Patent Application
20230203509
Transfer RNAs (tRNAs) are complex, naturally occurring RNA molecules that possess a number of functions including initiation and elongation of proteins.
The present disclosure features modified tRNA-based effector molecules (TREMs, e.g., a TREM, TREM core fragment, or TREM fragment), as well as related compositions and uses thereof. The inventors have discovered that a TREM composition can be used, inter alia, to: (1) modulate a production parameter of an RNA, or a protein encoded by an RNA, wherein the RNA has a contextually-rare codon (“con-rare codon”); and/or (2) to modulate tRNA pools in a cell or a subject. Accordingly, in an aspect, provided herein is a method of modulating a tRNA pool in a cell comprising an endogenous open reading frame (ORF), which ORF comprises a codon having a first sequence, comprising: optionally, acquiring knowledge of the abundance of one or both of (i) and (ii), e.g., acquiring knowledge of the relative amounts of: (i) and (ii) in the cell, wherein (i) is a tRNA moiety having an anticodon that pairs with the codon of the ORF having a first sequence (the first tRNA moiety) and (ii) is an isoacceptor tRNA moiety having an anticodon that pairs with a codon other than the codon having the first sequence (the second tRNA moiety) in the cell; contacting the cell with a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, wherein the TREM, TREM core fragment or TREM fragment has an anticodon that pairs with: (a) the codon having the first sequence; or (b) the codon other than the codon having the first sequence, in an amount and/or for a time sufficient to modulate the relative amounts of the first tRNA moiety and the second tRNA moiety in the cell, thereby modulating the tRNA pool in the cell.
In an embodiment, the TREM comprises an anticodon that pairs with (a). In an embodiment, the TREM comprises an anticodon that pairs with (b).
In another aspect, provided herein is a method of modulating a tRNA pool in a subject having an endogenous open reading frame (ORF), which ORF comprises a codon having a first sequence, comprising: optionally, acquiring knowledge of the abundance of one or both of (i) and (ii), e.g., acquiring knowledge of the relative amounts of: (i) and (ii) in the subject, wherein (i) is a tRNA moiety having an anticodon that pairs with the codon of the ORF having a first sequence (the first tRNA moiety) and (ii) is an isoacceptor tRNA moiety having an anticodon that pairs with a codon other than the codon having the first sequence (the second tRNA moiety) in the subject; contacting the subject with a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, wherein the TREM, TREM core fragment or TREM fragment has an anticodon that pairs with: (a) the codon having the first sequence; or (b) the codon other than the codon having the first sequence, in an amount and/or for a time sufficient to modulate the relative amounts of the first tRNA moiety and the second tRNA moiety in the subject, thereby modulating the tRNA pool in the subject.
In an embodiment, the TREM comprises an anticodon that pairs with (a). In an embodiment, the TREM comprises an anticodon that pairs with (b).
In an embodiment, the method comprises acquiring knowledge of (i). In an embodiment, the method comprises acquiring knowledge of (ii). In an embodiment, the method comprises acquiring knowledge of (i) and (ii).
In an embodiment, acquiring knowledge of (i) comprises acquiring a value for the abundance, e.g., relative amounts, of (i); and/or acquiring knowledge of (ii) comprises acquiring a value for the abundance, e.g., relative amounts, of (ii). In an embodiment, responsive to said value, the cell or subject is contacted with the TREM composition having an anticodon that pairs with (a) or (b).
In one aspect, the disclosure provides, a method of evaluating a tRNA pool in a cell or subject, comprising an endogenous open reading frame (ORF), which ORF comprises a codon having a first sequence, comprising acquiring, e.g., directly or indirectly acquiring, knowledge of the abundance of one or both of (i) and (ii), e.g., acquiring knowledge of the relative amounts of (i) and (ii) in the cell or subject wherein:
(i) is a tRNA moiety having an anticodon that pairs with the codon of the ORF having a first sequence (the first tRNA moiety); and
(ii) is an isoacceptor tRNA moiety having an anticodon that pairs with a codon other than the codon having the first sequence (the second tRNA moiety) in the cell or subject,
thereby evaluating the tRNA pool in the cell or subject.
In an embodiment, the method comprises acquiring knowledge of (i). In an embodiment, the method comprises acquiring knowledge of (ii). In an embodiment, the method comprises acquiring knowledge of (i) and (ii).
In an embodiment, acquiring knowledge of (i) comprises acquiring a value for the abundance, e.g., relative amount, of (i); and/or acquiring knowledge of (ii) comprises acquiring a value for the abundance, e.g., relative amount, of (ii). In an embodiment, responsive to said value, the method comprises administering a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, wherein the TREM, TREM core fragment or TREM fragment has an anticodon that pairs with: (a) the codon having the first sequence; or (b) the codon other than the codon having the first sequence, in an amount and for a time sufficient to modulate the relative amounts of the first tRNA moiety and the second tRNA moiety.
In yet another aspect, provided herein is a method of modulating a tRNA pool in a subject or cell having an endogenous open reading frame (ORF) comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC), comprising:
providing a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, wherein the TREM, TREM core fragment or TREM fragment comprises an isoacceptor tRNA moiety comprising an anticodon sequence that pairs with the SMC (the TREM);
contacting the subject or cell with the TREM composition in an amount and/or for a time sufficient to modulate the tRNA pool in the subject or cell,
thereby modulating the tRNA pool in the subject or cell.
In an embodiment, the method comprises acquiring knowledge of the abundance of one or both of (i) and (ii) e.g., acquiring knowledge of the relative amounts of (i) and (ii) wherein (i) is a tRNA moiety having an anticodon that pairs with the SMC (the first tRNA moiety) and (ii) is an isoacceptor tRNA moiety having an anticodon that pairs with a codon other than the SMC (the second tRNA moiety), in the subject or cell.
In one aspect, the disclosure provides a method of treating a subject having an endogenous open reading frame (ORF) comprising a codon having a first sequence, comprising:
(i) acquiring, e.g., directly or indirectly acquiring, a value for the status of the codon having the first sequence in the subject, wherein said value comprises a measure of the presence or absence of the codon having the first sequence in a sample from the subject; and identifying the subject as having the codon having the first sequence; and
(ii) responsive to said value, administering a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, wherein the TREM, TREM core fragment or TREM fragment comprises an isoacceptor tRNA moiety having an anticodon that pairs with the codon having the first sequence, to the subject,
thereby treating the subject.
In yet another aspect, provided herein is a method of treating a subject having an endogenous open reading frame (ORF) comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC), comprising:
(i) acquiring, e.g., directly or indirectly acquiring, a value for the SMC status of the subject, wherein said value comprises a measure of the presence or absence of SMC in a sample from the subject, and identifying the subject as having a SMC; and
(ii) responsive to said value, administering a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, wherein the TREM, TREM core fragment or TREM fragment comprises comprising an isoacceptor tRNA moiety having an anticodon that pairs with the SMC, to the subject,
thereby treating the subject.
In an aspect, the disclosure provides a method of evaluating a subject having an endogenous open reading frame (ORF) comprising a codon having a first sequence, comprising:
acquiring, e.g., directly or indirectly acquiring, a value for the status of the codon having the first sequence in the subject, wherein said value comprises a measure of the presence or absence of the codon having the first sequence in a sample from the subject; and
identifying the subject as having a codon having the first sequence,
thereby evaluating the subject.
In another aspect, provided herein is a method of evaluating a subject having an endogenous open reading frame (ORF) comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC), comprising:
acquiring, e.g., directly or indirectly acquiring, a value for the SMC status of the subject, wherein said value comprises a measure of the presence or absence of SMC in a sample from the subject; and
identifying the subject as having a SMC,
thereby evaluating the subject.
In an embodiment of any of the methods disclosed herein, the TREM, TREM core fragment or TREM fragment does not comprise an anticodon that pairs with a stop codon.
In an embodiment of any of the methods disclosed herein, (a) the ORF codon having the first sequence; or (b) the SMC; is other than a stop codon, e.g., TAA, TGA or TAG.
In an embodiment, (a) the ORF codon having the first sequence; or (b) the SMC; in the absence of contact with the composition comprising a TREM, is associated with a phenotype, e.g., an unwanted phenotype, e.g., a disorder or symptom, e.g., a disorder or symptom chosen from Table 1. In an embodiment, the disorder or symptom is chosen from a disease group provided in Table 1, e.g., cardiovascular, dermatology, endocrine, immunology, neurology, oncology, ophthalmology, or respiratory.
In one aspect, disclosed herein is a method of modulating a production parameter of an RNA, or a protein encoded by an RNA, in a target cell or tissue, comprising:
providing, e.g., administering, to the target cell or tissue, or contacting the target cell or tissue with, an effective amount of a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, which TREM, TREM core fragment or TREM fragment corresponds to a contextually-rare codon (“con-rare codon”) of the RNA,
thereby modulating the production parameter of the RNA, or protein encoded by the RNA in the target cell or tissue.
In an embodiment, the target cell or tissue is obtained from a subject.
In an embodiment, the method comprises administering the TREM composition to a subject.
In an embodiment, the method comprises contacting the TREM composition with the target tissue or cell ex vivo.
In an embodiment, the method comprises introducing the ex vivo-contacted target tissue or cell into a subject, e.g., an allogeneic or autologous subject.
In an embodiment, the target cell or tissue is a specific or selected target cell or tissue, e.g., a cell or tissue type in a particular developmental stage; a cell or tissue type in a particular disease state; or a cell present in a particular extracellular milieu.
In an embodiment of any of the methods disclosed herein, the production parameter comprises an expression parameter or a signaling parameter, e.g., as described herein. In an embodiment, the production parameter of the RNA is modulated, e.g., an RNA that can be translated into a polypeptide, e.g., a messenger RNA. In an embodiment, the production parameter of the RNA is increased or decreased. In an embodiment, the production parameter of the protein encoded by the RNA is modulated. In an embodiment, the production parameter of the protein is increased. In an embodiment, the production parameter of the protein is decreased.
In another aspect, provided herein is a method of determining the presence of a nucleic acid sequence, e.g., a DNA or RNA, having a contextually-rare codon (“con-rare codon nucleic acid sequence”), comprising:
acquiring knowledge of the presence of the con-rare codon nucleic acid sequence in a sample from a subject, e.g., a target cell or tissue sample,
wherein responsive to the acquisition of knowledge of the presence of the con-rare codon nucleic acid sequence:
(1) the subject is classified as being a candidate to receive administration of an effective amount of a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, which TREM, TREM core fragment or TREM fragment corresponds to a contextually-rare codon (“con-rare codon”) of the nucleic acid sequence; or
(2) the subject is identified as likely to respond to a treatment comprising the TREM composition.
In yet another aspect, the disclosure provides a method of treating a subject having a disease associated with a contextually-rare codon (“con-rare codon”), comprising:
acquiring knowledge of the presence of a nucleic acid sequence, e.g., a DNA or RNA, having the con-rare codon (“con-rare codon nucleic acid sequence”) in a target cell or tissue sample from the subject; and
administering to the subject an effective amount of a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, which TREM, TREM core fragment or TREM fragment corresponds to the con-rare codon of the nucleic acid sequence,
thereby treating the disease in the subject.
In an aspect, provided herein is a method of providing a tRNA effector molecule (TREM) to a subject, comprising:
providing, e.g., administering, to the subject, an effective amount of a TREM composition comprising a TREM, a TREM core fragment, or a TREM fragment described herein, which TREM, TREM core fragment or TREM fragment corresponds to a contextually-rare codon (“con-rare codon”) for a nucleic acid sequence in a target cell or tissue in the subject,
thereby providing a TREM composition to the subject.
In an embodiment of any of the methods disclosed herein, the method comprises acquiring a value for a con-rare codon in the nucleic acid sequence, e.g., DNA or RNA, wherein the value is a function of one or more of the following factors, e.g., by evaluating or determining one or more of the following factors:
(1) the sequence of the codon;
(2) the availability of a corresponding tRNA, e.g., charged tRNA, for that con-rare codon in a target cell or tissue, e.g., one or more iso-acceptor tRNA molecules;
(3) the expression profile (or proteomic properties) of the target cell or tissue (e.g., the abundance of expression of other proteins which include the con-rare codon);
(4) the proportion of the tRNAs corresponding to the con-rare codon which are charged; and
(5) the iso-decoder isotype of the tRNA corresponding to the con-rare codon;
In an embodiment, (1) comprises determining the presence or absence of a con-rare codon.
In an embodiment, a determination of the availability of a tRNA comprises acquiring a measure of one, two, three or all of the following parameters:
(a) level of a tRNA corresponding to the con-rare codon (“con-rare codon tRNA”) compared to a tRNA corresponding to a different codon;
(b) function, e.g., polypeptide chain elongation function, of a con-rare codon tRNA compared to a tRNA corresponding to a different codon;
(c) modification, e.g., aminoacylation or post-transcriptional modification, of a con-rare codon tRNA compared to a tRNA corresponding to a different codon; and/or
(d) sequence of a con-rare codon tRNA.
In an embodiment of any of the methods disclosed herein, the method comprises acquiring a value of the knowledge of the abundance of e.g., acquiring knowledge of the relative amounts of: (i) a tRNA moiety having an anticodon that pairs with the con-rare codon (the first tRNA moiety).
In an embodiment of any of the methods disclosed herein, the method comprises acquiring a value of the knowledge of the abundance of e.g., acquiring knowledge of the relative amounts of: (ii) an isoacceptor tRNA moiety having an anticodon that pairs with a codon other than the con-rare codon (the second tRNA moiety).
In an embodiment, the method comprises acquiring a value of the knowledge (i) and (ii).
In an embodiment of any of the methods disclosed herein, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs, TREM core fragments or TREM fragments in the TREM composition correspond to a con-rare codon.
In an embodiment of any of the methods disclosed herein, the TREM composition comprises TREMs, TREM core fragments or TREM fragments that correspond to a plurality of con-rare codons.
In an embodiment of any of the methods disclosed herein, the TREM composition comprises: a first TREM which corresponds to a first con-rare codon; and an additional TREM which corresponds to a different con-rare codon.
In an embodiment of any of the methods disclosed herein, the TREM composition comprises: a first TREM which corresponds to a first con-rare codon; and a second TREM which corresponds to a second con-rare codon.
In an embodiment of any of the methods disclosed herein, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs in the composition are charged.
In an embodiment of any of the methods disclosed herein, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs in the composition are of the same iso-decoder isotype.
In an embodiment of any of the methods disclosed herein, the cell is a host cell. In an embodiment, the cell is a host cell chosen from: a HeLa cell, a HEK293T cell (e.g., a Freestyle 293-F cell), a HT-1080 cell, a PER.C6 cell, a HKB-11 cell, a CAP cell, a HuH-7 cell, a BHK 21 cell, an MRC-S cell, a MDCK cell, a VERO cell, a WI-38 cell, or a Chinese Hamster Ovary (CHO) cell.
In an embodiment of any of the methods disclosed herein, the cell comprises an exogenous nucleic acid sequence. In an embodiment, the cell is autologous to the exogenous nucleic acid sequence. In an embodiment, the cell is allogeneic to the exogenous nucleic acid sequence. In an embodiment, the exogenous nucleic acid sequence (e.g., DNA or RNA) comprises a con-rare codon.
In an embodiment, administration of a TREM composition corresponding to the con-rare codon to the cell, modulates a production parameter, e.g., expression parameter or signaling parameter, of a product, e.g., RNA or polypeptide, of the exogenous nucleic sequence.
In an aspect, provided herein is a TREM comprising a sequence of Formula A:
[L1]-[ASt Domain1]-[L2]-[DH Domain]-[L3]-[ACH Domain]-[VL Domain]-[TH Domain]-[L4]-[ASt Domain2],
wherein independently, [L1] and [VL Domain], are optional; and one of [L1], [ASt Domain1], [L2]-[DH Domain], [L3], [ACH Domain], [VL Domain], [TH Domain], [L4], and [ASt Domain2] comprises a nucleotide having a non-naturally occurring modification.
In an embodiment, the TREM: (a) retains the ability to: support protein synthesis, be charged by a synthetase, be bound by an elongation factor, introduce an amino acid into a peptide chain, support elongation, or support initiation; (b) comprises at least X contiguous nucleotides without a non-naturally occurring modification, wherein X is greater than 10; (c) comprises at least 3, but less than all of the nucleotides of a type (e.g., A, T, C, G or U) comprise the same non-naturally occurring modification; (d) comprises at least X nucleotides of a type (e.g., A, T, C, G or U) that do not comprise a non-naturally occurring modification, wherein X=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50; (e) comprises no more than 5, 10, or 15 nucleotides of a type (e.g., A, T, C, G or U) that comprise a non-naturally occurring modification; and/or (f) comprises no more than 5, 10, or 15 nucleotides of a type (e.g., A, T, C, G or U) that do not comprise a non-naturally occurring modification.
In an embodiment, the TREM comprises feature (a). In an embodiment, the TREM comprises feature (b). In an embodiment, the TREM comprises feature (c). In an embodiment, the TREM comprises feature (d). In an embodiment, the TREM comprises feature (e). In an embodiment, the TREM comprises feature (f). In an embodiment, the TREM comprises all of features (a)-(f) or a combination thereof.
In an embodiment, the TREM Domain comprising the non-naturally occurring modification retains a function, e.g., a domain function described herein.
In an aspect, provided herein is a TREM core fragment comprising a sequence of Formula B:
[L1]y-[ASt Domain1]x-[L2]y-[DH Domain]y-[L3]y-[ACH Domain]x-[VL Domain]y-[TH Domain]y-[L4]y-[ASt Domain2]x,
wherein x=1 and y=0 or 1; and one of [ASt Domain1], [ACH Domain], and [ASt Domain2] comprises a nucleotide having a non-naturally occurring modification.
In an embodiment, the TREM retains the ability to support protein synthesis. In an embodiment, the TREM retains the ability to be able to be charged by a synthetase. In an embodiment, the TREM retains the ability to be bound by an elongation factor. In an embodiment, the TREM retains the ability to introduce an amino acid into a peptide chain. In an embodiment, the TREM retains the ability to support elongation. In an embodiment, the TREM retains the ability to support initiation.
In an embodiment, the [ASt Domain 1] and/or [ASt Domain 2] comprising the non-naturally occurring modification retains the ability to initiate or elongate a polypeptide chain.
In an embodiment, the [ACH Domain] comprising the non-naturally occurring modification retains the ability to mediate pairing with a codon.
In an embodiment, y=1 for any one, two, three, four, five, six, all or a combination of [L1], [L2], [DH Domain], [L3], [VL Domain], [TH Domain], [L4].
In an embodiment, y=0 for any one, two, three, four, five, six, all or a combination of [L1], [L2], [DH Domain], [L3], [VL Domain], [TH Domain], [L4].
In an embodiment, y=1 for linker [L1], and L1 comprises a nucleotide having a non-naturally occurring modification.
In an embodiment, y=1 for linker [L2], and L2 comprises a nucleotide having a non-naturally occurring modification.
In an embodiment, y=1 for [DH Domain (DHD)], and DHD comprises a nucleotide having a non-naturally occurring modification. In an embodiment, the DHD comprising the non-naturally occurring modification retains the ability to mediate recognition of aminoacyl-tRNA synthetase.
In an embodiment, y=1 for linker [L3], and L3 comprises a nucleotide having a non-naturally occurring modification.
In an embodiment, y=1 for [VL Domain (VLD)], and VLD comprises a nucleotide having a non-naturally occurring modification.
In an embodiment, y=1 for [TH Domain (THD)], and THD comprises a nucleotide having a non-naturally occurring modification. In an embodiment, the THD comprising the non-naturally occurring modification retains the ability to mediate recognition of the ribosome.
In an embodiment, y=1 for linker [L4], and L4 comprises a nucleotide having a non-naturally occurring modification.
In another aspect, the disclosure provides a TREM fragment comprising a portion of a TREM, wherein the TREM comprises a sequence of Formula A:
[L1]-[ASt Domain1]-[L2]-[DH Domain]-[L3]-[ACH Domain]-[VL Domain]-[TH Domain]-[L4]-[ASt Domain2], and wherein the TREM fragment comprises a non-naturally occurring modification.
In an embodiment, the TREM fragment comprises one, two, three or all or any combination of the following: (a) a TREM half (e.g., from a cleavage in the ACH Domain, e.g., in the anticodon sequence, e.g., a 5′ half or a 3′ half); (b) a 5′ fragment (e.g., a fragment comprising the 5′ end, e.g., from a cleavage in a DH Domain or the ACH Domain); (c) a 3′ fragment (e.g., a fragment comprising the 3′ end, e.g., from a cleavage in the TH Domain); or (d) an internal fragment (e.g., from a cleavage in any one of the ACH Domain, DH Domain or TH Domain).
In an embodiment, the TREM fragment comprise (a) a TREM half which comprises a nucleotide having a non-naturally occurring modification.
In an embodiment, the TREM fragment comprise (b) a 5′ fragment which comprises a nucleotide having a non-naturally occurring modification.
In an embodiment, the TREM fragment comprise (c) a 3′ fragment which comprises a nucleotide having a non-naturally occurring modification.
In an embodiment, the TREM fragment comprise (d) an internal fragment which comprises a nucleotide having a non-naturally occurring modification.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the TREM Domain comprises a plurality of nucleotides each having a non-naturally occurring modification.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the non-naturally occurring modification is a modification in a base or a backbone of a nucleotide, e.g., a modification chosen from any one of Tables 5, 6, 7, 8 or 9.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the non-naturally occurring modification is a base modification chosen from a modification listed in Table 5.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the non-naturally occurring modification is a base modification chosen from a modification listed in Table 6.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the non-naturally occurring modification is a base modification chosen from a modification listed in Table 7.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the non-naturally occurring modification is a backbone modification chosen from a modification listed in Table 8.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the non-naturally occurring modification is a backbone modification chosen from a modification listed in Table 9.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the TREM, TREM core fragment, or TREM fragment is encoded by a sequence provided in Table 4, e.g., any one of SEQ ID NOs 1-451.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the TREM, TREM core fragment, or TREM fragment is encoded by a consensus sequence chosen from any one of SEQ ID NOs: 562-621.
In another aspect, the disclosure provides a pharmaceutical composition comprising a TREM, a TREM core fragment, or a TREM fragment disclosed herein.
In another aspect, the disclosure provides a method of making a TREM, a TREM core fragment, or a TREM fragment disclosed herein, comprising linking a first nucleotide to a second nucleotide to form the TREM.
In an embodiment, the TREM, TREM core fragment or TREM fragment is synthetic.
In an embodiment, the TREM, TREM core fragment or TREM fragment is made by cell-free solid phase synthesis.
In an embodiment of any of the TREMs, TREM core fragments, or TREM fragments disclosed herein, the TREM Domain comprises a plurality of nucleotides each having a non-naturally occurring modification. In an embodiment, the non-naturally occurring modification comprises a nucleobase modification, a sugar (e.g., ribose) modification, or a backbone modification. In an embodiment, tbe non-naturally occurring modification is a sugar (e.g., ribose) modification. In an embodiment, tbe non-naturally occurring modification is 2′-ribose modification, e.g., a 2′-OMe, 2′-halo (e.g., 2′-F), 2′-MOE, or 2′-deoxy modification. In an embodiment, tbe non-naturally occurring modification is a backbone modification, e.g., a phosphorothioate modification.
Additional features of any of the aforesaid TREMs, TREM core fragments, TREM fragments, TREM compositions, preparations, methods of making TREM compositions and preparations, and methods of using TREM compositions and preparations include one or more of the following enumerated embodiments.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following enumerated embodiments.
Other features, objects, and advantages of the invention will be apparent from the Examples, Description, Figures, and Claims.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The present disclosure features uses of tRNA-based effector molecules (TREMs) comprising a non-naturally occurring modification to modulate tRNA pools in a cell or a subject. Also disclosed herein are methods of treating a disorder or ameliorating a symptom of a disorder by administering a composition comprising a TREM or a pharmaceutical composition comprising a TREM. Further disclosed herein are uses of TREMs comprising a non-naturally occurring modification to modulate a production parameter of an RNA, or a protein encoded by an RNA, wherein the RNA has a contextually-rare codon (“con-rare codon”).
As disclosed herein, TREMs are complex molecules which can mediate a variety of cellular processes. TREMs of the disclosure include TREMs, TREM core fragments and TREM fragments. TREMs, TREM core fragments or TREM fragments can be modified with non-naturally occurring modifications to, e.g., increase the level and/or activity (e.g., stability) of the TREM. Pharmaceutical TREM compositions, e.g., TREMs comprising a non-naturally occurring modification, can be administered to a cell, a tissue, or to a subject to modulate these functions, e.g., in vitro or in vivo. Disclosed herein are TREMs, TREM core fragments or TREM fragments comprising non-naturally occurring modifications, TREM compositions, preparations, methods of making TREM compositions and preparations, and methods of using the same.
A “contextually rare codon” or “con-rare codon”, as those terms are used herein, refer to a codon which, in a target cell or tissue, is limiting for a production parameter, e.g., an expression parameter, for a nucleic acid sequence having a con-rare codon (“con-rare codon nucleic acid sequence”), e.g., because the availability of a tRNA corresponding to the con-rare codon is limiting for a production parameter. Contextual rareness or con-rarity can be identified or evaluated by determining if the addition of a tRNA corresponding to a con-rare codon modulates, typically increases, a production parameter for a nucleic acid sequence, e.g., gene. By way of example, the method of Example 3, can be used, or adapted to be used, to evaluate con-rarity. Con-rarity as a property of a codon, is a function of, and can be identified or evaluated on the basis of, one, two, three, four, five, six, or all of the following factors:
(1) the sequence of the con-rare codon, or candidate con-rare codon;
(2) the availability of a corresponding tRNA for the con-rare, or candidate con-rare, -codon in a target cell or tissue Availability as a parameter can comprise or be a function of, one or both of the observed or predicted abundance or availability of a tRNA that corresponds to the con-rare codon. In an embodiment, abundance can be evaluated by quantifying tRNAs present in a target cell or tissue. See, e.g., Example 12;
(3) the contextual demand (the demand in a target cell or tissue) for a tRNA, e.g., a con-rare tRNA, or a candidate con-rare tRNA. This can be identified or evaluated by use of a parameter, a contextual demand-parameter, which comprises or is a function of, the demand or usage of a con-rare tRNA by one, some, or all of the nucleic acid sequences having con-rare codons in a target tissue or cell, e.g., the other nucleic acid sequences in a target cell or tissue which have a con-rare codon. A demand parameter can comprise of, or be a function of one or more, or all of:
(4) a parameter (or use-parameter) related to the con-rare codon usage in a con-rare codon nucleic acid sequence, and can include one or more of:
(5) the proportion of the tRNAs corresponding to the con-rare codon which are charged;
(6) the iso-decoder isotype of the tRNA corresponding to the con-rare codon; and
(7) one or more post-transcriptional modifications of the con-rare tRNA, or candidate con-rare tRNA; and
In an embodiment, a con-optimized nucleic acid sequence has one less or one more con-rare codon than a reference sequence, e.g., a parental sequence, a naturally occurring sequence, a wildtype sequence, or a conventionally optimized sequence.
In an embodiment, con-rarity can be identified or evaluated by: (i) direct determination of whether a con-rare codon or candidate con-rare codon is limiting for a production parameter, e.g., in an assay analogous to that of Example 3; (ii) whether a con-rare or candidate con-rare codon meets a predetermined value, e.g., a standard or reference value (e.g., as described herein), of one or more, or all of factors (1)-(7); or (i) and (ii).
In an embodiment, con-rarity can be identified or evaluated by a production parameter, e.g., an expression parameter or a signaling parameter, e.g., as described herein.
In an embodiment, con-rarity is a function of normalized proteome codon count and tRNA abundance in a target tissue or cell. In an embodiment, con-rarity is a measure of codon frequency that is contextually dependent on tRNA abundance levels in a target tissue or cell.
Thus, the identification of a codon as a con-rare codon can involve a multi-parameter function of (1)-(7). In an embodiment, the con-rare codon meets a reference value for at least one of (1)-(7). In an embodiment, the con-rare codon meets a reference value for at least one of (1)-(7). In an embodiment, the con-rare codon meets a reference value for at least two of (1)-(7). In an embodiment, the con-rare codon meets a reference value for at least three of (1)-(7). In an embodiment, the con-rare codon meets a reference value for at least four of (1)-(7). In an embodiment, the con-rare codon meets a reference value for at least five of (1)-(7). In an embodiment, the con-rare codon meets a reference value for at least six of (1)-(7). In an embodiment, the con-rare codon meets a reference value for at all of (1)-(7). In an embodiment the reference value is a pre-determined or pre-selected value, e.g., as described herein.
In an embodiment, the identity of a con-rare codon is the DNA sequence which encodes for the codon in the nucleic acid sequence, e.g., gene.
