In vitro production of heterologous polynucleotides and polypeptides in trypanosomes

Abstract

The invention relates to large-scale, in vitro production of biological molecules by genetically modified Trypanosomes. The invention has applications to various fields, such as medicine, human performance, and veterinary medicine. In various embodiments, the use of Trypanosomes may be purposefully engineered, altered, and designed to express foreign nucleic acid sequences that encode secreted proteins, polynucleotides, and / or pathways that can be used to treat disease or enhance specific aspects of host physiology to include metabolic dominance to correct or augment homeostatic imbalances when suitable. The engineered Trypanosomes may be used in the commercial production of enzymes, scavenger molecules, peptides and hormones, antibodies, nanobodies, signaling ligands, and polynucleotides in vitro under controlled, large-scale manufacturing conditions, which can then be recovered and administered to a host.

Description

IN VITRO PRODUCTION OF HETEROLOGOUS POLYNUCLEOTIDES AND POLYPEPTIDES IN TRYPANOSOMESSTATEMENT REGARDING FEDERALLY 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.BACKGROUND OF THE INVENTIONField of the Invention

[0001] The present invention relates to an improved method of in vitro production of polynucleotides and polypeptides, using genetically modified Trypanosomes.Background

[0002] The ability to identify new methods for producing biological molecules using in vitro systems is of critical importance to advancing biotechnology, medicine, and industrial applications. In vitro systems, which operate outside living organisms, provide a controlled environment for studying and synthesizing biological molecules such as proteins, nucleic acids, and small molecules. These systems enable precise manipulation of variables, fostering innovation in the development of novel therapeutics, diagnostics, and materials. By leveraging in vitro approaches, researchers can overcome limitations of traditional methods, such as dependency on bacterial or fugal systems. Furthermore, in vitro systems pave the way for creating biomolecules that are challenging or impossible to produce naturally, supporting breakthroughs in synthetic biology, personalized medicine, and sustainable manufacturing. Ultimately, these advancements hold the promise of transforming how we address global challenges in health, environment, and industry.

[0003] Thus, there is a continuing need for improved in vitro systems for producing substances, including proteins or polynucleotides that can be used, for example, 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 methods of making biological molecules at a lower cost of goods. The invention relates to in vitro generation of biological molecules by genetically modifying Trypanosomes to produce such biological molecules. The invention has applications in various fields, such as human medicine, human performance, and veterinary medicine.

[0005] As disclosed herein. Trypanosomes (either naturally occurring or derived in the laboratory) are purposefully engineered, altered, and designed to generate heterologous polynucleotides or polypeptides, including entire pathways in vitro and at high levels (e.g., scaled up for commercial purposes).

[0006] In vitro large-scale production of heterologous polynucleotides or polypeptides in Trypanosomes offers multiple advantages over existing bacterial, fungal or mammalian production systems. Inherent to Trypanosomes is both O- and N-linked glycosylation patterns consistent with mammalian systems. Moreover, the lack of endotoxins produced by Trypanosomes eliminates this downstream processing step. Additionally, large-scale production of Trypanosomes allows a xenofree culture system for feed which is substantially less costly than standard mammalian culture systems. Finally, focusing the production on secreted proteins eliminates the need for a cell lysing step. The combination of these features creates an unexpected opportunity for produce large quanties of sterile products with minimal downstream processing.

[0007] The Trypanosomes may be genetically altered so as to generate enzymes, scavenger molecules, peptides and hormones, antibodies, nanobodies, signaling ligands, and production of other therapeutic or novel synthetic agents, including precursors. The engineered Trypanosomes can be used for the sustained and commercial production of biological molecules to treat disease or enhance health in a host or other industrial purposes.

[0008] Moreover, Trypanosomes can be further modified to produce novel molecules to be used 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.

[0009] Specifically, a Trypanosome is engineered to express a heterologous polynucleotide, wherein said heterologous polynucleotide is the desired product or encodes for a heterologous polypeptide (as used herein, collectively a “biological molecule”). Examples of such Trypanosomes include, but are 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) expressed from a polynucleotide integrated in the Trypanosome ’s genome; (b) expressed from an episome; (c) 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; (d) 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); (e) encode for a polypeptide, such as an enzyme, an interleukin, a hormone, an antibody, a clotting factor, a growth factor, or a peptide; and / or (f) encode for an enzyme pathway.

