Systems, devices, and methods for generating substances in vivo using non-pathogenic microorganisms

Abstract

The present invention relates to the production of beneficial molecules in vivo using genetically modified non-pathogenic microorganisms. The invention can be applied to various fields such as medicine, human performance and veterinary medicine. In various embodiments, the use of a non-pathogenic microorganism transgenic trypanosoma rangeli can be purposefully engineered, altered, and designed such that it, after genetic modification, becomes a blood symbiont in humans and animals to express foreign nucleic acid sequences encoding proteins, polynucleotides, and / or pathways, such as proteins, polynucleotides, and / or pathways. Thereby treating a disease or enhancing specific aspects of host physiology, including the metabolic advantage of correcting or enhancing homeostatic imbalance in appropriate cases. The foreign nucleic acid sequences can be used to generate enzymes, scavenger molecules, peptides and hormones, antibodies, nanoantibodies, signaling ligands, and other therapeutic agents or novel synthetic agents.

Description

[0001] Statement regarding federally funded research or development

[0002] This invention was made in part with the support of the U.S. government, granted grant number 2123532 by the National Science Foundation. The U.S. government may hold certain rights in this invention.

[0003] Cross-reference with related applications

[0004] This application claims priority to U.S. Provisional Application 63 / 515854, filed July 27, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0005] This invention generally relates to the generation of substances in vivo. More specifically, this invention relates to the generation of substances in vivo using genetically modified non-pathogenic microorganisms. Background Technology

[0006] Over the past decade, numerous attempts have been made to deliver biologics to humans and animals using bacteria, viruses, and multicellular organisms from various sources. While bacterial and viral vectors offer the potential for continuous delivery of biologics and eliminate the need for repeated intravenous administration, their use has been fraught with problems, primarily due to the immunogenicity of these vectors. Similarly, although multicellular organism-based therapies (such as CAR-T cells and mesenchymal stem cells) have been designed to eradicate tumors or control inflammation and have been widely used in experiments to treat incurable diseases, these approaches have been problematic, hindering widespread clinical adoption. One of the key challenges and seemingly insurmountable obstacles facing these therapies is the limited bioavailability of multicellular therapeutic cells, primarily due to host immune rejection and the inability of these cells to adapt to the host's constantly changing and harsh microenvironment (i.e., resource and niche competition). In this regard, long-term therapeutic goals cannot be achieved simply by transplanting more cells, which not only increases immunogenicity and resource competition among the introduced therapeutic cells but also increases the cost of the therapy itself.

[0007] Therefore, there remains a persistent need for higher-performance systems, devices, and methods for producing and delivering biological products to humans and other animals. Summary of the Invention

[0008] This disclosure aims to meet the ongoing need for improved systems, devices, and methods for the generation and delivery of biomolecules.

[0009] This invention relates to the generation of substances in vivo by genetically modifying non-pathogenic microorganisms to produce desired substances in a host. This invention can be applied to various fields such as human medicine, human performance, and veterinary medicine.

[0010] In various embodiments, non-pathogenic microorganisms (naturally occurring or laboratory-derived) are purposefully engineered, altered, and designed to become blood symbionts after genetic modification, generating polynucleotides, proteins, and / or pathways that enhance specific aspects of host physiology, including metabolic advantages under appropriate conditions. These microorganisms can be genetically modified to not only produce enzymes, scavenging molecules, peptides and hormones, antibodies, nanobodies, signal transduction ligands, and other therapeutic agents or novel synthetic agents, including precursors, but also to enhance the function and effectiveness of cell therapy, gene therapy, synthetic vaccines, and probiotic and microbiome regulation.

[0011] The symbiont of this invention can act as a living drug or living pharmacy in human or animal hosts, producing biomolecules for the host's long-term use. The recombinant symbiont can be used to continuously produce beneficial biomolecules to treat diseases or enhance health. Furthermore, the recombinant symbiont can be engineered to sense and respond to changes in the host.

[0012] Furthermore, the recombinant symbiont can be further engineered to provide novel molecules to alleviate adverse conditions or enhance host performance and physiological function, including but not limited to improving muscle strength, endurance, cardiovascular health, metabolic efficiency, and cognitive function. The recombinant symbiont can be further engineered to confer disease-resistant phenotypes on the host, including but not limited to enhanced immunity to infectious diseases, reduced susceptibility to autoimmune diseases, and increased resistance to cancer growth. In other embodiments, the recombinant symbiont is engineered to (a) produce molecules that alleviate or combat pain in the host, including but not limited to endogenous opioids, neurotransmitter modulators, or anti-inflammatory agents; (b) produce molecules that enhance anti-infection capabilities in the host, including but not limited to antimicrobial peptides, antibodies, or proteins that interfere with pathogen invasion or replication; and (c) regulate tightly controlled homeostasis in the host, including but not limited to maintaining optimal levels of hormones, neurotransmitters, metabolites, or essential nutrients.

[0013] Specifically, the recombinant symbiont comprises trypanosomes engineered to express heterologous polynucleotides, wherein the heterologous polynucleotides encode therapeutic proteins or therapeutic polynucleotides (“beneficial molecules”). Examples of such trypanosomes include, but are not limited to, Trypanosoma runnifolia (…). Trypanosoma rangeli ), Trypanosoma mesnilbrimontii , Trypanosoma preguici , Trypanosoma myrmecophague , Trypanosoma mycetae , Trypanosoma diasi , Trypanosoma cebus , Trypanosoma saimiri or Trypanosoma advieri In some embodiments, the trypanosome is Trypanosoma runnifolia (…). Trypanosoma rangeli).

[0014] The heteropolynucleotide may be (a) an integrated foreign nucleic acid, artificial chromosome, or nucleic acid fragment containing specific genetic features for recombination and utilization of intrinsic genetic elements; (b) encoding 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) encoding a therapeutic protein selected from enzymes, interleukins, hormones, antibodies, coagulation factors, growth factors, or peptides; and / or (d) encoding an enzymatic pathway.

[0015] The heteropolynucleotide can also encode a signal sequence selected from SEQ ID NO: 1-15. Furthermore, the heteropolynucleotide can be operatively associated with a heteropromoter.

[0016] Examples of therapeutic proteins that can be expressed by the recombinant symbiont include proinsulin, GLP-1, GIP, or β-glucocerebrosidase.

