Patent Grant
11260081
The present invention, in some embodiments thereof, relates to a nucleic acid construct for the secretion of a pharmaceutical polypeptide fused to a Toxoplasma gondii secreted polypeptide, and Toxoplasma comprising same, and, more particularly, but not exclusively, to a pharmaceutical compositions and methods of using same for treating a subject.
The lack of robust methods for the delivery of protein therapeutics is to a great degree the current bottleneck in their translation into clinical treatment. Proteins often serve a highly specific and complex set of functions that cannot be mimicked by simple chemical compounds (Nat Rev Drug Discov. 2008; 7(1):21-39. Protein therapeutics: a summary and pharmacological classification. Leader B, Baca Q J, and Golan D E); however, owing to their macromolecular nature, the delivery of therapeutic protein to target sites in the body is extremely challenging. Low functional stability and rapid loss of activity following administration or during storage, along with low permeability through biological barriers, limit the delivery of active proteins. The ongoing development of chemical modifications, conjugates and carrier systems aided the delivery of some therapeutic proteins, but these are mostly still limited to targets within the “accessible target space”—in the vascular compartment or on the surface of cells (Mitragotri 2014, Nat. Rev. Drug. Discov. 13(9):655-72). Efficient delivery is particularly challenging in the field of neurological diseases due to the blood-brain barrier (BBB), which tightly regulates the transport of molecules to the brain, while further complexity is added when the target is intracellular.
In order to deliver supplementary proteins to cells within the CNS, it is necessary to develop mechanisms that penetrate the BBB and may target specific cells within the CNS.
Currently, the major approaches for treating conditions caused by a deficiency of a specific protein are gene therapy, stem cell therapy and enzyme replacement therapy.
Gene therapy is the insertion of a functional copy of a gene that encodes a therapeutic protein (Cox, D. B. T., et al., 2015) which aims to increase the expression of a functional copy of the protein. While gene therapy provides the correct sequence of the gene, it does not address loss of protein function that is caused by or accompanied by defects in translational mechanisms or post-translational modifications. In addition, it can bear the risk of cancer development due to insertional mutagenesis (Persons, D. A. & Baum, C. 2011), mobilization, germline transmission, immunogenicity and limited transgene capacity (Schambach, A., et al., 2013; Al-Dosari et al., 2009). To target the CNS, gene therapy is most commonly mediated by the use of viral vectors, including those derived from herpes simplex virus type 1 (HSV-1), adenovirus, AAV, lentiviruses such as HIV-1, feline immunodeficiency virus or equine infectious anemia virus, and more recently SV40-AAV and lentivirus which are currently the most common of those. While some viral vectors are safer than the others, they do not address protein processing defects, and clinical efficiency remains limited.
Stem cell therapy relies on the protein synthesis mechanisms of implanted cells which can supplement the defective endogenous synthesis of the protein. Despite numerous efforts to implement this approach clinically, the efficiency of such treatments is reduced by rejection of implanted cells, and the low cost-efficiency of using patient-specific stem cells. In addition, the risks involved in stem cell treatments include complications associated with immunosuppression required to reduce rejection of grafted cells, and the development of cancer (Dimmeler, S., et al., 2014).
Protein- or Enzyme replacement therapy (ERT), in which the supplementary protein is synthesized externally and then delivered into the CNS, must overcome the impermeability of the BBB, and develop precise targeting in order to be clinically relevant. One such approach to protein delivery is injection directly into the brain or into adjacent organs from which the protein may diffuse at a low efficiency. Thus, in addition to the risk of such approaches, the ability to target specific regions within the CNS with the necessary amount is limited. Another problem of such approaches is the requirement of repeated dosing, which is often clinically impractical (Abbott, N.J. 2013).
ERT can be combined with an increase in BBB permeability to allow the therapeutic protein to diffuse into the CNS with greater efficiency (Malhotra, M. & Prakash, S. 2011). This is achieved through the shrinking of the cells that compose the BBB, or manipulation of the transport mechanisms through the BBB. However, this approach is not efficient due to opsonization, and bears the risk of unwanted substances diffusing into the CNS along with the supplemented protein of interest (Bradbury, M. W. B. 2012).
An alternative approach is the attachment of the therapeutic protein to elements that enter the CNS to utilize their inherent ability to cross the BBB. Agents that may mediate trafficking across the BBB include fusion proteins, dendrimers, solid lipid nanoparticles, liposomes and nanoparticles (Solaro, R., 2010). However, these approaches require further development and have not reached clinical trials.
Toxoplasma gondii is a single-cell intracellular protozoan parasite of the phylum apicomplexa. Its primary hosts are felines, and only in them it can go through the sexual stages of its life cycle. However, as secondary hosts it can infect many warm-blooded organisms, including humans. In humans as in rodents, after the host contracts the parasite (typically after ingestion of infectious tissue cysts or oocysts), the parasite differentiates into the fast-replicating tachyzoite stage. Toxoplasma gondii tachyzoites invade nucleated host cells by active penetration and establish a vacuole within which they replicate by endodyogeny. The tachyzoites travel through the intestinal epithelium, and by both “hitch-hiking” on immune cells and by mobilizing itself autonomously, they reach distal tissues, breach the blood brain barrier and spread in the brain [Harker 2015, Parasite Immunol. 37(3):141-9]. Thus, the tachyzoites enter the circulation and disseminate to secondary tissues. Replication is critical for dissemination and for reaching distal tissues during the acute stage of infection. Once inside the unique environment of the brain, T. gondii penetrates cells in the brain (primarily neurons, but also glial cells in lower proportions) and following immune pressure, it differentiates to the latent bradyzoite stage, and resides in cytoplasmic intercellular cysts. This characterizes a chronic infection (Cabral et al. 2016, PLoS Pathog. 12(2):e1005447). Tissue cysts harbouring bradyzoites persist for the lifetime of the host, while multiplying very slowly and having a quiescent metabolic program (Parasite Immunol. 2015, 37(3):141-9. “Toxoplasma gondii dissemination: a parasite's journey through the infected host”).
Although T. gondii infects an estimated one third of the world population, infection remains asymptomatic in healthy humans (Montoya, J. G. & Liesenfeld, O. 2004) and is primarily a risk only for individuals with irregularly compromised immune systems. Most importantly, during infection T. gondii secretes proteins into the host's cells, both during cell invasion and while intracellular. T. gondii has three types of electrodense secretory organelles: micronemes, rhoptries, and dense granules. Host cell invasion is mediated by the sequential secretion of the contents of all three organelles, which are exocytosed from the apical region when the parasite invades the host cell and when it resides inside it (Dlugonska, H. 2008). Micronemes are involved in the attachment and penetration of T. gondii, while rhoptries are required for creating a transient structure, moving junction and then the establishment of the PV. Rhoptry proteins are secreted upon initial contact with the host in a process termed “kiss and spit” (Boothroyd, J. C. et al., 2008). Dense granules secrete proteins throughout most of the parasite stages. The secretion process coincides with the formation of intravacuolar network and continues during the intracellular residence of T. gondii (Dlugonska, H. 2008; Carruthers, V. B. & Sibley, L. D. 1997).
Additional background art includes Koshy, A. A. et al. 2010; U.S. Pat. No. 8,673,289; US20120045477 Al; Lodoen M. B., et al. 2010. Cellular Microbiology, 12: 55-66.
According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising a heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein in frame fused upstream to a second nucleic acid sequence encoding a pharmaceutical polypeptide, wherein the heterologous polynucleotide is operably linked to a promoter for directing transcription of the heterologous polynucleotide in a Toxoplasma, wherein the promoter is selected from the group consisting of: a constitutive promoter, an inducible promoter, a latent period-specific promoter, and a Toxoplasma endogenous promoter with the proviso that the promoter is not a Toxofilin promoter.
According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct system comprising at least two nucleic acid constructs, wherein a first nucleic acid construct of the at least two nucleic acid constructs is the nucleic acid construct of some embodiments of the invention, and a second nucleic construct of the at least two nucleic acid constructs comprises a polynucleotide encoding a selectable marker.
According to an aspect of some embodiments of the present invention there is provided a Toxoplasma transformed with the nucleic acid construct of some embodiments of the invention or with the nucleic acid construct system of some embodiments of the invention.
According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the Toxoplasma of some embodiments of the invention, and a pharmaceutically acceptable carrier.
According to an aspect of some embodiments of the present invention there is provided a method of administering a protein-of-interest into a central nervous system of a subject, the method comprising: administering to the subject the Toxoplasma of some embodiments of the invention or the pharmaceutical composition of some embodiments of the invention, thereby administering the protein-of-interest to the central nervous system of the subject.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need thereof, comprising administering to the subject the Toxoplasma of some embodiments of the invention or the pharmaceutical composition of some embodiments of the invention, wherein the subject is diagnosed with a pathology treatable by administration of the pharmaceutical polypeptide in a central nervous system of the subject, thereby treating the subject in need thereof.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need thereof, comprising administering to the subject a Toxoplasma comprising a nucleic acid construct which comprises a heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein in frame fused upstream to a second nucleic acid sequence encoding a pharmaceutical polypeptide, wherein the heterologous polynucleotide is operably linked to a promoter for directing transcription of the heterologous polynucleotide in a Toxoplasma, wherein the subject is diagnosed with a pathology treatable by administration of the pharmaceutical polypeptide in a central nervous system of the subject, thereby treating the subject in need thereof.
According to some embodiments of the invention, the promoter is selected from the group consisting of: a constitutive promoter, an inducible promoter, a latent period-specific promoter, and a Toxoplasma endogenous promoter with the proviso that the promoter is not a Toxofilin promoter.
According to some embodiments of the invention, the endogenous promoter is not of a rhoptry protein.
According to some embodiments of the invention, the Toxoplasma being not attenuated.
According to some embodiments of the invention, the Toxoplasma secreted protein is secreted from a rhoptry of the Toxoplasma.
According to some embodiments of the invention, the Toxoplasma secreted protein which is secreted from the rhoptry comprises Toxofilin and/or ROP1.
According to some embodiments of the invention, the Toxoplasma secreted protein which is secreted from the rhoptry comprises the amino acid sequence selected from the group consisting of SEQ ID NOs:344-465.
According to some embodiments of the invention, the Toxoplasma secreted protein is a non-rhoptry protein.
According to some embodiments of the invention, the Toxoplasma secreted protein is selected from the group consisting of a microneme protein and a dense granule protein.
According to some embodiments of the invention, the Toxoplasma secreted protein is secreted from a dense granule of the Toxoplasma.
According to some embodiments of the invention, the Toxoplasma secreted protein which is secreted from the dense granule comprises a GRA16, and/or GRA24.
According to some embodiments of the invention, the Toxoplasma secreted protein is secreted from a microneme of the Toxoplasma.
According to some embodiments of the invention, the protein secreted from the microneme comprises the amino acid sequence selected from the group consisting of SEQ ID NOs:280-322.
According to some embodiments of the invention, the protein secreted from the dense granule comprises the amino acid sequence selected from the group consisting of SEQ ID NOs:234-279.
According to some embodiments of the invention, the Toxoplasma secreted protein comprises Toxoplasma gondii macrophage migration inhibitory factor (TgMIF).
According to some embodiments of the invention, the heterologous polynucleotide further comprises a Toxoplasma untranslated region (UTR) nucleic acid sequence upstream and/or downstream of the Toxoplasma secreted protein open reading frame.
According to some embodiments of the invention, the Toxoplasma 5′-untranslated region (5′-UTR) is placed upstream of the Toxoplasma secreted protein open reading frame.
According to some embodiments of the invention, the Toxoplasma 3′-untranslated region (3′-UTR) is placed downstream of the Toxoplasma secreted protein open reading frame.
According to some embodiments of the invention, the Toxoplasma 3′ untranslated region (3′-UTR) nucleic acid sequence is the GRA2 3′-UTR, GRA16 3′-UTR, GRA24 3′-UTR, SAG1 3′-UTR, or the DHFR 3′-UTR.
According to some embodiments of the invention, the Toxoplasma 5′ untranslated region (5′-UTR) nucleic acid sequence is the GRA2 5′-UTR, GRA16 5′-UTR, GRA24 5′-UTR, SAG1 5′-UTR, or the DHFR 5′-UTR.