[0011] In preferred embodiments, the polypeptide produced as a secreted polypeptide, and therefore the heterologous polynucleotide will also encode a signal sequence. In some embodiments, the signal sequence is selected from SEQ ID NO: 1-15. Furthermore, the heterologous polynucleotide can also be operatively associated with a heterologous promoter.

[0012] Although an initial report published over 25 years ago demonstrated that a fluorescent protein was expressed in a Trypanosome, this experiment was designed to express the protein intracellularly for use as a research tool. See, e.g., Guevara et al., “Expression of fluorescent genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): its application to parasite-vector biology.” J Med Entomol. 2005 Jan;42(l):48-56. doi: 10.1093 / jmedent / 42.1.48. PMID: 15691008. Intracellular expression of a low amount of the fluorescent protein (e.g.. pg quantities) was then used as a tag to trace the life cycles of different strains of Trypanosomes. However, to be used as a tag requires that tag be expressed in amounts sufficient to be inherited while not disrupting the endogenous host pathways or causing cell death. Moreover, to be used as a tag, the tag is not secreted or recovered. Instead, the authors teach how the described research tools can be used “[o]verall, tagged parasites can be used to determine cell-to-cell interactions, quantify parasitepenetration, discriminate between previous infections and reinfection, and identify the presence of parasitic cells at chronic stages of infections in animal models.” See, e.g., page 55, last sentence of the paper. However, at no point do the authors suggest that the tagged Tiypanosomes could be used to produce and recover any quantities of the tag protein, either under large or small-scale conditions.

[0013] Large-scale manufacturing of biological molecules is characterized by its capacity for high-volume production, utilizing fermentors or bioreactors with capacities often ranging from 100 liters to tens of thousands of liters. These systems can yield kilograms of product per batch, with recovery amounts reaching hundreds of grams per liter of culture. In contrast, research-scale production, typically conducted in shake flasks or benchtop bioreactors under 10 liters, produces milligram to gram quantities, with recoveries often in the range of a few milligrams to tens of milligrams per liter. This substantial difference in yield reflects the focus of large-scale operations on maximizing efficiency and productivity, while small-scale setups prioritize flexibility and experimental optimization.

[0014] A significant distinction lies in the operational environment and regulatory requirements. Large-scale production is performed in GMP-compliant facilities with classified cleanrooms, rigorous environmental controls, and validated processes to ensure product consistency and regulatory compliance. These facilities rely on cost-optimized media and bulk raw materials designed to enhance yield while reducing costs. Conversely, small-scale production is carried out in standard laboratory environments using high-purity reagents and pre- sterilized equipment, with less emphasis on costefficiency and more on precision for experimental reproducibility.

[0015] The end goals and downstream processes further differentiate the scales. Large-scale operations produce material for therapeutic, industrial, or commercial applications, often over prolonged production schedules, with recovery systems tailored to maximize product purity and efficiency on an industrial scale. In contrast, research-scale production supports preclinical studies or protocol development, generating smaller quantities over shorter timeframes. The substantial disparity in product yield, equipment, quality control, and intended use underscores the gap between the high-efficiency focus of large-scale manufacturing and the experimental flexibility of small-scale production.

[0018] Examples of proteins that can be expressed by the engineered Trypanosomes include prepro-insulin, GLP-1, GIP, or P-Glucocerebrosidase.

[0019] Other examples of proteins or polynucleotides that can be expressed by the engineered Trypanosomes include, but are not limited to: (a) enzymes such as asparaginase, pancrelipase, collagenase from Clostridium histolyticum, alglucosidase alfa, imiglucerase, velaglucerase alfa, 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: (e) 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-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) 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.

[0020] Thus, as contemplated herein are methods of expressing a heterologous polynucleotide in a Trypanosome in vitro comprising: growing the recombinant Trypanosome under in vitro conditions to express the heterologous polynucleotide from the Trypanosomes. Additionally, the heterologous polynucleotide can be used to encode for a heterologous polypeptide. The heterologous polynucleotide or the heterologous polypeptide produced by the heterologous polynucleotide can then be recovered. Specifically contemplated is the large-scale production of the biological molecule from a Trypanosome.