[0017] 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, pancreatic lipase, Clostridium histolytica collagenase, α-glucosidase, imiglucosidase, α-vilasidase, α-talisidase, laronidase, idoosulfatase, thiodiase, or pegolone acetonide; (b) interleukins, such as those selected from interleukin-2, interleukin-6, interleukin-4, and interleukin-6. (c) Hormones, such as insulin, levothyroxine, adrenaline, glucagon, estrogen, progesterone, testosterone, human growth hormone, corticosteroids, desmopressin, parathyroid hormone, oxytocin, calcitonin, leuprorelin, goserelin, octreotide, or alpha-thyroid-stimulating hormone; (d) Antibodies, such as adalimumab, aflibercept, alenzab, atezolizumab, bailiximab, belimumab, bevacizumab. Monoclonal antibodies, bonnetuzumab, bentoxicumab, vedotin, cananulumab, caprilumab, cetoxicumab, cetuximab, daralimumab, denosumab, detoxicumab, durvalumab, duprexacumab, ikuzumab, erlotuzumab, imalumab, gemutuzumab, oxazolidin, golimumab, ibalizumab, oxalizumab, ipilimumab, ixazolizumab, lanalizumab, rotezip, mepolizumab Mogliflozin, Natalizumab, Nexitozumab, Nivolumab, Oxotozumab, Ofatozumab, Olazumab, Omalizumab, Panitumab, Pembrolizumab, Remoxitozumab, Rituximab, Salilluzumab, Secuciyu, Tocilizumab, Trastuzumab, Ustenozumab, Vedolizumab, Retizumab, Tesalenzab, Broluruzumab, Vitolasen, Idecabtagene (e) coagulation factors, such as factor VIII, factor IX, factor VIIa, 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 XIIIa, factor XIIIb, recombinant factor VIII Fc fusion protein, recombinant factor IX Fc fusion protein, emecizumab (Hemlibra) or Leyton factor V;(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-β (TGF-β), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), erythropoietin (… EPO or thrombopoietin (TPO); and / or (g) peptides, such as abapatide, angiotensin II, bivalirudin, bumelantide, busherin, carbetocin, cetrorexone, desmopressin, exenatide, glatiramer acetate, goserelin, liraglutide, lisiratide, nesiritide, oxytocin, pramlintide, smegglutide, semerapide, teriparatide, teduglutide, triptorelin, linaclotide, purcanatide, paretide, terlipressin, angiotensin, thymosin, sermorelin, leuprorelin, lanreotide, temorelin, degarelix, purcanatide, bivalirudin.

[0018] Methods for expressing heteropolynucleotides in multicellular hosts are also considered, the methods comprising: administering the recombinant symbiont described herein to the host and expressing the heteropolynucleotide in the host.

[0019] Furthermore, methods for preventing or treating hosts suffering from diseases or disorders are considered, including: administering the recombinant symbiont described herein to the host; and expressing the heteropolynucleotide to treat, alleviate, or prevent symptoms of the disease or disorder. In these methods, the method may further include translating the heteropolynucleotide into a therapeutic peptide.

[0020] If necessary, the claimed method can be reversed by applying an effective amount of an antiparasitic or antibiotic compound (such as arterolane).

[0021] Diseases that can be treated as described herein include, but are not limited to, cancer, genetic defects, infectious diseases, or autoimmune diseases. Specifically, the recombinant symbionts described herein can be used to treat diabetes, Gaucher disease, and / or obesity. Furthermore, the recombinant symbionts can be used to treat cystic fibrosis, sickle cell disease, hemophilia, Duchenne muscular dystrophy, Huntington's disease, β-thalassemia, macular degeneration, muscular dystrophy, Leber congenital amaurosis, severe combined immunodeficiency, immunodeficiency, retinitis pigmentosa, Fabry disease, Pompe disease, Wilson's disease, orphan diseases, amyotrophic lateral sclerosis, Allport syndrome, X-linked adrenoleukodystrophy, phenylketonuria, Marfan syndrome, or hereditary angioedema.

[0022] The method can also be used to modify the host's physiological and / or organ systems.

[0023] The recombinant symbiont can be delivered orally, intravenously, pulmonaryly, intramuscularly, subcutaneously, or intraperitoneally. Attached Figure Description

[0024] The accompanying drawings are included to illustrate various aspects of the invention, and not to limit the invention, in which: Figure 1 An exemplary method for converting non-pathogenic microorganisms into genetically engineered symbiotic microorganisms is shown.

[0025] Figure 2 AD shows the effect on Trypanosoma langsei ( T. rangeli Genetically engineered proinsulin to produce proinsulin and introduced it into the host demonstrated robust glycemic control without safety concerns regarding hypoglycemia or hyperglycemia. Various proinsulin-secreting symbionts were implanted into healthy mice to assess safety and tolerability. All animals tolerated the implantation and exhibited reduced glycemic levels and robust glycemic control during a 16-hour fasting glucose challenge, without the risk of hypoglycemia or hyperglycemia.

[0026] Figure 3 Candidate genomic integration sites are listed that can be used to integrate polynucleotides encoding beneficial molecules into the genome of the symbiont.

[0027] Figure 4 The non-limiting families of signal peptides used in this invention are listed.

[0028] Figure 5 Results are shown using a recombinant symbiont expressing proinsulin in non-obese diabetic (NOD) mice. Here, the recombinant symbiont (SM18) engineered to produce proinsulin delayed the onset of type 1 diabetes (TID) in genetically susceptible hosts, relative to control organisms engineered to express unrelated green fluorescent protein (GFP; control).

[0029] Figure 6 The in vitro and in vivo GCase activity of GBA1 D409V homozygous mutant mice (mutations in human Gaucher disease) after treatment with a recombinant symbiont expressing GBA1 (the gene encoding GCase) is shown. Figure 6 A indicates the in vitro GCase activity in three parallel wells after enzyme expression, secretion, and retention in the perfused livers of mice treated with either a control (mice 557) or a symbiont expressing GBA1 (mice 561 and 669). Figure 6B illustrates the accumulation levels of 22-carbon ceramide (left panel) and sphingosine (middle panel) in the liver and brain of D409V homozygous mutant mice treated with either a control or a symbiont expressing GBA1, compared to untreated wild-type mice. These data demonstrate that expression of GCase from the recombinant symbiont reduces tissue accumulation of lipids associated with Gaucher disease.

[0030] Figure 7 The study demonstrated that using a recombinant symbiont expressing GLP-1 prevented weight gain. The symbiont expressing GLP-1 or its parent protein (proglucagon) was implanted into healthy mice fed a Western diet (high-fat diet). Figure 7 A shows serum GLP-1 levels as measured by an ELISA for GLP-1. Figure 7 B shows the percentage of body weight gain during the 50-day observation period. These data demonstrate that GLP-1 expression in the recombinant symbiont leads to weight loss compared to control mice.

[0031] In the accompanying drawings and detailed description, the same or similar designations may represent the same or similar elements. It should be understood that, unless otherwise expressly stated or impossible, the implementations, features, etc., described with respect to the embodiments in a particular drawing may also be implemented with respect to other embodiments in other drawings. Detailed Implementation

[0032] This invention relates to the in vivo production of substances using genetically modified non-pathogenic microorganisms. This disclosure describes fundamental principles and know-how that those skilled in the art may need to consider when adequately designing non-pathogenic microorganisms to serve as recombinant symbionts and as biological carriers or devices for the long-term, sustained production of beneficial molecules within a host. Beneficial molecules may be deficient, defective, or heterologous proteins and polypeptides in the form of enzymes, interleukins, hormones, antibodies, coagulation factors, soluble receptors, growth factors, or ligands. These molecules can be used to treat diseases or disorders, such as cancer, genetic defects, infectious diseases, or autoimmune diseases, and may also serve as metabolically dominant molecules or toxicity scavengers, as well as other physiological regulators suitable within the scope of this invention. Beneficial molecules may also include polynucleotides, such as RNA. i .

