nucleic acid molecules can include a restriction endonuclease, a DNA polymerase, a plurality of nucleotides comprising at least one biotinylated nucleotide, and a ligase.
  • two or more restriction endonucleases are used. Any suitable method for carrying out proximity ligation may be used, as described herein.
  • Structural variants Provided herein are methods for detecting the presence or absence of a structural variant in a sample.
  • a method for detecting the presence or absence of a structural variant in a sample comprising analyzing sample nucleic acid from a subject, wherein the analyzing comprises: generating proximity ligated nucleic acid molecules; contacting the proximity ligated nucleic acid molecules with one or more oligonucleotide probes described herein, thereby generating enriched proximity ligated nucleic acid molecules; sequencing the enriched proximity ligated nucleic acid molecules, thereby generating sequences of the sample nucleic acid; and determining the presence or absence of a structural variant in the sample nucleic acid from the sequences.
  • the method includes comparing sequences of the sample nucleic acid to sequences of a reference genome to determine the presence or absence of a structural variant.
  • a structural variant may be referred to as a structural variation and/or a chromosomal rearrangement.
  • a structural variant may comprise one or more of a translocation, inversion, insertion, deletion, and duplication.
  • a structural variant comprises a microduplication and/or a microdeletion.
  • a structural variant comprises a fusion (e.g., a gene fusion where a portion of a first gene is inserted into a portion of a second gene).
  • Any type of structural variant, whether it be translocation, inversion, insertion, deletion, and/or duplication as described below, can be of any length, and in some embodiments, is about 1 base or base pair (bp) to about 250 megabases (Mb) in length. In some embodiments, a structural variation is about 1 base or base pair (bp) to about 50,000 kilobases (kb) in length (e.g., about 10 bp, 50 bp, 100 bp, 500 bp, 1 kb, 5 kb, 10kb, 50 kb, 100 kb, 500 kb, 1000 kb, AMG-1022 5000 kb or 10,000 kb in length).
  • kb kilobases
  • a Robertsonian translocation occurs when two non-homologous chromosomes become attached, meaning that given two healthy pairs of chromosomes, one of each pair sticks and blends together homogeneously.
  • a gene fusion may be created when a translocation joins two genes that are normally separate. Translocations may be balanced (i.e., in an even exchange of material with no genetic information extra or missing, sometimes with full functionality) or unbalanced (i.e., where the exchange of chromosome material is unequal resulting in extra or missing genes or fragments thereof).
  • a structural variant may comprise an inversion.
  • An inversion is a chromosome rearrangement in which a segment of a chromosome is reversed end-to-end.
  • An inversion may occur when a single chromosome undergoes breakage and rearrangement within itself. Inversions may be of two types: paracentric and pericentric. Paracentric inversions do not include the centromere, and both breaks occur in one arm of the chromosome. Pericentric inversions include the centromere, and there is a break point in each arm.
  • a structural variant may comprise an insertion.
  • An insertion may be the addition of one or more nucleotide base pairs into a nucleic acid sequence.
  • An insertion may be a microinsertion (generally a submicroscopic insertion of any length ranging from 1 base to about 10 megabases (e.g., about 1 megabase to about 3 megabases)).
  • an insertion comprises the addition of a segment of a chromosome into a genome, chromosome, or segment thereof. In certain embodiments an insertion comprises the addition of an allele, a gene, an intron, an exon, any non-coding region, any coding region, segment thereof or combination thereof into a genome or segment thereof. In certain embodiments an insertion comprises the addition (e.g., insertion) of nucleic acid of unknown origin into a genome, chromosome, or segment thereof. In certain embodiments an insertion comprises the addition (e.g., insertion) of a single base. A structural variant may comprise a deletion.
  • a deletion is a genetic aberration in which a part of a chromosome or a sequence of DNA is missing.
  • a deletion can, in certain embodiments, result in the loss of genetic material.
  • a deletion can be translocated to another portion of the genome (balanced translocation or unbalanced translocation), such as on the same chromosome (same arm of the chromosome or other arm of the chromosome) or on a different chromosome. Any number of nucleotides can AMG-1022 be deleted.
  • a deletion can comprise the deletion of one or more entire chromosomes, a segment of a chromosome, an allele, a gene, an intron, an exon, any non-coding region, any coding region, a segment thereof or combination thereof.