[0021] Using Trypanosomes for large-scale manufacturing of secreted polypeptides leverages many of the Trypanosomes properties while eliminating many post-processing steps. For example, Trypanosomes glycosylate proteins, similar to mammalian organisms, lack endotoxins, and can be grown in xenofree culture systems. The production in Trypanosomes of a secreted polypeptide eliminates the need for a cell lysing step, making the methods described herein even more efficient and improving recovery. The combination of these features creates an unexpected opportunity to produce large quantities of sterile products with minimal downstream processing.

[0022] Additionally, methods of preventing or treating a host suffering from a disease or disorder using the recovered heterologous polynucleotide or the heterologous polypeptide produced by the heterologous polynucleotide in the Trypanosome (as used herein “the recovered product”), comprising: administering to a host the heterologous polynucleotide or heterologous polypeptide produced by the heterologous polynucleotide expressed by the Trypanosomes to treat, reduce or prevent the symptoms of said disease or disorder.

[0023] 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 heterologous polynucleotides / polypeptides produced by the recombinant Trypanosomes can be used to treat diabetes, Gaucher’s Disease, and / or obesity. Additionally, the polynucleotides / polypeptides produced by the engineered Trypanosomes 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, orphandiseases, amyotrophic lateral sclerosis, alport syndrome, X-Linked adrenoleukodystrophy, phenylketonuria, Marfan’s Syndrome, or hereditary angioedema.

[0024] The recovered heterologous polynucleotides / polypeptides produced by the methods described herein could also be used to modify the host’s physiology and / or organ systems.

[0025] Delivery of the heterologous polynucleotides / polypeptides produced by the engineered Trypanosomes can be oral, intravenous, pulmonary, intramuscular, subcutaneous, or intraperitoneal routes.

[0026] In specific embodiments, the invention is as follows:1. A method of producing a heterologous polynucleotide in vitro, wherein said method comprises:a. expressing from a recombinant Trypanosome a heterologous polynucleotide; b. optionally translating a heterologous polypeptide from the polynucleotide; and c. recovering said heterologous polynucleotide or heterologous polypeptide.2. The method of claim 1 , wherein the heterologous polynucleotide encodes for the heterologous polypeptide.3. The method of either claim 1 or 2, wherein the heterologous polynucleotide encodes for a secreted polypeptide.4. The method of any one of the previous claims, wherein the heterologous polynucleotide further comprises a signal sequence selected from any one of SEQ ID NO: 1-15.5. The method of any one of the previous claims, wherein the heterologous polynucleotide is operatively associated with a heterologous promoter.6. The method of any one of the previous claims, wherein at least 0.5g of the heterologous polynucleotide or heterologous polypeptide is recovered.The method of any one of the previous claims, wherein said method produces at least 40% pure recovered heterologous polynucleotide or heterologous polypeptide.The method of any one of the previous claims, wherein said method:a. produces at least 1g, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g or 15g of the recovered heterologous polynucleotide or heterologous polypeptide;b. produces at least 50%, 60%, 70%. 80%, or 90% pure recovered heterologous polynucleotide or heterologous polypeptide; orc. is carried out in a 10L, 100L, WOOL, 5000L, or 10000L fermenter.The method of any one of the previous claims, wherein the recombinant Trypanosome is derived from Trypanosoma rangeli, Trypanosoma mesnilbrimontii, Trypanosoma preguici, Trypanosoma myrmecophague, Trypanosoma mycetae, Trypanosoma diasi, Trypanosoma cebus, Trypanosoma saimiri, or Trypanosoma advieri.The method of any one of the previous claims, wherein the heterologous polynucleotide encodes prepro-insulin.The method of any one of the previous claims, wherein the heterologous polynucleotide encodes GLP-1.The method of any one of the previous claims, wherein the heterologous polynucleotide encodes GIP.The method of any one of the previous claims, wherein the heterologous polynucleotide encodes P-Glucocerebrosidase.The method of any one of the previous claims, wherein the heterologous polynucleotide: a. is integrated into the Trypansome genome;b. is an extrachromosomal episome;c. is a foreign nucleic acid, artificial chromosome, or a nucleic acid fragment containing specific genetic features for recombination and utilization of native genetic elements; d. encodes for a ribonucleic acid, such as 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);e. encodes for an enzyme, an interleukin, a hormone, an antibody, a clotting factor, a growth factor, or a peptide; and / orf. encodes for an enzyme pathway.method 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;Q. 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 VUI / 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 / org. the peptide is selected from 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.The heterologous polynucleotide or heterologous polypeptide produced by the method of any one of the previous claims.Use of the heterologous polynucleotide or heterologous polypeptide produced by the method of any one of the previous claims.The use of claim 17 to treat a disease or a disorder selected from:a. cancer, a genetic deficiency, an infectious disease, or an autoimmune disease; b. diabetes, Gaucher’s Disease, or obesity; and / orc. 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.19. The use of either claim 17 or 18, wherein the heterologous polynucleotide or heterologous polypeptide is administered to a host.20. The use of claim 19, wherein the host is a human, canines, felines, equines, live-stock (e.g., cattle, goats, sheep, or pigs), chicken or other birds, fish, or any other animal, that is healthy or one that has compromised health.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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:

