DESIGN OF SYNTHETIC miRNAS TO RESTORE THE CORRECT GENE DOSAGE OF GENES ASSOCIATED WITH T LYMPHOCYTE EXHAUSTION

Abstract

The invention concerns synthetic miRNAs targeting the CISH gene and uses thereof. Pharmaceutical compositions comprising such miRNAs and uses thereof in the medical field, in particular in the treatment of cancer diseases and in immunotherapy, are also described.

Description

[0001] TITLE

[0002] DESIGN OF SYNTHETIC miRNAS TO RESTORE THE CORRECT GENE DOSAGE OF GENES ASSOCIATED WITH T LYMPHOCYTE EXHAUSTION

[0003] FIELD OF THE INVENTION

[0004] The invention concerns synthetic miRNAs targeting the CISH gene and uses thereof. Pharmaceutical compositions comprising such miRNAs and uses thereof in the medical field, in particular in the treatment of cancer diseases and immunotherapy, are also described.

[0005] STATE OF THE ART

[0006] CD8+ T lymphocytes are protagonists of the adaptive immune response, and their correct functioning is essential for the elimination of pathogenic cells such as infected and cancerous cells.

[0007] Physiologically, CD8+ T cells are activated upon recognition of specific antigens presented by MHC molecules via the T cell receptor (TCR), which triggers proliferation, maturation and cytotoxic response.

[0008] In addition to TCR activation, CD8+ T cells require co-stimulation of CD28 to be fully activated, and cytokine signaling to modulate this activation.

[0009] The TCR pathway is regulated by many inhibitory proteins, which are necessary to disrupt the cytotoxic response once the antigen has been eliminated. However, when T cells are chronically stimulated by an antigen (for example, during chronic infections or cancer), they enter a dysfunctional state in which they lose typical functional capabilities, such as cytotoxicity, known as exhaustion.

[0010] During exhaustion, the finely tuned balance of the immune response is disrupted by the overexpression of some of these inhibitory proteins, including CISH. CISH is usually upregulated in activated T cells, forming a negative feedback loop able to prevent T cell overactivation and the resulting deleterious effects. This increase becomes dramatic in exhausted T cells and contributes to overall dysfunction.

[0011] Exhaustion is particularly detrimental to the treatment of tumors. Exhausted T cells express an increased number of immune checkpoint inhibitory receptors and inhibitory cytokines, which compete with co-stimulatory signaling and Th1 cytokine signaling, respectively, due to the suppressive tumor microenvironment.

[0012] It has been previously reported that elimination of proteins that are upregulated during exhaustion through knock-out (KO) technology induces tumor regression and improved T cell avidity in vivo. However, the intrinsic and major limitation of such strategies is the permanent deletion of genes that are essential for T cell regulation, resulting in increased off-target response and cytotoxicity.

[0013] The present invention aims to restore the correct dosage of CISH, which is upregulated during exhaustion, to effectively counteract T cell dysfunction and ensure homeostasis of the immune response.

[0014] SUMMARY OF THE INVENTION

[0015] The engineering of CAR-T for specific targeting of tumor cells represented a breakthrough in the field of cancer immunotherapy. However, chronic exposure of T cells to antigens has been shown to lead to a dysfunction called exhaustion, a condition in which cells lose the ability to kill tumor cells. T cells progressing to exhaustion have been shown to exhibit specific phenotypic characteristics, such as overexpression of immune checkpoint receptors, inhibitory proteins involved in balanced T cell activation, such as CISH.

[0016] The present invention relates to novel synthetic microRNAs to modulate the CISH gene and obtain more active and persistent T cells.

[0017] In a first aspect, the present invention relates to a nucleic acid encoding for a synthetic microRNA (miRNA) targeting the CISH gene, said nucleic acid comprising the following elements from the 5’ end to the 3’ end: a. a 5’-UTR nucleotide sequence for miR-155; b. a nucleotide sequence antisense to the CISH gene sequence (siRNA); c. a loop nucleotide sequence of miR-155; and d. a nucleotide sequence complementary to the sequence of element b..

[0018] In a second aspect, a pharmaceutical composition comprising a nucleic acid encoding for a synthetic miRNA according to the invention and pharmaceutically acceptable excipients. In a third aspect, the invention relates to the use as a medicament of the nucleic acid encoding for a synthetic miRNA or the composition comprising it.

