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WO2025175173A1 - Cellules châssis à mémoire - Google Patents

Cellules châssis à mémoire

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Publication number
WO2025175173A1
WO2025175173A1 PCT/US2025/016038 US2025016038W WO2025175173A1 WO 2025175173 A1 WO2025175173 A1 WO 2025175173A1 US 2025016038 W US2025016038 W US 2025016038W WO 2025175173 A1 WO2025175173 A1 WO 2025175173A1
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WIPO (PCT)
Prior art keywords
seq
recombinase
probiotic composition
delivery system
synthetic
Prior art date
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Pending
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PCT/US2025/016038
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English (en)
Inventor
Corey J. Wilson
Brian David HUANG
Dowan Kim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Georgia Tech Research Institute
Georgia Tech Research Corp
Original Assignee
Georgia Tech Research Institute
Georgia Tech Research Corp
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Publication of WO2025175173A1 publication Critical patent/WO2025175173A1/fr
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • the present disclosure relates to programmable drug-delivery compositions, systems, and the methods of use thereof in preventing or treating disease and disorders.
  • Gut dysbiosis refers to an imbalance in the composition or function of the gut microbiota. It is often linked to a range of health issues, including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), obesity, metabolic disorders, and infections. Probiotics have long been explored as a therapeutic approach to restore a balance of the gut microbiota to address dysbiosis.
  • Traditional probiotic therapies typically employ live microorganisms, often derived from naturally occurring gut commensals or fermented foods, that demonstrated efficacy in reducing symptoms of gut-related diseases when administered in adequate amounts, albeit with limitations in colonization efficiency and specific targeting.
  • probiotics primarily manage symptoms of gut dysbiosis by restoring microbial diversity immune modulation, including inducing regulatory T cells, enhancing mucosal immunity, reducing inflammation, enhancing gut barrier function, and producing metabolites to maintain a healthy gut environment.
  • traditional probiotics face several challenges, such as off-target effects, and can only address simple factor conditions that result in limited survivability of the probiotics in the gastrointestinal tract, e.g., due to stomach acid and bile salts and difficulty in colonizing the gut or sustainability of their effects after treatment.
  • microorganism strain-specific effects lack personalization and do not address all aspects of dysbiosis.
  • the efficacy of the traditional probiotics is inconsistent across individuals due to differences in baseline microbiota composition.
  • An exemplary programmable drug-delivery platform, method of use, fabrications, and delivery are disclosed that combine decision-making, intercellular communication, and the equivalent of memory in an engineered chassis cell (referred to as Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)) that can facilitate discrete, precise and multi-input regulation of recombinase functions.
  • MEMORY Orthogonal Recombinase arraY
  • These recombinases can facilitate targeted and inheritable DNA modifications, including inversions, deletions, and insertions containing functional genomic elements - e.g., reading frames, promoters, terminators, or replication origins.
  • the exemplary MEMORY cells can achieve programmable and permanent changes (i.e., gain or loss) of functions either extrachromosomally or at a specific genomic locus without the loss or modification of the MEMORY platform.
  • the retention of the exemplary chassis cell platform facilitates the sequential programming and reprogramming of DNA circuits harbored within a given chassis cell, enabling dynamic modifications as needed.
  • the exemplary chassis cells are designed to embody all three core principles of biological intelligence: cellular decision-making, which allows cells to process inputs and make logical choices; inheritable memory, which ensures that genetic changes are inheritable and passed to progeny; and cell-to-cell communication, which enables coordinated interactions among cells including cell signaling. These features position MEMORY cells as a powerful tool for advancing synthetic biology and bioengineering applications.
  • a programmable drug-delivery system comprising a chassis cell, wherein the chassis cell (e.g., a Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)) comprises an inducible promoter, a modified ribosome-binding site (RBS), one or more genes expressing one or more orthogonal inducible recombinase(s), one or more transcription factor(s), wherein the one or more transcription factor(s) regulate the one or more orthogonal inducible recombinase(s) expression, one or more degradation tag(s), a variable start codon; and a terminator, wherein the terminator provides transcriptional insulation.
  • the chassis cell e.g., a Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)
  • RBS modified ribosome-binding site
  • the terminator provides transcriptional insulation.
  • the chassis cell is engineered to produce an orthogonal inducible recombinase. In some embodiments, the chassis cell facilitates discrete multi-input regulation of recombinase function to alter extrachromosomal nucleic acid, wherein chromosomal nucleic acid of the chassis cell is not altered.
  • the programmable drug-delivery system further comprises a drug.
  • the chassis cell is a non-colonizing bacterium (such as, for example, Escherichia coli).
  • the inducible promoter is selected from a group consisting of PLacIR, PPhlF, PCymRC, Pvan, PLuxB, cH OsymC*, dH OsymC*, fH OsymC*, bH OttaC*, cG OttaC*, pPhl, pBAD, pTet, and PvanBT.
  • the modified RBS is selected from a group consisting of phl3, cym2, lux2, van3, lac2, tet2, ara2, el, Al, BX, 13, 15, 18, and 112.
  • the one or more orthogonal inducible recombinase(s) is selected from a group consisting of Al 18, Bxbl, Int3, Int5, Int8, and Intl2.
  • Al 18 comprises at least 70% of SEQ ID NO: 73.
  • Al 18 comprises SEQ ID NO: 73.
  • Bxbl comprises at least 70% of SEQ ID NO: 74.
  • Bxbl comprises SEQ ID NO: 74.
  • Int3 comprises at least 70% of SEQ ID NO: 75.
  • Int3 comprises SEQ ID NO: 75.
  • Int5 comprises at least 70% of SEQ ID NO: 76.
  • Int5 comprises SEQ ID NO: 76.
  • Int8 comprises at least 70% of SEQ ID NO: 77.
  • Int8 comprises SEQ ID NO: 77.
  • Intl2 comprises at least 70% of SEQ ID NO: 78.
  • Intl2 comprises SEQ ID NO: 78.
  • the one or more orthogonal inducible recombinase(s) is induced by an inducer.
  • the inducer is selected from a group consisting of 2,4-Diacetylphloroglucinol (DAPG), aTc, L-Ara, cuminic acid, vanillic acid, Isopropyl [3- D-l -thiogalactopyranoside (IPTG), and 3OC6 Ahl.
  • the chassis cell comprises 1, 2, 3, 4, 5, 6 orthogonal inducible recombinases or a combination thereof.
  • the one or more transcription factor(s) is selected from a group consisting of PhlF, TetR, AraC, CymR, VanR, Lad, AraE, CelR (TAN), RbsR, and LuxR.
  • the one or more degradation tag(s) is selected from a group consisting of DAS tag, AAV tag, and LAA tag.
  • the terminator is selected from a group consisting of L3S1P11, L3S1P13, L3S2P21, L3S2P55, L3S3P00, L3S3P21, L3S3P22, L3S3P23, L3S3P41, ECK120010799, ECK120010818, ECK120010858-R, ECK120015170, ECK120015440, ECK120017009, ECK120033736, ECK120035133, rrnB Tl, BBa_B0014, BBa_B0053, BBa_B0062-R, BBa_B1006, and IOT.
  • the system is configured as a gain-of-function (GOF) memory circuit for both inversion and excision attachment site configuration.
  • the system is configured as a loss-of- function (LOF) memory circuit for both inversion and excision attachment site configuration.
  • a catalytically inactive Cas9 (dCas9) is employed to a recombinase attachment site to prevent recombination with high (-99%) efficiency.
  • extrachromosomal nucleic acid alteration comprises deletion, insertion, or inversion.
  • the insertion is a genomic insertion.
  • the genomic insertion is a functional element.
  • the functional element comprises a reading frame shift, a promoter, a terminator, or a replication origin.
  • a synthetic probiotic composition comprising a programmable drug-delivery system and a pharmaceutically acceptable carrier, wherein the programmable drug-delivery system comprising a chassis cell, wherein the chassis cell (e.g., a Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)) comprises, an inducible promoter, a modified ribosome-binding site (RBS), one or more genes expressing one or more orthogonal inducible recombinase(s), one or more transcription factor(s), wherein the one or more transcription factor(s) regulate the one or more orthogonal inducible recombinase(s) expression, one or more degradation tag(s), a variable start codon; and a terminator, wherein the terminator provides transcriptional insulation.
  • the chassis cell e.g., a Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)
  • RBS modified ribosome
  • the chassis cell is engineered to produce an orthogonal inducible recombinase. In some embodiments, the chassis cell facilitates discrete multi-input regulation of recombinase function to alter extrachromosomal nucleic acid, wherein chromosomal nucleic acid of the chassis cell is not altered.
  • the synthetic probiotic composition further comprises a drug.
  • the chassis cell is a non-colonizing bacterium (such as, for example, Escherichia coli).
  • the inducible promoter is selected from a group consisting of PLacIR, PPhlF, PCymRC, Pvan, PLuxB, cH OsymC*, dH OsymC*, fH OsymC*, bH OttaC*, cG OttaC*, pPhl, pBAD, pTet, and PvanBT.
  • the modified RBS is selected from a group consisting of phl3, cym2, lux2, van3, lac2, tet2, ara2, el, Al, BX, 13, 15, 18, and 112.
  • the one or more orthogonal inducible recombinase(s) is selected from a group consisting of Al 18, Bxbl, Int3, Int5, Int8, and Intl2.
  • Al 18 comprises at least 70% of SEQ ID NO: 73.
  • Al 18 comprises SEQ ID NO: 73.
  • Bxbl comprises at least 70% of SEQ ID NO: 74.
  • Bxbl comprises SEQ ID NO: 74.
  • Int3 comprises at least 70% of SEQ ID NO: 75.
  • Int3 comprises SEQ ID NO: 75.
  • Int5 comprises at least 70% of SEQ ID NO: 76.
  • Int5 comprises SEQ ID NO: 76.
  • Int8 comprises at least 70% of SEQ ID NO: 77.
  • Int8 comprises SEQ ID NO: 77.
  • Intl2 comprises at least 70% of SEQ ID NO: 78.
  • Intl2 comprises SEQ ID NO: 78.
  • the one or more orthogonal inducible recombinase(s) is induced by an inducer.
  • the inducer is selected from a group consisting of 2,4-Diacetylphloroglucinol (DAPG), aTc, L-Ara, cuminic acid, vanillic acid, Isopropyl [3- D-l -thiogalactopyranoside (IPTG), and 3OC6 Ahl.
  • the chassis cell comprises 1, 2, 3, 4, 5, 6 orthogonal inducible recombinases or a combination thereof.
  • the one or more transcription factor(s) is selected from a group consisting of PhlF, TetR, AraC, CymR, VanR, Lad, AraE, CelR (TAN), RbsR, and LuxR.
  • the one or more degradation tag(s) is selected from a group consisting of DAS tag, AAV tag, and LAA tag.
  • the terminator is selected from a group consisting of L3S1P11, L3S1P13, L3S2P21, L3S2P55, L3S3P00, L3S3P21, L3S3P22, L3S3P23, L3S3P41, ECK120010799, ECK120010818, ECK120010858-R, ECK120015170, ECK120015440, ECK120017009, ECK120033736, ECK120035133, rrnB Tl, BBa_B0014, BBa_B0053, BBa_B0062-R, BBa_B1006, and IOT.
