Systems, devices, and methods for in vivo generation of substances using nonpathogenic microorganisms

Abstract

The invention relates to in vivo generation of beneficial molecules using genetically modified, nonpathogenic microorganisms. The invention has application to various fields, such as medicine, human performance, and veterinary medicine. In various embodiments, the use of transgenic Trypanosoma rangeli, a nonpathogenic microorganism may be purposefully engineered, altered, and designed to be a blood symbiont in humans and animals after genetic modification to express foreign nucleic acid sequences that encode proteins, polynucleotides, and / or pathways to treat disease or enhance specific aspects of host physiology to include metabolic dominance to correct or augment homeostatic imbalances when suitable. The foreign nucleic acid sequences may be used to generate enzymes, scavenger molecules, peptides and hormones, antibodies, nanobodies, signaling ligands, and other therapeutic or novel synthetic agents.

Description

SYSTEMS, DEVICES, AND METHODS FOR IN VIVO GENERATION OF SUBSTANCES USING NONPATHOGENIC MICROORGANISMSSTATEMENT REGARDING FEDERAELY SPONSOREDRESEARCH OR DEVELOPMENTThis invention was made in part with United States government support under Award # 2123532 awarded by the National Science Foundation. The United States government may have certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to U.S. Provisional Application 63 / 515,854 filed July 27, 2023, hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTIONField of the Invention

[0001] The present invention generally relates to the in vivo substance generation. More specifically, the invention relates to in vivo generation of substances using genetically modified, nonpathogenic microorganisms.Background

[0002] Over the last decade, many attempts have been made to deliver biologies to humans and animals using bacteria and viruses as well as multicellular cells of various origins. While bacterial and viral vectors have the advantage of potentially delivering a continuous supply of biologies and eliminating the need for repeated intravenous administrations, their use has been extremely problematic, mostly stemming from the immunogenicity of these vectors. Similarly, although multicellular cell-based therapies such as CAR-T cells and mesenchymal stem cells that were designed to eradicate tumors or control inflammation and are experimentally popular for the treatment of incurable diseases, these approaches are fraught with problems resulting in the lack of widespread clinical adoption. One of the key challenges and seemingly insurmountable hurdles for these therapies is the limited bioavailability of multicellular therapeutic cells primarily due to immunerejection by the host and the inability of these cells to adapt to the changing hostile microenvironment inside the host (i.e. resource and niche competition). In this regard, long-term therapeutic objectives cannot be met by simply engrafting more cells, which will not only increase immunogenicity and resource competition amongst the input therapeutic cells but also increase the cost of the therapy itself.

[0003] As such, there is a continuing need for higher performance systems, devices, and methods to produce and deliver biological products to humans and other animals.BRIEF SUMMARY OF THE INVENTION

[0004] The present disclosure addresses the continuing need for improved biomolecule generation and delivery systems, devices, and methods.

[0005] The invention relates to in vivo generation of substances by genetically modifying nonpathogenic microorganisms to produce desired substances in a host. The invention has application to various fields, such as human medicine, human performance, and veterinary medicine.

[0006] In various embodiments, a nonpathogenic microorganism (naturally occurring or derived in the laboratory) is purposefully engineered, altered, and designed to be a blood symbiont after genetic modification to generate polynucleotides, proteins and / or pathways to enhance specific aspects of host physiology to include metabolic dominance when suitable. The microorganism may be genetically altered so as to not only generate enzymes, scavenger molecules, peptides and hormones, antibodies, nanobodies, signaling ligands, and production of other therapeutic or novel synthetic agents including precursors but also enhance the functions and effectiveness of cell therapies, gene therapies, synthetic vaccines, and probiotic and microbiomc modulation.

[0007] The inventive symbionts may serve as a living medicine or living pharmacy in a human or animal host that manufactures biomolecules for use by the host for extended periods of time. The recombinant symbiont can be used for the sustained production of beneficial biomolecules to treat disease or enhance health. The recombinant symbiont can also be engineered to sense and respond to changes in a host.

[0008] Moreover, the recombinant symbiont can be further modified to supply novel molecules to mitigate adverse conditions or enhance host performance and physiology, including but not limited to increasing muscle strength, endurance, cardiovascular fitness, metabolic efficiency, and cognitive function. The recombinant symbionts can be further engineered to confer disease-resistantphenotypes in the host, including but not limited to enhanced immunity against infectious diseases, reduced susceptibility to autoimmune disorders, and improved resistance to cancerous growth. In other embodiments, the recombinant symbiont is engineered (a) to produce molecules that alleviate or resist pain in the host, including but not limited to endogenous opioids, neurotransmitter modulators, or anti-inflammatory agents; (b) to produce molecules that enhance resistance to infection in the host, including but not limited to antimicrobial peptides, antibodies, or proteins that interfere with pathogen invasion or replication; (c) to regulate tightly controlled homeostasis in the host, including but not limited to maintaining optimal levels of hormones, neurotransmitters, metabolites, or essential nutrients.

[0009] Specifically, the recombinant symbiont comprises a Trypanosome microorganism engineered to express a heterologous polynucleotide, wherein said heterologous polynucleotide encodes for a therapeutic protein or a therapeutic polynucleotide (a “beneficial molecule”). Examples of such Trypanosome includes, but is not limited to Trypanosoma rangeli, Trypanosoma mesnilbrimontii, Trypanosoma preguici, Trypanosoma myrmecophague, Trypanosoma mycetae, Trypanosoma diasi, Trypanosoma cebus, Trypanosoma saimiri, or Trypanosoma advieri. In certain embodiments, the Trypanosome is Trypanosoma rangeli.

[0010] The heterologous polynucleotide can (a) be an integrated foreign nucleic acid, artificial chromosome, or a nucleic acid fragment containing specific genetic features for recombination and utilization of native genetic elements; (b) encode for a ribonucleic acid, including, but not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA); (c) encode for a therapeutic protein selected from an enzyme, an interleukin, a hormone, an antibody, a clotting factor, a growth factor, or a peptide; and / or (d) encode for an enzyme pathway.

[0011] The heterologous polynucleotide can also encode for a signal sequence selected from SEQ ID NO: 1-15. Furthermore, the heterologous polynucleotide can also be operatively associated with a heterologous promoter.

[0012] Examples of therapeutic proteins that can be expressed by the recombinant symbiont include prepro-insulin, GLP-1, GIP, or |3-Glucocerebrosidase.

