Reprogrammed dysfunctional natural killer cells and uses thereof

Abstract

The present disclosure relates to a modulator and / or a re-programmer for activating dysfunctional natural killer (NK) cells, and personalized therapeutic methods and uses thereof. The disclosed reprogramming agent comprises a compound that specifically inhibits the expression, activity and / or stability of at least one member of the EGR and / or the DGK family.

Description

[0001] REPROGRAMMED DYSFUNCTIONAL NATURAL KILLER CELLS AND USES THEREOF

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to the field of immune modulation. More specifically, the present disclosure relates to reprogramming dysfunctional natural killer (NK) cells, and uses thereof.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] 1. Morita, K., Okamura, T., Sumitomo, S., Iwasaki, Y., Fujio, K., & Yamamoto, K. (2016). Emerging roles of Egr2 and Egr3 in the control of systemic autoimmunity. Rheumatology (United Kingdom), 55, ii76-ii81. https: / / doi.org / 10.1093 / rheumatology / kew342

[0007] 2. Symonds, A. L. J., Miao, T., Busharat, Z., Li, S., & Wang, P. (2023). Egr2 and 3 maintain antitumour responses of exhausted tumour infiltrating CD8 + T cells. Cancer Immunology, Immunotherapy, 72(5), 1139-1151. https: / / doi.org / 10.1007 / s00262-022-03319-w.

[0008] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0009] BACKGROUND

[0010] Natural killer (NK) cells are lymphocytes of the innate immune system, providing the first line of immunosurveillance against viral infections and tumor growth. Most human NK cells express inhibitory receptors, such as the killer cell immunoglobulin-like receptors (KIRs) in humans and the heterodimeric inhibitory receptor CD94-NKG2A (hereafter referred to as NKG2A) in humans and mice, all of which recognize major histocompatibility complex class-I (MHC-I) molecules. The immune system employs intricate regulatory mechanisms to ensure that immune cells distinguish foreign invaders from healthy tissues. NK cells eliminate target cells lacking the expression of MHC-I molecules, whereas MHC-I-expressing cells are unaffected by NK cells, accounting for NK cell tolerance. In this regard, NK cells are mainly tuned by target cells expressing MHC-I molecules in their surroundings (Long et al, 2013, Annu Rev Immunol 31 :227- 258). This process of self-tuning, calibrated through “education” allows NK cells to acquire functional competence and host-specific adaptations (Orr and Lanier, 2010, Cell 142:847-856). Overall, NK cell education is determined by coordinating inhibitory, activating, and adhesion signals, through which inhibition and activation of NK cells are functionally linked (He and Tian, 2017, Cell Mol Immunol 14:321-330). This allows NK cells to assess the balance of activating versus inhibitory signals they receive.

[0011] The NK education models, including the licensing, disarming, rheostat, and confining models, propose that classical killing inhibitory receptors (KIR) play an instructive role in NK cell responsiveness (Boudreau and Hsu, 2018, Trends Immunol 39:222; Zhang et al, 2019, Nat Commun 10:5010). These “instructive” inhibitory receptors contain immune-receptor tyrosinebased inhibitory motifs (ITIM) in their cytoplasmic tails, with subsequent recruitment and activation of protein tyrosine phosphatases (PTP), including the Src homology region-2 domain (SH2)-containing phosphatase 1 / 2 (SHP-1 / 2), or SH2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), following binding to MHC-I. Recently, it was shown that SHP-1 expression levels and NK cell functional responsiveness are tightly linked, suggesting the involvement of SHP-1 in the molecular control of the rheostat determining NK cell responsiveness (Wu et al, 2021, Sci Signal 14:eabe5380). However, the molecular mechanisms underlying the control of NK cell responsiveness remain elusive. Due to the diversity in the affinity and the amount of self-MHC-I inhibitory receptors among NK cells, the strength of the educating signal differs from cell to cell (Brodin et al, 2009, Trends Immunol 30:143-149). Signaling mediated by inhibitory receptors, such as NKG2A and KIR, acts primarily at the early stages of Immunological synapse (IS) formation, to abolish the activating signals (Long et al, 2013, Annu Rev Immunol 31:227-258). Following education, NK cell reactivity increases with the number of different self- MHC-I-specific inhibitory receptors expressed (Thomas et al, 2013, J Immunol 191:3981-3985; Jaeger and Vivier, 2012, J Clin Investig 122:3053). If the target cell lacks MHC-I or expresses low levels of this surface marker, it results in NK cell activation and clearance of the target. However, failure to engage inhibitory receptors during development, due to lack of inhibitory receptor expression on the NK cell or lack of interaction with MHC-I, results in the generation of a subset of non-responsive peripheral NK cells termed “anergic cells” (Kim et al, 2005, Nature 436:709- 713; Fernandez et al, 2005, Blood 105:4416-4423). “Anergy” describes a state in which the NK cell is intrinsically functionally impaired (Joncker et al, 2010, J Exp Med 207:2065-2072). It was suggested that anergy might be an induced state, resulting from chronic exposure of NK cells to activating ligands without proper inhibitory signaling (Brodin et al, 2009, Trends Immunol 30:143-149; Tripathy et al, 2008, J Exp Med 205:1829-1841). On the other hand, in the context of cancer, inhibitory receptor signaling destabilizes the IS, and promotes NK cell detachment and migration (Burshtyn et al, 2000, Curr Biol 10:777-780). Upon encountering target cells expressing activating ligands but lacking MHC- I, NK cells are highly activated. However, if persistent, the over- activation of NK cells leads to their desensitization abolishing further interactions with additional targets such as cancer cells, leading to an “exhausted” NK cell state (Jaeger and Vivier, 2012, Cold Spring Harb Perspect Biol 4:a007229). Accordingly, NK cell dysfunction reflects different states, including anergy and exhaustion, each with a distinct etiology. “Anergic” NK cells are naturally unresponsive peripheral blood cells, constituting ~13 ± 6% of the entire NK cell population (Raulet et al, 2003, Annu Rev Immunol 19:291-330; Fernandez et al, 2005, Blood 105:4416-4423). They lack expression of MHC-I specific inhibitory receptors, and are not autoreactive, but rather tolerant to self (Anfossi et al, 2006, Immunity 25:331-342; Fernandez et al, 2005, Blood 105:4416-4423). In contrast, “exhausted” NK cells arise from chronic viral infections, inflammation, or cancer, because of their overstimulation by their targets.

[0012] NK cells play a complementary role to T cells in tumor immunity by recognizing tumors that downregulate MHC-I expression and escape CD8+ T cell-mediated tumor clearance (Fruci et al, 2013, J Transl Med 11 : 1M-; Eanier, 2008. Nat Immunol 9:495-502). Although the anti-tumor role of NK cells has been described in hematological malignancies, their role in the solid tumor milieu remains unclear due to their lack of activity in the tumor microenvironment (TME) (Kreisel et al, 2012, Cancer Res 72:4311-4317). Clinical observations highlight NK cells as a critical component in the anti-tumor response (Remark et al, 2013. Clin Cancer Res 19:4079-4091; Eckl et al, 2012, J Mol Med 90:55-66), but despite tumor infiltration, a functional NK-mediated anti-tumor response is often lacking in the TME (Childs and Carlsten, 2015, Nat Rev Drug Discov 14:487- 498; Schleinitz et al, 2010, Immunology 131:451-458; Kwon et al, 2017, Exp Mol Med 49:e311). While significant progress has been made in understanding the molecular mechanisms of T-cell dysfunction, the equivalent pathways remain relatively unexplored for NK cells. Despite their clinical relevance, the heterogeneity, molecular, and transcriptomic landscape underlying NK cell dysfunction remain poorly defined. Furthermore, both NK cell anergy and exhaustion states are typically characterized by decreased effector function or proliferation (Judge et al, 2020, Front Cell Infect Microbiol 10:49).

[0013] Early Growth Response (EGR) is a family of zinc finger transcription factors, including EGR1, EGR2, EGR3, and EGR4, that regulate gene expression in response to various cellular stimuli. EGR proteins play a pivotal role in the immune response by modulating T-cell activation, differentiation, and cytokine production.

[0014] Egr2 and Egr3 were shown to be important for preventing autoimmunity activity of immune cells [1]. Symonds et al [2] have shown that deficiency of Egr2 and Egr3 in T cells resulted in enhanced tumor growth and fewer tumor infiltrating T cells (TILs) in mouse models, thereby demonstrating the critical role of Egr2 and Egr3 in sustaining the anti-tumor activity of exhausted CD8+ TILs.

[0015] GENERAL DESCRIPTION

[0016] The present disclosure provides herein key intrinsic checkpoints in NK “anergy”, including early growth response (EGR)-2 and diacylglycerol kinase (DGK)-a , and reveal functional, phenotypic and transcriptional similarity with NK cell “exhaustion”. The inventors further demonstrate in situ reprogramming of “anergic” and “exhausted” NK cells by modulation of these key intrinsic regulators, DGKa and Egr2, via a nanoparticle (NP)-based drug delivery platform (Biber et al, 2021, EMBO Mol Med 14:el4073), both in vitro and in vivo, revealing that the identified markers are critical to reprogram both the dysfunctional states. In particular, the inventors' in vivo model of NK cell exhaustion reveals that gene silencing of Egr2 empowers NK cells to effectively control tumor growth and enhance NK cell effector functions for improved tumor lysis and clearance. Furthermore, the inventors' data suggest that “anergy” and “exhaustion” are not simply intrinsic non-responsive states, but that these newly identified targets can potentially enable these dysfunctional NK cells to be reprogrammed in their native environment, to become functional in diverse contexts, including cancer and viral infections.

[0017] A first aspect of the preset disclosure relates to a re -programming agent (also referred to herein as a modulator and / or re -programmer or an activator), comprising at least one compound that specifically inhibits the expression, activity and / or stability of at least one of: (i), at least one member of the Early Growth Response (EGR) family of transcription factors; and (ii), at least one member of the Diacylglycerol Kinase (DGK) family. In some embodiments the disclosed reprogramming agent may provided in a nano- or micro-particle, micellar formulation, vehicle, matrix, or a composition and thus the present disclosure further encompasses any nano- or microparticle, micellar formulation, vehicle, matrix, or composition comprising the disclosed reprogramming agent. In some embodiments, the disclosed re-programming agent activates, rewires and / or activates at least one lymphocyte. In more specific embodiments, the disclosed reprogramming agent rewires at least one dysfunctional lymphocyte. In some embodiments, the disclosed lymphocytes may be NK cells, specifically, dysfunctional NK cells. In yet some alternative or additional embodiments, the disclosed lymphocytes may be T cells, specifically, dysfunctional T cells. Thus, in some embodiments, the disclosed re -programming agent activates dysfunctional natural killer (NK) cells. In some embodiments, the present disclosure relates to a re-programming agent that comprises an effective amount of the at least one compound that specifically inhibits the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family. The effective amount as used herein is the amount sufficient for activation and / or re-wiring of the dysfunctional NK cells. A further aspect of the present disclosure relates to at least one nano- or micro-particle, micellar formulation, vehicle or matrix comprising at least one reprogramming agent comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and / or (ii), at least one member of the DGK family; wherein said reprogramming agent activates dysfunctional NK cells. Thus, in some embodiments, the at least one nano- or micro-particle, micellar formulation, vehicle or matrix disclosed herein may comprise an effective amount of the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix activators. In et some specific embodiments, the amount of the re-programming agent in the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix is the amount effective for activation of NK cells, specifically, activation of dysfunctional NK cells.

[0018] A further aspect of the present disclosure relates to a pharmaceutical composition comprising an effective amount of at least one re-programming agent comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix comprising the re-programming agent. More specifically, the re-programming agent of the disclosed composition activates dysfunctional NK cells. The composition further comprises at least one of pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s. It should be understood that in some embodiments, the disclosed composition comprises an effective amount of the disclosed re -programming agent that is sufficient for and effectively activate and / or for rewiring NK cells, specifically, dysfunctional NK cells.

[0019] A further aspect of the present disclosure relates to a method for activating and / or re-wiring at least one dysfunctional NK cell. The method comprising the step of contacting the dysfunctional NK cell with an activating effective amount of at least one re-programming agent comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent. In some embodiments, the re-programming agent activates and / or re-wires dysfunctional NK cells.

[0020] A further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof. The method comprising the step of administering to the subject a therapeutically effective amount of at least one re-programming agent comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent, wherein said re -programming agent activates dysfunctional NK cells.

[0021] A further aspect of the present disclosure relates to at least one re-programming agent or any nano- or micro-particle, micellar formulation, vehicle, matrix, cell or composition comprising said reprogramming agent, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof. The re-programming agent used herein comprises at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and / or (ii) at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising said reprogramming agent, wherein said re -programming agent activates dysfunctional NK cells.

[0022] A further aspect of the present disclosure relates to a kit comprising: (I), at least one reprogramming agent comprising at least one compound that specifically inhibits the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors, in a first dosage form; and / or (ii), at least one member of the DGK family, in a second dosage form; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising said re -programming agent, wherein said re-programming agent activates dysfunctional natural killer (NK) cells. The disclosed kit may optionally further comprise: (II), at least one additional immunomodulatory therapeutic agent, in a third dosage form. A further aspect of the present disclosure relates to a method for determining a personalized treatment regimen for a subject suffering from an immune-related disorder, and optionally for monitoring the effectiveness of a treatment regimen in the subject. The method comprising the steps of: In step (a), determining the levels of at least one of ERG2 and DGKa in at least one NK cell of at least one biological sample of said subject, to obtain the level value of said at least one of ERG2 and DGKa. In step (b), classifying said subject as: (i), a subject displaying a decreased NK cell functioning, (or having dysfunctional NK cells), if the level value determined in (a), for the at least one of: ERG2 and / or DGKa in the at least one sample is higher as compared to a predetermined standard value. Alternatively, the subject is classified as (ii), a subject displaying functioning NK cells, if the level value determined in (a) for the at least one of ERG2 and DGKa in the at least one sample is equal or lower as compared to a predetermined standard value. The next step (c), involves selecting for a subject displaying a decreased NK cell functioning (or dysfunctional NK cells), an activating treatment regimen that decreases the levels of at least one of ERG2 and DGKa in at least one NK cell of said subject.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0025] Figure 1A-1H. RNA seq analysis — gene and pathway enrichment

[0026] Fig. 1A. Graphical presentation of NK isolation process -gating strategy. Primary human NK cells were isolated from PBMCs using negative selection; following purity check by CD3-CD56+ expression, the cells were stained for NKG2A PE and panKIR PE and further sorted to anergic and responsive NK cell subsets based on PE (Phycoerythrin) expression (NKG2A-panKIR- (PE negative) anergic subset; NKG2A+ panKIR-i- (PE positive) responsive subset).

[0027] Fig. 1B-1C. Purified NK (pNK) anergic and responsive cells were subjected to incubation with 721.221 No HLA cell lines (E:T - 1:3) for 5 h at 37°C.

[0028] Fig. IB. For tumor lysis assay, the target HLA cells were labeled with S35and tumor lysis was measured (n = 3, where n is the number of healthy donors used to obtain the pNK cells). Fig. 1C. Degranulation was determined by measuring the mean fluorescence intensity (MFI) of CD107a-positive NK cells by FACS (n = 3, where n is the number of healthy donors used to obtain the pNK cells).

[0029] P values were calculated using a two-tailed paired t test and are represented within the graph presented as means ± SEM.

[0030] Fig. 1D-1E. Metascape analysis of key genes and the associated enriched pathways from KEGG and GO, upregulated in the responsive (Fig. ID), and anergic (Fig. IE) populations (n = 4, where n is the number of healthy donors used to obtain the purified NK cells).

[0031] Fig. 1F-1G. Metascape analysis of key genes and the enriched terms annotated to pathways from various datasets associated with the top-hit significant genes (Fig. IF), and the transcription factors (TFs) reflecting the top-significant genes upregulated in the anergic subset, obtained via enrichment analysis in TRRUST (Fig. 1G).

[0032] P values for (Fig. 1D-1G) were obtained using Hypergeometric test and Fisher’s exact test.

[0033] Fig. 1H. The interactome cluster of the enriched terms annotated to pathways obtained from the significant Differentially Expressed Genes (DEGs).

[0034] Figure 2A-2E. Gene expression profiling and analysis of anergic and responsive populations

[0035] Fig. 2A. PC A performed using DESeq2 on the anergic vs. responsive subset VST normalized RNA-seq data. The variance is depicted on the X and Y axes, and each individual donor is indicated. The anergic and responsive populations demonstrate high transcriptional differences reflected by a variance of 81%.

[0036] Fig. 2B. Heatmaps (transcripts per kilobase million normalization with Z score) showing expression of representative genes in anergic vs responsive populations from individual donors. The heatmaps illustrate subtle distinctions among the donors with noteworthy similarities.

[0037] Fig. 2C. Volcano plots showing differentially expressed genes in anergic vs. responsive NK cells (n = 4 healthy donors used to isolate pNK and sort anergic and responsive NK cell populations). Selected differentially expressed genes with an adjusted P value < 0.05 and log2 fold change >1 or -1 are indicated in the plot. Gene-specific t test was used for statistical analysis.

[0038] Fig. 2D-2E. The transcript levels of key genes expressed in the anergic, and responsive populations were normalized to log2 and are presented as line dot graphs. The P value was calculated using a two-paired tailed t test and is indicated within the graph (NC normalized counts). Genes in (Fig. 2D) reflect the markers of effector and functional maturity of NK cells — GZMB, Eomes, TBX21, and those in (Fig. 2E) represent the transcript levels of key signaling molecules involved in NK cell responses, PLCG1, PLCG2, PRKCQ, CBL, and MAPK1. The respective P values are indicated in the graph, and each line represents the values of a single donor (n = 4 healthy donors). Figure 3A-3I. Naturally induced “anergic” NK cells share a transcriptional program similar to “exhausted” NK / T cells with an overlap of key regulators

[0039] Fig. 3A. The transcript and protein levels of key genes reflecting NK cell activation expressed in anergic and responsive populations. RNA levels were normalized to log2 and are presented as line graphs (RNA), or bar graphs (protein- flow cytometry); FCGR3A (CD16), NCR1 (NKp46) (n = 4 donors for the RNA and for the FACS data, n = 3 healthy donors). The P value was calculated using a two-tailed paired t test and is indicated within the graph. The panels represent (from left to right): RNA transcript normalized count, relative MFI, raw MFI and percentage of parent.

[0040] Fig. 3B. Transcript and protein levels of DNAM-1 , 2B4, and CD160. Each line represents a single donor for RNA (n = 4 healthy donors), and dots represent individual donors for protein. The P value was calculated using a two-tailed paired t test and is indicated above the graph. For each marker, panels represent (from left to right): RNA transcript normalized count, relative MFI, raw MFI, and percentage of parent.

[0041] Fig. 3C. The transcript levels of key inhibitory surface checkpoint markers, PDCD1, HAVCR2, and TIGIT (n = 4, NK cells sourced from four healthy donors for RNA seq, left). Protein expression of the key inhibitory surface checkpoint markers was obtained from the number (n) of healthy donors thus mentioned, PD-1 (n = 6), Tim-3 (n = 6), and TIGIT (n = 5). The panels represent (from left to right): RNA transcript normalized count, relative MFI, raw MFI, and percentage of parent.

[0042] Fig. 3D. Gene Sequence Enrichment Analysis (GSEA) using gene sets from exhausted NK cells obtained from HB V-infected individuals vs healthy donors compared to the anergic and responsive population (Marotel et al, 2021, eLife 10: 1 - 47). The anergic NK cells showed positive correlation to the HBV-exhausted NK cells, whereas responsive NK cells showed positive correlation to the NK cells from healthy donors; NES value = 1.10, FDR q value = 0.26.

[0043] Fig. 3E. GSEA using gene sets from datasets of viral CD8 T-cell exhaustion (West et al, 2011, Immunity 35:285); NES value = -1.55, FDR q value = 0.033.

[0044] Fig. 3F. NFAT-induced T-cell exhaustion datasets from (Martinez et al, 2015, Immunity 42:265- 278). NES = 1.35, FDR q value = 0.014

[0045] Fig. 3G. HMG BOX mobility factors, TOX and TOX2 induced T-cell exhaustion datasets from (Scott et al, 2019, Nature 571:270-274). NES = -1.73, FDR q value = 0.02. The statistical tests for the GSEA (Fig. 2D-2G) were performed using the weighted Kolmogorov-Smirnov test. Fig. 3H. Transcript levels of key genes identified from the GSEA, DGKA, EGR2 (n = 4, NK cells sourced from four healthy donors for RNA seq).

[0046] Fig. 31. Purified responsive and anergic NK cells from healthy donors (n) were lysed and subjected to western blot with anti-DGKa (n = 18) and anti-Egr2 (n = 10) antibodies (left panel). Graphs showing the densitometric quantitative values of the western blots, with each dot representing an experimental repeat (right panel). Data are presented as mean ± SEM. P values were calculated using a two-tailed t test with matched data repeats and are indicated within the graph.

[0047] Figure 4A-4D. RNA seq analysis and DGKa expression levels

[0048] Fig. 4A-4B. Transcript level by RNA seq (left) (n = 4, healthy donors used to obtain the pNK) and protein expression (n = 3 healthy donors) via flow cytometry of NF ATI (NFATc2) (Fig. 4A) and NFAT2 (NF AT cl) (Fig. 4B), respectively. The P value was calculated using a two-tailed t test, and the number of repeats is indicated within the graph presented as means ± SEM.

[0049] Fig. 4C. The transcript levels of TOX and TOX2 in anergic vs responsive cells (n = 4, where n is the number of healthy donors used to obtain the pNK cells). P values were calculated using a two- tailed t test with pairing and are indicated within the graph; NC normalized counts.

[0050] Fig. 4D. Purified anergic and responsive NK cells were lysed and subjected to western blot analysis with DGKa antibody (n = 6, where n is the number of healthy donors used to obtain the pNK cells). P values were calculated using a two-tailed t test with pairing and are indicated within the graph presented as means ± SEM.

[0051] Figure 5A-5F. In vivo model exhibits shared intrinsic regulators governing tumor infiltrating “exhausted” NK cells and those controlling NK cell “anergy”

[0052] Fig. 5A. Experimental protocol. Timeline of in vivo experiment depicting tumor engraftment, effector administration, tumor excision, and ex vivo analysis. NOD / SCID IL-2Rynull (NRG) mice engrafted with 4 x 106aggressive human pancreatic ductal carcinoma cells (PANC-1) received a single infusion of primary human NK cells (i.t.). They were allowed to grow until the tumor size plateaued, indicating decreased ability of the NK to control the tumors. Tumor burden was assessed daily.

[0053] Fig. SB. Left panel: Graph illustrating progression of tumor growth (n = 6 mice). On day 14 these mice received single intratumor (i.t.) infusion of l.l*107human pNK from two healthy donors. The stripes-shaded region on the graph corresponds to the period during which pNK cells were present within the tumor post injection (with exception of tumor-only group). The groups treated with pNK cells exhibited a significant reduction in tumor size, although this effect was short-lived, lasting only 4 days. This suggests a decrease in the tumor lysis capacity of the pNK cells after 4- 5 days. Right panel: Graph based on data in left panel showing days 14-18 of the tumor growth and plateauing of the tumor size toward day 18 (experimental end point), suggesting the escape of the tumor cells from NK-mediated control. P values were calculated using one-way ANOVA with Tukeys post hoc multiple comparison test and are indicated within the graph.

[0054] Fig. 5C-5D. FACS analysis: Tumors excised on day 18 were dissociated to single-cell suspensions. NK cells were subjected to flow cytometry and were differentiated by hCD45 expression. Representative graphs showing: Left panel: Percentage of pNK expressing CD107a (Fig. 5C) and PD-1 (Fig. 5D) (n = 3 mice) in naive vs TINK (tumor infiltrating NK cells); Right panel: Relative MFI corresponding to the respective percentage values.

[0055] Fig. 5E-5F. FACS analysis: Tumors excised on day 18 were dissociated to single-cell suspensions. NK cells were subjected to flow cytometry and were differentiated by hCD45 expression. Representative graphs showing: Left panel: Percentage of pNK expressing Egr2 (Fig. 5F) and DGKa (Fig. 5E) (n = 6 mice) in naive vs TINK; Right panel: Relative MFI corresponding to the respective percentage values.

[0056] The P values were calculated using a two-tailed t test with pairing and are indicated within the graph (mean ± SEM) along with the number of experimental repeats. Each symbol represents an individual mouse.

[0057] Figure 6A-6C. Flow cytometry gating strategy

[0058] The gating strategy employed for the in vivo experiment.

[0059] Fig. 6A. NK cells were distinguished from the target PANC-1 cells according to FSC and SSC. CD45 (antibody specifically recognizing human (h)CD45) expression was used to further distinguish the pNK from the tumor and any other murine cells. NK cells, which are CD45+ (95.9%) referred as tumor infiltrating NK cells (TINK).

[0060] Fig. 6B. Flow cytometry analysis of intracellular staining was performed to measure pNK expression on cells from the mice with tumor-only (no NK administration) control and distinguished based on CD45 expression. The NK cells were distinguished from the target cells based on their volume and density (FSC and SSC) and were re-gated to CD45+ subsets. DGKa and Egr2 expressions were measured on the CD45+ gated population. Histogram offsets and MFI were used for graphical presentation. Graphs show fluorescence intensity. The Y axis indicates relative cell number, and the X axis indicates the MFI.

[0061] Fig. 6C. Representative histograms and contour plots including outliers for the data in Fig. 5C-5F. Figure 7A-7I. Knockdown of the intrinsic regulators reveals underlying molecular mechanism to reverse NK cell dysfunction

[0062] Fig. 7A. The indicated pNK cell subsets were either incubated with D5919, a pharmacological inhibitor of DGK, or DMSO. The cells were co-incubated with 721.221 HLA negative target cells for 5 h at 37°C. The graph shows the normalized pERK levels analyzed by intracellular staining from six independent experiments. Data are presented as means ± SEM. P value was calculated using one-way ANOVA and Tukeys’ post hoc test relative to the anergic NS treatment, and each experimental repeat is indicated by a dot (n = 6 healthy donors).

[0063] Fig. 7B-7C. The indicated pNK cell subsets were either transfected with DGKa, siRNA (Fig. 7B) (n = 6 obtained from three healthy donors), or Egr2 siRNA (Fig. 7C) (n = 10 obtained from 7 healthy donors) or NS siRNA as a control following incubation with 721.221 HLA-negative target cells for 5 h at 37 °C. Degranulation was determined by measuring the MFI of CD107a-positive NK cells by FACS. The NK cells were distinguished from the target cells according to FSC and SSC. Graph summarizing the normalized CD107a-positive cells from eight and six independent experiments, respectively. Data are presented as means ± SEM. P value was calculated using oneway ANOVA relative to the anergic cells treated with DMSO / NS siRNA, and multiple comparisons were performed by Tukeys’ post hoc test and are indicated within the graph. Fig. 7D. Purified anergic and responsive NK cells treated with Egr2 or NS siRNA and subjected to lysis and western blot analysis with Egr2 and DGKa antibodies on the same membrane (left panel). Graph summarizing data representative of three independent experiments (n = 3 healthy donors) (right panel). Data are presented as means ± SEM. P value was calculated using one-way ANOVA, and multiple comparisons were performed by Tukeys’ post hoc test and are indicated within the graph.

[0064] Fig. 7E. Purified anergic and responsive NK cells treated with Egr2 siRNA or NS siRNA as a control following incubation with 721.221 HLA-negative target cells for 5 h at 37°C. Phospho(p)SHP-l levels were measured via intracellular staining, and cells were subjected to flow cytometric analysis. The NK cells were distinguished from the target cells according to FSC and SSC (n = 3 healthy donors). Data are presented as means ± SEM. P value was calculated using one-way ANOVA relative to the anergic cells treated with DMSO / NS siRNA, and multiple comparisons were performed by Tukeys’ post hoc test and are indicated within the graph.

[0065] Fig. 7F. Anergic cells were treated with either Egr2 or NS siRNA, and responsive cells were treated with NS siRNA. The cells were stained with Indo-1 AM, activated by PMA / ionomycin activation cocktail, and intracellular calcium flux was measured as the ratio between the Ca2+ bound / unbound over 22 min. One experiment presented a representative of three. The traces represent each condition, anergic NS siRNA; anergic Egr2 siRNA; and responsive NS siRNA, as indicated in the figure.

[0066] Fig. 7G-7I. Isolated human pNK cell subsets were either transfected with Egr2 siRNA or NS siRNA. Surface expression of inhibitory checkpoints receptors was obtained from the number (n) of healthy donor thus mentioned, PD-1 (Fig. 7G) (n = 6), Tim-3 (Fig. 7H) (n = 5), and TIGIT (Fig. 71) (n = 6). Graph summarizing the normalized APC -positive cells from independent experiments. Data are presented as means ± SEM. The P value was calculated using one-way ANOVA with a Tukeys’ post hoc test, and is indicated within the graph, along with each experimental repeat.

[0067] Figure 8A-8F. Silencing efficiency and flow cytometry gating strategies

[0068] Fig. 8A. Gene silencing of DGKa. Anergic cells were treated with DGKa siRNA or NS siRNA and were then lysed and subjected to western blot analysis. One blot representative of three experiments is shown. The right panel shows a graph representing the quantified blots (n = 3, where n is the number of healthy donors used to obtain the pNK cells). Left panel: Data are presented as mean ± SEM. P value was calculated using a two-tailed paired t test and is indicated within the graph.

[0069] Fig. 8B. Gating strategy employed for the analysis of results in Fig. 7. The NK cells and the 221 HLA-Cw7 cells were differentiated based on size and granularity (FSC-SSC) and gated for PE to distinguish the anergic (PE-) versus the responsive population (PE+). They were subsequently gated for CD 107a, as indicated on the overlaid histograms.

[0070] Fig. 8C-8E. Purified responsive and anergic NK cells were stimulated with 721.221 target cells, lysed and subjected to FACS analysis with anti-pSHP-l(S591) (Fig. 8C), anti-pPLCyl (Y783) (Fig. 8D), and anti-pPLCy2 (Y1217) (Fig. 8E) antibodies. Graph summarizing the MFI of pSHP- 1 (n = 5), pPLCyl (n = 3), and pPLCy2 (n = 5) expression levels (where n is the number of healthy donors used to obtain the pNK cells). Data are presented as mean ± SEM. P value was calculated using a two-tailed t test with matched data repeats and is indicated within the graph.