In an embodiment, a con-rare codon is a function of the prevalence of the codon in the open reading frame (ORF) of protein coding genes in an organism, e.g., the proteome.
The availability, e.g., abundance, of tRNAs that correspond to a con-rare codon can be measured using an assay known in the art or as described herein, e.g., Nanopore sequencing, e.g., as described in Example 1. In an embodiment, a con-rare codon nucleic acid sequence has a low abundance of a tRNA corresponding to the con-rare codon, e.g., as compared to the abundance of a tRNA corresponding to a different/second codon.
The expression profile or proteomic property of a target cell or tissue refers to the protein expression, e.g., level of protein expression, from all of the protein coding genes in a target cell or tissue. The expression profile or proteomic property of a target cell or tissue can be measured using an assay known in the art or as described herein, e.g., a mass spectrometry based method, e.g., a SILAC based method as described in Example 13. In an embodiment, a protein coding gene in a target cell or tissue is a function of tissue or cell type specific regulation, e.g., a promoter element, an enhancer element, epigenetic regulation, and/or transcription factor control.
A “contextually-modified nucleic acid sequence” (sometimes referred to herein as a “con-modified nucleic acid sequence”) refers to a nucleic acid sequence in which the con-rarity of a codon of the con-modified nucleic acid sequence has been altered. E.g., a con-rare codon is replaced with a con-abundant codon and/or a con-abundant codon is replaced with a con-rare codon. In an embodiment, the con-modified nucleic acid sequence has one more or one less, e.g., two more or two lesser, con-rare codons, than a reference nucleic acid sequence. In an embodiment, the con-modified nucleic acid sequence has a codon with con-rarity that differs from the con-rarity of the corresponding codon in a reference nucleic acid sequence.
The reference nucleic acid sequence can be, e.g., any selected sequence, a parental sequence, a starting sequence, a wildtype or naturally occurring sequence that encodes the same amino acid at the corresponding codon, a wildtype or naturally occurring sequence that encodes the same polypeptide, or a conventionally codon-optimized sequence. In an embodiment, the reference nucleic acid sequence encodes the same polypeptide sequence as the con-modified nucleic acid sequence. In an embodiment, the reference nucleic acid sequence encodes a polypeptide sequence that differs from the con-modified nucleic acid sequence at a position other than the con-rare modified sequence. In an embodiment, a con-modified nucleic acid sequence results in a different production parameter, e.g., an expression parameter or signaling parameter, compared to that seen with expression of a reference nucleic acid sequence.
In an embodiment, a con-modified nucleic acid sequence refers to a nucleic acid sequence which has one more or one less, e.g., two more or two lesser, con-rare codons, than a reference sequence, wherein the con-modified nucleic acid sequence encodes a polypeptide that comprises the reference sequence.
A “contextually-rare tRNA” or “con-rare tRNA,” is a tRNA that corresponds to a con-rare codon.
A “contextually-abundant codon” or “con-abundant codon” as those terms are used herein, refer to a codon other than a con-rare codon.
A “con-rare codon nucleic acid sequence,” or a “nucleic acid sequence having a con-rare codon” as those terms are used herein, refer to a nucleic acid sequence, e.g., DNA, or RNA, or gene, comprising a con-rare codon. In an embodiment, in such con-rare codon nucleic acid sequences, modulation of a production parameter, e.g., an expression parameter or signaling parameter, can be mediated by altering the availability, e.g., abundance of a con-rare tRNA. In an embodiment, the con-rare codon is in a translated region of the con-rare codon nucleic acid sequence, e.g., in an open reading frame (ORF) or coding sequence (CDS).
A “con-rare codon RNA,” as that term is used herein, refers to an RNA sequence comprising a con-rare codon. In an embodiment, a con-rare codon RNA comprises a messenger RNA or an RNA that can be translated into a polypeptide or protein. In an embodiment, a con-rare codon RNA is transcribed from a complementary DNA sequence which comprises said con-rare codon. In an embodiment, the con-rare codon RNA is transcribed in vivo. In an embodiment, the con-rare codon RNA is transcribed in vitro.
A “codon-value” as that term is used herein, is a function of the con-rarity of a sequence-codon in a sequence. Con-rarity of a codon is a function of one or more factors as described in the definition of “con-rare codon” above. In an embodiment, a codon-value is the identity of a codon, e.g., a replacement codon selected to replace the sequence-codon. In an embodiment, when the replacement codon is a con-abundant codon, the sequence codon is a con-rare codon. In an embodiment, when the replacement codon is a con-rare codon, the sequence-codon is a con-abundant codon.
A “sequence-codon” as that term is used herein, refers to a codon in a nucleic acid sequence for which a codon-value is acquired.
A “production parameter,” refers to an expression parameter and/or a signaling parameter. In an embodiment a production parameter is an expression parameter. An expression parameter includes an expression parameter of a polypeptide or protein encoded by the con-rare codon nucleic acid sequence; or an expression parameter of an RNA, e.g., messenger RNA, encoded by the con-rare codon nucleic acid sequence. In an embodiment, an expression parameter can include:
(a) protein translation;
(b) expression level (e.g., of polypeptide or protein, or mRNA);
(c) post-translational modification of polypeptide or protein;
(d) folding (e.g., of polypeptide or protein, or mRNA),
(e) structure (e.g., of polypeptide or protein, or mRNA),
(f) transduction (e.g., of polypeptide or protein),
(g) compartmentalization (e.g., of polypeptide or protein, or mRNA),
(h) incorporation (e.g., of polypeptide or protein, or mRNA) into a supermolecular structure, e.g., incorporation into a membrane, proteasome, or ribosome,
(i) incorporation into a multimeric polypeptide, e.g., a homo or heterodimer, and/or
(j) stability.
In an embodiment, a production parameter is a signaling parameter. A signaling parameter can include:
(1) modulation of a signaling pathway, e.g., a cellular signaling pathway which is downstream or upstream of the protein encoded by the con-rare codon nucleic acid sequence;
(2) cell fate modulation;
(3) ribosome occupancy modulation;
(4) protein translation modulation;
(5) mRNA stability modulation;
(6) protein folding and structure modulation;
(7) protein transduction or compartmentalization modulation; and/or
(8) protein stability modulation.
An “isoacceptor,” as that term is used herein, refers to a plurality of tRNA molecule or TREMs wherein each molecule of the plurality comprises a different naturally occurring anticodon sequence and each molecule of the plurality mediates the incorporation of the same amino acid and that amino acid is the amino acid that naturally corresponds to the anticodons of the plurality.
A “tRNA pool,” as that term is used herein, refers to the pool of all species, e.g., endogenous tRNAs and TREMS, which can function as tRNAs. The endogenous tRNA pool for a cell or subject that has not been administered a TREM includes only endogenous tRNAs. A TREM can be added to modulate a tRNA pool comprising only endogenous tRNAs, but can also be administered to a cell or subject that has a tRNA pool that includes TREMs that have been administered previously. In an embodiment, the TREM which is administered to a cell or a subject, mediates initiation or elongation by incorporating the amino acid (the cognate amino acid) associated in nature with a particular anticodon. In an embodiment, the TREM which is administered has an anticodon other than a stop codon.
A “nucleotide,” as that term is used herein, refers to an entity comprising a sugar, typically a pentameric sugar; a nucleobase; and a phosphate linking group. In an embodiment, a nucleotide comprises a naturally occurring, e.g., naturally occurring in a human cell, nucleotide, e.g., an adenine, thymine, guanine, cytosine, or uracil nucleotide.
A “modification,” as that term is used herein with reference to a nucleotide, refers to a modification of the chemical structure, e.g., a covalent modification, of the subject nucleotide. The modification can be naturally occurring or non-naturally occurring. In an embodiment, the modification is non-naturally occurring. In an embodiment, the modification is naturally occurring. In an embodiment, the modification is a synthetic modification. In an embodiment, the modification is a modification provided in Tables 5, 6, 7, 8 or 9.
A “non-naturally occurring modification,” as that term is used herein with reference to a nucleotide, refers to a modification that: (a) a cell, e.g., a human cell, does not make on an endogenous tRNA; or (b) a cell, e.g., a human cell, can make on an endogenous tRNA but wherein such modification is in a location in which it does not occur on a native tRNA, e.g., the modification is in a domain, linker or arm, or on a nucleotide and/or at a position within a domain, linker or arm, which does not have such modification in nature. In either case, the modification is added synthetically, e.g., in a cell free reaction, e.g., in a solid state or liquid phase synthetic reaction. In an embodiment, the non-naturally occurring modification is a modification that is not present (in identity, location or position) if a sequence of the TREM is expressed in a mammalian cell, e.g., a HEK293 cell line. Exemplary non-naturally occurring modifications are found in Tables 5, 6, 7, 8 or 9.
A “non-naturally modified nucleotide,” as that term is used herein, refers a nucleotide comprising a non-naturally occurring modification on or of a sugar, nucleobase, or phosphate moiety.
A “non-naturally occurring sequence,” as that term is used herein, refers to a sequence wherein an Adenine is replaced by a residue other than an analog of Adenine, a Cytosine is replaced by a residue other than an analog of Cytosine, a Guanine is replaced by a residue other than an analog of Guanine, and a Uracil is replaced by a residue other than an analog of Uracil. An analog refers to any possible derivative of the ribonucleotides, A, G, C or U. In an embodiment, a sequence having a derivative of any one of ribonucleotides A, G, C or U is a non-naturally occurring sequence.
A “naturally occurring nucleotide,” as that term is used herein, refers to a nucleotide that does not comprise a non-naturally occurring modification. In an embodiment, it includes a naturally occurring modification.
A “tRNA-based effector molecule” or “TREM,” as that term is used herein, refers to an RNA molecule comprising a structure or property from (a)-(v) below, and which is a recombinant TREM, a synthetic TREM, or a TREM expressed from a heterologous cell. The TREMs described in the present invention are synthetic molecules and are made, e.g., in a cell free reaction, e.g., in a solid state or liquid phase synthetic reaction. TREMs are chemically distinct, e.g., in terms of primary sequence, type or location of modifications from the endogenous tRNA molecules made in cells, e.g., in mammalian cells, e.g., in human cells. A TREM can have a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9) of the structures and functions of (a)-(v).
In an embodiment, a TREM is non-native, as evaluated by structure or the way in which it was made.
In an embodiment, a TREM comprises one or more of the following structures or properties:
(a′) an optional linker region of a consensus sequence provided in the “Consensus Sequence” section, e.g., a Linker 1 region;
(a) an amino acid attachment domain that binds an amino acid, e.g., an acceptor stem domain (AStD), wherein an AStD comprises sufficient RNA sequence to mediate, e.g., when present in an otherwise wildtype tRNA, acceptance of an amino acid, e.g., its cognate amino acid or a non-cognate amino acid, and transfer of the amino acid (AA) in the initiation or elongation of a polypeptide chain. Typically, the AStD comprises a 3′-end adenosine (CCA) for acceptor stem charging which is part of synthetase recognition. In an embodiment the AStD has at least 75, 80, 85, 85, 90, 95, or 100% identity with a naturally occurring AStD, e.g., an AStD encoded by a nucleic acid in Table 4. In an embodiment, the TREM can comprise a fragment or analog of an AStD, e.g., an AStD encoded by a nucleic acid in Table 4, which fragment in embodiments has AStD activity and in other embodiments does not have AStD activity. (One of ordinary skill can determine the relevant corresponding sequence for any of the domains, stems, loops, or other sequence features mentioned herein from a sequence encoded by a nucleic acid in Table 4. E.g., one of ordinary skill can determine the sequence which corresponds to an AStD from a tRNA sequence encoded by a nucleic acid in Table 4.)
In an embodiment the AStD falls under the corresponding sequence of a consensus sequence provided in the “Consensus Sequence” section, or differs from the consensus sequence by no more than 1, 2, 5, or 10 positions;
In an embodiment, the AStD comprises residues R1-R2-R3-R4-R5-R6-R7 and residues R65-R66-R67-R68-R69-R70-R71 of Formula IZZZ, wherein ZZZ indicates any of the twenty amino acids;
In an embodiment, the AStD comprises residues R1-R2-R3-R4-R5-R6-R7 and residues R65-R66-R67-R68-R69-R70-R71 of Formula IIZZZ, wherein ZZZ indicates any of the twenty amino acids;
In an embodiment, the AStD comprises residues R1-R2-R3-R4-R5-R6-R7 and residues R65-R66-R67-R68-R69-R70-R71 of Formula IIIZZZ, wherein ZZZ indicates any of the twenty amino acids;
(a′-1) a linker comprising residues R8-R9 of a consensus sequence provided in the “Consensus Sequence” section, e.g., a Linker 2 region;
(b) a dihydrouridine hairpin domain (DHD), wherein a DHD comprises sufficient RNA sequence to mediate, e.g., when present in an otherwise wildtype tRNA, recognition of aminoacyl-tRNA synthetase, e.g., acts as a recognition site for aminoacyl-tRNA synthetase for amino acid charging of the TREM. In embodiments, a DHD mediates the stabilization of the TREM's tertiary structure. In an embodiment the DHD has at least 75, 80, 85, 85, 90, 95, or 100% identity with a naturally occurring DHD, e.g., a DHD encoded by a nucleic acid in Table 4. In an embodiment, the TREM can comprise a fragment or analog of a DHD, e.g., a DHD encoded by a nucleic acid in Table 4, which fragment in embodiments has DHD activity and in other embodiments does not have DHD activity.
In an embodiment the DHD falls under the corresponding sequence of a consensus sequence provided in the “Consensus Sequence” section, or differs from the consensus sequence by no more than 1, 2, 5, or 10 positions;
In an embodiment, the DHD comprises residues R10-R11-R12-R13-R14 R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28 of Formula IZZZ, wherein ZZZ indicates any of the twenty amino acids;
In an embodiment, the DHD comprises residues R10-R11-R12-R13-R14 R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28 of Formula IIZZZ, wherein ZZZ indicates any of the twenty amino acids;
In an embodiment, the DHD comprises residues R10-R11-R12-R13-R14 R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28 of Formula IIIZZZ, wherein ZZZ indicates any of the twenty amino acids;
(b′-1) a linker comprising residue R29 of a consensus sequence provided in the “Consensus Sequence” section, e.g., a Linker 3 region;
(c) an anticodon that binds a respective codon in an mRNA, e.g., an anticodon hairpin domain (ACHD), wherein an ACHD comprises sufficient sequence, e.g., an anticodon triplet, to mediate, e.g., when present in an otherwise wildtype tRNA, pairing (with or without wobble) with a codon; In an embodiment the ACHD has at least 75, 80, 85, 85, 90, 95, or 100% identity with a naturally occurring ACHD, e.g., an ACHD encoded by a nucleic acid in Table 4. In an embodiment, the TREM can comprise a fragment or analog of an ACHD, e.g., an ACHD encoded by a nucleic acid in Table 4, which fragment in embodiments has ACHD activity and in other embodiments does not have ACHD activity.
In an embodiment the ACHD falls under the corresponding sequence of a consensus sequence provided in the “Consensus Sequence” section, or differs from the consensus sequence by no more than 1, 2, 5, or 10 positions;
In an embodiment, the ACHD comprises residues —R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46 of Formula IZZZ, wherein ZZZ indicates any of the twenty amino acids;
In an embodiment, the ACHD comprises residues —R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46 of Formula IIZZZ, wherein ZZZ indicates any of the twenty amino acids;
In an embodiment, the ACHD comprises residues —R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46 of Formula IIIZZZ, wherein ZZZ indicates any of the twenty amino acids;
(d) a variable loop domain (VLD), wherein a VLD comprises sufficient RNA sequence to mediate, e.g., when present in an otherwise wildtype tRNA, recognition of aminoacyl-tRNA synthetase, e.g., acts as a recognition site for aminoacyl-tRNA synthetase for amino acid charging of the TREM. In embodiments, a VLD mediates the stabilization of the TREM's tertiary structure. In an embodiment, a VLD modulates, e.g., increases, the specificity of the TREM, e.g., for its cognate amino acid, e.g., the VLD modulates the TREM's cognate adaptor function. In an embodiment the VLD has at least 75, 80, 85, 85, 90, 95, or 100% identity with a naturally occurring VLD, e.g., a VLD encoded by a nucleic acid in Table 4. In an embodiment, the TREM can comprise a fragment or analog of a VLD, e.g., a VLD encoded by a nucleic acid in Table 4, which fragment in embodiments has VLD activity and in other embodiments does not have VLD activity.
In an embodiment the VLD falls under the corresponding sequence of a consensus sequence provided in the “Consensus Sequence” section.
In an embodiment, the VLD comprises residue —[R47]x of a consensus sequence provided in the “Consensus Sequence” section, wherein x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271);
(e) a thymine hairpin domain (THD), wherein a THD comprises sufficient RNA sequence, to mediate, e.g., when present in an otherwise wildtype tRNA, recognition of the ribosome, e.g., acts as a recognition site for the ribosome to form a TREM-ribosome complex during translation. In an embodiment the THD has at least 75, 80, 85, 85, 90, 95, or 100% identity with a naturally occurring THD, e.g., a THD encoded by a nucleic acid in Table 4. In an embodiment, the TREM can comprise a fragment or analog of a THD, e.g., a THD encoded by a nucleic acid in Table 4, which fragment in embodiments has THD activity and in other embodiments does not have THD activity.
In an embodiment the THD falls under the corresponding sequence of a consensus sequence provided in the “Consensus Sequence” section, or differs from the consensus sequence by no more than 1, 2, 5, or 10 positions;
In an embodiment, the THD comprises residues —R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64 of Formula IZZZ, wherein ZZZ indicates any of the twenty amino acids;
In an embodiment, the THD comprises residues —R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64 of Formula IIZZZ, wherein ZZZ indicates any of the twenty amino acids;
In an embodiment, the THD comprises residues —R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64 of Formula IIIZZZ, wherein ZZZ indicates any of the twenty amino acids;
(e′ 1) a linker comprising residue R72 of a consensus sequence provided in the “Consensus Sequence” section, e.g., a Linker 4 region;
(f) under physiological conditions, it comprises a stem structure and one or a plurality of loop structures, e.g., 1, 2, or 3 loops. A loop can comprise a domain described herein, e.g., a domain selected from (a)-(e). A loop can comprise one or a plurality of domains. In an embodiment, a stem or loop structure has at least 75, 80, 85, 85, 90, 95, or 100% identity with a naturally occurring stem or loop structure, e.g., a stem or loop structure encoded by a nucleic acid in Table 4. In an embodiment, the TREM can comprise a fragment or analog of a stem or loop structure, e.g., a stem or loop structure encoded by a nucleic acid in Table 4, which fragment in embodiments has activity of a stem or loop structure, and in other embodiments does not have activity of a stem or loop structure;
(g) a tertiary structure, e.g., an L-shaped tertiary structure;
(h) adaptor function, i.e., the TREM mediates acceptance of an amino acid, e.g., its cognate amino acid and transfer of the AA in the initiation or elongation of a polypeptide chain;
(i) cognate adaptor function wherein the TREM mediates acceptance and incorporation of an amino acid (e.g., cognate amino acid) associated in nature with the anti-codon of the TREM to initiate or elongate a polypeptide chain;
(j) non-cognate adaptor function, wherein the TREM mediates acceptance and incorporation of an amino acid (e.g., non-cognate amino acid) other than the amino acid associated in nature with the anti-codon of the TREM in the initiation or elongation of a polypeptide chain;
(k) a regulatory function, e.g., an epigenetic function (e.g., gene silencing function or signaling pathway modulation function), cell fate modulation function, mRNA stability modulation function, protein stability modulation function, protein transduction modulation function, or protein compartmentalization function;
(l) a structure which allows for ribosome binding;
(m) a post-transcriptional modification, e.g., a naturally occurring post-transcriptional modification;
(n) the ability to inhibit a functional property of a tRNA, e.g., any of properties (h)-(k) possessed by a tRNA;
(o) the ability to modulate cell fate;
(p) the ability to modulate ribosome occupancy;
(q) the ability to modulate protein translation;
(r) the ability to modulate mRNA stability;
(s) the ability to modulate protein folding and structure;
(t) the ability to modulate protein transduction or compartmentalization;
(u) the ability to modulate protein stability; or
(v) the ability to modulate a signaling pathway, e.g., a cellular signaling pathway.
In an embodiment, a TREM comprises a full-length tRNA molecule or a fragment thereof.
In an embodiment, a TREM comprises the following properties: (a)-(e).
In an embodiment, a TREM comprises the following properties: (a) and (c).
In an embodiment, a TREM comprises the following properties: (a), (c) and (h).
In an embodiment, a TREM comprises the following properties: (a), (c), (h) and (b).
In an embodiment, a TREM comprises the following properties: (a), (c), (h) and (e).
In an embodiment, a TREM comprises the following properties: (a), (c), (h), (b) and (e).
In an embodiment, a TREM comprises the following properties: (a), (c), (h), (b), (e) and (g).
In an embodiment, a TREM comprises the following properties: (a), (c), (h) and (m).
In an embodiment, a TREM comprises the following properties: (a), (c), (h), (m), and (g).
In an embodiment, a TREM comprises the following properties: (a), (c), (h), (m) and (b).
In an embodiment, a TREM comprises the following properties: (a), (c), (h), (m) and (e).
In an embodiment, a TREM comprises the following properties: (a), (c), (h), (m), (g), (b) and (e).
In an embodiment, a TREM comprises the following properties: (a), (c), (h), (m), (g), (b), (e) and (q).
In an embodiment, a TREM comprises:
(i) an amino acid attachment domain that binds an amino acid (e.g., an AStD, as described in (a) herein; and
(ii) an anticodon that binds a respective codon in an mRNA (e.g., an ACHD, as described in (c) herein).
In an embodiment the TREM comprises a flexible RNA linker which provides for covalent linkage of (i) to (ii).
In an embodiment, the TREM mediates protein translation.
In an embodiment a TREM comprises a linker, e.g., an RNA linker, e.g., a flexible RNA linker, which provides for covalent linkage between a first and a second structure or domain. In an embodiment, an RNA linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ribonucleotides. A TREM can comprise one or a plurality of linkers, e.g., in embodiments a TREM comprising (a), (b), (c), (d) and (e) can have a first linker between a first and second domain, and a second linker between a third domain and another domain.
In an embodiment, the TREM comprises a sequence of Formula A: [L1]-[ASt Domain1]-[L2]-[DH Domain]-[L3]-[ACH Domain]-[VL Domain]-[TH Domain]-[L4]-[ASt Domain2].
In an embodiment, a TREM comprises an RNA sequence at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical with, or which differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 ribonucleotides from, an RNA sequence encoded by a DNA sequence listed in Table 4, or a fragment or functional fragment thereof. In an embodiment, a TREM comprises an RNA sequence encoded by a DNA sequence listed in Table 4, or a fragment or functional fragment thereof. In an embodiment, a TREM comprises an RNA sequence encoded by a DNA sequence at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical with a DNA sequence listed in Table 4, or a fragment or functional fragment thereof. In an embodiment, a TREM comprises a TREM domain, e.g., a domain described herein, comprising at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical with, or which differs by no more than 1, 2, 3, 4, 5, 10, or 15, ribonucleotides from, an RNA encoded by a DNA sequence listed in Table 4, or a fragment or a functional fragment thereof. In an embodiment, a TREM comprises a TREM domain, e.g., a domain described herein, comprising an RNA sequence encoded by DNA sequence listed in Table 4, or a fragment or functional fragment thereof. In an embodiment, a TREM comprises a TREM domain, e.g., a domain described herein, comprising an RNA sequence encoded by DNA sequence at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical with a DNA sequence listed in Table 4, or a fragment or functional fragment thereof.
In an embodiment, a TREM is 76-90 nucleotides in length. In embodiments, a TREM or a fragment or functional fragment thereof is between 10-90 nucleotides, between 10-80 nucleotides, between 10-70 nucleotides, between 10-60 nucleotides, between 10-50 nucleotides, between 10-40 nucleotides, between 10-30 nucleotides, between 10-20 nucleotides, between 20-90 nucleotides, between 20-80 nucleotides, 20-70 nucleotides, between 20-60 nucleotides, between 20-50 nucleotides, between 20-40 nucleotides, between 30-90 nucleotides, between 30-80 nucleotides, between 30-70 nucleotides, between 30-60 nucleotides, or between 30-50 nucleotides.
In an embodiment, a TREM is aminoacylated, e.g., charged, with an amino acid by an aminoacyl tRNA synthetase.
In an embodiment, a TREM is not charged with an amino acid, e.g., an uncharged TREM (uTREM).
In an embodiment, a TREM comprises less than a full length tRNA. In embodiments, a TREM can correspond to a naturally occurring fragment of a tRNA, or to a non-naturally occurring fragment. Exemplary fragments include: TREM halves (e.g., from a cleavage in the ACHD, e.g., in the anticodon sequence, e.g., 5′ halves or 3′ halves); a 5′ fragment (e.g., a fragment comprising the 5′ end, e.g., from a cleavage in a DHD or the ACHD); a 3′ fragment (e.g., a fragment comprising the 3′ end, e.g., from a cleavage in the THD); or an internal fragment (e.g., from a cleavage in one or more of the ACHD, DHD or THD).
A “TREM core fragment,” as that term is used herein, refers to a portion of the sequence of Formula B: [L1]y-[ASt Domain1]x-[L2]y-[DH Domain]y-[L3]y-[ACH Domain]x-[VL Domain]y-[TH Domain]y-[L4]y-[ASt Domain2]x, wherein: x=1 and y=0 or 1.
A “TREM fragment,” as used herein, refers to a portion of a TREM, wherein the TREM comprises a sequence of Formula A: [L1]-[ASt Domain1]-[L2]-[DH Domain]-[L3]-[ACH Domain]-[VL Domain]-[TH Domain]-[L4]-[ASt Domain2].
A “cognate adaptor function TREM,” as that term is used herein, refers to a TREM which mediates initiation or elongation with the AA (the cognate AA) associated in nature with the anti-codon of the TREM.
“Decreased expression,” as that term is used herein, refers to a decrease in comparison to a reference, e.g., in the case where altered control region, or addition of an agent, results in a decreased expression of the subject product, it is decreased relative to an otherwise similar cell without the alteration or addition.
An “exogenous nucleic acid,” as that term is used herein, refers to a nucleic acid sequence that is not present in or differs by at least one nucleotide from the closest sequence in a reference cell, e.g., a cell into which the exogenous nucleic acid is introduced. In an embodiment, an exogenous nucleic acid comprises a nucleic acid that encodes a TREM.
An “exogenous TREM,” as that term is used herein, refers to a TREM that:
(a) differs by at least one nucleotide or one post transcriptional modification from the closest sequence tRNA in a reference cell, e.g., a cell into which the exogenous nucleic acid is introduced;
(b) has been introduced into a cell other than the cell in which it was transcribed;
(c) is present in a cell other than one in which it naturally occurs; or
(d) has an expression profile, e.g., level or distribution, that is non-wildtype, e.g., it is expressed at a higher level than wildtype. In an embodiment, the expression profile can be mediated by a change introduced into a nucleic acid that modulates expression or by addition of an agent that modulates expression of the RNA molecule. In an embodiment an exogenous TREM comprises 1, 2, 3 or 4 of properties (a)-(d).
A “GMP-grade composition,” as that term is used herein, refers to a composition in compliance with current good manufacturing practice (cGMP) guidelines, or other similar requirements. In an embodiment, a GMP-grade composition can be used as a pharmaceutical product.
As used herein, the terms “increasing” and “decreasing” refer to modulating that results in, respectively, greater or lesser amounts of function, expression, or activity of a particular metric relative to a reference. For example, subsequent to administration to a cell, tissue or subject of a TREM described herein, the amount of a marker of a metric (e.g., protein translation, mRNA stability, protein folding) as described herein may be increased or decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%, 2×, 3×, 5×, 10× or more relative to the amount of the marker prior to administration or relative to the effect of a negative control agent. The metric may be measured subsequent to administration at a time that the administration has had the recited effect, e.g., at least 12 hours, 24 hours, one week, one month, 3 months, or 6 months, after a treatment has begun.
“Increased expression,” as that term is used herein, refers to an increase in comparison to a reference, e.g., in the case where altered control region, or addition of an agent, results in an increased expression of the subject product, it is increased relative to an otherwise similar cell without the alteration or addition.
A “non-cognate adaptor function TREM,” as that term is used herein, refers to a TREM which mediates initiation or elongation with an AA (a non-cognate AA) other than the AA associated in nature with the anti-codon of the TREM. In an embodiment, a non-cognate adaptor function TREM is also referred to as a mischarged TREM (mTREM).
A “pharmaceutical TREM composition,” as that term is used herein, refers to a TREM composition that is suitable for pharmaceutical use. Typically, a pharmaceutical TREM composition comprises a pharmaceutical excipient. In an embodiment the TREM will be the only active ingredient in the pharmaceutical TREM composition. In embodiments the pharmaceutical TREM composition is free, substantially free, or has less than a pharmaceutically acceptable amount, of host cell proteins, DNA, e.g., host cell DNA, endotoxins, and bacteria.