According to some embodiments of the invention, the Toxoplasma endogenous promoter is the GRA2 promoter, GRA16 promoter, GRA24 promoter, SAG1 promoter, or the DHFR promoter.
According to some embodiments of the invention, the Toxoplasma untranslated region (UTR) nucleic acid sequence is the Toxofilin 3′-UTR.
According to some embodiments of the invention, the nucleic acid construct further comprises a third nucleic acid sequence encoding an inducible self-destruction element.
According to some embodiments of the invention, the third nucleic acid sequence encoding the inducible self-destruction element is comprised in the same nucleic acid construct which comprises the heterologous polynucleotide comprising the first nucleic acid sequence encoding the Toxoplasma secreted protein in frame fused upstream to the second nucleic acid sequence encoding the pharmaceutical polypeptide.
According to some embodiments of the invention, the third nucleic acid sequence encoding the inducible self-destruction element is comprised in a separate nucleic acid construct with respect to the nucleic acid construct which comprises the heterologous polynucleotide comprising the first nucleic acid sequence encoding the Toxoplasma secreted protein in frame fused upstream to the second nucleic acid sequence encoding the pharmaceutical polypeptide.
According to some embodiments of the invention, the nucleic acid construct does not comprise a Cre-recombinase coding sequence.
According to some embodiments of the invention, the nucleic acid construct does not comprise a beta (β)-lactamase (BLA) coding sequence.
According to some embodiments of the invention, the nucleic acid construct is suitable for integration into a genome of Toxoplasma.
According to some embodiments of the invention, the inducible self-destruction element is active in response to a drug.
According to some embodiments of the invention, the drug comprises an antibiotic.
According to some embodiments of the invention, the nucleic acid construct further comprises at least one in frame cleavage site which allows detachment of the pharmaceutical polypeptide from the Toxoplasma secreted protein.
According to some embodiments of the invention, the Toxoplasma is devoid of elements which facilitate propagation of the Toxoplasma in a host cell.
According to some embodiments of the invention, the Toxoplasma is devoid of virulence genes which are not necessary for delivery of the protein-of-interest into a CNS of a subject.
According to some embodiments of the invention, the heterologous polynucleotide further comprises a nucleic acid sequence encoding a selectable marker.
According to some embodiments of the invention, the selectable marker comprises chloramphenicol acetyltransferase (CAT), DHFR-TS, BLE, HXGPRT, UPRT, TK, CD, a fluorescent protein (such as GFP, YFP, RFP, mCherry or other) or an epitope tag (such as HA, Myc, Ty-1, FLAG or other).
According to some embodiments of the invention, the pharmaceutical composition is for treating a subject diagnosed with a pathology characterized by a deficient endogenous protein in a central nervous system of a subject.
According to some embodiments of the invention, the pharmaceutical composition is for treating a subject diagnosed with a pathology which is treatable by administration of the pharmaceutical polypeptide in a central nervous system of a subject.
According to some embodiments of the invention, the pharmaceutical composition further comprises an immunosuppression agent.
According to some embodiments of the invention, the method further comprises administering to the subject an immunosuppression drug prior to administration of the Toxoplasma and/or subsequent to administration of the Toxoplasma and/or concomitantly with administration of the Toxoplasma to the subject.
According to some embodiments of the invention, the method further comprises administering to the subject an immunosuppression drug prior to administration of the Toxoplasma to the subject.
According to some embodiments of the invention, the method further comprises administering to the subject an immunosuppression drug subsequent to administration of the Toxoplasma to the subject.
According to some embodiments of the invention, the method further comprises administering to the subject an immunosuppression drug concomitantly with administration of the Toxoplasma to the subject.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises a wild type amino acid sequence corresponding to the endogenous protein capable of treating the pathology.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises an antibody capable of treating the pathology.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises an antigen capable of treating the pathology.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises a toxin capable of treating the pathology.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises an enzyme, a structural polypeptide, a motility polypeptide, a regulatory polypeptide, a storage polypeptide, a signaling/ligand polypeptide, a receptor polypeptide, a sensory polypeptide, an antibody, a protein channel and/or a transport polypeptide.
According to some embodiments of the invention, administering is performed by peripheral administration.
According to some embodiments of the invention, the peripheral administration comprises intravenous administration.
According to some embodiments of the invention, the peripheral administration comprises oral administration.
According to some embodiments of the invention, administering is performed by direct administration to the central nervous system.
According to some embodiments of the invention, the deficient endogenous protein comprises a deletion, insertion, and/or substitution of at least one amino acid of the endogenous protein as compared to a wild type amino acid sequence of the endogenous protein.
According to some embodiments of the invention, the deficient endogenous protein comprises a reduced level of the endogenous protein as compared to a level of the endogenous protein in a healthy subject devoid of the pathology.
According to some embodiments of the invention, the deficient endogenous protein is absence of the endogenous protein in the subject diagnosed with the pathology.
According to some embodiments of the invention, the pharmaceutical polypeptide is Galactocerebrosidase (GALC).
According to some embodiments of the invention, the pharmaceutical polypeptide is Galactocerebrosidase (GALC) isoform 1, isoform 2, isoform 3, isoform 4 or isoform 5.
According to some embodiments of the invention, the pharmaceutical polypeptide is Methyl-CpG Binding Protein 2 (MECP2) isoform 1 or MECP2 isoform 2.
According to some embodiments of the invention, the pharmaceutical polypeptide is Glial Cell Derived Neurotrophic Factor (GDNF).
According to some embodiments of the invention, the pharmaceutical polypeptide is Glial Cell Derived Neurotrophic Factor (GDNF) isoform 1, isoform 2, isoform 3, isoform 4 or isoform 5.
According to some embodiments of the invention, the pharmaceutical polypeptide is Aspartoacylase (ASPA).
According to some embodiments of the invention, the pharmaceutical polypeptide is Survival Motor Neuron Protein (SMN1).
According to some embodiments of the invention, the pharmaceutical polypeptide is Survival Motor Neuron Protein isoform SMN, isoform SMN-delta5, isoform SMN-delta7, or isoform SMN-delta57.
According to some embodiments of the invention, the pharmaceutical polypeptide is Parkin (PARK2).
According to some embodiments of the invention, the pharmaceutical polypeptide is Parkin (PARK2) isoform 1, isoform 2, isoform 3, isoform 4, isoform 5, isoform 6, isoform 7, or isoform 8.
According to some embodiments of the invention, the pharmaceutical polypeptide is Transcription Factor E B (TFEB).
According to some embodiments of the invention, the pharmaceutical polypeptide is Transcription Factor E B (TFEB) isoform 1 or isoform 2.
According to some embodiments of the invention, the pharmaceutical polypeptide is a TALEN (TALE nuclease).
According to some embodiments of the invention, the pharmaceutical polypeptide is a TALE TF (TALE transcription factor).
According to some embodiments of the invention, the subject is diagnosed with Krabbe disease.
According to some embodiments of the invention, the subject is diagnosed with Rett syndrome.
According to some embodiments of the invention, the subject is diagnosed with Canavan disease.
According to some embodiments of the invention, the subject is diagnosed with Spinal Muscular Atrophy.
According to some embodiments of the invention, the subject is diagnosed with Parkinson's disease.
According to some embodiments of the invention, the subject is diagnosed with hypoxic/ischemic or neuroinflammatory CNS disorder.
According to some embodiments of the invention, the subject is diagnosed with Alzheimer's disease.
According to some embodiments of the invention, the subject is diagnosed with Amyotrophic Lateral Sclerosis.
According to some embodiments of the invention, the subject is diagnosed with Huntington's disease.
According to some embodiments of the invention, the subject is diagnosed with a lysosomal storage disease.
According to some embodiments of the invention, the subject is diagnosed with MECP2-duplication syndrome.
According to some embodiments of the invention, the method further comprising administering to the subject a drug capable of inducing the self-destruction element.
According to some embodiments of the invention, the method further comprising administering to the subject a molecule necessary for sustaining the Toxoplasma inside the host cell and/or body.
According to some embodiments of the invention, the molecule necessary for sustaining the Toxoplasma is an antibiotic.
According to some embodiments of the invention, the molecule necessary for sustaining the Toxoplasma is a small-molecule.
According to some embodiments of the invention, the molecule necessary for sustaining the Toxoplasma is a metabolite.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
The present invention, in some embodiments thereof, relates to a nucleic acid construct for secretion of Toxoplasma secreted protein fused to a pharmaceutical polypeptide, and Toxoplasma comprising same, and, more particularly, but not exclusively, to a pharmaceutical compositions and methods of using same for treating a subject.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
The present inventors have uncovered that Toxoplasma parasites transformed with genetic constructs encoding for a heterologous polypeptide synthesize and deliver the heterologous polypeptide to mammalian cells (
According to an aspect to some embodiments of the invention, there is provided a nucleic acid construct comprising a heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein in frame fused upstream to a second nucleic acid sequence encoding a pharmaceutical polypeptide, wherein the heterologous polynucleotide is operably linked to a promoter for directing transcription of the heterologous polynucleotide in a Toxoplasma, wherein the promoter is selected from the group consisting of: a constitutive promoter, an inducible promoter, a latent period-specific promoter, and a Toxoplasma endogenous promoter with the proviso that the promoter is not a Toxofilin promoter.
According to some embodiments of the invention, the nucleic acid construct is suitable for expression in a Toxoplasma.
According to some embodiments of the invention, the nucleic acid construct does not comprise a Cre-recombinase coding sequence.
According to some embodiments of the invention, the nucleic acid construct does not comprise a beta (β)-lactamase (BLA) coding sequence.
According to some embodiments of the invention, the nucleic acid construct is suitable for integration into a genome of Toxoplasma.
According to some embodiments of the invention, the Toxoplasma is devoid of elements which facilitate propagation of the Toxoplasma in a host cell (e.g., elements important for evading the immune system of the host, elements essential for the production or utilization of certain metabolites, elements important for counteracting certain toxins or antibiotics), yet including an endogenous functional CPSII.
According to some embodiments of the invention, the Toxoplasma is devoid of virulence genes which are not necessary for delivery of the protein-of-interest into a CNS of a subject.
The nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the Toxoplasma cell in a constitutive, transient, regulated or inducible manner.
As mentioned, the heterologous polynucleotide is operably linked to a promoter for directing transcription of the heterologous polynucleotide in a Toxoplasma.
A coding nucleic acid sequence is “operably linked” to a regulatory sequence (e.g., promoter) if the regulatory sequence is capable of exerting a regulatory effect on the coding sequence linked thereto.
As used herein, the term “promoter” refers to a region of DNA which lies upstream of the transcriptional initiation site of a gene to which RNA polymerase binds to initiate transcription of RNA. The promoter controls the timing and the intensity of expression of the gene, e.g., at which stage or condition in the lifetime of a parasite and/or of a host cell the gene is expressed.
According to some embodiments of the invention, the promoter is heterologous to the Toxoplasma used for expression of the nucleic acid construct.
According to some embodiments of the invention, the promoter is a constitutive promoter.
According to some embodiments of the invention, the promoter is an inducible promoter.
According to some embodiments of the invention, the promoter is a latent period-specific promoter.
According to some embodiments of the invention, the promoter is a Toxoplasma endogenous promoter with the proviso that the promoter is not a Toxofilin promoter.
According to some embodiments of the invention the promoter is not a Toxofillin endogenous promoter. It is noted that the predicted promoter of Toxofillin (predicted promoter of gene ID=TGME49_214080; SEQ ID NO:3689) contains the Toxofilin promoter used in some of the experiments of the Examples section (SEQ ID NO: 4482) and additional nucleotides upstream.
According to some embodiments the promoter does not comprise the nucleic acid sequence set forth by SEQ ID NO: 3689 and/or SEQ ID NO: 4482.
According to some embodiments of the invention, the endogenous promoter is not of a rhoptry protein.
According to some embodiments of the invention, the Toxoplasma endogenous promoter is the GRA2 promoter, GRA16 promoter, GRA24 promoter, SAG1 promoter, or the DHFR promoter.
According to some embodiments of the invention, the Toxoplasma endogenous promoter is the GRA2 promoter.
