Phenol-rich grapes

Abstract

The present invention relates to transgenic grape cells, compositions comprising such cells or extracts or fractions thereof, and methods for inhibiting cytokine release syndrome or cytokine storm or reducing the incidence of cytokine release syndrome or cytokine storm in subjects. Also disclosed herein are methods for preventing coronavirus infection or symptoms thereof, treating coronavirus infection or symptoms thereof, reducing the incidence of coronavirus infection or symptoms thereof, inhibiting coronavirus infection or symptoms thereof, or inhibiting coronavirus infection or symptoms thereof.

Description

Field of the Invention

[0001] Disclosed herein are transgenic grape cells, compositions comprising such cells or extracts or fractions thereof, and their use in inhibiting cytokine release syndrome or cytokine storm or reducing the incidence of cytokine release syndrome or cytokine storm in a subject. Also disclosed herein are methods for preventing coronavirus infection or symptoms thereof, treating coronavirus infection or symptoms thereof, reducing the incidence of coronavirus infection or symptoms thereof, inhibiting coronavirus infection or symptoms thereof, or inhibiting coronavirus infection or symptoms thereof.

[0002] background

[0003] The phenolic content in grape-based wines includes a large group of hundreds of compounds that affect the taste, color and mouthfeel of the wine. These compounds include phenolic acids, stilbenoids, flavonols, dihydroflavonols, anthocyanidins, flavanol monomers and flavanol polymers, and can be broadly divided into two categories: flavonoids and non-flavonoids. Flavonoids include anthocyanidins and tannins that contribute to the color and mouthfeel of wine, while non-flavonoids include stilbenoids, such as resveratrol and phenolic acids.

[0004] Stilbenes are a small group of phenylpropanoid compounds characterized by a 1,2-diphenylethylene backbone, most of which are derivatives of the monomer unit trans-resveratrol (Chong et al., 2009; Rimando et al., 2012). This unique group of secondary metabolites with phytoalexin characteristics is synthesized in a limited and unrelated number of plant species (Shen et al., 2009). Stilbenes have outstanding pharmacological and nutritional value, and many stilbene-producing plants are part of the human diet, including cranberries, peanuts, cocoa, and especially grapes (Counet et al., 2006; Yin et al., 2016). Grapes are the main source of stilbenes in human nutrition, accumulating more than one stilbene-derived compound, including isomers, polymers, and glycosylated forms.

[0005] Due to their antimicrobial properties, stilbenes accumulate in infected areas of plants after pathogen attack (Ahuja et al., 2012). Among the stilbenes, the resveratrol dehydrodimer piceid exhibits potent antifungal activity and is present in high concentrations in fungus-resistant varieties of grapevine. An example comparing susceptible and resistant grapevine varieties showed that fungal lesions of resistant cultivars (Botrytis cinerea and Plasmopara viticola) contained low concentrations of resveratrol and higher concentrations of its oligomers α-piceid and ε-piceid (Langcake, 1981). A second study on grapevine downy mildew showed that resveratrol production was induced in all varieties, but was glycosylated to the non-toxic stilbene, piceid (5,4'-dihydroxystilbene-3-O-β-pyranoglucopyranoside) in susceptible varieties, while in resistant varieties it was oxidized to toxic piceid (Pezet et al., 2004). Purified ε-viniferin was also shown to be a more potent phytoalexin against Plasmopara viticola and Botrytis cinerea than resveratrol (Chong et al., 2009; Schnee et al., 2013). Due to its strong antifungal properties compared to the well-studied resveratrol, viniferin content is becoming a criterion for selecting resistant grape varieties (Gindro et al., 2006).

[0006] Resveratrol has been shown to have activities that promote human health, including anticancer, antioxidant, anti-inflammatory and neuroprotective characteristics (Baur and Sinclair, 2006; Kalantari and Das, 2010). ε-Grapevine accumulates in grapes and wine to concentrations similar to resveratrol, and exhibits even higher pharmacological activity than resveratrol (Vitrac et al., 2005). One example is that in cancer prevention, grapevinevine has a stronger inhibitory effect on cytochrome P450 (CYP) enzyme activity than resveratrol (Piver et al., 2003). Another example is the inhibition of the onset of Alzheimer's disease by preventing the extracellular accumulation of amyloid β peptides aggregated in senile plaques: ε-Grapevinevine is more stable metabolically than resveratrol and is therefore more effective in preventing amyloid β aggregation and exerting anti-inflammatory and antioxidant activities (Vion et al., 2018). A third example is the protection of cardiovascular function: unlike resveratrol, vinblastine was found to inhibit angiotensin-converting enzyme (ACE) activity, an important therapeutic approach for lowering blood pressure and preventing heart failure, and improved cardiac mass in spontaneously hypertensive rats (Zghonda et al., 2012).

[0007] Resveratrol has more than one activity against harmful inflammatory cytokines and associated microRNAs. The anti-inflammatory properties of resveratrol have been studied in animal models, cell lines, and human subjects and have been shown to be very effective in reducing inflammatory cell production and pro-inflammatory cytokine accumulation. (Rafe et al., 2019).

[0008] Flavonoids are the most abundant polyphenols in the human diet and are considered health-promoting compounds due to their antioxidant and anti-inflammatory activities. High flavonoid intake has been associated with protection against cancer, cardiovascular disease, Alzheimer's disease, and atherosclerosis (Babu et al., 2009, Hollman and Katan, 1999, Kris-Etherton et al., 2004). There are three major flavonoid subgroups in grapes: flavonols, flavan-3-ols (tannins), and anthocyanins (Blancquaert et al., 2019). Many health-related properties of flavonoids have been attributed to their flavonol subclasses (Owens et al., 2008; Harborne et al., 2000). Among the health benefits of flavonols, kaempferol has been found to reduce the risk of chronic diseases including cancer (Chen et al., 2013), quercetin has been associated with increased lifespan in mammals (Haigis et al., 2010), and myricetin has been found to reduce the risk of cancer and diabetes (Feng et al., 2015).

[0009] Flavonol synthase (FLS) catalyzes the synthesis of three major flavonols, namely kaempferol, quercetin and myricetin, from their dihydroflavonols. Overexpression of FLS leads to increased flavonol levels. An example is that overexpression of Brassicanapus FLS in Arabidopsis leads to increased levels of kaempferol and quercetin (Vu et al., 2015). Overexpression of FLS in tobacco also leads to increased kaempferol levels (Jiang et al., 2020). On the other hand, inhibition of FLS in Eustoma plants prevents the biosynthesis of flavonols (Nielsen et al., 2002).

[0010] Unlike whole plants, plant cell suspensions provide a well-defined production system with rapid yield and relatively uniform quality, without geographical, environmental and seasonal restrictions (Davies and Deroles, 2014). These advantages of plant cell culture make grape (Vitis vinifera) cultivar Gamey cell suspension a promising material for the production of resveratrol and its derivatives.

[0011] Increased Phe availability has been shown to affect several metabolic pathways derived from Phe, including stilbenes. Overexpression of the feedback-insensitive bacterial form of DAHSP (3-deoxy-D-arabinoheptulonic acid 7-phosphate synthase), AroG*, led to increased production of aromatic amino acids, especially Phe, in Arabidopsis (Tzin et al., 2012), petunia (Oliva et al., 2015), tomato (Tzin et al., 2015) and cell suspensions of the grape cultivar Gamay Red (Manela et al., 2015). In all cases, AroG* overexpression caused an increase in the production of secondary metabolites, which varied from one plant to another. The only stilbene affected by AroG* overexpression in grape cell suspensions was resveratrol, whose concentration increased 20-fold (Manela et al., 2015).

[0012] The second way to increase stilbene is by overexpressing stilbene synthase (STS), which catalyzes the condensation of coumaroyl-CoA with three units of malonyl-CoA to produce resveratrol. Similarly, most studies overexpressing STS in plant cell cultures reported increased resveratrol production and identified only resveratrol (Aleynova et al., 2016; Chu et al., 2017; Hidalgo, 2017; Kiselev and Aleynova, 2016; Suprun et al., 2019). Only one study showed that viniferin levels were also increased (Suprun et al., 2019).

[0013] Diseases such as COVID-19 and influenza can be deadly due to an overreaction of the body's immune system called a cytokine storm. Cytokine release syndrome (CRS) or cytokine storm syndrome (CSS) is a form of systemic inflammatory response syndrome (SIRS) that can be triggered by a variety of factors, such as viral infection. Severe cases are called "cytokine storms." Cytokines are small proteins released by many different cells in the body, including those of the immune system, where they coordinate the body's response to infection and trigger inflammation. Sometimes the body's response to infection goes into overdrive. For example, when SARS-CoV-2—the virus behind the COVID-19 pandemic—enters the lungs, it triggers an immune response that attracts immune cells to the area to attack the virus, causing local inflammation. But in some patients, excessive or uncontrolled cytokine levels are released, which then activate more immune cells, leading to excessive inflammation. This can seriously injure or even kill the patient. Cytokine storms are common complications not only of COVID-19 and influenza, but also of other respiratory diseases caused by coronaviruses such as SARS and MERS. They have also been linked to non-infectious diseases such as multiple sclerosis and pancreatitis (https: / / www.newscientist.com / term / cytokine-storm / #ixzz6JhDBRH7E).

[0014] Currently, there is a high demand for stilbene due to its valuable pharmacological properties and role as a phytoalexin.

[0015] Overview

[0016] The present invention provides cells, compositions and methods of producing phenol-rich cells and compositions.Such phenol-rich products are useful in several areas of human therapy, as the health benefits of plant-based polyphenols have long been known.

[0017] In accordance with the principles of the present invention, methods are provided for enriching plant cells, particularly genetically modified plant cells, with beneficial polyphenolic compounds that far exceed the levels of polyphenols found in nature.

[0018] In one aspect, the present invention provides a double transgenic grape cell comprising at least one copy of an AroG* gene and at least one copy of a stilbene synthase (STS) gene or a flavonol synthase (FLS) gene.

[0019] In certain embodiments, the grape cells are Vitis vinifera cv. Gamay Red cells.

[0020] In certain embodiments, the AroG* gene encodes a 3-deoxy-D-arabinoheptulonic acid 7-phosphate (DAHP) synthase (DAHPS) enzyme.

[0021] In certain embodiments, the DAHPS enzyme is a feedback-insensitive DAHPS enzyme.

[0022] In certain embodiments, the DAHPS enzyme increases the availability of at least one amino acid in a cell.

[0023] In certain embodiments, the DAHPS enzyme increases the availability of phenylalanine in a cell.

[0024] In certain embodiments, the STS gene encodes an STS enzyme.

[0025] In certain embodiments, the STS enzyme produces stilbene.

[0026] In certain embodiments, the STS gene is a grapestilbene synthase (VvSTS) gene.

[0027] In certain embodiments, the STS gene is selected from the group consisting of VvSTS5, VvSTS10, and VvSTS28.

[0028] In certain embodiments, the FLS gene encodes a FLS enzyme.

[0029] In certain embodiments, the FLS enzyme produces flavonoids.

[0030] In certain embodiments, the FLS gene is a grape flavonol synthase (VvFLS) gene.

[0031] In certain embodiments, the FLS gene is VIT_07s0031g00100.

[0032] In certain embodiments, the AroG* gene or the STS gene is functionally linked to a constitutive promoter.

[0033] In certain embodiments, the constitutive promoter is the Cauliflower Mosaic Virus (CaMV) 35S RNA promoter (35S promoter).

[0034] In certain embodiments, both the AroG* gene and the STS gene are functionally linked to a constitutive promoter.

[0035] In certain embodiments, the AroG* gene and the STS gene are functionally linked to different constitutive promoters.

[0036] In certain embodiments, the AroG* gene or the FLS gene is functionally linked to a constitutive promoter.

[0037] In certain embodiments, both the AroG* gene and the FLS gene are functionally linked to a constitutive promoter.

[0038] In certain embodiments, the AroG* gene and the STS gene are functionally linked to different constitutive promoters.

[0039] In certain embodiments, the cells described above comprise higher levels of: at least one amino acid selected from the group consisting of phenylalanine, tryptophan and p-CA; at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine; at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1 and procyanidin B2; at least one anthocyanidin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin, malvidin, malvidin-3-o-glucoside, malvidin-3-coumaroyl-glucoside, malvidin-3-acetyl-glucoside, petunidin-3-coumaroyl-glucoside; or any combination thereof, as compared to a corresponding non-transgenic grape cell or as compared to a corresponding single transgenic grape cell.

[0040] In certain embodiments, the cells described above comprise trans-resveratrol at a concentration of at least 0.5 mg / g dry weight, cis-resveratrol at a concentration of at least 0.5 mg / g dry weight, resveratrol at a concentration of at least 0.8 mg / g dry weight, ε-glucose at a concentration of at least 0.6 mg / g dry weight, or any combination thereof.

[0041] In certain embodiments, the cells described above comprise similar or lower levels of: at least one flavonoid selected from the group consisting of myricetin, quercetin-3-glucoside, catechin, epicatechin, epigallocatechin, and proanthocyanidins; at least one anthocyanidin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin, and malvidin; or any combination thereof, compared to a corresponding non-transgenic grape cell or compared to a corresponding single transgenic grape cell.

[0042] In certain embodiments, the cells described above comprise resveratrol at a concentration of about 1.26 mg / g dry weight, ε-vinifera at a concentration of about 10.8 mg / g dry weight, or both.

