Patent Application
20220323521
Described herein are microbial probiotics that, in response to metabolite extracellular ATP (eATP) produced in the microenvironment of inflamed tissues detected, e.g., via an engineered mammalian P2Y purinoceptor 2 (P2Y2) receptor, secrete an anti-inflammatory protein, e.g., IL-2, IL-10, or the CD39-like eATP-degrading enzyme apyrase.
Inflammatory Bowel Disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract that includes Crohn's Disease and Ulcerative Colitis (1). Most available IBD therapies suppress the immune system systemically, increasing the risk of infections and some types of cancer (2). In addition, many patients with IBD do not respond to therapy or show loss of clinical response over time (2). Thus, there is a need for novel therapeutic approaches for IBD.
The microbiome controls immune processes relevant to the pathology of multiple human diseases including IBD (3-5). For example, IBD-associated single-nucleotide polymorphisms promote changes in the intestinal microbiota that result in the reduced production of anti-inflammatory microbial metabolites (6). Genetic polymorphisms associated to IBD also control the responsiveness to anti-inflammatory microbial metabolites (7). The role of the microbiome in disease pathogenesis, and in particular the anti-inflammatory effects of certain commensal microorganisms, supports the use of probiotic-based approaches for the treatment of IBD (8-10). However, therapies based solely on the intrinsic anti-inflammatory properties of un-manipulated probiotics may not be efficacious in controlling ongoing intestinal inflammation (10).
The development of therapeutic probiotics is a major area of IBD research. Previous attempts used engineered probiotics expressing therapeutic proteins in an uncontrolled manner, based on plasmid systems which require constant selection pressure and present the risk of horizontal transfer to other bacteria (80, 81). To overcome these limitations, the present inventors applied directed evolution and CRISPR/Cas9 to engineer microbes with a gene circuit that produces an anti-inflammatory in response to eATP levels, delivering a probiotic-based dynamic anti-inflammatory response in the inflamed tissue microenvironment. Provided herein are engineered therapeutic microbes designed to detect pathogenic gastrointestinal (GI) inflammation, and dynamically respond by delivery of a therapeutic protein, and methods of use thereof.
Thus, provided herein is an isolated Saccharomyces cell (or cells, e.g., a population of such cells) that has been engineered to express one, two, or all three exogenous proteins selected from: (i) a mammalian P2Y purinoceptor 2 (P2Y2) protein, preferably human P2Y2; (ii) a mutant Gpa1 protein comprising at least 5 C-terminal residues from a mammalian G alpha, preferably Gαi3, wherein the mutant Gpa1 protein couples the P2Y2 protein to the yeast mating pathway; and (iii) an anti-inflammatory protein, optionally wherein the anti-inflammatory protein is mammalian, preferably human, and wherein the anti-inflammatory protein is expressed under the control of a promoter activated downstream of P2Y2 activation, optionally a mating-responsive promoter, wherein the isolated Saccharomyces cell secretes the anti-inflammatory protein in the presence of extracellular adenosine triphosphate (eATP). Preferably the anti-inflammatory protein is secreted in the presence of eATP at pro-inflammatory concentrations (˜100 micromolar to high millimolar). Preferably, the anti-inflammatory protein is secreted in an eATP concentration-dependent manner, where a greater eATP concentration leads to a greater secretion of the anti-inflammatory protein within the dynamic range of the engineered P2Y2 receptor.
In some embodiments, the Saccharomyces cell has been engineered to reduce or remove expression of one or more endogenous proteins selected from the group consisting of: (i) a yeast GPCR, e.g., alpha-factor pheromone receptor STE2 (NP_116627.2); (ii) negative regulator of pathway function GTPase-activating protein SST2 (NP_013557.1); (iii) cell cycle regulator cyclin-dependent protein serine/threonine kinase inhibiting protein FAR1 (NP_012378.1); and (iv) yeast G alpha protein guanine nucleotide-binding protein subunit alpha GPA1 (NP_011868.1).
In some embodiments, the anti-inflammatory protein comprises a yeast-derived leader peptide that directs the protein to be secreted, and optionally lacks any signal or leader sequence endogenous to the anti-inflammatory protein.
In some embodiments, the anti-inflammatory protein comprises apyrase, interleukin 10 (IL-10), IL-2, IL-27, IL-22, or IFN-beta.
In some embodiments, at least one of the P2Y2 protein, mutant Gpa1, or anti-inflammatory protein are expressed from sequences codon-optimized for expression in the Saccharomyces cell.
In some embodiments, the P2Y2 comprises one or more mutations that increase expression of the anti-inflammatory protein. In some embodiments, the mutations are in residues peripheral to the ligand binding pocket (optionally A762.47, N1163.35, C1193.38, L1624.54, Q1654.57) and/or in residues in the intracellular facing side of the receptor (optionally F581.57, L591.58, C601.59, A229ICL3, K2406.31, F3077.54, G310C-term). In some embodiments, one or more mutations are in residues F581.57, N1163.35, F3077.54 and/or Q1654.57. In some embodiments, the one or more mutations comprise F58C, Q165H, F307S, and/or N116S. In some embodiments, the mutations comprise a mutation at N116. In some embodiments, the mutations comprise a mutation at N116 in combination with a mutation at F58 or F307. In some embodiments, the mutations comprise mutations N116S, optionally in combination with mutations F58I or F307S. In some embodiments, the P2Y2 further comprises mutations at L59 and/or C119. In some embodiments, the further mutations comprise L59I and/or C119S.
In some embodiments, the promoter activated downstream of P2Y2 activation is a mating-responsive promoter, e.g., pFUS1 or pFIG1
In some embodiments, the expression of the anti-inflammatory protein is driven by a synthetic transcription factor comprising a pheromone responsive domain and a DNA binding domain, binding to non-yeast DNA operator sequences upstream of the sequence encoding the anti-inflammatory protein.
In some embodiments, the isolated Saccharomyces cell is S. cerevisiae or S. boulardii.
Also provided herein are compositions that include the isolated Saccharomyces cells described herein, and optionally a physiologically-acceptable carrier. In some embodiments, the compositions are in a solid form for oral administration, e.g., tablets, pills, capsules, soft gelatin capsules, sugarcoated pills, orodispersing/orodispersing tablets, or effervescent tablets.
In some embodiments, the compositions are in a liquid form for oral administration, e.g., a drinkable solution.
In some embodiments, the compositions are nutritional compositions, optionally comprising liquid or solid food, feed or drinking water.
In some embodiments, the nutritional composition is selected from beverages (optionally smoothies or cultured beverages, flavored beverages, yogurt, drinking yogurt, set yogurt, fruit and/or vegetable juices or concentrates thereof, fruit and vegetable juice powders, reconstituted fruit products, powders, malt or soy or cereal based beverages, breakfast cereal such as muesli flakes, spreads, meal replacements, confectionary, chocolate, gels, ice creams, cereal, fruit, and/or chocolate bars, energy bars, snack bars, food bars, sauces, dips, and sports supplements including dairy and non-dairy based sports supplements.
Also provided herein are methods for reducing inflammation in a subject, the method comprising administering to the subject an effective amount of the isolated Saccharomyces cells or compositions as described herein. Further provided are the isolated Saccharomyces cells and the compositions for use in a method of reducing inflammation in a subject. In some embodiments, the subject has or is at risk of developing inflammatory bowel disease (IBD).
Additionally provided herein are engineered mammalian P2Y purinoceptor 2 (P2Y2) proteins comprising one or more mutations in residues peripheral to the ligand binding pocket (optionally A762.47, N1163.35, C1193.38, L16245.4, Q1654.57) and/or in residues in the intracellular facing side of the receptor (optionally F581.57, L591.58, C601.59, A229ICL3, K2406.31, F3077.54, G310C-term).
The engineered mammalian P2Y2 of claim 30, wherein one or more mutations are in residues F581.57, N1163.35, F3077.54 and/or Q1654.57.
The engineered mammalian P2Y2 of claim 31, wherein the one or more mutations comprise F58C, Q165H, F307S, and/or N116S.
The engineered mammalian P2Y2 of claim 30, wherein the mutations comprise a mutation at N116.
The engineered mammalian P2Y2 of claim 33, wherein the mutations comprise a mutation at N116 in combination with a mutation at F58 or F307.
The engineered mammalian P2Y2 of claim 34, wherein the mutations comprise mutations N116S, optionally in combination with mutations F58I or F307S.
The engineered mammalian P2Y2 of any of claims 30 to 35, wherein the P2Y2 further comprises mutations at L59 and/or C119.
The engineered mammalian P2Y2 of claim 36, wherein the further mutations comprise L59I and/or C119S.
An isolated nucleic acid sequence encoding the engineered mammalian P2Y2 of any of claims 30 to 37.
A host cell comprising the isolated nucleic acid sequence of claim 35, and optionally expressing the engineered mammalian P2Y2 of any of claims 30 to 37.
