Claims for Patent: 8,501,400

Summary for Patent: 8,501,400

Title:	Methods and compositions for determining the pathogenic status of infectious agents
Abstract:	The present invention relates to methods and compositions for improved detection of infectious agents and microbes. In particular, the present invention provides novel methods for detecting infectious agents, providing information about the viability status of such infectious agents and for determining drug susceptibility. In certain embodiments, the present invention employs techniques involving nucleic acid amplification-based microbial identification together with phage-based biological detection of drug resistance. The methods of the invention are suitable for all microbes and infectious agents, including bacterial agents such as Mycobacteria.
Inventor(s):	Mulvey; Matthew C. (Baltimore, MD), Einck; Leo (McLean, VA), Sacksteder; Katherine (Baltimore, MD)
Assignee:	Sequella, Inc. (Rockville, MD)
Application Number:	13/011,754
Patent Claims:	1. A vector encoding a surrogate marker locus (SML) generation module, the SML generation module comprising a nucleic acid sequence encoding a polypeptide or a catalytic nucleic acid and a source nucleic acid sequence from which the polypeptide or catlytic nucleic acid generates a distinct surrogate marker locus de novo, wherein the vector is a bacteriophage. 2. The vector of claim 1, wherein the polypeptide is selected from one or more of the following; a DNA recombinase, a RNA recombinase, a RNA polymerase, a DNA polymerase, a transcription factor, a sigma factor, a DNA methylase, a DNA demethylase, a DNA restriction endonuclease, a DNA ligase, a RNA ligase, a histone acetylase, a histone deacytlase, a uridine deaminase, a reverse transcriptase and a RNA maturase. 3. The vector of claim 1, wherein the catalytic nucleic acid is selected from one or more of the following; a RNA cyclase ribozyme, a group I intron, a group II intron, a riboswitch, a gene regulation ribozyme, and RNase P. 4. The vector of claim 1, wherein the source nucleic acid further encodes an isolation aptamer sequence for incorporation into the SML. 5. A method for creating a surrogate marker locus (SML) in cells comprising introducing into a cell a bacteriophage vector encoding a SML generation module, wherein the SML generation module comprises a nucleic acid sequence encoding a nucleic acid or nucleotide modifying function, wherein expression of the nucleic acid or nucleotide modifying function generates de novo, a distinct SML from a source nucleic acid sequence; and detecting the SML. 6. The method of claim 5, wherein the SML generation module further comprises a source nucleic acid from which the nucleic acid modifying function generates the SML. 7. The method of claim 5, wherein the nucleic acid or nucleotide modifying function is provided by a polypeptide or a catalytic nucleic acid. 8. The method of claim 7, wherein the polypeptide is selected from one or more of the following; a DNA recombinase, a RNA recombinase, a RNA polymerase, a DNA polymerase, a transcription factor, a sigma factor, a DNA methylase, a DNA demethylase, a DNA restriction endonuclease, a DNA ligase, a RNA ligase, a histone acetylase, a histone deacytlase, a uridine deaminase, a reverse transcriptase and a RNA maturase. 9. The method of claim 7, wherein the catalytic nucleic acid is selected from one or more of the following; a RNA cyclase ribozyme, a group I intron, a group II intron, a riboswitch, a gene regulation ribozyme, and RNase P. 10. A method for determining the viability of microbes comprising: introducing a bacteriophage vector encoding a SML generation module to a microbe, wherein the SML generation module comprises a nucleic acid sequence encoding a polypeptide or a catalytic nucleic acid, wherein expression of the polypeptide or catalytic nucleic acid generates, de novo, a distinct SML from a source nucleic acid sequence; and detecting generation of the SML, wherein detection of the surrogate marker locus indicates that the infectious agent is viable. 11. The method of claim 10, wherein the bacteriophage is specific for Mycobacterium. 12. The method of claim 10, wherein the polypeptide is selected from one or more of the following; a DNA recombinase, a RNA recombinase, a RNA polymerase, a DNA polymerase, a transcription factor, a sigma factor, a DNA methylase, a DNA demethylase, a DNA restriction endonuclease, a DNA ligase, a RNA ligase, a histone acetylase, a histone deacytlase, a uridine deaminase, a reverse transcriptase and a RNA maturase. 13. The method of claim 10, wherein the catalytic nucleic acid is selected from one or more of the following; a RNA cyclase ribozyme, a group I intron, a group II intron, a riboswitch, a gene regulation ribozyme, and RNase P. 14. The method of claim 10, wherein the source nucleic acid sequence further encodes an isolation aptamer. 15. The method of claim 10, further comprising determining the metabolic state of the microbe by determining a microbe genomic marker level using a nucleic acid detection method and comparing the microbe genomic marker level to the SML level, wherein detection of lower microbe genomic levels compared to SML levels indicates the microbe is metabolically active and wherein detection of higher microbe genomic levels compared to SML levels indicates the microbe is dormant. 