[0071] Fig. 8F. Representative overlaid histograms for Fig. 7G-7I.

[0072] Figure 9A-9J. Targeting Egr2 in situ using 3D organotypic spheroid (OTS) culture and in vivo model as a potential therapeutic approach

[0073] Fig. 9A-9B. Correlation of NK-DGKA-EGR2 signatures with the overall survival of AML patients (Fig. 9A), and glioma patients (Fig. 9B) comparing high and low quartiles. Left panels: Kaplan Meier curves are presented showing patient survival (obtained from Survival Genie (Dwivedi et al, 2022)) along with the P values. Right panels show the signature of immune cell infiltration in AML and glioma; and red squares represent positive correlation to immune cells observed in the cancer dataset. Bottom panels indicate the correlation of the survival score to the NK cell signatures. P values (Fig. 9A, 9B) were calculated using Log-rank t test.

[0074] Fig. 9C. Left panel: Schematic representation of the 3D OTS model using the NP delivery platform to reprogram anergic NK cells in a tumor milieu. Primary NK cells were used to obtain anergic and responsive subsets and were then seeded to preconstructed 3D cultures and administered with NP encapsulating Egr2 or NS siRNA. Next, the pNKs were subjected to an Incucyte-based killing assay. Right panel: Human chronic myeloid leukemia OTS 3D domes were established using Matrigel and cultured for 48 h in OTS media consisting of RPMI media supplemented with 1 pg / mL fibroblast growth factor (FGF), 0.18 pg / mL epidermal growth factor (EGF) and 500 lU / mL transforming growth factor (TGFP). After 48 h, the 3D cultures were incubated with either freshly isolated anergic or responsive NK cells. After 6 h of NK-tumor coincubation, NPs encapsulating Egr2 siRNA or NS siRNA were added (represented by the shaded region). The decrease in fluorescence intensity reflects target cell lysis by the respective NK cell population and the associated treatment. The analysis was performed for 25 individual field frames for each experimental condition (with approx, n = 30 cells each field). Statistical analysis was conducted for three independent experiments, and presented as means ± SEM (n = 3 healthy donors). P value was calculated using two-way ANOVA with a Tukey’s post hoc test for multiple comparison, and paired t test was performed between each time point and group to find significant changes in tumor lysis between the groups at specific time points. Anergic NS siRNA vs Anergic Egr2 siRNA (P value (*) = 0.0121, time point: 30 h).

[0075] Fig. 9D. Timeline of the in vivo experiment. PDAC-1 -xenograft NRG mice were established as previously described and received a single infusion of l.l*107human pNK from four healthy donors on day 9 once the tumors reached -250-300 cm2; “tumor only” control did not receive NK cells but received NS siRNA encapsulated NPs. NPs encapsulating Egr2 siRNA or NS siRNA were administered i.v. from day 12 for every 3 days until day 27.

[0076] Fig. 9E. Tumor volume (in mm3) was monitored and measured daily throughout the experiment. The pNK injection is depicted by a black arrow in a stripes-shaded region, and the NP injections are indicated by black arrows. Mice in the “tumor only” group (n = 6 mice) received no pNK treatment but were administered NP encapsulated NS siRNA. Mice that were administered with Egr2 siRNA (n = 8 mice) are depicted in triengles, while those receiving NS siRNA (n = 8 mice) are depicted in circles. The bold lines indicate the average, and the dashed lines represent individual mice within their respective experimental groups.

[0077] Fig. 9F. Tumor sizes (mm3) measured during the indicated days. Black graph represents the control group with tumor only. The gray and the white graphs represent groups of mice that received treatment with NP encapsulating NS siRNA or Egr2 siRNA, respectively. Tumor sizes at specific time points are indicated: Day 8 (one day before pNK injection) and Day 11 (2 days after pNK injection) are tumor sizes prior to NP injection; day 15 (6 days after pNK injection), day 19 (10 days after pNK injection) and day 27 (18 days after pNK injection and final day before tumor excision) represent tumor sizes following NP injection. Data are presented as mean ± SEM. P values are calculated using one-way ANOVA accompanied by a Tukeys’ post hoc multiple comparison test individually for each day presented and are indicated within the graph.

[0078] Fig. 9G. Tumor growth rate measured from day 12 (first NP administration) until day 27 (end point) shown for the three groups (Egr2 siRNA-NP vs NS siRNA-NP vs tumor only). Data are presented as mean ± SEM. P values are calculated using one-way ANOVA and are indicated within the graph (N = 4 healthy donors were used to obtain the pNK; Groups - tumor only (n = 6), NP Egr2 siRNA (n = 8), NP NS siRNA (n = 8), where n is the number of mice).

[0079] Fig. 9H-9I. Ex vivo analysis. The tumors were excised on day 27 and processed to single-cell suspensions by dissociation as described in the Materials and Methods. They were then stained for CD 107a (Fig. 9H) (n = 3, where n is the number of mice used to obtain the pNK cells) and PD-1 (Fig. 91) (n = 3, where n is the number of mice used to obtain the pNK cells). The pNK were distinguished based on hCD45 expression and NP incorporation (PE positive). Fluorescence is represented as both relative MFI (left panels) and percentage of pNK (right panel). P values were calculated using a two-tailed paired t test and are represented within the graph presented as means ± SEM.

[0080] Fig. 9J. Scheme depicting the proposed signaling pathway of anergic cells in accordance with the transcriptome and protein level profiling; PA phosphatidic acid, PLC phospholipase Cyl / 2, DAG diacylglycerol, DGK diacylglycerol kinase, Egr early growth response, MAPK mitogenactivated protein kinase, NF AT nuclear factor of activated T cells, PD-1 programmed cell death protein 1, PKC0 protein kinase C0, SHP-1 Src homology 2 domain-containing protein tyrosine phosphatase 1, pS591 phospho - S591. “Anergic” cells exhibit elevated EGR2 expression, which subsequently triggers an increase in DGKa. This leads to the conversion of DAG to PA, in turn, PA recruits more SHP-1 to the cellular membrane. As a result availability of DAG is restricted, which hampers PKC0 activity, rendering it incapable of modulating SHP-1 activity (Ben-Shmuel et al, 2022). This allows SHP-1 to dephosphorylate LAT and PLCyl / 2 (Matalon et al, 2016), preventing the initiation of a secondary cascade. DAG depletion also inhibits the activation of the DAG-mediated Ras-Raf-MEK-ERK pathway and IP3-mediated calcium flux. Consequently, there is no nuclear translocation of NF AT and its effector partners, such as AP-1, to initiate an effector response. This ultimately results in establishing an “anergy-associated gene transcription program”.

[0081] Figure 10A-10D. Incucyte analysis with representative images and ex vivo analysis

[0082] Fig. 10A. Anergic and responsive cells were treated with either Egr2 or NS siRNA, and Incucyte - based tumor lysis assay was performed on 721.221 HLA-Cw7 target cells expressing mCherry. Left panel: P value was cumulatively calculated for every time point using one-way ANOVA, and Tukeys’ post hoc test was used for multiple comparisons, as indicated in the graph (*P < 0.05). The analysis was performed for nine individual fields per image (16 images) (with ~N = 50 cells in each field), squares: anergic NS siRNA; circles: anergic Egr2 siRNA; left pointing triangles: responsive NS siRNA. Anergic and responsive cells were transfected with EGR2 siRNA or NS siRNA, seeded with 721.221 HLA-Cw7 expressing mCherry cells at an E:T ratio of 10:1, and subjected to Incucyte imaging and analysis; the decrease in fluorescence intensity was measured, and images from the indicated time points are shown. The values were normalized to 721.221 Cherry only — no effector control. Right panel: Images showing cell fluorescence at different time points (scale bar: 200 pm).

[0083] Fig. 10B. Human CML OTS prepared using K562 CFP cells in Matrigel (1 : l-v / v%), were seeded with anergic or responsive pNK cells at an E:T ratio of 5:1. After 6 h, NPs encapsulating Egr2 siRNA or NS siRNA were added to the OTS, and the decrease in fluorescence intensity was monitored and measured; the images along with the fluorescence intensity at the respective time points are shown (numbers on top right of each frame). The decrease in fluorescence over time shows the enhanced cytotoxic activity of anergic cells following Egr2 siRNA treatment (left panel: middle) similar to the responsive cells treated with NS siRNA (left panel: bottom). Whole well images of the OTS at 48 h are presented. Right panel: Images showing cell fluorescence at different time points (Scale bar: 200 pm).

[0084] Fig. 10C. The tumors were excised on day 27, a single-cell suspension was made, and cells were stained for intracellular Egr2. The pNK cells were distinguished based on FSC vs SSC, and PE content, reflecting nanoparticle incorporation. Representative histogram showing Egr2 expression levels, black line shows the EGR2 levels in the group treated with control NP (NS siRNA), and the dotted line shows the group receiving Egr2 siRNA- NP, as indicated in the graph. Fig. 10D. The dissociated tumors were then subjected to Annexin V staining for apoptotic cells. Representative histogram on the left panel shows the apoptosis of the tumors from each group (one mouse representative of three independent repeats). The right panel shows a graph summarizing the tumors obtained from three different mice. Data are presented as mean ± SEM. P values were calculated using one-way ANOVA with Tukeys post hoc test after normalization of the values to the “tumor only” group, which served as the control.

[0085] DETAILED DESCRIPTION OF EMBODIMENTS

[0086] The modulation of NK cell responsiveness represents a promising approach in cancer therapy due to their innate ability to target cancer cells. NK cells serve as the first line of immune defense against cancer cells, however, increasing evidence shows emergence of dysfunctional phenotypes. In this study, the inventors performed a thorough characterization and uncovered the underlying etiologies of the “anergic” and “exhausted” dysfunctional states. Although many surface markers have been suggested (Judge et al, 2020, Front Cell Infect Microbiol 10:49), the molecular and transcriptional wiring remains unexplored. The inventors demonstrate that the naturally existing “anergic” NK cell population shares phenotypic, transcriptomic, and functional similarity with the canonical NK cell “exhaustion” state arising from the tumor microenvironment (TME). DGKa and Egr2 were identified as key intrinsic regulators governing NK cell dysfunctional states of “anergy” and “exhaustion”. The inventors' findings indicate that Egr2 serves as the primary transcription factor accounting for the intrinsic non-functionality of these cells. Moreover, the transcriptome analysis reveals sharing of key genes between anergic / exhausted NK cells, and exhausted CD8+ T cells (Fig. 3D-3G), including NFAT2, EGR2, PDCD1, and TOX2, revealing transcriptional commonalities. In this regard, targeting a shared intrinsic regulator such as Egr2, identified here, for therapy can improve the response of the tumor-infiltrating lymphocytes (TILs) as a whole rather than only a specific immune cell population. The dysregulation of NK cell function is a significant hurdle in immunotherapeutic approaches, such as chimeric antigen receptors (CAR) and immune checkpoint blockade (ICB) including anti-CTLA-4 and anti-PD-1 treatments despite the great interest in NK cells as a candidate for immunotherapy (Ben-Shmuel et al, 2020, Immunol 11:275; Laskowski et al, 2022, Nat Rev Cancer 22:557-575). While these therapies have shown promise, cell evasion and exhaustion remain persistent challenges, limiting efficacy, especially of CAR- T and NK cells (Valeri et al, 2022, Front Immunol 13:4208; Kouro et al, 2022, J Transl Med 20:1-10; Titov et al, 2022, Cancers 14: 1078; Selli et al, 2023, Blood 141:3153-3165; Good et al, 2021, Cell 184:6081-6100.e26). Adoptive transfer of NK cells, a common approach, also leads to reduced cytotoxic potential due to loss of NK activity during ex vivo expansion of NK cells (Judge et al, 2020, Front Cell Infect Microbiol 10:49; Gill et al, 2012, Blood 119:5758). ICB therapies target one or more checkpoints such as the surface markers PD- 1, CTLA4, TIGIT, or NKG2A (Leach et al, 1996, Science 271:1734-1736; Ott et al, 2013, Clin Cancer Res 19:5300-5309; Pan et al, 2023, Adv Mater 35:2211370). Targeting these suppresses only some of the pathways that are associated with the particular checkpoint inhibitor used, and these checkpoint blockade strategies only extend the cytotoxic window (Barber et al, 2006, Nature 439:682-687), but do not reverse the exhaustion-associated transcriptional imprint (Pauken et al, 2016, Science 354:1160-1165). Along these lines, the inventors study highlights two key intrinsic checkpoints in NK cell dysfunction (Fig. 9J); (i) The transcription factor (TF) Egr2 establishes a molecular circuitry based on DGKa transcription leading to NK cell anergy. (ii) DGKa facilitates conversion of DAG to PA, leading to impaired signaling cascades through the recruitment of the phosphatase SHP-1 to the cell membrane, resulting in dephosphorylation of its targets, e.g., PLCyl / 2 (Fig. EV4C-E), LAT, and ZAP70, suppressing the signaling cascade, and resulting in the dysfunctional phenotype. Thus, the efficacy of EGR2 silencing observed in the inventors' results occurs through reduced DGKa expression limiting SHP-1 recruitment by PA, thereby enabling PLCyl / 2 enzymatic activity (Matalon et al, 2016, Sci Signal 9:1-16; Bradshaw and Dennis, 2010, Diacylglycerol kinases. In: Handbook of cell signaling. Vol 162. Academic Press, Elsevier, p 243-246; Frank et al, 1999, Biochemistry 38:11993-12002). This facilitates the initiation of the DAG-activated PKC0-IKK-NFkB axis and RasGRP-MAPK pathway (Chen et al, 2016, Front Cell Dev Biol 4:130; Krishna and Zhong, 2013, Front Immunol 4:52250) together with IP3-mediated calcium-calcineurin-NFAT signaling cascades (Vig and Kinet, 2009, Nat Commun 12:2782). In addition, EGR2 gene silencing shifted the dysfunctional phenotype from a PD-lhigh to PD-1 low profile (Fig. 7G), implying that the TF Egr2 induces PD-1 expression. Notably, the potent recovery of overall functionality and molecular wiring observed following EGR2 silencing, compared to the weaker effect of DGKa, silencing, reinforces the crucial role of Egr2 as a central regulator and a potent therapeutic target to restore NK cell functions.

[0087] The involvement of SHP-1 in NK cell signaling has been intensively studied by the inventors (Matalon et al, 2016, Sci Signal 9:1-16; Matalon et al, 2018, EMBO J 37:e96264; Ben-Shmuel et al, 2022, eLife ll:e73282; Biber et al, 2021, EMBO Mol Med 14:el4073; Ben-Shmuel et al, 2021, Cell Mol Immunol 18:1314-1316) and others (Wu et al, 2021, Sci Signal 14:eabe5380; Schmied et al, 2023, Sci Signal 16:eabq0752), yet the mechanism underlying its transcriptional regulation in anergy remains unclear. The inventors' data implies that EGR2 gene silencing downregulates SHP-1 activity, potentially via enhancing DAG availability by reducing DGKa levels. This, in turn, initiates the activation of PKC0, which modulates SHP-1 activity through serine 591, supported by the inventors' recent data (Ben-Shmuel et al, 2022, eLife l l:e73282).

[0088] Furthermore, the RNA sequencing analysis and the in vivo model shown here suggest that Egr2 potentially cooperates with other TFs, presumably NFAT2, to initiate the dysfunction-based transcriptional program along with high mobility group (HMG)-box TFs such as TOX2, and members of the NR4a family, which are validated targets of calcium-calcineurin regulated NFAT2 in the absence of partners such as AP-1 and NFkB (Wagle et al, 2021, Nat Commun 12:2782; Seo et al, 2019; Sekine et al, 2020, Sci Immunol 5:eaba7918). The involvement of Egr2 and potentially NFAT2 / TOX2 / other TFs in the terminal regulation of anergy highlights the complexity of the transcriptional program associated with dysfunctional NK cells. This finding establishes a functional link between the naturally induced “anergy” and the “exhausted” state of NK cells within the TME. Thus, targeting the intrinsic regulators of dysfunctional NK cells (i.e., Egr2), leads to the rewiring of NK cell functional circuitry, overcomes the dysfunction-associated transcriptional imprint, and potentially enhances the efficiency of immunotherapies. On this note, depletion of dysfunctional cells from the TME was suggested to improve therapeutic efficacy. The inventors study provides a novel strategy for in situ reprogramming of these dysfunctional cells rather than depleting them (Marcucci and Rumio, 2021, Cells 10:872; Cook and Whitmire, 2013, J Immunol 190:641-649). The inventors describe a potential platform for this approach using an NP drug delivery system (Biber et al, 2021, EMBO Mol Med 14:el4073) (Fig. 5C). In addition, the in vitro 3D OTS CLL model and in vivo PDAC model of NK cell exhaustion demonstrate that EGR2 silencing, not only restores the functionality of anergic cells but also enables these NK cells to potentially control solid tumor growth and influence disease outcomes. This potentially serves as proof of concept for the possibilities of reprogramming dysfunctional NK cells in situ, bypassing the need for adoptive transfer. While the majority of studies on anergic NK cells have focused on their presence in peripheral blood, evidence suggests that NK cells develop and mature in different tissues including spleen, liver, bone marrow, lymph node, and lung (Dogra et al, 2020, Cell 180:749; Hashemi and Malarkannan, 2020, Cancers 12: 1-23). This leads to the notion that the NK cells undergo education in the respective niche, resulting in the potential emergence of anergic phenotype. The characterization and the functional implications of anergic NK cells arising in different tissue contexts are still unknown. Further research is necessary to comprehensively describe the presence and functional characteristics of these potential population(s). Furthermore, the complex interplay between anergic cells and the TME requires in-depth investigation to unravel underlying mechanisms and their potential implications. In this regard, the inventors' NP- based approach to reprogramming these dysfunctional cells in their milieu holds great promise. Reversing both NK cell “anergy” and NK cell “exhaustion” is essential as it restores a crucial component of the immune system’s capacity to detect and eliminate cancer cells, potentially leading to more effective immunotherapeutic strategies and better patient outcomes.

[0089] In summary, the inventors' study not only elucidates the molecular framework of NK cell anergy but also highlights its functional, phenotypical, and transcriptional parallels with canonical NK cell exhaustion. In addition, the inventors identified shared intrinsic factors that can be targeted and reversed through immunotherapy. Furthermore, the inventors' results provide valuable insights into the mechanisms and signaling pathways responsible for NK cell dysfunctional states. Focusing on the regulation of NK cell anergy and exhaustion will potentially lead to the development of strategies to modulate the anergic state in settings of autoimmunity and transplantation or to enhance NK cell function in cancer and chronic viral infection.

[0090] The present disclosure provides in one aspect thereof re-programming agent (or modulator and / or re-programmer) of lymphocytes, for example, lymphocytes of the T lineage. In some embodiments, the disclosed modulator and / or re-programmer activates dysfunctional lymphocytes, specifically, dysfunctional lymphocytes of the T lineage. The disclosed reprogramming agents at least one of: (i), at least one member of the Early Growth Response (EGR) family of transcription factors; and (ii), at least one member of the Diacylglycerol Kinase (DGK) family. In some embodiments, the disclosed re-programming agent activates, rewires and / or activates at least one lymphocyte. In more specific embodiments, the disclosed re-programming agent rewires at least one dysfunctional lymphocyte. In yet some further embodiments, the disclosed re-programming agent rewires dysfunctional lymphocytes residing in a microenvironment of a diseased tissue. Such diseased tissue may be in some embodiment, a tumor tissue. Accordingly, in some embodiments the dysfunctional lymphocytes reprogramed and / or rewired and / or activated by the disclosed re-programming agent may be infiltrating lymphocytes of a tumor microenvironment. In some embodiments, the disclosed lymphocytes may be NK cells, specifically, dysfunctional NK cells. Thus, according to some embodiments, the disclosed reprogramming agent re-programs and / or rewires at least one dysfunctional NK cell. In some embodiments, the disclosed re-programming agent rewires and / or activates, and / or reprograms at least one NK cells that infiltrates into the tumor tissue, specifically, a least one TINK (tumor infiltrating NK cells). In yet some alternative or additional embodiments, the disclosed lymphocytes may be T cells, specifically, dysfunctional T cells. Thus, according to some embodiments, the disclosed re-programming agent re-program and / or rewires at least one dysfunctional T cell. In some embodiments, the disclosed re-programming agent rewires and / or activates, and / or reprograms at least one T cells that infiltrates into the tumor tissue, specifically, at lest one TIL (tumor infiltrating lymphocytes). More specifically, Tumor-Infiltrating Natural Killer (TINK) cells, refer to a subset of natural killer (NK) cells that are present within the tumor microenvironment and exhibit the ability to infiltrate solid tumors. These cells are a component of the innate immune system and play a crucial role in recognizing and eliminating malignant cells through mechanisms such as cytotoxic activity and cytokine production. TINKs are often phenotypically and functionally distinct from circulating NK cells, as they are influenced by the immunosuppressive tumor milieu, which can alter their activation status, cytotoxicity, and cytokine secretion profiles. Notably, TINKs may exhibit markers of exhaustion or dysfunction, reflecting tumor-induced immune evasion, while certain subsets retain potent anti-tumor activity. The tumor microenvironment (TME) refers to the complex and dynamic ecosystem surrounding a tumor, composed of cellular and non-cellular components that interact with the malignant cells to influence tumor progression, metastasis, and therapeutic response. Cellular components of the TME include immune cells (e.g., T cells, B cells, macrophages, and NK cells), fibroblasts, endothelial cells, and mesenchymal stromal cells, while non-cellular components encompass extracellular matrix (ECM) proteins, signaling molecules, cytokines, chemokines, and growth factors. The TME is characterized by hypoxia, altered pH, and nutrient deprivation, which promote tumor survival, immune evasion, and resistance to therapies. The interactions within the TME can lead to immunosuppressive conditions, enabling tumor growth and progression by impairing antitumor immune responses.

[0091] It should be understood that the definitions disclosed herein for the re -programming agent (or modulator and / or re-programmer) of the present disclosure are applicable for each and every aspect of the present disclosure.

[0092] A first aspect of the preset disclosure relates to a re -programming agent (also referred to herein as a modulator and / or re-programmer) comprising at least one compound that specifically inhibits the expression, activity and / or stability of at least one of: (i), at least one member of the Early Growth Response (EGR) family of transcription factors; and (ii), at least one member of the Diacylglycerol Kinase (DGK) family. In some embodiments the disclosed re-programming agent may provided in a nano- or micro-particle, micellar formulation, vehicle, matrix, or a composition and thus the present disclosure further encompasses any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the disclosed re-programming agent / s. More specifically, the disclosed re-programming agent activates and / or functionalize dysfunctional natural killer (NK) cells. In some embodiments, the present disclosure relates to a re-programming agent that comprises an effective amount of the at least one compound that specifically inhibits the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family. In some embodiments, the effective amount as used herein is the amount sufficient for activation and / or re-wiring of the dysfunctional NK cells.

[0093] Thus, in some embodiments, the present disclosure provides re -programming agents of NK cells, specifically of dysfunctional NK cells. A "re-programming agent' (also referred to herein as a modulator and / or re-programmer), as used herein, is defined as a compound, substance, material, or agent that reprograms, induces or enhances a specific biological, chemical, or physical effect in a targeted system. In the present case, the modulators and / or re-programmers disclosed herein act in functionalizing NK cells, and more specifically, in functionalizing dysfunctional NK cells. In some embodiments, the re-programming agents of the present disclosure act in rewiring the NK cells or reprograming the NK cells such that the re-wired, functionated, activated or reprogramed NK cells are functional NK cells, or even in some embodiments, displaying improved functionality. Rewiring or reprogramming of natural killer (NK) cells refers herein to the process of altering the gene expression profiles, signaling pathways and / or metabolic state of NK cells to enhance their activity, particularly in the context of immune responses against cancer, infections, or other diseases. Such rewiring or reprogramming improve NK cell functions, which may include for example cytotoxicity, cytokine production, and tumor infiltration, while overcoming immunosuppressive signals within the tumor microenvironment. In some embodiments of this disclosure, this process involves inhibiting the expression, activity and / or stability of at least one member of EGR family and / or at least one member of the DGK family. It should be further understood that "functionalizing dysfunctional NK cells" as used herein, refers to the process of restoring, enhancing, or even in some embodiments modifying (specifically enhancing), the functional capabilities of natural killer (NK) cells that have lost or reduced, specifically restoring the ability of the cells to perform their natural biological functions. The term "dysfunctional NK cells", as used herein, will be defined in more detail in the present disclosure, and also relates to NK cells that exhibit impaired activity, which can arise due to intrinsic factors (e.g., genetic or epigenetic defects, e.g., leading to anergy), external suppression by a diseased (tumor) microenvironment, or chronic stimulation leading to exhaustion. Functionalizing such cells by the disclosed re -programming agent encompass any action that rejuvenate or improve their activity, including, but not limited to Restoring cytotoxicity, enhancing proliferation and survival and / or reversing exhaustion. The NK natural biological functions are as defined in more detail herein after by the present disclosure and are encompassed by the present aspect.

[0094] The re-programming agent of the present disclosure thus may be interchangeably referred to herein as " re-programming agent ", "NK re-programming agent", "NK cell / s re-programming agent", "dysfunctional NK cell / s re -programming agent", and / or "re-programming agent of dysfunctional NK cells", "modulator and / or re -programmer", "NK modulator", "NK cell / s modulator ", "dysfunctional NK cell / s modulator ", and / or " modulator of dysfunctional NK cells", "re -programmer", "NK re-programmer ", "NK cell / s re-programmer", "dysfunctional NK cell / s reprogrammer", and / or " re-programmer of dysfunctional NK cells". The disclosed modulator and / or re-programmer, also acting as an activator or a reactivator promotes, initiates, or increases the activity, efficacy, or functionality of the NK cells. More specifically, the disclosed reprogramming agents provide activation of NK cells, as herein defined, as a process by which NK cells are stimulated to exert their immune functions, including cytotoxicity and cytokine production. More specifically, activation of dysfunctional NK cells involves restoring or enhancing their cytolytic potential and immunomodulatory functions. The activity of NK cells or activated NK cells may be characterized for example by altered receptor expression (e.g., activating receptors like NKG2D or CD 16) on the surface of NK cells, increased cytokine production, increased degranulation (e.g. increase in CD107), increased calcium flux and / or increased cytotoxic activity (lysis of target cells) as compared to a reference standard or a control sample. The reference standard or the control sample may comprise for example dysfunctional non-treated NK cells or NK cells of a healthy subject, that also naturally includes dysfunctional NK cells (e.g., anergic NK cells). The functionality or activity of NK cells may also be evaluated by improved disease parameters (e.g., reduced tumor size in animal models). In some embodiments, the disclosed re-programming agents increase the functionality of the NK cells or any population thereof in about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, as compared to NK cells, specifically, dysfunctional NK cells that were not activated or reprogramed or re- wired or functionated by the disclosed re-programming agents. Measurable indicators reflecting functionality of NK cells, specifically, that NK cells are functionally stimulated and engaged in their immune responses. These parameters can be assessed through various laboratory techniques, including flow cytometry, cytokine assays, and cytotoxicity tests.

[0095] The activation of NK cells can be assessed through a variety of parameters that reflect their functional and physiological changes. Surface marker expression, such as upregulation of early activation markers like CD69, prolonged activation markers like CD25, and degranulation indicators like CD107a (LAMP-1), are key indicators of activation. These parameters reflect normal functioning of NK cells. Downregulation of inhibitory receptors (e.g., KIRs, NKG2A), and also reflect normal function of NK cells. It should be understood that the term "functionalized" and / or "rewired" an / o "reactivated" as used herein refers to the natural functions of NK cells, for example, the ability to be activated, e.g., the ability of forming activating immunological synapse, and / or the ability of being deactivated, specifically, the ability of forming an inhibitory immunological synapse (NKIS).

[0096] Activated NK cells also produce key cytokines, including interferon-gamma (IFN-y), tumor necrosis factor-alpha (TNF-a), and granulocyte-macrophage colony-stimulating factor (GM- CSF), which enhance immune responses and recruit other immune cells. Their cytotoxic activity is reflected in their ability to lyse target cells, measured through assays like chromium release or fluorescent dye staining, as well as the release of cytotoxic molecules such as perforin and granzyme B.

[0097] Intracellular signaling pathways, including phosphorylation of proteins like STAT4, STATS, PI3K, and MAPK, as well as increased calcium flux, indicate activation at the molecular level. Metabolically, NK cells undergo a shift to increased glycolysis and exhibit enhanced mitochondrial activity to meet the energy demands of activation.

[0098] Gene expression changes, such as upregulation of genes like IFNG, GZMB, PRF1, and TNF, provide a molecular signature of activation and can be measured through PCR or RNA sequencing. Activation also drives NK cell proliferation, particularly in response to cytokines like IL-2 and IL-15, which can be tracked through cell counting or division assays.

[0099] Finally, the release of soluble factors, including cytotoxic granules (perforin, granzyme B) and molecules like Fas ligand (sFasL), along with elevated cytokines in the environment, serves as another hallmark of activation. These parameters collectively offer comprehensive insights into the activation state of NK cells in experimental and clinical settings. More specifically, degranulation of natural killer (NK) cells refers to the process by which NK cells release cytotoxic granules containing effector molecules, such as perforin and granzymes, to mediate the lysis of target cells, including virus-infected or malignant cells. Upon recognition of activating signals on the surface of target cells, NK cells form an immunological synapse, leading to the polarization and directed exocytosis of lytic granules toward the target cell membrane. Perforin creates pores in the target cell membrane, facilitating the entry of granzymes, which trigger apoptosis through caspase activation and other pathways. Degranulation is a hallmark of NK cell cytotoxic function and can be measured by the surface expression of markers such as CD107a (LAMP-1), which translocate to the NK cell membrane during granule release.

[0100] As indicated above, CD107, may be used as a marker for NK cell degranulation.

[0101] CD107 (also known as LAMP-1, or lysosome-associated membrane protein-1) is a transmembrane glycoprotein found in the membranes of cytolytic granules within natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). During degranulation, CD107 is transported to the cell surface as the cytolytic granules fuse with the plasma membrane, making it a key marker for evaluating the activation and cytotoxic activity of these immune cells. The surface expression of CD107a correlates with the release of effector molecules, such as perforin and granzymes, which are essential for target cell lysis. Its detection, commonly performed using flow cytometry, provides a reliable measure of immune cell degranulation and functional activity. Consequently, CD 107 plays a vital role as a biomarker in immunological research, particularly in studies of cancer immunotherapy, viral infections, and cell-mediated immune responses.