According to some embodiments of the invention, the Toxoplasma endogenous promoter is the GRA16 promoter.
According to some embodiments of the invention, the Toxoplasma endogenous promoter is the GRA24 promoter.
According to some embodiments of the invention, the Toxoplasma endogenous promoter is the SAG1 promoter.
According to some embodiments of the invention, the Toxoplasma endogenous promoter is the DHFR promoter.
Table 1 herein below lists suitable endogenous promoters which can be cloned into the nucleic acid construct of some embodiments of the invention in order to drive expression of the heterologous polynucleotide in the Toxoplasma.
Table 2 lists suitable constitutive and inducible promoters which can be cloned into the nucleic acid construct of some embodiments of the invention.
As mentioned, the heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein.
As used herein the phrase “Toxoplasma secreted protein” refers to at least a functional fragment (amino acid sequence) of a Toxoplasma polypeptide which is sufficient in order to be secreted to a host cell when infected in-vitro by the Toxoplasma.
According to some embodiments of the invention, the Toxoplasma secreted protein or the functional fragment thereof is capable of being secreted to a host cell when infected in-vivo by the Toxoplasma.
According to some embodiments of the invention, the first nucleic acid sequence encoding a functional fragment of the Toxoplasma secreted protein.
According to some embodiments of the invention, the first nucleic acid sequence encoding the full-length open reading frame (ORF) of the Toxoplasma secreted protein.
According to some embodiments of the invention, the Toxoplasma secreted protein is secreted from a rhoptry of the Toxoplasma.
According to some embodiments of the invention, the Toxoplasma secreted protein which is secreted from the rhoptry comprises Toxofilin and/or ROP1.
According to some embodiments of the invention, the Toxoplasma secreted protein which is secreted from the rhoptry comprises the amino acid sequence selected from the group consisting of SEQ ID NOs:344-465.
According to some embodiments of the invention, the Toxoplasma secreted protein is a non-rhoptry protein.
According to some embodiments of the invention, the Toxoplasma secreted protein is selected from the group consisting of a microneme protein and a dense granule protein.
According to some embodiments of the invention, the Toxoplasma secreted protein is secreted from a dense granule of the Toxoplasma.
According to some embodiments of the invention, the Toxoplasma secreted protein which is secreted from the dense granule comprises a GRA16, and/or GRA24.
According to some embodiments of the invention, the Toxoplasma secreted protein is secreted from a microneme of the Toxoplasma.
According to some embodiments of the invention, the protein secreted from the microneme comprises the amino acid sequence selected from the group consisting of SEQ ID NOs:280-322.
According to some embodiments of the invention, the protein secreted from the dense granule comprises the amino acid sequence selected from the group consisting of SEQ ID NOs:234-279.
According to some embodiments of the invention, the Toxoplasma secreted protein comprises Toxoplasma gondii macrophage migration inhibitory factor (TgMIF).
Table 3 below lists Toxoplasma-secreted proteins which can be cloned into the nucleic acid construct of some embodiments of the invention, organized according to the organelle to which the protein is localized or predicted to be localized to.
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
T. gondii
According to some embodiments of the invention, the heterologous polynucleotide further comprises a Toxoplasma untranslated region (UTR) nucleic acid sequence.
According to some embodiments of the invention, the Toxoplasma untranslated region (UTR) nucleic acid sequence is upstream and/or downstream of the open reading frame of the heterologous polynucleotide (e.g., which encodes the Toxoplasma secreted protein fused in frame with the pharmaceutical polypeptide).
According to some embodiments of the invention, the Toxoplasma 5′-untranslated region (5′-UTR) is placed upstream of the Toxoplasma secreted protein open reading frame.
According to some embodiments of the invention, the Toxoplasma 3′-untranslated region (3′-UTR) is placed downstream of the open reading frame encoding the Toxoplasma secreted protein fused in frame with the pharmaceutical polypeptide.
According to some embodiments of the invention, the Toxoplasma 3′ untranslated region (3′-UTR) nucleic acid sequence is the GRA2 3′-UTR, GRA16 3′-UTR, GRA24 3′-UTR, SAG1 3′-UTR, or the DHFR 3′-UTR.
According to some embodiments of the invention, the Toxoplasma 5′ untranslated region (5′-UTR) nucleic acid sequence is the GRA2 5′-UTR, GRA16 5′-UTR, GRA24 5′-UTR, SAG1 5′-UTR, or the DHFR 5′-UTR.
According to some embodiments of the invention, the Toxoplasma untranslated region (UTR) nucleic acid sequence is the Toxofilin 3′-UTR.
As mentioned, the heterologous polynucleotide comprises a second nucleic acid sequence encoding a pharmaceutical polypeptide being fused in frame downstream of the Toxoplasma secreted protein.
As used herein the phrase “pharmaceutical polypeptide” refers to a polypeptide having a therapeutic effect, e.g., capable of treating a pathology, when introduced into cell(s) of a subject in need thereof.
It should be noted that for certain diseases, the pharmaceutical polypeptide may be effective when reaching a certain organ, tissue, cell, cell compartment or cellular localization within the subject. For example, for treating a neurological disease, the pharmaceutical composition is preferably targeted to the nervous system, e.g., neurons, glia or other cells. Additionally or alternatively, for certain diseases, the pharmaceutical polypeptide should reach a certain cellular localization such as the cell nucleus in case the target of the pharmaceutical composition is situated (positioned) within the cell nucleus. For example, in the case of MECP2, the active protein binds to DNA within the nucleus and regulates gene expression.
According to some embodiments of the invention, the nucleic acid construct further comprises at least one in frame cleavage site, which allows detachment of the pharmaceutical polypeptide from the Toxoplasma secreted protein.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises a wild type amino acid sequence corresponding to the endogenous protein capable of treating the pathology.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises an antibody capable of treating the pathology.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises an antigen capable of treating the pathology.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises a toxin capable of treating the pathology.
According to some embodiments of the invention, the pharmaceutical polypeptide comprises an enzyme, a structural polypeptide, a motility polypeptide, a regulatory polypeptide, a storage polypeptide, a signaling/ligand polypeptide, a receptor polypeptide, a sensory polypeptide, an antibody, a protein channel and/or a transport polypeptide.
According to some embodiments of the invention, the pharmaceutical polypeptide is Galactocerebrosidase (GALC).
According to some embodiments of the invention, the pharmaceutical polypeptide is Galactocerebrosidase (GALC) isoform 1, isoform 2, isoform 3, isoform 4 or isoform 5.
According to some embodiments of the invention, the pharmaceutical polypeptide is Methyl-CpG Binding Protein 2 (MECP2).
According to some embodiments of the invention, the pharmaceutical polypeptide is Methyl-CpG Binding Protein 2 (MECP2) isoform 1 or MECP2 isoform 2.
According to some embodiments of the invention, the pharmaceutical polypeptide is Glial Cell Derived Neurotrophic Factor (GDNF).
According to some embodiments of the invention, the pharmaceutical polypeptide is Glial Cell Derived Neurotrophic Factor (GDNF) isoform 1, isoform 2, isoform 3, isoform 4 or isoform 5.
According to some embodiments of the invention, the pharmaceutical polypeptide is Aspartoacylase (ASPA).
According to some embodiments of the invention, the pharmaceutical polypeptide is Survival Motor Neuron Protein (SMN1).
According to some embodiments of the invention, the pharmaceutical polypeptide is Survival Motor Neuron Protein isoform SMN, isoform SMN-delta5, isoform SMN-delta7, or isoform SMN-delta57.
According to some embodiments of the invention, the pharmaceutical polypeptide is Parkin (PARK2).
According to some embodiments of the invention, the pharmaceutical polypeptide is Parkin (PARK2) isoform 1, isoform 2, isoform 3, isoform 4, isoform 5, isoform 6, isoform 7, or isoform 8.
According to some embodiments of the invention, the pharmaceutical polypeptide is Transcription Factor EB (TFEB).
According to some embodiments of the invention, the pharmaceutical polypeptide is Transcription Factor EB (TFEB) isoform 1 or isoform 2.
According to some embodiments of the invention, the pharmaceutical polypeptide is a TALEN (TALE nuclease).
According to some embodiments of the invention, the pharmaceutical polypeptide is a TALE TF (TALE transcription factor).
Table 4 hereinbelow, lists exemplary therapeutic proteins, which can be secreted to the subject by the Toxoplasma of some embodiments of the invention.
It should be noted that any of the sequences described herein (e.g., the Toxoplasma secreted protein and/or the therapeutic polypeptides) can be codon optimized for expression in a target cell (e.g., Toxoplasma). Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the target cell species of interest, and the removal of codons atypically found in the target cell a species commonly referred to as codon optimization.
The phrase “codon optimization” refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that approaches codon usage within the target cell of interest. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the target cell. The nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the target cell species determined using any suitable procedure. In this method, the standard deviation of codon usage, a measure of codon usage bias, may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed target cell genes, followed by a calculation of the average squared deviation. The formula used is: 1 SDCU=n=1N[(Xn−Yn)/Yn]2/N, where Xn refers to the frequency of usage of codon n in highly expressed target cell genes, where Yn to the frequency of usage of codon n in the gene of interest and N refers to the total number of codons in the gene of interest.
One method of optimizing the nucleic acid sequence in accordance with the preferred codon usage for a particular target cell type is based on the direct use, without performing any extra statistical calculations, of codon optimization Tables such as those provided on-line at the Codon Usage Database through the Codon usage tabulated from international DNA sequence databases: status for the year 2000. Y Nakamura, T Gojobori, T Ikemura—Nucleic acids research, 2000—Oxford Univ Press. The Codon Usage Database contains codon usage tables for a number of different species, with each codon usage Table having been statistically determined based on the data present in Genbank.
By using the above Tables to determine the most preferred or most favored codons for each amino acid in a particular species (for example, Toxoplasma gondii), a naturally-occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular target cell species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored. However, one or more less-favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5′ and 3′ ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively effect mRNA stability or expression.
The naturally-occurring encoding nucleotide sequence may already, in advance of any modification, contain a number of codons that correspond to a statistically-favored codon in a particular target cell species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically-favored with regards to a particular target cell, and modifying these codons in accordance with a codon usage table of the particular target cell to produce a codon optimized derivative. A modified nucleotide sequence may be fully or partially optimized for target cell codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Additionally or alternatively, codon optimized/usage sequence can fold better than the protein encoded by the corresponding naturally occurring or native gene. Additionally or alternatively, codon optimized/usage sequence is targeted better to the target organelle(s) than the protein encoded by the corresponding naturally occurring or native gene. Additionally or alternatively, codon optimized/usage sequence is less degraded than the protein encoded by the corresponding naturally occurring or native gene.
For example, the following Table 5 of Toxoplasma codon usage can be used.
According to some embodiments of the invention, the heterologous polynucleotide further comprises a nucleic acid sequence encoding a selectable marker.
According to some embodiments of the invention, the selectable marker comprises chloramphenicol acetyltransferase (CAT), DHFR-TS, BLE, HXGPRT, UPRT, TK, CD, a fluorescent protein (such as GFP, YFP, RFP, mCherry or other) or an epitope tag (such as HA, Myc, Ty-1, FLAG or other).
According to some embodiments of the invention, the selectable marker comprises HXGPRT.
Toxoplasma tachyzoites deficient in HXGPRT activity can be selected in the presence of 6-thioxanthine (6-TX), whereas strains expressing HXGPRT activity can be selected in mycophenolic acid (MPA) and/or xanthine (Pfefferkorn and Borotz, 1994, Pfefferkorn E. R., Borotz S. (1994) Exp. Parasitol. 79, 374-382).
According to some embodiments of the invention, the selectable marker comprises chloramphenicol acetyltransferase (CAT) (for positive selection using Chloramphenicol), DHFR-TS (for positive selection using Pyrimethamine), BLE (for positive selection using Phleomycin), HXGPRT (for positive selection using Mycophenolic acid+xanthine or negative selection using 6-thioxanthine, UPRT (for negative selection using 5′-fluo-2′-deoxyuridine), TK (for positive selection using Ganciclovir), CD (for positive selection using 5-fluorocytosine), a fluorescent protein (such as GFP, YFP, RFP, mCherry or other) or an epitope tag (such as HA, Myc, Ty-1, FLAG or other).