  • the system is configured as a gain-of-function (GOF) memory circuit for both inversion and excision attachment site configuration.
  • the system is configured as a loss-of- function (LOF) memory circuit for both inversion and excision attachment site configuration.
  • a catalytically inactive Cas9 (dCas9) is employed to a recombinase attachment site to prevent recombination with high (-99%) efficiency.
  • extrachromosomal nucleic acid alteration comprises deletion, insertion, or inversion.
  • the insertion is a genomic insertion.
  • the genomic insertion is a functional element.
  • the functional element comprises a reading frame shift, a promoter, a terminator, or a replication origin.
  • the chassis cell exchanges information with a stably colonizing species, wherein the stably colonizing species is found in the gut of a subject.
  • the stably colonizing species is Bacteroides thetaiotaomicron.
  • a method of preventing or treating gut microbiota dysbiosis in a subject in need thereof comprising administering a therapeutically effective amount of a synthetic probiotic composition comprising a programmable drug-delivery system and a pharmaceutically acceptable carrier, wherein the programmable drug-delivery system comprising a chassis cell, wherein the chassis cell (e.g., a Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)) comprises, an inducible promoter, a modified ribosome-binding site (RBS), one or more genes expressing one or more orthogonal inducible recombinase(s), one or more transcription factor(s), wherein the one or more transcription factor(s) regulate the one or more orthogonal inducible recombinase(s) expression, one or more degradation tag(s), a variable start codon; and a terminator, wherein the terminator provides transcriptional insulation.
  • a synthetic probiotic composition comprising a
  • the chassis cell is engineered to produce an orthogonal inducible recombinase. In some embodiments, the chassis cell facilitates discrete multi-input regulation of recombinase function to alter extrachromosomal nucleic acid, wherein chromosomal nucleic acid of the chassis cell is not altered.
  • the method further comprises a drug.
  • the chassis cell is a non-colonizing bacterium (such as, for example, Escherichia coli).
  • the inducible promoter is selected from a group consisting of PLacIR, PPhlF, PCymRC, Pvan, PLuxB, cH OsymC*, dH OsymC*, fH OsymC*, bH OttaC*, cG OttaC*, pPhl, pBAD, pTet, and PvanBT.
  • the modified RBS is selected from a group consisting of phl3, cym2, lux2, van3, lac2, tet2, ara2, el, Al, BX, 13, 15, 18, and 112.
  • the one or more orthogonal inducible recombinase(s) is selected from a group consisting of Al 18, Bxbl, Int3, Int5, Int8, and Intl2.
  • Al 18 comprises at least 70% of SEQ ID NO: 73.
  • Al 18 comprises SEQ ID NO: 73.
  • Bxbl comprises at least 70% of SEQ ID NO: 74.
  • Bxbl comprises SEQ ID NO: 74.
  • Int3 comprises at least 70% of SEQ ID NO: 75.
  • Int3 comprises SEQ ID NO: 75.
  • Int5 comprises at least 70% of SEQ ID NO: 76.
  • Int5 comprises SEQ ID NO: 76.
  • Int8 comprises at least 70% of SEQ ID NO: 77.
  • Int8 comprises SEQ ID NO: 77.
  • Intl2 comprises at least 70% of SEQ ID NO: 78.
  • Intl2 comprises SEQ ID NO: 78.
  • the one or more orthogonal inducible recombinase(s) is induced by an inducer.
  • the inducer is selected from a group consisting of 2,4-Diacetylphloroglucinol (DAPG), aTc, L-Ara, cuminic acid, vanillic acid, Isopropyl [3- D-l -thiogalactopyranoside (IPTG), and 3OC6 Ahl.
  • the chassis cell comprises 1, 2, 3, 4, 5, 6 orthogonal inducible recombinases or a combination thereof.
  • the one or more transcription factor(s) is selected from a group consisting of PhlF, TetR, AraC, CymR, VanR, Lad, AraE, CelR (TAN), RbsR, and LuxR.
  • the one or more degradation tag(s) is selected from a group consisting of DAS tag, AAV tag, and LAA tag.
  • the terminator is selected from a group consisting of L3S1P11, L3S1P13, L3S2P21, L3S2P55, L3S3P00, L3S3P21, L3S3P22, L3S3P23, L3S3P41, ECK120010799, ECK120010818, ECK120010858-R, ECK120015170, ECK120015440, ECK120017009, ECK120033736, ECK120035133, rrnB Tl, BBa_B0014, BBa_B0053, BBa_B0062-R, BBa_B1006, and IOT.
  • the system is configured as a gain-of-function (GOF) memory circuit for both inversion and excision attachment site configuration.
  • the system is configured as a loss-of- function (LOF) memory circuit for both inversion and excision attachment site configuration.
  • a catalytically inactive Cas9 (dCas9) is employed as a recombinase attachment site to prevent recombination with high (-99%) efficiency.
  • extrachromosomal nucleic acid alteration comprises deletion, insertion, or inversion.
  • the insertion is a genomic insertion.
  • the genomic insertion is a functional element.
  • the functional element comprises a reading frame shift, a promoter, a terminator, or a replication origin.
  • the chassis cell exchanges information with a stably colonizing species, wherein the stably colonizing species is found in the gut of a subject.
  • the stably colonizing species is Bacteroides thetaiotaomicron.
  • the synthetic probiotic composition is administered to the subject orally.
  • the synthetic probiotic composition is administered to the subject daily or multiple times a day.
  • the synthetic probiotic composition is administered to the subject weekly, monthly, or only once.
  • the synthetic probiotic composition is administered to the subject daily for at least 1, 2, 3, 4, 5, 6,7, or 8 weeks.
  • the subject is a human.
  • Figures 1A, IB, and 1C show components of an intelligent biological system.
  • Figure 1A shows the concept of decision-making illustrated on the left. Transient state changes are achieved by controlling gene expression with transcription factor-based gene circuits to achieve Boolean logic (middle, right).
  • Figure IB shows the concept of synthetic memory illustrated on the left. Permanent state changes are achieved through the manipulation of genetic components at the DNA level. Transiently expressed recombinases facilitate gain-of-function (middle) or loss-of-function (right) via genetic circuits.
  • Figure 1C shows the concept of communication illustrated on the left. Information transfer is achieved through inducible small-molecule production and subsequent sensing by sender and receiver cells, respectively (right).
  • Figures 2A, 2B, 2C, 2D, and 2E demonstrate the engineering of the MEMORY platform.
  • Figure 2A shows the variables involved in library generation for recombinase expression levels. Libraries contain randomized combinations of an inducible promoter, a modified RBS sequence, a variable start codon, and a C-terminal degradation tag. Each recombinase library is co-transformed with a reporter plasmid containing an inverted promoter upstream of green fluorescent protein (GFP). Individual transformants were screened for low levels of recombination (uninduced) and high levels of recombination (induced).
  • Figure 2B shows the performances of isolated optimized clones from a.
  • % GFP ON denotes the percentage of cells expressing GFP as measured by flow cytometry (See Methods).
  • Figure 2C shows the complete genetic schematic of the recombinase expression system. The transcription factors are those reported in [9] unaltered. The recombinases are inserted directly downstream of the ECK 120017009 terminator. Full sequences of parts are given in Table 2. The full sequence of the recombinase expression cluster is given in Table 2.
  • Figure 2D shows a map of ligand input to unique recombinase output via transcription factor- regulated expression for EcMem.
  • Figure 2E shows the orthogonality between the six recombinases.
  • the EcMem strain transformed with each of the six inversion GOF circuits is assayed with all single inducers.
  • the heatmap shows the percentage of cells with the reporter circuit recombined.
  • Source data are provided as a Source Data file.
  • Figures 3A, 3B, 3C, 3D, 3E, and 3F show the characterization of the MEMORY platform.
  • Figure 3 A shows the kinetics of recombinase activity. The time required for recombination is shown for the EcMem strain transformed with each of the six inversion GOF circuits (circles) and each of the six excision GOF circuits (squares).
  • Figure 3B shows the inversion GOF circuit architecture (left) along with the performance of 6 unique circuits corresponding to the 6 recombinases (right).
  • Figure 3C shows the inversion LOF circuit architecture (left) along with the performance of 6 unique circuits corresponding to the 6 recombinases (right).
  • Figure 3D shows the excision GOF circuit architecture (left) along with the performance of 6 unique circuits corresponding to the 6 recombinases (right).
  • Figure 3E shows the excision LOF circuit architecture (left) along with the performance of 6 unique circuits corresponding to the 6 recombinases (right).
  • Figure 3F shows the genetic stability of the MEMORY platform.
  • the EcMem strain transformed with each of the six inversion GOF circuits is cultured continuously for 11 days. Every other day, the cultures are used to seed media with inducers to assess for maintenance of recombinase functionality. Open circles represent no inducer, and filled circles represent induced cultures. A single biological time-course is shown. See Methods for additional information and Figure 13 for an additional biological replicate.
  • Source data are provided as a Source Data file.
  • Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 51, and 5J show MEMORY recording through genomic integration.
  • Figure 5A shows the first genomic safe harbor (aGSHl) recording circuit (left) and payload 1 (right).
  • Figure 5B shows the percentage of cells expressing GFP prior to recombinase induction.
  • Figure 5C shows the recombined genomic DNA (left) and plasmid DNA (middle) states after induction of Bxbl and Int8, followed by Int3 induction to erase the plasmids (right).
  • Figure 5D shows the percentage of cells expressing GFP after recombinase induction, representing the percentage of integration.
  • Figure 5E shows the new genomic sequence and aGSH2 (left) with payload 2 (right).
  • Figure 5F shows the percentage of cells expressing GFP and mKate prior to recombinase induction.
  • Figure 5G shows the recombined genomic DNA (left) and plasmid DNA (middle) states after induction of Int5 and Intl2, followed by Int3 induction (right).
  • Figure 5H shows the percentage of cells expressing GFP and mKate after recombinase induction.
  • Figure 51 shows representative colony PCR products from cells in Figure 5C. Primers are denoted by halffilled arrows.
  • Figure 5J shows representative colony PCR products from cells in Figure 5G.
  • Figures 6A, 6B, 6C, 6D, 6E, and 6F show the development of CRISPRp.
  • Figure 6A shows a representative CRISPRp program. Fad regulates sgRNA production, while dCas9 is constitutively expressed from the BAC. In the case of no IPTG present in the medium, the recombinase can recombine its target normally when induced. With IPTG in the medium, the sgRNA is produced and directs dCas9 to bind to an att site, preventing the recombinase from performing its catalysis.
  • Figure 6B shows a representative schematic of an att site with key features (left), along with a detailed schematic of the putative CRISPRp binding mechanisms (right).
  • Figure 6C shows an illustration of synthetic PAM addition to the inversion GOF circuit.
  • the positions P1-P4 were assigned based on 5’-3’ directionality, not based on specific attB/attP sites.
  • An illustration of the specific strand targeted by each synthetic PAM site is shown to the right.
