The ASCII text file named “047162-7374WO1(01839)_Seq Listing.xml” created on Mar. 1, 2023, comprising 1,207,787 bytes, is hereby incorporated by reference in its entirety.
The sequences and structures of 5′ untranslated regions (5′ UTRs) of mRNA molecules are known to affect translational efficiencies to a large extent. Specific sequence motifs in the 5′ UTR that increases and decreases translational efficiencies, called translational enhancers and translational silencers, however; remain largely unknown, and high-throughput methods that are able to identify translational enhancers and translational silencers are lacking.
Therefore, there is a need to develop high-throughput screening methods to identify translational enhancers and translational silencers, as well as a need to modulate mRNA translation with the identified enhancers/silencers and to construct mRNA molecules having desirable translational efficiencies. The present invention addresses these needs.
In some aspects, the present invention is directed to the following non-limiting embodiments: Method of modulating a translational efficiency of mRNA
In some aspects, the present invention is directed to a method of modulating a translational efficiency of a messenger RNA (mRNA) molecule.
In some embodiments the method includes: modifying a 5′-UTR of the mRNA molecule to include a translational enhancer or a translational silencer.
In some embodiments, modifying the 5′-UTR of the mRNA molecule includes modifying a sequence of a DNA molecule encoding the mRNA molecule.
In some embodiments, the translational enhancer or the translational silence is a translational enhancer or a translational silencer for the translational machinery of a yeast species, optionally a yeast species selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, and Schizosaccharomyces pombe.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational enhancer, and the translational enhancer includes at least one sequence selected from the group consisting of SEQ ID NOs: 1-3.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational silencer, and the translational silencer includes at least one sequence selected from the group consisting of SEQ ID NOs: 4-6.
In some embodiments, the translational enhancer or the translational silence is a translational enhancer or a translational silencer for the translational machinery of a mammal, optionally a human.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational enhancer, and the translational enhancer includes at least one sequence selected from the group consisting of SEQ ID NOs: 7-18, 29-51 and 56-1342.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational silencer, and wherein the translational silencer includes at least one sequence selected from the group consisting of SEQ ID NOs: 19-28.
In some embodiments, the 5′-UTR of the mRNA molecule is further modified to include a modified nucleobase.
In some embodiments, the modified nucleobase includes N6-methyladenosine, inosine, N1-propylpseudouridine, N1-methoxymethylpseudouridine, N1-ethylpseudouridine, 5-methoxycytidine, 5-hydroxyuridine, 5-carboxyuridine, 5-formyluridine, 5-hydroxycytidine, 5-hyrdoxymethylcytidine, 5-hydroxymethyluridine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, pseudoisocytidine, 2-thiocytidine, 4-thiocytidine, N1-methylpseudouridine, pseudouridine, 5-methyluridine, 5-methoxyuridine, dihydrouridine, 5-methylcytidine, 4-thiouridine, 2-thiouridine, uridine-5′-O-(1-thiophosphate), 5-aminoallyluridine, or 4-acetylcytidine.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational enhancer, and a ribosome retention score (RRS) of the modified mRNA molecule is 3× or higher than an RRS of the unmodified mRNA molecule.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational silencer, and wherein a ribosome retention score (RRS) of the modified mRNA molecule is 0.3× or lower than an RRS of the unmodified mRNA molecule.
mRNA Molecule
In some aspects, the present invention is directed to an mRNA molecule.
In some embodiments, the mRNA molecule includes a 5′-UTR, and the 5′-UTR includes: a translational enhancer including at least one sequence selected from the group consisting of SEQ ID NOs: 1-3, 7-18, 29-51 and 56-1342, or a translational silencer including at least one selected from the group consisting of SEQ ID NOs: 4-6 and 19-28; and a modified nucleobase.
In some embodiments, the modified nucleobase includes N6-methyladenosine, inosine, N1-propylpseudouridine, N1-methoxymethylpseudouridine, N1-ethylpseudouridine, 5-methoxycytidine, 5-hydroxyuridine, 5-carboxyuridine, 5-formyluridine, 5-hydroxycytidine, 5-hyrdoxymethylcytidine, 5-hydroxymethyluridine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, pseudoisocytidine, 2-thiocytidine, 4-thiocytidine, N1-methylpseudouridine, pseudouridine, 5-methyluridine, 5-methoxyuridine, dihydrouridine, 5-methylcytidine, 4-thiouridine, 2-thiouridine, uridine-5′-O-(1-thiophosphate), 5-aminoallyluridine, or 4-acetylcytidine.
In some embodiments, the 5′-UTR includes the translational enhancer, and wherein a ribosome retention score (RRS) of the mRNA molecule is 3× or higher than an RRS of an mRNA molecule that does not include the translational enhancer or the modified nucleobase but otherwise has the same sequence.
In some embodiments, the 5′-UTR includes at least one sequences selected from the group consisting of SEQ ID NOs: 29-50.
In some embodiments, the 5′-UTR includes the translational silencer, and wherein an RRS of the mRNA molecule is 0.3× or lower than an RRS of an mRNA molecule that does not include the translational silencer or the modified nucleobase but otherwise has the same sequence.
In some embodiments, the mRNA molecule encodes a therapeutic peptide or a therapeutic protein.
In some embodiments, the therapeutic peptide or the therapeutic protein includes a vaccine.
Method of Constructing an mRNA Molecule
In some aspects, the present invention is directed to a method of constructing an mRNA molecule for producing a therapeutic peptide or a therapeutic protein.
In some embodiments, the method includes a translational enhancer at a 5′ UTR to the 5′ side of a coding region encoding the therapeutic peptide or a therapeutic protein.
In some embodiments the therapeutic peptide or a therapeutic protein includes a vaccine.
In some aspects, the present invention is directed to a yeast cell for expressing a protein.
