siRNA-transfected platelets and their therapeutic use

Abstract

Disclosed is a therapeutic composition comprising platelets transfected with siRNA targeting a mutant form of the KRAS oncoprotein, and its use for the treatment of tumors in general, and pancreatic adenocarcinoma in particular.

Description

[Technical field]

[0001] The present invention relates to platelets transfected with siRNA designed to inhibit the mRNA of KRAS, a gene involved in oncogenesis.

[0002] In particular, the present invention provides a therapeutic composition comprising platelets transfected with siRNA targeting a mutant form of the KRAS oncoprotein.A further aspect of the present invention relates to the use of the therapeutic composition for the treatment of tumors in general, and pancreatic adenocarcinoma in particular. [Background technology]

[0003] KRAS oncoprotein is a GTPase that functions as an essential mediator of intracellular signaling pathways involved in tumor cell proliferation and survival. In normal cells, KRAS acts as a molecular switch, alternating between an inactive GDP-bound state and an active GTP-bound state. The transition between these states is facilitated by GTP hydrolysis catalyzed by guanine nucleotide exchange factors (GEFs), which load GTP and activate KRAS, and GTPase-activating proteins (GAPs), which inactivate KRAS. Binding of GTP to KRAS promotes binding of effectors that activate intracellular signaling pathways, such as RAF-MEK-ERK (MAPK). Mutations in the KRAS gene are common in pancreatic, lung, colorectal, gallbladder, thyroid, and biliary tract cancers. KRAS mutations are observed in approximately 94.1% of pancreatic tumors, with the KRASG12D mutation being the most frequent, with an incidence of 41%. Mutations in the GTPase KRAS have been found to be common in patients suffering from pancreatic ductal adenocarcinoma (PDAC), and these mutations are thought to be involved in the early stages of carcinogenesis, tumor progression, and metastatic spread. KRAS mutations at residues G12, G13, and Q61 are the most common mutations in solid tumors. Somatic mutations that activate KRAS are a hallmark of certain cancers, inhibiting its binding to GAPs, thereby stabilizing its binding to effectors, and consequently increasing the KRAS signaling pathway. Patients suffering from tumors with KRAS mutations have a significantly worse prognosis and poor prognosis. Although inhibitors of various proteins of the MAPK intracellular signaling pathway (MEK, BRAF, EGFR, etc.) have been approved for clinical use for other types of tumors, no molecules selective for mutant KRAS tumors are in clinical use to date. Several therapies targeting the MAPK pathway have proven clinically ineffective in treating tumors with KRAS mutations. Furthermore, therapies that do not selectively target specific tumor protein mutants may cause systemic toxicity because they inhibit MAPK-induced signaling in normal cells.

[0004] Therefore, there is a need to develop therapeutic approaches that selectively target tumors and minimize or eliminate drug distribution to healthy cells.

[0005] KRASG12D mutations are prevalent in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by high mortality. PDAC patients with KRASG12D tumors have a particularly poor prognosis compared with patients with other KRAS mutations. Therefore, to improve the survival of PDAC patients, it is important to develop novel therapeutic strategies that target KRASG12D, which are more efficient and safer.

[0006] MicroRNAs and their synthetic products, siRNAs, are small siRNA duplexes of non-coding RNA that play important roles in the post-transcriptional regulation of gene expression by suppressing the translation or inducing the degradation of specific mRNAs.

[0007] The therapeutic use of siRNA requires an efficient method for delivery to the bloodstream, as siRNA would otherwise be rapidly degraded and inactivated by plasma nucleases.

[0008] For this purpose, methods have been developed that use bacteria, viruses, synthetic vesicles (e.g. micelles, microsomes), and human cells (red blood cells, macrophages, lymphocytes, stem cells) as carriers.

[0009] Effective RNAi delivery to non-hepatic parenchymal organs, especially the pancreas, remains a challenge. Liposomes and nanoparticles offer advantages for RNAi delivery compared with viral systems, but they are less efficient and are rapidly cleared from the circulation.

[0010] Approaches based on RNA interference (RNAi) targeting wild-type KRAS and downstream effectors of the enzyme using nanoparticles as vectors have shown clear efficacy in the treatment of lung and several colon cancer models.