  • Figure 6D shows examples of CRISPRp applied to different recombinases.
  • Marionette-Wild cells transformed with the program described in Figure 6A were assayed for recombination with cognate inducer as well as in the presence of IPTG.
  • the specific sgRNA target is shown below each bar graph.
  • Figure 6E shows a schematic of EcMem with expanded memory capacity.
  • Figure 6F shows CRISPRp applied to Int8 with sgRNA regulation by RbsR (left) or CelR (right).
  • Figures 7A, 7B, 7C, and 7D show next-generation recombinase-based state machines.
  • Figure 7A shows a 3-input RSM with 9 possible states based on CRISPRp (left). The same RSM is shown without CRISPRp capability (right).
  • Figure 7B shows the RSM from Figure 7A as a genetic program.
  • the correct induction pattern (Int8, then Bxbl, then Int3) is required to deprotect and unlock the gfp output gene.
  • the EcMem strain transformed with the program was sequentially induced with each relevant inducer in all possible permutations. State transitions are shown by connecting arrows, and the state number is shown on the top left of each circled DNA arrangement. The percentage of cells expressing GFP is shown as a pie chart for each program state.
  • Figure 7C shows detailed state transitions for Figure 7B. A ’ indicates a synonymous state achieved by a different induction sequence.
  • Figure 7D shows data from Figure 7B as bar charts representing recombination percentages.
  • Figures 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, 81, 8J, and 8K show intercellular communication for programmed information exchange.
  • Figure 8A shows a representative autoinduction program.
  • Figure 8B shows a map of cognate interactions for a synthase pathway to activate a given recombinase.
  • Figure 8C shows the performance of the Al 18 autoinduction program for EcMem.
  • Figure 8D shows the performance of the Intl2 autoinduction program for EcMem.
  • Figure 8E shows the performance of the Int8 autoinduction program for EcMem.
  • Figure 8F shows a representative intercellular communication program (top) with a conceptual illustration (bottom).
  • FIG 8G shows the performance of the Al 18 intercellular communication program for EcMem.
  • Figure 8H shows the performance of the Intl2 intercellular communication program for EcMem.
  • Figure 81 shows the performance of the Int8 intercellular communication program for EcMem.
  • Figure 8J shows a cross-species communication program (top, bottom right) with a conceptual illustration (bottom left).
  • EcMem pro harbors an autoinduction program that controls the biosynthesis of vanillic acid, while B. thetaiotaomicron harbors a vanillic acid-responsive circuit that controls the production of Nanoluc.
  • Figure 8K shows the luminescence of the EcMem pro and B. thetaiotaomicron co-culture from Figure 8 J for different ligand conditions.
  • Figures 9A, 9B, 9C, and 9D show the performance of BAC -based recombinase circuits in Marionette-Wild.
  • Figure 9A shows the performances of the inversion GOF circuits when the individual recombinases are harbored on the BAC.
  • Figure 9B shows the performances of the inversion LOF circuits when the individual recombinases are harbored on the BAC.
  • Figure 9C shows the performances of the excision GOF circuits when the individual recombinases are harbored on the BAC.
  • Figure 9D shows the performances of the excision LOF circuits when the individual recombinases are harbored on the BAC. All data represent experiments performed using Marionette-Wild.
  • Figures 11A and 1 IB show orthogonality between Al 18 and Intl2.
  • Figure 11 A shows that PhlF is not induced by 3OC6 AHL.
  • Marionette-Wild was transformed with the Al 18-harboring BAC and Al 18 inversion GOF plasmid and grown in the presence and the absence of 3OC6 AHL.
  • Figure 1 IB shows that Intl2 does not recombine Al 18 att sites.
  • Marionette-Wild was transformed with the Intl2-harboring BAC and Al 18 inversion GOF plasmid and grown in the presence and absence of 3OC6 AHL.
  • Figures 12A, 12B, 12C, and 12D show GFP fluorescence in 24 recombinase circuits.
  • Figure 12A shows the FITC-H values for the inversion GOF circuits.
  • Figure 12B shows the FITC-H values for the inversion LOF circuits.
  • Figure 12C shows the FITC-H values for the excision GOF circuits.
  • Figure 12D shows the FITC-H values for the excision LOF circuits.
  • the dashed line represents the autofluorescence of wild-type E. coli harboring no plasmids.
  • the FITC-H values in Figure 12(A-D) correspond to the population data presented in Figure 3(B-E).
  • Source data are provided as a Source Data file.
  • Figure 13 shows additional data related to the genetic stability of EcMem. Data from a second evolutionary trajectory are shown from the experiment presented in Figure 3F. The EcMem strain transformed with each of the six inversion GOF circuits is cultured continuously for 11 days. Every other day, the cultures are used to seed media with inducers to assess for maintenance of recombinase functionality. Open circles represent no inducer, and filled circles represent induced cultures.
  • Figure 14 shows the cellular burden of recombinase expression. Growth curves of EcMem in minimal media during specific recombinase expression are shown. Each graph shows the growth curve for the EcMem without inducers (circles), with all inducers (small squares), and with the specific inducer of the indicated recombinase (large squares).
  • Figures 15A, 15B, and 15C show Al 18 excision gain-of-function with cellular reset.
  • Figure 15A shows the Al 18 excision GOF circuit with the Int3 origin excision (left). The percentage of recombined cells after growth in MM without inducer is shown (middle) along with an input-output table for this program (right).
  • Figure 15B shows the recombined circuit after Al 18 induction (left). The percentage of recombined cells after growth in MM with 2,4-Diacetylphloroglucinol (DAPG) is shown (middle). Cells were then grown with L- ara to induce Int3 expression and origin excision (right).
  • Figure 15C shows the efficiency of the cellular reset.
  • Figures 16A, 16B, 16C, and 16D show extended data related to Figure 4.
  • Figure 16A shows a recreated Figure 4A with flow cytometry dot plots.
  • Figure 16B shows a recreated Figure 4B with flow cytometry dot plots.
  • Figure 16C shows a recreated Figure 4D with flow cytometry dot plots.
  • FITC-H is shown on the X-axis, representing GFP expression
  • ECD-H is shown on the Y-axis, representing mKate expression.
  • a single representative dot plot is shown for each case.
  • Figure 16D shows the efficiency of the cellular reset. Reset cells from the “GFP + mKate” state in Figure 16B were serially diluted and plated on LB agar with and without kanamycin before and after Int3 induction to assess for pSClOl plasmid loss. Resultant colonies were counted, and colony-forming units (CFU) were determined.
  • Figures 17A, 17B, 17C, 17D, 17E, 17F, 17G, 17H, 171, 17J, 17K, and 17L show data related to inducible integration.
  • Figure 17A shows the first genomic safe harbor (aGSHl) recording site (left) and the memory sequence (right).
  • Figure 17B shows the percentage of cells expressing GFP prior to recombinase induction.
  • Figure 17C shows the recombined genomic DNA (left) and plasmid DNA (middle) states after induction of Bxbl and Int8, followed by Int3 induction to erase the plasmids (right).
  • Figure 17D shows the percentage of cells expressing GFP after recombinase induction, representing the percentage of integration.
  • Figure 17E shows the new genomic sequence and aGSH2 (left) with the second memory sequence (right).
  • Figure 17F shows the percentage of cells expressing GFP and mKate prior to recombinase induction.
  • Figure 17G shows the recombined genomic DNA (left) and plasmid DNA (middle) states after induction of Int5 and Intl2, followed by Int3 induction (right).
  • Figure 17H shows the percentage of cells expressing GFP and mKate after recombinase induction.
  • Figure 171 shows representative colony PCR products of 8 colonies from Figure 17C. Primers are denoted by half-filled arrows.
  • Figure 17J shows representative colony PCR products of 8 colonies from Figure 17G.
  • Figure 17K shows representative colony PCR products of 8 colonies from Figure 5C.
  • Figure 17E shows representative colony PCR products of 8 colonies from Figure 5G.
  • Source data are provided as a Source Data file.
  • Figure 18 shows additional demonstrations of CRISPR protection.
  • Figure 18A shows additional examples of successful CRISPR protection are shown.
  • Cells transformed with the circuit described in Figure 6A were assayed for recombination performance with cognate inducer as well as in the presence of IPTG.
  • the specific sgRNA target is shown below each bar graph.
  • Figures 19A and 19B show that dCas9 affects PhlF- and Al 18-based circuits.
  • Figure 19A shows the circuit described in Figure 6A tested with Al 18.
  • the top bar graphs show circuit performance when dCas9 is constitutively expressed from the BAC, while the bottom bar graphs show circuit performance when dCas9 is controlled by an IPTG-inducible promoter (Psym).
  • Figure 19B shows the genetic context for recombinase and dCas9 expression next to the appropriate bar graphs. Circuits were assayed in the EcMem strain.
  • Figures 20A, 20B, and 20C show controlling CRISPRp with additional transcription factors.
  • Figure 20A shows CRISPRp programs using transcription factors harbored on a pl5a plasmid.
  • RbsR or CelR regulates sgRNA production from the Ptta promoter of varying strengths (bH for RbsR and cG for CelR; see Tables 1 and 2 for details).
  • Figure 20B shows the performance of RbsR-controlled CRISPRp.
  • Figure 20C shows the performance of CelR-controlled CRISPRp. Circuits were assayed in the EcMem strain.
  • Figures 21A, 21B, and 21C show the expansion of memory capacity using CRISPRp.
  • Figures 22A and 22B show the next-generation RSM design.
  • Figure 22A shows a typical 2-input RSM designed by Roquet el al. (left).
  • the expanded RSM state diagram is shown if CRISPRp is applied (right).
  • “A” and “B” represent inputs for different recombinases.
  • a ‘ indicates a unique recombination event due to the ability to selectively use CRISPRp.
  • Figure 22B shows the genetic schematic for Figure 22A.
  • Red arrows indicate recombination by recombinase 1
  • blue arrows indicate recombination by recombinase 2.
  • a grey lobe covering an at site denotes the programmed CRISPRp of that site. This RSM assumes that CRISPRp can be applied to any at site independently.
  • Figures 23A, 23B, 23C, 23D, 23E, 23F, 23G, and 23H show performance of recombinase circuits in EcMem Pro .
  • Figure 23A shows the performances of the inversion GOF circuits assayed aerobically in EcMem Pro .
  • Figure 23B shows the performances of the inversion EOF circuits assayed aerobically in EcMem Pro .
  • Figure 23C shows the performances of the excision GOF circuits assayed aerobically in EcMcm Pro .
  • Figure 23D shows the performances of the excision LOF circuits assayed aerobically in EcMem Pro .
  • Figure 23E shows the performances of the inversion GOF circuits assayed anaerobically in EcMem Pro .
  • Figure 23F shows the performances of the inversion LOF circuits assayed anaerobically in EcMcm Pro .
  • Figure 23G shows the performances of the excision GOF circuits assayed anaerobically in EcMem Pro .
  • Figure 23H shows the performances of the excision LOF circuits assayed anaerobically in EcMem Pro .