[0028] FIG. 1 shows exemplary approaches for transforming a Trypanosome into a genetically engineered Trypanosome and shows an example of in vitro production rates of proteins by different signal peptides into the reaction mediums of engineered Trypanosome. These signal sequences are preferably used to produce secreted polypeptides.

[0029] FIG. 2 lists candidate genomic sites for integration that can be used to integrate the heterologous polynucleotide into the Trypanosome genome.

[0030] FIG. 3 lists non-limiting families of signal peptides for use in the present invention.

[0031] 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

[0032] The invention relates to the in vitro generation of biological molecules, such as heterologous polynucleotides or heterologous polypeptides encoded by said polynucleotides, using genetically modified Trypanosomes. Examples of biological molecules that can be produced by the engineered Trypanosomes include but are not limited to, 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 in 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. Biological molecules include polynucleotides, such as RNAi..

[0033] FIG. 2 lists non-limiting candidate genomic sites that can be used to integrate the heterologous polynucleotide in the Trypanosome.

[0034] The definitions herein provide guidance to the subject matter expertise, the broadest application examples, and the appended claims.OVERVIEW

[0035] As used herein, a “host” that can be administered a biological molecule produced by a recombinant Trypanosome as described herein, includes, but is not limited to a human, canines, felines, equines, live-stock (e.g., cattle, goats, sheep, or pigs), chicken or other birds, fish, or any other animal, that is healthy or one that has compromised health.

[0036] A “recombinant” or “engineered” Trypanosome as used herein is a Trypanosome that has been engineered and specifically designed to produce a heterologous, biological molecule (i.e., a heterologous polynucleotide and / or heterologous polypeptide - also referred to as “recovered product”). Such recovered products can be used for industrial or therapeutic purposes, such as to enhance or modify the host’s physiology.

[0037] Thus, described herein is a method for adapting a Trypanosome for the sustained production of biological molecules in vitro, comprising the steps of: (a) genetically modifying a Trypanosome, such as by integrating a foreign nucleic acid, an artificial chromosome, or a nucleic acid fragment comprising a polynucleotide encoding a biological molecule and also containing specific genetic features for recombination and utilization of native genetic elements into the Trypanosome to produce an engineered Trypanosome', (b) enabling the engineered Trypanosome toexpress the biological molecule (e.g., a heterologous protein or heterologous polynucleotide), and optionally recovering the biological molecule.

[0038] In embodiments, the engineered Trypanosome 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 engineered Trypanosome can be used to produce molecules that will modify host physiology, homeostasis and organ systems.

[0039] The biological molecules produced by the engineered Trypanosome can be administered to the host via oral, intravenous, intrapulmonary, intramuscular, subcutaneous, or intraperitoneal routes.

