Enhanced platforms for unnatural amino acid incorporation in mammalian cells

Abstract

To provide efficient platforms for expressing, in mammalian cells, proteins which site-specifically incorporate unnatural amino acids.SOLUTION: The present invention involves: 1) the ability to use a virus-assisted directed evolution platform to significantly improve the activity of engineered nonsense-suppressor tRNAs in mammalian cells; 2) the ability to provide mutants of archaeal pyrrolysyl and E. coli leucyl tRNAs that show remarkably improved Uaa incorporation efficiency in mammalian cells; and 3) the ability to use these tRNAs to express, in mammalian cells, recombinant proteins incorporating Uaas at significantly improved yields.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 864,570, filed Jun. 21, 2019, the entire disclosure of which is hereby incorporated by reference herein.

[0002] Government Support This invention was made with government support under R01 GM132471 awarded by the National Institutes of Health and CHE1900375 awarded by the National Science Foundation. The government has certain rights in the invention.

[0003] Incorporation of Material from ASCII Text File by Reference This application incorporates by reference the Sequence Listing contained in the following ASCII text file.

[0004] File Name: 0342_0008WO1_SL.txt; created on Jun. 19, 2020, size: 17,574 bytes.

[0005] Field of the Invention The present invention is directed to the field of biotechnology and focuses on the development of an efficient platform for expressing proteins with site-specifically incorporated unnatural amino acids in mammalian cells.

Background Art

[0006] Site-specific incorporation of non-natural amino acids (Uaa) holds great potential for exploring and manipulating mammalian cell biology. At the heart of this technology is a nonsense suppression aminoacyl-tRNA synthetase (aaRS) / tRNA pair that has been engineered to introduce the desired Uaa without cross-reacting with any of its host counterparts. Such "orthogonal" aaRS / tRNA pairs are typically brought into host cells from different domains of life. Since heterologous suppressor tRNAs must interact directly with the non-natural translation system, the performance of heterologous suppressor tRNAs is often not optimal in a new host. In fact, several studies have confirmed that the efficiency of Uaa incorporation in mammalian cells is limited by the poor performance of heterologous suppressor tRNAs, and that a large amount of heterologous suppressor tRNA needs to be overexpressed to achieve an acceptable Uaa incorporation efficiency. Such high levels of tRNA expression can be achieved by transient transfection in specific mammalian cell lines that exhibit high transfection efficiency, but it is difficult to do so in cells where transfection is difficult (e.g., primary cells, neurons, stem cells, etc.). Furthermore, it makes the generation of stable suppressor cell lines (expressing aaRS / tRNA engineered from the genome) very difficult. This is because hundreds of copies of the tRNA gene need to be inserted into the genome to reach sufficient nonsense suppression / Uaa incorporation efficiency. Overcoming the suboptimal performance of suppressor tRNAs would significantly improve the robustness of Uaa mutagenesis technology and facilitate advanced applications such as the facile generation of stable suppressor cell lines capable of Uaa incorporation and the simultaneous incorporation of Uaa into multiple sites within the same protein. Summary of the Invention Means for Solving the Problems

[0007] The origin of tRNA performance decline is often unclear, and it is difficult to address performance decline through rational design. However, directed evolution for Uaa incorporation in Escherichia coli (E. coli) has frequently generated improved orthogonal suppressor tRNAs. A clever selection system has been developed that can easily enrich active yet orthogonal suppressor tRNA variants from large-scale synthetic libraries. If similar tRNA evolution could be performed in mammalian cells, there is great potential to create improved suppression systems, but currently, there is no suitable platform available. Performing such directed evolution experiments in mammalian cells is important to ensure that tRNA variants are selected based on improved interactions with the mammalian-specific translation system.

[0008] Existing directed evolution strategies in mammalian cells almost exclusively rely on stably integrating the target gene into cell lines and then creating sequence diversity through non-targeted or targeted random mutagenesis. The associated low mutagenesis frequency is not suitable for tRNA evolution due to the small size of tRNAs (less than 100 bp). Furthermore, although the stem region of tRNAs is the most frequent target for genetic manipulation, to successfully evolve this tRNA stem region, any mutation must be accompanied by a corresponding mutation on the other side to maintain base pairing. Capturing such abundant sequence diversity within small tRNA genes is only achievable by using synthetic site-saturation mutant libraries. To enrich orthogonal and active suppressor tRNA variants within mammalian cells from such libraries, one needs to have: i) control over library delivery such that each cell receives a single variant, ii) a selection scheme to enrich active tRNA variants and remove cross-reactive ones, and iii) the ability to identify surviving variants. Currently, no selection system that meets these criteria exists.

[0009] Described herein are compositions comprising variant / mutant nonsense suppressor tRNA molecules (also referred to herein as suppressor tRNAs) having increased biological activity compared to the corresponding wild-type suppressor tRNA molecules for incorporation of non-natural amino acids (Uaa or UAA) into mammalian proteins; expression vectors (e.g., viral vectors) encoding these variant tRNAs, which are suitable for infecting mammalian cells; methods for producing suppressor tRNAs having increased biological activity using the virus-assisted directed evolution methods described herein; methods for producing proteins in which non-natural amino acids are site-specifically incorporated using these tRNAs having increased activity; and kits comprising such reagents containing the variant tRNAs and other reagents necessary for the production of such proteins.

[0010] In particular, the compositions of the invention include, for example, variant archaeal nonsense suppressor tRNA molecules or variant bacterial nonsense suppressor tRNA molecules, and this orthogonally active variant tRNA has increased activity for incorporating various non-natural amino acids (e.g., amino acid analogs) into mammalian proteins compared to its "wild-type" corresponding suppressor tRNA. As used herein, the term "wild-type" corresponding tRNA means a suppressor tRNA molecule that has not been subjected to the virus-assisted directed evolution methods described herein for generating (selecting and enriching) a population of suppressor tRNA molecules having increased biological activity for site-specifically incorporating Uaa into a protein of interest.

[0011] The activity of the variant tRNA included in the present invention is, for example, about 2.5 to about 200 times, about 2.5 to about 150 times, about 2.5 to about 100 times, about 2.5 to about 80 times, about 2.5 to about 60 times, about 2.5 to about 40 times, about 2.5 to about 20 times, about 2.5 to about 10 times, about 2.5 to about 5 times, about 5 to about 200 times, about 5 to about 150 times, about 5 to about 100 times, about 5 to about 80 times, about 5 to about 60 times, about 5 to about 40 times, about 5 to about 20 times, about 5 to about 10 times, about 10 to about 200 times, about 10 to about 150 times, about 10 to about 100 times, about 10 to about 80 times, about 10 to about 60 times, about 10 to about 40 times, about 10 to about 20 times, about 20 to about 200 times, about 20 to about 150 times, about 20 to about 100 times, about 20 to about 80 times, about 20 to about 60 times, about 20 to about 40 times, about 40 to about 200 times, about 40 to about 150 times, about 40 to about 100 times, about 40 to about 80 times, about 40 to about 60 times, about 60 to about 200 times, about 60 to about 150 times, about 60 to about 100 times, about 60 to about 80 times, about 80 to about 200 times, about 80 to about 150 times, about 80 to about 100 times, about 100 to about 200 times, about 100 to about 150 times, or about 150 to about 200 times greater than that of the wild-type tRNA.

[0012] The variant archaeal tRNA molecule is derived from, for example, the Methanosarcinacaea or Desulfitobacterium family, particularly any of the families of Methanosarcina barkeri (Mb), Methanosarcina alvus (Ma), Methanosarcina mazei (Mm), or Desulfitobacterium hafnisense (Dh). Specifically, the invention according to the claims is a pyrrolidyl tRNA (tRNA Pyl ) derived from a nucleic acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the full-length sequence of SEQ ID NO: 1 or any of SEQ ID NOs: 2 to 27 of the variant tRNA molecule shown in Table 1. More specifically, in certain embodiments, the variant tRNA Pylcomprises a sequence selected from the group consisting of nucleic acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NOs: 2-27, or the full-length SEQ ID NOs: 2-27. In certain embodiments, the tRNA Pyl contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., substitutions) compared to any one of SEQ ID NOs: 1-27. Suitable unnatural amino acids for incorporation by the variant archaeal-derived tRNAs described herein can be azidolysine (AzK) (Structure 1 in FIG. 18) or Nε-acetyllysine (AcK) (Structure 2 in FIG. 18), or any other lysyl analog such as Structures 3-6 shown in FIG. 18. Additionally, as described herein (e.g., see Example 9, (FIG. 19)), the incorporation efficiency of any other Uaa using engineered pyrrolysyl tRNA synthetase can also be improved by the use of these engineered tRNA Pyl variants.

