HK1262479A1 - Targeted delivery of therapeutic proteins bioencapsulated in plant cells to cell types of interest for the treatment of disease - Google Patents

Description

Targeted delivery of therapeutic proteins bio-encapsulated in plant cells to target cell types for treatment of disease

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No. 62/256,053 filed 11/16/2015, the entire disclosure of which is incorporated herein by reference as if fully set forth.

The U.S. government has rights in the invention which is accomplished by funds provided by the national institutes of health, the debit number: r01 GM 63879, R01 HL 109442 and R01 HL 107904.

Technical Field

The present invention relates to the field of transplastomic plants and low cost protein drug production and delivery. More specifically, the present invention provides plants that: it comprises a chloroplast expressed transgene encoding therapeutic proteins and peptides operably linked to a targeting sequence which directs the plant cell encapsulated fusion protein to the target tissue after treatment in the gut.

Background

Throughout this specification, several publications and patent documents are cited in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as if fully set forth.

Biopharmaceuticals produced in current systems are too expensive to be affordable by most of the world's population. In the united states, the average annual cost of protein drugs is 25 times higher than that of small molecule drugs. The cost of protein drugs ($ 1400 billion in 2013) exceeds GDP in over 75% of countries worldwide, making these protein drugs unbearable in these countries [1 ]. One third of the global population, which receives less than $ 2 per day, cannot afford any protein medication. Although recombinant insulin has been marketed for fifty years, it is still not affordable for the vast majority of the world population. This is because such drugs are too expensive to produce, often requiring expensive fermentors, multi-step purification procedures, refrigeration/shipping, sterile transport means. In addition, the short shelf life of these drugs is associated with increased costs. Oral delivery of protein drugs has been difficult to achieve for decades because they are degraded in the digestive system and cannot pass through the intestinal epithelium for delivery to target cells.

However, some recent studies have indirectly shown that plant cell walls protect expressed protein drugs from gastric acid and enzymes by bio-encapsulation [2,3 ]. Human digestive enzymes are not capable of breaking down the glycosidic bonds in the carbohydrates that make up the plant cell walls. However, when intact plant cells containing the protein drug reach the intestinal tract, the commensal microorganism can digest the plant cell wall and release the protein drug in the intestinal lumen. Bacteria that inhabit the human intestinal tract have evolved to utilize complex carbohydrates in plant cell walls and can utilize almost all plant glycans [4,5 ]. Fusions of the B subunit of the (avirulent) Cholera Toxin (CTB), expressed in chloroplasts and bio-encapsulated in plant cells, with Green Fluorescent Protein (GFP), are delivered across the intestinal epithelium via GM1 receptor, and GFP is released into the circulatory system [6 ]. The fusion of CTBs to therapeutic proteins facilitates their efficient oral delivery to induce oral tolerance [7-11], or to efficiently deliver functional proteins to serum [12-14], or even across the blood-brain barrier or retinal barrier [15,16 ].

Foreign proteins can also be delivered into living cells by fusion with Protein Transduction Domains (PTDs) with cell membrane penetrating properties that do not require specific receptors [17 ]. Peptide and Protein Transduction Domains (PTDs) are small cationic peptides containing 8-16 amino acids, most commonly used as transporters for delivery of macromolecules [17 ]. PTDs carry molecules into cells by receptor-independent fluid-phase macroendocytosis, a special form of endocytosis. Although different PTDs exhibit similar cellular uptake characteristics, they differ in their potency for the transport of protein molecules into cells. The efficacy of cellular uptake has been found to be closely related to the number of basic amino acid residues. Since PTDs have been demonstrated to deliver biologically active proteins in vivo and in vitro in cultured mammalian cells as well as in animal models [18-20], PTD fusion protein delivery methods should have great therapeutic drug delivery potential. T and B lymphocytes are the major cellular components of the adaptive immune response, but their activation and homeostasis is controlled by dendritic cells. B cells can directly recognize native Ag through B cell receptors on their surface and secrete antibodies. However, T cells are only able to recognize peptides displayed by MHC class I and class II molecules on the surface of APCs. Macrophages are a professional antigen presenting cell and play a number of important roles, including the removal of dead cells and cell debris from chronic inflammation and the initiation of immune responses [21,22 ]. Macrophages are involved in the orchestration of primary and secondary immune responses (organization). Mast cells are involved in the development of the first inflammatory response during infection, which is important for eliciting innate and adaptive immunity. When activated, mast cells rapidly release their characteristic granules and various hormonal mediators into the stroma. Thus, mast cells play an important role in wound healing, allergic diseases, anaphylaxis and autoimmunity.

Dendritic cells are important immunoregulatory cells. Dendritic cells form complexes with multifunctional APCs and are active against pathogensPlays a key role. In addition, the dendritic cells differentiate into different types of functional cells and induce humoral or cellular immunity, stimulated by different antigens. In contrast, DCs are also crucial for the homeostasis of regulatory T cells (tregs), the extrathymic induction of tregs and for the induction of immune tolerance in transplantation and the treatment of allergic or autoimmune diseases. Tissue microenvironment, activation signals and DC subsets are important parameters that determine whether antigen presentation by DC leads to immunity or tolerance [23-25 ]]. Thus, targeted in vivo delivery of antigens to DCs may not only be useful for inducing tumor-specific immune responses and establishing new strategies for vaccine development, cancer immunotherapy, but also for tolerance induction protocols [26-28]For example, Gut Associated Lymphoid Tissue (GALT) provides maximum surface area for antigen to enter the body and a very unique microenvironment with tolerogenic properties, including the expression of the immunosuppressive cytokines IL-10 and TGF- β [29-31 ]]. Intestinal epithelial cells and CX1CR5⁺Macrophages withdraw antigen from the intestinal lumen. In particular in the endothelium of Peyer's patches, the micropleated cells (M cells) endocytose and phagocytose antigens to direct these antigens to the DCs. CD1lc in intestinal tract⁺DC contains a high proportion of CD103⁺DCs expressing TGF- β preferentially induce Tregs [32]. Recently, we demonstrated that oral tolerance induction to clotting factors and CD103 in hemophilia mice following delivery of bio-encapsulated CTB fusion antigen⁺DC frequency increase, CD103⁺DC antigen uptake and induction of several Treg subsets [9,10]. Also added are plasma cell-like DCs, which also have important immunomodulatory functions [33]. DC peptides (DCpep) were developed as ligands for mucosal DCs [26]. The small peptides bind to DC-specific receptors and facilitate the transport of large molecules to the DC.

Disclosure of Invention

In accordance with the present invention, a method for targeted delivery of a therapeutic protein to a target tissue or cell in a subject in need thereof is disclosed. An exemplary method comprises administering an effective amount of a plant cell or remnant thereof comprising a plastid (plastic) expressed nucleic acid encoding the therapeutic protein operably linked to a fusion peptide sequence, the expression of the nucleic acid resulting in the production of a therapeutic fusion protein that selectively enters a target cell or tissue in vivo upon ingestion or administration of the plant or remnant thereof, thereby selectively delivering the therapeutic protein to the target cell or tissue of the subject. Preferably, the plant is a green leaf vegetable, including but not limited to lettuce, low nicotine tobacco, spinach, cabbage and kale. Other plants include eggplant, carrot and tomato. In one embodiment, the fusion peptide sequence is a PTD. In another embodiment, the fusion peptide sequence is a DC peptide. The fusion peptide may also be an antimicrobial peptide, such as PG1 or RC 101. In certain embodiments, the fusion peptide is a CTB peptide. In other embodiments of the invention, the use of CTB fusion peptides is excluded, particularly when targeting needs to be more specific. Cells to be targeted include, but are not limited to, immune cells, somatic cells, and dendritic cells. Tables 2,3 and 4 provide therapeutic molecules that can be used for the plastid-produced fusion proteins of the invention.

In one aspect, the therapeutic fusion protein is for treating an endocrine disease, and the protein is selected from the group consisting of insulin, a growth hormone, and an insulin-like growth factor.

The therapeutic fusion protein may augment an existing biological pathway and is selected from the group consisting of erythropoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, follicle stimulating hormone, chorionic gonadotropin, α interferon, interferon B, PDGF, keratinocyte growth factor, and bone morphogenic protein.

Another aspect of the invention includes a plant or plant cell comprising the therapeutic fusion protein described above. The plant may be freeze-dried. They may be in powder form and optionally encapsulated. In a preferred embodiment, the therapeutic fusion protein is stable for months at ambient temperature.

Drawings

FIGS. 1A-1F. Schematic representation of predicted protein structure and characterization of a transplastomic line expressing GFP fusion proteins. (FIG. 1A) interaction of CTB fusion protein with GM1 receptor, and predicted 3D structures of PTD and DCpep. The pentasaccharide moiety of the GM1 receptor establishes an interaction with the pentameric structure of CTB. A hinge sequence for avoiding steric hindrance and a furin cleavage site for releasing a tethered protein are placed between the CTB and the fusion protein. The three-dimensional structure of the computed predictions of the PTD and DCpep is obtained by an iterative thread assembly refinement (I-TASSER) server [40 ]. The structure is shown in iridescence, in which the residue gradually changes color from blue to red (blue-green-yellow-orange-red) from the N-terminus to the C-terminus. In the predicted structure, the model with the most reliable structure selected for each peptide is shown based on the combined results of the parametric calculations, e.g., confidence score (C-score), high resolution model with Root Mean Square Deviation (RMSD) value, and template modeling score (TM-score). (FIG. 1B) schematic representation of the expression cassette and flanking regions of the GFP fusion carrier protein. Vrrn, rRNA operon promoter; aadA, aminoglycoside 3' -adenylyltransferase gene; vpsbh, the promoter and 5' UTR of the psbA gene; CTB, the coding sequence of the avirulent cholera B subunit; PTD, coding sequence for a protein transduction domain; DCpep, dendritic cell binding peptide sequence; smGFP, a gene sequence of soluble modified green fluorescent protein; TpsbA, the 3' UTR of the psbA gene; trnl, isoleucyl-tRNA; trnh, alanyl-tRNA. The restriction enzyme used for Southern blot analysis was designated BamHHVBgUI for probe formation, and HindIII for digestion of genomic DNA. (FIG. 1C) Southern blot analysis of each of the transplastomic systems expressing GFP fusion tag proteins. HindIII-digested gDNA was probed with the above flanking region fragments. (FIG. 1D) GFP fluorescence signals from each of the transplastomic systems were confirmed under UV light. The photographs were taken 2 months after germination. The scale indicates 0.5 cm. (FIG. 1E) Western blot analysis of density quantitation was performed with GFP standard protein. The freeze-dried leaf material (10mg) and fresh leaf material (100mg) were extracted with 300. mu.L of extraction buffer. 1X represents 1. mu.L of homogenate resuspended in extraction buffer at a ratio of 100mg to 300. mu.L. (FIG. 1F) amount of GFP fusion protein in fresh (F) and lyophilized (L) leaves. Data are mean ± SD of three independent experiments.