According to some embodiments of the invention, the nucleic acid construct further comprises a third nucleic acid sequence encoding an inducible self-destruction element.
According to some embodiments of the invention, the inducible self destruction element is in the same frame as the therapeutic cassette.
According to some embodiments of the invention, the inducible self destruction element is in a separate reading frame, regulated by different regulatory elements. For instance, the therapeutic cassette can be driven by a latent-stage promoter, while the self-destruction cassette will be driven by a drug-inducible promoter.
According to some embodiments of the invention, the inducible self-destruction element is active in response to a drug.
According to some embodiments of the invention, the drug comprises an antibiotic.
According to some embodiments of the invention, the third nucleic acid sequence encoding the inducible self-destruction element is comprised in the same nucleic acid construct which comprises the heterologous polynucleotide comprising the first nucleic acid sequence encoding the Toxoplasma secreted protein in frame fused upstream to the second nucleic acid sequence encoding the pharmaceutical polypeptide.
According to some embodiments of the invention, the third nucleic acid sequence encoding the inducible self-destruction element is comprised in a separate nucleic acid construct with respect to the nucleic acid construct which comprises the heterologous polynucleotide comprising the first nucleic acid sequence encoding the Toxoplasma secreted protein in frame fused upstream to the second nucleic acid sequence encoding the pharmaceutical polypeptide.
According to some embodiments of the invention, the Toxoplasma untranslated region (UTR) nucleic acid sequence is upstream and/or downstream of the open reading frame of the selectable marker and/or of the self-destruction element.
According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct system comprising at least two nucleic acid constructs, wherein a first nucleic acid construct of the at least two nucleic acid constructs is the nucleic acid construct of some embodiments of the invention, and a second nucleic construct of the at least two nucleic acid constructs comprises a polynucleotide encoding a selectable marker.
Typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
Following is a non-limiting list of Toxoplasma nucleic acid constructs which can be used as a backbone to generate the nucleic acid construct of some embodiments of the invention: pGRA, pUPRT, pROP1, pTUB1, pTUB8, pLIC, pTOXO, pMIC2, pHX, pCAT, pDHFR, pBlueScript, pTetO7SAG1, pTetO7SAG4, pSAG1, pSAG4, pHTU, pTKO, pLoxP-DHFR, pminCAT/HXGPRT+, pminCAT/HXGPRT−, pDHFR-TSc3/M3, pDHFR-TSc3/M2M3, pminiHXGPRT, and pUC19. These vectors can be obtained from various sources, such as the nonprofit plasmid repository “addgene”; the NIH AIDS Reagent Program; Agilent Technologies; NEB; Thermo Fisher Scientific; Sigma Aldrich; GenScript; and MoBiTec GmbH.
Table 6 hereinbelow provides non-limiting exemplary vectors and catalogue numbers.
According to an aspect of some embodiments of the present invention there is provided a Toxoplasma transformed with the nucleic acid construct of some embodiments of the invention or with the nucleic acid construct system of some embodiments of the invention.
As used herein the term “Toxoplasma” refers to the intracellular, parasitic protozoan Toxoplasma gondii.
Non-limiting examples of a Toxoplasma gondii strains which can be used along with the construct and methods of some embodiments of the invention include:
(i) Type I—including but not limited to: GT1, RH, ENT, VEL, TgCatCo1 and CAST.
(ii) Type II—including but not limited to: ME49, Beverly, PDS, PLK, PTG, DEG, PIH, TgNmBr1 and PRU.
(iii) Type III—including but not limited to: VEG, C56, CTG, CEP, TgGoatUS4 and STRL.
(iv) Type Atypical—including but not limited to: TgCTPrC3, TgBbUS1, TgRabbitBr1 and TgPigUS15.
According to some embodiments of the invention, the Toxoplasma is not attenuated.
As is conventional in the art, the term “attenuated” refers to a weakened and/or less vigorous strain of T. gondii as compared to a native (wild type) T. gondii which is non-genetically modified. Usually, an attenuated mutant of T. gondii is capable of stimulating an immune response and creating immunity but not causing illness. Attenuation can be achieved by conventional methods including, but not limited, to γ-irradiation or the generation of a pyrimidine auxotroph.
Non-limiting examples of attenuated Toxoplasma are described in US 20120045477 Al and U.S. Pat. No. 8,673,289 B2, which are fully incorporated herein by reference.
It should be noted that T. gondii replication is essential for its ability to disseminate in the body and reach distal tissues during the acute stage of infection, its ability to persist in the infected cells and its ability to establish chronic cysts in the host.
According to some embodiments of the invention, the Toxoplasma of some embodiments of the invention is characterized by a higher proliferation rate as compared to the proliferation rate of an attenuated Toxoplasma.
According to some embodiments of the invention, the Toxoplasma of some embodiments of the invention is not an auxotroph.
According to some embodiments of the invention, the Toxoplasma of some embodiments of the invention comprises a functional endogenous pathway suitable for de-novo biosynthesis of pyrimidines.
According to some embodiments of the invention, the Toxoplasma of some embodiments of the invention is capable of differentiation into the Bradizoite stage.
According to some embodiments of the invention, the Toxoplasma of some embodiments of the invention is capable of RNA synthesis and/or DNA replication.
According to some embodiments of the invention, the Toxoplasma of some embodiments of the invention is capable of RNA synthesis.
According to some embodiments of the invention, the Toxoplasma of some embodiments of the invention is capable of DNA replication.
According to some embodiments of the invention, the Toxoplasma of some embodiments of the invention comprises an endogenous functional Carbamoyl phosphate synthetase II (CPSII) enzyme (as described in U.S. Pat. No. 8,673,289), e.g., as provided in any of SEQ ID NOs: 4612-4617.
The constructs and Toxoplasma of some embodiments of the invention can be used to deliver a protein-of-interest into the subject, e.g., into a specific tissue or cell type of the subject.
According to an aspect of some embodiments of the present invention there is provided a method of administering a protein-of-interest into a tissue-of-interest of a subject, the method comprising administering to the subject the Toxoplasma of some embodiments of the invention or the pharmaceutical composition of some embodiments of the invention, thereby administering the protein-of-interest to the tissue-of-interest of the subject.
Examples of tissue-of-interest include, but are not limited to, a central nervous system, muscles, parts of the eye, blood, lymph nodes, spleen, white blood cells, digestive system, and lamina propria.
It should be noted that since the Toxoplasma can cross the blood brain barrier it can deliver the protein-of-interest into the central nervous system of the subject in need thereof.
According to an aspect of some embodiments of the present invention there is provided a method of administering a protein-of-interest into a central nervous system of a subject, the method comprising administering to the subject the Toxoplasma of some embodiments of the invention or the pharmaceutical composition of some embodiments of the invention, thereby administering the protein-of-interest to the central nervous system of the subject.
Thus, the Toxoplasma of some embodiments of the invention can be used to treat a subject in need thereof by delivering a protein-of-interest (e.g., a therapeutic polypeptide) which is capable of treating the subject.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need thereof, comprising administering to the subject the Toxoplasma of some embodiments of the invention or the pharmaceutical composition of some embodiments of the invention, wherein the subject is diagnosed with a pathology treatable by administration of the pharmaceutical polypeptide in a central nervous system of the subject, thereby treating the subject in need thereof.
According to an aspect of some embodiments of the present invention there is provided a method of treating a subject in need thereof, comprising administering to the subject a Toxoplasma comprising a nucleic acid construct which comprises a heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein in frame fused upstream to a second nucleic acid sequence encoding a pharmaceutical polypeptide, wherein the heterologous polynucleotide is operably linked to a promoter for directing transcription of the heterologous polynucleotide in a Toxoplasma, wherein the subject is diagnosed with a pathology treatable by administration of the pharmaceutical polypeptide in a central nervous system of the subject, thereby treating the subject in need thereof.
The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
As used herein, the term “subject” includes mammals, preferably human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology.
According to some embodiments of the invention, the subject is diagnosed with a pathology characterized by a deficient endogenous protein in a central nervous system of a subject.
According to some embodiments of the invention, the deficient endogenous protein comprises a deletion, insertion, and/or substitution of at least one amino acid of the endogenous protein as compared to a wild type amino acid sequence of the endogenous protein.
According to some embodiments of the invention, the deficient endogenous protein comprises a reduced level of the endogenous protein as compared to a level of the endogenous protein in a healthy subject devoid of the pathology.
According to some embodiments of the invention, the deficient endogenous protein is absence of the endogenous protein in the subject diagnosed with the pathology.
According to some embodiments of the invention, the subject is diagnosed with Krabbe disease.
According to some embodiments of the invention, the subject is diagnosed with Rett syndrome.
According to some embodiments of the invention, the subject is diagnosed with Canavan disease.
According to some embodiments of the invention, the subject is diagnosed with Spinal Muscular Atrophy.
According to some embodiments of the invention, the subject is diagnosed with Parkinson's disease.
According to some embodiments of the invention, the subject is diagnosed with hypoxic/ischemic or neuroinflammatory CNS disorder.
According to some embodiments of the invention, the subject is diagnosed with Alzheimer's disease.
According to some embodiments of the invention, the subject is diagnosed with Amyotrophic Lateral Sclerosis.
According to some embodiments of the invention, the subject is diagnosed with Huntington's disease.
According to some embodiments of the invention, the subject is diagnosed with a lysosomal storage disease.
According to some embodiments of the invention, the subject is diagnosed with MECP2-duplication syndrome.
According to some embodiments of the invention the subject does not a have a compromised immune system. Examples of subjects with compromised immune system include, but are not limited to AIDS patients, subjects receiving chemotherapy, subjects receiving immunosuppressant drugs such as for cell, tissue and/or organ transplantation.
According to some embodiments of the invention, the method further comprising administering to the subject a drug capable of inducing the self-destruction element.
According to some embodiments of the invention, the method further comprising administering to the subject a molecule necessary for sustaining the Toxoplasma inside the host cell and/or body.
According to some embodiments of the invention, the molecule necessary for sustaining the Toxoplasma is an antibiotic.
According to some embodiments of the invention, the molecule necessary for sustaining the Toxoplasma is a small-molecule.
According to some embodiments of the invention, the molecule necessary for sustaining the Toxoplasma is a metabolite.
According to some embodiments of the invention, the method further comprises administering to the subject an immunosuppression drug prior to administration of the Toxoplasma and/or subsequent to administration of the Toxoplasma and/or concomitantly with administration of the Toxoplasma to the subject.
According to some embodiments of the invention, the method further comprises administering to the subject an immunosuppression drug prior to administration of the Toxoplasma to the subject.
According to some embodiments of the invention, the method further comprises administering to the subject an immunosuppression drug subsequent (or following) to administration of the Toxoplasma to the subject.
According to some embodiments of the invention, the method further comprises administering to the subject an immunosuppression drug concomitantly with administration of the Toxoplasma to the subject.
According to some embodiments of the invention, administering is performed by peripheral administration.
According to some embodiments of the invention, the peripheral administration comprises intravenous administration.
According to some embodiments of the invention, the peripheral administration comprises oral administration.
According to some embodiments of the invention, administering is performed by intraperitoneal injection.
According to some embodiments of the invention, administering is performed by intramuscular injection.
According to some embodiments of the invention, administering is performed by aerosols.
According to some embodiments of the invention, administering is performed by direct administration to the central nervous system or an adjacent tissue thereof.
According to some embodiments of the invention, the disease which is treated by the methods of some embodiments of the invention, e.g., using the Toxoplasma and/or the pharmaceutical composition comprising same is any of the diseases listed in Table 7 hereinabove.
Table 7 lists non-limiting diseases associated with deficient or abnormal expression or function of the endogenous proteins, which can be treated by administration of the therapeutic polypeptide of some embodiments of the invention.
According to some embodiments of the invention, the disease (pathology) is not cancer.
The nucleic acid construct, the nucleic acid construct system or the Toxoplasma transformed with the nucleic acid construct or with the nucleic acid construct system of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the Toxoplasma of some embodiments of the invention, and a pharmaceutically acceptable carrier.
