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JP-7854946-B2 - Nucleic acid artificial miniproteome library

JP7854946B2JP 7854946 B2JP7854946 B2JP 7854946B2JP-7854946-B2

Inventors

  • フリッチュ， エドワード エフ．

Assignees

  • ディオニス セラピューティクス インコーポレイテッド

Dates

Publication Date
20260507
Application Date
20210526
Priority Date
20200526

Claims (20)

  1. A method for enriching a library of in-frame coding region fragments from a collection of RNA transcripts, wherein the method is (a) Attaching a group of RNA transcripts to a puromycin-tagged linker polynucleotide, In the population of RNA transcripts, each RNA transcript is arranged in the order from 5' to 3', (i) A translation initiation site followed by any multiple of three nucleotides that do not code for a stop codon, (ii) RNA sequences transcribed from cDNA fragment sequences from a library of cDNA sequences from tumors, (iii) A nucleotide sequence encoding a polypeptide, having a length of a multiple of 3 nucleotides, and encoded by a reading frame beginning with the first 5' nucleotide of the nucleotide sequence, the reading frame lacking an in-frame stop codon, but each of the other two reading frames containing a stop codon, comprising: Each of the linker polynucleotides tagged with puromycin contains puromycin, The 3' end of the RNA transcript is bound to the puromycin-tagged linker polynucleotide to generate a puromycin-tagged RNA transcript; this involves binding. (b) Performing an in vitro translation reaction on the puromycin-tagged RNA transcript, wherein for each puromycin-tagged RNA fragment, the RNA sequence transcribed from the cDNA fragment sequence of the puromycin-tagged RNA transcript is in the frame together with the translation start site, does not have a stop codon in the reading frame, and is in the frame together with the nucleotide sequence encoding the polypeptide, the puromycin is used to covalently bind the translated polypeptide to the puromycin-tagged RNA transcript, thereby forming an RNA complex bound to the polypeptide. (c) A method comprising separating the RNA complex bound to the polypeptide from the RNA transcript which is not in such complex state, thereby enriching a library of in-frame coding region fragments from the population of RNA transcripts.
  2. A method for enriching a library of in-frame coding region fragments from a collection of RNA transcripts, wherein the method is (a) Attaching a group of RNA transcripts to a puromycin-tagged linker polynucleotide, In the population of RNA transcripts, each RNA transcript is arranged in the order from 5' to 3', (i) A translation initiation site followed by any multiple of three nucleotides that do not code for a stop codon, (ii) A nucleotide sequence encoding a polypeptide, having a length of a multiple of 3 nucleotides, encoded by a reading frame beginning with the first 5' nucleotide of the nucleotide sequence, and lacking an in-frame stop codon within the reading frame, (iii) RNA sequences transcribed from cDNA fragment sequences from a library of cDNA sequences from tumors, (iv) an adapter sequence having a length of a multiple of 3 nucleotides, and comprising an adapter sequence which lacks a stop codon in the reading frame beginning at the first 5' nucleotide of the adapter sequence, but contains a stop codon in the other two reading frames, Each of the linker polynucleotides tagged with puromycin contains puromycin, The 3' end of the RNA transcript is bound to the puromycin-tagged linker polynucleotide to generate a puromycin-tagged RNA transcript; this involves binding. (b) Performing an in vitro translation reaction on the puromycin-tagged RNA transcript, wherein for each puromycin-tagged RNA fragment, the RNA sequence transcribed from the cDNA fragment sequence of the puromycin-tagged RNA transcript is in the frame together with the translation start site, does not have a stop codon in the reading frame, and is in the frame together with the nucleotide sequence encoding the polypeptide, the puromycin is used to covalently bind the translated polypeptide to the puromycin-tagged RNA transcript, thereby forming an RNA complex bound to the polypeptide. (c) A method comprising separating the RNA complex bound to the polypeptide from the RNA transcript which is not in such complex state, thereby enriching a library of in-frame coding region fragments from the population of RNA transcripts.
  3. The process further includes the step of generating a library of RNA transcripts by performing a transcription reaction on a library of RNA expression constructs prior to step (a), wherein each RNA expression construct is (i) Transcription promoter, (ii) A translation initiation site followed by any multiple of three nucleotides that do not code for a stop codon, (iii) a cDNA fragment sequence from a library of cDNA fragment sequences, and (iv) a nucleotide sequence encoding a polypeptide, wherein the length is a multiple of 3 nucleotides, and the sequence is encoded by a reading frame beginning at the first 5' nucleotide of the nucleotide sequence, the reading frame lacking an in-frame stop codon, but each of the other two reading frames containing a stop codon, comprising: The method according to claim 1 or 2.