[0033] As described herein, a delivery system, biovector system, or platform for altering host physiology and / or performance is presented. Furthermore, the system can be further modified with feedback loop features that enable the recombinant symbiont to respond to physiological changes in the host to control the timing and conditions of the delivery of the beneficial molecules. For example, endogenous low-affinity glucose transporter channel sites, which are normally active when glucose is abundant in the environment, can be engineered to conditionally secrete insulin to mitigate the negative effects of uncontrolled hyperglycemia in the host. Thus, similar sites can be used to feedback and sense environmental changes in the host to coordinate the delivery of beneficial molecules. Figure 3 A list of non-restricted candidate genomic sites that can be used to continuously deliver beneficial molecules to the host through recombinant symbiotic microbial sensing and response (i.e., time and conditions).

[0034] This invention can be used to domesticate microorganisms into recombinant symbiotic microorganisms. The expertise defined herein, the broadest examples of applications, and the claims provide guidance.

[0035] Overview

[0036] The “host” as used in this article is a multicellular organism with tissues, organs and circulatory systems, such as humans, canines, felines, equines, livestock (e.g., cattle, goats, sheep or pigs), chickens or other poultry, fish or other animals, which may be healthy or impaired and can be colonized by microorganisms.

[0037] As used herein, the term “nonpathogenic microorganism” refers to a microorganism capable of colonizing a host to ensure its survival or as part of its natural life cycle. An example of a nonpathogenic microorganism that may be used as described herein includes trypanosomes. These microorganisms are typically found in the host’s gut, blood, or tissues. Such microorganisms are characterized, but are not limited to: (1) being non-toxic and non-pathogenic to the host; (2) being persistent and durable; (3) being culturable, engineerable, and modifiable in the laboratory; and (4) achieving universal immunocompatibility by evading the immune response. As used herein, the term “symbiont” refers to a microorganism that colonizes a host without causing harm. As used herein, “recombinant symbiont” refers to a symbiont that has been engineered and specifically designed to benefit the host by producing molecules (i.e., polynucleotides and / or polypeptides) (“beneficial molecules”) that enhance or alter host physiology in the long term as described herein.

[0038] Therefore, this paper describes a method for modifying a non-pathogenic microorganism into a “recombinant symbiotic microorganism” or “recombinant symbiont” for the continuous production of beneficial biomolecules in a host, the method comprising the steps of: (a) genetically modifying the non-pathogenic microorganism by integrating a foreign nucleic acid, artificial chromosome, or nucleic acid fragment containing specific genetic features for recombination and utilization of intrinsic genetic elements to produce a recombinant symbiont; (b) introducing the recombinant symbiont into a host; (c) allowing the recombinant symbiont to reside in the host without causing harm; and (d) enabling the recombinant symbiont to express beneficial biomolecules (e.g., proteins and peptides) that alter the physiology of the host.

[0039] In this embodiment, the recombinant symbiont has been engineered to produce enzymes, interleukins, interferons, hormones, antibodies, coagulation factors, soluble receptors, growth factors, peptides, or ligands for the treatment of diseases or disorders such as genetic defects or neurological, metabolic, endocrine, cancer, infectious, or autoimmune diseases. Furthermore, the recombinant symbiont can be used to regulate the host's physiology, homeostasis, and organ systems.

[0040] The recombinant symbiont can be administered to the host via oral, intravenous, intrapulmonary, intramuscular, subcutaneous, or intraperitoneal routes.

[0041] Other embodiments include a method for generating biomolecules in a host, the method 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 microorganism, and possibly including 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 genetically modified symbiont to generate biomolecules in the host; and (d) allowing the genetically modified symbiont to reside in the host and generate biomolecules. Alternatively, the beneficial molecule may be encoded by an episome that has not yet been integrated into the genome of the symbiont.

[0042] The non-pathogenic microorganism used to generate the recombinant symbiont is a trypanosome. For example, the recombinant symbiont can be derived from Trypanosoma runnifolia (…). Trypanosoma rangeli ("London trypanosoma") T. rangeli )”) Trypanosoma mesnilbrimontii , Trypanosoma preguici , Trypanosoma myrmecophague , Trypanosoma mycetae , Trypanosoma diasi , Trypanosoma cebus , Trypanosoma saimiri or Trypanosoma advieriIn other embodiments, Trypanosoma runnifolia (Ranunculus rondosa) is used to generate the recombinant symbiont. T. rangeli It can originate from laboratory strains ATCC #30032 or ATCC #30033.

[0043] One advantage of using trypanosomes to generate the recombinant symbiont is that the method described herein is reversible when using antimalarial drugs such as artemisinin (OZ277). For example, if it is necessary to stop the further expression of the biomolecule in the host, the host can be given artemisinin, which will eliminate the trypanosomes, including the recombinant symbiont.

[0044] General design of constructs

[0045] The term "recombinant" or "transgenic" symbiont refers to a symbiont whose genetic material has been modified to encode a beneficial molecule. This beneficial molecule may be encoded by a polynucleotide from an episome or integrated into the genome of the recombinant symbiont. When incorporated into the genome of the symbiont, the native genetic sequence may be subtracted during the addition of the foreign sequence conferring the desired beneficial trait or phenotype.

[0046] Methods of adding or subtracting sequences to modify the natural characteristics of the symbiont (which can endow the recombinant symbiont with unique features engineered in the laboratory but not found in nature) can involve homologous recombination or the addition of sequences such as... Figure 1 The artificial chromosome shown is engineered in the laboratory and is entirely within the scope of the art. As used herein, the term "homologous end joining or homologous recombination" refers to a method that can be used to target specific sites in the symbiont genome with a nucleotide fragment flanking a foreign nucleotide sequence carrying a naturally complementary nucleotide sequence originating from the symbiont. Homologous recombination can be performed using various techniques known in the art; see, for example, *Current Protocols in Molecular Biology* (1994), Greene Publishing Associates and John Wiley & Sons, NY.

[0047] For example, targeted or non-targeted genome integration systems can be used to target Trypanosoma mangiferum (Trekkissus 'London's'). Trypanosoma rangeli “ T. rangeliThe beneficial molecule can be engineered to stably integrate foreign nucleic acids, artificial chromosomes, or linear or circular nucleic acid fragments. Furthermore, the beneficial molecule can be expressed from a stable episodic polynucleotide. The heterologous polynucleotide encoding the beneficial molecule may contain specific genetic elements for constitutive, inducible, or targeted expression of the beneficial molecule. Alternatively, the beneficial molecule can also be expressed from polynucleotides already integrated into the genome of the recombinant symbiont.

[0048] The genetic modifications considered in this invention enable the transgenic symbiont (e.g., Trypanosoma runnifolia) to... T. rangeli The foreign genetic sequence differs from its naturally occurring counterpart in terms of metabolism, reproduction, immunity, or its tissue specificity (tropism), as determined by the purpose of the engineered modification, which aims to enhance host physiology and confer selectable characteristics that enable enrichment. In various embodiments, the foreign genetic sequence can be used to generate fluorescent colors or confer metabolic advantages against toxic chemicals such as antibiotics, thereby clearly identifying symbionts with successfully engineered characteristics. A non-limiting list of symbiotic characteristics resulting from the engineered foreign DNA sequence includes polypeptides that express (1) RNA, (2) proteins, or (3) end products that are free metabolites (e.g., carbohydrates, lipids, amino acids, steroids, fatty acids, vitamins, anti-infective agents) that alter host physiology.

[0049] As used herein, the term "protein or pathway" refers to the amino acid sequence encoding a functional polypeptide that acts as a single unit (i.e., a protein) or a series of units to treat or provide one or more specific biological responses (i.e., pathways). These polypeptides can be designed by introducing nucleic acid sequences into a starting symbiont using standard recombination methods, such as those disclosed in Sambrook et al. (2001).

