US 2026/0083787 A1 · SYSTEMS FOR PRODUCTION OF TRANSDUCING PARTICLES, METHODS, KITS, COMPOSITIONS AND USES THEREOF

Abstract

The present disclosure provides improved systems and methods for production of bacteriophage-based engineered transducing particles and uses thereof in manipulating bacterial populations to express any nucleic acid sequence of interest. The disclosed systems are based on inserting to producing cells nucleic acid molecules that comprise the nucleic acid sequence of interest and a protective regulated array, and regulators specific for the regulator array. The system further provides helper transducing particle that facilitates the propagation of the transducing particle.

Claims21

Dependent claims

1-60. (canceled)

A system for the preparation of a transducing particle for the delivery of at least one nucleic acid sequence of interest into a target host cell, the system comprising:

(a) at least one nucleic acid molecule comprising:
(i) at least one of said nucleic acid sequence of interest;
(ii) at least one CRISPR-associated (cas) gene;
(iii) a protection array comprising at least one clustered, regularly interspaced short palindromic repeat (CRISPR) array, wherein at least one spacer of said CRISPR array targets at least one proto-spacer comprised within at least one selective component, so as to specifically inactivate said selective component;
and
(iv) at least one nucleic acid sequence comprising at least one regulatory region for said protection array of (iii);
wherein said nucleic acid molecule is operably linked to at least one packaging signal;
and
(b) at least one nucleic acid molecule cassette and/or plasmid comprising:
(i) at least one nucleic acid sequence encoding at least one regulatory component specific for said regulatory region of (a)(iv);
and optionally,
(ii) at least one nucleic acid sequence encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, wherein said host recognition element is compatible with said target host cell such that the transducing particle is capable of delivering said nucleic acid sequence of interest to said target host cell;
the system optionally further comprises,
(c) a helper transducing particle, used for the propagation of said transducing particles.

11 more elements

A method for the preparation of a transducing particle for the delivery of at least one nucleic acid sequence of interest into a target host cell, the method comprising the step of:

(I) introducing into producing host cell/s:
(a) at least one nucleic acid molecule comprising:
(i) at least one of said nucleic acid sequence of interest;
(ii) at least one cas gene;
(iii) a protection array comprising at least one CRISPR array, wherein at least one spacer of said CRISPR array targets a proto-spacer comprised within at least one selective component so as to specifically inactivate said selective component;
and
(iv) at least one nucleic acid sequence comprising at least one regulatory region for said protection array of (iii);
wherein said nucleic acid molecule is operably linked to at least one packaging signal;
and
(b) at least one nucleic acid molecule comprising:
(i) at least one nucleic acid sequence encoding at least one regulatory component specific for said regulatory region of (a)(iv);
and optionally,
(ii) at least one nucleic acid sequence encoding at least one host-recognition element or any variant, mutant, protein or fragment thereof, wherein said host recognition element is compatible with said target host cell such that the transducing particle is capable of delivering said nucleic acid molecule of interest to said target host cell;
thereby obtaining producing host cell/s comprising nucleic acid molecule, cassette and/or plasmid of (a) and (b);
(II) contacting said producing host cell/s obtained in step (I) with (c), at least one helper transducing particle used for propagation of said transducing particles;
or contacting with said producing host cells a system comprising (a), (b) and (c);
and
(III) recovering from the producing host cell obtained in step (II), transducing particle/s comprising said nucleic acid molecule of interest, said protection array, and said regulatory region packaged therein, wherein said transducing particles comprise/s said host recognition element/s compatible with said target cell of interest.

15 more elements

A kit for the delivery of at least one nucleic acid sequence of interest into a target host cell of interest, the kit comprising:

(a) at least one transducing particle, or any cocktail or mixture of said at least one transducing particle/s, said at least one transducing particle/s comprising:
(i) at least one nucleic acid sequence of interest;
(ii) at least one cas gene;
(iii) at least one protection array comprising at least one CRISPR array, wherein at least one spacer of said CRISPR array targets at least one proto-spacer comprised within at least one selective component so as to specifically inactivate said selective component;
and
(iv) at least one nucleic acid sequence comprising at least one regulatory region for said protection array of (iii);
wherein said transducing particle comprises host recognition element/s compatible with said target host cell;
and
(b) at least one selective component comprising at least one transducing particle, wherein said selective component comprising at least one nucleic acid sequence encoding at least one factor affecting element/s essential for growth and/or survival and/or viability, and/or activity of said target host cell, said selective component comprises at least one protospacer targeted by at least one spacer of said protection array of (iii), such that said selective component is specifically inactivated by said protection array.

6 more elements

Dependencies are inferred from the claim text.

Systems for production of transducing particles, methods, kits, compositions and uses thereof

Life of this patent

Systems for production of transducing particles, methods, kits, compositions and uses thereof

Life of this patent

Abstract

Claims21