The host cell of claim 39, wherein the cell is a Saccharomyces cell, and the isolated nucleic acid sequence is codon-optimized for expression in the Saccharomyces cell.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
The convergence of efficient genetic manipulation (11, 12) and advanced synthetic gene circuit design (13, 14) paved the way for increasingly complex microbial engineering (15-18). In fact, recent advances in synthetic biology enabled the engineering of probiotics to deliver therapeutic proteins in response to disease-associated signals (19-22). One such signal relevant to IBD is extracellular adenosine triphosphate (eATP) which, upon release by activated immune cells and commensal bacteria, signals via purinergic receptors to trigger pro-inflammatory cytokine production, boost effector T cell activation, suppress regulatory T-cell responses and promote enteric neuron apoptosis among other biological responses thought to contribute to IBD pathology (23-27). eATP signaling is limited by the membrane-bound ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, also known as CD39), which hydrolyzes eATP into AMP; AMP is then metabolized by CD73 into immunosuppressive adenosine. CD39 limits eATP-driven pro-inflammatory responses, while it boosts the differentiation, stability and function of regulatory T cells (26). Further support for the physiological role of eATP and CD39 in the control of intestinal inflammation is provided by reports of dysregulated purinergic signaling in IBD patients resulting from increased eATP production and/or its decreased hydrolysis (25, 28).
Genetic polymorphisms that decrease CD39 expression have been associated with Crohn's disease (68). CD39 on Tregs suppresses effector T-cell generation and function in experimental and human IBD (26-28, 69). Indeed, increased CD39 levels are associated with disease remission induced by blocking antibodies against TNFα in IBD patients (70). Conversely, purinergic signaling driven by eATP promotes inflammation through multiple mechanisms including the modulation of antigen presenting cells (71), the boost of effector T-cell activation (23, 72) and the decreased function and stability of regulatory T cells (26, 27, 73). eATP also limits the production of immunoglobulin A (74), which protects the intestinal barrier and promotes the engraftment of anti-inflammatory commensal bacteria (75, 76). In addition, eATP also acts on non-immune cells to promote IBD pathogenesis by triggering the apoptosis of enteric neurons (24). Thus, the blockade of eATP-driven signaling is an attractive therapeutic approach for IBD.
eATP-depletion with apyrase has been shown to ameliorate intestinal inflammation (23). These anti-inflammatory effects of apyrase likely involve both eATP depletion through its conversion into AMP, and also the generation of immunosuppressive adenosine from AMP (29). Adenosine suppresses T-cell activation via the A2A adenosine receptor (29). Indeed, we recently reported that adenosine production driven by CD39 suppresses tumor-specific T cells in glioblastoma (77). Hence, the modulation of the eATP/adenosine balance is a potential approach to treat inflammation. However, the clinical application of this approach requires suitable methods for therapeutic agent administration and inducible systems that modulate the eATP/adenosine balance where and when needed to minimize unwanted side effects such as immunosuppression and fibrosis (26, 28, 77) and intestinal microbiome dysregulation (29, 30).
Utilizing the modularity of the S. cerevisiae mating pathway, with directed evolution (33) and synthetic biology (34) approaches, strains of this yeast were modified to express an engineered human G protein-coupled receptor (GPCR) that is activated by a pro-inflammatory signal, eliciting the secretion of a therapeutic protein. GPCRs function as biological sensors to detect a wide diversity of signals, including the detection of molecules indicative of disease (Marinissen, M. J. & Gutkind, J. S. G-protein-coupled receptors and signaling networks: emerging paradigms. Trends in pharmacological sciences 22, 368-376 (2001)). This ability makes GPCRs useful components of synthetic gene circuits, to elicit programed responses to specific disease cues (Heng, B. C., Aubel, D. & Fussenegger, M. G protein-coupled receptors revisited: therapeutic applications inspired by synthetic biology. Annu Rev Pharmacol Toxicol 54, 227-249 (2014)). The S. cerevisiae mating pathway provides a well characterized model for GPCR signaling that can be rewired to accommodate activation by human GPCRs (Ladds, G., Goddard, A. & Davey, J. Functional analysis of heterologous GPCR signalling pathways in yeast. Trends Biotechnol 23, 367-373 (2005)). Elevated extracellular adenosine triphosphate (ATP) is a major pro-inflammatory signal (Bours, M. J., Dagnelie, P. C., Giuliani, A. L., Wesselius, A. & Di Virgilio, F. P2 receptors and extracellular ATP: a novel homeostatic pathway in inflammation. Frontiers in bioscience 3, 1443-1456 (2011)), which increases over 100-fold in the gut in IBD (>100 μM) (Kurashima, Y., Kiyono, H. & Kunisawa, J. Pathophysiological role of extracellular purinergic mediators in the control of intestinal inflammation. Mediators of inflammation 2015, 427125 (2015)), and is specifically detected by the purinergic family of GPCRs (Burnstock, G. & Boeynaems, J. M. Purinergic signalling and immune cells. Purinergic signalling 10, 529-564 (2014)). The enzyme apyrase directly degrades ATP, converting it to immunosuppressive adenosine (Cekic, C. & Linden, J. Purinergic regulation of the immune system. Nature reviews. Immunology 16, 177-192 (2016)), and apyrase was shown to reduce GI inflammation in an animal model of IBD (Wan, P. et al. Extracellular ATP mediates inflammatory responses in colitis via P2 x 7 receptor signaling. Sci Rep 6, 19108 (2016)). The anti-inflammatory cytokine interleukin 10 (IL-10) is critical to limiting inflammation responses in the gut (Paul, G., Khare, V. & Gasche, C. Inflamed gut mucosa: downstream of interleukin-10. Eur J Clin Invest 42, 95-109 (2012)). Microbes have been engineered to constitutively secrete IL-10 (Braat, H. et al. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin Gastroenterol Hepatol 4, 754-759 (2006); Rottiers, P., Vandenbroucke, K. & Iserentant, D., Vol. EP1931762(B1). (ed. E.P. Office) 1-26 (Actogenix NV, Belgium; 2012)), but not in response to a pro-inflammatory signal, which have shown promise in a Phase I clinical trial for treating IBD (Braat, H. et al. (2006).).
Described herein are microbial probiotics that, in response to metabolite eATP produced in the microenvironment of inflamed tissues detected, e.g., via an engineered human P2Y2 receptor, secrete an anti-inflammatory protein, e.g., IL-2, IL-10, or the CD39-like eATP-degrading enzyme apyrase, which depletes pro-inflammatory eATP and promotes the generation of immunosuppressive adenosine. These engineered apyrase-expressing yeasts suppressed experimental intestinal inflammation in mice, reducing intestinal fibrosis and dysbiosis. The specific molecular pathways involved in purinergic signaling during inflammation are outlined in
The present data show that controlled eATP depletion by yeast probiotics engineered to produce apyrase in response to eATP-sensing minimize fibrosis induction. Moreover, the use of an inducible engineered yeast strain to modulate purinergic signaling also allowed the recovery of healthy microbiome, minimizing the dysbiosis thought to contribute to the pathology IBD and other human disorders (3, 4).
The inherent modularity of signaling pathways (78) enables engineering using exogenous proteins (79). Saccharomyces species are long-known for their use in foods, and certain Saccharomyces species have also been used as safe probiotics harboring engineered gene circuits to drive the controlled expression of proteins in response to stimuli of interest (15, 31, 32). In some embodiments, the present engineered microbes are made in S. cerevisiae. S. boulardii has been more commonly used as a probiotic than S. cerevisiae (89, 90), and the genetic tools to manipulate S. boulardii are available (91, 92). Thus, although S. cerevisiae is exemplified herein, the inducible system described herein can be established using other microbes, including S. boulardii.
In some embodiments, the microbes are generated by modifying the genome of the parental microbe, e.g., Saccharomyces, e.g., S. cerevisiae. The modifications can include (but are not limited to) introduction of the following proteins to the genome of the yeast: (i) engineered P2Y2, containing up to three mutations making it more responsive to eATP, e.g. under the control of a constitutive promoter (pTDH3); (ii) a mutant Gpa1 protein, e.g., containing the 5 C-terminal residues of a mammalian G alpha (Gαi3), which couples P2Y2 to the yeast mating pathway; and (4) potato apyrase or interleukin 10 (IL-10) containing a yeast-derived leader peptide that directs the apyrase to be secreted, controlled by a promoter downstream of GPCR activation, e.g., from the Fus1 gene. The modifications can also include (but are not limited to) deletion of one or more endogenous yeast proteins from the genome: (i) the natural yeast GPCR mating pathway receptor Ste2 (e.g., alpha-factor pheromone receptor STE2 (NP_116627.2); to avoid pathway activation by natural ligands), (ii) the negative regulator of pathway function Sst2 (e.g., negative regulator of pathway function GTPase-activating protein SST2 (NP_013557.1); to increase the pathway response when activated by P2Y2), (iii) the cell cycle regulator Far1 (e.g., cell cycle regulator cyclin-dependent protein serine/threonine kinase inhibiting protein FAR1 (NP_012378.1); to avoid cell cycle arrest upon mating pathway activation), and (iv) the yeast G alpha protein Gpa1 (e.g., yeast G alpha protein guanine nucleotide-binding protein subunit alpha GPA1 (NP_011868.1); to avoid competition for binding to other pathway components).