[0040] The engineered Trypanosome can be derived from a Trypanosoma rangeli (“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 engineered Trypanosome can be derived from laboratory strains ATCC #30032 or ATCC #30033.GENERAL DESIGN OF CONSTRUCT

[0041] The term “recombinant” or “engineered” Trypanosome refers to a Trypanosome having been modified to encode a biological molecule in vitro. The biological molecule can be encoded by the engineered Trypanosome from an episome or a heterologous polynucleotide that has been integrated into the Trypanosome’s genome. When incorporated into the Trypanosome’ s genome, natural genetic sequences may be subtracted during the addition of the foreign sequences that render the desired beneficial features or phenotypes.

[0042] As used herein, “heterologous” means a polynucleotide or polypeptide not naturally expressed by the Trypanosome from which the recombinant Trypanosome is derived. A heterologous polynucleotide can be derived from any other organism other than the species of Trypanosome used to generate the engineered Trypanosome.

[0043] Methods of adding or subtracting sequences to modify the Trypanosome’ s natural features enables the engineered Trypanosome to have unique features in the laboratory that are not present innature, and 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.

[0044] 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 Trypanosome are targeted by nucleotide fragments of foreign nucleotide sequences flanked by natural complementary nucleotide sequence native to the Trypanosomes. 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.

[0045] 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 a targeted or untargeted genomic integration system. Additionally, the biological molecule can be expressed in vitro from a stable episomal polynucleotide. The heterologous polynucleotide encoding the biological molecule may contain specific genetic elements for constitutive, inducible, or targeted expression of the biological molecule in vitro. Alternatively, the biological molecule can be expressed from a polynucleotide that has integrated into the engineered Trypanosome’s genome.

[0046] Genetic modifications contemplated in this invention renders the engineered Trypanosome distinct from its naturally-occurring counterpart to enhance the production of biological molecules in large scale systems. In various embodiments, the foreign genetic sequences may be used to render metabolic advantage against toxic chemicals like antibiotics to positively identify Trypanosomes with a successfully engineered feature. A non-limiting list of biological molecules that can be expressed from the recombinant Trypanosomes include (1) RNAs, (2) polypeptides, including secreted polypeptides, or (3) polypeptides that lead to free metabolites (e.g. carbohydrates, lipids, amino acids, steroids, fatty acids, vitamins, or anti-infectious agents) as an end-product that can, for example, modify host physiology.

[0047] 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 Trypanosome using standard recombinant methods, such as disclosed in Sambrook et al., 2001.

[0048] 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 asingle recombinant Trypanosomes can utilize cholesterol as feed to generate testosterone. Therefore, this pathway can be encoded by multiple different recombinant Trypanosomes grown as a mixture (to produce a mixture of proteins); expressed in a single recombinant Trypanosome or as individual processes in which the recovered proteins are combined.

[0049] As used herein, the term “native drivers” refers to endogenous regulatory elements allowing for the targeting at genomic integration sites located in the Trypanosome’ 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. A non-limiting list of candidate sites with variable exploitable features and chromatin activity may be found in FIG. 2. These locations are ideal for the insertion of a heterologous polynucleotide encoding a biological molecule as described herein as the transgene can be expressed at high, moderate or reduced levels depending on site of integration.

[0050] 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.

[0051] A coding sequence is "under the control of’ or "operatively associated with" an expression control sequence when a signal for 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 MAKING BIOLOGICAL MOLECULES USING TRYPANOSOMES

[0052] The engineered Trypanosomes of the present disclosure may be made using recombinant techniques as described herein or known in the art. For example, at least one heterologous polynucleotide could be cloned into an expression vector that would be transcribed when transfected or transformed into a Trypanosome. In embodiments, an expression vector may comprise a plasmid. The heterologous polynucleotide can be produced using standard molecular biology approaches, for example, by using polymerase chain reactions to produce the heterologous polynucleotide, which is then purified and cloned into an expression vector and transfected or transformed into the Trypanosome. Additional techniques useful in the practice of this disclosure may be found in CurrentProtocols in Molecular Biology 2007 by John Wiley and Sons, Inc.; Molecular Cloning: A Laboratory Manual (Third Edition) Joseph Sambrook, Peter MacCallum Cancer Institute, Melbourne, Australia; David Russell, University of Texas Southwestern Medical Center, Dallas, Cold Spring Harbor.