[0043] In another aspect, the present invention also provides a method of maintaining a grape cell, the cell optionally comprising at least one copy of an AroG* gene, optionally comprising at least one copy of a stilbene synthase (STS) gene, and optionally comprising at least one copy of a flavonol synthase (FLS) gene, the method comprising contacting the cell with a composition comprising: phenylalanine at a concentration of about 0.2 mM to about 5 mM, p-coumaric acid at a concentration of about 0.1 mM to about 0.3 mM, or any combination thereof.

[0044] In certain embodiments, the cells comprise at least one copy of the AroG* gene.

[0045] In certain embodiments, the cells comprise at least one copy of an STS gene.

[0046] In certain embodiments, the cells comprise at least one copy of the FLS gene.

[0047] In certain embodiments, the methods described above comprise contacting the cell with a composition comprising phenylalanine at a concentration of about 2 mM to about 5 mM.

[0048] In certain embodiments, the methods described above comprise contacting the cell with a composition comprising phenylalanine at a concentration of about 5 mM.

[0049] In certain embodiments, the methods described above comprise contacting the cell with a composition comprising p-coumaric acid at a concentration of about 0.3 mM.

[0050] In yet another aspect, the present invention also provides a pharmaceutical composition comprising the double transgenic grape cell or an extract or fraction thereof as described above.

[0051] In certain embodiments, the double transgenic grape cells are maintained by the methods described above.

[0052] In yet another aspect, the present invention also provides a pharmaceutical composition comprising a non-transgenic grape cell or a single transgenic grape cell containing at least one copy of the AroG* gene, or an extract or fraction thereof, wherein the cell is maintained by the method described above.

[0053] In certain embodiments, the pharmaceutical composition described above comprises: at least one amino acid selected from the group consisting of phenylalanine, tryptophan and p-CA; at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine; at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1 and procyanidin B2; at least one anthocyanidin selected from the group consisting of cyanidin, paeoniflorin, delphinidin, petunidin, malvidin, malvidin-3-o-glucoside, malvidin-3-coumaroyl-glucoside, malvidin-3-acetyl-glucoside and petunidin-3-coumaroyl-glucoside; or any combination thereof.

[0054] In certain embodiments, the pharmaceutical compositions described above comprise extracts of doubly transgenic grape cells or singly transgenic grape cells.

[0055] In certain embodiments, the pharmaceutical compositions described above comprise a cytoplasmic fraction of a doubly transgenic grape cell or a singly transgenic grape cell.

[0056] In certain embodiments, the pharmaceutical compositions described above comprise a polyphenolic fraction of a doubly transgenic grape cell or a single transgenic grape cell.

[0057] In certain embodiments, the pharmaceutical compositions described above comprise vacuoles of doubly transgenic grape cells or singly transgenic grape cells.

[0058] In certain embodiments, the pharmaceutical compositions described above are substantially dehydrated compositions.

[0059] In certain embodiments, the pharmaceutical compositions described above are substantially free of intact cells.

[0060] In certain embodiments, the pharmaceutical compositions described above are substantially free of disrupted cells.

[0061] In yet another aspect, the present invention also provides a method for preventing coronavirus infection or its symptoms, treating coronavirus infection or its symptoms, reducing the incidence of coronavirus infection or its symptoms, suppressing coronavirus infection or its symptoms, or inhibiting coronavirus infection or its symptoms in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition described above.

[0062] In certain embodiments, the symptom is a cytokine storm.

[0063] In certain embodiments, the pharmaceutical composition is administered systemically to a patient.

[0064] In certain embodiments, the coronavirus infection comprises a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

[0065] In certain embodiments, the pharmaceutical composition is administered orally to a patient.

[0066] In yet another aspect, the present invention also provides a crop plant or part thereof, comprising at least one copy of an AroG* gene and at least one copy of a stilbene synthase (STS) gene or a flavonol synthase (FLS) gene.

[0067] In certain embodiments, the crop plant is grapes.

[0068] In certain embodiments, the grape is of the Gamay Red cultivar.

[0069] On the other hand, the present invention also provides a method for treating, preventing, improving, inhibiting or reducing the incidence of cytokine release syndrome (CRS) or cytokine storm in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition described above.

[0070] In certain embodiments, CRS or cytokine storm is associated with coronavirus infection or a symptom thereof.

[0071] In certain embodiments, the coronavirus infection comprises a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

[0072] In yet another aspect, the present invention also provides a method for preventing or treating increased cytokine levels in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition described above.

[0073] In certain embodiments, the cytokine is selected from the group consisting of IL-6, IFN-γ, TNF-α, and IL-1-β.

[0074] In certain embodiments, increased cytokine levels are associated with coronavirus infection or symptoms thereof.

[0075] In certain embodiments, the increase in cytokine levels is measured in the patient's white blood cells (WBC) or in the patient's serum.

[0076] In yet another aspect, the present invention also provides a method for increasing the level of at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine in grape cells, the cells optionally comprising at least one copy of the AroG* gene and optionally comprising at least one copy of the stilbene synthase (STS) gene, the method comprising contacting the cells with a composition comprising: (a) phenylalanine at a concentration of about 0.2 mM to about 5 mM, (b) p-coumaric acid at a concentration of about 0.1 mM to about 0.3 mM, or any combination of (a) and (b).

[0077] In yet another aspect, the present invention also provides a method for increasing the level of at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1 and procyanidin B2 in grape cells, the cells optionally comprising at least one copy of the AroG* gene and optionally comprising at least one copy of the flavonol synthase (FLS) gene, the method comprising contacting the cells with a composition comprising: (a) phenylalanine at a concentration of about 0.2 mM to about 5 mM, (b) p-coumaric acid at a concentration of about 0.1 mM to about 0.3 mM, or (c) any combination of (a) and (b).

[0078] In yet another aspect, the present invention also provides an edible or drinkable composition comprising the pharmaceutical composition described above.

[0079] In yet another aspect, the present invention also provides a pharmaceutical composition according to the above description, which is formulated for slow release or extended release.

[0080] Other aspects and features of the present disclosure will become apparent to those of ordinary skill in the art after reading the following description of specific embodiments in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1. Generation of transgenic grape cv. Gamay cell cultures expressing either the AroG* gene alone or both the AroG* and STS genes. Figure 1A ) Schematic diagram of the transgene expression cassette in the pART27 vector. In the gene construct, transgene expression is controlled by the 35S promoter. 35S, CaMV 35S promoter; Ω, TMV; HA: HA tag sequence; AcV5, AcV5-tag sequence; T, OCS-terminator; KanR, kanamycin resistance marker; LB, T-DNA left border; RB, T-DNA right border; AroG*, feedback-insensitive bacterial form of 3-deoxy-D-arabinoheptulonic acid 7-phosphate synthase enzyme (DAHPS); STS, stilbene synthase. ( Figure 1B) The expression of AroG* was monitored by immunoblot analysis using anti-HA antibody (1:500). The major protein bands of AroG* were at 35 kDa and 45 kDa. Figure 1C ) The expression of STS was monitored by immunoblotting analysis using anti-AcV5 antibody (1:1000). The major protein band of STS was at 35 kDa. Figure 1D ) Growth curves of selected lines from each construction were measured by fresh weight gain from the day of re-cultivation (day 0) to the onset of cell death. Figure 1E ) On day 9, the cell morphology of each line was photographed using a Leica MZ FLII microscope.

[0083] Figure 2. Principal component analysis of phenylpropanoid metabolites detected in control, AroG* and AroG*+STS samples. The values ​​used for this analysis are normalized peak heights and then log transformed data. Each cloud represents 8 replicates for each strain.

[0084] Figure 3 .The co-expression of AroG* and STS has an effect on the biosynthetic precursors of stilbene in transformed grape cells ( Figure 3 A) and stilbene ( Figure 3 B) Effect of accumulation. The levels of metabolites are expressed as fold changes in transgenic lines compared to the control line (empty vector). The box plots are mixtures of metabolite content for each replicate. In the control n=24, including three lines, in the AroG* lines, n=32, including four lines, and in each AroG*+STS, n=24, including three lines. Statistical significance was analyzed by one-way ANOVA test followed by Dunnett's post hoc test. Black asterisks represent significant differences in metabolite content between the control (empty) and transgenic lines, and gray asterisks represent significant differences in metabolite content between single AroG* lines and double transgenic lines (p≤0.05; p≤0.01; p≤0.001).

[0085] Figure 4Effect of co-expression of both AroG and STS on flavonoid accumulation in grape transformed cells. The levels of metabolites are expressed as fold changes in transgenic lines compared to control lines (empty vector). Box plots are a mixture of metabolite content for each replicate. In the control, n=24, including 12 samples from two lines, in the AroG* lines, n=32, including 8 samples from four lines, and in each AroG*+STS, n=24, including 8 samples from three lines. Statistical significance was analyzed by one-way ANOVA test followed by Dunnett's post hoc test. Black asterisks represent significant differences in metabolite content between control (empty) and transgenic lines, and gray asterisks represent significant differences in metabolite content between single AroG* lines and double transgenic lines (p≤0.05; p≤0.01; p≤0.001).

[0086] Figure 5 Schematic representation of metabolic changes, where fold changes are normalized to control (empty vector). Levels represented are the median values ​​for each metabolite between different transgenic lines. Different transgenic lines are labeled as pentagons (single AroG* lines), diamonds (AroG*+STS5), triangles (AroG*+STS10), hexagons (AroG*+STS28).

[0087] Figure 6. Effects of different Phe and p-coumaric acid concentrations on growth and morphology. Fig. 6A ) Cell weights from 7-day-old samples treated with different Phe concentrations. Figure 6B ) Cell weights from 7-day-old samples treated with different p-coumaric acid concentrations. Figure 6C ) Microscope photo of AroG*+STS28 (line 16) cells on day 7 (taken by Leica MZ FLII microscope). Blue staining indicates dead cells stained with Evans blue.

[0088] Figure 7. Effects of Phe and p-CA feeding on stilbene production in AroG*+STS28 transformant strain (strain 16). Stilbene levels (mean ± SE, mg / g DW) were measured by feeding different concentrations of Phe ( Fig. 7A ) and p-CA( Figure 7B ) Samples after 7 (black) and 9 (grey) days of treatment. Letters represent significant differences between samples using two-way ANOVA (P<0.05) followed by Tukey HSD post hoc test (P≤0.05).

[0089] Figure 8Effect of Phe feeding on precursor metabolites and flavonoid production in AroG*+STS28 transformed lines (line 16). Metabolite levels (mean ± SE) after 7 (black) and 9 (grey) days of treatment with different concentrations of Phe are presented as peak heights. Letters represent significant differences between samples using two-way ANOVA (P < 0.05) followed by Tukey HSD post hoc test (P ≤ 0.05).

[0090] Fig. 9 Effect of p-CA feeding on the AroG*+STS28 transformant line (line 16) on the production of precursor metabolites and flavonoids. Metabolite levels (mean ± SE) after 7 (black) and 9 (grey) days of treatment with different concentrations of Phe are presented as peak heights. Letters represent significant differences between samples using two-way ANOVA (P < 0.05) followed by Tukey HSD post hoc test (P ≤ 0.05). Due to the exogenous nature of p-CA, p-CA levels in different samples were not compared.

[0091] Fig.10 . Schematic representation of changes in metabolite and gene expression levels between Phe-fed cells and controls collected after 9 days of treatment. Levels are expressed as fold changes normalized to controls.

[0092] Fig.11 Effect of Phe feeding on AroG*+STS28 transformed strain (strain 16) on CHS and STS gene expression. The levels of gene expression (mean ± SE) are presented as fold changes normalized to control samples. Stars represent significant differences (P < 0.05) in gene expression between control (black) and 5 mM Phe-fed samples (grey) using student t-test.

[0093] Fig.12 Induction of anti-inflammatory gene expressions in chicken WBCs at the mRNA level by LPS and their inhibition by the polyphenol extract (GCE) according to the invention.

[0094] Fig.13 Inhibition of mRNA expression levels of pro-inflammatory cytokines (INF-G, TNF, IL6) and induction of anti-inflammatory cytokine mRNA (IL10) in the spleen of chickens fed with grape cell powder (GCP) for 7 days and stimulated with LPS for 2 hours. Vertical lines indicate standard errors (n=10 in each group) *, P<0.05 (t-test).

[0095] Figure 14. Generation of transgenic grape cv. Gamay cell cultures expressing AroG* and FLS genes. Fig.14A) Proposed flavonoid and stilbene biosynthetic pathway designed by co-expression of AroG and FLS in grape cv. Gamay Red cell culture. ( Fig. 14B ) Schematic diagram of the transgene expression cassette in the pART27 vector. In the gene construct, transgene expression is controlled by the 35S promoter. 35S, CaMV 35S promoter; Ω, TMV; HA: HA tag sequence; AcV5, AcV5-tag sequence; T, OCS-terminator; KanR, kanamycin resistance marker; LB, T-DNA left border; RB, T-DNA right border; AroG*, feedback-insensitive bacterial form of 3-deoxy-D-arabinoheptulonic acid 7-phosphate synthase enzyme (DAHPS); FLS, flavonol synthase. ( Fig. 14C ) Accumulation of AroG* in transgenic lines. Immunoblotting analysis was performed using anti-HA antibody (1:500). The 35 kDa polypeptide represents the AroG* protein. Fig.14D ) Accumulation of FLS in transgenic lines. Immunoblotting analysis was performed using anti-AcV5 antibody (1:1000). The 35 kDa polypeptide represents the FLS protein. Fig.14E ) Using a Leica MZ FLII microscope, cell morphology of control (empty vector) and two AroG*+ FLS lines at day 9. Blue staining is for dead cells stained with Evans blue.