Thus the methods can include introducing a mutant G alpha protein where the 5 C-terminal amino acids of Gpa1 (KIGII) was replaced with the 5 C-terminal amino acids from the indicated mammalian Gα protein (Brown et al., Yeast. 2000 Jan. 15; 16(1):11-22. 2000) (e.g., a chimeric yeast Gpa1-human Gαi3 protein), introducing P2Y2 (e.g., a mutant P2Y2 optionally codon optimized for expression by yeast), and introducing an anti-inflammatory molecules such as apyrase or interleukin 10 (IL-10) controlled by a promoter activated downstream of P2Y2 activation (e.g. a mating pathway-responsive promoter). As shown herein, engineered variants of the GPCR P2Y2 responded to concentrations of eATP indicative of inflammation (˜100 micromolar to high millimolar). In addition, apyrase or IL-10 were secreted by engineered yeast strains in response to P2Y2 activation, in an ATP concentration dependent manner, and the apyrase functioned to degrade extracellular ATP. Finally, in a mouse model of IBD, treatment with engineered yeast strains that secrete apyrase directly improved disease outcomes and reduced pro-inflammatory cytokine production. The engineered yeast described herein can include, for example, a self-tunable P2Y2-RROP1 gene circuit responsive to pro-inflammatory eATP, which is itself hydrolyzed by the secreted apyrase encoded by RROP1 to dynamically control the eATP/adenosine balance in a time- and location-specific manner.
The exogenous sequences can be introduced into the microbe using molecular biological methods known in the art. In some embodiments, the engineered gene circuit is integrated into the yeast genome, e.g., using CRISPR-mediated integration, to avoid the use of antibiotic selection markers, while maintaining uracil auxotrophy for biocontainment, in agreement with Food and Drug Administration (FDA) guidelines on Live Biotherapeutic Organisms (docket number FDA-2010-D-0500). S. cerevisiae strains are present in healthy microbiomes and reduced during IBD (82-84), and have been associated with the physiological training the immune system (85-88).
P2Y Purinoceptor 2 (P2Y2)
The P2Y2 receptor is the most sensitive purinergic GPCR to eATP, and has previously been functionally linked to the S. cerevisiae mating pathway (Junger, W. G. Immune cell regulation by autocrine purinergic signalling. Nature reviews. Immunology 11, 201-212 (2011); Brown, A. J. et al. Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein alpha-subunit chimeras. Yeast 16, 11-22 (2000)). The present methods can include the use of yeast engineered to express a G protein-coupled receptor (GPCR) that is activated by a pro-inflammatory signal, e.g., a P2Y2 GPCR, e.g., human P2Y2.
An exemplary reference sequence for human P2Y2 protein is provided in GenBank at NP_002555.4. Exemplary reference sequences encoding human P2Y2 protein are provided in GenBank at NM_176072.3 (variant 1); NM_002564.4 (variant 2); and NM_176071.3 (variant 3). Transcript variants 1, 2 and 3 encode the same protein. The DNA sequence of human P2Y2 used in the exemplary engineered yeast strains presented here was codon optimized for expression in yeast, with a protein sequence as shown in NP_002555.4 (NP_002555.4), optionally with up to 2%, 5%, 10%, 15%, or 20% amino acids, e.g., including or in addition to the mutations described herein.
In some embodiments, an engineered human P2Y2 is used, wherein the mutations tune the response to physiological levels of eATP, i.e., by increasing G-protein signaling and expression of the anti-inflammatory protein. In some embodiments the mutations are in residues peripheral to the ligand binding pocket (A762.47, N1163.35, C1193.38, L1624.54, Q1654.57), or residues located in the intracellular facing side of the receptor (F581.57, L59158, C601.59, A229ICL3, K2406.31, F3077.54, G310C-term). In some embodiments, the P2Y2 includes one or more mutations in residues that contributed the most to the increase in eATP sensitivity (i.e. F581.57, N1163.35, F3077.54 and Q1654.57), e.g., one or more mutations in residues F58 (e.g., F58C), Q165 (e.g., Q165H), and F307 (e.g., F307S). In some embodiments, the mutations include a mutation at N116, e.g., N116S, optionally in combination with mutations at either F58, e.g., F58I, or F307, e.g., F307S. In some embodiments, the P2Y2 includes mutations at L59, e.g., L59I, and/or C119, e.g., C119S. In addition to the specific mutations described herein, mutations to other amino acids can also be used, e.g., F58 can be changed to any other amino acid. (Numbering corresponds to NP_002555.4—SEQ ID NO:13)
Anti-Inflammatory Agents
The microbes described herein are engineered to express one or more anti-inflammatory agents. Exemplary anti-inflammatory agents include apyrase, interleukin-10 (IL-10), IL-2, IL-27, IL-22, and IFN-beta. The anti-inflammatory agents are placed under the control of a promoter that is triggered by binding of eATP to the GPCR P2Y2, which (without wishing to be bound by theory) causes G protein mediated triggering of the MAP Kinases cascade and expression of the anti-inflammatory agents. Exemplary promoters include pFUS1 (defined as the 1636 bp immediately upstream of the Fus1 start codon; Gene ID 850330, GenBank Acc. No. NC_001135.5, Range 71803-73341), or pFIG1 (defined as the 500 bp immediately upstream of the Fig1 start codon; Gene ID 852328, GenBank Acc. No. NC_001134.8, Range 316968-317864). Alternatively a synthetic transcription factor containing a pheromone responsive domain and a DNA binding domain, paired with non-yeast DNA operator sequences upstream of the anti-inflammatory gene, similar to those described by Mukherjee et al., ACS Synth. Biol. 2015, 4, 12, 1261-1269 (2015) and Shaw et al. Cell. 177(3): 782-796.e27 (April 2019), can be used.
Apyrase (RROP)
In mouse models of IBD and chronic inflammation, intraperitoneal injection of apyrase reduces T cell activation, prevents the production of pro-inflammatory cytokines, and attenuates colitis (Wan, P. et al. Extracellular ATP mediates inflammatory responses in colitis via P2 x 7 receptor signaling. Sci Rep 6, 19108 (2016); Atarashi, K. et al. ATP drives lamina propria T(H)17 cell differentiation. Nature 455, 808-812 (2008); Cauwels, A., Rogge, E., Vandendriessche, B., Shiva, S. & Brouckaert, P. Extracellular ATP drives systemic inflammation, tissue damage and mortality. Cell death & disease 5, e1102 (2014)). Apyrase degrades pro-inflammatory ATP, assisting in its conversion to an anti-inflammatory signal, adenosine (Cekic, C. & Linden, J. Purinergic regulation of the immune system. Nature reviews. Immunology 16, 177-192 (2016)).
Apyrase isolated from the potato species S. tuberosum (RROP1; GenBank accession U58597.1) has the highest ATPase activity reported (115). The BlastPhyMe tool was employed for genome mining of homologous genes (116), using RROP1 as the initial input sequence. Apyrase from wild einkorn wheat Triticum urartu (named “TUAP1” and used in the examples described herein), was eventually selected as it had conserved domains known to be required for apyrase function (Knowles, Purinergic Signal. 2011 March; 7(1):21-45), and based on previous reports of wheat apyrase activity (Komoszynski Comp Biochem Physiol B Biochem Mol Biol. 1996 March; 113(3):581-91); see GenBank accession KD039156.1). The DNA sequences of S. tuberosum (RROP1) and Triticum urartu (TUAP1) used in the exemplary engineered yeast strains presented here were codon optimized for expression in yeast (see below). In addition, the endogenous apyrase N-terminal signal peptide (e.g., the first 30 nucleotides of U58597.1, or first 18 amino acids of KD039156.1) can be replaced by a yeast secretion signal, e.g., MFα1 signal peptide (first 85 or first 89 amino acids of NP_015137.1, depending on if Ste13 cut site is desired). Other signal sequences can alternatively be used, e.g., from pre-pro-α-factor, see, e.g., Wittke et al., Mol Biol Cell. 2002 July; 13(7): 2223-2232; Microb Cell Fact. 2014; 13: 125; or the BGL2 signal peptide (or the artificial BGL2 pre-Val7 variant) (see Achstetter et al., Gene 110(1): 25-21, 2 Jan. 1992); or the AGA2 or EXG1 signal peptide sequences (see Mori et al., J. Biosci. Bioeng. 2015; 120(5):518-525); or engineered peptide sequences not found in nature (see Rakestraw et al., Biotechnol. Bioeng. 2009; 103(6):1192-1201).