[0053] Selecting a particular heterologous polynucleotide is well within the skill in the art. For example, codon usage tables for a Trypanosome may be used to generate a reverse complement that encodes a heterologous polypeptide. See, for example, The Sequence Manipulation Suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28:1102-1104 (bioinformatics.org / sms2 / rev_trans.html) or the Codon Usage Table: From the codon usage database (http: / / www.kazusa.or.jp / codon / ): Homo sapiens [gbpri]: 93487 CDS's (40662582 codons). Alternatively, codon frequencies can be optimized for use in the Trypanosome using frequency data such as that available from various codon usage records. One such record is the Codon Usage Database. Y. Nakamura et al.. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000.” Nucl. Acids Res. 28, 292 (2000). Using these techniques, one skilled in the art can readily generate a “codon-optimized” polynucleotide sequence that encodes a desired heterologous polypeptide sequence.

[0054] In further embodiments, the polynucleotides can be operably joined to a promoter. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. In embodiments, the heterologous polynucleotide can further comprise transcriptional and translational regulatory sequences. The transcriptional and translational regulatory signals may be obtained or derived from viral sources, such as a retrovirus, adenovirus, bovine papilloma virus, simian virus, or the like. In preferred embodiments, the regulatory regions are derived from 'Trypanosomes.

[0055] In embodiments, at least one polynucleotide is inserted into a vector capable of integrating the desired sequences into the Trypansosome. Additional elements may also be needed for optimal synthesis of the mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals and are all within the art.

[0056] Methods of introduction of the heterologous polynucleotide into the Trypanosome can be performed by using well-known methods, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, and the like. These techniques are all within the art. See, for example, Current Protocols in Molecular Biology 2007 by John Wiley and Sons, Inc.; Molecular Cloning: A Laboratory Manual (Third Edition) JosephSambrook, Peter MacCallum Cancer Institute, Melbourne, Australia; David Russell. University of Texas Southwestern Medical Center, Dallas, Cold Spring Harbor.

[0057] Additionally, heterologous polynucleotides can be directly injected into the Trypanosomes or may be impelled through cell membranes after being adhered to microparticles or nanoparticles, such as synthetic nanocarriers.

[0058] In certain embodiments, the heterologous polynucleotide encodes a heterologous polypeptide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity to a sequence encoding an approved product or product in developments. Examples of such products of described herein. Unless otherwise described, variants (such as those above having less than 100% sequence identity) of the heterologous polynucleotide sequence or polypeptide sequence retain the ability of the wild type protein from which the variant was derived, although the activity may not be at the same level. In preferred embodiments, the variant polypeptide or polynucleotide has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% efficacy compared to the original sequence. In preferred embodiments, the variant has improved activity as compared to the original sequence. For example, variants with improved activity have at least about 105%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, or at least about 160% efficacy compared to the original sequence. Variants can be identified by the person skilled in the art by conducting BLAST searches using the disclosed sequences, or literature searches using gene, enzyme, substrate or product names, and those variants can be tested using the methods of the Examples disclosed herein.

[0059] Recovery of the biological molecule from the Trypanosome using the methods described herein provides high levels of purified protein. For example, the biological molecule may be separated from crude protein extracts by methods known in the art, such as affinity chromatography. Alternatively, the biological molecule can be recovered and purified from recombinant Trypanosome cell cultures by well-known large-scale production methods including ammonium sulfate or ethanol precipitation, Protein A, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography or combinations thereof. In particular embodiments, high performance liquid chromatography ("HPLC") is employed for purification. For example, a Protein A and a second chromatography (e.g. MEP) column can be used when the biological moleculeis, for an example, an antibody. See, for example, Zarrineh, M et al. Analytical Biochemistry 113909 (2020); or Rosenberg et al. PLoS One 8:e58724 (2013) or Diamos et al. Front. Bioeng. Biotechnol.7(472)1-15 (2020).

