Patent Application
20190264213
The present invention, in some embodiments thereof, relates to isolated polynucleotides and polypeptides and methods of using same for expressing an expression product of interest.
Recombinant DNA technologies, including inducible gene expression and genome editing technologies have provided opportunities for control of gene expression and precise, targeted modifications to genome sequences in many types of organisms, including plants and animals. Rational gene expression in general, and genome editing in particular, have an enormous potential across basic research, drug discovery and cell based medicine by inserting or removing a specific genetic trait. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, the removal of deleterious genes or genome sequences and alteration of plant genomes in order to generate improved crops. Existing methods for genome editing include, for example, the use of zinc finger nucleases, TALENs, and CRISPR-Cas9 systems.
According to an aspect of some embodiments of the present invention there is provided a method of expressing an expression product of interest, the method comprising:
(i) introducing into a cell a polynucleotide comprising an AimR responsive element operatively linked to a nucleic acid sequence encoding the expression product of interest, wherein the AimR comprises a DNA binding domain for binding the AimR responsive element, the AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain; and
(ii) contacting the cell with an AimP peptide comprising an amino acid sequence of XXXXGG/A, wherein the AimP peptide is capable of binding the AimR polypeptide and dissociating the AimR polypeptide from the AimR responsive element,
thereby expressing the expression product of interest.
According to an aspect of some embodiments of the present invention there is provided a method of expressing an expression product of interest, the method comprising introducing into a cell a polynucleotide comprising an AimR responsive element operatively linked to a heterologous nucleic acid sequence encoding the expression product of interest, wherein the AimR comprises a DNA binding domain for binding the AimR responsive element, the AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain, thereby expressing the expression product of interest.
According to an aspect of some embodiments of the present invention there is provided a method of expressing an expression product of interest, the method comprising introducing into a cell a polynucleotide comprising an AimR responsive element operatively linked to a nucleic acid sequence encoding the expression product of interest; and a nucleic acid construct comprising an AimR polynucleotide and a cis-acting regulatory element heterologous to the AimR for directing expression of the AimR polynucleotide,
wherein the AimR comprises a DNA binding domain for binding the AimR responsive element, the AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain, thereby expressing the expression product of interest.
According to some embodiments of the invention, the method comprising introducing into the cell a polynucleotide encoding the AimR.
According to some embodiments of the invention, the method comprising contacting the cell with an AimP peptide or a nucleic acid sequence encoding same, the AimP peptide comprising an amino acid sequence of XXXXGG/A, wherein the AimP peptide is capable of binding the AimR polypeptide and dissociating the AimR polypeptide from the AimR responsive element.
According to some embodiments of the invention, the method comprising contacting the cell with an agent capable of downregulating expression and/or activity of the AimR responsive element.
According to some embodiments of the invention, the expression product of interest is endogenous to the cell.
According to some embodiments of the invention, the expression product of interest is exogenous to the cell.
According to an aspect of some embodiments of the present invention there is provided an article of manufacture identified for expressing an expression product of interest comprising a packaging material packaging at least two of:
(i) a polynucleotide comprising an AimR responsive element, wherein the AimR comprises a DNA binding domain for binding the AimR responsive element, the AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain;
(ii) a polynucleotide encoding the AimR;
(iii) an AimP peptide comprising an amino acid sequence of XXXXGG/A or a nucleic acid sequence encoding same, wherein the AimP peptide is capable of binding the AimR polypeptide and dissociating the AimR polypeptide from the AimR responsive element; and/or
(iv) an agent capable of downregulating expression and/or activity of the AimR responsive element.
According to some embodiments of the invention, the polynucleotide encoding the AimR is comprised in a nucleic acid construct comprising a cis-acting regulatory element heterologous to the AimR for directing expression of the AimR polynucleotide.
According to some embodiments of the invention, the article of manufacture comprising a multiple cloning site (MCS).
According to some embodiments of the invention, the article of manufacture comprising a polynucleotide encoding the expression product of interest.
According to some embodiments of the invention, the expression product of interest is a DNA editing agent.
According to some embodiments of the invention, the AimR responsive element comprises a nucleic acid sequence for binding said AimR and an AimX polynucleotide, wherein the AimX polynucleotide or an AimX polypeptide encoded by the AimX polynucleotide is capable of inhibiting lysogeny of a temperate phage expressing the AimR in a host bacteria.
According to some embodiments of the invention, the expression product of interest is an AimX dependent DNA editing agent.
According to some embodiments of the invention, the method comprising introducing into the cell a nucleic acid sequence to be integrated into a genome of the cell by the DNA editing agent.
According to some embodiments of the invention, the DNA editing agent is an integrase.
According to some embodiments of the invention, the DNA editing agent is selected from the group consisting of zinc finger nuclease, an effector protein of Class 2 CRISPR/Cas (e.g. Cas9, Cpf1, C2c1, C2c3) and TALEN.
According to an aspect of some embodiments of the present invention there is provided an isolated AimP peptide comprising an amino acid sequence of XXXXGG/A, wherein the peptide is capable of binding an AimR polypeptide comprising a DNA binding domain for binding an AimR responsive element, the AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain; and dissociating the AimR polypeptide from an AimR responsive element.
According to some embodiments of the invention, there is provided an isolated polynucleotide encoding the peptide of the invention.
According to an aspect of some embodiments of the present invention there is provided an isolated AimR polypeptide comprising a DNA binding domain for binding an AimR responsive element, the AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain.
According to some embodiments of the invention, there is provided an isolated polynucleotide encoding the polypeptide of the invention.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising an AimR responsive element, wherein the AimR comprises a DNA binding domain for binding the AimR responsive element, the AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain.
According to some embodiments of the invention, AimR responsive element comprises a nucleic acid sequence for binding said AimR and an AimX polynucleotide, wherein the AimX polynucleotide or an AimX polypeptide encoded by the AimX polynucleotide is capable of inhibiting lysogeny of a temperate phage expressing the AimR in a host bacteria.
According to some embodiments of the invention, there is provided an isolated polypeptide encoded by the polynucleotide of the present invention.
According to some embodiments of the invention, there is provided an isolated polynucleotide comprising the polynucleotides of the present invention.
According to some embodiments of the invention, there is provided an isolated polynucleotide encoding an arbitrium system comprising the polynucleotides of the present invention, wherein the arbitrium system is capable of regulating lysogeny of a phage expressing the arbitrium system in a host bacteria.
According to some embodiments of the invention, there is provided a nucleic acid construct comprising the polynucleotide of the present invention and a multiple cloning site (MCS).
According to some embodiments of the invention, there is provided a nucleic acid construct comprising the polynucleotide of the present invention and a multiple cloning site (MCS).
According to some embodiments of the invention, there is provided a nucleic acid construct comprising the polynucleotide of the present invention or the nucleic acid construct of the present invention and a cis-acting regulatory element heterologous to the AimP, the AimR, the AimR responsive element and/or the AimX for directing expression of the polynucleotide.
According to some embodiments of the invention, the nucleic acid construct of the present invention being a nucleic acid construct system comprising at least two nucleic acid constructs each expressing at least one of the polynucleotides.
According to some embodiments of the invention, the construct encodes a polycistronic mRNA comprising the polynucleotide.
According to an aspect of some embodiments of the present invention there is provided an isolated nucleic acid agent capable of downregulating expression and/or activity of AimR responsive element, wherein the AimR comprises a DNA binding domain for binding the AimR responsive element, the AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain.
According to some embodiments of the invention, there is provided an article of manufacture identified for expressing an expression product of interest comprising a packaging material packaging at least one of:
(a)(i) the isolated polynucleotide of the present invention,
(a)(ii) a nucleic acid construct comprising the polynucleotide of the present invention and a multiple cloning site (MCS), and or
(a)(iii) a nucleic acid construct comprising the polynucleotide of the present invention and a cis-acting regulatory element heterologous to the AimR or the AimX for directing expression of the polynucleotide;
and at least one of:
(b)(i) the isolated peptide of the present invention,
(b)(ii) the isolated polynucleotide of the present invention,
(b)(iii) a nucleic acid construct comprising the polynucleotide of the present invention and a MCS,
(b)(iv) a nucleic acid construct comprising the polynucleotide of the present invention and a cis-acting regulatory element heterologous to the AimP for directing expression of the polynucleotide, and/or
(b)(v) the isolated agent of the present invention.
According to some embodiments of the invention, the isolated polynucleotide, the nucleic acid construct or the article of manufacture of the present invention comprising a nucleic acid sequence encoding an expression product of interest.
According to some embodiments of the invention, the expression product of interest is a DNA editing agent.
According to some embodiments of the invention, the AimP peptide is capable of leading to lysogeny of a temperate phage expressing the AimR in a host bacteria.
According to some embodiments of the invention, the AimP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 269-283 and 285-286.
According to some embodiments of the invention, the AimR comprises a nucleic acid sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NO: 1-113 and/or an amino acid sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 114-226.
According to some embodiments of the invention, the AimR comprises a nucleic acid sequence selected from the group consisting of 1-113 and/or an amino acid sequence selected from the group consisting of SEQ ID NOs: 114-226.
According to some embodiments of the invention, the AimR responsive element comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 287-334.
According to some embodiments of the invention, the AimR responsive element comprises SEQ ID NO: 378.
According to some embodiments of the invention, the AimX comprises a nucleic acid sequence as set forth in SEQ ID NO: 336.
According to some embodiments of the invention, the AimR and the AimP and/or the AimR responsive element are positioned sequentially 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same.
According to some embodiments of the invention, the AimP and the AimR responsive element are positioned sequentially 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same.
According to some embodiments of the invention, the AimR and the AimP and/or the AimR responsive element are positioned sequentially 5′ to 3′ in a genome of a temperate phage.
According to some embodiments of the invention, the AimP and the AimR responsive element are positioned sequentially 5′ to 3′ in a genome of a temperate phage.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
The present invention, in some embodiments thereof, relates to isolated polynucleotides and polypeptides and methods of using same for expressing an expression product of interest.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
Recombinant DNA technologies, including inducible gene expression and genome editing technologies developed in recent years allow control of gene expression and precise and targeted modifications to genome sequences in many types of organisms, including plants and animals.
Whilst conceiving embodiments of the invention and reducing to practice, the present inventors have devised a novel expression tool which is based on a newly uncovered communication system utilized by temperate viruses to coordinate lytic-lysogenic decisions.
As is illustrated hereinunder and in the Examples section, which follows, the present inventors show that during infection of its Bacillus host cell, the virus (spBeta family phage) produces a 6 amino acids long peptide that is released to the medium (Examples 1-2,
Taken together, the present teachings suggest that the arbitrium system and functional fragments thereof can be used for e.g. controlling expression of an expression product of interest in general and controlling the integration of DNA sequences into a target genome in particular. As is further described hereinbelow and in the Examples section that follows, AimR can bind an AimR responsive element thereby leading to activation of transcription of an expression product of interest operatively linked to the AimR responsive element (e.g. AimR binding site); while binding of AimP to AimR represses the transcription of the expression product of interest.