  4. The method according to claim 3, wherein each RNA expression construct further comprises an adapter sequence having a length of a multiple of 3 nucleotides, lacking a stop codon in a reading frame beginning at the first 5' nucleotide of the adapter sequence, but containing stop codons in the other two reading frames.
  5. The method according to claim 3 or 4, wherein the library of cDNA fragment sequences is enriched with cDNA fragment sequences containing exomes and/or mismatches.
  6. In step (a), (A) Contacting the RNA transcript with a sprint polynucleotide and the puromycin-tagged linker polynucleotide, The aforementioned sprint polynucleotides are arranged in the order from 3' to 5', (I) A sequence complementary to the 3' end of the nucleotide sequence encoding the polypeptide, and (II) Linker target sequence, Each of the puromycin-tagged linker polynucleotides is arranged in the order from 5' to 3', (1) A sequence complementary to the linker target sequence, and (2) comprising the above-mentioned puromycin, The nucleotide sequence encoding the polypeptide of the RNA transcript is hybridized to a sequence complementary to the 3' end of the nucleotide sequence encoding the polypeptide of the sprint polynucleotide, and the sequence complementary to the linker target sequence of the linker polynucleotide is hybridized to the linker target sequence of the sprint polynucleotide, by contact. (B) The method according to any one of claims 1 to 5, wherein a ligation reaction is carried out to ligate the 3' end of the RNA transcript to the 5' end of the puromycin-tagged DNA linker to produce a puromycin-tagged RNA transcript, thereby binding the group of RNA transcripts to the puromycin-tagged linker polynucleotide.
  7. A method for enriching a library of in-frame coding region fragments from a population of cellular RNA fragments from a tumor, wherein the method is (a) A population of tumor-derived cDNA fragments is generated by performing a strand-specific random priming nucleic acid amplification reaction on a population of cellular RNA fragments, (b) Contacting the collection of cDNA fragments with an exome capture probe, thereby enriching the collection of cDNA fragments with respect to cDNA fragments encoding exomes, to generate a library of exome-enriched cDNA fragments, (c) To generate an RNA expression construct comprising: (i) a transcription promoter; (ii) a translation start site followed by any multiple of three nucleotides that do not encode a stop codon; (iii) one of the exome-enriched cDNA fragments from a library of the exome-enriched cDNA fragments; and (v) a nucleotide sequence encoding a polypeptide, having a length of a multiple of three nucleotides, encoded by a reading frame beginning at the first 5' nucleotide of the nucleotide sequence, lacking an in-frame stop codon within that reading frame, but containing stop codons within the other two reading frames. (d) Performing a transcription reaction using the RNA expression construct to produce a library of RNA transcripts, wherein each RNA transcript is in the order of 5' to 3', (i) A translation initiation site followed by any multiple of three nucleotides that do not code for a stop codon, (ii) RNA sequences transcribed from cDNA fragment sequences of the exome-enriched cDNA fragment library, (iii) To generate a library of RNA transcripts comprising a nucleotide sequence encoding a polypeptide, having a length of a multiple of 3 nucleotides, encoded by a reading frame beginning at the first 5' nucleotide of the nucleotide sequence, lacking an in-frame stop codon within that reading frame, but containing a stop codon in each of the other two reading frames, (e) Attaching a group of RNA transcripts to a puromycin-tagged linker polynucleotide, wherein each of the puromycin-tagged linker polynucleotides contains the puromycin, and the 3' end of the RNA transcript is attached to the puromycin-tagged linker polynucleotide to produce a puromycin-tagged RNA transcript. (f) Performing an in vitro translation reaction on the puromycin-tagged RNA transcript, wherein, for each puromycin-tagged RNA fragment, the RNA sequence transcribed from the cDNA fragment sequence of the puromycin-tagged RNA transcript is in the frame together with the translation start site, does not have a stop codon in the reading frame, and is in the frame together with the nucleotide sequence encoding the polypeptide, the puromycin performs an in vitro translation reaction to covalently bind the translated polypeptide to the puromycin-tagged RNA transcript, thereby forming an RNA complex bound to the polypeptide. (g) A method comprising separating the RNA complex bound to the polypeptide from the RNA transcript which is not in such a complex state, thereby enriching a library of in-frame coding region fragments from a population of cellular RNA fragments.