In some embodiments, the yeast cell includes an mRNA molecule or a nucleotide encoding the mRNA molecule.
In some embodiments, the mRNA molecule includes: a 5′-UTR including at least one yeast translational enhancer; and a coding region encoding the protein, or a coding region for introducing a nucleotide sequence for encoding the protein.
In some embodiments, the 5′-UTR includes at least one sequence selected from the group consisting of SEQ ID NOs: 1-3.
In some embodiments, the yeast cell is a Saccharomyces cerevisiae cell, a Pichia pastoris cell, a Hansenula polymorpha cell, a Yarrowia lipolytica cell, an Arxula adeninivorans cell, a Kluyveromyces lactis cell, or a Schizosaccharomyces pombe cell.
The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the instant specification is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed.
In the first study described herein (“the first study”), a method capable of identifying translational enhancers and translational silencers in the 5′ untranslated regions (5′ UTRs) of mRNA molecules was developed. Specifically, in the first study, a library of mRNA molecules having similar coding regions but different 5′ UTR sequences were mixed with 80s ribosomes to form complexes. The complexes in the mixture were stabilized with cycloheximide and the mixture were then subjected to density gradient fractionation to isolate the ribosome-mRNA complexes and the mRNA. The ribosomal recruitment efficiencies of the 5′ untranslated regions were determined according to the relative abundances of the mRNA molecules in the complex.
In the second study described herein (“the second study”), using the method developed in the first study, translational enhancers and translational silencers in the 5′ UTR were determined in the budding yeast.
In the third study described herein (“the third study”), using the method developed in the first study, translational enhancers and translational silencers in the 5′ UTR were determined in humans.
Accordingly, in some aspects, the present invention is directed to a method of modulating a translational efficiency of a messenger RNA (mRNA) molecule.
In some aspects, the present invention is directed to a non-naturally occurring mRNA molecule. In some embodiments, the non-naturally occurring mRNA molecule has increased or decreased translational efficiencies in comparison to similar naturally occurring mRNA molecules.
In some embodiments, the present invention is directed to a method of constructing mRNA molecules for producing therapeutic proteins/peptide.
As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.
In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.
In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.”
“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of 20% or +10%, in certain embodiments ±5%, in certain embodiments 10%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
Method of Identifying Translational Enhancers and/or Translational Silencers in 5′ UTRs of mRNA Molecules
In some embodiments, the instant specification is directed to a method of identifying translational enhancers and/or translational silencers in 5′ untranslated regions (5′ UTRs) of mRNA molecules.
In some embodiments, the method includes: preparing a plurality of mRNA molecules, wherein each mRNA molecules including a 5′ UTR and a coding region to the 3′ end of the 5′ UTR; contacting the plurality of mRNA molecules with a ribosome; isolating the ribosome; and
In some embodiments, the 5′ UTRs of an mRNA molecule includes a translational enhancer if the mRNA molecule is more likely to be found in complex with the isolated ribosomes than average.
In some embodiments, the 5′ UTRs of an mRNA molecule includes a translational silencer if the mRNA molecule is less likely to be found in complex with the isolated ribosomes than average.
In some embodiments, the method further includes identifying one or more translational enhancers by determining one or more sequence motifs commonly found in 5′ UTRs of mRNA molecules that are more likely to be found in complex with the isolated ribosomes than average; or identifying one or more translational silencers by determining one or more sequence motifs commonly found in 5′ UTRs of mRNA molecules that are less likely to be found in complex with the isolated ribosomes than average.
In some embodiments, the plurality of mRNA molecules has the same sequence in the coding region. In some embodiments, the coding regions of the plurality of mRNA molecules have similar sequences. In some embodiments, the coding regions of the plurality of mRNA molecules affect translational efficiency at similar levels.
In some embodiments, isolating the ribosomes includes isolation according to the sedimentation coefficient of the ribosome.
In some embodiments, the ribosomes are isolated by density gradient fractionation.
In some embodiments, contacting the plurality of mRNA molecules with the ribosomes includes stabilizing complexes formed by mRNA molecules and ribosomes.
In some embodiments, stabilizing the complexes includes contacting the complexes with cycloheximide.
In some embodiments, the method includes calculating a ribosome retention score based on the relative abundance of the mRNA ribosome complex. As used herein, the term ribosome retention score or RRS refers to a numeric descriptor of the ability of a particular mRNA 5′ UTR to promote ribosome binding and thus translation of the 3′ coding region of the mRNA. The attached appendices describe non-limiting examples of methods for determining an RRS.
In some embodiments, the ribosome retention score mRNA molecules in complex with the isolated ribosomes is calculated based on the relative abundance of the mRNA molecule isolated in complex with ribosomes in comparison to an abundance of the mRNA molecule in the input pool.
In some embodiments, the ribosome retention score of an mRNA molecule in complex with the isolated ribosomes is calculated according to a relative abundance the mRNA molecule isolated in complex with ribosome in comparison to a sum of an abundance of the mRNA molecule isolated in complex with ribosome and an abundance of the mRNA molecule isolated without forming complex with ribosome.
In some embodiments, determining the sequences of 5′ UTRs of mRNA molecules includes sequencing the 5′ UTRs.
In some embodiments, the plurality of mRNA molecules further includes barcoding sequences.
In some embodiments, the barcoding sequences correspond to the sequences of the 5′ UTRs.
In some embodiments, determining the sequences of 5′ UTRs of mRNA molecules includes identifying the barcoding sequences of the mRNA molecules.
In some embodiments, the method further includes verifying the identified 5′ UTR translational enhancers and/or translational silencers.
In some embodiments, verifying the identified 5′ UTR translational enhancers and/or translational silencers includes constructing a first mRNA molecule including a first 5′ UTR and a coding region encoding a reporter protein.