The present inventors further show that an AimR-AimP system can function to control the expression of a GFP reporter gene operatively linked to AimR binding site in Bacillus subtilis BEST7003 in a phage-independent context (Example 5,
According to another aspect of the present invention, there is provided an isolated AimR polypeptide comprising a DNA binding domain for binding an AimR responsive element, said AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain.
As used herein the term “isolated” refers to at least partially separated from the natural environment, physiological environment e.g., a microorganism or e.g., phage.
As used herein, the term “AimR” refers to the polynucleotide and expression product e.g. polypeptide of the AimR gene. The product of the AimR gene contains a DNA binding domain for binding an AimR responsive element. The product of the AimR gene contains a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain.
As used herein, the phrase “DNA binding domain (DBD)” refers to a motif that can recognize and bind to a specific nucleic acid sequence (e.g. AimR responsive element). The DBD can recognize and bind to a double-stranded nucleic acid sequence (e.g. DNA) in a sequence specific manner. Typically, a DBD is a structural motif in a protein domain. According to specific embodiments the DBD comprises a helix-turn-helix (HTH) motif.
As used herein the term “helix-turn-helix (HTH) motif” refers to a well-known DNA binding motif which complements the shape of the DNA major groove, having the mfap number IPR000047 (see e.g. Ann Rev. of Biochem. (1984) 53:293 and Brunelle and Schleif J. Mol. Biol. (1989) 209:607). The domain can be illustrated by the sequence XXXPhoAlaXXPhoGlyPhoXXXXPhoXXPhoXX (SEQ ID NO: 335), wherein X is any amino acid and Pho is a hydrophobic amino acid.
As used herein, the term “tetratricopeptide repeat (TPR) domain” refers to a degenerate 34 amino acid consensus sequence found in tandem arrays of 3-16 motifs that is believed to mediate protein-protein binding Family: TPR_1 (PF00515). TPR domain forms two anti-parallel α-helices separated by a turn, to form a structure with an amphipathic groove [see e.g. Hirano et al. (1990), Cell, 60:319-328].
According to specific embodiments, the AimR polynucleotide has at least 80% identity to a nucleic acid selected from the group consisting of SEQ ID NO: 1-113.
According to specific embodiments, the AimR polynucleotide has at least 85%, at least 90%, at least 95% identity to a nucleic acid selected from the group consisting of SEQ ID NO: 1-113.
According to specific embodiments, the AimR polypeptide has at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 114-226.
According to specific embodiments, the AimR polypeptide has at least at least 85%, at least 90%, at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 114-226.
According to specific embodiments, the AimR polypeptide has at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or 100% identity to SEQ ID NO: 114.
Sequence identity can be determined using any protein alignment algorithm or any nucleic acid sequence alignment algorithm based on the polynucleotide sequence encoding the polypeptide such as Blast, ClustalW, MUSCLE, and HHpred.
According to specific embodiments, AimR amino acid sequence is selected from the group consisting of SEQ ID NOs: 114-226.
According to specific embodiments, AimR nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-113.
According to a specific embodiment, AimR comprises SEQ ID NO: 114.
The present inventors have uncovered that AimR binds the phage DNA as a dimer in the absence of the AimP peptide and functions as a transcriptional activator of e.g. AimX. Upon binding to the AimP peptide, the AimR changes its oligomeric state from the active dimer to the inactive monomer leading to a decrease in the transcription of e.g. AimX.
Hence, an AimR polypeptide according to some embodiments is capable of binding an AimP peptide comprising an amino acid sequence of XXXXGG/A (as further described hereinbelow); and in the absence of AimP binding DNA (i.e. AimR responsive element) and activating gene expression (i.e. a transcription factor).
Methods of determining binding of a transcription factor (e.g. AimR) to DNA (e.g. AimR Responsive element) are well known in the art and include, but not limited to, Chromatin Immunoprecipitation (ChIP) Assay, DNA Electrophoretic Mobility Shift Assay (EMSA), DNA Pull-down Assay and Microplate Capture and Detection Assay.
According to specific embodiments, the AimR binds DNA as a dimer. Methods of evaluating dimerization are well known in the art and are further described in the Examples section which follows and include migration on a gel filtration column.
According to specific embodiments, the term “AimR” refers to a full length AimR. According to other specific embodiments, the term “AimR” refers to a fragment of AimR which maintains the activity as described herein.
The term “AimR”, also refers to functional AimR homologues which exhibit the desired activity as described herein. Such homologues can be, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to the polypeptide SEQ ID NOs: 114-226, or 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polynucleotide encoding same (as further described hereinabove and below). The homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution.
Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm such as Blast, ClustalW, MUSCLE, and HHpred.
As used herein, the term “operatively linked” refers to a nucleic acid sequence having a functional relationship with another nucleic acid sequence. According to specific embodiments, a nucleic acid sequence is “operatively linked” to a regulatory sequence when the regulatory sequence (e.g. AimR binding site) controls and regulates the transcription and/or translation of that nucleic acid sequence. According to one embodiment, the regulatory element act in cis on the nucleic acid sequence it regulates. According to another embodiment, the regulatory element act in trans on the nucleic acid sequence it regulates. According to specific embodiments, the regulatory sequence is placed upstream to the nucleic acid sequence it regulates. According to specific embodiments, the term “operatively linked” includes having an appropriate start signal (e.g., ATG) upstream of the nucleic acid sequence to be expressed and maintaining the correct reading frame to allow expression of the nucleic acid sequence under the control of the regulatory sequence and expression of the desired product encoded by the nucleic acid sequence.
According to specific embodiments, the term “AimR responsive element” refers to a full length AimR responsive element. According to other specific embodiments, the term “AimR responsive element” refers to a fragment of AimR responsive element which maintains the activity as described herein. According to specific embodiments, the AimR responsive element is 4-100, 4-50, 4-30 or 4-10 nucleic acids long.
According to specific embodiment, the AimR responsive element comprises a nucleic acid sequence for binding AimR (i.e. AimR binding site).
According to specific embodiments, the AimR responsive element comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 287-334.
According to specific embodiments, the AimR responsive element is comprised in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 287-334.
According to specific embodiments, the AimR responsive element comprises SEQ ID NO: 378.
According to specific embodiments, the AimR responsive element is comprised in SEQ ID NO: 378.
According to specific embodiments, the AimR responsive element comprises a fragment of SEQ ID NOs: 287-334 or 378 which maintains SEQ ID NOs: 287-334 or 378 activity as a nucleic acid sequence for binding AimR (i.e. AimR binding site), as determined by e.g. Chromatin Immunoprecipitation (ChIP) Assay, DNA Electrophoretic Mobility Shift Assay (EMSA), Microplate Capture and Detection Assay, or DNA Pull-down Assay.
According to specific embodiments, the AimR responsive element comprises a nucleic acids sequence for binding AimR (i.e. AimR binding site) and AimX. Hence, specific embodiments of the present invention disclose that binding of AimR to its binding site controls expression of AimX, which in turn controls expression of an expression product of interest.
The term “AimR responsive element”, also refers to functional AimR responsive element homologues which exhibit the desired activity (i.e., binding an AimR). Such homologues can be, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to the polynucleotide SEQ ID NOs: 287-334 and 378. The homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution.
AimRs and their cognate sequences comprising AimR responsive elements which can be used in accordance with some embodiments are listed in Table 3 hereinbelow.
As used herein, the term “AimP” refers to a peptide having an amino acid sequence of XXXXGG/A, wherein X is any amino acid or a nucleic acid encoding same. According to specific embodiments, the AimP has an amino acid sequence of XXXX1GG/A, wherein X is any amino acid and wherein X1 is a positively charged amino acid. According to a specific embodiment, the AimP is the product of the AimP gene.
The peptide can be long e.g., more than 50 amino acids (e.g., 51-80 amino acids, 51-100 amino acids, 100-200 amino acids) or short e.g., 6-50 amino acids long. According to specific embodiments, the peptide is 6 amino acids long.
According to specific embodiments, AimP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 269-283 and 285-286.
According to specific embodiments, the AimP peptide comprises a SAIRGA (SEQ ID NO: 269) or GMPRGA (SEQ ID NO: 271) amino acid sequence.
According to specific embodiments, the AimP peptide comprises a SAIRGA (SEQ ID NO: 269) amino acid sequence.
A functional AimP peptide can bind an AimR polypeptide and dissociate the AimR polypeptide from DNA and specifically from an AimR responsive element.
As used herein, the terms “dissociating” and “dissociate” refer to at least 30% reduction in complexes comprising the AimR and AimR responsive element, as evidenced by an assay known in the art e.g., Chromatin Immunoprecipitation (ChIP) Assay, DNA Electrophoretic Mobility Shift Assay (EMSA), DNA Pull-down Assay and Microplate Capture and Detection Assay.
According to specific embodiments, a functional AimP can lead to lysogeny of a temperate phage expressing AimR in a host bacteria. Methods of analyzing phage lysogeny are described hereinbelow.
The term “AimP”, also refers to functional AimP homologues which exhibit the desired activity as described herein. Such homologues can be, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to the peptide SEQ ID NOs: 269-283 and 285-286. The homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution.
AimPs and their cognate AimRs which can be used in accordance with some embodiments are listed in Table 3 hereinbelow.
Binding assays for qualifying peptide binding to AimR are well known in the art and include e.g. western blot, BiaCore, high-performance liquid chromatography (HPLC) or flow cytometry.
Binding assays for qualifying the ability of the peptide to dissociate AimR from DNA are well known in the art and include and include, but not limited to, Chromatin Immunoprecipitation (ChIP) Assay, DNA Electrophoretic Mobility Shift Assay (EMSA), DNA Pull-down Assay and Microplate Capture and Detection Assay.
According to specific embodiments binding of the AimP peptide to AimR polypeptide inhibits dimerization of the AimR polypeptide.
As used herein, the terms “inhibit” and “inhibiting” refer to a decrease in activity (e.g. dimerization, binding, lysogeny). According to specific embodiments the decrease is at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, or at least 20 fold as compared to same in the absence of the AimP peptide or AimX.
According to other specific embodiments the decrease is by at least 5%, by at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 99% or 100% as compared to same in the absence of the AimP peptide or AimX.
A temperate phage is one capable of entering the lysogenic pathway, in which the phage becomes a dormant, passive part of the cell's genome through prior to completion of its lytic cycle.
According to specific embodiments the phage is capable of infecting a Bacillus bacteria.
According to specific embodiments the phage is a prophage.
Table 3 below indicates phages and prophages that can be used according to specific embodiments of the invention. According to a specific embodiment, the phage is a spBeta phage. According to a specific embodiment, the phage is Phi3T.