[0019] In a fourth and fifth aspect, the use of the nucleic acid encoding for a synthetic miRNA or the composition thereof in the treatment of cancer and immunotherapy is described herein.

[0020] The dependent claims describe specific embodiments of the invention.

[0021] DESCRIPTION OF THE FIGURES

[0022] The invention will now be described in detail and with reference to the attached Figures. Figure 1 : Mechanism of action of synthetic miRNAs. Schematic representation of plasmids encoding for synthetic miRNAs within the cell endogenous pathway.

[0023] Figure 2: Expression of the CISH target gene in HEK293 cells transfected with the designed siRNAs. Target gene expression is calculated versus the scrambled siRNA negative control. (n=2)

[0024] Figure 3: CISH target gene expression in Jurkat cells transfected with the designed siRNAs. Target gene expression is calculated versus the scrambled miRNA negative control (n=1 ).

[0025] Figure 4: CISH target gene expression in HEK293 cells transfected with miRNA (SYmiR) encoding Blockit plasmids. CISH target gene expression is calculated versus the scrambled miRNA negative control. Statistical analysis was performed on dCTs using the Paired t-student (p-value<0.05, n=4).

[0026] Figure 5: Functional and checkpoint receptor analysis of T cells transfected with Blockit miRNA plasmids. Histogram showing IL-2, TNF-a and GmzB production following stimulation with PMA / lonomycin in CD8+ T cells after CISH silencing, compared to the control treated with the C- miRNA negative control. For both C- miRNAs and CISH miRNAs, there is an additional negative control without stimulation with PMA / lonomycin. Figure 6: xCELLigence cytotoxicity assay showing Caspase-3 activation (Figure 6A) and confluence analysis of SKOV3 cells (Figure 6B) in co-culture with CD8+ T cells transfected with plasmids expressing scrambled miRNA (dashed line with diamonds) or anti-CISH miRNA (dashed line with triangles).

[0027] Figure 7: Flow cytometry of the functional assay. Percentage of Granzyme B in T cells with control scrambled miRNA (SYmiR C-, black bar) and CISH miRNA (SYmiR CISH, white bar) 48 hours post-electroporation. Representative dot plots show an example from a healthy donor. Data are presented as mean ± SEM, N = 3 biological replicates, *p-value < 0.05 using paired Student’s t-test.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] The invention presented herein centers around the development and application of novel microRNAs (miRNAs) to restore the correct dosage of upregulated genes in exhausted cells with the aim of neutralizing T-cell dysfunction and improving the longterm efficacy of adoptive T-cell therapy.

[0030] In a first aspect, a nucleic acid encoding for a synthetic miRNA targeting the CISH gene is described, said nucleic acid comprising the following elements from the 5’ end to the 3’ end: a. a 5’-UTR nucleotide sequence for miR-155; b. a nucleotide sequence antisense to the CISH gene sequence (siRNA); c. a loop nucleotide sequence of miR-155; and d. a nucleotide sequence complementary to the sequence of element b..

[0031] In particular, a miRNA targeting one of the key regulators of T cell exhaustion, namely CISH, was engineered. It was designed so that its expression is controlled by polymerase II (Pol II) promoters to regulate the strength and timing of activation, and to generate inducible systems.

[0032] In the present invention, the term “downregulation” or “downmodulation” is intended to encompass the ability of the miRNA or siRNA to modulate and / or reduce the expression of the target gene.

[0033] To date, most methods to turn off genes of interest are based on CRISPR-mediated knockout or on constitutive and non-modulable shRNAs expressed by RNA pol III.

[0034] With the present approach, we sought to achieve greater control over miRNA transcription with synthetic promoters that are inducible with drugs or proteins, thus allowing miRNA transcription in a more targeted manner, which is only possible with the Pol II promoter.

[0035] In a preferred embodiment said nucleic acid encoding for a synthetic miRNA has a length of 64 nucleotides.

[0036] The synthetic siRNA sequences are inserted into the miRNA155 support structure that maximizes the processivity to obtain the mature sequence directed against the miRNA target. The siRNA must have 100% complementarity to the target mRNA and can be between 21 and 23 nt in length. The siRNA length of 21 nt was selected by us. In a further embodiment said nucleic acid encoding for a synthetic miRNA has a nucleotide sequence of element b. having a length of 21 nucleotides, wherein said nucleotide sequence of element b. is a siRNA selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, preferably said nucleotide sequence of element b. is SEQ ID NO:7.

