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Publication Number
US-11166994-B2
Publication Date
2021-11-09
Expiration Date
Abstract
The invention relates in one aspect to a pharmaceutical composition comprising a nucleic acid delivery vehicle for delivering a deliverable nucleic acid into a bacterial cell, wherein the delivery vehicle comprises a deliverable nucleic acid packaged into one or more bacteriophage coat proteins, and wherein the delivery vehicle is capable of infecting the bacterial cell to introduce the deliverable nucleic acid into the cell, following which the deliverable nucleic acid is capable of forming a plasmid in the cell and being transmitted to one or more different bacterial cells by conjugation and not by infection. Compositions including a pharmaceutical composition comprising the delivery vehicle, and methods involving use or manufacture of the delivery vehicle, are also disclosed.
Core Innovation
The invention relates to a pharmaceutical composition that uses a non-lytic nucleic acid delivery vehicle to deliver a deliverable nucleic acid into a bacterial cell. The deliverable nucleic acid is packaged into one or more bacteriophage coat proteins and includes a vegetative replication origin and one or more rep genes that allow vegetative nucleic acid replication. The deliverable nucleic acid also includes a transmittal nucleic acid sequence with an origin of transfer and one or more tra genes encoding relaxasome functions required for plasmid mobilisation during conjugation.
The deliverable nucleic acid further includes one or more bacteriophage packaging signal sequences that allow packaging into the bacteriophage coat proteins. A selected nucleic acid of interest comprises a gene-inactivating nucleic acid sequence configured to inactivate an antibiotic resistance gene or a virulence gene. The gene-inactivating nucleic acid sequence includes a clustered regularly interspaced short palindromic repeat array nucleic acid sequence having or transcribing an RNA guide molecule with a spacer sequence complementary to a target sequence, enabling inactivation in the presence of a CRISPR associated DNA-binding polypeptide or a functional equivalent or modified version.
The system is arranged so that, after infecting the bacterial cell to introduce the deliverable nucleic acid, the deliverable nucleic acid forms a plasmid in the cell. The plasmid is then transmitted to one or more different bacterial cells by conjugation and not by infection. This requires either that the transmittal nucleic acid sequence further comprises one or more tra genes encoding conjugation functions required for plasmid conjugation, or that the bacterial cell comprises one or more tra genes encoding conjugation functions required for plasmid conjugation.
Claims Coverage
The provided claim content supports one independent claim with six inventive features centered on a non-lytic, bacteriophage-coat packaged delivery vehicle, plasmid formation and conjugative transmission, and a CRISPR-based gene-inactivating sequence.
Non-lytic bacteriophage coat packaged plasmid-conjugating delivery vehicle
A non-lytic nucleic acid delivery vehicle delivering a deliverable nucleic acid into a bacterial cell, where the deliverable nucleic acid is packaged into one or more bacteriophage coat proteins and is capable of infecting the bacterial cell to introduce the deliverable nucleic acid.
Vegetative replication origin and rep genes enabling plasmid formation
The deliverable nucleic acid comprises a vegetative replication origin and one or more rep genes that allow vegetative nucleic acid replication, such that the deliverable nucleic acid is capable of forming a plasmid in the cell.
Origin of transfer and relaxasome tra genes enabling conjugative transmittal
The deliverable nucleic acid comprises a transmittal nucleic acid sequence with an origin of transfer and one or more tra genes encoding relaxasome functions required for plasmid mobilisation during conjugation.
Bacteriophage packaging signals enabling packaging into coat proteins
The deliverable nucleic acid comprises one or more bacteriophage packaging signal sequences that allow packaging into the one or more bacteriophage coat proteins.
CRISPR/Cas gene-inactivating nucleic acid targeting antibiotic resistance or virulence genes
A selected nucleic acid of interest comprising a gene-inactivating nucleic acid sequence capable of inactivating an antibiotic resistance gene or virulence gene, wherein the gene-inactivating nucleic acid sequence comprises a clustered regularly interspaced short palindromic repeat array nucleic acid sequence having or transcribing an RNA guide molecule with a spacer sequence complementary to a target sequence for inactivation in the presence of a CRISPR associated DNA-binding polypeptide or a functional equivalent or modified version.
Conjugation without infection via tra function availability
Either the transmittal nucleic acid sequence further comprises one or more tra genes encoding conjugation functions required for plasmid conjugation, or the bacterial cell comprises one or more tra genes encoding conjugation functions required for plasmid conjugation, such that the plasmid is transmitted to one or more different bacterial cells by conjugation and not by infection.
The claims combine non-lytic bacteriophage coat protein packaging with deliverable nucleic acid elements for vegetative replication and conjugative transmittal, together with a CRISPR/Cas gene-inactivating sequence targeting antibiotic resistance or virulence genes. Transfer occurs by conjugation rather than infection after plasmid formation in the introduced bacterial cell.
Stated Advantages
The deliverable nucleic acid forms a plasmid in the bacterial cell and is transmitted to different bacterial cells by conjugation rather than infection.
The delivery system is capable of inactivating an antibiotic resistance gene or virulence gene.
In the disclosed context, inactivation of bacterial beta-lactamase (bla) genes restores ampicillin sensitivity.
The constructs are intended to reduce ampicillin resistance transfer.
Documented Applications
Delivery of CRISPR gene-inactivating nucleic acids intended to inactivate bla genes to restore ampicillin sensitivity, including coverage across multiple bla families.
Use in examples involving MDR E. coli ST131 and other experimental recipients.
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