According to some embodiments of the invention, the pharmaceutical composition is for treating a subject diagnosed with any of the pathologies described herein.
According to some embodiments of the invention, the pharmaceutical composition is for treating a subject diagnosed with a pathology characterized by a deficient endogenous protein in a central nervous system of a subject.
According to some embodiments of the invention, the pharmaceutical composition is for treating a subject diagnosed with a pathology which is treatable by administration of the pharmaceutical polypeptide in a central nervous system of a subject.
According to some embodiments of the invention, the pharmaceutical composition further comprises an immunosuppression agent.
As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term “active ingredient” refers to the nucleic acid construct, the nucleic acid construct system or the toxoplasma transformed with the nucleic acid construct or with the nucleic acid construct system of some embodiments of the invention accountable for the biological effect.
Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
The term “tissue” refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, culturing, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. For example, manufacturing of toxoplasma can be by culturing the toxoplasma in cell cultures.
Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, foodstuff, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g., the nucleic acid construct, the nucleic acid construct system or the toxoplasma transformed with the nucleic acid construct or with the nucleic acid construct system of some embodiments of the invention) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., a disorder affecting the central nervous system of the subject) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in-vitro assays, ex-vivo assays, cell culture assays, and/or animal models. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).
Dosage amount and interval may be adjusted individually to provide tissue levels (e.g., brain levels) of the active ingredient which are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC may vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma, tissue or CSF (cerebro-spinal fluid) concentrations.
Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks, or e.g., a lifetime in the case of chronic diseases, or until cure is effected or diminution of the disease state is achieved.
For example, for chronic diseases, the dosing can be adjusted until symptoms are managed, stabilized and/or ameliorated.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the immune state of the subject, the judgment of the prescribing physician, etc.
Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
As used herein the term “about” refers to ±10%
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., allelic variation, sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (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. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Toxoplasma gondii: the model apicomplexan—Perspectives and methods. L M Weiss, K Kim, 2011. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Toxoplasma gondii Culture
Parasites were grown on human foreskin fibroblasts (HFF) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-Glutamine, Penicillin and Streptomycin or Gentamicin antibiotic mix and 10% Fetal Bovine Serum (FBS), thereby referred to as “complete DMEM media”. Cultures were monitored daily, and the parasites were passaged by transferring a drop of the supernatant of a lysed dish or a drop of syringe-released intracellular parasites into a fresh dish with HFF.
Generation of T. gondii Expression Vectors for Expression of Heterologous Polypeptides Comprised of a Toxoplasma Secreted Protein Fused to a Pharmaceutical Polypeptide-of-Interest
In order to generate stable transgenic lines of T. gondii that express specific therapeutic proteins, the present inventors first generated T. gondii expression constructs by molecular cloning. The constructs include a long open reading frame (ORF), which consists of Toxofilin cDNA [SEQ ID NO: 4481] or GRA16 cDNA [SEQ ID NO: 4472] followed by an HA tag coding sequence (SEQ ID NO: 4474) followed by the therapeutic mammalian cDNA of interest, such as the murine MeCP2 isoform 1 [SEQ ID NO: 4476]; the human ASPA [SEQ ID NO: 4483], the human SMN1 [SEQ ID NO: 4479]; the human PARK2 [SEQ ID NO: 4478]; the human GDNF [SEQ ID NO: 4489]; the human TFEB (Transcription factor EB) isoform 1 [SEQ ID NO: 4600] sequence; the human GALC isoform 1 [SEQ ID NO:4486 or SEQ ID NO:4601 (GALC version 2); the GALC-TAT that includes a Glycine flexible linker (Gly) [SEQ ID NO:4490] followed by a protein transduction domain (TAT) that facilitates nonclassical trafficking across membranes in order to aid the targeting of GALC to the lysosome of the host cell (SEQ ID NO: 4488) or the “mutated GALC-TAT” (GALC-TAT that harbors a deletion) (SEQ ID NO: 4487). The cDNA of interest can also be codon-optimized according to the codon usage of T. gondii in order to increase efficiency of expression, stability and localization of the heterologous polypeptide in T. gondii (such as with the codon optimized murine MeCP2 isoform 1 [SEQ ID NO: 4477], 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: 4604] codon optimized for expression in T. gondii). The T. gondii expression vector also includes regulatory elements from the same or other Toxoplasma genes that guarantee its correct expression and targeting. Upstream of the ORF is the endogenous 5′ regulatory sequence which can be either the endogenous 5′ regulatory sequence of the Toxofilin gene, here referred to as the “Toxofilin promoter” or “Toxofilin 5′-UTR” (SEQ ID NO: 4482) or the endogenous 5′ regulatory sequence of the GRA16 gene, here referred to as the “GRA16 promoter” or “GRA16 5′-UTR” (SEQ ID NO: 4473). Downstream of the ORF is the 3′-UTR of the abundant dense granule protein GRA2 (SEQ ID NO: 4491;
Generation of Transgenic T. gondii
T. gondii of the type I strains RH or RHAHX or the type II strains Prugniaud, Prugniaud-GFP-Luciferase or Prugniaud ΔHPT (also known as Prugniaud ΔHX) were used for generating transgenic strains. Extracellular Tachyzoites were collected or mechanically egressed using a 22-26 gauge needle, filtered from cellular debris, pelleted and resuspended in 300 μl cytomix buffer (120 mM KCl, 0.15 mM CaCl2 10 mM K2HPO4/KH2PO4 pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 5 mM MgCl2) with freshly added 2 mM ATP and 5 mM GSH in 30 μl each (360 μl total). 20-60 μg plasmid DNA, or DNA linearized with the enzyme ScaI was transfected into the tachyzoites by electroporation with a BTX ECM electroporator. A few drops of the transfected cells were transferred onto IFA wells (immunofluorescence assay wells of a 24-well plate with HFF cells seeded on glass coverslips) and immunofluorescently stained for assessment of transfection efficiency and transient protein expression (expression from plasmid DNA). The rest of the transfected parasites were transferred onto a fresh flask with HFF in complete DMEM. If a drug-resistance selectable marker was used, the next day media was changed to fresh media containing the drug used for selection (1 μM pyrimethamine for DHFR-TS selection or 25 μg/ml mycophenolic acid+50 μg/ml xanthine for HXGPRT selection). Starting from the day the parasites started egressing from the HFF, drops of the supernatant containing extracellular parasites were passed every 1-2 days into a second flask of HFF in selective media containing the selection drug. When the parasites in the second flask started egressing from the HFF, drops of the supernatant were passed every 1-2 days from the second flask into a third flask, and so on. If a fluorescent protein selectable marker was used, when the parasites in the second flask egressed out of about 50%-90% of the HFF cells, the media with extracellular parasites was collected, filtered from cell debris and sorted using a FACS machine. Parasites emitting fluorescence corresponding to the fluorescent protein used for selection were collected and passed into the next flask of HFF in complete DMEM media. After the 3rd-5th flask started lysing (about 2-5 weeks), the parasites were considered a “stable pool” containing parasites that integrated the DNA construct into their genome. A drop of the stable pool was passed onto IFA (immuno fluorescent assay) wells and immunofluorescently stained to assess percentage of construct-positive parasites in the pool, protein expression and protein localization in the genome-integrated (“stable”) parasites (genomic expression). Clonal lines were established by manual sorting by limiting dilutions or by FACS sorting one parasite per well in 96 well plates. Plates were kept undisturbed for 5-10 days, after which they were screened by eye using a bright field microscope to detect wells with a single plaque which is assumed to originate from a single parasite. Wells with a single plaque were mixed vigorously with a pipette and 50 μl of the mixture was passed to a new dish and to a PCR tube. Lysates were made by pelleting the collected cells in the PCR tubes, resuspending them in 10 μl lysis buffer consisted of 10% proteinase K in TE buffer and incubation in 60° C. for 60 minutes followed by 95° C. for 10 minutes in a thermocycler. One microliter (1 μl) of the lysates were used as template for PCR screening using primers specific for the genetic construct. PCR-positive clones were passed onto IFA wells, fixed, immunofluorescently stained and analysed using a fluorescent microscope and polarized light microscope for protein expression and protein localization in each clone.
Antibodies
Antibodies and the respective concentrations they were used in, are as follows: anti-HA (Roche, IFA: 1:1000, WB: 1:1000), anti-IMC-1 (gift from Prof. Dominique Soldati-Favre, IFA: 1:1000-1:2000), anti-MeCP2 (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 594 (Invitrogen, IFA: 1:1000), Alexa Fluor anti-rabbit 488 and 594 (Invitrogen, IFA: 1:1000), Alexa Fluor anti-mouse 488 and 594 (Invitrogen, IFA: 1:1000).
Experimental Procedures
The constructs were transfected into RH (RHhxgprt) or Pru (Pruhxgprt) strain T. gondii parasites by electroporation. This is followed by a process of selection for parasites expressing the selection markers (either Pyrimethamine selection for a construct containing DHFR-TS (SEQ ID NO: 4484), which was co-transfected with the construct-of-interest), or using MPA+Xanthine for selection based on expression of HXGPRT (SEQ ID NO:4475), cloning by limiting dilutions or flow cytometry, and PCR-based screening. The positive clones were verified by Western Blot analysis and by immunofluorescence staining to characterize the expression and specific localization of the HA-tagged therapeutic protein in the secretory rhoptry organelles, dense granule organelles, parasitophorous vacuole space, parasitophorous vacuole membrane, or in the host cells. The verified positive lines were further maintained on HFF (human foreskin fibroblast) cells.
Experimental Results
Generation of Constructs and T. gondii Strains for Delivery of the Therapeutic Proteins Galactocerebrosidase, Galactocerebrosidase-TAT, GDNF, Aspartoacylase, MeCP2, Survival Motor Neuron Protein and E3 Ubiquitin Protein Ligase Parkin—
The results demonstrate that T. gondii can be engineered to express heterologous (mammalian, e.g., human) therapeutic proteins fused to an endogenous protein of the parasite. Using this approach, the present inventors have generated 3 novel stable T. gondii transgenic lines expressing the 3 human proteins associated with common neuropathologies, Galactocerebrosidase-TAT (the mutated GALC-TAT SEQ ID NO: 4487), MeCP2 (codon optimized SEQ ID NO: 4477) and GDNF (SEQ ID NO: 4489), and additional 4 mixed-population pools containing stable T. gondii transgenic parasites expressing the 3 human proteins associated with common neuropathologies Galactocerebrosidase (GALC, isoform 1, SEQ ID NO:4486), Galactocerebrosidase-TAT (the WT GALC-TAT SEQ ID NO: 4488), Aspartoacylase (ASPA SEQ ID NO:4483) and Survival Motor Neuron Protein (SMN1, SEQ ID NO: 4479).
Improving Secretion and Uptake of the Therapeutic Protein by a Viral Protein Transduction Domain—
In order to improve secretion of the Galactocerebrosidase protein from the host cell to the extracellular space, as well as the uptake of the protein by neighboring cells, the present inventors have fused Galactocerebrosidase to a viral protein transduction domain (PTD). Tat-PTD has been previously shown to significantly increase (by 6 fold) the cross-correction efficiency of Galactocerebrosidase through enhancing both the secretion and uptake of the enzyme in a cell culture model system (Meng, X.-L., et al., 2013).
Localization of the Therapeutic Proteins Galactocerebrosidase-TAT, Aspartoacylase, MeCP2 and Survival Motor Neuron Protein to T. gondii Rhoptries Secretory Organelles—
The results demonstrate that T. gondii can localize heterologous (mammalian, e.g., human) therapeutic proteins to the parasite's rhoptries secretory organelles (
Thus, the present inventors have succeeded in demonstrating that T. gondii may be engineered to express heterologous (mammalian, e.g., human) proteins fused to an endogenous protein of the parasite, and that these proteins localize to the parasite's rhoptries secretory organelles. The present inventors have engineered the human proteins to be fused to Toxofilin, an endogenous protein that is naturally secreted into host cells by the parasites, which allows to “ride” on the parasite's protein secretion machinery for the targeting and secretion of proteins of interest (e.g., a therapeutic protein). This is an important milestone on the path to suggesting T. gondii as a feasible basis for this technology.