“Post-transcriptional processing,” as that term is used herein, with respect to a subject molecule, e.g., a TREM, RNA or tRNAs, refers to a covalent modification of the subject molecule. In an embodiment, the covalent modification occurs post-transcriptionally. In an embodiment, the covalent modification occurs co-transcriptionally. In an embodiment the modification is made in vivo, e.g., in a cell used to produce a TREM. In an embodiment the modification is made ex vivo, e.g., it is made on a TREM isolated or obtained from the cell which produced the TREM.
A “subject,” as this term is used herein, includes any organism, such as a human or other animal. In embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In embodiments, the subject is a mammal, e.g., a human. In embodiments, the method subject is a non-human mammal. In embodiments, the subject is a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). The subject may be a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)). A non-human subject may be a transgenic animal.
A “synthetic TREM,” as that term is used herein, refers to a TREM which was synthesized other than in or by a cell having an endogenous nucleic acid encoding the TREM, e.g., a synthetic TREM is synthetized by cell-free solid phase synthesis. A synthetic TREM can have the same, or a different, sequence, or tertiary structure, as a native tRNA.
A “recombinant TREM,” as that term is used herein, refers to a TREM that was expressed in a cell modified by human intervention, having a modification that mediates the production of the TREM, e.g., the cell comprises an exogenous sequence encoding the TREM, or a modification that mediates expression, e.g., transcriptional expression or post-transcriptional modification, of the TREM. A recombinant TREM can have the same, or a different, sequence, set of post-transcriptional modifications, or tertiary structure, as a reference tRNA, e.g., a native tRNA.
A “tRNA”, as that term is used herein, refers to a naturally occurring transfer ribonucleic acid in its native state.
A “TREM composition,” as that term is used herein, refers to a composition comprising a plurality of TREMs, a plurality of TREM core fragments and/or a plurality of TREM fragments. A TREM composition can comprise one or more species of TREMs, TREM core fragments or TREM fragments. In an embodiment, the composition comprises only a single species of TREM, TREM core fragment or TREM fragment. In an embodiment, the TREM composition comprises a first TREM, TREM core fragment or TREM fragment species; and a second TREM, TREM core fragment or TREM fragment species. In an embodiment, the TREM composition comprises X TREM, TREM core fragment or TREM fragment species, wherein X=2, 3, 4, 5, 6, 7, 8, 9, or 10. In an embodiment, the TREM, TREM core fragment or TREM fragment has at least 70, 75, 80, 85, 90, or 95, or has 100%, identity with a sequence encoded by a nucleic acid in Table 4. A TREM composition can comprise one or more species of TREMs, TREM core fragments or TREM fragments. In an embodiment, the TREM composition is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% dry weight TREMs (for a liquid composition dry weight refers to the weight after removal of substantially all liquid, e.g., after lyophilization). In an embodiment, the composition is a liquid. In an embodiment, the composition is dry, e.g., a lyophilized material. In an embodiment, the composition is a frozen composition. In an embodiment, the composition is sterile. In an embodiment, the composition comprises at least 0.5 g, 1.0 g, 5.0 g, 10 g, 15 g, 25 g, 50 g, 100 g, 200 g, 400 g, or 500 g (e.g., as determined by dry weight) of TREM.
In an embodiment, at least X % of the TREMs in a TREM composition has a non-naturally occurring modification at a selected position, and X is 80, 90, 95, 96, 97, 98, 99, or 99.5.
In an embodiment, at least X % of the TREMs in a TREM composition has a non-naturally occurring modification at a first position and a non-naturally occurring modification at a second position, and X, independently, is 80, 90, 95, 96, 97, 98, 99, or 99.5. In embodiments, the modification at the first and second position is the same. In embodiments, the modification at the first and second position are different. In embodiments, the nucleotide at the first and second position is the same, e.g., both are adenine. In embodiments, the nucleotide at the first and second position are different, e.g., one is adenine and one is thymine.
In an embodiment, at least X % of the TREMs in a TREM composition has a non-naturally occurring modification at a first position and less than Y % have a non-naturally occurring modification at a second position, wherein X is 80, 90, 95, 96, 97, 98, 99, or 99.5 and Y is 20, 20, 5, 2, 1, 0.1, or 0.01. In embodiments, the nucleotide at the first and second position is the same, e.g., both are adenine. In embodiments the nucleotide at the first and second position are different, e.g., one is adenine and one is thymine.
“Pairs with” or “pairing,” as those terms are used herein, refer to the correspondence of a codon with an anticodon and includes fully complementary codon:anticodon pairs as well as “wobble” pairing, in which the third position need not be complementary. Fully complementary pairing refers to pairing of all three positions of the codon with the corresponding anticodon according to Watson-Crick base pairing. Wobble pairing refers to complementary pairing of the first and second positions of the codon with the corresponding anticodon according to Watson-Crick base pairing, and flexible pairing at the third position of the codon with the corresponding anticodon.
The terms modified, replace, derived and similar terms, when used or applied in reference to a product, refer only to the end product or structure of the end product, and are not restricted by any method of making or manufacturing the product, unless expressly provided as such in this disclosure.
Headings, titles, subtitles, numbering or other alpha/numeric hierarchies are included merely for ease of reading and absent explicit language to the contrary do not indicate order of performance, order of importance, magnitude or other value.
Disclosed herein, inter alia, is the observation that a production parameter of an RNA, or a protein encoded by an RNA having a con-rare codon, can be modulated by administration of a TREM composition comprising a TREM, TREM core fragment or TREM fragment, e.g., as described herein, corresponding to said con-rare codon. Accordingly, this disclosure provides, inter alia, methods of identifying a contextually rare codon (“con-rare codon”), compositions of TREMs corresponding to a con-rare codon and uses of said TREM compositions.
A con-rare codon is a codon that is limiting for a production parameter, e.g., an expression parameter or a signaling parameter, for a nucleic acid sequence, e.g., a DNA or an RNA, or a protein encoded by a nucleic acid sequence, e.g., a DNA or an RNA. Contextual rareness or con-rarity can be identified or evaluated by determining if the addition of a tRNA corresponding to a con-rare codon modulates, typically increases, a production parameter for a target nucleic acid sequence, e.g., target, e.g., gene. In an embodiment, con-rarity as a property of a codon, is a function of, one, two, three, four, all of the following factors:
(1) the sequence of the codon;
(2) the availability of a corresponding tRNA, e.g., charged tRNA, for that con-rare codon in a target cell or tissue, e.g., one or more iso-acceptor tRNA molecules;
(3) the expression profile (or proteomic properties) of the target cell or tissue (e.g., the abundance of expression of other proteins which include the con-rare codon);
(4) the proportion of the tRNAs corresponding to the con-rare codon which are charged; and
(5) the iso-decoder isotype of the tRNA corresponding to the con-rare codon.
In an embodiment, con-rarity is a function of normalized proteome codon count and tRNA abundance in a target tissue or cell. In an embodiment, con-rarity is a measure of codon frequency that is contextually dependent on tRNA abundance levels in a target tissue or cell. In an embodiment, con-rarity can be identified or evaluated by a production parameter, e.g., an expression parameter or a signaling parameter, e.g., as described herein.
An exemplary method of evaluating con-rarity and identifying a con-rare codon is provided in Example 3.
In an embodiment, con-rarity is a function of normalized proteome codon count and the tRNA profile, e.g., as described herein. In an embodiment, con-rarity is determined by dividing the normalized proteome codon count by the tRNA profile determined by Nanopore or other tRNA sequencing experiment. This provides a measure of codon usage that is contextually dependent on the tRNA profile, e.g., tRNA abundance levels.
In an embodiment, a codon is determined to be contextually rare (con-rare) if the con-rarity meets a reference value, e.g., a pre-determined or pre-selected reference value, e.g., a threshold, e.g., an internal threshold, e.g., as described herein. In an embodiment, the reference value is a value under which e.g., 1.5× sigma of the normally fit distribution to that codon frequency.
In an embodiment, a codon is con-rare if the value of a normalized proteome codon count divided by the tRNA profile value for a particular tRNA meets a reference value, e.g., a pre-determined or pre-selected reference value, e.g., a threshold, e.g., an internal threshold.
In an embodiment, a codon is con-rare if the value of a normalized proteome codon count divided by the tRNA profile value for a particular tRNA is in the top 5%, 10%, 20%, 30%, or 40% of values for normalized proteome codon count divided by the tRNA profile value for all codons measured, e.g., wherein all 64 codons are measured. In an embodiment, a codon is con-rare if the value of a normalized proteome codon count divided by the tRNA profile value for a particular tRNA is in the top 5% of values for normalized proteome codon count divided by the tRNA profile value for all codons measured. In an embodiment, a codon is con-rare if the value of a normalized proteome codon count divided by the tRNA profile value for a particular tRNA is in the top 10% of values for normalized proteome codon count divided by the tRNA profile value for all codons measured. In an embodiment, a codon is con-rare if the value of a normalized proteome codon count divided by the tRNA profile value for a particular tRNA is in the top 20% of values for normalized proteome codon count divided by the tRNA profile value for all codons measured. In an embodiment, a codon is con-rare if the value of a normalized proteome codon count divided by the tRNA profile value for a particular tRNA is in the top 30% of values for normalized proteome codon count divided by the tRNA profile value for all codons measured. In an embodiment, a codon is con-rare if the value of a normalized proteome codon count divided by the tRNA profile value for a particular tRNA is in the top 40% of values for normalized proteome codon count divided by the tRNA profile value for all codons measured.
In an embodiment, a codon is con-rare if for the value of a normalized proteome codon count divided by the tRNA profile value for a particular tRNA, the value for the normalized proteome codon count is below the value for all codons measured and the value for tRNA profile, is above the value for all codons measured, e.g., wherein all 64 codons are measured.
In an embodiment, a codon is a con-rare codon if it is in the upper left quadrant of a plot of normalized proteome codon count (y-axis) vs tRNA profile (x-axis), with equal number of codons in each quadrant, e.g., wherein all 64 codons are measured.
In an embodiment, a codon is a con-rare codon if it is in a quadrant other than the lower right quadrant of a plot of normalized proteome codon count (y-axis) vs tRNA profile (x-axis), with equal number of codons in each quadrant, e.g., wherein all 64 codons are measured.
Methods of Modulating a Production Parameter of an RNA, or a Protein Encoded by an RNA Having a Con-Rare Codon with a TREM Composition
A production parameter of an RNA, or a protein encoded by an RNA having a con-rare codon, can be modulated by administration of a TREM composition comprising a TREM, a TREM core fragment, or TREM fragment, e.g., as described herein, corresponding to said con-rare codon.
In an aspect, provided herein is a method of method of modulating a production parameter of an RNA, or a protein encoded by an RNA, in a target cell or tissue, comprising:
providing, e.g., administering, to the target cell or tissue, or contacting the target cell or tissue with, an effective amount of a TREM composition comprising a TREM, a TREM core fragment, or TREM fragment, which TREM, TREM core fragment or TREM fragment corresponds to a contextually-rare codon (“con-rare codon”) of the RNA,
thereby modulating the production parameter of the RNA, or protein encoded by the RNA in the target cell or tissue.
The TREM composition can be administered to the subject or the target cell or tissue can be contacted ex vivo with the TREM composition. In an embodiment, the target cell or tissue which has been contacted ex vivo with the TREM composition can be introduced into a subject, e.g., an allogeneic subject or an autologous subject.
Modulation of a production parameter of an RNA, or a protein encoded by an RNA having a con-rare codon by administration of a TREM composition (e.g., comprising a TREM, TREM core fragment or TREM fragment corresponding to the con-rare codon) comprises modulation of an expression parameter or a signaling parameter, e.g., as described herein.
For example, administration of a TREM composition to a target cell or tissue can result in an increase or decrease in any one or more of the following expression parameters for the con-rare codon RNA:
(a) protein translation;
(b) expression level (e.g., of polypeptide or protein, or mRNA);
(c) post-translational modification of polypeptide or protein;
(d) folding (e.g., of polypeptide or protein, or mRNA),
(e) structure (e.g., of polypeptide or protein, or mRNA),
(f) transduction (e.g., of polypeptide or protein),
(g) compartmentalization (e.g., of polypeptide or protein, or mRNA),
(h) incorporation (e.g., of polypeptide or protein, or mRNA) into a supermolecular structure, e.g., incorporation into a membrane, proteasome, or ribosome,
(i) incorporation into a multimeric polypeptide, e.g., a homo or heterodimer, and/or
(j) stability.
As another example, administration of a TREM composition to a target cell or tissue can result in an increase or decrease in any one or more of the following signaling parameters for the con-rare codon RNA:
(1) modulation of a signaling pathway, e.g., a cellular signaling pathway which is downstream or upstream of the protein encoded by the con-rare codon RNA;
(2) cell fate modulation;
(3) ribosome occupancy modulation;
(4) protein translation modulation;
(5) mRNA stability modulation;
(6) protein folding and structure modulation;
(7) protein transduction or compartmentalization modulation; and/or
(8) protein stability modulation.
A production parameter (e.g., an expression parameter and/or a signaling parameter) may be modulated, e.g., by at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 40%. 50%. 60%. 70%, 80%, 90%, 100%, 150%, 200% or more) compared to a reference nucleic acid sequence, e.g., parental, wildtype or conventionally optimized nucleic acid sequence.
Synonymous SNPs and Method of Modulating tRNA Pool
A single nucleotide polymorphism (SNP) is a mutation that is found in the genome. A SNP can occur anywhere in the genome, e.g., in a coding sequence (e.g., an exon), or in a regulatory region (e.g., in an intron, a promoter element, an enhancer), or in a non-coding sequence.
A SNP that occurs in a coding sequence, e.g., an exon, can affect the corresponding polypeptide by altering a codon to specify a different amino acid, e.g., a different amino acid compared to that specified by the non-mutated codon.
A SNP that occurs in a coding sequence which alters a codon but does not change the amino acid specified by said mutated codon will not change the amino acid that is incorporated into the corresponding polypeptide at that position. This is possible due to the degeneracy of the genetic codon (i.e. more than one codon specifying one amino acid). Codon degeneracy is supported by “wobble” base pairing at the first base of the tRNA anticodon. For example, if a wildtype CTT codon which specifies the amino acid leucine is mutated to a CTC codon which specifies the same amino acid Leucine, no change to the corresponding protein with respect to its composition at that particular position is expected. Both codons CTT and CTC are recognized by tRNAs that specify the amino acid Leucine. These different species of tRNAs are referred to as isoacceptor tRNAs.
A mutation which changes a codon but does not change the corresponding amino acid specified by the mutated codon is called a synonymous SNP. Synonymous SNPs are also known as silent SNPs.
Synonymous SNPs found in the human population are linked to certain diseases. Since synonymous SNPs are not expected to alter the composition of the polypeptide chain, without wishing to be bound by theory, is it believed that the effect of a synonymous SNP is linked to bias in codon usage. For example, a synonymous SNP may result in reduced protein translation, altered protein folding, altered protein localization or altered protein function. The relationship between codon usage and tRNA abundance is currently being investigated.
In an embodiment, the amount of a tRNA in a cell is correlated with codon usage. In an embodiment, a tRNA which pairs with a codon that is highly used is more abundant than a tRNA which pairs with a codon that is not highly used. In an embodiment, a tRNA which pairs with a codon that is not highly used is less abundant than a tRNA which pairs with a codon that is highly used.
As defined herein, the tRNA pool in a cell is the tRNA pool of all species, e.g., endogenous tRNAs and TREMS, which can function as tRNAs. The endogenous tRNA pool for a cell or subject that has not been administered a TREM includes only endogenous tRNAs. The tRNA pool for a cell or subject that has been administered a TREM includes endogenous tRNAs and the TREM.
Without wishing to be bound by theory, it is believed that the tRNA pool in a cell or subject can be altered by administering a composition comprising a TREM to the cell or subject. In an embodiment, the tRNA pool in a cell or subject that has been administered a Composition comprising a TREM comprises endogenous tRNAs and the administered TREM.
In an aspect, disclosed herein is a TREM composition comprising a TREM, a TREM core fragment or TREM fragment (e.g., a pharmaceutical composition comprising a TREM as described herein) for use in modulating a tRNA pool in a cell or subject, e.g., as described herein.
In embodiments, a TREM composition (e.g., a pharmaceutical composition comprising a TREM) described herein is contacted with a cell or tissue, or administered to a subject in need thereof, in an amount and for a time sufficient to modulate (increase or decrease) the tRNA pool. In embodiments, the tRNA pool comprises a first tRNA moiety and an additional tRNA moiety, e.g., a second tRNA moiety. In an embodiment, a tRNA moiety comprises an endogenous tRNA and/or a TREM.
In an embodiment, TREM composition described herein (e.g., a pharmaceutical composition comprising a TREM as described herein) can be used to treat a subject having an endogenous ORF comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC).
A TREM composition (e.g., a pharmaceutical composition comprising a TREM as described herein) can also be used to modulate a function in a cell, tissue or subject. In embodiments, a TREM composition (e.g., a pharmaceutical composition comprising a TREM) described herein is contacted with a cell or tissue, or administered to a subject in need thereof, in an amount and for a time sufficient to modulate (increase or decrease) one or more of the following parameters: adaptor function (e.g., cognate or non-cognate adaptor function), e.g., the rate, efficiency, robustness, and/or specificity of initiation or elongation of a polypeptide chain; ribosome binding and/or occupancy; regulatory function (e.g., gene silencing or signaling); cell fate; mRNA stability; protein localization; protein folding; protein stability; protein transduction; or protein compartmentalization.
A parameter may be modulated, e.g., by at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 40%. 50%. 60%. 70%, 80%, 90%, 100%, 150%, 200% or more) compared to a reference tissue, cell or subject (e.g., a healthy, wild-type or control cell, tissue or subject).
In an embodiment, a subject or a cell having a synonymous SNP has a tRNA pool which has a lower abundance of the tRNA that pairs with the SNP codon. In an embodiment, administration of a TREM that pairs with the SNP codon to the subject or cell, increases the amount of the isoaccepting tRNA pool in the subject or cell, e.g., increase the amount of amino acid specifying molecule that can pair with the SNP codon.
Exemplary synonymous SNPs and related genes are provided in Tables 1 and
In an embodiment, a cell or subject described in a method of treatment, a method of modulating a tRNA pool, or a method of evaluation disclosed herein has a SNP provided in Table 1 or
A “tRNA-based effector molecule” or “TREM” refers to an RNA molecule comprising one or more of the properties described herein. A TREM can comprise a non-naturally occurring modification, e.g., as provided in Tables 4, 5, 6 or 7.
In an embodiment, a TREM includes a TREM comprising a sequence of Formula A; a TREM core fragment comprising a sequence of Formula B; or a TREM fragment comprising a portion of a TREM which TREM comprises a sequence of Formula A.
In an embodiment, a TREM comprises a sequence of Formula A: [L1]-[ASt Domain1]-[L2]-[DH Domain]-[L3]-[ACH Domain]-[VL Domain]-[TH Domain]-[L4]-[ASt Domain2]. In an embodiment, [VL Domain] is optional. In an embodiment, [L1] is optional.
In an embodiment, a TREM core fragment comprises a sequence of Formula B: [L1]y-[ASt Domain1]x-[L2]y-[DH Domain]y-[L3]y-[ACH Domain]x-[VL Domain]y-[TH Domain]y-[L4]y-[ASt Domain2]x, wherein: x=1 and y=0 or 1. In an embodiment, y=0. In an embodiment, y=1.
In an embodiment, a TREM fragment comprises a portion of a TREM, wherein the TREM comprises a sequence of Formula A: [L1]-[ASt Domain1]-[L2]-[DH Domain]-[L3]-[ACH Domain]-[VL Domain]-[TH Domain]-[L4]-[ASt Domain2], and wherein the TREM fragment comprises: one, two, three or all or any combination of the following: a TREM half (e.g., from a cleavage in the ACH Domain, e.g., in the anticodon sequence, e.g., a 5′ half or a 3′ half); a 5′ fragment (e.g., a fragment comprising the 5′ end, e.g., from a cleavage in a DH Domain or the ACH Domain); a 3′ fragment (e.g., a fragment comprising the 3′ end, e.g., from a cleavage in the TH Domain); or an internal fragment (e.g., from a cleavage in any one of the ACH Domain, DH Domain or TH Domain). Exemplary TREM fragments include TREM halves (e.g., from a cleavage in the ACHD, e.g., 5′TREM halves or 3′ TREM halves), a 5′ fragment (e.g., a fragment comprising the 5′ end, e.g., from a cleavage in a DHD or the ACHD), a 3′ fragment (e.g., a fragment comprising the 3′ end of a TREM, e.g., from a cleavage in the THD), or an internal fragment (e.g., from a cleavage in one or more of the ACHD, DHD or THD).
In an embodiment, a TREM, a TREM core fragment or a TREM fragment can be charged with an amino acid (e.g., a cognate amino acid); charged with a non-cognate amino acid (e.g., a mischarged TREM (mTREM)); or not charged with an amino acid (e.g., an uncharged TREM (uTREM)). In an embodiment, a TREM, a TREM core fragment or a TREM fragment can be charged with an amino acid selected from alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, methionine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.
In an embodiment, the TREM, TREM core fragment or TREM fragment is a cognate TREM. In an embodiment, the TREM, TREM core fragment or TREM fragment is a non-cognate TREM. In an embodiment, the TREM, TREM core fragment or TREM fragment recognizes a codon provided in Table 2 or Table 3.
In an embodiment, a TREM comprises a ribonucleic acid (RNA) sequence encoded by a deoxyribonucleic acid (DNA) sequence disclosed in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4. In an embodiment, a TREM comprises an RNA sequence at least 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence provided in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4. In an embodiment, a TREM comprises an RNA sequence encoded by a DNA sequence at least 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a DNA sequence provided in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4.
In an embodiment, a TREM, a TREM core fragment, or TREM fragment comprises at least 5, 10, 15, 20, 25, or 30 consecutive nucleotides of an RNA sequence encoded by a DNA sequence disclosed in Table 4, e.g., at least 5, 10, 15, 20, 25, or 30 consecutive nucleotides of an RNA sequence encoded by any one of SEQ ID NOs: 1-451 disclosed in Table 4. In an embodiment, a TREM, a TREM core fragment, or TREM fragment comprises at least 5, 10, 15, 20, 25, or 30 consecutive nucleotides of an RNA sequence at least 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence provided in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4. In an embodiment, a TREM, a TREM core fragment, or TREM fragment comprises at least 5, 10, 15, 20, 25, or 30 consecutive nucleotides of an RNA sequence encoded by a DNA sequence at least 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a DNA sequence provided in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4.
In an embodiment, a TREM core fragment or a TREM fragment comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of an RNA sequence encoded by a DNA sequence provided in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4. In an embodiment, a TREM core fragment or a TREM fragment comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of an RNA sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence provided in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4. In an embodiment, a TREM core fragment or a TREM fragment comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of an RNA sequence encoded by a DNA sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a DNA sequence provided in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4.
In an embodiment, a TREM core fragment or a TREM fragment comprises at least 5 ribonucleotides (nt), 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt or 60 nt (but less than the full length) of an RNA sequence encoded by a DNA sequence disclosed in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4. In an embodiment, a TREM core fragment or a TREM fragment comprises at least 5 ribonucleotides (nt), 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt or 60 nt (but less than the full length) of an RNA sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an RNA sequence encoded by a DNA sequence provided in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4. In an embodiment, a TREM core fragment or a TREM fragment comprises at least 5 ribonucleotides (nt), 10 nt, 15 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt or 60 nt (but less than the full length) of an RNA sequence encoded by a DNA sequence with at least 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identity to a DNA sequence provided in Table 4, e.g., any one of SEQ ID NOs: 1-451 disclosed in Table 4.
In an embodiment, a TREM core fragment or a TREM fragment comprises a sequence of a length of between 10-90 ribonucleotides (rnt), between 10-80 rnt, between 10-70 rnt, between 10-60 rnt, between 10-50 rnt, between 10-40 rnt, between 10-30 rnt, between 10-20 rnt, between 20-90 rnt, between 20-80 rnt, 20-70 rnt, between 20-60 rnt, between 20-50 rnt, between 20-40 rnt, between 30-90 rnt, between 30-80 rnt, between 30-70 rnt, between 30-60 rnt, or between 30-50 rnt.
A TREM, a TREM core fragment or a TREM fragment described herein comprises a non-naturally occurring modification, e.g., a modification described in any one of Tables 5-9. A non-naturally occurring modification can be made according to methods known in the art. Exemplary methods of making non-naturally occurring modifications are provided in Examples 4-7.
In an embodiment, a non-naturally occurring modification is a modification that a cell, e.g., a human cell, does not make on an endogenous tRNA.
In an embodiment, a non-naturally occurring modification is a modification that a cell, e.g., a human cell, can make on an endogenous tRNA, but wherein such modification is in a location in which it does not occur on a native tRNA. In an embodiment, the non-naturally occurring modification is in a domain, linker or arm which does not have such modification in nature. In an embodiment, the non-naturally occurring modification is at a position within a domain, linker or arm, which does not have such modification in nature. In an embodiment, the non-naturally occurring modification is on a nucleotide which does not have such modification in nature. In an embodiment, the non-naturally occurring modification is on a nucleotide at a position within a domain, linker or arm, which does not have such modification in nature.
In an embodiment, a TREM, a TREM core fragment or a TREM fragment described herein comprises a non-naturally occurring modification provided in Table 5, or a combination thereof.
In an embodiment, a TREM, a TREM core fragment or a TREM fragment described herein comprises a modification provided in Table 6, or a combination thereof. The modifications provided in Table 6 occur naturally in RNAs, and are used herein on a synthetic TREM, a TREM core fragment or a TREM fragment at a position that does not occur in nature.
In an embodiment, a TREM, a TREM core fragment or a TREM fragment described herein comprises a non-naturally occurring modification provided in Table 7, or a combination thereof.
In an embodiment, a TREM, a TREM core fragment or a TREM fragment described herein comprises a non-naturally occurring modification provided in Table 8, or a combination thereof.
In an embodiment, a TREM, a TREM core fragment or a TREM fragment described herein comprises a non-naturally occurring modification provided in Table 9, or a combination thereof.
In an embodiment, a TREM, a TREM core fragment or a TREM fragment disclosed herein comprises an additional moiety, e.g., a fusion moiety. In an embodiment, the fusion moiety can be used for purification, to alter folding of the TREM, TREM core fragment or TREM fragment, or as a targeting moiety. In an embodiment, the fusion moiety can comprise a tag, a linker, can be cleavable or can include a binding site for an enzyme. In an embodiment, the fusion moiety can be disposed at the N terminal of the TREM or at the C terminal of the TREM, TREM core fragment or TREM fragment. In an embodiment, the fusion moiety can be encoded by the same or different nucleic acid molecule that encodes the TREM, TREM core fragment or TREM fragment.
In an embodiment, a TREM disclosed herein comprises a consensus sequence provided herein.
In an embodiment, a TREM disclosed herein comprises a consensus sequence of Formula IZZZ, wherein ZZZ indicates any of the twenty amino acids and Formula I corresponds to all species.
In an embodiment, a TREM disclosed herein comprises a consensus sequence of Formula IIZZZ, wherein ZZZ indicates any of the twenty amino acids and Formula II corresponds to mammals.
In an embodiment, a TREM disclosed herein comprises a consensus sequence of Formula IIIZZZ, wherein ZZZ indicates any of the twenty amino acids and Formula III corresponds to humans.
In an embodiment, ZZZ indicates any of the twenty amino acids: alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, methionine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.