Interleukin 10 (IL-10)
IL-10 is required for the proper regulation of inflammation, acting to downregulate pro-inflammatory genes (Paul, G., Khare, V. & Gasche, C. Inflamed gut mucosa: downstream of interleukin-10. Eur J Clin Invest 42, 95-109 (2012)). Delivery of IL-10 has been explored as a treatment for IBD, but its efficacy may be limited by a low concentration once it reaches the gut (Marlow, G. J., van Gent, D. & Ferguson, L. R. Why interleukin-10 supplementation does not work in Crohn's disease patients. World J Gastroenterol 19, 3931-3941 (2013)).
An exemplary reference sequence for human IL-10 protein is provided in GenBank at NP_000563.1 (interleukin-10 isoform 1 precursor) and for mouse IL-10 (mIL-10) NP_034678.1 (interleukin-10 precursor); exemplary DNA reference sequences encoding these two are provided in GenBank at NM_000572.3 and NM_010548.2, respectively. The DNA sequence of mIL-10 used in the exemplary engineered yeast strains presented here was codon optimized for expression in yeast. The endogenous IL-10 N-terminal signal peptide (first 21 amino acids of NP_034678.1) can be replaced by a yeast secretion signal, e.g., MFα1 signal peptide (first 85 or first 89 amino acids of NP_015137.1, depending on if Ste13 cut site is desired). Other signal sequences can alternatively be used, e.g., from pre-pro-α-factor, see, e.g., Wittke et al., Mol Biol Cell. 2002 July; 13(7): 2223-2232; Microb Cell Fact. 2014; 13: 125; or the BGL2 signal peptide (or the artificial BGL2 pre-Val7 variant) (see Achstetter et al., Gene 110(1): 25-21, 2 Jan. 1992); or the AGA2 or EXG1 signal peptide sequences (see Mori et al., J. Biosci. Bioeng. 2015; 120(5):518-525); or engineered peptide sequences not found in nature (see Rakestraw et al., Biotechnol. Bioeng. 2009; 103(6):1192-1201).
See also WO2007039586.
Interleukin-2 (IL-2)
Low dose IL-2 has been shown to expand Tregs and ameliorate disease in a humanized mouse model of experimental colitis. Goettel et al., Cell Mol Gastroenterol Hepatol. 2019; 8(2): 193-195.
An exemplary reference sequence for human IL-2 protein is provided in GenBank at NP_000563.1; an exemplary human reference sequence encoding IL2 is provided at NM_000586.4, optionally including a yeast secretion signal as described above.
IL-27
An exemplary reference sequence for human IL-27 protein is provided in GenBank at NP_663634.2; an exemplary human reference sequence encoding IL-27 is provided at NM_145659.3, optionally including a yeast secretion signal as described above. IL-27 therapy has been suggested as a treatment for IBD; see Andrews et al., Inflamm Bowel Dis. 2016 September; 22(9): 2255-2264.
IL-22
An exemplary reference sequence for human IL-27 protein is provided in GenBank at NP_065386.1; an exemplary human reference sequence encoding IL-27 is provided at NM_020525.5, optionally including a yeast secretion signal as described above. IL-22 therapy has been suggested as a treatment for IBD; see Li et al., World J Gastroenterol. 2014 Dec. 28; 20(48): 18177-18188.
Interferon Beta 1 (IFN-Beta)
An exemplary reference sequence for human IL-27 protein is provided in GenBank at NP_002167.1; an exemplary human reference sequence encoding IL-27 is provided at NM_002176.4, optionally including a yeast secretion signal as described above. Interferon β-1a is in clinical trials for IBD, e.g., in ulcerative colitis; see, e.g. Nikolaus et al., Gut. 2003 September; 52(9): 1286-1290.
Codon Optimization and Variants
In addition, the nucleic acid sequences used in the present methods and compositions are preferably codon-optimized for expression in a selected expression system, e.g., in S. cerevisiae. In order to optimize expression in non-mammalian cells, codon optimization specific for a selected host organism can be used. For example, in embodiments where S. cerevisiae is used as a host organism, the following Table A (source: kazusa.or.jp) can be used to select codons:
Saccharomyces cerevisiae codon frequency
In some embodiments, the methods include variants of a reference sequence as described herein. Thus, in some embodiments, the sequence can be at least 80%, 85%, 90%, 95%, or 99% identical to at least 60%, 70%, 80%, 90%, or 100% of a reference sequence; e.g., the sequence can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations, e.g., in addition to a mutation described herein, so long as the additional mutations don't significantly reduce a relevant activity of the protein (e.g., for P2Y2, the ability to sense eATP and trigger expression and secretion of the anti-inflammatory; for apyrase, the ability to degrade eATP; for IL-10, the ability to downregulate inflammatory genes, e.g., as shown in
For purposes of the present invention, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The gut microbiome plays central roles in health and disease (67). Based on the multiple functions performed by the microbiome, the use of engineered probiotics is considered an attractive therapeutic approach for inflammatory diseases, among other human disorders. The engineered microbes described herein can be used, e.g., in the treatment and prophylaxis of inflammatory conditions, e.g., by administering an effective amount of the engineered microbe to the GI tract of patients, e.g., by oral ingestion of a composition comprising the engineered microbes as described herein, sufficient to reduce inflammation and treat or reduce the risk of or delay development of an inflammatory condition.
The microbes can be used, e.g., in the treatment and prophylaxis of inflammatory conditions, e.g., inflammatory gut conditions including inflammatory bowel disease (IBD) by administering the engineered microbe to the GI tract of patients, e.g., by oral ingestion of a composition comprising the engineered microbes. IBD can include Crohn's disease; ulcerative colitis (UC); microscopic colitis; diverticulosis-associated colitis; collagenous colitis; lymphocytic colitis; and Behget's disease. The microbes can be used, e.g., in the treatment and prophylaxis of graft versus host disease (GVHD), or following anti-tumor therapy (e.g., chemotherapy, radiation therapy and checkpoint inhibitors, all of which induce GI inflammation). The microbes can be used, e.g., in the treatment and prophylaxis of GI inflammation.
eATP promotes intestinal inflammation in gut conditions including inflammatory bowel disease (IBD), as well as in other diseases besides IBD, such as graft versus host disease and irradiation-induced abdominal fibrosis (93, 94). Moreover, the intestinal microbiome controls inflammation at distant body sites such as the central nervous system (95-97). Thus, present methods can be used for the treatment and/or prophylaxis of inflammatory disorders targeting other tissues beyond the intestinal system, e.g., for the reduction of systemic inflammation.
Generally, the methods include administering an effective amount of engineered microbes as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. The methods can include administering the microbes as often as needed to reduce inflammation, e.g., once or twice per day, e.g., one, two, three, four, five, six, or seven days a week (e.g., daily); and administration can be continued for at least one, two, three, four, five, six, seven, eight or more weeks, or indefinitely.
As used in this context, to “treat” means to ameliorate (e.g., reduce severity or frequency of) at least one symptom of the disorder associated with inflammation; administration of a therapeutically effective amount of engineered microbes as described herein can result in a decrease in one or more symptoms of a disorders associated with inflammation. As noted above, in some embodiments, the disorder is IBD. For example, Crohn's often results in frequent diarrhea; occasional constipation; abdominal pain; fever; blood in the stool; fatigue; skin conditions; joint pain; malnutrition; weight loss; and/or fistulas. UC often results in abdominal pain; loose stools; bloody stool; urgency of bowel movement; fatigue; loss of appetite; weight loss; and/or malnutrition. Administration of a therapeutically effective amount of engineered microbes can result in a reduction in any one or more of these symptoms. Administration of a prophylactically effective amount of engineered microbes as described herein can result in decreased risk or delayed development of a disorders associated with inflammation. Subjects who have a disorder associated with inflammation can be identified by one of skill in the art, e.g., using imaging methods such as colonoscopy or a CT scan. In some embodiments, subjects treated using a method described herein include those who have a risk of developing a disorder associated with inflammation, e.g., that have a risk that is higher than the risk of the general population, e.g., as a result of genetics/family history, age, race, diet, or other risk factors.
See also WO2007039586.
Provided herein are compositions comprising the engineered microbes. Preferably the compositions are formulated for oral administration of the microbes, and include a physiologically-acceptable carrier or excipient, i.e., that is non-toxic and doesn't affect the activity of the engineered microbes.
In some embodiments, the compositions are solid forms, e.g., tablets, pills, capsules, soft gelatin capsules, sugarcoated pills, orodispersing/orodispersing tablets, effervescent tablets or other solids. In some embodiments, the compositions are in a liquid form, such as, for example, a drinkable solution.
Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
In some embodiments, the compositions are nutritional compositions comprising liquid or solid food, feed or drinking water. In some embodiments, the compositions are food products, such as, for example, beverages including dairy and non-dairy based drinks, plant- or animal-based milk products (e.g., almond, cashew, soy, or oat milk; or cow, goat, or sheep milk), milk powder, reconstituted milk, cultured milk, smoothies or cultured beverages (resulting from fermentation of the carbohydrate containing media), flavored beverages, yogurt, drinking yogurt, set yogurt, fruit and/or vegetable juices or concentrates thereof, fruit and vegetable juice powders, reconstituted fruit products, powders, or malt or soy or cereal based beverages, and sports supplements including dairy and non-dairy based sports supplements; or solid foods including breakfast cereal such as muesli flakes, spreads, meal replacements, confectionary, chocolate, gels, ice creams, cereal, fruit puree, and/or chocolate bars, energy bars, snack bars, food bars, sauces, dips. The compositions can also be additives, e.g., to be mixed into solid food, e.g., by sprinkling onto or mixing into a food; or to be mixed into a beverage, e.g., into water, juice, or milk, and can include flavors. As used herein, a smoothie is a drink made from pureed raw fruit and/or vegetables, typically using a blender. A smoothie typically comprises a liquid base such as water, fruit juice, plant and/or animal based milk products such as milk, yogurt, ice cream or cottage cheese. Smoothies can comprise additional ingredients, e.g., crushed ice, sweeteners (e.g., natural sweeteners such as agave syrup, maple syrup, honey or sugar, or artificial sweeteners), vinegar, protein supplements such as whey powder, chocolate, or nutritional supplements,
The microbes in the compositions should be viable, e.g., should either be alive or should be in a form that supports viability, e.g., in a dehydrated form that allows for the yeast to be viable when rehydrated, e.g., prepared as described in U.S. Pat. Nos. 3,843,800A1; 3,993,783A; 4,217,420A; 4,341,871A; 4,764,472; EP0616030A1; U.S. Pat. Nos. 6,033,887A; 6,372,481B1; US20050106287A1; US20050129808A1; US20100092611A1; WO2009130219A1; JP2010536360A; RU2444566C2; CN102803468A. See also WO2007039586.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Materials and Methods
The following materials and methods were used in the Examples below.
Reporter yeast strains. All genome modifications of initial yeast strains were conducted using homologous recombination of selectable markers, transformed using a standard lithium-acetate transformation method with at least 1 μg of linear insert DNA. The parent strain was either CB008, for constitutive overexpression of fluorescent reporter genes (98), or BS004 for the P2Y2-mCherry or P2Y2-apyrase gene circuit (99) (see Table 1 for detailed strain genotypes). pFUS1-mCherry was integrated at the MFA2 locus using plasmid pJW609 containing the KanR marker. pFUS1 was defined as the 1636 bp immediately upstream of the Fus1 start codon, the mCherry sequence used is from Keppler-Ross, Noffz and Dean (100), and ˜1 kb homology regions were used. Ste2 and Sst2 were targeted for deletion using Trp1 and HygB selectable markers respectively, each with 180 bp of flanking homology regions identical to the sequences flanking the ORF. The 5 C-terminal amino acids of Gpa1 (KIGII) were replaced with a Gpa1-Gα chimera containing the C-terminal amino acids from the indicated human Gα protein, using plasmid pBS600 containing selectable marker LEU2 and 800 bp homology regions. The C. albicans Adh terminator was used for the pFUS1-mCherry and Gpa1-Gα gene knock-ins. To create a strain that constitutively expresses mCherry, integration plasmid pJW609 was modified to replace the KanMX marker with HIS3 from C. glabrata, and pTDH3 mCherry was inserted at the PspOMI/BamHI sites. Linearized HIS3-pTDH3 mCherry cassette was transformed into strain CB008, and integrations selected by plating on SC-HIS. To create strains that contain the KanMX selectable marker and constitutively express GFP, an integration plasmid was constructed using the MoClo Yeast Toolkit (101). The resulting plasmid, pBS211, contained HO locus homology regions, the KanMX marker, and a yeast codon-optimized sfGFP gene downstream of pTDH3 (102). Linearized KanMX-pTDH3 sfGFP cassette was transformed into strains in Table 1, and integrations selected by plating on YPD-G418 sulfate (200 μg/mL). All strains were confirmed by PCR and flow cytometry.
Microscopy. Yeast strain BS016 expressing the endogenous yeast GPCR Ste2 or a yeast codon-optimized sequence of human P2Y2 (obtained from ATUM) C-terminally tagged with GFP were grown to log phase in SD-URA media. The centromere plasmid pRS316 was used, containing the endogenous Ste2 promoter for Ste2 expression, or pTDH3 for P2Y2 expression, and the GFP sequence used is from (103). Restriction enzyme sites introduced an amino acid linker (GGERGS) between the final GPCR residue and first GFP residue. Cells were plated on glass-bottomed dishes (Greiner Bio-One) that had been treated with concanavalin A (Sigma-Aldrich), then covered with 1 mL SD-URA media. Cells were imaged using a Leica TCS SP8 confocal microscope.
Flow cytometry evaluation of response to ATP and UTP. Yeast strain BS016 transformed with a human P2Y2 gene in the pRS316 pTDH3 vector was grown in SC-URA liquid media overnight. The same strain transformed with a plasmid not containing a P2Y2 sequence (Vector) was used as a negative control. Cells were diluted to OD600 0.05 in 600 μL SC-URA containing ATP (0-25.6 mM; pH 7.0, Bio Basic) or UTP (0-3.2 mM; pH 7.0, Sigma-Aldrich) and incubated for 6 hours at 30° C. Cells were then treated with cycloheximide to a final concentration of 10 μg/mL. The mCherry signal of at least 10,000 cells was measured for each sample with a Miltenyi Biotec MACSQuant VYB. The mean mCherry fluorescence was determined using FlowJo. For dose-response assays, data was fitted with the “log(agonist) vs. response—Variable slope (four parameters)” model in Prism (GraphPad). After subtracting the mCherry fluorescence signal of the Vector control, fluorescence values were normalized to the wild-type P2Y2 control used in the same experiment, to allow comparisons between experiments performed on different days.
Directed evolution of human P2Y2 receptor. Error-prone PCR mutagenesis was performed using the Agilent GeneMorph II Random Mutagenesis Kit with yeast codon-optimized human P2Y2 as a template, using previously described methods (104). 150 ng of template DNA and 30 cycles achieved the desired mutation rate of ˜3 mutations per P2Y2 gene, determined by sequencing 12 randomly selected plasmids (Table 2). The random mutants were inserted into pRS316 pTDH3 using AarI-based cloning and transformed into NEB 5-alpha competent E. coli cells (New England Biolabs) generating >15,000 individual colonies. Cells were scraped off agar plates, mixed together, and plasmid DNA was extracted (QIAQuick Spin Miniprep Kit, Qiagen) to create the final plasmid library. The library was transformed into yeast strain BS016 using a high-efficiency lithium acetate-based method (105), yielding at least 10-fold the number of colonies as the total library size, so that each mutant would be screened multiple times. Transformed cells were incubated overnight, then diluted to OD600 0.05 into fresh 100 mL SC-URA liquid media containing 100 μM ATP (pH 7.0, Biobasic), and incubated for either 18 or 6 hours at 30° C. (
Homology modeling of P2Y2. Modeling was conducted as described Rafehi, Neumann, Baqi, Malik, Wiese, Namasivayam and Muller (107) using the crystal structure of the human P2Y1 receptor (4XNW.pdb) bound to the nucleotide antagonist MRS2500 as a template. The sequences of human P2Y1 and P2Y2 were aligned using Clustal Omega. As only residues S38 to F331 of P2Y1 were visible in the crystal structure, these were used as the template to generate 500 models of the corresponding P2Y2 residues L20 to L313. Standard MODELLER 9.18 settings were used, maintaining MRS2500 in the models (108). The generated models were first analyzed based on the DOPE and GA341 scores, and the top five models were manually inspected to ensure the natural disulphide bonds were maintained (C25-C278, C106-C183). Next, models were evaluated by ProSA-WEB (109) and Ramachandran plots (110), and the final model was selected. ATP was docked to the wild-type P2Y2 homology model using the Galaxy7™ web server (111), which generated 10 docked models. The lowest energy model in which the adenine ring of ATP was oriented towards the key Y114 and F261 residues was selected (107). Publication quality images were generated with PyMOL (Schrödinger, Inc.).
Integration of P2Y2 mutants with CRISPR. The pCAS plasmid was obtained from AddGene, which expresses Cas9 and a yeast-optimized guide RNA (gRNA) (112). The gRNA sequence was replaced with an AarI-based multiple cloning site, to generate the pCAS AarI plasmid (
The forward oligos were ordered with CTTT 5′ overhang, and reverse oligos were ordered with AAAC 5′ overhang to facilitate ligation to plasmid pCAS AarI following digestion with AarI enzyme.
