Recombinant parasites for delivering proteins to the central nervous system (CNS)

Abstract

To provide nucleic acid constructs, a Toxoplasma having the same, pharmaceutical compositions comprising the same and methods for delivering a protein of interest to a tissue of interest of a subject, such as the CNS.SOLUTION: The invention provides a Toxoplasma transformed with a nucleic acid construct comprising a heterologous polynucleotide in which a first nucleic acid sequence encoding a Toxoplasma secreted protein is inframe fused upstream to a second nucleic acid sequence encoding a pharmaceutical polypeptide for transcriptional modulation and genome editing. The heterologous polynucleotide is operably linked to a promoter for directing transcription of the heterologous polynucleotide in the Toxoplasma, with the proviso that the promoter is not a Toxofilin promoter. The Toxoplasma secreted protein is a non-rhoptry protein and secreted into host cells. The Toxoplasma is for transcriptional modulation and genome editing in the host cells.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] In some embodiments, the present invention provides a method for treating Toxoplasma gondii. The composition of a pharmaceutical polypeptide fused to a polypeptide secreted by B. gondii and a nucleic acid construct for the secretion of Toxoplasma gondii, more particularly for treating a subject having the same. The present invention relates to, but is not limited to, pharmaceutical compositions for placing isn't it. [Background technology]

[0002] The lack of robust methods for the delivery of protein therapeutics poses a challenge in their translation into clinical treatment. Proteins cannot be mimicked by simple compounds, which is currently a major obstacle. They often perform a highly specific and complex set of functions that cannot be replicated (Nat Rev Drug Dis cov. 2008; 7(1):21-39. Protein therapeutics: a summary and pharmacological class ification. Leader B, Baca QJ, and Golan DE). However, due to its polymeric nature, Therefore, delivery of therapeutic proteins to target sites in the body is extremely challenging. In addition to low permeability to steroids, they also have low functional stability, resulting in rapid loss of activity after administration or during storage. Rapid loss of the active protein limits its delivery. Ongoing development of therapeutic proteins and carrier systems has contributed to the delivery of some therapeutic proteins, but these Many of these are located within the "accessible target space," i.e., in the vascular compartment or in the cellular remains limited to surface targets (Mitragotri 2014, Nat. Rev. Drug. Discov. 13(9):655-72). The blood-brain barrier (BBB) ​​tightly regulates the transport of molecules into the brain, Efficient delivery is particularly challenging in the field of transdermal disease, but when the target is intracellular, the complexity is minimal. will increase even more.

[0003] To deliver replacement proteins to cells in the CNS, it is necessary to cross the BBB and target specific proteins within the CNS. It is necessary to develop mechanisms that can target specific cells.

[0004] Currently, the main approach to treat pathologies caused by the deficiency of specific proteins is , gene therapy, stem cell therapy and enzyme replacement therapy.

[0005] Gene therapy aims to increase the expression of functional copies of therapeutic proteins. , a technique for inserting a functional copy of a gene that encodes a therapeutic protein (Cox, D. BT, et al., 2015). Gene therapy provides the exact sequence of a gene, but does not modify the translational machinery or or post-translational modification defects that result in or are associated with defects in protein function. Furthermore, gene therapy does not address the loss of a gene. Baum, C. 2011), mobility, germline transmission, immunogenicity, and limited transgene transformation potential (Schambach, A., et al., 2013; Al-Dosari et al., 2009) To target the CNS, gene therapy is most commonly performed using viral vectors. The viral vector is mediated by the use of herpes simplex virus type 1 ( HSV-1), adenovirus, AAV, lentivirus (e.g., HIV-1, feline immunoglobulin G1), virus or equine infectious anemia virus), and more recently SV40-AAV and lentiviruses are the most common of these. Although the virus vector is safer than others, it addresses defects in protein processing. However, clinical efficacy remains limited.

[0006] Stem cell therapy utilizes the protein synthesis mechanism of transplanted cells, and Much effort has been made to implement this approach clinically. Despite the benefits, the efficacy of such treatments is limited by the rejection of transplanted cells and This is hampered by the low cost-effectiveness of using patient-specific stem cells. Risks associated with this treatment include the immunosuppression required to reduce rejection of the transplanted cells. These include side effects associated with steroid use and the development of cancer (Dimmeler, S., et al., 2014).

[0007] Protein or enzyme replacement therapy, in which replacement proteins are synthesized outside the body and then delivered into the CNS For ERT to be clinically meaningful, it is necessary to overcome the impermeability of the BBB and Accurate targeting methods must be developed. One method involves direct injection into the brain or into adjacent organs where proteins may diffuse less efficiently. Thus, in addition to the risks of such a procedure, the required amount of Another problem with such approaches is that they are often ineffective in clinical trials. The need for repeated administration is often difficult to implement practically (Abbott, NJ 2013).

[0008] Combining ERT with increased BBB permeability allows therapeutic proteins to penetrate the CN more efficiently. It can be made to diffuse within S (Malhotra, M. & Prakash, S. 2011). This , achieved by shrinking the cells that make up the BBB or by manipulating transport mechanisms through the BBB However, this approach is not efficient due to opsonization and the target recruitment protein is not easily detected. There is a risk of unwanted substances spreading into the CNS along with the substance (Bradbury, MWB 2012).

[0009] An alternative approach is to conjugate the therapeutic protein to a component that enters the CNS and crosses the BBB. This utilizes their inherent ability to mediate transport across the BBB. Drugs that can be used include fusion proteins, dendrimers, solid lipid nanoparticles, liposomes, and Nanoparticles are one example (Solaro, R., 2010). However, these methods require further development. The drug requires further development and has not yet reached clinical trials.

[0010] Toxoplasma gondii is a unicellular intracellular parasitic protozoan of the phylum Apicomplexa. The primary host of Toxoplasma gondii is the feline family, and only in felines However, Toxoplasma gondii can undergo the sexual phase of its life cycle. , can infect many warm-blooded organisms, including humans, as secondary hosts. Similarly, after the host is infected with the parasite (typically by ingestion of infectious tissue cysts or oocysts), After the procedure, the parasite differentiates into the rapidly replicating tachyzoite stage. Zoites invade nucleated host cells by active penetration and establish a parasitophorous vacuole within which They replicate by endogenous binary fission. Tachyzoites migrate through the intestinal epithelium and bind to immune cells. They reach distal tissues by both "hitchhiking" and spontaneously transporting themselves, and It penetrates the brain barrier and spreads throughout the brain [Harker 2015, Parasite Immunol. 37(3):141-9]. Tachyzoites then invade the circulation and disseminate to secondary tissues. Replication is the process of dissemination and infection. Once in the unique environment of the brain, Soplasma gondii attacks cells in the brain (mainly neurons, but also a small percentage of glial cells). After immune pressure, they differentiate into latent bradyzoite stages and then into intracellular cysts in the cytoplasm. This characterizes chronic infection (Cabral et al. 2016, PLoS Pathog. 12(2):e The tissue cysts that house the bradyzoites grow very slowly and contain dormant metabolic processes. It persists for the life of the host while retaining its original program (Parasite Immunol. 2015, 37(3):141-9. “Toxoplasma gondii dissemination: a parasite's journey through the infected host”).

[0011] Toxoplasma gondii infects an estimated one-third of the world's population, but the infection In normal humans, the condition remains asymptomatic (Montoya, JG & Liesenfeld, O. 2004) and is not associated with regular It is only a risk to individuals with severely compromised immune systems. During cell invasion, Toxoplasma gondii secretes proteins into host cells, which contribute to the Upon entry, Toxoplasma gondii is an electron-dense secretory organelle. It is possible to have three types of micronemes, rhoptry, and dense granules. Entry into the host cell is mediated by sequential secretion of the contents of all three organelles, which They found that the apical membrane is formed when the parasite invades the host cell and when it resides inside it. Micronemes are exocytosed from the toxoplasmic reticulum (Dlugonska, H. 2008). Rhoptry is involved in the binding and penetration of Plasma gondii, while rhoptry is involved in the creation of transient structures, adhesion, and Rhoptry proteins are required for the migration of the apical septum and subsequent establishment of the PV. They are secreted upon initial contact with the host in a process called "spitting" (Boothroy d, JC et al., 2008). The dense granules secrete proteins throughout the various stages of the parasite. The secretory process coincides with the formation of an intravacuolar network, and It continues throughout its intracellular stay (Dlugonska, H. 2008, Carruthers, VB & Sibley, LD 19 97).

[0012] Further background art includes Koshy, AA et al. 2010, U.S. Pat. No. 8,673,288. No. 9, U.S. Patent Application Publication No. 20120045477, Lodoen MB, et al. . 2010. Cellular Microbiology, 12: 55-66. Summary of the Invention

[0013] According to an aspect of some embodiments of the present invention a method for producing a T. gondii gene encoding a Toxoplasma secretory protein A first nucleic acid sequence is located in frame upstream of a second nucleic acid sequence encoding a pharmaceutical polypeptide. A nucleic acid construct comprising a heterologous polynucleotide fused with , to a promoter for directing transcription of a heterologous polynucleotide in Toxoplasma gondii operably linked, and the promoter is a constitutive promoter, an inducible promoter, , a latency-specific promoter, and a Toxoplasma endogenous promoter. provided is a nucleic acid construct selected from the group consisting of: It is served.

[0014] According to an aspect of some embodiments of the present invention, a nucleic acid construct comprising at least two nucleic acid constructs. A nucleic acid system, wherein a first nucleic acid construct of the at least two nucleic acid constructs is a nucleic acid construct of the present invention. The nucleic acid construct of an embodiment, wherein a second of the at least two nucleic acid constructs is a nucleic acid construct comprising a selected A nucleic acid construct system is provided that includes a polynucleotide encoding the marker.

[0015] According to an aspect of some embodiments of the present invention, the nucleic acid construct of some embodiments of the present invention or Toxoplasma transformed with the nucleic acid construct system of some embodiments of the present invention is provided. can be.

[0016] According to an aspect of some embodiments of the present invention, the Toxoplasma gondii of some embodiments of the present invention and a pharmaceutically acceptable carrier.

[0017] According to an aspect of some embodiments of the present invention, a protein of interest is administered to the central nervous system of a subject. The method comprises administering to a subject an administration of Toxoplasma gondii or any of the embodiments of the present invention. administering the pharmaceutical composition of any one of the embodiments to administer the protein of interest to the central nervous system of the subject. A method is provided which includes:

[0018] According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need thereof. and administering to the subject Toxoplasma gondii of some embodiments of the present invention or and administering the pharmaceutical composition to the subject, wherein the subject is responsive to administration of the pharmaceutical polypeptide to the central nervous system. Thus, the administration of the compound to treat a subject who has been diagnosed with a treatable condition and is in need of treatment. A method is provided for placing a

[0019] According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need thereof. The first nucleic acid sequence encoding a Toxoplasma secretory protein is used to produce a pharmaceutical polypeptide. a heterologous polynucleotide fused in-frame upstream of a second nucleic acid sequence encoding administering to a subject Toxoplasma having a nucleic acid construct containing a heterologous polynucleotide a promoter for directing transcription of a heterologous polynucleotide in Toxoplasma gondii and wherein the subject is operatively linked to a pharmaceutical polypeptide that is administered to the central nervous system. and administering the compound to treat a subject in need of treatment who has been diagnosed with a condition treatable by the compound. A method is provided.

[0020] According to some embodiments of the present invention, the promoter may be a constitutive promoter, an inducible promoter, or The gene consists of a motor, a latency-specific promoter, and a Toxoplasma endogenous promoter. with the proviso that the promoter is not the toxophilin promoter.

[0021] According to some embodiments of the invention, the endogenous promoter is do not have.

[0022] According to some embodiments of the invention, the Toxoplasma is non-attenuated.

[0023] According to some embodiments of the invention, the Toxoplasma secreted protein is It is secreted from the rhoptry of the

[0024] According to some embodiments of the present invention, the Toxoplasma secretory protein secreted from the rhoptry The protein contains toxophilin and / or ROP1.

[0025] According to some embodiments of the present invention, the Toxoplasma secretory protein secreted from the rhoptry The protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 344 to 465.

[0026] According to some embodiments of the invention, the Toxoplasma secretory protein is a non-rhoptryta It is protein.

[0027] According to some embodiments of the present invention, the Toxoplasma secretory protein is a micronemucoprotein. The protein is selected from the group consisting of proteins and dense granule proteins.

[0028] According to some embodiments of the invention, the Toxoplasma secreted protein is It is secreted from dense granules of

[0029] According to some embodiments of the present invention, a Toxoplasma secretory protein secreted from dense granules is The protein contains GRA16 and / or GRA24.

[0030] According to some embodiments of the invention, the Toxoplasma secreted protein is It is secreted from the microneme.

[0031] According to some embodiments of the invention, the microneme-secreted protein has SEQ ID NO: The amino acid sequence is selected from the group consisting of Nos. 280 to 322.

[0032] According to some embodiments of the invention, the protein secreted from the dense granules is selected from the group consisting of SEQ ID NO: The amino acid sequence is selected from the group consisting of 234 to 279.

[0033] According to some embodiments of the invention, the Toxoplasma secreted protein is Contains Transgenic MIF (TgMIF) from Gondii.

[0034] According to some embodiments of the present invention, the heterologous polynucleotide is a Toxoplasma secretory protein. Toxoplasma gondii untranslated sequences upstream and / or downstream of the protein open reading frame It further comprises a unique repeat (UTR) nucleic acid sequence.

[0035] According to some embodiments of the present invention, the Toxoplasma 5'-untranslated region (5'-UTR) is located upstream of the Toxoplasma secretory protein open reading frame.

[0036] According to some embodiments of the present invention, the Toxoplasma 3'-untranslated region (3'-UTR) is located downstream of the Toxoplasma secretory protein open reading frame.

[0037] According to some embodiments of the present invention, the Toxoplasma 3'-untranslated region (3'-UTR) The nucleic acid sequences of GRA2 3'-UTR, GRA16 3'-UTR, GRA24 3' -UTR, SAG1 3'-UTR, or DHFR 3'-UTR.

[0038] According to some embodiments of the present invention, the Toxoplasma 5'-untranslated region (5'-UTR) The nucleic acid sequences of GRA2 5'-UTR, GRA16 5'-UTR, GRA24 5' -UTR, SAG1 5'-UTR, or DHFR 5'-UTR.

[0039] According to some embodiments of the present invention, the Toxoplasma endogenous promoter is the GRA2 promoter. promoter, GRA16 promoter, GRA24 promoter, SAG1 promoter , or the DHFR promoter.

[0040] According to some embodiments of the present invention, the nucleic acid sequence of the Toxoplasma untranslated region (UTR) is , toxophilin 3'-UTR.

[0041] According to some embodiments of the invention, the nucleic acid construct encodes an inducible self-destruct element. The nucleic acid sequence further comprises a third nucleic acid sequence.

[0042] According to some embodiments of the invention, a third nucleic acid encoding an inducible self-destruct element The sequence is selected from the group consisting of: a first nucleic acid sequence encoding a Toxoplasma secretory protein; a heterologous polynucleotide fused in frame upstream of a second nucleic acid sequence encoding a peptide; The gene is contained in the same nucleic acid construct as the gene that contains the target gene.

[0043] According to some embodiments of the invention, a third nucleic acid encoding an inducible self-destruct element The sequence is selected so that the first nucleic acid sequence encoding the Toxoplasma secretory protein is a pharmaceutical polypeptide. a heterologous polynucleotide fused in frame upstream of a second nucleic acid sequence encoding the is contained in a nucleic acid construct separate from the nucleic acid construct comprising

[0044] According to some embodiments of the invention, the nucleic acid construct comprises a Cre-recombinase coding sequence Does not include.

[0045] According to some embodiments of the invention, the nucleic acid construct encodes beta (β)-lactamase (BL A) Contains no coding sequence.

[0046] According to some embodiments of the invention, the nucleic acid construct is adapted for integration into the Toxoplasma genome. It is suitable for.

[0047] According to some embodiments of the present invention, the inducible self-destruction element is activated in response to a drug. This becomes:

[0048] According to some embodiments of the invention, the medication comprises an antibiotic.

[0049] According to some embodiments of the present invention, the nucleic acid construct is derived from said Toxoplasma secretory protein. at least one in-frame cleavage site that allows for the release of said pharmaceutical polypeptide from It further includes rank.

[0050] According to some embodiments of the invention, the Toxoplasma gondii is It does not contain elements that facilitate the proliferation of the vector.

[0051] According to some embodiments of the present invention, Toxoplasma gondii is a compound that delivers a protein of interest to the CNS of a subject. It does not contain virulence genes that are not necessary for delivery of the gene.

[0052] According to some embodiments of the present invention, the heterologous polynucleotide encodes a selectable marker. The present invention further includes a nucleic acid sequence comprising:

[0053] According to some embodiments of the invention, the selectable marker is chloramphenicol acetyltransferase. Transferase (CAT), DHFR-TS, BLE, HXGPRT, UPRT, TK , CDs, fluorescent proteins (e.g., GFP, YFP, RFP, mCherry, etc.) or contains an epitope tag (e.g., HA, Myc, Ty-1, FLAG, etc.).

[0054] According to some embodiments of the present invention, the pharmaceutical composition comprises a compound comprising: For treating a subject diagnosed with a condition characterized by a protein deficiency is.

[0055] According to some embodiments of the present invention, the pharmaceutical composition is a pharmaceutical polypeptide delivered to the central nervous system of a patient. The present invention is intended to treat a subject diagnosed with a condition treatable by administration of a steroid. do.

[0056] According to some embodiments of the invention, the pharmaceutical composition further comprises an immunosuppressant.

[0057] According to some embodiments of the invention, the method comprises administering to the subject prior to and / or after administration of Toxoplasma gondii to the subject. or administering immunization to the subject after and / or concurrently with the administration of Toxoplasma gondii. The method further comprises administering an immunosuppressant.

[0058] According to some embodiments of the invention, the method comprises administering to the subject Toxoplasma gondii prior to administering to the subject Further comprising administering an immunosuppressant.

[0059] According to some embodiments of the invention, the method comprises administering to the subject Toxoplasma gondii after administration to the subject. Further comprising administering an immunosuppressant.

[0060] According to some embodiments of the invention, the method comprises administering to the subject Toxoplasma gondii simultaneously with the administration of the The method further comprises administering an immunosuppressant to the elephant.

[0061] According to some embodiments of the present invention, the pharmaceutical polypeptide is capable of treating a medical condition. It contains the wild-type amino acid sequence corresponding to the endogenous protein.

[0062] According to some embodiments of the present invention, the pharmaceutical polypeptide is capable of treating a medical condition. This includes antibodies.

[0063] According to some embodiments of the present invention, the pharmaceutical polypeptide is capable of treating a medical condition. Contains antigens.

[0064] According to some embodiments of the present invention, the pharmaceutical polypeptide is capable of treating a medical condition. Contains toxins.

[0065] According to some embodiments of the present invention, the pharmaceutical polypeptide is an enzyme, a structural polypeptide, Motor polypeptides, regulatory polypeptides, storage polypeptides, signaling / ligand polypeptides Peptides, receptor polypeptides, sensory polypeptides, antibodies, protein channels and / or or a transport polypeptide.

[0066] According to some embodiments of the invention, the administration is performed by peripheral administration.

[0067] According to some embodiments of the invention, peripheral administration comprises intravenous administration.

[0068] According to some embodiments of the invention, peripheral administration comprises oral administration.

[0069] According to some embodiments of the invention, the administration is performed by direct administration into the central nervous system. do.

[0070] According to some embodiments of the invention, the endogenous defective protein is a region of the endogenous protein. Deletion of at least one amino acid from the endogenous protein when compared to the native amino acid sequence , insertions, and / or substitutions.

[0071] According to some embodiments of the present invention, the endogenous defective protein is a protein that is expressed in a healthy individual that does not exhibit a pathological condition. The endogenous protein level is decreased compared to the endogenous protein level in the subject. This includes those who are

[0072] According to some embodiments of the present invention, the endogenous defective protein is a protein that has been diagnosed as having a pathology. This includes endogenous proteins that are not present in the subject.

[0073] According to some embodiments of the present invention, the pharmaceutical polypeptide is galactocerebrosidase (GALC).

[0074] According to some embodiments of the present invention, the pharmaceutical polypeptide is galactocerebrosidase (GALC) isoform 1, isoform 2, isoform 3, isoform isoform 4 or isoform 5.

[0075] According to some embodiments of the present invention, the pharmaceutical polypeptide is a methyl-CpG binding protein. The two are MECP2 isoform 1 and MECP2 isoform 2.

[0076] According to some embodiments of the present invention, the pharmaceutical polypeptide is a glial cell line derived neurotrophic factor. Child (GDNF).