For the same culture conditions the extent of lysogeny is generally expressed in comparison to the lysogeny in bacteria of the same species but not contacted with the indicated agent (e.g. AimP, AimX) or contacted with a vehicle control, also referred to as control.
Methods of analyzing phage lysogeny are well known in the art and include, but not limited to, DNA sequencing and PCR analysis. As a temperate phage can employ the lysogenic pathway or the lytic pathway, the lysogenic activity of a phage can be assessed indirectly by determining reduction in the lytic activity of a phage by methods well known in the art including, but not limited to, optical density, plaque assay, and living dye indicators.
As used herein, the phrases “leading to lysogeny” and “lead to lysogeny” refers to an increase in lysogeny.
According to specific embodiments the increase is at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, or at least 20 fold as compared to same in the absence of the AimP peptide or AimX downregulating agent.
According to other specific embodiments the increase is by at least 5%, by at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 99% or 100% as compared to same in the absence of the AimP peptide or AimX downregulating agent.
According to specific embodiments, the bacteria can be infected with a temperate phage selected from the phages depicted in Table 3 below. According to specific embodiments, the bacteria can be infected with a spBeta phage. According to specific embodiments the bacteria is a Bacillus bacteria. According to specific embodiments the bacteria is selected from the group consisting of the bacteria listed in Table 3 below.
The present invention also contemplates isolated polynucleotides encoding the components of the arbitrium system and functional fragments thereof as described herein.
Thus, according to an aspect of the present invention, there is provided an isolated polynucleotide encoding an AimP peptide comprising an amino acid sequence of XXXXGG/A, wherein said peptide is capable of binding an AimR polypeptide comprising a DNA binding domain for binding an AimR responsive element, said AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain; and dissociating said AimR polypeptide from an AimR responsive element.
According to another aspect of the present invention, there is provided an isolated polynucleotide encoding an AimR polypeptide comprising a DNA binding domain for binding an AimR responsive element, said AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain.
According to specific embodiments, the polynucleotide encoding the AimR polypeptide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-113.
According to specific embodiments, the polynucleotide encoding the AimR polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-113.
According to another aspect of the present invention, there is provided an isolated polynucleotide comprising an AimR responsive element, wherein said AimR comprises a DNA binding domain for binding said AimR responsive element, said AimR comprises a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain.
According to specific embodiments, the AimR responsive element comprises a nucleic acid sequence for binding AimR and an AimX polynucleotide, wherein said AimX polynucleotide or an AimX polypeptide encoded by said AimX polynucleotide is capable of inhibiting lysogeny of a temperate phage expressing said AimR in a host bacteria.
According to another aspect of the present invention, there is provided an isolated AimX polynucleotide.
According to another aspect of the present invention, there is provided an isolated polypeptide encoded by the AimX polynucleotide.
As used herein, the term “AimX” refers to the polynucleotide and expression product e.g. polypeptide of the AimX gene. According to specific embodiments, the “AimX” refers to the polynucleotide of the AimX gene. According to specific embodiments, an AimR binding site is operatively linked to the AimX polynucleotide. A functional AimX is capable of inhibiting lysogeny of a temperate phage expressing the respective AimR in a host bacteria. According to specific embodiments, AimX and AimR binding site comprise an AimR responsive element.
According to specific embodiments, AimX comprises a nucleic acid sequence as set forth in SEQ ID NO: 336.
According to specific embodiments, the term “AimX” refers to a full length AimX. According to other specific embodiments, the term “AimX” refers to a fragment of AimX which maintains the activity as described herein.
The term “AimX”, also refers to functional AimX homologues which exhibit the desired activity as described herein. Such homologues can be, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical or homologous to the polynucleotide SEQ ID NO:336. The homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including an amino acid substitution.
AimRs and their cognate AimXs which can be used in accordance with some embodiments are listed in Table 3 hereinbelow.
According to another aspect of the present invention, there is provided an isolated polynucleotide comprising a polynucleotide encoding an AimP peptide, a polynucleotide encoding an AimR polypeptide, a polynucleotide comprising an AimR responsive element and/or an AimX polynucleotide and any combination of same.
According to another aspect of the present invention, there is provided an isolated polynucleotide encoding an arbitrium system comprising a nucleic acid sequence encoding an AimP peptide, a nucleic acid sequence encoding an AimR polypeptide and a nucleic acid sequence for binding AimR operatively linked to an AimX nucleic acid sequence, wherein said arbitrium system is capable of regulating lysogeny of a phage expressing said arbitrium system in a host bacteria.
As used herein “arbitrium system” or a “functional arbitrium”, refers to a multi-gene system which comprises AimP, AimR, and AimX as described herein and which activity controls phage lysogeny in its host genome.
The combinations of the arbitrium system components which can be used in accordance with some embodiments are listed in Table 3 hereinbelow.
As used herein, the term “components of the arbitrium system” refers to AimP, AimR, AimR responsive element, AimX and any combination of same or functional fragments thereof such as described hereinabove.
According to specific embodiments, the AimR is encoded by a gene positioned within 1-10 genes of a gene encoding AimP and/or AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) in a genome of a temperate phage.
According to specific embodiments, the AimP is encoded by a gene positioned within 1-10 genes of a gene encoding an AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) in a genome of a temperate phage. According to specific embodiments, the AimR and the AimP and/or the AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) are positioned sequentially 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same. According to specific embodiments, the AimR and the AimP and/or the AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) are positioned sequentially 5′ to 3′ in a genome of a temperate phage.
According to specific embodiments, the AimR and the AimP and/or the AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) are positioned contiguously 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same.
According to specific embodiments, the AimR and the AimP and/or the AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) are positioned contiguously 5′ to 3′ in a genome of a temperate phage.
According to specific embodiments, the AimP and the AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) are positioned sequentially 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same.
According to specific embodiments, the AimP and the AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) are positioned sequentially 5′ to 3′ in a genome of a temperate phage.
According to specific embodiments, the AimP and the AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) are positioned contiguously 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same.
According to specific embodiments, the AimP and the AimR responsive element (e.g. SEQ ID NOs: 287-334, 378) are positioned contiguously 5′ to 3′ in a genome of a temperate phage.
According to specific embodiments, the AimR is encoded by a gene positioned within 1-10 genes of a gene encoding AimX in a genome of a temperate phage.
According to specific embodiments, the AimP is encoded by a gene positioned within 1-10 genes of a gene encoding AimX in a genome of a temperate phage.
According to specific embodiments, the AimR and the AimP and/or the AimX are positioned sequentially 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same. According to specific embodiments, the AimR and the AimP and/or the AimX are positioned sequentially 5′ to 3′ in a genome of a temperate phage.
According to specific embodiments, the AimR and the AimP and/or the AimX are positioned contiguously 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same.
According to specific embodiments, the AimR and the AimP and/or the AimX are positioned contiguously 5′ to 3′ in a genome of a temperate phage.
According to specific embodiments, the AimP and the AimX are positioned sequentially 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same. According to specific embodiments, the AimP and the AimX are positioned sequentially 5′ to 3′ in a genome of a temperate phage.
According to specific embodiments, the AimP and the AimX are positioned contiguously 5′ to 3′ on a nucleic acid molecule of a temperate phage expressing same. According to specific embodiments, the AimP and the AimX are positioned contiguously 5′ to 3′ in a genome of a temperate phage.
Thus, according to specific embodiments, the polynucleotide of the present invention comprises:
(1) a polynucleotide encoding an AimP peptide;
(2) a polynucleotide comprising a nucleic acid sequence encoding an AimR polypeptide;
(3) a polynucleotide comprising an AimR responsive element nucleic acid sequence; (4) an AimX polynucleotide
(5) a polynucleotide comprising (2) and (3);
(6) a polynucleotide comprising (2) and (4);
(7) a polynucleotide comprising (1), (2) and (3);
(8) a polynucleotide comprising (1), (2) and (4);
(9) a polynucleotide comprising (1) and (2);
(10) a polynucleotide comprising (1) and (3); or
(11) a polynucleotide comprising (1) and (4)
According to specific embodiments, the AimR polynucleotide is upstream to the AimP polynucleotide.
According to specific embodiments, the AimR polynucleotide is upstream to the AimR responsive element polynucleotide.
According to specific embodiments, the AimR polynucleotide is upstream to the AimX polynucleotide.
According to specific embodiments, the AimR responsive element is upstream to the AimX polynucleotide.
According to specific embodiments, the AimP polynucleotide is upstream to the AimR responsive element polynucleotide.
According to specific embodiments, the AimP polynucleotide is upstream to the Aimx polynucleotide.
As used herein, the terms “peptide” and “polypeptide”, which are interchangeably used, encompass native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to N terminus modification, C terminus modification, peptide bond modification, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
Peptide bonds (—CO—NH—) within the peptide may be substituted, for example, by N-methylated amide bonds (—N(CH3)-CO—), ester bonds (—C(═O)—O—), ketomethylene bonds (—CO—CH2-), sulfinylmethylene bonds (—S(═O)—CH2-), α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl (e.g., methyl), amine bonds (—CH2-NH—), sulfide bonds (—CH2-S—), ethylene bonds (—CH2-CH2-), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds (—CS—NH—), olefinic double bonds (—CH═CH—), fluorinated olefinic double bonds (—CF═CH—), retro amide bonds (—NH—CO—), peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally present on the carbon atom.
These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) bonds at the same time.
Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted by non-natural aromatic amino acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe or O-methyl-Tyr.
The peptides of some embodiments of the invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
The term “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids.
Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non-conventional or modified amino acids (e.g., synthetic, Table 2), which can be used with some embodiments of the invention.
The peptides of some embodiments of the invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
The peptides of some embodiments of the invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
The amino acids of the peptides of the present invention may be substituted either conservatively or non-conservatively.
The term “conservative substitution” as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).
As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions.
For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.
When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.
The phrase “non-conservative substitutions” as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or —NH—CH [(—CH2)5—COOH]—CO— for aspartic acid. Those non-conservative substitutions which fall under the scope of the present invention are those which still constitute a peptide having anti-bacterial properties.
The N and C termini of the peptides of the present invention may be protected by function groups. Suitable functional groups are described in Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those that facilitate transport of the compound attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the compounds.
The peptides of the present invention may be attached (either covalently or non-covalently) to a penetrating agent.
As used herein the phrase “penetrating agent” refers to a heterologous agent which enhances translocation of any of the attached peptide across a cell membrane.
According to one embodiment, the penetrating agent is a peptide and is attached to the peptide (either directly or non-directly) via a peptide bond.
Typically, peptide penetrating agents have an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. Non-limiting examples of CPPs that can penetrate cells in a non-toxic and efficient manner and may be suitable for use in accordance with some embodiments of the invention include TAT (transcription activator from HIV-1), pAntp (also named penetratin, Drosophila antennapedia homeodomain transcription factor) and VP22 (from Herpes Simplex virus). Protocols for producing CPPs-cargos conjugates and for infecting cells with such conjugates can be found, for example L Theodore et al. [The Journal of Neuroscience, (1995) 15(11): 7158-7167], Fawell S, et al. [Proc Natl Acad Sci USA, (1994) 91:664-668], and Jing Bian et al. [Circulation Research. (2007) 100: 1626-1633].
