US 10,463,049 C1 (12,755th)
Altering microbial populations and modifying microbiota
Jasper Clube, London (GB); Morten Sommer, Hørsholm (DK); Christian Grøndahl, London (GB); Eric Van Der Helm, Hørsholm (DK); and Ruben Vasquez-Uribe, Hørsholm (DK)
Filed by SNIPR TECHNOLOGIES LIMITED, London (GB)
Assigned to SNIPR TECHNOLOGIES LIMITED, London (GB)
Reexamination Request No. 90/019,229, Jul. 19, 2023.
Reexamination Certificate for Patent 10,463,049, issued Nov. 5, 2019, Appl. No. 15/817,135, Nov. 17, 2017.
Application 90/019,229 is a continuation of application No. 15/460,962, filed on Mar. 16, 2017, granted, now 10,582,712.
Application 15/460,962 is a continuation of application No. 15/160,405, filed on May 20, 2016, granted, now 9,701,964.
Application 15/160,405 is a continuation of application No. PCT/EP2016/059803, filed on May 3, 2016.
Claims priority of application No. 1507773 (GB), filed on May 6, 2015; application No. 1507774 (GB), filed on May 6, 2015; application No. 1507775 (GB), filed on May 6, 2015; application No. 1507776 (GB), filed on May 6, 2015; application No. 1508461 (GB), filed on May 17, 2015; application No. 1509366 (GB), filed on May 31, 2015; application No. 1510891 (GB), filed on Jun. 20, 2015; application No. 1518402 (GB), filed on Oct. 17, 2015; application No. 1600417 (GB), filed on Jan. 10, 2016; and application No. 1600418 (GB), filed on Jan. 10, 2016.
Ex Parte Reexamination Certificate issued on Oct. 24, 2024.
Int. Cl. A01N 63/00 (2020.01); A01N 63/50 (2020.01); A01N 63/60 (2020.01); A61K 31/7105 (2006.01); A61K 31/711 (2006.01); A61K 35/74 (2015.01); A61K 45/06 (2006.01); A61K 48/00 (2006.01); C12N 1/20 (2006.01); C12N 7/00 (2006.01); C12N 9/16 (2006.01); C12N 15/10 (2006.01); C12N 15/113 (2010.01); C12N 15/70 (2006.01); C12N 15/74 (2006.01); C12N 15/90 (2006.01); A61K 35/00 (2006.01)
CPC A61K 31/7105 (2013.01) [A01N 63/00 (2013.01); A01N 63/50 (2020.01); A01N 63/60 (2020.01); A61K 31/711 (2013.01); A61K 35/74 (2013.01); A61K 45/06 (2013.01); A61K 48/005 (2013.01); C12N 1/20 (2013.01); C12N 7/00 (2013.01); C12N 9/16 (2013.01); C12N 15/102 (2013.01); C12N 15/113 (2013.01); C12N 15/70 (2013.01); C12N 15/746 (2013.01); C12N 15/902 (2013.01); A61K 2035/11 (2013.01); A61K 2300/00 (2013.01); C12N 2310/20 (2017.05); C12N 2320/31 (2013.01); C12N 2795/00032 (2013.01); C12N 2795/10132 (2013.01); Y02A 50/30 (2018.01)]
AS A RESULT OF REEXAMINATION, IT HAS BEEN DETERMINED THAT:
Claims 1-2 are cancelled.
Claims 3-9, 11-13 and 15-27 are determined to be patentable as amended.
Claims 10 and 14, dependent on an amended claim, are determined to be patentable.
New claims 28-39 are added and determined to be patentable.
3. The method of claim 1 [ 20] , wherein the Type II Cas is a Cas9.
4. The method of claim 1 [ 20] , wherein the Type II Cas is encoded by an engineered nucleic acid.
5. The method of claim 1 [ 20] , wherein the Type II Cas is a functional endogenous Cas of the host cells.
6. The method of claim 1 [ 20] , wherein the engineered nucleic acid for producing the plurality of HM-crRNAs is present in a phage, phagemid or plasmid.
7. The method of claim 1 [ 20] , wherein the engineered nucleic acid encoding the Type II Cas is present in a phage, phagemid or plasmid.
8. The method of claim 1 [ 20] , wherein the mixed population of bacteria is present in a human microbiota.
9. The method of claim 1 [ 18] , wherein the host cells are gram positive cells.
11. The method of claim 1 [ 20] , wherein the Type II Cas is a Streptococcus Cas9.
12. The method of claim 1 [ 20] , wherein the Type II HM-CRISPR/Cas system comprises an endogenous tracrRNA of the host cells.
13. The method of claim 1 [ 20] , wherein the Type II HM-CRISPR/Cas system comprises a tracrRNA, and wherein the tracrRNA is encoded by an engineered nucleic acid.