In an embodiment, a TREM disclosed herein comprises a property selected from the following:
a) under physiological conditions residue R0 forms a linker region, e.g., a Linker 1 region;
b) under physiological conditions residues R1-R2-R3-R4-R5-R6-R7 and residues R65-R66-R67-R68-R69-R70-R71 form a stem region, e.g., an AStD stem region;
c) under physiological conditions residues R8-R9 forms a linker region, e.g., a Linker 2 region;
d) under physiological conditions residues —R10-R11-R12-R13-R14 R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28 form a stem-loop region, e.g., a D arm Region;
e) under physiological conditions residue —R29 forms a linker region, e.g., a Linker 3 Region;
f) under physiological conditions residues —R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46 form a stem-loop region, e.g., an AC arm region;
g) under physiological conditions residue —[R47]x comprises a variable region, e.g., as described herein;
h) under physiological conditions residues —R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64 form a stem-loop region, e.g., a T arm Region; or
i) under physiological conditions residue R72 forms a linker region, e.g., a Linker 4 region.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IALA (SEQ ID NO: 562),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Ala is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIALA (SEQ ID NO: 563),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Ala is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIALA (SEQ ID NO: 564),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Ala is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IARG (SEQ ID NO: 565),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Arg is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIARG (SEQ ID NO: 566),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Arg is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIARG (SEQ ID NO: 567),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Arg is:
R18=is absent;
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IASN (SEQ ID NO: 568),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Asn is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIASN (SEQ ID NO: 569),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Asn is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIASN (SEQ ID NO: 570),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Asn is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula I ASP (SEQ ID NO: 571),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Asp is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula II ASP (SEQ ID NO: 572),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Asp is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula III ASP (SEQ ID NO: 573),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Asp is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula ICYS (SEQ ID NO: 574),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R1-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Cys is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IICYS (SEQ ID NO: 575),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R1-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Cys is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIICYS (SEQ ID NO: 576),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R1-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Cys is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IGLN (SEQ ID NO: 577),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Gln is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIGLN (SEQ ID NO: 578),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Gln is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIGLN (SEQ ID NO: 579),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Gln is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IGLU (SEQ ID NO: 580),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Glu is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIGLU (SEQ ID NO: 581),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R1-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Glu is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIGLU (SEQ ID NO: 582),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Glu is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IGLY (SEQ ID NO: 583),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Gly is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIGLY (SEQ ID NO: 584),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Gly is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIGLY (SEQ ID NO: 585),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Gly is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IHIS (SEQ ID NO: 586),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R1-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R3-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for His is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIHIS (SEQ ID NO: 587),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for His is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIHIS (SEQ ID NO: 588),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for His is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IILE (SEQ ID NO: 589),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Ile is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula II ILE (SEQ ID NO: 590),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Ile is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula III ILE (SEQ ID NO: 591),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Ile is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IMET (SEQ ID NO: 592),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Met is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIMET (SEQ ID NO: 593),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Met is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIMET (SEQ ID NO: 594),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Met is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula ILEU (SEQ ID NO: 595),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Leu is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IILEU (SEQ ID NO: 596),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Leu is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIILEU (SEQ ID NO: 597),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Leu is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula ILys (SEQ ID NO: 598),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Lys is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IILYS (SEQ ID NO: 599),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Lys is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIILYS (SEQ ID NO: 600),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Lys is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula I PHE (SEQ ID NO: 601),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Phe is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula II PHE (SEQ ID NO: 602),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Phe is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula III PHE (SEQ ID NO: 603),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Phe is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IPRO (SEQ ID NO: 604),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Pro is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIPRO (SEQ ID NO: 605),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Pro is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIPRO (SEQ ID NO: 606),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Pro is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula ISER (SEQ ID NO: 607),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Ser is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IISER (SEQ ID NO: 608),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Ser is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIISER (SEQ ID NO: 609),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R3-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Ser is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula ITHR (SEQ ID NO: 610),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Thr is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IITHR (SEQ ID NO: 611),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Thr is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIITHR (SEQ ID NO: 612),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Thr is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula ITRP (SEQ ID NO: 613),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R1-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R3-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Trp is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IITRP (SEQ ID NO: 614),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R1-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Trp is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIITRP (SEQ ID NO: 615),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Trp is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula ITYR (SEQ ID NO: 616),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R1-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R3-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R4-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Tyr is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IITYR (SEQ ID NO: 617),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Tyr is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIITYR (SEQ ID NO: 618),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Tyr is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IVAL (SEQ ID NO: 619),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R1-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Val is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIVAL (SEQ ID NO: 620),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Val is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises the sequence of Formula IIIVAL (SEQ ID NO: 621),
R0-R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R11-R12-R13-R14-R15-R16-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-R30-R31-R32-R33-R34-R35-R36-R37-R38-R39-R40-R41-R42-R43-R44-R45-R46-[R47]x-R48-R49-R50-R51-R52-R53-R54-R55-R56-R57-R58-R59-R60-R61-R62-R63-R64-R65-R66-R67-R68-R69-R70-R71-R72
wherein R is a ribonucleotide residue and the consensus for Val is:
wherein, e.g., x=1-271 (e.g., x=1-250, x=1-225, x=1-200, x=1-175, x=1-150, x=1-125, x=1-100, x=1-75, x=1-50, x=1-40, x=1-30, x=1-29, x=1-28, x=1-27, x=1-26, x=1-25, x=1-24, x=1-23, x=1-22, x=1-21, x=1-20, x=1-19, x=1-18, x=1-17, x=1-16, x=1-15, x=1-14, x=1-13, x=1-12, x=1-11, x=1-10, x=10-271, x=20-271, x=30-271, x=40-271, x=50-271, x=60-271, x=70-271, x=80-271, x=100-271, x=125-271, x=150-271, x=175-271, x=200-271, x=225-271, x=1, x=2, x=3, x=4, x=5, x=6, x=7, x=8, x=9, x=10, x=11, x=12, x=13, x=14, x=15, x=16, x=17, x=18, x=19, x=20, x=21, x=22, x=23, x=24, x=25, x=26, x=27, x=28, x=29, x=30, x=40, x=50, x=60, x=70, x=80, x=90, x=100, x=110, x=125, x=150, x=175, x=200, x=225, x=250, or x=271), provided that the TREM has one or both of the following properties: no more than 15% of the residues are N; or no more than 20 residues are absent.
In an embodiment, a TREM disclosed herein comprises a variable region at position R47. In an embodiment, the variable region is 1-271 ribonucleotides in length (e.g. 1-250, 1-225, 1-200, 1-175, 1-150, 1-125, 1-100, 1-75, 1-50, 1-40, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 10-271, 20-271, 30-271, 40-271, 50-271, 60-271, 70-271, 80-271, 100-271, 125-271, 150-271, 175-271, 200-271, 225-271, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 125, 150, 175, 200, 225, 250, or 271 ribonucleotides). In an embodiment, the variable region comprises any one, all or a combination of Adenine, Cytosine, Guanine or Uracil.
In an embodiment, the variable region comprises a ribonucleic acid (RNA) sequence encoded by a deoxyribonucleic acid (DNA) sequence disclosed in Table 10, e.g., any one of SEQ ID NOs: 452-561 disclosed in Table 10.
In vitro methods for synthesizing oligonucleotides are known in the art and can be used to make a TREM, a TREM core fragment or a TREM fragment disclosed herein. For example, a TREM, TREM core fragment or TREM fragment can be synthesized using solid state synthesis or liquid phase synthesis.
In an embodiment, a TREM, a TREM core fragment or a TREM fragment made according to an in vitro synthesis method disclosed herein has a different modification profile compared to a TREM expressed and isolated from a cell, or compared to a naturally occurring tRNA.
An exemplary method for making a synthetic TREM via 5′-Silyl-2′-Orthoester (2′-ACE) Chemistry is provided in Example 3. The method provided in Example 3 can also be used to make a synthetic TREM core fragment or synthetic TREM fragment. Additional synthetic methods are disclosed in Hartsel S A et al., (2005) Oligonucleotide Synthesis, 033-050, the entire contents of which are hereby incorporated by reference.
In an embodiment, a TREM composition, e.g., a TREM pharmaceutical composition, comprises a pharmaceutically acceptable excipient. Exemplary excipients include those provided in the FDA Inactive Ingredient Database
(https://www.accessdata.fda.gov/scripts/cder/iig/index.Cfm).
In an embodiment, a TREM composition, e.g., a TREM pharmaceutical composition, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or 150 grams of TREM, TREM core fragment or TREM fragment. In an embodiment, a TREM composition, e.g., a TREM pharmaceutical composition, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or 100 milligrams of TREM, TREM core fragment or TREM fragment.
In an embodiment, a TREM composition, e.g., a TREM pharmaceutical composition, is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99% dry weight TREMs, TREM core fragments or TREM fragments.
In an embodiment, a TREM composition comprises at least 1×106 TREM molecules, at least 1×107 TREM molecules, at least 1×108 TREM molecules or at least 1×109 TREM molecules.
In an embodiment, a TREM composition comprises at least 1×106 TREM core fragment molecules, at least 1×107 TREM core fragment molecules, at least 1×108 TREM core fragment molecules or at least 1×109 TREM core fragment molecules.
In an embodiment, a TREM composition comprises at least 1×106 TREM fragment molecules, at least 1×107 TREM fragment molecules, at least 1×108 TREM fragment molecules or at least 1×109 TREM fragment molecules.
In an embodiment, a TREM composition produced by any of the methods of making disclosed herein can be charged with an amino acid using an in vitro charging reaction as known in the art.
In an embodiment, a TREM composition comprise one or more species of TREMs, TREM core fragments, or TREM fragments. In an embodiment, a TREM composition comprises a single species of TREM, TREM core fragment, or TREM fragment. In an embodiment, a TREM composition comprises a first TREM, TREM core fragment, or TREM fragment species and a second TREM, TREM core fragment, or TREM fragment species. In an embodiment, the TREM composition comprises X TREM, TREM core fragment, or TREM fragment species, wherein X=2, 3, 4, 5, 6, 7, 8, 9, or 10.
In an embodiment, the TREM, TREM core fragment, or TREM fragment has at least 70, 75, 80, 85, 90, or 95, or has 100%, identity with a sequence encoded by a nucleic acid in Table 4.
In an embodiment, the TREM comprises a consensus sequence provided herein.
A TREM composition can be formulated as a liquid composition, as a lyophilized composition or as a frozen composition.
In some embodiments, a TREM composition can be formulated to be suitable for pharmaceutical use, e.g., a pharmaceutical TREM composition. In an embodiment, a pharmaceutical TREM composition is substantially free of materials and/or reagents used to separate and/or purify a TREM, TREM core fragment, or TREM fragment.
In some embodiments, a TREM composition can be formulated with water for injection. In some embodiments, a TREM composition formulated with water for injection is suitable for pharmaceutical use, e.g., comprises a pharmaceutical TREM composition.
A TREM, TREM core fragment, or TREM fragment, or a TREM composition, e.g., a pharmaceutical TREM composition, produced by any of the methods disclosed herein can be assessed for a characteristic associated with the TREM, TREM core fragment, or TREM fragment or the TREM composition, such as purity, sterility, concentration, structure, or functional activity of the TREM, TREM core fragment, or TREM fragment. Any of the above-mentioned characteristics can be evaluated by providing a value for the characteristic, e.g., by evaluating or testing the TREM, TREM core fragment, or TREM fragment, or the TREM composition, or an intermediate in the production of the TREM composition. The value can also be compared with a standard or a reference value. Responsive to the evaluation, the TREM composition can be classified, e.g., as ready for release, meets production standard for human trials, complies with ISO standards, complies with cGMP standards, or complies with other pharmaceutical standards. Responsive to the evaluation, the TREM composition can be subjected to further processing, e.g., it can be divided into aliquots, e.g., into single or multi-dosage amounts, disposed in a container, e.g., an end-use vial, packaged, shipped, or put into commerce. In embodiments, in response to the evaluation, one or more of the characteristics can be modulated, processed or re-processed to optimize the TREM composition. For example, the TREM composition can be modulated, processed or re-processed to (i) increase the purity of the TREM composition; (ii) decrease the amount of fragments in the composition; (iii) decrease the amount of endotoxins in the composition; (iv) increase the in vitro translation activity of the composition; (v) increase the TREM concentration of the composition; or (vi) inactivate or remove any viral contaminants present in the composition, e.g., by reducing the pH of the composition or by filtration.
In an embodiment, the TREM, TREM core fragment, or TREM fragment (e.g., TREM composition or an intermediate in the production of the TREM composition) has a purity of at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, i.e., by mass.
In an embodiment, the TREM (e.g., TREM composition or an intermediate in the production of the TREM composition) has less than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% TREM fragments relative to full length TREMs.
In an embodiment, the TREM, TREM core fragment, or TREM fragment (e.g., TREM composition or an intermediate in the production of the TREM composition) has low levels or absence of endotoxins, e.g., a negative result as measured by the Limulus amebocyte lysate (LAL) test.
In an embodiment, the TREM, TREM core fragment, or TREM fragment (e.g., TREM composition or an intermediate in the production of the TREM composition) has in-vitro translation activity, e.g., as measured by an assay described in Examples 12-13.
In an embodiment, the TREM, TREM core fragment, or TREM fragment (e.g., TREM composition or an intermediate in the production of the TREM composition) has a TREM concentration of at least 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, 0.1 ug/mL, 0.5 ug/mL, 1 ug/mL, 2 ug/mL, 5 ug/mL, 10 ug/mL, 20 ug/mL, 30 ug/mL, 40 ug/mL, 50 ug/mL, 60 ug/mL, 70 ug/mL, 80 ug/mL, 100 ug/mL, 200 ug/mL, 300 ug/mL, 500 ug/mL, 1000 ug/mL, 5000 ug/mL, 10,000 ug/mL, or 100,000 ug/mL.
In an embodiment, the TREM, TREM core fragment, or TREM fragment (e.g., TREM composition or an intermediate in the production of the TREM composition) is sterile, e.g., the composition or preparation supports the growth of fewer than 100 viable microorganisms as tested under aseptic conditions, the composition or preparation meets the standard of USP <71>, and/or the composition or preparation meets the standard of USP <85>.
In an embodiment, the TREM, TREM core fragment, or TREM fragment (e.g., TREM composition or an intermediate in the production of the TREM composition) has an undetectable level of viral contaminants, e.g., no viral contaminants. In an embodiment, any viral contaminant, e.g., residual virus, present in the composition is inactivated or removed. In an embodiment, any viral contaminant, e.g., residual virus, is inactivated, e.g., by reducing the pH of the composition. In an embodiment, any viral contaminant, e.g., residual virus, is removed, e.g., by filtration or other methods known in the field.
Any TREM composition or pharmaceutical composition described herein can be administered to a cell, tissue or subject, e.g., by direct administration to a cell, tissue and/or an organ in vitro, ex-vivo or in vivo. In-vivo administration may be via, e.g., by local, systemic and/or parenteral routes, for example intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, ocular, nasal, urogenital, intradermal, dermal, enteral, intravitreal, intracerebral, intrathecal, or epidural.
In some embodiments the TREM, TREM core fragment, or TREM fragment or TREM composition described herein, is delivered to cells, e.g. mammalian cells or human cells, using a vector. The vector may be, e.g., a plasmid or a virus. In some embodiments, delivery is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the virus is an adeno associated virus (AAV), a lentivirus, or an adenovirus. In some embodiments, the system or components of the system are delivered to cells with a viral-like particle or a virosome. In some embodiments, the delivery uses more than one virus, viral-like particle or virosome.
A TREM, a TREM composition or a pharmaceutical TREM composition described herein may comprise, may be formulated with, or may be delivered in, a carrier.
The carrier may be a viral vector (e.g., a viral vector comprising a sequence encoding a TREM, a TREM core fragment or a TREM fragment). The viral vector may be administered to a cell or to a subject (e.g., a human subject or animal model) to deliver a TREM, a TREM core fragment or a TREM fragment, a TREM composition or a pharmaceutical TREM composition.
A viral vector may be systemically or locally administered (e.g., injected). Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell. Viral genomes are known in the art as useful vectors for delivery because the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors include a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus, replication deficient herpes virus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology (Third Edition) Lippincott-Raven, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the teachings of which are incorporated herein by reference. In some embodiments the system or components of the system are delivered to cells with a viral-like particle or a virosome.
A TREM, a TREM core fragment or a TREM fragment, a TREM composition or a pharmaceutical TREM composition described herein can be administered to a cell in a vesicle or other membrane-based carrier.
In embodiments, a TREM, a TREM core fragment or a TREM fragment, or TREM composition, or pharmaceutical TREM composition described herein is administered in or via a cell, vesicle or other membrane-based carrier. In one embodiment, the TREM, TREM core fragment, TREM fragment, or TREM composition or pharmaceutical TREM composition can be formulated in liposomes or other similar vesicles. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.
Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for the TREM, TREM core fragment, TREM fragment, or TREM composition or pharmaceutical TREM composition described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of the SLN, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, may also be employed. These nanoparticles possess the complementary advantages of PNPs and liposomes. A PLN is composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell offers good biocompatibility. As such, the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390/nano7060122.
Exemplary lipid nanoparticles are disclosed in International Application PCT/US2014/053907, the entire contents of which are hereby incorporated by reference. For example, an LNP described in paragraphs [403-406] or [410-413] of PCT/US2014/053907 can be used as a carrier for the TREM, TREM core fragment, TREM fragment, or TREM composition or pharmaceutical TREM composition described herein.
Additional exemplary lipid nanoparticles are disclosed in U.S. Pat. No. 10,562,849 the entire contents of which are hereby incorporated by reference. For example, an LNP of formula (I) as described in columns 1-3 of U.S. Pat. No. 10,562,849 can be used as a carrier for the TREM, TREM core fragment, TREM fragment, or TREM composition or pharmaceutical TREM composition described herein.
Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example those described in Table 4 of WO2019217941, which is incorporated by reference, e.g., a lipid-containing nanoparticle can comprise one or more of the lipids in Table 4 of WO2019217941. Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of WO2019217941, incorporated by reference.
In some embodiments, conjugated lipids, when present, can include one or more of PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2′,3′-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypoly ethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing.
In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in WO2009/127060 or US2010/0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al (2020), incorporated herein by reference.
In some embodiments, the lipid particle comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticle comprises an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10:1 to about 30:1.
In some embodiments, the lipid to nucleic acid ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the lipid nanoparticle formulation's overall lipid content can range from about 5 mg/ml to about 30 mg/mL.
Some non-limiting example of lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA) described herein includes,
In some embodiments an LNP comprising Formula (i) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (ii) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (iii) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (v) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (vi) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (viii) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (ix) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
wherein X1 is O, NR1 or a direct bond, X2 is C2-5 alkylene, X3 is C(═O) or a direct bond, R1 is H or Me. R3 is Ci-3 alkyl, R2 is Ci-3 alkyl, or R2 taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2 form a 4-, 5-, or 6-membered ring, or X1 is NR1, R1 and R2 taken together with the nitrogen atoms to which they are attached from a 5- or 6-membered ring, or R2 taken together with R3 and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y is C2-12 alkylene. Y2 is selected from
n is 0 to 3, R4 is Ci-15 alkyl, Z1 is Ci-6 alkylene or a direct bond, Z2 is
(in either orientation) or absent, provided that if Z1 is a direct bond, Z2 is absent; R2 is C5-9 alkyl or C6-10 alkoxy, R6 is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7 is H or Me, or a salt thereof, provided that if R3 and R2 are C2 alkyls. X1 is O, X2 is linear C3 alkylene, X3 is C(═O), Y1 is linear Ce alkylene, (Y2)n-R4 is
R4 is linear C5 alkyl, Z1 is C2 alkylene, Z2 is absent, W is methylene, and R7 is H, then R5 and R6 are not Cx alkoxy.
In some embodiments an LNP comprising Formula (xii) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising Formula (xi) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprises a compound of Formula (xiii) and a compound of Formula (xiv).
In some embodiments, an LNP comprising Formula (xv) is used to deliver a TREM composition described herein to the liver and/or hepatocyte cells.
In some embodiments an LNP comprising a formulation of Formula (xvi) is used to deliver a TREM composition described herein to the lung endothelial cells.
In some embodiments, a lipid compound used to form lipid nanoparticles for the delivery of compositions described herein, e.g., a TREM described herein is made by one of the following reactions:
In some embodiments, a composition described herein (e.g., TREM composition) is provided in an LNP that comprises an ionizable lipid. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of U.S. Pat. No. 9,867,888 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01), e.g., as synthesized in Example 13 of WO2015/095340 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Di((Z)-non-2-en-1-yl) 9-((4-dimethylamino)-butanoyl)oxy)heptadecanedioate (L319), e.g. as synthesized in Example 7, 8, or 9 of US2012/0027803 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 1,1′-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl) amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO2010/053572 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, e.g., Structure (I) from WO2020/106946 (incorporated by reference herein in its entirety).
In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. In some embodiments, the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, a lipid nanoparticle may comprise a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a TREM described herein, encapsulated within or associated with the lipid nanoparticle. In some embodiments, the TREM is co-formulated with the cationic lipid. The TREM may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the TREM may be encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipid described herein, e.g., Formula (i), (ii), (ii), (vii) and/or (ix) encapsulates at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of a TREM.
Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016/0311759; I of US20150376115 or in US2016/0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-c of US20150140070; A of US2013/0178541; I of US2013/0303587 or US2013/0123338; I of US2015/0141678; II, III, IV, or V of US2015/0239926; I of US2017/0119904; I or II of WO2017/117528; A of US2012/0149894; A of US2015/0057373; A of WO2013/116126; A of US2013/0090372; A of US2013/0274523; A of US2013/0274504; A of US2013/0053572; A of WO2013/016058; A of WO2012/162210; I of US2008/042973; I, II, III, or IV of US2012/01287670; I or II of US2014/0200257; I, II, or III of US2015/0203446; I or III of US2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014/0308304; of US2013/0338210; I, II, III, or IV of WO2009/132131; A of US2012/01011478; I or XXXV of US2012/0027796; XIV or XVII of US2012/0058144; of US2013/0323269; I of US2011/0117125; I, II, or III of US2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011/0076335; I or II of US2006/008378; I of US2013/0123338; I or X-A-Y-Z of US2015/0064242; XVI, XVII, or XVIII of US2013/0022649; I, II, or III of US2013/0116307; I, II, or III of US2013/0116307; I or II of US2010/0062967; I-X of US2013/0189351; I of US2014/0039032; V of US2018/0028664; I of US2016/0317458; I of US2013/0195920; 5, 6, or 10 of U.S. Pat. No. 10,221,127; III-3 of WO2018/081480; I-5 or I-8 of WO2020/081938; 18 or 25 of U.S. Pat. No. 9,867,888; A of US2019/0136231; II of WO2020/219876; 1 of US2012/0027803; OF-02 of US2019/0240349; 23 of U.S. Pat. No. 10,086,013; cKK-E12/A6 of Miao et al (2020); C12-200 of WO2010/053572; 7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al; TS-P4C2 of U.S. Pat. No. 9,708,628; I of WO2020/106946; I of WO2020/106946.
In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 1Z)-heptatriaconta-6,9,28,3 1-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13, 16-dien-1-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety).
Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.Oc01386, incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS).
Other examples of non-cationic lipids suitable for use in the lipid nanoparticles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017/099823 or US patent publication US2018/0028664, the contents of which is incorporated herein by reference in their entirety.
In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018/0028664, incorporated herein by reference in its entirety. The non-cationic lipid can comprise, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).
In some embodiments, the lipid nanoparticles do not comprise any phospholipids.
In some aspects, the lipid nanoparticle can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-choiestanol, 53-coprostanol, choiesteryl-(2′-hydroxy)-ethyl ether, choiesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., choiesteryl-(4′-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT publication WO2009/127060 and US patent publication US2010/0130588, each of which is incorporated herein by reference in its entirety.
In some embodiments, the component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.
In some embodiments, the lipid nanoparticle can comprise a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.
Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2′,3′-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. Nos. 5,885,613, 6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, and US/099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018/0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, a PEG-lipid is of Formula II of US20150376115 or US2016/0376224, the content of both of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8′-(Cholest-5-en-3[beta]-oxy)carboxamido-3′,6′-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from:
In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.
Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289A9, the contents of all of which are incorporated herein by reference in their entirety.
In some embodiments, the PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. Molar ratios of the ionizable lipid, non-cationic-lipid, sterol, and PEG/conjugated lipid can be varied as needed. For example, the lipid particle can comprise 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non-cationic-lipid by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10-20% non-cationic-lipid by mole or by total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. The composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic-lipid by mole or by total weight of the composition. The composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition. The formulation may also be a lipid nanoparticle formulation, for example comprising 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1-20% cholesterol by mole or by total weight of the composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5:1.5.
In some embodiments, the lipid particle comprises ionizable lipid, non-cationic lipid (e.g. phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of non-cationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5.
In some embodiments, the lipid particle comprises ionizable lipid/non-cationic-lipid/sterol/conjugated lipid at a molar ratio of 50:10:38.5:1.5.
In an aspect, the disclosure provides a lipid nanoparticle formulation comprising phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.
In some embodiments, one or more additional compounds can also be included. Those compounds can be administered separately, or the additional compounds can be included in the lipid nanoparticles of the invention. In other words, the lipid nanoparticles can contain other compounds in addition to the nucleic acid or at least a second nucleic acid, different than the first. Without limitations, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.
In some embodiments, LNPs are directed to specific tissues by the addition of targeting domains. For example, biological ligands may be displayed on the surface of LNPs to enhance interaction with cells displaying cognate receptors, thus driving association with and cargo delivery to tissues wherein cells express the receptor. In some embodiments, the biological ligand may be a ligand that drives delivery to the liver, e.g., LNPs that display GalNAc result in delivery of nucleic acid cargo to hepatocytes that display asialoglycoprotein receptor (ASGPR). The work of Akinc et al. Mol Ther 18(7):1357-1364 (2010) teaches the conjugation of a trivalent GalNAc ligand to a PEG-lipid (GalNAc-PEG-DSG) to yield LNPs dependent on ASGPR for observable LNP cargo effect (see, e.g., FIG. 6 of Akinc et al. 2010, supra). Other ligand-displaying LNP formulations, e.g., incorporating folate, transferrin, or antibodies, are discussed in WO2017223135, which is incorporated herein by reference in its entirety, in addition to the references used therein, namely Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; and Peer and Lieberman, Gene Ther. 2011 18:1127-1133.
In some embodiments, LNPs are selected for tissue-specific activity by the addition of a Selective ORgan Targeting (SORT) molecule to a formulation comprising traditional components, such as ionizable cationic lipids, amphipathic phospholipids, cholesterol and poly(ethylene glycol) (PEG) lipids. The teachings of Cheng et al. Nat Nanotechnol 15(4):313-320 (2020) demonstrate that the addition of a supplemental “SORT” component precisely alters the in vivo RNA delivery profile and mediates tissue-specific (e.g., lungs, liver, spleen) gene delivery and editing as a function of the percentage and biophysical property of the SORT molecule.
In some embodiments, the LNPs comprise biodegradable, ionizable lipids. In some embodiments, the LNPs comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g, lipids of WO2019/067992, WO/2017/173054, WO2015/095340, and WO2014/136086, as well as references provided therein. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.
In some embodiments, the average LNP diameter of the LNP formulation may be between 10 s of nm and 100 s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.
A LNP may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a LNP may be from about 0.10 to about 0.20.
The zeta potential of a LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may be from about −10 mV to about +20 mV, from about −10 mV to about +15 mV, from about −10 mV to about +10 mV, from about −10 mV to about +5 mV, from about −10 mV to about 0 mV, from about −10 mV to about −5 mV, from about −5 mV to about +20 mV, from about −5 mV to about +15 mV, from about −5 mV to about +10 mV, from about −5 mV to about +5 mV, from about −5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.
The efficiency of encapsulation of a TREM describes the amount of TREM that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of TREM in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free TREM in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a TREM may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.
A LNP may optionally comprise one or more coatings. In some embodiments, a LNP may be formulated in a capsule, film, or table having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness or density.
Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020061457, which is incorporated herein by reference in its entirety.
In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoy_ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.
LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910, both incorporated by reference.
Additional specific LNP formulations useful for delivery of nucleic acids are described in U.S. Pat. Nos. 8,158,601 and 8,168,775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO.
Exosomes can also be used as drug delivery vehicles for the TREM, TREM core fragment, TREM fragment, or TREM compositions or pharmaceutical TREM composition described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; https://doi.org/10.1016/j.apsb.2016.02.001.
Ex vivo differentiated red blood cells can also be used as a carrier for a TREM, TREM core fragment, TREM fragment, or TREM composition, or pharmaceutical TREM composition described herein. See, e.g., WO2015073587; WO2017123646; WO2017123644; WO2018102740; wO2016183482; WO2015153102; WO2018151829; WO2018009838; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28): 10131-10136; U.S. Pat. No. 9,644,180; Huang et al. 2017. Nature Communications 8: 423; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28): 10131-10136.
Fusosome compositions, e.g., as described in WO2018208728, can also be used as carriers to deliver the TREM, TREM core fragment, TREM fragment, or TREM composition, or pharmaceutical TREM composition described herein.
Virosomes and virus-like particles (VLPs) can also be used as carriers to deliver a TREM, TREM core fragment, TREM fragment, or TREM composition, or pharmaceutical TREM composition described herein to targeted cells.
Plant nanovesicles, e.g., as described in WO2011097480A1, WO2013070324A1, or WO2017004526A1 can also be used as carriers to deliver the TREM, TREM core fragment, TREM fragment, or TREM composition, or pharmaceutical TREM composition described herein.
Delivery without a Carrier
A TREM, a TREM core fragment or a TREM fragment, a TREM composition or a pharmaceutical TREM composition described herein can be administered to a cell without a carrier, e.g., via naked delivery of the TREM, a TREM core fragment or a TREM fragment, a TREM composition or a pharmaceutical TREM composition.