The peptides of the present invention may also comprise non-amino acid moieties, such as for example, hydrophobic moieties (various linear, branched, cyclic, polycyclic or hetrocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptides; non-peptide penetrating agents; various protecting groups, especially where the compound is linear, which are attached to the compound's terminals to decrease degradation. Chemical (non-amino acid) groups present in the compound may be included in order to improve various physiological properties such; decreased degradation or clearance; decreased repulsion by various cellular pumps, improve immunogenic activities, improve various modes of administration (such as attachment of various sequences which allow penetration through various barriers, through the gut, etc.); increased specificity, increased affinity, decreased toxicity and the like.
Attaching the amino acid sequence component of the peptides of the invention to other non-amino acid agents may be by covalent linking, by non-covalent complexion, for example, by complexion to a hydrophobic polymer, which can be degraded or cleaved producing a compound capable of sustained release; by entrapping the amino acid part of the peptide in liposomes or micelles to produce the final peptide of the invention. The association may be by the entrapment of the amino acid sequence within the other component (liposome, micelle) or the impregnation of the amino acid sequence within a polymer to produce the final peptide of the invention.
The peptides of some embodiments of the invention may be synthesized and purified by any techniques known to those skilled in the art of peptide synthesis, such as, but not limited to, solid phase techniques and recombinant techniques such as further described hereinbelow.
As used herein, the term “polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
As the novel “arbitrium” system is involved in controlling integration of DNA into a genome of a host the present teachings suggest that components of the “arbitrium” system described herein can be used for controlling integration of expression product of interest into a target genome.
Thus, according to specific embodiments, the polynucleotides of the present invention comprise a nucleic acid sequence encoding an expression product of interest.
As used herein the term “expression product of interest” refers to a RNA and/or protein of interest.
According to specific embodiments, expression and/or activity of the expression product of interest is dependent on AimX; i.e. expression of AimX controls expression and/or activity of the expression product of interest.
According to specific embodiments, the expression product of interest is a therapeutic expression product such as an antibody, a growth factor, a cytokine etc.
According to specific embodiments, the expression product of interest is a DNA editing agent.
According to specific embodiments, the DNA editing agent expression and/or activity is dependent on AimX; i.e. expression of AimX controls expression and/or activity of the DNA editing agent.
As used herein, the term “DNA editing agent” refers to an agent capable of introducing sequence alterations in the genome of a cell. These targeted sequence alterations may involve loss-of function or gain of function alterations. Non-limiting examples of such alterations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby modulates the activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a protein with modulated activity; a readthrough mutation due to a frame-shift mutation or a modified stop codon mutation (i.e., when the stop codon is mutated into an amino acid codon), with a modulated activity; a deletion mutation, i.e., a mutation which deletes coding nucleic acids in a gene sequence and which may result in a frame-shift mutation or an in-frame mutation (within the coding sequence, deletion of one or more amino acid codons); an insertion mutation, i.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence, and which may result in a frame-shift mutation or an in-frame insertion of one or more amino acid codons; an inversion, i.e., a mutation which results in an inverted coding or non-coding sequence; a splice mutation i.e., a mutation which results in abnormal splicing or poor splicing; and a duplication mutation, i.e., a mutation which results in a duplicated coding or non-coding sequence, which can be in-frame or can cause a frame-shift.
According to specific embodiments DNA sequence alteration of a gene may comprise at least one allele of the gene.
According to other specific embodiments alteration of a gene sequence comprises both alleles of the gene. In such instances gene may be in a homozygous form or in a heterozygous form.
According to specific embodiments the DNA editing agent allows for the integration of exogenous nucleic acid sequences into the genome.
Thus, according to specific embodiments, the polynucleotides of some embodiments of the present invention comprise a nucleic acid sequence to be integrated into a genome of a cell by the DNA editing agent.
Methods of DNA genome editing are well known in the art [see for example Francisco Martin et al. “New Vectors for Stable and Safe Gene Modification” in Gene Therapy-Developments and Future Perspectives (2011) DOI: 10.5772/10622; Menke D. Genesis (2013) 51: —618; Capecchi, Science (1989) 244:1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; U.S. Pat. Nos. 8,771,945, 8,586,526, 6,774,279 and UP Patent Application Publication Nos. 20030232410, 20050026157, US20060014264; the contents of which are incorporated by reference in their entireties] and include, but not limited to integrases and engineered nucleases. Agents for DNA genome editing can be designed publically available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences.
According to specific embodiments, the DNA editing agent is an integrase.
As used herein, the term “integrase” refers to a recombinase that is capable of integrating a nucleic acid sequence into another nucleic acid sequence (e.g., a genome of a cell). According to specific embodiments, the integrase is a site-specific recombinase i.e. leads to integration of sequences between two nucleic acids, each nucleic acid comprising at least one recognition site for the recombinase. Such integrases are known in the art [see e.g. in Francisco Martin et al. Gene Therapy—Developments and Future Perspectives (2011) DOI: 10.5772/10622, Recchia A et al. Curr Gene Ther. (2011) 11(5): 399-405, Lim K I, BMB Rep. (2015) 48(1):6-12 and US Patent Application publication No. US20070190601; the contents of which are incorporated by reference in their entireties] and include for example a retroviral integrase (HIV integrase) and phage integrase (e.g. phi C31 integrase, lambda integrase).
According to specific embodiments the DNA editing agent is an engineered endonucleases.
Genome editing using engineered endonucleases such as meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system, is well known in the art and described for example in International Patent Application Publication No. WO2015/033343, the contents of which are incorporated herein by reference in their entirety.
According to other specific embodiments, the DNA editing agent is selected from the group consisting of zinc finger nuclease, an effector protein of Class 2 CRISPR/Cas (e.g. Cas9, Cpf1, C2c1, C2c3) and TALEN.
According to specific embodiments, the polynucleotides of the present invention are part of a nucleic acid construct (also referred to herein as an “expression vector”).
As used herein, the terms “nucleic acid construct” and “expression vector” refer to a nucleic acid vector designed to introduce specific expression products of interest (i.e. genes) in host cell. The expression can be transient or consistent, episomal or integrated into the chromosome of the host cell. According to specific embodiments, the expression is on a transmissible genetic element such as a plasmid.
Hence, the nucleic acid construct of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vector may contain regulatory elements e.g. promoters, enhancers, transcription and translation initiation sequence, transcription and translation terminator, polyadenylation signal transcription termination signals etc and at least one multiple cloning site (MCS) for cloning of expression products of interest. By way of example, such constructs will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof.
Thus, according to an aspect of the present invention, there is provided a nucleic acid construct comprising a polynucleotide encoding an AimP peptide, a polynucleotide encoding an AimR polypeptide, a polynucleotide comprising an AimR responsive element and/or an AimX polynucleotide and any combination of same; and a MCS.
According to another aspect of the present invention, there is provided a nucleic acid construct comprising a polynucleotide encoding an AimP peptide, a polynucleotide encoding an AimR polypeptide, a polynucleotide comprising an AimR responsive element and/or an AimX polynucleotide and any combination of same; and a cis-acting regulatory element heterologous to AimP, AimR, AimR responsive element and/or AimX for directing expression of the polynucleotide.
Thus, according to specific embodiments, the nucleic acid construct may comprise any of the polynucleotides (1)-(11) described hereinabove.
Teachings of the invention further contemplate that the polynucleotides are part of a nucleic acid construct system whereby the components of the arbitrium system are expressed from different constructs.
Thus, according to specific embodiments, the present invention provides for a nucleic acid construct system comprising at least two nucleic acid constructs each expressing at least one of the polynucleotides of the present invention.
Thus according to specific embodiments, the nucleic acid construct system comprises an individual nucleic acid construct for each polynucleotide of the present invention (i.e. AimP, AimR, AimR responsive element and AimX).
According to other specific embodiments a single construct comprises a number of polynucleotide of the present invention, as described hereinabove.
According to specific embodiments, the nucleic acid construct system comprises at least one construct allowing integration of the polynucleotide into the chromosome of the cell and at least one construct allowing episomal expression of the polynucleotide.
As used herein, the term “multiple cloning site (MCS)” refers to a nucleic acid sequence comprising at least one restriction site, and more typically a number of restriction sites, for the purpose of cloning nucleic acid sequences into an expression vector. The MCS is recognized and digested by a specific restriction enzyme, such that a target expression product of interest can be inserted into the digested MCS site. Any MCS known in the art can be used in the nucleic acid constructs of the present invention. It can be obtained from various commercially available vectors known in the art having MCS (e.g., pUC18, pUC19, etc.).
Cis acting regulatory sequences include those that direct constitutive expression of a nucleotide sequence as well as those that direct inducible expression of the nucleotide sequence only under certain conditions.
The cis regulatory sequences of the present invention are heterologous to the polynucleotides of the present invention (i.e. AimP, AimR, AimR responsive element and AimX).
As used herein, the term “heterologous” means derived from a different genetic location. For example, a polynucleotide may be placed by genetic engineering techniques into a vector derived from a different source, and is a heterologous polynucleotide; a promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
According to specific embodiments, the nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.
Preferably, the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed. Examples of cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264, 166).
Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.
According to specific embodiments, the promoter is a viral (e.g. phage) promoter.
According to specific embodiments the promoter is a bacterial promoter.
In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.
In addition to the elements already described, the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell. The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
The expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA and sequences for genomic integration of the promoter-chimeric polypeptide, as described in details herein above and below.
According to specific embodiments, the construct encodes a polycistronic mRNA comprising the polynucleotides of the present invention.
Various construct schemes can be utilized to express few genes from a single nucleic acid construct. According to specific embodiments, the construct encodes a polycistronic mRNA comprising the polynucleotides of the present invention; that is the polynucleotides can be co-transcribed as a polycistronic message from a single promoter sequence of the nucleic acid construct. To enable co-translation of all the genes from a single polycistronic message, the different polynucleotide segments can be transcriptionally fused via a linker sequence including an internal ribosome entry site (IRES) sequence which enables the translation of the polynucleotide segment downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule including the coding sequences of different combinations of the polynucleotides of the present invention will be translated from both the capped 5′ end and the internal IRES sequence of the polycistronic RNA molecule.
Alternatively, each two polynucleotide segments can be translationally fused via a protease recognition site cleavable by a protease expressed by the cell to be transformed with the nucleic acid construct. In this case, a chimeric polypeptide translated will be cleaved by the cell expressed protease.