[0037] During the design of the synthetic miRNAs, sequences that pair with the coding sequence of genes with perfect complementarity were favored to achieve more robust repression of target genes.

[0038] In another aspect, the nucleic acid encoding for a synthetic miRNA of the present invention has a nucleotide sequence of element d. complementary to the sequence of element b. in nucleotides 1 -8 and 1 1 -21 , and is selected from the group consisting of SEQ ID NO:1 1 , SEQ ID NO:12 and SEQ ID NO:13, preferably said nucleotide sequence of element d. is SEQ ID NO:1 1 .

[0039] It should be noted that the miRNA complementary strand lacks nucleotides 9 and 10, in order to make it easier to detach it from the mature miRNA sequence, to optimize the quality of repression.

[0040] In a preferred embodiment, the element a. of the nucleic acid encoding for a synthetic miRNA has a nucleotide sequence corresponding to TGCTG and a nucleotide sequence of element c. of SEQ ID NO:10.

[0041] In a further preferred embodiment, the nucleic acid encoding for a synthetic miRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:3 and SEQ ID NO:5, preferably said nucleotide sequence is SEQ ID NO:1 .

[0042] In a second aspect, a pharmaceutical composition comprising a nucleic acid encoding for a synthetic miRNA according to the invention and excipients suitable for use is described.

[0043] In a third aspect, the invention relates to the use of the nucleic acid encoding for a synthetic miRNA or the composition comprising it, as a medicament.

[0044] In a fourth and fifth aspect, the use of the nucleic acid encoding for a synthetic miRNA or the composition thereof in the development of T-lymphocyte-based therapies (e.g. CAR-T) for the treatment of tumors is described herein.

[0045] In a preferred embodiment, said tumors are solid tumors or blood tumors and may be selected from the group comprising cerebral astrocytoma, cerebellar astrocytoma, pineal gland astrocytoma, oligodendroglioma, pituitary adenoma, craniopharyngioma, sarcoma, multiform glioblastoma, grade II fibrillary astrocytoma, protoplasmic astrocytoma, grade III gemistocytic astrocytoma, anaplastic astrocytoma, including gliomatosis cerebri, pituitary adenoma, ependymoma, medulloblastoma, neural ectoderm tumor, neuroblastoma, hypothalamic glioma, breast cancer, lung cancer, colon cancer, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, esophageal cancer, basal cell carcinoma, cholangiocarcinoma, spleen carcinoma, osteosarcoma, intraocular melanoma, retinoblastoma, gastric carcinoma, cardiac carcinoma, liver carcinoma, hypopharyngeal carcinoma, laryngeal carcinoma, oral cavity carcinoma, nasal and paranasal carcinoma, salivary gland carcinoma, nasopharyngeal carcinoma, throat carcinoma, thyroid cancer, pancreatic cancer, kidney cancer, prostate cancer, bladder cancer, gastric and liver carcinoma, colorectal cancer, rectal cancer, testicular cancer, renal cell cancer, melanoma, sarcoma, mesothelioma, pheochromocytoma, hematological malignancies or chronic myeloid leukemia.

[0046] In a fifth aspect, the use of nucleic acid encoding for a synthetic miRNA or a composition thereof in immunotherapy is described herein.

[0047] In a preferred embodiment, said immunotherapy is mediated by T cells, NK cells or engineered lymphocyte cells selected from the group comprising CAR-T and CAR-NK. T and NK cells in the tumor microenvironment undergo a progressive loss of functionality due to the overexpression of proteins that act on the immune response as a brake. CISH is located upstream of the T cell activation pathway and tumor cells induce its overexpression as an evolutionary mechanism for survival and to evade the immune response. The same biological mechanism is also induced for NK cells. The latter, unlike T cells, exert an important cytotoxic response within the tumor microenvironment without the need to recognize the major histocompatibility complex. Tumor cells, as a mechanism of evasion of the immune response, elude the cytotoxic response by T cells by losing the major histocompatibility complex. To avoid the activation of the cytotoxic response by NK cells, tumor cells induce an overactivation of T cells that leads to the increase in the expression of CISH, which inhibits the cytotoxic response. CAR-T and CAR-NK exposed to the tumor microenvironment overexpress CISH with consequent loss of functionality. The creation of CAR-T and CAR-NK has a high manufacturing cost, and ensuring their functionality is of fundamental importance. The synthetic anti-CISH miRNA can be used in conjunction with CAR to create a new generation of immunotherapies that are more resistant to the tumor microenvironment. Furthermore, total suppression of CISH expression via CRISPR / CAS9 in T cells may have detrimental effects on patients, such as an increased risk of off-tumor cytotoxicity, autoimmunity phenomena and a higher probability of suffering severe (life-threatening) complications, such as a cytokine storm after the infusion of engineered T cells. These undesirable effects could be removed by reducing CISH levels only when T cells start to lose functionality.