[0077] According to some embodiments of the present invention, the pharmaceutical polypeptide is a glial cell line derived neurotrophic factor. GDNF isoform 1, isoform 2, isoform 3, and isoform isoform 4 or isoform 5.

[0078] According to some embodiments of the present invention, the pharmaceutical polypeptide is aspartoacylase (A SPA).

[0079] According to some embodiments of the invention, the pharmaceutical polypeptide is a survival motor neuron protein The protein is SMN1.

[0080] According to some embodiments of the invention, the pharmaceutical polypeptide is a survival motor neuron protein Protein isoform SMN, isoform SMN-delta5, isoform SMN -delta7, or the isoform SMN-delta57.

[0081] According to some embodiments of the present invention, the pharmaceutical polypeptide is Parkin (PARK2). be.

[0082] According to some embodiments of the present invention, the pharmaceutical polypeptide is Isoform 1, Isoform 2, Isoform 3, Isoform 4, Isoform isoform 5, isoform 6, isoform 7, or isoform 8.

[0083] According to some embodiments of the present invention, the pharmaceutical polypeptide is a transcription factor EB (TFEB) is.

[0084] According to some embodiments of the present invention, the pharmaceutical polypeptide is a transcription factor EB (TFEB) It is isoform 1 or isoform 2 of

[0085] According to some embodiments of the present invention, the pharmaceutical polypeptide is a TALEN (Takenyl Electron Leukocyte Enzyme Linker) Rease).

[0086] According to some embodiments of the present invention, the pharmaceutical polypeptide is a TALE TF (TALE transcription factor).

[0087] According to some embodiments of the invention, the subject has been diagnosed with Krabbe disease.

[0088] According to some embodiments of the invention, the subject has been diagnosed with Rett Syndrome.

[0089] According to some embodiments of the invention, the subject has been diagnosed with Canavan disease.

[0090] According to some embodiments of the invention, the subject has been diagnosed with spinal muscular atrophy.

[0091] According to some embodiments of the invention, the subject has been diagnosed with Parkinson's disease.

[0092] According to some embodiments of the invention, the subject is a patient suffering from a hypoxic / ischemic or neuroinflammatory CNS disorder. It has been diagnosed as a complication.

[0093] According to some embodiments of the invention, the subject has been diagnosed with Alzheimer's disease. .

[0094] According to some embodiments of the invention, the subject has been diagnosed with amyotrophic lateral sclerosis. do.

[0095] According to some embodiments of the invention, the subject has been diagnosed with Huntington's disease.

[0096] According to some embodiments of the invention, the subject has been diagnosed with a lysosomal storage disease. .

[0097] According to some embodiments of the invention, the subject has been diagnosed with MECP2 duplication syndrome. be.

[0098] According to some embodiments of the present invention, the method can induce a self-destruct element. The method further comprises administering to the subject a drug

[0099] According to some embodiments of the invention, the method comprises administering toxoplasmosis toxoids in and / or within a host cell. The method further comprises administering to the subject a molecule necessary to sustain the plasma.

[0100] According to some embodiments of the present invention, the molecule required for Toxoplasma persistence is an anti- It is a living substance.

[0101] According to some embodiments of the present invention, the molecules required for Toxoplasma persistence are It is a molecule.

[0102] According to some embodiments of the invention, the molecules required for Toxoplasma persistence are It is a product of gratitude.

[0103] Unless otherwise defined, all technical and / or scientific terms used herein are , as generally understood by a person having ordinary skill in the art to which the present invention pertains. Similar or equivalent methods and methods to those described herein have the same meaning as those described herein. Although exemplary materials may be used in the practice or testing of embodiments of the present invention, The methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, shall govern. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. It is not intended to be a diagram.

[0104] Some embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: Now, with particular reference to the drawings in detail, the features shown are illustrative and not restrictive of the invention. It is emphasized that the present invention is intended for a schematic discussion of the embodiments. The description provided will make it clear to those skilled in the art how to practice embodiments of the present invention. It is intended to be. [Brief explanation of the drawings]

[0105] [Figure 1] Schematic diagram of the clinical concept of some embodiments of the present invention: (1) a selected pharmaceutical polypeptide coding sequence is fused to a coding sequence of a secreted polypeptide of Toxoplasma gondii, (2) the nucleic acid construct is introduced into Toxoplasma, (3) the fusion protein is expressed inside the parasite and localized to the parasite's secretory organelles (shown here as an example, the rhoptry), (4) the parasite invades the subject's CNS and reaches the lesion, and (5) the protein is secreted into the subject's cells, reversing the pathological phenotype. [Figure 2]This is a schematic map of the pGRA backbone-based construct used to generate therapeutic transgenic Toxoplasma gondii strains. The nucleic acid construct contains an open reading frame (ORF) consisting of an in-frame fusion of the therapeutic polypeptide coding sequence with the coding sequence for Toxoplasma gondii toxophilin and an "HA" tag. Upstream of the ORF is the endogenous 5' untranslated region (UTR) of the toxophilin gene (the toxophilin 5'-UTR acts as a promoter), and downstream of the ORF is the 3' UTR of the abundant dense granule protein GRA2 (GRA2 3'-UTR). The construct also contains a selectable marker cassette consisting of the HXGPRT gene integrated between the endogenous 5' UTR and 3' UTR of DHFR-TS. A bacterial expression cassette containing selectable antibiotic resistance is also included in the construct. [Figure 3] This is a schematic map of the pGRA backbone-based construct used to generate therapeutic transgenic Toxoplasma gondii strains. The nucleic acid construct contains an ORF consisting of an in-frame fusion of the therapeutic polypeptide coding sequence with the coding sequence of Toxoplasma gondii GRA16 and an HA tag. Upstream of the ORF is the endogenous 5' UTR of the GRA16 gene (the GRA16 5'-UTR acts as a promoter), and downstream of the ORF is the 3' UTR of the abundant dense granule protein GRA2 (GRA2 3'-UTR). The construct also contains a selectable marker cassette consisting of the HXGPRT gene integrated between the endogenous 5' and 3' UTRs of DHFR-TS. A bacterial expression cassette containing selectable antibiotic resistance is also included in the construct. [Figure 4]Figure 4A-I shows a novel parasite strain in mammalian cells (HFF). The parasite strain expresses the toxophilin-fused therapeutic proteins aspartoacylase (ASPA), survival motor neuron protein (SMN1), methyl-CpG-binding protein 2 (MECP2), and galactocerebrosidase-TATΔ43 (also referred to as "mutant GALC-TAT" in the general Materials and Experimental Methods section), demonstrating specific localization to the parasite's secretory rhoptry organelles. Figure 4A: Schematic structure of intracellular Toxoplasma gondii, highlighting the rhoptry. Red indicates the inner membrane complex (IMC), blue indicates DNA (host cell nucleus and Toxoplasma gondii nucleus), and green indicates rhoptry proteins. Figure 4B-I: Fluorescence microscopy analysis of parasites grown on HFF cells endogenously expressing HA-tagged toxophilin-ASPA (Figures 4B and 4C), HA-tagged toxophilin-SMN1 (Figures 4D and 4E), HA-tagged toxophilin-MECP2 (Figures 4F and 4G), and HA-tagged toxophilin-GALC-TAT mutants (Figures 4H and 4I) using an anti-HA antibody (green in all panels) and co-staining for the inner membrane complex IMC1 with an anti-IMC1 antibody (red) and DAPI (blue), or overlaid on a polarized image (grayscale) of the cells. Parasites are shown in a mixed population; however, only parasites exhibiting green staining express the transgenic protein. Scale bar = 5 µM in all images shown in Figure 4B-I. [Figure 5]Figures 5A-I show a novel parasite strain in mammalian cells (HFFs). The parasite strain expresses the toxophilin-fused therapeutic proteins aspartoacylase (ASPA), survival motor neuron protein (SMN1), and methyl-CpG-binding protein 2 (MECP2), and exhibits specific localization to the parasitophorous vacuole (PV) space and the host cell nucleus. Figure 5A: Schematic diagram of intracellular Toxoplasma gondii parasites in parasitophorous vacuoles inside host cells (fibroblasts), highlighting the distribution of secreted dense granule effector proteins. Red indicates the inner membrane complex (IMC), blue indicates DNA (host cell nucleus and Toxoplasma gondii nucleus), yellow indicates dense granule-secreted effector proteins, and orange indicates the parasitophorous vacuole. Figure 5B-I: Fluorescence microscopy analysis of parasites grown on HFF cells endogenously expressing HA-tagged GRA16-ASPA (Figures 5B and 5C), HA-tagged GRA16-SMN1 (Figures 5D and 5E), or HA-tagged GRA16-MECP2 (Figures 5B-G at 100x magnification, Figures 5H-I at 40x magnification) using an anti-HA antibody (green in all panels) and co-staining for the inner membrane complex IMC1 with an anti-IMC1 antibody (red) and DAPI (blue), or overlaid on a polarized image (grayscale) of the cells. Parasites are shown in a mixed population; however, only parasites showing green staining express the transgenic protein. Scale bar = 5 µM (Figure 5B-G). Scale bar = 20 µM (Figure 5H-I). [Figure 6A]Schematic diagrams of TALE nucleases (FIG. 6A) and TALE transcription factors (FIG. 6B) designed for the "TACGTACG" (SEQ ID NO: 4505) target sequence are shown, as an example. TALE repeats are presented according to the order of nucleotides in the target sequence. Note that due to the nature of TALEs, the first nucleotide must always be a T (hence, no annotation is provided since it is incorporated at the N-terminus of the TALE), and the last nucleotide (in this case, a G) appears within a half-monomer (hence, its annotation is "half-repeat"). The open reading frames (ORFs) of these constructs are inserted into the nucleic acid constructs of some embodiments of the present invention for expression and secretion by Toxoplasma gondii of some embodiments of the present invention. For TALE_Nuc (TALE nuclease), the ORF is from the NLS to after FokI (e.g., nucleotides 2113-4104 of the polynucleotide set forth in SEQ ID NO: 4506). For TALE-TF (TALE transcription factor), the ORF is from the TALE N-terminus (N-terminus) to after EGFP (for example, nucleotides 2120 to 5005 of the polynucleotide shown in SEQ ID NO: 4507). "TALE-TF" = TALE transcription factor; "TALEN" = TALE nuclease. "NI", "NG", "NN", and "HD" = monomers described in Example 5 below. [Figure 6B]Schematic diagrams of TALE nucleases (FIG. 6A) and TALE transcription factors (FIG. 6B) designed for the "TACGTACG" (SEQ ID NO: 4505) target sequence are shown, as an example. TALE repeats are presented according to the order of nucleotides in the target sequence. Note that due to the nature of TALEs, the first nucleotide must always be a T (hence, no annotation is provided since it is incorporated at the N-terminus of the TALE), and the last nucleotide (in this case, a G) appears within a half-monomer (hence, its annotation is "half-repeat"). The open reading frames (ORFs) of these constructs are inserted into the nucleic acid constructs of some embodiments of the present invention for expression and secretion by Toxoplasma gondii of some embodiments of the present invention. For TALE_Nuc (TALE nuclease), the ORF is from the NLS to after FokI (e.g., nucleotides 2113-4104 of the polynucleotide set forth in SEQ ID NO: 4506). For TALE-TF (TALE transcription factor), the ORF is from the TALE N-terminus (N-terminus) to after EGFP (for example, nucleotides 2120 to 5005 of the polynucleotide shown in SEQ ID NO: 4507). "TALE-TF" = TALE transcription factor; "TALEN" = TALE nuclease. "NI", "NG", "NN", and "HD" = monomers described in Example 5 below. [Figure 7]This is a schematic map of the construct used to generate the therapeutic transgenic Toxoplasma gondii strain. The nucleic acid construct contains an open reading frame (ORF) consisting of an in-frame fusion of the therapeutic polypeptide coding sequence with the coding sequence of Toxoplasma gondii toxophilin and an "HA" tag. Upstream of the ORF is the endogenous 5' untranslated region (UTR) of the toxophilin gene ("Toxophilin 5'-UTR"), and downstream of the ORF is the 3' UTR of the abundant dense granule protein GRA2 ("GRA2 3'-UTR"). The construct also contains a selectable marker cassette consisting of the HXGPRT gene, DHFR-TS gene, or mCherry gene integrated between the endogenous 5' UTR and 3' UTR of DHFR-TS. A bacterial expression cassette containing selectable antibiotic resistance, used for molecular cloning, is also included in the construct. [Figure 8] This is a schematic map of the construct used to generate therapeutic transgenic Toxoplasma gondii strains. The nucleic acid construct contains an ORF consisting of an in-frame fusion of the therapeutic polypeptide coding sequence with the coding sequence of Toxoplasma gondii GRA16 and an HA tag. Upstream of the ORF is the endogenous 5' UTR of the GRA16 gene ("GRA16 5'-UTR"), and downstream of the ORF is the 3' UTR of the abundant dense granule protein GRA2 ("GRA2 3'-UTR"). The construct also contains a selectable marker cassette consisting of the HXGPRT gene, DHFR-TS gene, or mCherry gene integrated between the endogenous 5' UTR and 3' UTR of DHFR-TS. A bacterial expression cassette containing selectable antibiotic resistance, used for molecular cloning, is also included in the construct. [Figure 9]Figures 9A-N show transgenic parasite strains in mammalian cells (HFF) that express 12 novel toxophilin-fused therapeutic proteins. Figure 9A: Schematic structure of intracellular Toxoplasma gondii highlighting the rhoptry. Magenta indicates the inner membrane complex (IMC), cyan indicates DNA (host cell nucleus and Toxoplasma gondii nucleus), and yellow indicates rhoptry proteins. Figure 9B: Representative fluorescence microscopy images of Toxoplasma gondii grown in HFF cells immunostained for the rhoptry marker ROP2 / 4 (yellow). The left image is co-stained with DAPI (cyan), and the right image is overlaid on a polarized image (grayscale). Figures 9C-N: Images of transgenic parasites expressing HA-tagged toxophilin-fused therapeutic proteins. Figure 9C - aspartoacylase (ASPA), Figure 9D: codon-optimized aspartoacylase (ASPAopt), Figure 9E - galactocerebrosidase (GALC), Figure 9F: codon-optimized galactocerebrosidase (GALCopt), Figure 9G: galactocerebrosidase-TAT (GALC-TAT), Figure 9H: galactocerebrosidase-TATΔ43 ( GALC-TATΔ43 (also referred to as "mutant GALC-TAT" in the "General Materials and Experimental Methods" section), Figure 9I: glial cell line-derived neurotrophic factor (GDNF), Figure 9J: methyl-CpG binding protein 2 (MECP2), Figure 9K: codon-optimized methyl-CpG binding protein 2 (MECP2opt), Figure 9L: parkin (PARK2), Figure 9M - survival motor neuron protein (SMN1), and Figure 9N: codon-optimized transcription factor EB (TFEBopt). Example images of parasites expressing toxophilin-ASPAopt, toxophilin-GALC-TATΔ43, toxophilin-GDNF, toxophilin-MeCP2opt, toxophilin-PARK2, toxophilin-SMN1, and toxophilin-TFEB demonstrate the localization of these parasites to secretory rhoptry organelles. Toxophilin fusion proteins were immunostained using anti-HA antibody (yellow).Left: Co-stained with DAPI (cyan) and parasite marker anti-IMC-1 (magenta). Right: Overlaid on polarized image (grayscale). Parasites are shown in a mixed population, but only parasites showing yellow staining express the transgenic protein. Scale bar = 5 μM. [Figure 10]Figures 10A-J show transgenic parasite strains in mammalian cells (HFF). The parasite strains express eight novel toxophilin-fused therapeutic proteins. Figure 10A: Schematic structure of intracellular Toxoplasma gondii parasites in parasitophores within host cells, highlighting the distribution of secreted dense granule effector proteins. Magenta indicates the inner membrane complex (IMC), cyan indicates DNA (host cell nucleus and Toxoplasma gondii nucleus), yellow indicates dense granule-secreted effector proteins, and orange indicates the parasitophores. Figure 10B: Representative fluorescence microscopy images of Toxoplasma gondii grown in HFF cells expressing HA-tagged GRA16 protein and immunostained with anti-HA antibody (yellow). The left image is co-stained with DAPI (cyan), and the right image is overlaid on a polarized image (grayscale). Figures 10C-J: Exemplary images of transgenic parasites expressing HA-tagged GRA16-fusion therapeutic proteins: Figure 10C: aspartoacylase (ASPA), Figure 10D: survival motor neuron protein (SMN1), Figure 10E: galactocerebrosidase (GALC), Figure 10F: galactocerebrosidase-TAT (GALC-TAT), Figure 10G: codon-optimized aspartoacylase (ASPAopt), Figure 10H: codon-optimized galactocerebrosidase (GALCopt), Figure 10I: codon-optimized methyl-CpG binding protein 2 (MECP2opt), and Figure 10J: codon-optimized transcription factor EB (TFEBopt). GRA16-ASPA, GRA16-SMN1, GRA16-ASPAopt, GRA16-MECP2opt, and GRA16-TFEBopt demonstrate localization to the parasitophorous vacuole. GRA16-MECP2opt and GRA16-TFEBopt also show localization to the host cell nucleus. GRA16 fusion proteins were immunostained using an anti-HA antibody (yellow). On the left, they were co-stained with DAPI (cyan) and the parasite marker anti-IMC-1 (magenta), and on the right, they are overlaid on a polarized image (grayscale). Parasites are shown in a mixed population, but only those showing yellow staining express the transgenic protein. Scale bar = 5 μM. [Figure 11]Figure 1 shows the kinetics of protein delivery to the nuclei of HFF cells by tachyzoites of RH GRA16-HAstop, GRA16-MECP2opt, and GRA16-TFEBopt strains over time and multiplicity of infection (MOI). Infected cells and nuclear delivery were counted by automated image analysis using GE IN Cell Investigator software (GE Healthcare, Chicago, IL, USA). The graph represents the results for the three strains combined. [Figure 12] Figures 12A–C show representative images of in vitro differentiated LUHMES human neuronal cells 16–22 hours after infection with tachyzoites of Toxoplasma gondii strains RH GRA16-HAstop (Figure 12A), GRA16-MECP2opt (Figure 12B), and GRA16-TFEBopt (Figure 12C). GRA16 fusion proteins were immunostained using an anti-HA antibody (yellow) and co-stained with DAPI (cyan) and anti-NeuN (magenta), a marker for mature neurons. The insets on the left side of each figure show infected cells visualized with anti-HA alone (top, yellow), anti-HA and DAPI (middle, yellow and cyan), and anti-HA and NeuN (bottom, yellow and magenta). All strains demonstrate clear secretion of the fusion proteins and targeting to the nuclei of human neurons. [Figure 13]Figures 13A-D show representative immunohistochemical staining images of neuron-enriched primary cultures derived from the cerebral cortex and hippocampus of P1 mouse pups 12 hours after infection with tachyzoites of the transgenic strain RH GRA16-MECP2opt. GRA16-MeCP2 fusion protein was immunostained using an anti-HA antibody (yellow). Figure 13A: GRA16-MeCP2 alone (yellow). Figure 13B: DAPI alone (cyan). Figure 13C: Merged GRA16-MeCP2 and DAPI staining. Figure 13D: Merged GRA16-MeCP2 (yellow), DAPI (cyan), and NeuN (magenta) staining overlaid on a polarized image (grayscale). Delivered GRA16-MeCP2opt showed a characteristic pattern of colocalization with regions of enriched heterochromatic DNA in neuronal nuclei, suggesting effective binding of delivered MeCP2 to heterochromatin. [Figure 14] Western blot of nuclear extracts from R306C MeCP2 mutant human LUHMES neurons infected with the RH GRA16-MECP2opt transgenic Toxoplasma gondii strain at an MOI of 1, immunoprecipitated with an MeCP2-specific antibody, and blotted. The blot shows two bands corresponding to endogenous mutant MeCP2 and Toxoplasma gondii-delivered MeCP2. Toxoplasma gondii-delivered MeCP2 has a higher molecular weight due to its fusion with GRA16. [Figure 15]These are exemplary images of in vitro differentiated bradyzoites of the transgenic Toxoplasma gondii strain Pru GRA16-MECP2opt in HFF cells. The GRA16-MeCP2 fusion protein was immunostained using an anti-HA antibody (red). Figure 15A: GRA16-MeCP2 alone. Figure 15B: Co-stained with the bradyzoite cyst wall marker Dolichos Biflorus Agglutinin (DBA) (green) and DAPI (blue). Parasite cysts are shown in green, host fibroblast (HFF) nuclei are shown in blue, and the GRA16-MeCP2 fusion protein in the nucleus of the cyst-containing host fibroblast was stained with HA (red). Figure 15C: Co-stained with DBA and overlaid on a polarized image (grayscale). The displayed cysts demonstrate the continuous expression of GRA16-MeCP2, secretion, and targeting of the fusion protein to the host cell nucleus during the bradyzoite stage. The white arrowhead indicates the nucleus of a cell containing a bradyzoite cyst containing the delivered protein GRA16-MeCP2. DETAILED DESCRIPTION OF THE INVENTION

[0106] Some embodiments of the present invention provide a Toxoplasma secretory protein fused to a pharmaceutical polypeptide. The present invention relates more specifically to a nucleic acid construct for secretion of a protein, and to a Toxoplasma having the same. Specifically, the present invention relates to pharmaceutical compositions and methods of using the same for treating subjects. However, the present invention is not limited to the above.