  8. A method for enriching a library of in-frame coding region fragments from a population of cellular RNA fragments from a tumor, wherein the method is (a) A population of cDNA fragments is generated by performing a strand-specific random priming nucleic acid amplification reaction on a population of tumor-derived cell RNA fragments, (b) Contacting the collection of cDNA fragments with an exome capture probe, thereby enriching the collection of cDNA fragments with respect to cDNA fragments encoding exomes, to generate a library of exome-enriched cDNA fragments, (c) To generate an RNA expression construct comprising (i) a transcription promoter, (ii) a translation start site followed by any multiple of three nucleotides that do not encode a stop codon, (iii) a nucleotide sequence encoding a polypeptide, having a length of a multiple of three nucleotides, encoded by a reading frame beginning at the first 5' nucleotide of the nucleotide sequence, and lacking an in-frame stop codon within the reading frame, (iv) one of the exome-enriched cDNA fragments from a library of the exome-enriched cDNA fragments, and (v) an adapter sequence having a length of a multiple of three nucleotides, lacking a stop codon within the reading frame beginning at the first 5' nucleotide of the adapter sequence, and each of the other reading frames containing a stop codon, (d) Performing a transcription reaction using the RNA expression construct to produce a library of RNA transcripts, wherein each RNA transcript is in the order of 5' to 3', (i) A translation initiation site followed by any multiple of three nucleotides that do not code for a stop codon, (ii) A nucleotide sequence encoding a polypeptide, having a length of a multiple of 3 nucleotides, encoded by a reading frame beginning at the first 5' nucleotide of the nucleotide sequence, and lacking an in-frame stop codon within the reading frame, (iii) RNA sequences transcribed from cDNA fragment sequences of the exome-enriched cDNA fragment library, and (iv) adapter sequences having a length of a multiple of 3 nucleotides, and the adapter sequences having no stop codons in the reading frame starting at the first 5' nucleotide of the adapter sequence, and each of the other reading frames having a stop codon, to generate a library of RNA transcripts, (e) Attaching a group of RNA transcripts to a puromycin-tagged linker polynucleotide, wherein each of the puromycin-tagged linker polynucleotides contains the puromycin, and the 3' end of the RNA transcript is attached to the puromycin-tagged linker polynucleotide to produce a puromycin-tagged RNA transcript. (f) Performing an in vitro translation reaction on the puromycin-tagged RNA transcript, wherein, for each puromycin-tagged RNA fragment, the RNA sequence transcribed from the cDNA fragment sequence of the puromycin-tagged RNA transcript is in the frame together with the translation start site, does not have a stop codon in the reading frame, and is in the frame together with the nucleotide sequence encoding the polypeptide, the puromycin performs an in vitro translation reaction to covalently bind the translated polypeptide to the puromycin-tagged RNA transcript, thereby forming an RNA complex bound to the polypeptide. (g) A method comprising separating the RNA complex bound to the polypeptide from the RNA transcript which is not in such a complex state, thereby enriching a library of in-frame coding region fragments from a population of cellular RNA fragments.
  9. The method according to claim 7 or 8, wherein step (b) further comprises (1) contacting the collection of cDNA fragments with MutS protein to enrich the collection of cDNA fragments with cDNA fragments containing mismatches resulting from either mutation or single nucleotide polymorphism, or (2) contacting the library of exome-enriched cDNA fragments with MutS protein to enrich the library of exome-enriched cDNA fragments with cDNA fragments containing mismatches resulting from either mutation or single nucleotide polymorphism .
  10. In step (e), (A) Contacting the RNA transcript with a sprint polynucleotide and the puromycin-tagged linker polynucleotide, The aforementioned sprint polynucleotides are arranged in the order from 3' to 5', (I) A sequence complementary to the 3' end of the nucleotide sequence encoding the polypeptide, and (II) Linker target sequence, Each of the puromycin-tagged linker polynucleotides is arranged in the order from 5' to 3', (1) A sequence complementary to the linker target sequence, and (2) comprising the above-mentioned puromycin, The nucleotide sequence encoding the polypeptide of the RNA transcript is hybridized to a sequence complementary to the 3' end of the nucleotide sequence encoding the polypeptide of the sprint polynucleotide, and the sequence complementary to the linker target sequence of the linker polynucleotide is hybridized to the linker target sequence of the sprint polynucleotide, by contact. (B) The method according to any one of claims 7 to 9, wherein a ligation reaction is carried out to ligate the 3' end of the RNA transcript to the 5' end of the puromycin-tagged DNA linker to produce a puromycin-tagged RNA transcript, thereby binding the group of RNA transcripts to the puromycin-tagged linker polynucleotide.
  11. The method according to any one of claims 1 to 10, wherein the RNA complex bound to the polypeptide is separated from the RNA transcript that is not in the form of such complex by affinity purification of the RNA complex bound to the polypeptide using a reagent that binds to the polypeptide encoded by the nucleotide sequence encoding the polypeptide.