[0102] An additional hallmark parameter for activation of NK cells, is an increase in the calcium influx. Calcium influx or intracellular calcium flux refers to the process by which calcium ions (Ca2+) enter the cytoplasm of a cell, typically through calcium-permeable channels located in the plasma membrane or from intracellular stores such as the endoplasmic reticulum. This influx is a critical signaling event that regulates a wide range of cellular processes, including activation, proliferation, secretion, and apoptosis. In immune cells, such as natural killer (NK) cells and T lymphocytes, calcium influx is essential for initiating downstream signaling pathways that trigger degranulation, cytokine production, and cytotoxic activity. The entry of Ca2+often occurs following receptor stimulation, leading to the opening of store-operated calcium channels (e.g., CRAC channels) or voltage-gated calcium channels, resulting in a rise in intracellular calcium levels.

[0103] As indicated above, in some specific embodiments, activation of NK cells by the disclosed reprogramming agents may be evaluated by increased degranulation, that may be reflected in some embodiments by a measured increase in CD 107, increased calcium flux and / or increased lysis of target cells as compared to non-treated NK cells, specifically, cells that were not exposed or incubated with the re-programmer and / or modulator of the preset disclosure, to inactivated NK cells, to dysfunctional NK cells (e.g., exhausted or anergic NK cells as defined herein), or NK cells of a healthy subject that naturally contain a population of dysfunctional (e.g., anergic NK cells). Alternatively, or in addition, measurable parameters in immune-related disorders, e.g., reduced tumor size in animal models (of at least 10% or more, 20% or more of tumor size, volume or weight). More specifically, an increase as referred to herein is meant any increased elevated, enhanced, amplified, boosted, expanded, augmented, heightened, escalated, strengthened, intensified, maximized measurement in at least one of the indicated parameters. Activation, reactivation and functionalization of dysfunctional NK cells involves restoring or enhancing their cytolytic potential and immunomodulatory functions. The activity of NK cells or activated NK cells may be characterized for example by altered receptor expression (e.g., activating receptors like NKG2D or CD 16) on the surface of NK cells, increased cytokine production, increased degranulation (e.g. increase in CD107), increased calcium flux and / or increased cytotoxic activity (lysis of target cells) as compared to a reference standard or a control sample, as disclosed herein after. The reference standard or the control sample may comprise for example dysfunctional nontreated NK cells or NK cells of a healthy subject (that also includes dysfunctional cells (e.g., anergic cells). The activity of NK cells may also be evaluated by improved disease parameters (e.g., reduced tumor size in animal models). Increase as used herein encompasses any increase of about 1% to about 100%. More specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, as compared with dysfunctional functional NK cells that were not treated, contacted and / or exposed to the disclosed re-programming agent. It should be appreciated that 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.

[0104] As indicated above, the disclosed re-programming agents activate Dysfunctional natural killer (NK) cells. Specifically, dysfunctional natural killer (NK) cells refer to NK cells that exhibit impaired functionality, deviating from their normal cytotoxic and immunoregulatory roles in the immune system. These cells typically show diminished ability to recognize and eliminate aberrant cells, such as tumor cells or virus-infected cells, due to deficiencies in key molecular pathways. The dysfunction may result from intrinsic defects in activation or signaling pathways, such as reduced expression of activating receptors (e.g., NKG2D, NKp46), impaired signal transduction downstream of these receptors, or defective exocytosis of lytic granules containing perforin and granzymes.

[0105] Extrinsic factors can also contribute to NK cell dysfunction, including an immunosuppressive microenvironment, such as in tumors where cytokines (e.g., TGF-P) and metabolic stressors alter NK cell metabolism and suppress their activity. In some embodiments, dysfunctional NK cells may exhibit phenotypic markers of exhaustion, such as increased expression of inhibitory receptors (e.g., PD-1, TIM-3), and a skewed cytokine profile that reduces their efficacy.

[0106] As indicated above, dysfunction is typically assessed through assays measuring cytotoxic activity, cytokine production, and / or receptor expression profiles.

[0107] In some embodiments, dysfunctional NK cells display reduced activity, and in more specific embodiments reduced, attenuated, decreased, or inhibited activity of about 1% to 99.9%, as compared with functional NK cells. More specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, as compared with functional NK cells. It should be appreciated that 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. In some embodiments, dysfunctional NK cells display diminished activity, abolished activity, impaired activity, or no activity, specifically, when compared to functional NK cells, specifically, NK cells that display the natural functions of NK cells specified herein above.

[0108] As indicated above, in some embodiments, the disclosed modulators comprise at least one compound that inhibits or reduces the levels, expression, stability, and / or activity of at least one member of the Early Growth Response (EGR) family of transcription factors. The Early Growth Response (EGR) family of transcription factors constitutes a group of zinc finger proteins that play a pivotal role in regulating gene expression in response to a variety of extracellular and intracellular stimuli, including growth factors, stress signals, and neuronal activity. Members of this family, including EGR1, EGR2, EGR3, and EGR4, are characterized by the presence of highly conserved Cys2-His2-type zinc finger motifs within their DNA-binding domains, which facilitate specific interaction with GC-rich promoter regions of target genes. These transcription factors act as molecular switches, modulating the transcriptional activation or repression of genes implicated in critical cellular processes such as proliferation, differentiation, apoptosis, and immune responses. EGR proteins are rapidly inducible, often categorized as immediate-early genes, and their expression is tightly regulated at transcriptional and post- translational levels.

[0109] Still further, in some additional or alternative embodiments, the disclosed modulators and / or reprogrammers comprise at least one compound that inhibits or reduces the levels, expression, stability, and / or activity of at least one member of the Diacylglycerol Kinase (DGK) family. More specifically, the Diacylglycerol Kinase (DGK) family comprises a group of enzymes that catalyze the phosphorylation of diacylglycerol (DAG), a lipid second messenger, to produce phosphatidic acid (PA). Both DAG and PA serve as critical signaling molecules that regulate various cellular processes, including proliferation, differentiation, metabolism, and immune responses. DGKs play a central role in modulating the balance between DAG and PA, thereby influencing downstream signaling pathways mediated by protein kinase C (PKC) and RasGRP proteins, which depend on DAG, as well as PA-dependent pathways. The DGK family is highly conserved across species and is classified into ten isoforms (DGKa through DGKK) in mammals, each characterized by unique structural domains and regulatory properties. These isoforms share a conserved catalytic domain and two cysteine-rich Cl domains, which mediate DAG binding, but they differ in auxiliary domains such as EF-hand motifs, pleckstrin homology (PH) domains, and sterile alpha motifs (SAM), which confer specificity in their subcellular localization, lipid substrate preference, and interaction with other proteins.

[0110] As indicated above, the compounds used in the re -programming agents of the present disclosure specifically inhibit the expression, stability and / or activity of at least one member of the EGR family and / or at least one member of the DGK family. “Expression”, as used herein generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, according to the disclosure “expression” of a gene, specifically, may refer to transcription into a polynucleotide (also referred to herein as a transcript), and / or translation into a protein, or even posttranslational modification of the protein. Protein and / or transcript "stability", as used herein, refers to the physical (thermodynamic) stability, and chemical stability of the protein and / or transcript and relates to the net balance of forces, which determine whether a protein will be in its native folded conformation or a denatured state. More specifically, the levels of proteins and / or transcripts within cells are determined not only by rates of synthesis as discussed above, but also by rates of degradation and the half-lives of proteins and / or transcripts within cells that vary widely, from minutes to several days. In eukaryotic cells, two major pathways mediate protein degradation, the ubiquitin-proteasome pathway and lysosomal proteolysis.

[0111] As indicated above, the compounds used in the re -programming agents of the present disclosure and any compositions, kits and methods thereof, inhibit and / or reduce the expression, level, stability and / or activity of at least one of: at least one member of the EGR family and / or at least one member of the DGK family. More specifically, the terms "inhibition", "moderation", “reduction” or "attenuation" as referred to herein, relate to the retardation, restraining or reduction of the expression, levels, stability and / or activity of at least one EGR protein and / or at least one member of the DGK family by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%. It should be appreciated that 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. Therefore, the term inhibit or decrease refers to an inhibition of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 folds or more.

[0112] In some embodiments, the disclosed re-programming agent activates and / or functionalize dysfunctional NK cells that comprise anergic NK cells and / or exhausted NK cells. More specifically, in some embodiments, the dysfunctional cells may be anergic cells. In yet some embodiments the dysfunctional NK cells may be exhausted cells. In some embodiments, the dysfunctional NK cells may be exhausted cells and anergic cells.

[0113] Thus, in some embodiments, the dysfunctional NK cell referred to by the present disclosure may be anergic cells. As used herein, "Anergic NK cells" refer herein to a subset of non-responsive peripheral NK cells or dysfunctional NK cells that fail to engage inhibitory receptors during development, due to lack of inhibitory receptor expression on the NK cell. Anergy results primarily from insufficient or inappropriate activation signals during the priming of NK cells, often due to a lack of co- stimulatory cues or the dominance of inhibitory signals. In addition to the functional impairment indicated above, that characterize functionally anergic NK cells (that form a portion of about 13%+ / -6, or the entire NK cell population in a healthy subject), these cells may be further structurally characterized. In some embodiments, anergic NK cells comprise a phenotype of NKG2A“panKIR“. More specifically, anergic NK cell do not express the NKG2A protein, and do not express the panKIR. In some other embodiments, anergic NK cells do not express or express only undetectable levels of the NKG2A protein, and the panKIR proteins. More specifically, "panKIR" as used herein, refers to molecules that target Killer-cell Immunoglobulin-like Receptors (KIRs) broadly across their subtypes. The KIR family consists of multiple inhibitory and activating receptors, which interact with MHC class I molecules to regulate NK cell activity. KIR family is composed of multiple inhibitory and activating receptors, which interact with MHC class I molecules to regulate NK cell activity. In some embodiments, KIR proteins considered under the scope of panKIR include but are not limited to activating KIRs such as KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1 as well as to inhibitory KIRs such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3. It should be thus understood that according to some embodiments, anergic NK cells are NK cells that do not express at least one of the specified KIRs. Still further, as used herein, anergic NK cell is characterized in not expressing the NKG2A. More specifically, NKG2A is an inhibitory receptor expressed on NK cells and CD8+ T cells that interacts with HLA-E on target cells to suppress immune cell activation. NKG2A is a C-type lectin-like inhibitory receptor that pairs with CD94 to form a heterodimeric receptor complex known as CD94 / NKG2A. Thus, in some embodiments, anergic NK cells as used herein are cells that do not express the NKG2A, and any of the KIRs as specified above, and any combinations thereof.

[0114] In yet some additional or alternative embodiments, the dysfunctional NK cell referred to by the present disclosure may be exhausted NK cells. More specifically, “Exhausted” NK cells, refer herein to another subset of dysfunctional NK cells that arise because of chronic stimulation by their targets. This state is typically observed in settings such as chronic infections, cancer, or persistent inflammatory conditions, where continuous antigen exposure or signaling from inhibitory receptors leads to a dysregulated immune response. Exhausted NK cells exhibit a distinct phenotypic profile marked by the upregulation of inhibitory receptors (e.g., PD-1, TIM-3, LAG-3, TIGIT, and in some embodiments, NKG2A and KIRs) and downregulation of activating receptors (e.g., NKG2D, NKp30). Additionally, they often display altered transcriptional and epigenetic signatures that reinforce their dysfunctional state. As opposed to anergic NK cells, exhausted NK cells may comprise a phenotype of NKG2A+panKIR+' Unlike anergic NK cells, exhausted NK cells have a history of hyperactivation before transitioning to a dysfunctional state, whereas anergic cells may never achieve full activation. Still further, in some embodiments anergic cells do not achieve full activation.

[0115] In yet some further embodiments, the at least one compound that specifically inhibits the expression, activity and / or stability of at least one of at least one member of the EGR family of transcription factors; and / or at least one member of the DGK family comprises at least one nucleic acid molecule, at least one amino acid-based molecule, and / or at least one chemical inhibitor, each specific for one of: (i) at least one member of the EGR family of transcription factors; and / or (ii) at least one member of the DGK family. It should be understood that the disclosed re-programming agent (or modulator and / or re-programmer) may comprise several compounds, however, each of the compounds is specific for one target, for example, for one member of the ERG family or for one member of the DGK family. It should be further understood that the disclosed re-programming agent may comprise more than one compound (can be two or more different compounds specific for the same target), each specific for one member of the ERG family or for one member of the DGK family.

[0116] As indicated herein, the re-programming agent (or modulator and / or re -programmer) of the present disclosure comprises at least one compound that inhibits the expression, activity and / or stability of at least one member of the EGR family, or of at least one member of the DGK family. Such compound is also referred to herein as an "inhibitory compound". It should be therefore understood that the inhibitory action of the compound is specific for the target EGR family member or the target DGK family member, while it acts as a re -programming agent for NK cells, and specifically for dysfunctional NK cells. A "Compound" is used herein to refer to any substance, agent (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound may be any agent that can be represented by a chemical formula, chemical structure, or sequence. Example of compounds applicable for the present disclosure, include, e.g., nucleic acid molecules (e.g., RNAi agents, antisense oligonucleotide, gRNAs, aptamers), small molecules, amino acid based molecules, for example, polypeptides, peptides, antibodies specific for EGR and / or DGK, lipids, polysaccharides, etc., that inhibit or disturb the activity thereof. It should be understood that any compound described in connection to the present aspect is also applicable in all aspects of the disclosure. It should be further understood that the disclosure encompasses the use of any of the described compounds or any combinations or mixtures thereof, in any of the aspects disclosed by the present disclosure. In general, compounds may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the compound. A compound may be at least partly purified. In some embodiments a compound may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the compound, in various embodiments. In some embodiments a compound may be provided as a salt, ester, hydrate, or solvate. In some embodiments a compound is cell-permeable, e.g., within the range of typical compounds that are taken up by cells and acts intracellularly, e.g., within mammalian cells, to produce a biological effect. Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z- isomers, R- and S -enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-) - and (+)-isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated. Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates (e.g., with water (i.e. hydrates) or common solvents) and / or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable.

[0117] In yet some further embodiments, the inhibitory compound may be an antibody specifically directed against at least one member of the EGR family or at least one member of the DGK family, and affects, specifically, reduces the amount, stability and function of at least one member of the EGR family or at least one member of the DGK family. It should be noted that specific definition of the term antibodies as defined herein after in connection with other embodiments of the disclosure, is also relevant for these embodiments as well.

[0118] Still further, in certain embodiments, candidate compounds that specifically inhibit at least one of, the expression, activity and stability of at least one member of the EGR family, or of at least one member of the DGK family, can be screened from large libraries of synthetic or natural compounds. A compound to be tested may be referred to as a test compound or a candidate compound. Any compound may be used as a test compound in various embodiments. In some embodiments a library of FDA approved compounds that can be used by humans may be used. Compound libraries are commercially available from a number of companies including but not limited to Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Microsource (New Milford, CT), Aldrich (Milwaukee, WI), AKos Consulting and Solutions GmbH (Basel, Switzerland), Ambinter (Paris, France), Asinex (Moscow, Russia), Aurora (Graz, Austria), BioFocus DPI, Switzerland, Bionet (Camelford, UK), ChemBridge, (San Diego, CA), ChemDiv, (San Diego, CA), Chemical Block Lt, (Moscow, Russia), ChemStar (Moscow, Russia), Exclusive Chemistry, Ltd (Obninsk, Russia), Enamine (Kiev, Ukraine), Evotec (Hamburg, Germany), Indofine (Hillsborough, NJ), Interbio screen (Moscow, Russia), Interchim (Montlucon, France), Life Chemicals, Inc. (Orange, CT), Microchemistry Ltd. (Moscow, Russia), Otava, (Toronto, ON), PharmEx Ltd.(Moscow, Russia), Princeton Biomolecular (Monmouth Junction, NJ), Scientific Exchange (Center Ossipee, NH), Specs (Delft, Netherlands), TimTec (Newark, DE), Toronto Research Corp. (North York ON), UkrOrgSynthesis (Kiev, Ukraine), Vitas-M, (Moscow, Russia), Zelinsky Institute, (Moscow, Russia), and Bicoll (Shanghai, China). Combinatorial libraries are available and can be prepared. Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are commercially available or can be readily prepared by methods well known in the art. Compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, and marine samples may be tested for the presence of potentially useful pharmaceutical compounds. It will be understood that the agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. In some embodiments a library useful in the present disclosure may comprise at least 10,000 compounds, at least 50,000 compounds, at least 100,000 compounds, at least 250,000 compounds, or more.

[0119] In some specific embodiments, the compound used for the re-programming agents of the present disclosure, that specifically inhibit at least one of, the expression, activity and stability of at least one member of the EGR family, and / or of at least one member of the DGK family, may be a small molecule, and as indicated above, may be referred to herein as an inhibitory compound. A "small molecule" as used herein, is an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and / or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least one functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups.

[0120] In some embodiments, the re -programming agents of the present disclosure may comprise at least one compound that may comprise at least one nucleic acid molecule. In some embodiments, each nucleic acid molecule is specifically directed against, or is specific for at least one of: (i) at least one of EGR-2 and / or EGR-4; and (ii) at least one of DGK-a and / or DGK-^.

[0121] The term “nucleic acid', “nucleic acid sequence”, or "polynucleotide" and “nucleic acid molecule” refers to polymers of nucleotides, and includes but is not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA / RNA hybrids including polynucleotide chains of regularly and / or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an —OH, then and — H, then an —OH, then an — H, and so on at the 2' position of a sugar moiety), and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included. The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double- stranded polynucleotides. Preparation of nucleic acids is well known in the art. It should be appreciated that the disclosure may further refer to polyribonucleotide. The term "polyribonucleotide" refers to a polynucleotide comprising one or more modified or unmodified ribonucleotides and / or their analogs. The term "polyribonucleotide" is used interchangeably with the term "oligoribonucleotide”. In more specific embodiments a nucleic acid molecule according to the disclosure may be an iRNA molecule, more specifically, dsRNA molecule.

[0122] In some embodiments, the nucleic acid molecule of the disclosed re-programming agent (or modulator and / or re-programmer) is a ribonucleic acid (RNA) molecule, or any nucleic acid sequence encoding such RNA molecule. More specifically, in some embodiments, the disclosed re-programming agent comprises at least one RNA molecule that may be at least one of: a doublestranded RNA (dsRNA), an antisense RNA, a single-stranded RNA (ssRNA), guide RNA (gRNA) and a Ribozyme.

[0123] In yet some specific embodiments, the re-programming agent of the present disclosure may comprise at least one dsRNA molecule. In some embodiments, such dsRNA molecule is at least one of: a small interfering RNA (siRNA), endoribonuclease-prepared short interfering RNA (esiRNA), a MicroRNA (miRNA), a short hairpin RNA (shRNA) and a PIWI interacting RNAs (piRNAs). In certain embodiments, the re-programming agents of the present disclosure may comprise at least one dsRNA molecules, specifically, each directed against, or specific for, at least one of: (i) at least one of EGR-1 and EGR-2; and (ii) at least one of DGK-b, c-DGK and DGK-3. In yet more specific embodiments, such dsRNA molecules, may be at least one of small interfering RNA (siRNA), MicroRNA (miRNA), short hairpin RNA (shRNA) and PIWI interacting RNAs (piRNAs).

[0124] Thus, in some embodiments, the at least one compounds of the disclosure may be nucleic acid molecules that may comprise at least one of a small interfering RNA (siRNA), a short hairpin RNA (shRNA), microRNA (miRNA), antisense oligonucleotide (ASO), locked nucleic acid (LNA), as well as other nucleic acids derivatives.

[0125] In some embodiments, the at least one compounds of the disclosure may be dsRNA molecules participating in RNA interference. More specifically, the dsRNA encompassed by the disclosure may be selected from the group consisting of small interfering RNA (siRNA), MicroRNA (miRNA), short hairpin RNA (shRNA), PIWI interacting RNAs (piRNAs). RNA interference (RNAi) is a general conserved eukaryotic pathway which down regulates gene expression in a sequence specific manner. It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in its duplex region to the sequence of the silenced gene. Gene silencing is induced and maintained by the formation of partly or perfectly double-stranded RNA (dsRNA) between the target RNA and the siRNA / shRNA derived ‘guide” RNA strand. The expression of the gene is either completely or partially inhibited. As known in the art RNAi is a multistep process. In a first step, there is cleavage of large dsRNAs into 21-23 ribonucleotides-long double-stranded effector molecules called “small interfering RNAs” or “short interfering RNAs” (siRNAs). These siRNAs duplexes then associate with an endonuclease-containing complex, known as RNA-induced silencing complex (RISC). The RISC specifically recognizes and cleaves the endogenous mRNAs / RNAs containing a sequence complementary to one of the siRNA strands. One of the strands of the double-stranded siRNA molecule (the “guide” strand) comprises a nucleotide sequence that is complementary to a nucleotide sequence of the target gene, or a portion thereof, and the second strand of the doublestranded siRNA molecule (the passenger” strand) comprises a nucleotide sequence substantially similar to the nucleotide sequence of the target gene, or a portion thereof. After binding to RISC, the guide strand is directed to the target mRNA cleaved between bases 10 and 11 relative to the 5' end of the siRNA guide strand by the cleavage enzyme Argonaute-2 (AGO2). Thus, the process of mRNA translation can be interrupted by siRNA. Thus, in some specific embodiments, the re-programming agent (or modulator and / or reprogrammer) of the present disclosure may comprise at least one siRNA molecule that specifically target at least one member of the EGR family, and at least one siRNA molecule that specifically target at least one member of the DGK family. In more particular embodiments, siRNAs comprise a duplex, or double-stranded region, of about 5-50 or more, 10-50 or more, 15-50 or more, 5-45, 10-45, 15-45, 5-40, 10-40, 15-40, 5-35, 10-35, 15-35, 5-30, 10-30 and 15-30 or more nucleotides long. In yet some more particular embodiments, the siRNAs of the disclosure comprise a nucleic acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides.

[0126] Often, siRNAs contain from about two to four unpaired nucleotides at the 3' end of each strand. At least a portion of one strand of the duplex or double- stranded region of a siRNA is substantially homologous to or substantially complementary to a target sequence within the gene product (i.e. RNA) molecule as herein defined. The strand complementary to a target RNA molecule is the “antisense guide strand”, the strand homologous to the target RNA molecule is the “sense passenger strand” (which is also complementary to the siRNA antisense guide strand). siRNAs may also be contained within structured such as miRNA and shRNA which has additional sequences such as loops, linking sequences as well as stems and other folded structures.

[0127] More specifically, the strands of a double-stranded interfering RNA (e.g., siRNA) may be connected to form a hairpin or stem-loop structure (e.g., shRNA). Thus, as mentioned above the compounds of the present disclosure may also be short hairpin RNA (shRNA).

[0128] According to other embodiments the compounds according to the present disclosure may be a micro-RNA (miRNA). miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants. The primary transcript (termed the "pri-miRNA") is processed through various nucleolytic steps to a shorter precursor miRNA, or "pre-miRNA." The pre-miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA. The pre-miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. Unlike, siRNAs, miRNAs bind to transcript sequences with only partial complementarity and usually repress translation without affecting steady-state RNA levels. Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (RISC). More specific embodiments relate to the compounds of the re-programming agent (or modulator and / or re-programmer) of the present disclosure that may be at least one shRNA molecule. The term "shRNA", as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence. The degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions. The first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop.

[0129] An “antisense RNA” is a single strand RNA (ssRNA) molecule that is complementary to an mRNA strand of a specific target gene product. Antisense RNA may inhibit the translation of a complementary mRNA by base-pairing to it and physically obstructing the translation machinery. By "complementary" it is meant the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. Still further, in some embodiments, the inhibitory compound / s of the re-programming agent (or modulator and / or re-programmer) of the present disclosure may comprise an antisense oligonucleotide, or any derivatives thereof. In more specific embodiments such oligonucleotide is an antisense oligonucleotide (ASO). As used herein, "oligonucleotide" means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides. As used herein, "modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleoside and / or at least one modified internucleoside linkage. Still further, "Antisense oligonucleotide" (AON, or ASO / s as used herein interchangeably) means an oligomeric compound, at least a portion of which is at least partially complementary to a target nucleic acid to which it hybridizes, for example, a target sequence within the nucleic acid sequence encoding at least one of, at least one EGR protein, and at least one DGK protein. Such hybridization results in at least one antisense activity. In certain embodiments, the present disclosure provides antisense oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the present disclosure provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the disclosure provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides or nucleotides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and

[0130] Y are each independently selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 and more nucleosides or nucleotides; provided that X<Y. In some embodiments, the oligonucleotides provided and used by the disclosure may comprise DNA, RNA, any derivatives thereof or any combinations thereof. More specifically, the currently used antisense oligonucleotides are rarely regular RNA or DNA oligonucleotide, as alternative antisense oligonucleotide chemistries have been developed to improve affinity, boost stability in the circulation and in target cells, and enhance cell penetration and nuclear accumulation. The non-bridging oxygen in the phosphate backbone may be replaced with a sulfur atom, generating phosphorothioate (PS) AONs. This modification enhances cellular uptake and improves resistance to nucleases but reduces the affinity of the AON to the target RNA. Addition of a methyl or a methoxyethyl group to the 2'-0 atom of the ribose sugar (2'0Me and 2'0M0E, respectively) renders the AON-target RNA hybrid RNase H-resistant and increases the affinity of the AON for the target RNA. Most AONs have both the 2'0 and the phosphorothioate (PS) modification (2'OMe-PS and 2'OMOE-PS) since they have a good safety profile and their synthesis is relatively inexpensive. “2'-0Me” or “2'-OCH3” or “2'-O-methyl” each means a nucleoside comprising a sugar comprising an — OCH3 group at the 2' position of the sugar ring. “MOE” or “2'-M0E” or “2'-OCH2CH2OCH3” or “2'-O-methoxyethyl” each means a nucleoside comprising a sugar comprising a — OCH2CH2OCH3 group at the 2' position of the sugar ring. In a different available oligonucleotide chemistry, a methylene bridge connects the 2'-0 and the 4'-C of the ribose, forcing the nucleotide in the “endo” conformation, in what has been dubbed “locked nucleic acid” (LNA). This modification leads to a very high affinity for the target nucleic acid.

[0131] In addition to the described negatively charged oligonucleotides (2'OMe-PS, 2'OMOE-PS, and LNA), two more oligonucleotide chemistries may be used in attempts to inhibit at least one of the activity, expression and stability of at least one of, at least one EGR protein and at least one DGK protein, in accordance with some embodiments of the disclosure, specifically, peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs).

[0132] Still further, in some embodiments the inhibitory compounds acting as NK activating and / or functionalizing re -programming agent of the present disclosure may comprise at least one ribozyme. Ribozymes (ribonucleic acid enzymes) are RNA molecules that are capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes. As used herein, the term "ribozyme" refers to a catalytically active RNA molecule capable of site-specific cleavage of target mRNA. In certain embodiments, a ribozyme is a Varkud satellite ribozyme, a hairpin ribozyme, a hammerhead ribozyme, or a hepatitis delta ribozyme.

[0133] In yet some further embodiments, the inhibitory compounds used in the NK cell re-programming agent of the present disclosure, as well as in any of the aspects of the present disclosure, specifically, compound / s that inhibit the expression, activity and / or stability of at least one of, at least one EGR protein and at least one DGK protein, may be based on any gene editing system, specifically programmable system, that is specifically directed against nucleic acid sequences comprised within the nucleic acid sequence encoding at least one of, at least one EGR protein and at least one DGK protein. According to such embodiments, the inhibitory compound of the reprogramming agent of the present disclosure may comprise at least one nucleic acid sequence that targets a modifier protein, for example, a nuclease or any fusion proteins thereof, to a target sequence within the nucleic acid sequence encoding at least one of, at least one EGR protein and at least one DGK protein. Targeting the nucleic acid modifier to a specific target sequence, by a targeting molecule (such as a specific guide RNA), leads to specific binding to the target sequence and targeted manipulation (e.g., cleavage or any other modification), that leads to reduction in the expression, stability and / or activity of at least one of at least one EGR protein and at least one DGK protein. Still further, in some embodiments the inhibitory compound is at least one guide RNA that guides at least one programmable engineered nucleases (PEN) to the target nucleic acid sequence as specified herein. In some embodiments, the PEN comprises at least one clustered regulatory interspaced short palindromic repeat (CRISPR) / CRISPR associated (cas) protein. Thus, according to some embodiments, the inhibitory compound used by the disclosure comprises: first (a), at least one nucleic acid sequence comprising at least one gRNA, or any nucleic acid sequence encoding the gRNA; or any kit, composition, vector or vehicle comprising the gRNA or nucleic acid sequence encoding the gRNA. Optionally, the inhibitory compound may further comprise (b), at least one CRISPR / cas protein, or any nucleic acid molecule encoding the Cas protein, or any kit, composition, vector or vehicle comprising the CRISPR / cas protein or nucleic acid sequence encoding the CRISPR / cas protein, or any nucleic acid sequence encoding said gRNA; or any kit, composition or vehicle comprising at least one of (a) and (b).

[0134] Thus, in some embodiments, the Cas protein and the specific gRNA may be provided to and / or contacted with the target cell (e.g., hematopoietic cell, such as NK or T cell), or administered to the treated subject, either as a protein and gRNA, or alternatively, as nucleic acid sequences encoding these two elements, either in two separate nucleic acid molecules (e.g., two separate constructs), or in one nucleic acid molecule (e.g., a construct encoding both). The term "programmable engineered nucleases (PEN)" as used herein also known as "molecular DNA scissors", refers to enzymes either synthetic or natural, and used to replace, eliminate or modify target sequences in a highly targeted way. PEN target and cut specific genomic sequences (recognition sequences) such as DNA sequences. The at least one PEN may be derived from natural occurring nucleases or may be an artificial enzyme, all involved in DNA repair of double strand DNA lesions and enabling direct genome editing. In some alternative or additional embodiments the inhibitory compound according with the present disclosure encompasses also any nucleic acid molecule comprising at least one nucleic acid sequence encoding the PEN or any kit, composition or vehicle comprising the at least one PEN, or any nucleic acid sequence encoding the PEN.