[0107] Before describing at least one embodiment of the present invention in detail, it is important to note that the present invention is not limited to the above embodiments. It is understood that the present invention is not necessarily limited to the details set forth in the following description or illustrated by the examples. It is to be understood that the invention is capable of other embodiments and of being practiced or being carried out in various ways. It can be done.

[0108] The present inventors have developed a toxoplasmic reticulum (TRR) transformed with a gene construct encoding a heterologous polypeptide. We have found that the plasma membrane parasite synthesizes heterologous polypeptides and delivers them to mammalian cells. (Figures 4B-I, 5B-I, 9B-N, 10B-J, 11A-C, 12A-C, 13A 1-D, 14 and 15A-C, and the Examples below). The polypeptide is transported to its active site within the cell, where it binds to the known amino acid sequence of an endogenous pharmaceutical protein. For example, the present inventors have demonstrated that the cellular processes associated with the function of the toki Mammalian protein MeCP translationally fused with the Solanacearum secretory protein GRA16 Toxovirus types I and II expressing and delivering TFEB to mouse and human cells The Toxoplasma strain was produced using ex vivo Toxoplasma The fused therapeutic protein is delivered to the desired intracellular localization (nucleus) within the treated mammalian cells. The constructs, Toxoplasma gondii, and methods were used to secrete the proteins (Figures 13A-D). The feasibility of specific delivery and targeting of therapeutic proteins to elephants was demonstrated.

[0109] According to an aspect of some embodiments of the present invention a method for producing a T. gondii gene encoding a Toxoplasma secretory protein A first nucleic acid sequence is located in frame upstream of a second nucleic acid sequence encoding a pharmaceutical polypeptide. A nucleic acid construct comprising a heterologous polynucleotide fused with , to a promoter for directing transcription of a heterologous polynucleotide in Toxoplasma gondii The promoter may be a constitutive promoter, an inducible promoter, a latent promoter, or a a promoter selected from the group consisting of a phase-specific promoter, and a Toxoplasma endogenous promoter provided that the promoter is not a toxophilin promoter. will be done.

[0110] According to some embodiments of the invention, the nucleic acid construct is suitable for expression in Toxoplasma. is.

[0111] According to some embodiments of the invention, the nucleic acid construct comprises a coding sequence for Cre-recombinase. Does not include columns.

[0112] According to some embodiments of the invention, the nucleic acid construct encodes beta (β)-lactamase (BL A) does not contain the coding sequence.

[0113] According to some embodiments of the invention, the nucleic acid construct is adapted for integration into the Toxoplasma genome. It is suitable for.

[0114] According to some embodiments of the invention, the Toxoplasma gondii is Elements that facilitate proliferation (e.g., elements important for evading the host immune system) , elements essential for the production or utilization of certain metabolites, certain toxins or antibiotics It does not contain endogenous functional CPSII (elements important for combating the substance). .

[0115] According to some embodiments of the present invention, Toxoplasma gondii is a compound that delivers a protein of interest to the CNS of a subject. It does not contain virulence genes that are not necessary for delivery of the gene.

[0116] Nucleic acid constructs are used for the constitutive, transient, and tunable expression of polynucleotide sequences in Toxoplasma gondii cells. It contains a promoter sequence for directing transcription in a regulated or inducible manner.

[0117] As described, the heterologous polynucleotide is a heterologous polynucleotide in Toxoplasma gondii. The gene is operably linked to a promoter for directing transcription of the gene.

[0118] A coding nucleic acid sequence is a sequence in which a regulatory sequence exerts a regulatory effect on the coding sequence to which it is linked. A substance is "operably linked" to a regulatory sequence (e.g., a promoter) if it is capable of expressing itself in a specific manner.

[0119] As used herein, the term "promoter" refers to a promoter located upstream of the transcription start site of a gene. refers to the region of DNA where RNA polymerase binds and initiates RNA transcription. The target is the timing and intensity of gene expression, e.g., in the parasite and / or host cell. It controls at what stage or state during the lifespan of a cell the gene is expressed.

[0120] According to some embodiments of the invention, the promoter is a promoter used in the expression of the nucleic acid construct. It is heterologous to Xoplasma.

[0121] According to some embodiments of the invention, the promoter is a constitutive promoter.

[0122] According to some embodiments of the invention, the promoter is an inducible promoter.

[0123] According to some embodiments of the invention, the promoter is a latency-specific promoter. .

[0124] According to some embodiments of the invention, the promoter is a Toxoplasma endogenous promoter. with the proviso that the promoter is not the toxophilin promoter.

[0125] According to some embodiments of the invention, the promoter is the toxophilin endogenous promoter The predicted promoter of toxophilin (GeneID=TGME49_214 The predicted promoter of .080 (SEQ ID NO: 3689) is the promoter of the Toki gene used in some experiments in the Examples. Contains the sofilin promoter (SEQ ID NO: 4482) and additional nucleotides upstream Please note that

[0126] According to some embodiments, the promoter is SEQ ID NO: 3689 and / or SEQ ID NO: 4482 does not contain the nucleic acid sequence shown.

[0127] According to some embodiments of the invention, the endogenous promoter is that of a rhoptry protein. It's not that.

[0128] According to some embodiments of the present invention, the Toxoplasma endogenous promoter is the GRA2 promoter. promoter, GRA16 promoter, GRA24 promoter, SAG1 promoter , or the DHFR promoter.

[0129] According to some embodiments of the present invention, the Toxoplasma endogenous promoter is the GRA2 promoter. It is a motor.

[0130] According to some embodiments of the present invention, the Toxoplasma endogenous promoter is GRA16 He is a promoter.

[0131] According to some embodiments of the present invention, the Toxoplasma endogenous promoter is GRA24 He is a promoter.

[0132] According to some embodiments of the present invention, the Toxoplasma endogenous promoter is the SAG1 promoter. It is a motor.

[0133] According to some embodiments of the present invention, the Toxoplasma endogenous promoter is a DHFR promoter. It is a motor.

[0134] Table 1 below lists the vectors used to drive expression of heterologous polynucleotides in Toxoplasma gondii. Suitable endogenous genes that can be cloned into the nucleic acid constructs of some embodiments of the present invention. Promoters are listed.

[0135] [Table 1-1]

[0136] [Table 1-2]

[0137] [Table 1-3]

[0138] [Table 1-4]

[0139] [Table 1-5]

[0140] [Table 1-6]

[0141] [Table 1-7]

[0142] [Table 1-8]

[0143]

Table 1-9

[0144]

Table 1-10

[0145]

Table 1-11

[0146]

Table 1-12

[0147]

Table 1-13

[0148]

Table 1-14

[0149]

Table 1-15

[0150]

Table 1-16

[0151]

Table 1-17

[0152]

Table 1-18

[0153]

Table 1-19

[0154]

Table 1-20

[0155]

Table 1-21

[0156]

Table 1-22

[0157]

Table 1-23

[0158]

Table 1-24

[0159]

Table 1-25

[0160]

Table 1-26

[0161]

Table 1-27

[0162]

Table 1-28

[0163]

Table 1-29

[0164]

Table 1-30

[0165]

Table 1-31

[0166]

Table 1-32

[0167]

Table 1-33

[0168]

Table 1-34

[0169]

Table 1-35

[0170]

Table 1-36

[0171]

Table 1-37

[0172]

Table 1-38

[0173]

Table 1-39

[0174]

Table 1-40

[0175]

Table 1-41

[0176]

Table 1-42

[0177]

Table 1-43

[0178]

Table 1-44

[0179]

Table 1-45

[0180]

Table 1-46

[0181]

Table 1-47

[0182]

Table 1-48

[0183]

Table 1-49

[0184]

Table 1-50

[0185]

Table 1-51

[0186]

Table 1-52

[0187]

Table 1-53

[0188]

Table 1-54

[0189]

Table 1-55

[0190]

Table 1-56

[0191]

Table 1-57

[0192]

Table 1-58

[0193]

Table 1-59

[0194]

Table 1-60

[0195]

Table 1-61

[0196]

Table 1-62

[0197]

Table 1-63

[0198]

Table 1-64

[0199]

Table 1-65

[0200]

Table 1-66

[0201]

Table 1-67

[0202]

Table 1-68

[0203]

Table 1-69

[0204]

Table 1-70

[0205]

Table 1-71

[0206]

Table 1-72

[0207]

Table 1-73

[0208]

Table 1-74

[0209]

Table 1-75

[0210]

Table 1-76

[0211]

Table 1-77

[0212]

Table 1-78

[0213]

Table 1-79

[0214]

Table 1-80

[0215]

Table 1-81

[0216]

Table 1-82

[0217]

Table 1-83

[0218]

Table 1-84

[0219]

Table 1-85

[0220]

Table 1-86

[0221]

Table 1-87

[0222] Table 1-88

[0223]

Table 1-89

[0224]

Table 1-90

[0225]

Table 1-91

[0226]

Table 1-92

[0227]

Table 1-93

[0228]

Table 1-94

[0229]

Table 1-95

[0230]

Table 1-96

[0231]

Table 1-97

[0232]

Table 1-98

[0233]

Table 1-99

[0234]

Table 1-100

[0235]

Table 1-101

[0236]

Table 1-102

[0237]

Table 1-103

[0238]

Table 1-104

[0239]

Table 1-105

[0240]

Table 1-106

[0241]

Table 1-107

[0242]

Table 1-108

[0243]

Table 1-109

[0244]

Table 1-110

[0245]

Table 1-111

[0246]

Table 1-112

[0247]

Table 1-113

[0248]

Table 1-114

[0249]

Table 1-115

[0250]

Table 1-116

[0251]

Table 1-117

[0252]

Table 1-118

[0253]

Table 1-119

[0254]

Table 1-120

[0255]

Table 1-121

[0256] Table 1-122

[0257]

Table 1-123

[0258]

Table 1-124

[0259]

Table 1-125

[0260]

Table 1-126

[0261]

Table 1-127

[0262] Table 1-128

[0263]

Table 1-129

[0264] Table 1-130

[0265]

Table 1-131

[0266] Table 1-132

[0267]

Table 1-133

[0268] Table 1-134

[0269]

Table 1-135

[0270] Table 1-136

[0271] Table 1-137

[0272]

Table 1-138

[0273]

Table 1-139

[0274] Table 1-140

[0275]

Table 1-141

[0276] Table 1-142

[0277] Table 1-143

[0278] Table 1-144

[0279]

Table 1-145

[0280] Table 1-146

[0281]

Table 1-147

[0282] Table 1-148

[0283]

Table 1-149

[0284]

Table 1-150

[0285]

Table 1-151

[0286]

Table 1-152

[0287]

Table 1-153

[0288]

Table 1-154

[0289]

Table 1-155

[0290]

Table 1-156

[0291]

Table 1-157

[0292]

Table 1-158

[0293]

Table 1-159

[0294] Table 1-160

[0295]

Table 1-161

[0296] Table 1-162

[0297] Table 1-163

[0298] Table 1-164

[0299] Table 1-165

[0300]

Table 1-166

[0301]

Table 1-167

[0302] Table 1-168

[0303] Table 1-169

[0304] Table 1-170

[0305] Table 2 lists preferred nucleic acid sequences that can be cloned into nucleic acid constructs of some embodiments of the present invention. Suitable constitutive and inducible promoters are listed.

[0306] [Table 2]

[0307] As described, the heterologous polynucleotide encodes a Toxoplasma secretory protein. A first nucleic acid sequence is included.

[0308] As used herein, the phrase "Toxoplasma secretory protein" refers to a protein secreted by Toxoplasma. , sufficient to be secreted in infected host cells in vitro. It refers to at least a functional fragment (amino acid sequence) of a polypeptide.

[0309] According to some embodiments of the present invention, a Toxoplasma secretory protein or a functional fragment thereof The fragments are secreted by host cells during in vivo infection with Toxoplasma gondii. is.

[0310] According to some embodiments of the invention, the first nucleic acid sequence is a Toxoplasma secretory protein It encodes a functional fragment of

[0311] According to some embodiments of the invention, the first nucleic acid sequence is a Toxoplasma secretory protein It encodes the full-length open reading frame (ORF).

[0312] According to some embodiments of the invention, the Toxoplasma secreted protein is It is secreted from the rhoptry of the

[0313] According to some embodiments of the present invention, the Toxoplasma secretory protein secreted from the rhoptry The protein contains toxophilin and / or ROP1.

[0314] According to some embodiments of the present invention, the Toxoplasma secretory protein secreted from the rhoptry The protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 344 to 465.

[0315] According to some embodiments of the invention, the Toxoplasma secretory protein is a non-rhoptryta It is protein.

[0316] According to some embodiments of the present invention, the Toxoplasma secretory protein is a micronemucoprotein. The protein is selected from the group consisting of proteins and dense granule proteins.

[0317] According to some embodiments of the invention, the Toxoplasma secreted protein is It is secreted from dense granules of

[0318] According to some embodiments of the present invention, a Toxoplasma secretory protein secreted from dense granules is The protein contains GRA16 and / or GRA24.

[0319] According to some embodiments of the invention, the Toxoplasma secreted protein is It is secreted from the microneme.

[0320] According to some embodiments of the invention, the microneme-secreted protein has SEQ ID NO: The amino acid sequence is selected from the group consisting of Nos. 280 to 322.

[0321] According to some embodiments of the invention, the protein secreted from the dense granules is selected from the group consisting of SEQ ID NO: The amino acid sequence is selected from the group consisting of 234 to 279.

[0322] According to some embodiments of the invention, the Toxoplasma secreted protein is Contains Transgenic MIF (TgMIF) from Gondii.

[0323] Table 3 below lists toxoproteins that can be cloned into nucleic acid constructs of some embodiments of the present invention. The plasma secretory proteins are listed. The proteins are classified according to the location or The proteins are organized according to the organelles they are predicted to localize.

[0324] [Table 3-1]

[0325] [Table 3-2]

[0326] [Table 3-3]

[0327] [Table 3-4]

[0328] [Table 3-5]

[0329] [Table 3-6]

[0330] [Table 3-7]

[0331]

Table 3-8

[0332]

Table 3-9

[0333]

Table 3-10

[0334]

Table 3-11

[0335]

Table 3-12

[0336]

Table 3-13

[0337]

Table 3-14

[0338]

Table 3-15

[0339]

Table 3-16

[0340]

Table 3-17

[0341] [Table 3-18]

[0342] [Table 3-19]

[0343] [Table 3-20]

[0344] [Table 3-21]

[0345] [Table 3-22]

[0346] According to some embodiments of the present invention, the heterologous polynucleotide is a Toxoplasma untranslated region The present invention further includes the nucleic acid sequence of the untranslated transcribed region (UTR).

[0347] According to some embodiments of the present invention, the nucleic acid sequence of the Toxoplasma untranslated region (UTR) is , (e.g., Toxoplasma secretory proteins fused in-frame with pharmaceutical polypeptides upstream of the open reading frame of the heterologous polynucleotide (encoding a protein) and / or downstream.

[0348] According to some embodiments of the present invention, the Toxoplasma 5'-untranslated region (5'-UTR) is located upstream of the Toxoplasma secretory protein open reading frame.

[0349] According to some embodiments of the present invention, the Toxoplasma 3'-untranslated region (3'-UTR) The present invention relates to a method for producing a Toxoplasma secretory protein fused in-frame with a pharmaceutical polypeptide. It is placed downstream of the open reading frame it loads.

[0350] According to some embodiments of the present invention, the Toxoplasma 3' untranslated region (3'-UTR) The nucleic acid sequences are GRA2 3'-UTR, GRA16 3'-UTR, GRA24 3'- UTR, SAG1 3'-UTR, or DHFR 3'-UTR.

[0351] According to some embodiments of the present invention, the 5' untranslated region (5'-UTR) of Toxoplasma gondii The nucleic acid sequences are GRA2 5'-UTR, GRA16 5'-UTR, GRA24 5'- UTR, SAG1 5'-UTR, or DHFR 5'-UTR.

[0352] According to some embodiments of the present invention, the nucleic acid sequence of the Toxoplasma untranslated region (UTR) is , toxophilin 3'-UTR.

[0353] As described, the heterologous polynucleotide is inserted downstream of the Toxoplasma secretory protein. It includes a second nucleic acid sequence encoding a pharmaceutical polypeptide fused in frame.

[0354] As used herein, the phrase "pharmaceutical polypeptide" refers to a polypeptide that is administered to a subject in need of treatment. a polypeptide that has a therapeutic effect (e.g., capable of treating a disease state) when introduced into a Point.

[0355] For a particular disease, the pharmaceutical polypeptide may be administered to a particular organ, tissue, or cell in the subject. It may be effective when it reaches a cell, a cellular compartment, or is cellularly localized. For example, to treat a neurological disorder, the pharmaceutical composition may be administered to the nervous system, e.g. For example, it is preferably targeted to neurons, glial cells or other cells. Additionally or alternatively, for certain diseases, pharmaceutical polypeptides may be used as pharmaceuticals. If the target of the composition is located in the cell nucleus, it will be localized in a specific location, such as the cell nucleus. For example, in the case of MECP2, the active protein binds to DNA in the nucleus. and regulates gene expression.

[0356] According to some embodiments of the invention, the nucleic acid construct is derived from a Toxoplasma secretory protein and at least one in-frame cleavage site that allows for the release of the pharmaceutical polypeptide. This includes:

[0357] According to some embodiments of the present invention, the pharmaceutical polypeptide is capable of treating a medical condition. It contains the wild-type amino acid sequence corresponding to the endogenous protein.

[0358] According to some embodiments of the present invention, the pharmaceutical polypeptide is capable of treating a medical condition. This includes antibodies.

[0359] According to some embodiments of the present invention, the pharmaceutical polypeptide is capable of treating a medical condition. Contains antigens.

[0360] According to some embodiments of the present invention, the pharmaceutical polypeptide is capable of treating a medical condition. Contains toxins.

[0361] According to some embodiments of the present invention, the pharmaceutical polypeptide is an enzyme, a structural polypeptide, Motor polypeptides, regulatory polypeptides, storage polypeptides, signaling / ligand polypeptides Peptides, receptor polypeptides, sensory polypeptides, antibodies, protein channels and / or or a transport polypeptide.

[0362] According to some embodiments of the present invention, the pharmaceutical polypeptide is galactocerebrosidase (GALC).

[0363] According to some embodiments of the present invention, the pharmaceutical polypeptide is galactocerebrosidase (GALC) isoform 1, isoform 2, isoform 3, isoform 4 or isoform 5.

[0364] According to some embodiments of the present invention, the pharmaceutical polypeptide is a methyl-CpG binding protein. The most important protein is metastatic protein 2 (MECP2).

[0365] According to some embodiments of the present invention, the pharmaceutical polypeptide is a methyl-CpG binding protein. The two are mechanistically related to the regulation of cytokine 2 (MECP2) isoform 1 and MECP2 isoform 2.

[0366] According to some embodiments of the present invention, the pharmaceutical polypeptide is a glial cell line derived neurotrophic factor. Child (GDNF).

[0367] According to some embodiments of the present invention, the pharmaceutical polypeptide is a glial cell line derived neurotrophic factor. GDNF isoform 1, isoform 2, isoform 3, and isoform isoform 4 or isoform 5.

[0368] According to some embodiments of the present invention, the pharmaceutical polypeptide is aspartoacylase (A SPA).

[0369] According to some embodiments of the invention, the pharmaceutical polypeptide is a survival motor neuron protein The protein is SMN1.

[0370] According to some embodiments of the invention, the pharmaceutical polypeptide is a survival motor neuron protein Protein isoform SMN, isoform SMN-delta5, isoform SMN -delta7, or the isoform SMN-delta57.

[0371] According to some embodiments of the present invention, the pharmaceutical polypeptide is Parkin (PARK2). be.

[0372] According to some embodiments of the present invention, the pharmaceutical polypeptide is Isoform 1, Isoform 2, Isoform 3, Isoform 4, Isoform isoform 5, isoform 6, isoform 7, or isoform 8.

[0373] According to some embodiments of the present invention, the pharmaceutical polypeptide is a transcription factor EB (TFEB) is.

[0374] According to some embodiments of the present invention, the pharmaceutical polypeptide is a transcription factor EB (TFEB) It is isoform 1 or isoform 2 of

[0375] According to some embodiments of the present invention, the pharmaceutical polypeptide is a TALEN (Takenyl Electron Leukocyte Enzyme Linker) Rease).