[0013] Other examples of therapeutic proteins or therapeutic polynucleotides that can be expressed by the recombinant symbiont include, but are not limited to: (a) enzymes such as asparaginase, pancrelipase, collagenase Clostridium histolyticum, alglucosidase alfa, imiglucerase, velaglucerasealfa, taliglucerase alfa, laronidase, idursulfase, galsulfase, or pegloticase; (b) interleukins such as is selected from interleukin-2, interleukin-6, interleukin-4, interleukin-11, or interleukin- 12;; (c) hormones such as insulin, levothyroxine, epinephrine, glucagon, estrogen, progesterone, testosterone, human growth hormone, corticosteroids, desmopressin, parathyroid hormone, oxytocin, calcitonin, leuprolide, goserelin, octreotide, or thyrotropin alfa: (d) antibodies such as adalimumab, aflibercept, alemtuzumab, atezolizumab, basiliximab, belimumab, bevacizumab, blinatumomab, brentuximab vedotin, canakinumab, caplacizumab, certolizumab pegol, cetuximab, daratumumab, denosumab, dinutuximab, durvalumab, dupilumab, eculizumab, elotuzumab, emapalumab, gemtuzumab ozogamicin, golimumab, ibalizumab, inotuzumab ozogamicin, ipilimumab, ixekizumab, lanadelumab, luspatercept, mepolizumab, mogamulizumab, natalizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, ramucirumab, rituximab, sarilumab, secukinumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, reslizumab, tisagenlecleucel, brolucizumab, viltolarsen, idecabtagene vicleucel, dostarlimab, or satralizumab; (c) clotting factors such as Factor VIII, Factor IX, Factor Vila, Factor XIII, Fibrinogen (Factor I), Factor X, Factor XI, Factor XII, Von Willebrand Factor, Factor Vlll / von Willebrand Factor Complex, Prothrombin Complex Concentrate (PCC), Antithrombin III, Activated Prothrombin Complex Concentrate (aPCC), Factor Xllla, Factor Xlllb, Recombinant Factor VIII Fc Fusion Protein, Recombinant Factor IX Fc Fusion Protein, Emicizumab (Hemlibra) or Factor V Leiden; (f) growth factors such as Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Insulin-like Growth Factor-1 (IGF-1), Nerve Growth Factor (NGF), Platelet- Derived Growth Factor (PDGF), Transforming Growth Factor-Beta (TGF-P), Vascular Endothelial Growth Factor (VEGF), Keratinocyte Growth Factor (KGF), Bone Morphogenetic Proteins (BMPs), Hepatocyte Growth Factor (HGF), Erythropoietin (EPO), or Thrombopoietin (TPO); and / or(g) peptides such as abaloparatide, angiotensin II, bivalirudin, bremelanotide, buserelin, carbetocin, cetrorelix, desmopressin, exenatide, glatiramer acetate, goserelin, liraglutide, lixisenatide, nesiritide, oxytocin, pramlintide, semaglutide, setmelanotide, teriparatide, teduglutide, triptorelin, linaclotide, plecanatide, pasireotide, terlipressin, vasopressin, thymosin alpha- 1, sermorelin, leuprolide, lanreotide, tesamorelin, degarelix, plecanatide, bivalirudin.

[0014] Also contemplated are methods of expressing a heterologous polynucleotide in a multicellular host, comprising: administering to the host the recombinant symbiont as described herein and expressing the heterologous polynucleotide in the host.

[0015] Additionally, methods of preventing or treating a host suffering from a disease or disorder, comprising: administering to the host the recombinant symbiont as described herein; and expressing the heterologous polynucleotide to treat, reduce or prevent the symptoms of said disease or disorder. In these methods, the can further comprise translating the heterologous polynucleotide into the therapeutic polypeptide.

[0016] If necessary, the claimed methods can be reversed by the administration of an effective amount of antiparasitic or antibiotic compound, such as arterolane.

[0017] Diseases that can be treated as described herein include but are not limited to cancer, a genetic deficiency, an infectious disease, or an autoimmune disease. Specifically, the recombinant symbionts described herein can be used to treat diabetes, Gaucher’s Disease, and / or obesity. Additionally, the recombinant symbiont can be used to treat Cystic Fibrosis, Sickle Cell Disease, hemophilia, Duchenne Muscular Dystrophy, Huntington’s Disease, beta-thalassemia, macular degeneration, muscular atrophy, leber congenital amaurosis, severe combined immunodeficiency, immunodeficiency, retinitis pigmentosa, Fabry’s disease, Pompe’s disease, Wilson’s disease, orphan diseases, amyotrophic lateral sclerosis, alport syndrome, X-Linked adrenoleukodystrophy, phenylketonuria, Marfan’s Syndrome, or hereditary angioedema.

[0018] The methods could also be used to modify the host’s physiology and / or organ systems.

[0019] Delivery of the recombinant symbiont can be oral, intravenous, pulmonary, intramuscular, subcutaneous, or intraperitoneal routes.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are included for the purpose of exemplary illustration of various aspects of the present invention, and not for purposes of limiting the invention, wherein:

[0021] FIG. 1 shows exemplary approaches for transforming a nonpathogenic microorganism into a genetically engineered symbiotic microorganism.

[0022] FIG. 2A-D shows test data where T. rangeli is genetically engineered to produce prepro- insulin and introduced into a host, demonstrating tight glycemic control of blood glucose without the safety concern of hypoglycemia and hyperglycemia. Various symbionts secreting prepro-insulin were implanted into healthy mice to assess safety and tolerability. All animals tolerated implantation but also exhibited blood glucose level reduction and tight glycemic control without the risk of hypoglycemia or hyperglycemia in a 16 hour fasting glucose challenge.

[0023] FIG. 3 lists candidate genomic sites for integration that can be used to integrate the polynucleotide encoding a beneficial molecule into the genome of the symbiont.

[0024] FIG. 4 lists non-limiting families of signal peptides for use in the present invention.

[0025] FIG. 5 shows results from using recombinant symbiont expressing prepro-insulin in Non- Obese Diabetic (NOD) mice. Here, the recombinant symbiont engineered to produce prepro-insulin (SMI 8) delays the onset of Type I diabetes (TID) in a genetically susceptible host relative to the control organism engineered to express the irrelevant green-fluorescent protein (GFP; Control).

[0026] FIG. 6 shows ex vivo and in vivo GCase activity in GBA1 D409V homozygote mutant mice, the mutation in human Gaucher’s disease, after treatment with recombinant symbionts expressing GBA1 (the gene encoding for GCase). FIG. 6A indicates ex vivo GCase activity in triplicate wells after expression, secretion, and sequestration of enzyme in perfused livers from individual mice that were treated with control (mouse 557) or GBA1 -expressing (mouse 561 and 669) symbionts. FIG. 6B illustrates levels of 22-carbon chain ceramide (left panel) and sphingosine (middle panel) accumulation in the liver and 22-carbon chain ceramide in the brain (right panel) of D409V homozygote mutant mice upon treatment with control or GBA1 -expressing symbionts compared to untreated wild type mice. These data demonstrate that expression of GCase from a recombinant symbiont decreases the tissue accumulation of lipids associated with Gaucher’s disease.

[0027] FIG. 7 shows that use of a recombinant symbiont expressing GLP- 1 prevents weight gain.Symbionts expressing GLP-1 or its parent protein proglucagon were implanted into healthy mice on western diet chow (high fat diet). FIG. 7A shows serum levels of GLP-1 as measured by ELISA for GLP-1. FIG 7B shows percent weight gain across 50 days of observation. These data demonstrate that expression of GLP-1 from a recombinant symbiont results in weight loss as compared to control mice.

[0028] In the drawings and detailed description, the same or similar reference numbers may identify the same or similar elements. It will be appreciated that the implementations, features, etc. described with respect to embodiments in specific figures may be implemented with respect to other embodiments in other figures, unless expressly stated, or otherwise not possible.DETAILED DESCRIPTION OF THE INVENTION

[0029] The invention relates to in vivo generation of substances using genetically modified, nonpathogenic, microorganisms. The present disclosure describes the basic principles and technicalknow-how that a skilled artisan may need to consider to sufficiently design nonpathogenic microorganisms to be recombinant symbionts and serve as a biological carrier or device for the longterm and sustained production of beneficial molecules inside a host. Beneficial molecules may be missing, defective, or xeno proteins and polypeptides in the form of enzymes, interleukins, hormones, antibodies, clotting factors, soluble receptors, growth factors, or ligands. These molecules can be used to treat a disease or a disorder, such as for the treatment of cancer, genetic deficiencies, infectious diseases, or autoimmune diseases, as well as molecules of metabolic dominance or scavengers for toxicity and other physiology modifiers suitable within the scope of this invention. Beneficial molecules can also include polynucleotides, such as RNAi,.

