# US 2023/0054841 A1 · Methods for Directed Exaptation

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- Number: US 2023/0054841 A1
- Document ID: US20230054841A1
- Status: Published application
- Kind: A1
- Application number: 17/870,807
- Filed: 2022-07-21
- Published: 2023-02-23
- Earliest claimed: 2017-09-16
- Assignees: Massachusetts Institute of Technology
- Applicants: Massachusetts Institute of Technology
- Inventors: Andreas Mershin; Shannon Leigh Johnson
- Web page: https://patentgrep.com/patent/US20230054841A1

## CPC

- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- A61K 35/74: Bacteria (therapeutic use of a bacterial protein A61K38/00)
- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/205: Bacterial isolates
- C12Q 1/10: Enterobacteria
- C12R 2001/19: Escherichia coli
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

A method for directed exaptation includes dividing an original microorganism monoculture into subcultures that are subjected to different exaptation agents to obtain diversified substrains. At least one of the exaptation agents is selected to favor survival of sub strains exhibiting desired traits. The steps of dividing and subjecting may be iterated using at least some of the diversified substrains. Performance of diversified substrains is assessed and those that meet performance criteria for at least one desired trait are selected. Exaptation agents may include mutagenesis agents, training, horizontal gene transfer opportunities, and stressors. Substrains may be co-incubated with other living or dead microorganisms known to be preferentially adapted to have the desired trait. Diversified substrains may be combined into a multiculture microorganism population, to which microorganisms from the original monoculture may be added. The method may be used to create a treatment for a Multiple-Antibiotic Resistant Infection, preferably including a kill switch.

## Claims

### Claim 1

A method for creating a multiculture microorganism population exapted to meet at least one specific challenge condition, comprising the steps of:
dividing an original population comprising a monoculture of a plurality of the microorganisms into a plurality of subcultures having initially identical composition;
subjecting at least some of the plurality of subcultures to a plurality of different exaptation agents to obtain diversified substrains of the microorganism, wherein at least a first portion of the subcultures is subjected to different types or combinations of exaptation agents than are subcultures in other portions. and wherein at least one of the exaptation agents is selected to favor survival of substrains of the microorganism that meet the at least one specific challenge condition;
optionally iterating the steps of dividing and subjecting, using at least a subset of the diversified substrains in place of the original population, until a specified level of diversification of substrains is achieved; and
combining at least some of the diversified substrains with unexapted microorganisms from the original monoculture to create the multiculture microorganism population.

### Claim 2 (depends on 1)

The method of claim 1, wherein all of the diversified substrains are combined with unexapted microorganisms from the original monoculture to create the multiculture microorganism population.

### Claim 3 (depends on 1)

The method of claim 1, further comprising:
assessing the performance of each of the diversified substrains with respect to the at least one specific challenge condition; and
selecting only those of the diversified substrains that meet predetermined performance criteria for the at least one specific challenge condition for combination with the unexapted microorganisms from the original monoculture to form the multiculture microorganism population.

### Claim 4 (depends on 1)

The method of claim 1, wherein at least one of the exaptation agents is selected from the group consisting of mutagenesis agents, training, horizontal gene transfer opportunities, and stressors.

### Claim 5 (depends on 1)

The method of claim 1, further comprising the step of co-incubating at least a subset of the diversified substrains with at least one other living or dead microorganism condition before combining the diversified substrains with the unexapted microorganisms from the original monoculture to form the multiculture microorganism population, wherein the at least one other microorganism is known to be preferentially adapted to meet the specific challenge.

### Claim 6 (depends on 5)

The method of claim 5, wherein at least one of the exaptation agents is selected from the group consisting of mutagenesis agents, training, horizontal gene transfer opportunities, and stressors.

### Claim 7 (depends on 1)

The method of claim 1, wherein the step of subjecting further comprises the step of subjecting at least one portion of the plurality of subcultures to at least one adaptation-evoking agent.

### Claim 8 (depends on 1)

The method of claim 1, further comprising the step of co-incubating at least a subset of the diversified substrains with at least one other living or dead microorganism condition before combining the diversified substrains with the unexapted microorganisms from the original monoculture to form the multiculture microorganism population, wherein the at least one other microorganism is known to be preferentially adapted to have an adaptive advantage of fast growth.