[0096] Fig.15 Effect of AroG* and FLS co-expression on Phe, Trp and p-CA accumulation in transformed cells of grape cv. Gamay Red. Metabolite levels (mean ± SE, n = 8) are presented as fold changes in transgenic lines compared to control (empty vector). Statistical significance was analyzed by one-way ANOVA test followed by Dunnett's post hoc test. Black asterisks represent significant differences in metabolite content between control and transgenic lines, and grey asterisks represent significant differences in metabolite content between AroG* lines and AroG*+FLS transgenic lines (*P < 0.05; **P < 0.01; ***P < 0.001).

[0097] Fig.16Effect of AroG* and FLS co-expression on stilbene accumulation in transformed cells of grape cv. Gamay Red. Metabolite levels (mean ± SE, n = 8) are presented as fold change of transgenic lines compared to control (empty vector). Statistical significance was analyzed by one-way ANOVA test followed by Dunnett's post hoc test. Black asterisks represent significant differences in metabolite content between control and transgenic lines, and grey asterisks represent significant differences in metabolite content between AroG* lines and AroG*+FLS transgenic lines (*P < 0.05; **P < 0.01; ***P < 0.001).

[0098] Figure 17. Effect of co-expression of AroG* and FLS on the accumulation of flavonols (a), flavan-3-ols (b) and anthocyanins (c) in transformed cells of grape cv. Gamay Red. Metabolite levels (mean ± SE, n = 8) are presented as fold changes in transgenic lines compared to control lines (empty vector). Statistical significance was analyzed by one-way ANOVA test followed by Dunnett's post hoc test. Black asterisks represent significant differences in metabolite content between control and transgenic lines, and gray asterisks represent significant differences in metabolite content between AroG* lines and AroG*+FLS transgenic lines (*P<0.05; **P<0.01; ***P<0.001).

[0099] Figure 18. Summary of metabolite and gene expression changes in grape cv. Gamay Red cells due to transformation of AroG* and FLS and exogenous Phe feeding. Metabolites ( Fig.18A ) and gene expression ( Fig.18B ) levels are presented as fold changes in transgenic lines compared to non-fed controls. Gene expression is visualized in a heat map and differences are represented by log2 fold change values ​​centered and scaled per row. Abbreviations: acet, acetyl; glu, glucoside; coum, coumaroyl; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3'H and F3'5'H, flavonoid 3' and 3'5' hydroxylases; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; LDOX, leucocyanidin dioxygenase; LAR, leucocyanidin reductase; ANR, anthocyanidin reductase; UFGT, uridine diphosphate-glucose:flavonoid 3-O-glucosyltransferase; OMT, O-methyltransferase; STS, stilbene synthase.

[0100] Detailed Description

[0101] The present invention provides methods and compositions for increasing the production of flavonoids and stilbenes, and in particular resveratrol.

[0102] According to the principles of the present invention, the combination of increased Phe availability and STS overexpression or FLS overexpression diverts plant Phe metabolism to the stilbene pathway and / or the flavonoid pathway. Increased Phe availability was achieved by both overexpressing AroG* and feeding the cell culture with external Phe. These attempts resulted in increased production of several resveratrol-derived stilbenes, particularly viniferin, whose concentration was increased 600-fold compared to untreated cultures.

[0103] In one aspect, the invention provides a cell comprising at least one copy of an AroG* gene and at least one copy of a stilbene synthase (STS) gene.

[0104] In certain embodiments, the cell is a plant cell. In certain embodiments, the plant cell is a grape cell. In certain embodiments, the grape cell is a grape cultivar Gamay Red cell.

[0105] In certain embodiments, the AroG* gene is a plant gene. In certain embodiments, the AroG* gene is a grape gene. In certain embodiments, the AroG* gene encodes a 3-deoxy-D-arabinoheptulose 7-phosphate (DAHP) synthase (DAHPS) enzyme. In certain embodiments, the DAHPS enzyme catalyzes the chemical reaction: phosphoenolpyruvate + D-erythrose 7-phosphate + phosphate. In certain embodiments, the DAHPS enzyme is feedback insensitive. In certain embodiments, the DAHPS enzyme is phenylalanine insensitive. In certain embodiments, the DAHPS enzyme is tyrosine insensitive. In certain embodiments, the DAHPS enzyme is tryptophan insensitive.

[0106] In certain embodiments, the DAHPS enzyme increases the availability of at least one amino acid in a cell. In certain embodiments, the DAHPS enzyme increases the availability of at least one amino acid selected from the group consisting of phenylalanine, tyrosine, and tryptophan in a cell. In certain embodiments, the DAHPS enzyme increases the availability of phenylalanine in a cell. In certain embodiments, the DAHPS enzyme increases the availability of tyrosine in a cell. In certain embodiments, the DAHPS enzyme increases the availability of tryptophan in a cell.

[0107] In some embodiments, the STS gene is a plant gene. In some embodiments, the STS gene is a grape gene. In some embodiments, the STS gene encodes an STS enzyme. In some embodiments, the STS enzyme catalyzes the chemical reaction: 3-malonyl-CoA + 4-coumaryl-CoA

[0108] In certain embodiments, the STS enzyme increases the availability of at least one compound selected from the group consisting of dehydroepiandrosterone (DHEA), estrone, pregnenolone, and cholesterol in a cell. In certain embodiments, the STS enzyme increases the availability of DHEA in a cell. In certain embodiments, the STS enzyme increases the availability of estrone in a cell. In certain embodiments, the STS enzyme increases the availability of pregnenolone in a cell. In certain embodiments, the STS enzyme increases the availability of cholesterol in a cell. In certain embodiments, the STS enzyme increases the availability of resveratrol in a cell.

[0109] In certain embodiments, the STS enzyme produces stilbenes. In certain embodiments, the STS enzyme produces stilbenes. In certain embodiments, the stilbenes are resveratrol. In certain embodiments, the stilbenes are resveratrol derivatives. In certain embodiments, the resveratrol derivatives are resveratrol glycosides.

[0110] In certain embodiments, the STS gene is a grape stilbene synthase (VvSTS) gene. In certain embodiments, the STS gene is selected from the group consisting of VvSTS5, VvSTS10, and VvSTS28. In certain embodiments, the STS gene is VvSTS5. In certain embodiments, the STS gene is VvSTS10. In certain embodiments, the STS gene is VvSTS28.

[0111] In one aspect, the invention provides a cell comprising at least one copy of an AroG* gene and at least one copy of a flavonol synthase (FLS) gene.

[0112] In some embodiments, the FLS gene is a plant gene. In some embodiments, the FLS gene is a grape gene. In some embodiments, the FLS gene encodes a FLS enzyme. In some embodiments, the FLS enzyme catalyzes the chemical reaction:

[0113] In certain embodiments, the FLS enzyme produces a flavonoid. In certain embodiments, the FLS enzyme produces a flavonol. In certain embodiments, the flavonoid is a flavonol. In certain embodiments, the flavonoid is a flavan-3-ol. In certain embodiments, the flavonoid is an anthocyanidin. In certain embodiments, the flavonol is myricetin. In certain embodiments, the flavonol is quercetin-3-glucoside. In certain embodiments, the flavonol is kaempferol.

[0114] In certain embodiments, the FLS gene is a grape flavonol synthase (VvFLS) gene. In certain embodiments, the FLS gene is VIT_07s0031g00100.

[0115] In one aspect, the present invention provides a double transgenic grape cell comprising at least one copy of an AroG* gene and at least one copy of a stilbene synthase (STS) gene or a flavonol synthase (FLS) gene.

[0116] In certain embodiments, the AroG* gene or the STS gene is functionally linked to a constitutive promoter. In certain embodiments, both the AroG* gene and the STS gene are functionally linked to a constitutive promoter. In certain embodiments, the AroG* gene or the FLS gene is functionally linked to a constitutive promoter. In certain embodiments, both the AroG* gene and the FLS gene are functionally linked to a constitutive promoter. In certain embodiments, the constitutive promoter is the cauliflower mosaic virus (CaMV) 35S RNA promoter (also referred to as "35S promoter").

[0117] In certain embodiments, the AroG* gene and the STS gene are functionally linked to different constitutive promoters. In certain embodiments, the AroG* gene and the STS gene are functionally linked to the same constitutive promoter. In certain embodiments, the AroG* gene and the STS gene are functionally linked to the same constitutive promoter found upstream of the AroG* gene, and the AroG* gene is found upstream of the STS gene. In certain embodiments, the AroG* gene and the STS gene are functionally linked to the same constitutive promoter found upstream of the STS gene, and the STS gene is found upstream of the AroG* gene.

[0118] In certain embodiments, the AroG* gene and the FLS gene are functionally linked to different constitutive promoters. In certain embodiments, the AroG* gene and the FLS gene are functionally linked to the same constitutive promoter. In certain embodiments, the AroG* gene and the FLS gene are functionally linked to the same constitutive promoter found upstream of the AroG* gene, and the AroG* gene is found upstream of the FLS gene. In certain embodiments, the AroG* gene and the FLS gene are functionally linked to the same constitutive promoter found upstream of the FLS gene, and the FLS gene is found upstream of the AroG* gene.

[0119] In certain embodiments, the cells described herein comprise a higher level of at least one stilbenoid compound. In certain embodiments, the stilbenoid compound comprises resveratrol. In certain embodiments, the stilbenoid compound comprises trans-resveratrol glycosides. In certain embodiments, the stilbenoid compound comprises cis-resveratrol glycosides. In certain embodiments, the stilbenoid compound comprises ε-grapevine.

[0120] In certain embodiments, the cells described herein comprise a higher level of at least one flavonoid. In certain embodiments, flavonoids include flavonols. In certain embodiments, flavonoids include flavan-3-ols. In certain embodiments, flavonoids include anthocyanidins. In certain embodiments, flavonols include myricetin, quercetin-3-glucoside or kaempferol. In certain embodiments, flavonols include myricetin. In certain embodiments, flavonols include quercetin-3-glucoside. In certain embodiments, flavonols include kaempferol. In certain embodiments, flavan-3-ols include catechins, epicatechins, epigallocatechins, procyanidin B1 or procyanidin B2. In certain embodiments, anthocyanidin comprises cyanidin-3-glucoside, cyanidin-3-acetyl-glucoside, cyanidin-3-coumaroyl-glucoside, peonidin-3-glucoside, peonidin-3-acetyl-glucoside, peonidin-3-coumaroyl-glucoside, delphinidin-3-glucoside, delphinidin-3-acetyl-glucoside, delphinidin-3-coumaroyl-glucoside, malvidin-3-glucoside, malvidin-3-acetyl-glucoside, malvidin-3-coumaroyl-glucoside, petuniadin-3-glucoside, petuniadin-3-acetyl-glucoside, or petuniadin-3-coumaroyl-glucoside.

[0121] In certain embodiments, the cells described above comprise higher levels of: at least one amino acid selected from the group consisting of phenylalanine, tryptophan and p-CA (p-coumaric acid); at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine; at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin and procyanidin B2; at least one anthocyanidin selected from the group consisting of malvidin-3-o-glucoside, malvidin-3-coumaroyl-glucoside, malvidin-3-acetyl-glucoside and petunidin-3-coumaroyl-glucoside; or any combination thereof, compared to a corresponding non-transgenic grape cell or compared to a corresponding single transgenic grape cell.

[0122] In certain embodiments, the cells described herein comprise higher levels of: at least one amino acid selected from the group consisting of phenylalanine, tryptophan, and p-CA (p-coumaric acid) compared to a corresponding non-transgenic grape cell or compared to a corresponding single transgenic grape cell; at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol, and ε-grapevine; at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1, and procyanidin B2; at least one anthocyanidin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin, and malvidin; or any combination of the above.

[0123] In certain embodiments, the cells described above comprise higher levels of at least one amino acid selected from the group consisting of phenylalanine, tryptophan, and p-CA (p-coumaric acid) compared to a corresponding non-transgenic grape cell.

[0124] In certain embodiments, the cells described above comprise higher levels of at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol, and ε-grapevine compared to corresponding non-transgenic grapevine cells.

[0125] In certain embodiments, the cells described above comprise higher levels of at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1, and procyanidin B2 compared to corresponding non-transgenic grape cells.

[0126] In certain embodiments, the cells described above comprise higher levels of at least one anthocyanin selected from the group consisting of malvidin-3-o-glucoside, malvidin-3-coumaroyl-glucoside, malvidin-3-acetyl-glucoside, and petunidin-3-coumaroyl-glucoside compared to corresponding non-transgenic grape cells.

[0127] In certain embodiments, the cells described above comprise higher levels of at least one anthocyanin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin, and malvidin compared to corresponding non-transgenic grape cells.

[0128] In certain embodiments, the cells described above comprise higher levels of at least one amino acid selected from the group consisting of phenylalanine, tryptophan, and p-CA (p-coumaric acid) compared to corresponding single transgenic grape cells.

[0129] In certain embodiments, the cells described above comprise higher levels of at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol, and ε-grapevine compared to corresponding single transgenic grapevine cells.

[0130] In certain embodiments, the cells described above comprise higher levels of at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1, and procyanidin B2 compared to corresponding single transgenic grape cells.

[0131] In certain embodiments, the cells described above comprise higher levels of at least one anthocyanin selected from the group consisting of malvidin-3-o-glucoside, malvidin-3-coumaroyl-glucoside, malvidin-3-acetyl-glucoside, and petunidin-3-coumaroyl-glucoside compared to corresponding single transgenic grape cells.

[0132] In certain embodiments, the cells described above comprise higher levels of at least one anthocyanin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin, and malvidin compared to corresponding single transgenic grape cells.