  • Figure 25 shows an example gating strategy for flow cytometry. Representative gates used in flow cytometry analysis are shown. Cells were first gated by side scatter area vs. forward scatter area (left). This population was then gated by side scatter height vs. side scatter area to discriminate single cells (middle). In this example, cells with a FITC-H value greater than 3E3 were deemed GFP -positive while cells with a lower FITC-H value were deemed GFP-negative.
  • Figures 26A, 26B, 26C, and 26D show representative flow cytometry data for 24 recombinase circuits.
  • a representative flow cytometry plot is provided for each of the 24 recombinase circuits.
  • the uninduced and induced states are shown on the same plot with an arrow denoting the transition of the populations upon induction.
  • the y-axis is the cell count and the x-axis is the GFP intensity (FITC- H).
  • FIG. 27 shows relevant plasmid maps used in this study. Maps correspond to descriptions in Table 1.
  • the terms “about” and “approximately” are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%. [0062] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
  • composition refers to any agent that has a beneficial biological effect.
  • beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • composition includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • compositions, methods, etc. include the recited elements, but do not exclude others.
  • Consisting essentially of' when used to define compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • An "increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more, increase so long as the increase is statistically significant.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • "Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • reduce or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
  • the term “subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • a "promoter,” as used herein, refers to a sequence in DNA that mediates the initiation of transcription by an RNApolymerase. Transcription promoters may comprise one ormore of a number of different sequence elements as follows: 1) sequence elements present at the site of transcription initiation; 2) sequence elements present upstream of the transcription initiation site and; 3) sequence elements downstream of the transcription initiation site. The individual sequence elements function as sites on the DNA, where RNA polymerases and transcription factors that facilitate positioning of RNA polymerases on the DNA bind.
  • a “transcription factor” refers to a sequence- specific DNA-binding protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence.
  • a “transcription terminator” or a “terminator” refers to a segment of a nucleic acid sequence that marks the end of gene in genomic DNA during the transcription process, or gene expression. This sequence mediates or signals the end of transcription by providing signaling nucleotides in newly synthesized RNA transcripts that trigger an RNA polymerase to release the DNA and newly synthesized RNA.
  • the word “vector” refers to any vehicle that carries a polynucleotide into a cell for the expression of the polynucleotide in the cell.
  • the vector may be, for example, a plasmid, a virus, a phage particle, or a nanoparticle.
  • a “bacterial plasmid” is a small extrachromosomal DNA molecule that can be incorporated into another cell that is physically separated from the chromosomal DNA and is easily replicated. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself.
  • the vector is a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host cell.
  • control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control the termination of transcription and translation.
  • administer refers to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir.
  • parenteral includes subcutaneous, intravenous, intramuscular, intra- articular, intra- synovial, intrastemal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
  • a “host” refers to an organism or cell into which a heterologous component (polynucleotide, polypeptide, other molecule, cell) has been introduced.
  • a “host cell” refers to an in vivo or in vitro eukaryotic cell, prokaryotic cell (e.g., bacterial or archaeal cell), or cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, into which a heterologous polynucleotide or polypeptide has been introduced.
  • the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, an insect cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell.
  • the cell is in vitro.
  • the cell is in vivo.
  • An "effective amount" is an amount sufficient to affect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.
  • Effective amount encompasses, without limitation, an amount that can ameliorate, reverse, mitigate, prevent, or diagnose a symptom or sign of a medical condition or disorder (e.g., HIV-1 infection). Unless dictated otherwise, explicitly or by context, an “effective amount” is not limited to a minimal amount sufficient to ameliorate a condition.
  • the severity of a disease or disorder, as well as the ability of a treatment to prevent, treat, or mitigate, the disease or disorder can be measured, without implying any limitation, by a biomarker or by a clinical parameter.
  • microbiota refers to the range of microorganisms that may be commensal, symbiotic, or pathogenic found in and on all multicellular organisms, including plants and animals. These include bacteria, archaea, protists, fungi, and viruses and have been found to be crucial for immunologic, hormonal, and metabolic homeostasis of the host.
  • monitoring refers to the actions of observing and checking the progress or quality of a treatment or procedure over a period of time.
  • monitoring refers to the actions of observing and checking for changes to the GI tract microbiome following administration of a cell comprising a construct to (re)program to transcriptional regulation of the microbiome.
  • a “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA.
  • the four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule.
  • percent identity and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences and, therefore, achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST 2 Sequences a tool that is used for direct pairwise comparison of two nucleotide sequences.
  • Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.
  • a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
  • a “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250).
  • a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.
  • upstream refers to the relative position of a genetic sequence, either DNA or RNA. Upstream relates to the 5’ to 3’ direction relative to the start site of transcription, wherein upstream is usually closer to the 5’ end of a genetic sequence.
  • downstream refers to the relative position of a genetic sequence, either DNA or RNA. Downstream relates to the 5’ to 3’ direction relative the start site of transcription, wherein downstream is usually closer to the 3’ end of a genetic sequence.
  • Gene includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein, including regulatory sequences preceding (5’ noncoding sequences) and following (3’ non-coding sequences) the coding sequence.
  • “Native gene” refers to a gene as found in its natural endogenous location with its own regulatory sequences.
  • knock-out represents a DNA sequence of a cell that has been rendered partially or completely inoperative by targeting with a Cas protein; for example, a DNA sequence prior to knock-out could have encoded an amino acid sequence or could have had a regulatory function (e.g., promoter).
  • a regulatory function e.g., promoter
  • knock-in represents the replacement or insertion of a DNA sequence at a specific DNA sequence in cell by targeting with a Cas protein (for example, by homologous recombination (HR), wherein a suitable donor DNA polynucleotide is also used)
  • examples of knock-ins are a specific insertion of a heterologous amino acid coding sequence in a coding region of a gene, or a specific insertion of a transcriptional regulatory element in a genetic locus.
  • domain it is meant a contiguous stretch of nucleotides (that can be RNA, DNA, and/or RNA-DNA-combination sequence) or amino acids.
  • An “enhancer” is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue- specificity of a promoter. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature and/or comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.
  • Disclosed herein is an intelligent biotic system as one or more chassis cells capable of (i) decision-making, (ii) coupled memory development, (iii) and communication between chassis cells and/or the host. Accordingly, disclosed herein are programmable drugdelivery compositions, and the methods of use thereof, in preventing or treating gut microbiota dysbiosis.
  • a programmable drug-delivery system comprising a chassis cell.
  • a chassis cell is a self -replicating organism that serves as a foundation for engineered biological systems in synthetic biology. Chassis cells are used to produce specific chemicals and other bioproducts. Chassis cells provide a framework for genetic components, and they provide resources like transcription and translation machinery to support those components.
  • An exemplary chassis cell comprises a Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)).
  • MEMORY is designed to facilitate discrete multi-input regulation of recombinase functions enabling inheritable DNA inversions, deletions, and genomic insertions containing functional elements - e.g., reading frames, promoters, terminators, or replication origins.
  • extrachromosomal nucleic acid alteration comprises deletion, insertion, or inversion.
  • the insertion is a genomic insertion.
  • the genomic insertion is a functional element.
  • the functional element comprises a reading frame shift, a promoter, a terminator, or a replication origin.
  • MEMORY cells can achieve programmable and permanent gain (or loss) of functions extrachromosomally or from a specific genomic locus without the loss or modification of the MEMORY platform. Retention of the MEMORY platform facilitates the sequential programming and reprogramming of DNA circuits harbored within a given chassis cell. When fully deployed, MEMORY cells can achieve all three tenets of intelligence - i.e., cellular decision-making, inheritable memory, and communication between cells.
  • the chassis cell comprises an inducible promoter.
  • An inducible promoter is a molecular tool that can be used to control when and how strongly a gene is expressed. Inducible promoters are activated by a stimulus, such as a chemical, heat shock, osmotic stress, drought, or cold. Some exemplary inducers are IPTG, lactose, arabinose, tetracycline, anhydrotetracycline, doxycycline, rhamnose, galactose, estrogen, tamoxifen, heavy metals (Zn 2+ , Cu 2+ , Cd 2+ ), glucocorticoids.
  • a promoter can be made “inducible” or “regulated” by rationally modifying it to contain transcription factor operators.
  • PLacIR comprises at least 70% of SEQ ID NO: 2. In some embodiments, PLacIR comprises SEQ ID NO: 2. In some embodiments, PLacIR may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • PPhlF comprises at least 70% of SEQ ID NO: 3. In some embodiments, PPhlF comprises SEQ ID NO: 3. In some embodiments, PPhlF may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • PTet comprises at least 70% of SEQ ID NO: 4. In some embodiments, PTet comprises SEQ ID NO: 4. In some embodiments PTet may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 4.
  • pBAD comprises at least 70% of SEQ ID NO: 5. In some embodiments, pBAD comprises SEQ ID NO: 5. In some embodiments, pBAD may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 5.
  • PCymRC comprises at least 70% of SEQ ID NO: 6. In some embodiments, PCymRC comprises SEQ ID NO: 6. In some embodiments PCymRC may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • Pvan comprises at least 70% of SEQ ID NO: 7. In some embodiments, Pvan comprises SEQ ID NO: 7. In some embodiments Pvan may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 7.
  • PLuxB comprises at least 70% of SEQ ID NO: 8. In some embodiments, PLuxB comprises SEQ ID NO: 8. In some embodiments PLuxB may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 8.
  • cH OsymC* comprises at least 70% of SEQ ID NO: 9. In some embodiments, cH OsymC* comprises SEQ ID NO: 9. In some embodiments cH OsymC* may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 9.
  • dH OsymC* comprises at least 70% of SEQ ID NO:
  • dH OsymC* comprises SEQ ID NO: 10.
  • dH OsymC* may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 10.
  • fH OsymC* comprises at least 70% of SEQ ID NO:
  • fH OsymC* comprises SEQ ID NO: 11.
  • fH OsymC* may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 11.
  • bH OttaC* comprises at least 70% of SEQ ID NO: 12.
  • cG OttaC* comprises at least 70% of SEQ ID NO: 13. In some embodiments, cG OttaC* comprises SEQ ID NO: 13. In some embodiments cG OttaC* may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 13.
  • the chassis cell comprises a modified ribosome-binding site (RBS).
  • a ribosome binding site (RBS) is a sequence of nucleotides in messenger RNA (mRNA) that helps ribosomes bind to the mRNA and start translation.
  • the RBS is located upstream of the start codon.
  • a modified RBS such as, for example, phl3, cym2, lux2, van3, lac2, tet2, ara2, el, Al, BX, 13, 15, 18, and 112.
  • phl3 comprises at least 70% of SEQ ID NO: 15. In some embodiments, phl3 comprises SEQ ID NO: 15. In some embodiments, phl3 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 15. [0121] In some embodiments, cym2 comprises at least 70% of SEQ ID NO: 16. In some embodiments, cym2 comprises SEQ ID NO: 16.
  • cym2 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 16.
  • lux2 comprises at least 70% of SEQ ID NO: 17. In some embodiments, lux2 comprises SEQ ID NO: 17. In some embodiments, lux2 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 17. [0123] In some embodiments, van3 comprises at least 70% of SEQ ID NO: 18. In some embodiments, van3 comprises SEQ ID NO: 18.
