EP4680739A1

EP4680739A1 - Selective delivery of onco-suppressive mirna (mir126) to metastatic melanoma cells resistant to therapies

Info

Publication number: EP4680739A1
Application number: EP24712594.1A
Authority: EP
Inventors: Nadia Felli; Federica FELICETTI; Mauro Biffoni; Michela Flego; Maria Beatrice ARASI; Cleofe Palocci
Original assignee: Istituto Superiore di Sanita ISS; Universita degli Studi di Roma La Sapienza
Current assignee: Istituto Superiore di Sanita ISS; Universita degli Studi di Roma La Sapienza
Priority date: 2023-03-14
Filing date: 2024-03-14
Publication date: 2026-01-21
Also published as: WO2024189572A1; JP2026509509A

Abstract

The present invention discloses carriers with an onco-suppressive agent and selectively directed to a tumoral target for the treatment of metastatic melanoma resistant to a target therapy.

Description

“Selective delivery of onco-suppressive miRNA (miR126) to metastatic melanoma cells resistant to therapies”

DESCRIPTION

Melanoma is a cancer that originates from the malignant transformation of melanocytes, pigmented cells in the epidermis (Situm M et al, 2014). According to World Health Organization (WHO), there are an estimated 300,000 new cases and 60,000 deaths worldwide each year. Melanoma is a rapidly increasing and aggressive cancer, with a 75% increase in the last 30 years (Saginala K et al, 2021 ). The incidence and mortality vary among countries, with high rates in Australia, New Zealand, Europe, and North America (Bolick NL et al., 2020). Recent research has focused on improving therapeutic strategies for advanced melanoma, with the use of immunological checkpoint inhibitors showing promise (Guo W et al., 2021 ). Effective treatment for metastatic melanoma has also been achieved through targeting specific molecular pathways (Ostrowski SM et al, 2020). However, new biomarkers and drug targets are needed to improve diagnosis and treatment (Davis LE et al, 2019).

Molecular analysis during melanoma development has shown that over 50% of melanomas have activating mutations in the BRAF gene, with the most common mutation being a substitution of valine for glutamic acid at codon 600 (V600E). Drugs such as vemurafenib and dabrafenib can selectively inhibit the mutated BRAF protein (Wellbrock C et al, 2016). The clinical use of these drugs have shown promising results, but early recurrence and resistance to treatment remains a limitation. To overcome this, new therapies focused on combining multiple compounds to inhibit various pathways crucial to cancer cell survival have been developed (Long GV et al, 2014). On these bases, the simultaneous inhibition of the mitogen-activated protein kinase (MAPK/ERK) and phosphatidylinositol-3-kinase-protein/AKT (PI3K/AKT) pathways represents a valid therapeutic strategy. PIK-75, an inhibitor of the PI3K/AKT pathway, has been identified as a potential drug for combined therapy with vemurafenib or dabrafenib to inhibit both pathways and overcome resistance (Pedini F et al, 2019).

Non-coding RNAs, such as microRNAs (miRs), play important roles in various cellular processes. MiR126, which has been shown to act as a tumor suppressor in several types of cancer, has been demonstrated to inhibit the growth and spread of metastatic melanoma by regulating the expression of oncogenic molecules (Felli N et al, 2013), particularly the PI3K regulatory subunit p850 (Guo C et al, 2008). MiR126 has been shown to suppress tumor growth in metastatic melanoma and its potential to enhance other treatments was evaluated. This includes combining it with PIK-75 and vemurafenib or dabrafenib. The enforced expression of miR126 reduces tumor growth and enhances the effectiveness of PIK-75 alone or in combination with vemurafenib or dabrafenib and it has also been effective against early passage cell lines derived from patient’s biopsies and on melanoma cell lines resistant to either vemurafenib or dabrafenib, suggesting that it has the potential to overcome drug resistance (Pedini F et al, 2019).