[0013] The variant bacterial tRNA molecules of the present invention are derived from, for example, E. coli tRNA. Specifically, the invention according to the claims encompasses variant tRNAs that are leucyl tRNAs (tRNA Leu ) derived from SEQ ID NO: 28. Specifically, in certain embodiments, the variant tRNA Leu comprises a nucleic acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 29-45, or any one of the full-length SEQ ID NOs: 29-45. In certain embodiments, the tRNA LeuIt contains a mutation (e.g., substitution) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 as compared with any one of SEQ ID NOs: 28 to 45. Any suitable unnatural amino acid / analog can be used together with the methods described herein for incorporation into the protein of interest. In particular, the unnatural amino acids suitable for incorporation by the variant bacterial-derived tRNAs described herein can be Uaas having structures 7 to 12 shown in FIG. 18, or structures that are structurally and functionally similar to structures 7 to 12. In addition, as described herein (e.g., see FIG. 19), the incorporation efficiency of any other Uaa using the engineered Escherichia coli leucyl-tRNA synthetase can also be improved by these engineered tRNAs Leu can be improved by the use of variants.

[0014] An expression vector (e.g., a viral vector) containing a variant archaeal suppressor tRNA or a variant bacterial suppressor tRNA, wherein the variant tRNA has an increased activity for incorporating an unnatural amino acid into a mammalian protein as compared to its wild-type corresponding tRNA as described herein is also encompassed by the present invention. Viruses suitable for the present invention include any virus that integrates or does not integrate into the mammalian cell genome. Such viruses include adenovirus, adeno-associated virus, baculovirus, lentivirus, and retrovirus. More specifically, as described herein, any serotype of adeno-associated virus can be used in the present invention, particularly adeno-associated virus serotype 2. The expression vectors (e.g., viral vectors) of the present invention can also encode a reporter gene such as mCherry, GFP or EGFP, or other suitable detector molecules.

[0015] Cells (one or more) containing the expression vectors (e.g., viral vectors) described herein, and stable cell lines of these cells are also encompassed by the present invention. In certain embodiments, the cells are mammalian cells, and the stable mammalian cells contain genomically integrated (or episomally maintained) engineered tRNA.

[0016] The cells of the present invention can further contain one or more additional expression vectors (e.g., plasmids) encoding genes necessary for viral replication of the viral vector in the cells. More specifically, the cells of the present invention may contain an expression vector (e.g., plasmid) encoding all the genetic components essential for viral replication, in which a nonsense codon is inserted into the protein sequence so that viral replication depends on the activity of the variant suppressor tRNA.

[0017] In one embodiment, the essential viral protein can be the VP1 capsid protein (Cap) of a non-enveloped virus such as adeno-associated virus AAV2 SEQ ID NO: 46, or an amino acid sequence having about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 46. The suppressor codon can be inserted at a selected site in the capsid protein (e.g., TAG - amber; TAA - ochre or TGA - opal) (Agnew. Chem. Int. Ed. 2016, 55, 10645; Agnew. Chem. Int. Ed. 2017, 56, 4234). As described herein, the essential viral protein for adeno-associated virus can be Cap having a TAG codon at position 454. The cells can be cultured in the presence of the cognate Uaa RNA synthetase (UaaRS) and Uaa. In one embodiment, the UaaRS is, for example, Mb Pyl RS, and the Uaa is, for example, AzK or AcK. In another embodiment, the variant tRNA is Escherichia coli leucyl tRNA (tRNALeu ) where aaRS is E. coli LeuRS and Uaa is a leucine analog as shown in FIG. 18.

[0018] Also included in the present invention is a method for virus-assisted directed evolution of a suppressor tRNA variant having increased biological activity compared to wild-type suppressor tRNA. Replication of a virus in mammalian cells requires the expression of an essential protein that depends on the activity of the target tRNA variant.

[0019] The method includes encoding a library of the target suppressor tRNA variant in a viral genome, infecting a population of mammalian host cells with a viral vector at a low multiplicity of infection (MOI), and maintaining this cell population under conditions suitable for viral replication in the cells, wherein viral replication in mammalian cells requires the expression of an essential protein that depends on the activity of the target tRNA variant, collecting and selectively amplifying viral progeny encoding the active tRNA variant to remove cross-reactive tRNA molecules, whereby an orthogonal suppressor tRNA variant having increased biological activity is recovered. The amplified tRNA variant can then be sequenced to determine its nucleic acid sequence and be subjected to further evaluation. Next-generation sequencing of the virus-encoded tRNA library before and after selection can be performed to confirm the enrichment level of each possible variant in the library. This enrichment factor can be used as an indicator of tRNA activity, and the most enriched tRNA variant can be constructed and tested to confirm its activity. In certain embodiments, the disclosed method results in an enrichment of 10,000 to 30,000-fold or 20,000 to 30,000-fold of the virus encoding the active tRNA over the virus carrying the inactive tRNA.

[0020] In more specific embodiments, as described herein, the sequences of the targeted nonsense-suppressing tRNAs are randomized to create libraries and are encoded in suitable expression vectors (e.g., viral vectors). The tRNA variant libraries will include inactive tRNA molecules, active orthogonal tRNA molecules, and active but cross-reactive tRNA molecules.

[0021] Certain contemplated methods involve the use of a population of transformation-competent host cells. Such cells are typically mammalian cells, more specifically immortalized human cells. This suitable host cell can be infected with the viral vector at a very low to low multiplicity of infection (MOI). In certain embodiments, the MOI is from about 0.1 to about 15, from about 0.1 to about 10, from about 0.1 to about 5, from about 0.1 to about 3, from about 0.1 to 1, from about 1 to about 15, from about 1 to about 10, from about 1 to about 5, from about 1 to about 3, from about 3 to about 15, from about 3 to about 10, from about 3 to about 5, from about 5 to about 15, from about 5 to about 10, or from about 10 to about 15. In certain embodiments, the MOI is 0.1 to 5. In certain embodiments, the MOI is less than 15, less than 10, less than 5, less than 3, less than 1, or less than 0.1. In certain embodiments, a single viral vector encoding the variant tRNA is all that is required for the expression of essential viral proteins and the production of viral progeny within the cell. More specifically, each cell receives a single virus-encoded tRNA variant.

[0022] The cells are then transfected (typically within a few hours of viral infection) with one or more expression vectors (e.g., plasmids), where the expression vector (e.g., plasmid) contains all the genetic components essential for viral replication and nonsense codons are inserted into the protein sequence such that viral replication is made to depend on the activity of variant suppressor tRNAs. In one embodiment, the essential viral protein is Cap (SEQ ID NO: 46) having a TAG codon at position 454. In certain embodiments, the expression vector (e.g., plasmid) also encodes a cognate Uaa RNA synthetase (UaaRS). For example, if the tRNA library is a pyrrolidyl tRNA (tRNA Pyl ) library, the UaaRS is Mb Pyl RS. During transfection, Uaa may be added to the culture medium at an appropriate concentration. In certain embodiments, the Uaa is AzK. Alternatively, the variant tRNA is leucyl tRNA (tRNA Leu ), the aaRS is E. coli Leu RS, and the Uaa is AzK or any one of structures 7 - 12 in FIG. 18. Additional expression vectors (e.g., plasmids) encoding the genetic components necessary for viral replication may also be transfected into the host cells as described herein.

[0023] In the contemplated method, infected / transfected cells are maintained (i.e., cultured) in a medium containing Uaa under conditions suitable for the expression of variant tRNAs, the expression of essential viral proteins, and viral replication. In certain embodiments, the cells are harvested, viral progeny are isolated, and subjected to further enrichment to remove cross-reactive but active tRNA molecules, and an orthogonal suppressor tRNA variant having increased biological activity is recovered. The enriched tRNA variant can be sequenced to obtain its nucleic acid sequence. Next-generation DNA sequencing of the viral-encoded tRNA library before and after selection can be performed to measure the abundance of each tRNA variant and the changes due to selection. The tRNA variants that undergo the strongest enrichment during selection are likely to have the highest activity.