[0133] In certain embodiments, the corresponding single transgenic grapevine cells comprise at least one copy of the AroG* gene. In certain embodiments, the corresponding single transgenic grapevine cells comprise the same number of copies of the AroG* gene.

[0134] In certain embodiments, the cells described above comprise trans-resveratrol at a concentration of at least 0.3 mg / g dry weight, cis-resveratrol at a concentration of at least 0.3 mg / g dry weight, resveratrol at a concentration of at least 0.1 mg / g dry weight, ε-glucose at a concentration of at least 0.02 mg / g dry weight, or any combination thereof.

[0135] In certain embodiments, the cells described above comprise trans-resveratrol glycosides at a concentration of at least 0.3 mg / g dry weight. In certain embodiments, the cells described above comprise cis-resveratrol glycosides at a concentration of at least 0.3 mg / g dry weight. In certain embodiments, the cells described above comprise resveratrol at a concentration of at least 0.1 mg / g dry weight. In certain embodiments, the cells described above comprise ε-grapevine at a concentration of at least 0.02 mg / g dry weight.

[0136] In certain embodiments, the cells described above comprise trans-resveratrol at a concentration of at least 0.3 mg / g dry weight, cis-resveratrol at a concentration of at least 0.3 mg / g dry weight, resveratrol at a concentration of at least 0.1 mg / g dry weight, and ε-glucose at a concentration of at least 0.02 mg / g dry weight.

[0137] In certain embodiments, the cells described above comprise trans-resveratrol at a concentration of at least 0.5 mg / g dry weight, cis-resveratrol at a concentration of at least 0.5 mg / g dry weight, resveratrol at a concentration of at least 0.8 mg / g dry weight, ε-glucose at a concentration of at least 0.6 mg / g dry weight, or any combination thereof.

[0138] In certain embodiments, the cells described above include trans-resveratrol glycosides at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above include trans-resveratrol glycosides at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above include trans-resveratrol glycosides at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above include trans-resveratrol glycosides at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above include trans-resveratrol glycosides at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above include trans-resveratrol glycosides at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above include trans-resveratrol glycosides at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above include trans-resveratrol glycosides at a concentration of at least 1.2 mg / g dry weight.

[0139] In certain embodiments, the cells described above include cis-resveratrol glycosides at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above include cis-resveratrol glycosides at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above include cis-resveratrol glycosides at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above include cis-resveratrol glycosides at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above include cis-resveratrol glycosides at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above include cis-resveratrol glycosides at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above include cis-resveratrol glycosides at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above include cis-resveratrol glycosides at a concentration of at least 1.2 mg / g dry weight.

[0140] In certain embodiments, the cells described above include resveratrol at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above include resveratrol at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above include resveratrol at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above include resveratrol at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above include resveratrol at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above include resveratrol at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above include resveratrol at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above include resveratrol at a concentration of at least 1.2 mg / g dry weight. In certain embodiments, the cells described above include resveratrol at a concentration of at least 1.3 mg / g dry weight.

[0141] In certain embodiments, the cells described above comprise ε-grapevine at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above comprise ε-grapevine at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above comprise ε-grapevine at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above comprise ε-grapevine at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above comprise ε-grapevine at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above comprise ε-grapevine at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above comprise ε-grapevine at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above comprise ε-grapevine at a concentration of at least 1.2 mg / g dry weight.

[0142] In certain embodiments, the cells described above include myricetin at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above include myricetin at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above include myricetin at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above include myricetin at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above include myricetin at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above include myricetin at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above include myricetin at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above include myricetin at a concentration of at least 1.2 mg / g dry weight.

[0143] In certain embodiments, the cells described above include quercetin-3-glucoside at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above include quercetin-3-glucoside at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above include quercetin-3-glucoside at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above include quercetin-3-glucoside at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above include quercetin-3-glucoside at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above include quercetin-3-glucoside at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above include quercetin-3-glucoside at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above comprise quercetin-3-glucoside at a concentration of at least 1.2 mg / g dry weight.

[0144] In certain embodiments, the cells described above comprise catechins at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above comprise catechins at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above comprise catechins at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above comprise catechins at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above comprise catechins at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above comprise catechins at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above comprise catechins at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above comprise catechins at a concentration of at least 1.2 mg / g dry weight.

[0145] In certain embodiments, the cells described above comprise epicatechin at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above comprise epicatechin at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above comprise epicatechin at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above comprise epicatechin at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above comprise epicatechin at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above comprise epicatechin at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above comprise epicatechin at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above comprise epicatechin at a concentration of at least 1.2 mg / g dry weight.

[0146] In certain embodiments, the cells described above comprise epigallocatechin at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above comprise epigallocatechin at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above comprise epigallocatechin at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above comprise epigallocatechin at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above comprise epigallocatechin at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above comprise epigallocatechin at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above comprise epigallocatechin at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above comprise epigallocatechin at a concentration of at least 1.2 mg / g dry weight.

[0147] In certain embodiments, the cells described above comprise procyanidin B1 at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B1 at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B1 at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B1 at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B1 at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B1 at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B1 at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B1 at a concentration of at least 1.2 mg / g dry weight.

[0148] In certain embodiments, the cells described above comprise procyanidin B2 at a concentration of at least 0.5 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B2 at a concentration of at least 0.6 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B2 at a concentration of at least 0.7 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B2 at a concentration of at least 0.8 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B2 at a concentration of at least 0.9 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B2 at a concentration of at least 1.0 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B2 at a concentration of at least 1.1 mg / g dry weight. In certain embodiments, the cells described above comprise procyanidin B2 at a concentration of at least 1.2 mg / g dry weight.

[0149] In certain embodiments, the cells described above comprise similar or lower levels of: at least one flavonoid selected from the group consisting of myricetin, quercetin-3-glucoside, catechin, epicatechin, epigallocatechin, and proanthocyanidins; at least one anthocyanidin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin, and malvidin; or any combination thereof, compared to a corresponding non-transgenic grape cell or compared to a corresponding single transgenic grape cell.

[0150] In certain embodiments, the cells described above comprise similar or lower levels of at least one flavonoid selected from the group consisting of myricetin, quercetin-3-glucoside, catechin, epicatechin, epigallocatechin, and proanthocyanidins compared to a corresponding non-transgenic grape cell or compared to a corresponding single transgenic grape cell.

[0151] In certain embodiments, the cells described above comprise similar or lower levels of at least one anthocyanin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin, and malvidin as compared to a corresponding non-transgenic grape cell or as compared to a corresponding single transgenic grape cell.

[0152] In certain embodiments, the corresponding single transgenic grapevine cells comprise at least one copy of the AroG* gene. In certain embodiments, the corresponding single transgenic grapevine cells comprise the same number of copies of the AroG* gene.

[0153] In certain embodiments, the cells described above comprise resveratrol at a concentration of about 1.26 mg / g dry weight, ε-glucose at a concentration of about 10.8 mg / g dry weight, or both. In certain embodiments, the cells described above comprise resveratrol at a concentration of about 1.26 mg / g dry weight. In certain embodiments, the cells described above comprise ε-glucose at a concentration of about 10.8 mg / g dry weight.

[0154] In another aspect, the present invention also provides a method of maintaining a cell, which optionally comprises at least one copy of an AroG* gene, optionally comprises at least one copy of a stilbene synthase (STS) gene, and optionally comprises at least one copy of a flavonol synthase (FLS) gene, the method comprising contacting the cell with a composition comprising: phenylalanine at a concentration of about 0.2 mM to about 5 mM, p-coumaric acid at a concentration of about 0.1 mM to about 0.3 mM, or any combination thereof.

[0155] In another aspect, the present invention also provides a method of maintaining a cell, optionally comprising at least one copy of an AroG* gene and optionally comprising at least one copy of a stilbene synthase (STS) gene, the method comprising contacting the cell with a composition comprising: phenylalanine at a concentration of about 0.2 mM to about 5 mM, p-coumaric acid at a concentration of about 0.1 mM to about 0.3 mM, or any combination thereof.

[0156] In another aspect, the present invention also provides a method of maintaining a cell, optionally comprising at least one copy of an AroG* gene and optionally comprising at least one copy of a flavonol synthase (FLS) gene, the method comprising contacting the cell with a composition comprising: phenylalanine at a concentration of about 0.2 mM to about 5 mM, p-coumaric acid at a concentration of about 0.1 mM to about 0.3 mM, or any combination thereof.

[0157] In certain embodiments, the cell is a transgenic cell. In certain embodiments, the cell is a plant cell. In certain embodiments, the cell is a transgenic plant cell. In certain embodiments, the plant cell is a grape cell. In certain embodiments, the cell comprises at least one copy of the AroG* gene. In certain embodiments, the cell comprises at least one copy of the STS gene. In certain embodiments, the cell comprises at least one copy of the FLS gene. In certain embodiments, the cell comprises a single copy of the AroG* gene. In certain embodiments, the cell comprises a single copy of the STS gene. In certain embodiments, the cell comprises a single copy of the FLS gene.

[0158] In certain embodiments, the methods described above include contacting a cell with a composition comprising phenylalanine at a concentration of at least about 0.2 mM. In certain embodiments, the methods described above include contacting a cell with a composition comprising phenylalanine at a concentration of at least about 0.5 mM. In certain embodiments, the methods described above include contacting a cell with a composition comprising phenylalanine at a concentration of at least about 1.0 mM. In certain embodiments, the methods described above include contacting a cell with a composition comprising phenylalanine at a concentration of at least about 2.0 mM. In certain embodiments, the methods described above include contacting a cell with a composition comprising phenylalanine at a concentration of at least about 5.0 mM.

[0159] In certain embodiments, the methods described above include contacting a cell with a composition comprising phenylalanine at a concentration of about 0.2 mM to about 5 mM. In certain embodiments, the methods described above include contacting a cell with a composition comprising phenylalanine at a concentration of about 0.5 mM to about 5 mM. In certain embodiments, the methods described above include contacting a cell with a composition comprising phenylalanine at a concentration of about 1 mM to about 5 mM. In certain embodiments, the methods described above include contacting a cell with a composition comprising phenylalanine at a concentration of about 2 mM to about 5 mM.

[0160] In certain embodiments, the methods described above include contacting the cell with a composition comprising phenylalanine at a concentration of about 0.2 mM. In certain embodiments, the methods described above include contacting the cell with a composition comprising phenylalanine at a concentration of about 0.5 mM. In certain embodiments, the methods described above include contacting the cell with a composition comprising phenylalanine at a concentration of about 1 mM. In certain embodiments, the methods described above include contacting the cell with a composition comprising phenylalanine at a concentration of about 2 mM. In certain embodiments, the methods described above include contacting the cell with a composition comprising phenylalanine at a concentration of about 5 mM.

[0161] In certain embodiments, the methods described above comprise contacting a cell with a composition comprising p-coumaric acid at a concentration of at least about 0.1 mM. In certain embodiments, the methods described above comprise contacting a cell with a composition comprising p-coumaric acid at a concentration of at least about 0.3 mM.

[0162] In certain embodiments, the methods described above comprise contacting the cell with a composition comprising p-coumaric acid at a concentration of about 0.1 mM to about 0.3 mM.

[0163] In certain embodiments, the methods described above comprise contacting the cell with a composition comprising p-coumaric acid at a concentration of about 0.1 mM. In certain embodiments, the methods described above comprise contacting the cell with a composition comprising p-coumaric acid at a concentration of about 0.3 mM.

[0164] In yet another aspect, the present invention also provides a pharmaceutical composition comprising the double transgenic grape cell or an extract or fraction thereof as described above.

[0165] In certain embodiments, the double transgenic grape cells are maintained by the methods described above.

[0166] In yet another aspect, the present invention also provides a pharmaceutical composition comprising non-transgenic grape cells or single transgenic grape cells containing at least one copy of the AroG* gene, or an extract or fraction thereof, wherein the cells are maintained by the method described above.

[0167] In certain embodiments, the pharmaceutical composition described above comprises: at least one amino acid selected from the group consisting of phenylalanine, tryptophan and p-CA; at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine; at least one flavonoid selected from the group consisting of quercetin-3-glucoside and procyanidin B2; at least one anthocyanidin selected from the group consisting of malvidin-3-o-glucoside, malvidin-3-coumaroyl-glucoside, malvidin-3-acetyl-glucoside, petunidin-3-coumaroyl-glucoside, procyanidin B2 and myricetin; or any combination thereof.

[0168] In certain embodiments, the pharmaceutical composition described above comprises: at least one amino acid selected from the group consisting of phenylalanine, tryptophan and p-CA; at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine; at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1 and procyanidin B2; at least one anthocyanidin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin and malvidin; or any combination thereof.

[0169] In certain embodiments, the pharmaceutical composition described above comprises at least one amino acid selected from the group consisting of phenylalanine, tryptophan and p-CA. In certain embodiments, the pharmaceutical composition described above comprises at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine. In certain embodiments, the pharmaceutical composition described above comprises at least one flavonoid selected from the group consisting of quercetin-3-glucoside and proanthocyanidin B2. In certain embodiments, the pharmaceutical composition described above comprises at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, myricetin, catechin, epicatechin, epigallocatechin, proanthocyanidin B1 and proanthocyanidin B2. In certain embodiments, the pharmaceutical composition described above comprises at least one anthocyanidin selected from the group consisting of malvidin-3-o-glucoside, malvidin-3-coumaroyl-glucoside, malvidin-3-acetyl-glucoside and petunia-3-coumaroyl-glucoside. In certain embodiments, the pharmaceutical composition described above comprises at least one anthocyanin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin, and malvidin.