FIGS. 2A-2B. Oral delivery efficiency and biodistribution of GFP fused to different tags. Serum (fig. 2A) and tissue (fig. 2B) GFP levels in mice fed leaf material expressing CTB-GFP, PTD-GFP and DCpep-GFP (N ═ 6 per group). Adult mice were orally fed with leaf material of transgenic tobacco plants for three consecutive days, the amount of which was adjusted to the GFP expression level. The control group (N ═ 6) remained unfed. Blood samples were collected at 2 and 5 hours after the last gavage, at which time the mice were sacrificed and tissue samples were collected for protein isolation. The concentration of GFP in serum and tissues was determined by ELISA. Data are shown as mean ± SEM. Statistical significance was determined by paired student t-test, with p-values less than 0.05 considered significant. P <0.05, P <0.01, P <0.05 or P <0.01(CTD, PTD and DCpep relative to nature)

FIGS. 3A-3L. Visualization of GFP in ileum and liver cells of mice following oral delivery of plant cells. GFP delivery to the small intestine (left panel). Shown are cross sections stained with anti-GFP (green signal; Alexa Fluor 488), UEA-1 (which stains M cells as well as other cells, red signal, rhodamine) and DAPI (nuclear stain, blue). (FIGS. 3A-C). PTD-GFP delivery. (FIG. 3B) none of the primary antibodies (NC: negative control). (FIGS. 3D-E) CTB-GFP delivery. (FIG. 3F) DCpep delivery. Original magnification: 200x (inset in fig. 3A, B, D-F, C) or 40x (fig. 3C). GFP staining in frozen sections of liver (right panel) is shown with the same exposure time during image capture. The following compositions were used: 1000 primary antibody: rabbit anti-GFP antibody, and secondary antibody: the GFP staining was performed with AlexaFluor 488 donkey anti-rabbit IgG. (G, I and K) liver sections of mice fed untransformed lyophilized plant cells. (FIGS. 3H, J and L) GFP signal of liver sections of mice fed lyophilized plant cells expressing DCpep-GFP (FIG. 3H), PTD-GFP (FIG. 3J) and CTB-GFP (FIG. 3L). Original magnification: 100 x.

FIGS. 4A-4F. Characterization of purified GFP fusion proteins. (FIG. 4A) quantification of purified GFP fusion proteins, and Coomassie staining and fluorescence images. Densitometry of western blot images was performed using known amounts of GFP standard protein to quantify purified tag-fused GFP protein. The purified proteins were electrophoresed on SDS-PAGE and immunodetected with anti-GFP antibodies. The loading amounts are shown in the figure. Purity was calculated as the percentage of total amount detected in the immunoblot assay to total loading. (FIG. 4B) Coomassie staining of purified GFP-tagged proteins. M, protein molecular weight markers; lane 1, PTD-GFP (10. mu.L, 2.37. mu.g); lane 2, Dcpep-GFP (40. mu.l, 3.12. mu.g), lane 3, CTB-GFP (10. mu.L, 32.8. mu.g), and lane 4, GFP (400 ng). (FIG. 4C) non-denaturing SDS-PAGE of purified GFP fusion proteins to determine GFP fluorescence. Lane 1 (loaded PTD-GFP 10. mu.l, 9.17. mu.g TSP), lane 2 (loaded DCpep-GFP 15. mu.l, 4.6. mu.g TSP) and lane 3 (loaded CTB-GFP 20. mu.l, 33. mu.g TSP). (FIGS. 4D and 4E). The binding affinity of the purified GFP-tagged protein to the GM1 receptor was examined. anti-CTB (fig. 4D) and anti-GFP (fig. 4E) antibodies were used to detect the interaction between GM1 and the GFP fusion protein. The amounts of protein used for the assay were as follows. CTB, 10 pg; CTB-GFP, 1.25 ng; PTD-GFP10 ng; DCpep-GFP 10 ng; GFP, 10ng and UT, untransformed wild type total protein, 100 ng. (FIG. 4F) native Tris-Tris glycine PAGE of purified CTB-GFP to determine pentamer structure. The pentameric structure of purified CTB-GFP was immunodetected using anti-CTB antibody (1 to 10,000). The loading of CTB-GFP is shown.

FIGS. 5A-5B. Fused by different labelsGFP is taken up by human immune and non-immune cells. (FIG. 5A) translocation of purified GFP fusion proteins in human cell lines. With purified GFP fusion proteins: CTB-GFP (8.8. mu.g/100. mu.L LPBS), PTD-GFP (13. mu.g/100. mu.L PBS), DCpep-GFP (1.3. mu.g/100. mu.L PBS) and commercial standard GFP (2.0. mu.g/100. mu.L PBS) were incubated at 37 ℃ for 2X 10⁴Cultured human Dendritic Cells (DCs), B cells, T cells and mast cells were cultured for 1 hour. After PBS washing, B cells, T cells and mast cells were stained with DAPI diluted 1:3000 and fixed with 2% paraformaldehyde. The cells were then sealed on a glass slide and examined by confocal microscopy. Live DCs were stained with Hoechst diluted 1:3000 and examined directly under confocal microscopy. For 293T, pancreatic cells (PANC-1 and HPDE) and macrophages, 8-well chamber slides were used for overnight cell culture at 37 ℃. After incubation with purified CTB-GFP (8.8. mu.g/100. mu.L PBS), PTD-GFP (13. mu.g/100. mu.L PBS), DCpep-GFP (1.3. mu.g/100. mu.L PBS) and commercial standard GFP (2.0. mu.g/100. mu.L PBS), respectively, for 1 hour at 37 ℃, the nuclei were washed with PBS and stained with 1:3000 DAPI. (FIG. 5B) Nuclear localization of PTD-GFP in human pancreatic ductal epithelial cells (HPDE). Green fluorescence indicates GFP expression; blue fluorescence indicates that nuclei were labeled with DAPI. The image was observed at 100x magnification. The scale indicates 10 μm. All image studies were analyzed in triplicate.

Fig. 6A and 6B. GFP fused with different tags was taken up by different human somatic and stem cell types. (FIG. 6A) in vitro evaluation of purified fusion proteins CTB-GFP, PTD-GFP, DCpep-GFP and GFP-protein-1 (GFP-PG1) and GFP-recycle 101(GFP-RC 101) in human pancreatic cells (PANC-1 and HPDE), human periodontal ligament stem cells (DLHPS), maxilla mesenchymal stem cells (MMS), human head and neck squamous cell carcinoma cells (squamomous cell carcinoma cells, SCC), retinal pigment epithelial cells (RPE), gingival derived mesenchymal stromal cells (GMSC), adult human keratinocyte (adult keratinocyte) cells (AGK) and osteogenic cells (AGK) using confocal microscopyosteoplast cell, OBC). 2X 10 of human cell lines PANC-1, HPDE, HPDLS, MMS, SCC, RPE, GMSC, AGK and OBC⁴Individual cells were cultured overnight at 37 ℃ in 8-well chamber slides (Nunc) and subsequently purified GFP fusion proteins: CTB-GFP (8.8. mu.g), PTD-GFP (13. mu.g), DCpep-GFP (1.3. mu.g), GFP-PG1 (1.2. mu.g), GFP-RC101 (17.3. mu.g) in 100. mu.L of PBS supplemented with 1% FBS were incubated at 37 ℃ for 1 hour. After washing the wells three times with PBS, the cells were stained with 1:3000DAPI for 10 minutes at room temperature and then fixed with 2% paraformaldehyde for 10 minutes at room temperature. For negative control wells, cells were incubated with commercial GFP (2 μ g) in PBS containing 1% FBS or untreated. After 1 hour incubation, cells were washed three times with PBS. All fixed cells were imaged using confocal microscopy. Green fluorescence indicates GFP expression; blue fluorescence indicates that nuclei were labeled with DAPI. The image was viewed under a 100x objective. The scale indicates 10 μm. All image studies were analyzed in triplicate. FIG. 6B: the purified fusion proteins CTB-GFP, PTD-GFP, DCpep-GFP, PG1-GFP and antimicrobial peptides (PG1-GFP and RC101-GFP) were evaluated in vitro by confocal microscopy for transformation in human pancreatic cells (PANC-1 and HPDE), human periodontal ligament stem cells (HPDLS), maxillary mesenchymal stem cells (MMS), human head and neck squamous cell carcinoma cells (SCC), retinal pigment epithelial cells (RPE), gingiva-derived mesenchymal stromal cells (GMSC), Adult Gingival Keratinocytes (AGK), Osteoblasts (OBC).

Detailed Description

Targeted oral delivery of GFP fused to GM1 receptor binding protein (CTB) or human cell penetrating Peptide (PTD) or dendritic cell peptide (DCpep) was investigated. Intervillous GFP of ileum⁺The presence of intact plant cells confirms its protection from acid/enzymes in the digestive system. GFP was efficiently delivered to intestinal epithelial cells by PTD or CTB, and to M cells by all these fusion tags, confirming GFP uptake in the small intestine. PTD fusion delivers GFP to most tissues or organs more efficiently than the other two tags. GFP is efficiently tagged by all fusionsDelivered to the liver, possibly through the entero-hepatic axis. In confocal imaging studies of human cell lines using purified GFP fused to different tags, GFP signal of DCpep-GFP was detected only in dendritic cells. PTD-GFP was detected only in kidney or pancreatic cells, but not in immunoregulatory cells (macrophages, dendritic cells, T cells, B cells, or mast cells). In contrast, CTB-GFP was detected in all cell types tested, confirming that the GM1 receptor is ubiquitous. Low cost oral delivery of such protein drugs to serum, immune system or non-immune cells should significantly reduce their cost and increase patient compliance by eliminating overly expensive fermentation, protein purification refrigeration/transportation.

Defining:

as used herein, the term "administering" or "administration" an agent, drug, or peptide to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration may be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), rectal or topical. Administration includes self-administration and administration by others.

As used herein, the term "disease," "disorder," or "complication" refers to any deviation from a normal state in a subject.

As used herein, the term "effective amount" refers to an effective amount of a dose and period of time necessary to achieve a desired result.

As used herein, the term "inhibit" or "treatment" refers to the lack of worsening or progression of clinical symptoms of a disease state, e.g., inhibiting the onset of a disease in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or exhibit symptoms of the disease state.

As used herein, the term "expression" in the context of a gene or polynucleotide relates to the transcription of the gene or polynucleotide into RNA. The term may also, but need not, relate to the subsequent translation of RNA into polypeptide chains, and their assembly into proteins.

By "transgenic plant" is meant a plant whose genome has been altered by the introduction of at least one heterologous nucleic acid molecule.