[0024] In the method of the present invention, only the active and orthogonal tRNA variants enable the incorporation of Uaa into essential viral gene proteins and viral replication in cells. However, in certain embodiments, viruses containing active and cross-reactive tRNAs can also replicate, so additional steps are required to remove the viral population encoding the cross-reactive tRNAs and enrich the viral population encoding the desired tRNAs. The isolated viral progeny can be enriched for tRNA variants with increased biological activity. For example, the isolated viral progeny can be chemically selectively labeled with a purified handle / tag attached through a photocleavable moiety such as a photocleavable linker. In one embodiment, this moiety is a photocleavable DBCO-sulfo-biotin conjugate. The reaction mixture contains viruses incorporating the Uaa protein, viruses not containing the Uaa protein, the photocleavable biotin label, and an excess of Uaa as a quencher. Viruses labeled with the biotin conjugate are recovered using streptavidin-coated beads, and the viruses are eluted from the beads using an appropriate wavelength (e.g., 365 nm). The recovered virus particles (virions) contain the desired suppressor tRNA with increased biological activity compared to wild-type suppressor tRNA.

[0025] After lysing the recovered virus and amplifying the tRNA, it can be sequenced to obtain its nucleic acid sequence. Alternatively, the recovered virus can be lysed, the tRNA amplified, and cloned as described above using an appropriate vector. Then, colonies can be selected and sequenced to obtain the nucleic acid sequence of the desired suppressor tRNA. In addition, next-generation DNA sequencing (e.g., Illumina) of the virus-encoded tRNA library before and after selection can be performed to measure the abundance of each tRNA variant and the changes due to selection. The tRNA variants that undergo the strongest enrichment during selection are likely to have the highest activity. Next, the identified variants can be constructed and tested.

[0026] A method for producing (generating) a protein of interest in mammalian cells, wherein the protein of interest has one or more amino acid analogs at specific positions in the protein of interest, is further encompassed by the present invention. In one embodiment, the steps of the method include culturing the mammalian cells in a culture medium under conditions suitable for growth, wherein the cells contain a nucleic acid encoding a protein having one or more selector codons, and the cells also contain a variant archaeal-derived pyrrolidyl tRNA having increased biological activity that recognizes the selector codon, and its cognate aminoacyl-tRNA synthetase. The cell culture medium is contacted (added at an appropriate concentration) with one or more lysine analogs under conditions suitable for incorporation of the one or more lysine analogs into the protein in response to the selector codon, whereby a protein of interest (desired protein) having one or more lysine analogs may be produced (generated).

[0027] In one embodiment, the variant tRNA is a pyrrolidyl tRNA (tRNA Pyl ) derived from a nucleobase sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or any of SEQ ID NOs: 2-27 in full length. In certain embodiments, the variant tRNA Pyl comprises a sequence selected from the group consisting of SEQ ID NOs: 2-27 or a nucleic acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 2-27 in full length. The lysine analog can be any lysine analog, in particular azidolysine (AzK) or acetyllysine (AcK) or any of Structures 3-6 in FIG. 18. In addition, as described in Example 9 (FIG. 19), the incorporation efficiency of any other Uaa using an engineered pyrrolidyl tRNA synthetase can also be improved by the use of these engineered tRNA Pyl variants.

[0028] In another embodiment of the method, the variant suppressor tRNA is an Escherichia coli-derived leucyl-tRNA having increased biological activity that recognizes the selector codon, and its cognate aminoacyl-RNA synthetase incorporates one or more leucine analogs into the protein of interest in response to its selector codon, thereby producing a protein having one or more leucine analogs. In one embodiment, the variant tRNA is leucyl-tRNA (tRNA Leu ) derived from SEQ ID NO: 28. In certain embodiments, the variant tRNA Leu comprises a nucleic acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 29-45, or any one of the full-length SEQ ID NOs: 29-45. Non-natural amino acids suitable for incorporation by the variant bacterium-derived tRNAs described herein can be Structures 7-12 shown in FIG. 18. Additionally, as described in Example 9 (FIG. 19), the incorporation efficiency of any other Uaa using engineered Escherichia coli leucyl-tRNA synthetase can also be improved by the use of these engineered tRNA Leu variants.

[0029] The method of the present invention is a method for site-specifically incorporating one or more azidolysine (AzK) or acetyllysine (AcK) residues into a protein or peptide in a cell, comprising culturing the cell in a culture medium under conditions suitable for growth, wherein the cell comprises a nucleic acid encoding a protein or peptide of interest having one or more amber, ochre, or opal selector codons at a specific site in the protein or peptide of interest, and the cell comprises a variant archaeal-derived pyrrolidyl-tRNA having increased biological activity that recognizes the selector codon PylFurther includes and further encompasses a method further comprising an archaeal Pyl-tRNA synthetase. Next, the cell culture medium may be contacted with one or more AzK or AcK residues under conditions suitable for incorporating one or more AzK or AcK residues into a protein or peptide at one or more sites of the selector codon(s), thereby producing a protein or peptide of interest having one or more site-specifically incorporated AzK or AcK residues.

[0030] In one embodiment, the variant pyrrolidyl tRNA (tRNA Pyl ) is derived from SEQ ID NO: 1. For example, the variant tRNA Pyl comprises a sequence selected from the group consisting of nucleic acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 2-27, or the full-length SEQ ID NOs: 2-27. See, e.g., FIG. 18, Structures 1-6.

[0031] In another embodiment, the method site-specifically incorporates one or more leucine analog residues into a protein or peptide in a cell, wherein the cell comprises a variant E. coli-derived tRNA Leu having increased biological activity that recognizes the selector codon and further comprises an E. coli Leu-tRNA synthetase. The cell culture medium may be contacted with one or more leucine analog residues under conditions suitable for incorporating one or more leucine analog residues into a protein or peptide at the site of the selector codon(s), thereby producing a protein or peptide of interest having one or more site-specifically incorporated leucine analog residues.

[0032] Specifically, in certain embodiments, the variant tRNA is a leucyl tRNA (tRNA Leu ) derived from SEQ ID NO: 28. For example, the variant tRNA LeuIt includes any one of SEQ ID NOs: 29 to 45, or a nucleic acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the full-length SEQ ID NOs: 29 to 45. The unnatural amino acids suitable for incorporation by the variant bacterial-derived tRNA described herein can be Structures 7 to 12 shown in FIG. 18. In addition, as described in Example 9 (FIG. 19), the incorporation efficiency of any other Uaa using the engineered Escherichia coli leucyl-tRNA synthetase can also be improved by these engineered tRNAs Leu by the use of the variants.

[0033] A kit for producing a protein or peptide of interest in a cell, wherein the protein or peptide contains one or more lysine analogs, and the kit comprises a variant archaeal-derived tRNA having increased biological activity that recognizes a selector codon in the nucleic acid of interest in the cell Pyl A kit comprising a container containing a polynucleotide sequence encoding the same is also encompassed by the present invention. Specifically, in certain embodiments, the variant tRNA Pyl comprises a sequence selected from the group consisting of SEQ ID NOs: 2 to 27 (see, for example, FIG. 18, Structures 1 to 6), or a nucleic acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the full-length SEQ ID NOs: 2 to 27. The kit can further comprise a container containing a nucleotide sequence encoding an archaeal Pyl-tRNA synthetase. The kit can further comprise one or more lysine analogs such as azidolysine (AzK) or acetyllysine (AcK). The kit can also comprise instructions for producing the protein or peptide of interest.

[0034] In another embodiment, the kit is directed to producing a protein or peptide of interest in a cell, the protein or peptide comprising one or more leucine analogs, and the kit comprises a container comprising a polynucleotide sequence encoding a variant E. coli-derived tRNALeu having increased biological activity that recognizes a selector codon in a nucleic acid of interest in the cell, the variant tRNA Leu comprises a nucleic acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 29-45, or any one of the full-length SEQ ID NOs: 29-45. The kit can also comprise a container comprising a polynucleotide sequence encoding an E. coli Leu-tRNA synthetase and one or more leucine analogs such as Structures 7-12 of FIG. 18. The kit can comprise instructions for producing the protein or peptide of interest.