[0135] In yet some further specific embodiments, such nucleases may include RNA guided nucleases such as CRISPR-Cas. However, it should be understood that in some alternative embodiments, other nucleases such as ZFN, TALEN, Homing endonuclease, Meganuclease, Mega-TALEN may be used by the methods of the disclosure for targeting at least one target nucleic acid sequence comprised within the nucleic acid sequence that encodes at least one of: at least one EGR protein and at least one DGK protein.

[0136] More specifically, in some embodiments, the at least one PEN may be at least one of a mega nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR / Cas) system. In some embodiments, the at least one PEN may be a mega nuclease. Mega nucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs); such that this site generally occurs only once in any given genome. Meganucleases are specific naturally occurring restriction enzymes and include among others, the LAGLID ADG family of homing endonucleases, mostly found in the mitochondria and chloroplasts of eukaryotic unicellular organisms.

[0137] In some embodiments, the at least one PEN may be a megaTAL. MegaTALs are fusion proteins that combine homing endonucleases, such as LAGLIDADG family, with the modular DNA binding domains of TALENs.

[0138] In some alternative embodiments, the at least one PEN may be a zinc finger nuclease (ZFN). ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA- cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences, enabling ZFN to target the target sequences within the target transcripts specified by the disclosure, thereby inhibiting the expression, activity and / or stability of at least one of, at least one EGR protein and at least one DGK protein.

[0139] In yet some other embodiments, the at least one PEN may be a transcription activator-like effectorbased nuclease (TALEN). TALEN are restriction enzymes that can be engineered to cut specific sequences of DNA. TALEN are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands).

[0140] In some specific embodiments, the targeting of the target nucleic acid sequence that is comprised within the nucleic acid sequence that encodes at least one of, at least one EGR protein and at least one DGK protein, may be mediated by a PEN that may comprise at least one clustered regulatory interspaced short palindromic repeat (CRISPR) / CRISPR associated (cas) protein system. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a bacterial immune system that has been modified for genome engineering. CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single large Cas protein for the same purpose. More specifically, Class 1 may be divided into types I, III, and IV and class 2 may be divided into types II, V, and VI (that target mRNA targets). Thus, in some embodiments, the Cas protein may be a member of at least one of CRISPR-associated system of Class 1 and Class 2. In some embodiments, the cas protein may be a member of at least one of CRISPR-associated system of any one of type II, type I, type III, type IV, type V and type VI from E. coli, Mycobacterium tuberculosis, Haloferax mediterranei, Methanocaldococcus jannaschii, Thermotoga maritima and other bacteria and archaea. It should be understood that the disclosure contemplates the use of any of the known CRISPR systems, particularly any of the CRISPR systems disclosed herein. The CRISPR-Cas system, targets DNA molecules based on short homologous DNA sequences, called spacers that exist between repeats. These spacers guide CRISPR-associated (Cas) proteins to matching sequences within the target DNA, called proto-spacers, which are subsequently cleaved. The spacers can be rationally designed to form guide RNAs (gRNAs) that target any target DNA sequence, for example, the target sequence within the nucleic acid sequence that encodes at least one of, at least one EGR protein and / or at least one DGK protein. It should be noted that the inhibitory compounds of the NK re-programming agent of the present disclosure may comprise in some embodiments at least one gRNA targeted against at least one nucleic acid target that is comprised within at least one nucleic acid sequence that encodes at least one of, at least one EGR protein and at least one DGK protein. Alternatively, the inhibitory compound acting as NK activating and / or functionalizing re-programming agent of the present disclosure may comprise any nucleic acid sequence encoding such gRNA.

[0141] In some specific embodiment, the RNA guided DNA binding protein nuclease used by the disclosure may be a CRISPR Class 2 system. In yet some further particular embodiments, such class 2 system may be a CRISPR type II system. The type II CRISPR-Cas systems include the ' HNH’-typc system (Streptococcus-like; also known as the Nmeni subtype, for Neisseria meningitidis serogroup A str. Z2491, or CASS4), in which Cas9, a single, very large protein, seems to be sufficient for generating crRNA and cleaving the target DNA, or mRNA, in addition to the ubiquitous Casl and Cas2. Cas9 contains at least two nuclease domains, a RuvC-like nuclease domain near the amino terminus and the HNH (or McrA-like) nuclease domain in the middle of the protein, but the function of these domains remains to be elucidated. However, as the HNH nuclease domain is abundant in restriction enzymes and possesses endonuclease activity responsible for target cleavage. It should be appreciated that any type II CRISPR-Cas systems may be applicable in the present disclosure, specifically, any one of type II-A, typell-B or typell-C. In more particular embodiments, at least one cas protein of type II CRISPR system used by the disclosure may be the cas9 protein, or any fragments, mutants, fusion proteins, variants or derivatives thereof (e.g., Cas9 / Cpfl / CTc(l / 2 / 3), SpCas9, SaCas9, engineered Cas9, and any mutants or fusion proteins thereof, for example, dCas9-Fokl, and the like). The CRISPR- associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to a target site (proto-spacer). After recognition between Cas9 and the target sequence double stranded DNA (dsDNA) cleavage occur, creating the double strand brakes (DSBs). Still further, CRISPR type II system as used herein requires the inclusion of two essential components: a “guide” RNA (gRNA), that is comprised within the inhibitory compound / s of the NK re-programming agent (or modulator and / or re-programmer) of the present disclosure, and a non-specific CRISPR-associated endonuclease (Cas9). Guide RNA (gRNA), as used herein refers to a synthetic fusion of the endogenous tracrRNA with a targeting sequence (also named crRNA), providing both scaffolding / binding ability for Cas9 nuclease and targeting specificity. Also referred to as “single guide RNA” or “sgRNA”. In some embodiments, the gRNA of the disclosure may comprise between about 15 to about 50 nucleotides, specifically, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides. More specifically, spacers, or gRNA may comprise between about 20-35 nucleotides. In some specific embodiments, the gRNA that specifically targets EGR2 comprises the nucleic acid sequence as denoted as SEQ ID NO: 15 and / or SEQ ID NO: 16. In other specific embodiments, the gRNA that specifically targets DGKa comprises a nucleic acid sequence as denoted as SEQ ID NO: 17 and / or SEQ ID NO: 18.

[0142] In yet some further embodiments, where the inhibitory compounds of the re-programming agent of the present disclosure comprise at least one nucleic acid sequence encoding the gRNA, such encoding sequence may be designed to target at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

[0143] 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

[0144] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

[0145] 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

[0146] 94, 95, 96, 97, 98, 99, 100 or more target protospacers (target sequences recognized by the gRNA) within the at least one nucleic acid sequence that encodes at least one of, at least one EGR protein and at least one DGK protein. In CRISPR systems based on PAM (protospacer adjacent motif) sequence recognition like CRISPR Type II, the PAM is absolutely necessary for target binding and the exact sequence is dependent upon the species of Cas9 used. In certain embodiments, Cas9 from S. pyogenes may be used in the methods, cells, compositions, and kits of the disclosure. Nevertheless, it should be appreciated that any known Cas9 may be applicable. Non-limiting examples for Cas9 useful in the present disclosure include but are not limited to Streptococcus pyogenes (SP), also indicated herein as SpCas9, Staphylococcus aureus (SA), also indicated herein as SaCas9, Neisseria meningitidis (NM), also indicated herein as NmCas9, Streptococcus thermophilus (ST), also indicated herein as StCas9 and Treponema denticola (TD), also indicated herein as TdCas9. In some specific embodiments, the Cas9 of Streptococcus pyogenes Ml GAS. Still further, it should be appreciated that type V CRISPR / Cas, including Casl2a, Cpfl (type VI), C2C1 (type V-B), Casl3 (type VI), specifically, C2C2 and CasRx and CasX, as well as any variants or fusion proteins thereof, is also applicable in the methods of the disclosure. In more specific embodiments, the gRNA comprised within the inhibitory compounds acting as NK activating and / or functionalizing re -programming agent of the present disclosure, targets the specific target sequence as disclosed by the disclosure and guides the CRISPR / Cas-protein, specifically, Cas9 to cleave, or perform other modification in the target site. The end result of Cas9-mediated DNA cleavage is a double strand break (DSB) within the target DNA. The resulting DSB may be then repaired by one of two general repair pathways, the efficient but error-prone Non-Homologous End Joining (NHEJ) pathway and the less efficient but high-fidelity Homology Directed Repair (HDR) pathway. In some specific embodiments, the targeted nucleic acid sequences specified above are repaired through the NHEJ pathway, resulting in most cases in alteration of the target sequence (deletions / insertions / non-sense mutations etc.), thereby inhibiting the expression, activity and / or stability of at least one of, at least one EGR protein and at least one DGK protein.

[0147] As indicated above, the gene editing system used as the inhibitory compounds of the reprogramming agent of the present disclosure may be provided as nucleic acid molecules, specifically in a delivery vector or vehicle. However, it should be appreciated that any of the gene editing systems used, may be also administered as a protein complex, or alternatively, as a ribonucleoprotein complex. More specifically, when gene editing system is used by the disclosure such system may be delivered either as nucleic acid sequences encoding the components of this system, e.g., constructs comprising nucleic acid sequences that encode the CRISPR / Cas protein, for example, Cas9 and the specific gRNAs. However, it should be appreciated that the present disclosure further encompasses in some embodiments thereof the option of using Cas9 / gRNA Ribonucleoprotein complexes (Cas9 RNPs), that comprise purified Cas9 and purified gRNAs delivered as functional complexes. In some particular embodiments, purified gRNAs can be generated by PCR amplification of annealed gRNA oligos or in vitro transcription of a linearized gRNA containing plasmid. Cas9 (or any variant of Cas9) can be purified from bacteria through the use of bacterial Cas9 expression plasmids. In yet some further embodiments, the Cas9 RNP delivery to target cells may be carried out in some specific and non-limiting embodiments, via lipid-mediated transfection or electroporation.

[0148] Still further, in some embodiments, the inhibitory compounds acting as NK activating and / or functionalizing re-programming agent (or modulator and / or re-programmer) of the present disclosure may comprise PIWI interacting RNAs (piRNAs). PlWI-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs, typically 24-32 nucleotides in length, that associate specifically with PIWI proteins, a subfamily of the Argonaute protein family. These RNA-protein complexes play a critical role in the regulation of gene expression, particularly within the context of genomic stability and transposon silencing in germline cells. piRNAs are primarily transcribed from discrete genomic loci known as piRNA clusters, which often comprise repetitive and transposable element-derived sequences. The biogenesis of piRNAs is uniquely characterized by a processing pathway independent of the Dicer enzyme, contrasting with the production of other small RNAs such as microRNAs and small interfering RNAs.

[0149] In some embodiments, the disclose re-programing agent comprise compounds such as gRNA or siRNA that target the mRNA encoding at least one member of the EGR family and / or at least member of the DGK family. It should be understood that in some embodiments, targeting the mRNA enables a temporary effect. It should be understood that in some embodiments the inhibition of the expression, activity and / or stability of at least one of a member of the EGR or a member of the DGK family is transient or temporary. In some alternative embodiments the inhibition of the expression, activity and / or stability of at least one of a member of the EGR or a member of the DGK family is permanent.

[0150] In more specific embodiments, the disclosed NK re-programming agent (or modulator and / or reprogrammer) comprise or are composed of at least one compound that comprises at least one siRNA and / or esiRNA molecule specific for at least one member of the EGR family, and / or at least one siRNA and / or esiRNA molecule specific for at least one member of the DGK family. Each of such siRNA molecule / s is specific for one of: (i) one member of the EGR family of transcription factors; or (ii) one member of the DGK family. Accordingly, in some specific embodiments, the disclosed re -programming agent may comprise at least one siRNA molecule, each siRNA and / or esiRNA molecule is specific for at least one member of the EGR family. In yet some further embodiments, the disclosed re-programming agent may comprise at least one siRNA and / or esiRNA molecule, each siRNA molecule is specific for at least one member of the DGK family. In more specific embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for one member of the EGR family and / or at least one siRNA molecule specific for at least one member of the DGK family.

[0151] In more specific embodiments, the EGR family of transcription factors comprises at least one of EGR2, EGR4, EGR1 and EGR3. Accordingly, the disclosed re-programming agent (or modulator and / or re-programmer) may comprise at least one compound, each specific for one of EGR2, EGR4, EGR1 and EGR3. Specifically, the disclosed re-programming agent may comprise at least one siRNA molecule specific for EGR2 and / or at least one siRNA molecule specific for EGR4, and / or at least one siRNA molecule specific for EGR1 and / or at least one siRNA molecule specific for EGR3.

[0152] EGR2 (Early Growth Response 2), as used herein, is a transcription factor encoded by the EGR2 gene and is part of the zinc-finger protein family that regulates gene expression in response to cellular signals. It plays a critical role in the development and function of the peripheral nervous system, particularly in Schwann cells, where it is essential for the formation and maintenance of myelin sheaths. Mutations in the EGR2 gene are associated with inherited neuropathies such as Charcot-Marie-Tooth disease types ID and 4E and Dejerine-Sottas syndrome, which result in defective nerve conduction and progressive muscle weakness. In some embodiments, the EGR 2, as referred to herein relates to the human EGR 2. In yet some further embodiments, the human EGR 2 is as denoted by genebank accession number nm_000399. Still further, in some embodiments, the EGR 2 as used herein refers to a protein comprising the amino acid sequence as denoted by SEQ ID NO: 1, or any variants or isoforms thereof. In yet some further embodiments, the EGR 2 as used herein refers to a protein encoded by a nucleic acid molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 2, or any variants or isoforms thereof.

[0153] Still further, in some embodiments, the EGR4 (Early Growth Response 4) as used herein, is a transcription factor belonging to the EGR family, characterized by zinc-finger domains that regulate gene expression in response to external stimuli such as growth factors and stress. It plays critical roles in various biological processes, particularly in the reproductive system, where it is essential for spermatogenesis and male fertility due to its high expression in the testes. EGR4 is also expressed in the brain, contributing to neuronal function, plasticity, and potentially learning and memory processes. Dysregulation of EGR4 has been associated with reproductive disorders, cancer, and potential roles in neurodegenerative diseases, highlighting its importance in both development and disease contexts.

[0154] In some embodiments, the EGR 4, as referred to herein relates to the human EGR 4. In yet some further embodiments, the human EGR 4 is as denoted by genebank accession number nm_001964. Still further, in some embodiments, the EGR 4 as used herein refers to a protein comprising the amino acid sequence as denoted by SEQ ID NO: 3, or any variants or isoforms thereof. In yet some further embodiments, the EGR 4 as used herein refers to a protein encoded by a nucleic acid molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 4, or any variants or isoforms thereof.

[0155] Still further, in some embodiments, DGKa (Diacylglycerol Kinase Alpha), as used herein, is an enzyme that plays a critical role in lipid signaling by catalyzing the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), both of which are important secondary messengers in various cellular processes. DGKa is involved in the regulation of key signaling pathways, including those controlling cell proliferation, differentiation, and survival. It has a significant role in T-cell activation and function, influencing immune responses by modulating the levels of DAG, which activates signaling proteins like protein kinase C (PKC) and RasGRPs. DGKa is also implicated in cancer biology, as its dysregulation can alter cell growth and migration, contributing to tumor progression.

[0156] In some embodiments, the DGKa, as referred to herein relates to the human DGKa. In yet some further embodiments, the human DGKa is as denoted by genebank accession number NM_001345. Still further, in some embodiments, the DGKa as used herein refers to a protein comprising the amino acid sequence as denoted by SEQ ID NO: 5, or any variants or isoforms thereof. In yet some further embodiments, the DGKa as used herein refers to a protein encoded by a nucleic acid molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 6, or any variants or isoforms thereof.

[0157] Still further, DGK (Diacylglycerol Kinase Zeta), as used herein, is an isoform of the diacylglycerol kinase (DGK) family of enzymes, which catalyze the phosphorylation of diacylglycerol (DAG) to phosphatidic acid (PA), key lipid signaling molecules involved in various cellular processes. DGK^ plays a pivotal role in regulating intracellular signaling pathways by modulating the balance between DAG and PA. It is particularly important in T-cell biology, where it helps regulate immune responses by terminating DAG-mediated activation of pathways like protein kinase C (PKC) and RasGRPs, thus ensuring proper immune cell activation and function. DGK^ also contributes to neuronal function and synaptic plasticity, with roles in learning and memory. Furthermore, it is implicated in cancer progression, as its dysregulation can affect processes like cell migration, survival, and proliferation. Due to its diverse functions in immune regulation, neurobiology, and tumor biology, DGK^ is an emerging target of interest for therapeutic development in autoimmune diseases, neurological disorders, and cancer.

[0158] In some embodiments, the DGK^, as referred to herein relates to the human DGKq. In yet some further embodiments, the human DGK^ is as denoted by genebank accession number NM_001105540. Still further, in some embodiments, the DGK^ as used herein refers to a protein comprising the amino acid sequence as denoted by SEQ ID NO: 7, or any variants or isoforms thereof. In yet some further embodiments, the DGK^ as used herein refers to a protein encoded by a nucleic acid molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 8, or any variants or isoforms thereof.

[0159] In yet some further specific embodiments of the disclosed re-programming agent (or modulator and / or re-programmer), the DGK family comprises at least one of DGKa, DGKq, DGK5, DGK0 and DGKy. Accordingly, the disclosed re -programming agent may comprise at least one compound, each specific for one of DGKa, DGK^, DGK5, DGK0 and DGKy. Specifically, the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa and / or at least one siRNA molecule specific for DG Kq and / or at least one siRNA molecule specific for DGK5, and / or at least one siRNA molecule specific for DGK0 and / or at least one siRNA molecule specific for DGK5, and / or at least one siRNA molecule specific for DGKy. In some embodiments, the disclosed re-programming agent comprises at least one compound that inhibits the expression and / or the stability and / or the activity of at least on member of the EGR family, with the proviso that such EGR family member is not the EGR3. Still further, in some embodiments, the disclosed re-programming agent comprises at least one compound that inhibits the expression and / or the stability and / or the activity of at least on member of the EGR family, with the proviso that such EGR family member is not the EGR1. In yet some further embodiments, the disclosed re-programming agent comprises at least one compound that inhibits the expression and / or the stability and / or the activity of at least on member of the EGR family, with the proviso that such EGR family member is not the EGR3 and not the EGR1.

[0160] In some specific embodiments, the at least one member of the EGR family of transcription factors comprises at least one of EGR2 and EGR4; and the at least one member of the DGK family comprises at least one of DGKa and DGK^. Accordingly, in some embodiments, the disclosed NK re-programming agent may comprise at least one compound, each specific for one of EGR2 and EGR4, and / or at least one compound, each specific for one of DGKa and DGK^. For example, in case the compound of the disclosed re-programming agent is at least one siRNA molecule, the disclosed re-programming agent may comprise at least one siRNA molecule specific for EGR2, and / or at least one siRNA molecule specific for EGR4, and / or at least one siRNA molecule specific for DGKa, and / or at least one siRNA molecule specific for DGK^. For example, at least one siRNA molecule specific for EGR2 and at least one siRNA molecule specific for EGR4. In some other embodiments, at least one siRNA molecule specific for EGR4 and at least one siRNA molecule specific for DGKa. In some alternative embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for EGR2. In yet some further embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for EGR2 and at least one siRNA molecule specific for DGK^. Still further, in some embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGK^ and at least one siRNA molecule specific for EGR4. Still further, in some embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for EGR4, and at least one siRNA molecule specific for EGR2 and at least one siRNA molecule specific for DGKa. In some embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for EGR4, and at least one siRNA molecule specific for EGR2 and at least one siRNA molecule specific for DGK^. In some embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for DGK^. In some other embodiments, the disclosed re -programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for DGK^, and at least one siRNA molecule specific for EGR2. In some other embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for DGK^, and at least one siRNA molecule specific for EGR4. In some other embodiments, the disclosed re-programming agent may comprise at least one siRNA molecule specific for DGKa, and at least one siRNA molecule specific for DGK^, and at least one siRNA molecule specific for EGR2, and at least one siRNA molecule specific for EGR4. It should be understood that all combinations of the disclosed siRNA molecules as disclosed herein for all the optional NK cell re -programming agents that functionalize NK cell (e.g., functionalize and / or rewire dysfunctional NK cells), is also applicable for any of the aspects of the present disclosure, as detailed herein after or before.

[0161] It should be understood that the inhibiting compounds (e.g., the disclosed siRNA molecules) for at least one member of the EGR family and / or at least one member of the DGK family, may provide in some embodiments a synergistic effect.

[0162] Thus, in some embodiments, the re-programming agents of the present disclosure may be also referred to as synergistic re-programming agents, that synergistically functionalize and / or rewire, and / or reprogram, and / or activate dysfunctional NK cells. It should be understood that any of the combinations of siRNA molecules disclosed herein above is considered in some embodiments as a synergistic combination. For example, the combination of siRNA specific for EGR2 and an siRNA molecule specific for EGR4, that may be used herein as the re -programming agent that functionalize dysfunctional NK cells (e.g., exhausted or anergic NK cells). In yet some further embodiments, a re-programming agent comprising a combination of siRNA specific for EGR2 and an siRNA molecule specific for DGKa, that may be used herein as the re -programming agent (or modulator and / or re -programmer) that functionalize dysfunctional NK cells (e.g., exhausted or anergic NK cells). A synergistic combination as used herein in connection with the disclosed reprogramming agents, refers to the interaction between two or more components, or the cumulative action of the two components (e.g., siRNA specific for EGR2 and an siRNA molecule specific for EGR4) that produce a combined effect greater than the sum of their individual effects.

[0163] The re-programming agents disclosed herein may comprise any of the inhibitory compounds (e.g., siRNA molecules) at any ratio, for example, 1:1, 1:1:1, 1 : 1 : 1 : 1 , 1:2 to 1: 106, 2:2:1, 3:1:1, 4: 1 : 1 : 1 , or between 0.0001 to 106: between 0.0001 to 106: and between 0.0001 to 106. The effective amount of the NK re-programming agents (e.g., siRNAs) may be in some embodiments, the amount of the compounds sufficient to inhibit the expression, stability and / or activity of any one of the at least one EGR protein and / or of the at least one DGK protein as discussed above. In some embodiments, the active ingredient in the compositions of the disclosure may be provided in an amount effective for inhibiting the expression, stability and activity of at least one EGR protein and / or of the DGKs proteins. Percentage of such inhibitory effect is as indicated herein before in connection with the re-programming agents of the present disclosure. Such inhibitory effect is in extent for activating hematopoietic cells, specifically NK cells, more specifically, dysfunctional NK cells (e.g., anergic or exhausted NK cells).

[0164] Thus, in some embodiments, the disclosed re-programming agent comprises an effective amount of at least one of: (i) at least one siRNA or esiRNA molecule specific for EGR2; (ii) at least one siRNA or esiRNA molecule specific for EGR4; (iii) at least one siRNA or esiRNA molecule specific for DGKa and (iv) at least one siRNA or esiRNA molecule specific for DGK^; and / or any combinations of (i), (ii), (iii) and (iv).

[0165] It should be understood that the disclosed siRNA sequences are only non-limiting embodiments and working examples for nucleic acid-based inhibitors (e.g., siRNA, or esiRNA, gRNAs, Oligonucleotides, etc.), of the expression of at least one of the EGR family and / or at least one of the DGK family. More specifically, it should be understood that any siRNA that target the nucleic acid sequence encoding the EGR2 molecule, specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 2, or any fragments or parts thereof, may be used herein as a suitable siRNA molecule. Similarly, it should be understood that any siRNA that target the nucleic acid sequence encoding the EGR4 molecule, specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 4, or any fragments or parts thereof, may be used herein as a suitable siRNA molecule. Still further, in some embodiments, it should be understood that any siRNA that target the nucleic acid sequence encoding the DGKa molecule, specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 6, or any fragments or parts thereof, may be used herein as a suitable siRNA molecule. In some embodiments, it should be understood that any siRNA that target the nucleic acid sequence encoding the DGKq molecule, specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 8, or any fragments or parts thereof, may be used herein as a suitable siRNA molecule.

[0166] In some particular embodiments, a non-limiting example of an siRNA molecule specific for EGR2, may be any siRNA molecule or a plurality of siRNA molecules, or specifically, an esiRNA stands, specific for the EGR2 molecule. More specifically, esiRNA for "endoribonuclease-prepared small interfering RNA", is a form of small interfering RNA (siRNA) generated through enzymatic cleavage of long double- stranded RNA (dsRNA) by endoribonucleases such as RNase III or Dicer, thereby randomly generating a pool of plurality of various siRNA and / or esiRNA molecules targeting various target sequences within the target member of the EGR family, at the coding and / or the non-coding regions thereof. Still further, in some embodiments, the present disclosure provides siRNA and / or esiRNA molecules targeting EGR 2 at exon 2, and / or at the 3' UTR , for example, from position 2242 of the cDNA sequence as denoted by SEQ ID NO: 2 (NM_000399). In some specific embodiments an esiRNA molecule specific for EGR2 may comprise the nucleic acid sequence as denoted by SEQ ID NO: 9, or any complementary sequnce thereof. It should be therefore appreciated that in some embodiments, the siRNA used herein for targeting EGR2 comprise any optional 21 to 25 mers of said esiRNA sequence and thus, at least one fragment of the nucleic acid sequence as denoted by SEQ ID NO: 9, or any complementary sequence thereof. In some particular and non-limiting embodiments, an esiRNA molecule useful in the present disclosure for specifically targeting EGR2 may be the esiEgr2 MISSION Sigma Aldrich Cat no. EHU124311, as denoted by SEQ ID NO: 9, or any complementary sequence thereof, in more specific embodiments, the siRNA used herein for targeting EGR2 comprise any fragment between about 21 to 25 mers of the nucleic acid sequence as denoted by SEQ ID NO: 9, or any complementary sequence thereof. Therefore, the siRNA used for targeting EGR2 may comprise for example bases 1-21, and / or bases 2-22, and / or bases 1-23, and / or bases 2-24, and / or bases 1- 25 and / or bases 2-26 and / or bases 30-34 and / or bases 60-81, and so on of the nucleic acid sequence as denoted by SEQ ID NO: 9. In yet some further specific embodiments of the disclosed modulator and / or re-programmers, an esiRNA molecule specific for EGR4. Still further, in some embodiments, the present disclosure provides siRNA and / or esiRNA molecules targeting EGR 4 at exon 2, and / or at the 3' UTR , for example, from position 1385 of the cDNA sequence as denoted by SEQ ID NO: 4 (NM_001965). In some specific embodiments an esiRNA molecule specific for EGR4 may comprise the nucleic acid sequence as denoted by SEQ ID NO: 10. It should be therefore appreciated that in some embodiments, the siRNA used herein for targeting EGR4 comprise at least one fragment (e.g., any optional 21 to 25 mers) of the nucleic acid sequence as denoted by SEQ ID NO: 10, or any complementary sequence thereof. In some particular and nonlimiting embodiments, an esiRNA molecule useful in the present disclosure for specifically targeting EGR4 may be the esiEgr4 MISSION Sigma Aldrich Cat no. EHU135811, as denoted by SEQ ID NO: 10, or any complementary sequence thereof. It should be therefore appreciated that in some embodiments, the siRNA used herein for targeting EGR4 may comprise any fragment between about 21 to 25 mers of the nucleic acid sequence as denoted by SEQ ID NO: 10, or any complementary sequence thereof. Therefore, the siRNA used for targeting EGR4 may comprise for example bases 1-21, and / or bases 2-22, and / or bases 1-23, and / or bases 2-24, and / or bases 1- 25 and / or bases 2-26 and / or bases 30-34 and / or bases 60-81, and so on of the nucleic acid sequence as denoted by SEQ ID NO: 10.

[0167] As noted above, the present disclosure further encompasses any siRNA molecule that targets the nucleic acid molecules that encode any one of EGR2, as denoted by SEQ ID NO: 2, or any variants or homologs thereof or of the EGR4 molecule as denoted by SEQ ID NO: 4, or any variants or homologs thereof.

[0168] In yet some further specific embodiments of the disclosed modulator and / or re -programmers, an siRNA molecule specific for DGKa. Still further, in some embodiments, the present disclosure provides siRNA and / or esiRNA molecules targeting DGKa at exon 21, from position 2158 of the cDNA sequence as denoted by SEQ ID NO: 6. In yet some further specific embodiments of the disclosed re -programming agent, an siRNA molecule specific for DGKa may comprise the nucleic acid sequence as denoted by SEQ ID NO: 13, or any complementary sequnce thereof. In yet some further specific embodiments of the disclosed modulator and / or re -programmers, an siRNA molecule specific for DGK^. Still further, in some embodiments, the present disclosure provides siRNA and / or esiRNA molecules targeting DGK^ at exon 15, from position 1624 of the cDNA sequence as denoted by SEQ ID NO: 8. Still further, in some embodiments of the disclosed reprogramming agent, an siRNA molecule specific for DGK^, may comprise the nucleic acid sequence as denoted by SEQ ID NO: 14, or any complementary sequnce thereof, or of any variants, homologs or derivatives thereof. In some particular and non-limiting embodiments, an siRNA molecule useful in the present disclosure may be the DGKq siRNA Sigma Aldrich SASI_HS02_00324291.

[0169] As noted above, the present disclosure further encompasses any siRNA molecule that targets the nucleic acid molecules that encode any one of EGR2, as denoted by SEQ ID NO: 2, or any variants or homologs thereof or of the EGR4 molecule as denoted by SEQ ID NO: 4, or any variants or homologs thereof.

[0170] As noted above, the present disclosure further encompasses any siRNA molecule that targets the nucleic acid molecules that encode any one of DGKa, as denoted by SEQ ID NO: 6, or any variants or homologs thereof or of the DGK^ molecule as denoted by SEQ ID NO: 8, or any variants or homologs thereof. In some embodiments, the disclosed re -programming agent (or re -programmer or modulator) may comprise any combination of the siRNA and / or esiRNA nuclei acid molecules as denoted by SEQ ID NO: 9, any fragment between about 21 to 25 mers (nucleotides) of the nucleic acid sequence as denoted by SEQ ID NO: 9, SEQ ID NO: 10, any fragment between about 21 to 25 mers of the nucleic acid sequence as denoted by SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, or any combinations thereof, for example, SEQ ID NO: 9, and SEQ ID NO: 10; or SEQ ID NO: 9 and SEQ ID NO: 13; or SEQ ID NO: 9 and SEQ ID NO: 14; or SEQ ID NO: 10 and SEQ ID NO: 13; or SEQ ID NO: 10 and SEQ ID NO: 14; or SEQ ID NO: 13 and SEQ ID NO: 14; or SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13; or SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 14, or SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 9; or SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 10, or SEQ ID NO: 9; or SEQ ID NO: 10, SEQ ID NO: 13 and SEQ ID NO: 14.