  12. The method according to claim 11, further comprising performing an RT-PCR amplification reaction on a library of RNA complexes bound to the purified polypeptide to produce an amplified product containing the sequence of the cDNA fragment.
  13. The method according to claim 1 or 2, further comprising contacting the amplification product with a MutS protein to enrich the amplification product with respect to a cDNA fragment containing a mismatch resulting from either a mutation or a single nucleotide polymorphism.
  14. The method according to claim 12 or 13 , further comprising inserting the amplification product into a vector to generate a vector containing the sequence of the cDNA fragment.
  15. The method according to claim 14 , wherein the vector is a cloning vector, an expression vector, or a vector encoding a vaccine, and the vaccine encoded by the vaccine encoding vector is produced from the amplification product.
  16. The method according to claim 14 or 15, wherein the vector is a vector encoding a vaccine, and the method further comprises (1) inserting the vector encoding the vaccine into a bacterium or yeast and incubating the bacterium or yeast under conditions such that the bacterium or yeast expresses the vaccine encoded by the vector encoding the vaccine, or ( 2 ) subjecting the vector encoding the vaccine to an in vitro translation reaction to produce the vaccine encoded by the vector encoding the vaccine.
  17. The method described above is (1) Transfect or transduce the vector into mammalian cells and incubate the mammalian cells under conditions such that the vaccine encoded by the vector is expressed in the mammalian cells. (2) The method according to claim 14 or 15 , further comprising transfecting or transfecting human cells with the vector ex vivo.
  18. The method according to claim 17 , wherein the mammalian cell is a human cell.
  19. A pharmaceutical composition comprising a vector or its translation product produced by the method of claim 14 or 15 , and a pharmaceutically acceptable carrier.
  20. A method for producing a tumor vaccine, (a) Generating cell RNA fragments from the target tumor sample, (b) A cDNA fragment is generated by performing a strand-specific random priming nucleic acid amplification reaction on the RNA fragment, (c) Contacting the cDNA fragment with an exome capture probe, thereby enriching the cDNA fragment with respect to the cDNA fragment encoding the exome, and generating a library of exome-enriched cDNA fragments. (d) To generate an RNA expression construct comprising: (i) a transcription promoter; (ii) a translation start site followed by any multiple of three nucleotides that do not encode a stop codon; (iii) one of the exome-enriched cDNA fragments from a library of the exome-enriched cDNA fragments; and (iv) a nucleotide sequence encoding a polypeptide, having a length of a multiple of three nucleotides, encoded by a reading frame beginning at the first 5' nucleotide of the nucleotide sequence, lacking an in-frame stop codon within the reading frame, but containing a stop codon in each of the other two reading frames. (e) Performing a transcription reaction using the RNA expression construct to produce a library of RNA transcripts, wherein each RNA transcript is in the order of 5' to 3', (i) A translation initiation site followed by any multiple of three nucleotides that do not code for a stop codon, (ii) RNA sequences transcribed from cDNA fragment sequences of the exome-enriched cDNA fragment library, (iii) Generating a library of RNA transcripts comprising a polypeptide encoding nucleotide sequence having a length of a multiple of 3 nucleotides, encoded by a reading frame beginning at the first 5' nucleotide of the nucleotide sequence, lacking an in-frame stop codon within that reading frame, but containing a stop codon in each of the other two reading frames, (f) Binding the RNA transcript to a puromycin-tagged linker polynucleotide, wherein each of the puromycin-tagged linker polynucleotides contains puromycin, and the 3' end of the RNA transcript is bound to the puromycin-tagged linker polynucleotide to produce a puromycin-tagged RNA transcript. (g) Performing an in vitro translation reaction on the puromycin-tagged RNA transcript, wherein, for each puromycin-tagged RNA fragment, the RNA sequence transcribed from the cDNA fragment sequence of the puromycin-tagged RNA transcript is in the frame together with the translation start site, does not have a stop codon in the reading frame, and is in the frame together with the nucleotide sequence encoding the polypeptide, the puromycin is used to covalently bind the translated polypeptide to the puromycin-tagged RNA transcript, thereby forming an RNA complex bound to the polypeptide, in an in vitro translation reaction. (h) Purifying the RNA complex bound to the polypeptide with affinity using a reagent that binds to the polypeptide encoded by the nucleotide sequence encoding the polypeptide, thereby generating a library of the purified polypeptide-bound RNA complex, (i) An amplification reaction is carried out on the library of RNA complexes bound to the purified polypeptide to produce an amplified product containing the sequence of the cDNA fragment, (j) A method comprising generating a tumor vaccine from one or more of the amplification products of step (i).