As used herein, "nucleic acid" or "nucleic acid molecule" refers to any single-or double-stranded DNA or RNA molecule, and if single-stranded, molecules of complementary sequence that are linear or circular. In discussing nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule can be described herein in the 5 'to 3' direction according to conventional conventions that provide sequences. The term "isolated nucleic acid" is sometimes used in relation to a nucleic acid of the invention. When applied to DNA, the term refers to a DNA molecule that is separated from directly adjacent sequences in the naturally occurring genome of the organism from which the DNA molecule originates. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector (e.g., a plasmid or viral vector) or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.

The term "isolated nucleic acid" when applied to RNA refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it is associated in its native state (i.e., in a cell or tissue). An "isolated nucleic acid" (DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present in its production process.

The terms "percent similarity", "percent identity" and "percent homology" when referring to a particular sequence are used as specified in the university of wisconsin GCG software program.

The term "substantially pure" refers to a formulation that contains at least 5060% by weight of a given material (e.g., nucleic acids, oligonucleotides, proteins, etc.). More preferably, the formulation comprises at least 75%, and most preferably 9095% by weight of a given compound. Purity is measured by methods appropriate for a given compound (e.g., chromatography, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

A "replicon" is any genetic element capable of replicating under its own control, such as a plasmid, cosmid, bacmid, phage or virus. Replicons may be either RNA or DNA, and may be single-stranded or double-stranded.

A "vector" is any vehicle to which another genetic sequence or element (DNA or RNA) can be linked in order to bring about replication of the linked sequence or element.

An "expression operon" refers to a segment of nucleic acid that may have transcriptional and translational control sequences, such as promoters, enhancers, translational initiation signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitates expression of the polypeptide coding sequence in a host cell or organism.

The term "oligonucleotide" as used herein refers to the sequences, primers and probes of the invention and is defined as a nucleic acid molecule consisting of two or more ribonucleotides or deoxyribonucleotides (preferably more than three). The exact size of the oligonucleotide will depend on various factors and the particular application and use of the oligonucleotide.

The phrase "specifically hybridizes" refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to allow such hybridization (sometimes referred to as "substantially complementary") under predetermined conditions commonly used in the art. In particular, the term refers to hybridization of an oligonucleotide to a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of an oligonucleotide to a single-stranded nucleic acid of non-complementary sequence.

The term "promoter region" refers to the 5 'regulatory region of a gene (e.g., a 5' UTR sequence (e.g., a psbA sequence, a promoter (e.g., a common Prnn promoter or a psbA promoter endogenous to the plant to be transformed and optionally an enhancer element).

As used herein, the term "reporter", "reporter system", "reporter gene" or "reporter gene product" shall refer to the manipulation of a genetic system in which the nucleic acid comprises a gene encoding: when expressed, the product produces a reporter signal that is readily measurable, for example, by bioassay, immunoassay, radioimmunoassay, or by calorimetry, fluorescence, chemiluminescence, or other means. The nucleic acid may be RNA or DNA, linear or circular, single or double stranded, of antisense or sense polarity, and is operably linked to control elements necessary for expression of the reporter gene product. The control elements required will vary depending on the nature of the reporter system and whether the reporter gene is in DNA or RNA form, but may include, but are not limited to, elements such as promoters, enhancers, translational control sequences, poly A addition signals, transcription termination signals, and the like.

The terms "transformation", "transfection", "transduction" shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism, and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion, and the like.

The term "selectable marker gene" refers to a gene that: when expressed, the gene confers a selectable phenotype, such as antibiotic resistance, on the transformed cell or plant.

The term "operably linked" refers to the placement of regulatory sequences necessary for the expression of a coding sequence in a DNA molecule at the appropriate position relative to the coding sequence to achieve expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g., enhancers) in expression vectors.

The term "DNA construct" refers to a gene sequence used to transform a plant and produce a progeny transgenic plant. These constructs can be administered to plants in the form of viral or plasmid vectors. However, most preferred for use in the present invention are plastid transformation vectors. Other delivery methods, such as Agrobacterium T-DNA mediated transformation and transformation using biolistic processes, are also contemplated as being within the scope of the invention. Transformation DNA can be prepared according to standard Protocols, such as those set forth in Current Protocols in molecular biology, edited by Frederick M.Ausubel et al, John Wiley & Sons, 1995.

The phrase "double-stranded RNA-mediated gene silencing" refers to a process of inhibiting expression of a target gene in a plant cell by introducing a nucleic acid construct encoding: this molecule forms a double-stranded RNA structure with mRNA encoding the target gene, which is then degraded.

The phrase "consisting essentially of," when referring to a particular nucleotide or amino acid, refers to a sequence having the characteristics of a given SEQ id no. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the basic and novel characteristics of the sequence.

The terms "tag", "tag sequence" or "protein tag" refer to a chemical moiety, either an oligonucleotide, or more preferably a peptide or other chemical, that when added to another sequence provides additional utility or imparts a useful property, such as specifically targeting a target protein to a desired cell type. Such tags may also be used to isolate and purify fusion proteins containing them. Protein tags, such as those described herein or other protein tags commonly used in the art, may be added to the amino or carboxy terminus of the protein of interest.

By "immune response" is meant any response by an antigen (e.g., a viral or plant antigen) in a host with a functional immune system. The immune response may be humoral, i.e., involving the production of immunoglobulins or antibodies, or cellular, involving various types of B and T lymphocytes, dendritic cells, macrophages, antigen presenting cells, etc., or both. The immune response may also involve the production or processing of various effector molecules, such as cytokines, lymphokines, and the like. Immune responses can be measured in vitro and in various cell or animal systems.

An "antibody" or "antibody molecule" is any immunoglobulin that binds a particular antigen, including antibodies and fragments thereof. Several of the drugs listed in table 4 are antibodies. The term includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies and bispecific antibodies. As used herein, an antibody or antibody molecule contemplates both intact immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as those portions referred to in the art as Fab, Fab ', F (ab')2, and F (v).

As used herein, a fusion peptide increases the ability of a protein to enter a cell by fusing with the cell membrane without the need for a specific receptor. Other fusion proteins may target cellular receptors. Certain fusion proteins specifically target immune cells. Other fusion proteins target dendritic cells, such as DC peptides. Some fusion proteins are quite non-specific and can be used to deliver therapeutic proteins to a variety of target cell types.

Plant residues may include one or more molecules derived from a plant in which the protein of interest is expressed (such as, but not limited to, proteins and fragments thereof, minerals, nucleotides and fragments thereof, plant structural components, and the like). Thus, compositions related to whole plant material (e.g., all or part of a plant leaf, stem, fruit, etc.) or crude plant extracts must contain high concentrations of plant residues, as well as compositions comprising purified proteins of interest with one or more detectable plant residues. In a particular embodiment, the plant residue is ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco).

In another embodiment, the invention relates to an administrable composition for treating or preventing a disease by administering a therapeutic fusion protein that is produced in a plant chloroplast and comprises a tag that directs the therapeutic fusion protein to a target cell or tissue of interest. The composition comprises a therapeutically effective amount of a fusion protein expressed by plants and plant residues.

Methods, vectors and compositions for transforming plants and plant cells are described, for example, in WO 01/72959; WO 03/057834; and WO 04/005467. WO 01/64023 discusses the use of marker-free gene constructs.

Proteins expressed according to certain embodiments of the teachings herein may be used in vivo by administration to a subject, human or animal in various ways. The pharmaceutical composition may be administered orally or parenterally, i.e. subcutaneously, intramuscularly or intravenously, although oral administration is preferred. A list of therapeutic proteins of interest is provided in example II.

The oral compositions produced by embodiments of the present invention may be administered by eating a food product made with a transgenic plant that produces a plastid-derived therapeutic fusion protein. Edible parts of plants or parts thereof are used as dietary components. The therapeutic compositions may be formulated in a classical manner using solid or liquid vehicles, diluents and additives appropriate to the mode of administration desired. The composition can be administered orally in the form of tablets, capsules, granules, powders, etc., with at least one vehicle such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. The formulation may also be emulsified. The active immunogenic or therapeutic ingredient is typically mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents or adjuvants. In a preferred embodiment, the edible plant, juice, grain, leaf, tuber, stem, seed, root or other plant part of the transgenic plant from which the drug is produced is ingested by a human or animal, thereby providing a very inexpensive means of treating or immunizing against disease.

In a specific embodiment, a plant material comprising chloroplasts capable of expressing a therapeutic fusion protein (e.g., lettuce, tomatoes, carrots, low-nicotine tobacco material, and the like) is homogenized and encapsulated. In a particular embodiment, an extract of lettuce material is encapsulated. In an alternative embodiment, the lettuce material is powdered prior to encapsulation.

In an alternative embodiment, the composition may be provided in the form of the sap of the transgenic plant, for ease of application. For said purpose, the plant to be transformed is preferably selected from edible plants consisting of tomato, carrot and apple, etc., which are usually consumed in the form of juice.

According to another embodiment, the invention relates to a transformed chloroplast genome that has been transformed with a vector comprising a heterologous gene expressing a therapeutic fusion protein or peptide as disclosed herein.

Reference herein to a protein sequence relates to the known full-length amino acid sequence, as well as at least 12, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, or 265 consecutive amino acids selected from these amino acid sequences, or biologically active variants thereof. Typically, the polypeptide sequence relates to a known human version of the sequence.

Variants with biological activity are those which activate T cells and/or induce Th2 cell responses in the case of oral tolerance, which are characterized by upregulation of immunosuppressive cytokines (e.g. IL10 and IL4) and serum antibodies (e.g. IgG1), or, in the case of the desired natural function of the protein, are variants which retain the natural function of the protein. Preferably, the amino acid sequence of a naturally or non-naturally occurring polypeptide variant has at least about 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, or 98% identity to the full-length amino acid sequence or fragment thereof. Percent identity between the putative polypeptide variant and the full-length amino acid sequence was determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic code).

The change in percent identity may be due to, for example, an amino acid substitution, insertion, or deletion. An amino acid substitution is defined as an amino acid substitution to one. Substituted amino acids are conserved in nature when they have similar structural and/or chemical properties. Examples of conservative substitutions are the replacement of leucine with isoleucine or valine, aspartic acid with glutamic acid, or threonine with serine.

Amino acid insertions or deletions are changes in the amino acid sequence or within it. They generally fall within the range of about 1 to 5 amino acids. Computer programs well known in the art, such as DNASTAR software, can be used to find guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing the biological or immunological activity of the polypeptide. For example, as described in the specific examples below, it can be readily determined whether amino acid changes result in a biologically active therapeutic fusion polypeptide by assaying for natural activity.

Reference herein to a genetic sequence refers to a single or double stranded nucleic acid sequence and comprises the coding sequence of a polypeptide of interest or the complement of the coding sequence. Degenerate nucleic acid sequences encoding polypeptides, as well as homologous nucleotide sequences having at least about 50, 55, 60, 65, 60, preferably about 75, 90, 96, or 98% identity to a cDNA, polynucleotides may be used in accordance with the teachings herein. Percent sequence identity between two polynucleotide sequences is determined using a computer program, such as ALIGN using the FASTA algorithm, using an affine gap search (affine gap search) with a gap open penalty of-12 and a gap extension penalty of-2. Complementary dna (cdna) molecules, species homologs, and variants of nucleic acid sequences encoding biologically active polypeptides are also useful polynucleotides.