A gene cassette containing an engineered P2Y2 mutant downstream of the pTDH3 promoter was assembled in the plasmid pBS600, flanked by 800 bp homology arms for the SST2 locus. The cassettes were amplified by PCR and transformed into strain BSO21 along with plasmid pCAS AarI HygB 1143 as described Ryan, Skerker, Maurer, Li, Tsai, Poddar, Lee, DeLoache, Dueber, Arkin and Cate (112). Colonies were screened for mCherry expression in response to ATP, and P2Y2 integration was confirmed by sequencing.
Apyrase genome mining. Apyrase isolated from the potato species S. tuberosum (RROP1; GenBank accession U58597.1) has the highest ATPase activity reported (115). The BlastPhyMe tool was employed for genome mining of homologous genes (116), using RROP1 as the initial input sequence. Apyrase from wild einkom wheat Triticum urartu (named “TUAP1” in our study; GenBank accession KD039156.1) was selected as it had conserved domains known to be required for apyrase function (115), and based on previous reports of wheat apyrase activity (117). Yeast codon optimized RROP1 and TUAP1 were modified to contain a N-terminal alpha factor signal peptide (first 85 amino acids of the yeast MFα1 gene, lacking Ste13 cut site) and a C-terminal HA tag (gene synthesis by ATUM).
Integration of apyrase genes into the genome of P2Y2 strains. A gene cassette containing one of the apyrase genes downstream of the pFUS1 promoter was assembled in the plasmid pBS600. The cassettes were amplified by PCR and transformed into a strain where P2Y2 had previously been integrated, along with plasmid pCAS AarI mCherry g664 as outlined by Ryan, Skerker, Maurer, Li, Tsai, Poddar, Lee, DeLoache, Dueber, Arkin and Cate (112). The promoter and terminator of the cassette functioned as homology arms, as mCherry had previously been inserted with pFUS1 and the C. albicans Adh terminator at the MFA2 locus. Colonies were screened for mCherry expression in response to ATP, and apyrase integration was confirmed by sequencing colonies that did not express mCherry. A second group of gene cassettes was assembled using plasmid pBS603 (pBS600 containing a HIS3 selection marker), with one of the apyrase genes downstream of the pTDH3 promoter, flanked by 1 kb homology arms for the MFA2 locus. The cassettes were amplified by PCR and transformed into strain CB008, before plating on selective media, to create strains BS029 (pTDH3 RROP1) and BSO30 (pTDH3 TUAP1).
Western blot. Overnight cultures were diluted to OD600 0.05 in 50 mL YPD. ATP was added to 400 μM to induce apyrase expression in the initial media, and again at hour 6, and at hour 22 (final concentration of 1200 μM assuming no ATP was degraded). All cultures were incubated for 24 hours at 30° C. with shaking (225 rpm). Samples of lysed cells were resolved on a 10% SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane using a Bio-Rad Trans-Blot Turbo. Membranes were blocked overnight with Odyssey® Blocking Buffer (TBS) (LI-COR Biosciences). The following primary antibodies were used: rabbit anti-HA tag (C29F4, Cell Signaling Technology), mouse anti-PGK (459250, Invitrogen). After washing, the following secondary antibodies were used: IRDye® 680LT Goat anti-Mouse IgG (926-68020, LI-COR Biosciences), IRDye® 800CW Goat anti-Rabbit IgG (926-32211, LI-COR Biosciences). Bands were visualized with a Licor Odyssey CLx infrared imaging system (LI-COR Biosciences).
Induction of apyrase secretion with ATP. Yeast strains containing a P2Y2 mutant gene and pFUS1 regulating the expression of RROP1 apyrase were incubated overnight in YPD media. Cells were diluted to OD600 0.05 in 2 mL fresh YPD, with 0-500 μM ATP (pH 7.0) added. After incubation for 16 hours at 30° C. with shaking (225 rpm) to OD600 3.5, 500 μL samples were pipetted into 1.5 mL tubes and centrifuged at 2000×g for 5 minutes to pellet cells. Culture supernatants were then evaluated for ATPase activity.
Quantification of secreted ATPase activity. The amount of ATP remaining following incubation with apyrase was determined by KinaseGlo Plus luminescence as previously described (118). In a white 96-well microplate (#655075, Greiner Bio-One) 5 μL of raw supernatant from yeast cultures at OD600 3.5, where ATP had been added at the start of culturing, was mixed with 50 μM ATP (pH 7.0) in assay buffer (60 mM HEPES pH 6.0, 2 mM MgCl2, 2 mM CaCl2), 1 mM dithiothreitol, 0.1 mg/mL bovine serum albumin, 0.1 mM EDTA, and 0.01% Tween-20) to a final volume of 50 μL. The reaction was incubated for 30 minutes at 30° C., and quenched by addition of 50 μL KinaseGlo Plus (Promega). Luminescence was measured with a Fluoroskan Ascent FL microplate reader (Thermo Fisher Scientific). ATPase activity was compared to that of commercial potato apyrase (A6410, Sigma-Aldrich), incubated with ATP under the same conditions. “Percent ATP degraded” was calculated by comparing to 50 μM ATP incubated in YPD media and assay buffer under the same conditions.
Yeast cultures for in vivo testing. Yeast strains were cultured in 550 mL or 1 L YPD media (BioShop Canada) at 30° C. with shaking (225 rpm). 200 μg/mL G418 sulfate antibiotic (BioShop Canada) was added to media when culturing strains containing the KanMX resistance marker. After 24 hours, cultures were centrifuged and yeast were resuspended in fresh YPD to an OD600 of 92, or approximately 2×109 cfu/mL, and colony density was confirmed by plating. Yeast were stored as 800 μL aliquots at −80° C. for up to one year.
Mice. C57BL/6J female (for DSS model) or males (for TNBS model) mice between 8-10 weeks of age were used throughout the study. Mice were obtained from the Jackson Laboratory. All experiments were carried out in accordance with guidelines prescribed by the Institutional Animal Care and Use Committee (IACUC) at Brigham and Women's Hospital and Harvard Medical School.
Dextran sodium sulfate (DSS)-induced mouse colitis model. IBD was induced by adding 4% of dextran sulfate sodium salt (DSS colitis grade; MP Biomedicals) in the drinking water. Treatment was maintained for 7 days and two cycles were performed with a week without treatment in between. After the second cycle of DSS, DSS was removed and mice were sacrificed. Animal body weight was evaluated daily throughout the study.
Trinitrobenzenesulfonic acid (TNBS)-induced mouse colitis model. To induce TNBS colitis in C57BL/6J, males were pre-sensitized one week before the colitis induction by applying 150 μL of pre-sensitization TNBS solution (64% acetone (#179124, Sigma Aldrich), 16% olive oil (Sigma Aldrich #01514), 20% of 50 mg/mL TNBS (Picrylsulfonic acid solution 5% Sigma Aldrich #P2297)) on their preshaved back. One week after, pre-sensitized mice were fasted for 4 hours and subsequently 100 μL of TNBS induction solution (50% ethanol, 50% 50 mg/mL TNBS). Was administered rectally. Control group was treated only with 50% Ethanol. Mice weight was monitored daily until the day of the euthanasia 72 hours after the colitis induction at the peak of the disease.
Mice treatment with yeasts: Both DSS and TNBS mice were given 2×108 cfu of the corresponding yeast strain by oral gavage for the whole length of the experiment meaning from day 0 for DSS mice and from the day of pre-sensitization for TNBS mice. For yeasts culture from feces studies, mice were gavaged once. For mCherry and ATP measurements studies mice were gavages for 3 days before the study with 2×108 cfu of the corresponding yeasts.
Yeast culture from mice feces: CB008, BS029 and AP TM-3 yeasts expressing the resistance gene to the antibiotic G418 were administered by oral gavage as above. Feces were collected 2, 4 and 6 hours after the gavage, weighted, homogenized in PBS and cultured at 30° C. in YPD agar (cat number #Y1500—Sigma Aldrich) containing 500 μg/mL of G418 (cat number #A1720—Sigma Aldrich). Colony Forming Units (CFUs) were quantified after 72 hours.
ATP measurement in fecal content: In order to evaluate the ATP amount in the fecal content, feces from duodenum, jejunum, ileum, cecum and colon of TNBS mice treated with the corresponding yeast strain was collect 72 hours after TNBS induction, 2 hours after the last gavage the yeasts. The fecal content of the corresponding part of the gut was homogenized in PBS and the ATP measurement was performed using ATP determination kit (#A22066, Molecular Probes) following manufacturer's instructions. Data was normalized to weight of the fecal content and to the control sample.
mCherry Reporter Yeast Strains Detection In Vivo
To confirm the response to ATP of our engineered P2Y2 mutant in vivo, reporter yeasts expressing the mCherry under the control of the most efficient P2Y2 mutant (see above) and constitutive GFP were administered as above to TNBS colitis mice at the peak of the disease when we expect more ATP to be present in the gut. Content from the specified section of the gut was collected 2 hours after the gavage, homogenized in YPD media (#Y1375, Sigma-Aldrich) and cultured overnight. GFP and mCherry expression was measured by flow cytometry in a Fortessa flow cytometer (BD Biosciences) and the data analysis were performed at using FlowJo 10.6.1. software.