[0050] For example, converting cholesterol into testosterone involves four enzymes in five steps: CYP11A1 > CYP17A1 > 3. -HSD>17 -HSD. Therefore, the expression of these enzymes in a single symbiont can utilize the host's cholesterol as a raw material to generate testosterone for the host.

[0051] As used herein, the term "intrinsic driver" refers to endogenous regulatory elements that allow targeting of genomic integration sites located within the symbiont genome. These regions are known to regulate the expression of specific sequences, proteins, and / or pathways. In certain embodiments of the invention, these intrinsic driver elements can be targeted as preferred sites for homologous recombination to confer specific sensing and response characteristics in the transgenic symbiont. A non-limiting list of candidate sites with variable available characteristics and chromatin activity may be provided. Figure 3These sites are ideal for inserting heterologous polynucleotides encoding beneficial molecules as described herein, because the introduced gene can be expressed at high, medium, or low levels depending on the integration site.

[0052] Figure 1 A demonstrates a representative construct designed to express secreted peptides using intrinsic driver factors. Specifically, it is anticipated that strong promoters can also be used instead of intrinsic driver factors, such as early or late promoters of SV40, CMV, vaccinia virus, polyomavirus, adenovirus, herpesvirus, and other sequences known to control gene expression in multicellular organisms.

[0053] When the signal of RNA polymerase transcribes the coding sequence into RNA (especially mRNA), and then the RNA is spliced ​​(if it contains introns) and translated into a polypeptide encoded by the coding sequence, the coding sequence is either "under the control" of the expression control sequence or "operably associated" with the expression control sequence.

[0054] Treatment

[0055] Treatment methods using the recombinant symbiont have also been disclosed. For example, one method of treating a host with diabetes includes: (a) introducing a linear or circular DNA sequence encoding proinsulin into Trypanosoma lancifolium (… T. rangeli In the genetic sequence of the *Treatisena rankie*, so that the *Treatisena rankie* ( T. rangeli ) produces proinsulin in the host, which is effective against the aforementioned Trypanosoma rankie ( T. rangeli (a) genetically modifying the genetically modified Trypanosoma langurenis; (b) genetically modifying the Trypanosoma langurenis ( T. rangeli (c) Allowing the genetically modified Trypanosoma languren to be introduced into the host; T. rangeli (d) allows the genetically modified Trypanosoma langurenis to remain within the host without causing harm; and (d) allows the genetically modified Trypanosoma langurenis to remain within the host without causing harm. T. rangeli It can produce proinsulin in the host.

[0056] The transgenic symbiont can be programmed to sense changes in the host and respond to unwanted changes by providing a substance, or thereby enhancing host performance. One example of such a sensing and response system is a transgenic symbiont sensing high concentrations of glucose in the host's blood via an endogenous glucose transporter, subsequently producing potent insulin to mitigate the adverse effects of hyperglycemia in the host. Alternatively, the transgenic symbiont can be programmed to sense lower levels of hormones (such as vasopressin) to improve performance and allow adaptation to harsh environments.

[0057] This invention can be used to sustainably supply a diverse group of molecules with both therapeutic and physiologically enhancing effects, which are unavailable using naturally occurring microorganisms. It should be understood that the technology of this invention can be used in any host described herein. Those skilled in the art will also recognize that this invention can be used to prepare molecules for sustained delivery and in vivo manufacturing, such as… Figure 1 The apparatus shown is for a substance with therapeutic value.

[0058] For example, the recombinant symbiont could be used to treat lysosomal storage diseases. This invention is expected to allow for the treatment and / or improvement of lysosomal storage diseases currently managed through enzyme replacement therapy. A common feature of lysosomal diseases is the accumulation of unprocessed cellular products from lysosomes, which leak from cells into tissues and blood in the form of sugars and lipids, causing complex symptoms. Currently, these cellular products are treated by exogenously supplementing enzymes to eliminate the disease-mediating cellular products.

[0059] Similarly, recombinant symbionts can be used for hormone replacement and health enhancement. For example, these recombinant symbionts are intended to allow for the continuous production and delivery of hormones and their precursors as a substitute in the absence of biomolecules, or to elevate certain proteins above normal levels, thereby enabling the host to heal, tolerate, manage pain, or optimize desired performance and fitness goals. Therefore, the present invention contemplates the use of recombinant symbionts to optimize human performance and resilience.

[0060] As demonstrated in this paper, glucose homeostasis management in diabetes can also be controlled using the recombinant symbiont described herein. Figure 2 A representative scenario is presented, in which Trypanosoma runnifolia ( T. rangeli Genetic engineering was used to modify it to produce the insulin precursor, proinsulin.

[0061] The transgenic Trypanosome Langerhans was introduced into laboratory mice via a single dose. T. rangeli ).like Figure 2 As shown in A, for two different test samples SM18 (SEQ ID NO: 11) and SM22 (SEQ ID NO: 12), the transgenic Trypanosoma lanchomatis (… T. rangeli It produced sufficient proinsulin, surprisingly achieving 70 days of low and stable glycemic homeostasis. Furthermore, as... Figure 2 As shown in B, the levels remained unchanged during fasting, indicating that the risk of hypoglycemia is also manageable.

[0062] like Figure 2As shown in Figure C, in the glucose tolerance test, test samples SM18 (SEQ ID NO: 11) and SM22 (SEQ ID NO: 12) also showed slightly higher and faster glucose clearance from the blood. Figure 5 As shown, the onset of type 1 diabetes was also delayed in the test sample SM18 (SEQ ID NO: 11) compared to the control sample. Therefore, the transgenic symbiont can be genetically engineered to produce proinsulin with potential therapeutic value for patients in vivo.

[0063] The recombinant symbionts described herein can also be used for neurological applications. For example, the present invention may be able to treat, improve, and / or enhance neurological conditions for which current interventions are limited. The transgenic symbionts can sustainably deliver beneficial molecules that can cross the blood-brain barrier, offering therapeutic benefits by reducing pathogenic physiology or enhancing neuronal function and proliferation. By administering the symbiont, loss of neuronal function can be restored or the pathological accumulation of molecules leading to neurological disorders can be eliminated. The present invention also contemplates a delivery device designed to mitigate the effects of neurotransmitter dysregulation on mental disorders by expressing or eliminating neurotransmitters.

[0064] The recombinant symbionts described herein can also be used for immunomodulation, such as in the treatment of autoimmune diseases, allergies, vaccination, and / or cancer. This invention is expected to allow for the treatment of autoimmune diseases, the sensitization of subjects to allergens, and the delivery of biomolecules to treat cancer. A common feature of autoimmune and allergic diseases is the overactivation of immune responses and immune cells against harmless antigens. Here, the recombinant symbiont can continuously supply an agent that modulates the immune response, in the form of an anti-inflammatory molecule or an agent that restricts cell migration or activation. The recombinant symbiont can deliver low doses of allergens to sensitize the subject to the molecule and prevent allergic reactions. Furthermore, the recombinant symbiont can also be used for the continuous delivery of proteins used in vaccination strategies. Similarly, the recombinant symbiont can also be engineered to express molecules with antitumor activity to treat, manage, and / or improve cancer. Currently, efforts to utilize common characteristics of cancer, such as evading growth inhibition, avoiding immune destruction, resisting cell death, and deregulating cellular energy, have successfully and effectively controlled these diseases. The recombinant symbionts described herein can continuously supply therapeutic molecules to not only treat the disease, but also to investigate, monitor, and / or diagnose based on an expanding range of cancer biomarkers.