Variants and homologues of the above-mentioned nucleic acid sequences are also useful nucleic acid sequences. Generally, homologous polynucleotide sequences can be identified by hybridizing candidate polynucleotides to known polynucleotides under stringent conditions, as is known in the art. For example, the following washing conditions were used: 2 XSSC (0.3M NaCl, 0.03M sodium citrate, pH 7.0), 0.1% SDS at room temperature twice for 30 minutes each; then 2 XSSC, 0.1% SDS, once at 50 ℃ for 30 minutes; homologous sequences containing up to about 25-30% base pair mismatches can then be identified in 2 XSSC, twice at room temperature for 10 minutes each. More preferably, homologous nucleic acid strands contain 15-25% base pair mismatches, and even more preferably, contain 5-15% base pair mismatches.

Species homologues of the polynucleotides mentioned herein may also be identified by preparing suitable probes or primers and screening cDNA expression libraries. It is well known that for every 1% decrease in homology, the Tm of double-stranded DNA decreases by 1-1.5 deg.C (Bonner et al, J.mol.biol.81,123 (1973)). Nucleotide sequences that hybridize to the target polynucleotide or its complement under stringent hybridization and/or wash conditions are also useful polynucleotides. Stringent washing conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: laborotorymoleculer CLONING, Alborory MANUAL, second edition, 1989, pages 9.50-9.51.

In general, for stringent hybridization conditions, a combination of temperature and salt concentration should be selected which is greater than the calculated T for the hybrid under study_mAbout 12-20 deg.c lower. T of the hybrid between the target polynucleotide or its complement and the polynucleotide sequence can be calculated_mSaid polynucleotide sequence having at least about 50, preferably about 75, 90, 96 or 98% identity to one of those nucleotide sequences, said calculation being performed, for example, using the equations of Bolton and McCarthy, proc.natl.acad.sci.u.s.a.48,1390 (1962):

tm 81.5 ℃ -16.6(logl0[ Na + ]) +0.41 (% G + C) -0.63 (% formamide) -600/l),

where l is the length of the hybrid in base pairs. Stringent washing conditions include, for example, 4 XSSC at 65 ℃ or 50% formamide, 4 XSSC at 42 ℃ or 0.5 XSSC, 0.1% SDS at 65 ℃. Highly stringent wash conditions include, for example, 0.2 XSSC at 65 ℃. The following materials and methods are provided to facilitate the practice of the present invention.

Creation of a Transplasty System expressing GFP fusion proteins of different markers

Transplastomic plants expressing CTB-GFP and PTD-GFP were created as described in the previous study [6,34 ]. DC-specific peptides identified from the Ph.D.12-mer phage display library screen [26] were bound to the C-terminus of GFP and cloned into chloroplast transformation vectors, and a transformant system expressing DCpep-GFP was created and confirmed using Southern blot analysis (homoplasmic line) as previously described [35 ]. In addition, the expression of GFP-tagged proteins was confirmed by visualizing the green fluorescence of the leaves of each construct under UV irradiation.

Freeze-drying

The harvested mature leaves were stored at-80 ℃ and freeze-dried using a freeze-dryer (Genesis 35XL, VirTis SPScientific), the frozen and ground small leaves were thus subjected to sublimation under vacuum (400mTorr) with the chamber temperature gradually increasing from-40 ℃ to 25 ℃ for 3 days. The freeze-dried leaves were then ground 3 times (10 seconds each) at maximum speed in a coffee grinder (hamilton beach). Powdered plant cells were stored under airtight and anhydrous conditions with silica gel at room temperature.

Quantification of GFP fusion proteins and GM1 binding assay

Densitometry to quantify GFP fusion proteins as well as GM1ELISA assays were performed according to the previous method [14], except that GFP standard protein (Vector laboratories MB-0752-100) and mouse monoclonal anti-GFP antibody (EMDMILLIPORE MAB3580) were used. Non-denaturing Tris-Glycine gels were performed according to previous studies [36] to identify the pentameric structure of CTB-GFP.

Purification of tag-fused GFP proteins

Protein purification of GFP fusion peptides PTD-GFP and DCpep-GFP was performed by an organic extraction/FPLC based method [37] as described previously. Approximately 200mg of freeze-dried plant cells were homogenized in10 ml of plant extraction buffer (100mM NaCl, 10mM EDTA, 200mM Tris-Cl pH 8.0, 0.2% TritonX, 400mM sucrose, 2% v/v PMSF, 1 tablet of protease inhibitor, total volume 10 ml). After sonication, the homogenate was centrifuged and the supernatant collected. The supernatant was transferred to a 50ml falcon tube and organic extraction was performed as previously described [37 ]. The plant extract was treated with saturated ammonium sulfate to a final concentration of 70% in the extract. 1/4 total extraction volume of 100% ethanol was then added, mixed vigorously for 2 minutes, and then centrifuged. The resulting organic phase (upper phase) was collected into a new 50ml falcon tube. To the remaining aqueous phase was added 1/16 total volume of 100% ethanol, shaken vigorously for 2 minutes, and then centrifuged again. The organic phases from both centrifuges were combined together, a total volume of 1/3 of 5M NaCl and a volume of n-butanol obtained at 1/4 were added, and shaken vigorously for 2 minutes and centrifuged. The resulting organic extract layer (lower phase) at the bottom of the tube was collected and then desalted by passing it through a 7KDa MWCO desalting column (Thermo scientific zebaspin column 89893). The organic extract was loaded onto a desalting column and centrifuged according to the manufacturer's instructions. The desalted organic extract (about 5mL volume) was then loaded onto an FPLC column (LKB-FPLC purification System, Pharmacia; 48mL column volume). During purification, the sample was washed with 3.5 column volumes of buffer A (10mM Tris HCl, 10mM EDTA and 291gm ammonium sulfate, pH7.8) containing 20% saturated ammonium sulfate. The column was then stepwise increased in buffer B (10mM Tris HCl, 10mM EDTA sulfate, pH7.8) to elute the GFP fusion. The protein was detected by measuring the absorbance at 280nm, which corresponds to a single peak plotted on the recorder. Fractions corresponding to the peaks were collected in a single tube with a total volume of 9 ml. The purified fraction was then dialyzed three times against 2L of 0.01 XPBS and then lyophilized (Labconco lyophilizer). The lyophilized purified GFP fusion protein was then quantified by western blot/densitometry.

For the purification of CTB-GFP, the freeze-dried leaf material (400mg) was resuspended in 20ml of extraction buffer (50mM Na-P, pH 7.8; 300mM NaCl; 0.1% Tween-20; 1tb EDTA-free protease inhibitor cocktail). The suspension was sonicated and then centrifuged at 10,000rpm for 10 minutes at 4 ℃. The supernatant was mixed with 1ml of His60Ni resin (Clonetech, 635657) and purified according to the manufacturer's instructions.

Purity measurement and Coomassie staining

Total protein purified from each GFP-tagged fraction was quantified using the Bradford assay and then densitometry was performed as described in the quantification section to quantify the amount of GFP fusion protein in the fractions. Purity was then assessed by calculating the percentage of the amount of GFP fusion protein relative to the total amount of protein obtained from the Bradford assay. Non-denaturing SDS-PAGE was also performed to check the fluorescence of GFP fusion proteins by running a 10% SDS gel under non-denaturing conditions.

Assessment of GFP expression

GFP expression in serum and tissues was quantified by our internal GFP ELISA. Blood and tissue samples were collected 2 and 5 hours after the last oral gavage and serum was stored at-80 ℃ as we previously described [6,16 ]. Tissues were homogenized in RTPA buffer and supernatants were collected for GFP ELISA assay. We established an internal ELISA protocol and calibrated standards based on the GFP ELISA kit (AKR 121; Cell Biolab). Briefly, 96-well Maxisorp plates (Nunc) were coated with goat polyclonal GFP antibody (2.5mg/ml, Rockland) coating buffer (pH 9.6) at 4 ℃. Plates were blocked in PBS containing 3% BSA for 2 hours at 37 ℃. Serial dilutions of 2-fold serum in PBS containing 1% BSA were added in duplicate and incubated overnight at 4 ℃. Plates were then incubated with biotin-conjugated rabbit polyclonal anti-GFP 1:5000(Rockland) overnight at 4 ℃ followed by addition of streptavidin-peroxidase (Rockland) diluted 1: 2000. After further incubation at 37 ℃ for 1 hour, the plates were washed and substrate solution was added and incubated for 10 minutes at room temperature. The reaction was stopped by adding 100 μ L of 2N sulfuric acid per well and absorbance was measured at 450nm using an ELISA reader. Results are shown as mean ± SEM.

Mouse and oral delivery experiments

8-week-old female C57BL/6 mice (Jackson Laboratories, Bar Harbor, MI) were randomly divided into four groups (n ═ 6 per group) and each lyophilized bio-encapsulated CTB-GFP, PTD-GFP and DCpep-GFP plant cells were orally gavaged for 3 consecutive days at 20 mg/mouse/day. All lyophilized material was suspended in 200 μ L PBS. On day 3, blood samples were collected 2 and 5 hours after the last oral gavage. At the 5 hour time point, all mice were sacrificed, organs (liver, kidney, lung, brain, tibialis anterior) harvested and stored at-80 ℃. Control groups (n-6) were fed with untransformed freeze-dried plant cells. Mice were housed in animal facilities at the university of florida and university of pennsylvania under controlled humidity and temperature conditions, and experiments were conducted according to the guidelines of the animal care and use committee.

Immunofluorescence staining

As we previously described [8,16], C57BL/6 mice were orally fed with GFP expressing plant cells twice at 2-hour intervals. Two hours after the last feeding, the mice were sacrificed and the liver and intestine were removed. The intestine was cut longitudinally and washed with PBS, then rolled up and fixed in 4% paraformaldehyde overnight at 4 ℃. Liver tissue was similarly fixed. Subsequently, the fixed tissue was further incubated in 30% sucrose in PBS at 4 ℃ and embedded in OCT. The sections were cut into a thickness of 10 μm.

To analyze GFP expression, sections were permeabilized with 0.1% Triton X-100 and blocked with 5% donkey serum in PBS for 30 minutes, followed by incubation with 1:1000 rabbit anti-GFP antibody (ab290, Abcam,) overnight at 4 ℃. The sections were then incubated with Alexa Fluor 488 donkey anti-rabbit IgG (Jackson ImmunoResearch) for 30 minutes or rhodamine-labeled vitellin (Ulex europaeus agglutinin) (UEA-1; Vector Labs; 10. mu.g/mL) for 10 minutes, then washed and either with or without DAPI (4, 6-diamidino-2-phenylindole) mounting. Images were captured using a Nikon Eclipse 80i fluorescence microscope and a reiga 2000R digital camera (QImaging) and analyzed with Nikon Elements software.

