Bacteriophage variants having extended host-range, methods for preparation and uses thereof in transducing nucleic acids into hosts of interest

Abstract

The invention relates to platform for of the preparation of improved nucleic acid delivery vehicles, specifically, vehicles having an extended host recognition ability. The invention further provides improved vehicles, compositions and uses thereof.

1 . A modified bacteriophage and/or transducing particle comprising:
 a. at least one modified host recognition element or any variant, mutant, protein or fragment thereof, wherein said modified host recognition element is compatible to a target cell of interest; and optionally   b. at least one nucleic acid molecule of interest.   
     
     
         2 . The modified bacteriophage and/or transducing particle according to  claim 1 , wherein said target cell/s of interest is any one of a prokaryotic and eukaryotic host cell/s. 
     
     
         3 . The modified bacteriophage and/or transducing particle according to  claim 2 , wherein said prokaryotic cell is a bacterial cell of at least one of  Escherichia coli  spp, ( E. coli ),  Pseudomonas  spp,  Staphylococcus  spp,  Streptococcus  spp.  Salmonella  spp.  Shigella  spp.  Clostridium  spp,  Enterococcus  spp,  Klebsiella  spp.  Acinetobacter  spp,  Yersinia  spp and  Enterobacter  spp or any mutant, variant isolate or any combination thereof. 
     
     
         4 . The modified bacteriophage and/or transducing particle according to  claim 1 , wherein bacteriophage is at least one T7like-virus. 
     
     
         5 . The modified bacteriophage and/or transducing particle according to  claim 1 , wherein said host recognition element comprises at least one protein residing in the tail region of said bacteriophage. 
     
     
         6 . The modified bacteriophage and/or transducing particle according to  claim 5 , wherein said at least one protein residing in the tail region of said bacteriophage is at least one of a tail protein and a fiber protein. 
     
     
         7 . The modified bacteriophage and/or transducing particle according to  claim 1 , wherein said at least one host recognition element is prepared by a method comprising the steps of:
 a. providing a plurality of nucleic acid molecules encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, wherein said nucleic acid molecules further comprise at least one packaging signal and at least one nucleic acid sequence encoding a selectable element;   b. contacting first host cell/s comprising said plurality of nucleic acid molecules with a delivery vehicle that carries nucleic acid sequence/s encoding at least one defective host recognition element or any protein or fragment thereof, under conditions that allow packaging and/or propagation of said delivery vehicle, and recovering the resultant delivery vehicle variants propagated and/or packaged in said first host cell/s;   c. contacting second host cell/s with the delivery vehicle variants recovered in step (b);   d. selecting for host cells obtained in step (c) that comprise said selectable element; and   e. isolating and/or characterizing the at least one host recognition element/s or any nucleic acid sequence encoding the same, from the host cell/s selected in step (d), to obtain at least one host recognition element compatible with at least one of said second host cell and said target cell/s of interest, or a nucleic acid sequence encoding the same; optionally, said method further comprises mutagenizing said plurality of nucleic acid molecules provided in step (a), thereby obtaining a plurality of nucleic acid molecules encoding at least one mutated host-recognition element.   
     
     
         8 . The modified bacteriophage and/or transducing particle according to  claim 1 , wherein said bacteriophage has an extended or modified host range and is prepared by a method comprising the steps of:
 a. providing a plurality of nucleic acid molecules encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, wherein said nucleic acid molecules further comprise at least one packaging signal and at least one nucleic acid sequence encoding a selectable element;   b. contacting first host cell/s comprising said plurality of nucleic acid molecules with at least one delivery vehicle that carries nucleic acid sequence/s encoding at least one defective host recognition element or any protein or fragment thereof, under conditions that allow packaging and/or propagation of said delivery vehicle, and recovering the resultant delivery vehicle variants packaged and/or propagated in said first host cells;   c. contacting second host cells with the delivery vehicle variants recovered in step (b);   d. selecting for host cell/s obtained in step (c) that comprise said selectable element;   e. isolating and/or characterizing the at least one host recognition element or any nucleic acid sequence encoding the same from the host cells selected in step (d), to obtain at least one of said host recognition element compatible with said second host cells or any nucleic acid sequence encoding the same;   f. introducing into third host cells at least one nucleic acid sequence comprising at least one sequence encoding the compatible host recognition element obtained in step (e) and contacting said third host cell/s with a delivery vehicle that carries nucleic acid sequence/s encoding at least one defective host recognition element or any protein or fragment thereof, thereby obtaining a delivery vehicle variant comprising at least one of said compatible host recognition element that is capable of delivering a nucleic acid molecule of interest to a target cell of interest; optionally, wherein said method further comprises mutagenizing said plurality of nucleic acid molecules provided in step (a), thereby obtaining a plurality of nucleic acid molecules encoding at least one mutated host-recognition element.   
     