Still alternatively, the nucleic acid construct of some embodiments of the invention can include at least two promoter sequences each being for separately expressing a distinct polynucleotide. These at least two promoters which can be identical or distinct can be constitutive, tissue specific or regulatable (e.g. inducible) promoters functional in one or more cell types.
When secretion of the polypeptides is desired the polynucleotides of the invention can be expressed as fusion polypeptides comprising the nucleic acid sequence encoding e.g. the components of the “arbitrium” system (e.g. AimP) ligated in frame to a nucleic acid sequence encoding a signal peptide that provides for secretion. According to specific embodiments, the signal sequence is an N-terminal signal sequence. According to specific embodiments, the signal peptide is cleaved upon peptide secretion.
DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) (Masui et al. (1983) FEBS Lett. 151(1):159-164; Ghrayeb et al. (1984) EMBO J. 3:2437-2442) and the E. coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212). Other prokaryotic signals include, for example, the signal sequence from penicillinase, Ipp, or heat stable enterotoxin II leaders and signal sequences of the Phr family of quorum sensing systems [described e.g. in Pottathil, M. & Lazazzera, B. A. Front. Biosci. 8, d32-45 (2003)].
According to specific embodiments, the signal sequence is as set forth in SEQ ID NO: 342.
Selectable marker genes that ensure maintenance of the vector in the cell can also be included in the expression vector. Preferred selectable markers include those which confer resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev. Microbiol. 32:469). Selectable markers can also allow a cell to grow on minimal medium or in the presence of toxic metabolite and can include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed polypeptide. Thus, for example, the peptide of some embodiments of the invention (e.g. AimP) may be a pro-peptide containing a sequence processed by extracellular proteases to produce the mature peptide. According to specific embodiments the signal for extracellular processing is as set forth in SEQ ID NO: 348.
Or, for example, the expression of a fusion protein or a cleavable fusion protein comprising the protein of some embodiments of the invention (e.g. AimP) and a heterologous protein can be engineered. Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein. Where a cleavage site is engineered between the protein of some embodiments of the invention and the heterologous protein, the protein of some embodiments of the invention can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site [e.g., see Booth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].
When needed, recovery of the recombinant polypeptide is effected following an appropriate time in culture. The phrase “recovering the recombinant polypeptide” refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification. Notwithstanding the above, polypeptides of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.
Where appropriate, the polynucleotides may be optimized for increased expression in the transformed organism. For example, the polynucleotides can be synthesized using preferred codons for improved expression.
It will be appreciated that the individual elements comprised in the expression vector can be arranged in a variety of configurations. For example, enhancer elements, promoters and the like, and even the polynucleotide sequence(s) encoding components of the “arbitrium” system can be arranged in a “head-to-tail” configuration, may be present as an inverted complement, or in a complementary configuration, as an anti-parallel strand. While such variety of configuration is more likely to occur with non-coding elements of the expression vector, alternative configurations of the coding sequence within the expression vector are also envisioned.
Examples for mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
Examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).
In yeast, a number of vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. No. 5,932,447. Alternatively, vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.
In cases where plant expression vectors are used, the expression of the coding sequence can be driven by a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter to TMV [Takamatsu et al. (1987) EMBO J. 6:307-311] can be used. Alternatively, plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley et al. (1986) Mol. Cell. Biol. 6:559-565] can be used. These constructs can be introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.
Other expression systems such as insects and mammalian host cell systems which are well known in the art can also be used by some embodiments of the invention.
Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. Thus, the type of vector used by some embodiments of the invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein. Recombinant viral vectors are useful for in vivo expression of the polynucleotides and polypeptides of some embodiments of the present invention since they offer advantages such as lateral infection and targeting specificity. Various methods can be used to introduce the polynucleotides, expression vectors and polypeptides of some embodiments of the invention into cells. The polynucleotides and nucleic acid construct described herein can be introduced into cells by any method known in the art, as further described in details hereinbelow. Alternatively or additionally, the polypeptides described herein can be contacted with the cell per se.
It will be appreciated that the cell may be comprised inside a particular organism, for example inside a mammalian body or inside a plant.
Thus, according to a specific embodiment introducing and/or contacting is effected in-Vivo.
According to another specific embodiment introducing and/or contacting is effected ex-vivo or in-vitro.
Various methods can be used to introduce the polynucleotides and expression vectors of some embodiments of the invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)]. The most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct. Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
Regardless of the method of introduction, the present teachings provide for an isolated cell which comprises the polynucleotides, the nucleic acid constructs and/or the polypeptides, as described herein.
As used herein, the term “cell” refers to a prokaryotic or a eukaryotic cell. Non-limiting examples of cells that can be used in some embodiments of the present invention include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence.
According to specific embodiments the cell is a bacteria.
According to specific embodiments the cell is a mammalian cell.
According to specific embodiments, the mammalian cell is selected from the group consisting of a Chinese Hamster Ovary (CHO), HEK293, PER.C6, HT1080, NS0, Sp2/0, BHK, Namalwa, COS, HeLa and Vero cell.
According to another specific embodiment, the cell is a primary cell.
According to a specific embodiment, the cell is a cell line.
The cell may be derived from a suitable tissue including but not limited to blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testis, ovary, hair, skin, bone, breast, uterus, bladder, spinal cord, or various kinds of body fluids. According to specific embodiment the cell does not express the polynucleotides and/or the polypeptides of the present invention endogenously.
According to specific embodiments, the cell expresses AimR endogenously.
The term “endogenous” as used herein, refers to the expression of the native gene in its natural location and expression level in the genome of an organism.
As shown in the Examples section which follows the arbitrium system is a genetic system that uses peptides to control the integration of specific DNA into a specific target site. Thus, the present teachings suggest the use of the polynucleotides, the nucleic acid construct and/or the polypeptides described hereinabove for inducible gene expression in general and to control genome editing in particular.
Thus, according to an aspect of the present invention, there is provided a method of expressing an expression product of interest, the method comprising:
(i) introducing into a cell a polynucleotide comprising an AimR responsive element operatively linked to a nucleic acid sequence encoding the expression product of interest, wherein said AimR comprises a DNA binding domain for binding said AimR responsive element, said AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain; and
(ii) contacting said cell with an AimP peptide comprising an amino acid sequence of XXXXGG/A, wherein said AimP peptide is capable of binding said AimR polypeptide and dissociating said AimR polypeptide from an AimR responsive element,
thereby expressing the expression product of interest.
According to another aspect of the present invention, there is provided a method of expressing an expression product of interest, the method comprising introducing into a cell a polynucleotide comprising an AimR responsive element operatively linked to a heterologous nucleic acid sequence encoding the expression product of interest, wherein said AimR comprises a DNA binding domain for binding said AimR responsive element, said AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain, thereby expressing the expression product of interest.
According to specific embodiments, the method comprising introducing into the cell a polynucleotide encoding AimR.
According to another aspect of the present invention, there is provided a method of expressing an expression product of interest, the method comprising introducing into a cell a polynucleotide comprising an AimR responsive element operatively linked to a nucleic acid sequence encoding the expression product of interest; and a nucleic acid construct comprising an AimR polynucleotide and a cis-acting regulatory element heterologous to said AimR for directing expression of said AimR polynucleotide,
wherein said AimR comprises a DNA binding domain for binding said AimR responsive element, said AimR comprising a helix-turn-helix (HTH) motif and a tetratricopeptide repeat (TPR) domain, thereby expressing the expression product of interest.
According to specific embodiments, the method comprising contacting the cell with an AimP peptide or a nucleic acid sequence encoding same.
According to specific embodiments, the method comprising contacting the cell with an agent capable of downregulating expression and/or activity of said AimR responsive element.
As used herein the phrase “dowregulating expression” refers to dowregulating the expression at the genomic (e.g. homologous recombination and site specific endonucleases) and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression) or on the protein level (e.g., aptamers, small molecules and inhibitory peptides, antagonists, enzymes that cleave the polypeptide, antibodies and the like).
According to specific embodiments, the downregulating agent is a nucleic acid agent.
According to specific embodiments, the downregulating agent is an effector protein of Class 2 CRISPR/Cas (e.g. Cas9, Cpf1, C2c1, C2c3).
According to specific embodiments, the downregulating agent is a crRNA or a sgRNA of a CRISPR/Cas system.
According to specific embodiments, the downregulating agent is an RNAi.
According to specific embodiments, down regulating expression refers to the absence of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively.
According to other specific embodiments down regulating expression refers to a decrease in the level of mRNA and/or protein, as detected by RT-PCR or Western blot, respectively as compared to same in the absence of the downregulating agent. The reduction may be by at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% reduction.
As the CRISPR/Cas system used by the present inventors for downregulating expression and/or activity of AimX disclosed in the Examples section which follows is novel; according to an aspect of the present invention there is provided an isolated nucleic acid agent capable of downregulating expression and/or activity of an AimR responsive element.
According to specific embodiments, the downregulating agent is capable of downregulating expression and/or activity of an AimX.
As used herein, “expressing” or “expression” refers to gene expression at the nucleic acid and/or protein level. Expression can be determined using methods known in the art e.g. but not limited to selectable marker gene, Northern blot analysis, PCR analysis, DNA sequencing, RNA sequencing, Western blot analysis, and Immunohistochemistry.
When expression of the expression product of interest results in modulated activity, qualifying efficacy of DNA integration can also be determined by determining activity.
In addition, one ordinarily skilled in the art can readily design a knock-in/knock-out construct including positive and/or negative selection markers for efficiently selecting transformed cells that underwent DNA integration event with the polynucleotide or construct. Positive selection provides a means to enrich the population of clones that have taken up foreign DNA. Non-limiting examples of such positive markers include Human influenza hemagglutinin (HA) tag, glutamine synthetase, dihydrofolate reductase (DHFR), His-tag, FLAG peptide, and markers that confer antibiotic resistance, such as neomycin, hygromycin, puromycin, and blasticidin S resistance cassettes. Negative selection markers are necessary to select against random integrations and/or elimination of a marker sequence (e.g. positive marker). Non-limiting examples of such negative markers include the herpes simplex-thymidine kinase (HSV-TK) which converts ganciclovir (GCV) into a cytotoxic nucleoside analog, hypoxanthine phosphoribosyltransferase (HPRT) and adenine phosphoribosytransferase (ARPT).
According to specific embodiments, the expression product of interest is endogenous to the cell.
According to other specific embodiments, the expression product of interest is exogenous to the cell.
As mentioned above the expression product of interest may be a DNA editing agent. Thus, according to specific embodiments the method comprising introducing into the cell a nucleic acid sequence to be integrated into a genome of the cell by the DNA editing agent. Methods for qualifying efficacy and detecting integration of a nucleic acid sequence into the genome of the cell are well known in the art and include, but not limited to, DNA sequencing, electrophoresis, an enzyme-based mismatch detection assay and a hybridization assay such as PCR, RT-PCR, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis.