In some embodiments, the first 5′ UTR includes the identified translational enhancers and/or translational silencers; constructing a second mRNA molecule including a second 5′ UTR and the coding region encoding the reporter protein.
In some embodiments, the second 5′ UTR does not include the identified translational enhancers and/or translational silencers, but is otherwise the same as the first 5′ UTR.
In some embodiments, verifying the identified 5′ UTR translational enhancers and/or translational silencers further includes contacting the first mRNA molecule and the second mRNA molecule with ribosome.
In some embodiments, verifying the identified 5′ UTR translational enhancers and/or translational silencers further includes measuring a first level of reporter signal from the translation of the first mRNA molecule.
In some embodiments, verifying the identified 5′ UTR translational enhancers and/or translational silencers further includes measuring a second level of reporter signal from the translation of the second mRNA molecule.
In some embodiments, verifying the identified 5′ UTR translational enhancers and/or translational silencers further includes comparing the first level and the second level.
Method of Modulating Translational Efficiency of mRNA Molecules
In some aspects, the present invention is directed to a method of modulating a translational efficiency of an mRNA molecule.
In some embodiments, the method includes modifying a 5′-UTR of the mRNA molecule to include a translational enhancer or a translational silencer.
In some embodiments, modifying the 5′-UTR of the mRNA molecule includes modifying a sequence of a DNA molecule encoding the mRNA molecule.
In some embodiments, the translational enhancer or the translational silence is a translational enhancer or a translational silencer for the translational machinery of yeasts. It is worth noting that, although the working examples described herein were tested in Saccharomyces cerevisiae, since the translation machinery is highly conserved among yeast species, the translational enhancers or translational silencers identified in Saccharomyces cerevisiae are expected to work in yeast in general. In some embodiments, the translational enhancer or the translational silence is a translational enhancer or a translational silencer for the translational machinery of Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, or Schizosaccharomyces pombe.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational enhancer, and wherein the translational enhancer includes at least one sequence selected from the group consisting of SEQ ID NOs: 1-3.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational silencer, and wherein the translational silencer includes at least one sequence selected from the group consisting of SEQ ID NOs: 4-6.
In some embodiments, the translational enhancer or the translational silence is a translational enhancer or a translational silencer for the translational machinery of a mammal, such as a human. It is worth noting that, although the working examples described herein were tested in human cells, the translational machinery among mammals are conserved. As such, translational enhancer and translational silencers identified for humans are expected to work in other mammals.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational enhancer, and wherein the translational enhancer includes at least one sequence selected from the group consisting of SEQ ID NOs: 7-18, 29-51, and 56-1342.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational silencer, and wherein the translational silencer includes at least one sequence selected from the group consisting of SEQ ID NOs: 19-28.
In some embodiments, the 5′-UTR of the mRNA molecule is further modified to include a modified nucleobase. In some embodiments, the modified nucleobase includes N6-methyladenosine, inosine, N1-propylpseudouridine, N1-methoxymethylpseudouridine, N1-ethylpseudouridine, 5-methoxycytidine, 5-hydroxyuridine, 5-carboxyuridine, 5-formyluridine, 5-hydroxycytidine, 5-hyrdoxymethylcytidine, 5-hydroxymethyluridine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, pseudoisocytidine, 2-thiocytidine, 4-thiocytidine, N1-methylpseudouridine, pseudouridine, 5-methyluridine, 5-methoxyuridine, dihydrouridine, 5-methylcytidine, 4-thiouridine, 2-thiouridine, uridine-5′-O-(1-thiophosphate), 5-aminoallyluridine, or 4-acetylcytidine.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational enhancer, and wherein a ribosome retention score (RRS) of the modified mRNA molecule is 2× or higher, such as 3× or higher, 10× or higher, 100× or higher or 1000× or higher, than an RRS of the unmodified mRNA molecule.
In some embodiments, the 5′-UTR of the mRNA molecule is modified to include the translational silencer, and wherein a ribosome retention score (RRS) of the modified mRNA molecule is 0.5× or lower, such 0.3× or lower, 0.1× or lower, 0.0 × or lower or 0.00 × or lower, than an RRS of the unmodified mRNA molecule.
In some embodiments, the 5′-UTR is modified to include the translational enhancer and a modified nucleobase that is able to enhance translation. In some embodiments, the 5′-UTR is modified to include the translational silencer and a modified nucleobase that is able to suppress translation. Whether a modified nucleobase in the 5′-UTR enhances or suppresses translation of an mRNA molecule is context dependent (i.e., a modified nucleobase that enhances translation in one position of one 5′UTR sequence might suppress translation if placed in another position, or in another 5′UTR sequence).
mRNA Molecule
In some embodiments, the present invention is directed to an mRNA molecule.
In some embodiments, the mRNA molecule does not exist in the nature. In some embodiments, the mRNA molecule does not exist in the nature because the mRNA molecule includes a modified nucleobase. As used herein, the term “modified nucleobase” refers to nucleobases other than the four common nucleobases adenine (A), cytosine (C), uracil (U), and guanine (G). The term “modified nucleobase” does not exclude naturally occurring nucleobases that are found in non-mRNA molecules. For example, nucleobases like pseudouridine, 5-methylcytosine, N1-methylpseudouridine and 2′-O-methylated exist in nature. These nucleobases are considered to be examples of modified nucleobases herein.
In some embodiments, the mRNA molecule includes a 5′-UTR.