The use of nucleic acids (including miRs) in therapy involves the need to overcome some difficulties, especially related to their rapid degradation or elimination. An effective strategy in these critical issues is the development of cancer nanomedicine, which is the practical application of nanotechnology in cancer therapy. Nanoparticles (NPs), thanks to their specific features, including sustained and controlled drug release, excellent availability and biocompatibility, ability to maintain in the human body their physico-chemical properties over a long period of time, have been considered as efficient candidates for cancer therapy (Ojha A et al, 2022; Arasi MB et al. 2020). More generally the traditional use of nanotechnology in cancer therapeutics has been to improve the pharmacokinetics and reduce the systemic toxicities of chemotherapies through the selective targeting and delivery of these anticancer drugs to tumor tissues.

Chitosan based nanoparticles (CSs) have been identified as a promising candidate for drug nucleic acids delivery due to their positive charge in acid environment, biocompatibility and stability (Dahlman JE et al, 2014; Sargazi S et al, 2022).

Summary of the invention

The inventors of the present application have developed a new therapeutic agent for the treatment of tumors and particularly metastatic melanoma resistant to therapies. In particular, they have surprisingly found how to deliver a mature miR-126-3p directly to tumor cells. The system is based on the use of particles functionalized with a targeting moiety against a specific tumor marker. These nanoparticles have proven to be extremely effective in the treatment of metastatic melanoma resistant to targeted therapy in preclinical models. Brief description of the figures

Figure 1. Chemical modification in the OMe-miR126 do not change its functionality making it more stable in human plasma. A: Chemical modifications to the canonical sequence of miR126-3p: OMe-miR126; B (left panel ) Immunoblotting analysis: comparable down-modulation of target protein (p850) by OMe-miR126 and commercial miR126-3p sequence (Dharm-miR126); B (right panel) Immunoblotting analysis: time-course evaluation of p85-0 targeting by OMe-miR126; C: OMe-miR126 and Dharm-miR126 sequences stability in PBS containing 50% human plasma at 37 °C; D: Cell growth inhibition after OMe-miR-126 over-expression. The experiments reported in B and D are performed using A375M-VR cell line.

Figure 2. Synergistic combination of OMe-miR126 sequence and BRAF or PI3K inhibitors.

A. A375M growth inhibition mediated by OMe-miR126 or Dharm-miR126-3 sequences, compared to OMe- CTRL, in combination with vemurafenib (upper, left panel), dabrafenib (upper, middle panel), PIK-75 (upper, right panel), vemurafenib+PIK-75 (bottom, left panel) and dabrafenib+PIK-75 (bottom, right panel) B A375M- DR growth inhibition mediated by OMe-miR126 or Dharm-miR126-3 sequences, compared to OMe-CTRL, in combination with PIK-75. IC50 values are reported for each treatment.

Figure 3. Synergistic combination of 0Me-miR126 sequence and BRAF or PI3K inhibitors. The Excess Over Bliss (EOB) score. The values obtained indicated that OMe-miR126 is synergistic with single drug (vemurafenib, dabrafenib or PIK-75) or with their combination (vemurafenib + PIK-75 or dabrafenib + PIK-75) in A375M WT and A375M-DR cell lines.

Figure 4. Synthesis and analysis of single chain antibody 9.2.27 (scFv-9.2.27). A: Schematic representation and western blot of purified scFv-9.2.27. B Flow cytometry analysis in A375M-DR shows that scFv 9.2.27 can selectively interact with melanoma cell surfaces; C No toxic effect was observed up to 1 pg/ml of scFv-9.2.27 treatment on A375M-DR.

Figura 5. Synthesis and characterization of nanoparticles. A: Schematic diagram of the scFv-CS126 nanoparticles synthesis (left); different scFv:CS weight ratios have been studied and scFv:CS; weight ratio of 1 :10 was selected for Ab-CS126 synthesis (right). B: The average (^-potential value was +37,8 ± 4,3 mV for CS126s and it was +33,3 ± 1 ,2 mV for Ab-CS126s. C: NanoSight evaluation of size distribution and concentration of nanoparticles.