[0035] Mammalian cells stably incorporated with variant tRNA-Pyl or variant tRNA-Leu for Uaa incorporation are also encompassed by the present invention. In one embodiment, the mammalian cell comprises variant tRNA-Pyl-Leu, the sequence of variant tRNA-Pyl-Leu is selected from the group consisting of SEQ ID NOs: 2-27, and Uaa is a pyrrolidyl residue selected from the group consisting of any one of Structures 1-7. In another embodiment, the cell comprises variant tRNA-Leu selected from the group consisting of SEQ ID NOs: 29-45, and Uaa is a leucine analog selected from the group consisting of any one of Structures 7-12.

[0036] More specifically, the present invention encompasses engineered mammalian cells containing less than 250, less than 200, less than 150, less than 100, less than 75, or less than 50 copies of a gene encoding a variant suppressor tRNA capable of incorporating unnatural amino acids into a preselected protein expressed intracellularly (e.g., a protein expressed from a gene containing a premature stop codon). It is contemplated that the cells may contain 25 to 250, 25 to 200, 25 to 150, 25 to 100, 25 to 75, 25 to 50, 50 to 250, 50 to 200, 50 to 150, 50 to 100, 50 to 75, 75 to 250, 75 to 200, 75 to 150, 75 to 100, 100 to 250, 100 to 200, or 100 to 150 copies of the gene encoding the suppressor tRNA. Using variant tRNAs developed using the VADER approach improves the efficiency of amino acid incorporation into the target protein, so that a desired protein expression level can be obtained by introducing fewer tRNAs into the cell than wild-type tRNAs. It is expected that the fewer the number of exogenous tRNAs introduced into the cell, the less disruptive the effect on the structure, function, or viability of the host cell.

[0037] The present invention demonstrates features and advantages that will be apparent to those skilled in the art upon reading the accompanying detailed description.

[0038] The above and other features of the present invention, including various novel details of construction and combinations of parts, as well as other advantages, will now be described in more detail with reference to the accompanying drawings and pointed out in the claims. It will be understood that the specific methods and apparatuses shown for the purpose of illustration are not intended to limit the present invention. The principles and features of the present invention may be employed in a variety of numerous embodiments without departing from the scope of the present invention.

Brief Description of the Drawings

[0039] In the accompanying drawings, reference characters refer to the same parts throughout different views. The drawings are not necessarily drawn to scale, but rather emphasis is placed on explaining the principles of the present invention. The patent file or application file includes at least one drawing executed in color. A copy of this patent or patent application publication that includes a color drawing is provided by the appropriate office upon payment of the fee for the claims and the necessary fees.

[0040]

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[0041] The present invention describes a novel strategy, virus-assisted directed evolution of tRNA (VADER) in mammalian cells, for obtaining highly efficient active orthogonal tRNA variant molecules. The tRNA variants described herein, produced by the methods described herein, are characterized by an increase (enhancement) in the biological activity of nonsense codon suppression and the incorporation of unnatural amino acids in a site-specific manner in the protein of interest. The method for selecting these highly efficient tRNA variants couples the activity of a suppressor tRNA to the replication of a human virus (e.g., adeno-associated virus, or AAV). In certain embodiments, the method comprises: i) encoding a library of tRNA variants in a viral genome to enable controlled delivery into mammalian cells; ii) inserting a nonsense codon into an essential viral protein to render viral replication dependent on the activity of the suppressor tRNA, thereby facilitating the selective amplification of viral particles (virions) encoding the active tRNA variant; and iii) isolating and sequencing the genome of the newly amplified virus to readily retrieve the enriched tRNA sequences.

[0042] The method of the present invention is particularly shown to have the ability to enrich an AAV population encoding an active suppressor tRNA by a factor of about 10,000 to 50,000-fold, typically more than about 30,000-fold, compared to an AAV population encoding an inactive tRNA, and thus provides a powerful selection scheme for enriching active variants from a naive tRNA library. In particular, for variant tRNAs identified and isolated by the methods described herein, the activity is increased 2.5-fold to 80-fold.

[0043] Next-generation sequencing of the virus-encoded tRNA library (such techniques are known to those of skill in the art; see, for example, kits / reagents commercially available from Illumina) before and after the VADER selection method described herein can be performed to assess and confirm the enrichment of each possible variant / variant in the library.

[0044] The technology of the present invention can be further applied to evolve different suppressor tRNAs commonly used for Uaa incorporation in mammalian cells, including, for example, pyrrolidyl tRNA derived from archaea and leucyl tRNA derived from Escherichia coli. Subjecting the synthetic variant libraries of both tRNAs results in the identification of variants with significantly improved activity for Uaa incorporation in mammalian cells. These variants, when expressed at low levels, are particularly more efficient compared to their wild-type counterparts, and their essential efficiency improvement is further confirmed.

[0045] As a result of the present invention, a general strategy for evolving the efficiency of any engineered suppressor tRNA for Uaa incorporation in mammalian cells is now available. Encoding the tRNA library in a viral genome and subjecting the resulting library to the VADER selection scheme enables the selective enrichment of those encoding active tRNA variants.

[0046] As a result of the present invention, a method that can be used to evolve the efficiency of other biological moieties in mammalian cells is now available if the activity of these other biological moieties can be coupled to the expression of the AAV capsid protein. Such biological moieties include, but are not limited to, promoter sequences (promoter elements), internal ribosome entry sites (IRES), novel transcription factors, receptor proteins (e.g., GPCR), gene or mRNA editing proteins (e.g., Cas / CRISPR), mammalian two-hybrid systems, etc.

[0047] Variant suppressor tRNAs (e.g., pyrrolidyl and leucyl) generated through the VADER selection scheme described herein enable highly efficient Uaa incorporation in mammalian cells. These tRNA variants can be used to improve the yields of Uaa-incorporated proteins (e.g., antibodies and other therapeutically relevant proteins) in mammalian cells.

[0048] Improved suppressor tRNAs (e.g., pyrrolyl and leucyl) generated through the VADER selection scheme can be used to create improved expression vectors (e.g., viral vectors) that deliver the genetic machinery for Uaa incorporation into mammalian cells and tissues. Importantly, because these tRNAs are more efficient, fewer copies of the tRNA variant need to be encoded per genome. Currently, including multiple tRNA copies (to achieve sufficient high expression of tRNA) often leads to instability of the genome of the expression vector (e.g., viral vector).

[0049] Improved suppressor tRNAs (e.g., pyrrolyl and leucyl) generated through the VADER selection scheme can be used to create stable cell lines for protein expression incorporating Uaa. Currently, it is difficult to stably encode the Uaa incorporation mechanism in the mammalian genome because a large number of tRNA copies per genome are required. If the efficiency of the new tRNAs is increased, far fewer copies can be used.

[0050] The present invention establishes a unique virus-assisted directed evolution platform in mammalian cells that can improve the activity of tRNAs and other biological moieties for biotechnological applications. Suppressor tRNA variants that exhibit significantly improved activity in mammalian cells are also described.

[0051] Although the present invention has been particularly shown and described with reference to its preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made to the above preferred embodiments without departing from the scope of the invention as encompassed by the appended claims.

[0052] Without further elaboration, it is believed that one of ordinary skill in the art can, based on the above description, make maximum use of the present invention. Therefore, the following specific embodiments and examples are to be construed as merely illustrative and not limiting the remainder of the disclosure in any way.

Example

[0053] The following examples are provided to illustrate embodiments of the present invention and are in no way intended to limit its scope.

[0054] The examples described herein will be understood by those of ordinary skill in the art as exemplary protocols. Those of ordinary skill in the art may appropriately modify the following procedures as necessary.

[0055] Materials and Methods Cell culture. HEK293T cells (ATCC) were maintained in DMEM-high glucose (HyClone) supplemented with penicillin / streptomycin (HyClone, final concentration 100 U / mL penicillin and 100 μg / mL streptomycin) and 10% fetal bovine serum (Corning) at 37 °C and 5% CO2. All references to DMEM hereinafter refer to the complete medium described herein.