In some embodiments, the 5′-UTR includes:
In some embodiments, the modified nucleobase is the nucleobases of N6-methyladenosine, inosine, N1-propylpseudouridine, N1-methoxymethylpseudouridine, N1-ethylpseudouridine, 5-methoxycytidine, 5-hydroxyuridine, 5-carboxyuridine, 5-formyluridine, 5-hydroxycytidine, 5-hyrdoxymethylcytidine, 5-hydroxymethyluridine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, pseudoisocytidine, 2-thiocytidine, 4-thiocytidine, N1-methylpseudouridine, pseudouridine, 5-methyluridine, 5-methoxyuridine, dihydrouridine, 5-methylcytidine, 4-thiouridine, 2-thiouridine, uridine-5′-O-(1-thiophosphate), 5-aminoallyluridine, or 4-acetylcytidine.
In some embodiments, the 5′-UTR includes the translational enhancer, and the modified nucleobase is able to enhance translation. In some embodiments, the 5′-UTR includes the translational silencer, and the modified nucleobase is able to suppress translation.
In some embodiments, the 5′-UTR includes the translational enhancer. In some embodiments, a ribosome retention score (RRS) of the mRNA molecule is 2× or higher, such as 3× or higher, 10× or higher, 100× or higher or 1000× or higher, than an RRS of an mRNA molecule that does not include the translational enhancer or the modified nucleobase but otherwise has the same sequence.
In some embodiments, the 5′-UTR includes the translational silencer. In some embodiments, an RRS of the mRNA molecule is 0.5× or lower, such as 0.3× or lower, 0.1× or lower, 0.01× or lower or 0.001× or lower, than an RRS of an mRNA molecule that does not include the translational silencer or the modified nucleobase but otherwise has the same sequence.
In some embodiments, the mRNA molecule encodes a therapeutic peptide or a therapeutic protein.
In some embodiments, the therapeutic peptide or the therapeutic protein includes a vaccine.
Method of Constructing mRNA Molecules for Producing Therapeutic Protein/Peptide
In some embodiments, the present invention is directed to a method of constructing an mRNA molecule for producing a therapeutic peptide or a therapeutic protein.
In some embodiments, the method includes including a translational enhancer at a 5′ UTR to the 5′ side of a coding region encoding the therapeutic peptide or a therapeutic protein.
In some embodiments, the therapeutic peptide or a therapeutic protein includes a vaccine.
In some embodiments, the present invention is directed to a yeast cell, such as a yeast cell for expressing a protein. In some embodiments, the yeast cell is a bioengineered yeast cell. In some embodiments, the protein to be expressed by the yeast cell is a protein exogenous or endogenous to the yeast cell. In some embodiments, the protein is a secreted protein.
In some embodiments, the yeast cell is a recombinant yeast cell having a sequence encoding a 5′-UTR translational enhancer engineered into either the genomic DNA or a plasmid/expression vector.
In some embodiments, the nucleic acid encoding the protein to be expressed is already in the yeast cell and is located downstream of the 5′-UTR translational enhancer such that the expression of the protein is enhanced by the 5′-UTR translational enhancer. In some embodiments, the nucleic acid encoding the protein to be expressed has not been engineered into the yeast cell, such that any desired nucleic acid sequences for encoding a protein can be placed downstream of the 5′-UTR translational enhancer.
In some embodiments, the 5′-UTR translational enhancers for yeasts are the same as or similar to those as detailed elsewhere herein.
In some embodiments, the yeast cell is Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, or Schizosaccharomyces pombe.
The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
In the study described in Example 1 (“the first study”), a method to quantify translation initiation on thousands of 5′ UTRs in parallel was developed. The first study uncovered sequence-specific motifs that control translation by varied mechanisms and establish a platform for systematic interrogation of 5′ UTR variants that can be used to engineer mRNAs for optimized protein output.
Translational control shapes the proteome in normal and pathophysiological conditions. Current high-throughput approaches reveal large differences in mRNA-specific translation activity but cannot identify the causative mRNA features. The first study developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 5′ untranslated regions that confer 1,000-fold differences in ribosome recruitment in biochemically accessible cell lysates. Using DART, the first study determined a functional role for most alternative 5′ UTR isoforms expressed in yeast, revealed a general mode of increased translation via direct binding to a core translation factor, and identified numerous translational control elements including C-rich silencers that are sufficient to repress translation both in vitro and in vivo. DART enables systematic assessment of the translational regulatory potential of 5′ UTR variants, whether native or disease-associated, and will facilitate engineering of mRNAs for optimized protein production in various systems.
Translation initiation is an important step in eukaryotic gene expression, the dysregulation of which is linked to heritable human diseases and cancer. Systematic characterization has shown that mRNA-specific translational activity varies by orders of magnitude under normal growth conditions and is extensively regulated in response to a wide range of physiological signals. Despite growing interest in the mRNA features responsible for this widespread translational control, current high-throughput methods, which rely on quantification of the average number of translating ribosomes per transcript, have revealed only partial correlations (R=0.3-0.8) that leave many differences in translation activity unexplained. In contrast, in-depth genetic and biochemical analysis has revealed detailed regulatory mechanisms for certain mRNAs.
Genome-wide association studies (GWASs) show diverse disease phenotypes associated with non-coding single-nucleotide polymorphisms (SNPs) found within 5′ UTRs. However, current efforts to identify causal variants, which are potential targets for new therapies, are largely restricted to non-synonymous changes within protein coding sequences. A richer understanding of mRNA sequence-specific translation mechanisms is needed to predict which 5′ UTR variants are likely to do damage by dysregulating protein levels. The first study developed direct analysis of ribosome targeting (DART) technology as a bridge between high-throughput, but mechanistically difficult to parse, in vivo approaches and mechanistically precise, but low-throughput, in vitro translation reconstitution assays. In DART, thousands of synthetic mRNAs initiate translation in vitro. Differential abundance analysis of ribosome-bound and input mRNA reveals differences in translation activity. By massively parallel testing of defined sequences, both endogenous and mutated, the approach moves beyond correlation analysis to enable rapid, hypothesis-driven dissection of the causative role of specific 5′ UTR elements to determine their mechanisms of action.