[0030] As described herein, a delivery system, biological carrier system, or platform to alter the physiology and / or performance of a host is demonstrated. Furthermore, the system can be further modified with feedback loop features that enable recombinant symbiont to control the time and condition of the delivery of the beneficial molecules in response to physiological changes of the host. For instance, the endogenous low affinity glucose transporter channel loci, which is generally active during glucose abundance in the environment, can be engineered to conditionally secret insulin to mitigate the negative effects of uncontrolled high blood glucose levels in the host. As a result, similar loci can be used for feedback and the sensing of environmental changes in the host to coordinate beneficial molecule delivery. FIG. 3 lists non-limiting candidate genomic sites that can be utilized for either a sense and respond (i.e. time and condition) or continuous delivery of beneficial molecules to the host by a recombinant symbiotic microorganism.

[0031] The present invention may be used to domesticate microorganisms into recombinant symbiotic microorganisms. The definitions herein provide guidance to the subject matter expertise, the broadest application examples, and the appended claims.OVERVIEW

[0032] As used herein, a “host” is a multicellular organism with tissues, organs, and a circulatory system, such as a human, canines, felines, equines, live-stock (e.g., cattle, goats, sheep, or pigs), chicken or other birds, fish, or other animal, that is healthy or one that has compromised health that can be inhabited by a microorganism.

[0033] As used herein, the term “nonpathogenic microorganism” refers to a microorganism that is capable of inhabiting a host in order to ensure its survival or as part of its natural life cycle. One example of nonpathogenic microorganisms that can be used as described herein include subclasses ofTrypanosomes. These microorganisms are often found residing in the gut, blood, or tissues of hosts. Characteristics of such microorganisms include, but are not limited to: (1) avirulence and nonpathogenicity to host; (2) persistence and durability; (3) culturablility, engineerability, and tunability in the laboratory; and (4) universal immune compatibility via immune response evasion. As used herein, the term “symbiont” refers to a microorganism that inhabits a host without causing harm. A “recombinant symbiont” as used herein is a symbiont that has been engineered and specifically designed to benefit the host by producing molecules (i.e., polynucleotides and / or polypeptides) to enhance or modify the host’s physiology long-term (“beneficial molecules”) as described herein.

[0034] Thus, described herein is a method for adapting a non-pathogenic microorganism to become a “recombinant symbiotic microorganism” or “recombinant symbiont” for the sustained production of beneficial biomolecules in a host, comprising the steps of: (a) genetically modifying a non-pathogenic microorganism by integrating foreign nucleic acids, artificial chromosomes, or nucleic acid fragments containing specific genetic features for recombination and utilization of native genetic elements to produce a recombinant symbiont; (b) introducing the recombinant symbiont into a host; (c) allowing the recombinant symbiont to reside within the host without causing harm; and (d) enabling the recombinant symbiont to express the beneficial biomolecules (e.g., proteins and peptides) that modify the physiology of the host.

[0035] In embodiments, the recombinant symbiont has been engineered to produce an enzyme, an interleukin, an interferon, a hormone, an antibody, a clotting factor, a soluble receptor, a growth factor, a peptide or a ligand for the treatment of a disease or a disorder, such as a genetic deficiency or a neurologic, metabolic, endocrine, cancer, infectious or autoimmune disease. Additionally, the recombinant symbiont can be used to modify host physiology, homeostasis and organ systems.

[0036] The recombinant symbiont can be administered to the host via oral, intravenous, intrapulmonary, intramuscular, subcutaneous, or intraperitoneal routes.

[0037] Further embodiments include a method of generating biomolecules in a host, comprising: (a) genetically modifying a non-pathogenic microorganism by adding at least one foreign nucleic acid, artificial chromosome, or nucleic acid fragment to the genetic sequence of the non-pathogenic organism, and may include removing genetic material from the genetic sequence of the non- pathogenic microorganism; (b) introducing the genetically modified symbiont into the host; (c) the modified genetic sequence enabling the generation of biomolecules by the genetically modified symbiont in a host; and (d) allowing the genetically modified symbiont to reside within the host andgenerate biomolecules. Alternatively, the beneficial molecule can be encoded by an episome that has not integrated into the symbiont’s genome.

[0038] The non-pathogenic microorganism that is used to generate the recombinant symbiont is a Trypanosome. For example, the recombinant symbiont can be derived from a Trypanosoma range li (“T. rangeli”), Trypanosoma mesnilbrimontii, Trypanosoma preguici, Trypanosoma myrmecophague, Trypanosoma mycetae, Trypanosoma diasi, Trypanosoma cebus, Trypanosoma saimiri, or Trypanosoma advieri. In further embodiments, the T. rangeli used to create the recombinant symbiont can be derived from laboratory strains ATCC #30032 or ATCC #30033.

[0039] One benefit of using a Trypanosome to generate the recombinant symbiont is the reversibility of the methods described herein using antimalaria drags, such as Arterolane (OZ277). For example, if it is needed to stop further expression of the biological molecule in the host, the host can be given Arterolane which will eliminate Trypanosomes, including the recombinant symbionts.GENERAL DESIGN OF CONSTRUCT

[0040] The term “recombinant” or “transgenic” symbiont refers to a symbiont whose genetic material has been modified to encode a beneficial molecule. The beneficial molecule can be encoded by the recombinant symbiont from an episome or a polynucleotide that has been integrated into the symbiont’s genome. When incorporating into the symbiont’s genome, natural genetic sequences may be subtracted during the addition of the foreign sequences that render the desired beneficial features or phenotypes.

[0041] Methods of adding or subtracting sequences to modify the symbiont’s natural features, which enable recombinant symbionts to have unique features engineered in the laboratory not present in nature, can involve homologous recombination or addition of artificial chromosomes engineered in the laboratory as illustrated in FIG. 1 and is well within the knowledge of the art. As used herein, the term “homologous end joining or homologous recombination” refers to methods that may be used whereby specific sites in the genome of a symbiont are targeted by nucleotide fragments of foreign nucleotide sequences flanked by natural complementary nucleotide sequence native to the symbiont. Homologous recombination may be performed using various techniques known in the art, see, e.g., Current Protocols in Molecular Biology (1994) Greene Publishing Associates and John Wiley & Sons, NY.

[0042] For example, Trypanosoma rangeli ("T. rangeli”) may be engineered to stably integrate foreign nucleic acids, artificial chromosomes, or linear or circular nucleic acid fragments using atargeted or untargeted genomic integration system. Additionally, the beneficial molecule can be expressed from a stable episomal polynucleotide. The heterologous polynucleotides encoding the beneficial molecule may contain specific genetic elements for constitutive, inducible, or targeted expression of the beneficial molecule. Alternatively, the beneficial molecule can be expressed from a polynucleotide that has integrated into the recombinant symbiont’s genome.