Figure 6. Characterization of CS126 and Ab-CS126 nanoparticles. A: Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) of CS126 and Ab-CS126; B: WES analysis of bound scFv-9.2.27 allowed us to estimate an amount of 250 pg of scFv/6.8X10^A7 Ab-CS126; C: The integrity of the OMe-miR126 sequences entrapped in CS126s in 50% of human plasma was demonstrated until 48 hours of incubation.

Figure 7. Evaluation of internalization of CSs and Ab-CSs in A375M-DR cells. A: Fluorescent microscopy visualization (left) and FACS analysis shows that after 20 min. of incubation with 1 ,6* 10⁸ particles about 100% of the cells were FITC positive; B: qRT-PCR reveals that CS126 and Ab-CS126 intrapped quantity of OMe-miR126 is 1 .2x10^-8 and 1 .7x10^-10 pMole/nanoparticles respectively; C: qRT-PCR analysis of OMe- miR126 transduction in A375M-DR cell line after being exposed to the same number of the different types of nanoparticles; D: Delivering efficiency (DE) of Ab-CS126 is approximately 9.5 times higer compared to CS126.

Figure 8. CS-FITCs and Ab-CS-FITCs stability: in vitro and in vivo experiments. A: In vivo plasma analysis of mice injected with fluorescent nanoparticles showed fluorescence below detection limit after 60 minutes (upper panel). In vitro experiments showed CSFITC and Ab-CSFITC fluorescence retention for up to 6 days (lower panel). B: Analysis of nanoparticles distribution showed that a significant portion of FITC signal is present in lung, liver, kidney and spleen of injected mice. Figure 9. Evaluation of tumor suppressor role played by nanoparticles contained OMe-126 in in vivo experiments. A: Schematic animal model. B: Statistical analysis of tumor growth C: Fluorescent I VIS images of one representative mouse of six treatment arms at different times. In the right panel, liver and lung from each mouse are imaged individually in the I VIS Spectrum.

Figure 10. Apoptotis detection in liver and lung mice cryopreserved tissue sections. A. Tunel Assay to detect apopotosis was performed on liver and lung cryopreserved tissue sections of NSG mice. As shown, it has been observed a very intense fluorescent signal in mice injected with A357M-DR cell lines, treated with dabrafenib, PIK-75 and Ab-CS126, compared to the control ones, due to advanced apoptosis specifically induced in the tumor infiltrated compartment by the combined treatment. B Immunostaining with anti-scFv- 9.2.27 and relative negative control of mouse kidney sections treated with CS126 or Ab-CS126 was performed to verify the distribution of Ab-CS126 in the animal's organs.

Figure 11 . CSPG4 expression in ovarian (SK-OV3 and A2780) and lung (H1975 and HCC827) cancer cell lines.

Figure 12. Synergistic combination of OMe-miR126 sequence and PI3K inhibitors in ovarian cancer cell line SK-OV3.

Object of the invention

In a first object, the present invention describes a double strand RNA compriseing the mature onco- suppressive miR-126-3p sequence.

In a second object, the present invention discloses nanocarrier systems with the double strand RNA containing the mature onco-suppressive miR-126-3p sequence.

In a particular embodiment, the nanocarrier systems are linked to a targeting moiety.

In a third object, the invention double strand RNA, the invention mature onco-suppressive miR-126- 3p sequence and the invention nanocarrier systems are disclosed for the medical use.

In a preferred aspect, the medical use is disclosed for the treatment of tumors expressing the tumor marker CSPG4.

In more preferred aspect, the medical use is disclosed in the treatment of tumors comprising: melanoma, ovarian cancer and lung cancer.

In a even more preferred aspect, the medical use is disclosed in the treatment of melanoma.

In a still more preferred aspect, the nanocarrier systems of the invention are disclosed for the medical use in the treatment of metastatic melanoma resistant to targeted therapy. The invention double strand RNA, the invention mature onco-suppressive miR-126-3p sequence and the invention nanocarrier systems are disclosed for the medical use in the treatment of tumors in combination with PI3K/AKT inhibitors.