Here, the first study demonstrates the broad power of DART to illuminate the mechanisms underlying translational control. Overall, the first study tested 4,354 full-length alternative mRNA isoforms from 2,064 yeast genes, which revealed widespread translational control by alternative 5′ UTRs. The first study also exploited the throughput of the DART approach to systematically interrogate the effects of RNA secondary structure, start codon context, and protein binding motifs on translation initiation. The results establish a broad stimulatory role for eIF4G1 binding sequences and also demonstrate highly context-dependent control by inhibitory 5′ UTR structures. This illustrates the potential of DART to illuminate the function of putative RNA regulatory elements identified by other high-throughput approaches. The first study further leveraged the alternative 5′ UTR isoform comparisons to discover hundreds of previously uncharacterized translational enhancers and silencers, short sequence elements that are sufficient to promote or repress ribosome recruitment. The study validated several C-rich silencers as sufficient to repress translation both in vitro and in vivo. Together, the results reveal thousands of previously unidentified functional elements within 5′ UTRs that substantially affect translation. This study establishes DART as a powerful new high-throughput method that can be broadly applied to both discover and interrogate regulatory features within 5′ UTRs.
5′ UTRs directly contact the translation initiation machinery and can strongly influence translation activity (
Next, the first study used these designed pools to generate dsDNA templates for in vitro transcription, followed by enzymatic mRNA capping to produce the pooled RNA substrates for in vitro translation initiation. RNAs were enzymatically biotinylated at their 3′ ends to facilitate quantitative recovery. RNA pools were incubated in yeast translation extracts (Gilbert et al., 2007; Hodgman and Jewett, 2013; Iizuka et al., 1994) for 30 min, a time point that maximizes quantitative differences in ribosome recruitment between 5′ UTRs while yielding enough initiated mRNA for robust library preparation. Cycloheximide was included in the translation reaction to freeze recruited ribosomes at initiation codons and prevent ribosome run-off during ultracentrifugation to separate 80S ribosome-mRNA complexes from untranslated mRNAs. Ribosome-bound mRNA was isolated and sequenced, and the relative abundance of each sequence was compared to its abundance in the input pool to determine a ribosome recruitment score (RRS) for each 5′ UTR (
A caveat to the 80S/input calculation used to assess translation initiation activity is that sequences that destabilize 5′ UTRs in extracts could also lead to a reduction in 80S-bound RNA compared to the input pool. The first study therefore tested an alternative method of calculating RRSs by sequencing the untranslated mRNA from the top of the gradient in addition to the 80S fraction, using the formula RRS0=80S/(80S+mRNP). These two metrics, RRS and RRS0, produced correlated, but not identical, results (R=0.61; p<2.2e-16). Notably, each of the main conclusions reported below is consistent between calculation methods. Because changes in translation initiation can indirectly affect mRNA levels, the 80S/input calculation (RRS) is preferred.
It was hypothesized that 5′ UTR elements that bind preferentially to eukaryotic initiation factors (eIFs) may function as translational enhancers. Consistent with this model, diverse viruses rely on high-affinity interactions between their 5′ UTRs and cellular initiation factors and/or ribosomes for efficient translation. The eIF4G subunit of the cap binding complex is a prime candidate to mediate the activity of translational enhancer sequences. eIF4G contains three RNA binding domains that directly interact with mRNA and are essential for yeast growth, although specific functional interactions between eIF4G and cellular mRNAs had not been characterized. A high-throughput approach, RNA Bind-n-Seq (RBNS), was used to interrogate the RNA binding specificity of eIF4G1. Using a library of random 20-mer RNA to test ˜87,380 distinct RNA 7-mer motifs for binding to different concentrations of recombinant eIF4G1, the first study uncovered a preference for RNA sequences containing oligo-uridine (U). Consistently, it was found that inserting oligo(U) into an unstructured RNA increased binding to eIF4G1 by 20-fold.
Here, the first study sought to comprehensively test the impact of endogenous oligo(U) motifs on translation initiation. Hundreds of native yeast 5′ UTRs contain oligo(U) sequences, which are evolutionarily conserved among budding yeast species and enriched in genes with regulatory roles. The first synthesized a pool of capped mRNAs consisting of all yeast 5′ UTR sequences ≤94 nt long that contain U37 (168 in total), together with their start codons and some coding sequence. The pool included matched controls for each 5′ UTR in which the oligo(U) motif was replaced with a CA repeat of equal length (
Many eukaryotic genes express multiple mRNA isoforms that differ in their 5′ UTRs, which complicates the analysis of ribosome profiling data to identify translational control elements. Because ribosome-protected footprints within coding sequences cannot be correctly assigned to translation initiation by specific 5 UTRs, the mRNA-specific translational efficiency inferred from ribosome profiling data may not accurately reflect the actual translation activity of any mRNA isoform (
The first study analyzed RNA sequencing (RNA-seq) data from wild-type yeast (STAR Methods) and identified 4,354 alternative mRNA isoforms expressed from 2,064 genes that we tested for isoform-specific translation activity by using DART. Notably, 1,639 isoform pairs with reproducible differences in ribosome recruitment were identified (p<0.05, Bonferroni corrected two-tailed t test), of which 843 differed by more than 3-fold. These results provide significantly evidence that alternative 5′ UTRs differ in ribosome association in vivo. DART establishes the direct contribution of differences in 5′ UTR sequences to differences in translation initiation and eliminates potentially confounding effects of co-occurring alternative 3′-UTR sequences.