[0056] General cloning. For all cloning, Escherichia coli TOP10 strain was used for transformation and plasmid propagation, and the bacteria were grown using LB for both solid and liquid cultures. All PCR reactions were carried out using Phusion Hot Start II DNA Polymerase (Thermo Scientific) according to the manufacturer's protocol. Restriction enzymes and T4 DNA ligase were from New England Biolabs (NEB). All DNA oligos were purchased from Integrated DNA Technologies (IDT). Sanger sequencing was performed by Eton Bioscience.

[0057] The unnatural amino acid azidolysine (AzK) was purchased from Iris Biotech GMBH (Germany). Nε-acetyllysine (AcK) was purchased from Bachem.

[0058] Mock and library tRNA packaging and titration into AAV (wild-type capsid). To package various cargos into AAV-2, 8 million HEK293T cells were seeded in a 10 cm tissue culture dish. The next day, the cells were transfected with 8 μg each of the appropriate cargo plasmid (pAAV-ITR-tRNA-fluorescent protein), pHelper, and pAAV-RC2 using polyethyleneimine (PEI) (Sigma). The medium was changed to fresh DMEM 24 hours after transfection. 72 hours after transfection, the cells were resuspended, pelleted, and lysed by freeze / thaw as previously described. The virus was concentrated and semi-purified by PEG precipitation, resuspended in 1 mL DMEM containing 1% FBS, and snap-frozen.

[0059] The sequences described in the examples The wild-type and derived sequences described throughout the examples and this application are shown in a table.

Table 1(1)

Table 1(2)

Table 1(3)

[0060] Example 1: Positive Selection Eight million HEK293T cells each were seeded into three 10-cm tissue culture dishes. The next day, the cells were infected with virus containing the tRNAPyl library at an apparent MOI of 5 (the actual MOI is significantly reduced in the presence of the transfection reagent PEI). Four hours after infection, the cells were transfected with 22 μg of pHelper and 10 μg of pIDTSmart-RC2(T454TAG)-PylRS per dish using PEI. At this point, 1 mM of AzK was also added. One day after transfection, the culture medium was replaced with fresh DMEM containing 1 mM AzK. Three days after transfection, the cells were harvested and lysed as in virus isolation. The culture medium was saved, clarified lysate was recombined with it, and this mixture was treated with 500 U of universal nuclease (Thermo Scientific) for 30 minutes. The virus was recovered by PEG precipitation using 11% polyethylene glycol (Fisher) as described above and resuspended in 3 mL of PBS. Small-scale mock positive selection was performed in 12-well plates. 0.7 million cells were seeded per well and the next day, infected with AAV having the tRNAPyl-mCherry cargo. Four hours later, the cells were transfected as described for the above selection, but the transfection mix and AzK were scaled down to 1 / 15. In wells with PEI only, the cells received an equal amount of the transfection reagent but no plasmid. The next day after transfection, the medium was replaced with fresh DMEM containing 1 mM AzK. Three days after transfection, the virus was harvested and PEG precipitation was performed as in the above selection. Confluent cells in a 12-well plate were infected with the total output of one mock selection well and analyzed by flow cytometry.

[0061] Example 2: Negative Selection Virus from positive selection (3 mL) was labeled with photocleavable DBCO-sulfo-biotin (Jena Biosciences) at a concentration of 5 μM for 1 hour in the dark with mixing. Immediately after labeling, excess DBCO-biotin was quenched with AzK (final concentration 1 mM), and the reaction mixture was dialyzed overnight against 1 L of PBS at 4 °C using a Slide-A-Lyzer 100 kDa MWCO device (Thermo Scientific). The dialyzed virus mixture was divided into three 2 mL tubes and rotated overnight at 4 °C using 400 μL of streptavidin agarose resin (Thermo Scientific) in each. The next day, each tube of beads was washed 8 times with 1 mL of PBS with added NaCl (final concentration 300 mM) with mixing between washes. Finally, the washed beads were resuspended in 8 mL of PBS (300 mM NaCl), and the virus was eluted from the resin by irradiating 4 times for 30 seconds using a 365 nm UV diode array (Larson Electronics) with mixing during irradiation.

[0062] Example 3: Recovery, Amplification, and Cloning of Viral DNA The eluted virus was concentrated from 3 mL to 300 μL using an Amicon Ultra-4 100 kDa MWCO centrifugal concentrator (Millipore). This mixture was heated at 100 °C for 10 minutes to denature the viral capsid protein and expose the DNA. Next, the viral DNA was washed and concentrated by ethanol precipitation using yeast tRNA (Ambion) and resuspended in a final volume of 50 μL. 20 μL of this mixture was added to a 200 μL PCR reaction and amplified using tRNAAmp-F and R primers. The resulting DNA was digested with KpnI and NcoI and cloned into the library cloning vector using the same protocol as for original library preparation.

[0063] Example 4: Mock Selection Using Pyl-mCherry and Tyr-GFP The mock selection shown in Figure 1 was performed according to the same protocol as above, except that the starting library virus was a 1:10,000 mixture of the virus made from pAAV-ITR-PytR-mCherry and the virus made from pAAV-ITR-EcYtR-GFP. The results of the mock selection were analyzed by flow cytometry as described for virus titration, where cells in a 12-well plate were infected with 200 μL of the above virus pool after either positive or negative selection. Red and green fluorescent cells were counted to determine the virus ratio.

[0064] Example 5: Hit Sequencing and Characterization For each library, 30 - 50 colonies were selected from the transformation plates generated above and sent for Sanger sequencing (Eton Bioscience). Sequences with all randomized bases paired were all treated as hit candidates, and these tRNAs were subcloned into pAAV - ITR - PytR - mCherry for analysis. The initial hit analysis was performed by transfecting HEK293T cells in 24 - well plates with 0.5 μg each of the potential hit pAAV - ITR - PytR - mCherry plasmid, pIDTSmart - MbPylRS, and pAcBac1 - GFP(39TAG) in the presence and absence of 1 mM AzK. Two days after transfection, the cells were lysed with CelLytic M buffer (Sigma), and the fluorescence of EGFP and mCherry was measured with an aMolecular Devices SpectraMax M5 microplate reader. The values of untransfected wells were subtracted, and for each well, EGFP - fluorescence was normalized to mCherry fluorescence. The best hit, Ac2.1(GGG / CCU), was selected for further analysis using other stop codons and different synthetases, as well as Uaa, AcKRS3, and AcK. HEK293T cells in 12 - well plates were transfected with 0.375 μg of pIDTSmart - PytR containing wild - type or evolved tRNA, 0.375 μg of pIDTSmartaaRS containing the appropriate synthetase, and 0.75 μg of pAcBac1 - EGFP containing one or two of the appropriate stop codons. Wild - type EGFP control wells used pIDTSmart - PytR(TAG, wild - type), pIDTSmart - MbPylRS, and pAcBac1 - EGFP(wild - type) in the same ratio. Two days after transfection, the cells were lysed and the EGFP fluorescence was measured with a microplate reader. The values from untransfected wells were subtracted.

[0065] Example 6: Further evolution of pyrrolidyl tRNA using a custom - randomized mutant library In the first-generation VADER experiment, only short segments of tRNA (3 base pairs at a time) were randomized at a time to create a small mutant library, which was then subjected to selection. This led to the identification of improved mutants, but the inventors speculated that randomizing and selecting a broader sequence space might lead to even more efficient mutant identification. However, randomizing large segments of tRNA exponentially increases the size of the library. For example, randomizing one additional base pair in the stem region increases the number of library members by 16-fold. Due to technical constraints, currently, it is difficult for the inventors' VADER platform to process library sizes exceeding 10 5 and ensure complete coverage of all possible mutants. This size limitation restricts the complete randomization of up to 4 base pairs in the stem region of tRNA to manipulate its activity. However, when the base pairs in the tRNA-stem region are completely randomized, out of the 16 resulting mutants, only 6 can maintain the base-pairing interactions (either A:T, G:C, or G:U) essential for the stability of the stem region. Most of the mutants unable to form base pairs create "bubbles" without base pairing in the center of the tRNA stem, which generally reduces the performance of tRNA. Another method of synthesizing a tRNA library was devised where each base pair is randomized only to the desired base pair sequences. This approach utilizes recent advancements in DNA synthesis technology to enable the synthesis of large quantities of different DNA oligonucleotides of considerable length (up to 300 nucleotides). This allows for the synthesis of a DNA library encoding the entire tRNA gene, where each position of each library member can be specified, enabling the inclusion of only mutants that form base pairs and avoiding mutants that do not.