[0065] As described above, the recombinant symbionts described herein consider a wide range of applications as delivery systems for the continuous supply of proteins and pathways within a host. In addition to transgenic symbionts designed for the continuous expression of one or more genes of interest, recombinant symbionts can also be designed to sense host stimuli and autoregulate the expression of desired molecules in response to these stimuli. Furthermore, recombinant symbionts can be designed to interact with the host via receptor-ligand interactions, home to specific tissues, and perform enzyme-substrate dynamic regulation.

[0066] Once generated, the recombinant symbiont can be used to achieve pharmacokinetic and pharmacodynamic goals of molecules continuously supplied to the host by the transgenic symbiont. The methods listed below are not limiting, but rather examples of strategies to optimize the effectiveness of the symbiont as a delivery device in altering host physiology. The extension of the half-life of therapeutic substances produced by the symbiont can be achieved through several methods, including increasing the rate of therapeutic substance production without compromising the stability and effectiveness of the transgenic symbiont. Alternatively, the ultrastable design of the cargo molecules can be employed to achieve specific pharmacokinetic and pharmacodynamic goals in the host.

[0067] The third strategy involves using "variable immunogenicity control," thereby engineering the host's immune regulatory factors into the symbiont, making the symbiont more stable and less susceptible to common threats from the host. Another option is to design tissue-homing signatures. Some species associated with the symbiont naturally exhibit tissue specificity, which can be engineered into selected symbionts. Therefore, the ease of adding or subtracting foreign nucleotide sequences allows those skilled in the art to generate well-optimized delivery devices. Alternatively, the beneficial molecule can be expressed by an episome.

[0068] Delivery and formulation of recombinant symbionts

[0069] The delivery of transgenic symbionts can be achieved using standard techniques known in the art. This includes, but is not limited to, administration of the symbiont to the host via intramuscular, subcutaneous, pulmonary, or intravenous injection, as well as other parenteral, enteric, or dermal routes suitable for customized applications.

[0070] The recombinant symbiont can be formulated with ingredients compatible with pharmaceutical and microbiological applications and procedures. For example, the formulations of the present invention can incorporate a sterile recombinant symbiont into a suitable carrier solution that is harmless to the recombinant symbiont or the host, such as, but not limited to, saline, glycol, oil, gel, or hydronaphthalene. The present invention considers local, intravenous, subcutaneous, or intraperitoneal administration routes. The quantity or effective concentration of the recombinant symbiont to be delivered will vary depending on a variety of factors, including indication, host species, route of administration, disease severity, relative bioactivity of the delivered molecule, and overall host health status.

[0071] Equivalent scheme

[0072] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the above embodiments should in all respects be regarded as illustrative rather than limiting of the invention described herein.

[0073] Some implementations may be described in conjunction with thresholds in this document. When used in this document, satisfying a threshold can refer to a value greater than, exceeding, higher than, greater than or equal to, less than, less than, lower than, less than or equal to, or equal to the threshold.

[0074] The foregoing disclosure provides examples, descriptions, and illustrations of the invention, but is not intended to be exhaustive or to limit implementations to the specific forms disclosed. Modifications and variations are possible based on the foregoing disclosure, or may be obtained from practice of the described implementations. These and other variations and modifications of the invention are possible and contemplated, and it is intended that such modifications and variations be covered by the foregoing description and claims.

[0075] Although specific 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 can be combined in ways not specifically recited in the claims and / or not disclosed in the specification. Although each dependent claim listed below may be directly dependent on only one claim, the disclosure of possible implementations includes combinations of each dependent claim with each other claim in the group of claims.

[0076] Unless explicitly stated otherwise, no element, action, or instruction used herein should be considered critical or essential. Furthermore, references without a specific number as used herein are intended to include one or more references and may be used interchangeably with “one or more.” Additionally, the term “group” as used herein is intended to include one or more items and may be used interchangeably with “one or more.” When referring to only one item, “one” or similar language is used. Furthermore, the terms “having” and others as used herein are intended to be open-ended terms. Additionally, the phrase “based on” is intended to mean “at least partially based on” unless explicitly stated otherwise.

[0077] The presence or absence of an overview, abstract, or claims in this application should in no way be construed as a limitation on the scope of any invention disclosed herein.

[0078] Example

[0079] The practice of the present invention will be more fully understood from the following embodiments, which are set forth herein for illustrative purposes only and should not be construed as limiting the invention in any way.

[0080] Example 1: General construction and application of recombinant symbionts

[0081] like Figure 1 As shown, various methods and tools can be used to incorporate polynucleotides encoding beneficial molecules into symbionts. In this embodiment, Trypanosoma runnifolia (…) is used. T. rangeli To generate recombinant symbionts. Figure 1 A depicts a close-up of a representative gene segment added to the organism's genome. HEJ stands for Homologous End Join, ETS for External Transcriptional Spacer, and ITS for Internal Transcriptional Spacer. In this embodiment, intrinsic driver factors specific to this symbiont (including both intrinsic promoters and intrinsic signal peptides) are used to ensure payload secretion. However, other strong promoters can be used interchangeably.

[0082] After construction, electroporation was used to permanently modify *Treatisena lancifolium* by random integration through homologous end-connection. T. rangeli The genetic composition of ). Figure 1 B describes a transgenic symbiote introduced into experimental mammals.

[0083] Figure 3 A non-limiting list of candidate sites with variable chromatin states is provided, which can be used for expression regulation, in this invention for controlling the timing and conditions of delivery of beneficial molecules, thereby achieving genetic modification through incorporation of polynucleotides into episomes or site-directed integration. For example, when using laboratory-derived Trypanosoma langurii (… T. rangeli These sites were considered when generating recombinant symbionts from Trypanosoma runnifolia (Trekkerus rankii). T. rangeli The intrinsic signal peptide selected from naturally encoded proteins is integrated into the expression cassette, and the variable proinsulin production of the expression cassette is measured over 24 hours.

[0084] Figure 4 A non-restrictive list of intrinsic signal peptide families that can be used to promote secretion is provided.

[0085] To generate recombinant symbionts, Amaxa from Lonza Biosciences can be used. TM Nucleofector TM Methods including systemic parasite or T-cell nuclear transfection kits (using U-033, X-001, or D-023 procedures) are used to introduce circular DNA into non-pathogenic organisms such as Trypanosoma runnifolia (…). T. rangeliIn ) molecular cloning methods and recombinant Trypanosoma rankiensis ( T. rangeli The generation of ) was carried out using recombination methods well known to those skilled in the art, as disclosed in Sambrook et al., (2001), Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (“Sambrook et al., 2001”).

[0086] Then, antibiotic resistance screening was used to identify *Treatisea langurii* (…). T. rangeli Clones of beneficial molecules were incorporated into the genome, and the expression and secretion of the molecules of interest were confirmed in vitro before being implanted into subjects.