[0011] However, targeting KRAS in the treatment of pancreatic adenocarcinoma has so far been limited to administering inhibitors via direct electroporation or biopolymer implantation in pancreatic cancer xenograft models.

[0012] cutting edge technology The use of human platelets to release siRNA is described in the international application WO2014 / 118817 in the name of the applicant. Several siRNA sequences have been reported that target mutations of the KRAS oncogene, but not against the G12D mutation. In particular, the use of platelets loaded with siRNA against KRASG12D for the treatment of pancreatic adenocarcinoma has not been described or suggested.

[0013] Patent Publications WO2017 / 127473 and US2015 / 0307885 describe inhibition of expression of mutant KRAS by RNA interference, and dsRNA sequences useful for this purpose.

[0014] WO2010 / 001325 describes polymeric systems suitable for the delivery of therapeutic agents, including siRNA. Summary of the Invention

[0015] We now show that platelets transfected with siRNA specific for the KRAS G12D mutant, or microparticles derived from them, effectively inhibit the growth of pancreatic cancer tumours in vivo in a mouse model.

[0016] Platelets transfected with siRNA targeting KRASG12D prepared according to the present invention are capable of reducing tumor burden in animal models of tumors expressing KRASG12D, and have proven particularly effective in studies conducted in an animal model of pancreatic ductal adenocarcinoma (PDAC).

[0017] Thus, a first aspect of the present invention relates to a therapeutic composition comprising platelets transfected with siRNA capable of inhibiting the mRNA of KRAS having a G12D mutation (KRASG12D), or platelet microparticles derived therefrom, for use in the treatment of KRASG12D-expressing tumors, preferably pancreatic adenocarcinoma, more preferably pancreatic Duchenne adenocarcinoma.

[0018] In a preferred embodiment, the sequence of the antisense strand of the siRNA is complementary to the KRASG12D mRNA sequence containing the mutant codon.

[0019] The siRNA molecule can contain from 15 to 30, preferably 21 base pairs.

[0020] In a particularly preferred embodiment, the siRNA molecule comprises a sense strand and an antisense strand, (i) the sense strand consists of or comprises the sequence 5'-GUUGGAGCUGAUGGCGUAGTT-3' (SEQ ID NO:1); and (ii) the antisense strand consists of or comprises the sequence 5'-CUACGCCAUCAGCUCCAACTT-3' (SEQ ID NO:2);

[0021] The sequence of the platelet-transfected siRNA recognizes the nucleotide substitution G → A in the sequence of the mutant KRAS gene (KRASG12D) and contains a 3'TT overhang to promote silencing efficiency. The central position of the mutant nucleotide increases the specificity of the siRNA, thus preventing silencing of the wild-type KRAS gene and genes with other types of mutations such as KRAS G12C, KRAS G12N, and KRAS G12V.

[0022] The transfection of platelets with siRNA is carried out according to the method described in EP2951292 (WO2014 / 118817) in the name of the applicant. Briefly, platelets isolated from peripheral blood are contacted with siRNA in a medium containing ethyl alcohol and a polyamine selected from polyethyleneimine and polylysine. To obtain microparticles, after transfection, the platelets are activated with a suitable stimulant, for example by adding thrombin in the presence of calcium salts, as described in EP2951292.

[0023] In transfusion medicine, separation of platelets and plasma from a donor allows the platelets to be harvested, transfected, resuspended in plasma, and retransfused. Furthermore, the short time required for platelet transfection allows the transfected platelets to be reinfused intravenously in a single dose into the same individual from whom they were harvested, eliminating the risk of alloimmunization or rejection of the transfected platelets.

[0024] Thus, in a preferred embodiment, a therapeutic treatment according to the invention involves harvesting platelets from a cancer patient or a healthy donor, transfecting them with siRNA, optionally activating them, and reintroducing the transfected platelets and / or microparticles derived therefrom into the patient.

[0025] The dosage, route, and frequency of administration of KRASG12D siRNA-transfected platelets were determined in an animal model of PDAC.

[0026] In addition to the intravenous route of administration used in the in vivo experimental studies, intramuscular, intradermal or subcutaneous routes, and / or in situ administration may be employed for clinical use in humans.

[0027] In the in vivo test, the drug was administered a total of six times over a 14-day period, but in clinical applications, the drug can be administered one to six times.