  • van3 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 18.
  • lac2 comprises at least 70% of SEQ ID NO: 19. In some embodiments, lac2 comprises SEQ ID NO: 19.
  • lac2 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 19.
  • tet2 comprises at least 70% of SEQ ID NO: 20.
  • tet2 comprises SEQ ID NO: 20.
  • tet2 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 20.
  • ara2 comprises at least 70% of SEQ ID NO: 21. In some embodiments, ara2 comprises SEQ ID NO: 21.
  • ara2 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 21.
  • el comprises at least 70% of SEQ ID NO: 22. In some embodiments, el comprises SEQ ID NO: 22.
  • el may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 22.
  • Al comprises at least 70% of SEQ ID NO: 23. In some embodiments, Al comprises SEQ ID NO:23.
  • Al may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 23.
  • BX comprises at least 70% of SEQ ID NO: 24. In some embodiments, BX comprises SEQ ID NO: 24. In some embodiments, BX may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 24.
  • 13 comprises at least 70% of SEQ ID NO: 25. In some embodiments, 13 comprises SEQ ID NO: 25. In some embodiments, 13 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 25.
  • 15 comprises at least 70% of SEQ ID NO: 26. In some embodiments, 15 comprises SEQ ID NO: 26. In some embodiments, 15 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 26.
  • 18 comprises at least 70% of SEQ ID NO: 27. In some embodiments, 18 comprises SEQ ID NO: 27. In some embodiments, 18 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 27.
  • 112 comprises at least 70% of SEQ ID NO: 28. In some embodiments, 112 comprises SEQ ID NO: 28. In some embodiments, 112 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 28.
  • the chassis cell as disclosed herein comprises one or more genes expressing one or more orthogonal inducible recombinase(s).
  • Orthogonal inducible recombinases are systems that use multiple recombinases to control gene expression in a cell or organism, are site- specific enzymes that mediate DNA recombination in response to a specific inducer.
  • the inducer is selected from a group consisting of 2,4- Diacetylphloroglucinol (DAPG), aTc, L-Ara, cuminic acid, vanillic acid, Isopropyl -D-1- thiogalactopyranoside (IPTG), and 3OC6 Ahl.
  • the chassis cell is engineered to produce an orthogonal inducible recombinase.
  • the chassis cell facilitates discrete multi-input regulation of recombinase function to alter extrachromosomal nucleic acid, wherein the chromosomal nucleic acid of the chassis cell is not altered.
  • the one or more orthogonal inducible recombinase(s) is selected from a group consisting of Al 18, Bxbl, Int3, Int5, Int8, and Intl2.
  • Al 18 comprises at least 70% of SEQ ID NO: 73. In some embodiments, Al 18 comprises SEQ ID NO: 73. In some embodiments, Al 18 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • Bxbl comprises at least 70% of SEQ ID NO: 74. In some embodiments, Bxbl comprises SEQ ID NO: 74. In some embodiments, Bxbl may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • Int3 comprises at least 70% of SEQ ID NO: 75. In some embodiments, Int3 comprises SEQ ID NO: 75. In some embodiments, Int3 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 75. [0139] In some embodiments, Int5 comprises at least 70% of SEQ ID NO: 76. In some embodiments, Int5 comprises SEQ ID NO: 76.
  • Int5 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 76.
  • Int8 comprises at least 70% of SEQ ID NO: 77. In some embodiments, Int8 comprises SEQ ID NO: 77.
  • Int8 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 77.
  • Intl2 comprises at least 70% of SEQ ID NO: 78. In some embodiments, Intl2 comprises SEQ ID NO: 78.
  • Intl2 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 78.
  • the chassis cell comprises 1, 2, 3, 4, 5, 6 orthogonal inducible recombinases or a combination thereof. In some embodiments, the chassis cell comprises 1 orthogonal inducible recombinase. In some embodiments, the chassis cell comprises two orthogonal inducible recombinases. In some embodiments, the chassis cell comprises 3 orthogonal inducible recombinases. In some embodiments, the chassis cell comprises 4 orthogonal inducible recombinases. In some embodiments, the chassis cell comprises 5 orthogonal inducible recombinases. In some embodiments, the chassis cell comprises 6 orthogonal inducible recombinases. In some embodiments, the chassis cell comprises more than 6 orthogonal inducible recombinase.
  • the chassis cell as disclosed herein comprises one or more transcription factor(s), wherein the one or more transcription factor(s) regulate the one or more orthogonal inducible recombinase(s) expression.
  • the one or more transcription factor(s) is selected from a group consisting of PhlF, TetR, AraC, CymR, VanR, LacI, AraE, CelR (TAN), RbsR, and LuxR.
  • PhlF comprises at least 70% of SEQ ID NO: 60. In some embodiments, PhlF comprises SEQ ID NO: 60. In some embodiments, PhlF may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 60. [0145] In some embodiments, CymR comprises at least 70% of SEQ ID NO: 61. In some embodiments, CymR comprises SEQ ID NO: 61.
  • CymR may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 61.
  • LuxR comprises at least 70% of SEQ ID NO: 62. In some embodiments, LuxR comprises SEQ ID NO: 62.
  • LuxR may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • VanR comprises at least 70% of SEQ ID NO: 63. In some embodiments, VanR comprises SEQ ID NO: 63. In some embodiments, VanR may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • LacI comprises at least 70% of SEQ ID NO: 64. In some embodiments, LacI comprises SEQ ID NO: 64. In some embodiments, LacI may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 64.
  • TetR comprises at least 70% of SEQ ID NO: 65. In some embodiments, TetR comprises SEQ ID NO: 65.
  • TetR may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • AraC comprises at least 70% of SEQ ID NO: 66. In some embodiments, AraC comprises SEQ ID NO: 66. In some embodiments, AraC may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • CelR (TAN) comprises SEQ ID NO: 68.
  • CelR (TAN) may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 68.
  • AAV tag comprises at least 70% of SEQ ID NO: 58. In some embodiments, AAV tag comprises SEQ ID NO: 58. In some embodiments, AAV tag may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • the chassis cell comprises a terminator, wherein the terminator provides transcriptional insulation.
  • the terminator is selected from a group consisting of L3S1P11, L3S1P13, L3S2P21, L3S2P55, L3S3P00, L3S3P21, L3S3P22, L3S3P23, L3S3P41, ECK120010799, ECK120010818, ECK120010858-R, ECK120015170, ECK120015440, ECK120017009, ECK120033736, ECK120035133, rrnB Tl, BBa_B0014, BBa_B0053, BBa_B0062-R, BBa_B1006, and IOT.
  • L3S1P11 comprises at least 70% of SEQ ID NO: 30.
  • L3S3P00 comprises at least 70% of SEQ ID NO: 34.
  • L3S3P21 comprises at least 70% of SEQ ID NO: 35.
  • L3S3P22 comprises at least 70% of SEQ ID NO: 36.
  • L3S3P22 comprises SEQ ID NO: 36.
  • L3S3P22 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • L3S3P23 comprises at least 70% of SEQ ID NO: 37.
  • L3S3P41 comprises at least 70% of SEQ ID NO: 38. In some embodiments, L3S3P41 comprises SEQ ID NO: 38. In some embodiments, L3S3P41 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 38.
  • ECK120010799 comprises at least 70% of SEQ ID NO: 39. In some embodiments, ECK120010799 comprises SEQ ID NO: 39. In some embodiments, ECK120010799 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 39.
  • ECK120010818 comprises at least 70% of SEQ ID NO: 40. In some embodiments, ECK120010818 comprises SEQ ID NO: 40. In some embodiments, ECK120010818 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 40.
  • ECK120010858-R comprises at least 70% of SEQ ID NO: 41. In some embodiments, ECK120010858-R comprises SEQ ID NO: 41. In some embodiments, ECK120010858-R may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 41.
  • ECK120015170 comprises at least 70% of SEQ ID NO: 42. In some embodiments, ECK120015170 comprises SEQ ID NO: 42. In some embodiments, ECK120015170 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 42.
  • ECK120015440 comprises at least 70% of SEQ ID NO: 43. In some embodiments, ECK120015440 comprises SEQ ID NO: 43. In some embodiments, ECK120015440 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 43.
  • ECK120017009 comprises at least 70% of SEQ ID NO: 44. In some embodiments, ECK120017009 comprises SEQ ID NO: 44. In some embodiments, ECK120017009 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 44.
  • ECK120033736 comprises at least 70% of SEQ ID NO: 45. In some embodiments, ECK120033736 comprises SEQ ID NO: 45. In some embodiments, ECK120033736 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 45.
  • rrnB Tl comprises at least 70% of SEQ ID NO: 47. In some embodiments, rrnB Tl comprises SEQ ID NO: 47. In some embodiments, rrnB Tl may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO:
  • BBa_B0014 comprises at least 70% of SEQ ID NO:
  • BBa_B0053 comprises SEQ ID NO: 49.
  • BBa_B0053 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 49.
  • BBa_B0062-R comprises at least 70% of SEQ ID NO:
  • BBa_B0062-R comprises SEQ ID NO: 50.
  • BBa_B0062-R may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 50.
  • BBa_B1006 comprises at least 70% of SEQ ID NO:
  • BBa_B1006 comprises SEQ ID NO: 51.
  • BBa_B1006 may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 51.
  • IOT comprises at least 70% of SEQ ID NO: 52. In some embodiments, IOT comprises SEQ ID NO: 52. In some embodiments, IOT may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to SEQ ID NO: 52. [0184] In some embodiments, the programmable drug-delivery system further comprises a drug.
  • the term "drug” refers to any biologically active compound, molecule, or composition that is capable of exerting a therapeutic, diagnostic, prophylactic, or pharmacological effect in a subject.
  • the term includes, but is not limited to: Small molecules - Organic or inorganic compounds, including synthetic and naturally occurring substances, that modulate biological pathways. Biologies - Proteins, peptides, antibodies (monoclonal, polyclonal, and recombinant), nucleic acids (DNA, RNA, aptamers, CRIS PR-based), gene therapy vectors, and cellular therapies.
  • compositions Any formulation containing an active drug substance along with excipients, carriers, stabilizers, or other formulation agents suitable for administration.
  • Vaccines and Immunomodulators Any antigenic compositions, adjuvants, or immune- modulating agents used for disease prevention or treatment.
  • Controlled Substances and Regulatory Compounds Any compound classified as a therapeutic agent by regulatory authorities, including prescription drugs, over-the-counter medications, and investigational new drugs.
  • drug encompasses agents intended for use in humans, animals, or other biological systems via any route of administration, including but not limited to oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, intranasal, or topical delivery.
  • the term also includes derivatives, salts, isomers, analogs, metabolites, and polymorphic forms of active compounds, where applicable.
  • the drug can be a genetically encoded drug.
  • the chassis cell is a non-colonizing bacterium (such as, for example, Escherichia coli, strain Nissle 1917 or K12).
  • a study engineered Escherichia coli strains to harbor a genome-integrated array of six orthogonal inducible recombinases - forming the Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY).