[0060] In some embodiments, expression level of the biological molecule produced in the Trypanosome are in the range 100-2000 mg / kg per biomass, such as 200-2000 mg / kg per biomass, eg, 400-1400 mg / kg per biomass, or at least 500 mg / kg per biomass based on the signal peptide employed for the expression. In some embodiments, the recovered product is at 0.2% w / w to 2%w / w. In other embodiments, affinity chromatography is conducted using passage over a column to purify the biological molecule at >95% purity. In other preferred embodiments, the recovered biological molecule is obtained at >60%, >70% purity, >75% purity, >80% purity, >85% purity, >90% purity. >95% purity, >96% purity, >97% purity, >98% purity, >99% purity, and / or at 100% purity. The large scale production of the biological molecule from the Trypanosome, may result, for example, in bulk product of at least 0.3g / L, of at least 0.4g / L, of at least 0.5g / L, of at least 0.6g / L, of at least 0.7g / L, of at least 0.8g / L, of at least 0.9g / L, of at least l.Og / L, of at least l.lg / L, of at least 1.2g / L, of at least 1.3g / L, of at least 1.4g / L, or of at least 1.5g / L.

[0061] In some embodiments the biological molecule is recovered and is maintained in a liquid, frozen or powdered form.

[0062] Moreover, the biological molecule may be differentially modified during or after translation, e.g., by acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH.sub.4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

[0063] Additional post-translational modifications encompassed by the present disclosure include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue.METHODS OF TREATMENT

[0064] Methods of treating using the biological molecules produced by the in vitro methods described herein using the engineered Trypanosome are also disclosed.

[0065] For example, the engineered Trypanosomes can be used to produce biological molecules, such as those that 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.

[0066] FIG. 1C illustrates a representative scenario where T. rangeli was genetically engineered to produce various levels of prepro-insulin, an insulin precursor.

[0067] Thus, an engineered Trypanosome may be genetically engineered to produce prepro-insulin, GLP-1, GIP, or GCase (or any other of the therapeutic molecules described herein) in vitro that has the potential for therapeutic value to patients.EQUIVALENTS

[0068] 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.

[0069] 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.

[0070] The foregoing disclosure provides examples, illustrations and descriptions of the embodiments, 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.

[0071] 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.

[0072] 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.

[0073] 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

[0074] 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 of Engineered Trypanosomes

[0075] As depicted in FIG. 1, various methods and tools may be used to incorporate a polynucleotide encoding a biological molecule into a Trypanosome. For example, T. rangeli was used to generate an engineered Trypanosome. 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 Trypanosome that includes both a native promoter and a native signal peptide was used to ensuresecretion of payload. However, other strong promoters or signal sequences could be used interchangeably.

[0076] Once constructed, electroporation can be used to modify the genetic composition of T. rangeli permanently using a random integration with homologous end joining.

[0077] FIG. 2 provides a non-limiting list of candidate sites with variable chromatin state for for use in the present invention 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 engineered Trypanosome. Native signal peptides selected from proteins naturally encoded by T. rangeli were incorporated into the expression cassette.

[0078] FIG. 3 provides a non-limiting list of families of native signal peptides that can be used to facilitate secretion.

[0079] To create an engineered Trypanosome, a circular DNA can be introduced into a 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”).

[0080] Clones incorporating the biological molecule in the T. rangeli genome were then selected using antibiotic resistance and confirmed forexpression and secretion of molecules of interest in vitro.

[0081] A simple and inexpensive method for culturing the engineered 'Trypanosome can be 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 et 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 engineered Trypanosome include ATCC #30032 and ATCC #30033.

[0082] 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.

[0083] One method of manipulating DNA content is by amplifying or synthesizing long DNA fragments comprised of heterologous DNA sequences containing expression modifiers such signal peptides, beneficial features (e.g., enzymes and receptors) as well as a selectable features flanked byrecombination target sequences specific to T. rangeli for the purpose of preferred- site specific integration. For example, FIG. 2 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.

[0084] 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 engineered 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.Example 2: Engineered Trypanosomes Used to Produce Prepro-Insulin

[0085] 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 carboxypeptidase 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.

[0086] The therapeutic efficacy of prepro-insulin expressed from an engineered Trypanosome was assessed for activity. We created an engineered Trypanosome as described in Example 1 to express prepro-insulin while also testing seven distinct signal peptides in vitro.

[0087] 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.

[0088] 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 SM00 (SEQ ID NO:4) promoting intermediate secretion (FIG. 1C).Example 3: Engineered Trypanosomes Used to Produce GCase

[0089] Mutations in the GBA1 gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), 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 GBA1 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) into the engineered Trypanosome. To this end, efforts were directed to the design, select, and laboratoryscale produce GCase from the Trypanosome.