[0066] Using the DNA synthesis service provider TWIST bioscience, a pyrrolidyl-tRNA library as depicted in Figure 11 was created in which six base pairs of the acceptor stem were randomized to the desired combination of base sequences. The resulting library was packaged into AAV2 and subjected to the dual VADER selection scheme as described above. The library packaged in AAV2 was sequenced using the Illumina platform for next-generation sequencing before and after being subjected to VADER selection (Figure 12). The enrichment level of each variant was calculated using the abundance before and after the selection process, and the variants were ranked based on the degree of enrichment. The variant that showed the highest degree of enrichment during selection was resynthesized and its activity was benchmarked using the EGFP-39-TAG expression assay as described above (Figure 13). As shown in Figure 13, 26 tRNA-Pyl variants showed at least 250% higher activity than wild-type tRNA-Pyl, and the most active variant showed 540% activity relative to WT-tRNA-Pyl.

[0067] Example 7: Evolution of Escherichia coli leucyl tRNA to enhance nonsense suppression activity in mammalian cells The application of the VADER selection scheme for manipulating tRNA activity in mammalian cells is not limited to only pyrrolysyl tRNA. It can also be used to improve the activity of other tRNAs suitable for Uaa incorporation in mammalian cells. The use of the VADER method described herein was also used to improve the activity of Escherichia coli leucyl tRNA (tRNA-Leu), which was previously used for Uaa incorporation in mammalian cells (J. Am. Chem. Soc. 2004, 126, 14306; Biochemistry 2018, 57, 441), together with the cognate Escherichia coli leucyl tRNA synthetase (EcLeuRS). As shown by the work described herein regarding tRNA-Pyl, since the acceptor stem is involved with many components of the translation system, manipulating this region is often very attractive. To design a "smart" library, an alignment of the sequences of 120 known bacterial tRNA sequences was performed to create a consensus sequence of the acceptor stem (Figure 14). This consensus sequence can be used as a guide to predict which parts of the acceptor stem are important for tRNA-aaRS interaction (identity elements) and which regions have room for modification. Based on this approach, a custom randomization library of the tRNA-Leu acceptor stem (Figure 14) was designed. This library was packaged into AAV2 and subjected to the VADER selection scheme as described above. A previously developed multi-specific EcLeuRS mutant (Biochemistry 2018, 57, 441) that can introduce azide-containing (azide-modified) Uaa AzK was used in the VADER scheme for introducing tRNA mutants. Next-generation Illumina DNA sequencing was used to measure the enrichment of each library member before and after selection as described above, and those showing the greatest enrichment were resynthesized and characterized using the EGFP-39-TAG reporter expression assay described above. As shown in Figure 15, 17 tRNA-Leu mutants demonstrated at least 1,000% higher activity compared to wild-type tRNA-Leu, and the most active mutant demonstrated approximately 13,000% improved activity compared to WT-tRNA-Leu.These tRNA sequences containing WT-tRNA-Leu contain U at position 33, which is the first nucleotide of the anticodon loop. Mutation of this U to C usually improves the context of the anticodon of the nonsense suppressor, so the nonsense suppression activity may be enhanced. To examine whether this is the case, 33-U of mutant 29 (SEQ ID NO: 29) was mutated to C to create mutant 30 (SEQ ID NO: 30). Indeed, this mutant shows significantly improved suppression activity compared to 29 (Figure 15). Introduction of this mutation into other identified tRNA-Leu mutants (SEQ ID NOs: 31-45) should also result in further improvement of their activity.

[0068] Example 8: Engineered tRNA mutants show further improvement compared to their wild-type counterparts when their expression levels are restrictively controlled So far, all evaluations of all tRNA activities have been performed by transiently transfecting plasmids encoding tRNA, aaRS, and reporter into mammalian cells. In transient transfection of mammalian cell cultures, it is well established that unregulated and heterogeneous levels of DNA delivery occur, such that some cells take up and overexpress the relevant plasmid at very high levels while others do not. As a result, it has previously been shown that overexpression of encoded tRNA and aaRS can compensate for their poor intrinsic activity and inflate the estimated values of their intrinsic efficiency (ACS Synth. Biol. 2017, 6, 13). As described herein, it is further demonstrated that, as a result, when comparing two different Uaa incorporation systems by transient transfection, inaccurate estimates may be obtained where the efficiency of the weaker system is overestimated. It was speculated that the efficiency differences observed using the transient transfection assay may underestimate the actual improvement in the intrinsic efficiency of different tRNA mutants relative to their wild-type counterparts.

[0069] To overcome this challenge, we used a previously developed baculovirus vector (ACS Synth. Biol. 2017, 6, 13) that facilitates more homogeneous and controlled delivery of transgenes into mammalian cells. By systematically varying the virus-to-cell ratio, the expression level of the transgene can be easily controlled. Using this delivery system, two different gene systems for Uaa incorporation can be compared over a wide spectrum of different expression levels, thereby more accurately revealing the differences in their essential performance. Using this approach, to compare the activities of engineered tRNA-Pyl and tRNA-Leu mutants compared to wild-type, a baculovirus vector encoding a wild-type mCherry reporter and one of four tRNAs, WT tRNA-Pyl, WT-tRNA-Leu, tRNA-Pyl-2 (SEQ ID NO: 2), or tRNA-Leu-30 (SEQ ID NO: 30) was constructed. A second baculovirus was developed to deliver the EGFP-39-TAG reporter and the necessary aaRS (MbPylRS for tRNA-Pyl or EcLeuRS for tRNA-Leu). HEK293T cells were transduced with the aaRS / EGFP-39-TAG baculovirus at a constant MOI of 1 (multiplicity of infection, or the number of infectious virus particles added per cell), while increasing the MOI (0.3 - 15) of either the WT or engineered tRNA virus. The expression of mCherry (to confirm delivery of the desired level of tRNA-baculovirus) and EGFP-39-TAG (representing the Uaa incorporation efficiency in response to the TAG) was recorded 48 hours after transfection using their characteristic fluorescence in cell-free extracts. As shown in Figure 16, the engineered tRNA-Pyl promotes the expression of EGF-39-TAG at a much lower expression level (low MOI) compared to WT tRNA-Pyl.For example, when WT tRNA-Pyl virus is used at an MOI of 1, the expression of EGFP-39-TAG is only 0.5% relative to the wild-type EGFP control, while the virus encoding engineered tRNA-Pyl (SEQ ID NO: 2) results in 9.3% EGFP-39-TAG expression at the same MOI, indicating that the latter is more than 18-fold more efficient at this expression level. At a higher MOI of 3, the engineered tRNA shows approximately 14-fold higher activity compared to the wild type, revealing how higher expression can underestimate the true difference in intrinsic activity. The activity of wild-type tRNA-Leu and one of its engineered counterparts (SEQ ID NO: 30) was also compared in the same way. As shown in Figure 17, the engineered tRNA provides nearly 29-fold improved EGFP-39-TAG expression at the highest MOI tested (15). The difference was even more pronounced when the tRNA was expressed at lower levels. For example, at an MOI of 5, WT tRNA-Leu does not give any detectable EGFP-39-TAG expression, while tRNA-Leu-30 enables its expression at a level of 16% relative to the wild-type reporter. It is very important that engineered tRNAs provide significantly higher efficiency at low expression levels. This is because it greatly facilitates the generation of stable mammalian cell lines with genomically integrated aaRS / tRNAs that provide high Uaa incorporation efficiency.

[0070] Example 9: Engineered tRNA Variants for More Efficient Incorporation of Multiple Uaas Without being bound by theory, the improved activities of engineered tRNAs are not fully understood, but they are likely to interact much better with the mammalian translation system than their wild-type counterparts borrowed from different domains of life. As a result, if the activities of these tRNAs are improved, they should be able to incorporate more efficiently all Uaas that can be incorporated by engineered variants of cognate aaRSs. To test this hypothesis, several Uaas that can be incorporated using engineered MbPylRS (structures 1 - 6; Figure 18) or EcLeuRS (structures 7 - 12; Figure 18) were evaluated for their incorporation efficiency using the EGFP-39-TAG expression assay described above. Indeed, each of these Uaas was incorporated into the reporter with significantly higher efficiency by the engineered tRNAs, by the two engineered tRNAs, compared to their wild-type counterparts (Figure 19).