15. The method of claim 1 [ 18] , wherein the second bacterial species is C. difficile, E. coli or a Salmonella species.
16. The method of claim 1 [ 18] , wherein the mixed population of bacteria comprises E. coli and a bacterial species selected from the group consisting of Lactobacillus and Streptococcus.
17. The method of claim 1 [ 20] , wherein the method inhibits growth of the host cells on a surface.
18. The method of claim 1 [ A method for modifying a mixed population of bacteria, wherein the mixed population of bacteria comprises a first bacterial sub-population and a second bacterial sub-population, wherein the first bacterial sub-population comprises a first bacterial species and the second bacterial sub-population comprises a population of host cells of a second bacterial species, wherein the second bacterial species is a different species than the first bacterial species, the method comprising:
(a) contacting the mixed population of bacteria with an engineered nucleic acid for producing a plurality of different host modifying crRNAs (HM-crRNAs), and
(b) producing the plurality of different HM-crRNAs in the host cells, wherein the plurality of different HM-crRNAs comprises a first nucleotide sequence that hybridizes to a first target sequence in the host cells; and a second nucleotide sequence that hybridizes to a second target sequence in the host cells, wherein the second target sequence is different from the first target sequence; and wherein:
1) the first target sequence is comprised by a first antibiotic resistance gene or RNA thereof and the second target sequence is comprised by a second antibiotic resistance gene or RNA thereof;
2) the first target sequence is comprised by an antibiotic resistance gene or RNA thereof and the second target sequence is comprised by an essential gene or a virulence gene or RNA thereof;
3) the first target sequence is comprised by a first essential gene or RNA thereof and the second target sequence is comprised by a second essential gene or a virulence gene or RNA thereof; or
4) the first target sequence is comprised by a first virulence gene or RNA thereof and the second target sequence is comprised by an essential gene or a second virulence gene or RNA thereof;
wherein:
(i) the plurality of HM-crRNAs is operable with a Type II Cas in the host cells, wherein the engineered nucleic acid and the Type II Cas are comprised by a Type II HM-CRISPR/Cas system in the host cells; and
(ii) the plurality of HM-crRNAs guide the Type II Cas to modify the target sequences in the host cells,
wherein the host cells are modified by the Type II HM-CRISPR/Cas system] , wherein the first bacterial species has a 16s ribosomal RNA-encoding DNA sequence that is at least about 80% identical to a 16s ribosomal RNA-encoding DNA sequence of the second bacterial species, and
wherein the growth of the first bacterial species in the mixed population is not inhibited [ , and
wherein the host cells are killed or growth of the host cells is reduced] .
19. The method of claim 1 [ A method for modifying a mixed population of bacteria, wherein the mixed population of bacteria comprises a first bacterial sub-population and a second bacterial sub-population, wherein the first bacterial sub-population comprises a first bacterial species and the second bacterial sub-population comprises a population of host cells of a second bacterial species, wherein the second bacterial species is a different species than the first bacterial species, the method comprising:
(a) contacting the mixed population of bacteria with an engineered nucleic acid for producing a plurality of different host modifying crRNAs (HM-crRNAs), and
(b) producing the plurality of different HM-crRNAs in the host cells, wherein the plurality of different HM-crRNAs comprises a first nucleotide sequence that hybridizes to a first target sequence in the host cells; and a second nucleotide sequence that hybridizes to a second target sequence in the host cells, wherein the second target sequence is different from the first target sequence; and wherein:
1) the first target sequence is comprised by a first antibiotic resistance gene or RNA thereof and the second target sequence is comprised by a second antibiotic resistance gene or RNA thereof;
2) the first target sequence is comprised by an antibiotic resistance gene or RNA thereof and the second target sequence is comprised by an essential gene or a virulence gene or RNA thereof;
3) the first target sequence is comprised by a first essential gene or RNA thereof and the second target sequence is comprised by a second essential gene or a virulence gene or RNA thereof; or
4) the first target sequence is comprised by a first virulence gene or RNA thereof and the second target sequence is comprised by an essential gene or a second virulence gene or RNA thereof;
wherein:
(i) the plurality of HM-crRNAs is operable with a Type II Cas in the host cells, wherein the engineered nucleic acid and the Type II Cas are comprised by a Type II HM-CRISPR/Cas system in the host cells; and
(ii) the plurality of HM-crRNAs guide the Type II Cas to modify the target sequences in the host cells,
wherein the host cells are modified by the Type II HM-CRISPR/Cas system] , wherein the first bacterial species is a Firmicutes and the second bacterial species is a Firmicutes [ ,
wherein the growth of the first bacterial species in the mixed population is not inhibited, and
wherein the host cells are killed or growth of the host cells is reduced] .