16. A method for determining drug suceptiblity of a microbes comprising: exposing a test sample to a drug composition; incubating the test sample with a bacteriophage encoding a SML generation module, wherein the SML generation module comprises a nucleic acid sequence encoding a catalytic nucleic acid, wherein expression of the catalytic nucleic acid generates a SML, and detecting the presence of the SML, wherein detection of the SML indicates the test sample contains a microbe that is resistant to the drug composition. 17. The method of claim 16, wherein the microbe is a Mycobacterium. 18. The method of claim 16, wherien the sample is a body fluid sample, an industrial sample, or an environmental sample. 19. The method of claim 16, wherein incubating the test sample comprises incubating the test sample with multiple SML generation modules. 20. A vector encoding a surrogate marker locus (SML) generation module, the SML generation module comprising a nucleic acid sequence encoding a recombinase and a source nucleic acid, the source nucleic acid comprising a first recombinase recognition site, a second recombinase recognition site, and one or more signature tag sequences between the first recombinase recognition sequence and the second recombinase recognition sequence, wherein the first and second recobinase recognition sites are specific to the recombinase. 21. A vector encoding a surrogate marker locus (SML) generation module, the SML generation module comprising a nucleic acid sequence encoding a RNA polymerase and a source nucleic acid, the source nucleic acid comprising a sequence encoding a first portion of a RNA cyclase ribozyme, a sequence encoding a second portion of a RNA cyclase ribozyme, and two signature tag sequences located between the sequence encoding a first portion of a RNA cyclase ribozyme and the sequence encoding a second portion of a RNA cylcase ribozyme, and wherein the source nucleic acid is under the transcriptional control of a promoter specific to the RNA polymerase. 22. A vector encoding a surrogate marker locus (SML) generation module, the SML generation module comprising a sequence encoding a Intron Encoded Protein (IEP) under the control of a first promoter, a group II intron sequence lacking an internal IEP under the control of a second promoter, a first and second exon sequence, a first copy of the first and second exon sequences flanking either side of the group II intron sequence, and an inverted second copy of the first and second exon sequences located adjacent to each other and upstream of the second promoter. 23. The vector of claim 20, wherein the source nucleic acid further encodes an isolation apatamer sequence for incorporation into the SML. 24. The vector of claim 21, wherein the source nucleic acid further encodes an isolation apatamer sequence for incorporation into the SML. 25. The vector of claim 22, wherein the SML generation module further comprises an isolation aptamer sequence for incorporation into the SML. 26. A method for creating a surrogate marker locus (SML) in a cell comprising: introducing into the cell the vector of claim 20, 21, or 22, wherein expression of the nucleic acid sequence encoded by the SML generation module of the vector generates de novo, a distinct SML; and detecting the SML. 27. The method of claim 26, wherein the vector is a bacteriophage. 28. The method of claim 27, wherein the bacteriophage is specific for Mycobacterium. 29. The method of claim 26, wherein the vector further encodes an isolation aptamer for incorporation into the SML. 30. A method for determining the viability of microbes comprising: introducing into a microbe the vector of claim 20, 21, or 22, wherein expression of the nucleic acid sequence encoded by the SML generation module of the vector generates de novo, a distinct SML; and detecting generation of the SML, wherein detection of the surrogate marker locus indicates that the infectious agent is viable. 31. The method of claim 30, wherein the vector is a bacteriophage. 32. The method of claim 31, wherein the bacteriophage is specific for Mycobacterium. 33. The method of claim 30, wherein the vector further encodes an isolation aptamer for incorporation into the SML. 34. The method of claim 30, further comprising: determining the metabolic state of the microbe by determining a microbe genomic marker level using a nucleic acid detection method; and comparing the microbe genomic marker level to the SML level, wherein detection of lower microbe genomic levels compared to SML levels indicates the microbe is metabolically active and wherein detection of higher microbe genomic levels compared to SML levels indicate the microbe is dormant. 35. A method for determining the drug susceptibility of a microbes drugs comprising: exposing a test sample to a drug composition; incubating the test sample with the vector of claim 21, 22, or 23, wherein expression of the nucleic acid sequence encoded by the SML generation module of the vector generates de novo, a distinct SML; and detecting the presence of the SML, wherein detection of the SML indicates the test sample contains a microbe, that is resistant to the drug compositions. 36. The method of claim 35, wherein the vector is a bacteriophage. 37. The method of claim 36, wherein the bacteriophage is specific for Mycobacterium. 38. The method of claim 35, wherein the microbe is a Mycobacterium. 39. The method of claim 35, wherein the test sample is a body fluid sample, an industrial sample, or an environmental sample. 40. The method of claim 35, where the incubating step comprising incubating the test sample with multiple SML generation modules.

Details for Patent 8,501,400

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2027-04-05
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2027-04-05
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2027-04-05