     
         9 . The modified bacteriophage and/or transducing particle according to  claim 1 , wherein said nucleic acid sequence of interest comprise any one of:
 a. at least one sensitizing component comprising at least one cas gene and at least one CRISPR array, wherein at least one spacer of said CRISPR targets a proto-spacer comprised within a pathogenic or undesired gene of a target cell of interest so as to specifically inactivate said pathogenic or undesired gene, and at least one spacer of said CRISPR targets a proto-spacer comprised within a selective component so as to specifically inactivate said selective component; or   b. at least one nucleic acid sequence comprising at least one protospacer.   
     
     
         10 . A composition comprising at least one modified bacteriophage and/or transducing particle as defined in  claim 1 , or any cocktail or mixture of modified bacteriophages and/or transducing particle, said bacteriophage and/or transducing particle comprises:
 a. at least one modified host recognition element; and optionally   b. at least one nucleic acid molecule of interest.   
     
     
         11 . The composition according to  claim 10 , wherein said at least one host recognition element is prepared by a method comprising the steps of:
 a. providing a plurality of nucleic acid molecules encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, wherein said nucleic acid molecules further comprise at least one packaging signal and at least one nucleic acid sequence encoding a selectable element;   b. contacting first host cell/s comprising said plurality of nucleic acid molecules with a delivery vehicle that carries nucleic acid sequence/s encoding at least one defective host recognition element or any protein or fragment thereof, under conditions that allow packaging and/or propagation of said delivery vehicle, and recovering the resultant delivery vehicle variants propagated and/or packaged in said first host cell/s;   c. contacting second host cell/s with the delivery vehicle variants recovered in step (b);   d. selecting for host cells obtained in step (c) that comprise said selectable element; and   e. isolating and/or characterizing the at least one host recognition element/s or any nucleic acid sequence encoding the same, from the host cell/s selected in step (d), to obtain at least one host recognition element compatible with at least one of said second host cell and said target cell/s of interest, or a nucleic acid sequence encoding the same; optionally, said method further comprises mutagenizing said plurality of nucleic acid molecules provided in step (a), thereby obtaining a plurality of nucleic acid molecules encoding at least one mutated host-recognition element.   
     
     
         12 . The composition according to  claim 10 , wherein said nucleic acid sequence of interest comprise any one of:
 a. at least one sensitizing component comprising at least one cas gene and at least one CRISPR array, wherein at least one spacer of said CRISPR targets a proto-spacer comprised within a pathogenic or undesired gene of a target cell of interest so as to specifically inactivate said pathogenic or undesired gene, and at least one spacer of said CRISPR targets a proto-spacer comprised within a selective component so as to specifically inactivate said selective component: or   b. at least one nucleic acid sequence comprising at least one protospacer.   
     
     
         13 . A kit comprising:
 a. a plurality of nucleic acid molecules encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, wherein said nucleic acid molecules further comprise at least one packaging signal and at least one nucleic acid sequence encoding a selectable element;   b. at least one delivery vehicle that carries nucleic acid sequence/s encoding at least one defective host recognition element/s or any fragment thereof; and optionally   c. at least one compound for selecting cells that carry said selectable element.   
     
