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

A method for manipulating a population of cells by transducing at least one nucleic acid sequence of interest into target cell/s comprised within said population of cells, the method comprising the step of contacting said population of cells in at least one of a subject, a tissue, an organ, a surface, a substance and an article containing said target cell/s with an effective amount at least one delivery vehicle or any kit, system or composition comprising the same, wherein said delivery vehicle comprises:

• i. at least one host recognition element compatible with said target cell/s, or any variant, mutant, protein or fragment thereof;
• and
• ii. at least one of said nucleic acid molecule of interest;
• and
• 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, and introducing said plurality of nucleic acid molecules into first host cell/s, 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 said 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, wherein said delivery vehicle is a bacteriophage and/or a transducing particle, and wherein at least one of said host-recognition element or any variant, mutant, protein or fragment thereof encoded by said plurality of nucleic acid molecules, is provided in trans to said delivery vehicle;
• 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,
• wherein at least one of said second host cells and said target cells of interest is a prokaryotic host cell;
• and
• f. introducing into third host cell/s 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 comprising at least one of said compatible host recognition element that is capable of delivering a nucleic acid molecule of interest into said target cell of interest, wherein said delivery vehicle is a bacteriophage and/or a transducing particle.

A method for preparing, identifying and/or isolating host recognition element/s compatible for a target cell of interest, the method comprising:

• a. contacting first host cell/s comprising a plurality of nucleic acid molecules encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, 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, wherein said delivery vehicle is a bacteriophage and/or a transducing particle, wherein said nucleic acid molecules further comprise at least one packaging signal and at least one nucleic acid sequence encoding a selectable element, and wherein at least one of said host-recognition element or any variant, mutant, protein or fragment thereof encoded by said plurality of nucleic acid molecules, is provided in trans to said delivery vehicle;
• b. contacting second host cell/s with the delivery vehicle variants recovered in step (a);
• c. selecting for host cells obtained in step (b) that comprise said selectable element;
• and
• d. 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 (c), 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, wherein at least one of said second host cells and said target cells of interest is a prokaryotic host cell.

A method for the preparation of a nucleic acid delivery vehicle capable of delivering a nucleic acid molecule of interest into a target cell of interest, the method comprising the steps of:

• a. contacting first host cell/s comprising a plurality of nucleic acid molecules encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, 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, wherein said delivery vehicle is a bacteriophage and/or a transducing particle, wherein said nucleic acid molecules further comprise at least one packaging signal and at least one nucleic acid sequence encoding a selectable element, and wherein at least one of said host-recognition element or any variant, mutant, protein or fragment thereof encoded by said plurality of nucleic acid molecules, is provided in trans to said delivery vehicle;
• b. contacting second host cells with the delivery vehicle variants recovered in step (a);
• c. selecting for host cell/s obtained in step (b) that comprise said selectable element;
• d. 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 (c), to obtain at least one of said host recognition element compatible with said second host cells or any nucleic acid sequence encoding the same;
• e. 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 (d) 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 comprising at least one of said compatible host recognition element that is capable of delivering a nucleic acid molecule of interest to said target cell of interest, wherein at least one of said second host cells and said target cells of interest is a prokaryotic host cell.