  • MEMORY Orthogonal Recombinase arraY
  • a study engineered a probiotic MEMORY strain capable of programmable information exchange between Nissle 1917 and the gastrointestinal commensal Bacteroid.es thetaiotaomicron.
  • the system is configured as a gain-of-function (GOF) memory circuit for both inversion and excision attachment site configuration.
  • the system is configured as a loss-of-function (LOF) memory circuit for both inversion and excision attachment site configuration.
  • a catalytically inactive Cas9 (dCas9) is employed to a recombinase attachment site to prevent recombination with high (-99%) efficiency.
  • Cas9 refers to CRIS PR-associated protein 9, an RNA-guided endonuclease derived from Streptococcus pyogenes or other bacterial species, which functions as a component of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immune system. Cas9 is capable of recognizing and cleaving specific DNA sequences in a programmable manner when guided by a complementary single-guide RNA (sgRNA) or a dual crRNA:tracrRNA complex.
  • sgRNA complementary single-guide RNA
  • a synthetic probiotic composition comprising a programmable drug-delivery system and a pharmaceutically acceptable carrier, wherein the programmable drug-delivery system comprising a chassis cell, wherein the chassis cell (e.g., a Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)) comprises, an inducible promoter, a modified ribosome-binding site (RBS), one or more genes expressing one or more orthogonal inducible recombinase(s), one or more transcription factor(s), wherein the one or more transcription factor(s) regulate the one or more orthogonal inducible recombinase(s) expression, one or more degradation tag(s), a variable start codon; and a terminator, wherein the terminator provides transcriptional insulation, as in any of the preceding aspects.
  • the chassis cell e.g., a Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)
  • the chassis cell exchanges information with a stably colonizing species, wherein the stably colonizing species is found in the gut of a subject.
  • the stably colonizing species is Bacteroides thetaiotaomicron.
  • a method of preventing or treating gut microbiota dysbiosis in a subject in need thereof comprising administering a therapeutically effective amount of a synthetic probiotic composition comprising a programmable drug-delivery system and a pharmaceutically acceptable carrier, wherein the programmable drug-delivery system comprising a chassis cell, wherein the chassis cell (e.g., a Molecularly Encoded Memory via an Orthogonal Recombinase arraY (MEMORY)) comprises, an inducible promoter, a modified ribosome-binding site (RBS), one or more genes expressing one or more orthogonal inducible recombinase(s), one or more transcription factor(s), wherein the one or more transcription factor(s) regulate the one or more orthogonal inducible recombinase(s) expression, one or more degradation tag(s), a variable start codon; and a terminator, wherein the terminator provides transcriptional insulation
  • the chassis cell is engineered to produce an orthogonal inducible recombinase as in any of the preceding aspects.
  • the chassis cell facilitates discrete multi-input regulation of recombinase function to alter extrachromosomal nucleic acid, wherein chromosomal nucleic acid of the chassis cell is not altered, as described herein.
  • the chassis cell is a non-colonizing bacterium (such as, for example, Escherichia coli), as in any of the preceding aspects.
  • the system is configured as a gain-of-function (GOF) memory circuit for both inversion and excision attachment site configuration as in any of the preceding aspects.
  • GAF gain-of-function
  • LEF loss-of-function
  • a catalytically inactive Cas9 (dCas9) is employed as a recombinase attachment site to prevent recombination with high (-99%) efficiency as in any of the preceding aspects.
  • extrachromosomal nucleic acid alteration comprises deletion, insertion, or inversion as in any of the preceding aspects.
  • the insertion is a genomic insertion.
  • the genomic insertion is a functional element.
  • the functional element comprises a reading frame shift, a promoter, a terminator, or a replication origin.
  • the subject is a human.
  • the synthetic probiotic composition is administered to the subject orally. In some embodiments, the synthetic probiotic composition is administered to the subject daily or multiple times a day. In some embodiments, the synthetic probiotic composition is administered to the subject weekly, monthly, or only once. In some embodiments, the synthetic probiotic composition is administered to the subject daily for at least 1, 2, 3, 4, 5, 6,7, or 8 weeks.
  • the synthetic probiotic composition may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the synthetic probiotic composition will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the gut microbiota dysbiosis, the particular synthetic probiotic composition, its mode of administration, its mode of activity, and the like.
  • the synthetic probiotic composition is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the synthetic probiotic composition will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors, including the severity of the gut microbiota dysbiosis; the activity of the synthetic probiotic composition employed; the specific synthetic probiotic composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific synthetic probiotic composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific synthetic probiotic composition employed; and like factors well known in the medical arts.
  • the synthetic probiotic composition may be administered by any route.
  • the synthetic probiotic composition is administered orally, nasally, buccal, enterally, sublingually, or by tablet, liquid, or oral spray forms.
  • the most appropriate route of administration will depend upon a variety of factors, including the nature of the synthetic probiotic composition (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.
  • the exact amount of synthetic probiotic composition required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects, identity of the particular compound(s), mode of administration, and the like.
  • the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • the concentration of active agent(s) can vary widely and will be selected primarily based on the activity of the active ingredient(s), body weight, and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages ranging from about 0.1 or 1 mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosages range from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably from about 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about 7.2 mg/kg/day to about 11.0 mg/kg/day, and most preferably from about 11.0 mg/kg/day to about 15.0 mg/kg/day.
  • dosages range from about 10 mg/kg/day to about 50 mg/kg/day. In certain embodiments, dosages range from about 20 mg to about 50 mg, and they are given orally twice daily. It will be appreciated that such dosages may be varied to optimize a therapeutic and/or prophylactic regimen in a particular subject or group of subjects.
  • a synthetic probiotic composition of any preceding aspect and a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, and a cream.
  • the synthetic probiotic composition can be administered if desired in the form of salts, esters, amides, prodrugs, or a derivative that is pharmacologically suitable. Salts, esters, amides, prodrugs, and other derivatives of the active agents can be prepared using standards procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced. Organic Chemistry; Reactions, Mechanisms, and Structure, 4 th Ed. N.Y. Wiley-Interscience.
  • a synthetic probiotic composition can be prepared as a “concentrate,” e.g., in a storage container of a premeasured volume and/or a predetermined amount ready for dilution or in a soluble capsule ready for addition to a specified volume of water, saline, alcohol, hydrogen peroxide, or other diluent.
  • the synthetic probiotic composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
  • the synthetic probiotic composition is administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more.
  • the synthetic probiotic composition is administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more.
  • the instant study developed a novel Escherichia coli chassis cell with a genomically integrated memory array composed of six orthogonal, inducible recombinases - regulated by a set of transcription factors commonly used in the Marionette biosensing array (z.e., PhlF, TetR, AraC, CymR, VanR, and LuxR).
  • the expression level of each recombinase was carefully optimized to achieve near digital switching of cell genotype when induced to perform a specific recombination function.
  • the instant study developed 24 fundamental gain-of-function (GOF) and loss-of-function (LOF) memory circuits for both inversion and excision attachment site configurations.
  • GAF gain-of-function
  • LEF loss-of-function
  • CRISPRp CRISPR-Cas9- mediated protection of recombinase action
  • CRISPRp of a given att site could be programmed with fundamental decision-making via T-Pro transcription factors - with concurrent MEMORY operation.
  • CRISPRp was used to develop a nextgeneration recombinase-based state machine (ngRSM) to demonstrate an application of this posttranslational control mechanism.
  • ngRSM nextgeneration recombinase-based state machine
  • the engineered chassis cells can be used to program information exchange between a probiotic E.
  • Inheritable synthetic memory Decision-making is composed of one or more INPUT(s) mapped to an OUTPUT, such that the system can be reset upon the removal of the INPUT(s).
  • a synthetic memory operation is not reset upon the removal of cognate INPUT(s) - i.e., memory operations retain changes in the OUTPUT state upon the removal of the cognate INPUT(s).
  • Canonical synthetic memory type-I is achieved by way of the regulation of a given recombinase, which is typically induced by a small molecule. Once matured (folded and assembled), the recombinase attaches to DNA elements attb and attp, resulting in the reconfiguration of DNA.
  • interception is defined as the controlled blocking of any protein-DNA interaction (other than RNA polymerase) via a transcription factor (TF) that interacts with a cognate DNA operator pair in situ. Interception was achieved by strategically replacing a small segment of a recombinase attachment site with a DNA operator. The study posits that mechanistically, this results in the TF - when bound to operator DNA - sterically hindering a given recombinase from binding to a cognate attachment site.
  • TF transcription factor
  • this iteration of synthetic memory requires two parts: (i) an operation that regulates recombinase attachment post-translation and (ii) a genetic circuit to define the memory function - i.e., the orientation and positioning of recombinase attachment sites allb and allp.
  • Each recombinase library consisted of an inducible promoter, a degenerate ribosome binding site (RBS) sequence [43], a degenerate start codon, and two degradation tags of variable strength. These libraries were cloned into a single-copy bacterial artificial chromosome (BAC) with the intent of mimicking genomic expression levels [44].
  • BAC bacterial artificial chromosome
  • output circuits were designed where a strong, inverted promoter (Pj23i 19) was flanked by anti-aligned att sites followed by a green fluorescent protein (GFP') gene, harbored on a low-copy (3-5 copy) pSClOl plasmid ( Figure 2a).
  • M9 minimal medium MM
  • the study designed the recombinase expression system to be implemented at the single-copy level at the outset of this study.
  • the study Prior to genomic integration, the study used the BAC as a testbed for the design of an insulated locus for the six recombinases. The study anticipated that the induction of one recombinase could lead to the unintended expression of a different recombinase if its coding sequence was in frame with an active promoter.
  • the study incorporated strong terminators [63] upstream and downstream of each recombinase, as well as alternated the direction of transcription of each successive gene to provide further transcriptional insulation.
  • the study cloned an initial version of this insulated locus into the BAC and used Marionette- Wild to perform the memory assay with each of the six inversion GOF reporter plasmids.
  • all sets of inducers for each of the six circuits were used to assess for cross-induction of recombinases.
  • the study saw evidence of transcriptional readthrough and cryptic promoter activity causing unintended activation of certain recombinases ([0091], also see Figure 10).
  • the study characterized the relative rate of recombination for each recombinase by measuring the amount of time required for recombination under ideal growth conditions in a minimal medium with inducer (Figure 3a).
  • Each genome-integrated recombinase was tested for its recombination rate using the inversion GOF circuit as well as an excision-based version of GOF synthetic memory. All recombinases showed complete recombination after approximately 12 hours when maintained in exponential growth.
  • the study posited that engineering a diverse set of optimized (z.e., near digital) circuits capable of both inversion and excision would allow for the accelerated design and development of complex genetic programs that utilize the strategic arrangement of att sites (z.e., nesting) that rearrange transcriptionally regulating elements such as promoters, terminators, or noncoding RNAs.
  • the study developed the complementary inversion LOF circuit by flanking an in-frame promoter with anti-aligned att sites upstream of the gfp gene (Figure 3c).
  • the excision GOF circuit was designed to have a strong constitutive promoter upstream of aligned att sites flanking two terminators in series, followed by the gfp gene ( Figure 3d).