[0376] According to some embodiments of the present invention, the pharmaceutical polypeptide is a TALE TF (TALE transcription factor).

[0377] Table 4 below shows examples of compounds that may be secreted into a subject by Toxoplasma in some embodiments of the present invention. Representative therapeutic proteins are listed below.

[0378] [Table 4-1]

[0379] [Table 4-2]

[0380] [Table 4-3]

[0381] [Table 4-4]

[0382] [Table 4-5]

[0383] [Table 4-6]

[0384] [Table 4-7]

[0385] The sequences described herein (e.g., Toxoplasma secretory proteins and / or therapeutic polypeptide) are engineered for expression in target cells (e.g., Toxoplasma gondii). It should be noted that codon optimization can be performed. Examples of such sequence modifications include Vary the G / C content to approximate sequences typically found in the target cell type of interest and the use of codons that are atypically found in target cell types, commonly referred to as codon optimization. This includes, but is not limited to, removal.

[0386] The term "codon optimization" refers to the process of optimizing a construct to approximate codon usage in a desired target cell. It refers to the selection of DNA nucleotides appropriate for use within a gene or fragment thereof. Therefore, an optimized gene or nucleic acid sequence is a nucleic acid sequence that is identical to the original or naturally occurring gene's nucleotide sequence. The nucleotide sequence is a codon that is statistically preferred or statistically favored in the target cell. The nucleotide sequence is typically a sequence of a gene that has been modified to utilize the gene. The coding region, tested in vitro and optimized for expression in the target cell type, can be expressed in any suitable manner. This method uses a codon usage index (Codon Usage Index), which is a measure of codon usage bias. The standard deviation of the usage can be calculated as follows: First, the usage of each codon in the original gene is calculated. The proportional deviation squared for each codon usage in the highly expressed target cell genes is Then calculate the mean square deviation. The formula used is: 1SDCU=n = 1N[(Xn-Yn) / Yn] / N, where Xn is the number of highly expressed target cellular genes. represents the frequency of codon n in the gene of interest, and Yn represents the frequency of codon n in the gene of interest. represents the degree of codon division, and N represents the total number of codons in the gene of interest).

[0387] One method is to optimize nucleic acid sequences based on preferred codon usage for a particular target cell type. The method directly uses the codon optimization table without performing any additional statistical calculations. The codon optimization table is, for example, generated from the International DNA Sequence Database of 2000. Codon usage tables are provided online in the Codon Usage Database. (Y Nakamura, T Gojobori, T Ikemura - Nucleic acids research, 2000 - Oxford Un iv Press). The codon usage database contains codon usage tables for a variety of different species, Each codon usage table is statistically determined based on data provided by Genbank. is.

[0388] Use the table above to identify the most common genotypes in a particular species (e.g., Toxoplasma gondii). The preferred or most favored codon for each amino acid is determined to determine the target sequence. The naturally occurring nucleotide sequence encoding the protein is then transfected into the target cell type. This allows for codon optimization of sequences that have a statistically low occurrence within the genome of a particular species. The codons that are considered to be unfavorable are replaced by the corresponding codons for the amino acids that are statistically more favored. However, if one or more less preferred codes are Select a gene to delete existing restriction sites or to remove potentially useful junctions (signal peptides). 5' and 3' ends for adding a sequence or stop cassette, or to generate the correct full-length sequence. new internal sites that can be used for segment cleavage and splicing to Nucleotides that create new restriction sites or that may negatively affect mRNA stability or expression are also introduced. The nucleotide sequence can be removed.

[0389] The naturally occurring coding nucleotide sequence is designed to encode a gene that encodes a target gene in a particular target cell type prior to any modification. However, the nucleotide sequence may already contain some codons that correspond to statistically preferred codons. Therefore, codon optimization of the original nucleotide sequence involves optimizing which codons in the original nucleotide sequence are used. Determine whether the gene is statistically disfavored in a particular target cell, and then select a gene for the particular target cell. By modifying the determined codons according to the codon usage table, a codon-optimized derivative is obtained. The modified nucleotide sequence may comprise a nucleotide sequence encoding the Proteins are encoded by corresponding naturally occurring or native genes. The modified nucleotide sequence is capable of inducing a cellular response to the target protein, so long as it is produced at a higher level than the target protein. It can be fully or partially optimized for codon usage. Alternatively, the codon-optimized / usage sequence may be a sequence similar to that of the corresponding naturally occurring protein or The protein may be better folded than the protein encoded by the original gene. Additionally or alternatively, the codon-optimized / usage sequence may be codon-optimized / usage-matched to the corresponding naturally occurring protein. It binds to the target organelle better than the protein or protein encoded by the original gene. Additionally or alternatively, codon-optimized / usage sequences may be used. The sequence is the sequence of the corresponding naturally occurring protein or protein encoded by the native gene. It is less easily broken down than protein.

[0390] For example, Table 5 below showing codon usage in Toxoplasma can be used.

[0391] [Table 5-1]

[0392] [Table 5-2]

[0393] According to some embodiments of the present invention, the heterologous polynucleotide encodes a selectable marker. The present invention further includes a nucleic acid sequence comprising:

[0394] According to some embodiments of the invention, the selectable marker is chloramphenicol acetyltransferase. Transferase (CAT), DHFR-TS, BLE, HXGPRT, UPRT, TK , CDs, fluorescent proteins (e.g., GFP, YFP, RFP, mCherry, etc.) or contains an epitope tag (e.g., HA, Myc, Ty-1, FLAG, etc.).

[0395] According to some embodiments of the invention, the selectable marker comprises HXGPRT.

[0396] Toxoplasma tachyzoites lacking HXGPRT activity were found to express 6-thioxanthine (6-T X), strains expressing HXGPRT activity can be selected for in the presence of mycophenone. It can be selected among methylparaben (MPA) and / or xanthine (Pfefferkorn and d Borotz, 1994, Pfefferkorn ER, Borotz S. (1994) Exp. Parasitol. 79, 374-382 ).

[0397] According to some embodiments of the invention, the selectable marker is chloramphenicol acetyltransferase (CAT) (for positive selection), DHFR-TS (for positive selection using pyrimethamine), BLE (for positive selection using mycophenolic acid plus xanthine), or or 6-thioxanthine for negative selection), HXGPRT (5'-fluoro- UPRT (for negative selection using 2'-deoxyuridine), (for positive selection using 5-fluorocytosine) TK (for positive selection using 5-fluorocytosine) CDs, fluorescent proteins (e.g., GFP, YFP, RFP, mCherry, etc.) or Contains an epitope tag (e.g., HA, Myc, Ty-1, FLAG, etc.).

[0398] According to some embodiments of the invention, the nucleic acid construct encodes an inducible self-destruct element. The nucleic acid sequence further comprises a third nucleic acid sequence.

[0399] According to some embodiments of the present invention, the inductive self-destruct element is the same as the therapeutic cassette. It is included in the same reading frame.

[0400] According to some embodiments of the invention, the inducible self-destruction element is contained in a separate reading frame. and are regulated by different regulatory elements. For example, the therapeutic cassette is The self-destruct cassette is driven by a drug-inducible promoter.

[0401] According to some embodiments of the present invention, the inducible self-destruction element is activated in response to a drug. This becomes:

[0402] According to some embodiments of the invention, the medication comprises an antibiotic.

[0403] According to some embodiments of the invention, a third nucleic acid encoding an inducible self-destruct element The sequence is selected so that the first nucleic acid sequence encoding the Toxoplasma secretory protein is a pharmaceutical polypeptide. a heterologous polynucleotide fused in frame upstream of a second nucleic acid sequence encoding the is contained in the same nucleic acid construct as that containing

[0404] According to some embodiments of the invention, a third nucleic acid encoding an inducible self-destruct element The sequence is selected so that the first nucleic acid sequence encoding the Toxoplasma secretory protein is a pharmaceutical polypeptide. a heterologous polynucleotide fused in frame upstream of a second nucleic acid sequence encoding the is contained in a nucleic acid construct separate from that containing

[0405] According to some embodiments of the invention, the Toxoplasma untranslated region (UTR) nucleic acid sequence is Open reading frames upstream of selectable markers and / or self-destruct elements and / or downstream.

[0406] According to an aspect of some embodiments of the present invention, a nucleic acid construct comprising at least two nucleic acid constructs. A nucleic acid system, wherein a first nucleic acid construct of at least two nucleic acid constructs is a nucleic acid construct that is part of the present invention. The nucleic acid construct of any one of the embodiments, wherein the second of the at least two nucleic acid constructs is a selected A nucleic acid construct system is provided that includes a polynucleotide encoding a selectable marker.

[0407] A typical cloning vector contains transcription and translation initiation sequences, transcription and translation termination sequences, and as well as a polyadenylation signal.

[0408] The following may be used as a backbone to generate nucleic acid constructs of some embodiments of the present invention: A non-limiting list of Toxoplasma nucleic acid constructs that can be used include: pGRA, pUPRT , pROP1, pTUB1, pTUB8, pLIC, pTOXO, pMIC2, pHX, pCAT, pDHFR, pBlueScript, pTetO7SAG1, pTetO7 SAG4, pSAG1, pSAG4, pHTU, pTKO, pLoxP-DHFR, pm inCAT / HXGPRT+, pminCAT / HXGPRT-, pDHFR-TSc3 / M3, pDHFR-TSc3 / M2M3, pminiHXGPRT, and pUC19 These vectors are available from the non-profit plasmid repository "addgene"; H AIDS Reagent Program;Agilent Technolog ies; NEB; Thermo Fisher Scientific; Sigma a Aldrich; GenScript; and MoBiTec GmbH It can be obtained from a variety of sources.

[0409] Table 6 below provides exemplary vectors and catalog numbers, but is not limited to these. It's not something like that.

[0410] [Table 6]

[0411] According to an aspect of some embodiments of the present invention, the nucleic acid construct of some embodiments of the present invention or Toxoplasma transformed with the nucleic acid construct system of some embodiments of the present invention is provided. can be.

[0412] As used herein, the term "Toxoplasma" refers to the intracellular parasitic protozoan Toxoplasma gondii. Refers to Ma Gondi.

[0413] Toxoplasma gondii that can be used with the constructs and methods of some embodiments of the present invention M. gondii strains include, but are not limited to: (i) Type I: GT1, RH, ENT, VEL, TgCatCo1, and CAST These include, but are not limited to: (ii) Type II, including ME49, Beverly, PDS, PLK, PTG, and DEG; These include, but are not limited to, PIH, TgNmBr1 and PRU. (iii) Type III, including VEG, C56, CTG, CEP, TgGoatUS4, and Examples of such proteins include, but are not limited to, STRL and STRL. (iv) Atypical TgCTPrC3, TgBbUS1, TgRabbitBr1, and Examples of such vectors include, but are not limited to, TgPigUS15 and TgPigUS15.

[0414] According to some embodiments of the invention, the Toxoplasma is non-attenuated.

[0415] As is conventionally known in the art, the term "attenuated" refers to a genetically modified Weakened and / or less potent than native (wild-type) Toxoplasma gondii Refers to a reduced virulence strain of Toxoplasma gondii. Usually, the attenuation of Toxoplasma gondii Mutants can stimulate an immune response and produce immunity, but they may not cause disease. Attenuation can be achieved by conventional methods such as gamma irradiation or the creation of pyrimidine auxotrophs. This can be achieved by, but is not limited to, the following.

[0416] Examples of attenuated Toxoplasma include those described in U.S. Pat. Patent Application Publication No. 2012 / 0045477 and U.S. Patent No. 8,673,289 Examples of the present invention include, but are not limited to, those described in the specification of US Pat.

[0417] Toxoplasma gondii replication disseminates throughout the body during the acute phase of infection, reaching distal tissues. the ability to infect the host, to persist in infected cells, and to establish chronic cysts in the host. It should be noted that this is essential for the ability to

[0418] According to some embodiments of the present invention, the Toxoplasma of some embodiments of the present invention is attenuated It is characterized by a high growth rate compared to that of immunized Toxoplasma gondii.

[0419] According to some embodiments of the present invention, the Toxoplasma gondii of some embodiments of the present invention is It is not a demand stock.

[0420] According to some embodiments of the present invention, the Toxoplasma gondii of some embodiments of the present invention is It has a functional endogenous pathway suitable for the de novo biosynthesis of thymidin.

[0421] According to some embodiments of the present invention, the Toxoplasma gondii of some embodiments of the present invention is They can differentiate into the dizoite stage.

[0422] According to some embodiments of the present invention, the Toxoplasma gondii of some embodiments of the present invention is A synthesis and / or DNA replication can occur.

[0423] According to some embodiments of the present invention, the Toxoplasma gondii of some embodiments of the present invention is A synthesis can be performed.

[0424] According to some embodiments of the present invention, the Toxoplasma gondii of some embodiments of the present invention is A Replication can be performed.

[0425] According to some embodiments of the present invention, the Toxoplasma gondii of some embodiments of the present invention is, for example, For example, an endogenous functional carbamoyltransferase such as that shown in any of SEQ ID NOs: 4612 to 4617. The enzyme carboxyphosphate synthetase II (CPSII) (U.S. Pat. No. 8,673,289 (described in the above).

[0426] Constructs of some embodiments of the present invention and Toxoplasma can be used to express proteins of interest. Delivery can be to a subject, for example, to a particular tissue or cell type in a subject.

[0427] According to an aspect of some embodiments of the present invention, a method is provided for administering a protein of interest to a tissue of interest in a subject. The method comprises administering to a subject an administration of Toxoplasma gondii or any of the embodiments of the present invention. A method for administering a target protein to a target tissue of a subject by administering the pharmaceutical composition of the embodiment of is provided.

[0428] Examples of tissues of interest include the central nervous system, muscle, parts of the eye, blood, lymph nodes, spleen, and white blood cells. These include, but are not limited to, the globe, the digestive system, and the lamina propria.

[0429] Toxoplasma can cross the blood-brain barrier, so it can deliver target proteins to It should be noted that delivery to the central nervous system of a subject in need thereof is possible.

[0430] According to an aspect of some embodiments of the present invention, a protein of interest is administered to the central nervous system of a subject. The method includes administering to a subject a Toxoplasma gondii of some embodiments of the present invention or a Administering the pharmaceutical composition of some embodiments to administer the protein of interest to the central nervous system of the subject. A method is provided that includes:

[0431] Thus, some embodiments of the present invention can be used to treat subjects with Toxoplasma gondii. by delivering a target protein (e.g., a therapeutic polypeptide) to a subject in need of treatment. Subjects can be treated.

[0432] According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need thereof. Toxoplasma gondii of some embodiments of the present invention or pharmaceutical compositions of some embodiments of the present invention administering to a subject, wherein the subject receives a pharmaceutical polypeptide in the central nervous system of the subject. have been diagnosed with a condition treatable by the administration of Methods for treating a subject suffering from rheumatoid arthritis are provided.

[0433] According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need thereof. The first nucleic acid sequence encoding a Toxoplasma secretory protein is used to produce a pharmaceutical polypeptide. a heterologous polynucleotide fused in-frame upstream of a second nucleic acid sequence encoding administering to the subject Toxoplasma gondii having a nucleic acid construct containing a heterologous polynucleotide; The protease acts as a promoter for directing the transcription of heterologous polynucleotides in Toxoplasma gondii. a motor, the motor being operably linked to a central nervous system, the subject being able to administer a pharmaceutical polypeptide to the central nervous system; and has been diagnosed with a condition treatable by administration of the Methods for treating a subject are provided.

[0434] The term "treat" refers to the process of inhibiting, preventing, or treating a condition (disease, disorder, or pathological state). to stop the progression of the disease and / or cause the alleviation, remission, or regression of the disease Those skilled in the art will appreciate that a variety of methods and assays can be used to assess the occurrence of a disease state. and similarly, various methods and assays can be used to determine the alleviation, remission or progression of disease. will understand that regression can be assessed.

[0435] As used herein, the term "prevent" refers to preventing a person from having the disease, even though they may be at risk for the disease. Preventing a disease, disorder, or condition from occurring in a subject who has not yet been diagnosed with the condition. This refers to the following.

[0436] As used herein, the term "subject" refers to a mammal, preferably any subject suffering from a medical condition. The term preferably includes individuals at risk of developing a condition. Desirable.

[0437] According to some embodiments of the invention, the subject is provided with a method for detecting endogenous proteins in the subject's central nervous system. have been diagnosed with a medical condition characterized by a deficit in quality.

[0438] According to some embodiments of the invention, the deficiency of the endogenous protein is a defect in the endogenous protein. Deletion of at least one amino acid from the endogenous protein when compared to the native amino acid sequence , insertions, and / or substitutions.

[0439] According to some embodiments of the present invention, the deficiency of the endogenous protein is not a pathological condition associated with a healthy A decrease in the level of the endogenous protein when compared to the level of the endogenous protein in the subject. Includes reductions.

[0440] According to some embodiments of the present invention, a deficiency in an endogenous protein is diagnosed as having a pathology. The absence of endogenous protein in the subject.

[0441] According to some embodiments of the invention, the subject has been diagnosed with Krabbe disease.

[0442] According to some embodiments of the invention, the subject has been diagnosed with Rett Syndrome.

[0443] According to some embodiments of the invention, the subject has been diagnosed with Canavan disease.

[0444] According to some embodiments of the invention, the subject has been diagnosed with spinal muscular atrophy.

[0445] According to some embodiments of the invention, the subject has been diagnosed with Parkinson's disease.

[0446] According to some embodiments of the invention, the subject is a patient suffering from a hypoxic / ischemic or neuroinflammatory CNS disorder. It has been diagnosed as a complication.

[0447] According to some embodiments of the invention, the subject has been diagnosed with Alzheimer's disease. .

[0448] According to some embodiments of the invention, the subject has been diagnosed with amyotrophic lateral sclerosis. do.

[0449] According to some embodiments of the invention, the subject has been diagnosed with Huntington's disease.

[0450] According to some embodiments of the invention, the subject has been diagnosed with a lysosomal storage disease. .

[0451] According to some embodiments of the invention, the subject has been diagnosed with MECP2 duplication syndrome. be.

[0452] According to some embodiments of the invention, the subject is not immunocompromised. Examples include AIDS patients, subjects undergoing chemotherapy, cells, tissues and / or organs. This includes, but is not limited to, subjects receiving immunosuppressive drugs for transplantation or other reasons. isn't it.

[0453] According to some embodiments of the present invention, the method can induce a self-destruct element. The method further comprises administering to the subject a drug

[0454] According to some embodiments of the invention, the method comprises administering toxoplasmosis toxoids in and / or within a host cell. The method further comprises administering to the subject a molecule necessary to sustain the plasma.

[0455] According to some embodiments of the present invention, the molecule required for Toxoplasma persistence is an anti- It is a living substance.

[0456] According to some embodiments of the present invention, the molecules required for Toxoplasma persistence are It is a molecule.

[0457] According to some embodiments of the invention, the molecules required for Toxoplasma persistence are It is a product of gratitude.

[0458] According to some embodiments of the invention, the method comprises administering to the subject prior to and / or after administration of Toxoplasma gondii to the subject. or administering immunization to the subject after and / or concurrently with the administration of Toxoplasma gondii. The method further comprises administering an immunosuppressant.

[0459] According to some embodiments of the invention, the method comprises administering to the subject Toxoplasma gondii. The method further comprises administering an immunosuppressant to the patient.

[0460] According to some embodiments of the invention, the method comprises administering Toxoplasma gondii to the subject (or subsequently), further comprising administering to the subject an immunosuppressant.

[0461] According to some embodiments of the invention, the method includes, concurrently with administering to the subject Toxoplasma: The method further comprises administering to the subject an immunosuppressant.

[0462] According to some embodiments of the invention, the administration is performed by peripheral administration.

[0463] According to some embodiments of the invention, peripheral administration comprises intravenous administration.

[0464] According to some embodiments of the invention, peripheral administration comprises oral administration.

[0465] According to some embodiments of the invention, the administration is performed by intraperitoneal injection.

[0466] According to some embodiments of the invention, the administration is performed by intramuscular injection.

[0467] According to some embodiments of the invention, administration is performed by aerosol.

[0468] According to some embodiments of the invention, administration is by direct administration to the central nervous system or adjacent tissues. This is done by administration.

[0469] According to some embodiments of the present invention, the method of some embodiments of the present invention may, for example, Diseases that can be treated using Toxoplasma gondii and / or pharmaceutical compositions containing same The disease is any of the diseases listed in Table 7 above.

[0470] Table 7 shows the effects of treatment by administration of a therapeutic polypeptide of some embodiments of the present invention. Non-limiting examples of diseases associated with defects or abnormalities in the expression or function of endogenous proteins include: This section lists some examples.