[0043] Genetic modifications contemplated in this invention renders the transgenic symbiont (e.g., recombinant T. rangeli) distinct from its naturally-occurring counterpart metabolically, reproductively, immunologically, or in its tissue specificity (tropism) as directed by the purpose of the engineering to enhance host physiology and also render a selectable feature that enables enrichment. In various embodiments, the foreign genetic sequences may be used to produce fluorescent color or render metabolic advantage against toxic chemicals like antibiotics to positively identify symbionts with a successfully engineered feature. A non-limiting list of symbiont features that result from engineering foreign DNA sequences include the expression of (1) RNAs, (2) proteins, or (3) polypeptides that lead to free metabolites (e.g. carbohydrates, lipids, amino acids, steroids, fatty acids, vitamins, anti-infectious agents) as an end-product that modify host physiology.

[0044] As used herein, the term “protein or pathway” refers to amino acid sequences coding a functional polypeptide as a single unit (i.e. protein) or series of units that process or render one or more specific biological reactions (i.e. pathway). These polypeptides may be designed by introducing nucleic acid sequences into the starting symbiont using standard recombinant methods, such as disclosed in Sambrook et al., 2001.

[0045] For instance, the conversion of cholesterol to testosterone involves 4 enzymes CYP11A1>CYP17A1>3|3-HSD>17|3-HSD in 5 steps. Thus, the expression of such enzymes in a single symbiont can utilize host cholesterol as feed to generate testosterone for the host.

[0046] As used herein, the term “native drivers” refers to endogenous regulatory elements allowing for the targeting at genomic integration sites located in the symbiont’s genome. These regions are known to regulate the expression of specific sequences, proteins, and / or pathways. In certain embodiments of the invention, these natural drivers may be targeted as preferred sites for homologous recombination to render specific sense and response features in the transgenic symbiont. A non-limiting list of candidate sites with variable exploitable features and chromatin activity may be found in FIG. 3. These locations are ideal for the insertion of a heterologous polynucleotide encoding a beneficial molecule as described herein as the transgene can be expressed at high, moderate or reduced levels depending on site of integration.

[0047] FIG. 1A illustrates a representative construct designed to express a secreted polypeptide using a natural driver. It is specifically contemplated that a strong promoter, such as for example, the early or late promoters of SV40, CMV, vaccinia, polyoma, adenovirus, herpes virus and other sequences known to control the expression of genes of multicellular cells can also be used instead of a natural driver.

[0048] A coding sequence is "under the control of’ or "operatively associated with" an expression control sequence when a signal for a RNA polymerase to transcribe the coding sequence into RNA, particularly mRNA, which is then spliced (if it contains introns) and translated into the polypeptide encoded by the coding sequence.METHODS OF TREATMENT

[0049] Methods of treating using the recombinant symbiont are also disclosed. For example, a method of treating a host suffering from diabetes comprises (a) genetically modifying T. rangeli by introducing a linear or circular DNA sequence coding into the genetic sequence of the T. rangeli, so as the T. rangeli will generate prepro-insulin in a host; (b) introducing the genetically modified T. rangeli into the host; (c) allowing the genetically modified T. rangeli to reside within the host without causing harm; and (d) enabling the genetically modified T. rangeli to generate prepro-insulin in the host.

[0050] The transgenic symbiont may be designed to be able to sense changes in the host and respond by supplying a substance to counter the undesired changes in the host or enhance host performance. An example of such a sense-and-response system is the sensing of a high concentration of glucose in the blood of the host by the transgenic symbiont via endogenous glucose transporter and the subsequent production of potent insulin to mitigate undesired effects of high glucose in the host. Alternatively, a transgenic symbiont can be designed to sense lower levels of hormones like vessopressin to improve performance and allow for adaptation to austere environments.

[0051] The present invention may be used to deliver a sustained supply of a diverse set of molecules both with therapeutic value and for enhancement of physiology, that are not available using naturally occurring microorganisms. It will be understood that the present art can be used for applications in any host as described herein. The skilled artisan will also realize the present invention may be used to prepare devices for sustained delivery and in vivo manufacturing of a therapeutically significant substance per illustration in FIG. 1.

[0052] For example, the recombinant symbionts can be used to treat Lysosomal Storage Disease. It is anticipated that the present invention will permit the treatment and / or amelioration of lysosomal storage diseases currently managed by enzyme replacement therapy. A common feature of lysosomal diseases is the accumulation of unprocessed cellular products from the lysosome, which leak out from cells in the form of sugars and lipids into the tissues and blood stream causing complicated symptoms. These cellular products are currently treated with exogenous supplementation of enzymes in order to eliminate the cellular products that mediate disease.

[0053] Similarly, recombinant symbionts can be used for hormone replacement and health augmentation. For example, the recombinant symbionts are expected to permit the continued production and delivery of hormones and their precursors for replacement in cases of missing biomolecules or the augmentation of proteins beyond normal levels to enable host to heal, tolerate, manage pain, or optimize desired performance and fitness goals. Thus, the present invention contemplates the use of recombinant symbionts to optimize human performance and resilience.

[0054] As demonstrated herein, glucose homeostasis management in diabetes can also be controlled using the recombinant symbionts described herein. FIG. 2 illustrates a representative scenario where T. rangeli was genetically engineered to produce prepro-insulin, an insulin precursor.

[0055] The transgenic T. rangeli was introduced via a single dose to laboratory mice. The transgenic T. rangeli produced sufficient prepro-insulin to manage a lower and tight glucose homeostasis surprisingly for 70 days as shown in FIG. 2A for two different test samples, SM18 (SEQ ID NO: 11) & SM22 (SEQ ID NO: 12). In addition, the levels persisted during times of fasting, as shown in FIG. 2B, suggesting the risk of hypoglycemia is also manageable.

[0056] Test samples SM18 (SEQ ID NO: 11) & SM22 (SEQ ID NO: 12) also exhibited somewhat higher and faster glucose clearance from the blood in glucose tolerance assay as shown in FIG. 2C. The onset of Type 1 diabetes in test sample SMI 8 (SEQ ID NO: 11) was also delayed relative to the control sample as shown in FIG. 5. Thus, a transgenic symbiont may be genetically engineered to produce prepro-insulin in vivo that has the potential for therapeutic value to patients.

[0057] The recombinant symbionts described herein can also be used in neurological applications . For example, the present invention may enable the treatment, amelioration, and / or augmentation of neurological conditions that currently have limited interventions. Transgenic symbionts are able to deliver a sustainable supply of beneficial molecules that could permeate through the blood brain barrier with the therapeutic benefit of reducing disease causing physiology or augmenting neuronal functions and proliferation. Restoring the loss of function of neuronal cells or removing thepathological accumulation of molecules that lead to neurological disorders can be achieved by administering symbionts. The present invention also contemplates the delivery device to be designed to mitigate the effects of psychological disorders that have deregulated neurotransmitters by expressing or scavenging these neurotransmitters.