[0048] Synthetic miRNAs can be integrated into any mammalian cell wherein regulation is needed, as well as into immune cells such as T cells, NK cells and engineered biological therapeutic derivatives thereof, such as CAR-T, CAR-NK, wherein their modulation is associated with a different effector phenotype to develop more effective cell-based anticancer immunotherapies.

[0049] Table 1 reports the Forward (Fwd) and Reverse (Rev) oligonucleotide sequences inserted into the plasmids and encoding for single-stranded miRNAs that will correspond to the sequence of the Fwd strand, in which the thymine “t” base will be replaced by uracil “u”. Furthermore, the sequences of siRNAs, loops and complementary siRNAs, in which the uracil “u” base is represented as thymine “t” base, are reported in compliance with the requirements of the WIPO standard ST26 Annex 1 .

[0050] Table 1 :

[0051] The sequence characteristics of the miRNA Fwd strand are as follows:

[0052] - Nucleotides 1 to 5: overhang of the insert + G (element a.);

[0053] - Nucleotides 6 to 26: antisense sequence to the CISH gene sequence (element b.);

[0054] - Nucleotides 27 to 45: loop sequence (element c.);

[0055] - Nucleotides 46 to 64: complementary sequence to the sequence of element b. (1 -8 / 1 1 -21 siRNA) (element d.).

[0056] The sequence characteristics of the miRNA Rev strand (64 nt) are the mirror image of those of the Fwd strand for the first 64nt and have, in addition, the nucleotides 65 to 68, corresponding to the miR-155 overhang nucleotide sequence.

[0057] Below are Examples of the miRNA implementation of the present invention provided for illustrative purposes. EXAMPLES

[0058] The synthetic miRNAs (SYmiR) were first tested in model cell lines (HEK293) to probe the repression of target mRNAs by qPCR and flow cytometry which quantify mRNA and protein levels, respectively. Furthermore, according to the hypothesis that synthetic miRNAs improve functionality and cytotoxicity of T cells, cytotoxicity and functional phenotype tests were carried out. The results reported in the following examples indicate an improvement in T cell functioning.

[0059] Example 1 : miRNA design

[0060] Figure 1 shows how synthetic miRNAs are involved in the RNA interference (RNAi) pathway. Once transfected, plasmids are expressed by RNA polymerase II in a pri- miRNA-like structure, which is recognized by DROSHA and transformed into a pre- miRNA. The latter is exported out of the nucleus into the cytosol, where it is further processed by DICER into a siRNA, the final active structure that is complexed into the RISC complex to induce RNA silencing through mRNA degradation.

[0061] Novel synthetic miRNA sequences were developed to achieve significant downregulation of key regulatory genes of T cell exhaustion.

[0062] MiRNAs are non-coding RNAs and are present in mammalian cell genes embedded in a double-stranded region (stem) which is connected to a single-stranded region (loop) that together form a short hairpin called a pre-miRNA.

[0063] First, the designed pre-miRNA sequences were cloned into the miRNA expression plasmid pcDNA™6.2-GW / EmGFP-miR (Thermo Fisher Scientific).

[0064] In order to make polymerase II transcribe a siRNA sequence, it is necessary to incorporate the siRNA sequence into a pri-miRNA structure. The miR-155 architecture was used for this purpose.

[0065] Each pre-miRNA is 68 nucleotides long and contains a 21 nt siRNA that binds to the exon of the target gene and is made of a backbone derived from miR-155 into which the CISH siRNA sequence was integrated.

[0066] Table 1 reports the 3 Fwd and Rev miRNA sequences tested in a preliminary assay (SEQ ID NO:1 - SEQ ID NO:6).