According to another aspect there is provided an article of manufacture identified for expressing an expression product of interest comprising a packaging material packaging at least two of:
(i) a polynucleotide comprising an AimR responsive element;
(ii) a polynucleotide encoding said AimR;
(iii) an AimP peptide; and/or
(iv) an agent capable of downregulating expression and/or activity of said AimR responsive element.
The article of manufacture may comprise two, three or all; i.e. (i)+(ii), (i)+(iii), (i)+(iv), (ii)+(iii), (ii)+(iv), (i)+(ii)+(iii), (ii)+(iii)+(iv), (i)+(ii)+(iv), (i)+(iii)+(iv) or (i)+(ii)+(iii)+(iv).
According to another aspect there is provided an article of manufacture identified for expressing an expression product of interest comprising a packaging material packaging:
According to specific embodiments, the polynucleotide encoding the AimR is comprised in a nucleic acid construct comprising a cis-acting regulatory element heterologous to the AimR for directing expression of the AimR polynucleotide.
According to specific embodiment, the article of manufacture comprises a multiple cloning site (MCS).
According to specific embodiment, the article of manufacture comprises a polynucleotide encoding the expression product of interest.
According to specific embodiments of these aspects of the present invention the (i), (ii) (iii) and/or (iv) or the (a) and (b) are packaged in separate containers.
According to yet other specific embodiments of these aspects of the present invention the (i), (ii) (iii) and/or (iv) or the (a) and (b) are in c-formulation.
It is expected that during the life of a patent maturing from this application many relevant DNA editing agents will be developed and the scope of the term DNA editing agent is intended to include all such new technologies a priori.
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
Bacillus
amyloli-
quefaciens
plantarum
Bacillus
subtilis
Bacillus
Bacillus
subtilis
subtilis
Bacillus
subtilis
Bacillus
subtilis
Bacillus
subtilis
Bacillus
subtilis
subtilis
Bacillus
amyloli-
quefaciens
amyloli-
quefaciens
Bacillus
subtilis
Bacillus
murimartini
Bacillus
subtilis
Bacillus
subtilis
subtilis
Bacillus
subtilis
subtilis
Bacillus
subtilis
Bacillus
subtilis
subtilis
Bacillus
subtilis
subtilis
Bacillus
subtilis
inaquosorum
Bacillus
subtilis
subtilis
Bacillus
subtilis
Bacillus
subtilis
Bacillus
subtilis
subtilis
Bacillus
subtilis
Bacillus
subtilis
subtilis
Bacillus
subtilis
Bacillus
subtilis
Bacillus
subtilis
subtilis
Bacillus
subtilis
Bacillus
subtilis
subtilis
Bacillus
glycini-
fermentans
Bacillus
amyloli-
quefaciens
Bacillus
pumilus
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
subtilis
Paeni-
bacillus
polymyxa
Bacillus
atrophaeus
Bacillus
atrophaeus
Bacillus
atrophaeus
globigii
Bacillus
atrophaeus
globigii
Bacillus
atrophaeus
globigii
Bacillus
atrophaeus
Bacillus
atrophaeus
Bacillus
atrophaeus
globigii
Bacillus
amyloli-
quefaciens
plantarum
Bacillus
amyloli-
quefaciens
Bacillus
subtilis
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
methylo-
trophicus
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
plantarum
Bacillus
amyloli-
quefaciens
plantarum
Bacillus
subtilis
Bacillus
subtilis
subtilis
Bacillus
methylo-
trophicus
Bacillus
methylo-
trophicus
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
plantarum
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
plantarum
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
lichen-
iformis
Bacillus
Bacillus
sonorensis
Bacillus
lichen-
iformis
Bacillus
lichen-
iformis
Bacillus
Bacillus
lichen-
iformis
Bacillus
Bacillus
lichen-
iformis
Bacillus
lichen-
iformis
Bacillus
subtilis
Bacillus
atrophaeus
Bacillus
subtilis
Bacillus
amyloli-
quefaciens
Bacillus
subtilis
Bacillus
subtilis
inaquosorum
Bacillus
subtilis
Bacillus
aerophilus
Bacillus
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
subtilis
Bacillus
cellulo-
silyticus
Bacillus
subtilis
subtilis
Bacillus
amyloli-
quefaciens
Bacillus
subtilis
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
planatarum
Bacillus
amyloli-
quefaciens
planatarum
Bacillus
amyloli-
quefaciens
planatarum
Bacillus
subtilis
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
amyloli-
quefaciens
Bacillus
vallis-
mortis DV1-
Bacillus
atrophaeus
Bacillus
atrophaeus
Bacillus
atrophaeus
Bacillus
atrophaeus
Bacillus
atrophaeus
Bacillus
subtilis
Bacillus
subtilis
Bacillus
atrophaeus
Bacillus
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells— A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Oligos and Reagents—
All oligos were purchased from Sigma (St. Louis, Mo.) or Integrated DNA Technologies (IDT, San Jose, Calif.). Synthetic peptides were purchased from Peptide 2.0 Inc. (Chantilly, Va.), at 98% purity, desalted.
Preparation of Conditioned Media—
A schematic representation of the procedure is shown in
Proteinase K Treatment—
7.5 mg (per reaction) of Proteinase K-Agarose from Tritirachium album (Sigma, CAT # P9290) were washed twice with 750 μl of sterile water and then resuspended with 750 μl of LB supplemented with 0.1 mM MnCl2 and 5 mM MgCl2. Following, the tubes were centrifuged again and the supernatant was discarded. 1.5 ml of phi3T-derived conditioned medium or control medium was added to a tube containing the washed proteinase K. The media were incubated for 2 hours at 37° C. with the proteinase K. The media were centrifuged and the supernatants were collected for the infection assay.
Growth Dynamics of Phage-Infected Cultures—
Overnight cultures of bacteria were diluted 1:100 in LB media and incubated at 37° C. with shaking until reaching O.D 600 nm=0.1. The bacterial culture was centrifuged at 4000 rpm for 10 minutes at room temperature. The supernatant was discarded and the pellet was resuspended in LB medium supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 at 10% of the initial volume. The concentrated bacterial culture was added to conditioned medium or medium supplemented with synthesized arbitrium peptide in a ratio of 1:9 (bacteria to medium) and incubated for 1 hour at room temperature. Following, the culture was infected with phages at MOI=0.1. Optical density measurements at a wavelength of 600 nm were taken using a TECAN Infinite 200 μlate reader in a 96-wells plate. For infection experiments that did not include conditioned medium or addition of a synthesized peptide, the diluted overnight culture was grown to early-logarithmic phase and then infected as described above.
Semi Quantitative PCR Assay for Lysogeny—
An overnight culture of bacteria was diluted 1:100 until reaching O.D 600 nm=0.1. Medium was replaced (with conditioned medium or control medium) as described above, and the culture was incubated for 1 hour at room temperature. Bacteria were infected by phi3T at MOI=5. Cell pellets were collected at 0, 15, 30, 40 and 60 minutes post infection in the presence of conditioned or control medium. DNA was extracted using DNeasy blood and tissue kit (CAT #69504). Multiplex PCR assays to detect phage phi3T DNA, B. subtilis DNA, and the junction between integrated phage and bacterial genome were performed as previously described at Goldfarb et al29.
Mass Spec—
Conditioned media was filtered using 3 kDa MW cutoff filters (Millipore) and the low molecular weight fraction was desalted using the Oasis HLB uElution plates (Waters Corp.). Samples were dried and stored at −80° C. until analysis. ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nano-Ultra Performance Liquid Chromatography (10 kpsi nanoAcquity; Waters, Milford, Mass., USA). The mobile phase was: A) H2O+0.1% formic acid and B) acetonitrile+0.1% formic acid. Samples' desalting was performed online using a reversed-phase C18 trapping column (180 μm internal diameter, 20 mm length, 5 μm particle size; Waters). Following, the peptides were separated using a T3 HSS nano-column (75 μm internal diameter, 250 mm length, 1.8 μm particle size; Waters) at 0.35 μL/min. The peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 35% B in 65 min, 35% to 90% B in 5 min, maintained at 90% for 5 min and then back to initial conditions. The nanoUPLC was coupled online through a nanoESI emitter (10 μm tip; New Objective; Woburn, Mass., USA) to a quadrupole orbitrap mass spectrometer (Q Exactive Plus, Thermo Scientific) using a Flexlon nanospray apparatus (Proxeon). Data was acquired in parallel reaction monitoring (PRM) mode, targeting precursor masses 574.33 and 287.67, the singly and doubly charged forms of peptide SAIRGA (SEQ ID NO: 269). MS2 resolution was set to 35,000 and the maximum injection time set to 200 msec, automatic gain control was set to 2e5. Raw data was imported to Skyline software30 version 3.5. Product ion intensities were extracted and the total area under the curve was calculated.
AimR Purification—
AimR (SEQ ID NO: 1) was cloned into the expression vector pET28a (Novagen) using Transfer-PCR (TPCR)31, using the following primers:
Following, AimR was expressed in E. coli BL21(DE3) cells with a C-terminal 6× His-tag. Expression was performed at 15° C. for about ˜18 hours using 200 μM IPTG (Isopropyl β-D-1-thiogalactopyranoside) as an inducer. The cells pellet was resuspended in lysis buffer [50 mM Tris pH 8, 0.3M NaCl, 20 mM Imidazole, 2 mM DTT, 0.2 mg/ml Lysozyme, 1 μg/ml DNAse, protease inhibitor cocktail (Calbiochem)], disrupted by a cell disrupter at 4° C. and clarified at 15,000 g for 30 minutes. The clarified lysate was loaded onto a HisTrap_FF_5 ml column (GE Healthcare) and washed with buffer containing 50 mM Tris pH 8, 0.3 M NaCl, 20 mM imidazole and 2 mM DTT. AimR was eluted from the column in one step with the same buffer containing 0.5 M imidazole. Fractions containing AimR were pooled and injected to a size exclusion column (HiLoad_16/60_Superdex_200_prepgrade,GE_Healthcare) equilibrated with 20 mM Tris pH 8, 0.3 M NaCl, 2 mM TCEP. Fractions containing pure AimR were pooled and flash frozen in aliquots using liquid nitrogen.