In some embodiments, naked delivery as used herein refers to delivery without a carrier. In some embodiments, delivery without a carrier, e.g., naked delivery, comprises delivery with a moiety, e.g., a targeting peptide.
In some embodiments, a TREM, a TREM core fragment or a TREM fragment, or TREM composition, or pharmaceutical TREM composition described herein is delivered to a cell without a carrier, e.g., via naked delivery. In some embodiments, the delivery without a carrier, e.g., naked delivery, comprises delivery with a moiety, e.g., a targeting peptide.
All references and publications cited herein are hereby incorporated by reference.
1. A TREM comprising a sequence of Formula A:
[L1]-[ASt Domain1]-[L2]-[DH Domain]-[L3]-[ACH Domain]-[VL Domain]-[TH Domain]-[L4]-[ASt Domain2],
wherein:
independently, [L1] and [VL Domain], are optional;
one of [L1], [ASt Domain1], [L2]-[DH Domain], [L3], [ACH Domain], [VL Domain], [TH Domain], [L4], and [ASt Domain2] comprises a nucleotide having a non-naturally occurring modification; and
wherein:
[L1]y-[ASt Domain1]x-[L2]y-[DH Domain]y-[L3]y-[ACH Domain]x-[VL Domain]y-[TH Domain]y-[L4]y-[ASt Domain2]x,
wherein:
x=1 and y=0 or 1;
one of [ASt Domain1], [ACH Domain], and [ASt Domain2] comprises a nucleotide having a non-naturally occurring modification; and
the TREM retains the ability to: support protein synthesis; be able to be charged by a synthetase, be bound by an elongation factor, introduce an amino acid into a peptide chain, support elongation, or support initiation.
23. The TREM core fragment of embodiment 22, wherein AStD1 and AStD2 comprise an ASt Domain (AStD).
24. The TREM core fragment of embodiment 22, wherein the [ASt Domain 1], and/or [ASt Domain 2] comprising the non-naturally occurring modification retains the ability to initiate or elongate a polypeptide chain.
25. The TREM core fragment of embodiment 22, wherein the [ACH Domain] comprising the non-naturally occurring modification retains the ability to mediate pairing with a codon.
26. The TREM core fragment of embodiment 22, wherein y=1 for any one, two, three, four, five, six, all or a combination of [L1], [L2], [DH Domain], [L3], [VL Domain], [TH Domain], [L4].
27. The TREM core fragment of embodiment 22, wherein y=0 for any one, two, three, four, five, six, all or a combination of [L1], [L2], [DH Domain], [L3], [VL Domain], [TH Domain], [L4].
28. The TREM core fragment of embodiment 22, wherein y=1 for linker [L1], and L1 comprises a nucleotide having a non-naturally occurring modification.
29. The TREM core fragment of embodiment 22, wherein y=1 for linker [L2], and L2 comprises a nucleotide having a non-naturally occurring modification.
30. The TREM core fragment of embodiment 22, wherein y=1 for [DH Domain (DHD)], and DHD comprises a nucleotide having a non-naturally occurring modification.
31. The TREM core fragment of embodiment 30, wherein the DHD comprising the non-naturally occurring modification retains the ability to mediate recognition of aminoacyl-tRNA synthetase.
32. The TREM core fragment of embodiment 22, wherein y=1 for linker [L3], and L3 comprises a nucleotide having a non-naturally occurring modification.
33. The TREM core fragment of embodiment 22, wherein y=1 for [VL Domain (VLD)], and VLD comprises a nucleotide having a non-naturally occurring modification.
34. The TREM core fragment of embodiment 22, wherein y=1 for [TH Domain (THD)], and THD comprises a nucleotide having a non-naturally occurring modification.
35. The TREM core fragment of embodiment 34, wherein the THD comprising the non-naturally occurring modification retains the ability to mediate recognition of the ribosome.
36. The TREM core fragment of embodiment 22, wherein y=1 for linker [L4], and L4 comprises a nucleotide having a non-naturally occurring modification.
37. A TREM fragment comprising a portion of a TREM, wherein the TREM comprises a sequence of Formula A:
[L1]-[ASt Domain1]-[L2]-[DH Domain]-[L3]-[ACH Domain]-[VL Domain]-[TH Domain]-[L4]-[ASt Domain2], and wherein:
the TREM fragment comprises:
a non-naturally occurring modification; and
one, two, three or all or any combination of the following:
acquiring knowledge of (i) comprises acquiring a value for the abundance, e.g., relative amount, of (i); and/or
acquiring knowledge of (ii) comprises acquiring a value for the abundance, e.g., relative amount, of (ii).
132. The method of embodiment 131, wherein responsive to said value, the method comprises administering a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, wherein the TREM has an anticodon that pairs with: (a) the codon having the first sequence; or (b) the codon other than the codon having the first sequence, in an amount and for a time sufficient to modulate the relative amounts of the first tRNA moiety and the second tRNA moiety.
133. A method of modulating a tRNA pool in a cell comprising an endogenous open reading frame (ORF), which ORF comprises a codon having a first sequence, comprising:
optionally, acquiring knowledge of the abundance of one or both of (i) and (ii), e.g., acquiring knowledge of the relative amounts of: (i) and (ii) in the cell, wherein (i) is a tRNA moiety having an anticodon that pairs with the codon of the ORF having a first sequence (the first tRNA moiety) and (ii) is an isoacceptor tRNA moiety having an anticodon that pairs with a codon other than the codon having the first sequence (the second tRNA moiety) in the cell;
contacting the cell with a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, wherein the TREM, TREM core fragment or TREM fragment has an anticodon that pairs with: (a) the codon having the first sequence; or (b) the codon other than the codon having the first sequence, in an amount and/or for a time sufficient to modulate the relative amounts of the first tRNA moiety and the second tRNA moiety in the cell, thereby modulating the tRNA pool in the cell.
134. The method of embodiment 133, wherein the TREM comprises an anticodon that pairs with (a).
135. The method of embodiment 134, wherein the TREM comprises an anticodon that pairs with (b).
136. A method of modulating a tRNA pool in a subject having an endogenous open reading frame (ORF), which ORF comprises a codon having a first sequence, comprising:
optionally, acquiring knowledge of the abundance of one or both of (i) and (ii), e.g., acquiring knowledge of the relative amounts of: (i) and (ii) in the subject, wherein (i) is a tRNA moiety having an anticodon that pairs with the codon of the ORF having a first sequence (the first tRNA moiety) and (ii) is an isoacceptor tRNA moiety having an anticodon that pairs with a codon other than the codon having the first sequence (the second tRNA moiety) in the subject;
contacting the subject with a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, wherein the TREM, TREM core fragment or TREM fragment has an anticodon that pairs with: (a) the codon having the first sequence; or (b) the codon other than the codon having the first sequence, in an amount and/or for a time sufficient to modulate the relative amounts of the first tRNA moiety and the second tRNA moiety in the subject,
thereby modulating the tRNA pool in the subject.
137. The method of embodiment 136, wherein the TREM comprises an anticodon that pairs with (a).
138 The method of embodiment 136, wherein the TREM comprises an anticodon that pairs with (b).
139. The method of any one of embodiments 136-138, comprising acquiring knowledge of (i).
140. The method of any one of embodiments 136-138, comprising acquiring knowledge of (ii).
141. The method of any one of embodiments 136-138, comprising acquiring knowledge of (i) and (ii).
142. The method of any one of embodiments 136-139 or 141, wherein acquiring knowledge of (i) comprises acquiring a value for the abundance, e.g., relative amounts, of (i).
143. The method of any one of embodiments 136-138 or 140-141, wherein acquiring knowledge of (ii) comprises acquiring a value for the abundance, e.g., relative amounts, of (ii).
144. The method of embodiment 18 or 19, wherein responsive to said value, the cell or subject is contacted with the TREM composition having an anticodon that pairs with (a) or (b).
145. A method of modulating a tRNA pool in a subject having an endogenous open reading frame (ORF) comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC), comprising:
providing a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, wherein the TREM, TREM core fragment or TREM fragment comprises an isoacceptor tRNA moiety comprising an anticodon sequence that pairs with the SMC (the TREM);
contacting the subject with the TREM composition in an amount and/or for a time sufficient to modulate the tRNA pool in the subject,
thereby modulating the tRNA pool in the subject.
146. A method of modulating a tRNA pool in a cell comprising an endogenous open reading frame (ORF) comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC), comprising:
providing a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, wherein the TREM, TREM core fragment or TREM fragment comprises an isoacceptor tRNA moiety comprising an anticodon sequence that pairs with the SMC (the TREM);
contacting the cell with the TREM composition in an amount and/or for a time sufficient to modulate the tRNA pool in the cell,
thereby modulating the tRNA pool in the cell.
147. The method of embodiment 145 or 146, comprising acquiring knowledge of the abundance of one or both of (i) and (ii) e.g., acquiring knowledge of the relative amounts of (i) and (ii) wherein (i) is a tRNA moiety having an anticodon that pairs with the SMC (the first tRNA moiety) and (ii) is an isoacceptor tRNA moiety having an anticodon that pairs with a codon other than the SMC (the second tRNA moiety), in the subject or cell.
148. The method of embodiment 147, comprising acquiring knowledge of (i).
149. The method of embodiment 147, comprising acquiring knowledge of (ii).
150. The method of embodiment 147, comprising acquiring knowledge of (i) and (ii).
151. The method of embodiment 147, wherein acquiring knowledge of (i) comprises acquiring a value for the abundance, e.g., relative amounts, of (i).
152. The method of embodiment 147, wherein acquiring knowledge of (ii) comprises acquiring a value for the abundance, e.g., relative amounts, of (ii).
153. The method of embodiment 151 or 152, wherein responsive to said value, the cell or subject is contacted with the TREM composition.
154. A method of treating a subject having an endogenous open reading frame (ORF) which comprises a codon having a first sequence, comprising:
providing a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, wherein the TREM, TREM core fragment or TREM fragment comprises an isoacceptor tRNA moiety having: an anticodon that pairs with the codon of the ORF having the first sequence; or an anticodon that pairs with a codon other than the codon having the first sequence,
contacting the subject with the TREM composition in an amount and/or for a time sufficient to treat the subject,
thereby treating the subject.
155. A method of treating a subject having an endogenous open reading frame (ORF) comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC), comprising:
providing a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, wherein the TREM, TREM core fragment or TREM fragment comprises an isoacceptor tRNA moiety having an anticodon that pairs with the SMC (the TREM);
contacting the subject with the TREM composition in an amount and/or for a time sufficient to treat the subject,
thereby treating the subject.
156. The method of embodiment 154 or 155, comprising acquiring knowledge of the abundance of one or both (i) and (ii), e.g., acquiring knowledge of the relative amounts of:
(i) a tRNA moiety having an anticodon that pairs with the codon having a first sequence or the SMC (the first tRNA moiety); and/or
(ii) an isoacceptor tRNA moiety having an anticodon that pairs with a codon other than a codon having the first sequence, or pairs with a codon other than the SMC (the second tRNA moiety).
157. The method of embodiment 156, comprising acquiring knowledge of (i).
158. The method of embodiment 156, comprising acquiring knowledge of (ii).
159. The method of embodiment 156, comprising acquiring knowledge of (i) and (ii).
160. The method of embodiment 156, wherein acquiring knowledge of (i) comprises acquiring a value for the abundance, e.g., relative amounts, of (i).
161. The method of embodiment 156, wherein acquiring knowledge of (ii) comprises acquiring a value for the abundance, e.g., relative amounts, of (ii).
162. The method of embodiment 160 or 161, wherein responsive to said value, the subject is contacted with the TREM composition.
163. A method of treating a subject having an endogenous open reading frame (ORF) comprising a codon having a first sequence, comprising:
(i) acquiring, e.g., directly or indirectly acquiring, a value for the status of the codon having the first sequence in the subject, wherein said value comprises a measure of the presence or absence of the codon having the first sequence in a sample from the subject; and identifying the subject as having the codon having the first sequence; and
(ii) responsive to said value, administering a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, wherein the TREM, TREM core fragment or TREM fragment comprises an isoacceptor tRNA moiety having an anticodon that pairs with the codon having the first sequence, to the subject,
thereby treating the subject.
164. A method of treating a subject having an endogenous open reading frame (ORF) comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC), comprising:
(i) acquiring, e.g., directly or indirectly acquiring, a value for the SMC status of the subject, wherein said value comprises a measure of the presence or absence of SMC in a sample from the subject, and identifying the subject as having a SMC; and
(ii) responsive to said value, administering a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, wherein the TREM, TREM core fragment or TREM fragment comprises comprising an isoacceptor tRNA moiety having an anticodon that pairs with the SMC, to the subject,
thereby treating the subject.
165. A method of selecting a therapy for a subject having an endogenous open reading frame (ORF) which comprises a codon having a first sequence, comprising:
acquiring, e.g., directly or indirectly acquiring, a value for the status of the codon having the first sequence in the subject, wherein said value comprises a measure of the presence or absence of the codon having the first sequence in a sample from the subject; and
wherein the presence of the codon having the first sequence is indicative that said subject is likely to be a responder to the therapy, or said subject will respond, or will likely respond, to the therapy,
thereby selecting the therapy.
166. A method of selecting a therapy for a subject having an endogenous open reading frame (ORF) comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC), comprising:
acquiring, e.g., directly or indirectly acquiring, a value for the SMC status of the subject, wherein said value comprises a measure of the presence or absence of SMC in a sample from the subject, and
wherein the presence of SMC is indicative that said subject is likely to be a responder to the therapy, or said subject will respond, or will likely respond, to the therapy,
thereby selecting the therapy.
167. A method of evaluating a subject having an endogenous open reading frame (ORF) comprising a codon having a first sequence, comprising:
acquiring, e.g., directly or indirectly acquiring, a value for the status of the codon having the first sequence in the subject, wherein said value comprises a measure of the presence or absence of the codon having the first sequence in a sample from the subject; and
identifying the subject as having a codon having the first sequence,
thereby evaluating the subject.
168. A method of evaluating a subject having an endogenous open reading frame (ORF) comprising a codon comprising a synonymous mutation (a synonymous mutation codon or SMC), comprising:
acquiring, e.g., directly or indirectly acquiring, a value for the SMC status of the subject, wherein said value comprises a measure of the presence or absence of SMC in a sample from the subject; and
identifying the subject as having a SMC,
thereby evaluating the subject.
169. The method of any one of embodiments 126-168, wherein the TREM, TREM core fragment or TREM fragment does not comprise an anticodon that pairs with a stop codon.
170. The method of any one of embodiments 126-169, wherein: (a) the ORF codon having the first sequence; or (b) the SMC; is other than a stop codon, e.g., TAA, TGA or TAG.
171. The method of any one of embodiments 126-170, wherein the TREM, TREM core fragment or TREM fragment comprises a canonical anti-codon/charging site combination.
172. The method of any one of embodiments 126-171, wherein: (a) the ORF codon having the first sequence; or (b) the SMC; has a mutation, e.g., a SNP, in the first position of said codon.
173. The method of any one of embodiments 126-171, wherein: (a) the ORF codon having the first sequence; or (b) the SMC; has a mutation, e.g., a SNP, in the second position of said codon.
174. The method of any one of embodiments 126-171, wherein: (a) the ORF codon having the first sequence; or (b) the SMC; has a mutation, e.g., a SNP, in the third position of said codon.
175. The method of any one of embodiments 126-174, wherein the first tRNA moiety comprises an endogenous tRNA, and the second tRNA moiety comprises an endogenous tRNA, e.g., wherein the cell or subject has not been contacted with a composition comprising a TREM.
176. The method of any one of embodiments 126-174, wherein one of the first tRNA moiety and the second tRNA moiety comprises an endogenous tRNA and a TREM.
177. The method of any one of embodiments 126-176, wherein: (a) the ORF codon having the first sequence; or (b) the SMC; in the absence of contact with the composition comprising a TREM, is associated with a phenotype, e.g., an unwanted phenotype, e.g., a disorder or symptom, e.g., a disorder or symptom chosen from Table 1.
178. The method of embodiment 177, wherein the disorder or symptom is chosen from a disease group provided in Table 1, e.g., cardiovascular, dermatology, endocrine, immunology, neurology, oncology, ophthalmology, or respiratory.
179. A method of modulating a production parameter of an RNA, or a protein encoded by an RNA, in a target cell or tissue, comprising:
providing, e.g., administering, to the target cell or tissue, or contacting the target cell or tissue with, an effective amount of a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, which TREM, TREM core fragment or TREM fragment corresponds to a contextually-rare codon (“con-rare codon”) of the RNA,
thereby modulating the production parameter of the RNA, or protein encoded by the RNA in the target cell or tissue.
180. The method of embodiment 179, wherein the target cell or tissue is obtained from a subject.
181. The method of embodiment 179, comprising administering the TREM composition to a subject.
182. The method of embodiment 181, comprising contacting the TREM composition with the target tissue or cell ex vivo.
183. The method of embodiment 182, comprising introducing the ex vivo-contacted target tissue or cell into a subject, e.g., an allogeneic or autologous subject.
184. The method of any one of embodiments 179-183, wherein the target cell or tissue is a specific or selected target cell or tissue, e.g., a cell or tissue type in a particular developmental stage; a cell or tissue type in a particular disease state; or a cell present in a particular extracellular milieu.
185. The method of any one of embodiments 179-184, wherein the production parameter comprises an expression parameter or a signaling parameter, e.g., as described herein.
186. The method of any one of embodiments 179-185, wherein the production parameter of the RNA is modulated, e.g., an RNA that can be translated into a polypeptide, e.g., a messenger RNA.
187. The method of embodiment 186, wherein the production parameter of the RNA is increased or decreased.
188. The method of any one of embodiments 179-187, wherein the production parameter of the protein encoded by the RNA is modulated.
189. The method of embodiment 188, wherein the production parameter of the protein is increased.
190. The method of embodiment 188, wherein the production parameter of the protein is decreased.
191. A method of determining the presence of a nucleic acid sequence, e.g., a DNA or RNA, having a contextually-rare codon (“con-rare codon nucleic acid sequence”), comprising:
acquiring knowledge of the presence of the con-rare codon nucleic acid sequence in a sample from a subject, e.g., a target cell or tissue sample,
wherein responsive to the acquisition of knowledge of the presence of the con-rare codon nucleic acid sequence:
(1) the subject is classified as being a candidate to receive administration of an effective amount of a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, which TREM, TREM core fragment or TREM fragment corresponds to a contextually-rare codon (“con-rare codon”) of the nucleic acid sequence; or
(2) the subject is identified as likely to respond to a treatment comprising the TREM composition.
192. A method of treating a subject having a disease associated with a contextually-rare codon (“con-rare codon”), comprising:
acquiring knowledge of the presence of a nucleic acid sequence, e.g., a DNA or RNA, having the con-rare codon (“con-rare codon nucleic acid sequence”) in a target cell or tissue sample from the subject; and
administering to the subject an effective amount of a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, which TREM, TREM core fragment or TREM fragment corresponds to the con-rare codon of the nucleic acid sequence,
thereby treating the disease in the subject.
193. A method of providing a tRNA effector molecule (TREM) to a subject, comprising:
providing, e.g., administering, to the subject, an effective amount of a TREM composition comprising a TREM of any one of embodiments 1-8, the TREM core fragment of embodiment 22, or the TREM fragment of embodiment 37, which TREM, TREM core fragment or TREM fragment corresponds to a contextually-rare codon (“con-rare codon”) for a nucleic acid sequence in a target cell or tissue in the subject,
thereby providing a TREM composition to the subject.
194. A method of manufacturing a tRNA effector molecule (TREM) composition comprising:
identifying a TREM corresponding to a contextually-rare (con-rare) codon;
combining the TREM with a component, e.g., a carrier or excipient.
thereby manufacturing a TREM composition.
195. The method of any one of embodiments 179-194, wherein the method comprises acquiring a value for a con-rare codon in the nucleic acid sequence, e.g., DNA or RNA, wherein the value is a function of one or more of the following factors, e.g., by evaluating or determining one or more of the following factors:
(1) the sequence of the codon;
(2) the availability of a corresponding tRNA, e.g., charged tRNA, for that con-rare codon in a target cell or tissue, e.g., one or more iso-acceptor tRNA molecules;
(3) the expression profile (or proteomic properties) of the target cell or tissue (e.g., the abundance of expression of other proteins which include the con-rare codon);
(4) the proportion of the tRNAs corresponding to the con-rare codon which are charged; and
(5) the iso-decoder isotype of the tRNA corresponding to the con-rare codon;
196. The method of embodiment 195, wherein (1) comprises determining the presence or absence of a con-rare codon.
197. The method of embodiment 196, wherein a determination of the availability of a tRNA comprises acquiring a measure of one, two, three or all of the following parameters:
(a) level of a tRNA corresponding to the con-rare codon (“con-rare codon tRNA”) compared to a tRNA corresponding to a different codon;
(b) function, e.g., polypeptide chain elongation function, of a con-rare codon tRNA compared to a tRNA corresponding to a different codon;
(c) modification, e.g., aminoacylation or post-transcriptional modification, of a con-rare codon tRNA compared to a tRNA corresponding to a different codon; and/or
(d) sequence of a con-rare codon tRNA.
198. The method of embodiment 197, wherein a measure of availability (e.g., level) of a con-rare codon tRNA comprises a measure of the con-rare codon tRNA that is charged, e.g., aminoacylated, compared to: (1) the proportion of the con-rare codon tRNA that is not charged; or (2) the proportion of charged tRNA corresponding to a different codon.
199. The method of any one of embodiments 195-198, wherein responsive to said value, the target cell, or tissue, is identified as having a nucleic acid sequence having a con-rare codon (“con-rare codon nucleic acid sequence”) or an RNA having a con-rare codon (“con-rare codon RNA”).
200. The method of any one of embodiments 195-198, wherein responsive to said value, the RNA is identified as, an RNA having a con-rare codon.
201. The method of any one of embodiments 179-200, wherein the target cell or tissue is identified as having an RNA having a con-rare codon.
202. The method of any one of embodiments 179-201, wherein the nucleic acid sequence, e.g., DNA or RNA, is identified as, a nucleic acid sequence having a con-rare codon (“con-rare codon nucleic acid sequence”) or an RNA having a con-rare codon (“con-rare codon RNA”).
203. The method of any one of embodiments 179-202, wherein the nucleic acid sequence (e.g., DNA or RNA) is, or is identified as, a nucleic acid sequence (e.g., DNA or RNA) having a plurality of con-rare codons.
204. The method of any one of embodiments 179-203, wherein the nucleic acid sequence (e.g., DNA or RNA) is, or is identified as, a nucleic acid sequence (e.g., DNA or RNA) having a plurality of occurrences of a con-rare codon.
205. The method of any one of embodiments 179-204, wherein the nucleic acid, e.g., RNA is, or is identified as, a nucleic acid, e.g., RNA, having a first tRNA which corresponds to a first con-rare codon; and an additional tRNA, e.g., a second tRNA, which corresponds to a different, e.g., a second, con-rare codon.
206. The method of any one of embodiments 179-205, wherein the nucleic acid sequence (e.g., DNA or RNA) is, or is identified as, a nucleic acid sequence (e.g., DNA or RNA), having multiple occurrences of the first tRNA which corresponds to a first con-rare codon.
207. The method of embodiment 205 or 206, wherein the nucleic acid sequence (e.g., DNA or RNA) is, or is identified as, a nucleic acid sequence (e.g., DNA or RNA) having multiple occurrences of the additional tRNA, e.g., a second tRNA, which corresponds to a different, e.g., a second, con-rare codon.
208. The method of any one of embodiments 179-207, wherein modulation of a production parameter of the con-rare codon RNA comprises increasing a production parameter, e.g., an expression parameter or signaling parameter of the protein encoded by the con-rare codon RNA, e.g., increasing the expression level of the protein encoded by the con-rare codon RNA.
209. The method of any one of embodiments 179-208, wherein modulation of a production parameter of the con-rare codon RNA comprises decreasing a production parameter, e.g., an expression parameter or signaling parameter, of the protein encoded by the con-rare codon RNA, e.g., decreasing the expression level of the protein encoded by the con-rare codon RNA.
210. The method of any one of embodiments 195-198, wherein a determination of the expression profile (or proteome codon count) of the target cell or tissue, comprises a measure of:
(a) the abundance (e.g., expression) of proteins in a target cell or tissue; and
(b) a protein codon count for expressed proteins in a target cell or tissue.
211. The method of any one of embodiments 179-210, wherein the target cell or tissue is identified as comprising a con-rare-codon nucleic acid, e.g., RNA.
212. The method of any one of embodiments 179-211, wherein, the con-rare codon meets a reference value for one or more of the following:
(1) the sequence of the codon;
(2) the availability of a corresponding tRNA, e.g., charged tRNA, for that con-rare codon in a target cell or tissue, e.g., one or more iso-acceptor tRNA molecules;
(3) the expression profile (or proteomic properties) of the target cell or tissue (e.g., the abundance of expression of other proteins which include the con-rare codon);
(4) the proportion of the tRNAs corresponding to the con-rare codon which are charged; and
(5) the iso-decoder isotype of the tRNA corresponding to the con-rare codon;
213. The method of embodiment 212, wherein the con-rare-codon meets a reference value for two of (1)-(5).
214. The method of embodiment 212, wherein the con-rare-codon meets a reference value for three of (1)-(5).
215. The method of embodiment 212, wherein the con-rare-codon meets a reference value for four of (1)-(5).
216. The method of embodiment 212, wherein the con-rare-codon meets a reference value for all of (1)-(5).
217. The method of embodiment 212, wherein the con-rare-codon meets a reference value for (1).
218. The method of embodiment 212, wherein the con-rare-codon meets a reference value for (2).
219. The method of embodiment 212, wherein the con-rare-codon meets a reference value for (3).
220. The method of embodiment 212, wherein the con-rare-codon meets a reference value for (4).
221. The method of embodiment 212, wherein the con-rare-codon meets a reference value for (5).
222. The method of embodiment 212, wherein the reference value is a pre-determined or pre-selected reference value.
223. The method of embodiment 212, wherein the reference value is determined according to a method described herein.
224. The method of any of embodiments 179-223, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs, TREM core fragments or TREM fragments in the TREM composition correspond to a con-rare codon.
225. The method of any one of embodiments 179-224, wherein the TREM composition comprises TREMs, TREM core fragments or TREM fragments that correspond to a plurality of con-rare codons.
226. The method of any of embodiments 179-225, wherein the TREM composition comprises: a first TREM which corresponds to a first con-rare codon; and an additional TREM which corresponds to a different con-rare codon.
227. The method of any of embodiments 179-226, wherein the TREM composition comprises: a first TREM which corresponds to a first con-rare codon; and a second TREM which corresponds to a second con-rare codon.
228. The method of any of embodiments 179-227, wherein the TREM composition comprises: a first TREM which corresponds to a first con-rare codon; a second TREM which corresponds to a second con-rare codon; and a third TREM which corresponds to a third con-rare codon.
229. The method of any of embodiments 179-228, wherein the TREM composition comprises: a first TREM which corresponds to a first con-rare codon; a second TREM which corresponds to a second con-rare codon; a third TREM which corresponds to a third con-rare codon; and a fourth TREM which corresponds to a fourth con-rare codon.
230. The method of any of embodiments 179-229, wherein the TREM composition comprises: a first TREM which corresponds to a first con-rare codon; a second TREM which corresponds to a second con-rare codon; a third TREM which corresponds to a third con-rare codon; a fourth TREM which corresponds to a fourth con-rare codon; and a fifth TREM which corresponds to a fifth con-rare codon.
231. The method of any one of embodiments 226-230, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs in the TREM composition correspond to the first con-rare codon.
232. The method of any one of embodiments 226-231, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs in the TREM composition correspond to the additional, e.g., second, third, fourth or fifth, con-rare codon.
233. The method of any of embodiments 179-232, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs in the composition are charged.
234. The method of any of embodiments 179-233, wherein the TREM composition comprises a first TREM which corresponds to a first con-rare codon and an additional TREM, e.g., a second, third, fourth, or fifth TREM, which corresponds to a different, e.g., second, third, fourth, or fifth, con-rare codon, and wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the first TREM in the composition is charged.
235. The method of embodiment 234, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the additional TREM e.g., second, third, fourth, or fifth TREM, in the composition is charged.
236. The method of any of embodiments 179-235, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs in the preparation are of the same iso-decoder isotype.
237. The method of any one of embodiments 179-236, wherein the TREM composition comprises: a first TREM which corresponds to a first con-rare codon; and an additional TREM which corresponds to the first con-rare codon, e.g., the first TREM and the additional TREM are of the same iso-decoder isotype.
238. The method of any one of embodiments 179-237, wherein the TREM composition comprises: a first TREM which corresponds to a first con-rare codon; and a second TREM which corresponds to the first con-rare codon, e.g., the first TREM and the second TREM are of the same iso-decoder isotype.