Description

Related Application This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/030,056, filed May 26, 2020, which is incorporated herein by reference in its entirety. The availability of nucleic acid artificial miniproteome libraries enriched with sequences encoding open reading frames would have many different potential applications. For example, such libraries would be valuable in the production of vaccines, particularly cancer vaccines. Vaccines have a long history in cancer treatment. Cancer vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., cytokines or TLR ligands) that work together to activate antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. Such vaccines often contain either co-represented or patient-specific tumor antigens or whole tumor cell preparations. Co-represented tumor antigens are immunogenic proteins that are selectively expressed in tumors across many individuals and are generally delivered to patients as synthetic peptides, recombinant proteins, RNA, or DNA vectors. Patient-specific tumor antigens used in vaccines consist of proteins with tumor-specific mutations that result in changes in the amino acid sequence. Such mutated proteins have the potential to (a) uniquely mark tumors (compared to non-tumor cells) for recognition and destruction by the immune system, and (b) evade central, and occasionally peripheral, T cell resistance, and thus be recognized by more effective high-avidity T cell receptors. Whole tumor cell preparations contain all potential antigens present in tumor cells and can be delivered to the patient as autoirradiated cells, cell lysates, cell fusions, heat shock protein preparations, or total mRNA (or a cDNA/DNA vector corresponding to total mRNA). When whole tumor cells are isolated from an autologous patient, the cells express patient-specific tumor antigens as well as co-existing tumor antigens. Total mRNA from cells is used to prepare cancer vaccines based on the total cellular proteome. However, such mRNA samples are often fragmented, especially when obtained from paraffin-embedded (FFPE) samples. A problem with using fragmented mRNA from tumor cells as a cancer vaccine is that most of the RNA fragments are not within a suitable reading frame for effective translation. Therefore, there remains a need for improved nucleic acid miniproteome libraries enriched with open reading frame fragments, which are useful for cancer vaccine production. In particular, there remains a need for the preparation of improved nucleic acid miniproteome libraries for the preparation of personalized vaccines based on the proteomic composition of each individual. This is a schematic diagram illustrating the synthesis of chained, double-stranded (ds) cDNA. If necessary, the same library may be used to capture open reading frames (ORFs) from antisense RNA, but an exome capture mixture in the opposite direction is required, and the strand specificity of the primers is reversed in subsequent steps.This is a schematic diagram illustrating the enrichment (arbitrary selection) and exome capture of MutS.This is a schematic diagram illustrating the preparation of RNA for display. A sequence encoding a small protein can be added to the 5' upstream or 3' downstream region of a cDNA fragment sequence from a cDNA library.This is a schematic diagram illustrating RNA display.This is a schematic diagram illustrating the capture and recovery of polypeptide-bound RNA (AMPL-NA library fragments).This is a schematic diagram illustrating an exemplary cloning process for a film surface display.This is a schematic diagram illustrating the transformation, proliferation, and surface presentation of an in-frame library member according to certain exemplary embodiments disclosed herein.This is a schematic diagram illustrating affinity enrichment and DNA recovery of an in-frame library according to certain exemplary embodiments disclosed herein.This is a schematic diagram showing the structure of an exemplary exome capture transcription library. RBS is the E. coli ribosome binding site, ATG is the start codon for protein translation, Read1 and Read2 are Illumina TrSeq sequences, the Twin-Strep-tag is the coding sequence of a 28-amino acid peptide used for binding and purification, and the peptide is the coding sequence of the peptide spacer segment.The results of comparing the full-length insertions within the target reading frame of the construct with the complete ORF after exome capture ("before RNA display") and subsequent RNA display ("after RNA display") are shown. Overview In certain embodiments, the foregoing provides a method for enriching a library of in-frame coding region fragments from a population of RNA transcripts or from a population of cellular RNA fragments. In some embodiments, the foregoing provides a method for generating a tumor vaccine or for treating a patient with a tumor using the generate