[0067] If the miRNA sequence is not inserted into a plasmid designed to express miRNAs such as pcDNA™6.2-GW / EmGFP-miR miRNA (Thermo Fisher Scientific), the entire pri- miRNA sequence must be cloned downstream of an inducible or constitutive promoter. The complete sequences to be inserted into a normal expression plasmid, which include the regions flanking miRNA 155, are described in Table 2.

[0068] Table 2:

[0069] In this case, the characteristics of the miRNA Fwd strand sequence are as follows:

[0070] - Nucleotides -23 to 0: scaffold structure of pri-miRNA 155;

[0071] - Nucleotides 1 to 5: overhang of insert + G (element a.);

[0072] - Nucleotides 6 to 26: antisense sequence to the CISH gene sequence (element b.);

[0073] - Nucleotides 27 to 45: loop sequence (element c.);

[0074] - Nucleotides 46 to 64: complementary sequence to the sequence of element b. (1 -8 / 1 1 -21 siRNA) (element d.);

[0075] - Nucleotides 65 to 68: miR-155 overhang nucleotide sequence; and

[0076] - Nucleotides 69 to 109: scaffold structure of pri-miRNA 155.

[0077] The sequence characteristics of the miRNA Rev strand are mirrored by those of the Fwd strand. The DNA duplex formed by Fwd and Rev has 132 nucleotides in total, while in the strategy used by us the DNA duplex formed by Fwd and Rev has 64 nt with 4 nt single-stranded flanking at the 5’ and 3’ ends. The 68 nt double-stranded pre-miRNA is created once inserted into the pcDNA™6.2-GW / EmGFP-miR plasmid (Thermo Fisher Scientific).

[0078] During the design of synthetic miRNAs, sequences pairing with perfect complementarity to the coding sequence of the genes were favored in order to obtain a more robust downregulation of the target genes.

[0079] Furthermore, the complementary strand of the synthetic miRNA lacks nucleotides 9 and 10, making it easier to detach it from the mature synthetic miRNA sequence. Proteins upregulated during exhaustion were identified from the literature and subsequently validated in the laboratory through flow cytometry and RNA-seq analyses.

[0080] Example 2: Design and evaluation of miRNAs in cell lines

[0081] Based on the human gene sequence, three siRNAs targeting the CISH gene were designed and tested (Table 3).

[0082] Table 3. Synthetic siRNA sequences tested in the preliminary experiments.

[0083] The selection of the siRNA directed against the target mRNA followed three steps. The first one is the computational analysis of potential OFF-targets by comparing the sequences with the human genome. The second one is the analysis of the target gene repression in HEK293 cells. The expression of the target mRNA was quantified by RT q-PCR (Figure 2). The third step was the analysis of the desired gene repression in a cellular model similar to the corresponding primary T cells, using Jurkat cell lines (Figure 3), and the target mRNA repression was assessed. Overall, the analyses indicate that the siRNAs robustly repress the target gene in different cellular models.

[0084] The best performing siRNA, whose sequence is reported in Table 4, was cloned as a synthetic miRNA and expressed downstream of a CMV promoter (BlockIT miRNA expression plasmid, Thermo Fisher Scientific) for further testing. EmGFP co-expressed in the same plasmid was used to probe the success of transfection.

[0085] Table 4. Mature siRNA sequences that gave the best downregulation of target genes.

[0086] To verify the activity of the selected synthetic miRNAs, experiments were performed in HEK293 cells to verify whether the selected synthetic miRNA induces a significant and reproducible knockdown of the target mRNA. To this end, synthetic miRNA plasmids were transfected, and the transfection efficiency was measured by flow cytometry (FC), while repression was assessed by qPCR. A scrambled miRNA plasmid, that does not bind to and does not reduce the expression of any human genetic sequence, was used as a negative control.

[0087] Figure 4 shows the efficient downregulation of target genes following transfection of synthetic miRNAs.

[0088] Example 3: CD8 T lymphocyte assay

[0089] The anti-CISH synthetic miRNA was tested in CD8+ T cells. Specifically, plasmids expressing the synthetic miRNA were electroporated onto primary CD8+ T cells (purified from PBMC) 1 day after CD3 / CD28 stimulation. 2 days after transfection, CD8+ T cells were subjected to a functional assay and an immuno-checkpoint receptor analysis by flow cytometry. The RNA was extracted to verify RNA-downregulation of the target.