239. The method of any one of embodiments 179-238, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs in the TREM composition correspond to the first con-rare codon.
240. The method of any one of embodiments 179-239, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs in the TREM composition correspond to the additional, e.g., second or third, con-rare codon, e.g., the first TREM and the additional TREM are of the same iso-decoder isotype.
241. The method of any one of embodiments 179-240, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the TREMs in the composition are charged.
242. The method of any one of embodiments 179-241, wherein the TREM composition comprises a first TREM which corresponds to a first con-rare codon, and an additional TREM, e.g., a second or third TREM, which corresponds to the first con-rare codon, and wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the first TREM in the composition is charged.
243. The method of embodiment 242, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% (by weight or number) of the additional TREM e.g., second or third TREM, in the composition is charged.
244. The method of any one of embodiments 179-243, wherein the cell is a host cell.
245. The method of any one of embodiments 179-244, wherein the cell is a mammalian cell, e.g., a human cell, a murine cell, or a rodent cell.
246. The method of any one of embodiments 179-245, wherein the cell is a non-mammalian cell, e.g., a bacterial cell, an insect cell or a yeast cell.
247. The method of any one of embodiments 179-246, wherein the cell is a host cell chosen from: a HeLa cell, a HEK293T cell (e.g., a Freestyle 293-F cell), a HT-1080 cell, a PER.C6 cell, a HKB-11 cell, a CAP cell, a HuH-7 cell, a BHK 21 cell, an MRC-S cell, a MDCK cell, a VERO cell, a WI-38 cell, or a Chinese Hamster Ovary (CHO) cell.
248. The method of any one of embodiments 179-247, wherein the cell comprises an exogenous nucleic acid sequence.
249. The method of any one of embodiments 179-248, wherein the cell is autologous to the exogenous nucleic acid sequence.
250. The method of any one of embodiments 179-249, wherein the cell is allogeneic to the exogenous nucleic acid sequence.
251. The method of any one of embodiments 248-250, wherein the exogenous nucleic acid sequence (e.g., DNA or RNA) comprises a con-rare codon.
252. The method of any one of embodiments 248-251, wherein administration of a TREM composition corresponding to the con-rare codon to the cell, modulates a production parameter, e.g., expression parameter or signaling parameter, of a product, e.g., RNA or polypeptide, of the exogenous nucleic sequence.
253. The method of any one of embodiments 248-251, wherein administration of a TREM composition corresponding to the con-rare codon to the cell, increases a production parameter, e.g., expression parameter or signaling parameter, of a product, e.g., RNA or polypeptide, of the exogenous nucleic sequence.
254. The method of any one of embodiments 248-251, wherein administration of a TREM composition corresponding to the con-rare codon, to the cell decreases a production parameter, e.g., expression parameter or signaling parameter, of a product, e.g., RNA or polypeptide, of the exogenous nucleic sequence.
255. The method of any one of embodiments 179-254, wherein the modulation, increase or decrease in production parameter, is compared to an otherwise similar cell, which: (1) is not contacted with the TREM composition; (2) does not comprise an exogenous nucleic acid sequence; or (3) comprises an exogenous nucleic acid sequence which does not comprise a con-rare codon.
256. A method of modulating a production parameter of an RNA, or a protein encoded by the RNA, in a cell, comprising:
providing to the cell an effective amount of a tRNA corresponding to the con-rare codon RNA,
thereby modulating the production parameter of the RNA, or the protein encoded by the RNA in the cell.
257. A method of modulating a production parameter of an RNA, or a protein encoded by an RNA, in a cell, comprising:
optionally, acquiring knowledge of the presence of an RNA having a contextually-rare codon (“con-rare codon RNA”) in the cell,
modulating a culture parameter such that a production parameter of the RNA or protein encoded by the RNA is modulated.
258. The method of embodiment 256 or 257, wherein acquiring knowledge of the con-rare codon RNA comprises acquiring a value for a con-rare codon in the RNA, wherein the value is a function of one or more of the following factors, e.g., by evaluating or determining one or more of the following factors:
(1) the sequence of the codon;
(2) the availability of a corresponding tRNA, e.g., charged tRNA, for that con-rare codon in a target cell or tissue, e.g., one or more iso-acceptor tRNA molecules;
(3) the expression profile (or proteomic properties) of the target cell or tissue (e.g., the abundance of expression of other proteins which include the con-rare codon);
(4) the proportion of the tRNAs corresponding to the con-rare codon which are charged;
(5) the iso-decoder isotype of the tRNA corresponding to the con-rare codon;
259. The method of embodiment 257 or 258, wherein modulating a culture parameter comprises any one or all of the following:
The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
This example describes the synthesis of guanosine 2′-O-MOE phosphoramidite. Guanosine 2′-O-MOE phosphoramidite is prepared and purified according to previously published procedures (Wen K. et al. (2002) The Journal of Organic Chemistry, 67(22), 7887-7889).
Briefly, guanosine and imidazole are dried by co-evaporation with pyridine, dissolved in dry DMF, and treated with bis(diisopropylchlorosilyl) methane added dropwise at 0° C. The temperature is gradually increased to 25° C. and then held for 5 h. The reaction mixture is poured into ice water, and the precipitated white solid filtered to afford compound 1.
To a solution of compound 1, BrCH2CH2OCH3, and TBAI in DMF at −20° C. is added with sodium bis (trimethylsilyl)amide, and the mixture is stirred for 4 hours under argon. After the reaction is quenched with methanol, the THF is evaporated and the residue is precipitated in ice to furnish compound 2.
TBAF is added to a solution of compound 2 at 25° C. and then the mixture is stirred at 35° C. for 5 hours. The solvent is then evaporated under reduced pressure, and the residue is filtered in a short pad of silica gel using 10% methanol in dichloromethane to afford guanosine 2′-O-MOE phosphoramidite.
This example describes the synthesis of 5,6 dihydrouridine. 5,6 dihydrouridine phosphoramidite is prepared and purified according to previously published procedures (Hanze A R. et al., (1967) Journal of the American Chemical Society, 89(25), 6720-6725).
Briefly, oxygen is bubbled through a solution uridine in the presence of platinum black. The reaction is followed by spotting the reaction mixture on silica gel thin layer chromatographic plates and developing in methanol-chloroform (1:1). After 1 hour, the mixture is cooled and centrifuged and the clear liquid lyophilized to yield the 5,6 dihydrouridine product.
This example describes the synthesis of a TREM via 5′-Silyl-2′-Orthoester (2′-ACE) Chemistry summarized from (Hartsel S A et al., (2005) Oligonucleotide Synthesis, 033-050).
5′-O-silyl-2′-O-ACE protected phosphoramidites are prepared and purified according to previously published procedures (Hartsel S A et al., (2005) Oligonucleotide Synthesis, 033-050). Briefly, monomer synthesis begins from standard base-protected ribonucleosides [rA(ibu), rC(acetyl), rG(ibu) and U]. Orthogonal, 5′-silyl-2′-ACE protection and amidite preparation is then accomplished in five general steps:
The fully protected, phosphitylated monomer is an oil. For ease of handling and dissolution, the phosphoramidite solution is evaporated to dryness in a tared flask to enable quantitation of yields. The phosphoramidite oil is then dissolved in anhydrous acetonitrile, distributed into synthesis vials in 1.0-mmol aliquots, and evaporated to dryness under vacuum in the presence of potassium hydroxide (KOH) and P2O5.
5′-silyl-2′-ACE oligoribonucleotide synthesis begins with the appropriately modified 3′-terminal nucleoside attached through the 3′-hydroxyl to a polystyrene support. The solid support contained in an appropriate reaction cartridge is then placed on the appropriate column position on the instrument. A synthesis cycle is created using the delivery times and wait steps outlined in Table 11.
A two-stage rapid deprotection strategy is employed to remove phosphate backbone protection, release the oligonucleotide from the solid support, and remove the exocyclic amine protecting groups on A, G, and C. The treatment also removes the acetyl moiety from the acetoxyethyl orthoester, resulting in the 2′-bis-hydroxyethyl protected intermediate that is now 10 times more labile to final acid deprotection. In the first deprotection step, S2Na2 is used to selectively remove the methyl protection from the internucleotide phosphate, leaving the oligoribonucleotide attached to the polystyrene support. This configuration allows any residual reagent to be thoroughly washed away before proceeding. Alternatively, a multicolumn, manifold approach can also be used.
This example describes the synthesis of an Arg TREM having one 2′-O-MOE modification. The 2′-O-MOE modification can be placed on a nucleotide on any domain or linker of the Arg TREM, or at any position in said domain or linker.
A 2′-ACE RNA oligoribonucleotide synthesis is performed on a modified Applied Biosystems 394 DNA/RNA synthesizer or similar instrument. 2′-O-MOE amidites are synthesized as in example 1. An oligonucleotide sequence: GGCUCCGUGGCGCAAUGGAUAGCGCAUUGGACUUCUAAUUCAAAGGUUCCGGGUU CG(A-MOE)GUCCCGGCGGAGUCG is synthesized following the protocol described in example 3. A similar method can be used to add a 2′-O-MOE modification on a TREM specifying any one of the other 19 amino acids.
This example describes the synthesis of an Arg TREM having a pseudouridine and 2′-O-MOE modification. The modification can be placed on a nucleotide on any domain or linker of the Arg TREM, or at any position in said domain or linker.
A 2′-ACE RNA oligoribonucleotide synthesis is performed on a modified Applied Biosystems 394 DNA/RNA synthesizer or similar instrument. 2′-O-MOE amidites are synthesized as in example 1. Pseudouridine (P) amidites are obtained from Glen Research or similar provider. An oligonucleotide sequence: GGCUCCGUGGCGCAAUGGAUAGCGCAPUGGACUUCUAAUUCAAAGGUUCCGGGUU CG(A-MOE)GUCCCGGCGGAGUCG is synthesized following the protocol described in example 3. A similar method can be used to add a pseudouridine and 2′-O-MOE modification on a TREM specifying any one of the other 19 amino acids.
This example describes the synthesis of a Gln TREM having a dihydrouridine modification. The modification can be placed on a nucleotide on any domain or linker of the Gln TREM, or at any position in said domain or linker.
A 2′-ACE RNA oligoribonucleotide synthesis is performed on a modified Applied Biosystems 394 DNA/RNA synthesizer or similar instrument. Dihydrouridine (D) is synthesized as in example 2. An oligonucleotide sequence: GGUUCCAUGGUGUAAUGGDAAGCACUCUGGACUCTGAAUCCAGCGAUCCGAGUUC GAGUCUCGGUGGAACCUCCA is synthesized following the protocol described in example 3. A similar method can be used to add a dihydrouridine modification on a TREM specifying any one of the other 19 amino acids.
This example describes the synthesis of a Gln TREM having a pseudouridine modification. The modification can be placed on a nucleotide on any domain or linker of the Gln TREM, or at any position in said domain or linker.
A 2′-ACE RNA oligoribonucleotide synthesis is performed on a modified Applied Biosystems 394 DNA/RNA synthesizer or similar instrument. Pseudouridine (P) amidites are obtained from Glen Research or similar provider. An oligonucleotide sequence: GGUUCCAUGGUGPAAUGGUAAGCACUCUGGACUCTGAAUCCAGCGAUCCGAGUUC GAGUCUCGGUGGAACCUCCA is synthesized following the protocol described in example 3.
A similar method can be used to add a pseudouridine modification on a TREM specifying any one of the other 19 amino acids.
This example describes the quality control of a synthesized TREM via Mass Spectrometry Analysis.
Using the Perceptive Biosystems Voyager-DE BioSpectrometry Workstation, the referenced protocol for mass spectrometry analysis (4-Van Ausdall) is followed. Briefly, a 3-hydroxy picolinic acid matrix is used for sample crystallization. It is prepared by mixing (10:1:1) 3-HPA:picolinic acid:ammonium hydrogen citrate where each component is dissolved in 30% aqueous acetonitrile at a concentration of 50 mg/mL. One optical density unit (ODU) of oligonucleotide is dissolved in the matrix and heated at 55° C. for 10 min. The sample is spotted on a MALDI plate, allowed to dry, and analyzed accordingly. This method allows confirmation of oligonucleotide identity and detection of low-level impurities present in synthetic oligonucleotide samples.
This example describes the quality control of a synthesized TREM via anion-exchange HPLC.
Using the Dionex DNA-Pac-PA-100 column, a gradient is employed using HPLC buffer A and HPLC buffer B. 0.5 ODUs of a sample that has been dissolved in H2O or Tris buffer, pH 7.5 is injected onto the gradient. The gradient employed is based on oligonucleotide length and can be applied according to Table 12. The parameters provided in Table 13 can be used to program a linear gradient on the HPLC analyzer.
This example describes the quality control of a synthesized TREM via PAGE Purification and Analysis. Gel purification and analysis of 2′-ACE protected RNA follows standard protocols for denaturing PAGE (Ellington and Pollard (1998) In Current Protocols in Molecular Biology, Chanda, V). Briefly, the 2′-ACE protected oligo is resuspended in 200 mL of gel loading buffer. Invitrogen™ NuPAGE™ 4-12% Bis-Tris Gels or similar gel is prepared in gel apparatus. Samples are loaded and gel ran at 50-120 W, maintaining the apparatus at 40° C. When complete, the gel is exposed to ultraviolet (UV) light at 254 nm to visualize the purity of the RNA using UV shadowing. If necessary, the desired gel band is excised with a clean razor blade. The gel slice is crushed and 0.3M NaOAc elution buffer is added to the gel particles, and soaked overnight. The mixture is decanted and filtered through a Sephadex column such as Nap-10 or Nap-25.
This example describes the deprotection of a TREM made according to an in vitro synthesis method, e.g., as described in Example 3. The 2′-protecting groups are removed using 100 mM acetic acid, pH 3.8. The formic acid and ethylene glycol byproducts are removed by incubating at 60° C. for 30 min followed by lyophilization or SpeedVac-ing to dryness. After this final deprotection step, the oligonucleotides are ready for use.
This Example describes the quantification of tRNA levels in a cell line or tissue type. Transfer RNA levels are determined using Oxford Nanopore direct RNA sequencing, as previously described in Sadaoka et al., Nature Communications (2019) 10, 754.
Briefly, cells transfected with a tRNA molecule are lysed and total RNA is purified using a method such as phenol chloroform. RNAs smaller than 200 nucleotides are separated from the lysate using a small RNA isolation kit per manufacturer's instructions, to generate a small RNA (sRNA) fraction.
The sRNA fraction is de-acylated using 100 mM Tris-HCl (pH 9.0) at 37° C. for 30 minutes. The solution is neutralized by the addition of an equal volume of 100 mM Na-acetate/acetic acid (pH 4.8) and 100 mM NaCl, followed by ethanol precipitation. Deacylated sRNA is dissolved in water, and its integrity verified by agarose gel electrophoresis. Deacylated sRNA is then polyadenylated using yeast poly(A) tailing kit per manufacturer's instructions to generate a sRNA polyadenylated pool. Following polyadenylation, a reverse transcription reaction is performed to generate cDNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) or a thermostable group II intron RT (TGIRT, InGex LLC) that is less sensitive to RNA structure and modifications. A sequencing adapter is ligated onto the cDNA mixture by incubating the cDNA mixture with RNA adapter, T4 ligase and ligation buffer following the standard protocol for Oxford Nanopore resulting in a cDNA library. Nanopore sequencing is then performed on the libraries and the sequences are mapped to a genomic database, in this example to the genomic tRNA database, GtRNAdb. The methods described in this example can be adopted for use to evaluate the tRNA pool across cell lines or tissue types.
This Example describes the quantification of protein expression levels across cell lines or tissue types.
The protein expression levels are monitored using SILAC based mass-spectrometry proteomics, as previously described in Geiger et al., Molecular and Cellular Proteomics (2012) 10, 754.
Briefly, populations of cells are cultured either in media containing isotope-labeled amino acids, such as Lys8 (e.g., 13C615N2-lysine) and Arg10 (e.g., 13C615N4-arginine); or in media containing natural amino acids. The media is further supplemented with 10% dialyzed serum. Cell cultured in media containing isotope-labeled amino acids incorporate the isotope-labeled amino acids into all of the proteins translated after incubation with said isotope-labeled amino acids. For example, all peptides containing a single arginine will be 6 Da heavier in cells cultured in the presence of instead of isotope-labeled amino acid compared to cells cultured with natural amino acids. Cultured are lysed and sonicated. Cell lysates (e.g., about 100 g) are diluted in 8 M urea in 0.1 M Tris-HCl followed by protein digestion with trypsin according to the FASP protocol (Wisniewski, J. R., et al. (2009) Universal sample preparation method for proteome analysis. Nat. Methods 6, 359-362). After an overnight digestion, peptides are eluted from the filters with 25 mM ammonium bicarbonate buffer. From each sample, about 40 ug of peptides are separated into six fractions by strong anion exchange as described previously (Wisniewski, J. R., et al. (2009) Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J. Proteome Res. 8, 5674-5678).
Eluted peptides are concentrated and purified on C18 StageTips, e.g., as described in Rappsilber et al., Nature Protocols (2007).
Peptides are separated by reverse-phase chromatography using a nano-flow HPLC (Easy nanoLC, Thermo Fisher Scientific). The high performance liquid chromatography (HPLC) is coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Peptides are loaded onto the column with buffer A (0.5% acetic acid) and eluted with a 200 min linear gradient from 2 to 30% buffer B (80% acetonitrile, 0.5% acetic acid). After the gradient the column is washed with 90% buffer B and re-equilibrated with buffer A.
Mass spectra are acquired in a data-dependent manner, with an automatic switch between MS and MS/MS scans using a top 10 method. MS spectra are acquired in the Orbitrap analyzer, with a mass range of 300-1650 Th and a target value of 106 ions. Peptide fragmentation is performed with the HCD method and MS/MS spectra is acquired in the Orbitrap analyzer and with a target value of 40,000 ions. Ion selection threshold is set to 5000 counts. Two of the data sets are acquired with a high field Orbitrap cell in which the resolution is 60,000 instead of 30,000 (at 400 m/z) for the MS scans. In the first of the two replicates with the high field Orbitrap MS/MS scans are acquired with 15,000 resolution, and in the second with 7500 resolution, which is the same as in the standard Orbitrap, but with shorter transients.
Raw MS files are analyzed by MaxQuant using standard metrics, e.g., as described in Table 2 of Tyanova S et al. (2016) Nat. Protocols 11(12) pp. 2301-19. Categorical annotation is supplied in the form of Gene Ontology (GO) biological process, molecular function, and cellular component, the TRANSFAC database as well as participation in a KEGG pathway and membership in a protein complex as defined by CORUM.
The methods described in this example can be adopted for use to evaluate the protein expression levels across cell lines or tissue types.
This example describes the method used to determine components of contextual rarity (con-rarity). This method utilizes the cell line or tissue protein expression level determined by proteomics described in Example 13 or taken from literature. This method also utilizes the tRNA profile determined by Nanopore or other tRNA sequencing platform described in the Example 1 or taken from literature.
Using the coding DNA sequence (CDS) defined using National Center for Biotechnology Information (NCBI https://www.ncbi.nlm.nih.gov/) or other database, the protein-coding sequence is segmented into codons and summed per codon to give a codon count per nucleic acid sequence for each codon encoded in the protein-coding sequence.
The codon count per nucleic acid sequence is then multiplied by the corresponding cell line or tissue protein expression level determined by proteomics to give a cell type normalized proteome codon count across the cell line or tissue.
Con-rarity is a function of normalized proteome codon count and the tRNA expression level. In an embodiment, the con-rarity is determined by dividing the normalized proteome codon count by the tRNA expression level determined by Nanopore or other tRNA sequencing experiment. This provides a measure of codon usage that is contextually dependent on the tRNA profile, e.g., tRNA abundance levels. A codon is determined to be contextually rare (con-rare) if the con-rarity meets a reference value, e.g., a pre-determined or pre-selected reference value, e.g., a threshold. In an embodiment, a codon is con-rare if the value of a normalized proteome codon count divided by the tRNA expression level for a particular tRNA meets a pre-determined reference. In an embodiment, the reference value is a value under e.g., 1.5× sigma of the normally fit distribution to that codon frequency.
This Example describes the identification of a nucleic acid sequence having con-rare codons. Con-rare codons are identified as described in Example 14.
Using the coding DNA sequences (CDS) defined using National Center for Biotechnology Information (NCBI https://www.ncbi.nlm.nih.gov/) or other database, all human gene sequences are segmented into codons and summed per codon to give a codon count per nucleic acid sequence, e.g., gene.
Each codon, per nucleic acid sequence, is classified as a con-rare codon or a con-abundant codon. The counts for all con-rare codons, for each nucleic acid sequence, are summed and normalized to the sequence length.
The con-rare codon count is fit to a normalized distribution. A nucleic acid sequence that meets a reference value, e.g., a pre-determined reference value, is classified as a nucleic acid sequence having con-rare codons. In an embodiment, a nucleic acid sequence is classified as having con-rare codons if it falls above a reference value, e.g., in the upper 3sigma of the normalized distribution. In an embodiment, a nucleic acid sequence having con-rare codons can have one, two, or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 200, 500) of the same con-rare codon or different con-rare codons.
This Example describes the identification of a nucleic acid sequence having con-rare codons. Con-rare codons are identified as described in the Example 14.
Using the coding DNA sequences (CDS) defined using National Center for Biotechnology Information (NCBI https://www.ncbi.nlm.nih.gov/) or other database, all human gene sequences are segmented into codons and summed per codon to give a codon count per nucleic acid sequence, e.g., gene.
Each codon, per nucleic acid sequence, is classified as a con-rare codon or a con-abundant codon. For each con-rare codon, the counts per nucleic acid sequence is fit to a normalized distribution. A nucleic acid sequence that meets a reference value, e.g., a pre-determined reference value, is classified as a nucleic acid sequence having con-rare codons. In an embodiment, a nucleic acid sequence is classified as having con-rare codons, e.g., specified con-rare codons, if it falls e.g., in the upper 3sigma of the normalized distribution. In an embodiment, a nucleic acid sequence having con-rare codons can have one, two, or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 200, 500) of the same con-rare codon or different con-rare codons.
This Example describes an exemplary nucleic acid sequence having con-rare codons.
The GRK2 nucleic acid sequence encodes the GRK2 protein (G-protein coupled receptor kinase 2). The method of Examples 4 or 5 was used to identify the GRK2 nucleic acid sequence as having con-rare codons. The GRK2 nucleic acid sequence has a coding sequence that has con-rare codons AAG and CTG. The AAG codon codes for lysine and the CTG codon codes for leucine. In an embodiment, under certain cellular conditions, the expression of the GRK2 protein can be affected by the frequency of tRNAs corresponding to one or more con-rare codons in the GRK2 nucleic acid sequence, e.g., CUU-tRNA which corresponds to con-rare codon AAG, and/or CAG-tRNA which corresponds to con-rare codon CTG.
This Example describes the computational pipeline that can be utilized to codon modify a nucleic acid sequence.
Con-rarity (determined using the method described in Example 14) is read into the algorithm. Con-rare codons are identified as described in Example 14. For example, a codon is determined to be contextually rare (con-rare) if the con-rarity meets a reference value, e.g., a pre-determined or pre-selected reference value, e.g., a threshold. A corresponding contextually abundant (con-abundant) codon is identified as the most contextually frequent codon that encodes the same amino acid as the con-rare codon (e.g., an isoacceptor or an isodecoder). In an embodiment, a con-rare codon can have more than one corresponding con-abundant codon. In an embodiment, the corresponding con-abundant codon can be utilized to replace a con-rare codon.
Each sequence to be modified is read in and segmented into codons. Each codon is then evaluated to determine if it is a con-rare codon. If the codon is identified as a con-rare codon, the codon is replaced, e.g., with a corresponding con-abundant codon. A con-abundant codon is a codon other than a con-rare codon. This process can be repeated for two, three, four, or a portion of, or all of the con-rare codons found in the sequence. The resultant con-rare modified sequence (e.g., also referred to as contextually modified nucleic acid sequence) is then outputted.
This Example describes administration of a TREM to modulate expression levels of a protein encoded by a nucleic acid sequence having a con-rare codon in its coding sequence (CDS).
To create a system in which to study the effects of TREM administration on protein expression levels of a protein encoded by a nucleic acid sequence having a con-rare codon in its CDS, the sequence for the GRK2 gene (GRK2-CCDS8156.1 sequence) is inserted into a plasmid. The plasmid is transfected in the normal human hepatocyte cell line THLE-3. A TREM is delivered to the CCDS8156.1 containing cells. As a control, a population of cells prior to the delivery of the TREM is set aside. In this example, the tRNA-LysCUU containing an CUU anticodon, that base pairs to the AAG codon, i.e. with the sequence GCCCGGCUAGCUCAGUCGGUAGAGCAUGGGACUCUUAAUCCCAGGGUCGUGGGUU CGAGCCCCACGUUGGGCG is used. A time course is performed ranging from 30 minutes to 6 hours with hour-long interval time points. At each time point, a population of cells that have been delivered the TREM, and a population of cells that have not been exposed to the TREM are trypsinized, washed and lysed. Cell lysates are analyzed by Western blotting and blots are probed with antibodies against the GRK2 protein. A total protein loading control, such as GAPDH, actin or tubulin, is also used.
The methods described in this example can be adopted to evaluate the expression levels of the GRK2 protein in cells endogenously expressing CCDS8156.1.
This example describes the manufacturing of a TREM produced in mammalian host cells.
To generate a plasmid comprising a TREM which comprises a tRNA gene, in this example, tRNAiMet, a DNA fragment containing the tRNA gene (chr6.tRNA-iMet(CAT) with genomic location 6p22.2 and sequence AGCAGAGTGGCGCAGCGGAAGCGTGCTGGGCCCATAACCCAGAGGTCGATGGATCG AAACCATCCTCTGCTA) is PCR-amplified from human genomic DNA using the following primer pairs: 5′-TGAGTTGGCAACCTGTGGTA and 5′-TTGGGTGTCCATGAAAATCA. This fragment is cloned into the pLKO.1 puro backbone plasmid with a U6 promoter (or any other RNA polymerase III recruiting promoter) following the manufacturer's instructions.
1 mg of plasmid described above is used to transfect a 1 L culture of suspension-adapted HEK293T cells (Freestyle 293-F cells) at 1×105 cells/mL. Cells are harvested at 24, 48, 72, or 96 hours post-transfection to determine the optimized timepoint for TREM expression as determined by Northern blot, or by quantitative PCR (q-PCR).
At the optimized harvest cell density point, the TREM is purified as previously described in Cayama et al., Nucleic Acids Research. 28 (12), e64 (2000). Briefly, short RNAs (e.g., tRNAs) are recovered from cells by phenol extraction and concentrated by ethanol precipitation. The total tRNA in the precipitate is then separated from larger nucleic acids (including rRNA and DNA) under high salt conditions by a stepwise isopropanol precipitation. The elution fraction containing the TREM is further purified through probe binding. The TREM fraction is incubated with annealing buffer and the biotinylated capture probe corresponding to a DNA probe or a 2′-OMe nucleic acid that is complementary to a unique region of the TREM being purified, in this example, a probe conjugated to biotin at the 3′ end with the sequence UAGCAGAGGAUGGUUUCGAUCCAUCA, is used to purify the TREM comprising tRNA-Lys-UUU. The mixture is incubated at 90° C. for 2-3 minutes and quickly cooled down to 45° C. and incubated overnight at 45° C. The admixture is then incubated with binding buffer previously heated to 45° C. and streptavidin-conjugated RNase-free magnetic beads for 3 hours to allow binding of the DNA-tRNA complexes to the beads. The mixture is then added to a pre-equilibrated column in a magnetic field separator rack and washed 4 times. The TREM retained on the beads are eluted three times by adding elution buffer pre-heated to 80° C. and then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
One microgram of the test TREM preparation and a control agent are contacted by transfection, electroporation or liposomal delivery, with a cultured cell line, such as a HEP-3B or HEK293T, a tissue or a subject, for a time sufficient for the TREM preparation to modulate a translation level or activity of the cell, relative to the control agent.
This example describes the manufacturing of a TREM produced in mammalian host cells.
To generate a plasmid comprising a TREM which comprises a tRNA gene, in this example, tRNA-iMet-CAT, a DNA fragment containing at least one copy of the tRNA gene with the sequence AGCAGAGTGGCGCAGCGGAAGCGTGCTGGGCCCATAACCCAGAGGTCGATGGATCG AAACCATCCTCTGCTA is synthesized and cloned into the pLKO.1 puro backbone plasmid with a U6 promoter (or any other RNA polymerase III recruiting promoter) following the manufacturer's instructions and standard molecular cloning techniques.