Using this approach, the present inventors have generated a novel stable T. gondii transgenic line, expressing and localizing to the rhoptries the human protein associated with Krabbe disease Galactocerebrosidase-TAT (the mutated GALC-TAT SEQ ID NO: 4487), and additional 4 mixed-population pools containing stable T. gondii transgenic parasites expressing and localizing to the rhoptries the 3 human proteins associated with common neuropathologies Galactocerebrosidase-TAT (the WT GALC-TAT SEQ ID NO: 4488), Aspartoacylase (ASPA SEQ ID NO:4483), MeCP2 (SEQ ID NO:4477) and Survival Motor Neuron Protein (SMN1, SEQ ID NO: 4479).
Localization of the Therapeutic Proteins Aspartoacylase, MeCP2 and Survival Motor Neuron Protein to T. gondii Dense Granules Secretory Organelles and Parasitophorous Vacuolar Space—
The results demonstrate that T. gondii can localize the heterologous (mammalian) human therapeutic proteins to the parasite's Dense granules secretory organelles and parasitophorous vacuolar space (
Thus, the present inventors have succeeded in demonstrating that T. gondii may be engineered to express heterologous (mammalian) proteins fused to an endogenous protein of the parasite, and that these proteins localize to the parasite's Dense granules secretory organelles and parasitophorous vacuolar space. The present inventors have engineered the human proteins to be fused to GRA16, an endogenous protein that is naturally secreted into host cells by the parasites, which allows to “ride” on the parasite's protein secretion machinery for the targeting and secretion of proteins of interest (e.g., a therapeutic protein). This is an important milestone on the path to suggesting T. gondii as a feasible basis for this technology.
Using this approach, the present inventors have generated a novel stable T. gondii transgenic line, expressing and localizing to the Dense granule secretory organelles and parasitophorous vacuolar space the human protein associated with Rett Syndrome MeCP2, and additional 2 mixed-population pools containing stable T. gondii transgenic parasites expressing and localizing to the Dense granule secretory organelles and parasitophorous vacuolar space the two human proteins associated with common neuropathologies Aspartoacylase and Survival Motor Neuron Protein.
Secretion of the Therapeutic Protein MeCP2 from the Transgenic Parasite into the Host Cell, and Localization in the Region of Activity in the Host Cell, the Nucleus—
The results demonstrate that protein fusions consisting of a pharmaceutical polypeptide fused to a Toxoplasma gondii secreted polypeptide (and specifically here, the therapeutic protein MeCP2 fused to the T. gondii dense granule protein GRA16), can be released from the parasite into the host cell and translocate to the therapeutic protein's target site in the host cell (here, the nucleus). The fusion protein containing MeCP2 localized to the host cell nucleus as shown by immunofluorescence staining (IFAs) (
Using this approach, the present inventors have generated novel stable T. gondii transgenic lines, expressing and localizing to the host cell nucleus the human protein associated with Rett Syndrome, MeCP2 (
The Galactocerebrosidase enzymatic activity assay which is based on the enzymatic breakup of a synthetic fluorescent substrate of Galactocerebrosidase named 6-Hexadecanoylamino-4-methylumbelliferyl-beta-D-galactoside (6HMU-beta-D-galactoside) is based on the manufacturer's protocol [Otto P. van Diggelen. MOSCERDAM SUBSTRATES, Laboratory protocol for enzyme analysis for Krabbe disease; Galactocerebrosidase]. Using Krabbe patient-derived fibroblasts, the assay is used to evaluate the ability of the GALC- and GALC-TAT-expressing parasite's to rescue Krabbe disease phenotypes in culture. Untreated Krabbe disease cells (Krabbe patient-derived fibroblasts), Krabbe disease cells transfected with sham vector (empty human expression vector) and/or Krabbe disease cells infected with T. gondii parasites not expressing GALC are used as negative controls. Positive controls are wild type (WT) cell lines, or the Krabbe disease cells transfected with human expression vectors driving the expression of either the standalone (HA-tagged) GALC or GALC-TAT gene or a Toxofilin/GRA16-fused GALC or GALC-TAT gene, identical to the fusion expressed by the transgenic parasites tested. This allows ruling out the possibility that any negative results could be caused by a lack of sufficient effect of the protein itself, or from disruption of the therapeutic protein's activity by the fusion of Toxofilin or GRA16 to the N-terminus of the therapeutic protein, and thus allows quantifiable and direct inference of the efficiency of the parasitic delivery system.
The assay is used to characterize the Galactocerebrosidase-expressing parasites' ability to rescue the Galactocerebrosidase-deficiency shown in Krabbe disease patient-derived fibroblasts. This experiment demonstrates the transgenic parasites' ability to rescue Krabbe disease by delivering the supplementary protein to cells in culture.
Neural and glial cells derived from mouse models of Rett syndrome that harbor a mutation or deletion of the MECP2 gene present an array of abnormal morphological and molecular characteristics. [Percy, A. Rett syndrome: coming to terms with treatment. Adv. Neurosci. (2014), [de la Torre-Ubieta et al. Advancing the understanding of autism disease mechanisms through genetics. Nat Med. 2016].
Using a Rett Syndrome cell culture model, the quantitative scoring of the molecular and morphological characteristic phenotypes of Rett syndrome is used to evaluate the ability of the MeCP2-expressing parasite's to rescue Rett Syndrome phenotypes in culture. Untreated Rett Syndrome model cells, Rett Syndrome model cells transfected with sham vector and/or Rett Syndrome model cells infected with T. gondii parasites not expressing MeCP2 are used as negative controls. Positive controls are WT cell lines and Rett Syndrome model cells transfected with human expression vectors driving the expression of either the standalone (HA-tagged) MECP2 coding sequence or a Toxofilin/GRA16-fused MECP2 coding sequence identical to the fusion expressed by the transgenic parasites tested. This allows ruling out the possibility that any negative results could be caused by a lack of sufficient effect of the protein itself, or from disruption of the therapeutic protein's activity by the fusion of Toxofilin or GRA16 to the N-terminus of the therapeutic protein, and thus allows quantifiable and direct inference of the efficiency of the parasitic delivery system.
The assay is used to characterize the MeCP2-expressing parasites' ability to rescue the MeCP2-deficiency shown in a Rett Syndrome cell culture model. This experiment demonstrates the transgenic parasites' ability to rescue a Rett Syndrome by delivering the supplementary protein to cells in culture.
The transgenic lines of the parasite are tested for their ability to affect phenotypes in whole organism. Testing the transgenic parasites in mice allows the assessment of the system in the presence of an immune system and a functional blood brain barrier (BBB). The transgenic T. gondii parasites are administered to the respective animal model of the condition being tested intranasally, enterally (such as orally), subcutaneously, intra-muscularly, intravenously, intradermally, intraperitoneally, intracranially or intracerebrally. The transgenic T. gondii parasites are administered in the form of tachyzoites, bradyzoite tissue cysts or oocysts.
Following inoculation with the parasites, the animal model are characterized and assessed for the manifestation of phenotypes related to the condition studied (such as pathological phenotypes of the disease). Phenotypes include behavioral, morphological and molecular phenotypes. The effects of the transgenic parasites are revealed by comparison to animals treated with parasites not expressing the therapeutic polypeptide and to sham-treated animals.
For instance, mice model of Rett Syndrome treated with T. gondii parasites expressing the therapeutic protein MeCP2 are assessed by known phenotypes of the disease which include impaired social interaction, increased scent marking, hindlimb clasping, reduced lifspan, anxiety, respiratory problem, hypoactivity, impaired motor coordination, miRNA-mediated IGF1 deficit, decreased level of BDNF, impaired cortical plasticity, glial dysfunction, GABAergic neuronal dysfunction, decreased soma size, decreased dendritic spine density, decreased synapse number, decreased activity (Ca2+ imaging), decreased sEPSC and sIPSC frequency and amplitude, decreased Tujl+ cell number, decreased PAX6, SCN1A/1B expression, decreased dendrite complexity in cortical neurons, decreased AP frequency in cortical neurons, decreased total transcription in cortical neurons, decreased expression of neuronal, synaptic, highly-expressed genes, immediate early genes and mitochondrial genes in cortical neurons, decreased mRNA translation in cortical neurons, decreased BDNF secretion from cortical neurons, decreased AKT/mTOR pathway activity in cortical neurons and decreased oxygen consumption and maximal respiration in cortical neurons [de la Torre-Ubieta et al. Advancing the understanding of autism disease mechanisms through genetics. Nat Med. 2016.]
T. gondii are engineered to express and secrete customized Transcription activator-like effectors (TALE). Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. The DNA recognition and binding region of TALEs are made up of tandem repeats of 34-aa sequences (monomers) which determine the sequence of the recognized DNA sequence. There are 4 types of monomers which differ in their repeat variable diresidues (RVDs) which determines the nucleotide they recognize. The monomer termed “NI” (SEQ ID NO: 4508) is specific for the “A” nucleotide; the monomer termed “HD” (SEQ ID NO: 4495) is specific to the “C” nucleotide”; the monomer termed “NG” (SEQ ID NO: 4509) is specific to the “T” nucleotide and the monomer termed “NN” (SEQ ID NO: 4494) is specific the “G” or “A” nucleotide. More recently, the additional RVD NH (SEQ ID NO:4504) was shown to provide higher G-specificity. The tandem repeat DNA-binding domain always ends with a half-length repeat (0.5 repeat). Customized TALE DNA-binding domains can be used to generate custom TALE transcription factors (TALE-TFs) and modulate the transcription of endogenous genes from the genome, by fusion to the synthetic VP64 transcriptional activator. Customized TALE DNA-binding domains can also be used to generate custom TALE nucleases (TALENs) and can be used to generate site-specific double-strand breaks to facilitate genome editing through nonhomologous repair or homology directed repair, by fusion to the catalytic domain of FokI endonuclease. The TALEs are made by constructing a DNA-binding domain from the monomers (SEQ IDs NO:4508, 4509, 4494, 4495 and 4504), which is inserted into a TALEN backbone or TALE-TF backbone. Because the backbones also include the 0.5 repeat at the end of the DNA binding domain, there are 4 types of TALEN backbones (e.g., SEQ ID NOs:4500, 4501, 4502, and 4503), as well as 4 TALE-TF backbones (e.g., SEQ ID NO:4496, 4497, 4498, and 4499). The backbones also contain cytomegalovirus promoter (CMV), nonrepetitive N terminus from the Hax3 TALE (N-term), nonrepetitive C terminus from the Hax3 TALE (C-term), type IIs restriction sites used for the insertion of custom TALE DNA-binding domains (such as BsaI), negative selection cassette containing the ccdB negative selection gene and chloramphenicol resistance gene (ccdB+CmR), nuclear localization signal (NLS), and either a catalytic domain from the FokI endonuclease (FokI) or synthetic transcriptional activator derived from VP16 protein of herpes simplex virus (VP64) followed by 2A self-cleavage linker (2A) and enhanced green fluorescent protein (EGFP). All are followed by a polyadenylation signal (polyA signal) (Sanjana et al., A transcription activator-like effector toolbox for genome engineering, 2012). The resulting TALE sequences are inserted downstream of the coding sequence of a T. gondii endogenous secreted polypeptide as described in the GENERAL MATERIALS AND EXPERIMENTAL METHODS hereinabove, and the resulting constructs are transfected into T. gondii to produce transgenic T. gondii strains that express and secreted the TALE.
For example, in order to target the TACGTACG (SEQ ID NO: 4505) sequence in a genome of a subject in need thereof, the TALE nuclease construct or the TALE transcription factor construct can be used. In the open reading frame of these constructs the monomers which are specific for targeting the exemplary “target sequence” (SEQ ID NO: 4505) are arranged one after the other, as shown schematically in
In order to generate T. gondii parasites that express mammalian proteins and target them to their rhoptry secretory organelles the present inventors transfected type I RHΔRX tachyzoites or RH (co-transfected with the pDHFR selection plasmid) T. gondii with plasmids containing sequences coding for the protein of interest fused to the rhoptry protein Toxofilin. The mammalian proteins tested in the Toxofilin fusion system were: glial derived neurotrophic factor (GDNF), parkin (PARK2), galactocerebrosidase (GALC), galactocerebrosidase fused to a TAT protein transduction domain (GALC-TAT; SEQ ID NO: 4487), methyl CpG binding protein (MeCP2), aspartoacylase (ASPA) and Survival of motor neuron (SMN1). In an attempt to improve protein expression and targeting, the present inventors also tested codon-optimized versions of some of the proteins, including GALC, MECP2, ASPA and the additional gene TFEB which was tested in the codon-optimized form (SEQ ID NO: 4604).