20. The method of claim 1 [ A method for modifying a mixed population of bacteria, wherein the mixed population of bacteria comprises a first bacterial sub-population and a second bacterial sub-population, wherein the first bacterial sub-population comprises a first bacterial species and the second bacterial sub-population comprises a population of host cells of a second bacterial species, wherein the second bacterial species is a different species than the first bacterial species, the method comprising:
(a) contacting the mixed population of bacteria with an engineered nucleic acid for producing a plurality of different host modifying crRNAs (HM-crRNAs), and
(b) producing the plurality of different HM-crRNAs in the host cells, wherein the plurality of different HM-crRNAs comprises a first nucleotide sequence that hybridizes to a first target sequence in the host cells; and a second nucleotide sequence that hybridizes to a second target sequence in the host cells, wherein the second target sequence is different from the first target sequence; and wherein:
1) the first target sequence is comprised by a first antibiotic resistance gene or RNA thereof and the second target sequence is comprised by a second antibiotic resistance gene or RNA thereof;
2) the first target sequence is comprised by an antibiotic resistance gene or RNA thereof and the second target sequence is comprised by an essential gene or a virulence gene or RNA thereof;
3) the first target sequence is comprised by a first essential gene or RNA thereof and the second target sequence is comprised by a second essential gene or a virulence gene or RNA thereof; or
4) the first target sequence is comprised by a first virulence gene or RNA thereof and the second target sequence is comprised by an essential gene or a second virulence gene or RNA thereof;
wherein:
(i) the plurality of HM-crRNAs is operable with a Type II Cas in the host cells, wherein the engineered nucleic acid and the Type II Cas are comprised by a Type II HM-CRISPR/Cas system in the host cells; and
(ii) the plurality of HM-crRNAs guide the Type II Cas to modify the target sequences in the host cells,
wherein the host cells are modified by the Type II HM-CRISPR/Cas system] , wherein the first bacterial species is a gram positive species and the second bacterial species is a gram positive species [ ,
wherein the growth of the first bacterial species in the mixed population is not inhibited, and
wherein the host cells are killed or growth of the host cells is reduced] .
21. The method of claim 1 [ 20] , wherein the mixed population of bacteria comprises a third bacterial species.
22. The method of claim 1 [ 18] , wherein (i) the first bacterial species is a Firmicutes, the second bacterial species is a Firmicutes and the third bacterial species is a human gut commensal species or a human gut probiotic species; or (ii) wherein the first bacterial species is a gram positive species, the second bacterial species is a gram positive species and the third bacterial species is a human gut commensal species or a human gut probiotic species.
23. The method of claim 1 [ 20] , wherein the host cells are wild-type cells.
24. The method of claim 1 [ 20] , wherein the method reduces the growth of the host cells by at least 5-fold.
25. The method of claim 1 [ 20] , wherein the mixed population of bacteria is present in a subject, and wherein the host cells cause a disease in the subject.
26. The method of claim 1 [ 20] , wherein the mixed population of bacteria is a gut microbiota of a human or an animal.
27. The method of claim 1 [ 20] , wherein the mixed population of bacteria is present in an industrial or medical fluid, an apparatus, a container, a waterway, water, a beverage, a foodstuff, or a cosmetic, and wherein the host cells cause contamination of the industrial or medical fluid, apparatus, container, waterway, water, beverage, foodstuff or cosmetic.
[ 28. The method of claim 18, wherein the first bacterial species is a Firmicutes and the second bacterial species is a Firmicutes.]
[ 29. The method of claim 20, wherein the first bacterial species is a Firmicutes and the second bacterial species is a Firmicutes.]
[ 30. The method of claim 18, wherein the first target sequence is comprised by a first essential gene or RNA thereof.]
[ 31. The method of claim 19, wherein the first target sequence is comprised by a first essential gene or RNA thereof.]
[ 32. The method of claim 20, wherein the first target sequence is comprised by a first essential gene or RNA thereof.]
[ 33. The method of claim 19, wherein the mixed population of bacteria comprises E. coli and a bacterial species selected from the group consisting of Lactobacillus and Streptococcus.]
[ 34. The method of claim 20, wherein the mixed population of bacteria comprises E. coli and a bacterial species selected from the group consisting of Lactobacillus and Streptococcus.]
[ 35. The method of claim 19, wherein the mixed population of bacteria comprises a third bacterial species, wherein the third bacterial species is a human gut commensal species or a human gut probiotic species.]
[ 36. The method of claim 20, wherein the mixed population of bacteria comprises a third bacterial species, wherein the third bacterial species is a human gut commensal species or a human gut probiotic species.]
[ 37. The method of claim 18, wherein the first target sequence is comprised by a first essential gene or RNA thereof and the second target sequence is comprised by an essential gene or RNA thereof.]
[ 38. The method of claim 19, wherein the first target sequence is comprised by a first essential gene or RNA thereof and the second target sequence is comprised by an essential gene or RNA thereof.]
[ 39. The method of claim 20, wherein the first target sequence is comprised by a first essential gene or RNA thereof and the second target sequence is comprised by an essential gene or RNA thereof.]