[0058] The recombinant symbionts described herein can also be used in immunomodulation, such as to treat autoimmune diseases, allergy, vaccination, and / or cancer. It is expected that the present invention will permit the treatment of autoimmune diseases, sensitization of subjects to allergens, and delivery of biomolecules for the treatment of cancer. A common feature of autoimmune and allergic diseases is the over activation of immune responses and immune cells to harmless antigens. Here, the recombinant symbionts can deliver a sustained supply of an agent that can modulate immune responses in the form of anti-inflammatory molecules or agents that limit cellular migration or activation. The recombinant symbiont can deliver low doses of an allergen to sensitize the subject to a molecule and avoid the onset of an allergic response. Furthermore, the recombinant symbionts can be used for continued delivery of proteins for vaccination strategics. In a similar fashion, the recombinant symbionts can also be designed to express molecules with anti-tumor activity to permit the treatment, management, and / or amelioration of cancer. Current efforts to exploit common features of cancers such as evading growth suppression, avoiding immune destruction, resisting cell death, and deregulating cellular energetics have been successful at effectively controlling the disease. The recombinant symbionts described herein can deliver a sustained supply of therapeutic molecules to not only treat the disease but also to survey, monitor, and / or diagnose based on the expanding hallmarks of cancer.

[0059] As stated above, the recombinant symbionts described herein contemplate the broad application as a delivery system for the continued supply of proteins and pathways in hosts. In addition to transgenic symbionts designed to continually express a gene or genes of interest, the recombinant symbionts can also be designed to sense and autoregulate the expression of the desired molecules in response to host stimuli. Recombinant symbionts may also be designed to interact with the host through receptor-ligands, home to specific tissues, and perform enzyme-substrate dynamic regulation.

[0060] Once generated, the recombinant symbiont can be used to achieve pharmacokinetic and pharmacodynamic objectives for molecules continuously supplied to a host by transgenic symbionts. The methods listed below are non-limiting but rather examples of strategies to optimize the effectiveness of the symbiont as a delivery device in altering host physiology. A longer half-life oftherapeutic substances produced by symbionts may be achieved by several methods including increasing the production rate of therapeutic substances without compromising the stability and effectiveness of the transgenic symbiont. Alternatively, hyperstable designs of cargo molecules can be employed to achieve specific pharmacokinetic and pharmacodynamic objectives in the host.

[0061] A third strategy involves using “variable immunogenicity control” whereby immune regulators of the host are engineered into the symbiont to render the symbiont more stable and less susceptible to common threats from the host. Another option involves designing tissue homing signatures. Some species related to symbionts naturally exhibit tissue specificity, which can be engineered into the symbiont of choice. Thus, the ease of which the addition or subtraction of foreign nucleotide sequences enables anyone with ordinary skill in the art to generate a well-optimized delivery device. Alternatively, the beneficial molecule can be expressed by an episome.DELIVERY AND FORMULATION OF RECOMBINANT SYMBIONTS

[0062] The method of transgenic symbiont delivery may be achieved by standard techniques that are well-known in the art. This includes but is not limited to, administration of symbionts to hosts via intramuscular, subcutaneous, pulmonary, or intravenous injection as well as other parenteral, enteral, or dermal routes suitable for customized applications.

[0063] The recombinant symbionts can be formulated with ingredients compatible with pharmacy and microbiology applications and procedures. For example, the formulations of the present invention may combine a sterile recombinant symbiont in a suitable carrier solution such as, but not limited to, saline, glycols, oils, gels, or hydrogenated naphthalenes that are not harmful to the recombinant symbiont or the host. The present invention contemplates a topical, intravenous, subcutaneous, or intraperitoneal route of administration. The number or effective concentration of recombinant symbiont to be delivered will vary depending upon a number of factors including indication of use, host species, route of administration, extent of disease, relative biological activity of molecule being delivered, and the overall health status of the host.EQUIVALENTS

[0064] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than limiting to the invention described herein.

[0065] Some implementations may be described herein in connection with thresholds. As used herein, satisfying a threshold may refer to a value being greater than the threshold, more than the threshold, higher than the threshold, greater than or equal to the threshold, less than the threshold, fewer than the threshold, lower than the threshold, less than or equal to the threshold, equal to the threshold, etc.

[0066] The foregoing disclosure provides examples, illustrations and descriptions of the present invention, but is not intended to be exhaustive or to limit the implementations to the precise form disclosed. Modifications and variations are possible in light of the above disclosure or may be acquired from practice of the implementations. These and other variations and modifications of the present invention are possible and contemplated, and it is intended that the foregoing specification and the following claims cover such modifications and variations.

[0067] Even though particular combinations of features are recited in the claims and / or disclosed in the specification, these combinations are not intended to limit the disclosure of possible implementations. In fact, many of these features may be combined in ways not specifically recited in the claims and / or disclosed in the specification. Although each dependent claim listed below may directly depend on only one claim, the disclosure of possible implementations includes each dependent claim in combination with every other claim in the claim set.

[0068] No element, act, or instruction used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles “a” and “an” are intended to include one or more items and may be used interchangeably with “one or more.” Furthermore, as used herein, the term “set” is intended to include one or more items and may be used interchangeably with “one or more.” Where only one item is intended, the term “one” or similar language is used. Also, as used herein, the terms “has,” “have,” “having,” or the like are intended to be open-ended terms. Further, the phrase “based on” is intended to mean “based, at least in part, on” unless explicitly stated otherwise.

[0069] The presence or absence of a summary, abstract, or claims in this application should in no way be considered as limiting on the scope of any inventions disclosed herein.EXAMPLES

[0070] Practice of the invention is more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.Example 1: General Construction and Administration of Recombinant Symbionts

[0071] As depicted in FIG. 1, various methods and tools may be used to incorporate a polynucleotide encoding a beneficial molecule into symbiont. In this example, T rangeli was used to generate the recombinant symbiont. FIG. 1A depicts a close-up of a representative gene segment being added to an organism gene. HEJ is short for Homologous End Joining, ETS for External transcribed spacer, ITS for Internal transcribed spacer. In this example, a native driver to the symbiont that includes both a native promoter and a native signal peptide was used to ensure secretion of pay load. However, other strong promoters can also be used interchangeably.

[0072] Once constructed, electroporation was used to modify the genetic composition of T. rangeli permanently using a random integration with homologous end joining. FIG. IB depicts the transgenic symbiont being introduced in a lab mammal.

[0073] FIG. 3 provides a non-limiting list of candidate sites with variable chromatin state for expression modulation for use in the present invention to control timing and condition of the delivered beneficial molecule to achieve genetic modification by incorporation into episomal or site directed integration of polynucleotides. For example, these sites were considered when a laboratory derived T. rangeli was used to create the recombinant symbiont. Native signal peptides selected from proteins naturally encoded by T. rangeli were incorporated into the expression cassette with variable prepro- insulin production as measured over 24hrs.

[0074] FIG. 4 provides a non-limiting list of families of native signal peptides that can be used to facilitate secretion.

[0075] To create a recombinant symbiont, circular DNA can be introduced into a non-pathogenic organism, such as T. rangeli, usings methods including the Amaxa™ Nucleofector™ system by Lonza Biosciences parasite or T cell nucleofector kits with U-033, X-001 or D-023 programs. The molecular cloning methods and recombinant T. rangeli generation was performed using recombinant methods familiar to those in the art as disclosed in Sambrook et al., (2001) Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, NY (“Sambrook et al., 2001”).

[0076] Clones incorporating the beneficial molecule in the T. rangeli genome were then selected using antibiotic resistance and confirmed for expression and secretion of molecules of interest in vitro before implantation into subjects.

[0077] A simple and inexpensive method for culturing the recombinant symbiont was employed. A T. rangeli derivative Tejara El cells cultured in LIT (Liver infusion tryptose) supplemented with 10% (v / v) fetal bovine serum was performed as described in Koerich, LB el al. (2002) Differentiation of Trypanosoma rangeli'. high production of infective trypanomastigote forms in vitro. Parasitol Res 88: 21-25. The T. rangeli strains that can be used to derive the recombinant symbiont include ATCC #30032 and ATCC #30033.