[0471] [Table 7-1]

[0472] [Table 7-2]

[0473] [Table 7-3]

[0474] [Table 7-4]

[0475] [Table 7-5]

[0476] According to some embodiments of the invention, the disease (condition) is not cancer.

[0477] The nucleic acid construct, nucleic acid construct system, or nucleic acid construct or Toxoplasma transformed with the nucleic acid construct system is administered to the organism as it is. or can be administered as a pharmaceutical composition mixed with a suitable carrier or excipient. Cut.

[0478] According to an aspect of some embodiments of the present invention, the Toxoplasma gondii of some embodiments of the present invention and a pharmaceutically acceptable carrier.

[0479] According to some embodiments of the invention, the pharmaceutical composition is for use in treating any of the conditions described herein. It is intended to treat a diagnosed subject.

[0480] According to some embodiments of the present invention, the pharmaceutical composition comprises a compound comprising: for treating a subject diagnosed with a condition characterized by a deficiency of a protein .

[0481] According to some embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutical polypeptide for delivery to the central nervous system of a subject. for treating a subject diagnosed with a condition treatable by administration of a peptide be.

[0482] According to some embodiments of the invention, the pharmaceutical composition further comprises an immunosuppressant.

[0483] As used herein, a "pharmaceutical composition" refers to a pharmaceutical composition containing one or more of the active ingredients described herein. refers to a formulation containing one or more of the above together with other chemical ingredients such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0484] As used herein, the term "active ingredient" refers to a compound of the present invention that is capable of producing a biological effect. Nucleic acid constructs, nucleic acid construct systems, or nucleic acid constructs or nucleic acid constructs of some embodiments This refers to Toxoplasma transformed by the microbial system.

[0485] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutical A "virtually acceptable carrier" is a compound that can be administered without causing significant irritation to the organism. An adjuvant refers to a carrier or diluent that does not abolish the biological activity and properties of a substance. These words are included in the phrase.

[0486] As used herein, the term "excipient" refers to a pharmaceutical agent that further facilitates administration of an active ingredient. It refers to an inactive substance added to a composition. Examples of excipients include calcium carbonate, calcium phosphate, etc. calcium, various sugars and starches, cellulose derivatives, gelatin, vegetable oils and polyesters Examples of suitable solvents include, but are not limited to, polyethylene glycol.

[0487] Techniques for drug formulation and administration are described in "Reming Drugs," which is incorporated herein by reference. Mack Publishing Co., Easton, PA, latest edition can be found in.

[0488] Suitable routes of administration include, for example, oral, rectal, transmucosal (especially nasal), intestinal or parenteral. Parenteral delivery includes intramuscular, subcutaneous, and intramedullary injections, and intrathecal delivery. , directly intraventricular, intracardiac (e.g., intraright or left ventricular), common coronary, This includes intravenous, intraperitoneal, intranasal, or intraocular injection.

[0489] Traditional approaches for drug delivery to the central nervous system (CNS) include neurosurgical strategies (e.g., In an attempt to exploit one of the endogenous transport pathways of the BBB, Molecular manipulation of drugs within the body (e.g., drugs that cannot cross the BBB themselves and drugs that cannot cross the BBB) and a transport peptide having affinity for a cell surface molecule. generation), pharmacological strategies designed to increase the lipid solubility of drugs (e.g., lipid or or binding of water-soluble drugs to cholesterol carriers), and disruption of the BBB by hyperosmotic disruption Transient total destruction (infusion of mannitol solution or angiotensin peptide into the carotid artery) These include the use of bioactive agents such as

[0490] Alternatively, the pharmaceutical composition can be administered locally rather than systemically. Thus, local administration can be achieved, for example, by injection directly into a tissue area of ​​a patient.

[0491] The term "tissue" refers to a tissue composed of cells designed to perform one or more functions. Refers to parts of an organism. Examples include brain tissue, retina, skin tissue, liver tissue, pancreatic tissue, bone, and cartilage. , connective tissue, blood tissue, muscle tissue, heart tissue, vascular tissue, kidney tissue, lung tissue, gonad tissue, These include, but are not limited to, hematopoietic tissues.

[0492] The pharmaceutical compositions of some embodiments of the present invention can be prepared by processes well known in the art, e.g., by known mixing methods. Compounding, dissolving, culturing, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping or lyophilizing For example, the production of Toxoplasma gondii can be carried out by a cell culture process. This can be done by culturing Toxoplasma gondii in

[0493] Thus, pharmaceutical compositions for use according to some embodiments of the present invention may comprise a pharmaceutical One or more ingredients containing excipients and adjuvants that facilitate processing into formulations that can be used commercially. The compositions can be formulated in a conventional manner using any number of physiologically acceptable carriers. The exact formulation will depend on the route of administration chosen.

[0494] For injection, the active ingredients of the pharmaceutical composition are dissolved in an aqueous solution, preferably Hank's solution, Lysine solution, or the like. The drug may be formulated in a physiologically compatible buffer, such as saline buffer, or in an aqueous solution of 100 mg of ethanol. For transmucosal administration, penetrants appropriate to the barrier to be permeated can be used. Such penetrants are generally known in the art.

[0495] For oral administration, the active compound is combined with a pharmaceutically acceptable carrier well known in the art. By using such a carrier, it is possible to easily formulate a pharmaceutical composition. The pharmaceutical composition may be administered as a tablet, pill, dragee, capsule, liquid, gel, or the like for oral ingestion by a patient. It can be formulated into syrups, foods, slurries, suspensions, etc. Medicines for oral use The pharmaceutical preparations can be made using solid excipients, and if desired, The resulting mixture is milled, and suitable auxiliaries are added as needed, followed by processing the granular mixture to Tablets or dragee cores can be obtained. Suitable excipients are, inter alia, lactose, sucrose, fillers such as sugars, including corn starch, mannitol, or sorbitol; Wheat starch, rice starch, potato starch, gelatin, tragacanth gum, ethylcellulose, hydroxypropyl methylcellulose, sodium carbomethylcellulose and / or biodegradable materials such as polyvinylpyrrolidone (PVP). Optionally, cross-linked polyvinylpyrrolidone, agar, or Alternatively, a disintegrating agent such as alginic acid or a salt thereof, such as sodium alginate, may be added. stomach.

[0496] Dragee cores are provided with suitable coatings. A sugar solution can be used, and a concentrated sugar solution can be optionally added with gum arabic, talc, etc. , polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, It may contain a lacquer solution and a suitable organic solvent or solvent mixture. For the identification or characterization of various combinations of doses of a substance, tablets or dragee coatings A dye or pigment may be added to the composition.

[0497] Orally usable pharmaceutical compositions include push-type capsules made from gelatin. Capsules and capsules made from gelatin and plasticizers such as glycerol or sorbitol Push-fit capsules include soft, sealed capsules containing fillers such as lactose. , a binder such as starch, a lubricant such as talc or magnesium stearate, and The active ingredient may optionally be contained in a mixture with a stabilizer. The active ingredient may be a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. The formulation may be dissolved or suspended in the body. In addition, a stabilizer may be added. All formulations for the administration of the compounds of the present invention should be in dosages suitable for the chosen route of administration.

[0498] For buccal administration, the compositions take the form of tablets or lozenges formulated in conventional manner. That's fine.

[0499] For administration by nasal inhalation, some embodiments of the present invention may be administered by use of a suitable propellant. The active ingredient for use by the The propellant can be, for example, dichlorodifluoromethane. fluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide. In the case of pressurized aerosols, a valve may be provided to deliver a metered amount. Amount units can be determined. Capsules and cartridges made from PEG-400 (PVC) may contain the compound and a suitable filling such as lactose or starch. The formulation may comprise a powder mixture with a suitable powder base.

[0500] The pharmaceutical compositions described herein are intended for parenteral administration, e.g., by bolus injection or continuous infusion. Injectable formulations can be formulated in unit doses, optionally with added preservatives. The composition may be provided in dosage form, for example, in ampoules or multi-dose containers. The composition may be a suspension, solution or emulsion in a solvent or aqueous vehicle, and may contain suspending agents, stabilizers, Formulating agents such as dispersants and / or dispersing agents may also be included.

[0501] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredient in water-soluble form. Suspensions of the drug may be prepared as injectable suspensions in appropriate oily or aqueous bases. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or oleic acid. synthetic fatty acid esters such as ethyl acetate, triglycerides, or liposomes Aqueous injection suspensions may be prepared using sodium carboxymethyl cellulose, sorbitol or dextran. It may also contain substances that increase the viscosity of the suspension, such as xanthran. The liquid may also contain suitable stabilizers or active ingredient solubility enhancers to allow for the preparation of highly concentrated solutions. It may also contain an agent that increases

[0502] Alternatively, the active ingredient may be dissolved in a suitable vehicle (e.g., sterile, pyrolytic, or ethanol-containing solution) before use. It may also be in powder form for constitution with a phenylephrine-free water-based solution.

[0503] Pharmaceutical compositions of some embodiments of the present invention may also contain, for example, cocoa butter or other glycerides. It can be formulated into rectal compositions such as suppositories or retention enemas using conventional suppository bases such as tetracycline. It is also possible to do so.

[0504] Suitable pharmaceutical compositions in the context of some embodiments of the present invention include compositions containing an active ingredient in a dosage form suitable for its intended purpose. More specifically, a therapeutically effective amount includes a composition containing an amount effective to achieve the objective of the present invention. prevents, reduces, or ameliorates the symptoms of a disorder (e.g., a disorder affecting the central nervous system of a subject) or an active ingredient (e.g., a compound of the present invention) effective to prolong the survival of the subject being treated. Some embodiments of the nucleic acid construct, nucleic acid construct system, or nucleic acid construct or nucleic acid construct This means the amount of Toxoplasma transformed with the construct system.

[0505] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. within the capabilities of the individual.

[0506] For any formulation used in the methods of the present invention, a therapeutically effective amount or dose is in vitro assays, ex vivo assays, cell culture assays, and / or For example, the concentration or titer can be estimated from a clinical study or animal model. Such information can be used to formulate a dose in animal models so that the efficacy and safety of the drug can be assessed. can be used to more accurately determine useful doses in humans.

[0507] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro, in cell cultures or These can be determined by standard pharmaceutical procedures in experimental animals. Data obtained from in vitro assays, cell culture assays, and animal studies It can be used to formulate a range of doses for use in humans. The exact formulation, route of administration and The dosage and dosage can be selected by the individual physician taking into account the patient's condition (e.g., Fingl, See, for example, S. et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1. I want to be.

[0508] Tissue levels (e.g., brain levels) of active ingredients may induce or inhibit biological effects. The dose and administration interval should be individually adjusted to achieve a dose sufficient for the patient to achieve the minimum effective concentration (MEC). The MEC may vary for each formulation, but is based on in vitro The dose necessary to achieve the MEC can be estimated based on individual characteristics. and route of administration. Detection assays may be used to measure the activity of the compound in plasma, tissue, or CSF. (cerebrospinal fluid) can be determined.

[0509] Depending on the severity and responsiveness of the condition being treated, the dosage may be for single or multiple administrations. The course of treatment may last from a few days to a few weeks (e.g., lifelong in the case of chronic disease), or or until a cure is achieved or a diminution of the disease state is achieved.

[0510] For example, for chronic conditions, symptoms are controlled, stabilized, and / or improved. Doses can be adjusted until

[0511] The amount of composition administered will, of course, depend on the subject being treated, the severity of the affliction, the manner of administration, the subject's This will depend on immune status and the prescribing physician's judgment.

[0512] Compositions of some embodiments of the present invention may be approved by the FDA (U.S. Food and Drug Administration) as appropriate. may be provided in a pack or dispenser device such as a kit approved by the Such a pack or device may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, be a blister pack comprising metal or plastic foil. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be used to facilitate the manufacture, use, or sale of the drug. There may be an accompanying notice attached to the container in a form prescribed by the regulating government agency; This notice indicates that the composition form has been approved by the agency for administration to humans or animals. Such notices reflect, for example, the U.S. Food and Drug Administration's (FDA) guidelines for prescription drugs. It may be in the form of labeling approved by the Department of Pharmacy or in the form of an approved product insert. It may also be in the form of a drink containing the formulation of the present invention, formulated in a compatible pharmaceutical carrier. The encapsulated composition is prepared and placed in a suitable container and administered to the patient for the indicated condition, as detailed above. The composition may be labeled for the treatment of

[0513] As used herein, the term "about" refers to ±10%.

[0514] The terms "comprises," "comprising," "including" includes, including, having and its cognates mean "including but not limited to" (not limited to)"

[0515] The term "consisting of" means "including, limited to" means "using and limited to."

[0516] The term "consisting essentially of" defines that the composition, method, or structure may include additional components, steps, and / or moieties. However, additional components, steps and / or parts may be incorporated into the claimed compositions, methods or compositions. is limited to those which do not materially alter the basic and novel characteristics of the structure.

[0517] As used herein, the singular forms "a," "an," and "the" are used unless the context clearly indicates otherwise. Unless otherwise indicated, plurals are used, e.g., "a compound" or "At least one compound" includes multiple compounds and may also include mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. Ranges The formal description is merely for convenience and brevity and does not limit the scope of the invention in any flexible manner. It should be understood that the description of a range is not limiting, and therefore includes all possible subranges, as well as ranges should be considered to specifically disclose the range and each individual value within that range. The description of a range such as 6 is a partial range such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. but also specifically discloses individual values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the size of the scope.

[0518] Whenever a numerical range is given herein, it is understood that it is always the case that any cited number (fraction) within the given range is included. or integers). A "range between" a first designated number and a second designated number is intended to include The phrases "from" a first designated number to a second designated number are interchangeable in this specification. It can be used to calculate the first and second indicated numbers and all the fractions and integers between them. It is intended to include parts.

[0519] As used herein, the term "method" refers to a manner, means, or technique for accomplishing a given task. It refers to techniques and procedures and is used by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine. Known or readily developable by practitioners from known methods, means, techniques and procedures These include, but are not limited to:

[0520] As used herein, "treating" means arresting, substantially inhibiting, slowing, or otherwise improving the progression of a disease state. or reversal, substantial amelioration of the clinical or cosmetic symptoms of the condition, or clinical or cosmetic improvement of the condition. This includes substantially preventing cosmetic deterioration.

[0521] When a particular sequence listing is referred to, such reference substantially corresponds to the complementary sequence thereof. sequence, e.g., allelic variation, sequencing error, cloning error, or salt Small sequence variations due to base substitutions, deletions, or other alterations resulting in base additions However, the frequency of such conversion is less than 1 in 50 nucleotides. Alternatively, there is less than 1 in 100 nucleotides, or less than 1 in 200 nucleotides. or less than 1 in 500 nucleotides, or less than 1 in 1000 nucleotides, or is less than 1 in 5,000 nucleotides or less than 1 in 10,000 nucleotides I want you to understand something.

[0522] Features of the invention which are, for clarity, described as separate embodiments, may also be combined. It should be understood that they may be provided together as a single embodiment. Various features of the invention that are described in the context of a single embodiment may be used individually or in any suitable combination. provided in any suitable subcombination or in any suitable combination with other embodiments described herein. Features described in connection with various embodiments may be practiced without those features. Unless features are inoperable, they are not considered essential to those embodiments.

[0523] The present invention as described hereinabove and as claimed in the claims below Various embodiments and aspects are experimentally supported by the following examples. . [Example]

[0524] Reference is now made to the following examples, which together with the above descriptions illustrate some of the embodiments of the present invention. Although shown in detail, it is not intended to limit the invention.

[0525] The nomenclature used herein and the experimental procedures used in the present invention generally include: These include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are well explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989), "Current Protocols in Molecular Biol ogy" Volumes I-III Ausubel, RM, ed. (1994), Ausubel et al., "Current Protocol s in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989), Perbal , "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988), Watson et al., "Recombinant DNA", Scientific American Books, New York, Birren et al. al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998), U.S. Patent No. 4,666,828 , No. 4,683,202, No. 4,801,531, No. 5,192,65 9 and 5,272,057, "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994), "Current Protocol ls in Immunology" Volumes I-III Colligan JE, ed. (1994), Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (19 94), Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH See Freeman and Co., New York (1980). Available immunoassays are available under patent. and has been widely described in the scientific literature, e.g., U.S. Pat. No. 3,791,932 , Specification No. 3,839,153, Specification No. 3,850,752, No. 3,850,57 Specification No. 8, Specification No. 3,853,987, Specification No. 3,867,517, No. 3,8 Specification No. 79,262, Specification No. 3,901,654, Specification No. 3,935,074 , Specification No. 3,984,533, Specification No. 3,996,345, No. 4,034,07 Specification No. 4, Specification No. 4,098,876, Specification No. 4,879,219, No. 5,0 Nos. 11,771 and 5,281,521, "Oligonucleotide Syntheses" "Nucleic Acid Hybridization" Gait, MJ, ed. (1984), "Nucleic Acid Hybridization" Hames, BD, and H iggins SJ, eds. (1985), "Transcription and Translation" Hames, BD, and Hig. gins SJ, Eds. (1984), "Animal Cell Culture" Freshney, RI, ed. (1986), "Imm obilized Cells and Enzymes" IRL Press, (1986), "A Practical Guide to Molecular C loning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press ss, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Di. ego, CA (1990), Marshak et al., "Strategies for Protein Purification and Charact" See "Chemical Analysis - A Laboratory Course Manual" CSHL Press (1996). All of the references thereto are hereby incorporated by reference as if fully set forth herein. Ru. Toxoplasma gondii: the model apicomplexan - Perspectives and methods. LM Wei ss, K Kim, 2011. Other general references are provided throughout this document. The procedures described therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0526] General Materials and Experimental Methods Toxoplasma gondii culture 2 mM L-glutamine, penicillin, and streptomycin or gentamicin Dulbecco's modified broth supplemented with a bronchodilator antibiotic mixture and 10% fetal bovine serum (FBS) Human follicles cultured in Eagle's Medium (DMEM) (also known as "complete DMEM medium") Parasites were grown on human dermal fibroblasts (HFFs). Cultures were monitored daily and lysed. Droplets of supernatant from the plate or droplets of free intracellular parasites in a syringe were transferred to a new culture containing HFFs. Parasites were passaged by transferring to fresh plates.

[0527] Heterologous polypeptides containing Toxoplasma gondii secretory proteins fused to pharmaceutical polypeptides of interest 1. Preparation of Toxoplasma gondii Expression Vectors for Expression of Peptides Stable Transgenic Toxoplasma gondii Expressing Specific Therapeutic Proteins To generate the T. gondii strain, we first isolated T. gondii by molecular cloning. A M. gondii expression construct was generated. This construct contains the toxophilin cDNA [SEQ ID NO: 4481] or GRA16 cDNA [SEQ ID NO: 4472], followed by the HA tag coding sequence (SEQ ID NO: 4474), then a therapeutic mammalian cDNA of interest, e.g., mouse MeCP2 Isoform 1 [SEQ ID NO: 4476], human ASPA [SEQ ID NO: 4483], human SM N1 [SEQ ID NO: 4479], human PARK2 [SEQ ID NO: 4478], human GDNF [SEQ ID NO: No. 4489], human TFEB (transcription factor EB) isoform 1 [SEQ ID NO: 4600] Sequence, human GALC isoform 1 [SEQ ID NO: 4486 or SEQ ID NO: 4601] (G ALC version 2), a glycine flexible linker (Gly) [SEQ ID NO: 4490] and followed by a protein transduction domain (TAT) (SEQ ID NO: 4488) TAT, or "mutant GALC-TAT" (GALC-TAT with a deletion) (SEQ ID NO: It contains a long open reading frame (ORF) consisting of the protein The transduction domain (TAT) targets GALC to the lysosomes of the host cell. It facilitates non-classical transport across the membrane to aid in the translation of the cDNA of interest. A is codon-optimized according to the codon usage of Toxoplasma gondii, Increasing the efficiency, stability, and localization of heterologous polypeptide expression in M. gondii It can also be codon-optimized for expression in Toxoplasma gondii (e.g., codon-optimized mouse MeCP2 isoform 1 [SEQ ID NO: 4477], codon-optimized codon-optimized human ASPA [SEQ ID NO: 4602], codon-optimized human GALC isoform 1 [ SEQ ID NO: 4603] and codon-optimized human TFEB isoform 1 [SEQ ID NO: 460 4]). Toxoplasma gondii expression vectors also allow for accurate expression and transcription. Regulatory elements derived from the same or different Toxoplasma genes ensure targeting. The ORF also includes an endogenous 5' regulatory sequence upstream of the ORF. As used herein, the term "toxophilin promoter" or "toxophilin 5'-UTR" refers to (SEQ ID NO: 4482), or or, as used herein, "GRA16 promoter" or "GRA16 5'-UTR" The endogenous 5' regulatory sequence of the GRA16 gene is designated as (SEQ ID NO: 4473). Downstream is the 3′-UTR of the abundant dense granule protein GRA2 (SEQ ID NO: 4491; Figure 1 The expression vector may further comprise a separate ORF, which may be a DH 5'-UTR (upstream) of DHFR-TS [SEQ ID NO: 4492] and 3'U of DHFR-TS Selectable marker HXG surrounded by TR (downstream) [SEQ ID NO: 4493 or 4609] PRT [SEQ ID NO: 4475] or DHFR-TS [SEQ ID NO: 4484 or 4606] ] or mCherry [SEQ ID NO: 4608].