[0170] In certain embodiments, the pharmaceutical compositions described above comprise extracts of doubly transgenic grape cells or single transgenic grape cells. In certain embodiments, the pharmaceutical compositions described above comprise extracts of doubly transgenic grape cells. In certain embodiments, the pharmaceutical compositions described above comprise extracts of single transgenic grape cells.

[0171] In certain embodiments, the extract is a polyphenol extract. In certain embodiments, the extract is in the form of a dry powder. In certain embodiments, the extract is a grape cell polyphenol extract (GCE). In certain embodiments, the extract is in the form of a dry grape cell powder (GCP).

[0172] In certain embodiments, the fraction is a polyphenol fraction. In certain embodiments, the fraction is in the form of a dry powder. In certain embodiments, the fraction is a grape cell polyphenol fraction (GCF). In certain embodiments, the fraction is in the form of a dry grape cell powder.

[0173] In certain embodiments, the pharmaceutical composition described above comprises the cytoplasmic fraction of a doubly transgenic grape cell or a single transgenic grape cell. In certain embodiments, the pharmaceutical composition described above comprises the cytoplasmic fraction of a doubly transgenic grape cell. In certain embodiments, the pharmaceutical composition described above comprises the cytoplasmic fraction of a single transgenic grape cell.

[0174] In certain embodiments, the pharmaceutical composition described above comprises a polyphenolic fraction of a doubly transgenic grape cell or a single transgenic grape cell. In certain embodiments, the pharmaceutical composition described above comprises a polyphenolic fraction of a doubly transgenic grape cell. In certain embodiments, the pharmaceutical composition described above comprises a polyphenolic fraction of a single transgenic grape cell.

[0175] In certain embodiments, the pharmaceutical composition described above comprises the vacuole of a double transgenic grape cell or a single transgenic grape cell. In certain embodiments, the pharmaceutical composition described above comprises the vacuole of a double transgenic grape cell. In certain embodiments, the pharmaceutical composition described above comprises the vacuole of a single transgenic grape cell.

[0176] In certain embodiments, the pharmaceutical composition described above is a substantially dehydrated composition.In certain embodiments, the pharmaceutical composition described above comprises 0% to 50% water by weight.

[0177] In certain embodiments, the pharmaceutical composition described above comprises 0% to 40% water by weight. In certain embodiments, the pharmaceutical composition described above comprises 0% to 30% water by weight. In certain embodiments, the pharmaceutical composition described above comprises 0% to 20% water by weight. In certain embodiments, the pharmaceutical composition described above comprises 0% to 10% water by weight. In certain embodiments, the pharmaceutical composition described above comprises 0% to 5% water by weight. In certain embodiments, the pharmaceutical composition described above comprises 0% to 1% water by weight.

[0178] In certain embodiments, the pharmaceutical compositions described above are substantially free of intact cells. In certain embodiments, the pharmaceutical compositions described above include 0% to 50% intact cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 40% intact cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 30% intact cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 20% intact cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 10% intact cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 5% intact cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 1% intact cells by weight.

[0179] In certain embodiments, the pharmaceutical compositions described above are substantially free of ruptured cells. In certain embodiments, the pharmaceutical compositions described above include 0% to 50% ruptured cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 40% ruptured cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 30% ruptured cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 20% ruptured cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 10% ruptured cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 5% ruptured cells by weight. In certain embodiments, the pharmaceutical compositions described above include 0% to 1% ruptured cells by weight.

[0180] In certain embodiments, disclosed herein are methods of preventing, treating, reducing the incidence of, suppressing, or inhibiting a viral infection, disease, disorder, or symptom thereof in a subject.

[0181] In certain embodiments, disclosed herein are methods for preventing, treating, reducing the incidence of, suppressing, or inhibiting a viral infection, disease, disorder, or symptom thereof in a subject, comprising the step of administering to the subject a pharmaceutical composition described in detail herein.

[0182] In certain embodiments, the term "viral disease" may encompass pathological conditions in a subject caused directly or indirectly by the presence of a virus. The term "viral disease" may also encompass clinical manifestations or symptoms caused by or associated with a viral infection, including but not limited to viral diseases caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0183] In certain embodiments, the terms "virus" and "viral" can encompass pathogens of infection including coronavirus (CoV), severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, and influenza virus.

[0184] In certain embodiments, the method for preventing viral infection, disease, disorder or its symptoms, treating viral infection, disease, disorder or its symptoms, reducing viral infection, disease, disorder or its symptom incidence, suppressing viral infection, disease, disorder or its symptoms or suppressing viral infection, disease, disorder or its symptoms in a subject includes reducing cytokine release syndrome (CRS) or cytokine storm in the subject. In certain embodiments, the method includes reducing cytokine release syndrome (CRS) in a subject. In certain embodiments, the method includes reducing cytokine storm in a subject.

[0185] In certain embodiments, the viral infection comprises a coronavirus (CoV) infection, a severe acute respiratory syndrome (SARS) infection, a Middle East respiratory syndrome (MERS) infection, or an influenza virus infection.

[0186] In certain embodiments, the coronavirus infection comprises a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In certain embodiments, the viral infection comprises a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

[0187] In one embodiment, the term "SARS-CoV-2", also referred to as "2019 novel coronavirus (2019-nCoV)", "severe acute respiratory syndrome-related coronavirus (SARSr-CoV)", "COVID-19 virus" or "coronavirus", is a positive-sense single-stranded RNA (+ssRNA) virus belonging to the Coronaviridae family of viruses, known as coronaviruses. SARS-CoV-2 was first identified in December 2019. In certain embodiments, SARS-CoV-2 is spread from person to person, typically through respiratory droplets such as sneezing, coughing, or exhaling. Those skilled in the art will recognize that SARS-CoV-2 is a member of the Sarbecovirus subgenus and has an RNA sequence of approximately 30,000 bases in length. The present disclosure encompasses methods for treating all coronavirus variants. Those skilled in the art will recognize that seven coronaviruses are known to infect humans. In certain embodiments, the coronavirus comprises human coronavirus 229E (HCoV-229E). In certain embodiments, the coronavirus comprises human coronavirus OC43 (HCoV-OC43). In certain embodiments, coronaviruses include severe acute respiratory syndrome-associated coronaviruses (SARS-CoV). In certain embodiments, coronaviruses include human coronavirus NL63 (HCoV-NL63, New Haven coronavirus). In certain embodiments, coronaviruses include human coronavirus HKU1. In certain embodiments, coronaviruses include Middle East respiratory syndrome-associated coronaviruses (MERS-CoV), previously known as novel coronavirus 2012 and HCoV-EMC. In certain embodiments, coronaviruses include SARS-CoV-2.

[0188] In certain embodiments, the disease comprises Coronavirus Disease-2019 (COVID-19).

[0189] In certain embodiments, SARS-CoV-2 viral infection causes a respiratory disease called "coronavirus disease 2019" (COVID-19), also known as "new coronavirus pneumonia (NCP)", "SARS-CoV-2 acute respiratory disease", and "2019-nCoV acute respiratory disease". In certain embodiments, COVID-19 symptoms appear after an incubation period of between 2 days and 14 days. In certain embodiments, coronaviruses primarily affect the lower respiratory tract. In certain embodiments, coronaviruses primarily affect the upper respiratory tract. In certain embodiments, COVID-19 symptoms include fever, cough, shortness of breath, muscle pain, fatigue, pneumonia, acute respiratory distress syndrome, sepsis, septic shock, death, or any combination thereof.

[0190] In certain embodiments, the viral disease is caused by SARS-CoV-2. In certain embodiments, the viral disease is caused by coronavirus. In certain embodiments, the viral disease is caused by SARS virus. In certain embodiments, the viral disease is caused by MERS virus. In certain embodiments, the viral disease is caused by influenza virus.

[0191] In certain embodiments, "cytokine release syndrome" or "CRS" may encompass systemic inflammatory response syndrome (SIRS) or cytokine storm syndrome (CSS), which may be caused by a variety of factors such as infection and certain drugs. In certain embodiments, CRS includes activation of leukocytes that release inflammatory cytokines. In certain embodiments, CRS includes elevated levels of various cytokines such as MCP-1, IL-8, IL-6, TNF-α, IFN-γ, and IL-10. Those skilled in the art will appreciate that "cytokine storm" may encompass CRS of immediate onset. In certain embodiments, CRS or cytokine storm may occur as a result of infectious or non-infectious diseases, including coronavirus disease 2019 (COVID-19).

[0192] In one embodiment, the term "elevated" may encompass an increased amount or level, for example, "elevated cytokine levels" may refer to cytokine levels that are higher than the cytokine levels measured in a blood sample of a healthy individual.

[0193] In certain embodiments, the pharmaceutical compositions disclosed herein are used to treat or prevent viral infections. In certain embodiments, the pharmaceutical compositions disclosed herein are used to treat or prevent coronaviruses. In certain embodiments, the pharmaceutical compositions disclosed herein are used to treat or prevent SARS. In certain embodiments, the pharmaceutical compositions disclosed herein are used to treat or prevent MERS. In certain embodiments, the pharmaceutical compositions disclosed herein are used to treat or prevent influenza.

[0194] In certain embodiments, the present disclosure provides methods for preventing or treating a viral disease (e.g., COVID-2019) in a subject, comprising administering any of the compositions disclosed herein.

[0195] In certain embodiments, the virus comprises a coronavirus (CoV), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), influenza virus, or a mutant thereof. In certain embodiments, the coronavirus comprises severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In certain embodiments, the virus comprises severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0196] On the other hand, the present invention also provides a method for treating a coronavirus infection or its symptoms in a patient in need, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition as described above. In certain embodiments, disclosed herein is a method for preventing coronavirus infection or its symptoms, treating coronavirus infection or its symptoms, reducing the incidence of coronavirus infection or its symptoms, suppressing coronavirus infection or its symptoms, or inhibiting coronavirus infection or its symptoms in a patient in need, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition. In certain embodiments, the symptom is a cytokine storm. In certain embodiments, the symptom includes elevated cytokine levels. In another embodiment, the symptom includes elevated IL-6 levels. In certain embodiments, the symptom includes elevated IL-8 levels. In another embodiment, the symptom includes elevated IL-17A levels.

[0197] In certain embodiments, disclosed herein is a method for treating, preventing, improving, suppressing cytokine release syndrome (CRS) or cytokine storm or reducing the incidence of cytokine release syndrome (CRS) or cytokine storm in a subject. In certain embodiments, a method for treating, preventing, improving, suppressing cytokine release syndrome (CRS) or cytokine storm caused by viral infection, disease or disorder or reducing the incidence of cytokine release syndrome (CRS) or cytokine storm caused by viral infection, disease or disorder in a subject includes administering a pharmaceutical composition described in detail herein to a subject.

[0198] In certain embodiments, the method for treating, preventing, ameliorating, inhibiting, or reducing the incidence of cytokine release syndrome (CRS), cytokine storm, further comprises the step of administering to the subject one or more additional compositions comprising a therapeutic agent or an antiviral agent.

[0199] In certain embodiments, the pharmaceutical composition is administered systemically to a patient. In certain embodiments, the pharmaceutical composition is administered orally to a patient. According to some embodiments, the pharmaceutical composition is formulated for oral administration.

[0200] In yet another aspect, the present invention also provides a crop plant or part thereof, comprising at least one copy of an AroG* gene.

[0201] In yet another aspect, the present invention also provides a crop plant or part thereof, comprising at least one copy of an AroG* gene and at least one copy of a stilbene synthase (STS) gene.

[0202] In yet another aspect, the present invention also provides a crop plant or part thereof, comprising at least one copy of an AroG* gene and at least one copy of a stilbene synthase (STS) gene or a flavonol synthase (FLS) gene.

[0203] In yet another aspect, the present invention also provides a crop plant or part thereof, comprising at least one copy of an AroG* gene and at least one copy of a flavonol synthase (FLS) gene.

[0204] In certain embodiments, the part of a crop plant is not an isolated cell.

[0205] In certain embodiments, the crop plant is grapes.

[0206] In certain embodiments, the grape is of the Gamay Red cultivar.

[0207] In yet another aspect, the present invention also provides a method for preventing or treating cytokine storm in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition described above.

[0208] In yet another aspect, the present invention also provides a method for treating, preventing, improving, inhibiting or reducing the incidence of cytokine release syndrome (CRS) or cytokine storm in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition described above.

[0209] In certain embodiments, the methods of the invention are prophylactic and are used for prevention. In certain embodiments, the methods of the invention are therapeutic and are used for treatment.

[0210] In certain embodiments, CRS or cytokine storm is associated with coronavirus infection or its symptoms. In certain embodiments, CRS or cytokine storm is associated with coronavirus infection. In certain embodiments, CRS or cytokine storm is associated with symptoms of coronavirus infection. In certain embodiments, coronavirus infection includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

[0211] In yet another aspect, the present invention also provides a method for preventing or treating increased cytokine levels in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition described above.

[0212] In certain embodiments, the level of cytokines is measured in blood or serum. In certain embodiments, the level of cytokines is measured in whole blood. In certain embodiments, the level of cytokines is measured in serum. In certain embodiments, the level of cytokines is measured in blood cells.

[0213] In certain embodiments, the cytokine is selected from the group consisting of IL-6, IFN-γ, TNF-α, and IL-1-β. In certain embodiments, the cytokine is IL-6. In certain embodiments, the cytokine is IFN-γ. In certain embodiments, the cytokine is TNF-α. In certain embodiments, the cytokine is IL-1-β.