  • the excision LOF circuit was designed by flanking an in-frame constitutive promoter with aligned at sites, followed by the gfp gene ( Figure 3e).
  • Extrachromosomal MEMORY programming, erasing, and reprogramming In principle, the EcMem chassis cell can be used to execute bespoke memory programs supplied on extrachromosomal DNA or via genome-integrated circuits.
  • the study aimed to demonstrate MEMORY programming by way of extrachromosomal (plasmid DNA) circuits.
  • the study posited that functional memory circuits with an additional feature could be designed that would enable the complete removal of the extrachromosomal DNA at any point on cue - effectively erasing the plasmid-based circuit - while retaining the genomically integrated MEMORY platform.
  • a putative synthetic memory eraser was designed via an aligned pair of all sites flanking the origin of replication of the pSClOl plasmid.
  • the addition of the L-arabinose inducer should result in the origin of replication being excised, preventing extrachromosomal DNA propagation - i.e., erasing the corresponding memory circuit and resetting the EcMem chassis cell ( Figure 15).
  • the erasable DNA plasmid contained a two-output memory circuit, designed for independent inducible GOF by way of the expression of GFP or mKate - via transient exposure to small molecules vanillic acid or aTc, respectively ( Figure 4a).
  • the initial recombination event incorporates the entire plasmid.
  • the authors used the FLP recombinase and cognate attachment sites to minimize the footprint of the insert.
  • the recombinases are supplied via plasmid DNA and are unregulated.
  • Santos et al. demonstrated in an earlier study that the Cre recombinase can be used to insert a specific DNA fragment into the E. coli genome using two sets of orthogonal att sites - again unregulated [64].
  • the study introduces the next iteration of recombinase-based genomic insertion technology that leverages the MEMORY platform for the programmed insertion of DNA into the genome of EcMem chassis cells.
  • MEMORY chassis cells can accomplish the programmed genomic insertion of an exact pay load in a single step - z.e., without the need to remove unwanted integrated DNA - and can support programmed serial genomic integrations.
  • the study created an artificial genomic safe harbor (aGSH) using a nonsynonymous pair of attP sites corresponding to two recombinases (Methods).
  • aGSH can be integrated with genetic information stored on a plasmid between a set of complementary attB sites ( Figure 5a).
  • the study positioned a promoter upstream of the aGSH in the genome of the EcMem chassis cell and paired the engineered safe harbor with a set of complementary attB sites directing a pay load (Payload 7) containing the gfp gene and a kanamycin resistance gene (kanR) flanked by a second pair of nonsynonymous attP sites - creating a new aGSH upon genomic integration ( Figure 5a-c). Additionally, the pSClOl donor vector was equipped with the memory eraser to remove the “empty” plasmid after integration, as well as the sacB gene to provide a counterselection and eliminate integrants receiving the entire plasmid.
  • Payment 7 pay load
  • kanR kanamycin resistance gene
  • dCas9 could be repurposed to bind (within or in proximity to) specific recombinase att sites and protect the DNA from programmed recombination.
  • Shur and Murray presented a proof-of-concept of unregulated dCas9-mediated protection of a single att site cognate to Bxbl in a cell-free (TX-TL) environment [65], which is distinct from canonical CRISPR interference (CRISPRi) of transcription [66].
  • DNA registers were designed to adopt a distinct DNA state (predicated on the concurrent recombination of all pairs of cognate att sites) for every possible permuted substring of inputs (Figure 21). For example, a 2-input system mapped to a register containing two sets of orthogonal attachment sites - i.e., where recombinase 1 corresponds to an inversion att configuration, and recombinase 2 corresponds to two sets of att sites distinguished by variation in the central dinucleotide - resulting in 5 unique states.
  • a limitation of current RSM technology is that DNA registers require complete sets (i.e., even-numbered pairs) of att sites to function - such that all att sites are recombined in the presence of a cognate recombinase.
  • a canonical 2-input 5- state RSM can be expanded to a 2-input 16- state RSM via CRISPRp of single att sites ( Figure 22).
  • the number of inputs that the EcMem chassis can sense and respond to includes the six recombinase inducers, and any additional T-Pro signals corresponding to CRISPRp regulators - e.g., TFs regulating sgRNAs for CRISPRp - including multiple-input T-Pro operations (Figure 6e).
  • CRISPRp regulators e.g., TFs regulating sgRNAs for CRISPRp - including multiple-input T-Pro operations
  • 5 signal-distinct synthetic repressors [4] and 5 signal-distinct anti-repressors [5], [50], [67] have been developed.
  • this system of network-capable transcription factors can be used to develop more than 100 2-input T-Pro operations that can be used to regulate and program multiple CRISPRp operations.
  • GRSM gene-regulatory RSM
  • the register of the ngGRSM contains 2 sets of odd-numbered attachment sites (z.e., an attB/attP set with a duplicated all site) corresponding to Bxbl and Int3, and one even-numbered set corresponding to Int8.
  • a functional circuit is formed (recombined) - resulting in the constitutive production of green fluorescent protein.
  • near-perfect protection was observed from recombination for several of the tested sgRNA targets (Figure 6d, also see Figure 18).
  • each regulated biosynthetic pathway was designed to function as an inducible input for its cognate biosensor present in the genome- integrated MEMORY system, such that induction would cause a corresponding recombinase to activate an inversion GOF circuit located on the same plasmid containing the synthase pathway ( Figure 8a-b).
  • the design goal for each autoinduction program was to achieve near digital recombination performance - i.e., successful recombination of an inversion GOF target with >95% efficiency while exhibiting ⁇ 5% recombination in the uninduced state.
  • the study was able to achieve the desired autoinduction effect - i.e., near digital recombination performance quantified by flow cytometry (Figure 8c-e).
  • EcMem pro the recombinase-based MEMORY platform could be useful for engineering advanced functionalities into the probiotic E. coli Nissle 1917. Therefore, the study transferred the MEMORY system into the genome of the Nissle chassis cell to create a probiotic memory strain (EcMem pro ).
  • GI gastrointestinal
  • the performance of certain circuits varied unpredictably under anaerobic conditions, but the majority behaved as expected.
  • the EcMem pro strain could be exogenously regulated to produce up to six separate therapeutic modalities by mapping them to orthogonal recombinase circuits.
  • E. coli strains used were NEB® 10-beta (for routine cloning), TransforMaxTM EPI 300TM (for BAC amplification), TransforMaxTM EC100D pir+ (for R6K plasmid propagation), S17-1 pir (for conjugation), MG1655 Marionette-Wild9, and Nissle 1917 (Mutaflor).
  • coli were routinely cultured aerobically in LB Miller Medium (Fisher BP9723) at 37°C (unless otherwise specified) with shaking, on LB Miller agar (Fisher BP1425), or in M9 Minimal Medium (MM) (MM contains 3 g/L KH2PO4, 0.5 g/L NaCl, 6.78 g/L Na 2 HPO 4 , 1 g/L NHrCl, 0.1 mM CaCL, 2 mM MgSCh, 1 mM thiamine hydrochloride, 0.4% D-glucose, and 0.2% casamino acids).
  • MM M9 Minimal Medium
  • thetaiotaomicron (ATCC 29148) was routinely cultured anaerobically at 37°C in TYG broth or BHI Agar (Difco), unless otherwise specified.
  • TYG broth contains: [10 g tryptone, 5 g yeast extract, 2.5 g D-glucose, 0.5 g L-cysteine, 13.6 g KH2PO4, 9.2 mg MgSO 4 , 1 g NaHCOs, 80 mg NaCl, 8 mg CaCh, 1 mg menadione, 0.218 mg FeSO4, 5 pg vitamin B12, and 1 ml histidine hematin solution (1.2 mg/ml hematin in 0.2 M histidine, pH 8.0)].
  • L- cysteine was resuspended in water and sterile filtered (0.2 pm VWR 28145-477). Menadione was resuspended in 100% ethanol. L-cysteine and menadione were prepared and added to autoclaved media immediately prior to inoculation. Anaerobic culturing was performed in a Whitley DG250 anaerobic chamber with an atmosphere of 10% H2, 10% CO2, and 80% N2 (Airgas X03NI80C2000511). Antibiotics for plasmid selection in E.
  • coli were used at the following concentrations: carbenicillin (Goldbio C- 103-25)- 100 pg/ml; chloramphenicol (Goldbio C- 105-25)- 25 pg/ml; kanamycin (Goldbio K- 120-25)- 35 pg/ml.
  • Antibiotics for Bacteroides were used as appropriate: erythromycin (Alfa Aesar J62279)-25 pg/ml and gentamycin (VWR 0304-500G)- 200 pg/ml.
  • the final concentrations used for each inducer were: 1 mM IPTG; 25 pM DAPG; 100 ng/ml aTc; 5 mM L-ara; 100 pM cuminic acid; 100 pM vanillic acid; 10 pM 3OC6 AHL; 10 mM Ribose; 10 mM Cellobiose.
  • BAC backbone vector was a kind gift from J. J. Collins (MIT) and J. W. Lee (POSTECH). Recombinase genes were synthesized as gene fragments and subcloned using standard molecular biology techniques. All BAC constructs were created using Golden Gate assembly69. pSClOl constructs were created using Golden Gate assembly, inverse PCR, and Gibson cloningvo. Q5 polymerase (NEB M0491L) was used for PCR. T4 DNA ligase (NEB M0202L), BsmBLv2 (R0739L), and BsaI-HFv2 (NEB R3733L) were used for Golden Gate cloning.
  • NEBuilder HiFi DNA Assembly Master Mix (NEB E2621X) was used for Gibson cloning. All DNA primers were synthesized by Eurofins Genomics. The DNA sequences of all constructs were verified by Sanger sequencing (Eurofins Genomics). Relevant plasmid maps are given in Figure 27. [0247] Conjugation of Bacteroides. E. coli S17-1 pir was used for conjugation of plasmids into Bacteroides. The pNBU2 vector harbors intN2 which mediates site-specific recombination of the attN2 site of pNBU2 and one of two attB2 sites located at the 3' ends of tRNA-Ser genes in Bacteroides genomes.
  • Donor cultures of E. coli S17-1 pir transformed with the appropriate pNBU2 construct and recipient cultures of Bacteroides were separately grown to OD600-0.5. 1 ml of donor culture and 1 ml of recipient culture were pelleted by centrifugation (5000 x g, 5 min.) separately and resuspended in 1 ml of PBS. This step was then repeated for a second wash. The cultures were then mixed at a ratio of 1:10 (donor: receiver) and pelleted again by centrifugation.
  • Recombinase memory assay Cells harboring a specific recombinase in the genome or on a BAC were transformed with the desired pSClOl output plasmid and plated on LB agar supplemented with chloramphenicol and kanamycin. After overnight incubation, three colonies were picked into separate 200 pL LB cultures supplemented with chloramphenicol and kanamycin in a flat-bottom 96-well plate (Coming 3370) and sealed with a Breathe Easier membrane (Electron Microscopy Sciences 70536-20).