16S microbiome sequencing and analysis: Fecal samples were collected from control and TNBS colitis mice from each respective yeast treatment at the end of the study. DNA was extracted using the DNeasy PowerLyzer PowerSoil kit (#12855, Qiagen), following manufacturer's instructions. 16S rRNA gene V4 region was amplified and barcoded by PCR using HotMaster Taq DNA Polymerase and Hotmastermix (#10847-708, VWR) and a primer library that contain adaptors for MiSeq sequencing and dual index barcodes so that the PCR products can be pooled. DNA was then quantified using Quant-iT™ PicoGreen™ dsDNA Assay Kit (#P11496, Thermo Scientific) and 100 ng of each sample were pooled and cleaned-up using the QIAquick PCR Purification Kit (#28104, Qiagen). DNA was re quantified after clean-up by Qubit Fluorometric Quantification kit (Thermo Scientific) and submitted for paired-end 151 base-pair reads sequencing on the Illumina MiSeq instrument at the Harvard Medical School Biopolymer Facility as described (119). Quantitative insights for microbial ecology software 2 (QIIME2) was used for quality filtering and downstream analysis for Apha and Beta diversity, and compositional analysis following standardized protocols (120) (More detail here? Laurie?: Quality sequences were filtered by trimming reads below a of q20 and discarding reads shorter than 75% percent of the original length). Operational Taxonomic Units (OTUs) were picked and taxonomy was assigned. Distances between samples (β-diversity), were calculated using the phylogenetic based distance UniFrac (121). Statistical testing for differential clustering of samples on the PCoA plots was performed using the Permanova test using 999 permutations. Significant differences in taxa modulated by control or active yeast treatment was determined by linear discriminant analysis effect size (LEfSe) (122).
Cytokine quantification by ELISA. 2 cm of distal colon were extracted, thoroughly washed and cultured in RPMI supplemented with 10% FBS, 100 I.U./ml penicillin, 100 ug/ml streptomycin, 100 ug/ml of ampicillin and 50 ug/ml of kanamycin. Supernatants were collected for later ELISA analysis. ELISAs were performed following manufacturer's instructions (eBioscience).
Histological evaluation of colitis. Colonic tissue was removed and assessed for histological evaluation blindly upon Bouin's solution (Sigma-Aldrich) fixation. Paraffin-embedded tissues were sectioned, stained with hematoxylin and eosin and examined for evidence of colitis. Histology score (range: 0-6) was calculated based on the presence of lymphomononuclear cell infiltrate (‘0’: absence of inflammatory foci; ‘1’: mild presence of inflammatory foci in mucosa; ‘2’: presence of multiple inflammatory foci in mucosa and submucosa; ‘3’: evidence of transmural infiltration) and intestinal architecture disruption (‘0’: normal architecture; ‘1’: presence of focal erosions; ‘2’: erosions and focal ulcerations; ‘3’: extended ulceration, granulation of tissue and or pseudopolys) as previously described (Erben et al int J Clin Exp Pathol 2014).
Flow cytometry staining and acquisition. Cell suspensions were prepared from mesenteric lymph nodes. Antibodies for flow cytometry were purchased from eBioscience or BD Pharmingen and used at a concentration of 1:200 unless recommended otherwise by the manufacturer. Cells were then analyzed on a Fortessa flow cytometer (BD Biosciences and Miltenyi Biotec, respectively). Treg cells were defined as CD3+CD4+IFN-γ-IL-17-IL-10-FOXP3+.
RNA extraction and qPCR. 20 mg of the distal colon was flash frozen and later disrupted in Trizol (Invitrogen). RNA was extracted following manufacturer's instructions for miRNAeasy kit (Qiagen). When needed, to remove DSS from the RNA we further purified the mRNA using Oligotex kit (Qiagen). cDNA was prepared using High capacity RT kit (Applied Biosystems) and used for qPCR. Results were normalized to Gapdh. All primers and probes were from Applied Biosystems. Gapdh Mm99999915_g1, I117a Mm00439618_m1, Ifng Mm00801778_m1, Foxp3 Mm00475162_ml, Ccl2 Mm00441242_m1, Nos2 Mm00440502_m1, Il1b Mm00434228_m1.
Gene expression analysis by Nanostring. 100 ng of total RNA from colon tissue was analyzed using nCounter Mouse Immunology Panel expression code sets according to manufacturer's instructions (NanoString Technologies). Data were analyzed using nSolver Analysis software and plotted with Heatmapper (123). Functional pathway enrichment analysis was conducted using Enrichr. The combined score was calculated as c=ln(p)*z where p is the p-value computed using Fisher's exact test and z is the rank score or z-score computed using a modification to Fisher's exact test in which a z-score for deviation from an expected rank is computed (124).
Gene expression analysis by RNA sequencing: 5 ng of total RNA form colon tissue was were sent for SMARTseq sequencing by the Broad Technology Labs and the Broad Genomics Platform. Processed RNA-Seq data was filtered, removing genes with low read counts. Read counts were normalized using TMM normalization and CPM (counts per million) were calculated to create a matrix of normalized expression values. The fastq files of each RNA-seq data sample were aligned to Mus musculus GRCm38 transcriptome using Kallisto (v0.46.1), and the same software was used to quantify the alignment results. The differential expression analysis was used to conduct using DESeq2, and the log 2 fold change was adjusted using apeGLM for downstream analysis. The Benjamini-Hochberg method was used for multiple hypothesis testing correction. The GSEA analysis was performed using the apeGLM adjusted differential expression analysis results. Genes that were differentially expressed with adjusted p values<0.05 were analyzed with the Ingenuity® Pathway Analysis (IPA) tool to determine significantly regulated pathways.
The P2Y2 receptor is a G protein-coupled receptor (GPCR) that senses eATP and also extracellular uridine triphosphate (eUTP) (29). We first engineered the human P2Y2 receptor to increase its sensitivity to eATP when expressed in yeast. To establish a platform amenable to directed evolution, we coupled the human P2Y2 receptor to the yeast mating pathway via a chimeric yeast Gpa1-human Gαi3 protein and monitored pathway activation using a fluorescent mCherry reporter controlled by the mating-responsive FUS1 promoter (pFUS1) (
Physiological eATP levels associated to inflammation have been detected in the 100 μM to high mM range (35). However, yeast expressing wild-type (WT) P2Y2 show a weak response to 100 μM eATP as determined by the analysis of mCherry expression by flow cytometry (
We performed multiple iterative rounds of FACS-based selection (36) to isolate mutants displaying the desired increase in eATP sensitivity (
We focused on human P2Y2 receptor mutants that showed an enhanced response to eATP, and a high eATP/eUTP response ratio concomitant with no constitutive expression of mCherry. The sequencing of these human P2Y2 receptor mutants revealed a diverse range of genotypes, with up to three non-synonymous mutations (Table 3). Eight of the 19 human P2Y2 mutants harbored a mutation at site F581.57, as defined by the Ballesteros-Weinstein convention in which the first number is the transmembrane helix, followed by a conserved position across all family A GPCRs (37). We also detected mutations at nearby residues L591.58 and C601.59, and at Q1654.57 and F3077.54.
We selected 10 P2Y2 mutants for detailed characterization, each mutant was named using a unique identifier based on the location of the mutated residue(s) (Table 4). In dose-response studies, the engineered P2Y2 receptors were more responsive to both eATP and eUTP (
Only unique mutants that consistently improved upon WT response/sensitivity to ATP were selected for detailed characterization.
Maximum response values were normalized to the maximum mating pathway activation provided by the WT human P2Y2 receptor incubated with eATP. Dynamic range was the ratio of the highest fluorescence obtained in the presence of the indicated ligand versus 10% signal saturation. Linear range was the series of ligand concentrations for which a change in signal can be detected. The minimum limit of the linear range was estimated as the ligand concentration corresponding to 10% signal saturation. Data represents the mean of six colonies for eATP, three colonies for eUTP.