[0071] While the invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A composition comprising a variant archaeal suppressor tRNA or a variant bacterial suppressor tRNA, wherein the variant tRNA has increased activity for incorporating an unnatural amino acid into a mammalian protein compared to its wild-type counterpart tRNA.

2. 2. The composition of claim 1, wherein the activity of the variant tRNA is increased by about 2.5 to 80 fold relative to the wild-type tRNA.

3. The composition of claim 1 or claim 2, wherein the variant archaeal tRNA is from the family Methanosarcina or Desulfitobacterium.

4. 4. The composition of claim 3, wherein the variant archaeal tRNA is selected from the group consisting of Methanosarcina barkeri (Mb), Methanosarcina albus (Ma), Methanosarcina mazei (Mm) or Desulfitobacterium hafniens (Dh).

5. The variant tRNA is a pyrrolysyl-tRNA (tRNA Ｐｙｌ 5. The composition according to claim 4, wherein

6. The variant tRNA Ｐｙｌ The composition of claim 5, wherein said nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 2-27, or a nucleic acid sequence having at least 90% sequence identity to the full length sequence of any of SEQ ID NOs: 2-27.

7. 3. The composition of claim 1 or claim 2, wherein the variant bacterial tRNA is derived from an E. coli tRNA.

8. The variant tRNA is leucyl-tRNA (tRNA Ｌｅｕ 8. The composition according to claim 7, wherein

9. The variant tRNA Ｌｅｕ The composition of claim 8, wherein said nucleic acid sequence has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 29-45, or a nucleic acid sequence having at least 90% sequence identity to the full length sequence of any of SEQ ID NOs: 29-45.

10. The composition of claim 5 or claim 6, wherein the unnatural amino acid conforms to any of structures 1-6.

11. 10. The composition of claim 8 or claim 9, wherein the unnatural amino acid conforms to any of structures 7-12.

12. A viral vector comprising a variant archaeal suppressor tRNA or a variant bacterial suppressor tRNA, wherein the variant tRNA has increased activity for incorporating an unnatural amino acid into a mammalian protein compared to its wild-type counterpart tRNA.

13. The viral vector of claim 12, wherein the activity of the variant tRNA is increased by about 2.5 to 80 times compared to the wild-type tRNA.

14. The viral vector of claim 13 , wherein the variant archaeal tRNA is from the family Methanosarcina or Desulfitobacterium.

15. The viral vector of claim 14, wherein the variant archaeal tRNA is selected from the group consisting of Methanosarcina barkeri (Mb), Methanosarcina albus (Ma), Methanosarcina mazei (Mm) or Desulfitobacterium hafniens (Dh).

16. The variant tRNA is a pyrrolysyl-tRNA (tRNA Ｐｙｌ 16. The viral vector according to claim 15 .

17. The variant tRNA Ｐｙｌ The viral vector of claim 16, comprising a nucleic acid sequence having at least 90% sequence identity to any of the sequences selected from the group consisting of SEQ ID NOs: 2 to 27, or the full-length sequences of SEQ ID NOs: 2 to 27.

18. The viral vector of claim 12 , wherein the variant bacterial tRNA is derived from an E. coli tRNA.

19. The variant tRNA is leucyl-tRNA (tRNA Ｌｅｕ 19. The viral vector of claim 18 .

20. The variant tRNA Ｌｅｕ The viral vector of claim 19, comprising a nucleic acid sequence having at least 90% sequence identity to any of the sequences selected from the group consisting of SEQ ID NOs: 29-45, or the full-length sequences of SEQ ID NOs: 29-45.

21. The viral vector of claim 16 or claim 17, wherein the unnatural amino acid conforms to any of structures 1-6.

22. The viral vector of claim 19 or claim 20, wherein the unnatural amino acid conforms to any of structures 7-12.

23. 23. The viral vector of any one of claims 12 to 22, wherein the virus is an adeno-associated virus (AAV).

24. A cell comprising a viral vector according to any one of claims 12 to 23.

25. The cell of claim 24, wherein the cell is a mammalian cell.

26. The cells, a) a protein essential for viral replication, wherein a nonsense codon has been inserted into the sequence of said protein, making viral replication dependent on the activity of said variant suppressor tRNA; b) the cognate Uaa RNA synthetase (UaaRS), and c) Genetic components necessary for viral replication 26. The mammalian cell of claim 24 or claim 25, further comprising a plasmid encoding

27. 27. The mammalian cell of claim 26, wherein the viral vector is an adeno-associated virus and the essential viral protein is a TAG mutant of Cap (SEQ ID NO: 46).

28. The cognate aaRS is Mb Ｐｙｌ 28. The mammalian cell of claim 27, wherein the Uaa is any one of structures 1 to 6.

29. The homologous aaRS is Ｌｅｕ The mammalian cell of claim 26, wherein the Uaa is any one of structures 7 to 12.

30. 1. A method for virus-assisted directed evolution of an orthogonal suppressor tRNA variant of interest having increased biological activity compared to a wild-type suppressor tRNA, comprising: a) encoding a library of suppressor tRNA variants of interest into the viral genome; b) infecting a population of mammalian host cells with a viral vector at a low multiplicity of infection (MOI) and maintaining said cell population under conditions suitable for viral replication in said cells, where viral replication in mammalian cells requires expression of essential proteins that are dependent on the activity of the tRNA variant of interest; c) harvesting and selectively amplifying viral progeny encoding active tRNA variants to remove cross-reactive tRNA molecules, thereby recovering orthogonal suppressor tRNA variants with increased biological activity; The method includes:

31. 1. A method for virus-assisted directed evolution of an orthogonal suppressor tRNA variant of interest having increased biological activity compared to a wild-type suppressor tRNA, wherein replication of the virus in a mammalian cell requires expression of an essential protein that is dependent on the activity of the tRNA variant of interest, the method comprising: a) encoding a library of suppressor tRNA variants into the viral genome; b) infecting a population of mammalian host cells with a viral vector at a low multiplicity of infection (MOI); c) thereafter transfecting the population of mammalian host cells with the plasmid, said plasmid comprising: i) a protein essential for viral replication, in which a nonsense codon has been inserted into the sequence of said protein, making viral replication dependent on the activity of said variant suppressor tRNA; ii) the cognate Uaa RNA synthetase (UaaRS), and iii) Genetic components necessary for viral replication and d) substantially simultaneously adding an appropriate unnatural amino acid to the culture medium; e) maintaining the infected / transfected cells in a medium under conditions suitable for replication of the virus; f) harvesting the cells and isolating viral progeny; g) labeling the virus isolated in step f) with a purification handle attached via a photocleavable linker; h) recovering the labeled viruses by concentration and then releasing them using irradiation at an appropriate wavelength; i) lysing the recovered viruses and amplifying the tRNA variants contained in the lysate, thereby recovering orthogonal suppressor tRNA variants with increased biological activity; The method includes:

32. 32. The method of claim 30 or claim 31, wherein the suppressor tRNA variants of the library are sequenced prior to encoding into a virus.

33. 32. The method of claim 30 or claim 31, further comprising the step of sequencing the amplified tRNA to obtain the nucleic acid sequence of the suppressor tRNA of interest.

34. 32. The method of claim 30 or claim 31, wherein the virus is an adeno-associated virus.

35. The method of claim 30 or claim 31, wherein the essential viral protein is the adeno-associated virus capsid protein (CAP) (SEQ ID NO: 46) and is mutated to contain a stop codon at position 454 of the protein.

36. 32. The method of claim 30 or claim 31, wherein only a single virion infects the host mammalian cell.

37. The tRNA library is pyrrolysyl-tRNA (tRNA Ｐｙｌ ) library, wherein the UaaRS is Mb Ｐｙｌ The method of claim 30 or claim 31, wherein the Uaa is any one of structures 1 to 6.

38. The variant tRNA is leucyl-tRNA (tRNA Ｌｅｕ ) wherein the aaRS is Escherichia coli Ｌｅｕ The method of claim 30 or claim 31, wherein the Uaa is any one of structures 7 to 12.