[0214] In certain embodiments, the present invention provides methods for reducing the production of inflammatory cytokines. In certain embodiments, inflammatory cytokines include IL-6. In certain embodiments, inflammatory cytokines include IFN-γ. In certain embodiments, inflammatory cytokines include TNF-α. In certain embodiments, inflammatory cytokines include IL-1-β.

[0215] In certain embodiments, the increase in cytokine levels is associated with a coronavirus infection or a symptom thereof. In certain embodiments, the increase in cytokine levels is associated with a coronavirus infection. In certain embodiments, the increase in cytokine levels is associated with a symptom of a coronavirus infection. In certain embodiments, a coronavirus infection includes a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

[0216] In certain embodiments, the increase of cytokine levels is measured in the patient's white blood cells (WBC) or the patient's serum. In certain embodiments, the increase of cytokine levels is measured in the patient's WBC. In certain embodiments, the increase of cytokine levels is measured in the patient's serum.

[0217] In yet another aspect, the present invention also provides a method for increasing the level of at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine in grape cells, the cells optionally comprising at least one copy of an AroG* gene and optionally comprising at least one copy of a stilbene synthase (STS) gene, the method comprising contacting the cells with a composition comprising: (a) phenylalanine at a concentration of about 0.2 mM to about 5 mM, (b) p-coumaric acid at a concentration of about 0.1 mM to about 0.3 mM, or (c) any combination of (a) and (b).

[0218] In certain embodiments, at least one stilbene is trans-resveratrol glycoside. In certain embodiments, at least one stilbene is cis-resveratrol glycoside. In certain embodiments, at least one stilbene is resveratrol. In certain embodiments, at least one stilbene is ε-grapevine.

[0219] In yet another aspect, the present invention also provides a method for increasing the level of at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1 and procyanidin B2 in grape cells, the cells optionally comprising at least one copy of the AroG* gene and optionally comprising at least one copy of the flavonol synthase (FLS) gene, the method comprising contacting the cells with a composition comprising: (a) phenylalanine at a concentration of about 0.2 mM to about 5 mM, (b) p-coumaric acid at a concentration of about 0.1 mM to about 0.3 mM, or (c) any combination of (a) and (b).

[0220] In certain embodiments, at least one flavonoid is quercetin-3-glucoside. In certain embodiments, at least one flavonoid is myricetin. In certain embodiments, at least one flavonoid is catechin. In certain embodiments, at least one flavonoid is epicatechin. In certain embodiments, at least one flavonoid is epigallocatechin. In certain embodiments, at least one flavonoid is procyanidin B1. In certain embodiments, at least one flavonoid is procyanidin B2.

[0221] In certain embodiments, the grape cell optionally comprises at least one copy of the AroG* gene and optionally comprises at least one copy of the stilbene synthase (STS) gene. In certain embodiments, the grape cell optionally comprises at least one copy of the AroG* gene and optionally comprises at least one copy of the stilbene synthase (STS) gene. In certain embodiments, the grape cell optionally comprises at least one copy of the AroG* gene and optionally comprises at least one copy of the stilbene synthase (STS) gene. In certain embodiments, the grape cell optionally comprises at least one copy of the AroG* gene and optionally comprises at least one copy of the stilbene synthase (STS) gene.

[0222] In certain embodiments, the grape cell optionally comprises at least one copy of the AroG* gene and optionally comprises at least one copy of the flavonol synthase (FLS) gene. In certain embodiments, the grape cell optionally comprises at least one copy of the AroG* gene and optionally comprises at least one copy of the flavonol synthase (FLS) gene. In certain embodiments, the grape cell optionally comprises at least one copy of the AroG* gene and optionally comprises at least one copy of the flavonol synthase (FLS) gene. In certain embodiments, the grape cell optionally comprises at least one copy of the AroG* gene and comprises at least one copy of the flavonol synthase (FLS) gene.

[0223] In yet another aspect, the present invention also provides an edible or drinkable composition comprising the pharmaceutical composition described above.

[0224] In yet another aspect, the present invention also provides the pharmaceutical composition described above, which is formulated for slow release or extended release.

[0225] In certain embodiments, the pharmaceutical composition is formulated for slow release.According to some embodiments, the pharmaceutical composition is formulated for extended release.

[0226] definition

[0227] Unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" as used herein include plural referents. For example, the term "molecule" also includes more than one molecule. As used herein, the term "about" can encompass deviations between 0.0001%-5% from the indicated number or numerical range. In one embodiment, the term "about" can encompass deviations between 1%-10% from the indicated number or numerical range. Throughout this application, various embodiments of the present invention can be presented in the form of ranges. It should be understood that the description in the range format is only for convenience and brevity and should not be interpreted as an unchangeable limitation on the scope of the present invention. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, the description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers such as 1, 2, 3, 4, 5 and 6 within the range. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fraction or integer) within the indicated range. The phrases "ranging / ranges between" a first indicated number and a second indicated number "between" and "ranging / ranges from" a first indicated number "to" a second indicated number are used interchangeably herein and are meant to include the first indicated number and the second indicated number and all fractions and integers therebetween.

[0228] As used herein, the terms "treat," "treatment," or "therapy" (and their various forms) refer to therapeutic treatment, including prophylactic or preventative measures, in which the object is to prevent or slow (mitigate) the undesirable physiological changes associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease or condition, stabilization of the disease or condition (i.e., no worsening of the disease or condition), delay or slowing of the progression of the disease or condition, improvement or alleviation of the disease or condition, and remission (whether partial or complete), whether detectable or undetectable, of the disease or condition. Those in need of treatment include those already suffering from the disease or condition, as well as those predisposed to suffering from the disease or condition or those in which the disease or condition is to be prevented.

[0229] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a human or non-human animal to whom treatment with a composition or formulation according to the invention is provided.

[0230] The embodiments described above are intended to be examples only. Those skilled in the art may make changes, modifications and variations to the specific embodiments. The scope of the claims should not be limited to the specific embodiments set forth herein, but should be interpreted in a manner consistent with the entire specification. Example

[0231] The following materials and methods were used in the examples:

[0232] Plant materials and cell growth

[0233] Vitis vinifera cv. GamayRed cell suspensions were established from young grape berries as previously described (Kiselev et al., 2013). Wild-type (wt) cells were maintained in solid B5 medium supplemented with 250 mg / L casein hydrolysate, 100 mg / L inositol, 0.2 mg / L kinetin and 0.1 mg / L NAA, 2% sucrose (w / v). 11,27 Liquid cell suspension cultures were prepared with the same nutrient and hormone composition and maintained at 25 ± 1 °C under constant light conditions (25 μmol m-2 s-1) with continuous gentle shaking. 50 mL suspension cell stocks were maintained by subculturing 5 g of cells into fresh medium once a week.

[0234] Plasmid generation and preparation of stably transformed cell lines

[0235] Binary vector constructs for ectopic expression of AroG* and FLS or STS were prepared by using a previously described vector and cloning system (Wang et al., 2021). The full-length FLS cDNA of VIT_07s0031g00100 (NCBI: XM_002283156) or the STS cDNAs of VvSTS5, VvSTS10, and VvSTS28 were cloned into Easy vector system (Promega, Madison, WI, USA).

[0236] The ORFs of FLS and STS were cloned into the pART7 vector under the control of the double cauliflower mosaic virus (CaMV) 35S promoter and fused to the C-terminal AcV5 tag. The AroG* gene was PCR amplified using the Gibson assembly cloning system (Gibson et al., 2009) and assembled into an empty pART27 vector together with the above-mentioned FLS and STS gene cassettes. The construct contained a kanamycin selection marker. These constructs were used to transform Agrobacterium tumefaciens EHA105 using the freeze-thaw method. Agrobacterium-mediated transformation was performed as previously described (Wang et al., 2021).

[0237] Immunoblot analysis

[0238] Immunoblotting was performed using the following antibodies: monoclonal anti-HA antibody (sc-7392; 1:500 dilution; Santa Cruz Biotechnology, Inc. Dallas, Texas, USA) to detect AroG* protein, and mouse monoclonal anti-Acv5 antibody (sc65499; 1:500 dilution; Santa Cruz Biotechnology, Inc. Dallas, Texas, USA) for FLS and STS proteins.

[0239] Metabolite extraction and analysis by LC-MS

[0240] Grape cells were collected on day 9 and washed three times with cold dH2O. After lyophilization, metabolites of 40 mg of cells were extracted according to the method described in reference 40. Separation and identification of metabolites were performed by using ultra-high performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer (UPLC-QTOF-MS, Waters, MA, USA). Analytical standards: Resveratrol and resveratrol glycosides were obtained from Sigma-Aldrich (St. Louis, MO, USA), and ε-grapevine was obtained from Extrasynthese (ZI Lyon Nord, Genay Cedex, France).

[0241] Phe feeding

[0242] As previously described (Wang et al., 2021), triplicate feeding experiments were performed 4 h after subculturing with 5 mM Phe (Merck Darmstadt, Germany). After 9 days, samples were collected for LC-MS and gene expression analysis.

[0243] Real-time PCR

[0244] Total RNA was isolated from grapevine cells by ZR Plant RNA MiniprepTM (Zymo Research, Irvine, USA), followed by DNase treatment (Qiagen, Valencia, CA, USA). First-strand cDNA was synthesized from 2 μg RNA using RevertAid reverse transcriptase (Thermo Fisher Scientific Inc, Waltham, MA, USA), and RT-qPCR was performed according to the protocol described in Wang et al., 2021. Relative expression was determined by normalizing to reference gene values ​​for ubiquitin and β-actin. PAL, CHS, F3'H, F3'5H, DFR members were identified based on grapevine PN 40024 12X V2 coverage.

[0245] Statistical analysis

[0246] LC-MS data are normalized to internal standard and sample weight. Metabolic profiles between control and transgenic samples or between AroG* strain and AroG*+FLS strain or AroG*+STS strain are compared by one-way ANOVA, followed by Dunnett's test. Tukey's HSD test is then performed to analyze the difference in gene expression before and after feeding in different strains by two-way ANOVA. All statistical analyses are performed using JMP 14.0 (SAS Institute, Inc, Cary, NC). Gene expression levels are shown by log2 fold change values, which are centered and scaled by row, and visualized in heat maps using "ggplot2" package (R v4.0.1 in RStudio).

[0247] Example 1. Generation of transgenic grape cv. Gamay Red cell cultures overexpressing AroG* and AroG*+STS genes.

[0248] Previous studies revealed that overexpression of AroG* in Gamay Red cell cultures resulted in elevated levels of Phe and its derived metabolites, including resveratrol (Manela et al., 2015). This suggests that the availability of Phe is a limiting factor for resveratrol production. To further increase stilbene levels, Gamay Red cell cultures were transformed with both (a) AroG*, a feedback-insensitive bacterial form of 3-deoxy-D-arabinoheptulonic acid 7-phosphate (DAHP) synthase (DAHPS) for increasing the availability of amino acids and, in particular, Phe, and (b) STS for directing carbon flow toward stilbene production.

[0249] Among the 48 grape stilbene synthase (VvSTS) genes in grape, these three STS genes were selected and cloned based on the results that the levels of resveratrol glycosides after transient expression of VvSTS5, VvSTS10 and VvSTS28 in N. benthamiana leaves were higher than those of other selected STS genes (Parage et al., 2012). Four constructs were designed, including AroG*, AroG*+STS5, AroG*+STS10, and AroG*+STS28 genes, all of which were expressed under the control of the 35S promoter ( Figure 1A ).

[0250] These four constructs were expressed in grape cv. Gamay Red cell culture using Agrobacterium tumefaciens-mediated transformation. The control was a wild-type cell line transformed with an empty vector containing only the kanamycin resistance cassette. Figure 1B ) and STS, and selected independent transformed strains for each construct. There were some differences in protein accumulation between strains ( Figure 1C ).

[0251] AroG* protein accumulation in transgenic lines revealed two polypeptides of ∼35 and 45 kDa ( Figure 1B ), which is consistent with the predicted sizes of the mature polypeptide and uncleaved protein, respectively, and similar to previous reports (Tzin, Oliva, Manela).

[0252] Prior to the experiments, transgenic cultures were grown in liquid medium for three weeks to increase the growth rate and maintain a homogeneous environment for the cells.

[0253] Four AroG* lines and three lines of each dual construct (AroG* and STS5, STS10, or STS28) were selected for metabolic analysis based on their stable viability. The growth rate of the AroG*+STS line in liquid culture (determined by the increase in culture fresh weight) was approximately 1.8-fold slower than that of the control and single AroG* lines ( Figure 1D The visual phenotype of transgenic cells was almost identical to that of control cells ( Figure 1E ).

[0254] Example 2. Effect of co-expression of AroG* and STS genes on metabolic profile of grape cell culture.

[0255] Metabolomic comparisons were performed between the control, AroG*, and AroG*+STS transgenic lines. Figure 1D ) were subjected to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) analysis to detect metabolites differentially accumulated in the transgenic lines.

[0256] Principal component analysis (PCA) was performed on the annotated metabolite data ( Figure 2A The changes in metabolite profiles produced a clear separation between the control and transgenic lines on the PCA plot ( Figure 2B The metabolite data between AroG* and AroG*+STS showed little difference. The PCA profiles showed that shikimate-derived metabolites and stilbenes contributed significantly to the differences between the control and transgenic lines (Table 1).

[0257] Table 1. Eigenvector values ​​of metabolites (in descending order) calculated by the PCA algorithm components.

[0258]

[0259] To obtain a comprehensive view of the effects of AroG* and STS transgenic expression on grape metabolism, the data were subjected to two metabolite comparison analyses: (i) comparison between control and transgenic lines and (ii) comparison between AroG* alone and double transgenic lines, where all lines from the same construct were treated as one group / pool ( Figure 3 and Figure 4 ).