These mRNA isoforms were leveraged comparisons to identify previously undetected translational control elements within 5′ UTRs. Enhancer and silencer elements were operationally defined as sequences present in a longer 5′ UTR variant that showed a higher RRS than a shorter alternative 5′ UTR of the same gene (enhancer) or lower RRS than the corresponding shorter alternative 5′ UTR (silencer). 658 enhancer regions and 541 silencer regions were identified by the criteria that their inclusion reproducibly changed RRS more than 2-fold (p<0.001, n=3 replicates). Of these, 72 enhancers and 67 silencers changed RRS by more than 10-fold (
To illuminate the mechanisms of translational control, the first study looked for overrepresented motifs within enhancers and silencers using DREME (Bailey et al., 2015). Long 5′ UTR isoforms with higher ribosome recruitment activity were enriched for AU-rich sequences, which are distinct from the previously identified eIF4G-binding U7 motif. Intriguingly, the silencer regions were enriched in CCH motifs. The first study observed similar C-rich motifs in the bottom 10% of all 5′ UTRs (
The first study verified the repressive effects of C-rich silencer motifs on the translational output of full-length mRNAs by using luciferase reporters. For selected genes containing the motif of interest in the longer 5′ UTR isoform, mutations were introduced to disrupt the putative silencer in the long isoform, thereby testing its necessity for translation repression. In parallel, the motif was added to the short isoform, thereby testing its sufficiency. In each case, disruption of the motif from the longer isoform resulted in an increase in protein production whereas addition of the motif reduced luciferase activity from the short isoform (
Here DART is presented as a new approach for high-throughput functional testing of endogenous and engineered 5′ UTR variants. The first study applied DART to more than 8,000 endogenous 5′ UTRs from the model eukaryote S. cerevisiae and identified C-rich motifs as translational silencers present within hundreds of 5′ UTRs. The study also used DART to systematically probe the effect of oligo(U) motifs and thereby established a general stimulatory role for this 5′ UTR element in endogenous mRNAs. The results illustrate the power of the DART method to uncover the regulatory elements underlying mRNA-specific differences in protein output, which are anticipated will be broadly applicable to the study of translation initiation, including by human mRNAs.
′ UTR sequences that increased or decreased ribosome recruitment by more than 1,000-fold in cell lysates were identified. Specific sequences, such as C-rich motifs, that affected ribosome recruitment as measured by DART similarly affected protein synthesis from luciferase reporters in cells (e.g.,
The yeast strain YWG1245 (MATa trplDleu2-3,112 ura3-52 gcn2D::hisG PGAL1-myc-UBR1::TRP1::ubr1, pRS316 <URA3>) used in this assay was cultured in liquid YPAD (1% yeast extract, 2% peptone, 2% glucose, 0.01% adenine hemisulfate) at 30° C. with constant shaking.
Each sequence in the pool consisted of approximately 122 nucleotides of 5′ UTR sequence followed by at least 24 nucleotides of coding sequence followed by a randomized 10 nucleotide unique identifier barcode and an adaptor sequence used for priming reverse transcription and Illumina sequencing (
Yeast translation extracts were made as described (Rojas-Duran and Gilbert, 2012) from YWG1245 (MATa trp1Dleu2-3,112 ura3-52 gcn2D::hisG PGAL1-myc-UBR1::TRP1::ubr1, pRS316 <URA3>) cultured in liquid YPAD (1% yeast extract, 2% peptone, 2% glucose, 0.01% adenine hemisulfate). Cultures were grown to mid-log phase, harvested and then washed two times in mannitol buffer (30 mM HEPES pH 7.4, 100 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT, 0.1 mM PMSF, 8.5% mannitol). Cells were then resuspended in 1.5× volume of mannitol buffer and subsequently lysed by bead beating with 5× pellet volume of glass beads. Six 1 min rounds of bead beating were performed with intermittent one minute cooling periods on ice. Beads were pelleted and the supernatant transferred to a fresh tube. Cell debris was pelleted by a 30 min spin at 16,000 rcf Supernatant was removed and dialyzed using Slide-A-lyzer, 3-12 mL, 3,500 MWCO (Pierce #66110) for four h (30 mM HEPES pH 7.4, 100 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT), changing the buffer once. Approximately 40 pmoles of mRNA were added per 2 mL in vitro translation reaction (22 mM HEPES pH 7.4, 120 mM KOGln, 3 mM MgOGln, 3.75 mM ATP, 0.5 mM GTP, 25 mM creatine phosphate, 1.6 mM DTT, 400U RNaseIn, 2 mM PMSF, 650 mg creatine phosphokinase, 1× cOmplete EDTA-free protease inhibitor) containing 50% yeast extract. Reactions were incubated with 0.5 mg/mL cycloheximide at 26° C. for 30 min in a shaking thermomixer.
To measure translation activity in vivo YWG1245 was transformed with pGAL-5′ UTR-Fluc plasmids and grown to log phase in media containing 2% raffinose. 5′ UTR sequences were generated by gBlock (Twist Bioscience) mRNA expression was induced by addition of galactose to a final concentration of 2%. Whole-cell lysates were prepared by vortexing with glass beads in 1×PBS with protease inhibitors (2 mM phenylmethanesulfonyl fluoride, and 1 3protease inhibitor cocktail (Roche). Luciferase activity was measured using the Luciferase Bright-Glo Assay System (Promega) on a Berthold Centro XS Luminometer. Luciferase values were normalized to both FLUC mRNA levels and total protein. Fluc mRNA levels were determined by Northern blot on total cellular RNA isolated from whole-cell lysates by hot phenol extraction and normalized to U1 RNA levels. Fluc mRNA levels were determined by Northern blot on total cellular RNA isolated from whole-cell lysates by hot phenol extraction and normalized to U1 RNA levels. Probes made using radiolabeled PCR products from the following primer pairs: 5′-AAGGCGTGTTTGCTGACGTTTC-3′ (SEQ ID NO: 52), 5′-CACCCGTTCCTACCAAGACC-30 (U1) (SEQ ID NO: 53); 5′-TGGGCGCGTTATTTATCGGAGTTGC-3′ (SEQ ID NO: 54), 5′-GAGCCCATATCCTTGCCTGATACC-3′ (Fluc) (SEQ ID NO: 55).