Uptake of purified tag-fused GFP protein by human cell lines

To determine the uptake of the three tag CTB, PTD and DC peptides in immunoregulatory cells, mature dendritic cells, human T cells (Jurkat cells), human B cells (BCBL1), macrophages (m0), mast cells and non-immunoregulatory cells, human kidney cells (293T), human pancreatic epithelial-like cancer cells (PANC-1) and human pancreatic ductal epithelial cells (UPDE) were cultured and used for in vitro transformation of purified tag fusion proteins. Subjecting the cells to cell culture(2×10⁴) Incubate in 100 μ L PBS supplemented with 1% FBS, along with purified CTB (8.8 μ g), PTD (13.5 μ g), and DCpep (1.3 μ g) fusion proteins, for 1 hour at 37 ℃. After PBS washing, the cell pellet was stained with DAPI diluted 1:3000 and fixed with 2% paraformaldehyde for 10 min at room temperature. Cells were then sealed on slides with cytoseal and examined by confocal microscopy. For in vivo image studies, dendritic cells were loaded on glass-bottom microwell dishes (MatTek) after incubation with purified GFP fusion protein and observed under a confocal microscope followed by nuclear staining with DAPI. For 293T, PANC-1 and macrophages, cells were cultured overnight at 37 ℃ in 8-well chamber slides (Nunc) and then incubated with purified CTB-GFP, PTD-GFP and DCpep-GFP (same as above) for 1 hour at 37 ℃. After washing the wells with PBS, the cells were stained with 1:3000DAPI and fixed with 2% paraformaldehyde for 10 min at room temperature. For the negative control group, cells were incubated with commercial GFP (2 μ g) in PBS containing 1% FBS or untreated. After 1 hour incubation, cells were washed once with PBS. Live, unfixed cells were imaged using confocal microscopy.

To determine the efficiency of uptake of purified GFP fusion proteins in different human cell lines, the number of cells showing GFP signal was counted and expressed as a percentage of the total number of cells observed. Under a confocal microscope, a total of 15-20 images of three independent samples of each cell line were recorded at 100x magnification.

The following examples are provided to illustrate certain embodiments of the present invention. They are not intended to limit the invention in any way.

Example I

Uptake of GFP by human cells fused with different tags

In this study, we investigated oral administration of plant cells expressing GFP fused with different tags in chloroplasts and evaluated cell targeting and its biodistribution. PTD, DCpep and CTB fusions are delivered across the intestinal epithelium using different routes, resulting in systemic delivery, biodistribution, and perhaps most importantly, different uptake patterns by non-immune or immunoregulatory cells. These peptides can be used to deliver therapeutic proteins to serum, immunoregulatory cells, or specific tissues.

The interaction of three different transmucosal vectors (transmucosal carriers) CTB pentamers with the GM1 receptor has been well studied as shown in fig. 1A. the pentasaccharide structure of GM1 receptor interacts with the amino acids of CTB through hydrogen bonds [38 ]. in contrast, the mechanism of the structure shown by PTD (fig. 1A) or DCpep (fig. 1A) has not been well studied the secondary structure of cell-penetrating peptides (CPP) has been studied through Circular Dichroism (CD) the exact role of such peptides in interacting with negatively charged phospholipid vesicles, leading to the induction of secondary structure, Small Unilamellar Vesicles (SUV) have been used in these studies, however, the exact role of any secondary structure of CPP during translocation has been difficult to determine, changing its structure from α helix folding to β according to experimental conditions, even if it is in a simple-lamellar vesicle, SUV, the model of which is a model of a high-resolution, the calculated as a high-resolution predictive model of the results of the structural similarity of the Protein score in the model, I-map, the model is 0.14. the approximate model of the topological structure of the map is shown to be a high-score as calculated as a high-linear model, the approximate model of the approximate of the map of the model of the map of the Protein model, the map of the map, the map of the model of the map of the model of the map of the.

All three tags were fused to green fluorescent protein (smGFP) to assess their potency and specificity. CTB (MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLEAKVEKLCVWNNKTPHAIAAISMAN; SEQ ID NO: 1) is fused N-terminally to GFP via the furin cleavage site Pro-Arg-Ala-Arg-Arg (SEQ ID NO: 2) [6 ]. Sixteen amino acids (RHIKIWFQNRRMKWKK; SEQ ID NO: 3) [41] derived from pancreatic and duodenal homeobox factor-1 (PDX-1) were fused N-terminally to GFP and were referred to in this study as PTD-GFP. For nuclear targeting, an additional localization signal is required. 6 amino acids (RH, RR and KK) of a 16 amino acid PTD are critical for the nuclear localization of PDX-1 [42 ]. Human dendritic cell-specific peptide ligands (FYPSYHSTPQRP (SEQ ID NO: 4)) [26] identified by screening 12-mer phage display libraries were fused to the C-terminus of GFP. Both PTD and DC-peptides were engineered (without the use of furin cleavage sites) to study entry and tissue distribution. All three of these fusion constructs were cloned into a chloroplast transformation vector (pLD) for chloroplast transformation, as described in the materials methods section.

To create plants expressing GFP fusion proteins, tobacco chloroplasts were transformed using a biolistic particle delivery system. As shown in FIG. 1B, each tag fused GFP was driven by the same regulatory sequences, the psbA promoter and 5'UTR, which were regulated by light, and the transcribed mRNA was stabilized by the 3' psbA UTR. The PsbA gene is the most highly expressed chloroplast gene, and thus the psbA regulatory sequences were used in our laboratory for transgene expression [7,35 ]. To facilitate integration of the expression cassette into the chloroplast genome, two flanking sequences, an isoleucyl-tRNA synthetase (trnI) and an alanyl-tRNA synthetase (tRNA) gene, were inserted flanking the expression cassette, which flanking sequences were identical to the native chloroplast genome sequence. For shoots that newly appeared in the selection medium, the specific integration of the transgene cassette in the trnI and trnA spacers was investigated by Southern blot analysis with Dig-labeled probes containing trnI and trnA flanking sequences, followed by investigation of the transformation of all chloroplast genomes in individual plant cells (without untransformed wild type chloroplast genomes) (FIG. 1C). As shown in FIG. 1C, for CTB-GFP, PTD-GFP and DCpep-GFP, the HindIII digested gDNA from three lines of each GFP plant showed transformed large DNA fragments of 7.06, 6.79 and 6.78kbp in size, respectively, and no untransformed smaller fragments (4.37kbp) when hybridized with the probe. Thus, stable integration of three different GFP expression cassettes and the homogeneity of the chloroplast genome to the transgene were confirmed. Furthermore, GFP expression was phenotypically monitored by observing green fluorescence under UV light (fig. 1D).

To amplify the biomass of each GFP-tagged plant leaf material, each homogenous system was grown in an automated greenhouse with controlled temperature and humidity. Fully grown mature leaves were harvested at night to maximize the accumulation of GFP fusion protein driven by light-regulated control sequences. To further increase the content of fusion protein on a dry weight basis, the frozen leaves were freeze-dried under vacuum at-40 ℃. In addition to the concentration effects of proteins, lyophilization can increase the shelf life of therapeutic proteins expressed in plants over a year at room temperature [13 ]. Thus, in this example, lyophilized powder plant cells expressing GFP fusion tag protein were used for oral delivery to mice. Immunoblot analysis of the GFP fusion tag protein showed the same size protein in fresh and 4-month-old lyophilized leaves (fig. 1E), confirming the stability of the fusion protein during lyophilization and long-term storage at room temperature. In the immunoblot images, dimers of PTD-GFP (58.4kDa) and DCpep-GFP (56.6kDa) were detected in addition to the 39.5kDa, 29.2kDa and 28.3kDa monomers for CTB-GFP, PTD-GFP and DCpep-GFP, respectively (FIG. 1E). Homodimerization is one of the physiochemical characteristics of GFP, occurring in solution and in crystals. The contact between monomers is very tight due to a broad interaction consisting of a hydrophobic side chain core from each monomer and many hydrophilic contacts [43 ]. Furthermore, CTB monomers can self-assemble into pentameric structures that are very stable and resistant to heat and denaturants due to intersubunit interactions within the pentameric structure (which are mediated by hydrogen bonds, salt bridges, and hydrophobic interactions) [44 ].

The concentrations of GFP protein in the powdered freeze-dried leaf material were 5.6. mu.g/mg, 24.1. mu.g/mg and 2.16. mu.g/mg for CTB-GFP, PTD-GFP and DCpep-GFP, respectively (FIG. 1C). For CTB-GFP, PTD-GFP and DCpep-GFP, the lyophilized GFP protein concentrations increased 17.1-fold, 12.7-fold and 18.8-fold, respectively (FIG. 1F). Removal of water from fresh leaves by freeze-drying reduced the weight by 90-95%. The effect then appeared to be a 10-20 fold increase in protein per gram of dry leaf [13,15,45 ].

GFP uptake in different tissues following oral delivery of plant cells

For oral delivery, lyophilized plant cells (20mg) were rehydrated in consistent volumes (200 μ L) and similar durations. Since the mature plant cells are of uniform size, there is no change in the size of the dispersed plant cells. As shown in fig. 2, the biodistribution of GFP did not show any significant change. Following oral delivery of lyophilized plant cells expressing GFP (fused to PTD or CTB or DC peptides), animals fed PTD-GFP had systemic GFP levels higher than any other test tag (fig. 2A). The biodistribution of the liver and lungs is significantly higher than that of other tissues (skeletal muscle, kidney). For PTD fusion proteins, GFP levels were consistently highest in these tissues (fig. 2B). Immunohistochemical studies using GFP-specific antibodies provided further insight into the route of delivery. As shown in fig. 3A (3C inset), a sensitive method of detecting GFP using Alexa Flour 488-labeled secondary antibody showed that the PTD tag directed intestinal epithelial cells to take up some GFP. When no primary antibody was used, or the tissues were from mice fed untransformed tobacco cells, no GFP was detected (FIG. 3B). To more easily find the region of the intestine where the plant cells are located and where antigen uptake can be observed, the small intestine was rolled up before fixation so that the proximal and distal portions were visible on the same slide (fig. 3C). The presence of plant cells expressing GFP between the villi of the ileum (fig. 3C and E) provides the first direct evidence of protection of plant cells from the digestive system. When CTB tags were used (fig. 3D), a broader delivery of GFP to endothelial cells was also observed, due to the efficient targeting of the CTB pentamer to GM1 receptor. In addition to delivery to epithelial cellsIn addition, we found evidence of GFP uptake by M cells by all fusion tags (solid arrows in fig. 3C-F). Again, these observations provide direct evidence of protein uptake in the upper intestine following lysis in the intestine. FIG. 3E particularly illustrates the presence of GFP in CTB-GFP transplastomic plants in the vicinity of the site of delivery of released GFP to ileal epithelial cells ("EC") and M cells (solid arrows)⁺Plant cells ("PCs"). For DCpep-GFP, no GFP was observed⁺An epithelial cell. However, we found an example of co-localization of GFP and M cells (fig. 3F), suggesting that systemic delivery of DCpep-GFP is possible due to transport from the intestinal lumen via M cells. As expected, GFP delivered with all three tags showed aggregation in the liver (fig. 3H, J and L) as blood could carry antigen from the gut to the liver through the portal vein ("gut-liver-axis").