  • a deletion can comprise a microdeletion (generally a submicroscopic deletion of any length ranging from 1 base to about 10 megabases (e.g., about 1 megabase to about 3 megabases)).
  • a deletion can comprise the deletion of a single base.
  • a structural variant may comprise a duplication.
  • a duplication is a genetic aberration in which a part of a chromosome or a sequence of DNA is copied and inserted back into the genome.
  • a duplication is any duplication of a region of DNA.
  • a duplication is a nucleic acid sequence that is repeated, often in tandem, within a genome or chromosome.
  • a duplication can comprise a copy of one or more entire chromosomes, a segment of a chromosome, an allele, a gene, an intron, an exon, any non-coding region, any coding region, segment thereof or combination thereof.
  • a duplication can comprise a microduplication (generally a submicroscopic duplication of any length ranging from 1 base to about 10 megabases (e.g., about 1 megabase to about 3 megabases)).
  • a duplication sometimes comprises one or more copies of a duplicated nucleic acid.
  • a duplication may be characterized as a genetic region repeated one or more times (e.g., repeated 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times). Duplications can range from small regions (thousands of base pairs) to whole chromosomes in some instances. Duplications may occur as the result of an error in homologous recombination or due to a retrotransposon event.
  • a structural variant may include a plurality of chromosomal rearrangements (e.g., translocations, inversions, insertions, deletions, duplications). For example, a structural variant may include a plurality of intra-chromosomal rearrangements.
  • a structural variant may include a plurality of inter-chromosomal rearrangements. In certain instances, a structural variant may include a plurality of intra-chromosomal rearrangements and inter- chromosomal rearrangements.
  • a structural variant may be defined according to one or more breakpoints.
  • a breakpoint generally refers to a genomic position (i.e., genomic coordinate) where a structural variant occurs (e.g., translocation, inversion, insertion, deletion, or duplication).
  • a breakpoint may refer to a genomic position where an ectopic portion of genomic material is inserted (e.g., a recipient site for an insertion or a translocation).
  • a breakpoint may refer to a genomic position where a portion of genomic material is deleted (e.g., a donor site for an insertion or a translocation).
  • a breakpoint may refer to a pair of genomic positions (i.e., genomic coordinates) that have become flanking (i.e., adjacent) to one another as a result of a structural variant (e.g., translocation, inversion, insertion, deletion, or duplication).
  • a breakpoint may be defined in terms of a position or positions in a reference genome.
  • a breakpoint may be defined in terms of a position or positions in a human reference genome (e.g., HG38 human reference genome).
  • genomic positions discussed herein are in reference to an HG38 human reference AMG-1022 genome, and corresponding and/or equivalent positions in any other human reference genome are contemplated herein.
  • a breakpoint may be defined in terms mapping to a position or positions in a reference genome.
  • a breakpoint may be defined in terms of mapping to a position or positions in a human reference genome (e.g., HG38 human reference genome).
  • a breakpoint may map to a position in a reference genome when a nucleic acid sequence located upstream, downstream, or spanning the breakpoint aligns with a corresponding sequence in a reference genome.
  • Any suitable mapping method e.g., process, algorithm, program, software, module, the like or combination thereof
  • mapping processes are described hereafter.
  • Mapping a nucleic acid sequence may comprise mapping one or more nucleic acid sequence reads (e.g., sequence information from a fragment whose physical genomic position is unknown), which can be performed in a number of ways, and often comprises alignment of the obtained sequence reads with a matching sequence in a reference genome.
  • sequence reads generally are aligned to a reference sequence and those that align are designated as being "mapped", "a mapped sequence read” or “a mapped read”.
  • the terms “aligned”, “alignment”, or “aligning” generally refer to two or more nucleic acid sequences that can be identified as a match (e.g., 100% identity) or partial match.
  • Alignments can be done manually or by a computer (e.g., a software, program, module, or algorithm), non- limiting examples of which include the Efficient Local Alignment of Nucleotide Data (ELAND) computer program distributed as part of the Illumina Genomics Analysis pipeline.
  • Alignment of a sequence read can be a 100% sequence match. In some cases, an alignment is less than a 100% sequence match (e.g., non-perfect match, partial match, partial alignment).
  • an alignment is about a 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76% or 75% match.
  • an alignment comprises a mismatch (i.e., a base not correctly paired with its canonical Watson-Crick base partner (e.g., A or T incorrectly paired with C or G).
  • an alignment comprises 1, 2, 3, 4 or 5 mismatches. Two or more sequences can be aligned using either strand.