1 mg of plasmid described above is used to transfect a 1 L culture of suspension-adapted HEK293T cells (Freestyle 293-F cells) at 1×105 cells/mL. Cells are harvested at 24, 48, 72, or 96 hours post-transfection to determine the optimized timepoint for TREM expression as determined by Northern blot, or by quantitative PCR (q-PCR) or Nanopore sequencing.
At the optimized harvest timepoint, the cells are lysed and separation from the lysate of RNAs smaller than 200 nucleotides is performed using a small RNA isolation kit per manufacturer's instructions, to generate a small RNA (sRNA) fraction.
To prepare the affinity purification reagents, streptavidin-conjugated RNase-free magnetic beads are incubated at room temperature for 30 min with 200 mM of biotinylated oligonucleotides corresponding to a DNA probe or a 2′-OMe nucleic acid that is complementary to a unique region of the TREM being purified. In this example, a probe with the sequence 5′biotin-TAGCAGAGGATGGTTTCGATCCATCA is used to purify the TREM comprising tRNA-iMet (CAT). The beads are washed and heated for 10 min at 75° C.
The sRNA fraction is heated for 10 min at 75° C. and then mixed with the affinity purification reagent described above. The admixture is incubated at room temperature for 3 hours to allow binding of the TREMs to the bead-bound DNA probe in a sequence specific manner.
The beads are then washed until the absorbance of the wash solution at 260 nm is close to zero. Alternatively, the beads are washed three times and the final wash is examined by UV spectroscopy to measure the amount of nucleic acid present in the final wash. The TREM retained on the beads are eluted three times using RNase-free water which can be pre-heated to 80° C., and then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
One microgram of the test TREM preparation and a control agent are contacted by transfection, electroporation or liposomal delivery, with a cultured cell line, such as HeLa, HEP-3B or HEK293T, a tissue or a subject, for a time sufficient for the TREM preparation to modulate a translation level or activity of the cell, relative to the control agent.
This example describes the manufacturing of a TREM in mammalian host cells modified to overexpress myc.
To make the production host cells for this example, HeLa cells (ATCC® CCL-2™) or HEP-3B cells (ATCC® HB-8064™) are transfected with a plasmid containing the gene sequence coding for the c-myc oncogene protein (e.g., pcDNA3-cmyc (Addgene plasmid #16011)) using routine molecular biology techniques. The resulting cell line is referred to herein as HeLamyc+ host cells or HEP-3Bmyc+ host cells.
To prepare a TREM expressing lentivirus, HEK293T cells are co-transfected with 3 μg of each packaging vector (pRSV-Rev, pCMV-VSVG-G and pCgpV) and 9 μg of the plasmid comprising a TREM as described in Example 9, using Lipofectamine 2000 according to manufacturer's instructions. After 24 hours, the media is replaced with fresh antibiotic-free media and after 48 hours, virus-containing supernatant is collected and centrifuged for 10 min at 2000 rpm before being filtered through a 0.45 m filter.
Transduction of host cells with TREM expressing lentivirus 2 mL of virus prepared as described above is used to transduce 100,000 HeLamyc+ host cells or HEP-3Bmyc+ host cells, in the presence of 8 μg/mL polybrene. Forty-eight hours after transduction, puromycin (at 2 μg/mL) antibiotic selection is performed for 2-7 days alongside a population of untransduced control cells.
The TREMs are isolated, purified, and formulated as described in Example 20 or 21 to result in a composition comprising a TREM or preparation comprising a TREM.
This example describes the preparation of Hek293Maf-/TRM1 cells for the production of a TREM.
Maf1 is a repressor of tRNA synthesis. A Maf1 knockout HEK293T cell line is generated using standard CRISPR/Cas knockout techniques, e.g., a CRISPR/Cas system can be designed to introduce a frameshift mutation in a coding exon of Maf1 to reduce the expression of Maf1 or knockout Maf1 expression, to generate a Hek293Maf-cell line that has reduced expression level and/or activity of Maf1. This cell line is then transfected with an expression plasmid for modifying enzyme Trm1 (tRNA (guanine26-N2)-dimethyltransferase) such as pCMV6-XL4-Trm1, and selected with a selection marker, e.g., neomycin, to generate a stable cell line overexpressing Trm1 (Hek293Maf-/TRM1 cells).
Hek293Maf-/TRM1 cells can be used as production host cells for the preparation of a TREM as described in any of Examples 20-22.
This Example describes the manufacturing of a TREM in mammalian host cells modified to overexpress Myc and Trm1.
In this example, a plasmid comprising a TREM is generated as described in Example 20 or 21.
A human cell line, such as HEK293T, stably overexpressing Myc oncogene is generated by transduction of retrovirus expressing the myc oncogene from the pBABEpuro-c-mycT58A plasmid into HEK293T cells. To generate myc-expressing retrovirus, HEK293T cells are transfected using the calcium phosphate method with the human c-myc retroviral vector, pBABEpuro-c-mycT58A and the packaging vector, ψ2 vector. After 6 hours, transfection media is removed and replaced with fresh media. After a 24-hour incubation, media is collected and filtered through a 0.45 um filter. For the retroviral infection, HEK293T cells are infected with retrovirus and polybrene (8 ug/ml) using spin infection at 18° C. for 1 hour at 2500 rpm. After 24 hours, the cell culture medium is replaced with fresh medium and 24 hours later, the cells are selected with 2 μg/mL puromycin. Once cells stably overexpressing the oncogene myc are established, they are transfected with a Trm1 plasmid, such as the pCMV6-XL4-Trm1 plasmid, and selected with a selection marker, in this case with neomycin, to generate a stable cell line overexpressing Trm1, in addition to Myc. In parallel, lentivirus to overexpress TREM is generated as described in Example 3 with HEK293T cells and PLKO.1-tRNA vectors. 1×105 cells overexpressing Myc and Trm1 are transduced with the TREM virus in the presence of 8 μg/mL polybrene. Media is replaced 24 hours later. Forty-eight hours after transduction, antibiotic selection is performed with 2 μg/mL puromycin for 2-7 days alongside a population of untransduced control cells. The TREMs are isolated, purified and formulated using the method described in Example 20 or 21 to produce a TREM preparation.
This example describes assays to evaluate the ability of a TREM to be incorporated into a nascent polypeptide chain.
A test TREM is assayed in an in-vitro translation reaction with an mRNA encoding the peptide FLAG-XXX-His6x, where XXX are 3 consecutive codons corresponding to the test TREM anticodon.
A tRNA-depleted rabbit reticulocyte lysate or human cell lysate (Jackson et al. 2001. RNA 7:765-773) is incubated 1 hour at 30° C. with 10-25 ug/mL of the test TREM in addition to 10-25 ug/mL of the tRNAs required for the FLAG and His tag translation. A different mammalian lysate such as a HEK293T human cell-derived lysate can also be used in this assay. In this example, the TREM used is tRNA-Ile-GAT, therefore the peptide used is FLAG-LLL-His6x and the tRNAs added are tRNA-Ile-GAT, in addition to the following, which are added for translate the peptide FLAG and HIS tags: tRNA-Asp-GAC, tRNA-Tyr-TAC, tRNA-Lys-AAA, tRNA-Lys-AAAG, tRNA-Asp-GAT, tRNA-His-CAT. To determine if the test TREM is functionally able to be incorporated into a nascent peptide, an ELISA capture assay is performed. Briefly, an immobilized anti-His6X antibody is used to capture the FLAG-LLL-His6x peptide from the reaction mixture. The reaction mixture is then washed off and the peptide is detected with an enzyme-conjugated anti-FLAG antibody, which reacts to a substrate in the ELISA detection step. If the TREM produced is functional, the FLAG-LLL-His6 peptide is produced and detection occurs by the ELISA capture assay. The methods described in this example can be adopted for use to evaluate the functionality of the TREM.
This assay describes a test TREM having translational adaptor molecule function by rescuing a suppression mutation and allowing the full protein to be translated. The test TREM, in this example tRNA-Ile-GAT, is produced such that it contains the sequence of the tRNA-Ile-GAT body but with the anticodon sequence corresponding to CUA instead of GAT. HeLa cells are co-transfected with 50 ng of TREM and with 200 ng of a DNA plasmid encoding a mutant GFP containing a UAG stop codon at the S29 position as described in Geslain et al. 2010. J Mol Biol. 396:821-831. HeLa cells transfected with the GFP plasmid alone serve as a negative control. After 24 hours, cells are collected and analyzed for fluorescence recovery by flow cytometry. The fluorescence is read out with an emission peak at 509 nm (excitation at 395 nm). The methods described in this example can be adopted for use to evaluate the functionality of the TREM, or if the TREM can rescue the stop mutation in the GFP molecule and can produce the full-length fluorescent protein.
This assay describes a test TREM having translational adaptor molecule function by successfully being incorporated into a nascent polypeptide chain in an in vitro translation reaction. First, a rabbit reticulocyte lysate that is depleted of the endogenous tRNA using an antisense or complimentary oligonucleotide which (i) targets the sequence between the anticodon and variable loop; or (ii) binds the region between the anticodon and variable loop is generated (see, e.g., Cui et al. 2018. Nucleic Acids Res. 46(12):6387-6400). 10-25 ug/mL of the test TREM is added in addition to 2 ug/uL of a GFP-encoding mRNA to the depleted lysate. A non-depleted lysate with the GFP mRNA and with or without test TREM added are used as a positive control. A depleted lysate with the GFP mRNA but without the test TREM added is used as a negative control. The progress of GFP mRNA translation is monitored by fluorescence increase on a microplate reader at 37° C. for 3-5 h using λex485/λem528. The methods described in this example can be adopted for use to evaluate if the test TREM can complement the depleted lysate and is thus likely functional.
This example describes the production of a TREM in mammalian host cells.
To generate a plasmid comprising a TREM which comprises a tRNA gene, in this example, tRNA-Ser-AGA, a DNA fragment containing at least one copy of the tRNA gene with the sequence GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTAGAAATCCATTGGGGTTTCCCCGC GCAGGTTCGAATCCTGCCGACTACG is synthesized and cloned into the pLKO.1 puro backbone plasmid with a U6 promoter (or any other RNA polymerase III recruiting promoter) following the manufacturer's instructions and standard molecular cloning techniques.
One (1) mg of plasmid described above is used to transfect a 1 L culture of suspension-adapted HEK293T cells (Freestyle 293-F cells) at 1×105 cells/mL. Cells are harvested at 24, 48, 72, or 96 hours post-transfection to determine the optimized timepoint for TREM expression as determined by a quantitative method such as Northern blot, quantitative PCR (q-PCR) or Nanopore sequencing.
At the optimized harvest timepoint, the cells are lysed, and total RNA is purified using a method such as phenol chloroform. RNAs smaller than 200 nucleotides are separated from the lysate using a small RNA isolation kit per manufacturer's instructions, to generate a small RNA (sRNA) fraction.
The sRNA fraction is incubated with annealing buffer and the biotinylated capture probe corresponding to a DNA probe that is complementary to a unique region of the TREM being purified, in this example, a probe with the sequence 3′ biotin-CCAATGGATTTCTATCCATCGCCTTAACCACTCGGCCACGACTACAAAA is used to purify the TREM comprising tRNA-Ser-AGA. The mixture is incubated at 90° C. for 2-3 minutes and quickly cooled down to 45° C. and incubated overnight at 45° C. The admixture is then incubated with binding buffer previously heated to 45° C. and streptavidin-conjugated RNase-free magnetic beads for 3 hours to allow binding of the DNA-tRNA complexes to the beads. The mixture is then added to a pre-equilibrated column in a magnetic field separator rack and washed 4 times. The TREM retained on the beads are eluted three times by adding elution buffer pre-heated to 80° C. and then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
This example describes the production of a TREM in bacterial host cells.
To generate a plasmid to produce a TREM in bacteria, a tRNA gene, in this example, a DNA fragment containing at least one copy of the tRNA-Lys-UUU gene with the sequence GCCCGGATAGCTCAGTCGGTAGAGCATCAGACTTTTAATCTGAGGGTCCAGGGTTCA AGTCCCTGTTCGGGCG is synthesized and cloned into a bacterial tRNA expression vector as previously described in Ponchon et al., Nat Protoc 4, 947-959 (2009).
1×109 bacteria grown from TREM expression plasmid transformed competent bacteria will be harvested at different cell density points, in this example OD(600)=0.5, OD(600)=0.7, OD(600)=0.9 to determine the optimal point of TREM expression as determined by a quantitative method such as Northern blot, quantitative PCR (q-PCR) or Nanopore sequencing.
At the optimized harvest cell density point, the TREM is purified as previously described in Cayama et al., Nucleic Acids Research. 28 (12), e64 (2000). Briefly, short RNAs (e.g., tRNAs) are recovered from cells by phenol extraction and concentrated by ethanol precipitation. The total tRNA in the precipitate is then separated from larger nucleic acids (including rRNA and DNA) under high salt conditions by a stepwise isopropanol precipitation. The elution fraction containing the TREM is further purified through probe binding. The TREM fraction is incubated with annealing buffer and the biotinylated capture probe corresponding to a DNA probe that is complementary to a unique region of the TREM being purified, in this example, a probe conjugated to biotin at the 3′ end with the sequence CAGAUUAAAAGUCUG, is used to purify the TREM comprising tRNA-Lys-UUU. The mixture is incubated at 90° C. for 2-3 minutes and quickly cooled down to 45° C. and incubated overnight at 45° C. The admixture is then incubated with binding buffer previously heated to 45° C. and streptavidin-conjugated RNase-free magnetic beads for 3 hours to allow binding of the DNA-tRNA complexes to the beads. The mixture is then added to a pre-equilibrated column in a magnetic field separator rack and washed 4 times.
The TREM retained on the beads are eluted three times by adding elution buffer pre-heated to 80° C. and then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
This example describes production of a TREM using chemical synthesis.
The TREM, in this example, tRNA-Thr-CGT, is chemically synthesized with the sequence GGCUCUAUGGCUUAGUUGGUUAAAGCGCCUGUCUCGUAAACAGGAGAUCCUGGG UUCGACUCCCAGUGGGGCCUCAA. This TREM is produced by solid-phase chemical synthesis using phosphoroamedite chemistry as previously described, for example as in Zlatev et. al. (2012) Current Protocols, 50 (1), 1.28.1-1.28.16. Briefly, protected RNA phorphoroamedites are sequentially added in a desired order to a growing chain immobilized on a solid support (e.g. controlled pore glass). Each cycle of addition has multiple steps, including: (i) deblocking the DMT group protecting the 5′-hydroxyl of the growing chain, (ii) coupling the growing chain to an incoming phosphoramidite building block, (iii) capping any chain molecules still featuring a 5′-hydroxyl, i.e. those that failed to couple with the desired incoming building block, and (iv) oxidation of the newly formed tricoordinated phosphite triester linkage. After the final building block has been coupled and oxidized, the chain is cleaved from the solid support and all protecting groups except for the DMT group protecting the 5′-hydroxyl are removed. The chain is then purified by RP-HPLC (e.g., DMT-on purification) and the fraction containing the chain is subjected to deprotection of the DMT group under acidic conditions, affording the final TREM. The TREM will feature a 5′-phosphate and a 3′-OH. The TREM is then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
If the TREM needs to be charged, the TREM produced by the chemical synthesis reaction is then aminoacylated in vitro using aminoacyl tRNA synthetase, as previously described in Stanley, Methods Enzymol 29:530-547 (1974). Briefly, the TREM is incubated for 30 min at 37° C. with its synthetase and its cognate amino, in this example, with threonyl-tRNA synthetase and threonine, respectively, and then phenol extracted, filtered using a Nuc-trap column, and ethanol precipitated. The TREM is then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
This example describes production of a TREM using in vitro transcription (IVT).
The TREM, in this example, tRNA-Leu-CAA, is produced using in vitro transcription with the sequence GUCAGGAUGGCCGAGUGGUCUAAGGCGCCAGACUCAAGUUCUGGUCUCCGUAUG GAGGCGUGGGUUCGAAUCCCACUUCUGACA as previously described in Pestova et al., RNA 7(10):1496-505 (2001). Briefly, a DNA plasmid containing a bacteriophage T7 promoter followed by the tRNA-Leu-CAA gene sequence is linearized and transcribed in vitro with T7 RNA polymerase at 37° C. for 45 min and then phenol extracted, filtered using a Nuc-trap column, and ethanol precipitated. The TREM is then admixed with a pharmaceutically acceptable excipient to make a test TREM product. Optionally, before admixing with a pharmaceutically acceptable excipient, the TREM is heated and cooled to refold the TREM.
If the TREM needs to be charged, the TREM produced by the IVT reaction is then aminoacylated in vitro using aminoacyl tRNA synthetase, as previously described in Stanley, Methods Enzymol 29:530-547 (1974). Briefly, the TREM is incubated for 30 min at 37° C. with its synthetase and its cognate amino, in this example, with leucyl-tRNA synthetase and leucine, respectively, and then phenol extracted, filtered using a Nuc-trap column, and ethanol precipitated. The TREM is then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
This example describes an assay to test an uncharged TREM for ability to modulate, e.g., induce, autophagy, e.g., the ability to activate GCN2-dependent stress response (starvation) pathway signaling, inhibit mTOR or activate autophagy.
A test uncharged TREM (uTREM) preparation is delivered to HEK293T or HeLa cells through transfection or liposomal delivery. Once the uTREM is delivered, a time course is performed ranging from 30 minutes to 6 hours with hour-long interval time points. Cells are then trypsinized, washed and lysed. The same procedure is executed with a charged control TREM as well as random RNA oligos as controls. Cell lysates are analyzed by Western blotting and blots are probed with antibodies against known readouts of GCN2 pathway activation, mTOR pathway inhibition or autophagy induction, including but not limited to phospho-eIF2a, ATF4, phospho-ULK1, phospho-4EBP1, phospho-eIF2a, phospho-Akt and phospho-p70S6K. A total protein loading control, such as GAPDH, actin or tubulin, as well as the non-modified (i.e. non-phosphorylated) signaling protein, i.e. using eIF2a as a control for phospho-eIF2a, are probed as loading controls. The methods described in this example can be adopted for use to evaluate activation of GCN2 starvation signaling pathway, autophagy pathway and/or inhibition of the mTOR pathway upon TREM delivery.
This example describes an assay to test the functionality of a mTREM produced in a cell system using plasmid transfection followed by in vitro mischarging.
In this example, an mTREM can translate a mutant mRNA into a wild type (WT) protein by incorporation of the WT amino acid in the protein despite an mRNA containing a mutated codon. GFP mRNA molecules with either a T203I or E222G mutation, which prevent GFP excitation at the 470 nm and 390 nm wavelengths, respectively, are used for this example. GFP mutants which prevent GFP fluorescence could also be used as reporter proteins in this assay. Briefly, an in vitro translation assay is used, using a rabbit reticulocyte lysate containing the GFP E222G mutated mRNA (GAG→GGG mutation) and an excess of the mTREM, in this case tRNA-Glu-CCC. As a negative control, no mischarged TREM is added to the reaction. The methods described in this example can be adopted for use to evaluate the functionality of the mTREM.
This example describes SMC-containing protein target selection for TREM-based therapy. SMCs can be understood as mutations that are informationally silent, they change the codon sequence to a synonymous codon but may have an effect on a translational or post-translational property. The selection method was segmented into three progressive selection steps (1) SMC identification, (2) examination of tRNA frequency and (3) annotation of disease relevance. These steps are described in further detail below.
A curated inclusive list of all known SNPs was utilized as a starting point for SMC selection. In this example, the dbSNP NCBI mutation database (https://www.ncbi.nlm.nih.gov/and FTP site ftp://ftp.ncbi.nih.gov/snp/organisms/) was filtered to select for a SMC, also known as synonymous SNPs (i.e. single nucleotide changes in the coding sequence not causing a change in the amino acid). Briefly, the mutated sequences were aligned to the human genome (here GRCh38p7) and the SNPS were classified into variant and mutation types, such as: non-coding-variant or coding-variant; and synonymous or non-synonymous mutations. Those classified as coding variants with synonymous mutations were designated as SMCs and taken forward into the next selection.
Examination of tRNA Frequency
For each SMC, the corresponding tRNA to each wildtype and mutated codon (SMC) was identified. The abundance of the tRNA for each of the wildtype and mutated codon (SMC) was determined from tRNA-sequencing data. In this example, the tRNA-seq previously determined from HEK293T cells (Zheng et al., Nature Methods 12, 835-837 (2015)) was utilized. SNPs that have differences, e.g., large differences, such as >10× change, in the tRNA abundance are prioritized into the next selection.
SNP IDs were mapped to a collection of known disease associated SNPs to determine which SNPs have disease correlation. In this example we utilize the GWAS (Genome Wide Associate Studies) (https://www.ebi.ac.uk/gwas/) or a similar resource to determine which SNPs have known disease correlations. Those with therapeutically relevant disease correlations (e.g., oncogenic, or relevant to a neurological disorder) were taken forward to the next step.
The filtered list of SMCs contains SMCs in coding regions that: (1) do not alter the coding sequence of an amino acid; (2) have difference, e.g., large difference, in tRNA population; and (3) have disease relevancy. In this example, the final selection is done based upon a disease of interest, e.g., pancreatic cancer. The BCAR1 gene is, e.g., known to be associated with pancreatic cancer, and has a SNP (rs7190458) that causes a change from codon CUC to CUU. This coding sequence change results in a corresponding change in incorporated TREMs. In some embodiments, the mutated incorporated TREMs has, e.g., about a 100× fold decrease in abundance making it a potential target for upregulation and/or amelioration of the disease phenotype.
The method of Example 32 was used to identify an SMC in the PNPL3A gene. The PNPL3A gene has a rs738408 polymorphism that was identified as a predisposing factor for nonalcoholic fatty liver disease, fibrosis and elevation of serum alanine transaminase in the human. The rs738408 polymorphism is a SMC as it is located in an ORF and changes the codon from CCC to CCU. Both the CCC and CCU codons code for the proline amino acid, resulting in an identical polypeptide sequence at that position of the chain as that of the wildtype PNPL3A ORF. This polypeptide chain is the adiponutrin protein.
The method of Example 32 was used to identify an SMC in the TERT gene. The TERT gene has a rs2736098 polymorphism that was identified as a susceptibility factor for pancreatic cancer and non-small cell lung carcinoma in the human. The rs2736098 polymorphism is a SMC as it is located in an ORF and changes the codon from GCG to GCA. Both the GCG and GCA codons code for the alanine amino acid, resulting in an identical polypeptide sequence at that position of the chain as that of the wildtype TERT ORF. This polypeptide chain is the telomerase reverse transcriptase protein.
The method of Example 32 was used to identify an SMC in the ACHE gene. The ACHE gene has a rs7636 polymorphism that was identified as a susceptibility factor for Type 2 Diabetes in Asian populations. The rs7636 polymorphism is a SMC as it is located in an ORF and changes the codon from CCC to CCT. Both the CCC and CCT codons code for the proline amino acid, resulting in an identical polypeptide sequence at that position of the chain as that of the wildtype ACHE ORF. This polypeptide chain is the acetylcholinesterase (AChE) protein, which is the primary enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine (ACh) into choline and acetate.
The method of Example 32 was used to identify an SMC in the CFTR gene. The CFTR gene has a rs1042077 polymorphism that is present in patients with CFTR-related disorders. The rs1042077 polymorphism is a SMC as it is located in an ORF and changes the codon from ACT to ACG. Both the ACT and ACG codons code for the threonine amino acid, resulting in an identical polypeptide sequence at that position of the chain as that of the wildtype CFTR ORF. This polypeptide chain is the cystic fibrosis transmembrane conductance regulator (CFTR).
The method of Example 32 was used to identify an SMC in the MAP3K1 gene. The MAP3K1 gene has a rs2229882 polymorphism that was identified as a susceptibility factor for the early onset of breast cancer. The rs2229882 polymorphism is a SMC as it is located in an ORF and changes the codon from ACC to ACT. Both the ACC and ACT codons code for the threonine amino acid, resulting in an identical polypeptide sequence at that position of the chain as that of the wildtype MAP3K1 ORF. This polypeptide chain is the Mitogen-Activated Protein Kinase Kinase Kinase 1 (MAP3K1), which is serine/threonine kinase that regulates the ERK and JNK MAPK pathways as well as the transcription factor NF-kappa-B pathway.
This example describes the production of a TREM in mammalian host cells.
To generate a plasmid comprising a TREM which comprises a tRNA gene, in this example, tRNA-Ser-AGA, a DNA fragment containing at least one copy of the tRNA gene with the sequence GTAGTCGTGGCCGAGTGGTTAAGGCGATGGACTAGAAATCCATTGGGGTTTCCCCGC GCAGGTTCGAATCCTGCCGACTACG is synthesized and cloned into the pLKO.1 puro backbone plasmid with a U6 promoter (or any other RNA polymerase III recruiting promoter) following the manufacturer's instructions and standard molecular cloning techniques.
One (1) mg of plasmid described above is used to transfect a 1 L culture of suspension-adapted HEK293T cells (Freestyle 293-F cells) at 1×105 cells/mL. Cells are harvested at 24, 48, 72, or 96 hours post-transfection to determine the optimized timepoint for TREM expression as determined by a quantitative method such as Northern blot, quantitative PCR (q-PCR) or Nanopore sequencing.
At the optimized harvest timepoint, the cells are lysed, and total RNA is purified using a method such as phenol chloroform. RNAs smaller than 200 nucleotides are separated from the lysate using a small RNA isolation kit per manufacturer's instructions, to generate a small RNA (sRNA) fraction.
The sRNA fraction is incubated with annealing buffer and the biotinylated capture probe corresponding to a DNA probe that is complementary to a unique region of the target TREM being purified, in this example, a probe with the sequence 3′ biotin-CCAATGGATTTCTATCCATCGCCTTAACCACTCGGCCACGACTACAAAA is used to purify the TREM comprising tRNA-Ser-AGA. The mixture is incubated at 90° C. for 2-3 minutes and quickly cooled down to 45° C. and incubated overnight at 45° C. The admixture is then incubated with binding buffer previously heated to 45° C. and streptavidin-conjugated RNase-free magnetic beads for 3 hours to allow binding of the DNA-tRNA complexes to the beads. The mixture is then added to a pre-equilibrated column in a magnetic field separator rack and washed 4 times. The TREM retained on the beads are eluted three times by adding elution buffer pre-heated to 80° C. and then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
This example describes the production of a TREM in bacterial host cells.
To generate a plasmid to produce a TREM in bacteria, a tRNA gene, in this example, a DNA fragment containing at least one copy of the tRNA-Lys-UUU gene with the sequence GCCCGGATAGCTCAGTCGGTAGAGCATCAGACTTTTAATCTGAGGGTCCAGGGTTCA AGTCCCTGTTCGGGCG is synthesized and cloned into a bacterial tRNA expression vector as previously described in Ponchon et al., Nat Protoc 4, 947-959 (2009).
1×109 bacteria grown from TREM expression plasmid transformed competent bacteria will be harvested at different cell density points, in this example OD(600)=0.5, OD(600)=0.7, OD(600)=0.9 to determine the optimal point of TREM expression as determined by a quantitative method such as Northern blot, quantitative PCR (q-PCR) or Nanopore sequencing.
At the optimized harvest cell density point, the TREM is purified as previously described in Cayama et al., Nucleic Acids Research. 28 (12), e64 (2000). Briefly, short RNAs (e.g., tRNAs) are recovered from cells by phenol extraction and concentrated by ethanol precipitation. The total tRNA in the precipitate is then separated from larger nucleic acids (including rRNA and DNA) under high salt conditions by a stepwise isopropanol precipitation. The elution fraction containing the TREM is further purified through probe binding. The TREM fraction is incubated with annealing buffer and the biotinylated capture probe corresponding to a DNA probe that is complementary to a unique region of the target TREM being purified, in this example, a probe conjugated to biotin at the 3′ end with the sequence CAGAUUAAAAGUCUG, is used to purify the TREM comprising tRNA-Lys-UUU. The mixture is incubated at 90° C. for 2-3 minutes and quickly cooled down to 45° C. and incubated overnight at 45° C. The admixture is then incubated with binding buffer previously heated to 45° C. and streptavidin-conjugated RNase-free magnetic beads for 3 hours to allow binding of the DNA-tRNA complexes to the beads. The mixture is then added to a pre-equilibrated column in a magnetic field separator rack and washed 4 times. The TREM retained on the beads are eluted three times by adding elution buffer pre-heated to 80° C. and then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
This example describes production of a TREM using chemical synthesis.