[0028] The dose used in animal models was approximately 45 million platelets per dose. The human dose was 5 × 10 11 Thus, the dosage range for clinical application is preferably 45×10 6 From 5×10 11 Between transfected platelets.

[0029] The invention is further illustrated in the following examples and accompanying drawings. [Brief description of the drawings]

[0030] [Figure 1] Figure 1 shows the efficiency of inhibition of KRAS G12D by platelets transfected with siRNA targeting KRAS G12D in PANC-1 cells. When platelets transfected with siRNA for KRASG12D were co-cultured with the pancreatic adenocarcinoma cell line PANC-1 in vitro for 24 and 48 hours, intracellular KRASG12D mRNA expression was specifically decreased. Data are shown as the reduced level of KRASG12D in PANC-1 cells compared to untreated PANC-1 cells. Plts-NC: platelets transfected with siRNA Scrambled Negative Control DsiRNA (IDT). Plts-KRAS: platelets transfected with siRNA KRASG12D. [Diagram 2] Tumor mass in vivo-early treatment. Analysis performed after euthanasia of animals; n = 6 mice per group. Control group: human platelets transfected with siRNA Scrambled Negative Control DsiRNA (IDT). K-ras group: human platelets transfected with siRNA KRASG12D. A. Images of each excised tumor. B. Tumor volume. C. Tumor weight. [Diagram 3] Pharmacodynamics: A. Percentage of human platelets in the total platelet population during treatment; n = 6 mice per group. B. Percentage change in mouse body weight during treatment (endpoint); n = 6 mice per group. EXAMPLES

[0031] Materials and Methods Preparation of human platelets with KRASG12D siRNA Peripheral venous blood is collected and collected in a test tube containing an anticoagulant. 1) Centrifuge the sample at 120 g for 10 minutes to obtain PRP (platelet-rich plasma). 2) Platelets are separated from plasma by standard methods (platelet washing or gel filtration). 3) The platelets thus isolated were cultured in RPMI 1640 supplemented with antibiotics (100 U penicillin and 100 U streptomycin) or in donor plasma at a concentration of 2 × 10 5 ~1×10 6 Platelets were resuspended at a concentration of 100 platelets / microliter and 1 milliliter aliquots were transferred to wells of 24-well plates and incubated at 37°C in a controlled atmosphere of 5% CO2. 4) Next, 32.4 microliters of absolute ethyl alcohol, 32 microliters of a polyamine selected from polylysine and polyethyleneimine, and 168 microliters of RPMI1640 are placed in a test tube so that the total volume is 200 microliters, to prepare a transfection medium. 5) After a 5 minute pause, 200 nM concentration of siRNA KRASG12D is added to the mixture and after 15 minutes of incubation at room temperature, this solution is transferred to the well in which 1 ml of platelet suspension has already been introduced. 6) Transfection by centrifugation of platelets (10 min at 1000 g in the presence of 0.2 μM PGI2, then resuspended in 3 ml of RPMI1640 medium) is interrupted after a 5 min incubation at 37°C.

[0032] Real-time PCR After purification of RNA with Trizol (Invitrogen), RNA was reverse transcribed using iScript Reverse Transcription Supermix for RT-qPCR (Bio-rad Laboratories). Quantitative PCR (qPCR) analysis was performed using SYBR Green Master Mix (Applied Biosystems) with the AriaDX Real-time PCR System (Agilent). Relevant transcripts were normalized to RNA 18S transcript levels. Each reaction contained three technical replicates, the average of which was calculated to represent the biological replicates. Experiments were repeated three times on different days, and each experiment was defined as a biological replicate. Statistical analysis was performed on the ΔCt of biological replicates, and the results were expressed as the variation of the relative increase. The primer sequences were as follows: Human KRASG12D: forward 5'-ACTTGTGGTAGTTGGAGCAGA-3' (SEQ ID NO:3), reverse 5'-TTGGATCATTCGTCCACAA-3' (SEQ ID NO:4).

[0033] 18S: forward 5′-GCTTAATTTGACTCAACACGGGA-3′ (SEQ ID NO: 5), reverse 5′-AGCTATCAATCTGTCAATCCTGT-3′ (SEQ ID NO: 6).