[0260] All transgenic lines had significantly higher levels of two of the three aromatic amino acids derived from the shikimate pathway: Phe, tryptophan (Trp), and p-CA (p-coumaric acid) derived from Phe, which is a common precursor for both the stilbene and flavonoid pathways ( Figure 3 , top). These results are consistent with previous findings in Arabidopsis, tomato, petunia, and grape cell cultures (Manela et al., 2015; Oliva et al., 2015; Tzin et al., 2012; Tzin et al., 2015), in which AroG* overexpression led to increased production of amino acids and phenolic acids.

[0261] In the double transgenic line (AroG*+STS), the Phe level of the STS10 gene was significantly higher compared to AroG* alone, and the Trp and p-CA levels of the double transgenic lines AroG*+STS10 and AroG*+STS28 were significantly increased compared to AroG*. Figure 3 , above).

[0262] The transformed lines had significant effects on four annotated and quantified stilbenes, namely trans-resveratrol, cis-resveratrol, resveratrol, and ε-viniferin. In the AroG* line, resveratrol and ε-viniferin were significantly increased in the transgenic lines compared to the control ( Figure 3 The following figure, Figure 5 Compared with AroG* alone, stilbene levels were further increased in the double-transformed lines (AroG*+STS): trans- and cis-resveratrol glycosides were increased in AroG*+STS5 and AroG*+STS28, and ε-viniferin was increased in AroG*+STS5 and AroG*+STS10 ( Figure 3 The following figure, Figure 5 Interestingly, resveratrol levels were not significantly increased by overexpression of any of the three STS genes, except AroG* ( Figure 3 The following figure, Figure 5 ).

[0263] Of the four identified stilbenes in grape cell cultures, the only one that was significantly increased in all transgenic lines tested was ε-viniferin (Table 2). The line with the highest ε-viniferin levels was AroG*+STS10-182, which increased 74-fold compared to the control to a concentration of 0.74 mg / g dry weight (Table 2). In this same line, resveratrol levels were 9-fold higher than the control. The doubly transformed cell lines increased amino acid production and directed carbon flux toward the stilbene biosynthetic pathway, accumulating higher concentrations of additional stilbenes, and particularly ε-viniferin.

[0264] Table 2. Stilbene content (mg / g dry weight) in each cell line of AroG* and AroG*+STS. Values ​​are presented as mean ± SE (n=8). In the control n=24, including three lines. Bold numbers indicate significant increases in transgenic lines compared to the control lines (P<0.05, Dunnett's test).

[0265]

[0266] In contrast, the levels of flavonoids and anthocyanins (which have the same precursors as stilbenes, namely Phe and p-CA), were only slightly affected by overexpression of AroG* or AroG*+STS, and in most cases their levels were decreased compared to the control ( Figure 4 , Figure 5 The AroG*+STS28 line was exceptional, with increased levels of several anthocyanins and flavonoids, and the AroG*+STS5 line had higher levels of two flavonoids, quercetin-3-glucoside and proanthocyanidin B2 ( Figure 4 , Figure 5 ).

[0267] Example 3. Effects of Phe and p-CA feeding on the metabolic profile of the AroG*+STS transformed strain.

[0268] Metabolomic analysis of transgenic AroG* and AroG*+STS lines clearly showed that substrate availability was the bottleneck for stilbene production. To test whether further increases in precursors for stilbene production would lead to even higher levels of stilbene, transgenic grape cell lines were fed Phe or p-CA. The AroG*+STS28 transgenic line had the most consistent effect on stilbene levels, with all three lines having significantly higher levels of viniferin, resveratrol, and t-resveratrol glycosides (Table 2). Therefore, the strongest AroG*+STS28 (line 16) was selected to test the potential to further increase stilbene accumulation by feeding precursors of these metabolites.

[0269] The concentrations of Phe and p-CA selected for the feeding experiments were those at which cell viability was not affected but cell growth was slower. To compare growth rates, the fresh weight (FW) of cells per ml of growth medium was measured on day 7 of growth. On day 7, the weights of cells treated with 0.2 mM and 0.5 mM Phe were similar to the controls, while the weights of cells grown in 1 mM, 2 mM, and 5 mM Phe were significantly lighter ( Fig. 6A Since p-CA is not water soluble, the effects of the treatment were compared to cells grown in 0.2% ethanol; cells grown in 0.3 mM p-CA grew slower ( Figure 6B Even though the viability of cells treated with 0.5-5 mM Phe and 0.1-0.3 mM p-CA was similar, their growth patterns were different, forming larger clumps in liquid culture ( Figure 6C ).

[0270] Feeding Phe at 2mM and 5mM concentrations caused a significant increase in the content of resveratrol and ε-viniferin in the double-transformed line AroG*+STS28-16 ( Fig. 7A When treated with 5 mM Phe, the most significant increase in both stilbenes occurred on day 9, when cell cultures are known to accumulate the most polyphenols (Manela et al., 2015). Resveratrol levels increased 6-fold, resulting in a concentration of 1.26 mg / g dry weight, while ε-viniferin levels increased 30-fold, up to 10.8 mg / g dry weight ( Fig. 7A Feeding the AroG*+STS28-16 strain with p-CA significantly increased the levels of stilbenes, and especially ε-glucose, and the most significant increase in ε-glucose was seen with 0.3 mM p-CA treatment on day 7 ( Figure 7B ).

[0271] Phe and p-CA feeding of strain AroG*+STS28-16 also caused a significant increase in several flavonoids ( Figure 8 and Fig. 9 ). The levels of most anthocyanins and their derivatives were significantly increased in cells fed with 5 mM Phe compared to the control ( Figure 8 The AroG*+STS28-16 line fed with 0.3 mM p-CA had higher levels of two flavonoids, epigallocatechin-3-O-glucoside and petunidin-3-O-glucoside ( Fig. 9 ).

[0272] In summary, the strongest effect on stilbene production due to transformation and feeding with Phe was an increase of approximately 24-fold and 600-fold in resveratrol and naringenin concentrations, respectively, in line AroG*+STS28-16. This precursor-based feeding strategy in transgenic lines did not induce changes in the expression of STS and chalcone synthase or naringenin-chalcone synthase (CHS) genes ( Fig.10 and Fig.11 ).

[0273] Example 4. Attenuation of LPS-induced cytokine storm in chicken white blood cells (WBC) by GCE.

[0274] Rationale: Since WBCs play a major role in the onset of cytokine storm, this part of the study provides basic data for more focused in vivo experiments (Example 5).

[0275] Experimental design: Grape cells were collected on day 9 and washed twice with cold dH2O, lyophilized and frozen at -80°C (GCP). Polyphenols were extracted from GCP as follows: Samples were homogenized in a frozen mixer mill with metal beads. Then, pre-cooled 70% methanol was added to the test tube. Samples were incubated on an orbital shaker at room temperature for 20 minutes and centrifuged at full speed for 10 minutes, and the supernatant was transferred to a new tube (70% methanol in GCE). The supernatant was vacuum dried in a Speed ​​Vac concentrator at room temperature and dissolved in 50% DMSO. The final solution was stored at -80°C for further analysis and use (50% DMSO in GCE).

[0276] Male laying hens were purchased and raised. At day 14 of age, blood was drawn and WBCs were purified and treated with GCE and LPS, as Fig.12 The GCE formulation was described in the previous article, but with a larger GCE dose range (corresponding to 0.1 mg / ml to 1 mg / ml powder), loner and shorter pre-intubation time (0 to 16 hours), and a more complete probe of multiple cytokines. Several experiments were performed to optimize GCE for the improvement of cytokine storm in triplicate. The optimized protocol was further used to compare the efficiency of the new formulation of GCE with the old formulation used in in vivo studies (in rats), thereby helping to estimate the effective dose range for in vivo studies.

[0277] Results: Based on preliminary results, these experiments allowed for detailed characterization of the effects of GCE on leukocytes, laying the foundation for in vivo studies.

[0278] Example 5. Attenuation of LPS-induced cytokine storm in chickens by feeding with GCP.

[0279] Rationale: LPS challenge is a well-characterized model of cytokine storm in mammals and chickens, thus providing a good assay system for inhibition of cytokine storm by GCE in vivo. In the experiments of Example 6, this set of experiments enabled more focused studies with fewer unknown variants.

[0280] Experimental design: 20 broiler type chickens at weeks of age (purchased from Brown and sons, LTD) were randomly divided into 2 groups, 10 in each group. The treated chickens were fed grape cell powder (GCP; ~170 mg / Kg BW / day) for 7 days. The control group was fed a conventional formula. On the 7th day, 2 hours before killing, lipopolysaccharide (LPS; 1 mg / Kg BW) was injected into the wing vein of all chickens. The spleen was removed immediately after killing by cervical dislocation and stored in RNA-Later. RNA was extracted using a total RNA micro kit (Geneaid) and mRNA expression levels were analyzed by qPCR according to standard procedures.

[0281] Results: GCP feeding in chickens ameliorated the induction of cytokine storm surge. Fig.13 Shown is suppression of mRNA expression levels of pro-inflammatory cytokines (INF-G, TNF, IL-6), and induction of anti-inflammatory cytokine mRNA (IL-10) in spleens of chickens fed with grape cell powder for 7 days and stimulated with LPS for 2 hours compared to spleens from LPS-fed chickens.

[0282] In conclusion, feeding with grape cell powder (GCP) reduced proinflammatory cytokines and induced anti-inflammatory cytokines. These results support the use of GCP to prevent CRS or cytokine storm.

[0283] Example 6. Attenuation of IBV-induced cytokine storm and disease symptoms by feeding with GCP.

[0284] Experimental Design: Male chicks were purchased and maintained. At 14 days of age, these chicks were infected with an attenuated IBV vaccine (H-120, Biovac, Or-Akiva, Israel) at doses 4 and 8 times higher than conventional vaccination, directly inoculated into the alveoli on both sides of the chicks. On days 5 and 10 after the virulent IBV challenge, clinical signs of IBV infection (coughing, rattling, body temperature) were recorded daily until recovery or death, and the viral load in tracheal swab samples was determined by RT-qPCR. After selecting the optimal infection conditions, experiments using GCP to treat IBV infection were performed with a similar experimental design described in Example 5, but only using the optimized GCP dose and treatment schedule. The number of chicks / group was tripled to include a group for long-term tracking of disease symptoms in addition to the characteristics of cytokine storm improvement. Tissue sampling was as in Example 5, but with the addition of lungs and trachea. All tissue samples were split into 3 for RNA analysis by qPCR, as well as protein mass spectrometry and RNA-seq, and these were used only for experiments with the best cytokine storm suppression.

[0285] Results: High-dose vaccination induced disease symptoms highly similar to those of the human SARS-CoV family of human coronaviruses, and feeding with GCP reduced the severity of cytokine storm and disease symptoms.

[0286] Example 7. Attenuation of IBV-induced cytokine storm (CS) and disease symptoms by GCE applied via Alzet osmotic pumps.

[0287] Rationale: One of the advantages of the present invention is that grape cell lines overexpress stilbenes, mainly epsilon viniferin, which is known to have strong anti-inflammatory activity. In order to ensure the maximum bioavailability of the unique stilbenes mixture and its synergistic effect, the impact of application by feeding into a slow release system using an Alzet osmotic pump was tested.

[0288] Experimental design: The osmotic pump ( 2ML4 pump, allowing a flow rate of 2.5μl / hour), was implanted subcutaneously at the 7th cervical vertebra area. The timing of implantation and IBV infection was determined based on the optimization experiments in Examples 5 and 6.

[0289] Results: GCE had a higher improvement effect on CS.

[0290] Example 8. Generation of transgenic grape cv. Gamay Red cell cultures co-expressing AroG* and FLS genes.

[0291] To divert carbon flux towards flavonoid production, grape cells of the Vitis vinifera cv. Gamay Red were transformed with a construct containing both AroG* and flavonol synthase (FLS) under the control of the 35S promoter ( Fig.14A , Fig. 14B Of the six FLS genes identified in grapevine (Anesi et al., 2015; Fugita et al., 2006), the one selected for transformation was VIT_07s0031g00100 (NCBI: XM_002283156) because it was the only FLS gene expressed in grapevine Gamay Red cell culture. This FLS gene was cloned to prepare a construct containing both AroG* and FLS ( Fig. 14B ), used for transformation of grape cell cultures.

[0292] Analysis of AroG* and FLS protein accumulation in independently transformed cell lines ( Fig. 14C , Fig.14D Based on their activity, four of the ten transgenic lines expressing these two proteins were selected for extensive metabolomic analysis ( Figure 14C-14E ).

[0293] Example 9. Effect of co-expression of AroG* and FLS genes on metabolic profile of grape cell culture.

[0294] Similar to the AroG* line, the transformed AroG*+FLS line accumulated higher levels of Phe and p-coumaric acid (p-CA) compared to the control ( Fig.15 ). These two metabolites are common precursors of stilbenes and flavonoids. In addition, several AroG*+FLS lines had even higher levels of Phe and p-CA than the AroG* lines, and additionally accumulated high levels of tryptophan (Trp) ( Fig.15 ).