[0078] The cells were incubated at 28°C in an incubator with access to room temperature circulating oxygen. The cells were enumerated using a manual hemocytometer in normal culture medium. The cell growth rate was optimized and determined for transfection protocol.

[0079] One method of manipulating DNA content of the symbiont is by amplifying or synthesizing long DNA fragments comprised of foreign DNA sequences containing expression modifiers such signal peptides, beneficial features (e.g., enzymes and receptors) as well as a selectable features flanked by recombination target sequences specific to T. rangeli for the purpose of preferredsite specific integration. For example, FIG. 3 discloses a non-limiting list of genomic sites selected for specific features and the flanking sequences to guide the insertion of DNA fragments at these sites. In order to confirm that fragments retained the desired functional elements of value (i.e. enzyme or receptor proteins), a minimal T7 promoter can be added upstream of the fragment during synthesis for confirmation by amplification.

[0080] Large fragments can be introduced into T rangeli by using published trypanosome specific reagents and kits for the Amaxa™ Biosystems Nucleofection™ technology platform. Enrichment of symbiont T. rangeli with the desired incorporation of DNA sequences may be performed using a neomycin resistance based antibiotic selection method over four weeks. Secreted protein and peptides are monitored by suitable assays (i.e. enzyme activity, westerns, ELISA, etc.). The skilled artisan will appreciate that these ordinary molecular biology techniques and methods are common art and the example here is not to be interpreted as limiting.

[0081] Recombinant symbionts can be further expanded to larger numbers and inoculated into naive mice or other suitable host intravenously for the purposes of testing. For example, weekly venous blood draw can be performed to monitor glucose homeostasis. However, the skilled artisanappreciates that various designs of the recombinant symbionts will require suitable assays to monitor various physiological endpoints.Example 2: Treatment of Diabetes Using Recombinant Symbiont

[0082] Insulin stands as a cornerstone in endocrinology, for having been the inaugural peptide hormone identified in the study hormones. Originating as a prepro-insulin polypeptide of 110 amino acids, it encompasses an A-chain of twenty-one amino acids, a B-chain comprising thirty amino acids, a C-chain of thirty-five amino acids, and a distinct signal peptide. The maturation of prepro-insulin is intricate, necessitating multiple enzymatic processes to finally yield the insulin hormone. The first pivotal step occurs within the lumen of the rough endoplasmic reticulum, where a signal peptidase excises the signal peptide, culminating in the formation of proinsulin. Subsequently, an endoprotease enzyme reminiscent of trypsin further refines proinsulin by severing the C-chain, an intermediary peptide linking the A and B chains, typically ranging between 30-35 amino acids in length. The final protein processing is overseen by an exopeptidase carboxypcptidasc B, which meticulously removes any residual basic amino acids after the C-chain's excision. Prepro-insulin holds profound implications in the realm of immunology. It is instrumental in fostering self-tolerance in individuals devoid of autoimmune conditions, highlighting its therapeutic potential. Specifically, this potential can be attributed to immunological pathways that either purge reactive cells or spawn natural regulatory T cells, effectuating tolerance via sustained prepro-insulin exposure.

[0083] The therapeutic efficacy of prepro-insulin expressed from a recombinant symbiont was assessed for activity. We created a recombinant symbiont as described in Example 1 to express prepro-insulin while also testing seven distinct signal peptides, intending to modulate the secretion rate of the hormone in test subjects.

[0084] FIG. 1C shows data from in vitro data from seven different T. rangeli cultures that have been modified to secrete prepro-insulin as described herein. Naturally occurring T. rangeli is not shown as it does not secrete prepro-insulin. In these experiments, the following procedure and gene segments were used: Murine prepro-insulin (Genbank Accession No. X04725) was flanked by alpha- tublin sequences for Homologous End Joining (HEJ) in such a way that the native alpha-tublin promoter was able to drive expression. Random nucleic acid sequences were integrated externally and internally (ETS & ITS) to preserve translation frame.

[0085] Intriguingly, in vitro assays showcased that four out of the seven signal peptides augmented secretion, with signal peptides SM14 (SEQ ID NO:9), SM18 (SEQ ID NO: 11), and SM22(SEQ ID NO: 12) emerging as the most effective, and signal peptide SMOO (SEQ ID NO:4) promoting intermediate secretion (FIG. 1C).

[0086] For a safety and tolerability study of symbionts with a therapeutic payload, healthy wild type mice were implanted (IV injections) with symbionts SMOO (SEQ ID NO:4), SM14 (SEQ ID NO:9), SM18 (SEQ ID NO: 11), SM22 (SEQ ID NO: 12) expressing prepro-insulin at 13 weeks of age (n=8). Interestingly, animals implanted with SM22 (SEQ ID NO: 12) & SM14 (SEQ ID NO:9), the signal peptides that promoted secretion of the highest levels of prepro-insulin consistently exhibited lower blood glucose levels compared to control animals implanted with GFP-expressing symbionts (FIG. 2). Over the following weeks SM22 (SEQ ID NO:12) and SM18 (SEQ ID NO: 11) exhibited Tight Glycemic Control (TGC) while SMOO (SEQ ID NO:4) and SM14 (SEQ ID NO:9) had effects similar to control symbionts. Over the 10-week observation period, it was obvious that SM18 (SEQ ID NO: 11) and SM22 (SEQ ID NO: 12) had statistically significant TGC (p<0.05) consistently, with SM22 (SEQ ID NO: 12) being the most profound. To probe the safety and efficacy of these symbionts with a therapeutic payload of the first kind, wild type mice were implanted with these symbionts at 13 weeks of age (n=8 / group). This outcome was encouraging, with no hypoglycemia documented across all 4 experimental groups and control suggesting the system is unperturbed by symbionts of various therapeutic load. Additionally, in assessing the host's physiological response to hyperglycemia, glucose tolerance assays with SM18 (SEQ ID NO: 11) and SM22 (SEQ ID NO: 12) were executed. The results indicated an enhanced blood glucose clearance rate in subjects treated with SM18 (SEQ ID NO: 11) and SM22 (SEQ ID NO: 12), signifying an augmented capacity (i.e., increased efficiency) to metabolize substantial glucose loads.

[0087] Given the successful safety and tolerability of prepro-insulin production by the recombinant symbionts, we next evaluated the impact of prepro-insulin to overcome the beta-cell loss (i.e. immune-mediated destruction of the insulin-secreting cells of the pancreas) in Non-Obese Diabetic (NOD) mice, a spontaneous model of Type I diabetes. SM18 (SEQ ID NO: 11) expressing symbionts were transferred to NOD mice groups around 19 weeks of age. SM18 (SEQ ID NO: 11) treated mice exhibited a significantly delayed onset of Type I diabetes compared to the GFP-symbiont treated control group (FIG. 5). Here, one can speculate that the therapeutic effects of symbiont- delivered prepro-insulin are two-fold: 1) providing an insulin precursor that is processed into active insulin; and 2) delivering a molecule that regulates mechanisms of self-tolerance to pancreas antigens.