[0171] In some specific embodiments, the siRNA and / or the esiRNA used herein for inhibiting the expression of at least one of: at least one member of the EGR family, and / or at least one member of the DGK family, specifically, at least one of EGR-2 and / or EGR-4; derived from the nucleic acid sequence as denoted by SEQ ID NO: 9 and / or SEQ ID NO: 10, and any complementary sequence thereof, including also any variants, homologs or derivatives thereof. Still further, in some embodiments, the siRNAs used herein for at least one of DGK-a and / or DGK-^, as denoted by Catalog number Sigma Aldrich SASI_HSS01_00072301 (SEQ ID NO: 13) and Sigma Aldrich SASI_HS02_00324291 (SEQ ID NO: 14). In yet some further embodiments, the disclosed reprogrammer of the present disclosure may comprise a nucleic acid-based inhibitor of at least one of the EGR family and / or of at least one of the DGK family, that may be at least one gRNA molecule, or any nucleic acid sequence encoding such gRNA molecule, to thereby applying in some embodiments the CRISPR / Cas system. In some particular and non-limiting embodiments, the disclosed re-programmer of the present disclosure may comprise the gRNA molecule that directs the CAS9 protein to a target sequence within the EGR2, or the EGR4, or the DGKa, or the DGK^, or any nucleic acid molecule encoding such gRNA. In some embodiments, the disclosed specification provides any of the disclosed siRNA and / or eiRNA molecules, as well as any complementary sequence of at least one of SEQ ID NO: 9, 10, 13 and 14, of any variants, homologs or derivatives thereof.

[0172] More specifically, it should be understood that any gRNA that target the nucleic acid sequence encoding the EGR2 molecule, specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 2, or any fragments or parts thereof, may be used herein as a suitable gRNA molecule. Similarly, it should be understood that any gRNA that target the nucleic acid sequence encoding the EGR4 molecule, specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 4, or any fragments or parts thereof, may be used herein as a suitable gRNA molecule. Still further, it should be understood that any gRNA that target the nucleic acid sequence of the DGKa molecule, specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 6, or any fragments or parts thereof, may be used herein as a suitable gRNA molecule. In some embodiments, it should be understood that any gRNA that target the nucleic acid sequence encoding the DGK^ molecule, specifically, any target sequence at the nucleic acid sequence as denoted by SEQ ID NO: 8, or any fragments or parts thereof, may be used herein as a suitable gRNA molecule.

[0173] In some particular and non-limiting embodiments, the disclosed re -programming agent (or modulator or re -programmer), comprises at least one gRNA molecule specific for EGR2. In some embodiments, the gRNA molecule comprises the nucleic acid sequence as denoted by SEQ ID NO: 15 (sense), and / or the gRNA molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 16 (antisense). Still further, in some particular and non-limiting embodiments, the disclosed modulator or re-programmer, comprises at least one gRNA molecule specific for DGKa. In some embodiments, the gRNA molecule comprises the nucleic acid sequence as denoted by SEQ ID NO: 17 (sense), and / or the gRNA molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 18 (antisense). In yet some further embodiments, the disclosed reprogrammer or modulator may comprise any combination of the gRNAs of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0174] Variants of the polynucleotides of the present disclosure may have at least 80% sequence similarity to the entire sequence, often at least 85% sequence similarity, 90% sequence similarity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarity or identity at the nucleic acid level, with the nucleic acid sequence of interest, such as the various polynucleotides of the disclosure. The term "derivative" is used to define nucleic acid sequence variants, and covalent modifications of a polynucleotide made use of in the present disclosure, e.g. of a specified sequence. The functional derivatives of any of the polynucleotides utilized according to the present disclosure, e.g. of a specified sequence of any one of the polynucleotides of SEQ ID NOs: 9, 10, 13, 14, 15, 16, 17 and 18, or any complementary sequence thereof, preferably have at least about 65%, more preferably at least about 75%, even more preferably at least about 85%, most preferably at least about 95% overall sequence homology with the nucleic acid sequence of the polynucleotide as structurally defined above, e.g. of a specified sequence, more specifically, the entire nucleic acid sequence of the polynucleotides as denoted by any one of SEQ ID NOs: 9, 10, 13, 14, 15, 16, 17 and 18, or any complementary sequence thereof, specifically, any homolog that retains the inhibitory effect on stability, expression and / or activity of any one of the EGR family member / s and the DGK family members, as specified herein, that act as re-programming agents of NK cells, specifically, reprogramming agents of dysfunctional NK cells. "Homology" with respect to a native polynucleotide and its functional derivative is defined herein as the percentage of nucleic acid bases in the sequence that are identical with the bases of a corresponding polynucleotide. Methods and computer programs for the alignment are well known. It should be appreciated that by the terms "insertions" or "deletions", as used herein it is meant any addition or deletion, respectively, of nucleic acid bases to the polynucleotides used by the disclosure, of between 1 to 10 nucleic acid bases, specifically, between 1 to 10 nucleic acid bases. More particularly, insertions or deletions may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleic acids, of any of the sequences disclosed herein. Still further, any derivatives or variants that retain the inhibitory effect of any of the specified sequences.

[0175] The terms "identical", "substantial identity", "substantial homology" or percent "identity", in the context of two or more nucleic acids or polynucleotide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleic acid bases or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, specifically, over the whole sequence.

[0176] In some specific embodiments, the disclosed re -programming agent (or re -programming agent) may be comprised within at least one of a nano- or micro-particle, a micellar formulation, a vehicle, a matrix, or a composition.

[0177] In some specific embodiments, the re-programming agents of the present disclosure may be comprised within, encapsulated or enveloped by or within, at least one of a nano- or micro-particle, a micellar formulation, any vehicle, matrix, or a composition. It should be noted that vehicle, matrix, nano- or micro-particle, a micellar formulation or composition applicable in this aspect are in some embodiments those described herein after in connection with other aspects of the disclosure.

[0178] According to the aspect of the present disclosure, provided is at least one nano- or micro-particle or micellar formulation or any vehicle, or matrix, comprising at least one re-programming agent comprising at least one compounds that specifically inhibit at least one of, the expression, activity and stability of at least one Transcription factor (e.g., of the EGR family), and of at least one Kinase (e.g., of the DGK family). In some specific embodiments, the at least one vehicle, matrix, nano- or micro-particle or micellar formulation of the disclosure may comprise at least one reprogramming agent comprising at least one compound that specifically inhibit at least one of, the expression, activity and stability of at least one member of the EGR family and / or at least one member of the DGK family.

[0179] Thus, a further aspect of the present disclosure relates to at least one nano- or micro-particle, micellar formulation, vehicle or matrix comprising at least one re-programming agent (or modulator and / or re -programmer) comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and / or (ii), at least one member of the DGK family. The re-programming agent activates and / or functionalizes dysfunctional NK cells. Thus, in some embodiments, the at least one nano- or micro-particle, micellar formulation, vehicle or matrix disclosed herein may comprise an effective amount of the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix re-programming agents. In yet some specific embodiments, the amount of the re-programming agent in the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix is the amount effective for activation of NK cells, specifically, activation of dysfunctional NK cells.

[0180] In some embodiments, the at least one nano- or micro-particle, micellar formulation, vehicle or matrix disclosed herein, may comprise at least one of any of the NK re-programming agent (or modulator and / or re -programmer) of the present disclosure, specifically, any of the modulator and / or re-programmers described herein above.

[0181] Still further, in some further embodiments of the disclosed at least one nano- or micro-particle, micellar formulation, vehicle or matrix, the at least one re -programming agent (or modulator and / or re -programmer), or any compound of the re -programming agent is encapsulated within the intra-nanoparticle core or cavity of the nano- or micro-particle, micellar formulation, vehicle or matrix.

[0182] In yet some further embodiments, the outer nanoparticle surface of the nano- or micro-particle, micellar formulation, vehicle or matrix disclosed herein, is associated directly or indirectly with at least one targeting moiety. Accordingly, the present disclosure provides at least one nano- or micro-particle, micellar formulation, vehicle or matrix with an outer nanoparticle surface that is associated directly or indirectly with at least one targeting moiety.

[0183] In some embodiments, the at least one targeting moiety of the at least one nano- or micro-particle, micellar formulation, vehicle or matrix of the present disclosure is, or may comprise at least one of: an antibody, an aptamer, a ligand or any combinations thereof, that specifically recognizes and binds at least one molecule expressed on the surface of at least one NK cell. In more specific embodiments, the targeting moiety on the surface of the disclosed nanoparticles, specifically recognizes and binds at least one dysfunctional NK cells. It should be understood that in some embodiments, where the re -programming agent activates at least one dysfunctional lymphocyte, the targeting moiety (e.g., antibody, an aptamer, a ligand or any combinations thereof), specifically recognizes and binds at least one dysfunctional lymphocyte.

[0184] In some embodiments of the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix, the at least one targeting moiety comprises at least one antibody that specifically recognizes and binds at least one molecule expressed on the surface of at least one NK cell. In some specific embodiments of the disclosed nano- or micro-particle, micellar formulation, vehicle or matrix, the at least one targeting moiety comprises at least one antibody that specifically recognizes and binds at least one molecule expressed on the surface of at least one dysfunctional NK cell.

[0185] In yet some further embodiments, the at least one vehicle, matrix, nano- or micro-particle or micellar formulation of the disclosure may comprise any of the re-programming agent (or modulator and / or re-programmer) described by the disclosure. It should be noted that in some embodiments, the compounds of the re-programming agents of the present disclosure may be any inhibitory nucleic acid molecules, SMCs, aptamers, peptide, or any combinations thereof, that specifically inhibit at least one of: (i) at least one of EGR-1 and EGR-2; and (ii) at least one of DGK-b, c-DGK and DGK-3. More specifically, in some embodiments, the re-programming agent comprised within the vehicle, matrix, nano- or micro-particle or micellar formulation of the disclosure may comprise at least one compound that comprise at least one nucleic acid molecule. More specifically, each of the nucleic acid molecules is specific for or specifically directed against one of at least one of: (i) at least one of EGR-2 and / or EGR-4; and / or (ii) at least one of DGK-a and / or DGK-^.

[0186] In yet some further embodiments, the nucleic acid molecule of the re-programming agent (or modulator and / or re-programmer) comprised within the nano- or micro-particle or micellar formulation or vehicle or matrix of the present disclosure, may be RNA molecules or any nucleic acid sequence encoding the RNA molecules. In more specific embodiments, such RNA molecules may be at least one of a dsRNA, an antisense RNA, a ssRNA, a Ribozyme and a guide RNA.

[0187] In certain embodiments, the re-programming agents comprised within the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure may comprise at least one dsRNA molecules. More specifically, each of the dsRNA molecules may be directed against or specific for one of at least one of: (i) at least one of EGR-1 and EGR-2; and (ii) at least one of: (i) at least one of EGR-2 and / or EGR-4; and / or (ii) at least one of DGK-a and / or DGK-^. In yet more specific embodiments, such dsRNA molecules, may be at least one of siRNA, miRNA, shRNA and piRNAs.

[0188] In yet some further embodiments, the re-programming agents comprised within the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure may comprise at least one of: at least one siRNA molecule specifically directed against, or specific for EGR-2, and / or at least one siRNA molecule specifically directed against EGR-4; and / or at least one siRNA molecule specifically directed against DGK-a, and / or at least one siRNA molecule specific for, or specifically directed against DGK-^.

[0189] Still further, in some embodiments, the re-programming agents comprised within the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure may comprise at least one siRNA molecule specifically directed against, or specific for EGR-2, and / or at least one siRNA molecule specifically directed against, or specific for DGK-a. It should be understood that any of the disclosed nano- or micro-particle or micellar formulation or vehicle or matrix, are applicable for any aspect of the present disclosure.

[0190] As indicated above, in yet some further embodiments of the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure, the at least one re-programming agent (or modulator and / or re -programmer) may be encapsulated within the intra-nanoparticle core and / or cavity of the nanoparticle of the disclosure. More specifically, in some embodiments, the compounds of the modulator and / or re -programmers of the present disclosure, specifically, the siRNAs of the disclosure may be surrounded, enveloped, encapsulated, entrapped and comprised within a nanoparticle, specifically, within the inner core and / or cavity of a nano-or microparticle. In more specific embodiments, at least one targeting moiety is connected and / or associated directly, or indirectly, with the outer nanoparticle surface of the nano-micro particle, vehicle, matrix, microparticle or micellar formulation of the disclosure.

[0191] In more specific embodiments, the at least one targeting moiety of the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure, may be any affinity molecule, for example, at least one of an antibody, an aptamer, a ligand (e.g., for an activating receptor, or alternatively, a ligand for inhibitory receptor) or any combinations thereof, that specifically recognizes and binds at least one molecule expressed on the surface of at least one hematopoietic cell. As indicated above, in some embodiments, the targeting moiety used by the nano-particles of the disclosure may comprise an antibody or any antigen binding fragments thereof. In yet some further embodiments, the antibody used as a targeting moiety for the vehicle, matrix, nano- or micro-particle or micellar formulation of the disclosure, may be any one of: full length antibody, antibody fragment, single-chain variable fragment (scFv), bi-specific antibody, tri-specific antibody and variable new antigen receptor antibody (V-NAR).

[0192] The term "antibody" as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Exemplary categories of antigen-binding domains that can be used in the context of the present disclosure include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen or antigen-binding scaffolds. The antigen binding domains in accordance with the disclosure may recognize and bind a specific antigen or epitope. It should be therefore noted that the term “binding specificity”, ’’specifically binds to an antigen”, “specifically immuno-reactive with”, “specifically directed against” or “specifically recognizes”, when referring to an antigen or particular epitope, refers to a binding reaction which is determinative of the presence of the epitope in a heterogeneous population of proteins and other biologies.

[0193] The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. Still further, as indicated above, an "antigen-binding domain" can comprise or consist of an antibody or antigen-binding fragment of an antibody. Still further, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein. An antigenbinding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH (e.g., VHH nanobodies), VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0194] The antibody suitable for the targeting moiety of the nanoparticle of the disclosure may also be a bi-specific antibody (such as Bi-specific T-cell engagers-BiTEs) or a tri-specific antibody.

[0195] The antibody suitable for the disclosure may also be a variable new antigen receptor antibody (V-NAR). VNARs are a class of small, immunoglobulin-like molecules from the shark immune system. Humanized versions of VNARs could be used to bind protein epitopes that are difficult to access using traditional antibodies. As noted above, in some embodiments, the targeting moiety of the nanoparticle of the disclosure may comprise aptamers that specifically recognize and bind at least one molecule expressed on the surface of at least one hematopoietic cell. As used herein the term "aptamer” or “specific aptamers” denotes single-stranded nucleic acid (DNA or RNA) molecules which specifically recognizes and binds to a target molecule. The aptamers according to the disclosure may fold into a defined tertiary structure and can bind a specific target molecule with high specificities and affinities. Aptamers are usually obtained by selection from a large random sequence library, using methods well known in the art, such as SELEX and / or Molinex. In various embodiments, aptamers may include single-stranded, partially single-stranded, partially double-stranded or doublestranded nucleic acid sequences; sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branch points and non-nucleotide residues, groups or bridges; synthetic RNA, DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes; and any ribonucleotide, deoxyribonucleotide or chimeric counterpart thereof and / or corresponding complementary sequence. In certain specific embodiments, aptamers used by the disclosure are composed of deoxyribonucleotides .

[0196] In some embodiments, the aptamer that may be applicable herein may optionally comprise a spacer between the nucleic acid sequence and the reactive group. The spacer may be an alkyl chain such as (CH2)e / i2, namely comprising six to twelve carbon atoms.

[0197] In some embodiments, the at least one target moiety of the nano-particles of the disclosure, specifically recognizes and binds and thereby targets at least one molecule expressed on the surface of at least one hematopoietic cell.

[0198] "Hematopoietic cells " are cellular blood components all derived from hematopoietic stem cells in the bone marrow. It should be appreciated that in certain embodiments, hematopoietic cells as used herein include cells of the myeloid and the lymphoid lineages of blood cells. More specifically, myeloid cells include monocytes, (macrophages and dendritic cells (DCs)), granulocytes (neutrophils), basophils, eosinophils, erythrocytes, and megakaryocytes or platelets. The Lymphoid cells include T cells, B cells, and natural killer (NK) cells. Thus, in certain embodiments, the cells targeted by the modulator and / or re-programmers of the present disclosure may be any hematopoietic cell described herein. Generally, blood cells are divided into three lineages: red blood cells (erythroid cells) which are the oxygen carrying, white blood cells (leukocytes that are further subdivided into granulocytes, monocytes and lymphocytes) and platelets (thrombocytes). In yet some embodiments, the at least one target moiety of the nano-particles of the disclosure, specifically recognizes and binds and thereby targets at least one molecule expressed on the surface of a lymphocyte.

[0199] "Lymphocytes" as used herein, are mononuclear nonphagocytic leukocytes found in the blood, lymph, and lymphoid tissues. They comprise the body's immunologically competent cells and their precursors. They are divided on the basis of ontogeny and function into two classes, B and T lymphocytes, responsible for humoral and cellular immunity, respectively. Most are small lymphocytes 7-10 pm in diameter with a round or slightly indented heterochromatic nucleus that almost fills the entire cell and a thin rim of basophilic cytoplasm that contains few granules. When "activated" by contact with antigen, small lymphocytes begin macromolecular synthesis, the cytoplasm enlarges until the cells are 10-30 pm in diameter, and the nucleus becomes less completely heterochromatic; they are then referred to as large lymphocytes or lymphoblasts. These cells then proliferate and differentiate into B and T memory cells and into the various effector cell types: B cells into plasma cells and T cells into helper, cytotoxic, and suppressor cells. In yet some further embodiments, the hematopoietic cell recognized or targeted by the targeting moiety of the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure, may be a natural killer (NK) cell. Still further, in some embodiments, the nanoparticle, vehicle, matrix, microparticle or micellar formulation of the disclosure, may be associated directly or indirectly by the outer nanoparticle surface thereof with at least one antibody, at least one aptamer or any combinations thereof, that specifically recognize and bind at least one of at least one NK cell activating receptor and at least one NK cell inhibitory receptor or any combinations thereof. Natural killer (NK) cells are a type of cytotoxic lymphocytes that are critical to the innate immune system in providing rapid responses to viral-infected cells and tumor formation. In contrast to CTLs, NK cells do not express T cell antigen receptors (TCR) or pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, instead they express the surface markers CD16 (FcyRIII) and CD56 in humans (NK1.1 or NK1.2 in mice), up to 80% of human NK cells also express CD8. Further, NK cells are effectors of innate immunity in expressing activating and inhibitory NK receptors, which play an important function in self-tolerance and in sustaining NK activity.

[0200] Natural killer cell cytolysis of target cells and cytokine production is controlled by a balance of inhibitory and activating signals, which are facilitated by NK cell receptors. NK cell inhibitory receptors are part of either the immunoglobulin-like (IgSF) superfamily or the C-type lectin-like receptor (CTLR) superfamily. Members of the IgSF family comprise the human killer cell immunoglobulin-like receptor (KIR) and the Immunoglobulin-like transcripts (ILT). Killer-cell immunoglobulin-like receptors (KIRs), are a family of type I transmembrane glycoproteins expressed on the plasma membrane of natural killer (NK) cells and a minority of T cells. They regulate the killing function of these cells by interacting with major histocompatibility (MHC) class I molecules, which are expressed on all nucleated cell types. KIR receptors can distinguish between major histocompatibility (MHC) class I allelic variants, which allows them to detect virally infected cells or transformed cells. Most KIRs are inhibitory, meaning that their recognition of MHC molecules suppresses the cytotoxic activity of their NK cell. Only a limited number of KIRs are activating, meaning that their recognition of MHC molecules activates the cytotoxic activity of their cell.

[0201] Inhibitory receptors recognize self-MHC class I molecules on target self-cells, causing the activation of signaling pathways that stop the cytolytic function of NK cells. Self-MHC class I molecules are always expressed under normal circumstance. According to the missing-self hypothesis, inhibitory KIR receptors recognize the downregulation of MHC class I molecules in virally-infected or transformed self-cells, leading these receptors to stop sending the inhibition signal, which then leads to the lysis of these unhealthy cells. Because natural killer cells target virally infected host cells and tumor cells, inhibitory KIR receptors are important in facilitating self-tolerance.

[0202] KIR inhibitory receptors signal through their immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic domain. When inhibitory KIR receptors bind to a ligand, their ITIMs are tyrosine phosphorylated and protein tyrosine phosphatases, including SHP-1, are recruited. In some embodiments, inhibitory NK cells receptors may include but are not limited to at least one of Killer-cell immunoglobulin-like receptor (KIR), Programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Ly49, NKRP1A, CD94, KIRNKG2A, T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), Lymphocyte-activation gene 3 (LAG-3), Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), Leukocyte-associated immunoglobulin- like receptor 1 (LAIR-1), Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), and any derivatives, splice variants, homologs and orthologs thereof or any combinations thereof.

[0203] Activating receptors recognize ligands that indicate host cell aberration, including induced-self antigens (which are markers of infected self-cells and comprise MICA, MICB, and ULBP, all of which are related to MCH class 1 molecules), altered-self antigens (MHC class I antigens laden with foreign peptide), and / or non-self (pathogen encoded molecules). The binding of activating KIR receptors to these molecules causes the activation of signaling pathways that cause NK cells to lyse virally infected or transformed cells.

[0204] Activating receptors do not have the immunoreceptor tyrosine-base inhibition motif (ITIM) characteristic of inhibitory receptors, and instead contain a positively charged lysine or arginine residue in their transmembrane domain (with the exception of KIR2B4) that helps to bind DAP12, an adaptor molecule containing a negatively charged residue as well as immunoreceptor tyrosinebased activation motifs (IT AM). Activating KIR receptors include KIR2DS, KIR2DL1, KIR3DS and CD244 (Cluster of Differentiation 244), also known as Natural Killer Cell Receptor 2B4. In some embodiments, activating NK cells receptors may include but are not limited to at least one of Natural cytotoxicity triggering receptor 1 (NCR1, NKp46), Natural cytotoxicity triggering receptor 2 (NCR2, NKp44), Natural cytotoxicity triggering receptor 3 (NCR3, NKp30), tumor necrosis factor receptor superfamily 7 (TNFRSF7, CD27), Lymphocyte function-associated antigen 1 (LFA-1), cluster of differentiation 16 (CD16), NKG2D, Cytotoxic And Regulatory T Cell Molecule (CRTAM), DNAX Accessory Molecule-1)(DNAM-1), NKp80 (NKp80, also known as killer cell lectin-like receptor subfamily F, member 1 (KLRF1)), and any derivatives, splice variants, homologs and orthologs thereof.

[0205] NK cells express receptors for MHC class I molecules comprising the C-type lectin-like receptors, CD94 / NKG2. CD94 / NKG2 receptors are expressed on majority of NK cells and a subset of CD8+ T cells. Five different molecular species of NKG2 (NKG2A, B, C, E and H) have been reported to form disulfide-linked heterodimers with invariant CD94. NKG2A and B, which are products from a single gene by alternative splicing, have two immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic domains and form inhibitory receptors complexed with CD94. NKG2C, E and H, the latter two of which are also products from a single gene, as well as NKG2C, have positively charged residues within their transmembrane regions. NKG2C and possibly NKG2E and H interact with the adapter molecule DAP 12, and act as activating receptors, when heterodimerized with CD94.

[0206] NK cells also play a role in adaptive immune response in their ability to readily adjust to the immediate environment and formulate antigen-specific immunological memory, fundamental for responding to secondary infections with the same antigen. Thus NK cells are acting in both the innate and adaptive immunity, which makes them particularly useful targets for the modulator and / or re-programmers of the present disclosure. In more specific embodiments, the NK cell activating receptor targeted by the one or more targeting moieties of the disclosed nanoparticles, may be at least one of Natural cytotoxicity triggering receptor 1 (NCR1, NKp46), Natural cytotoxicity triggering receptor 2 (NCR2, NKp44), Natural cytotoxicity triggering receptor 3 (NCR3, NKp30), tumor necrosis factor receptor superfamily 7 (TNFRSF7, CD27), Lymphocyte function-associated antigen 1 (LFA-1), cluster of differentiation 16 (CD16), NKG2D, Cytotoxic And Regulatory T Cell Molecule (CRTAM), DNAX Accessory Molecule-1) (DNAM-1), NKp80, 2B4 also known as CD244 (Cluster of Differentiation 244) and any derivatives, splice variants, homologs and orthologs thereof. In more specific embodiments, the NK cell inhibitory receptor targeted by the one or more targeting moieties of the disclosed nanoparticles, may be at least one of Killer-cell immunoglobulin-like receptor (KIR), Programmed cell death protein 1 (PD-1), CTLA-4, Ly49, NKRP1A, CD94, NKG2A, T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), Lymphocyteactivation gene 3 (LAG-3), Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), Leukocyte- associated immunoglobulin-like receptor 1 (LAIR-1), Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), and any derivatives, splice, homologs and orthologs variants thereof or any combinations thereof.

[0207] In yet some further specific embodiments, the NK cell activating receptor may be at least one of NKp46 (denoted by the human RefSeqs: NM_001145457, NM_001145458, NM_001242356, NM_001242357, NM_004829, also known as Natural cytotoxicity triggering receptor 1 (NCR1), CD335, LY94, NK-p46, NKP46, ), NKp44 (denoted by the human RefSeqs: NM_004828, NM_001199509, NM_001199510, also known as Natural cytotoxicity triggering receptor 2 (NCR2), CD336, LY95, NK-p44), NKp30 (denoted by the human RefSeqs: NM_001145466, NM_001145467, NM_147130, also known as Natural cytotoxicity triggering receptor 3 (NCR3), 1C7, CD337, LY117, MALS), NKp80 (RefSeq. NM_016523.3, encoding the protein as denoted by NP_057607.1 the also known as KLRF1), CD27 (denoted by the human RefSeq: NM_001242, also known as S152, S152. LPFS2, T14, TNFRSF7, Tp55, CD27 molecule), LFA-1 (RefSeq. NM_002209.3, encoding the protein as denoted by NP_002200.2), CD16 (denoted by the human RefSeqs: NM_000569, NM_000570also known as FCGR3, FCG3, FcyRIII), NKG2D (denoted by the human RefSeq: NM_007360, also known as KLRK1, CD314, D12S2489E, KLR, NKG2-D, NKG2D, killer cell lectin like receptor KI), CRTAM, DNAM-1 (denoted by the human RefSeqs: NM_001303618, NM_001303619, NM_006566, also known as CD226, DNAM-1, DNAM1, PTA1, TLiSAl, CD226 molecule), 2B4 (denoted by the human RefSeqs: NM_001166663, NM_001166664, NM_016382, also known as CD244, 2B4, NAIL, NKR2B4, Nmrk, SLAMF4, CD244 molecule), and any derivatives and splice variants thereof. In yet some further embodiments, the NK cell inhibitory receptor may be at least one of KIR (RefSeq: NM_014218.3, encoding the protein as denoted by NP_055033.2, also referred to herein as KIR2DL1, killer cell immunoglobulin-like receptors), PD-1 (denoted by the human RefSeq: NM_005018, also known as PDCD1, CD279, PD-1, PD1, SLEB2, hPD-1, hPD-1, hSLEl, Programmed cell death 1), Ly49 (denoted by the human RefSeqs: NM_008462.5 and NM_006611, also known as Klra2, Ly49b; Klra30, and as KLRA1; Ly49; LY49L; Ly-49L, KLRAP1, respectively), NKRP1A (denoted by the human RefSeq: NM_002258, also known as KLRB1, CD161, CLEC5B, NKR, NKR-P1, NKR-P1A, hNKR-PlA, killer cell lectin like receptor Bl), CD94 (denoted by the human RefSeqs: NM_001114396, NM_002262, NM_007334, NM_001351060, NM_001351062, also known as KLRD1), NKG2A (also known as CD94), TIGIT (Gene ID: 201633), CD96 (denoted by the human RefSeqs: NM_005816, NM_198196, NM_001318889, also known as TACTILE, CD96 molecule), TIM-3 (Gene ID: 84868, also known as TIM3; CD366; KIM-3; SPTCL; TIMD3; Tim-3; TIMD-3; HAVcr-2), LAG-3 (denoted by the human RefSeq: NM_002286, also known as LAG3, CD223, lymphocyte activating 3), CEACAM1 (denoted by the human RefSeqs: NM_001024912, NM_001184813, NM_001184815, NM_001184816, NM_001205344 also known as CECAM, BGP, BGP1, BGPI, AND CEA cell adhesion molecule 1), LAIR-1 (denoted by the human RefSeq: NM_001289023, NM_001289025, NM_001289026, NM_001289027, NM_002287 also known as CD305, LAIR1), LILRB1 (denoted by the human RefSeq: NM_001081637, NM_001081638, NM_001081639, NM_001278398, NM_001278399, also known as ILT2, CD85J, ILT-2, ILT2, LIR-1, LIR1, MIR- 7, MIR7, PIR-B, PIRB, leukocyte immunoglobulin like receptor Bl), BTLA (denoted by the human RefSeq: NM_001085357, NM_181780, also known as BTLA, BTLA1, CD272, B and T lymphocyte), CTLA4 (denoted by the human RefSeq: NM_001037631, NM_005214, also known as ALPS5, CD, CD152, CELIAC3, CTLA-4, GRD4, GSE, IDDM12). It should be understood that the NK activating or inhibitory receptors as specified herein are also applicable for all other aspects of the disclosure.

[0208] In some specific embodiments, the at least one nano- or micro-particle, micellar formulation, vehicle or matrix of the present disclosure, comprise at the outer nanoparticle surface thereof (e.g., associated or bound to the outer surface), at least one antibody. In some embodiments, such antibody may be an antibody specific for or against at least one member of the natural cytotoxicity receptor (NCR) family.