[0087] A simple and low-cost method was used to culture the recombinant symbiont. For example, Koerich, LB et al. (2002), “Differentiation of Trypanosoma lancifolium: Mass production of infective trypanosome flagellates in vitro”. Trypanosoma rangeli : high production of infectious trypanomastigote forms in ex vitro As described in Parasitol Res 88: 21-25, the study of Trypanosomiasis Langerhans strain in LIT (liver extract tryptone) supplemented with 10% (v / v) fetal bovine serum was performed. T. rangeli The culture of Tejara E1 cells derived from the recombinant symbiont can be used to derive Trypanosoma langsei (Trypanosoma langsei). T. rangeli The strains include ATCC #30032 and ATCC #30033.

[0088] The cells were incubated at 28°C in an incubator with circulating oxygen at room temperature. Cell counts were performed in normal culture medium using a manual hemocytometer. Cell growth rates were optimized for the transfection protocol and determined.

[0089] One method for regulating the DNA content of the symbiont is by amplifying or synthesizing long DNA fragments composed of foreign DNA sequences containing expression modifiers (such as signal peptides), beneficial features (such as enzymes and receptors), and selectable features flanked by Trypanosomes of Langerhans designed for specific integration at preferred sites. T. rangeli ) Specific recombination target sequences. For example, Figure 3A non-restricted list of genomic sites selected based on specific characteristics is disclosed, along with flanking sequences that guide the insertion of DNA fragments at these sites. To confirm that the fragment retains the desired valuable functional element (i.e., an enzyme or receptor protein), a minimal T7 promoter can be added upstream of the fragment during synthesis for confirmation via amplification.

[0090] By using published trypanosome-specific reagents and for Amaxa TM Biosystems Nucleofection TM The platform's reagent kits can introduce large fragments into Trypanosoma mansoni (London trypanosoma mansoni). T. rangeli To achieve the desired integration of the DNA sequence, a neomycin-based antibiotic screening method can be used to screen the symbiotic Trypanosoma ranunculus (Treatisea rankie). T. rangeli The culture was enriched for four weeks. Secreted proteins and peptides were monitored using suitable assays (i.e., enzyme activity, Western blotting, ELISA, etc.). Those skilled in the art will understand that these are standard molecular biology techniques and methods, and the examples herein should not be construed as limiting.

[0091] The recombinant symbiont can be further expanded to larger quantities and intravenously inoculated into naïve mice or other suitable hosts for testing purposes. For example, weekly intravenous blood sampling can be performed to monitor glucose homeostasis. However, those skilled in the art will understand that the various designs of the recombinant symbiont will require appropriate assays to monitor a variety of physiological endpoints.

[0092] Example 2: Treatment of diabetes with recombinant symbionts

[0093] Insulin is a cornerstone of endocrinology because it was the first peptide hormone identified in hormone research. It originates from a 110-amino acid proinsulin polypeptide, comprising a 21-amino acid A chain, a 30-amino acid B chain, a 35-amino acid C chain, and various signal peptides. The maturation of proinsulin is intricate, requiring multiple enzymatic processes to ultimately produce insulin. The first crucial step occurs within the lumen of the rough endoplasmic reticulum, where a signal peptidase cleaves the signal peptide, ultimately leading to proinsulin formation. Subsequently, an endopeptidase similar to trypsin further refines proinsulin by cleaving the C chain (the intermediate peptide connecting the A and B chains, typically between 30 and 35 amino acids in length). The final protein processing is supervised by the exopeptidase carboxypeptidase B, which meticulously removes any remaining basic amino acids after C chain cleavage. Proinsulin has profound implications in immunology. It plays a role in promoting self-tolerance in individuals without autoimmune diseases, highlighting its therapeutic potential. Specifically, this potential can be attributed to immune pathways that either clear reactive cells or generate natural regulatory T cells to achieve tolerance through sustained pre-proinsulin exposure.

[0094] The therapeutic efficacy of the proinsulin expressed from the recombinant symbiont was evaluated. The inventors generated a recombinant symbiont to express proinsulin as described in Example 1, and also tested seven different signal peptides to modulate the secretion rate of the hormone in test subjects.

[0095] Figure 1 C shows samples from seven different Trypanosoma langurii species ( T. rangeli In vitro data for cultures of *Treatisena rankie*, which have been modified as described herein to secrete proinsulin. Naturally occurring *Treatisena rankie* is not shown. T. rangeli Because it does not secrete proinsulin. In these experiments, the following procedures and gene segments were used: mouse proinsulin (Genbank accession number X04725) was used to flank the α-tubulin sequence of the homologous end join (HEJ), enabling the intrinsic α-tubulin promoter to drive expression. Random nucleic acid sequences were integrated into the outer and inner (ETS and ITS) to maintain the translation frame.

[0096] Interestingly, in vitro assays showed that four of the seven signal peptides enhanced secretion, with signal peptides SM14 (SEQ ID NO: 9), SM18 (SEQ ID NO: 11), and SM22 (SEQ ID NO: 12) being the most effective, while signal peptide SM00 (SEQ ID NO: 4) promoted moderate secretion. Figure 1 C).

[0097] To investigate the safety and tolerability of symbionts with therapeutic payloads, 13-week-old healthy wild-type mice were implanted (IV-injected) with symbionts expressing proinsulin SM00 (SEQ ID NO: 4), SM14 (SEQ ID NO: 9), SM18 (SEQ ID NO: 11), and SM22 (SEQ ID NO: 12) (n=8). Interestingly, compared with control animals implanted with symbionts expressing GFP, animals implanted with the signal peptides SM22 (SEQ ID NO: 12) and SM14 (SEQ ID NO: 9), which promote the highest levels of proinsulin secretion, consistently exhibited lower blood glucose levels. Figure 2 Over the following weeks, SM22 (SEQ ID NO: 12) and SM18 (SEQ ID NO: 11) exhibited strong total glycemic control (TGC), while SM00 (SEQ ID NO: 4) and SM14 (SEQ ID NO: 9) showed similar effects to the control symbiont. Throughout the 10-week observation period, SM18 (SEQ ID NO: 11) and SM22 (SEQ ID NO: 12) consistently demonstrated statistically significant TGC (p<0.05), with SM22 (SEQ ID NO: 12) showing the most significant TGC. To investigate the safety and efficacy of these symbionts with the first therapeutic payload, these symbionts were implanted into 13-week-old wild-type mice (n=8 / group). The results were encouraging; no hypoglycemia was recorded in any of the four experimental groups and the control group, indicating that the system was unaffected by the various therapeutic payloads of the symbionts. In addition, glucose tolerance tests were performed using SM18 (SEQ ID NO: 11) and SM22 (SEQ ID NO: 12) to assess the host's physiological response to hyperglycemia. The results showed that subjects treated with SM18 (SEQ ID NO: 11) and SM22 (SEQ ID NO: 12) had improved glucose clearance, indicating an enhanced ability to metabolize large glucose loads (i.e., improved efficiency).

[0098] Given the successful safety and tolerability of proinsulin production via recombinant symbionts, the inventors next evaluated the effect of proinsulin on overcoming β-cell loss (i.e., immune-mediated destruction of pancreatic insulin-secreting cells) in non-obese diabetic (NOD) mice (a spontaneous model of type 1 diabetes). Symbionts expressing SM18 (SEQ ID NO: 11) were transferred to approximately 19-week-old NOD mice. Mice treated with SM18 (SEQ ID NO: 11) showed a significantly delayed onset of type 1 diabetes compared to the control group treated with GFP symbionts. Figure 5It can be inferred that the therapeutic effects of proinsulin delivered by the symbiont are dual: 1) providing insulin precursors that are processed into active insulin; and 2) delivering molecules that regulate the self-tolerance mechanism against pancreatic antigens.