ASPA (SEQ ID NO: 4483), ASPA codon optimized (SEQ ID NO: 4602), GALC (SEQ ID NOs: 4486 and 4601), GALC-TAT (SEQ ID NO: 4488), PARK2 (SEQ ID NO: 4478), SMN1 (SEQ ID NO: 4479), GDNF (SEQ ID NO:4489), MeCP2 (SEQ ID NO: 4476) and MeCP2 codon optimized (SEQ ID NO:4477) showed variable targeting of the fusion proteins in the parasites, which included rhoptry targeting as well as other patterns of targeting, resembling endoplasmic reticulum, golgi, nucleus, cytoplasm, apicoplast, microneme and other localizations in the parasites (
GALC-TAT mutated form generated primarily rhoptry-like fusion protein localization. From GALC-TAT mutated the present inventors also generated clonal lines that strongly expressed the fusion protein and targeted it to a rhoptries (
In order to generate T. gondii parasites that express mammalian proteins and target them to their dense granule secretory organelles type I RHAHX T. gondii were transfected with plasmids containing sequences coding for the protein of interest fused to the dense granule protein GRA16. The mammalian proteins-coding genes tested in the GRA16 fusion system were GALC [human forms (SEQ ID NOs: 4486 and 4601) and codon-optimized (SEQ ID NO: 4603), GALC-TAT (human form SEQ ID NO: 4488), MECP2 (codon-optimized, SEQ ID NO:4477), ASPA [human form (SEQ ID NO:4483) and codon-optimized (SEQ ID NO:4602)], SMN1 (human form, SEQ ID NO: 4479) and TFEB (codon-optimized, SEQ ID NO:4604).
GALC, GALC codon optimized and GALC-TAT were expressed in the transfected parasites (
The present inventors have engineered six Type I and Type II Toxoplasma clonal lines which in addition to secreting the fusion protein into the parasitophorous vacuole, were also able to release it from the parasitophorous vacuole and target it to the nucleus of the host cell in amounts detectable by immunofluorescence staining. These clonal lines are: RH GRA16-MECP2 codon optimized (RH GRA16-MECP2opt), RH GRA16-TFEB codon optimized (RH GRA16-TFEBopt), Prugniaud-GFP-Luciferase DHFR-TS GRA16-MECP2 codon optimized (Pru-GFP-LUC GRA16-MECP2opt), Prugniaud-GFP-Luciferase DHFR-TS GRA16-TFEB codon optimized (Pru-GFP-LUC GRA16-TFEBopt), Prugniaud GRA16-MECP2 codon optimized (Pru GRA16-MECP2opt), and Prugniaud GRA16-TFEB codon optimized (Pru GRA16-TFEBopt). In addition to the transgenic lines expressing the fusion proteins, the present inventors also generated 2 control lines expressing the carrier protein GRA16 fused to the HA epitope tag alone: RH GRA16-HA (RH GRA16-HAstop) and Prugniaud GRA16-HA (Pru GRA16-HAstop). As MeCP2 and TFEB are both nuclear proteins, host cell nuclear targeting means they were targeted to their site of activity in the cell. Secretion and host cell nuclear targeting of the proteins in all the lines described above was detected in HFF human fibroblasts by infection of HFF cells in complete DMEM with tachyzoites of the respective line, fixation, immunofluorescence staining and analysis by fluorescence and polarized light microscopy (
In order to quantify the efficiency and the dynamics of GRA16-mediated protein delivery in vitro, the present inventors quantified delivery over time and multiplicity of infection (MOI) for tachyzoites of the three type I lines RH GRA16-HAstop, RH GRA16-MECP2opt and RH GRA16-TFEBopt, as summarized in
Immortalized Human Dopaminergic Neuronal Precursor Cells, also known as Lund Human Mesencephalic (LUHMES) cells, were grown in media containing F12 advanced DMEM media supplemented with L-glutamine, N-2 serum-free supplement and βFGF (beta-fibroblast growth factor). The cells were differentiated in culture into morphologically and biochemically mature dopamine-like neurons using F12 advanced DMEM media supplemented with L-glutamine, N-2 serum-free supplement, tetracycline, GDNF and cAMP (Cyclic adenosine monophosphate). On day 6-9 of differentiation (at which point the cells are mature neurons), tachyzoites of the clonal lines RH GRA16-MECP2opt, RH GRA16-TFEBopt and RH GRA16-HAstop were used to infect the neurons. 16-22 hours post infection the neurons were fixed and immunofluorescently stained and analysed by fluorescence and polarized light microscopy. All lines tested presented clear secretion of the fusion proteins, which were targeted to the nuclei of the human neurons (
In addition, human neurons were infected with RH GRA16-MECP2opt tachyzoites and collected 24 hours post infection. Infected neurons were washed with PBS, scraped using a sterile cell scraper and pelleted by centrifugation, residual PBS was removed and the pellets were snap-frozen in liquid nitrogen. Nuclear proteins extracted from the infected neurons were immunoprecipitated using magnetic beads bound to an MeCP2 antibody. Immunoprecipitated proteins were immunoblotted with the same MeCP2 antibody following a standard Western blot protocol. The resulting blot presented 2 strong bands, of approximately 75 kDa and approximately 130 kDa. The ˜75 kDa band represents the endogenous untruncated MeCP2 from the human neurons (predicted size: 75 kDa) and the ˜130 kDa band represents the parasite-delivered GRA16-MeCP2 fusion protein (GRA16 predicted size: 55 kDa. Fusion protein predicted size: 75+55=130 kDa) (
Neuron-enriched primary cultures from the cortices and hippocampi of P1 (day 1) mice pups were cultured for 5 days and infected with tachyzoites of the transgenic line RH GRA16-MECP2opt. Twelve (12), 24 and 48 hours post infection, the neurons were fixed, immunofluorescently stained and analysed by fluorescence and polarized light microscopy. In all listed time points, the GRA16-MECP2 fusion protein was secreted and targeted to the nuclei of the neurons, and it also appeared in foci corresponding to areas of dense heterochromatic DNA, recognizes by intense DAPI staining (
During the normal course of infection, after the initial acute phase in which the parasite replicates and disseminates in the body as tachyzoites, immune pressure causes T. gondii tachyzoites to differentiate into bradyzoites which resides in the long-lasting quiescent cysts in tissues, and mostly in the brain (Carruthers V B, Suzuki Y, 2007. Schizophr Bull. 33(3):745-51. Effects of Toxoplasma gondii infection on the brain). Therefore the present inventors investigated whether the transgenic T. gondii continue to express the GRA16-fused therapeutic proteins after they differentiate into the bradyzoite form. An in vitro differentiation of the transgenic T. gondii into bradyzoites was induced by stress with alkaline media for 3 to 5 days. Alkaline media contained DMEM medium adjusted to pH 8.1 with 10 mM HEPES and 1% fetal bovine serum supplemented with penicillin-streptomycin (Tomita T, Bzik D J, et al. 2013. PLoS Pathog. 9(12):e1003823. The Toxoplasma gondii cyst wall protein CST1 is critical for cyst wall integrity and promotes bradyzoite persistence). The present inventors tested differentiation of the strains Pru GRA16-HAstop and Pru GRA16-MeCP2. Bradyzoite cysts, identified by DBA (Dolichos Biflorus Agglutinin) staining of the cyst wall, continue to express both the GRA16-HAstop (Data not shown) and GRA16-MeCP2 proteins (
By performing co-immunoprecipitation on cell lysates from infected cultures and from infected animals, the present inventors can decipher molecular interactions between the polypeptides delivered by the transgenic T. gondii and endogenous proteins and nucleic acids.
For example, in mammalian cells MeCP2 binds both DNA and proteins from the SMRT/NCoR and SIN3A co-repressor complexes. Binding to DNA can be probed by immunofluorescent stainings and by chromatin-immunoprecipitation, and binding to other proteins can be probed by immunofluorescent stainings and by protein co-immunoprecipitation. The added epitope tags on the delivered fusion proteins can be used to distinguish between copies of the protein produced by the host cell and copies of the protein delivered by the parasites, while antibodies against the protein itself can be used to probe both the endogenous and the parasite-delivered protein (e.g. for comparative assays).
The transgenic lines of the parasite are tested for their ability to affect phenotypes in whole organism. Testing the transgenic parasites in animal models allows the assessment of the system in the presence of an immune system and a functional blood brain barrier (BBB). This is done by following the standard infection protocols established in the field to infect mice with the transgenic T. gondii (Cabral C M et al., 2016, PLoS Pathog. 12(2):e1005447. Neurons are the Primary Target Cell for the Brain-Tropic Intracellular Parasite Toxoplasma gondii; Berenreiterová M et al., 2011. PLoS One. 6(12):e28925. The distribution of Toxoplasma gondii cysts in the brain of a mouse with latent toxoplasmosis: implications for the behavioral manipulation hypothesis; Tait E D et al. 2010. J. Immunol. 185(3):1502-12. Virulence of Toxoplasma gondii is associated with distinct dendritic cell responses and reduced numbers of activated CD8+ T cells; Koshy A A et al. 2012. PLoS Pathog. 8(7):e1002825. Toxoplasma co-opts host cells it does not invade; Jensen K D et al. 2015. MBio. 6(2):e02280. Toxoplasma gondii superinfection and virulence during secondary infection correlate with the exact ROP5/ROP18 allelic combination). The transgenic T. gondii parasites are administered to the animal model intranasally, enterally (such as orally), subcutaneously, intra-muscularly, intravenously, intradermally, intraperitoneally, intracranially or intracerebrally. The transgenic T. gondii parasites are administered in the form of tachyzoites, bradyzoite tissue cysts or oocysts. Animal models can be wild type animals or model animals bearing a condition treatable or otherwise affected by delivery of the protein of interest expressed in the transgenic parasites. After the initial stage of acute infection, the animal develops chronic bradyzoite tissue cysts, primarily in the brain. This mode of delivery relies on continuous expression and secretion the protein of interest from the transgenic T. gondii at the chronic stage of infection.
At different stages during development of the chronic infection (first 3 weeks to 2 months after infection, depending on strain and route of infection), as well as in different time points after the chronic infection has been established and stabilized (time-scale of months to years after infection), brain histological stainings are used to assess the percentage of cells infected, distribution of parasites and the delivered protein in the brain and in other tissues and to quantify the levels of delivered protein. Co-immunoprecipitation and cellular morphologies can be used to confirm molecular interactions between the delivered proteins and endogenous counterparts in the receiving cells and to provide evidence for their function.
The condition of the treated animal model is monitored and characterized at different stages of the chronic infection and at different stages of the disease progression using physiological, behavioural, cellular and molecular measures in order to assess manifestations of disease phenotypes and efficacy of the treatment in alleviating them as well as any potential toxicity arising from the treatment. The effects of the transgenic parasites are assessed by comparison to animals treated with parasites not expressing the pharmaceutical polypeptide and/or to sham-treated animals.
For instance, mice model of Rett Syndrome treated with T. gondii parasites expressing the therapeutic protein MeCP2 are assessed by known phenotypes of the disease which include impaired social interaction, increased scent marking, hindlimb clasping, reduced lifspan, anxiety, respiratory problem, hypoactivity, impaired motor coordination, miRNA-mediated IGF1 deficit, decreased level of BDNF, impaired cortical plasticity, glial dysfunction, GABAergic neuronal dysfunction, decreased soma size, decreased dendritic spine density, decreased synapse number, decreased activity (Ca2+ imaging), decreased sEPSC and sIPSC frequency and amplitude, decreased Tujl+ cell number, decreased PAX6, SCN1A/1B expression, decreased dendrite complexity in cortical neurons, decreased AP frequency in cortical neurons, decreased total transcription in cortical neurons, decreased expression of neuronal, synaptic, highly-expressed genes, immediate early genes and mitochondrial genes in cortical neurons, decreased mRNA translation in cortical neurons, decreased BDNF secretion from cortical neurons, decreased AKT/mTOR pathway activity in cortical neurons and decreased oxygen consumption and maximal respiration in cortical neurons (de la Torre-Ubieta et al. Advancing the understanding of autism disease mechanisms through genetics. Nat Med. 2016, 22:345-61).