[0090] As shown in Figure 4, the identified synthetic miRNA sequence has the ability to downregulate the target sequence in CD8+ T cells. CISH levels were assessed after treatment with synthetic miRNAs by qPCR. CISH downregulation showed a strong change in functional and exhaustion phenotypes (Figure 5). Specifically, repression of CISH in CD8+ T cells strongly induces the expression of IL-2, TNF-a and GmzB upon stimulation with Phorbol myristate acetate (PMA) / lonomycin, as compared to the scrambled control. PMA / lonomycin are chemical compounds commonly used to activate T cells and induce cytokine secretion, bypassing the T cell receptor and directly activating phosphokinase C (PKC) and the Ca2+ / calmodulin pathway.

[0091] The observed effects are beneficial for T cell fitness and cytotoxic potential against tumor cells.

[0092] Example 4: Co-culture test of CD8+ T cells with SKOV3 tumor cells

[0093] Subsequently, co-culture assays were performed using anti-HER2 CAR CD8+ T cells transfected with the synthetic anti-CISH miRNA containing the siRNA of SEQ ID NO:7 and the human cervical cancer cell line SKOV3 expressing HER2. The same experiment was conducted using anti-HER2 CAR CD8+ T cells transfected with scrambled miRNA. As shown in Figure 6 from the xCELLigence cytotoxicity assay, anti-HER2 CAR CD8+ T cells with CISH knockdown exhibited increased cytotoxicity against the HER2- expressing human cervical cancer cell line SK0V3 in at least three independent experiments.

[0094] In addition, to assess the percentage of Granzyme B in T cells with control scrambled miRNA (SYmiR C) and CISH miRNA (SYmiR CISH), a flow cytometry-based functional assay was performed 48 hours post-electroporation (Figure 7).

[0095] As shown in Figure 7, CISH repression in CD8+ T cells strongly induces Granzyme B expression.

[0096] Materials and Methods siRNA Transfection

[0097] Cells were seeded in a 24-well culture plate at a density of 120,000 cells per well. Jurkat cells were transfected with 25 pmol of siRNA using the Neon® electroporation device (Thermofisher). After 48 hours, the cells were harvested in a 1 .5 ml tube, washed once with PBS1X, and stored at -80 °C as a dry pellet. HEK293 cells were plated in a 24-well culture plate at a density of 60,000 cells per well. After 24 hours, 25 pmol of siRNA were transfected into the cells using Lipofectamine®2000 (Thermofisher) as a transfection reagent. After 48 hours, the cells were harvested in a 1 .5 ml tube, washed once with PBS1 X, and stored at -80 °C as a dry pellet.

[0098] Plasmid Transfection

[0099] Cells were seeded in a 24-well culture plate at a density of 120,000 cells per well. Jurkat cells were transfected with 2 pg of synthetic miRNA plasmid using the Neon® electroporation device (Thermofisher). HEK293 cells were plated in a 24-well culture plate at a density of 60,000 cells per well. After 24 hours, 600 ng of synthetic miRNA plasmids were transfected into cells using PEI MAX™ - Transfection Grade Linear (POLYSCIENCES) as a transfection reagent (PELDNA ratio 3:1 ). After 48 hours, the cells were collected in a 1 .5 ml tube, washed once with PBS1 X and stored at -80°C as a dry pellet.

[0100] RNA Extraction, Reverse Transcription, and qPCR

[0101] The dry pellet of transfected cells was used to extract RNA according to the manufacturer’s protocol, using the EZNA TOTAL RNA KIT (Omega Bio-tek). Reverse transcription was performed with the PrimeScript RT reagent Kit (Takara) using the oligo-dT strategy and according to the manufacturer’s instructions. cDNA samples were stored in multiple aliquots at -20°C for later use. Amplifications were performed with Brilliant Applied Biosystems Fast SYBR Green master mix. Following the manufacturer’s instructions, the reactions were performed in a final volume of 10 pl with 1 pl of cDNA and 1 pl of each primer at the optimized concentration. Amplifications were performed as described above for conventional PCR assays. A dissociation step was included for all the reactions to confirm the amplification of a single specific PCR product and define the Tm of each amplicon. A negative control (no-plate control) was included in each primer assay to verify primer-dimer formation. Thermal cycling was performed on a QuantStudio3 RT-PCR thermal cycler (Applied Biosystems) with the following cycling parameters: 50 °C for 2 min, 95 °C for 10 min, 35 cycles of 95 °C for 15 s and 60 °C for 1 min for the amplification step, 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s for the melting curve step.