[0295] Interestingly, co-expression of AroG*+FLS resulted in an increase in stilbene levels in grape cell cultures, similar to that caused by transformation with AroG* alone ( Fig.16 This was true even though the FLS gene is part of the flavonoid biosynthetic pathway. In most cases, the levels of the four different stilbenes identified in the cell cultures (i.e., resveratrol, trans- and cis-resveratrol glycosides, and ε-viniferin) were similar to those in the individual AroG* transformed lines and were higher than in the controls. Resveratrol levels increased approximately 6-fold (0.3 mg / g DW) and viniferin levels increased approximately 30-fold (0.3 mg / g DW), while the other stilbenes increased to lower levels ( Fig.16 ). Obviously, the co-expression of AroG*+FLS did not reduce the metabolic flux toward stilbene.

[0296] AroG*+FLS transformation also resulted in an increase in flavonoid levels in grape cell cultures. Levels of the two direct products of the FLS enzyme, flavonols myricetin and quercetin-3-O-glucose, increased significantly (Figure 17, Figure (a)). Compared to the control and a single AroG* line, two of the four AroG*+FLS lines accumulated significantly higher levels of flavonols (up to 3.5 times). Additional flavonoids increased due to co-expression, including flavan-3-ols and anthocyanins, reaching higher levels than the AroG* and control lines (Figure 17).

[0297] Flavonoid levels in the AroG* lines were lower or the same compared to the control, with the exception of malvidin, which was higher in the AroG* lines. The AroG*+FLS lines had significantly higher levels of flavonoids, including 5 identified flavan-3-ols and 15 anthocyanidin glucosides, compared to AroG*, and in some cases compared to the control (Figure 17 panels (b), (c)). Of the four AroG*+FLS lines, line 2 stood out with very low levels of several flavan-3-ols and anthocyanidins. Interestingly, this line had exceptionally high levels of Phe, p-CA, and viniferin ( Fig.15 , Fig.16 ).

[0298] These metabolomics results demonstrated that transformation of grape cv. Gamay Red grape cell cultures with AroG*+FLS increased total carbon flux toward phenylpropanoid production compared to the AroG* line. Specifically, stilbenes accumulated to similar levels in both cases, in addition to increased levels of flavonoids in the AroG*+FLS line.

[0299] Example 10. Effect of Phe feeding on stilbene and flavonoid levels in AroG*+FLS transformed lines.

[0300] The availability of Phe is the rate-limiting factor for stilbene production in Gamay Red cell cultures. The AroG*+FLS line accumulated high levels of both stilbene and flavonoids. To test further increases in the production of both phenylpropanoid subgroups, cell cultures were fed with Phe.

[0301] The AroG*+FLS line selected for the Phe feeding experiment was the line with the highest levels of both stilbenes and flavonoids (line 22). In the control, AroG* and AroG*+FLS-22 lines, Phe feeding (5 mM) increased both stilbenes and flavonoids in all three lines and particularly in the AroG*+FLS-22 line (Table 3).

[0302] Table 3. Effects of Phe feeding on phenylpropanoid, flavonoid, and stilbene levels in control, AroG*, and AroG*+ FLS.

[0303]

[0304] Values ​​(mean ± SE, n = 3) are fold changes compared to unfed controls. Shaded metabolites indicate that these values ​​were significantly increased (P < 0.05) using one-way ANOVA followed by Dunnett's test. Bold metabolites are metabolites with higher levels in the AroG*+FLS line compared to the AroG* line, analyzed by student's t-test (P < 0.05). Abbreviations: acet, acetyl; glu, glucoside; coum, coumaroyl.

[0305] In all three strains, Phe levels in cells increased as a result of exogenous feeding. Both Trp and p-CA in the AroG*+FLS-22 strain increased to significantly higher levels than both the control and AroG* strains (Table 3). Stilbene and especially resveratrol and vinblastine increased significantly in all strains as a result of Phe feeding, with similar and significant increases in the AroG* and AroG*+FLS-22 strains (Table 3, Fig.18A ).

[0306] Phe feeding caused a significant increase in the two products of the FLS enzyme, the flavonols myricetin and quercetin-3-glucoside, in AroG*+FLS-22, and a smaller increase in flavan-3-ols in the AroG*+FLS-22 line (Table 3). In addition, anthocyanidin levels also increased in all three lines, mainly in delphinidin-based anthocyanidins (Table 3, Fig.18A ). The fold change increase of anthocyanins was significantly higher in line AroG*+FLS-22.

[0307] The AroG* lines had significantly higher levels of stilbenes (further enhanced by Phe feeding). The only anthocyanidin with higher levels in the AroG* lines, malvidin-based anthocyanidins, was slightly increased by Phe feeding ( Fig.18A In the AroG*+FLS-22 line, stilbene levels were increased compared to the control and similar to the AroG* line, and anthocyanin levels were significantly increased compared to both lines. Phe feeding of the AroG*+FLS-22 line enhanced this effect and resulted in a further increase in both stilbene and flavonoids ( Fig.18A ).

[0308] Example 11. Examination of whether metabolomic changes due to AroG*+FLS conversion and Phe feeding are dependent on changes in gene expression levels.

[0309] The effects of transformation with AroG*, AroG*+FLS-22, and Phe feeding on gene expression levels in grape cell cultures were tested by quantitative real-time PCR analysis. The expression levels of genes along the stilbenoid and flavonoid biosynthetic pathways as well as transcription factors known to affect these pathways were determined. Of the 48 V. grape V. Vitis vinifera V., three were selected for gene expression analysis (VvSTS5, VvSTS10, and VvSTS28) based on the fact that these three genes caused the highest increase in several stilbenes when they were transiently expressed in Nicotiana benthamiana leaves (Parage et al., 2012). In addition, these three STS genes represent the three major VvSTS subphylogenetic families (Vannozzi et al., 2012). Of the six V. grape V. Vitis vinifera FLS genes, only one was expressed in grape cell cultures, the same FLS gene overexpressed in the transgenic AroG*+FLS line. The expression levels of four R2R3-MYB transcription factors were analyzed: MYB14 and MYB15, which regulate stilbene biosynthesis (Holl et al., 2013), and MYBPA and MYBA, which regulate flavonoid biosynthesis in grapevine (Czemmel et al., 2012).

[0310] AroG* overexpression had relatively minor effects on gene expression levels, with several genes such as STS10 and MYB14 having decreased expression ( Fig.18B One gene that was significantly induced (15-20 fold) by the introduction of the AroG* transgene was F3'5'H, which directs flavonoid biosynthesis toward delphinidin-related anthocyanidins. This induction was associated with an increase in malvidin anthocyanidin levels in the transgenic line ( Fig.18A ). The expression levels of the three STS genes were lower in the AroG* strain.

[0311] The gene expression pattern differed with the additional overexpression of FLS. Co-expression of AroG* and FLS resulted in a significant induction of genes along the phenylpropanoid pathway, including PAL, C4H, and 4CL, as well as many genes along the anthocyanin biosynthesis pathway ( Fig.18B This was associated with an increase in flavonoids and anthocyanins in this line. The induction in LAR was directly associated with an increase in the levels of several flavan-3-ols in the AroG*+FLS-22 line ( Fig.18B Here too, apart from a small increase in MYB15, no STS genes were significantly induced despite a clear increase in stilbene levels.

[0312] Feeding with exogenous Phe had a slight effect on gene expression levels in the control lines, including induction of the F3'5'H gene, associated with an increase in delphinidin-related anthocyanins due to this feeding ( Fig.18B The most dramatic effect of Phe feeding was on the AroG* line, resulting in the induction of PAL, C4H and 4CL as well as several genes along the flavonoid and anthocyanin pathways to levels similar to those of unfed AroG*+ FLS cells.

[0313] These results demonstrate that both stilbenes and flavonoids were significantly increased in grape cell cultures by transforming cells with AroG* (for increased Phe production) and FLS (for diverting carbon flux toward flavonoid biosynthesis). This co-expression and addition of exogenous Phe resulted in grape cells enriched for both groups of health-promoting compounds ( Fig.18A When Phe availability was high in cultures of grape cv. Gamay Red cells by overexpression of AroG* plus external Phe feeding, stilbene levels increased significantly, whereas the increase in flavonoids was minimal ( Fig.18A ). However, co-expression of AroG*+FLS resulted in similar stilbene levels, but additionally increased accumulation of flavonoids compared to the cell line transformed with AroG* alone. This demonstrates that the total carbon flux towards both stilbene and flavonoid biosynthesis is increased in the AroG*+FLS line compared to the AroG* line (Figure 18, Table 3).

[0314] In conclusion, increasing Phe availability and overexpressing FLS increased the overall carbon flow towards phenylpropanoid production, leading to enhanced levels of both stilbenes and flavonoids in cell suspensions of grape cv. Gamay Red that promote a healthy metabolome.

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Claims

1. A method for maintaining a doubly transgenic grape cell comprising at least one copy of AroG * gene and at least one copy of stilbene synthase STS gene, or at least one copy of AroG * gene and at least one copy of flavonol synthase FLS gene, the method comprising contacting the cell with a composition comprising: (a) Phenylalanine concentrations between 0.2 mM and 5 mM, (b) p-coumaric acid at a concentration between 0.1 mM and 0.3 mM, or (c) any combination of (a) and (b), The FLS gene is VIT_07s0031g00100, NCBI: XM_002283156, and the STS gene is selected from: VvSTS5, VvSTS10 or VvSTS28.

2. The method of claim 1, comprising contacting the cell with a composition comprising phenylalanine at a concentration between 2 mM and 5 mM.

3. The method of claim 2, comprising contacting the cell with a composition comprising phenylalanine at a concentration of 5 mM.

4. The method of claim 1, comprising contacting the cell with a composition comprising p-coumaric acid at a concentration of 0.3 mM.

5. A pharmaceutical composition comprising at least one copy of the AroG* gene and at least one copy of stilbene synthase STS gene, or at least one copy of AroG * gene and at least one copy of flavonol synthase FLS The double transgenic grape cell containing the gene, wherein the FLS gene is VIT_07s0031g00100, NCBI: XM_002283156, and the STS gene is selected from: VvSTS5, VvSTS10 or VvSTS28.

6. The pharmaceutical composition according to claim 5, wherein the double transgenic grape cells are maintained by the method according to any one of claims 1 to 4.

7. The pharmaceutical composition according to claim 5 or 6, comprising: (i) at least one amino acid selected from the group consisting of phenylalanine, tryptophan and p-CA; (ii) at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine; (iii) at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1 and procyanidin B2; (iv) at least one anthocyanin selected from the group consisting of cyanidin, peonidin, delphinidin, petunidin and malvidin; or (v) any combination of (i), (ii), (iii) and (iv).

8. The pharmaceutical composition according to claim 5 or 6, which is a dehydrated composition.

9. The pharmaceutical composition according to claim 5 or 6, which does not contain intact cells.

10. The pharmaceutical composition according to claim 5 or 6, which does not contain disrupted cells.

11. The pharmaceutical composition of any one of claims 5 or 6 for use in the preparation of a medicine for preventing coronavirus infection or its symptoms, treating coronavirus infection or its symptoms, reducing coronavirus infection or its symptom incidence, suppressing coronavirus infection or its symptoms in a patient in need, wherein the symptom is a cytokine storm.

12. The use according to claim 11, wherein the pharmaceutical composition is for systemic administration.

13. The use according to claim 11, wherein the pharmaceutical composition is for oral administration.

14. The method of claim 11, wherein the coronavirus infection comprises severe acute respiratory syndrome coronavirus 2 infection.

15. Use of the pharmaceutical composition according to any one of claims 5 or 6 in the preparation of a medicament for treating, preventing, ameliorating, inhibiting cytokine release syndrome or cytokine storm or reducing the incidence of cytokine release syndrome or cytokine storm in a patient in need thereof.

16. The use according to claim 15, wherein the cytokine release syndrome or cytokine storm is associated with coronavirus infection.

17. The method of claim 16, wherein the coronavirus infection comprises severe acute respiratory syndrome coronavirus 2 infection.

18. Use of the pharmaceutical composition of any one of claims 5 or 6 in the preparation of a medicament for preventing or treating increased cytokine levels in a patient in need thereof.

19. The use according to claim 18, wherein the cytokine is selected from the group consisting of IL-6, IFN-γ, TNF-α and IL-1-β.

20. The use according to claim 18 or claim 19, wherein the increase in the cytokine level is associated with coronavirus infection.

21. The use according to claim 20, wherein the increase in the level of the cytokine is measured in the leukocytes of the patient or in the serum of the patient.

22. A method for increasing the level of at least one stilbene selected from the group consisting of trans-resveratrol glycosides, cis-resveratrol glycosides, resveratrol and ε-grapevine in a grape cell, the cell comprising at least one copy of AroG * gene and at least one copy of stilbene synthase STS gene, the method comprising contacting the cell with a composition comprising: (a) phenylalanine at a concentration between 0.2 mM and 5 mM, (b) p-coumaric acid at a concentration between 0.1 mM and 0.3 mM, or (c) any combination of (a) and (b), The STS gene is selected from: VvSTS5, VvSTS10 or VvSTS28.

23. A method for increasing the level of at least one flavonoid selected from the group consisting of quercetin-3-glucoside, myricetin, catechin, epicatechin, epigallocatechin, procyanidin B1 and procyanidin B2 in grape cells, the cells comprising at least one copy of the AroG* gene and at least one copy of a flavonol synthase FLS gene, the method comprising contacting the cell with a composition comprising: (a) phenylalanine at a concentration between 0.2 mM and 5 mM, (b) p-coumaric acid at a concentration between 0.1 mM and 0.3 mM, or (c) any combination of (a) and (b), The FLS gene is VIT_07s0031g00100, NCBI: XM_002283156.

24. An edible or drinkable composition comprising the pharmaceutical composition of claim 5 or 6.

25. The pharmaceutical composition of claim 5 or 6, formulated for slow release or extended release.

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