Pure AimR was injected to an analytical gel filtration column (Superdex_200_Increase_10/30 GL, GE Healthcare) equilibrated with buffer containing 20 mM Tris pH 8, 0.3 M NaCl, 2 mM TCEP. The migration position of pure AimR was compared to that of AimR-peptide mixtures at the following molar ratios: AimR and SAIRGA (SEQ ID NO: 269) peptide (1:2), AimR and GMPRGA (SEQ ID NO: 271) peptide (1:1). The column was calibrated (inset of
Microscale Thermophoresis (MST)—
Two-step purified 6×His-tagged AimR stored in Tris/NaCl buffer (50 mM Tris pH 8.0, 150 mM NaCl, 2 mM TCEP) at −80° C. was thawed on ice and centrifuged at 21,000 g for 10 minutes at 4° C. prior to analysis. Peptides [SAIRGA (SEQ ID NO: 269) and GMPRGA (SEQ ID NO: 271)] were solubilized in 50 mM Tris-HCl pH 8.0, 150 mM NaCl to a final concentration of 100 μM. AimR was diluted to 200 nM and was incubated with 16 different peptide concentrations varying between 9-4000 nM, which were prepared in Tris/NaCl buffer containing 0.1% [v/v] Pluronic acid (NanoTemper). Roughly 3 μl were loaded into NT.LabelFree Zero-Background Premium Coated Capillaries (NanoTemper) and inserted into a Monolith NT.LabelFree device (NanoTemper). MST experiments were performed at 60% MST power (infra-red laser) and 20% LED power at 23° C. using the Monolith NT.LabelFree instrument (Nanotemper). Ratios between normalized initial fluorescence and post-temperature-jump and thermophoresis were calculated and averaged from 3 independent runs (runs were incubated for 20 minutes at room temperature before the measurement). A plot of fluorescence ratios versus peptide concentration was used to assess the binding capacity of the phage protein and its cognate peptide ligand.
ChIP-Seq—
For the ChIP-seq experiments, B. subtilis cell cultures were grown in 100 ml to O.D of 0.1 in LB at 37° C. Following, 50 ml of culture was centrifuged at 4000 rpm for 10 minutes at room temperature. The pellets were then suspended with 25 ml LB either containing or lacking the peptide (SAIRGA, SEQ ID NO: 269) at a final concentration of 1 μM. The cultures were placed for an additional incubation hour at room temperature with shaking. The cultures were then infected with phage (MOI=0.5) having a final plaque forming units (PFU) of 5×107 PFU/ml phages. 15 minutes post phage infection the cultures were centrifuged at 3000 g for 5 minutes at 4° C. The supernatant was discarded and the pellets were resuspended with 1 ml of ice-cold 1×PBS (10 mM Phosphate, 137 mM NaCl, 2.7 mM KCl, pH of 7.4).
For the formaldehyde fixation of protein to DNA, the 1 ml PBS resuspended pellets were mixed with 62.5 μl formaldehyde (Thermoscientific 16% formaldehyde solution (w/v) methanol-free ampule) yielding a final formaldehyde concentration of 1% (w/v) within the solution. The formaldehyde-containing cell suspension was incubated at room temperature for 10 minutes with mild agitation. Following, 75 μl 2M Glycine (f.c. 150 mM) was added to quench residual formaldehyde. Glycine-containing samples were kept on ice for an additional 10 minutes followed by centrifugation at 5500 g for 1 minute at 4° C. The supernatants were discarded and the pellets were washed with a 1 ml of ice-cold 1×PBS. Centrifugation and wash were repeated 3 times for each sample.
The cell pellets were suspended with 600 μl lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl and a protease inhibitor mix (cOmplete ULTRA Tablets Roche). The lysis-buffer-containing cells were applied on a lysing matrix B (MP Biomedicals) 0.1 mm silica beads. The mixture and beads were placed in a FastPrep-24 (MP Biomedicals) apparatus and shaken aggressively for 20 seconds 6 m/sec at 4° C. The beads were then separated from lysed cells by centrifugation at 10,000 g for 1 minutes at 4° C. according to manufacturer's instructions. Following, 300 μl of the supernatant, containing the lysed cell mixture, were transferred into a 1.5 ml Bioruptor® Plus TPX microtubes (diagenode) and kept on ice for 10 minute (according to manufacturer's instructions). The samples were sonicated at 4° C. with full power for 15 minutes (30 seconds off/on cycles) using the BioRuptor plus (Diagenode) apparatus. Sonication sheared the DNA to an average size of ˜500 bp. Following sonication, samples were centrifuge at 20,000 g for 10 minutes at 4° C.
For the IP experiments, supernatant containing the lysis buffer and cellular content was mixed with Triton X-100 and deoxycholate yielding a final IP-buffer composition containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% [vol/vol] Triton X-100, 0.1% [wt/vol] sodium deoxycholate supplemented with proteases inhibitors (cOmplete ULTRA Tablets Roche). Anti-6×His Tag® ChIP-grade antibody (abcam (ab9108)) was then added to the sonicated samples and gently mixed over night at 4° C. In parallel, Protein G Dynabeads (100.04 D; Invitrogen) were washed three times with IP buffer.
DNA-protein-antibody complexes (300 μl) were captured with a 100 μl Dynabeads protein G by mixing them for 1 hour at room temperature with rotation. 0.72 μl of 0.5M EDTA (f.c. 1 mM) was added to that mixture to prevent DNase activity at room temperature. Beads were applied to a magnetic stand (Qiagen) and washed three times with IP buffer (200 μl) at room temperature. Two elution steps were applied with 100 μl and 50 μl of elution buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% [wt/vol] SDS) for 15 minutes at 65° C. on a rocking platform. The eluate (100 μl) was incubated with 5 μl of proteinase K (20 mg ml−1) for 1 hour at 50° C. followed by 6 hours at 65° C. Immunoprecipitated DNA was recovered using a QIAquick PCR Purification kit (Qiagen). Immunoprecipitated DNA was then converted into NGS libraries using an existing protocol32 and was sequenced on a NextSeq500 Illumina machine generating 75nt-long reads.
Sequencing-Based Assay for Lysogeny—
An overnight culture of bacteria was diluted 1/100 until reaching O.D 600 nm=0.1. Medium was replaced as described above [LB supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 with or without SAIRGA (SEQ ID NO: 269)], and incubated for 1 hour at room temperature. Bacteria were infected by phi3T at multiplicity of infection (MOI)=2. Cell pellets were collected at 5, 10, 20 and 60 minutes post infection in the presence or absence of SAIRGA (SEQ ID NO: 269) peptide at 1 μM final concentration in the medium. DNA was extracted using QIAGEN DNeasy blood and tissue kit (CAT #69504) and subjected to Illumina-based whole genome sequencing on NextSeq500. The relative abundance of lysogens in each sample was estimated using the number of reads mapped to the uninterrupted integration site versus reads mapped to the integration junction spanning the prophage DNA on one end and the bacterial DNA on the other end.
CRISPRI Experiments—
Construction of strains silencing phage genes was done by inserting a dCas9 construct controlled by a xylose promoter22, into the lacA region in B. subtilis 168 genome, and sgRNA with spacers targeting the gene of choice under constitutive promoter to thrC region [Spacer targeting aimR: ACCATTACTTTTCATAAC (SEQ ID NO: 358), spacer targeting aimX: TTTCCGCTTCATCTCAAGA (SEQ ID NO: 359)]. Infection assays in CRISPRi strains were performed in LB supplemented with 0.1 mM MnCl2, 5 mM MgCl2 and 0.2% xylose.
For complementation assays of aimR on the background of aimR-silenced CRISPRi strain, aimR was amplified from the phi3T genome using the following primers:
These primers amplify aimR together with 158 bases from it upstream region, with a 6×His at its C-terminus. The amplified fragment was cloned into the pBS1C plasmid (received from BGSC). Following, the native protospacer adjacent motif in the complemented aimR gene was changed by a synonymous point mutation (C->A at codon #20 of the aimR gene) using a primer set containing the point mutation and Gibson assembly. The modified gene was then integrated into the amyE locus in the B. subtilis genome. The aimX complementation was constructed on the background of aimR-silenced CRISPRi strain. For this, aimX was amplified from the phi3T genome using the following primers:
These primers amplify aimX together with 60 bases from its upstream region and 107 bases from its downstream region (containing the gene terminator). The amplified aimX was cloned into a pBS1C plasmid modified to contain a xylose promoter, and was then integrated into the amyE locus in the B. subtilis genome.
RNA-Seq—
To determine the difference in gene expression with and without the peptide, bacteria were incubated for 1 hour in LB medium supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 in the presence or absence of 1 μM synthesized SAIRGA (SEQ ID NO: 269) peptide. Following, the bacteria were infected with phi3T (MOI=0.1); and cell pellets were collected at 0, 5, 10 and 20 minutes post infection.
RNA extraction and RNA-seq was performed as described in Dar et al.33. Briefly, pellets were lysed using the Fastprep homogenizer (MP Biomedicals, Santa Ana, Calif.) and RNA was extracted with the FastRNA PRO blue kit (MP Biomedicals, 116025050) according to manufacturer's instructions. RNA samples were treated with TURBO deoxyribonuclease (DNase) (Life technologies, AM2238) and fragmented with fragmentation buffer (Ambion) in 72° C. for 1:45 minutes. The reactions were cleaned by adding×2.5 SPRI beads. The beads were washed twice with 80% EtOH, and air dried for 5 minutes. The RNA was eluted using H2O. rRNA was depleted by using the Ribo-Zero rRNA Removal Kit (epicenter, MRZB12424). Strand-specific RNA-seq was performed using the NEBNext Ultra Directional RNA Library Prep Kit (NEB, E7420) with the following adjustments: all cleanup stages were performed using×1.8 SPRI beads, and only one cleanup step was performed after the end repair step.
To determine the effect of CRISPRi silencing of the aimR gene, bacteria at early logarithmic stage were infected with phi3T (MOI=0.1) and cell pellets were collected 20 minutes post infection. RNA-seq libraries were prepped as described above.
RNA-seq libraries were sequenced using the Illumina NextSeq500 platform. Sequenced reads were demultiplexed and adapters were trimmed using fastx_clipper with default parameters. Reads were mapped to the reference genomes (gene annotation and sequences were downloaded from Genbank: NC_000964 for Bacillus subtilis str. 168, AP012496 for Bacillus subtilis BEST7003) using NovoAlign (Novocraft) V3.02.02 with default parameters. All downstream analyses and normalized genome-wide RNA-seq coverage maps were generated as described in Dar et al33.
Differential Expression Analysis—
Reads per gene were calculated for each biological replicate at 20 minutes post infection with and without the synthetic peptide and normalized relative to the total mapped reads hitting the phage genome in each replicate. Log 10 transformation of the average of 3 replicates per gene in each condition was used to plot
Identification of AimR Homologs and the Arbitrium Peptide Code—
Homologs for the phi3T AimR receptor were searched for using the BLAST option in the Integrated Microbial Genomes (IMG) web server (img(dot)jgi(dot)doe(dot)gov/cgi-bin/mer/main(dot)cgi). The phi3T AimR (SEQ ID NO: 114) was provided as a query sequence and was searched against all isolated genomes with an e-value threshold of 1e-35. The gene neighborhood for each AimR homolog was visually inspected via the IMG “gene neighborhood” representation, and genes found located next to proteins annotated as phage proteins were considered as found in a prophage. The immediate downstream gene for each AimR homolog was considered the respective AimP gene if it contained a signal peptide as predicted by the IMG web server. If no immediate downstream gene was annotated, the intergenic region immediately downstream to the AimR homolog was translated using the Expasy Translate Tool (web(dot)expasy(dot)org/translate/), and short translated ORFs were inspected for the AimP signature. Results of this analysis are presented in Table 3 above.