[0102] Isolation of Human CD8+ T Cells by Electroporation

[0103] Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy donors by Ficoll-Paque density gradient centrifugation. CD8+ T cells were purified using the Human CD8+ T Cell Isolation Kit (Miltenyi Biontec) and stimulated with CD3 / CD8 Dynabeads™ (Gibco) at a 1 :1 ratio (day 0) and maintained at 1 million cells / ml. On day 3, 3 hours before electroporation, the stimulating beads were removed with DynaMyg™-2 Magnet (Thermofischer). 2 million T cells were electroporated with 20 pg of plasmid containing the synthetic mi RNA using the program #24 of the Neon® electroporator (1600 V, 10 ms, 3 pulses). Cells were left at a concentration of 1 .5 million cells / ml in R10 medium for 48 hours.

[0104] Functional Assay

[0105] 48 hours after electroporation with the plasmid containing the synthetic miRNA, human CD8+ T cells were incubated with phorbol myristate acetate and lonomycin (PMA / I) to induce activation. Golgistop (Monensin) and Golgiplug (brefeldin A) were added to block cytokine release. After 6 hours, the expression of interleukin 2 (IL-2), granzyme B (GzmB) and tumor necrosis factor alpha (TNF-a) was measured by flow cytometry.

[0106] Checkpoint Receptor Assay 48 hours after electroporation, human CD8+ T cells were analyzed by flow cytometry and expressed as median fluorescence intensity (MFI). FMO controls were used to set the gating of positive events.

[0107] These examples collectively provide compelling evidence for the efficacy of synthetic miRNAs in influencing target gene expression and preventing T cell exhaustion.

Claims

CLAIMS1 . A nucleic acid encoding for a synthetic miRNA targeting the CISH gene, said nucleic acid comprising the following elements from the 5’ end to the 3’ end: a. a 5’-UTR nucleotide sequence of miR-155; b. a nucleotide sequence antisense to the CISH gene sequence (siRNA); c. a loop nucleotide sequence of miR-155; d. a nucleotide sequence complementary to the sequence of element b..

2. The nucleic acid encoding for a synthetic miRNA according to claim 1 , wherein said nucleic acid encoding for a synthetic miRNA has a length of 64 nucleotides.

3. The nucleic acid encoding for a synthetic miRNA according to any one of claims 1 or2, wherein said nucleotide sequence of element b. has a length of 21 nucleotides, and wherein said nucleotide sequence of element b. is a siRNA selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, preferably said nucleotide sequence of element b. is SEQ ID NO:7.

4. The nucleic acid encoding for a synthetic miRNA according to any one of claims 1 to3, wherein said nucleotide sequence of element d. is complementary to the sequence of element b. in nucleotides 1 -8 and 1 1 -21 and is selected from the group consisting of SEQ ID NO:1 1 , SEQ ID NO:12 and SEQ ID NO:13, preferably said nucleotide sequence of element d. is SEQ ID NO:11 .

5. The nucleic acid encoding for a synthetic miRNA according to any one of claims 1 to4, wherein said nucleotide sequence of element a. is TGCTG and the nucleotide sequence of element c. is SEQ ID NQ:10.

6. The nucleic acid encoding for a synthetic miRNA according to any one of claims 1 to5, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:3 and SEQ ID NO:5, preferably said nucleotide sequence is SEQ ID7. A pharmaceutical composition comprising a nucleic acid encoding for a synthetic miRNA according to any one of claims 1 to 6 and excipients suitable for use.

8. A nucleic acid encoding for a synthetic miRNA according to any one of claims 1 to 6 or a composition according to claim 7, for use as a medicament.

9. A nucleic acid encoding for a synthetic miRNA according to any one of claims 1 to 6, or a composition according to claim 7, for use in the treatment of cancer.

10. A nucleic acid encoding for a synthetic miRNA according to any one of claims 1 to 6 or a composition according to claim 7, for use in immunotherapy.

11. The nucleic acid for use according to claim 10, wherein said immunotherapy is mediated by T cells, NK cells or engineered lymphocyte cells selected from the group comprising CAR-T and CAR-NK.

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