Purification of GFP fused to different tags

To study GFP uptake by different cell types, the GFP fusion proteins were purified using toyopearl butyl columns for PTD-GFP and DCpep-GFP, and Ni for CTB-GFP²⁺Column purification of GFP fusion proteins. To check for purity, densitometry was performed using western blots and GFP standards (fig. 4A). The purity of each tag-fused GFP was-95% for PTD-GFP,. -52% for DCpep-GFP and-13% for CTB-GFP. The change in purity level was attributed to the difference in expression level of each tag, which was reflected on the GFP fusion protein recovered after purification. Since proteins are purified based on hydrophobic interactions, other hydrophobins may be present in the purified fractions. With Ni²⁺The affinity column purified CTB-GFP. When the pentameric structure of CTB is formed, a histidine cluster is generated, and then the imidazole ring in the histidine cluster is reacted with Ni²⁺Interaction [46 ]]. The low purity of CTB-GFP was attributed to less stringent washing steps, but increased stringency with higher loss of fusion protein.

The purified GFP fusion proteins showed different bands at the expected sizes for each fusion protein in SDS-PAGE and Coomassie stained gels, 29.2kDa, 28.3kDa and 39.5kDa for PTD-GFP, DCpep-GFP and CTB-GFP, respectively (FIG. 4B). In the case of PTD-GFP, there were two bands around the expected size, 29.2kDa and 28.3 kDa. The C-terminal tail of GFP (His-Gly-Met-Asp-Glu-Tyr-Lys) is reported to be very sensitive to proteolytic cleavage by carboxypeptidases and unspecific proteases, including proteinase K and pronase, with the various isoforms resulting from partial proteolytic cleavage, produced by ion exchange chromatography, isoelectric focusing and native gel electrophoresis [47 ]. To determine GFP fluorescence, non-denaturing SDS-PAGE was performed, with the strongest fluorescence intensity of PTD-GFP (lane 1) (FIG. 4C). In DCpep-GFP (lane 2), 3 different proteins were observed, with the top band most likely representing dimerized GFP [47], and the same band was observed in PTD-GFP, however at higher intensity. The CTB-GFP fusion protein showed a large set of fluorescent bands starting at 190kDa (lane 3), 190kDa corresponding to pentamerization of CTB-fused GFP, and other higher bands likely representing multimers. On the same lane, a slightly lower smaller fragment than the GFP standard was observed, which may be a differently folded product, similar to that observed for PTD-GFP and DCpep-GFP.

To assess the correct formation of pentameric structures of purified CTB-GFP, GM1 binding assays were performed with anti-CTB and anti-GFP antibodies. It is well known that the pentameric structure of CTB has strong binding affinity to the ganglioside GM1 receptor, which is widely present on the surface of mammalian cells [48 ]. As shown in fig. 4D, only CTB and CTB-GFP showed binding affinity, indicating that a complex was formed between CTB and GM 1. Furthermore, the interaction of GM1-CTB-GFP was confirmed again using an anti-GFP antibody. Only CTB-GFP could be detected (FIG. 4E). To further confirm the pentameric structure of CTB-GFP, purified CTB-GFP was run on modified Tris-trihydroxymethylglycine gel under non-denaturing conditions [36] and probed using anti-CTB antibodies. The expected CTB-GFP pentamer form was detected at-200 kDa and the monomeric form was detected at 39.5kDa (FIG. 4F). Due to SDS (which is added to the gel and running buffer), monomers may separate from oligomeric structures during running. Thus, the CTB-GFP fusion protein forms a pentameric structure and retains the ability to bind to GM1 receptor, whereas the PTD-GFP or DCpep-GFP fusion protein has no binding affinity for GM 1.

Uptake of GFP fused with different tags by human immune and non-immune cells

The purified GFP fusion protein was incubated with human cultured cells. Blood monocyte-derived mature DCs, T cells (Jurkat cells), B cells (BCBL1), differentiated macrophages and mast cells were cultured for in vitro studies. Human kidney cells (HEK293T) and human pancreatic epithelial-like cancer cells (PANC-1) were tested in parallel as examples of non-immune cells. Mixing cells (2X 10)⁴) GFP fused to purified CTB, PTD and DC target peptides was incubated at 37 ℃ for 1 hour. After incubation with DCpep-GFP, intracellular GFP signal was detected only in DCs, but not in any other cell type, confirming its specificity (fig. 5A). PTD-GFP entered either kidney cells or pancreas cells, but failed to enter any immunoregulatory cells (fig. 5A). The PTD sequence is derived from PDX1, PDX1 induces insulin expression in pancreatic cells, and exogenous PDX1 can penetrate mouse insulinoma cell line and activate insulin gene [41]. As expected, a strong GFP signal was observed in PANC-1 cells when incubated with purified PTD-GFP. In addition, PTD-GFP was observed in the nuclei of pancreatic ductal epithelial cells (FIG. 5B). The cell penetrating ability of PTDs was also evident in human renal cell lines (fig. 5A). In sharp contrast, GFP signal was detected in all cell types after incubation with CTB-GFP, consistent with the ubiquitous presence of GM1 receptor (fig. 5A). Bone marrow-derived murine mast cells, a cell type that plays an important role in wound healing, pathogen defense, and allergic responses, did not show internalization of GFP delivered by DCpep or PTD, whereas CTB-fused GFP was efficiently taken up (fig. 5A). Although only one representative image is shown here, uptake studies were performed in triplicate and 15-20 images were recorded for each cell line under confocal microscopy. CTB-GFP was observed in 70-92% of all cell types examined, and these differences were due to differences in cell density, resulting in lower availability of GFP for its uptake. In the case of PTD-GFP, exceptNo uptake (0%) was observed in any other cell type, except kidney and pancreas cells. DCpep-GFP (0%) was not observed in any cell type other than dendritic cells (Table 1). This study provides specificity for delivery of protein drugs to the serum, immune system, or specific organs or tissues, thus facilitating further development of this new concept.

Table 1: efficiency of uptake of purified GFP fusion proteins in human cell lines. The relative delivery efficiency of GFP to human cell lines by three different tags was compared by counting the number of cells showing GFP signal at 100x magnification under confocal microscopy. A total of 15-20 images were observed for each cell line. All cell lines were tested in triplicate.

4. Discussion of the related Art

In our previous publications [6-16], we have shown that plant cell-derived proteins fused to CTBs can be delivered across the intestinal epithelium for a variety of applications, including delivery of functional proteins to the circulatory system and delivery of protein antigens to the immune system (for oral tolerance induction or as booster vaccines). This is probably because CTB pentamer binds to GM1 receptor on the surface of intestinal epithelial cells and M cells, and then undergoes transcytosis (transcytosis). However, GM1 is widely expressed by many different cell types and is difficult to target to a particular cell type. Therefore, in this study, we sought to develop alternative tags that resulted in higher cell type specificity and/or more efficient transmucosal delivery. We selected DCpep as an example of specific delivery to professional antigen presenting cells, and we were able to demonstrate its specificity. PTDs not only delivered GFP cargo more efficiently to the circulation by penetrating intestinal epithelial cells (fig. 3A and 3C), but also completely avoided delivery to immune cells (fig. 5A). We believe that the data presented here regarding PTDs are a paradigm shift in drug delivery, in stark contrast to previous assumptions that they are non-specific.

This is a very timely contribution to the field in view of recent controversy in the literature [49] regarding the mechanism of PTD cell penetration (direct transmission of macropinocytosis vs) and regarding the effect of PTD on its cargo carrying capacity. Our data show that the body's immune system can be excluded from protein drug delivery by specific selection of PTDs, while delivering efficiently to other cell types and circulation. Thus, this study provides a new approach for increasing the specific targeting of therapeutic agents to selected tissue types using pre-selected PTDs. Combining sequence and structural knowledge, custom designed PTDs can be generated. We used a defined in vitro system to demonstrate differences in protein transduction between different tags as a function of target cell type. This method can also be used to assess immunological consequences when using disease-specific antigens rather than GFP and corresponding animal models.

To reveal the pathway by which GFP fusion proteins are delivered across the epithelial lining of the intestine to the circulation and biodistribution to other organs, immunostaining studies were performed on small intestine tissue sections following oral delivery of leaf material expressing three GFP-tagged proteins. CTB was demonstrated to target GM1 receptor on epithelial and M cells. Our studies found that PTD was able to penetrate non-immune cells, explaining why this tag would also transfer GFP to epithelial cells. In contrast, DC peptides are specific ligands for dendritic cells and therefore should not target epithelial cells. However, our data show that M cell uptake is the mechanism of DCpep fusion protein entry, thus explaining how DCpep-GFP is delivered systemically by the oral route. Dendritic cells are also directly targeted by DCpep through their processes between epithelial cells. However, the amount of intestinal luminal antigen collected by this mechanism is too small to be visualized.

We expect that protein antigens released from CTBs after transcytosis are taken up by DCs in the intestinal immune system (and to a lesser extent by macrophages) as we have published in large numbers, and DCpep-GFP, which is displaced by M cells, is also taken up by DCs. However, for preliminary studies on the interaction with immune cells, we used cultured human cells, using a more sensitive and well-defined in vitro method.

CTB fusions delivered GFP to all tested tissues and cell types, including non-immune cells and immune cells. It is well known that CTB specifically binds to GM1 ganglioside, and various CTB fusion proteins expressed in chloroplasts in our laboratory also show strong binding affinity to GM1 [6,8,9,13-16 ]]. Once CTB binds to GM1 abundant on the membrane lipid rafts of intestinal epithelial cells, CTB retrograde enters the Endoplasmic Reticulum (ER) through the trans-Golgi network to enter cells [50,51]. Indeed, CTB has been widely used as a probe for quantitative studies of GM1 and its cellular and subcellular distribution [52]. Using CTB as a transmucosal vehicle could pass through large mucosal areas with GM1 and human intestine (approximately 1.8-2.7m for body weight)²[53]) To facilitate transport of the conjugated protein into the circulation. Up to 15,000 CTB molecules can bind to one intestinal epithelial cell simultaneously [54]And GM1 receptor rapidly flips over the cell surface [55]. In addition, GM1 ganglioside is also present in the plasma membrane of many other cell types, with particular enrichment in the nervous system and retina [56,57]Thereby directing efficient uptake of CTB fusion proteins in these cells.