[0209] In more specific embodiments, the member of the NCR family targeted by the antibody of the nanoparticles of the present disclosure, may comprise Natural Killer Cell Protein 46 (NKp46). Thus, in some embodiments, the nano- or micro-particle, micellar formulation, vehicle or matrix of the present disclosure may be associated directly or indirectly at the outersurface thereof, with at least one targeting moiety (e.g., antibody, an aptamer, a ligand or any combinations thereof), specifically, antibody, that recognizes and binds NKp46.

[0210] The disclosure relates to the nano- or micro-particle or micellar formulation or vehicle or matrix of the disclosure targeting (by way of having a targeting moiety targeted at) any of the activating and / or inhibitory receptors of NK cells or T cells, and to any derivatives, splice, homologs and orthologs variants thereof or any combinations thereof. Thus, in some embodiments, the activating or inhibitory receptors of the NK cells targeted by the targeting moiety of the nano-particles of the disclosure may be any of the molecules described herein that may comprise 'equivalent amino acid residues'. This term refers to an amino acid residue capable of replacing another amino acid residue in a polypeptide without substantially altering the structure and / or functionality of the polypeptide. Equivalent amino acids thus have similar properties such as bulkiness of the sidechain, side chain polarity (polar or non-polar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral or basic) and side chain organization of carbon molecules (aromatic / aliphatic). As such, equivalent amino acid residues can be regarded as conservative amino acid substitutions.

[0211] In the context of the present disclosure, within the meaning of the term 'equivalent amino acid substitution' as applied herein, is meant that in certain embodiments one amino acid may be substituted for another within the groups of amino acids indicated herein below:

[0212] (i) Amino acids having polar side chains (Asp, GIu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys); (ii) Amino acids having non-polar side chains (Gly, Ala, Vai, Leu, lie, Phe, Trp, Pro, and Met); (iii) Amino acids having aliphatic side chains (Gly, Ala Vai, Leu, ile); (iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro); (v) Amino acids having aromatic side chains (Phe, Tyr, Trp); (vi) Amino acids having acidic side chains (Asp, GIu); (vii) Amino acids having basic side chains (Lys, Arg, His); (viii) Amino acids having amide side chains (Asn, Gin); (ix) Amino acids having hydroxy side chains (Ser, Thr); (x) Amino acids having sulphur-containing side chains (Cys, Met); (xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr); (xii) Hydrophilic, acidic amino acids (Gin, Asn, GIu, Asp), and (xiii) Hydrophobic amino acids (Leu, lie, Vai).

[0213] Still further, the activating or inhibitory NK receptors targeted by the targeting moiety of the nanoparticles of the disclosure may have secondary modifications, such as phosphorylation, acetylation, glycosylation, sulfhydryl bond formation, cleavage and the likes, as long as said modifications retain the functional properties of the original protein, specifically, act as inhibitory or activating receptors. Secondary modifications are often referred to in terms of relative position to certain amino acid residues. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide. The disclosure further encompasses any derivatives, enantiomers, analogues, variants or homologues of any of the NK activating and or inhibitory receptors disclosed herein. The term "derivative" is used to define amino acid sequences (polypeptide), with any insertions, deletions, substitutions and modifications to the amino acid sequences (polypeptide) that do not alter the activity of the original polypeptides. By the term “derivative” it is also referred to homologues, variants and analogues thereof, as well as covalent modifications of a polypeptides made according to the present disclosure. Still further, any splice variants of the indicated receptors are also encompassed by the present disclosure.

[0214] In some particular embodiments, the nanoparticles of the disclosure may be connected and / or associated directly or indirectly in the outer nanoparticle surface thereof with at least one antibody directed against NKp46 or any derivative, splice variant, homolog, ortholog or valiant thereof. In yet some specific embodiments, the nanoparticles of the disclosure may be associated as a targeting moiety, with any of the anti-NKp46 antibodies disclosed by the Examples section.

[0215] In yet some further embodiments, the nanoparticle of the disclosure may be associated with a plurality of antibodies, aptamers or any combinations thereof. It should be noted that each of the plurality of antibodies or aptamers specifically recognizes and binds an NK cell inhibitory or activating receptor of a plurality of inhibitory or activating receptors expressed by NK cells of a subject suffering from an immune-related disorder.

[0216] In some embodiments, the targeting moiety, for example, antibodies and / or aptamers, may be directed against a variety of molecules expressed on a target cell, specifically, any hematopoietic cell, more specifically, NK cell. NK cells within the tumor microenvironment often display a decrease in expression of activating receptors, an increased expression of inhibitory receptors, and a decrease in cytokine secretion and cytotoxic ability. Thus, in some embodiments, the present nanoparticles of the disclosure may offer personalized therapy, by efficient targeting of the reprogramming agents of the present disclosure to the particular NK present in the diseased subject, for example, NK cells obtained from a tumor of a particular patient. Therefore, in yet some further embodiments, the targeting moiety may target receptors, specifically activating and / or inhibitory receptors expressed on an NK cell isolated or obtained from the treated subject or a subject suffering from an immune-related disorder. According to such embodiments, the profiling of the specific molecules (e.g., activating and / or inhibitory receptors) in the patient's NK cells should be determined in order to design specific targeting moieties for the nanoparticles of the disclosure. More specifically, in some specific and non-limiting embodiments, the nano-particles of the disclosure may be connected to any commercial antibody specific for any of the inhibitors and / or activating NK cell receptor. Examples for NKp46 antibody may be the LS-C662543 by LSBio. In yet some further embodiments, the nano-particles of the disclosure may be connected to any commercially available antibody specific for any one of CD57, NKG2A, CD96, the natural cytotoxicity receptors (NCRs) NKp30, NKp44, NKp80, CD16, NKG2D, NKG2C, DNAX Accessory Molecule-1 (DNAM-1), and 2B4, or any of the receptors disclosed by the disclosure. In some embodiments, the NK cells obtained from the patient may be used for screening antibodies libraries or alternatively, aptamer libraries to design the most appropriate targeting moieties that may be used for a specific patient. As indicated above, in some embodiments, the targeting moieties may be aptamers.

[0217] Aptamers are produced by a combinatorial procedure named SELEX (Systematic Evolution of Ligands by Exponential enrichment), that are emerging as promising diagnostic and therapeutic tools. Among selection strategies, procedures using living cells as complex targets (referred as “cell-SELEX”) have been developed as an effective mean to generate aptamers for heavily modified cell surface proteins, assuring the binding of the target in its native conformation. A major advantage of the cell-based procedures is that they may be employed for the targeting of a specific cell type, without any prior knowledge of the specific target, leading to identify multiple aptamers able to recognize specific cell phenotypes and discover new cell biomarkers. To date, aptamers have been developed for different cell types and other complex systems, especially for live cancer cells, and thus, may be also developed for NK cells of a subject suffering from an immune-related disorder. The SELEX procedure involves repeated cycles of: 1. Incubation of the high complexity library with the targets (binding); 2. Removal of unbound sequences and recovery of the bound oligonucleotides (partitioning); 3. Amplification of the bound sequences by PCR (for DNA library) or RT-PCR and transcription (for RNA library). In Whole-Cell SELEX strategy, a fundamental aspect is the inclusion of a counter-selection step, for example, using NK cells obtained from a healthy subject or a subject that is not suffering from the specific immune-related disorder, to avoid the parallel enrichment of aptamers for unwanted targets. The negative selection step is introduced before the positive selection at each round, allowing filtering out sequences against those molecules commonly expressed on both the target and control cell lines. Specifically, an alternative cell-SELEX strategy (referred as “differential cell- SELEX”) has been developed to isolate aptamers able to recognize a specific cell phenotype, rather than a single specific target of interest. This strategy offers the possibility to select multiple ligands discriminating between even closely related cell types, without any prior knowledge of the target. Briefly, the procedure consists of the incubation of the starting library on a non-target cell line (with undesired phenotype, negative selection step) followed by the recovery of unbound oligonucleotides that are, then, incubated on cells with the desired phenotype (positive selection step). Several screening methods have emerged to optimize the selection process, some examples are briefly described below.

[0218] Fluorescence-Activated Cell Sorting (FACS)-SELEX: An extension of the cell-SELEX strategy is a Fluorescence-Activated Cell Sorting (FACS)-based protocol that allows to select aptamers targeting a specific subpopulation. In this strategy, once a fluorescently labeled aptamer library is incubated on target cells, a cell-sorting device is used to differentiate and separate the cell subpopulations that are bound or unbound to the aptamers. Bound aptamers are, then, eluted and amplified. The protocol permits to eliminate dead cell population that, absorbing single-stranded nucleic acid molecules, may negatively influence the selection procedure. In addition, such an approach has two additional advantages. First, it allows the reduction of experimental steps, incorporating in one round both positive and negative selection. Second, it allows to simultaneously monitor the selection process during the rounds without the need of additional binding assays.

[0219] Cell Internalization SELEX: Aptamers are emerging as one of the most promising tools for the specific deliver to diseased cells of secondary reagents. Indeed, it has been shown that upon binding to their targets, aptamers can be rapidly internalized, allowing the tissue specific internalization of active therapeutic substances, including nanoparticles, anti-cancer therapeutics, small interfering RNAs (siRNAs), microRNAs, and anti-microRNAs. This permits the exposition to secondary reagents only of target cells, increasing the efficacy and reducing the toxicity of the therapy. Based on these considerations, modified cell-based selection approaches was developed to isolate internalizing aptamers, eliminating those sequences that do not, or very slowly, internalize. In yet some further embodiments, the targeting moieties of the nanoparticles of the disclosure may be antibodies, each antibody recognizes and binds at least one of a plurality of antigens expressed on the surface of a particular immune-cell (e.g., NK cell) of a patient suffering from an immune-related disorder, or cancer. Thus, in some embodiments, any similarly to the approach discussed above for aptamers, immune-cells of a patient may be used for screening antibody libraries, to select appropriate plurality of antibody targeting moieties for a specific relevant subject. It should be understood that the disclosure further encompasses the use of any combination of antibodies and aptamers that recognize and target to the plurality of receptors expressed by the patient's immune cell (e.g., NK cell).

[0220] The targeting moiety may be connected, linked, conjugated, or associated either directly to the outer surface of the nano-particle of the disclosure, or any matrix or micellar formulation described herein, or indirectly, for example, via at list one linker. In some embodiments, the at least one targeting moiety is connected (conjugated) to the carrier surface via chemical or physical bonding as described herein below. The association of the at least one targeting moiety with the carrier may be direct or may be via a linker. The linker can be inert, or the linker can have biological activity. The linker must be at minimum bivalent; however, in some embodiments, the linker can be bound to more than one active agent, in which case, the linker is polyvalent. The linker can be composed of any assembly of atoms, including oligomeric and polymeric chains, which functions to connect one or more of the targeting moieties (e.g., antibodies, aptamers), to the nanoparticles. In some cases, the linker may be an oligomeric and polymeric chain, such as an oligo- or polyethylene glycol chain, or an oligo- or poly(amino acid) chain. In some cases the linker is a non-polymeric organic functional group, such as an alkyl group or an alkylaryl group. In certain embodiments, the linker may be hydrophilic to facilitate passage of the nanoparticles across biological membranes. In many cases, the linker is a linear chain; however, in some embodiments, the linker / s may contain one or more branch points. In the case of branched linker, the terminus of each branch point can be functionalized with the targeting moiety. Still further, it should be noted that the targeting moiety may be associated or linked to the nano-or microparticles of the disclosure either directly or indirectly, for example, via a linker or any adaptor molecule, for example, an adaptor molecule that is based on affinity interactions, for example, avidin-biotin, leucine zipper adaptor and the like. The use of adaptor molecules may enable the use of common or universal nanoparticles loaded with the inhibitory compounds of the re-programming agents of the present disclosure (e.g., the siRNAs of the disclosure), and adapted for personalized treatments by connecting to the affinity adaptor variety of antibodies that are specific for receptors expressed by infiltrating NK cells isolated from the particular patient and profiled by the present disclosure. In yet some further embodiments, a linker that links the targeting moiety to the nano-particle of the disclosure may be used. The term "linker" in the context of the disclosure concerns an amino acid sequence of from about 1 to about 10 or more amino acid residues positioned on the outer layer of the nanoparticles of the disclosure. The linker is covalently linked or joined to the amino acid residues in its vicinity. For example, a linker in accordance with the disclosure may be of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid residues long. Linkers are often composed of flexible amino acid residues, for example but not limited to glycine and serine so that the adjacent targeting moiety (e.g., an antibody or any other affinity molecule) are free to move relative to one another. In more specific embodiments, the targeting moieties of the nano- or micro-particle or micellar formulation, or vehicle or matrix of the disclosure, recognize and bind NK cells of a subject suffering from an immune -related disorder, for example, at least one of a cancer, a proliferative disorder, an infectious disease, a graft versus host disease, an inflammatory disorder, an immunecell mediated disorder and an autoimmune disorder. It should be noted that the immune-related disorders relevant for this aspect are those as described herein after in connection with other aspects of the disclosure.

[0221] As indicated by the present aspect, of particular relevance are formulations of the NK reprogramming agent (or modulator and / or re-programmer) of the present disclosure (e.g., siRNA molecules) are combinations encompassed within a nano- or micro-particles. Nanoscale drug delivery systems using liposomes and nanoparticles are emerging technologies for the rational drug delivery, which offers improved pharmacokinetic properties, controlled and sustained release of drugs and, more importantly, lower systemic toxicity. A particularly desired solution allows for externally triggered release of encapsulated compounds. Externally controlled release can be accomplished if drug delivery vehicles, such as liposomes or polyelectrolyte multilayer capsules, incorporate nanoparticle (NP) actuators.

[0222] More specifically, Controlled drug delivery systems (DDS) have several advantages compared to the traditional forms of drugs. A drug is transported to the place of action, hence, its influence on vital tissues and undesirable side effects can be minimized. Accumulation of therapeutic compounds in the target site increases and, consequently, the required doses of drugs are lower. This modern form of therapy is especially important when there is a discrepancy between the dose or the concentration of a drug and its therapeutic results or toxic effects. Cell-specific targeting as described above, can be accomplished by attaching drugs to specially designed carriers. Various nanostructures, including liposomes, polymers, dendrimers, silicon or carbon materials, and magnetic nanoparticles are applicable in the present disclosure. Polymeric nanoparticles are one technology being developed to enable clinically feasible oral delivery.

[0223] The term "nanostructure" or "nanoparticle" is used herein to denote any microscopic particle smaller than about 100 nm in diameter. In some other embodiments, the carrier is an organized collection of lipids. When referring to the structure forming lipids, specifically, micellar formulations or liposomes, it is to be understood to mean any biocompatible lipid that can assemble into an organized collection of lipids (organized structure). In some embodiments, the lipid may be natural, semi-synthetic or fully synthetic lipid, as well as electrically neutral, negatively or positively charged lipid. In some embodiments, the lipid may be a naturally occurring phospholipid. Examples of lipids forming glycerophospholipids include, without being limited thereto, glycerophospholipid. phosphatidylglycerols (PG) including dimyristoyl phosphatidylglycerol (DMPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), l-palmitoyl-2- oleoylphosphatidyl choline (POPC), hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC); phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS). Examples of cationic lipids may include, for example, 1 ,2-dimyristoyl- 3 -trimethylammonium propane (DMTAP) l,2-dioleyloxy-3-(trimethylamino) propane (DOTAP); N-[l-(2,3,- ditetradecyloxy )propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide

[0224] (DMRIE); N-[l-(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethyl- ammonium bromide (DORIE); N-[l-(2,3-dioleyloxy) propyl]-N,N,N- trimethylammonium chloride (DOTMA); 3p[N- (N',N'- dimethylaminoethane) carbamoly] cholesterol (DC-Chol); and dimethyl-dioctadecylammonium (DD AB ), N- [2- [ [2 , 5 -bis [3 - aminopropyl) amino] - 1 - oxopentyl]amino]ethyl]-N,N-dimethyl-2,3-bis[(l-oxo-9-octadecenyl)oxy]-l propanaminium (DOSPA), and ceramide carbamoyl spermine (CCS), or the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) derivatized with polylysine to form a cationic lipopolymer.

[0225] The lipids may be combined with other lipid compatible substances, such as, sterols, lipopolymers etc. A lipopolymer may be a lipid modified by inclusion in its polar headgroup a hydrophilic polymer. The polymer headgroup of a lipopolymer may be preferably water-soluble. In some embodiments, the hydrophilic polymer has a molecular weight equal or above 750Da. There are numerous polymers which may be attached to lipids to form such lipopolymers, such as, without being limited thereto, polyethylene glycol (PEG), poly sialic acid, polylactic (also termed polylactide), poly glycolic acid (also termed poly glycolide), apolylactic-poly glycolic acid, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polyaspartamide, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyvinylmethylether, polyhydroxyethyl acrylate, derivatized celluloses such as hydroxymethylcellulose or hydroxy ethylcellulose. The polymers may be employed as homopolymers or as block or random copolymers. The lipids derivatized into lipopolymers may be neutral, negatively charged, as well as positively charged. The most commonly used and commercially available lipids derivatized into lipopolymers are those based on phosphatidyl ethanolamine (PE), usually, distearoylphosphatidylethanolamine (DSPE).

[0226] In some embodiments, the structure forming lipids may be combined with other lipids, such as a sterol. Sterols and in particular cholesterol are known to have an effect on the properties of the lipid's organized structure (lipid assembly), and may be used for stabilization, for affecting surface charge, membrane fluidity. In some embodiments, a sterol, e.g. cholesterol is employed in order to control fluidity of the lipid structure. The greater the ratio steroklipids (the structure forming lipids), the more rigid the lipid structure is. Liposomes are often distinguished according to their number of lamellae and size. The liposomes employed in the context of the present disclosure may be multilamellar vesicles (MLVs), multivesicular vesicles (MVVs), small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs) or large multivesicular vesicles (LMVV). It should be appreciated that the re -programming agents of the present disclosure may be encapsulated or associated with any of the nanostructures described above, specifically, any of the micellar formulations, liposomes, polymers, dendrimers, silicon or carbon materials, polymeric nanoparticles and nanoparticles disclosed herein above. The term “association” may be used interchangeably with the term “entrapped” , “attachment” , “linked”, “embedded”, “absorbed” and the like, and contemplates any manner by which the compounds of the invention is held. This may include for example, physical or chemical attachment to the carrier. Chemical attachment may be via a linker, such as polyethylene glycol. The association provides capturing of the at least one compounds of the disclosure by the nanostructure such that the release of the at least one compound used by the re-programming agents of the present disclosure may be controllable. As indicated above, it should be appreciated that in some embodiments, the nanostructure in accordance with the present disclosure may further comprise at least one targeting moiety on the surface. Such targeting moiety, may facilitate targeting the compound-nanostructures of the disclosure into a particular target cell, target tissue, target organ or particular cellular organelle target. In some embodiments, that targeting moiety targets the nano-particles to NK cells. It should be noted that the transporting or targeting moiety may be attached directly or indirectly via any linker, and may comprise affinity molecules, for example, antibodies or aptamers or any other affinity molecule, as described herein above, that specifically recognize target antigen on specific hematopoietic cells.

[0227] Still further, in some specific and non-limiting embodiments, the nanoparticles of the disclosure may be composed of phosphatidylcholine (PC), dipalmitoylphosphatidylethanolamine (DPPE), and cholesterol (Choi). In yet some further specific embodiments, the nanoparticles of the disclosure may comprise PC, DPPE and Choi at molar ratios of 3:1:1 (PC:DPPE:Chol). Still further, the nanoparticles of the disclosure may be prepared by a lipid-film method.

[0228] In yet some further embodiments, the nanoparticles of the disclosure, also referred to herein as unilamellar nano-scale liposomes (ULNL), are further surface-modified with high molecular weight glycosaminoglycan hyaluronic acid (HA), thereby obtaining HA-NPs. Still further, in some embodiments, the HA-NPs of the disclosure are coated with monoclonal anti-NKp46 antibody. Natural killer cell p46-related protein (NKp46) is also known as Natural cytotoxicity triggering receptor 1 (NCR1), Lymphocyte antigen 94 homolog (LY94), CD335 Antigen (cluster of differentiation 335, NKP46, NKp46, NK-p46, and CD33. NKp46 is a member of the natural cytotoxicity receptor (NCR) family and was identified as an important regulator of NK cell function. Engagement of the CD335 receptor on NK cells results in increased cellular activation, manifesting as increased cytokine production and release of cytolytic granules. NKp46, as used herein, refers to any one of: the human NKp46 isoform b precursor as denoted by NP_001138929, the isoform c precursor as denoted by NP_001138930.2, isoform e precursor as denoted by NP_001229286, as well as other isoporms as denoted by any one of NP_001229285, NP_001229286 and NP_004820.

[0229] In yet some further embodiments, the nanoparticles of the disclosure may be conjugated or covered by at least one anti-NKp44 antibody. Natural cytotoxicity triggering receptor 2 is a protein that in humans is encoded by the NCR2 gene is a transmembrane glycoprotein characterized by a single extracellular V-type Ig-like domain and a cytoplasmic tail containing an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) and no known activating signaling motifs. In some embodiments, the NKp44, as used herein, refers to any one of: the human NKp44 as denoted by any one of NP_001186438, NP_001186439 and NP_004819.

[0230] In some non-limiting embodiments, the disclosed nanoparticles that encapsulate the modulators of the present disclosure (e.g., esiRNA, siRNA molecules), are composed of Phosphatidylcholine(PC), cholesterol (Choi), and 1, 2-dihexadecanoyl-snglycero-3- phosphoethanolamine (DPPE), were prepared by a method previously described by the present inventors (Biber G, et al., 2021, EMBO Mol Med 14:el4073). More specifically, four lipids were mixed at 6:2:1.9:0.1 molar ratios: (i) Phosphatidy lcholine(PC), (ii) cholesterol (Choi), (iii) 1, 2- dihexadecanoyl-snglycero-3-phosphoethanolamine (DPPE), and (iv) DPPE labeled with rhodamine red (DPPE-PE, excitation / emission: 560 / 583 nm;Avanti). Liposomes were coated with 6 mg hyaluronic acid(HA; high molecular weight, R&D) and 40 mg of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDAC; Sigma). For each 50 pg of liposomes, 400 mM of EDAC and 100 mM of NHydroxysuccinimide (NHS) were added (Sigma). The solution was incubated with 50 pg of NKp46 antibodies.

[0231] It should be understood that although in some embodiments and aspects disclosed herein, the reprogramming agents (or modulators and / or re-programmers) of the present disclosure are delivered using nanoparticles, the present disclosure further encompasses the option of using any other vector or vehicle for delivery of the re -programming agents of the present disclosure, specifically any combination of inhibitory compounds that inhibit the expression, stability and / or activity of at least one EGR protein and at least one DGK proteins, that are based on nucleic acid molecules, for example, the siRNAs of the present disclosure, or any oligonucleotide or even any gene editing system as described herein before. In more specific embodiments, such vector may be any one of a viral vector, a non- viral vector and a naked DNA vector.

[0232] Vectors, as used herein, are nucleic acid molecules of particular sequence can be incorporated into a vector that is then introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art, including promoter elements that direct nucleic acid expression. Many vectors, e.g. plasmids, cosmids, minicircles, phage, viruses, etc., useful for transferring nucleic acids into target cells may be applicable in the present disclosure. The vectors comprising the nucleic acid(s) may be maintained episomally, e.g. as plasmids, minicircle DNAs, viruses such cytomegalovirus, adenovirus, etc., or they may be integrated into the target cell genome, through homologous recombination or random integration, e.g. retrovirus-derived vectors such as AAV, MMLV, HIV-1, ALV, etc.

[0233] Vectors may be provided directly to the subject cells. In other words, the cells are contacted with vectors comprising the oligonucleotides of the disclosure such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. DNA can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV).

[0234] More specifically, in some embodiments, a vector useful in the preset disclosure may be a viral vector. In yet some particular embodiments, such viral vector may be any one of recombinant adeno associated vectors (rAAV), single stranded AAV (ssAAV), self-complementary rAAV (scAAV), Simian vacuolating virus 40 (SV40) vector, Adenovirus vector, helper-dependent Adenoviral vector, retroviral vector and lentiviral vector. As indicated above, in some embodiments, viral vectors may be applicable in the present disclosure. The term "viral vector" refers to a replication competent or replication-deficient viral particle which are capable of transferring nucleic acid molecules into a host.

[0235] Still further, in some embodiments, the vector may be a naked DNA vector. More specifically, such vector may be for example, a plasmid, minicircle or linear DNA.

[0236] Naked DNA alone may facilitate transfer of a gene (2-19 kb) into skin, thymus, cardiac muscle, and especially skeletal muscle and liver cells when directly injected. It enables also long-term expression. Although naked DNA injection is a safe and simple method, its efficiency for gene delivery is quite low.

[0237] In yet some further aspects thereof, the present disclosure provides at least one cell comprising the re-programming agent (or modulator and / or re-programmers) of the present disclosure or any nano-particles comprising these re-programming agents, specifically as described by the disclosure, as well as any cell population comprising at least 10% or more of the cells of the disclosure.

[0238] In some specific embodiments, the cell of the disclosure may comprise any of the re-programming agents disclosed by the disclosure, specifically, the cell in accordance with some embodiments of the disclosure may comprise any inhibitory nucleic acid molecules, SMCs, aptamers, peptide, or any combinations thereof, that specifically inhibit the expression and / or stability and / or activity of at least one of: (i) at least one of EGR-2 and / or EGR-4; and (ii) at least one of DGK-a and / or DGK

[0239] More specifically, in some embodiments, the re -programming agents comprised within the cell of the disclosure may comprise at least one compounds that comprise at least one nucleic acid molecule. More specifically, each of the nucleic acid molecules is specific for or specifically directed against one of at least one of: (i) at least one of EGR-2 and / or EGR-4; and / or (ii) at least one of DGK-a and / or DGK-^.

[0240] In yet some further embodiments, the nucleic acid molecule of the re-programming agent comprised within the cell of the present disclosure, may be RNA molecules or any nucleic acid sequence encoding the RNA molecules. In more specific embodiments, such RNA molecules may be at least one of a dsRNA, an antisense RNA, a ssRNA, a gRNA and a Ribozyme.

[0241] In certain embodiments, the re-programming agents comprised within the cell of the present disclosure may comprise at least one dsRNA molecules. More specifically, each of the dsRNA molecules may be directed against or specific for one of at least one of: (i) at least one of EGR-2 and / or EGR-4; and / or (ii) at least one of DGK-a and / or DGK-^. In yet more specific embodiments, such dsRNA molecules, may be at least one of siRNA, miRNA, shRNA and piRNAs.

[0242] In yet some further embodiments, the re-programming agents comprised within and / or functionalizing and / or activating the cell of the present disclosure may comprise at least one of: at least one siRNA molecule specifically directed against, or specific for EGR-2, and / or at least one siRNA molecule specifically directed against DGK-a. In yet some further embodiments, the reprogramming agents comprised within and / or functionalizing and / or activating the cell of the present disclosure may comprise least one siRNA molecule specifically directed against, or specific for EGR-2, and / or at least one siRNA molecule specifically directed against EGR-4.

[0243] It should be noted that in some embodiments, the cell of the present disclosure may comprise any of the nano- or micro-particle or micellar formulation or vehicle or matrix as disclosed by the disclosure. In some embodiments, the cell of the present disclosure may be any hematopoietic cell. In some embodiments, the cell is a lymphocyte of the T lineage. In yet some further particular embodiments, the cell of the present disclosure may be an NK cell. Still further, in some embodiments, the cell of the present disclosure may be a cell obtained from a subject suffering from an immune related disorder, specifically from a proliferative disorder. In some embodiments, the cells or population of cells may be obtained from at least one allogeneic healthy subject or from a pool of at least two allogeneic subjects. As indicated above, the present disclosure further encompasses any population of the cells disclosed herein. Specifically, such population of cells may be an enriched population of cells comprising the re-programming agents of the present disclosure, or any nano-particles thereof, or cells activated and / or functionalized and / or rewired, and / or reprogramed by the re-programming agents of the present disclosure, i.e. a population of cells in which a high percentage of cells among the total number of cells in the population of cells comprise the re-programming agents of the present disclosure. Specifically, such a percentage of cells may be 10%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,

[0244] 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,

[0245] 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,

[0246] 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

[0247] 96%, 97%, 98%, 99 %, 99.9%, or 100% of the total population of cells obtained by the disclosure. In some specific embodiments, at least 50% of the cells of the population may be cells that were manipulated to comprise the re -programming agents of the present disclosure or any nano-particles or any vectors or vehicles thereof, and / or cells (NK cells) that were activated and / or functionalized and / or rewired, and / or reprogramed by the re -programming agents of the present disclosure. In some embodiments, "activated by the re-programming agents of the present disclosure" is meant, changed from dysfunctional to functional cells. It should be understood that the present disclosure further encompasses any composition or preparation of NK cells or cell population thereof activated by the disclosed re-programming agents (being functional). Such composition contains a population of cells having an increased proportion of activated NK cells. In yet some further embodiments, the disclosure provides a population of NK cells activated by the re-programming agents of the present disclosure, that display reduced proportion of dysfunctional NK cells. Specifically, the disclosed NK cell population is an improved cell population as it is composed of less than 20% of dysfunctional cells, specifically, less than 20% of the NK cells in the NK cell population of the present disclosure or of any composition thereof are anergic NK cells. In yet some further embodiments, less than 20% of the NK cells in the cell population of the present disclosure or of any composition thereof, is exhausted NK cells. In yet some further embodiments, the disclosed NK cell population is an improved cell population as it is composed of less than 13% of dysfunctional cells, specifically, less than 13% of the NK cells in the NK cel population of the present disclosure or of any composition thereof are anergic NK cells. In yet some further embodiments, less than 13% of the NK cells in the cell population of the present disclosure or of any composition thereof, is exhausted NK cells. In some embodiments, the disclosed NK cell population contain 80% or more functional NK cells (cells that can be activated, and / or cells that are not exhausted cells, or cells that are not anergic cells). Still further in this context, less than 20% as used herein, and also in context with all other aspects of the present disclosure, is meant, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, and also for the term "less than 20%, or "less than 13%), I is meant: 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less, 0.009% or less, 0.008% or less, 0.007% or less, 0.006% or less, 0.005% or less, 0.004% or less, 0.003% or less, 0.002% or less, and 0.001% or less, of the NK cells in the NK cell population provided in the present disclosure or any composition or preparation thereof are dysfunctional cells, specifically, anergic NK cells or exhausted cells.

