Modulation of microbiota function by gene therapy of the microbiome to prevent, treat or cure microbiome-associated diseases or disorders

Patent metadata

Field	Value
Publication number	US-11534467-B2
Application number	US-202217716313-A
Country	US
Kind code	B2
Filing date	Apr 8, 2022
Priority date	Apr 8, 2020
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

The invention encompasses compositions, kits and methods for modifying bacteria, preferably naturally occurring bacteria, in situ. These can be used to treat, prevent or cure microbiome-associated diseases or disorders by modulating the molecules expressed and/or secreted by bacterial populations of the microbiome in a specific manner. The genomic modifications can modify the interactions between part or all of these populations and the host in a way that decreases their deleterious potential on host health. The compositions, kits and methods of the invention do not result in the direct death of these populations or a direct significant inhibition of their growth. The invention further includes methods for screening for genetic modifications in the bacteria, for determining the efficiency of vectors at inducing these genetic mutations, and for determining the effects of these mutations on bacterial growth.

First claim

We claim:

1. A method of modifying a naturally occurring bacteria in situ in a subject comprising:
administering to the subject a vector in a delivery vehicle, wherein the vector encodes a gene editing enzyme or system targeting a specific nucleotide sequence in the bacteria;
genetically modifying a DNA sequence to generate at least one change in the targeted nucleotide sequence in the naturally occurring bacteria in situ without introducing a double strand break in the DNA sequence,
wherein said genetic modification does not lead to the death of bacteria.

2. The method of claim 1 , wherein said vector is a phagemid.

3. The method of claim 1 , wherein the vector is a packaged phagemid.

4. The method of claim 3 , wherein the phagemid comprises a nucleic acid sequence encoding a dCas9 (dead-Cas9).

5. The method of claim 3 , wherein the phagemid comprises a nucleic acid sequence encoding an nCas9 (nickase Cas9).

6. The method of claim 4 , wherein the phagemid comprises a nucleic acid sequence encoding a fusion protein of the dCas9 and a deaminase domain.

7. The method of claim 5 , wherein the phagemid comprises a nucleic acid sequence encoding a fusion protein of the nCas9 and a deaminase domain.

8. The method of claim 1 , wherein the vector further comprises a conditional origin of replication which is inactive in the targeted naturally occurring bacteria.

9. The method of claim 1 , wherein the gene editing enzyme further comprises one or two uracil DNA glycosylase inhibitor domain(s) (UGI).

10. The method of claim 1 , wherein the gene editing enzyme further comprises Mu-GAM.

11. The method of claim 1 , wherein the gene editing enzyme is a dual base editor.

12. The method of claim 1 , wherein the gene editing enzyme further comprises a reverse transcriptase domain.

13. The method of claim 1 , wherein the gene editing enzyme further comprises an inhibitor of base repair.

14. The method of claim 1 , wherein the gene editing system is a retron based system.

15. The method of claim 1 , wherein said genetic modification is a point mutation.

16. The method of claim 1 , wherein said genetic modification is a point mutation leading to gene disruption.

17. The method of claim 1 , wherein the genetic modification is in a bacterial toxin gene.

18. The method of claim 1 , wherein the vector generates a genetic modification in a DNA sequence coding for an immunogenic component expressed or secreted by the bacteria.

19. The method of claim 1 , wherein the genetic modification is a point mutation that results in a change of an amino acid in a mimic peptide, wherein said mimic peptide is a bacterial antigen that mimics a human protein.

20. The method of claim 1 , wherein the bacteria with the genetic modification does not have a reduced in vivo growth rate as compared to the same bacteria without the genetic modification.

21. The method of claim 1 , wherein said naturally occurring bacteria is involved in a microbiome associated disorder or disease.

22. The method of claim 1 , wherein the naturally occurring bacteria is Bacteroides faecis or Bacteroides thetaiotaomicron and the genetic modification is in the Bacteroides faecis or Bacteroides thetaiotaomicron beta-galactosidase gene.

23. The method of claim 22 , wherein the genetic modification in the beta-galactosidase gene results in a change in the amino acid sequence of the beta-galactosidase protein and results in lower identity with human MYH6 cardiac peptide (SEQ ID NO: 2324) as compared to the Bacteroides faecis or Bacteroides thetaiotaomicron beta-galactosidase protein without the genetic modification, wherein the changed amino acid corresponds to Leu9, Leu11, Met12, Ala13, Leu15, Thr18, Ala20, Ser21, and Ala22 of SEQ ID NO: 2322.

Assignees

• Eligo Bioscience

Inventors

• Duportet Xavier
• Decrulle Antoine
• Gil-Cruz Cristina Del Carmen
• Pérez-Shibayama Christian Ivan
• Ludewig Burkhard
• Fernandez Rodriguez Jesus
• Brodel Andreas

Classifications

• C12N15/1079 Primary

  Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086) · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• C12N15/902

  using homologous recombination · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title