Translation reactions were loaded onto 10%-50% sucrose gradients in polysome lysis buffer (20 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate, 0.1 M potassium acetate, 0.1 mg/mL cycloheximide, 1% Triton X-100) and centrifuged at 27,000×g in a Beckman SW28 rotor for 3 h. Gradients were fractionated from the top down using a Biocomp Gradient Station (Biocomp Instruments) with continual monitoring of absorbance at 254 nm. Fractions corresponding to the 80S peak were pooled and RNA extracted using phenol/chloroform followed by isopropanol precipitation.
RNA pellets from the input pool, total lysate, and gradient fractions were resuspended in binding buffer (0.5M NaCl, 20 mM Tris-HCl pH 7.5, 1 mM EDTA) and biotinylated 5′ UTRs were recovered using Hydrophilic Streptavidin Beads (NEB S1421S). Isolated RNA was reverse transcribed using the barcoded primer OWG921 and Superscript III (Invitrogen 18080093). Gel-purified cDNA products were ligated to the adaptor OWG920 using T4 RNA ligase 1 (NEB M0437M). cDNA cleanup was performed using 10 ml MyOne Silane beads (Thermo Scientific 37002D) per sample. Libraries were then PCR amplified with primers RP1 and OBC and sequenced on a HiSeq 2500.
In the second study described herein (“the second study”), using the method developed in the first study, translational enhancers and translational silencers in the 5′ UTR were determined in the budding yeast. These enhancers and silencers are shown in
In the third study described herein (“the third study”), using the method developed in the first study, translational enhancers and translational silencers in the 5′ UTR were determined in humans.
Specifically, a human 5′ UTR pool was designed and synthesized. In this 5′-UTR pool, each sequence in the human 5′ UTR pool consisted of a T7 promoter followed by between 10-230 nucleotides of 5′ UTR sequence followed by at least 24 nucleotides of GFP coding sequence followed by an adaptor sequence used for priming reverse transcription and Illumina sequencing. 5′ UTR sequences were derived from Ensembl annotations. Designed oligos were purchased as pools (Twist Bioscience) and PCR amplified. RNAs were produced by runoff T7 transcription from gel-purified DNA template. RNAs were 5′ capped using the Vaccinia Capping System (NEB M2080S).
In the human translation assays, 20 pmoles of pool RNA were added per 500 microliter translation reaction (16 mM HEPES KOH pH 7.4, 40 mM KOGln, 2 mM MGOGln, 0.8 mM ATP, 0.1 mM GTP, 20 mM creatine phosphate, 0.1 mM spermidine, 1.6 mM DTT, 2 mM PMSF, 140U RNasin Plus, 165 mg creatine phosphokinase, 1× cOmplete EDTA-free protease inhibitor) containing 50% Hela cytoplasmic extract (Ipracell). Reactions were incubated with 0.5 mg/mL cycloheximide at 37° C. for 30 min in a shaking thermomixer.
The human 80S was isolated according to the following: translation reactions were loaded onto 10%-50% sucrose gradients in polysome lysis buffer (20 mM HEPES-KOH pH 7.4, 2 mM magnesium glutamate, 0.1 M potassium glutamate, 0.5 mg/mL cycloheximide, 10% Triton X-100) and centrifuged at 35,000×g in a Beckman SW41 rotor for 3 h. Gradients were fractionated from the top down using a Biocomp Gradient Station (Biocomp Instruments) with continual monitoring of absorbance at 254 nm. Fractions corresponding to the 80S peak were pooled and RNA extracted using phenol/chloroform followed by isopropanol precipitation.
The study confirmed that the DART analysis developed in the first study is highly reproduceable (
One batch of identified enhancers and silencers are shown in
The study further shows that the incorporation of modified nucleobases, such as the NJ-methylpseudouridine, in the 5′ UTRs resulted in in the change of translational efficiencies (
A further screening identified more than a thousand short 5′-UTR sequences that, when combined with N1-methylpseudouridine, resulted in higher translational efficiency than the 5′-UTR used in Moderna mRNA-based vaccine. The short 5′-UTR sequences in this batch are listed below in Table 4 below:
In some aspects, the present invention is directed to the following non-limiting embodiments:
Embodiment 1: A method of modulating a translational efficiency of a messenger RNA (mRNA) molecule, the method comprising: modifying a 5′-UTR of the mRNA molecule to include a translational enhancer or a translational silencer.
Embodiment 2: The method of Embodiment 1, wherein modifying the 5′-UTR of the mRNA molecule comprises modifying a sequence of a DNA molecule encoding the mRNA molecule.
Embodiment 3: The method of any one of Embodiments 1-2, wherein the translational enhancer or the translational silence is a translational enhancer or a translational silencer for the translational machinery of a yeast species, optionally a yeast species selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Hansenda polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, and Schizosaccharomyces pombe.
Embodiment 4: The method of Embodiment 3, wherein the 5′-UTR of the mRNA molecule is modified to include the translational enhancer, and wherein the translational enhancer includes at least one sequence selected from the group consisting of SEQ ID NOs: 1-3.
Embodiment 5: The method of Embodiment 3, wherein the 5′-UTR of the mRNA molecule is modified to include the translational silencer, and wherein the translational silencer includes at least one sequence selected from the group consisting of SEQ ID NOs: 4-6.
Embodiment 6: The method of any one of Embodiment 1-2, wherein the translational enhancer or the translational silence is a translational enhancer or a translational silencer for the translational machinery of a mammal, optionally a human.