[0099] As illustrated herein, the expression of beneficial molecules such as insulin, GLP-1, or GIP from the recombinant symbiont allows for the treatment of diabetes or the introduction of insulin through periodic, semi-continuous, or continuous injection or generation of proinsulin or GLP-1 or GIP in the host. Current practices utilize processed "modified" insulin or GLP-1 or GIP for frequent administration as daily or weekly injections. It is generally found that injection of proinsulin into muscle or adipose tissue is ineffective for treating diabetes. However, the inventors have discovered that the continuous or semi-continuous production and secretion of proinsulin in the host system using the recombinant symbiont exhibits unique therapeutic properties, such as… Figure 2 As shown, its usefulness in treating diabetes has been demonstrated. This invention can be used to deliver precursor compounds that can be processed by the body to generate compounds that can be used to treat other conditions.

[0100] Example 3: Treatment of Gaucher Disease with Recombinant Symbionts – An Example of Large Payload

[0101] Mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), have been identified as a key contributing factor to Gaucher disease (GD), a lysosomal storage disorder. Furthermore, these mutations are also associated with Parkinson's disease (PD). More than three hundred different GBA1 mutations have been documented, collectively representing the most potent known genetic determinants of both GD and idiopathic PD. GCase plays a crucial role in lysosomal glycolipid metabolism. Insufficient GCase activity leads to the accumulation of specific glycosphingolipid (GSL) substrates, particularly glucosceramide (GlcCer) and glucosphosine (GlcSph). These accumulated GSLs are symptomatic markers of GD. Most rodent models of GD are based on GBA1 gene knockout or characterized by the GBA1 D409V point mutation. The latter is particularly important because it leads to a sharp decline in GCase enzyme function and the subsequent accumulation of certain GSL substrates in target organs such as the spleen and brain. The GBA1 D409V KI mouse model exhibits significantly reduced GCase activity and GSL accumulation in peripheral organs, making it a valuable tool for studying interventions for gastrointestinal disorders (GD) and peripheral dysplasia (PD). By 3 months of age, these mutant mice exhibit physiological defects in the form of lipid accumulation in selected organs, including the brain and liver, and cognitive deficits worsen over the following year.

[0102] This embodiment demonstrates the platform's ability to deliver large payloads (>50 kDa) and evaluates the efficacy of symbiotic-delivered GCases against the accumulation of GlcCer and GlcSph in the liver and brain of GBA1-deficient mice. To this end, D409V heterozygous breeding pairs were obtained to acquire homozygous GBA1 D409V mutants for efficacy studies. Simultaneously, efforts are being made to design, select, and scale up the production of chassis adept at GCase secretion.

[0103] The autosomal recessive inheritance of the GBA1 mutation requires sustained breeding over a long period to ensure a sufficient number of qualified animals are available for research. The proportion of homozygous mutant offspring produced by the resulting breeding pairs is lower than expected according to Mendelian laws of inheritance, requiring more time to accumulate the desired number for the research. Therefore, continuous recruitment of qualified animals from matched pairs is considered necessary to ensure consistency in age and disease state across experiments.

[0104] Furthermore, after detecting significant sequence differences between human and mouse GBA1, the inventors constructed mouse GBA1 (GenBank accession number M24119.1) with highly active signal peptide 22 (SEQ ID NO: 12), thereby obtaining the prototype SM22 (SEQ ID NO: 12) mGBA (referred to as GBA in the study). In vitro tests using the 4MU-β-glucosidase assay (standard GCase activity assay) showed that the GCase secreted by the symbiont had significant enzymatic activity.

[0105] After ensuring a sufficient number of subjects with homozygous D409V mutants, 8-week-old mice were administered either a symbiont expressing GFP (as a negative control) or a symbiont expressing SM22 (SEQ ID NO: 12) mGBA. Four weeks post-implantation, GCase activity in the liver was measured in vitro as initial treatment quality control prior to lipidomics studies. Figure 6 As shown in A, higher but variable GCase activity was detected in the perfused livers of subjects 561 and 669 who received SM22 (SEQ ID NO: 12) mGBA implants compared to the GFP control implant, suggesting that enzyme replacement strategies may be an effective therapy to reduce the accumulation of ceramide and sphingosine substances in affected organs.

[0106] Then, lipid accumulation in the liver and brain was assessed by mass spectrometry. Figure 6 B-6C). Notably, lipidomics analysis of ceramide and sphingosine lipids revealed that, compared to control GFP symbiont-treated mice, mice implanted with GBA-expressing Trypanosoma lancifolium (B-6C) showed significantly higher levels of ceramide and sphingosine lipids. T. rangeliIn mice, several lipids, including large lipids such as 22-carbon glucose ceramide C22-GlcCer and total glucose sphingosine GlcSph, showed statistically significant reductions. More notably, small amounts of homologous lipids that typically accumulate in the brain were reduced; these lipids usually take an unusually long time (up to 1 year) to show their accumulation effect, suggesting that this delivery method may also be suitable for delivering neurotherapies. Figure 6 D).

[0107] Example 4: Treatment of Obesity with Recombinant Symbionts – Sustained Expression

[0108] GLP-1 drugs (i.e., Ozempic) TM Wagovy TM Trulicity TM Mountaro TM And the recently approved EliLilly's Zepbound TM Injectable GLP-1 inhibitors have become a cornerstone therapy for type 2 diabetes (T2D), offering a range of benefits, including weight management and cardiovascular benefits. Despite the successful transition from daily to weekly dosing with GLP-1 inhibitors, including GIPs, patient adherence remains a significant challenge. A delicate challenge with GLP-1 therapy is maintaining consistent, albeit low, levels of bioavailable drug concentrations, typically achieved through once-weekly administration of a stable, high dose of GLP-1. Unfortunately, this dosing regimen can trigger serious side effects, including post-injection nausea and vomiting or paralytic ileus with prolonged use, often leading to poor treatment adherence.

[0109] The inventors have surprisingly discovered that the use of the recombinant symbiont produces a significant sustained release of GLP-1 for up to three months. Using the GLP-1-expressing recombinant symbiont described herein offers dual advantages: firstly, it eliminates the side effects associated with current therapies, and secondly, it significantly reduces the frequency of high-dose injections, from once weekly to once quarterly or even once a year, thus facilitating easy integration into various lifestyles. Given the biological characteristics of GLP-1 (a breakdown product of proglucagon), the prototype symbiont was engineered to express either proglucagon or GLP-1.

[0110] In this embodiment, the inventors operatively associated polynucleotides encoding proglucagon and GLP-1 (both derived from GenBank accession number Z46845.1) with highly active signal peptide 22 (SM22 – SEQ ID NO: 12), as described in Example 1. Proglucagon and GLP-1 secreted into growth medium or serum were tested in vitro and in vitro using a commercially available ELISA assay.

[0111] In this experiment, healthy mice were implanted with symbionts expressing GFP, proglucagon, or GLP-1 and fed a high-fat diet (Western diet model rat food) to promote weight gain, and the effect of the recombinant symbionts on weight gain was assessed. The inventors also confirmed the secretion of the target payload in the serum of all mice on day 21 post-implantation (i.e., 3 weeks).