  • the inducible recombinase cassettes were serially integrated using the lambda red recombineering met hod? i. Briefly, the Al 18, Intl2, Bxbl, and Int8 genes were cloned into an R6K vector along with a kanamycin resistance cassette, upstream homology to araE, and downstream homology to the glvC pseudogene. The Int3 and Int5 genes were cloned into a second R6K vector along with a chloramphenicol resistance cassette, upstream homology to Int8, and downstream homology to the glvC pseudogene. Bsal sites were incorporated upstream and downstream of the homology regions to allow for linearization of the DNA to be integrated.
  • Marionette -Wild was transformed with pKD46?i and made recombineering- ready. Briefly, transformants were selected on LB agar with carbenicillin at 30°C. A single colony was used to inoculate LB medium with carbenicillin and grown at 30°C overnight. The following morning this culture was diluted 1:200 into 50 ml fresh LB and grown for 1.5 hours. At this point, L-arabinose was added (5 mM) to induce recombineering proteins. Cells were grown for approximately 3 more hours until an OD600-0.5.
  • Resultant colonies were screened for the correct genomic insertion by colony PCR and sequencing of the inserted DNA region. These cells were then made recombineering-ready and the insertion process was repeated with the last two recombinase cassettes, conferring chloramphenicol resistance. This EcMem strain was also modified to remove the chloramphenicol resistance through Flp-mediated excision of the resistance cassette. EcMempro was created in an analogous fashion, but the Marionette transcription factor operons were inserted first. Integration was performed at the LacI locus of EcN. The only difference between the EcMem and EcMempro memory arrays is that the Al 18 gene has a GTG start codon in EcMem pro .
  • MM cultures were then used to seed fresh 500 pL MM cultures with the appropriate inducer for a given circuit (1:100 dilution), in deep-well plates (Greiner 780271) sealed with a Breathe Easier membrane.
  • an additional set of MM cultures (without inducers) was seeded (1:200 dilution).
  • These uninduced cultures were grown for 12 hours and analyzed by flow cytometry to assess recombination levels (designated as the 0-hour time point).
  • the inducing cultures were grown for a total of 16 hours. To maintain cells in exponential phase, the inducing cultures were used to seed fresh inducer containing media (1:100 dilution) at 8 hours. Every 4 hours during the induction period, a fresh set of MM cultures without inducers was inoculated using the inducing cultures (1:200 dilution). These inducer- free cultures were all grown for 12 hours prior to being analyzed by flow cytometry.
  • CRISPR protection assay For IPTG-inducible sgRNA CRISPRp circuits, cells transformed with a given circuit were precultured in LB medium supplemented with chloramphenicol and kanamycin with and without IPTG for 8 hours in a flat-bottom 96-well plate sealed with a Breathe Easier membrane. After 8 hours, the IPTG-free preculture was used to seed MM with and without the cognate inducer of a given recombinase (sealed with a Breathe Easy membrane). The IPTG-containing preculture was used to seed MM with IPTG or IPTG plus the cognate inducer of a given recombinase.
  • the first genomic safe harbor (aGSHl) based on the attP sites of Bxbl and Int8 was integrated into EcMem through the recombineering method described above.
  • the promoter and attP sites of Bxbl and Int8 were cloned into the R6K vector along with the kanamycin resistance cassette, upstream homology to Int5, and downstream homology to the glvC pseudogene.
  • the desired insert was digested with Bsal and electroporated into recombineering-ready EcMem cells. Confirmation of genomic insertion was performed as described above.
  • the kanamycin resistance gene was then removed using FLP recombination.
  • EcMem with aGSHl was transformed with a pSClOl plasmid (equipped with the origin eraser) containing the first gene to be integrated and a kanamycin resistance gene flanked by Int5 and Inti 2 attP sites (aGSH2), all nested between Bxbl and Int8 attB sites.
  • Inducible integration was achieved by performing the Memory Assay in MM with kanamycin and inducing cells for 24 hours with aTc and vanillic acid. A second 24-hour induction with L-arabinose in the absence of kanamycin was performed to erase the pSClOl plasmid.
  • Colony PCR for inducible integration genotyping Colony PCR was conducted to confirm the insertion of the memory circuit and the deletion of the pSClOl plasmid after inducible integration. After pSClOl deletion with L-arabinose, cells were streaked on LB agar plates and individual colonies were randomly selected for colony PCR. Each colony was diluted in 100 pL DI H2O and 1 pL was added directly to the PCR reaction as a template. One set of primers was designed to specifically bind upstream and downstream of the inserted region to determine if the integration worked correctly. A second set of primers was designed to check the presence of pSClOl, testing the deletion of residual DNA sequences.
  • the colony PCR reaction was performed with Q5 polymerase. A 7-minute incubation at 98°C (5 minutes for lysis, and 2 minutes for denaturation of DNA) was followed by a 30-second annealing step, a 30-second extension step, and a 30-second denaturation step (25 cycles). After the PCR, gel electrophoresis was performed with 1:12 diluted PCR products on 0.8% agarose gel with Ikb DNA Ladder (NEB #N3232). The gel bands are imaged by ChemiDoc XRS+ System (Bio-Rad).
  • the primers named CPlINT_fwd and CPlINT_rev bind to Bxbl attP and Int8 attP, respectively, checking the length of integration.
  • Primers CPlERA_fwd and CPlERA_rev bind to Bxbl atB and Int3 atB, respectively, checking the presence of pSClOl plasmid.
  • the primers named CP2INT_fwd and CP2INT_rev bind downstream of the Int5 gene and downstream of glvC gene, respectively, checking the whole length of the insertion.
  • Primers CP2ERA_fwd and CP2ERA_rev bind to the kanR gene, linearizing the pSClOl plasmid.
  • the sequences of colony PCR primers are as follows: CPHNT.fwd: GTCGGGGTTTGTACCG TACACCAC, CPlINT_rev: TTAATAAACTATGGAAGTATGTACAGTCTTGC, CPlERA.fwd: GCCCGGATGATCCTGACGAC, CPlERA.rev: TTTGTAAAGGAGAC TGATAATGGC, CP2INT_fwd: ATCCGCAGGCAAGCGAAGATG. CP2INT_rev: GTTGAGGATTTTCGCATTCGG, CP2ERA_fwd: TGGATACTTTCTCGGCAGGAG, CP2ERA_rev: TCATGGCTGATGCAATGCG.
  • E. coli intercellular communication assay Sender cells (lacking chloramphenicol resistance) were transformed with the appropriate synthase plasmid while receiver cells (having chloramphenicol resistance) were transformed with the appropriate inversion GOF circuit. Individual 200 pL LB precultures were inoculated for the sender and receiver cells using single colonies. These cultures were grown for 8 hours in a flat-bottom 96-well plate sealed with a Breathe Easier membrane. Following this, the receiver cells were diluted with fresh MM to the following degrees: 1:100 for the Al 18 and Inti 2 circuits, and 1:20 for the Int8 circuit.
  • thetaiotaomicron cells were diluted 1:10 with fresh TYG. 5 pL of the diluted B. thetaiotaomicron cells and 5 pL of the precultured EcMem pro cells were then used to seed 1 ml of TYG cultures without ligand, with IPTG, or with vanillic acid. These cultures were grown anaerobically for 16 hours, and then gently mixed with pipetting. 500 pL of cells was then pelleted by centrifugation (10,000xg for 5 minutes) and the supernatant was carefully aspirated. The cell pellet was then resuspended in 30 pL Bugbuster Mastermix (Millipore 71456) and incubated at room temperature for 15 minutes to facilitate cell lysis. Nanoluc production was then quantified using a luminescence assay.
  • Luminescence assay The Promega Nano-Gio assay kit was used to determine expression of NanoLuc. Assay buffer and substrate were mixed as per the manufacturer recommendation (1:50 ratio of substrate to buffer). 30 pL of this mixture was transferred to a well of a flat-bottom white 96-well microplate (Costar 3912) containing 40 pL DI water. Following cell lysis, 30 pL of lysate was added to the microplate well and mixed by pipetting. After 5 minutes of incubation, the luminescence was measured with a Spectramax M2e plate reader (Molecular devices) with 800 v gain and 30 reads per well. Data was collected with SoftMax Pro Software. Background luminescence generated from an equal mix of EcMempro and wildtype B.
  • CFUs B. thetaiotaomicron colony forming units
  • Cytometry analysis Fluorescence analysis was performed with a Beckman Coulter Cytoflex S flow cytometer. Cells were diluted 1:50 into PBS with 2 mg/ml kanamycin and incubated for at least 1 hour at room temperature. Cells were processed at 10- 30 pL/min and monitored through the FITC channel for GFP expression and the ECD channel for mKate expression. Events were gated by forward scatter area vs. side scatter area to eliminate debris and then gated by side scatter height vs. side scatter area to discriminate doublets. More than 10,000 events were collected for final analysis. A representative gating schematic is shown in Figure 26.
  • CRISPRp can be used concurrently with interception - even on a shared att site - which should provide a powerful tool to expand the capabilities of MEMORY chassis cells.
  • CRISPRp can be used concurrently with CRISPRi, enabling the coordination of synthetic memory with transient gene knock-down(s), which can be used to increase the versatility of MEMORY chassis cells.
  • the scale-up of recombinasebased genetic programs has been limited by the number of independently inducible recombinases, as well as the cellular burden they impose when overexpressed from multicopy plasmids [20].
  • EcMem and EcMem pro chassis cells double the number of inducible recombinases that can be used in a single E. coli cell compared to previous studies, they have significantly expanded the capacity for recombinase-based programming.
  • the platform technology is operational with minimal metabolic burden.
  • CRISPRp provides the ability to couple Transcriptional Programming with recombination events, and intercellular communication allowing for multicellular applications; the instant study has provided a platform technology for advanced control over cellular behavior.
  • the instant study demonstrated this capability by programming information exchange between the EcMem Pro strain and Bacteroides thetaiotaomicron, presenting consortium-based living therapeutic technologies. This is the first system that allows for the direct integration of decision-making, memory, and communication in living cells. While the study have shown how to apply this platform in the area of living therapeutics, the combined technologies can be used to guide cellular processes in countless ways. The study shows that MEMORY strains are of great use in diverse applications in the areas of metabolic engineering, biosecurity, DNA information storage, and human health.
  • Triassi A. J. et al. Redesign of an Escherichia coli Nissle treatment for phenylketonuria using insulated genomic landing pads and genetic circuits to reduce burden. Cell Systems 14, 512-524.e512 (2023).
  • Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012).

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Abstract

L'invention concerne un système d'administration de médicament programmable comprenant une cellule châssis, (par exemple, une mémoire codée moléculairement via un réseau de recombinases orthogonales (MÉMOIRE)) modifiée pour produire une recombinase inductible orthogonale pour modifier l'acide nucléique extrachromosomique. L'invention concerne également une composition probiotique synthétique comprenant le système d'administration de médicament programmable, décrit ici, et un support pharmaceutiquement acceptable, et leurs procédés d'utilisation dans la prévention ou le traitement de la dysbiose du microbiote intestinal chez un sujet en ayant besoin.
PCT/US2025/016038 2024-02-16 2025-02-14 Cellules châssis à mémoire Pending WO2025175173A1 (fr)

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