### Claim 9 (depends on 1)

The method of claim 1, wherein the specific challenge condition is the ability to outcompete a Multiple-Antibiotic Resistant Infection, the exaptation agents are selected to favor survival of substrains exhibiting an ability to outcompete the Multiple-Antibiotic Resistant Infection, the specified level of diversification of substrains is the ability to outcompete the Multiple-Antibiotic Resistant Infection, and the predetermined performance criteria include the ability to outcompete the Multiple-Antibiotic Resistant Infection, such that the multiculture microorganism population is an antiMARI microorganism population.

### Claim 10

A method for producing a treatment for a Multiple-Antibiotic Resistant Infection, comprising the steps of:
creating an antiMARI microorganism by the steps of :
dividing an original population comprising a monoculture of a plurality of microorganisms into a plurality of subcultures having initially identical composition;
subjecting at least some of the plurality of subcultures to a plurality of different exaptation agents to obtain diversified substrains of the microorganism, wherein the exaptation agents are selected to favor survival of sub strains, or sets of substrains, wherein at least a first portion of the subcultures is subjected to different types or combinations of exaptation agents than are subcultures in other portions, and wherein at least one of the exaptation agents is selected to favor survival of substrains of the microorganism that exhibiting an ability to outcompete the Multiple-Antibiotic Resistant Infection;
optionally iterating the steps of dividing and subjecting, using at least a subset of the diversified substrains in place of the original population, until an ability to outcompete the Multiple-Antibiotic Resistant Infection is achieved; and
combining at least some of the diversified substrains with microorganisms from the original monoculture to create the Multiple-Antibiotic Resistant Infection treatment.

### Claim 11 (depends on 10)

The method of claim 10, further comprising the step of incorporating at least one kill switch into the antiMARI microorganism.

### Claim 12 (depends on 11)

The method of claim 11, wherein the kill switch is a requirement of the antiMARI microorganism for at least one component not available in nature, wherein the antiMARI microorganism is unable to adapt or exapt around the absence of the component.

### Claim 13 (depends on 12)

The method of claim 12, wherein the component is a synthetic nutrient that must be supplied to a patient or a non-canonical amino acid.

### Claim 14 (depends on 10)

The method of claim 10, wherein all of the diversified substrains are combined with unexapted microorganisms from the original monoculture to create the Multiple-Antibiotic Resistant Infection treatment.

### Claim 15 (depends on 10)

The method of claim 10, further comprising:
assessing the performance of each of the diversified substrains with respect to ability to outcompete the Multiple-Antibiotic Resistant Infection treatment; and
selecting only those of the diversified substrains that have the ability to outcompete the Multiple-Antibiotic Resistant Infection for combination with the microorganisms from the original monoculture to form the Multiple-Antibiotic Resistant Infection treatment.

### Claim 16 (depends on 10)

The method of claim 10, wherein at least one of the exaptation agents is selected from the group consisting of mutagenesis agents, training, horizontal gene transfer opportunities, and stressors.

### Claim 17 (depends on 10)

The method of claim 10, further comprising the step of co-incubating at least a subset of the diversified substrains with at least one other living or dead microorganism condition before combining the diversified substrains with the microorganisms from the original monoculture to form the Multiple-Antibiotic Resistant Infection treatment, wherein the at least one other microorganism is known to be preferentially adapted to outcompete the Multiple-Antibiotic Resistant Infection.

### Claim 18 (depends on 17)

The method of claim 17, wherein at least one of the exaptation agents is selected from the group consisting of mutagenesis agents, training, horizontal gene transfer opportunities, and stressors.

### Claim 19 (depends on 10)

The method of claim 10, wherein the step of subjecting further comprises the step of subjecting at least one portion of the plurality of subcultures to at least one adaptation-evoking agent.

### Claim 20 (depends on 10)

The method of claim 10, further comprising the step of co-incubating at least a subset of the diversified substrains with at least one other living or dead microorganism condition before combining the diversified substrains with the unexapted microorganisms from the original monoculture to form the Multiple-Antibiotic Resistant Infection treatment, wherein the at least one other microorganism is known to be preferentially adapted to have an adaptive advantage of fast growth.