[0248] In this connection, the present disclosure provides in some other aspects thereof, a method for improving a population of cells, specifically, hemopoietic cells, and more specifically a population of cells comprising NK cells, by contacting the cells in the population of cells (that can be of either an autologous source, or alternatively, of an allogeneic source), with an activating effective amount of any of the re -programming agents of the present disclosure or any nano-or micro-particles thereof, or any composition or preparation thereof, thereby providing a population of cells having less than 20% thereof or even less than 13% thereof, or less than 10% thereof dysfunctional cells, as defined above. In some embodiments, less than 20% of the NK cells in the cell population prepared by the disclosed methods are exhausted NK cells. In some embodiments, less than 20% of the NK cells in the cell population prepared by the disclosed methods are anergic NK cells. This method therefore provides an improved population of cells (of either an autologous source or an allogeneic source) that contain more functional NK cells, and / or less dysfunctional NK cells as specified above. The improved cell population provided by be disclosed methods is rewired and reprogramed thereby functionated.

[0249] A further aspect of the present disclosure relates to a pharmaceutical composition comprising an effective amount of at least one re -programming agent (or modulator and / or re -programmer) comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix comprising the re-programming agent. More specifically, the re-programming agent of the disclosed composition activates and / or functionalize and / or rewire, and / or reprogram dysfunctional NK cells. The composition further comprises at least one of pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s. It should be understood that in some embodiments, the disclosed composition comprises an effective amount of the disclosed reprogramming agent (or modulator and / or re-programmer) that is sufficient for and effectively activate and / or functionalize and / or rewire, and / or reprogram NK cells, specifically, dysfunctional NK cells.

[0250] In some embodiments, the re-programming agent of the disclosed compositions is any of the reprogramming agents as defined by the present disclosure, defined above, and in connection with other aspects of the present disclosure. Still further, in some embodiments, the nano- or microparticle, micellar formulation, vehicle or matrix of the compositions of the present disclosure is any of the nano- or micro-particles as disclosed and defined herein by the present disclosure.

[0251] In yet some other specific embodiments the effective amount of the re-programming agents of the present disclosure, specifically, the siRNA molecule specific for EGR-2, the siRNA molecule specific for EGR-4, the siRNA molecule specific for DGK-a, and / or the siRNA molecule specific for DGK-^, may range between 0.001 mg to 10,000mg each, specifically, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 8000, 9000, 10000 mg a day or more. In yet some further embodiments, the effective amount of the re-programming agents of the present disclosure, specifically, the siRNA molecule specific for EGR-2, the siRNA molecule specific for EGR-4, the siRNA molecule specific for DGK-a, and / or the siRNA molecule specific for DGK-^ may range between about 0.1 to 100 microgram / kg, specifically, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 microgram / kg and more. In some specific embodiments, an effective amount for in vitro treatment may be about 1 to 10 micrograms, specifically, 6.6 micrograms. Still further, in some embodiments the effective amount for in vivo treatment may range between about 1 to 50 microgram / kg, specifically, about 20 microgram / kg. As indicated by the examples, the present siRNA and / or esiRNA molecules of the disclosed modulator or re-programmer, were injected to mice at an amount of 1500pmole of each, specifically, an amount of about 0.0025 mg / per dose (mouse). Accordingly, in some specific embodiments, the effective amount of the siRNA and / or esiRNA molecules of the disclosed modulator or re-programmer may be about 0.125 mg per kilogram of body weight / dose.

[0252] Still further, in some specific embodiments where the disclosed modulator or re-programmer is provided in nano-particles (e.g., the disclosed liposomes), about 300 micrograms of liposomes were injected to each mouse (300 microgram per dose), in addition to the siRNA molecules as discussed above. Accordingly, in some specific embodiments, the effective amount of the liposomes of the present disclosure that encompass the disclosed modulator or re -programmer (e.g., siRNA and / or esiRNA molecules), may be about 15 mg per kilogram of body weight / dose. In some embodiments, the subject is treated with 1 dose each day. In yet some further embodiments, the subject is treated using one dose each 3 days. In yet some further embodiments, the subject is treated using one dose every 3 days for 21 days period.

[0253] The pharmaceutical compositions of the disclosure can be administered and dosed by the methods of the disclosure, in accordance with good medical practice, systemically, for example by parenteral, e.g. intravenous, intraperitoneal or intramuscular injection. In another example, the pharmaceutical composition can be introduced to a site by any suitable route including intravenous, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e.g. oral, intranasal, or intraocular administration. Of particular interest are injectable compositions comprising any of the disclosed re-programming agents or any nano- or microparticles thereof (e.g., liposomes), an any formulation thereof. Local administration to the area in need of treatment may be achieved by, for example, by local infusion during surgery, topical application, direct injection into the specific organ, etc. More specifically, the re -programming agents of the present disclosure or any nanoparticles or compositions thereof, described herein after, may be adapted for administration by parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). It should be noted that any of the administration modes discussed herein, may be applicable for any of the methods of the disclosure as described in further aspects of the disclosure herein after.

[0254] Compositions and formulations for oral administration may include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0255] Pharmaceutical compositions used to treat subjects in need thereof according to the disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. The pharmaceutical compositions of the present disclosure also include, but are not limited to, emulsions and liposome-containing formulations. It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0256] The compositions of the disclosure may also be administered directly to the eye or ear, typically in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose or methyl cellulose or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis. Formulations for ocular and aural administration may be formulated to be immediate and / or modified release. Modified release includes delayed, sustained, pulsed, controlled, targeted, and programmed release.

[0257] In specific embodiments, the unit dosage formulations are those containing a daily dose or subdose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

[0258] In yet a further aspect, the disclosure relates to a method for activating, and / or functioning, and / or rewiring, and / or reprograming at least one hematopoietic cells, specifically, dysfunctional cells (e.g., anergic or exhausted). In yet some further specific embodiments, the methods of the disclosure may be suitable for activating at least one of dysfunctional NK cells, dysfunctional T cells and dysfunctional B cells. Still further, the methods of the disclosure may be particularly suitable for activating dysfunctional NK cells (e.g., anergic or exhausted). In more specific embodiments, the method may comprise the step of contacting the NK cell (specifically, the dysfunctional NK cell) with an activating effective amount of at least one re-programming agent (or modulator and / or re-programmer) comprising at least one compound that specifically inhibit at least one of, the expression, activity and stability of at least one transcription factor, specifically, at least one member of the EGR family and of at least one Kinase, specifically, at least one member of the DGK family, or any vehicle, matrix, nano- or micro-particle, micellar formulation or composition comprising the re-programming agents of the present disclosure.

[0259] A further aspect of the present disclosure relates to a method for activating and / or functionalizing, and / or re-wiring, and / or reprograming at least one dysfunctional NK cell. The method comprising the step of contacting the dysfunctional NK cell with an activating effective amount of at least one re-programming agent (or modulator and / or re-programmer) comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent. In some embodiments, the re-programming agent activates and / or re- wires dysfunctional NK cells.

[0260] In this context, activating NK cells, is meant, changing the cells from dysfunctional cells (either anergic or exhausted), to functional NK cells. In some embodiments, activating the NK cells in the context of the present disclosure is meant enhancing the functional properties of the cells, such that they are able to function and be activated.

[0261] Rewiring NK cells or reprogramming NK cells refers to the deliberate modification of the signaling pathways, molecular networks, or genetic programs within natural killer (NK) cells to alter or enhance their functional properties. These processes aim to improve the cells’ ability to recognize, target, and eliminate abnormal cells, such as tumor cells or virally infected cells, or to modulate their interactions with the immune system.

[0262] In some embodiments, the dysfunctional NK cells are anergic NK cells and / or exhausted NK cells. Thus, in some embodiments, the present disclosure provides methods for activating and / or rewiring dysfunctional NK cells that may be anergic NK cells and / or exhausted NK cells. Specifically, in some embodiments the present disclosure provide method for functionalizing, and / or rewiring and / or reprograming and / or activating anergic NK cells. In some embodiments, the present disclosure provide method for functionalizing, and / or rewiring and / or reprograming and / or activating exhausted NK cells.

[0263] In some embodiments of the disclosed methods, the at least one compound that specifically inhibits the expression, activity and / or stability of at least one of: at least one member of the EGR family of transcription factors; and / or at least one member of the DGK family, may comprise at least one nucleic acid molecule, at least one amino acid-based molecule, and / or at least one chemical inhibitor. Each of these molecules is specific for one of: (i) at least one member of the EGR family of transcription factors; and / or (ii) at least one member of the DGK family.

[0264] In some embodiments of the disclosed methods, the nucleic acid molecule is a ribonucleic acid (RNA) molecule or any nucleic acid sequence encoding said RNA molecule, said RNA molecule is at least one of a double-stranded RNA (dsRNA), an antisense RNA, a single- stranded RNA (ssRNA), guide RNA (gRNA) and a Ribozyme. Still further, in some embodiment of the disclosed methods, dsRNA is at least one of siRNA, esiRNA, miRNA, shRNA and piRNAs. Accordingly, in some embodiments, the disclosed methods use at least one of these dsDNA molecules for activating and / or rewiring and / or functioning or functionalizing dysfunctional NK cells.

[0265] In some embodiments, the compound of the re-programming agent used in the disclosed methods, may comprise at least one siRNA and / or esiRNA molecule, each siRNA and / or esiRNA molecule is specific for one of: (i) one member of the EGR family of transcription factors; and / or (ii) one member of the DGK family.

[0266] In some embodiments, the EGR family of transcription factors comprises at least one of EGR2, EGR4, EGR1 and EGR3. Thus, in some embodiments, the disclosed methods may use at least one siRNA molecule, each siRNA molecule is specific for at least one of EGR2, EGR4, EGR1 and EGR3.

[0267] In more specific embodiments of the disclosed methods, the DGK family comprises at least one of DGKa, DGK^, DGK5, DGK0 and DGKy. Thus, in some embodiments, the disclosed methods may use at least one siRNA molecule, each siRNA molecule is specific for at least one of DGKa, DGK^ DGK5, DGK0 and DGKy.

[0268] In some embodiments of the disclosed methods, the at least one member of the EGR family of transcription factors comprises at least one of EGR2 and EGR4; and the at least one member of the DGK family comprises at least one of DGKa and DGK^. Accordingly, in some embodiments, the disclosed methods use at least one siRNA molecule, each siRNA molecule is specific for at least one of EGR2 and / or EGR4, and / or DGKa and DGK^. It should be understood that the disclosed methods may use any combination of the disclosed siRNA molecules as disclosed herein above in connection with other aspects of the present disclosure.

[0269] In some embodiments, the re -programming agent of the disclosed methods, comprises an effective amount of at least one of: (i) at least one siRNA molecule specific for EGR2; (ii) at least one siRNA molecule specific for EGR4; (iii) at least one siRNA molecule specific for DGKa and (iv) at least one siRNA molecule specific for DGK^; and / or any combinations of (i), (ii), (iii) and (iv). Still further, in some embodiments of the methods of the present disclosure, the esiRNA molecule specific for EGR2 that comprise the nucleic acid sequence as denoted by SEQ ID NO: 9, and any complementary sequence thereof, is randomly fragmented to generate a plurality of siRNA molecules targeting EGR2 and / or the esiRNA molecule specific for EGR4 comprising the nucleic acid sequence as denoted by SEQ ID NO: 10, and any complementary sequence thereof, is randomly fragmented to generate a plurality of siRNA molecules targeting EGR4, and / or the siRNA molecule specific for DGKa comprises the nucleic acid sequence as denoted by SEQ ID NO: 13, and / or the siRNA molecule specific for DGK^ comprises the nucleic acid sequence as denoted by SEQ ID NO: 14, or of any variants, homologs or derivatives thereof.

[0270] In some embodiments of the disclosed methods, the re-programming agent is comprised within at least one of a nano- or micro-particle, a micellar formulation, a vehicle, a matrix, or a composition.

[0271] In some embodiments of the disclosed methods, the at least one targeting moiety of the nano-or micro particles used in the methods, is at least one of an antibody, an aptamer, a ligand or any combinations thereof, that specifically recognizes and binds at least one molecule expressed on the surface of at least one NK cell. In more specific embodiments, an antibody, an aptamer, a ligand or any combinations thereof, that specifically recognizes and binds at least one molecule expressed on the surface of at least one dysfunctional NK cell.

[0272] In some embodiments of the disclosed methods, the at least one antibody comprises antibody against at least one member of the NCR family.

[0273] Still further, in some embodiments, the member of the NCR family comprises NKp46, and the targeting moiety comprises at least one antibody that specifically recognizes and binds NKp46.

[0274] In some embodiments, the disclosed methods are particularly useful for activating and / or rewiring dysfunctional NK cell in a subject suffering from an immune-related disorder.

[0275] In more specific embodiments of the disclosed methods, the immune-related disorder may be at least one of a cancer, a proliferative disorder, an infectious disease, a graft versus host disease, an inflammatory disorder, an immune-cell mediated disorder and an autoimmune disorder. In some specific embodiments, the disorder is cancer. In yet some further embodiments, the cancer is at least one of AML and glioma. In yet some further embodiments, the NK cells are Tumor infiltrating NK cells (TINK).

[0276] The methods of the present disclosure are thus applicable in some embodiments to a lymphocyte cell that is a NK cell, specifically, a dysfunctional NK cell. More specifically, Natural killer cells or NK cells are a type of cytotoxic lymphocyte critical to the innate immune system. NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL). The role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to viral-infected cells, acting at around three days after infection, and respond to tumor formation. Typically, immune cells detect major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named "natural killers" because of the initial notion that they do not require activation to kill cells that are missing "self" markers of MHC class 1. Still further, in a broader sense, immunological synapse ("NKIS") denotes the dynamic interface formed between an NK cell and its target cell. Formation of NKIS involves several distinct stages, beginning with the initiation of contact with a target cell and culminating in the directed delivery of lytic granule contents to lyse the target cell. Progression through the individual stages is methodical and underlies the precision with which NK cells select and kill susceptible target cells (including virally infected cells and cancerous cells) that they encounter during their routine surveillance of the body.

[0277] It should be therefore understood that in some embodiments, the methods of the present disclosure that provide 'functionalizing', "rewiring", "reprograming" and / or "activating" of NK cell, led to the creation of functional NK cell population, or functional NK cell that having the ability to perform all functions of NK cells. In more specific embodiments, functions of NK cells comprise the formation of activating NK cell immunological synapse (IS), that is activating NKIS, or alternatively, the formation of inhibitory NK cell immunological synapse (IS), that is inhibitory NKIS. More specifically, the formation of a mature and functional NKIS can be divided into a series of sequential (nonparallel) stages: the recognition and initiation stage, the effector stage and the termination stage. Together, these processes enable the delivery of lytic granules to the synapse followed by their close association with the NK cell membrane to which they can fuse and release their contents onto the target cell. Because lytic granules exist in resting NK cells before activation, each stage must be controlled to prevent accidental release of cytotoxic mediators and to enable rapid directed secretion at the appropriate moment. In some embodiments, molecules related to the above processes, can be used as markers for evaluating activity and effectivity of the reprogramming agents of the present disclosure and any nano-particles thereof. Specifically: the initial stage is characterized by formation of a close association between the NK cell and a target cell, initial signaling and adherence of NK cell to its target cell. This stage is facilitated by a number of molecules, including, although not limited to, members of the selectin family, the CD2 receptor, and receptors from the integrin family of adhesion molecules in particular such as the integrins lymphocyte function-associated antigen 1 (LFA1; CDl la / CD18) and MAC1 (CDl lb / CD18). Importantly, this initial stage is rapid and occurs before molecular patterning is evident. The decision whether NK cell progresses to maturation and molecular reorganization at NKIS depends on the level of signals through inhibitory receptors (KIRs, Killer-cell Immunoglobulin-like Receptors), which can establish a so-called inhibitory synapse. Such regulation ensures that NK cells effectively carry out their surveillance function, by leaving most cells undisturbed, while being poised to destroy those that are diseased. The inhibitory NKIS is especially elegant in that it directly interferes with the ability of the lytic synapse to progress past the initiation stage. The effector stage is characterized by a number of processes, most prominent of which are (1) formation of a stable NK cell-target cell interface with a 'cleft' into which cytolytic molecules are secreted; (2) recruitment of lytic granules to the synapse; (3) clearance of a conduit in the NK cell cortex through which lytic granules could be directed to the cell membrane; and (4) fusion of the lytic-granule membrane with plasma membrane for release of lytic-granule contents. Parallel events include receptor clustering, lipid-raft aggregation, further activation signaling and lytic- granule redistribution. Among receptors that undergo clustering, the most important for both, adhesion and triggering of cytotoxicity, are CDl la, CDl lb and CD2. Another requirement for effector function is polarization of lytic granules to NKIS, or in other words movement of the granules along the microtubules to the microtubule-organizing centre (MTOC). Signals required for MTOC polarization include ERK (extracellular-signal-regulated kinase) phosphorylation, VAV1 activation and PYK2 (protein tyrosine kinase 2) activities. Still further, granule docking to the synapse requires members of the RAB family of small GTPases, which are important regulators of vesicle trafficking and compartmentalization. RAB27a also performs this function in docking lytic granules in CTLs. Of further relevance are Muncl3-4 (putative vesicle priming factor) and SNAREs (N-ethylmaleimide-sensitive fusion protein attachment protein receptors) and their regulators acting in a coordinated manner to facilitate membrane fusion and providing a fine- tuning of NKIS; SHP-1 (a SH2 domain containing tyrosine phosphatase), yet another regulator of the effector function.

[0278] Termination stages of NKIS refer to those that occur after the lytic-granule contents have been secreted. Those include a period of inactivity and down modulation of the accumulated activating receptors followed by NK cell detachment from the target cell and recycling of cytolytic capacity. Once NK cell has carried out its cytolytic function, it can detach from the target cell and restore its ability to kill another susceptible cell. At the inhibitory synapse, detachment may result from reduced integrity of interactions between the F-actin cortex and the plasma membrane through dephosphorylation of ERM protein targets. The signals initiating the process of recycling NK cytolytic capacity are largely unknown, apart from activation of the nuclear factor-KB (NF-KB) which has been shown to serve as a transcription factor for expression of the lytic granule component perforin. As mentioned above, secretion of cytolytic granules in response to increased intracellular Ca2+flux is characteristic of 'Termination stage' of a lymphocyte responding to an activating stimulus at IS, a which is common to NK cells and CTLs, i.e. innate or adaptive immune response. When CTL or NK cells kill infected or cancerous cells they secrete cytolytic proteins (perforin and granzymes) into the target cell. These “death factors” are pre-stored in cytolytic granules within the CTL until an increase in the intracellular Ca2+drives granule to exocytosis. Secretion of cytolytic granules and increased intracellular Ca2+flux are measurable and can serve as markers for evaluating the activity of the presently conceived compounds.

[0279] In some embodiments, the lymphocyte cell modulated, specifically activated by the reprogramming agents of the present disclosure, or any nano-particles thereof, may be at least one of an NK cell, a T cell and a B cell forming an inhibitory IS, specifically, NK cells.

[0280] A further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof. The method comprising the step of administering to the subject a therapeutically effective amount of at least one re-programming agent (or modulator and / or re-programmer) comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and (ii), at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent, wherein said reprogramming agent activates dysfunctional NK cells. In some additional or alternative embodiments, the disclosed methods may comprise an ex vivo step of functionalizing and / or activating and / or rewiring the NK cells ex vivo or in vitro, by contacting the cells with the effective amount of the re -programming agents of the preset disclosure. The cells are then transferred back to the subject, for example, by adoptive transfer. The cells may be of an autologous or of allogeneic source. Accordingly, in some embodiments of the disclosed methods, the subject is administered with at least one NK cell, or a population of NK cells or any composition or preparation thereof, that were functionalized, activated and / or rewired. The NK cells may be in some embodiments of an autologous source. In yet some further embodiments, the cells may be of an allogeneic source (either of a single donor or a pool of two or more donors).

[0281] In some embodiments, the disclosed therapeutic methods may use any of the re-programming agents disclosed by the present disclosure, and any of the nano- or micro-particle, micellar formulation, vehicle, matrix as defined by the present disclosure.

[0282] In some embodiments, the subject treated by the disclose therapeutic methods is suffering from an immune-related disorder. More specifically, the immune-related disorder applicable for the disclosed methods is at least one of a cancer, a proliferative disorder, primary or secondary immunodeficiency, a graft versus host disease, an inflammatory disorder, an immune-cell mediated disorder, an autoimmune disorder and a viral infection.

[0283] In some embodiments of the disclosed therapeutic methods, the subject is further treated with at least one immunomodulatory therapeutic agent prior to, after or simultaneously with the at least one re-programming agent of the present disclosure.

[0284] In yet some further embodiments, the at least one immunomodulatory therapeutic agent comprises at least one of: (i) at least one checkpoint inhibitor; (ii) at least one cytokine; and (iii) chimeric antigen receptor (CAR) T cells.

[0285] A further aspect of the present disclosure relates to at least one re-programming agent (or modulator and / or re-programmer) or any nano- or micro-particle, micellar formulation, vehicle, matrix, cell or composition comprising said re-programming agent, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune- related disorder in a subject in need thereof. The re-programming agent used herein comprises at least one compound that specifically inhibit the expression, activity and / or stability of at least one of: (i), at least one member of the EGR family of transcription factors; and / or (ii) at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising the re-programming agent. The re -programming agent activates and / or functionalize and / or rewire, and / or reprogram dysfunctional NK cells.

[0286] In some embodiments of the disclosed at least one re-programming agent for use, the compound used herein is any of the compounds as defined by the present disclosure, and the nano- or microparticle, micellar formulation, vehicle or matrix is as defined by the present disclosure, and the compositions used herein are any of the compositions disclosed y the present disclosure.

[0287] As noted above, the methods and uses of the disclosure may be relevant for treating any immune- related disorder, for example, an infectious disease, specifically, a viral infection, cancer or any other proliferative disorder, a graft versus host disease, an inflammatory disorder, an immune-cell mediated disorder and an autoimmune disorder. An "Immune-related disorder" or "Immune- mediated disorder", as used herein encompasses any condition that is associated with the immune system of a subject, more specifically through inhibition of the immune system, or that can be treated, prevented or ameliorated by reducing degradation of a certain component of the immune response in a subject, such as the adaptive or innate immune response. More specifically, an 'immune-related disorder', as meant herein, encompasses a range of dysfunctions of the innate and adaptive immune systems. In more specific terms, immune-related disorder can be characterized, for example, (1) by the component(s) of the immune system; (2) by whether the immune system is overactive or underactive; (3) by whether the condition is congenital or acquired, as will be specified herein after.

[0288] In some specific embodiments, the methods of the disclosure may be used for treating cancer or any other proliferative disorders. As used herein to describe the present disclosure, “proliferative disorder”, “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include nonsolid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the methods of the present disclosure may be applicable for treatment of a patient suffering from any one of non-solid and solid tumors.

[0289] Malignancy, as contemplated in the present disclosure may be any one of carcinomas, melanomas, lymphomas, leukemias, myeloma and sarcomas. Carcinoma as used herein, refers to an invasive malignant tumor consisting of transformed epithelial cells. Alternatively, it refers to a malignant tumor composed of transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges.

[0290] Melanoma as used herein, is a malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin, but are also found in other parts of the body, including the bowel and the eye. Melanoma can occur in any part of the body that contains melanocytes.

[0291] Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic).

[0292] Sarcoma is a cancer that arises from transformed connective tissue cells. These cells originate from embryonic mesoderm, or middle layer, which forms the bone, cartilage, and fat tissues. This is in contrast to carcinomas, which originate in the epithelium. The epithelium lines the surface of structures throughout the body, and is the origin of cancers in the breast, colon, and pancreas. Myeloma as mentioned herein is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies. Collections of abnormal cells accumulate in bones, where they cause bone lesions, and in the bone marrow where they interfere with the production of normal blood cells. Most cases of myeloma also feature the production of a paraprotein, an abnormal antibody that can cause kidney problems and interferes with the production of normal antibodies leading to immunodeficiency. Hypercalcemia (high calcium levels) is often encountered. Lymphoma is a cancer in the lymphatic cells of the immune system. Typically, lymphomas present as a solid tumor of lymphoid cells. These malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs in which case it is referred to as extranodal lymphoma. Non limiting examples for lymphoma include Hodgkin's disease, non-Hodgkin's lymphomas and Burkitt's lymphoma.

[0293] Still further, in some embodiments, the disclosed modulator or re-programmer, methods, compositions and kits of the present disclosure are applicable for any type and / or stage and / or grade of any metastasis, metastatic cancer or status of any of the cancerous conditions disclosed herein. As used herein the term "metastatic cancer" or "metastatic status" refers to a cancer that has spread from the place where it first started (primary cancer) to another place in the body. A tumor formed by metastatic cancer cells originated from primary tumors or other metastatic tumors, that spread using the blood and / or lymph systems, is referred to herein as a metastatic tumor or a metastasis. Further malignancies that may find utility in the present invention can comprise but are not limited to hematological malignancies (including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma. The invention may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extrahepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma, Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma / Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma / malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas / carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Cerebral astrocytoma / Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas, Non- Hodgkin (an old classification of all lymphomas except Hodgkin's); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease; Macroglobulinemia, Waldenstrom; Malignant Fibrous Histiocytoma of Bone / Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic / Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (Surface epithelial-stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituitary adenoma; Plasma cell neoplasia / Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma - see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); Testicular cancer; Throat cancer; Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer; Thyroid cancer, childhood; Transitional cell cancer of the renal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureter and renal pelvis, transitional cell cancer; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, childhood; Vulvar cancer; Waldenstrom macroglobulinemia and Wilms tumor (kidney cancer).

[0294] In some embodiments, the disclosed methods and uses are particularly applicable for AML. Acute Myeloid Leukemia (AML) is an aggressive form of blood and bone marrow cancer characterized by the rapid proliferation of abnormal myeloid cells, which interfere with the production of healthy blood cells. It originates in the bone marrow but often spreads quickly to the blood and, in advanced stages, to other parts of the body such as the lymph nodes, liver, and spleen. AML leads to symptoms like fatigue, frequent infections, easy bruising or bleeding, and anemia due to the overcrowding of abnormal cells in the bone marrow. It is most common in adults but can occur in children as well. Diagnosis typically involves blood tests, bone marrow biopsies, and genetic studies to classify the subtype, which is crucial for treatment planning. Management of AML often includes chemotherapy, targeted therapy, and in some cases, stem cell transplantation. The prognosis varies depending on factors such as age, genetic mutations, and the patient’s overall health

[0295] Still further, in some embodiments, the disclosed methods and uses are particularly applicable for glioblastoma. Glioblastoma is an aggressive and malignant form of brain cancer that originates in the glial cells, which support and protect neurons. It is the most common and lethal primary brain tumor in adults, often arising in the cerebral hemispheres but capable of occurring in other brain regions or the spinal cord. Glioblastoma is classified as a Grade IV astrocytoma by the World Health Organization due to its rapid growth, infiltration into surrounding brain tissue, and resistance to treatment. Symptoms vary depending on the tumor's location but commonly include headaches, seizures, cognitive impairment, and neurological deficits such as weakness or speech difficulties. Diagnosis is typically confirmed through imaging techniques like MRI and a biopsy. Treatment involves a combination of surgery, radiation therapy, and chemotherapy, although complete surgical removal is usually not possible due to the tumor's invasive nature. Despite aggressive treatment, glioblastoma has a poor prognosis, with a median survival of 12-18 months after diagnosis. Research into novel therapies, including immunotherapy and targeted treatments, is ongoing.

[0296] In some embodiments, the methods of the disclosure may be used to treat a proliferative disorder, cancer, tumor and malignancy by activating / enhancing antitumor immunity. The term “antitumor immunity” refers to innate and adaptive immune responses which may lead to tumor control.

[0297] The immune system can be activated by tumor antigens and, once primed, can elicit an antitumor response. Natural Killer (NK) cells are a front-line defense against drug-resistant tumors and can provide tumoricidal activity to enhance tumor immune surveillance. Cytokines like IFN-y or TNF play a crucial role in creating an immunogenic microenvironment and therefore are key players in the fight against metastatic cancer. Critical aspects in the tumor-immune system interface include the processing and presentation of released antigens by antigen-presenting cells (APCs), interaction with T lymphocytes, subsequent immune / T-cell activation, trafficking of antigen-specific effector cells, and, ultimately, the engagement of the target tumor cell by the activated effector T cell.

[0298] Nevertheless, although often successful in preventing tumor outgrowth, this “cancer-immunity cycle” can be disrupted by artifices involved in immune escape and development of tolerance, culminating with the evasion and proliferation of malignant cells. Furthermore, the tumor microenvironment induces suppression and reduced activity of NK and T cells, through the secretion of inhibitory factors suppressing the anti-tumor response, a phenomena known as exhaustion. Using the re-programming agents of the present disclosure or any nanoparticles or compositions thereof, provides methods and compositions for activating and / or functionalization and / or rewiring, and / or reprograming of lymphocytes, specifically, NK cells, in particular dysfunctional NK cells for enhancing anti-tumor immunity.

[0299] In yet other embodiments, the methods, as well as the re -programming agents, nanoparticles, compositions and kits of the disclosure may be also applicable for treating a subject suffering from an infectious disease. More specifically, such infectious disease may be any one of viral diseases, protozoan diseases, bacterial diseases, parasitic diseases, fungal diseases and mycoplasma diseases.

[0300] It should be appreciated that an infectious disease as used herein also encompasses any infectious disease caused by a pathogenic agent. Pathogenic agents include viruses, prokaryotic microorganisms, lower eukaryotic microorganisms, complex eukaryotic organisms, fungi, prions, parasites, yeasts, toxins and venoms. Of particular relevance are infectious diseases caused by a bacterial pathogen. A prokaryotic microorganism includes bacteria such as Gram positive, Gram negative and Gram variable bacteria and intracellular bacteria. Examples of bacteria contemplated herein include the species of the genera Treponema sp., Borrelia sp., Neisseria sp., Legionella sp., Bordetella sp., Escherichia sp., Salmonella sp., Shigella sp., Klebsiella sp., Pseudomonas sp., Yersinia sp., Vibrio sp., Hemophilus sp., Rickettsia sp., Chlamydia sp., Mycoplasma sp., Staphylococcus sp., Streptococcus sp., Bacillus sp., Clostridium sp., Corynebacterium sp., Proprionibacterium sp., Mycobacterium sp., Ureaplasma sp. and Listeria sp.