[0088] As shown herein, expressing a beneficial molecule, such as insulin, GLP-1, or GIP, from the recombinant symbiont allows for the treatment of diabetes or the introduction of insulin by theperiodic, semi-continuous, or continuous injection or generation of prepro-insulin or GLP-1 or GIP in the host. Current practice utilizes frequent administration of processed “modified and mature” insulin or GLP-1 or GIP either as a daily or weekly injectable. The injection of prepro-insulin into muscle or fat tissue has been generally found to be ineffective for treating diabetes. However, the inventors have found that the use of recombinant symbionts to manufacture and secrete prepro-insulin continuously or semicontinuously in the host system exhibits unique therapeutic properties as shown in FIG. 2 demonstrating usefulness in treating diabetes. The present invention may be useful for the delivery of precursor compounds that the body can process to generate compounds useful in the treatment of other conditions.Example 3: Treatment of Gaucher’s Disease Using Recombinant Symbiont - Example of a Large Payload

[0089] Mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCasc), have been identified as pivotal contributors to the lysosomal storage disorder GD. Furthermore, these mutations are also implicated in Parkinson’s disease (PD). Over three hundred distinct GB A 1 mutations have been documented, and they collectively stand as the most potent known genetic determinants for developing GD and idiopathic PD. GCase plays a vital role in lysosomal glycolipid metabolism. A deficiency in its activity culminates in the buildup of specific glycosphingolipid (GSL) substrates, notably glucocermide (GlcCer) and glucosphingosine (GlcSph). These accumulated GSLs are pathognomonic markers for GD. Most rodent models for GD are based on the GBA1 gene knockout or feature the GBA1 D409V point mutation. The latter is especially significant as it precipitates a dramatic decrease in GCase enzymatic function and a subsequent accumulation of certain GSL substrates in target organs like spleen and brain. The GBA1 D409V KI mouse model exhibits pronounced GCase activity reduction and GSL accretion in peripheral organs, making it an invaluable tool for studying interventions for GD and PD. By the age of 3 months, these mutant mice exhibit physiological defects in the form of accumulation of lipids in select organs, including the brain and liver, with cognitive deficits that intensify over the following year.

[0090] This example demonstrates platform's capability of delivering large payloads (>50 kDa) and evaluates the efficacy of symbiont-delivered GCase on the accumulation of GlcCer and GlcSph in the liver and brain of mice with GBA1 deficiency. To this end, D409V heterozygote breeding pairs were obtained in order to obtain homozygous GBA1 D409V mutants for therapeutic efficacy studies.Concurrently, efforts were directed to the design, selection, and laboratory- scale production of a chassis proficient in GCase secretion.

[0091] The autosomal recessive nature of GBA1 mutations required sustained breeding for ample duration to ensure the availability of a sufficient number of eligible animals for the study. The breeding pairs obtained yielded homozygote mutant offsprings at a rate below Mendelian expectations, demanding more time to accrue the requisite numbers for the intended research. Consequently, continuous enrollment of eligible animals in matched pairs was deemed necessary to ensure age and disease status consistency across experiments.

[0092] Moreover, upon detecting significant sequence differences between the human and murine GBA1, we formulated the murine GBA1 (GenBank Accession No. M24119.1) under the highly active signal peptide 22 (SEQ ID NO: 12), yielding the prototype SM22 (SEQ ID NO: 12) mGBA (referred to GBA in our studies). In vitro tests using the 4MU- Beta-glucosidase assay (standard GCase activity assay) demonstrated significant enzymatic activity of symbiont- secreted GCase.

[0093] Upon securing enough D409V homozygotc mutant subjects, 8-wcck-old mice were administered with either a GFP expressing symbiont (as a negative control) or the SM22 (SEQ ID NO: 12) mGBA expressing symbiont. Four weeks post-implantation, GCase activity in the liver was examined ex vivo, serving as an initial treatment quality control prior to lipidomics studies. As depicted in FIG. 6A, higher but variable GCase activity in perfused livers was detected in SM22 (SEQ ID NO: 12) mGBA implanted subjects 561 and 669 compared to the GFP control implant, suggesting the enzyme replacement strategy may be an effective therapeutic to mitigate the accrual of ceramide and sphingosine species in affected organs.

[0094] Lipid accumulation was then assessed in the liver and brain by mass spectrometry (FIG. 6B- 6C). Notably, lipidomics for ceramide and sphingosine lipids revealed a statistically significant reduction of several species of lipids including large lipids like the 22-carbon chain Glucosylceramide C22-GlcCer and total Glucosylsphingosine GlcSph in mice implanted with GBA-expressing T. rangeli compared to control GFP-symbiont treated mice. Far more striking is the reduction of the same species of small amount of lipids accumulated in the brain, which normally take an exceptionally long time (up to 1 year) to manifest, suggesting this method of delivery may also suitable for delivering neurological therapies (FIG. 6D).Example 4 - Treatment of Obesity using a Recombinant Symbiont - Lasting Expression

[0095] GLP-1 drugs (i.e., Ozempic™, Wagovy™, Trulicity™, Mounjaro™ and the recently approved Zepbound™ injection by Eli Lilly) have become cornerstone therapies for Type II diabetes (T2D), offering multiple benefits, including weight management and cardiovascular benefits. Although advancements in GLP-1 drugs, including GIP, have successfully transitioned from daily to weekly dosing, patient compliance remains a significant issue. One nuanced challenge in GLP-1 therapy is maintaining consistent but low levels of bioavailable drug, often achieved by administering a stable, high dose of GLP-I once a week. Regrettably, this regimen can induce severe side effects, including nausea and vomiting after injections or gut paralysis from extended use, often leading to poor treatment adherence.

[0096] We surprisingly found that use of a recombinant symbiont produced a remarkable three-month sustained release of GLP-1. Use of a recombinant symbiont described herein expressing GLP-1 has the dual advantage of eliminating the side effects associated with current therapies while dramatically reducing the frequency of high-dose injections from weekly to quarterly or even yearly, thus facilitating effortless integration into various lifestyles. Given the biology of GLP-1, which is the breakdown product of proglucagon, prototype symbionts were designed to express proglucagon or GLP-1.

[0097] In this example, we operatively associated a polynucleotide encoding proglucagon and GLP- 1, both derived from GenBank Accession No. Z46845.1 under the highly active signal peptide 22 (SM22 - SEQ ID NO: 12) as described in Example 1. Commercially available ELISA assays were used both for the in vitro and ex vivo testing of proglucagon and GLP1 secretion into growth medium or serum.

[0098] In this experiment, healthy mice were implanted with GFP, proglucagon, or GLP-1 expressing symbionts and feed a high-fat diet (Western diet model chow) to facilitate the gain of weight and evaluate the effect of the recombinant symbionts on weight gain. We also confirmed the secretion of target pay load on day 21, 3 weeks after implantation, in the serum of all mice.

[0099] Interestingly, as shown in FIG. 7, mice implanted with GLP-1 secreting symbiont exhibited reduced weight gain by nearly 40% as compared to the controls or the GLP-1 parent protein proglucagon. Furthermore, the average serum concentration of GLP-1 in these mice was on average less than control payload consistent with the biology of this molecule (i.e. higher levels of availability of the protein means higher amounts of it is used by the body). Interestingly the parent protein proglucagon symbionts exhibited a higher GLP-1 concentration in the blood with normal weight gain indicating the GLP-1 ELISA used here is unable to distinguish between the parent proteinproglucagon and GLP-1. Furthermore, this also indicates the accumulation of unprocessed proglucagon in the blood stream from the symbionts, which was confirmed by ELISA for proglucagon itself, indicating that in the event of a stable protein secretion, the protein accumulates in the blood. This suggests that hyperstable design of payload can improve therapeutic outcome in hosts.