Our oral tolerance induction strategies for autoantigens and therapeutic proteins used in the replacement therapy of genetic disorders (e.g., hemophilia and lysosomal storage disorders) rely in part on the efficient targeting of CTB to intestinal epithelial cells, followed by transmucosal delivery and proteolytic cleavage resulting in the release of antigen from the CTB tag and uptake by the DC [7,9,10,11,33 ]. However, DCs and macrophages also sample antigens directly in the intestinal lumen, and M cells can pass intact antigens across epithelial cells to DC-rich regions. Our new data show that CTB fusions are efficiently taken up by DCs, macrophages and other immune cells, providing additional explanation for the effectiveness of CTB fusion antigens in plant-based immunomodulation protocols.

Specific targeting of dendritic cells by DCpep is an ideal option for delivery to the immune system

Here we introduce an alternative and more specific peptide sequence with high immunotherapeutic potential. DCpep specifically targets DCs, but does not target any other immune or non-immune cells. To assess transformation outcomes (translational imaging), we used mainly human immune cells to differentiate the targeting characteristics of the fusion tag. DCpep only delivers intact GFP antigen to DCs, but not to any other APCs or immune or non-immune cells. Consistent with this finding, DCpep-GFP was unable to target intestinal epithelial cells in vivo. Systemic delivery is likely to be caused by M cell uptake. Looking to the future, one can now design immune tolerance and vaccine protocols based on specific delivery to DCs based on activation signals, where DCs have a key function in Treg induction and immune stimulation.

It is well known that the delivery of antigens to lymph nodes is of great importance for immunotherapy. However, GFP signal cannot directly demonstrate this because proteins taken up by DCs in lamina propria (lamina propria) or peyer's patches are fragmented into small peptides and loaded onto MHC molecules as the DCs migrate to the lymph nodes to present antigen to T cells. Consistent with the notion that Mesenteric Lymph Nodes (MLNs) are critical for their response to ingested antigens, we recently demonstrated the increase of different DC subsets and the induction of tregs in MLNs of mice orally delivered transplastomic plant cells [10 ].

In addition to the immune system, PTD is an ideal candidate for effective systemic delivery via the oral route

Although CTB fusions effectively target the intestinal immune system and are therefore useful for tolerance induction, another strategy to avoid immune complications is to minimize interactions with the immune system. The protein transduction domain of PDX-1 shows unique selectivity in the transfer of GFP to different cell types. PTD-GFP is completely unable to deliver antigen to APCs and lymphocytes, but is able to transfer GFP to non-immune cells (including intestinal epithelial cells in vivo). Because bone marrow and lymphocytes are hematopoietic cells, PDX-1 may not be able to transduce this particular cell lineage. PDX-1 induces insulin expression when protein transduction is carried out by megakaryokinesis, a special form of endocytosis, as opposed to receptor-mediated uptake [59,60 ]. Megalocytosis is also the major mechanism of macromolecular uptake in the kidney, so that the observation of GFP signal in HEK293T after incubation with purified PTD-GFP is likely the result of PTD-induced endocytosis. The lack of GFP signal in immune cells following incubation with PTD-GFP cannot be explained by the enhanced degradation following uptake, but reflects the inability of these cells to transduce proteins, since i) they also lack binding to the cell surface, and ii) PTD of HIV tat also exploits the mechanism of megalocytosis, allowing the complete GFP delivery to human DCs and other APCs through PTD of HIVtat [61-63 ]. The PDX-1 derived PTD clearly shows unique selectivity for cell transduction, possibly related to the surface properties of the target cell membrane. Although both PTDs enter cells through megakaryokinesis, their amino acid sequences are very different, which may affect cell surface binding. Although lymphocyte infection is a critical step in the life cycle of HIV, insulin expression needs to be tightly regulated and responsive to environmental stimuli [64], which may explain in part the selectivity of PTDs derived from PDX-1. At the same time, PTD-GFP is superior in vivo for systemic protein delivery and can be used in therapies requiring certain protein levels in the blood, such as our disclosed examples of therapies for treating hypertension and hormones or cytokines [9,11,14,15 ]. Interestingly, despite significant differences in systemic delivery, the CTB-tag, DCpep-tag and PTD-tag all resulted in very similar GFP antigen levels in the liver as demonstrated by immunohistochemistry and more quantitatively by ELISA. The link between the intestinal tract and the response in the liver has long been known, commonly referred to as the "gut-hepatic axis". After being ingested by the gut, the antigen can be delivered indirectly to the liver via migratory DCs that route mesenteric lymph nodes. Alternatively, blood can carry antigens from the intestine to the liver via the portal vein. The latter explanation seems to be more applicable to our delivery system given the wide distribution of quantifiable levels of GFP in the liver. These data indicate that the liver ingests orally delivered antigen to saturation levels, which is less dependent on the label.

Unique advantages of protein delivery for bio-encapsulation in plant cells

Lyophilization of plant cells has several advantages. Freeze-dried powdered leaves can be stored at room temperature for years, eliminating the costly refrigeration and transportation required for protein pharmaceuticals for injection [13,65 ]. Furthermore, the concentration effect of the therapeutic protein increases, causing the size of the capsule containing the lyophilized plant cells to decrease by a factor of 10-20. The pharmaceutical industry widely uses freeze-drying techniques to preserve protein drugs, including preserving blood coagulation factors. Therefore, the freeze-drying process does not denature the protein. In fact, we again show that freeze-drying retains the correct folds and disulfide bonds (11,13-16, 66). Following oral delivery, the lyophilized plant cells reach the intestinal tract, and the bio-encapsulated proteins are released by intestinal microbial digestion of the plant cell walls. At that time, the released proteins as well as the plant cell walls can be degraded by intestinal microbes. However, it is possible that intestinal microorganisms are enriched by anaerobic bacteria, which release more enzymes to degrade plant cell walls rather than proteins. Indeed, bacteria that inhabit the human gut have developed the ability to utilize complex carbohydrates in plant cell walls and can utilize almost all plant glycans [4,5 ]. Our previously published work identified enzymes required to break down plant cell walls [67,68 ]. The delivery of several functional proteins suggests that they are protected in the gut lumen or that they release sufficient amounts of protein drug to survive in the gut luminal proteases.

DCpep-GFP content was found to be the lowest of the three fusion proteins, 2.16. mu.g/mg, 10-fold lower than PTD-GFP. In general, due to the high copy number of the chloroplast genome, the chloroplast expression level of the foreign protein can reach very high levels, up to 70% of the total leaf protein [7 ]. However, the expression level varies depending on the protein, the N-terminal fusion protein, proteolytic cleavage and stability. In this study, all chimeric genes were driven by the psbA promoter and the psbA 5'UTR and were stabilized by the psbA3' UTR. Since the GFP sequence was also identical in all three constructs, the contributing factor to the difference in expression levels was probably due to the N-terminal sequence of the fusion construct. In contrast to the PTD and CTB tags, DCpep was fused to the C-terminus of GFP. Thus, one possible explanation for the lower level of GFP expression of DCpep fusions is insufficient protection of the N-terminus.

Conclusion

As mentioned above, the bioavailability of oral delivery of protein drugs expressed in transgenic plant cells is now becoming a new concept for inducing tolerance against autoimmune disorders [7], or eliminating toxicity of injected protein drugs [8-10], or delivering functional blood proteins to treat diabetes [12,13], hypertension [14], prevent retinopathy [15], or remove plaques in the brain of alzheimer's disease [16 ]. These new approaches can improve patient compliance in addition to significantly reducing healthcare costs, as seen in diabetes studies where oral delivery using insulin or sialoprotein-4 (exendin-4) is as effective as injection delivery at reducing blood glucose levels [12,13 ].

This study enabled the use of different fusion tags for delivery to immunoregulatory cells or non-immune cells or directly to serum without interfering with the immune system. This opens the potential for low cost oral delivery of proteins to enhance or suppress immunity, or delivery of functional proteins to modulate metabolic pathways.

The cost of protein drugs now exceeds GDP in over 75% of countries, making them unaffordable. This is because they are produced, purified, refrigerated/transported in fermenters, are too expensive, have a short shelf life and are delivered aseptically. Using Green Fluorescent Protein (GFP) as a model, we demonstrated in this study that plant cells protect GFP from the digestive system and release it into the intestinal lumen where they are taken up by epithelial cells. Based on delivery tags fused to GFP, they are able to reach the circulatory system, immune or non-immune cells, specific organs or tissues. Such low cost oral delivery of protein drugs should increase patient compliance and significantly reduce costs by eliminating the overly expensive methods/injections currently used.

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Example II

Uptake of GFP fused with different tags was assessed in additional human cell lines as follows: CTB-GFP, PTD-GFP, PG1-GFP and RC101-GFP

To expand the cell types that can be successfully targeted using the protein therapeutics described herein, we evaluated the use of human somatic and stem cell lines. To produce targeted delivery of therapeutic proteins to benign and malignant tissues, six cell lines were selected. These cell lines include normal and cancer cell lines from human teeth, head and neck, and soft tissues. To develop functional protein-based therapies for developmental defects and immature/stem cell tumors, three human stem cell lines were selected.

FIGS. 6A and 6B show results obtained with different cell types and tag studies. These human somatic cells include pancreatic cancer cells (PANC-1); human pancreatic ductal epithelial cells (HPDE); human head and neck squamous cell carcinoma cell (SCC-1); retinal pigment epithelial cells (RPE); adult Gingival Keratinocytes (AGK); osteoblasts (OBC). Certain types of human stem cells have also been evaluated. These include human periodontal ligament stem cells (HPDLSCs); maxilla Mesenchymal Stem Cells (MMSCs) and gingiva-derived mesenchymal stromal cells (GMSCs).

CTB, PTD and DC-peptide fusion tags are described in example I. Protegrin (PG) and Retrocyclin (RC) are antimicrobial peptides and are described in detail in Plant Biotechnology Journal 9 of Lee et al: 100, 115 (2011).

Protegrin 1：Arg-Gly-Gly-Arg-Leu-Cys-Tyr-Cys-Arg-Arg-Arg-Phe-Cys-Val-Cys-Val-Gly-Arg(SEQ ID NO：5)

Protegrin 2：Arg-Gly-Gly-Arg-Leu-Cys-Tyr-Cys-Arg-Arg-Arg-Phe-Cys-Val-Cys-Val(SEQ ID NO：6)

Protegrin-3 replaces arginine with glycine at position 4, which also has a less positive charge, Protegrin-4 replaces valine with phenylalanine at position 14, and the sequence in the β -turn differs, making Protegrin-4 less polar than other peptides and less positively charged, Protegrin-5 replaces arginine with proline, which has a less positive charge.

Additional retrocyclin variants are provided in PCT/US2002/012353, which is incorporated herein by reference.

Results

PANC-1 cells: in human pancreatic cancer cells, CTB-GFP showed moderate accumulation on the cell membrane and scattered plaques in the cytoplasm (scattered foci). PTD-GFP is similarly concentrated on the cell membrane, but the signal in the cytoplasm is stronger than CTB-GFP. PG1-GFP showed a very strong accumulation on the cell membrane, but only a diffuse, weak signal in the cytoplasm. RC101-GFP shows the strongest GFP levels in both the cell membrane and the cytoplasm.