[0091] 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. In vitro tests using the 4MU-bc / o-glucosidase assay (standard GCase activity assay) demonstrated significant enzymatic activity of symbiont-secreted GCase.Example 4 - Use of Engineered Trypanosome to produce GLP-1.

[0092] 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-1 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.

[0093] 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 for the in vitro testing of proglucagon and GLP1 secretion into growth medium or serum.

[0094] 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.

[0095] 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 method of producing a heterologous polynucleotide in vitro, wherein said method comprises:a. expressing from a recombinant Trypanosome a heterologous polynucleotide; b. optionally translating a heterologous polypeptide from the polynucleotide; and c. recovering said heterologous polynucleotide or heterologous polypeptide.

2. The method of claim 1 , wherein the heterologous polynucleotide encodes for the heterologous polypeptide.

3. The method of either claim 1 or 2, wherein the heterologous polynucleotide encodes for a secreted polypeptide.

4. The method of any one of the previous claims, wherein the heterologous polynucleotide further comprises a signal sequence selected from any one of SEQ ID NO: 1-15.

5. The method of any one of the previous claims, wherein the heterologous polynucleotide is operatively associated with a heterologous promoter.

6. The method of any one of the previous claims, wherein at least 0.5g of the heterologous polynucleotide or heterologous polypeptide is recovered.

7. The method of any one of the previous claims, wherein said method produces at least 40% pure recovered heterologous polynucleotide or heterologous polypeptide.

8. The method of any one of the previous claims, wherein said method:a. produces at least 1g, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g. 14g or 15g of the recovered heterologous polynucleotide or heterologous polypeptide;b. produces at least 50%, 60%, 70%, 80%, or 90% pure recovered heterologous polynucleotide or heterologous polypeptide; orc. is carried out in a 10L, 100L, WOOL, 5000L, or 10000L fermenter.

9. The method of any one of the previous claims, wherein the recombinant Trypanosome is derived from Trypanosoma rangeli, Trypanosoma mesnilbrimontii, Trypanosoma preguici, Trypanosoma myrmecophague, Trypanosoma mycetae, Trypanosoma diasi, Trypanosoma cebus, Trypanosoma saimiri, or Trypanosoma advieri.

10. The method of any one of the previous claims, wherein the heterologous polynucleotide encodes prepro-insulin.

11. The method of any one of the previous claims, wherein the heterologous polynucleotide encodes GLP-1.

12. The method of any one of the previous claims, wherein the heterologous polynucleotide encodes GIP.

13. The method of any one of the previous claims, wherein the heterologous polynucleotide encodes P-Glucocerebrosidase.

14. The method of any one of the previous claims, wherein the heterologous polynucleotide: a. is integrated into the Trypansome genome;b. is an extrachromosomal episome;c. is a foreign nucleic acid, artificial chromosome, or a nucleic acid fragment containing specific genetic features for recombination and utilization of native genetic elements; d. encodes for a ribonucleic acid, such as 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);Q. encodes for an enzyme, an interleukin, a hormone, an antibody, a clotting factor, a growth factor, or a peptide; and / orf. encodes for an enzyme pathway.

15. The method 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 / org. the peptide is selected from 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.

16. The heterologous polynucleotide or heterologous polypeptide produced by the method of any one of the previous claims.

17. Use of the heterologous polynucleotide or heterologous polypeptide produced by the method of any one of the previous claims.

18. The use of claim 17 to treat a disease or a disorder selected from:a. cancer, a genetic deficiency, an infectious disease, or an autoimmune disease; b. diabetes, Gaucher’s Disease, or obesity; and / orc. 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-Einked adrenoleukodystrophy, phenylketonuria, Marfan’s Syndrome, or hereditary angioedema.

19. The use of either claim 17 or 18, wherein the heterologous polynucleotide or heterologous polypeptide is administered to a host.

20. The use of claim 19, wherein the host is a human, canines, felines, equines, live-stock (e.g., cattle, goats, sheep, or pigs), chicken or other birds, fish, or any other animal, that is healthy or one that has compromised health.

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