The TREM, in this example, tRNA-Thr-CGT, is chemically synthesized with the sequence GGCUCUAUGGCUUAGUUGGUUAAAGCGCCUGUCUCGUAAACAGGAGAUCCUGGG UUCGACUCCCAGUGGGGCCUCAA. This TREM is produced by solid-phase chemical synthesis using phosphoroamedite chemistry as previously described, for example as in Zlatev et. al. (2012) Current Protocols, 50 (1), 1.28.1-1.28.16. Briefly, protected RNA phorphoroamedites are sequentially added in a desired order to a growing chain immobilized on a solid support (e.g. controlled pore glass). Each cycle of addition has multiple steps, including: (i) deblocking the DMT group protecting the 5′-hydroxyl of the growing chain, (ii) coupling the growing chain to an incoming phosphoramidite building block, (iii) capping any chain molecules still featuring a 5′-hydroxyl, i.e. those that failed to couple with the desired incoming building block, and (iv) oxidation of the newly formed tricoordinated phosphite triester linkage. After the final building block has been coupled and oxidized, the chain is cleaved from the solid support and all protecting groups except for the DMT group protecting the 5′-hydroxyl are removed. The chain is then purified by RP-HPLC (e.g., DMT-on purification) and the fraction containing the chain is subjected to deprotection of the DMT group under acidic conditions, affording the final TREM. The TREM will feature a 5′-phosphate and a 3′-OH. The TREM is then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
If the TREM needs to be charged, the TREM produced by the chemical synthesis reaction is then aminoacylated in vitro using aminoacyl tRNA synthetase, as previously described in Stanley, Methods Enzymol 29:530-547 (1974). Briefly, the TREM is incubated for 30 min at 37° C. with its synthetase and its cognate amino, in this example, with threonyl-tRNA synthetase and threonine, respectively, and then phenol extracted, filtered using a Nuc-trap column, and ethanol precipitated. The TREM is then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
This example describes production of a TREM using in vitro transcription (IVT).
The TREM, in this example, tRNA-Leu-CAA, is produced using in vitro transcription with the sequence GUCAGGAUGGCCGAGUGGUCUAAGGCGCCAGACUCAAGUUCUGGUCUCCGUAUG GAGGCGUGGGUUCGAAUCCCACUUCUGACA as previously described in Pestova et al., RNA 7(10):1496-505 (2001). Briefly, a DNA plasmid containing a bacteriophage T7 promoter followed by the tRNA-Leu-CAA gene sequence is linearized and transcribed in vitro with T7 RNA polymerase at 37° C. for 45 min and then phenol extracted, filtered using a Nuc-trap column, and ethanol precipitated. The TREM is then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
If the TREM needs to be charged, the TREM produced by the IVT reaction is then aminoacylated in vitro using aminoacyl tRNA synthetase, as previously described in Stanley, Methods Enzymol 29:530-547 (1974). Briefly, the TREM is incubated for 30 min at 37° C. with its synthetase and its cognate amino, in this example, with leucyl-tRNA synthetase and leucine, respectively, and then phenol extracted, filtered using a Nuc-trap column, and ethanol precipitated. The TREM is then admixed with a pharmaceutically acceptable excipient to make a test TREM product.
This example describes administration of a TREM to a cell to modulate tRNA pools in the cell.
TREMs produced as in Examples 38-41 are delivered to a cell through electroporation, as previously described in Nature Methods 3, 67-68 (2006). Briefly, 106-107 cells, in this example the human epithelial MCF10A cells, are transferred in an electroporation cuvette and mixed gently after the addition of 1-30 ug of TREM, in this example tRNA-Thr-CGT with the sequence GGCUCUAUGGCUUAGUUGGUUAAAGCGCCUGUCUCGUAAACAGGAGAUCCUGGG UUCGACUCCCAGUGGGGCCUCAA. The cuvette is transferred to the electroporator and the device is discharged (a voltage of 200-350V is used). Place cuvette on ice and transfer the electroporated cells to a culture dish with complete medium and transfer to an incubator for 24-48 hrs.
Once delivered, the change in tRNA pools can be quantified by methods such as Nanopore sequencing, tRNA-sequencing, Northern blotting or quantitative RT-PCR. In this example, the tRNA pool changes are monitored using Oxford Nanopore direct RNA sequencing, as previously described in Sadaoka et al., Nature Communications (2019) 10, 754.
Briefly, the TREM-transfected cells are lysed and total RNA is purified using a method such as phenol chloroform. RNAs smaller than 200 nucleotides are separated from the lysate using a small RNA isolation kit per manufacturer's instructions, to generate a small RNA (sRNA) fraction.
The sRNA fraction is de-acylated using 100 mM Tris-HCl (pH 9.0) at 37° C. for 30 minutes. The solution is neutralized by the addition of an equal volume of 100 mM Na-acetate/acetic acid (pH 4.8) and 100 mM NaCl, followed by ethanol precipitation. Deacylated sRNA is dissolved in water, and its integrity verified by agarose gel electrophoresis. Deacylated sRNA is then polyadenylated using yeast poly(A) tailing kit per manufacturer's instructions to generate a sRNA polyadenylated pool. Following polyadenylation, a reverse transcription reaction is performed to generate cDNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) or a thermostable group II intron RT (TGIRT, InGex LLC) that is less sensitive to RNA structure and modifications. A sequencing adapter is ligated onto the cDNA mixture by incubating the cDNA mixture with RNA adapter, T4 ligase and ligation buffer following the standard protocol for Oxford Nanopore. Nanopore sequencing is then performed on the libraries and the sequences are mapped to a genomic database, in this example to the genomic tRNA database, GtRNAdb. The methods described in this example can be adopted for use to evaluate the tRNA pool in the cells administered with a TREM compared to those not administered with a TREM.
This example describes administration of a TREM to a cell using liposome vesicles to modulate tRNA pools in the cell.
TREMs produced as in Examples 38-41 are delivered to a cell in a vesicle or other lipid-based carrier, such as liposomes or lipid nanoparticles. In this example, a liposome kit (from Sigma or other vendor) is used to prepare liposomes containing the TREM, in this example tRNA-Thr-CGT with the sequence GGCUCUAUGGCUUAGUUGGUUAAAGCGCCUGUCUCGUAAACAGGAGAUCCUGGG UUCGACUCCCAGUGGGGCCUCAA following manufacturer's directions. The human cell line, HEK293T, is used in this example. Cells are seeded to obtain 70-80% confluency the day of the transfection. The media is replaced 30 minutes prior to the transfection with serum-free media after which the liposomes are added to the cell media.
Once delivered, the change in tRNA pools can be quantified by methods such as Nanopore sequencing, tRNA-sequencing (Zheng et al., Nature Methods 12, 835-837 (2015)), Northern blotting or quantitative RT-PCR. In this example, the tRNA pool changes are monitored using tRNA-sequencing. Briefly, the TREM-transfected cells are lysed and total RNA is purified using a method such as phenol chloroform. RNAs smaller than 200 nucleotides are separated from the lysate using a small RNA isolation kit per manufacturer's instructions, to generate a small RNA (sRNA) fraction.
The sRNA fraction is treated with a demethylase mixture to remove m1A, m1G and m3C modifications located at the Watson-Crick face. Following demethylation of the tRNA pool, a cDNA library is generated from the tRNAs using a thermostable group II intron RT (TGIRT) that is less sensitive to tRNA structure. This reverse transcriptase adds RNA-sequencing adaptors to the tRNAs by template-switching without requiring RNA ligation. Illumina sequencing is then performed on the libraries generated from the tRNAs and the sequencing reads are mapped to a genomic database, in this example to the genomic tRNA database, GtRNAdb. The methods described in this example can be adopted for use to evaluate the tRNA pool in the cells administered with a TREM compared to those not administered with a TREM.
This example describes delivery of TREM-encoding plasmid to a cell to modulate tRNA pools in the cell.
A TREM is expressed in cells through delivery of a TREM-encoding plasmid using a vesicle-based carrier. To express a TREM in human cells, a plasmid is created, which contains a tRNA gene, in this example, tRNA-Gly-GCC, with the sequence GCATTGGTGGTTCAGTGGTAGAATTCTCGCCTGCCACGCGGGAGGCCCGGGTTCGAT TCCCGGCCAATGCA. The plasmid is generated using seamless assembly of DNA fragments, in this example using NEBuilder HiFi Assembly Master Mix, where a linearized mammalian expression vector of interest, in this example pLKO.1-puro-turboGFP linearized by PpuMI enzyme restriction, is fused with a DNA fragment that contains the tRNA gene. The DNA fragment in this example includes the following elements in 5′ to 3′ order: a 25 nucleotide-long sequence from the 3′ end of the vector linearization site, a U6 promoter, the tRNA sequence, a RNA polymerase III termination signal, a 25 nucleotide-long sequence from the 5′ end of the vector linearization site.
Once the plasmid is made, the human cell line, in this example HEK293T, is transfected with TREM-encoding plasmid, using Lipofactamine 3000 following manufacturer's directions. Once delivered, the change in tRNA pools can be quantified by methods such as Nanopore sequencing, tRNA-sequencing (Zheng et al., Nature Methods 12, 835-837 (2015)), Northern blotting or quantitative RT-PCR. In this example, the tRNA pool changes are monitored using tRNA-sequencing. Briefly, the TREM-transfected cells are lysed and total RNA is purified using a method such as phenol chloroform. RNAs smaller than 200 nucleotides are separated from the lysate using a small RNA isolation kit per manufacturer's instructions, to generate a small RNA (sRNA) fraction.
The sRNA fraction is treated with a demethylase mixture to remove m1A, m1G and m3C modifications located at the Watson-Crick face. Following demethylation of the tRNA pool, a cDNA library is generated from the tRNAs using a thermostable group II intron RT (TGIRT) that is less sensitive to tRNA structure. This reverse transcriptase adds RNA-sequencing adaptors to the tRNAs by template-switching without requiring RNA ligation. Illumina sequencing is then performed on the libraries generated from the tRNAs and the sequencing reads are mapped to a genomic database, in this example to the genomic tRNA database, GtRNAdb. The methods described in this example can be adopted for use to evaluate the tRNA pool in the cells administered with a TREM compared to those not administered with a TREM.
This example describes delivery of a TREM-encoding viral vector to a cell to modulate tRNA pools in the cell.
A TREM is expressed in cells through delivery of a TREM-encoding viral vector. In this example, a lentivirus packaging and delivery system encoding a TREM is used. Briefly, the TREM-encoding viral vector is built by first generating a plasmid comprising a TREM, in this example, tRNA-Gly-GCC, with the sequence GCATTGGTGGTTCAGTGGTAGAATTCTCGCCTGCCACGCGGGAGGCCCGGGTTCGAT TCCCGGCCAATGCA. The plasmid is generated using seamless assembly of DNA fragments where the pLKO.1-puro-turboGFP linearized vector is ligated to a DNA fragment containing the tRNA sequence as described in Example 25. To prepare a TREM expressing lentivirus, HEK293T cells are co-transfected with 3 μg of each packaging vector (pRSV-Rev, pCMV-VSVG-G and pCgpV) and 9 μg of the plasmid comprising a TREM, using Lipofectamine 3000 according to manufacturer's instructions. After 24 hours, the media is replaced with fresh antibiotic-free media and after 48 hours, virus-containing supernatant is collected and centrifuged for 10 min at 2000 rpm before being filtered through a 0.45 m filter.
The cell of interest is then infected with the virus. In this example, 2 mL of virus prepared is used to transduce 100,000 HeLa cells, in the presence of 8 μg/mL polybrene. Forty-eight hours after transduction, puromycin (at 2 μg/mL) antibiotic selection is performed for 2-7 days alongside a population of untransduced control cells to select for cells that integrated the TREM in their genome for expression.
The change in tRNA pools can be quantified by methods such as Nanopore sequencing, tRNA-sequencing (Zheng et al., Nature Methods 12, 835-837 (2015)), Northern blotting or quantitative RT-PCR. In this example, the tRNA pool changes are monitored using quantitative RT-PCR (Korniy et al., Nucleic Acids Research (2019), gkz202). Briefly, the TREM-transfected cells are lysed and total RNA is purified using a method such as phenol chloroform. RNAs smaller than 200 nucleotides are separated from the lysate using a small RNA isolation kit per manufacturer's instructions, to generate a small RNA (sRNA) fraction.
The sRNA fraction is treated with a demethylase mixture to remove m1A, m1G and m3C modifications located at the Watson-Crick face. Following demethylation, the pool is reverse transcribed into cDNA using stem-loop adapters complimentary to the 3′-ends of the tRNAs of interest. In this step, reverse transcription (RT) is performed using the Superscript III first strand synthesis system (ThermoFisher Scientific). Quantitative PCR is then performed using the QuantiTect SYBR Green Kit (Qiagen) according to the manufacturer's protocol with forward primers complimentary to the region of cDNA encoded by the tRNAs of interest and a universal primer complimentary to the stem-loop adapter appended during RT. The methods described in this example can be adopted for use to evaluate the levels of glycine specifying molecule that can pair with the CGT codon in the cells administered with a TREM compared to those not administered with a TREM.
This example describes a system, in this example a cell line, that expresses an SMC-containing ORF to study the effects of TREM administration.
To study the effects of TREM administration on a SMC-containing ORF, in this example on the rs2229882 polymorphism of the MAP3K1 gene, an established cell line, in this example human breast epithelial cells, such as MCF10A or 184A1 cells, are genomically edited by CRISPR-Cas to knock out the expression of the endogenous gene of interest, in this example the MAP3K1 gene. MAP3K1 knockout cells were generated using the CRISPR-Cas9 system to insert 1 bp in a coding exon of MAP3K1 to cause a frameshift mutation as previously described (for example, Bauer et al., J. Vis. Exp., (95), doi:10.3791/52118 (2015)). Briefly, an online design tool that predicts the most effective guide RNA to use for genome editing, for example, https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design, is used to select a high-score guide RNA (gRNA) containing a 20-base pair (bp) target sequence that minimizes genomic matches to reduce the risk of off-target site cleavage. In this example, the targeting sequence is CAGTGTGTGAAGACGGCTGC. The targeting sequence is cloned into pSpCas9 (BB) plasmid (pX330) (Addgene plasmid ID 42230). HEK293T cells are transiently transfected with the CRISPR/Cas9 construct targeting MAP3K1 and a puromycin expression construct for clone selection. The next day, cells are selected with puromycin for 2 days and subcloned to form single colonies. MAP3K1 KO clones are identified by PCR screen. The obtained clones are validated by qPCR and immunoblot using an antibody against MAP3K1.
Once created, this cell line is used to overexpress the WT or SMC-containing mRNA through transient plasmid transfection or through stable lentivirus transduction methods. The TREM of interest is then administered to each cell line and its effect on the SMC-containing ORF compared to the WT ORF is assessed using assays such as the ones described in Examples 25, 30 or 31.
This example describes administration of a TREM to alter expression levels of an SMC-containing ORF.
To create a system in which to study the effects of TREM administration on protein expression levels of an SMC-containing protein, in this example, from the PNPL3A gene coding for adiponutrin, a plasmid containing the PNPL3A rs738408 ORF sequence is transfected in the normal human hepatocyte cell line THLE-3, edited by CRISPR/Cas to contain a frameshift mutation in a coding exon of PNPLA3 to knock out endogenous PNPLA3 (THLE-3_PNPLA3KO cells). As a control, an aliquot of THLE-3_PNPLA3KO cells are transfected with a plasmid containing the wildtype PNPL3A ORF sequence.
An arginine TREM is synthesized as described in Examples 3-7 and quality control methods as described in Examples 8-10 are performed. To ensure proper folding, the TREM is heated at 85° C. for 2 minutes and then snap cooled at 4° C. for 5 minutes.
A TREM is delivered to the THLE-3_PNPLA3KO cells containing the rs738408 ORF sequence as well as to the THLE-3_PNPLA3KO cells containing the wildtype PNPL3A ORF sequence. In this example, the TREM contains a proline isoacceptor containing an AGG anticodon, that base pairs to the CCT codon, i.e. with the sequence GGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUAGGGUGCGAGAGGUCCCGGGUU CAAAUCCCGGACGAGCCC. A time course is performed ranging from 30 minutes to 6 hours with hour-long interval time points. At each time point, cells are trypsinized, washed and lysed. Cell lysates are analyzed by Western blotting and blots are probed with antibodies against the adiponutrin protein. A total protein loading control, such as GAPDH, actin or tubulin, is also probed as a loading control.
The methods described in this example can be adopted for use to evaluate the expression levels of the adiponutrin protein in rs738408 ORF containing cells.
This example describes administration of a TREM to alter the rate of protein translation of an SMC-containing ORF.
To monitor the effects of TREM addition on translation elongation rates, an in vitro translation system, in this example the RRL system from Promega, is used in which the fluorescence change over time of a reporter gene, in this example GFP, is a surrogate for translation rates.
An arginine TREM is synthesized as described in Examples 3-7 and quality control methods as described in Examples 8-10 are performed. To ensure proper folding, the TREM is heated at 85° C. for 2 minutes and then snap cooled at 4° C. for 5 minutes.
Evaluation of protein translation rate of SMC-containing ORF First, a rabbit reticulocyte lysate that is depleted of the endogenous tRNA using an antisense oligonucleotide targeting the sequence between the anticodon and variable loop is generated (see, e.g., Cui et al. 2018. Nucleic Acids Res. 46(12):6387-6400). In this example, a TREM comprising an alanine isoacceptor containing an UGC anticodon, that base pairs to the GCA codon, i.e. with the sequence GGGGAUGUAGCUCAGUGGUAGAGCGCAUGCUUUGCAUGUAUGAGGUCCCGGGUU CGAUCCCCGGCAUCUCCA is added to the in vitro translation assay lysate in addition to 0.1-0.5 ug/uL of mRNA coding for the wildtype TERT ORF fused to the GFP ORF by a linker or an mRNA coding for the rs2736098 TERT ORF fused to the GFP ORF by a linker. The progress of GFP mRNA translation is monitored by fluorescence increase on a microplate reader at 37° C. using λex485/λem528 with data points collected every 30 seconds over a period of 1 hour. The amount of fluorescence change over time is plotted to determine the rate of translation elongation of the wildtype ORF compared to the rs2736098 ORF with and without TREM addition. The methods described in this example can be adopted for use to evaluate the translation rate of the rs2736098 ORF and the wildtype ORF in the presence or absence of TREM.
This example describes administration of a TREM to alter expression levels of an SMC-containing ORF.
To create a system in which to study the effects of TREM administration on protein expression levels of SMC-containing protein, in this example, from the PNPL3A gene coding for adiponutrin, a plasmid containing the PNPL3A rs738408 ORF sequence is transfected in the normal human hepatocyte cell line THLE-3, edited by CRISPR/Cas to contain a frameshift mutation in a coding exon of PNPLA3 to knock out endogenous PNPLA3 (THLE-3_PNPLA3KO cells). As a control, an aliquot of THLE-3_PNPLA3KO cells are transfected with a plasmid containing the wildtype PNPL3A ORF sequence.
A TREM is delivered to the THLE-3_PNPLA3KO cells containing the rs738408 ORF sequence as well as to the THLE-3_PNPLA3KO cells containing the wildtype PNPL3A ORF sequence. In this example, the TREM contains a proline isoacceptor containing an AGG anticodon, that base pairs to the CCT codon, i.e. with the sequence GGCUCGUUGGUCUAGGGGUAUGAUUCUCGCUUAGGGUGCGAGAGGUCCCGGGUU CAAAUCCCGGACGAGCCC. A time course is performed ranging from 30 minutes to 6 hours with hour-long interval time points. At each time point, cells are trypsinized, washed and lysed. Cell lysates are analyzed by Western blotting and blots are probed with antibodies against the adiponutrin protein. A total protein loading control, such as GAPDH, actin or tubulin, is also probed as a loading control.
The methods described in this example can be adopted for use to evaluate the expression levels of the adiponutrin protein in rs738408 ORF containing cells.
This example describes administration of a TREM to alter the rate of protein translation of an SMC-containing ORF.
To monitor the effects of TREM addition on translation elongation rates, an in vitro translation system, in this example the RRL system from Promega, is used in which the fluorescence change over time of a reporter gene, in this example GFP, is a surrogate for translation rates. First, a rabbit reticulocyte lysate that is depleted of the endogenous tRNA using an antisense oligonucleotide targeting the sequence between the anticodon and variable loop is generated (see, e.g., Cui et al. 2018. Nucleic Acids Res. 46(12):6387-6400). In this example, a TREM comprising an alanine isoacceptor containing an UGC anticodon, that base pairs to the GCA codon, i.e. with the sequence GGGGAUGUAGCUCAGUGGUAGAGCGCAUGCUUUGCAUGUAUGAGGUCCCGGGUU CGAUCCCCGGCAUCUCCA is added to the in vitro translation assay lysate in addition to 0.1-0.5 ug/uL of mRNA coding for the wildtype TERT ORF fused to the GFP ORF by a linker or an mRNA coding for the rs2736098 TERT ORF fused to the GFP ORF by a linker. The progress of GFP mRNA translation is monitored by fluorescence increase on a microplate reader at 37° C. using λex485/λem528 with data points collected every 30 seconds over a period of 1 hour. The amount of fluorescence change over time is plotted to determine the rate of translation elongation of the wildtype ORF compared to the rs2736098 ORF with and without TREM addition. The methods described in this example can be adopted for use to evaluate the translation rate of the rs2736098 ORF and the wildtype ORF in the presence or absence of TREM.
This example describes administration of a TREM to change the function of an SMC-containing ORF.
Using an in vitro translation (IVT) system (such as the RRL system from Promega), the wildtype and SMC containing mRNAs are translated in the presence and absence of a TREM. In this example the SMC-containing gene is AChE, coding for the acetylcholinesterase protein, and the TREM contains a proline isoacceptor containing an AGG anticodon, that base pairs to the CCU codon, i.e. with the sequence GGCUCGUUGGUCUAGGGGUAUGAUCUCGCUUAGGGUGCGAGAGGUCCCGGGUUC AAAUCCCGGACGAGCCC.
To determine if addition of the TREM changes the functional activity of the SMC-containing protein, in this example the acetylcholinesterase protein, a functional assay that uses DTNB to quantify the thiocholine produced from the hydrolysis of acetylthiocholine by AChE is used. Briefly, the translation reactions are incubated at room temperature for 10-30 minutes with the kit AChE reaction mixture, after which the absorption intensity of DTNB adduct at OD 410 nm is used to measure the amount of thiocholine formed, which is proportional to the AChE activity. The methods described in this example can be adopted for use to evaluate AChE activity of the protein resulting from the translation of the rs7636 AChE mRNA or the wildtype AChE mRNA.
This example describes administration of a TREM to alter the localization of an SMC-containing ORF.
To create a system in which to study the effects of TREM administration on protein localization of an SMC-containing ORF, a plasmid containing the CFTR rs1042077 ORF sequence tagged with a reporter, such as GFP or myc, is transfected in the human lung epithelial cell line MRC-5. As a control, a plasmid containing the wildtype CFTR ORF sequence tagged with a reporter is also transfected in parallel in MRC-5 cells.
To determine if TREM addition changes the localization of CFTR, the cells are seeded on coverslips and 24 hours later are transfected with a TREM complementary to the CFTR SMC or a control TREM. In this example the TREM complementary to the CFTR SMC comprises a threonine isoacceptor containing an CGU anticodon, that base pairs to the ACG codon, i.e. with the sequence GGCUCUGUGGCUUAGUUGGCUAAAGCGCCUGUCUCGUAAACAGGAGAUCCUGGG UUCGAAUCCCAGCGGGGCCU. The control TREM consists of either a scrambled sequence or the threonine sequence where the 5′ end of the TREM has been changed to prevent charging. After 24 hours, the cells are fixed, stained for CFTR and its reporter and visualized under a microscope. The methods described in this example can be adopted for use to evaluate the localization of wildtype CFTR and rs1042077 CFTR.
This example describes administration of a TREM to alter the folding of an SMC-containing ORF.
To identify SMCs that result in protein misfolding, the SMC-ORF containing protein, in this example the rs7190458 BCAR1 ORF is synthesized and cloned into a plasmid containing a CMV promoter (or any other mammalian promoter) and a purification tag, in this example a FLAG tag (DYKDDDDK epitope), following the manufacturer's instructions and standard molecular cloning techniques. Here, the pFLAG-CMV-1 plasmid is used. The plasmid is transfected in the human HeLa cell line. A TREM, in this example comprising a leucine isoacceptor containing an UUG anticodon, that base pairs to the CUU codon, i.e. with the sequence GGUAGCGUGGCCGAGCGGUCUAAGGCGCUGGAUUAAGGCUCCAGUCUCUUCGGA GGCGUGGGUUCGAAUCCCACCGCUGCCA is also transfected into the HeLa cells. As a control, the BCAR1 KO cells are transfected with the SMC BCAR1 ORF containing plasmid alone and separately with a plasmid containing the wildtype BCAR1 ORF sequence.
At the optimized harvest timepoint, in this example 72 hours post-transfection, the cells are lysed, and centrifuged at 12,000×g for 10 minutes. The supernatant is loaded under gravity flow onto a pre-packed and equilibrated anti-flag packed M2-agarose column. The column is washed with 10-20 column volumes of TBS (Tris HCl, NaCl) or with a salt containing buffer. To elute the FLAG-tagged protein from the beads, the beads are incubated with FLAG-tag peptide. The eluate is run on an SDS-PAGE gel for purity quality control. This purification is performed on cells transfected with the WT BCAR1 ORF and the SMC BCAR1 ORF in the presence and absence of a TREM.
To examine the effects of protein folding, the stability of the purified proteins derived from the WT and SMC containing ORFs are monitored using thermal melting. In this example, Differential Scanning Fluorimetry (DSF) with a fluorescent dye (Sypro Orange), which measures the changes of binding of the intercalator dye to the unfolding protein is used. Alterations in protein folding results in variations of the thermal melting curves. Using this methodology, the SMC ORF-derived protein with and without TREM addition is compared to the control wildtype BCAR1. The methods described in this example can be adopted for use to evaluate the thermal melting curve of proteins derived from SMC-containing ORFs.
This example describes administration of a TREM to alter the cellular phenotype of an SMC-containing ORF.
To create a system in which to study the effects of TREM administration on cellular processes, in this example on cell migration, a plasmid containing the SMC-containing ORF, in this example the rs7190458 BCAR1 ORF sequence, is transfected in the human pancreatic cancer cell line PANC-1, in which BCAR1 has been knocked out using CRISPR/Cas. As a control, the PANC-1 BCAR1 KO cells are transfected with a plasmid containing the WT BCAR1 ORF sequence.
A TREM, in this example comprising a leucine isoacceptor containing an UUG anticodon, that base pairs to the CUU codon, i.e. with the sequence GGUAGCGUGGCCGAGCGGUCUAAGGCGCUGGAUUAAGGCUCCAGUCUCUUCGGA GGCGUGGGUUCGAAUCCCACCGCUGCCA is delivered to the PANC-1 cells. Delivery of a control TREM containing either a scrambled sequence or the leucine sequence where the 5′ end of the TREM has been changed to prevent charging are used as a control. The cells are grown to 80% confluency in a monolayer and scratched with a new 1 ml pipette tip across the center of the well. The cells are rinsed twice to remove floating cells and the media is replenished. After 48 hours, the cells are fixed and stained with crystal violet. The stained monolayer is photographed, and the gap distance quantified. The methods described in this example can be adopted for use to evaluate the migratory phenotype of cells administered a TREM.
This example describes increasing TREM levels to ameliorate a disease state resulting from an SMC-containing ORF.
To create a system in which to study the effects of TREM administration on disease state, in this example on breast cancer onset, a plasmid containing the SMC-containing ORF, in this example the rs2229882 MAP3K1 ORF sequence, is transfected in the human non-transformed breast cell line MCF10A, in which MAP3K1 has been knocked out using CRISPR/Cas. As a control, the MCF10A MAP3K1 KO cells are transfected with a plasmid containing the wildtype MAP3K1 ORF sequence.
A TREM, in this example comprising a threonine isoacceptor containing an AGU anticodon, that base pairs to the ACU codon, i.e. with the sequence GGCGCCGUGGCUUAGUUGGUUAAAGCGCCUGUCUAGUAAACAGGAGAUCCUGGG UUCGAAUCCCAGCGGUGCCU is delivered to the MCF10A cells. Delivery of a control TREM containing either a scrambled sequence or the threonine sequence where the 5′ end of the TREM has been changed to prevent charging are used as a control. The cells are monitored for increased MAPK signaling by Western blotting using antibodies against the phosphorylation state of the ERK and JNK kinases. A total protein loading control, such as GAPDH, actin or tubulin, as well as the non-modified (i.e. non-phosphorylated) signaling protein, i.e. using ERK as a control for phospho-ERK, are probed as loading controls. The cells are also monitored for cell proliferation and invasion using standard proliferation and transwell invasion assays. To monitor breast cancer progression, the cells are injected subcutaneously or in the mammary fat pad of SCID mice and tumor volume is monitored daily using calipers to measure the length, width and height of the tumor(s). The methods described in this example can be adopted for use to evaluate tumor phenotype.
This application claims priority to U.S. Provisional Application No. 63/032,008, filed on May 29, 2020, the entire contents of which is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/035015 | 5/28/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63032008 | May 2020 | US |