Key residues that participate in the binding of nucleotides and the activation of human P2Y2 receptor have been identified (38, 39), but the mutations detected in the ten human P2Y2 receptor mutants that we analyzed did not involve previously identified key residues. Instead, the novel human P2Y2 receptor mutants we identified involved residues peripheral to the ligand binding pocket (A762.47, N1163.35, C1193.38, L1624.54, Q1654.57), or residues located in the intracellular facing side of the receptor (F581.57, L591.58, C601.59, A229ICL3, K2406.31, F3077.54, G310C-term) (
To determine the molecular mechanisms responsible for the increased sensitivity of the selected human P2Y2 receptor mutants generated by directed evolution, we first analyzed their expression levels by microscopy and flow cytometry using receptor mutants tagged with C-terminal GFP. Human P2Y2 receptor expression in yeast was increased when F581.57 was mutated to a smaller hydrophobic residue (C/I/L, “H1” mutants), and also in TM-1 and TM-2 mutants (
We detected increased responsiveness and signaling in the absence of agonist (constitutive activity) in P2Y2 mutants harboring the N116S3.35 and F307S7.54 mutations (TM-3, H7-1, H7-2 mutants) (
The F7.54 residue is located immediately after the highly conserved D/NPxxY (SEQ ID NO:18) motif required for G protein activation (44). Indeed, mutations at F7.54 in the P2Y12 receptor result in constitutive activity (45). The human P2Y2 receptor lacks the conserved F8.50 residue in helix 8, which in other GPCRs interacts with Y7.53 to stabilize the inactive conformation (46). In the human P2Y2 receptor, F3077.54 may instead form this interaction with Y7.53, in addition to conserved contacts with helix 8 in the inactive state (47). Taken together, our findings suggest that the F307S7.54 mutation facilitates the rotation of Y7.53 into the active conformation, resulting in constitutive activity and increased eATP sensitivity.
To further investigate the mechanisms responsible for the differential activity of human P2Y2 receptor mutants, we evaluated the effects of each mutation alone or in combination with other mutations in P2Y2 receptor activation by eATP. Certain mutations (C60Y, K240N, G310A) did not cooperate to further increase P2Y2 receptor responsiveness to eATP, while others showed deleterious effects (A76T/A229V, L162I, S359P) or exhibited positive epistasis when combined (N116S with F58I or F307S, L59I/C119S) (
In summary, none of the tested combinations of mutations was superior to the original set of 10 selected human P2Y2 receptor mutants, which provided a range of improved sensitivity to physiological concentrations of eATP associated to inflammation. Mutations at F581.57, N1163.355, F3077.54 and Q1654.57 contributed the most to the increased sensitivity to eATP of the human P2Y2 receptor via independent mechanisms involving increased receptor expression (F581.57), stabilization of the active receptor conformation (N1163.35 and F3077.54) and improved interactions with ATP (Q1654.57). All 20 amino acids were later tested at residue F581.57, which revealed a diversity of eATP-induced signaling phenotypes (
We next incorporated a therapeutic response element in the yeast synthetic gene circuit responsive to eATP. We focused on apyrases, which hydrolyze pro-inflammatory eATP and participate in its conversion into immunosuppressive adenosine (26). We selected the apyrase encoded by RROP1 in potato (Solanum tuberosum) (
We first incorporated each modified apyrase gene under the control of a strong constitutive promoter into the yeast genome. The analysis of protein expression detected multiple protein bands, suggesting that the apyrases are partially degraded when expressed in yeast (
Culture supernatants from yeasts expressing RROP1 (BS029) showed higher ATPase activity than those of TUAP1-expressing yeasts (BSO30) (
We then co-introduced sensing (P2Y2) and responding (RROP1) elements into the genome of the same yeast strains using a CRISPR/Cas9-based approach. We selected 6 human P2Y2 receptor mutants for integration into the yeast genome based on their low EC50, high dynamic range, and high maximum activation. We also removed the HygB selection marker from the yeast genome, to ensure that the final strain would not contain an antibiotic resistance gene while retaining uracil auxotrophy, an important consideration for the biocontainment and safety of an engineered microbe.
eATP induced, in a dose-dependent manner, ATPase enzymatic activity in culture supernatants from yeast strains containing the P2Y2-RROP1 gene circuit (
We used a yeast strain constitutively overexpressing RROP1 (strain BS029) to estimate the theoretical maximum of secreted ATPase. At 500 μM ATP, strains harboring engineered human P2Y2 receptor mutants showed 45%-69% of the ATPase activity detected with the BS029 constitutively secreting strain; yeast strains harboring the human WT P2Y2 receptor showed only 27% ATPase activity. Collectively, these findings show that through the combination of directed evolution and gene-circuit engineering we generated yeast strains that secrete functional ATPase in response to physiological levels of eATP.
Represented as fold-differences versus strain AP-P4. Apyrase data measured as % ATP degraded (ATPase activity), with data from
We then evaluated the anti-inflammatory activity of the engineered yeast probiotics using a murine experimental model of IBD. Specifically, we tested the AP TM-3 engineered yeast strain that expresses apyrase in an eATP-dependent manner. We selected this yeast strain because it secretes low levels of apyrase when not stimulated, and because the increased responsiveness to eATP can be directly connected to a single mutation in P2Y2 with a known mechanism of action (N116S3.35). Moreover, when AP TM-3 was stimulated with eATP the ATPase activity detected was greater than or similar to the one detected in other engineered P2Y2-RROP1 strains.
We first evaluated the viability of the engineered yeasts in the murine digestive tract. To address this point, we incorporated an antibiotic resistance cassette to the CB008, BS029 and AP TM-3 engineered yeast strains, to generate kanamycin-resistant CB008 KG, BS029 KG and AP TM-3 KG strains which can be easily quantified in fecal cultures. Six hours after the administration of CB008 KG, BS029 KG or AP TM-3 KG yeasts by gavage (of 2×108 cfu) we detected viable antibiotic-resistant yeasts in feces (
Increased local eATP levels are associated to intestinal inflammation (24, 25) (
We first evaluated the therapeutic value of the engineered yeasts in the experimental model of TNBS-induced colitis, in which C57BL/6J mice are pre-sensitized and colitis is induced by rectal injection of TNBS 7 days later. We administered the AP TM-3 engineered yeast strain in which apyrase is induced following the activation of mutant TM-3 P2Y2 by eATP daily by gavage (2×108 cfu) starting on the day of topical sensitization with TNBS; the parent CB008 yeast strain and the BS029 engineered yeast strain that expresses apyrase constitutively were used as controls. AP TM-3 administration ameliorated TNBS-induced colitis, as indicated by the evaluation of weight loss, colon shortening and the histological analysis of intestinal pathology (
The analysis of colon samples by RNA-Seq detected decreased expression of pro-inflammatory genes in mice treated with apyrase-producing yeast strains BS029 and APTM-3; these effects were more pronounced in the AP TM-3 group (
To further evaluate the therapeutic potential of engineered apyrase-expressing yeasts we used the model of colitis induced with two rounds, seven days apart, of dextran sodium sulfate (DSS) administered in drinking water (53). We administered yeast orally starting on the day in which DSS administration was initiated. Treatment with AP TM-3, but not with BS029, interfered with the weight loss associated to DSS-induced colitis (
Fibrosis contributes to the pathogenesis of IBD (56-58). Although adenosine produced by the metabolism of eATP dampens inflammation, chronic activation of purinergic signaling driven by adenosine can promote fibrosis (26, 29). Thus, although yeast strains constitutively expressing apyrase show anti-inflammatory effects, they may also promote additional pathogenic responses avoidable by the use of yeast strains that produce apyrase in response local eATP levels. Indeed, we detected fibrotic lesions in the colon of mice treated with control CB008 and also with the constitutive apyrase-expressing BS029 yeast strains. However, we detected a significant reduction in fibrosis in mice treated with the eATP-inducible APTM3 engineered yeast strain (
The microbiome plays an important role in intestinal physiology in health and disease (5). Moreover, purinergic signaling participates in gut microbiota-host communication (28, 30). Thus, probiotics engineered to act on an inducible and localized manner are likely to minimize disturbances on the gut microbiome. To investigate whether constitutive versus inducible apyrase production by engineered yeast strains differ on their effects on the gut microbiome, we performed 16S rRNA sequencing in fecal samples. In agreement with previous reports (59), the induction of colitis with TNBS reduced microbiome diversity within each sample as indicated by the analysis of the Shannon entropy index of alpha-diversity (
We then analyzed beta-diversity, which measures the differences in microbiome composition between samples, using the unweighted UniFrac distance metric that evaluates qualitative differences on microbial taxa, taking into account their phylogenetic relationship. Principal coordinate analysis (PCoA) visualization of permanova testing (
Finally, we analyzed the taxonomic composition of the microbiome in the different treatment groups. Several taxa of commensal bacteria have been shown to be decreased in IBD and ameliorate intestinal inflammation (4, 5, 60, 61). For example, Clostridium cluster XIVa, associated with the induction of regulatory T cells (Tregs), is consistently depleted in people with IBD and acute colitis (62-64). We found that the Lachnospiraceae family, which is part of Clostridium cluster XIVa, was significantly reduced in TNBS mice treated with the CB008 and BS029 yeast strains, but not in TNBS mice treated with the AP TM-3 strain expressing inducible apyrase (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/891,603, filed on 26 Aug. 2019. The entire contents of the foregoing are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/048049 | 8/26/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62891603 | Aug 2019 | US |
