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

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.

We claim: 
     
         1 . A method of modifying a naturally occurring bacteria in situ in a subject comprising:
 genetically modifying a DNA 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 , comprising contacting said naturally occurring bacteria with a vector. 
     
     
         3 . The method of  claim 1 , comprising contacting said naturally occurring bacteria with a vector located inside a delivery vehicle. 
     
     
         4 . The method of  claim 3 , wherein said vector located inside a delivery vehicle is a phagemid. 
     
     
         5 . The method of  claim 1 , comprising transducing said naturally occurring bacteria with a packaged phagemid. 
     
     
         6 . The method of  claim 2 , wherein the vector further comprises a conditional origin of replication which is inactive in the targeted naturally occuring bacteria but is active in a donor bacterial cell. 
     
     
         7 . The method of  claim 1 , wherein said genetic modification is a point mutation. 
     
     
         8 . The method of  claim 1 , wherein said genetic modification is a point mutation leading to gene disruption. 
     
     
         9 . 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. 
     
     
         10 . The method of  claim 1 , wherein the genetic modification is in a bacterial toxin gene. 
     
     
         11 . The method of  claim 2 , wherein said vector encodes a gene editing enzyme or system targeting a specific nucleotide sequence in the naturally occurring bacteria. 
     
     
         12 . The method of  claim 11 , wherein the gene editing enzyme further comprises one or two uracil DNA glycosylase inhibitor domain(s) (UGI). 
     
     
         13 . The method of  claim 11 , wherein the gene editing enzyme further comprises Mu-GAM. 
     
     
         14 . The method of  claim 11 , Wherein the gene editing enzyme is a base editor or a prime editor. 
     
     
         15 . The method of  claim 11 , wherein the gene editing enzyme is a dual base editor. 
     
     
         16 . The method of  claim 11 , wherein the gene editing enzyme further comprises a reverse transcriptase domain. 
     
     
         17 . The method of  claim 11 , wherein the gene editing enzyme further comprises an inhibitor of base repair. 
     
     
         18 . The method of  claim 11 , wherein the gene editing system is a retron based system. 
     
     
         19 . The method of  claim 2 , wherein the vector comprises a nucleic acid sequence encoding a dCas9 (dead-Cas9), an nCas9 (nickase Cas9), a fusion protein of a dCas9 and a deaminase domain, or a fusion protein of the nCas9 and a deaminase domain. 
     
     
         20 . The method of  claim 1 , wherein the naturally occurring bacteria are Cutibacterium  acnes  bacteria,  Escherichia coli  bacteria,  Staphylococcus aureus  bacteria,  Clostridium difficile  bacteria,  Porphyromonas gingivalis  bacteria,  Streptococcus  bacteria, or  Pseudomonas  bacteria. 
     
     
         21 . A method of modulating host-microbiome interaction by genetically modifying naturally occurring bacteria in situ wherein said naturally occurring bacteria is involved in microbiome associated disorder or disease comprising:
 genetically modifying a DNA sequence responsible for the microbiome associated disorder or disease in the naturally occurring bacteria in situ without introducing a double strand break in the DNA sequence,   wherein said genetic modification reduces the effects of the microbiome associated disorder or disease, and   wherein said genetic modification does not lead to the death of bacteria.