  • a nucleic acid sequence is aligned with the reverse complement of another nucleic acid sequence.
  • extra or missing bases within a sequence are expressed as gaps in an alignment and may or may not be factored into a percent identity calculation.
  • a percent identity calculation may include a number of mismatches and gaps or may include a number of mismatches only.
  • Various computational methods can be used to map and/or align sequence reads to a reference genome.
  • Non-limiting examples of computer algorithms that can be used to align sequences include, without limitation, BLAST, BLITZ, BWA, FASTA, BOWTIE 1, BOWTIE 2, ELAND, MAQ, PROBEMATCH, SOAP or SEQMAP, or variations thereof or combinations AMG-1022 thereof.
  • sequence reads can be aligned with reference sequences and/or sequences in a reference genome.
  • the sequence reads can be found and/or aligned with sequences in nucleic acid databases known in the art including, for example, GenBank, dbEST, dbSTS, EMBL (European Molecular Biology Laboratory) and DDBJ (DNA Databank of Japan).
  • BLAST or similar tools can be used to search the identified sequences against a sequence database.
  • a structural variant may be defined in terms of a receiving site and a donor site.
  • a receiving site may be referred to as a first partner or “partner 1” and a donor site may be referred to as a second partner or “partner 2.”
  • a structural variant may be defined in terms of comprising an ectopic portion of genomic DNA (i.e., a portion of genomic DNA at a receiving site from a different region of a chromosome or from a different chromosome).
  • the ectopic portion may be referred to as a donor portion.
  • a structural variant may comprise an ectopic portion of genomic DNA (i.e., a portion of genomic DNA at a receiving site from a different region of a chromosome or from a different chromosome).
  • the ectopic portion may be referred to as a donor portion. If the ectopic portion (donor portion) is from the same chromosome as the structural variant, the ectopic portion may be from a location outside of the position ranges provided herein for certain structural variants.
  • the ectopic portion may comprise genomic DNA from a genomic coordinate window provided herein, or part thereof.
  • the ectopic portion may comprise genomic DNA from a genomic coordinate window provided herein, or part thereof, and may further comprise genomic DNA from a region outside of a genomic coordinate window provided herein.
  • an ectopic portion of genomic DNA is characterized by its location (e.g., observed location for a given sample or samples) at a receiving site (e.g., at a structural variant site).
  • an ectopic portion is characterized by its location (e.g., observed location for a given sample samples) relative to a coding region of a gene and/or oncogene.
  • a coding region of a gene and/or oncogene generally refers to a part of the gene and/or oncogene that is transcribed and translated into protein (i.e., the sum total of its exons).
  • an ectopic portion is within a coding region of a gene and/or oncogene. In some embodiments, an ectopic portion is not within a coding region of a gene and/or oncogene.
  • an ectopic portion may be located in an intronic region, an intergenic region, or within another gene.
  • an ectopic portion is located at a position in proximity to a coding region for a gene and/or oncogene.
  • the term “in proximity” may refer to spatial proximity and/or linear proximity.
  • a structural variant may comprise extrachromosomal DNA (ecDNA) (see e.g. Dong et al., Extrachromosomal DNA (ecDNA) in cancer: mechanisms, functions, and clinical implications, Front. Oncol.(2023) 13: 1194405).
  • Spatial proximity generally refers to 3-dimensional chromatin proximity, which may be assessed according to a method that preserves spatial-proximity relationships, such as a AMG-1022 method described herein or any suitable method known in the art.
  • An ectopic portion may be located at a position in spatial proximity to a coding region for a gene and/or oncogene when an ectopic portion and a gene and/or oncogene (or a fragment thereof) are ligated in a proximity ligation assay or are bound by a common solid phase in a solid substrate-mediated proximity capture (SSPC) assay, for example.
  • Linear proximity generally refers to a linear base-pair distance, which may be assessed according to mapped distances in a reference genome, for example.

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Abstract

La technologie concerne en partie des procédés et des compositions servant à la préparation d'acides nucléiques de vésicules extracellulaires qui préservent des informations de proximité spatiale et leurs applications.
PCT/US2024/035061 2023-06-21 2024-06-21 Procédés et compositions servant à la préparation d'acides nucléiques de vésicules extracellulaires qui préservent des informations de proximité spatiale et leurs applications Pending WO2024263946A2 (fr)

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