[0528] Generation of transgenic Toxoplasma gondii Toxoplasma gondii type I strain RH or RHΔHX, or type II strain Prug niaud, Prugniaud-GFP-luciferase or Prugniaud ΔHPT (also known as Prugniaud ΔHX) was used to create the transgenic strain. Extracellular tachyzoites were collected or extracted using a 22- to 26-gauge needle. The cytoplasmic cytoplasmic vesicles were mechanically released, filtered from cellular debris, pelleted, and then transferred to a 300 μl cytoplasmic membrane. Mix buffer (120 mM KCl, 0.15 mM CaCl, 10 mM K HPO4 / KH2PO4pH7.6, 25mM HEPES pH7.6, 2mM E 30 μl of 2 mM ATP and 5 mM GTA (5 mM MgCl) in situ The plasmid was resuspended in 360 μl of M GSH (20-60 μg total). DNA or DNA linearized with ScaI enzyme was electroporated using a BTX ECM electroporator. Tachyzoites were transfected by electroporation using a few drops of Transfected cells were placed in IFA wells (HFF cells seeded on glass coverslips). Immunofluorescence assay wells of a 24-well plate were used to determine the transfection efficiency. and for the assessment of transient protein expression (expression from plasmid DNA), immunofluorescence was used. The remaining transfected parasites were transferred to the HFF culture medium in complete DMEM. If a drug resistance selection marker was used, the medium was , drugs used for selection (1 μM pyrimethamine for selection of DHFR-TS, HXGP RT selection includes 25 μg / ml mycophenolic acid + 50 μg / ml xanthine Starting from the day when parasites began to release from the HFFs, the medium was replaced with fresh medium containing extracellular Droplets of the parasite-containing supernatant were transferred every 1-2 days to the HFFs in selective medium containing the selective drug. When the parasites in the second flask began to release from the HFF, Subculture was continued by transferring a drop of the supernatant to a third flask every 1-2 days. When a protein selection marker was used, approximately 50%–90% of the parasites in the second flask were H Upon release from the FF cells, the medium containing the extracellular parasites was collected, filtered from the cell debris, and purified by FA. The CS system was used for selection. The fluorescent proteins used for selection were selected. Parasites were collected and transferred to the next flask containing HFFs in complete DMEM medium. After the fifth flask of parasites begins to lyse (approximately 2-5 weeks), DNA constructs are transfected into the parasites. The stable pool contained the parasites integrated into the genome. The droplets were transferred to IFA (immunofluorescence assay) wells and immunofluorescently stained to identify the constructs in the pool. Percentage of positive parasites, proteins expressed by parasites integrated into the genome ("stable"). Protein expression and protein localization (genomic expression) were assessed. Manual selection by limiting dilution or FACS sorting, sorting one parasite per well in a 96-well plate. After the plates were left undisturbed for 5-10 days, single cells were isolated. Wells containing single plaques presumed to be of viable origin were identified using bright-field microscopy. Wells containing a single plaque were visually screened and detected by vigorously pipetting. Mix thoroughly and transfer 50 μl of the mixture to a new plate and PCR tube. The collected cells in the tube were pelleted and placed in a TE buffer containing 10% proteinase K. Resuspend in 10 μl lysis buffer consisting of 10 μl of lysate and incubate at 60 °C for 60 min in a thermocycler. Lysates were prepared by incubating at 95°C for 1 minute and then at 95°C for 10 minutes. A microliter (1 μl) of lysate was analyzed using primers specific for the gene construct. PCR-positive clones were used as templates for PCR screening. The cells were transferred to a plate, fixed, and immunofluorescently stained to determine the protein expression and protein activity in each clone. Localization was analyzed using fluorescence and polarized light microscopy.

[0529] antibody The concentrations of each antibody used were as follows: anti-HA (Roche, IF A:1:1000, WB:1:1000), anti-IMC-1 (Dominique Sol donated by Professor dati-Favre, IFA: 1:1000-1:2000), anti-MeC P2 (manufactured by Cell Signaling, IFA: 1:200, WB: 1:1000, IP:1:26), anti-NeuN (Abcam, IFA:1:500), anti-NCoR1 (Bethyl Laboratories, WB: 1:1000), anti-TBL1( Abcam, WB: 1:1000), Alexa Fluor anti-rat 488 and and 594 (Invitrogen, IFA: 1:1000), Alexa Fluo r Anti-rabbit 488 and 594 (Invitrogen, IFA: 1:1000) , Alexa Fluor anti-mouse 488 and 594 (Invitrogen, IFA:1:1000).

[0530] Example 1 Expression of the therapeutic protein of interest, its localization to parasite secretory organelles, and its interaction with the host cell Preparation of Toxoplasma gondii constructs leading to secretion into the host

[0531] Experimental procedure The constructs were transfected into the RH ( The vectors were transfected into the Pru (Pruhxgprt) or Pru ​​(RHhxgprt) strains. This is followed by a selection process for parasites expressing the selection marker [co-transfected with the construct of interest]. Constructs containing the modified DHFR-TS (SEQ ID NO: 4484) were selected with pyrimethamine or For selection based on expression of HXGPRT (SEQ ID NO: 4475), MPA + xanthine cloned by limiting dilution or flow cytometry, and PCR Positive clones were identified by Western blot analysis and immunofluorescence. Staining confirmed secretory rhoptry organelles, dense granular organelles, and parasitophorous vacuole spaces. Expression and specificity of HA-tagged therapeutic proteins in the parasite vacuole, or host cells The confirmed positive strains were further analyzed on HFF (human foreskin fibroblast) cells. Maintained.

[0532] Experimental results Therapeutic protein galactocerebrosidase, galactocerebrosidase-TA T, GDNF, aspartoacylase, MeCP2, survival motor neuron protein and Constructs and toxoplasmin for delivery of the E3 ubiquitin protein ligase Parkin Construction of Toxoplasma gondii strains: Results show that Toxoplasma gondii can be engineered to express the parasite Expression of heterologous (mammalian, e.g., human) therapeutic proteins fused to endogenous proteins Using this approach, we demonstrate that it is possible to identify genes associated with common neuropathologies. Three human proteins, namely, galactocerebrosidase-TAT (mutant GALC- TAT, SEQ ID NO: 4487), MeCP2 (codon-optimized SEQ ID NO: 4477) and Three novel, stable strains of Toxoplasma gondii expressing T. gondii and GDNF (SEQ ID NO: 4489) ditransgenic lines and three human proteins associated with common neuropathologies, viz. , galactocerebrosidase (GALC, isoform 1, SEQ ID NO: 4486), Gal ... ctocerebrosidase-TAT (WT GALC-TAT, SEQ ID NO: 4488), Asparagus Glutoacylase (ASPA, SEQ ID NO: 4483) and survival motor neuron protein ( Stable Toxoplasma gondii transformants expressing SMN1 (SEQ ID NO: 4479) Four mixed population pools containing virulent parasites were generated.

[0533] Improving the Secretion and Uptake of Therapeutic Proteins with Viral Protein Transduction Domains Good: Secretion of galactocerebrosidase protein from host cells into the extracellular space, and To improve uptake of the protein by neighboring cells, the inventors have The levulosidase was fused to a viral protein transduction domain (PTD). PTDs enhance both the secretion and uptake of enzymes in cell culture model systems. This significantly increases (6-fold) the cross-correction efficiency of galactocerebrosidase. has already been shown (Meng, X.-L., et al., 2013).

[0534] Therapeutic proteins into rhoptry secretory organelles of Toxoplasma gondii Galactocerebrosidase-TAT, aspartoacylase, MeCP2 and survival motility Neuronal protein localization: Results show that Toxoplasma gondii is a heterologous (mammalian) , e.g., human) therapeutic proteins are delivered to the parasite's rhoptry-secretory organelles (Figure 4A ), from where it is released into the cytoplasm of the host cell and localizes to the target site within the infected cell. Galactocerebrosidase-TAT, A fusion protein containing spartoacylase, MeCP2, and survival motor neuron protein Proteins have been reported for conventional toxophilin fusions that were able to enter host cells. As shown in Fig. 1, the parasite was localized to the rhoptry, as evidenced by immunofluorescence staining (IFA). This was explained (Figure 4B to I).

[0535] Thus, the inventors have developed a heterologous (mammalian, e.g., Toxoplasma gondii can be engineered to express a protein (e.g., human). These proteins were localized to the rhoptry secretory organelles of the parasite. The present inventors have succeeded in demonstrating that a human protein can be fused to toxophilin. Toxophilin is a protein naturally secreted by the parasite into host cells. It is an endogenous protein that is expressed in the target protein (e.g., These are drugs that can "pick up" proteins (e.g., therapeutic proteins) onto the protein secretion machinery of the parasite. This suggests that Toxoplasma gondii may be a feasible basis for this technique. This is an important matter to be addressed.

[0536] Using this approach, we identified Gastrin, a human protein associated with Krabbe disease. Expressing lactocerebrosidase-TAT (mutant GALC-TAT, SEQ ID NO: 4487) A novel, stable Toxoplasma gondii transgenic vector that localizes to the rhoptry strains and three human proteins associated with common neuropathologies: galactocerebromocriptine (GCL) and galactocerebromocriptine (GCL). GALC-TAT (WT GALC-TAT, SEQ ID NO: 4488), aspartoacyl ase (ASPA, SEQ ID NO: 4483), MeCP2 (SEQ ID NO: 4477) and survival motility Neuronal protein (SMN1, SEQ ID NO: 4479) was expressed and localized to the rhoptry. A four-species mixture containing stable Toxoplasma gondii transgenic parasites A population pool was created.

[0537] Therapeutic proteins aspartoacylase, MeCP2 and motor neuron survival Proteins in Toxoplasma gondii dense granule-secreting organelles and parasitophorous vacuoles Localization between the xenogeneic (mammalian) and human therapeutic targets: The results show that Toxoplasma gondii The protein was localized in the dense granule-secreting organelles and parasitophorous vacuole space of the parasite (Fig. 5 A), from where it is released into the host cell cytoplasm and localized at the target site within the infected cell ( 5B-I) demonstrates that it is possible to separate target proteins from the high-density granules of parasites. This approach, using sequences from high-density granule proteins to drive secretion, is unprecedented. Fusion with the dense granule protein (GRA16) allows mammalian proteins to be transported into the parasite vacuole. This is the first case of secretion of erythropoietin during the ovarian period. This was demonstrated by immunofluorescence staining (IFA). The fusion protein then crossed the parasite vacuole membrane and entered the host cell (Fig. 5B-I). This prediction is based on the known location of the native GRA16 protein, which is the fusion partner. It is based on localization (Bougdour et al. 2013).

[0538] Thus, the inventors have developed a heterologous (mammalian, e.g., Toxoplasma gondii can be engineered to express a protein (e.g., human). These proteins are secreted into the dense granule-secreting organelles and parasitophorous vacuoles of the parasite. The present inventors have succeeded in demonstrating that GRA16 is fused to GRA16 and localizes it in space. GRA16 is a human protein naturally secreted by the parasite into host cells. It is an endogenous protein that is used to target and secrete a protein of interest (e.g., These are drugs that can "pick up" proteins (e.g., therapeutic proteins) onto the protein secretion machinery of the parasite. This suggests that Toxoplasma gondii may be a feasible basis for this technique. This is an important matter to be addressed.

[0539] Using this approach, we identified M, a human protein associated with Rett syndrome. New granule-secreting organelles expressing eCP2 and localizing it to the parasitophorous vacuole space. Novel and stable Toxoplasma gondii transgenic strains and their association with common neuropathologies Two human proteins, namely aspartoacylase and survival motor neuron protein, Proteins are stably expressed and localized to dense granule-secreting organelles and the parasitophorous vacuole space. Two species mixed population pools containing Toxoplasma gondii transgenic parasites and were produced.

[0540] Secretion of the therapeutic protein MeCP2 from transgenic parasites into host cells, and Toxoplasma gondii secretion and localization in the nucleus, the active site in the host cell: Protein fusions (referred to herein as protein fusions) comprise a pharmaceutical polypeptide fused to a polypeptide. Specifically, a therapeutic tag fused to the Toxoplasma gondii dense granule protein GRA16 was The protein MeCP2 is released from the parasite into the host cell and then transduced into the host cell as a therapeutic protein. Immunofluorescence staining (I) shows that the nuclei can be transported to the target site (here, the nucleus) of the plasma membrane. Fusion proteins containing MeCP2 localized to the host cell nucleus, as shown by FA. This demonstrates that Toxoplasma gondii is a viable basis for this technique (Figure 5F-I). This is an important point that suggests that

[0541] Using this approach, we identified M, a human protein associated with Rett syndrome. A novel, stable Toxoplasma gondii strain expressing eCP2 and localizing to the host cell nucleus Transgenic strains were generated (Figure 5F-I).

[0542] Example 2 Amelioration of galactocerebrosidase deficiency by parasite delivery 6-Hexadecanoylamino-4-methylumbelliferyl-beta-D-galactosyl The synthesis of galactocerebrosidase, named 6HMU-beta-D-galactoside, Galactocerebrosidase enzyme activity assays based on the enzymatic destruction of a synthetic fluorogenic substrate are available. Otto P. van Diggelen, MOSCERDAM SUBSTRATES, Laboratory protocol for enzyme analysis for Krabbe disease; Galactocerebrosidase]. Using fibroblasts derived from Krabbe disease patients, this assay revealed that GALC was expressed Parasites expressing GALC-TAT and GALC-TAT ameliorated Krabbe disease phenotypes in culture. The ability of the cells to improve the growth of Krabbe disease was evaluated using untreated Krabbe disease cells (fibroblasts derived from Krabbe disease patients), sham Krabbe disease cells transfected with vector (empty human expression vector) and / or Krabbe disease cells infected with Toxoplasma gondii parasites that do not express GALC , used as a negative control. Positive controls were wild-type (WT) cell lines or human expression vectors. The Krabbe disease cells were transfected with the expression vector, and the expression vector was The transgenic parasites express a free-standing (HA-tagged) GALC or The gene encoding the GALC-TAT gene, or the GALC fused to toxophilin / GRA16, This drives the expression of either the GALC-TAT gene or the GALC-TAT gene. The result may be a lack of sufficient efficacy of the protein itself or a toxic effect of the N-terminus of the therapeutic protein. These results suggest that the disruption of therapeutic protein activity due to fusion with GRA16 or GRA16 may be a cause of death. This allows excluding the possibility that the parasite may be delivered to the host, thus making it possible to quantify the efficiency of the parasite delivery system. and direct inference becomes possible.

[0543] This assay was performed to identify Galactocerebrosidae, which are found in fibroblasts derived from Krabbe disease patients. To characterize the ability of galactocerebrosidase-expressing parasites to ameliorate galactocerebrosidase deficiency, This experiment demonstrates that transgenic parasites recruit proteins to cells in culture. This demonstrates the ability to ameliorate Krabbe disease by delivery of a soluble protein.

[0544] Example 3 Amelioration of MECP2 deficiency by parasite delivery Neurons derived from mouse models of Rett syndrome harboring mutations or deletions in the MECP2 gene Lung and glial cells exhibit a number of abnormal morphological and molecular features [Percy, A. Rett syndrome: coming to terms with treatment. Adv. Neurosci. (2014), [de la To rre-Ubieta et al. Advancing the understanding of autism disease mechanisms through gh genetics. Nat Med. 2016].

[0545] Using a Rett syndrome cell culture model to characterize the molecular and morphological features of Rett syndrome Quantitative scoring of the phenotype revealed that MeCP2-expressing parasites were The ability to ameliorate the Rett syndrome phenotype was assessed using untreated Rett syndrome model cells, mock vectors, and genomic DNA. Rett syndrome model cells transfected with vectors and / or expressing MeCP2 Rett syndrome model cells infected with Toxoplasma gondii parasites that do not express Positive controls were the WT cell line and the WT cell line transfected with the human expression vector. The expression vector is a model cell of Rett syndrome that was tested. The standalone (HA-tagged) MECP2 coding sequence or the fusion identical to that expressed by the avian parasite It drives expression of either the MECP2 coding sequence fused to GRA16 or toxophilin / GRA16. This allows negative results to be attributed to the lack of sufficient effect of the protein itself, or is a therapeutic protein produced by fusing toxophilin or GRA16 to the N-terminus of the therapeutic protein. It is possible to exclude the possibility that this may result from the disruption of the activity of the protein, thus This allows for a quantifiable and direct estimation of the efficiency of the parasite delivery system.

[0546] This assay was performed on MeCP2-expressing parasites found in a cell culture model of Rett syndrome. This experiment was used to characterize the ability of transgenic worms to ameliorate MeCP2 deficiency. Transgenic parasite ameliorates Krabbe disease by delivering replacement proteins to cells in culture Demonstrates the ability to improve.

[0547] Example 4 Testing the therapeutic potential of transgenic parasite strains in animal models Testing transgenic strains of parasites for their ability to affect the phenotype of the whole organism Testing of the transgenic parasite in mice has shown that it alters the immune system and functional blood This allows for evaluation of the system in the presence of the blood-brain barrier (BBB). The Plasma gondii parasite is administered by the test condition, i.e., intranasal, enteral (e.g., oral), subcutaneous, or intramuscular route. It is administered to animal models intramuscularly, intravenously, intradermally, intraperitoneally, intracranially, or intracerebrally. Transgenic Toxoplasma gondii parasites develop into tachyzoites, bradyzoites, tissue cysts, or or in the form of oocytes.

[0548] After inoculation with the parasite, the animal model is adapted to produce a phenotype associated with the pathology being tested (pathological manifestation of the disease). The phenotypes include behavioral, morphological, and The effects of transgenic parasites expressing therapeutic polypeptides include genetic phenotypes. Comparison with animals treated with parasites that do not cause leukemia and comparison with sham-treated animals .

[0549] For example, treatment with Toxoplasma gondii parasites expressing the therapeutic protein MeCP2 The mouse model of Rett syndrome developed is characterized by known phenotypes of the disease, including the following phenotypes: Impaired social interaction, increased scent-finding behavior, hind-limb hugging, decreased survival, Anxiety, respiratory problems, hypoactivity, impaired motor coordination, miRNA-mediated IGF1 deficiency, BDN Decreased F levels, impaired cortical plasticity, glial dysfunction, and dysfunction of GABA-producing neurons , decreased soma size, decreased dendritic spine density, decreased synapse number, decreased activity (Ca2+ imaging), decreased sEPSC and sIPSC frequency and amplitude, Tuj1+ cells Decreased number, PAX6, and SCN1A / 1B expression, and dendrites in cortical neurons Decreased complexity, decreased AP frequency in cortical neurons, and generalized turnover in cortical neurons Decrease in transcription, neurons, synapses, highly expressed genes, immediate early genes in cortical neurons and decreased expression of mitochondrial genes and mRNA translation in cortical neurons. , decreased BDNF secretion from cortical neurons, AKT / mTOR in cortical neurons Decreased activity of the pathway and decreased oxygen consumption and maximal respiration in cortical neurons (de la Torre-Ubieta et al. Advancing the understanding of autism disease mechanisms thr ough genetics. Nat Med. 2016).