     
         14 . The kit according to  claim 13 , wherein said kit is adapted for use in a method for preparing, identifying and/or isolating host recognition element/s compatible for a target cell of interest, the method comprising:
 a. providing a plurality of nucleic acid molecules encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, wherein said nucleic acid molecules further comprise at least one packaging signal and at least one nucleic acid sequence encoding a selectable element;   b. contacting first host cell/s comprising said plurality of nucleic acid molecules with a delivery vehicle that carries nucleic acid sequence/s encoding at least one defective host recognition element or any protein or fragment thereof, under conditions that allow packaging and/or propagation of said delivery vehicle, and recovering the resultant delivery vehicle variants propagated and/or packaged in said first host cell/s;   c. contacting second host cell/s with the delivery vehicle variants recovered in step (b);   d. selecting for host cells obtained in step (c) that comprise said selectable element; and   e. isolating and/or characterizing the at least one host recognition element/s or any nucleic acid sequence encoding the same, from the host cell/s selected in step (d), to obtain at least one host recognition element compatible with at least one of said second host cell and said target cell/s of interest, or a nucleic acid sequence encoding the same.   
     
     
         15 . The kit according to  claim 14 , wherein said method further comprises mutagenizing said plurality of nucleic acid molecules provided in step (a), thereby obtaining a plurality of nucleic acid molecules encoding at least one mutated host-recognition element, optionally, wherein at least one of steps (a), (b), (c), (d) and (e) is repeated at least one more time. 
     
     
         16 . The kit according to  claim 13 , wherein said delivery vehicle is at least one bacteriophage and/or transducing particle. 
     
     
         17 . The kit according to  claim 13 , wherein said host recognition element comprises at least one protein residing in the tail region of said bacteriophage. 
     
     
         18 . The kit according to  claim 13 , adapted for extending or modulating host range of a nucleic acid delivery vehicle. 
     
     
         19 . The kit according to  claim 18 , wherein said delivery vehicle is prepared by a method comprising the steps of:
 a providing a plurality of nucleic acid molecules encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, wherein said nucleic acid molecules further comprise at least one packaging signal and at least one nucleic acid sequence encoding a selectable element;   b. contacting first host cell/s comprising said plurality of nucleic acid molecules with at least one delivery vehicle that carries nucleic acid sequence/s encoding at least one defective host recognition element or any protein or fragment thereof, under conditions that allow packaging and/or propagation of said delivery vehicle, and recovering the resultant delivery vehicle variants packaged and/or propagated in said first host cells;   c. contacting second host cells with the delivery vehicle variants recovered in step (b);   d. selecting for host cell/s obtained in step (c) that comprise said selectable element;   e. isolating and/or characterizing the at least one host recognition element or any nucleic acid sequence encoding the same from the host cells selected in step (d), to obtain at least one of said host recognition element compatible with said second host cells or any nucleic acid sequence encoding the same;   f. introducing into third host cells at least one nucleic acid sequence comprising at least one sequence encoding the compatible host recognition element obtained in step (e) and contacting said third host cell/s with a delivery vehicle that carries nucleic acid sequence/s encoding at least one defective host recognition element or any protein or fragment thereof, thereby obtaining a delivery vehicle variant comprising at least one of said compatible host recognition element that is capable of delivering a nucleic acid molecule of interest to a target cell of interest; optionally, the method further comprises mutagenizing said plurality of nucleic acid molecules provided in step (a), thereby obtaining a plurality of nucleic acid molecules encoding at least one mutated host-recognition element.   
     
     
         20 . A method for preparing, identifying and/or isolating host recognition element/s compatible for a target cell of interest, the method comprising:
 a. providing a plurality of nucleic acid molecules encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, wherein said nucleic acid molecules further comprise at least one packaging signal and at least one nucleic acid sequence encoding a selectable element;   b. contacting first host cell/s comprising said plurality of nucleic acid molecules with a delivery vehicle that carries nucleic acid sequence/s encoding at least one defective host recognition element or any protein or fragment thereof, under conditions that allow packaging and/or propagation of said delivery vehicle, and recovering the resultant delivery vehicle variants propagated and/or packaged in said first host cell/s;   c. contacting second host cell/s with the delivery vehicle variants recovered in step (b);   d. selecting for host cells obtained in step (c) that comprise said selectable element; and   e. isolating and/or characterizing the at least one host recognition element/s or any nucleic acid sequence encoding the same, from the host cell/s selected in step (d), to obtain at least one host recognition element compatible with at least one of said second host cell and said target cell/s of interest, or a nucleic acid sequence encoding the same, optionally, said method further comprises mutagenizing said plurality of nucleic acid molecules provided in step (a), thereby obtaining a plurality of nucleic acid molecules encoding at least one mutated host-recognition element.