39. 1. A method for producing a protein in a mammalian cell, said protein having one or more amino acid analogs at specific positions in said protein, said method comprising: a. culturing the mammalian cells in a culture medium under conditions suitable for growth, the cells comprising a nucleic acid encoding a protein having one or more selector codons, the cells further comprising a variant archaeal pyrrolysyl-tRNA with enhanced biological activity that recognizes the selector codon, and its cognate aminoacyl-RNA synthetase; b. contacting the cell culture medium with one or more lysine analogs under conditions suitable for incorporation of the one or more lysine analogs into the protein in response to the selector codon, thereby producing a protein having one or more lysine analogs; The method includes:

40. The variant tRNA is a pyrrolysyl-tRNA (tRNA Ｐｙｌ 40. The method of claim 39, wherein

41. The variant tRNA Ｐｙｌ The method of claim 39 or claim 40, wherein said nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 2-27, or a nucleic acid sequence having at least 90% sequence identity to any of SEQ ID NOs: 2-27.

42. 42. The method of claim 41, wherein the lysine analog is any one of structures 1 to 6.

43. 1. A method for producing a protein in a mammalian cell, said protein having one or more amino acid analogs at specific positions in said protein, said method comprising: a. culturing the mammalian cells in a culture medium under conditions suitable for growth, the cells comprising a nucleic acid encoding a protein having one or more selector codons, the cells further comprising a variant E. coli-derived leucyl-tRNA and its cognate aminoacyl-RNA synthetase with enhanced biological activity that recognizes the selector codon; b. contacting the cell culture medium with one or more leucine analogs under conditions suitable for incorporation of the one or more leucine analogs into the protein in response to the selector codon, thereby producing a protein having one or more leucine analogs; The method includes:

44. The variant tRNA is a leucyl-tRNA (tRNA Ｌｅｕ 44. The method of claim 43, wherein

45. The variant tRNA Ｌｅｕ The method of claim 43 or claim 44, wherein said nucleic acid sequence has at least 90% sequence identity to any one of SEQ ID NOs: 29-45, or the full length sequence of any of SEQ ID NOs: 29-45.

46. 46. ​​The method of claim 45, wherein the leucine analog is any of structures 7-12.

47. 1. A method for site-specifically incorporating one or more pyrrolidyl residues into a protein or peptide in a cell, comprising: a. culturing the cells in a culture medium under conditions suitable for growth, the cells comprising a nucleic acid encoding a protein or peptide of interest having one or more amber, ochre or opal selector codons at a specific site in the protein or peptide of interest, the cells further comprising a variant archaeal-derived pyrrolysyl-tRNAPyl with increased biological activity that recognizes the selector codon, and further comprising an archaeal PylRNA synthetase; b. contacting the cell culture medium with one or more pyrrolidyl residues under conditions suitable for incorporating one or more pyrrolidyl residues into the protein or peptide at the site of the selector codon, thereby producing a protein or peptide of interest having one or more site-specifically incorporated pyrrolidyl residues. The method includes:

48. The variant pyrrolysyl-tRNA (tRNA Ｐｙｌ 48. The method of claim 47, wherein the sequence is derived from SEQ ID NO:

1.

49. The variant tRNA Ｐｙｌ The method of claim 47 or claim 48, wherein said nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 2-27, or a nucleic acid sequence having at least 90% sequence identity to the full length sequence of any of SEQ ID NOs: 2-27.

50. 50. The method of claim 49, wherein the pyrrolidyl residue is any of structures 1 to 6.

51. 1. A method for site-specifically incorporating one or more leucine analog residues into a protein or peptide in a cell, comprising: a. culturing the cells in a culture medium under conditions suitable for growth, the cells comprising a nucleic acid encoding a protein or peptide of interest having one or more amber, ochre or opal selector codons at specific sites in the protein or peptide of interest, the cells expressing a variant E. coli-derived tRNA with enhanced biological activity that recognizes the selector codon; Ｌｅｕ and further comprising Escherichia coli Leu RNA synthetase; b. contacting the cell culture medium with one or more leucine analog residues under conditions suitable for incorporating one or more leucine analog residues into the protein or peptide at the site of the selector codon, thereby producing a protein or peptide of interest having one or more site-specifically incorporated leucine residues. The method includes:

52. The variant tRNA is a leucyl-tRNA (tRNA Ｌｅｕ 52. The method of claim 51 .

53. The variant tRNA Ｌｅｕ The method of claim 51 or claim 52, wherein said nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 29-45, or a nucleic acid sequence having at least 90% sequence identity to the full length sequence of any of SEQ ID NOs: 29-45.

54. 54. The method of claim 53, wherein the leucine analog is any of structures 7-12.

55. 1. A kit for producing a protein or peptide of interest in a cell, the protein or peptide comprising one or more lysine analogues, the kit comprising: a. A variant archaeal-derived tRNA with increased biological activity that recognizes a selector codon in a nucleic acid of interest in a cell. Ｐｙｌ A container containing a polynucleotide sequence encoding the variant tRNA Ｐｙｌ a container containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-27, or a nucleic acid sequence having at least 90% sequence identity to the full length sequence of any of SEQ ID NOs: 2-27; b. a container containing a polynucleotide sequence encoding an archaeal Pyl-tRNA synthetase; Kit including:

56. 56. The kit of claim 55, further comprising one or more lysine analogues.

57. The kit of claim 56, wherein the lysine analog is an analog according to any of structures 1-7.

58. 58. The kit of claim 57, further comprising instructions for producing the protein or peptide of interest.

59. 1. A kit for producing a protein or peptide of interest in a cell, the protein or peptide comprising one or more leucine analogues, the kit comprising: a. A variant E. coli-derived tRNA with increased biological activity that recognizes a selector codon in a nucleic acid of interest in a cell. Ｌｅｕ A container containing a polynucleotide sequence encoding the variant tRNA Ｌｅｕ a container containing any one of SEQ ID NOs:29-45, or a nucleic acid sequence having at least 90% sequence identity to the full length sequence of any one of SEQ ID NOs:29-45; b. a container containing a polynucleotide sequence encoding Escherichia coli Leu-tRNA synthetase; Kit including:

60. 60. The kit of claim 59, further comprising one or more leucine analogs according to any of structures 7-12.

61. 61. The kit of claim 60, further comprising instructions for producing the protein or peptide of interest.

62. Mammalian cells stably incorporating variant tRNA-Pyl or variant tRNA-Leu for Uaa incorporation.

63. 63. The cell of claim 62, wherein the variant tRNA-Pyl-Leu is selected from the group consisting of SEQ ID NOs: 2-27, and the Uaa is a pyrrolidyl residue.

64. The cell of claim 63, wherein the pyrrolidyl residue is any one of structures 1 to 7.

65. 63. The cell of claim 62, wherein the variant tRNA-Leu is selected from the group consisting of SEQ ID NOs: 29-45, and the Uaa is a leucine analog.

66. The cell of claim 65, wherein the leucine analog is any of structures 7-12.

67. An engineered mammalian cell comprising fewer than 250, fewer than 200, fewer than 150, fewer than 100, fewer than 75, fewer than 50 copies of a gene encoding a variant suppressor tRNA capable of incorporating an unnatural amino acid into a protein of interest.

68. 68. The cell of claim 67, wherein the cell comprises 25-250 copies, 25-200 copies, 25-150 copies, 25-100 copies, 25-75 copies, 25-50 copies, 50-250 copies, 50-200 copies, 50-150 copies, 50-100 copies, 50-75 copies, 75-250 copies, 75-200 copies, 75-150 copies, 75-100 copies, 100-250 copies, 100-200 copies, 100-150 copies of the gene encoding the suppressor tRNA.

69. The cell of claim 67 or claim 68, wherein the variant tRNA-Pyl-Leu is selected from the group consisting of SEQ ID NOs: 2-27, and the Uaa is a pyrrolidyl residue.

70. The cell of claim 69, wherein the pyrrolidyl residue is any one of structures 1 to 7.

71. The cell of claim 67 or claim 68, wherein the variant tRNA-Leu is selected from the group consisting of SEQ ID NOs: 29-45, and the Uaa is a leucine analog.

72. The cell of claim 71, wherein the leucine analog is any one of structures 7 to 12.

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