[0550] Example 5 Constructs for delivery of customized transcription activator-like effectors (TALEs) Preparation of substrates and Toxoplasma gondii strains Express and secrete customized transcription activator-like effectors (TALEs) To achieve this, we performed recombinant T. gondii. The customized TALE was It can be used for a variety of genome engineering applications, including transcriptional regulation and genome editing. The DNA recognition and binding domain of ALE is a sequence-determining 34-nucleotide adenine dinucleotide (ALE) fragment in the recognized DNA sequence. They are made up of tandem repeats of amino acid sequences (monomers). There are four types that differ in the repeat variable 2 residue (RVD) that determines the nucleotide that is recognized. The monomer designated "NI" (SEQ ID NO: 4508) is specific for the "A" nucleotide. The monomer designated "HD" (SEQ ID NO: 4495) is specific for the "C" nucleotide. The monomer designated "NG" (SEQ ID NO: 4509) is a "T" nucleotide. The monomer designated "NN" (SEQ ID NO: 4494) is specific and can be either "G" or "A" More recently, a further RVD NH (SEQ ID NO: 450) 4) was shown to provide higher G specificity. The in always ends with a half-length repeat (0.5 repeat). Use the LE DNA-binding domain to create custom TALE transcription factors (TALE-TFs) 1. Preparation and Transcription of Endogenous Genes from the Genome by Fusion with the Synthetic VP64 Transcription Activator It can also regulate the expression of TALE using a customized DNA-binding domain. To this end, custom TALE nucleases (TALENs) were created and used to perform site-specific A site-specific double-stranded break can be created by inserting a FokI endonuclease into the FokI endonuclease. Fusion of the catalytic domain of a ribosomal enzyme with the ribosomal enzyme allows for gene repair by non-homologous or homology-directed repair. Facilitates genome editing. Monomers inserted into TALEN or TALE-TF scaffolds (SEQ ID NOs: 4508, 4509, 4494, 4495 and 4504) The TALE is created by constructing a scaffold. Four TALEN scaffolds (e.g., SEQ ID NO: 4500) were used to contain the terminal 0.5 repeats. , 4501, 4502 and 4503), and four TALE-TF scaffolds (e.g., SEQ ID NOs: 4496, 4497, 4498 and 4499). Tomegalovirus promoter (CMV), non-repetitive N-terminus (N) from Hax3 TALE end), non-repetitive C-terminus from Hax3 TALE (C-terminus), custom TALE DNA binding Type II restriction sites (e.g., BsaI) used for insertion of domains, ccdB negative selection Negative selection containing the gene and chloramphenicol resistance gene (ccdB+CmR) The cassette contains a nuclear localization signal (NLS), and a FokI endonuclease (Fok I) catalytic domain or synthetic transcriptional activity derived from herpes simplex virus VP16 protein The activating factor (VP64) is then attached to the 2A self-cleaving linker (2A) and the sensitive green It also contains an enhanced fluorescent protein (EGFP). All of this is followed by a polyadenylation signal. (polyA signal) (Sanjana et al., A transcription activator-like effect The resulting TALE sequences were analyzed using the general method described above. Methods and Experimental Methods for Encoding Toxoplasma gondii Endogenous Secreted Polypeptides Insert the sequence downstream of the nucleotide sequence and transfect the resulting construct into Toxoplasma gondii. 2. Generating a Transgenic Toxoplasma gondii Strain that Expresses and Secretes TALE do.

[0551] For example, the TACGTACG sequence (SEQ ID NO: 4505) in the genome of a subject in need of treatment ) using TALE nuclease constructs or TALE transcription factor constructs. In the open reading frame of these constructs, the following sequences can be used: Monomers specific for targeting the "target sequence" (SEQ ID NO: 4505) are shown in Figure 1. 6A (for TALE nucleases) and 6B (for TALE transcription factors). The sequence information for these constructs is provided in the sequence listing (Sequences Nos. 4506 and 4507).

[0552] Example 6 Expressing mammalian therapeutic proteins and localizing them to rhoptry secretory organelles 1. Preparation of Toxoplasma gondii Parasites Expressing mammalian proteins and targeting them to rhoptry secretory organelles To generate Toxoplasma gondii parasites, we used type I RHΔHx Tachyzoites or RH (co-transfected with pDHFR selection plasmid) , containing a sequence encoding a protein of interest fused to the rhoptry protein toxophilin. Toxoplasma gondii was transfected with a plasmid carrying the The mammalian proteins tested in the lysin fusion line were glial-derived neurotrophic factor (GDN) and F), Parkin (PARK2), galactocerebrosidase (GALC), TAT protein Galactocerebrosidase fused to a gene transduction domain (GALC-TAT; SEQ ID NO: No. 4487), methyl-CpG binding protein (MeCP2), aspartoacylase (A The protein expression and survival of motor neuron protein (SMN1) were In an attempt to improve targeting, we also investigated the expression of GALC, MECP2, Codon-optimized versions of various proteins, including ASPA, will be tested and additional genetic A codon-optimized form of TFEB (SEQ ID NO: 4604) was tested.

[0553] ASPA (SEQ ID NO: 4483), codon-optimized ASPA (SEQ ID NO: 4602), GAL C (SEQ ID NOs: 4486 and 4601), GALC-TAT (SEQ ID NO: 4488), PA RK2 (SEQ ID NO: 4478), SMN1 (SEQ ID NO: 4479), GDNF (SEQ ID NO: 44 89), MeCP2 (SEQ ID NO: 4476) and codon-optimized MeCP2 (SEQ ID NO: 44 77) demonstrated various targeting of fusion proteins in parasites, including the Not only targeting the endoplasmic reticulum, but also targeting the Golgi apparatus, nucleus, cytoplasm, apicoplast, and mitochondria. Other patterns of targeting similar to clonames and other localizations were also included (Fig. 9A~N).

[0554] The GALC-TAT mutant resulted in predominantly rhoptry-like localization of the fusion protein. From the GALC-TAT mutant, we have demonstrated that the fusion protein is strongly expressed and expressed in the troponin-1 gene. A clonal line targeted to the rhesus monkey was also generated (Fig. 9H).

[0555] Example 7 Expressing mammalian therapeutic proteins and localizing them to dense granule secretory organelles 1. Preparation of Toxoplasma gondii Parasites that Secrete Them into Parasitophorous Vacuoles Express mammalian proteins and express them in the dense granule secretory organelle type I RHΔH To generate Toxoplasma gondii parasites targeting X. gondii, In Zuma gondii, encoding a protein of interest fused to the dense granule protein GRA16 The plasmid containing the sequence for the GRA16 fusion strain was transfected. The mammalian protein-coding genes selected are GALC [human type (SEQ ID NOs: 4486 and 4487)]. 601) and codon-optimized (SEQ ID NO: 4603), GALC-TAT (human, SEQ ID NO: No. 4488), MECP2 (codon optimized, SEQ ID NO: 4477), ASPA [human type (sequence number sequence number 4483) and codon-optimized (sequence number 4602)], SMN1 (human, sequence No. 4479) and TFEB (codon optimized, SEQ ID NO: 4604).

[0556] GALC, codon-optimized GALC, and GALC-TAT were transfected was expressed in the parasites (Fig. 10E, 10F and 10H). ASPA, codon-optimized AS PA, SMN1, codon-optimized MeCP2, and codon-optimized TFEB are expressed in dense granules. It was expressed by the IL-1 receptor and secreted into the parasitophorous vacuole (Fig. 10C, 10G, 10D, 10I, 10J). .

[0557] Example 8 Transgenic Toxoplasma gondii and its feeding to cultured human fibroblasts Animal therapeutic protein delivery The present inventors have not only secreted the fusion protein into the parasite vacuole, but also secreted it from the parasite vacuole. and targeting them to the nucleus of the host cell in amounts detectable by immunofluorescence staining. Six clonal strains of Toxoplasma gondii that can be used for both type I and type II infection were generated by recombination. These clones express RH GRA16-codon-optimized MECP2 (RH GRA16- MECP2opt), RH GRA16-codon optimized TFEB (RH GRA16-T FEBopt), Prugniaud-GFP-luciferase DHFR-TS GRA 16-codon optimized MECP2 (Pru-GFP-LUC GRA16-MECP2op t), Prugniaud-GFP-luciferase DHFR-TS GRA16-code Pru-GFP-LUC GRA16-TFEBopt, Pru niaud GRA16-codon-optimized MECP2 (Pru GRA16-MECP2o pt), and Prugniaud GRA16-codon optimized TFEB (Pru GR A16-TFEBopt) was added to a transgenic line expressing the fusion protein. In addition, the present inventors have synthesized the carrier protein GRA16 fused only to the HA epitope tag. Two control strains expressing RH GRA16-HA (RH GRA16-HAstop) and Also produced is Prugniaud GRA16-HA (Pru GRA16-HAstop). Both MeCP2 and TFEB are nuclear proteins, and therefore act as nuclear targets in the host cell. Targeting means that they are targeted to their active site in the cell. Infection of HFF cells in complete DMEM with tachyzoites of each strain, fixation, and immunofluorescence Light staining and analysis by fluorescence and polarized light microscopy revealed that all the above strains Secretion and host cell nuclear targeting of proteins detected in HFF human fibroblasts (Figures 10B, 10I, and 10J).

[0558] To quantify the efficiency and kinetics of GRA16-mediated protein delivery in vitro, To this end, the present inventors investigated the three type I strains RH GRA16- HAstop, RH GRA16-MECP2opt and RH GRA16-TFEB The time course of delivery and multiplicity of infection (MOI) of opt tachyzoites were quantified.

[0559] Example 9 Transgenic Toxoplasma gondii for the induction of peritoneal adenocarcinoma in cultured human neurons Mammal Therapeutic Protein Delivery Immortalized human dopaminergic cells, also known as Lund human midbrain (LUHMES) cells The neural progenitor cells were cultured in a medium containing L-glutamine, N-2 serum-free supplement, and βFGF (beta-irradiation). A medium containing F12 advanced DMEM medium supplemented with fibroblast growth factor Grown in a medium containing L-glutamine, N-2 serum-free supplement, tetracycline, and GDNF. and F12 advanced DM supplemented with cAMP (cyclic adenosine monophosphate). EM medium was used to culture morphologically and biochemically mature dopaminergic cells. The cells were differentiated into mature neurons from day 6 to day 9 of differentiation (at this point the cells were mature neurons). ) and the clone strains RH GRA16-MECP2opt and RH GRA16-TFEBo Infecting neurons using pt and rh GRA16-HAstop tachyzoites After 16-22 hours of infection, neurons were fixed, immunofluorescently stained, and analyzed by fluorescence microscopy. All strains tested showed clear secretion of the fusion protein. These were targeted to the nuclei of human neurons (Fig. 12A-C).

[0560] Furthermore, human neurons were infected with RH GRA16-MECP2opt tachyzoites. The infected neurons were washed with PBS and then harvested 24 hours after infection. Remove the residual PBS by scraping off the pellet using a paper towel and pelleting by centrifugation. The infected neurons were then flash frozen in liquid nitrogen. Nuclear proteins extracted from the infected neurons were analyzed using MeC Immunoprecipitated proteins were analyzed using magnetic beads conjugated to P2 antibody. , immunoblotting using the same MeCP2 antibody according to standard Western blot protocols. The resulting blot showed two strong bands at approximately 75 kDa and 130 kDa. The band of approximately 75 kDa was the endogenous non-truncated form derived from human neurons. The band at approximately 130 kDa represents MeCP2 (estimated size: 75 kDa), and the band at approximately 130 kDa is expressed by the parasite. The GRA16-MeCP2 fusion protein (estimated size of GRA16: 55 kDa, the estimated size of the fusion protein: 75 + 55 = 130 kDa) (Figure 14).

[0561] Example 10 Transgenic Toxoplasma gondii into cultured primary mouse cortex and cultured mouse hippocampal cultures Delivery of Mammalian MECP2 Using Zuma gondii and into Heterochromatic DNA Binding of delivered MECP2 Neuron-enriched primary cultures derived from the cortex and hippocampus of P1 (1-day-old) mouse pups were cultured for 5 days. tachyzoites of the transgenic strain RH GRA16-MECP2opt were cultured for 1 day. Neurons were fixed and immunofluorescently stained 12, 24, and 48 hours after infection. At all listed time points, GRA The 16-MECP2 fusion protein is secreted and targeted to the nucleus of neurons, where it They also appear in foci corresponding to regions of dense heterochromatin DNA, suggesting that This was recognized by the clear DAPI staining (Fig. 13A-D). This suggests that it is a useful marker for heterochromatin and binds well to it. In particular, in culture, Toxoplasma gondii attacks both neurons and glial cells. Primary cultures were pure (by NeuN staining) and capable of infecting bacteria and other cells. Because the neurons were not pure, we used transgenic Toxoplasma gondii Non-neuronal cells (probably mostly glial cells) infected with M. gondii also express MeCP2 fusions. It was possible to see that the cells received the protein and displayed the same characteristic lesion pattern.

[0562] Example 11 GRA16 and GRA1 in bradyzoite cysts of Toxoplasma gondii Expression of 6-MECP2 fusion protein During normal infection, the parasite replicates and disseminates within the body as tachyzoites during an early acute phase. After the sexual stage, Toxoplasma gondii tachyzoites produce a prolonged viral load in tissues, primarily the brain. Differentiation into bradyzoites present within the maintained resting cysts is induced by immune pressure. (Carruthers VB, Suzuki Y, 2007. Schizophr Bull. 33(3):745-51. Effects of Toxoplasma gondii infection on the brain). Toxoplasma gondii continues to express GRA16 even after differentiation into bradyzoites. We investigated whether the fused therapeutic protein was continuously expressed. In vitro differentiation of Toxoplasma gondii into bradyzoites is achieved using alkaline The induction was performed by stressing the cells with alkaline medium for 3 to 5 days. DMEM medium adjusted to pH 8.1 with 1 M HEPES and penicillin-streptomycin It contained 1% fetal bovine serum supplemented with tomycin (Tomita T, Bzik DJ, et al. 2013. PLoS Pathog. 9(12):e1003823. The Toxoplasma gondii cyst wall protein CST1 is cri (tical for cyst wall integrity and promotes bradyzoite persistence). The results are from the Pru GRA16-HAstop and Pru GRA16-MeCP2 strains. DBA (Dolichos Biflorus agglutinin) staining of the cyst wall was examined. Bradyzoite cysts, identified by color, express the GRA16-HAstop protein ( Both the GRA16-MeCP2 protein and the GRA16-MeCP2 protein were continuously expressed (data not shown) (Fig. 15A-C). This finding suggests that transgenic parasites, after differentiation to the bradyzoite stage, This indicates that GRA16 and GRA16 fusion proteins can be continuously expressed in the same manner. This is important for continuous secretion and protein delivery from the cyst during chronic infection.

[0563] Example 12 Heterologous Polypeptides Delivered by Toxoplasma gondii and Recipient Cells Probing molecular interactions with endogenous proteins within Co-immunoprecipitation was performed on infected cultures and cell lysates from infected animals. We have demonstrated that the IL-16 receptor agonist (IL-16) is delivered by transgenic Toxoplasma gondii. The molecular interactions of polypeptides with endogenous proteins and nucleic acids can be elucidated. do.

[0564] For example, in mammalian cells, MeCP2 acts as a SMRT / NCoR corepressor Both DNA and proteins derived from the complex and the SIN3A corepressor complex Binding to DNA was probed by immunofluorescence staining and chromatin immunoprecipitation. Binding to other proteins was detected by immunofluorescence staining and protein co-immunoprecipitation. The epitope tag attached to the fusion protein to be delivered can be used. They use the ATP to synthesize copies of proteins produced by the host cell and proteins delivered by the parasite. It is possible to distinguish between copies of the protein and the protein itself using antibodies against the protein itself. Endogenous proteins and proteins delivered to the parasite (e.g., for comparative assays) It is also possible to probe both.

[0565] Example 13 Transgenic Toxoplasma gondii expresses heterologous proteins of interest in animal models. Testing the ability to deliver proteins continuously (or chronically) Testing the ability of transgenic strains of parasites to affect the phenotype of the whole organism Testing of transgenic parasites in animal models has demonstrated alterations in the immune system and functional blood This allows for the evaluation of strains in the presence of the liquid-brain barrier (BBB). Nick the industry-established standard infection for infecting Toxoplasma gondii. The procedure will be performed according to the protocol (Cabral CM et al., 2016, PLoS Pathog. 12(2):e100544 7. Neurons are the Primary Target Cell for the Brain-Tropic Intracellular Parasi te Toxoplasma gondii, Berenreiterova M et al., 2011. PLoS One. 6(12):e28925. The distribution of Toxoplasma gondii cysts in the brain of a mouse with latent tox oplasmosis: implications for the behavioral manipulation hypothesis, Tait ED et al. 2010. J. Immunol. 185(3):1502-12. Virulence of Toxoplasma gondii is associat ed with distinct dendritic cell responses and reduced numbers of activated CD8+ T cells, Koshy AA et al. 2012. PLoS Pathog. 8(7):e1002825. Toxoplasma co-opts ho st cells it does not invade, Jensen KD et al. 2015. MBio. 6(2):e02280. Toxoplasm a gondii superinfection and virulence during secondary infection correlate with Transgenic Toxoplasma gondii (the exact ROP5 / ROP18 allelic combination). The parasite can be administered intranasally, enterally (orally, etc.), subcutaneously, intramuscularly, intravenously, intradermally, intraperitoneally, or intracranially. Intravenous or intracerebral administration to transgenic Toxoplasma gondii animal models The parasites are administered in the form of tachyzoites, bradyzoites, tissue cysts or oocysts. The model involves the delivery of a protein of interest expressed in wild-type animals or transgenic parasites. These are animal models with pathologies that can be treated or affected by After the initial stage of acute infection, animals develop chronic bradyzoite tissue formation, primarily in the brain. This mode of delivery allows for the maintenance of transgenic Toxoplasma gondii during the chronic phase of infection. It relies on the continuous expression and secretion of a protein of interest by Zyma gondii.

[0566] Various stages in the development of chronic infection (depending on the strain and route of infection) occur from the first 3 weeks after infection up to 2 months), as well as at various time points after chronic infection was established and stabilized (several months post-infection). Over time scales of months to years, brain tissue staining was used to measure the percentage of infected cells, parasitemia, and The distribution of the worms and the delivered proteins in the brain and other tissues will be evaluated, and the delivered proteins will be analyzed. Quantify protein levels in recipient cells using co-immunoprecipitation and cell morphology. Identify molecular interactions between proteins and their endogenous counterparts, providing evidence for their function It is possible.

[0567] Symptoms of the disease phenotype and the efficacy of treatments to alleviate them, as well as potential toxicity associated with treatments. To assess the efficacy of the treatment, the status of the treated animal model was monitored at various stages of chronic infection and disease progression. Monitoring the various stages of the process using physiological, behavioral, cellular and molecular means The efficacy of transgenic parasites was assessed by comparing the expression of the pharmaceutical polypeptides with the control parasites. The disease is assessed by comparison with animals treated with non-infectious parasites and / or sham treated animals.

[0568] For example, treatment with Toxoplasma gondii parasites expressing the therapeutic protein MeCP2 The mouse model of Rett syndrome developed is characterized by known phenotypes of the disease, including the following phenotypes: Impaired social interaction, increased scent-finding behavior, hind-limb hugging, decreased survival, Anxiety, respiratory problems, hypoactivity, impaired motor coordination, miRNA-mediated IGF1 deficiency, BDN Decreased F levels, impaired cortical plasticity, glial dysfunction, and dysfunction of GABA-producing neurons , decreased soma size, decreased dendritic spine density, decreased synapse number, decreased activity (Ca2+ imaging), decreased sEPSC and sIPSC frequency and amplitude, Tuj1+ cells Decreased number, PAX6, and SCN1A / 1B expression, and dendrites in cortical neurons Decreased complexity, decreased AP frequency in cortical neurons, and generalized turnover in cortical neurons Decrease in transcription, neurons, synapses, highly expressed genes, immediate early genes in cortical neurons and decreased expression of mitochondrial genes and mRNA translation in cortical neurons. , decreased BDNF secretion from cortical neurons, AKT / mTOR in cortical neurons Decreased pathway activity and decreased oxygen consumption and maximal respiration in cortical neurons (de la Torre-Ubieta et al. Advancing the understanding of autism disease mechanisms thr 2016, 22:345-61).

[0569] Example 14 Transgenic Toxoplasma gondii expresses heterologous proteins of interest in animal models. Testing the ability to transiently ( / acutely) deliver proteins As in Example 13 above, transgenic strains of the parasite affect the phenotype of the whole organism. Testing transgenic parasites in animal models will This allows for the evaluation of strains in the presence of the immune system and a functional blood-brain barrier (BBB). Transgenic cells in the form of tachyzoites, bradyzoites, tissue cysts or oocytes Xoplasma gondii: nasal, enteral (oral, etc.), subcutaneous, intramuscular, intravenous, intradermal, and intraperitoneal routes The animal model can be administered intravenously, intracranially, or intracerebrally. can be treated by delivery of a protein of interest expressed by a transgenic parasite, or The experimental design may be an animal model for a pathological condition that can be affected by the does not rely on differentiation into bradyzoites and establishment of chronic infection and / or chronic cysts; These focus on evaluating transient delivery. Some examples include: inability to differentiate into bradyzoites and / or inability to replicate, and and / or unable to persist in vivo for longer than 2-3 weeks or chronic Delivery by attenuated parasites that are unable to establish an infection; target tissue or target group Delivery by local administration to an area close to the tissue, followed by clearance or inactivation of the parasite. These tests can also be repeated multiple times for multiple doses. and to aid in the reinfection of animals previously infected with Toxoplasma gondii. Immunosuppressants may be administered before, during, or after infection with the parasite. The plan also includes transgenic Toxoplasma gondii capable of establishing chronic infection. The virus is delivered by the immunization system, but the collection or analysis of samples is performed during the acute phase of infection before the establishment of a chronic infection. This form of delivery involves administering the virus to the target organs, primarily via tachyzoites of the transgenic strain. The pathogenicity of the virus depends on the secretion of specific proteins. Brain tissue staining was used to measure the percentage of infected cells over a period of time (from the first few days to approximately 2 months after infection). Assessment of stage, distribution of parasites and characterization of delivered proteins in brain and other tissues The delivered protein levels in recipient cells were then quantified. To confirm the molecular interactions between proteins and their endogenous counterparts and provide evidence for their functions, To investigate this, we used co-immunoprecipitation and cell morphology.