Embodiment 7: The method of Embodiment 6, wherein the 5′-UTR of the mRNA molecule is modified to include the translational enhancer, and wherein the translational enhancer includes at least one sequence selected from the group consisting of SEQ ID NOs: 7-18, 29-51 and 56-1342.
Embodiment 8: The method of Embodiment 6, wherein the 5′-UTR of the mRNA molecule is modified to include the translational silencer, and wherein the translational silencer includes at least one sequence selected from the group consisting of SEQ ID NOs: 19-28.
Embodiment 9: The method of any one of Embodiment 1-8, wherein the 5′-UTR of the mRNA molecule is further modified to comprise a modified nucleobase.
Embodiment 10: The method of Embodiment 9, wherein the modified nucleobase comprises N6-methyladenosine, inosine, N1-propylpseudouridine, N1-methoxymethylpseudouridine, N1-ethylpseudouridine, 5-methoxycytidine, 5-hydroxyuridine, 5-carboxyuridine, 5-formyluridine, 5-hydroxycytidine, 5-hyrdoxymethylcytidine, 5-hydroxymethyluridine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, pseudoisocytidine, 2-thiocytidine, 4-thiocytidine, N1-methylpseudouridine, pseudouridine, 5-methyluridine, 5-methoxyuridine, dihydrouridine, 5-methylcytidine, 4-thiouridine, 2-thiouridine, uridine-5′-O-(1-thiophosphate), 5-aminoallyluridine, or 4-acetylcytidine.
Embodiment 11: The method of any one of Embodiment 1-10, wherein the 5′-UTR of the mRNA molecule is modified to include the translational enhancer, and wherein a ribosome retention score (RRS) of the modified mRNA molecule is 3× or higher than an RRS of the unmodified mRNA molecule.
Embodiment 12: The method of any one of Embodiments 1-10, wherein the 5′-UTR of the mRNA molecule is modified to include the translational silencer, and wherein a ribosome retention score (RRS) of the modified mRNA molecule is 0.3× or lower than an RRS of the unmodified mRNA molecule.
Embodiment 13: An mRNA molecule comprising a 5′-UTR, wherein the 5′-UTR comprises: a translational enhancer comprising at least one sequence selected from the group consisting of SEQ ID NOs: 1-3, 7-18, 29-51 and 56-1342, or a translational silencer comprising at least one selected from the group consisting of SEQ ID NOs: 4-6 and 19-28; and a modified nucleobase.
Embodiment 14: The mRNA molecule of Embodiment 13, wherein the modified nucleobase comprises N6-methyladenosine, inosine, N1-propylpseudouridine, N1-methoxymethylpseudouridine, N1-ethylpseudouridine, 5-methoxycytidine, 5-hydroxyuridine, 5-carboxyuridine, 5-formyluridine, 5-hydroxycytidine, 5-hyrdoxymethylcytidine, 5-hydroxymethyluridine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, pseudoisocytidine, 2-thiocytidine, 4-thiocytidine, N1-methylpseudouridine, pseudouridine, 5-methyluridine, 5-methoxyuridine, dihydrouridine, 5-methylcytidine, 4-thiouridine, 2-thiouridine, uridine-5′-O-(1-thiophosphate), 5-aminoallyluridine, or 4-acetylcytidine.
Embodiment 15: The mRNA molecule of any of Embodiments 13-14, wherein the 5′-UTR comprises the translational enhancer, and wherein a ribosome retention score (RRS) of the mRNA molecule is 3× or higher than an RRS of an mRNA molecule that does not comprise the translational enhancer or the modified nucleobase but otherwise has the same sequence.
Embodiment 16: The mRNA molecule of any of Embodiments 13-15, wherein the 5′-UTR comprises at least one sequences selected from the group consisting of SEQ ID NOs: 29-50.
Embodiment 17: The mRNA molecule of any of Embodiment 13-14, wherein the 5′-UTR comprises the translational silencer, and wherein an RRS of the mRNA molecule is 0.3× or lower than an RRS of an mRNA molecule that does not comprise the translational silencer or the modified nucleobase but otherwise has the same sequence.
Embodiment 18: The mRNA molecule of any one of Embodiment 13-17, wherein the mRNA molecule encodes a therapeutic peptide or a therapeutic protein.
Embodiment 19: The mRNA molecule of Embodiment 18, wherein the therapeutic peptide or the therapeutic protein comprises a vaccine.
Embodiment 20: A method of constructing an mRNA molecule for producing a therapeutic peptide or a therapeutic protein, the method comprising including a translational enhancer at a 5′ UTR to the 5′ side of a coding region encoding the therapeutic peptide or a therapeutic protein.
Embodiment 21: The method of Embodiment 20, wherein the therapeutic peptide or a therapeutic protein comprises a vaccine.
Embodiment 22: A yeast cell for expressing a protein, comprising an mRNA molecule or a nucleotide encoding the mRNA molecule, wherein the mRNA molecule comprises: a 5′-UTR comprising at least one yeast translational enhancer; and a coding region encoding the protein, or a coding region for introducing a nucleotide sequence for encoding the protein.
Embodiment 23: The yeast cell of Embodiment 22, wherein the 5′-UTR comprises at least one sequence selected from the group consisting of SEQ ID NOs: 1-3.
Embodiment 24: The yeast of any one of Embodiment 22-23, wherein the yeast cell is a Saccharomyces cerevisiae cell, a Pichia pastoris cell, a Hansenula polymorpha cell, a Yarrowia lipolytica cell, an Arxula adeninivorans cell, a Kluyveromyces lactis cell, or a Schizosaccharomyces pombe cell.
The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and/or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.
The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/317,363, filed Mar. 7, 2022, which is incorporated herein by reference in its entirety.
This invention was made with government support under CA246118, ES031525, GM101316, GM125955, GM132358 and NS118616 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/063837 | 3/7/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63317363 | Mar 2022 | US |