Similarly to Example 13 hereinabove, the transgenic lines of the parasite are tested for their ability to affect phenotypes in whole organism. Testing the transgenic parasites in animal models allows the assessment of the system in the presence of an immune system and a functional blood brain barrier (BBB), and can be conducted by intranasal, enteral (such as oral), subcutaneous, intra-muscular, intravenous, intradermal, intraperitoneal, intracranial or intracerebral administration of the transgenic T. gondii in the form of tachyzoites, bradyzoite tissue cysts or oocysts. Animal models can be wild type animals or model animals bearing a condition treatable or otherwise affected by delivery of the protein of interest expressed in the transgenic parasites. This experimental design focuses on assessment of transient delivery that does not rely on differentiation into bradyzoites and establishment of chronic infection and/or chronic cysts. Some examples include: delivery by attenuated parasites that cannot differentiate into bradyzoites and/or cannot replicate and/or cannot persist in vivo longer than a few weeks, or otherwise cannot establish a chronic infection; delivery by local administration to the target tissue or to an area close to the target tissues followed by clearance or inactivation of the parasites. These can also be repeated multiple times for repeated dosing. Immunosupressive agents can be administered before, concomitantly or after infection with the parasites to enhance infection and to aid re-infection of animals that have already been infected with T. gondii before. This experimental design also includes delivery by transgenic T. gondii capable of establishing chronic infection, but in which the collection of samples or analysis are performed at the acute stage of infection before establishment of chronic infection. This form of delivery relies mostly on secretion of the protein of interest by tachyzoites of the transgenic lines. At different stages during the acute infection (first days to about 2 months after infection, depending on strain and route of infection) brain histological stainings are used to assess the percentage of cells infected, characterize the distribution of parasites and the delivered protein in the brain and in other tissues and quantify the levels of delivered protein. Co-immunoprecipitation and cellular morphologies are used to confirm molecular interactions between the delivered proteins and endogenous counterparts in the receiving cells and to provide evidence for their function.
The condition of the treated animal model is monitored and characterized at different time points after initiation of treatment and at different stages of the disease progression using physiological, behavioural, cellular and molecular measures in order to assess manifestations of disease phenotypes and efficacy of the treatment in alleviating them as well as any potential toxicity arising from the treatment. The effects of the transgenic parasites are assessed by comparison to animals treated with parasites not expressing the therapeutic polypeptide and/or to sham-treated animals.
Although the parasite is generally considered harmless in healthy humans, additional modifications to the parasites may be incorporated in the therapeutic strains in order to further attenuate their virulence. As the model organism of its phylum, there is a wide range of highly developed genetic tools for modifying the parasite (Jiménez-Ruiz E, Wong E H, Pall G S, Meissner M. Parasitology. 2014, 141:1390-8. “Advantages and disadvantages of conditional systems for characterization of essential genes in Toxoplasma gondii”) which may be used to generate attenuated and exogenously-controllable parasites (Moe-Behrens G H, Davis R, Haynes K A. Front Microbiol. 2013 4:5. “Preparing synthetic biology for the world”). One approach may include engineered auxotrophy. Secondly, attenuated parasites may be developed by targeting and disrupting specific virulence mechanisms of the parasite which are not essential to its ability to deliver the protein drugs. Another approach may include active control through engineered induced lethality such as inducible “suicide switches” (self-destruction element) that eliminate the parasite upon administration of an activating molecule. Some examples may include utilizing the widely-used tetracyclin-inducible promoter for drug-inducible activation of lethal or apoptotic pathways in the parasites in vivo, or the rapamycin-dimerizing diCre system for inducible knock-out of essential genes or for inducible activation of lethal or apoptotic pathways. Such drug-inducible control may also allow differential clearance of the parasite from specific tissues, regions or cell types due to differential permeability of the tissues or cells to the drug and its different distribution in the body.
Analysis and Discussion
The invention is a protein delivery platform that utilizes the machinery of the brain parasite T. gondii, which allows it to penetrate the blood-brain barrier and delivery proteins to cells, and especially neurons, within the CNS.
To enable delivery of therapeutic proteins to the CNS, the present inventors reappropriate the parasite Toxoplasma gondii for the synthesis and delivery of proteins of interest to cells in the CNS. The present inventors thus establish a versatile platform for specific and controlled delivery of proteins to cells within the CNS. The engineered parasite has the following novel capabilities:
(1) It can be easily modified to synthesize a range of therapeutic heterologous proteins and secrete them with specificity into target cells within the CNS.
(2) It can contain modification that reduce or exclude elements of the parasite that can mediate deleterious effects on the infected host.
(3) It can contain regulatory and/or self-destruction elements that allow control of its distribution and spatiotemporal activity once in the host.
Following is a non-limiting summary of alterations in the parasite's genome that are described herein:
(i) Fusion of the selected therapeutic protein to a specific endogenous secreted protein of the parasite. Additional accessory elements and modifications may be added to the fusion gene/protein that may increase the effectiveness of the therapeutic fusion protein, such as cleavage sites that may allow detachment of the therapeutic protein from the endogenous parasite protein, after it is no longer needed for its targeting or activity, or mediate detection and assessment of efficiency and distribution of the Toxoplasma and of the delivered protein, such as epitope tags and fluorescent proteins.
(ii) Removal of virulence genes that are not necessary for the therapeutic role of the parasite. These modifications reduce the potential deleterious effects of the parasite on the patient, and makes it relevant for safe clinical use.
(iii) Insertion of regulatory elements that can be induced to cause the parasite to self-destruct in response to an externally administered drug, or to manipulate the parasite's activity. These elements augment the safety features of the technology, and also provide increased control of its spatiotemporal activity.
The initial strains which were developed with the method of some embodiments of the invention were based on the Galactocerebrosidase and Galactocerebrosidase-TAT proteins for Krabbe disease, GDNF protein for a range of neurodegenerative diseases and other conditions (among them Parkinson's disease, Supranuclear Palsy, Multiple sclerosis, Alzheimers disease, Amyotrophic Lateral Sclerosis, Huntington's disease, retinal degenerations, Traumatic Brain Injury and hypoxic/ischaemic CNS disorders), Aspartoacylase protein for Canavan disease, MeCP2 protein for Rett Syndrome, SMN protein for Spinal Mascular Atrophy, E3 ubiquitin-protein ligase parkin for Parkinson's Disease and TFEB for lysosomal storage diseases and neurodegenerative diseases. Though the present inventors have selected a number of therapeutic proteins that are currently being tested clinically and have known supplementary functions, the platform may be adapted to a wide range of proteins.
The possible applications of the technology include, but are not limited to:
1. The treatment of conditions through the delivery of therapeutic proteins;
2. The supply of proteins to the brain in healthy individuals for the purpose of augmenting brain functions or promoting neuro-regeneration;
3. A general solution for highly specific and efficient targeted protein delivery for supplementing protein synthesis.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
This application is a National Phase of PCT Patent Application No. PCT/IL2017/050731 having International filing date of Jun. 29, 2017, which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application No. 62/355,898 filed on Jun. 29, 2016. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety. The ASCII file, entitled 74636SequenceListing.txt, created on Dec. 24, 2018, comprising 17,070,469 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. The sequence listing submitted herewith is identical to the sequence listing forming part of the international application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2017/050731 | 6/29/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/002938 | 1/4/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5693325 | Kahn | Dec 1997 | A |
| 8293224 | Bzik et al. | Oct 2012 | B2 |
| 8673289 | Bzik et al. | Mar 2014 | B2 |
| 20120045477 | Bzik et al. | Feb 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2016046321 | Mar 2016 | WO |
| WO 2018002938 | Jan 2018 | WO |
| Entry |
|---|
| Notice of Reason(s) for Rejection dated Jul. 20, 2021 From the Japan Patent Office Re. Application No. 2018-568203 and Its Translation Into English. (14 Pages). |
| Sommerville et al. “Biochemical and Immunological Characterization of Toxoplasma Gondii Macrophage Miigration Inhibitory Factor”, Journal of Biological Chemistry, 288(18):12733-12741, May 2013. |
| Wang et al. “Research Advances in Microneme Protein 3 of Toxoplasma Gondii”, Parasites & Vectors, 8(Art.384):l-12, Jul. 22, 2015. |
| International Preliminary Report on Patentability dated Jan. 10, 2019 From the International Bureau of WIPO Re. Application No. PCT/IL2017/050731. (10 Pages). |
| International Search Report and the Written Opinion dated Aug. 22, 2017 From the International Searching Authority Re. Application No. PCT/IL2017/050731. (15 Pages). |
| Boothroyd et al. “Kiss and Spit: The Dual Roles of Toxoplasma Rhoptries”, Nature Reviews Microbiology, 6(1): 79-88, Published Online Dec. 3, 2007. |
| Carruthers et al. “Effects of Toxoplasma Gondii Infection on the Brain”, Schizophrenia Bulletin, 33(3): 745-751, Advance Access Publication Feb. 23, 2007. |
| Dlugonska “Toxoplasma Rhoptries: Unique Secretory Organelles and Source of Promising Vaccine Proteins for Immunoprevention of Toxoplasmosis”, Journal of Biomedicine and Biotechnology, 12008(Art.ID 632424): 1-7, Jul. 22, 2008. |
| Feustel et al. “Toxoplasma Gondii and the Blood-Brain Barrier”, Virulence, 3(2): 182-192, Mar. 1, 2012. |
| Kim et al. “Toxoplasma Gondii: The Model Apicomplexan”, International Journal of Parasitology, 34(3): 423-432, Mar. 9, 2004. Chaps.11, 15. |
| Koshy et al. “Toxoplasma Secreting Cre Recombinase for Analysis of Host-Parasite Interactions”, Nature Methods, 7(4): 307-309, Apr. 2010. Electronic Version, p. 2. 2nd & 3rd Para. Fig.1a. |
| Sibley “Invasion and Intracellular Survival by Protozoan Parasites”, Immunological Reviews, 240(1): 72-91, Mar. 2011. Electronic Version, p. 3, 2nd Para, p. 9, 2nd Para, Ref.112. |
| Stanislaus et al. “Genetically Engineered Self-Destruction: An Alternative to Herbicides for Cover Crop Systems”, Weed Science, 50(6): 794-801, Nov.-Dec. 2002. Abstract, p. 795, Left col. Last Para—Right col. First Para, p. 797, Right col. 2nd Para, Figs.l. 2. |
| Van Diggelen “Laboratory Protocol for Enzyme Analysis for Krabbe Disease: Galactocerebrodiase: HMU-BetaGal”. Protocol KRB, Moscerdam Substrates, p. 1-3. Dec. 2009. |
| Supplementary Partial European Search Report and the European Provisional Opinion dated Dec. 17, 2019 From the European Patent Office Re. Application No. 17819507.9. (13 Pages). |
| Bracha et al. “Engineering Brain Parasites for Intracellular Delivery of Therapeutic Proteins”, BioRxiv, XP055649990, 481192-1-481192-45, Jan. 2018. |
| Gissot et al. “Epigenomic Modifications Predict Active Promoters and Gene Structure in Toxoplasma Gondii”, PLoS One Pathogens, XP055650151, 396): e77-0709-e77-0719, Published Online Jun. 8, 2007. |
| Zou et al. “Evaluation of Toxoplasma Gondii as A Live Vaccine Vector in Susceptible and Resistant Hosts”, Parasites & Vectors, XP021109662, 4(1): 168-1-168-10, Aug. 28, 2011. |
| Supplementary European Search Report and the European Search Opinion dated Mar. 23, 2020 From the European Patent Office Re. Application No. 17819507.9. (12 Pages). |
| Number | Date | Country | |
|---|---|---|---|
| 20200121731 A1 | Apr 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62355898 | Jun 2016 | US |
