Non-replicative transduction particles and transduction particle-based reporter systems

• US 9,388,453 B2
• Filed: 11/21/2014
• Issued: 07/12/2016
• Est. Priority Date: 03/13/2013
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: GeneWEAVE BioSciences, Inc. (Roche Holding AG)
• Inventors: Rey, Diego Ariel
• Primary Examiner(s): Joike, Michele K
• Assistant Examiner(s): Brown, Mindy G
• Application Number:
  US14/550,335
• Publication Number:
  US 20150104787A1
• Time in Patent Office: 599 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 2795/10143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2795/10152 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 2795/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2795/10352 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/14 Streptococcus; Staphylococcus
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Y 113/12007 Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
• CPC Class Codes: G01N 2333/31 from Staphylococcus (G)
• CPC Class Codes: G01N 2333/90241 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Abstract

Methods and systems are provided for packaging reporter nucleic acid molecules into non-replicative transduction particles for use as reporter molecules. The non-replicative transduction particles can be constructed from viruses and use viral transduction and replication systems. The reporter nucleic acid molecules include a reporter gene, such as a reporter molecule or selectable marker, for detecting target genes or cells. Methods and systems are provided for detection of cells and target nucleic acid molecules using the non-replicative transduction particles as reporter molecules.

Claims

1. A bacterial cell packaging system for packaging a reporter nucleic acid molecule into a non-replicative transduction particle (NRTP) for introduction into a cell, the packaging system comprising:

a host bacteria cell;

a first nucleic acid construct inside the host bacteria cell, comprising of a bacteriophage genome having a non-functional packaging initiation site sequence, wherein the non-functional packaging initiation site sequence prevents packaging of the bacteriophage genome into the NRTP; and

a second nucleic acid construct inside the host bacteria cell and separate from the first nucleic acid construct, comprising of the reporter nucleic acid molecule having a reporter gene and a functional packaging initiation site sequence for facilitating packaging of a replicon of the reporter nucleic acid molecule into the NRTP,

wherein the functional packaging initiation site sequence on the second nucleic acid construct complements the non-functional packaging initiation site sequence in the bacteriophage genome on the first nucleic acid construct.

2. The system of claim 1, wherein the reporter gene is luxAB.

3. The system of claim 1, wherein the reporter gene is operatively linked to a constitutive promoter.

4. The system of claim 3, wherein the constitutive promoter is a Staphylococcus aureus (S. aureus) clpB promoter.

5. The system of claim 4, wherein the S. aureus clpB promoter comprises the sequence of SEQ ID NO:

12.

6. The system of claim 1, wherein the functional packaging initiation site sequence comprises the sequence of SEQ ID NO:

9.

7. The system of claim 1, wherein the reporter nucleic acid molecule is a plasmid comprising at least one origin of replication.

8. The system of claim 7, wherein the at least one origin of replication is a pT181 origin of replication.

9. The system of claim 7, wherein the at least one origin of replication comprises the sequence of SEQ ID NO:

5.

10. The system of claim 1, wherein the reporter nucleic acid molecule is a plasmid comprising the sequence of SEQ ID NO:

14.

11. The system of claim 1, wherein the functional packaging initiation site sequence is a φ

80α

small terminase (terS) gene.

12. The system of claim 1, wherein the bacteriophage is φ

80α

.

13. The system of claim 1, wherein the non-functional and the functional packaging initiation site sequences are small terminase (terS) genes.

14. A bacterial cell packaging system for packaging a reporter plasmid into a non-replicative transduction particle (NRTP) for introduction into a cell, the packaging system comprising:

a host bacteria cell;

a first nucleic acid construct inside the host bacteria cell, comprising of a φ

80α

bacteriophage genome having a non-functional φ

80α

small terminase (terS) gene, wherein the non-functional φ

80α

terS gene prevents packaging of the φ

80α

bacteriophage genome into the NRTP; and

a second nucleic acid construct inside the host bacteria cell and separate from the first nucleic acid construct, comprising of the reporter plasmid having a luxAB reporter gene operatively linked to a S. aureus clpB promoter comprising the sequence of SEQ ID NO;

12, a pT181 origin of replication comprising the sequence of SEQ ID NO;

5, and a functional φ

80α

terS gene comprising the sequence of SEQ ID NO;

9, the functional φ

80α

terS gene facilitating packaging of a replicon of the reporter nucleic acid molecule into the NRTP,

wherein the functional φ

80α

terS gene on the second nucleic acid construct complements the non-functional φ

80α

terS gene in the φ

80α

bacteriophage genome on the first nucleic acid construct.