[0034] Animal models of pancreatic adenocarcinoma 4-6 week old male NSG mice Cg-Prkdc scid Il2rg tm1Wjl Mice were housed in ventilated cages at 21–23°C and 40–60% humidity with a 12-h light:12-h dark cycle. Mice had free access to food and sterile water. PANC-1 (10 6 The cells were injected subcutaneously using a 27-gauge syringe under general anesthesia. Tumor volume analysis was performed using Living Image version 4.4 (Caliper Life Sciences). Exposure conditions (time, aperture, stage position, binning) were kept the same for all measurements within each experiment. Tumor mass was measured with an electronic balance (BL 224 Touch).

[0035] Mice were heparinized and blood was collected from the tail. Blood (10 μl per mouse) was then diluted with 100 μl of PBS. Samples were then incubated with human CD41-FITC antibody (BD Biosciences) and corresponding controls and analyzed on a Cytoflex flow cytometer (Beckman Coulter). All control samples were analyzed together with the experimental samples. result Platelets transfected with siRNA targeting KRASG12D were cocultured with PANC-1 cells for 24 and 48 h, and the expression levels of KRASG12D mRNA in human PANC-1 cells were assessed by qPCR (Fig. 1 ).

[0036] This was followed by the first set of in vivo efficacy data in mouse models of KRAS-mutated pancreatic cancer.

[0037] Platelets transfected with siRNA for KRASG12D suppressed tumor growth in vivo, whereas platelets transfected with siRNA designed not to recognize the target (siRNA NC) did not.

[0038] NSG mice (NOD scid gamma mice) were treated with 1 × 10 6 Human PANC-1 cells (a PDAC cell line) were injected subcutaneously, followed by intravenous administration of human platelets starting on day 7 (early treatment start).

[0039] 45×10 6human platelets were injected intravenously, a total of six times into each mouse, every 2–3 days from day 7 to day 21. Human platelets were isolated from six healthy donors.

[0040] Tumor burden was quantified at the end of the study. Human platelet counts in the mouse blood and animal body weights were monitored throughout the experiment.

[0041] A reduction in tumor burden was observed in mice injected with platelets transfected with siRNA against KRASG12D compared with mice injected with platelets transfected with siRNA NC, as indicated by the statistically significant reduction in tumor volume and tumor burden shown in Figure 2 .

[0042] Furthermore, no significant variation was observed in the number of human platelets circulating in the blood of mice (measured as percentage of human platelets / total number of platelets in mouse blood), suggesting that the transfected platelets were not different between the two experimental groups and were efficiently circulating in the host (Fig. 3A).

[0043] Finally, no significant differences were observed between the two experimental groups regarding the weight changes of the mice after treatment, confirming that this treatment was safe and did not cause any unwanted side effects (Figure 3B).

Claims

1. A therapeutic composition for use in treating KRASG12D-expressing tumors, comprising platelets transfected with siRNA capable of inhibiting the expression of mRNA encoding a KRAS protein having a G12D mutation (KRASG12D).

2. The composition for use according to claim 1, wherein the KRASG12D-expressing tumor is pancreatic adenocarcinoma.

3. A composition for use as described in claim 1, wherein the KRASG12D-expressing tumor is pancreatic duodenal adenocarcinoma.

4. The composition for use according to claim 1 or 2, wherein the siRNA targets a KRASG12D mRNA sequence containing a mutant codon.

5. The composition for use according to claim 1 or 2, wherein the siRNA consists of 15 to 30 base pairs.

6. the siRNA comprises a sense strand and an antisense strand; (i) the sense strand consists of or comprises the sequence 5'-GUUGGAGCUGAUGGCGUAGTT-3' (SEQ ID NO:1), and (ii) the antisense strand consists of or comprises the sequence 5'-CUACGCCAUCAGCUCCAACTT-3' (SEQ ID NO:2); A composition for use according to claim 1 or 2.

7. 3. The composition for use according to claim 1, wherein the platelets are obtained by a transfection method comprising contacting platelets isolated from peripheral blood with siRNA in a transfection medium comprising ethyl alcohol and a polyamine selected from polyethyleneimine and polylysine.

8. The composition for use according to claim 7, wherein the platelets are activated to produce microparticles.

9. 3. The composition for use according to claim 1 or 2, wherein the tumor treatment comprises (i) collecting platelets from a tumor patient or donor, (ii) transfecting the platelets with siRNA, and (iii) (re)introducing the platelets into the patient.

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