[0112] Interestingly, such as Figure 7 As shown, mice implanted with GLP-1-secreting symbionts exhibited nearly 40% less weight gain compared to controls or the parent GLP-1 proglucagon. Furthermore, the mean serum concentration of GLP-1 in these mice was lower than that of the control payload, consistent with the molecule's biological characteristics (i.e., higher protein availability means greater utilization by the body). Notably, the parent proglucagon symbionts showed higher blood GLP-1 concentrations with normal weight gain, indicating that the GLP-1 ELISA used in this experiment could not distinguish between parent proglucagon and GLP-1. This also indicates the accumulation of unprocessed proglucagon from the symbiont in the bloodstream, confirmed by ELISA of proglucagon itself, demonstrating protein accumulation in the blood under stable secretion conditions. This suggests that ultra-stable payload design can improve treatment outcomes in the host.

[0113] Although the present invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention can be devised without departing from the true spirit and scope of the invention. The claims are intended to be construed as encompassing all such embodiments and equivalent variations.

[0114] Every patent, patent application, and publication cited in this document is incorporated herein by reference in its entirety.

Claims

1. A recombinant symbiont comprising a trypanosome microorganism engineered to express a heteropolynucleotide, wherein the heteropolynucleotide encodes a therapeutic protein or therapeutic polynucleotide.

2. The recombinant symbiont according to claim 1, wherein the trypanosome is Trypanosoma runnifolia (…). Trypanosoma rangeli ), Trypanosoma mesnilbrimontii , Trypanosoma preguici , Trypanosoma myrmecophague , Trypanosoma mycetae , Trypanosoma diasi , Trypanosoma cebus , Trypanosoma saimiri or Trypanosoma advieri .

3. The recombinant symbiont according to claim 2, wherein the trypanosome is Trypanosome langurii.

4. The recombinant symbiont according to any one of claims 1-3, wherein the heteropolynucleotide encodes proinsulin.

5. The recombinant symbiont according to any one of claims 1-3, wherein the heteropolynucleotide encodes GLP-1.

6. The recombinant symbiont according to any one of claims 1-3, wherein the heteropolynucleotide encodes GIP.

7. The recombinant symbiont according to any one of claims 1-3, wherein the heteropolynucleotide encodes β-glucocerebroside lipase.

8. The recombinant symbiont according to any one of claims 1-3, wherein the heteropolynucleotide: a. It is an integrated foreign nucleic acid, artificial chromosome, or nucleic acid fragment containing specific genetic features for recombination and utilization of inherent genetic elements; b. Encoding 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. A therapeutic protein encoding an enzyme, interleukin, hormone, antibody, coagulation factor, growth factor, or peptide; and / or d. Encoding enzyme pathway.

9. The recombinant symbiont according to any one of the preceding claims, wherein the heteropolynucleotide encodes a signal sequence selected from SEQ ID NO: 1-15.

10. The recombinant symbiont according to any one of the preceding claims, wherein the heteropolynucleotide is operatively associated with a heteropromoter.

11. The recombinant symbiont according to any one of the preceding claims, wherein: a. The enzyme is selected from asparaginase, pancreatic lipase, Clostridium histolytica collagenase, α-glucosidase, imiglucosidase, α-vilasidase, α-talidase, laronidase, idoosulfatase, thioglucosidase, or pegolone acetonide. b. The interleukin is selected from interleukin-2, interleukin-6, interleukin-4, interleukin-11 or interleukin-12; c. The hormones are selected from insulin, levothyroxine, adrenaline, glucagon, estrogen, progesterone, testosterone, human growth hormone, corticosteroids, desmopressin, parathyroid hormone, oxytocin, calcitonin, leuprorelin, goserelin, octreotide, or α-thyroid-stimulating hormone. d. The antibody is selected from adalimumab, aflibercept, alenmab, atezolizumab, baliximab, belimumab, bevacizumab, bonatumab, ventoxicum, vedotin, cananulumab, caprilumab, cetoxicum, cetuximab, daralimumab, denosumab, detoxicum, durvalumab, dupilumab, iculizumab, erlotuzumab, imalumab, gemutuzumab, oxazolidin, golimumab, ibalizumab, oxalizumab, ipilimumab, and ixazolizumab. Monoclonal antibodies, lanalidomide, rotezip, mepolizumab, moglizumab, natalizumab, nexitozumab, nivolumab, oxotuzumab, oftatuzumab, oxotuzumab, oxotuzumab, oxotuzumab, panitumab, pembrolizumab, ramosinidomide, rituximab, saliluzumab, secuciyu, tocilizumab, trastuzumab, ustekinumab, vedocilizumab, reticulizumab, tesalenzab, broluzumab, vetolasen, idekiolenzab, dotalizumab, or satolizumab; e. The coagulation factor is selected from factor VIII, factor IX, factor VIIa, 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 XIIIa, factor XIIIb, recombinant factor VIII Fc fusion protein, recombinant factor IX Fc fusion protein, emecizumab (Hemlibra), or Leyton factor V; 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-β (TGF-β), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), erythropoietin (EPO), or thrombopoietin (TPO); and / or g. The peptides are selected from abapatide, angiotensin II, bivalirudin, bumelantide, seserolelin, carbetocin, cetrorex, desmopressin, exenatide, glatiramer acetate, goserelin, liraglutide, lisiratide, nesiritide, oxytocin, pramlintide, smegglutide, semeprazole, teriparatide, teduglutide, triptorelin, linaclotide, pucanatide, paretide, terlipressin, angiotensin, thymosin, sermorelin, leuprorelin, lanreotide, temorelin, degarelix, pucanatide, and bivalirudin.

12. A method for expressing heteropolynucleotides in a host, the method comprising: a. Applying the recombinant symbiont according to any one of claims 1-11 to the host; and b. Express the heteropolynucleotide in the host.

13. A method for preventing or treating a host suffering from a disease or disorder, the method comprising: a. Applying the recombinant symbiont according to any one of claims 1-11 to the host; and b. Express the heteropolynucleotide to treat, alleviate or prevent symptoms of the disease or disorder.

14. A method for modifying the physiology or homeostasis of a host, the method comprising: a. Applying the recombinant symbiont according to any one of claims 1-11 to the host; and b. Express the heteropolynucleotide in the host to modify the host's physiology or homeostasis.

15. The method according to any one of claims 12-14, wherein the method further comprises translating the heteropolynucleotide into a therapeutic peptide.

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

17. The method according to any one of claims 12-16, wherein: a. The host suffers from cancer, genetic defects, infectious diseases, or autoimmune diseases; b. The method modifies the host's physiological and / or organ systems; c. The host suffers from diabetes, Gaucher disease, or obesity; and / or d. The host suffers from cystic fibrosis, sickle cell disease, hemophilia, Duchenne muscular dystrophy, Huntington's disease, β-thalassemia, macular degeneration, muscular dystrophy, Leber congenital amaurosis, severe combined immunodeficiency, immunodeficiency, retinitis pigmentosa, Fabry disease, Pompe disease, Wilson's disease, orphan disease, amyotrophic lateral sclerosis, Allport syndrome, X-linked adrenoleukodystrophy, phenylketonuria, Marfan syndrome, or hereditary angioedema.

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

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