[0570] Symptoms of the disease phenotype and the efficacy of treatments to alleviate them, as well as potential toxicity associated with treatments. The status of the treated animal model was monitored at various times after the start of treatment and at Monitor using physiological, behavioral, cellular and molecular measures at various stages of disease progression. The efficacy of the transgenic parasites was assessed by the pharmacological polypeptides. The effect of the worms on the expression of the IL-1 gene is assessed by comparison with animals treated with non-expressing parasites and / or sham-treated animals. do.

[0571] Example 15 Increasing the safety and controllability of transgenic Toxoplasma gondii Introducing further modifications The parasite is generally considered harmless in healthy humans, but its virulence can be further enhanced by Therapeutic strains may incorporate further modifications to the parasite to make it more attenuated. Being a model organism for that phylum, it is possible to develop an attenuated, externally controllable parasite. (Moe-Behrens GH, Davis R, Haynes KA. Front Microbiol. 2013 4:5. "Preparing synt Various high-level methods for modifying parasites that can be used to create "parasitic biology for the world" There are well-developed genetic tools available (Jimenez-Ruiz E, Wong EH, Pall GS, Meissner M. Parasitology. 2014, 141:1390-8. "Advantages and disadvantages of conditional sys "Mems for characterization of essential genes in Toxoplasma gondii" Second, the ability to deliver protein drugs is not essential. Attenuated parasites by targeting and disrupting specific virulence mechanisms of the parasite Another approach involves active control through engineered inducible lethality, e.g. For example, an inducible "suicide switch" (self-destruct enzyme) can be developed that eliminates the parasite by administering an activating molecule. Some examples include in vivo lethality or is a widely used tetracycline-mediated pathway for drug-induced activation of apoptotic pathways. Use of gene-inducible promoters, inducible knockout of essential genes, or lethal or The rapamycin-dimerized diCre system is responsible for the inducible activation of apoptotic pathways. Such drug-induced regulation also varies depending on the tissue or cell. Differences in drug permeability and distribution in the body can affect specific tissues, regions, or cell types. This may allow differential clearance of parasites from the

[0572] Analysis and Discussion This invention utilizes the mechanism of Toxoplasma gondii, a brain parasite, to deliver proteins. The platform is capable of crossing the blood-brain barrier and intracellular delivery within the CNS. This allows for the delivery of proteins to cells, particularly neurons.

[0573] To enable delivery of therapeutic proteins to the CNS, the present inventors have developed a method for transfecting the target protein. Toxoplasma gondii parasites for protein synthesis and delivery to cells within the CNS Thus, the present inventors have demonstrated the specific and controlled delivery of proteins to cells within the CNS. Establish a versatile platform for controlled delivery.

[0574] The recombinant parasite has the following novel capabilities: (1) Synthesize various therapeutic heterologous proteins and deliver them specifically into target cells within the CNS. The parasite can be easily engineered to secrete (2) reduce or eliminate elements of the parasite that can mediate harmful effects on the infected host; The parasite may contain modifications to eliminate it. (3) Regulation and / or regulation that allows control of its distribution and spatiotemporal activity after it enters the host The parasite may contain a self-destructing element.

[0575] The following is a summary of the genomic changes in the parasites described herein, including but not limited to: It's not something like that. (i) Fusion of a selected therapeutic protein to a specific endogenously secreted protein of the parasite. Additional auxiliary elements and modifications that may increase the efficacy of the therapeutic fusion protein may be incorporated into the fusion protein. Such auxiliary elements and modifications can be added to the synthetic gene / protein. Therapeutic proteins that are no longer required for targeting or activity are then transferred to endogenous parasite proteins. a cleavage site that allows the protein to be detached from the Toxoplasma Epitope tags and fluorescent proteins that can mediate detection and assessment of the efficacy and distribution of proteins Proteins, etc. (ii) Removal of virulence genes that are not necessary for the therapeutic role of the parasite. The modifications will reduce the parasite's potential harmful effects on patients, making the parasite safe for clinical use. Make it appropriate for the situation. (iii) to induce parasite self-destruction in response to exogenously administered drugs; or the insertion of inducible regulatory elements to manipulate the activity of the parasite. These elements increase the safety of this technology and also provide increased control over its spatiotemporal activity. To provide.

[0576] The primary strains developed using the methods of some embodiments of the present invention are the molluscs responsible for Krabbe disease. Lactocerebrosidase and galactocerebrosidase-TAT proteins, various gods Degenerative diseases and other conditions (especially Parkinson's disease, supranuclear palsy, multiple sclerosis, Alzheimer's disease, Immer's disease, amyotrophic lateral sclerosis, Huntington's disease, retinal degeneration, traumatic brain injury and hypoxia GDNF protein involved in neuropathic / ischemic CNS disorders, aspartame involved in Canavan disease The seroconvertible phospholipase protein, the MeCP2 protein involved in Rett syndrome, and the protein involved in spinal muscular atrophy SMN protein, an E3 ubiquitin-protein ligase involved in Parkinson's disease and TFEB-based proteins involved in lysosomal storage disorders and neurodegenerative diseases. The inventors have identified several compounds currently being clinically tested and with known auxiliary functions. We selected a therapeutic protein for this purpose, but this platform can be adapted to a variety of proteins. It is possible.

[0577] Potential applications of this technology include, but are not limited to: do not have. 1. Treatment of disease states through delivery of therapeutic proteins. 2. Delivery of proteins to the brain of healthy individuals to increase brain function or promote neuronal regeneration. supply. 3. For highly specific and efficient targeted protein delivery to supplement protein synthesis A common solution to this problem.

[0578] While this invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications and variations are possible. It will be apparent to those skilled in the art and therefore fall within the spirit and broad scope of the appended claims. It is intended to embrace all such alternatives, modifications and variations.

[0579] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. The same references are hereby incorporated by reference as if the patent applications were specifically and individually set forth. Further, the citation or disclosure of any reference in this application is hereby incorporated by reference. Neither citation nor any statement hereof shall be construed as an admission that such reference is prior art to the instant application. The use of section headings should not necessarily be construed as limiting. You shouldn't.

[0580] References 1. Pardridge, WM Targeted delivery of protein and gene medicines through the blood-brain barrier. Clin. Pharmacol. Ther. 97, 347-61 (2015). 2. Cox, DBT, Platt, RJ & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121-131 (2015). 3. Persons, DA & Baum, C. Solving the problem of γ-retroviral vectors contai ning long terminal repeats. Mol. Ther. 19, 229-31 (2011). 4. Schambach, A., Zychlinski, D., Ehrnstroem, B. & Baum, C. Biosafety features o f lentiviral vectors. Hum. Gene Ther. 24, 132-42 (2013). 5. Gray, S. J., Nagabhushan Kalburgi, S., McCown, T. J. & Jude Samulski, R. Glob al CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathe cal AAV administration in non-human primates. Gene Ther. 20, 450-9 (2013). 6. Templeton, N. Gene and cell therapy: therapeutic mechanisms and strategies. ( 2008). 7. Dimmeler, S., Ding, S., Rando, T. A. & Trounson, A. Translational strategies and challenges in regenerative medicine. Nat. Med. 20, 814-21 (2014). 8. Abbott, N. J. Blood-brain barrier structure and function and the challenges f or CNS drug delivery. J. Inherit. Metab. Dis. 36, 437-49 (2013). 9. Malhotra, M. & Prakash, S. Targeted Drug Delivery Across Blood-Brain-Barrier Using Cell Penetrating Peptides Tagged Nanoparticles. Curr. Nanosci. 7, 81-93 (2 011). 10. Bradbury, M. W. B. Physiology and Pharmacology of the Blood-Brain Barrier. ( Springer Science & Business Media, 2012). 11. Solaro, R., Chiellini, F. & Battisti, A. Targeted Delivery of Protein Drugs by Nanocarriers. Materials (Basel). 3, 1928-1980 (2010). 12. Sanecka, A. & Frickel, E.-M. Use and abuse of dendritic cells by Toxoplasma gondii. Virulence 3, 678-89 (2012). 13. Carruthers, V. B. & Suzuki, Y. Effects of Toxoplasma gondii infection on the brain. Schizophr. Bull. 33, 745-51 (2007). 14. Feustel, S. M., Meissner, M. & Liesenfeld, O. Toxoplasma gondii and the bloo d-brain barrier. Virulence 3, 182-92 15. Montoya, J. G. & Liesenfeld, O. Toxoplasmosis. Lancet 363, 1965-76 (2004). 16. Dlugonska, H. Toxoplasma rhoptries: unique secretory organelles and source o f promising vaccine proteins for immunoprevention of toxoplasmosis. J. Biomed. B iotechnol. 2008, 632424 (2008). 17. Carruthers, V. B. & Sibley, L. D. Sequential protein secretion from three di stinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts . Eur. J. Cell Biol. 73, 114-23 (1997). 18. Boothroyd, J. C. & Dubremetz, J.-F. Kiss and spit: the dual roles of Toxopla sma rhoptries. Nat. Rev. Microbiol. 6, 79-88 (2008). 19. Koshy, A. A. et al. Toxoplasma secreting Cre recombinase for analysis of hos t-parasite interactions. Nat. Methods 7, 307-9 (2010). 20. Fox, B. A., Sanders, K. L. & Bzik, D. J. Non-replicating Toxoplasma gondii r everses tumor-associated immunosuppression. Oncoimmunology 2, e26296 (2013). 21. Fox, B. A., Sanders, K. L., Chen, S. & Bzik, D. J. Targeting tumors with non replicating Toxoplasma gondii uracil auxotroph vaccines. Trends Parasitol. 29, 4 31-7 (2013). 22. Elliott, D. E. & Weinstock, J. V. Helminth-host immunological interactions: prevention and control of immune-mediated diseases. Ann. N. Y. Acad. Sci. 1247, 83-96 (2012). 23. Rothman, J. & Paterson, Y. Live-attenuated Listeria-based immunotherapy. Exp ert Rev. Vaccines 12, 493-504 (2013). 24. Reeves, A. Z. et al. Engineering Escherichia coli into a protein delivery sy stem for mammalian cells. ACS Synth. Biol. 4, 644-54 (2015). 25. Meng, X.-L., Eto, Y., Schiffmann, R. & Shen, J.-S. HIV Tat Domain Improves C ross-correction of Human Galactocerebrosidase in a Gene- and Flanking Sequence-d ependent Manner. Mol. Ther. Nucleic Acids 2, e130 (2013). 26. Otto P. van Diggelen. MOSCERDAM SUBSTRATES, Laboratory protocol for enzyme a nalysis fir Krabbe disease; Galactocerebrosidase. MOSCERDAM SUBSTRATES, Laborato ry protocol for enzyme analysis fir Krabbe disease; Galactocerebrosidase. [Sequence List Free Text]

[0581] SEQ ID NO: 67: n is a, c, g or t SEQ ID NO: 206: n is a, c, g or t SEQ ID NO: 300: Xaa is any naturally occurring amino acid SEQ ID NO: 439: Xaa is any naturally occurring amino acid SEQ ID NO: 1462: n is a, c, g or t SEQ ID NO: 2112: n is a, c, g or t SEQ ID NO: 2292: n is a, c, g or t SEQ ID NO: 3004: n is a, c, g or t SEQ ID NO: 3270: n is a, c, g or t SEQ ID NO: 3326: n is a, c, g or t SEQ ID NO: 3586: n is a, c, g or t SEQ ID NO: 4031: 5RT70 constitutive promoter SEQ ID NO: 4032: DHFR constitutive promoter SEQ ID NO: 4033: MIC2 constitutive promoter SEQ ID NO: 4034: MIC8 constitutive promoter SEQ ID NO: 4035: SAG1 constitutive promoter SEQ ID NO: 4036: TetO7SAG1 inducible promoter SEQ ID NO: 4037: TetO7SAG4 inducible promoter SEQ ID NO: 4038: TUB1 constitutive promoter, where n is a, c, g, or t be SEQ ID NO: 4039: TUB8 constitutive promoter SEQ ID NO: 4472: Nucleic acid sequence encoding GRA16 SEQ ID NO: 4473: Nucleic acid sequence of GRA16_5'UTR SEQ ID NO: 4474: nucleic acid sequence encoding HA tag SEQ ID NO: 4475: nucleic acid sequence encoding HXGPRT SEQ ID NO: 4476: Nucleic acid sequence encoding MeCP2 SEQ ID NO: 4477: nucleic acid encoding MECP2_codon_optimized array SEQ ID NO: 4478: Nucleic acid sequence encoding PARK2 SEQ ID NO: 4479: Nucleic acid sequence encoding SMN1 SEQ ID NO: 4480: Nucleic acid sequence encoding TAT SEQ ID NO: 4481: Nucleic acid sequence encoding toxophilin SEQ ID NO: 4482: Nucleic acid sequence of Toxofilin_5'UTR SEQ ID NO: 4483: Nucleic acid sequence encoding ASPA SEQ ID NO: 4484: Nucleic acid sequence encoding DHFR-TS_cDNA SEQ ID NO: 4485: nucleic acid sequence of DHFR-TS_genomic SEQ ID NO: 4486: Nucleic acid sequence encoding GALC SEQ ID NO: 4487: Nucleic acid sequence encoding mutated GALC-TAT SEQ ID NO: 4488: Nucleic acid sequence encoding GALC-TAT SEQ ID NO: 4489: Nucleic acid sequence encoding GDNF SEQ ID NO: 4490: nucleic acid sequence of Gly_linker SEQ ID NO: 4491: Nucleic acid sequence of GRA2_3'UTR SEQ ID NO: 4492: Nucleic acid sequence of DHFR-TS_5'UTR SEQ ID NO: 4493: Nucleic acid sequence of DHFR-TS_3'UTR SEQ ID NO: 4494: Nucleic acid sequence of TALE monomer template pNN SEQ ID NO: 4495: Nucleic acid sequence of TALE monomer template pHD SEQ ID NO: 4496: nucleic acid sequence of pTALE-TF(NI) SEQ ID NO: 4497: nucleic acid sequence of pTALE-TF(NG) SEQ ID NO: 4498: nucleic acid sequence of pTALE-TF(NN) SEQ ID NO: 4499: nucleic acid sequence of pTALE-TF(HD) SEQ ID NO: 4500: nucleic acid sequence of pTALEN(NI) SEQ ID NO: 4501: nucleic acid sequence of pTALEN(NG) SEQ ID NO: 4502: nucleic acid sequence of pTALEN(NN) SEQ ID NO: 4503: nucleic acid sequence of pTALEN(HD) SEQ ID NO: 4504: Nucleic acid sequence of TALE NH monomer SEQ ID NO: 4505: Nucleic acid sequence of target sequence SEQ ID NO: 4506: nucleic acid sequence of TALE_Nuc_TACGTACG Numbers 2 to 805 are pUC_ori; Numbers 1454 to 2054 are CMV_Promoter, Numbers 2113 to 2232 are NLS, 2233~2640 are TAL_N-Term, 2641~2742 are Tandem_Repeats "Repeat 1:N I”, 2743~2844 are Tandem_Repeats "Repeat 2:H D”, 2845~2946 are Tandem_Repeats "Repeat 3:N N”, 2947~3048 are Tandem_Repeats "Repeat 4:N G”, 3049~3150 are Tandem_Repeats "Repeat 5:N I”, 3151~3252 are Tandem_Repeats "Repeat 6:H D”, Numbers 3253 to 3312 are Half_Repeat, Nos. 3313 to 3501 are TAL_C-term, Numbers 3502 to 4104 are Fok1, Nos. 4342 to 4685 are SV40_ori, Nos. 4731 to 5756 are hygromycin; Nos. 5884 to 6014 are SV40_pA_Signal, 6069-6917 is ampicillin (complementary strand) SEQ ID NO: 4507: TALE_TF_TACGTACG; Numbers 2 to 805 are pUC_ori; Numbers 1454 to 2054 are: CMV_Promoter, 2120~2839 are TAL_N-Term, 2840~2941 are Tandem_Repeats "Repeat 1:N I”, 2942~3043 are Tandem_Repeats "Repeat 2:H D”, 3044~3145 are Tandem_Repeats "Repeat 3:N N”, 3146~3247 are Tandem_Repeats "Repeat 4:N G”, 3248~3349 are Tandem_Repeats "Repeat 5:N I”, 3350~3451 are Tandem_Repeats "Repeat 6:H D”, Numbers 3452 to 3511 are Half_Repeat, Nos. 3512 to 4045 are TAL_C-terms, Numbers 4049 to 4051 are NLS_(SPKKKRKVEA), Numbers 4052 to 4222 are VP64_AD, Nos. 4229 to 4291 are 22A, Nos. 4292 to 5005 are EGFP, 5641 to 6666 are hygromycin; Nos. 6794 to 6924 are SV40_pA_Signal, 6979-7827 are ampicillin (complementary strand) SEQ ID NO: 4508: Nucleic acid sequence of TALE monomer template pNI SEQ ID NO: 4509: Nucleic acid sequence of TALE monomer template pNG SEQ ID NO: 4602: ASPA codon optimized SEQ ID NO: 4603: Human GALC isoform 1 codon-optimized SEQ ID NO: 4604: Human TFEB (transcription factor EB) isoform 1 codon optimized done SEQ ID NO: 4605: DHFR (version 2) amino acid sequence SEQ ID NO: 4606: DHFR (version 2) nucleic acid sequence SEQ ID NO: 4607: mCherry mammalian codon optimized SEQ ID NO: 4608: mCherry mammalian codon optimized SEQ ID NO: 4609: 3'UTR DHFR (version 2) nucleic acid sequence

Claims

1. Toxoplasma transformed with a nucleic acid construct comprising a heterologous polynucleotide in which a first nucleic acid sequence encoding a Toxoplasma secretory protein is fused in frame upstream of a second nucleic acid sequence encoding a pharmaceutical polypeptide for transcription regulation or genome editing, wherein the heterologous polynucleotide is operably linked to a promoter for directing transcription of the heterologous polynucleotide in Toxoplasma, the promoter being selected from the group consisting of a constitutive promoter, an inducible promoter, a latency-specific promoter, and a Toxoplasma endogenous promoter, with the proviso that the promoter is not a toxophilin promoter, the Toxoplasma secretory protein is a non-rhoptry protein and is secreted into host cells, and the Toxoplasma is for transcription regulation or genome editing in the host cell.

2. The Toxoplasma of claim 1, wherein the pharmaceutical polypeptide is a TALEN (TALE nuclease) for genome editing.

3. The Toxoplasma gondii according to claim 1, wherein the pharmaceutical polypeptide is a TALE-TF (TALE transcription factor) for transcriptional regulation.

4. The Toxoplasma according to any one of claims 1 to 3, wherein the non-rhoptry protein is selected from a protein secreted by micronemes and Toxoplasma gondii macrophage migration inhibitory factor (TgMIF).

5. The Toxoplasma of claim 4, wherein the protein secreted from the microneme comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 280 to 322.

6. The Toxoplasma according to any one of claims 1 to 5, wherein the Toxoplasma persists in the infected host cells.

7. The Toxoplasma gondii of any one of claims 1 to 6, wherein the nucleic acid construct further comprises a third nucleic acid sequence encoding an inducible self-destruction element.

8. The Toxoplasma gondii of claim 7 , wherein the inducible self-destruct element becomes active in response to a drug.

9. The Toxoplasma gondii according to any one of claims 1 to 8, provided that the nucleic acid construct does not contain a Cre-recombinase coding sequence.

10. The Toxoplasma of any one of claims 1 to 9, wherein the nucleic acid construct further comprises at least one in-frame cleavage site that allows detachment of the pharmaceutical polypeptide from the Toxoplasma secretory protein.

11. The Toxoplasma gondii according to any one of claims 1 to 10, which is not attenuated.

12. The Toxoplasma according to any one of claims 1 to 10, which does not contain Toxoplasma elements that facilitate the proliferation of the Toxoplasma in a host cell.

13. The Toxoplasma of claim 12, which does not contain virulence genes that are not necessary for delivery of the pharmaceutical polypeptide to the host cell.

14. The Toxoplasma of claim 12, which does not contain virulence genes that are not necessary for delivery of the pharmaceutical polypeptide to the central nervous system (CNS) or muscle tissue.

15. A pharmaceutical composition comprising the Toxoplasma gondii according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. a first container containing the pharmaceutical composition of claim 15; a second container containing an immunosuppressant; Kit including:

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