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

• US 9,771,622 B2
• Filed: 06/13/2016
• Issued: 09/26/2017
• 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, de Forest, Nikol, Cox, Heather, Shukla, Soni
• Primary Examiner(s): Brown, Mindy G
• Application Number:
  US15/180,790
• Publication Number:
  US 20160281179A1
• Time in Patent Office: 470 Days
• Field of Search: None
• 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 method for producing and collecting a plurality of non-replicative transduction particles (NRTPs), comprising:

(a) inducing a lytic phase of the bacterial cell packaging system, wherein said bacterial cell packaging system comprises;

a host bacteria cell;

a first nucleic acid construct inside the host bacteria cell, comprising of a bacteriophage genome having a first bacteriophage gene that contains 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 a reporter nucleic acid molecule having a reporter gene and a second bacteriophage gene that contains 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 within the bacteriophage gene on the second nucleic acid construct complements the non-functional packaging initiation site sequence in the bacteriophage genome on the first nucleic acid construct;

(b) allowing the replicon of the reporter nucleic acid molecule to be packaged to produce the NRTPs; and

(c) collecting a lysate comprising the plurality of NRTPs.

2. The method of claim 1, wherein the lysate is stable at room temperature.

3. The method of claim 1, wherein the reporter gene is luxAB.

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

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

6. The method of claim 4, wherein the constitutive promoter is an Enterobacteriaceae β

-lactamase gene bla promoter.

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

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

9. The method of claim 7, wherein the at least one origin of replication is a pBBR1-derived origin of replication.

10. The method of claim 1, wherein the bacteriophage is φ

80α

.

11. The method of claim 1, wherein the bacteriophage is P1.

15. The method of claim 1 wherein the second bacteriophage gene is a terminase gene.

19. The method of claim 15, wherein the terminase gene is a φ

80α

small terminase (terS) gene.

20. The method of claim 15, wherein the terminase gene is a P1 pacA terminase gene.

12. A method for detecting a presence or an absence of a bacterial cell in a sample, comprising:

(a) contacting the sample with a lysate comprised of a plurality of non-replicative transduction particles (NRTPs) such that the plurality of NRTPs transduces one or more of the bacterial cells in the sample,

wherein the plurality of NRTPs are produced by (i) inducing a lytic phase of a bacterial cell packaging system, wherein said bacterial cell packaging system comprises;

a host bacteria cell;

a first nucleic acid construct inside the host bacteria cell, comprising of a bacteriophage genome having a first bacteriophage gene that contains 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 a reporter nucleic acid molecule having a reporter gene and a second bacteriophage gene that contains 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 within the bacteriophage gene on the second nucleic acid construct complements the non-functional packaging initiation site sequence in the bacteriophage genome on the first nucleic acid construct;

(ii) allowing the replicon of the reporter nucleic acid molecule to be packaged to produce the NRTPs;

(b) providing conditions for expression of the reporter gene; and

(c) detecting the presence or absence of a signal produced by the reporter gene, wherein the presence of the signal indicates the presence of the bacteria cell, and wherein the absence of the signal indicates the absence of the bacteria cell.

13. The method of claim 12, wherein the bacterial cell is Staphylococcus aureus (S. aureus).

14. The method of claim 12, further comprising providing an antimicrobial agent to the sample and detecting for the presence or absence of a signal produced by the reporter gene to determine whether the sample contains a cell that is resistant or susceptible to the antimicrobial agent.

16. The method of claim 12 wherein the second bacteriophage gene is a terminase gene.

17. The method of claim 16 wherein the terminase gene is a φ

80α

small terminase (terS) gene.

18. The method of claim 16 wherein the terminase gene is a P1 pacA terminase gene.