[0301] A lower eukaryotic organism includes a yeast or fungus such as but not limited to Pneumocystis carinii, Candida albicans, Aspergillus, Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Trichophyton and Microsporum.

[0302] A complex eukaryotic organism includes worms, insects, arachnids, nematodes, aemobe, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Trypanosoma brucei gambiense, Trypanosoma cruzi, Balantidium coli, Toxoplasma gondii, Cryptosporidium or Leishmania.

[0303] In yet some further relevant diseases are infectious diseases caused by a viral pathogen. The term “viruses” is used in its broadest sense to include viruses of the families adenoviruses, papovaviruses, herpesviruses: simplex, varicella-zoster, Epstein-Barr, CMV, pox viruses: smallpox, vaccinia, hepatitis B, rhinoviruses, coronaviruses, retroviruses, zika virus, ebola virus, hepatitis A, poliovirus, rubella virus, hepatitis C, arboviruses, rabies virus, influenza viruses A and B, measles virus, mumps virus, HIV, HTLV I and II.

[0304] The term "fungi" includes for example, fungi that cause diseases such as ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidio-idoiny cosis, and candidiasis.

[0305] The term "parasite" includes, but not limited to, infections caused by somatic tapeworms, blood flukes, tissue roundworms, ameba, and Plasmodium, Trypanosoma, Leishmania, and Toxoplasma species.

[0306] Still further, in certain embodiments, the methods, as well as the re-programming agent (or modulator and / or re-programmers), nanoparticles, compositions and kits and compositions of the disclosure may be applicable for treating disorders associated with immunodeficiency.

[0307] In some specific embodiments wherein the immune -related disorder or condition may be a primary or a secondary immunodeficiency. It should be understood that any of the immune-related disorders described herein after in connection with other aspects of the disclosure is also applicable or the present aspect as well. 'Immunodeficiency', primary or secondary, meaning inherited or acquired, respectively. The term 'immunodeficiency' is intended to convey a state of an organism, wherein the immune system's ability for immuno-surveillance of infectious disease or cancer is compromised or entirely absent. According to the International Union of Immunological Societies, more than 150 primary immunodeficiency diseases (PIDs) have been characterized, and the number of acquired (or secondary) immuno-deficiencies exceeds the number of PIDs. PIDs are those caused by inherited genetic mutations. Secondary immuno-deficiencies are caused by various conditions, aging or agents such as viruses or immune suppressing drugs. A number of notable examples of PIDs include Severe combined immunodeficiency (SCID), DiGeorge syndrome, Hyperimmunoglobulin E syndrome (also known as Job’s Syndrome), Common variable immunodeficiency (CVID): B- cell levels are normal in circulation but with decreased production of IgG throughout the years, so it is the only primary immune disorder that presents onset in the late teens. Chronic granulomatous disease (CGD): a deficiency in NADPH oxidase enzyme, which causes failure to generate oxygen radicals. Classical recurrent infection from catalase positive bacteria and fungi. Wiskott-Aldrich syndrome (WAS); autoimmune lymphoproliferative syndrome (ALPS); Hyper IgM syndrome: X- linked disorder that causes a deficiency in the production of CD40 ligand on activated T-cells. This increases the production and release of IgM into circulation. The B-cell and T-cell numbers are within normal limits. Increased susceptibility to extracellular bacteria and opportunistic infections. Leukocyte adhesion deficiency (LAD); NF-KB Essential Modifier (NEMO) Mutations; Selective immunoglobulin A deficiency: the most common defect of the humoral immunity, characterized by a deficiency of IgA. Produces repeating sino-pulmonary and gastrointestinal infections. X-linked agammaglobulinemia (XLA; also known as Bruton type agammaglobulinemia): characterized by a deficiency in tyrosine kinase enzyme that blocks B-cell maturation in the bone marrow. No B-cells are produced to circulation and thus, there are no immunoglobulin classes, although there tends to be a normal cell-mediated immunity. X-linked lymphoproliferative disease (XLP); and Ataxia-telangiectasia.

[0308] Thus patients' populations diagnosed with one of PIDs can particularly benefit from methods and compositions of the re-programming agents according to the present disclosure.

[0309] With respect to secondary immunodeficiencies, those can be manifested in both the young and the elderly. Under normal conditions immune responses are beginning to decline at around 50 years of age, what is called immunosenescence. The term 'immunosenescence' refers to the gradual deterioration of the immune system brought on by natural age advancement. It involves both the host’s capacity to respond to infections and the development of long-term immune memory. Additional common causes of secondary immunodeficiency include severe burns, malnutrition, certain types of cancer, and chemotherapy in cancer patients.

[0310] More specifically, in developed countries, obesity, alcoholism, and drug use are common causes of poor immune function. However, malnutrition is the most common cause of immunodeficiency in developing countries. Diets lacking sufficient protein are associated with impaired cell-mediated immunity, complement activity, phagocyte function, IgA antibody concentrations, and cytokine production. Additionally, the loss of the thymus at an early age through surgical removal, for example, results in severe immunodeficiency and high susceptibility to infections.

[0311] Of particular relevance to the present context are cellular immunodeficiencies associated with cancer and certain viral pathogens. A cellular immunodeficiency refers to a deficiency the count or function of T lymphocytes, which are the main type of cells responsible for the cellular adaptive immune response in attacking viruses, cancer cells and other parasites. Extensive research has reasonably well established the role of immunodeficiency in cancers of the head and neck, lung, esophagus and breast. Among virally induced immunodeficiencies, the most notable example is AIDS (Acquired Immunodeficiency Syndrome) cause by HIV infection. The role of HIV as a direct cause of cellular immunodeficiency, particularly the deficiency of the CD4+ T helper lymphocyte population, has been well established. Additional examples of viral- or pathogen- induced immunodeficiencies include, although not limited to chickenpox, cytomegalovirus, German measles, measles, tuberculosis, infectious mononucleosis (Epstein-Barr virus), chronic hepatitis, lupus, and bacterial and fungal infections. One of the most recent examples is virus- induced Severe Acute Respiratory Syndrome (SARS). These and additional examples of disorders related to cellular immunodeficiency may include Aplastic anemia, Leukemia, Multiple myeloma, Sickle cell disease, chromosomal disorders such as Down syndrome, infectious diseases caused by pathogens such as Cytomegalovirus, Epstein-Barr virus, Human immunodeficiency virus (HIV), Measles and certain bacterial infections. Chronic kidney disease, Nephrotic syndrome, Hepatitis, Liver failure and other conditions caused by Malnutrition, alcoholism and burns.

[0312] Thus patients' populations diagnosed with one of the secondary immunodeficiencies, and particularly one of the cellular immunodeficiencies as above, can particularly benefit from methods, as well as the re -programming agents, nanoparticles, cells, compositions and kits of the present disclosure. Differential diagnosis of such immunodeficient patients is routinely performed in various clinical settings.

[0313] Additional secondary immunodeficiencies applicable for the disclosed methods may result following bone marrow (BM) transplantation, gene therapy or adaptive cell transfer. Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient's own stem cells are used), allogeneic (the stem cells come from a donor) or syngeneic (from an identical twin). Performance of this medical procedure usually requires the destruction of the recipient's immune system using radiation or chemotherapy before the transplantation. To limit the risks of transplanted stem cell rejection or of severe graft-versus- host disease in allogeneic HSCT, the donor should preferably have the same human leukocyte antigens (HLA) as the recipient. In the case of a bone marrow transplant, the HSC are removed from a large bone of the donor, typically the pelvis, through a large needle that reaches the center of the bone. Peripheral blood stem cells are now the most common source of stem cells for HSCT. They are collected from the blood through a process known as apheresis. The donor's blood is withdrawn through a sterile needle in one arm and passed through a machine that removes white blood cells. The red blood cells are returned to the donor. The peripheral stem cell yield is boosted with daily subcutaneous injections of Granulocyte-colony stimulating factor (G-CSF), serving to mobilize stem cells from the donor's bone marrow into the peripheral circulation. It should be noted that amniotic fluid as well as umbilical cord blood may be also used as a source of stem cells for HSCT.

[0314] Of further relevance are patients' populations diagnosed with stress-induced immune-related diseases. Many studies have shown that exposure to physical or psychological stress can affect disease outcomes in immune-related disorders such as viral and bacterial infections, contact dermatitis and allergy. Stress, being it acute and short, or chronic and persistent have been shown to influence and modify various components of the immune system, in particular stress has be related to leukocytosis, increased NK cell cytotoxicity and reduced proliferative response to mitogens.

[0315] Of further relevance are patients' populations diagnosed with one of hypersensitivities of an immune response or allergies. Hypersensitivities are divided into four classes (Type I- IV) based on the mechanisms involved and the time course of the hypersensitive reaction. Type I is an immediate or anaphylactic reaction, often associated with allergy; it is mediated by IgE antibodies that trigger degranulation of mast cells and basophils. Type II (also called antibody-dependent or cytotoxic) occurs when antibodies bind to antigens on the patient's own cells, marking them for destruction; it is mediated by IgG and IgM antibodies. Type III and Type IV (also known as cell- mediated or delayed type) are mediated by T cells, monocytes, and macrophages; Type IV reactions are involved in many autoimmune and infectious diseases. A partial list including the most common allergies includes but not limited to Seasonal allergy, Mastocytosis, Perennial allergy, Anaphylaxis, Food allergy, Allergic rhinitis and Atopic dermatitis.

[0316] Of further relevance are patients diagnosed with splenomegaly (enlargement of spleen) or hypersplenism. Splenomegaly of between 11-20 cm greater than 20 cm in the size of spleen has been associated with hemolytic anemias, and other diseases involving abnormal red blood cells being destroyed in the spleen, as well as with other disorders, including congestion due to portal hypertension, and infiltration by leukemias and lymphomas.

[0317] In further specific embodiments, compositions and methods of the present disclosure can be applied to prevent an immunodeficiency and / or GvHD in immunocomprimised cancer patients, being it a result of cancer itself (as mentioned above) or an adverse effect of high doses of chemotherapy or radiotherapy (which may induce burns).

[0318] A number of human diseases were specifically related to NK cell deficiency and dysfunctionality. Those include certain PIDs characterized by genetic aberrations that impair NK cells function. Several of these diseases induce a specific blockade in the stages leading to the formation of a functional lytic synapse. Most of these diseases can result in haemophagocytic lymphohistiocytosis (HLH), i.e. an inappropriately robust immune response to infection (typically with herpesviruses), which results in a persistent systemic inflammatory syndrome. This leads to the physiological symptoms of septic shock, but is also associated with the pathological finding of haematophagocytosis (the ingestion of red blood cells by phagocytes). The NK cells are most relevant to the HLH phenotype, given their localization to marginal zones in lymphoid organs after viral infection, their innate function early in the course of infection and their inherent ability to eliminate hyperactivated macrophages.

[0319] It is thus meant that the re-programming agents of the present disclosure or any nanoparticles or compositions or methods thereof, are particularly applicable to patients diagnosed with one of the disorders related to NK cell or NKIS deficiency, or abnormal NK lytic granule trafficking. Notable examples of disorders belonging to this group are detailed below.

[0320] In some embodiments, Leukocyte adhesion deficiency type I (LAD-I) results from a defect in the CD 18 (P-integrin) component of leukocyte integrin heterodimers. Thus, LAD-I leukocytes do not appropriately adhere to inflamed or activated cells and cannot localize effectively to tissues and sites of inflammation. This leads to increased numbers of leukocytes in the blood and susceptibility to infectious diseases. Because early steps in NK-cell synapse formation - adhesion and activation signaling depend on integrins, NK cells from patients with LAD-I do not adhere to their target cells, resulting in defective cytotoxicity. LAD-I is also distinguished from other diseases discussed here because it does not lead to HLH.

[0321] In yet some further embodiments, Wiskott-Aldrich syndrome (WAS) results from a hematopoietic- cell-specific defect in actin reorganization and cell signaling due to WASP deficiency. Patients lacking WASP expression or expressing abnormal WASP have NK cells with decreased cytolytic capacity. Clinically, patients with WAS are susceptible to herpesviruse and can develop HLH, thereby demonstrating the functional relevance of WASP deficiency for the NK-cell lytic synapse. Formation of the lytic synapse is abnormal in NK cells from WAS patients and includes decreased F-actin accumulation and adhesion-receptor clustering at the synapse.

[0322] Still further, in some embodiments, Chediak-Higashi syndrome (CHS) and Hermansky-Pudlak syndrome type II (HPS2), both affect the normal formation of lytic granules and lead to the presence of “giant” lytic granules. Both are also associated with albinism, which is caused by aberrant functioning of melanocytes, which pigment skin via secretion of melanosomes (an equivalent of lytic granules). CHS and HPS2 are similar in that they represent a failure in generation of the NK-cell lytic synapse at the end of the effector stages, as the abnormal lytic granules will not migrate along the microtubules to the MTOC.

[0323] In some embodiments, Familial erythrophagocytic lymphohistiocytosis (FHL) types 3 and 4 are similar to CHS and HPS2, but are not associated with albinism demonstrating that the affected genes are not essential in melanocytes. FHL3 is caused by mutation in the UNC13D gene, which encodes MUNC13-4. FHL4 is caused by mutations in the STX11 gene, which encodes syntaxin- 11.

[0324] Still further, of particular relevance for the methods of the disclosure are patients' populations diagnosed with one of autoimmune disorders, also referred to as disorders of immune tolerance, when the immune system fails to properly distinguish between self and non-self-antigens. It has been well established that T cells lymphocytes, and the NK cells in particular, play a pivotal role in the control of immune tolerance under normal conditions, and in T- and B-cell mediated human autoimmune disorders. The NK cells have been further implicated in rheumatoid arthritis, systemic lupus erythematosus, and in multiple sclerosis.

[0325] Thus, according to some embodiments, the methods of the disclosure, as well as any reprogramming agents, nanoparticles, cells, compositions and kits of the disclosure may be used for the treatment of a patient suffering from any autoimmune disorder. In some specific embodiments, the methods as well as any re-programming agents, nanoparticles, cells, cell populations, compositions and kits of the disclosure may be used for treating an autoimmune disease such as for example, but not limited to, inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, fatty liver disease, Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Behcet's syndrome, Indeterminate colitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), Eaton-Lambert syndrome, Goodpasture's syndrome, Greave's disease, Guillain-Barr syndrome, autoimmune hemolytic anemia (AIHA), Idiopathic thrombocytopenic purpura (ITP), hepatitis, insulin-dependent diabetes mellitus (IDDM) and NIDDM, multiple sclerosis (MS), myasthenia gravis, plexus disorders e.g. acute brachial neuritis, polyglandular deficiency syndrome, primary biliary cirrhosis, scleroderma, thrombocytopenia, thyroiditis e.g. Hashimoto's disease, Sjogren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, arthritis, alopecia areata, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, dermatitis herpetiformis, psoriatic arthritis, reactive arthritis, and ankylosing spondylitis, inflammatory arthritis, including juvenile idiopathic arthritis, gout and pseudo gout, as well as arthritis associated with colitis or psoriasis, Pernicious anemia, some types of myopathy and Lyme disease (Late).

[0326] Of particular interest to the present context is a condition denoted Graft versus Host Disease (GvHD) that may occur after an allogeneic transplant, wherein the donated transplant cells view the recipient’s body as foreign. GvHD is a possible complication of high dose cancer treatment. It also happens after an allogeneic bone marrow or stem cell transplant that use very high doses of chemotherapy, sometimes with radiotherapy. The term 'GvHD' as meant herein encompasses all known form of GvHD, namely the acute GvHD (aGvHD), the chronic GvHD (cGvHD), and the late acute GVHD and overlap syndrome (with features of both aGvHD and cGvHD).

[0327] More specifically, the pa...

Claims

CLAIMS:

1. A re-programming agent comprising at least one compound that specifically inhibits the expression, activity and / or stability of at least one of:(i) at least one member of the Early Growth Response (EGR) family of transcription factors; and(ii) at least one member of the Diacylglycerol Kinase (DGK) family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising said re-programming agent, wherein said re-programming agent activates and / or functionalizes dysfunctional natural killer (NK) cells.

2. The re -programming agent according to claim 1, wherein said dysfunctional NK cells comprise anergic NK cells and / or exhausted NK cells.

3. The re-programming agent according to any one of claims 1 to 2, wherein said at least one compound that specifically inhibits the expression, activity and / or stability of at least one of at least one member of the EGR family of transcription factors; and / or at least one member of the DGK family comprises at least one nucleic acid molecule, at least one amino acid-based molecule, and / or at least one chemical inhibitor, each specific for one of: (i) at least one member of the EGR family of transcription factors; and / or (ii) at least one member of the DGK family.

4. The re-programming agent according to claim 3, wherein said nucleic acid molecule is a ribonucleic acid (RNA) molecule, or any nucleic acid sequence encoding said RNA molecule, said RNA molecule is at least one of: a double- stranded RNA (dsRNA), an antisense RNA, a singlestranded RNA (ssRNA), guide RNA (gRNA) and a Ribozyme.

5. The re-programming agent according to claim 4, wherein said dsRNA is at least one of: a small interfering RNA (siRNA), a MicroRNA (miRNA), a short hairpin RNA (shRNA) and a PIWI interacting RNAs (piRNAs).

6. The re-programming agent according to any one of claims 1 to 5, wherein said compound comprises at least one siRNA molecule or an endoribonuclease-prepared short interfering RNA (esiRNA), each siRNA and / or esiRNA molecule is specific for one of: (i) at least one member of the EGR family of transcription factors; and / or (ii) at least one member of the DGK family.

7. The re -programming agent according to any one of claims 1 to 6, wherein said EGR family of transcription factors comprises at least one of EGR2, EGR4, EGR1 and EGR3.

8. The re -programming agent according to any one of claims 1 to 7, wherein said DGK family comprises at least one of DGKa, DGK^, DGK5, DGK0 and DGKy.

9. The re-programming agent according to any one of claims 1 to 8, wherein said at least one member of the EGR family of transcription factors comprises at least one of EGR2 and EGR4; and wherein said at least one member of the DGK family comprises at least one of DGKa and DGK^.

10. The re-programming agent according to any one of claims 1 to 9, wherein said reprogramming agent comprises an effective amount of at least one of: (i) at least one siRNA or esiRNA molecule specific for EGR2; (ii) at least one siRNA or esiRNA molecule specific for EGR4; (iii) at least one siRNA or esiRNA molecule specific for DGKa and (iv) at least one siRNA or esiRNA molecule specific for DGK^; and / or any combinations of (i), (ii), (iii) and (iv).

11. The re-programming agent according to any one of claims 5 to 10, wherein said esiRNA molecule specific for EGR2 comprises the nucleic acid sequence as denoted by SEQ ID NO: 9, said esiRNA molecule specific for EGR4 comprises the nucleic acid sequence as denoted by SEQ ID NO: 10, wherein said siRNA molecule specific for DGKa comprises the nucleic acid sequence as denoted by SEQ ID NO: 13, said siRNA molecule specific for DGK^ comprises the nucleic acid sequence as denoted by SEQ ID NO: 14; or of any complementary sequence of at least one of SEQ ID NO: 9, 10, 13 and 14, of any variants, homologs or derivatives thereof.

12. The re-programming agent according to any one of claims 1 to 11, wherein said reprogramming agent is comprised within at least one of a nano- or micro-particle, a micellar formulation, a vehicle, a matrix, or a composition.

13. At least one nano- or micro-particle, micellar formulation, vehicle or matrix comprising at least one re-programming agent comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of:(i) at least one member of the EGR family of transcription factors; and(ii) at least one member of the DGK family; wherein said re-programming agent activates and / or functionalizes dysfunctional NK cells.

14. The at least one nano- or micro-particle, micellar formulation, vehicle or matrix according to claim 13, comprising at least one compound as defined by any one of claims 1 to 12.

15. The at least one nano- or micro-particle, micellar formulation, vehicle or matrix according to any one of claims 13 and 14, wherein said at least one compound is encapsulated within the intra-nanoparticle core or cavity of said nano- or micro-particle, micellar formulation, vehicle or matrix.

16. The at least one nano- or micro-particle, micellar formulation, vehicle or matrix according to any one of claims 13 to 15, wherein the outer nanoparticle surface of said nano- or microparticle, micellar formulation, vehicle or matrix is associated directly or indirectly with at least one targeting moiety.

17. The at least one nano- or micro-particle, micellar formulation, vehicle or matrix according to claim 16, wherein said at least one targeting moiety is at least one of an antibody, an aptamer, a ligand or any combinations thereof, that specifically recognizes and binds at least one molecule expressed on the surface of at least one NK cell.

18. The at least one nano- or micro-particle, micellar formulation, vehicle or matrix according to claim 17, wherein said at least one targeting moiety comprises at least one antibody that specifically recognizes and binds at least one molecule expressed on the surface of at least one NK cell.

19. The at least one nano- or micro-particle, micellar formulation, vehicle or matrix according to claim 18, wherein said at least one antibody comprises an antibody specific for at least one member of the natural cytotoxicity receptor (NCR) family.

20. The at least one nano- or micro-particle, micellar formulation, vehicle or matrix according to claim 19, wherein said member of the NCR family comprises Natural Killer Cell Protein 46 (NKp46).

21. A pharmaceutical composition comprising an effective amount of at least one a reprogramming agent comprising at least one compound that specifically inhibits the expression, activity and / or stability of at least one of:(i) at least one member of the EGR family of transcription factors; and(ii) at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, comprising said reprogramming agent, wherein said re-programming agent activates and / or functionalize dysfunctional NK cells, wherein said composition further comprises at least one of pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

22. The composition according to claim 21, wherein said re-programming agent is as defined by any one of claims 1 to 12, and wherein said nano- or micro-particle, micellar formulation, vehicle or matrix is as defined by any one of claims 13 to 20.

23. A method for activating at least one dysfunctional NK cell, the method comprising the step of contacting said dysfunctional NK cell with an activating effective amount of at least one re-programming agent comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of:(i) at least one member of the EGR family of transcription factors; and(ii) at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising said re-programming agent, wherein said re-programming agent activates and / or functionalize dysfunctional NK cells.

24. The method according to claim 23, wherein said dysfunctional NK cells comprise anergic NK cells and / or exhausted NK cells.

25. The method according to any one of claims 23 to 24, wherein said at least one compound that specifically inhibits the expression, activity and / or stability of: at least one of at least one member of the EGR family of transcription factors; and / or at least one member of the DGK family; comprises at least one nucleic acid molecule, at least one amino acid-based molecule, and / or at least one chemical inhibitor, each specific for one of: (i) at least one member of the EGR family of transcription factors; and / or (ii) at least one member of the DGK family.

26. The method according to claim 25, wherein said nucleic acid molecule is a ribonucleic acid (RNA) molecule or any nucleic acid sequence encoding said RNA molecule, said RNA molecule is at least one of a dsRNA, an antisense RNA, a ssRNA, a gRNA and a Ribozyme.

27. The method according to claim 25, wherein said dsRNA is at least one of: siRNA, esiRNA, miRNA, shRNA and piRNAs.

28. The method according to any one of claims 23 to 27, wherein said compound comprises at least one siRNA and / or esiRNA molecule, each siRNA or esiRNA molecule is specific for one of: (i) at least one member of the EGR family of transcription factors; and / or (ii) at least one member of the DGK family.

29. The method according to any one of claims 23 to 28, wherein said EGR family of transcription factors comprises at least one of EGR2, EGR4, EGR1 and EGR3.

30. The method according to any one of claims 23 to 29, wherein said DGK family comprises at least one of DGKa, DGK^ DGK5, DGK0 and DGKy.

31. The method according to any one of claims 23 to 30, wherein said at least one member of the EGR family of transcription factors comprises at least one of: EGR2 and EGR4; and wherein said at least one member of the DGK family comprises at least one of: DGKa and DGK^.

32. The method according to any one of claims 22 to 30, wherein said re-programming agent comprises an effective amount of at least one of: (i) at least one esiRNA molecule specific forEGR2; (ii) at least one esiRNA molecule specific for EGR4; (iii) at least one siRNA molecule specific for DGKa and (iv) at least one siRNA molecule specific for DGK^; and / or any combinations of (i), (ii), (iii) and (iv).

33. The method according to any one of claims 23 to 32, wherein said re-programming agent is comprised within at least one of a nano- or micro-particle, a micellar formulation, a vehicle, a matrix, or a composition.

34. The method according to claim 33, wherein said at least one targeting moiety is at least one of an antibody, an aptamer, a ligand or any combinations thereof, that specifically recognizes and binds at least one molecule expressed on the surface of at least one NK cell.

35. The method according to claim 34, wherein said at least one antibody comprises antibody specific for at least one member of the NCR family.

36. The method according to claim 35, wherein said member of the NCR family comprises NKp46, and wherein said targeting moiety comprises at least one antibody that specifically recognizes and binds NKp46.

37. The method according to any one of claims 23 to 36, for activating and / or functionalizing dysfunctional NK cell in a subject suffering from an immune-related disorder.

38. The method according to claim 37, wherein said immune-related disorder is at least one of a cancer, a proliferative disorder, an infectious disease, a graft versus host disease, an inflammatory disorder, an immune-cell mediated disorder and an autoimmune disorder.

39. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune -related disorder in a subject in need thereof, said method comprises administering to said subject a therapeutically effective amount of at least one re-programming agent comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of:(i) at least one member of the EGR family of transcription factors; and(ii) at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising said re -programming agent, wherein said re-programming agent activates and / or functionalize dysfunctional NK cells.

40. The method according to claim 39, wherein said activator is as defined by any one of claims 1 to 12, and wherein said nano- or micro-particle, micellar formulation, vehicle, matrix is as defined by any one of claims 13 to 20.

41. The method according to any one of claims 39 to 40, wherein said subject is suffering from an immune-related disorder, said immune-related disorder is at least one of: a cancer, aproliferative disorder, primary or secondary immunodeficiency, a graft versus host disease, an inflammatory disorder, an immune-cell mediated disorder, an autoimmune disorder and a viral infection.

42. The method according to any one of claims 39 to 41, wherein said subject is further treated with at least one immunomodulatory therapeutic agent prior to, after or simultaneously with said at least one re-programming agent.

43. The method according to claim 42, wherein said at least one immunomodulatory therapeutic agent comprises at least one of: (i) at least one checkpoint inhibitor; (ii) at least one cytokine; and (iii) chimeric antigen receptor (CAR) T cells.

44. At least one re -programming agent or any nano- or micro-particle, micellar formulation, vehicle, matrix, cell or composition comprising said re-programming agent, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof, said re-programming agent comprising at least one compound that specifically inhibit the expression, activity and / or stability of at least one of:(i) at least one member of the EGR family of transcription factors; and(ii) at least one member of the DGK family; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising said re-programming agent, wherein said re-programming agent activates dysfunctional NK cells.

45. The at least one re-programming agent for use according to claim 44, wherein said compound is as defined by any one of claims 2 to 12, and wherein said nano- or micro-particle, micellar formulation, vehicle or matrix is as defined by any one of claims 13 to 20, and wherein said composition is as defined in any one of claims 21 to 22.

46. A kit comprising:(I) at least one re-programming agent comprising at least one compound that specifically inhibits the expression, activity and / or stability of at least one of:(i) at least one member of the EGR family of transcription factors, in a first dosage form; and(ii) at least one member of the DGK family, in a second dosage form; and / or any nano- or micro-particle, micellar formulation, vehicle, matrix, or composition comprising said re -programming agent, wherein said re-programming agent activates and / or functionalize dysfunctional natural killer (NK) cells; the kit optionally further comprises(II) at least one additional immunomodulatory therapeutic agent, in a third dosage form.

47. The kit according to claim 46, wherein said re-programming agent is as defined in any one of claims 1 to 12.

48. The kit according to any one of claims 46 and 47, wherein said kit comprises:(a) at least one of:(i) at least one siRNA and / or esiRNA molecule specific for EGR2;(ii) at least one siRNA and / or esiRNA molecule specific for EGR4; and(b) at least one of:(i) at least one siRNA and / or esiRNA molecule specific for DGKa; and(ii) at least one siRNA and / or esiRNA molecule specific for DGK^.

49. The kit according to any one of claims 46 to 48, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of an immune-related disorder in a subject in need thereof.

50. The kit according to any one of claims 46 to 49, wherein said at least one immunomodulatory therapeutic agent comprises at least one of: (i) at least one checkpoint inhibitor; (ii) at least one cytokine; and (iii)chimeric antigen receptor (CAR) T cells.

51. A method for determining a personalized treatment regimen for a subject suffering from an immune-related disorder, and optionally for monitoring the effectiveness of a treatment regimen in said subject, the method comprising the steps of:(a) determining the levels of at least one of ERG2 and DGKa in at least one NK cell of at least one biological sample of said subject, to obtain the level value of said at least one of ERG2 and / or DGKa;(b) classifying said subject as:(i) a subject displaying a decreased NK cell functioning, if the level value determined in (a) for said at least one of: ERG2 and / or DGKa in said at least one sample is higher as compared to a predetermined standard value; or(ii) a subject displaying functioning NK cells, if the level value determined in (a) for said at least one of ERG2 and / or DGKa in said at least one sample is equal or lower as compared to a predetermined standard value; and(c) selecting for a subject displaying a decreased NK cell functioning, an activating treatment regimen that decreases the levels of at least one of ERG2 and DGKa, in at least one NK cell of said subject.

52. The method according to claim 51 , wherein said activating treatment regimen selected in (c), comprises at least one re-programming agent as defined in any one of claims 1 to 12.

53. The method according to any one of claims 51 and 52, wherein the subject is a subject treated with at least one immunomodulatory therapeutic compound.

54. The method according to claim 53, said at least one immunomodulatory therapeutic agent comprises at least one of: (i) at least one checkpoint inhibitor; (ii) at least one cytokine; and (iii) chimeric antigen receptor (CAR) T cells.

55. The method according to any one of claims 51 to 54, wherein selecting for a subject displaying a decreased NK cell functioning, an activating treatment regimen comprises one of:(a) ceasing a treatment regimen with said at least one immunomodulatory therapeutic agent, and replacing said treatment with a treatment regimen comprising said re-programming agent as defined in any one of claims 1 to 12; or(b) administering a re -programming agent as defined in any one of claims 1 to 12, prior to, after or concomitantly with said immunomodulatory therapeutic agent.

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