Plasmid Construction—
The vectors were constructed using the following primers and oligonucleotides:
Construct #1 comprises 2 components: the Bacteriophage Phi3T virus aimR-aimP-aimX locus and a Fluorescent reporter gene (Superfolder Green Fluorescent Protein (sfGFP(sp)), denoted herein as GFP. Both components were inserted into a target shuttle plasmid (pDR111) that enabled propagation of the plasmid in E. coli followed by transformation into the Bacillus subtilis BEST7003 genome. pDR111 contains two sequences, each matching either the 5′-end or the 3′-end of the target gene (amyE) which through homologous recombination allows insertion of Construct #1. In addition, pDR111 also includes a Spectinomycin antibiotic resistance (spec) gene that allows for selection of the desired insertion, namely Construct #1.
Specifically, the aimR-aimP-aimX locus (SEQ ID NO: 372) was directly amplified from bacteriophage Phi3T genome, using primers A+B, which contains the aimR, aimP and aimX coding genes, including their intergenic spaces (Erez Z, et al., Nature. 2017; 541(7638):488-493).
The reporter GFP gene (SEQ ID NO: 373) was amplified, using primers C+D, from plasmid pDR111-sfGFP(sp) which contains a Bacillus subtilis-optimized superfolder-GFP (Overkamp W, et al., Appl Environ Microbiol. 2013 October; 79(20):6481-90).
The reporter gene GFP was genetically fused to the Phi3T aimR-aimP-aimX locus by inserting 3 STOP codons followed by a ribosome-binding-site (denoted herein as rbs) immediately downstream of the 37 bp long 3′UTR of aimX (see
The construct was inserted into the shuttle plasmid pDR111 between the restriction sites EcoRI and BamHI using the NEB builder HIFI DNA assembly reaction kit (NEB, MA, USA), resulting in the plasmid pDR111-Construct #1 (
The pDR111-Construct #1 plasmid was propagated in Escherichia coli and then used as a shuttle vector to insert the Construct #1 operon into the Bacillus subtilis genome within the amyE gene together with a spec gene on the opposite strand upstream of the RPX operon (
In order to create a minimized expression system the pDR111-Construct #1 plasmid was amplified using primers E+F followed by DNA assembly using the NEB builder HIFI DNA assembly reaction kit (NEB, MA, USA), resulting in the plasmid pDR111-Construct #1 with a deleted aimP gene. This construct was further amplified using primers G+H followed by DNA assembly leading to deletion of the aimX gene as well. The end product, pDR111-Construct #2 (SEQ ID NO: 377), contained the double gene deletion Δ aimP/Δ aimX variant of Construct #1 [i.e. Construct #2 (SEQ ID NO: 376)]. pDR111-Construct #2 was used as a shuttle plasmid in a similar manner to pDR111-Construct #1. This led to insertion of Construct #2 into the bacterial genome as described above for Construct #1 (
Growth Dynamics of Plasmid-Containing Cultures—
Starter growths of wild type (WT) Bacillus subtilis BEST7003 strain and of Bacillus subtilis BEST7003 strains containing Construct #1 or Construct #2 were cultured in 3 ml Luria-Bertani (LB) broth until the growth curve indicated stationary phase. Following, the bacterial cells were diluted in a ratio of 1/100 in LB broth or LB broth supplemented with a SAIRGA (SEQ ID NO: 269) peptide at a concentration range of 62.5 nM-1000 nM. Optical density (O.D. at 600 nm) and GFP fluorescence levels (488 nm excitation/518 nm emission) were measured over time in a 96 wells plate using a TECAN Infinite 200 μlate reader.
Cultures of Bacillus subtilis str. 168 were infected by 1 of the four different phages: phi29 (Podoviridae, obligatory lytic), phi105 (Siphoviridae, temperate, lambda-like), rho14 (Siphoviridae, temperate, lambda-like) or phi3T (Siphoviridae, temperate, spBeta-like) in control and in phage-derived conditioned media (see
Taken together, these results imply that a small molecule is released to the medium during infection of B. subtilis by phi3T and this molecule can affect infection dynamics of downstream infections of this phage.
It is known that quorum sensing (QS) in Bacilli and other Firmicutes is typically based on short peptides that are secreted to the medium and sensed by intra-cellular or membrane bound receptors1-3. Thus, to test whether the active substance in the medium is proteinaceous the conditioned medium was treated with proteinase K. As shown in
A close examination of the phage infection dynamics in the oppD mutant showed increased culture lysis in both control and conditioned media as compared to infection of wild type bacteria (
Taken together, these results suggest that during phi3T infection a short peptide is released to the medium and, as this peptide accumulates, it acts as a communication agent affecting the lysis/lysogeny decision of later generations of the phage progeny. This newly discovered putative communication molecule is denoted herein as arbitrium (the Latin word for “decision”).
Phi3T was isolated 4 decades ago and was characterized as belonging to the spBeta family of phages, although to date its genome was not sequenced6. To search for the possible genetic system encoding the arbitrium peptide, the genome of phi3T was sequenced and analyzed. This genome assembled into a single 128 kbps contig containing 185 predicted genes. To search for proteins likely to be secreted into the medium all of the open reading frames (ORFs) in phi3T were screened for the presence of an N-terminal signal peptide using the signalP software8. Three ORFs were predicted to have a signal peptide, suggesting that they are secreted or membrane-localized. While two of these genes seemed irrelevant (one was an integral membrane protein and the other was a large nuclease), the third gene exhibited features reminiscent of Bacillus quorum sensing peptides (
To test whether the predicted mature peptide is indeed the arbitrium molecule that influences the phage lysogeny decision, bacteria were infected with phi3T in LB medium supplemented with increasing amounts of synthesized SAIRGA (SEQ ID NO: 269) peptide. A clear concentration-dependent effect on the phage infection dynamics was observed, such that reduced culture lysis was apparent when the medium contained higher concentrations of the synthesized peptide (
To verify that the observed effect of the SAIRGA peptide on the dynamics of the infected culture was the result of increased lysogeny, total DNA of bacteria collected from a time course experiment during infection by phi3T with and without the peptide was directly sequenced. By comparing the fraction of sequencing reads passing through the intact phage integration site in the bacterial genome to reads demonstrating phage integration at that site, the fraction of lysogenized bacteria at each time point was directly quantified. Remarkably, a consistently elevated lysogeny in the presence of the SAIRGA (SEQ ID NO: 269) peptide was observed, such that 48% (±7.9%) of the bacteria were lysogenized at 60 minutes post infection, as compared to 18% (±3.3%) of bacteria grown without the peptide (
The aimP gene is located immediately downstream of a gene (SEQ ID NO: 1) encoding a 378 amino acids long open reading frame (SEQ ID NO: 114), suggesting that these two genes may be co-transcribed from the phage genome as a polycistron. This upstream gene encodes a predicted tetratricopeptide repeat (TPR) domain, typical of intracellular peptide receptors of the RRNPP family in QS systems of Gram positive bacteria9-11 (
To appreciate the abundance of this system in nature, a homology search was conducted to find homologs of the aimR gene in available sequenced genomes. Using this search 112 instances of AimR homologs were detected, virtually all of them in Bacillus phages or in prophages found integrated within Bacilli genomes, suggesting that this gene primarily fulfills a phage-related function (
Taken together, these results demonstrate a sequence-specific peptide code that guides phage lysogeny in a phage species-specific manner.
In communication systems of Gram positive bacteria, the binding of the communication peptide to its receptor usually leads to reprogramming of the transcriptional response. This can occur either directly, when the receptor is a transcription regulator such as in the cases of the PrgX12,13 in Enterococci, the PlcR14-16 of the Bacillus cereus group, and in other systems17,18; or indirectly, as in the case of Rap/Phr systems of Bacilli, in which the receptor is a phosphatase that regulates downstream transcriptional regulators by dephosphorylation19,20, or steric interference21. The presence of a predicted helix-turn-helix (HTH) motif in the N-terminus of AimR suggested that the receptor of the arbitrium system directly binds DNA. To test whether AimR binds the phage DNA in vivo, a His-tagged aimR gene was engineered into a B. subtilis 168 strain in which a dCas9 (CRISPRi) technology22 was used to silence the expression of the phage AimR gene, but not the cloned His-tagged AimR (see Materials and Methods hereinabove). Following, a ChIP-seq assay was performed 15 minutes after phage infection with and without the presence of the arbitrium peptide. Sequencing of the DNA bound to AimR clearly showed that AimR binds a single site in the phage genome, directly downstream of the aimP gene (
During the process of AimR purification it was noticed that the protein migrates as homodimer in a gel filtration column. Upon addition of the phi3T-derived SAIRGA (SEQ ID NO: 269) peptide, however, the protein strictly migrated as a monomer (
To examine whether binding of the arbitrium peptide to its AimR receptor leads to a transcriptional response in the phage genome RNA-seq was applied to RNA extracted from bacteria during a time course of infection with and without the peptide. As shown in
These results suggest that AimR, when bound to the phage DNA as a dimer in the absence of the arbitrium peptide, is a transcriptional activator of AimX. Indeed, when AimR was silenced using dCas9, the expression of AimX was dramatically reduced (
Since the AimR knockdown did not lead a dramatic transcriptional effect for any gene except AimX at 20 minutes post infection (
Taken together, the present inventors have shown that a large family of phages uses communication peptides in order to decide whether to enter a lytic cycle or lysogenize the infected bacterium. In a sense, the communication mechanism described allows an offspring phage particle to communicate with its ancestors, i.e., measure the amount of the predecessor phages that completed successful infections in prior cycles. The biological logic behind this strategy is clear: when a single phage encounters a bacterial colony, there is ample prey for the progeny phages that are produced from the first cycles of infection, and hence a lytic cycle is preferred. In later stages of the infection dynamics the number of bacterial cells is reduced to a point that progeny phages are at risk of no longer having a new host to infect. Then, it is logical for the phage to switch into lysogeny to preserve chances for viable reproduction.
The arbitrium system provides an elegant mechanism for a phage particle to estimate the amount of recent prior infections and hence decide whether to employ the lytic or lysogenic cycle. Without being bound by theory, the results point to the following model (
To demonstrate that the AimR-AimP system can function as a heterologous expression system in a phage-independent context, it was utilized to control the expression of a GFP reporter gene in Bacillus subtilis BEST7003.
To this end, the arbitrium locus (AimR-AimP-AimR binding site-AimX) was engineered such that the AimX gene was fused to a GFP reporter gene (Construct #1,
Following, a minimized expression system containing only the AimR gene, AimR binding site and a GFP downstream to the AimR binding site was engineered (Construct #2,
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2017/051293 | 11/28/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62426669 | Nov 2016 | US |