HPDE cells: in human pancreatic ductal epithelial cells, CTB-fused GFP showed relatively strong dense signals in the cell membrane and cytoplasm. PTD-GFP gives no clear signal on the cell membrane but has dense moderate signals in the cytoplasm and nucleus. PG1-GFP showed a strong accumulation on the cell membrane, but only scattered plaques in the cytoplasm. RC101-GFP shows only a punctate GFP localization on the cell membrane.

HPDLSC cells: in human periodontal ligament stem cells, CTB-GFP shows a relatively strong signal only on the cell membrane. PTD-GFP showed very weak accumulation on the cell membrane, but had a dispersed medium-intensity GFP signal in the cytoplasm. PG1-GFP showed strong localization in both the cell membrane and the cytoplasm. RC101-GFP cannot localize to the cell membrane and cytoplasm of these cells.

MMSC cells: in the maxilla mesenchymal stem cells, CTB-GFP showed relatively strong dense localization on the cell membrane and scattered spots in the cytoplasm. PTD-GFP did not accumulate on the cell membrane, but showed moderate intensity plaques in the cytoplasm. PG1-GFP showed a very strong GFP signal on the cell membrane and a scattered strong signal in the cytoplasm. RC101-GFP does not appear to localize anywhere in these cells.

SSC-1 cells: in human head and neck squamous cell carcinoma cells, CTB-GFP shows weak GFP signal both on the cell membrane and in the cytoplasm. PTD-GFP shows a relatively strong localization on the cell membrane and a strong signal in the cytoplasm. PG1-GFP showed moderate accumulation on the cell membrane and a diffuse signal in the cytoplasm. RC101-GFP shows weak GFP localization on the cell membrane and small scattered plaques in the cytoplasm.

RPE cells: in retinal pigment epithelial cells, CTB-GFP shows a continuous (rather than punctate) localization on the cell membrane and relatively strong GFP plaques in the cytoplasm. PTD-GFP showed relatively strong accumulation on the cell membrane and strong accumulation in the cytoplasm. PG1-GFP showed the strongest signal of any peptide in these cells, both on the cell membrane and in the cytoplasmic pool (foci).

GMSC cell: in gingiva-derived mesenchymal stromal cells, CTB-GFP localizes to the cell membrane and cytoplasm. PTD-GFP showed relatively strong accumulation of GFP in the cell membrane and strong aggregated plaques in the cytoplasm. PG1-GFP showed a moderate accumulation of GFP on the cell membrane and a strong aggregation signal in the cytoplasm.

AGK cells: in adult gingival keratinocytes, CTB-GFP shows many relatively strong dense signals in the cytoplasm. PTD-GFP also showed a relatively strong signal in the cytoplasm, but had relatively few plaques. PG1-GFP showed much stronger accumulation in the cytoplasm.

OBC cells: in osteoblasts, CTB-GFP shows a strong spot-like localization in the cytoplasm. PTD-GFP also localizes strongly to the cytoplasm. PG1-GFP only showed dense, continuous GFP localization across the cell membrane.

Discussion of the related Art

GFP signal was detected in all cell lines when incubated with CTB-GFP, and the expression pattern in these cells was similar, consistent with the ubiquitous presence of GM1 receptor on these cell membranes. Protein Transduction Domains (PTDs) also effectively facilitate GFP delivery to these cell lines, even transferring GFP into the nucleus of HPDE cells. These results demonstrate that PTDs can directly transfer foreign proteins without membrane receptors.

Protegrin 1(PG1) fused GFP was shown to localize strongly to the cell membrane and cytoplasm of most cell lines. There are three expression patterns:

1. both the cell membrane and the cytoplasm have strong GFP signals (MMSC, RPE), indicating that PG-1 can penetrate the cell membrane.

2. There was a strong GFP signal on the cell membrane, but a weak or absent cytoplasmic signal (PANC-1, HPDE and OBC), indicating that PG-1 was unable to penetrate the membranes of these cells.

3. There is a weak GFP signal on the cell membrane, but a strong signal in the cytoplasm (SCC-1, HPDLSC, GMSC, and AGK). This pattern indicates that PG1 most easily and efficiently penetrates the membranes of these cells and efficiently transports protein drugs into the cytoplasm. These results indicate that the efficiency of PG1-GFP penetration varies from cell type to cell type.

Protein drug delivery targets using fusion tags

The fusion between CTB and the therapeutic protein facilitates efficient oral delivery of the therapeutic protein to induce oral tolerance, delivery to serum or even crossing the blood brain barrier or retinal barrier. Exogenous proteins can be delivered into living cells by fusing them to Protein Transduction Domains (PTDs) that can penetrate the cell membrane independent of the specific receptor. PTDs can deliver biologically active proteins into cultured mammalian cells and animal models in vivo and in vitro, giving PTD fusion proteins a great potential for therapeutic drug delivery.

Although antimicrobial peptides are known to kill microorganisms, human cell-specific targeting remains a novel and unexpected observation. Here we show that antimicrobial peptides can perform this dual function, making them ideal candidates for peptide antibiotics and for the efficient delivery of other protein drugs in a cell-specific manner. Some examples of other protein drugs are provided below. The GFP reporter amino acid sequence can be replaced with a nucleic acid encoding a therapeutic protein listed in table 4, including anti-inflammatory functional drugs, for novel therapies for pancreatitis, periodontal inflammation and periodontitis, pancreatic cancer, and head and neck squamous cell carcinoma.

In the area of periodontal regeneration, several protein-based therapeutics have been demonstrated to have efficacy. The fusion of such proteins with the tags of the present invention, followed by the application of plant cells or plant parts expressing such fusion proteins, should provide efficacy in the treatment of various dental conditions.

TABLE 2 candidate growth factor peptides for periodontal regeneration

See also J.Penodontol.86: S134-S152,2015.

Table 3 provides a list of cells that can be selectively targeted by fusion with different tags of the present invention.

In addition, the methods described herein can be used to deliver drugs useful for the listed medical indications.

Replacement of defective proteins of said disorders

Endocrine disorders (hormone deficiency)

Insulin (analogs with faster action, longer duration)

Growth hormone (growth hormone) (somatotropin)

Insulin-like growth factor

Hemostasis and thrombosis (post-translational changes)

Blood coagulation factors (VII, VIII, IX)

Antithrombin (operation)

Protein C (venous thrombosis)

Tissue plasminogen activator (embolism, apoplexy)

Substitution of defective enzyme

Enzyme deficiency

β glucocerebrosidase

α -glucosidase

Disorders of the lung/gastrointestinal tract

Protease inhibitors (antitrypsin deficiency)

Lactase

Lipase enzyme

Amylase

Protease enzyme

The therapeutic fusion proteins of the present invention may also be used to enhance existing pathways, such as those involved in hematopoiesis and fertility.

Hormones such as parathyroid hormone and exenatide (exenatide) may also be produced as selectively targeted fusion proteins according to the present invention.

Bone morphogenic proteins for the treatment of spinal fusion, gonadotropin releasing hormone (gonatropin releasing hormone) for puberty, keratinocyte growth factor for chemotherapy-related oral mucositis, and platelet-derived growth factor for wound healing can be used.

Other protein-based drugs may also be fused to the fusion peptides of the invention to improve targeted delivery. These include, but are not limited to, botulinum toxin for dystonia and cosmetic use, collagenase for severe burn treatment, DNase for cystic fibrosis, papain for burns and ulcers, asparaginase for leukemia, hirudin for coronary angioplasty and streptokinase for DVT or embolization.

Other effective agents may also be used in the fusion proteins of the present invention and are listed in table 4.

Table 42013 best-selling 20 biopharmaceutical products

^aFinancial data is from LaMerie Business intelligence&J,Johnson&Johnson

For more information on this table and other protein drugs, please see Walsh, g. (2014) Biopharmaceutical benchmark 2014.nat. biotechnol.32, 992-1000.

While certain preferred embodiments of the present invention have been described and specifically exemplified above, the present invention is not intended to be limited to these examples. Various modifications may be made thereto without departing from the scope and spirit of the invention as set forth in the following claims.

Claims (19)

1. A method of targeted delivery of a therapeutic protein to a target tissue or cell of a subject to treat a disease or disorder in the subject, comprising:

a) administering an effective amount of a plant cell or residue thereof comprising a plastid-expressed nucleic acid encoding the therapeutic protein operably linked to a fusion peptide sequence, the expression of the nucleic acid resulting in the production of a therapeutic fusion protein in the plastid; and

b) ameliorating or alleviating a symptom associated with the disease or condition, the amelioration or alleviation being caused by: the therapeutic fusion protein selectively permeates into the target tissue or cell in vivo to substantially exclude non-target cells.

2. The method of claim 1, wherein the plant is selected from the group consisting of lettuce, tobacco, spinach, kale, cabbage, eggplant, carrot and tomato.

3. The method of claim 1, wherein the peptide is a PTD peptide.

4. The method of claim 1, wherein the peptide is a DC peptide.

5. The method of claim 1, wherein the peptide is an antimicrobial peptide.

6. The method of claim 5, wherein the peptide is PG1 peptide or RC101 peptide.

7. The method of claim 1, wherein the peptide is a CTB peptide.

8. The method of claim 1, wherein the cell is an immune cell.

9. The method of claim 1, wherein the cell is a somatic cell.

10. The method of claim 1, wherein the cell is a dendritic cell.

11. The method of any one of claims 1-10, wherein the therapeutic fusion protein is for treating an endocrine disorder and is selected from the group consisting of: insulin, somatotropin and insulin-like growth factors.

12. The method of any one of claims 1-10, wherein the therapeutic fusion protein is for maintaining hemostasis or preventing thrombosis, and the protein is selected from the group consisting of: coagulation factors, antithrombin, protein C and tissue plasminogen activator.

13. The method of any one of claims 1-10, wherein the subject has an enzyme deficiency and the therapeutic fusion protein is selected from the group consisting of β glucocerebrosidase, α -glucosidase, lactase, lipase, amylase, protease, and protease inhibitor.

14. The method of any one of claims 1-10, wherein the therapeutic fusion protein enhances an existing treatment regimen and is selected from the group consisting of erythropoietin, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, follicle stimulating hormone, chorionic gonadotropin, α interferon, interferon B, PDGF, keratinocyte growth factor, and bone morphogenic protein.

15. The method of any one of claims 1-10, wherein the therapeutic protein is provided in table 4.

16. A plant or plant cell comprising the therapeutic fusion protein of any one of the preceding claims.

17. The plant cell of claim 16, which has been freeze-dried, and optionally in powder form.

18. The plant cell of claim 16, which has been encapsulated.

19. The plant cell of claim 16, wherein the therapeutic fusion protein is stable at ambient temperature.