[0100] While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0101] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A recombinant symbiont comprising a Trypanosome microorganism engineered to express a heterologous polynucleotide, wherein said heterologous polynucleotide encodes for a therapeutic protein or a therapeutic polynucleotide.

2. The recombinant symbiont of claim 1, wherein the Trypanosome is Trypanosoma rangeli, Trypanosoma mesnilbrimontii, Trypanosoma preguici, Trypanosoma myrmecophague, Trypanosoma mycetae, Trypanosoma diasi, Trypanosoma cebus, Trypanosoma saimiri, or Trypanosoma advieri.

3. The recombinant symbiont of claim 2, wherein the Trypanosome is Trypanosoma rangeli.

4. The recombinant symbiont of any one of claims 1-3, wherein the heterologous polynucleotide encodes prepro-insulin.

5. The recombinant symbiont of any one of claims 1-3, wherein the heterologous polynucleotide encodes GLP-1.

6. The recombinant symbiont of any one of claims 1-3, wherein the heterologous polynucleotide encodes GIP.

7. The recombinant symbiont of any one of claims 1-3, wherein the heterologous polynucleotide encodes P-Glucocerebrosidase.

8. The recombinant symbiont of any one of claims 1-3, wherein the heterologous polynucleotide: a. is an integrated foreign nucleic acid, artificial chromosome, or a nucleic acid fragment containing specific genetic features for recombination and utilization of native genetic elements;b. encodes for a ribonucleic acid, including, but not limited to, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, guide RNA, microRNA (miRNA), small interfering RNA (siRNA), and cell-free RNA (cfRNA); c. encodes for a therapeutic protein selected from an enzyme, an interleukin, a hormone, an antibody, a clotting factor, a growth factor, or a peptide; and / or d. encodes for an enzyme pathway.

9. The recombinant symbiont of any one of the previous claims, wherein the heterologous polynucleotide encodes for a signal sequence selected from SEQ ID NO: 1-15.

10. The recombinant symbiont of any one of the previous claims wherein the heterologous polynucleotide is operatively associated with a heterologous promoter.

11. The recombinant symbiont of any one of the previous claims, wherein: a. the enzyme is selected from asparaginase, pancrelipase, collagenase Clostridium histolyticum, alglucosidase alfa, imiglucerase, velaglucerase alfa, taliglucerase alfa, laronidase, idursulfase, galsulfase, or pegloticase; b. the interleukin is selected from interleukin-2, interleukin-6, interleukin-4, interleukin- 11, or interleukin- 12; c. the hormone is selected from insulin, levothyroxine, epinephrine, glucagon, estrogen, progesterone, testosterone, human growth hormone, corticosteroids, desmopressin, parathyroid hormone, oxytocin, calcitonin, leuprolide, goserelin, octreotide, or thyrotropin alfa: d. the antibody is selected from adalimumab, aflibercept, alemtuzumab, atezolizumab, basiliximab, belimumab, bevacizumab, blinatumomab, brentuximab vedotin, canakinumab, caplacizumab, certolizumab pegol, cetuximab, daratumumab, denosumab, dinutuximab, durvalumab, dupilumab, eculizumab, elotuzumab, emapalumab, gemtuzumab ozogamicin, golimumab, ibalizumab, inotuzumab ozogamicin, ipilimumab, ixekizumab, lanadelumab, luspatercept, mepolizumab, mogamulizumab, natalizumab, necitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, ramucirumab,rituximab, sarilumab, secukinumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, reslizumab, tisagenlecleucel, brolucizumab, viltolarsen, idecabtagene vicleucel, dostarlimab, or satralizumab; e. the clotting factor is selected from Factor VIII, Factor IX, Factor Vila, Factor XIII, Fibrinogen (Factor I), Factor X, Factor XI, Factor XII, Von Willebrand Factor, Factor VIII / von Willebrand Factor Complex, Prothrombin Complex Concentrate (PCC), Antithrombin III, Activated Prothrombin Complex Concentrate (aPCC), Factor Xllla, Factor Xlllb, Recombinant Factor VIII Fc Fusion Protein, Recombinant Factor IX Fc Fusion Protein, Emicizumab (Hemlibra) or Factor V Leiden; f. the growth factor is selected from Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte- Macrophage Colony-Stimulating Factor (GM-CSF), Insulin-like Growth Factor- 1 (IGF-1), Nerve Growth Factor (NGF), Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor-Beta (TGF-0), Vascular Endothelial Growth Factor (VEGF), Keratinocyte Growth Factor (KGF), Bone Morphogenetic Proteins (BMPs), Hepatocyte Growth Factor (HGF), Erythropoietin (EPO), or Thrombopoietin (TPO); and / or g. the peptide is selected from abaloparatidc. angiotensin II, bivalirudin, bremelanotide, buserelin, carbetocin, cetrorelix, desmopressin, exenatide, glatiramer acetate, goserelin, liraglutide, lixisenatide, nesiritide, oxytocin, pramlintide, semaglutide, setmelanotide, teriparatide, teduglutide, triptorelin, linaclotide, plecanatide, pasireotide, terlipressin, vasopressin, thymosin alpha- 1, sermorelin, leuprolide, lanreotide, tesamorelin, dcgarclix, plecanatide, bivalirudin.

12. A method of expressing a heterologous polynucleotide in a host, comprising: a. administering to the host the recombinant symbiont of any one of claims 1-11; and b. expressing the heterologous polynucleotide in the host.

13. A method of prevent or treating a host suffering from a disease or disorder, comprising: a. administering to the host the recombinant symbiont of any one of claims 1-11; and b. expressing the heterologous polynucleotide to treat, reduce or prevent the symptoms of said disease or disorder.

14. A method of modifying the physiology or homeostasis of a host, wherein said method comprises a. administering to the host the recombinant symbiont of any one of claims 1-11; and b. expressing the heterologous polynucleotide in the host to modify the physiology or homeostatis of a host.

15. The method of any one of claims 12-14, wherein the method further comprises translating the heterologous polynucleotide into the therapeutic polypeptide.

16. The method of any one of claims 12-15 wherein the method further comprises administering an effective amount of antimicrobial such as arterolane to reduce or eliminate the recombinant symbiont from the host.

17. The method of any one of the claims 12-16, wherein: a. the host suffers from cancer, a genetic deficiency, an infectious disease, or an autoimmune disease; b. the method modifies the host’s physiology and / or organ systems; c. the host suffers from diabetes, Gaucher’s Disease, or obesity; and / or d. the host suffers from Cystic Fibrosis, Sickle Cell Disease, hemophilia, Duchenne Muscular Dystrophy, Huntington’s Disease, beta-thalassemia, macular degeneration, muscular atrophy, leber congenital amaurosis, severe combined immunodeficiency, immunodeficiency, retinitis pigmentosa, Fabry’s disease, Pompe’s disease, Wilson’s disease, orphan diseases, amyotrophic lateral sclerosis, alport syndrome, X-Linked adrenoleukodystrophy, phenylketonuria, Marfan’s Syndrome, or hereditary angioedema.

18. The method of any one of the claims 12-17, wherein the recombinant symbiont is administered to the host via oral, intravenous, pulmonary, intramuscular, subcutaneous, or intraperitoneal routes.

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