In particular, the invention double strand RNA, the invention mature onco-suppressive miR-126-3p sequence and the invention nanocarrier systems are disclosed for the medical use in the treatment of metastatic melanoma resistant to targeted therapy in combination with PI3K/AKT inhibitors. Detailed description of the invention

According to a first object, the present invention describes a double strand RNA comprising a mature onco-suppressive miRNA sequence.

For the purposes of the present invention, the doble-strand miRNA comprises the mature onco- suppressive miR-126-3p sequence.

More in particular, said double strand RNA is represented by a double stranded oligonucleotide characterized by the sense strand sequence corresponding to SEQ. ID. No. 6 and by the anti-sense strand sequence corresponding to SEQ ID. No. 7 as reported below:

According to a preferred embodiment, the invention double strand RNA containing the mature oncosuppressive miR-126-3p sequence is chemically modified.

In particular, all the pyrimidine residues of the sense strand and one of the uridine residue in the antisense strand are modified by O-methylation at the 2’-O pentose (2’-0Me) (see Figure 1 A).

More in particular, in the anti-sense strand, the O-methylation is at the fourth uridine residue, corresponding to the fourteenth nucleidic residue.

Such a modification has the effect of improving the thermal stability.

In addition, both the sense strand and the anti-sense strand have been further modified with the addition of two 2’-deoxythymidine residues at their respective 3’-terminal of each strand.

Such a modification has the effect to protect the molecule from nuclease cleavage.

The active onco-suppressive molecule is finally represented by the canonical miR-126-3p in anti-sense strand having the following structure: According to a second object, the present invention discloses nanocarrier systems with the double strand RNA containing the mature onco-suppressive miR-126-3p sequence.

According to an embodiment of the present invention, the nanocarrier systems of the invention are targeted to tumoral cells.

For said purposes, the nanocarrier systems are linked to a targeting moiety.

According to an embodiment, the nanocarrier system may be represented by nanoparticles loaded with the double strand RNA containing the mature onco-suppressive miR-126-3p sequence.

In a particular embodiment, the nanoparticles are represented by chitosan nanoparticles.

For said purposes, the chitosan nanoparticles can be prepared by mixing a solution of chitosan with a solution comprising a double strand RNA containing the mature onco-suppressive miR-126-3p sequence.

Preferably, the solution of chitosan has a concentration of 1 mg/ml.

According to an embodiment of the present invention, the chitosan may have a molecular weight of 30-40 KDa.

Preferably, the double strand RNA is diluted in a solution of distilled water.

The two solutions are preferably mixed in a 1 :1 volume ratio.

After mixing, the mixture is left under constant agitation for about 15 minutes and then left at room temperature to allow the spontaneous formation of the nanoparticles (NPs).

Once formed, the chitosan nanoparticles are preferably stored at from about -25°C to about -15°C.

For the purposes of the present invention, the chitosan nanoparticles are characterized by a size within the range of 130 nm to 160 nm

As said above, the chitosan nanoparticles are loaded with a double strand RNA containing a mature onco-suppressive miRNA.

According to a preferred embodiment, said double strand RNA comprises the mature onco- suppressive miR-126-3p sequence as above disclosed.

According to another embodiment of the invention, the nanocarrier systems are represented by lipid based vesicles represented by liposomes (LPNs).

Lipid based vesicles can be loaded with the mature onco-suppressive miR-126-3p sequence above disclosed or formulated in multiple lipidic compositions able to complex nucleic acids such as the mature onco- suppressive miR-126-3p sequence above disclosed. According to an embodiment of the invention, the disclosed nanocarrier systems with a double strand RNA containing the mature onco-suppressive miR-126-3p are targeted to tumoral cells.

In an embodiment, said tumoral cells are selected from melanoma, ovarian cancer and lung cancer cells.

In a preferred embodiment, said tumoral cells are represented by metastatic melanoma cells.

In particular, the nanocarrier systems of the invention can be suitably targeted by conjugation with a targeting moiety.

More in particular, said targeting moiety is covalently linked to the nanocarrier system.

For the purposes of the present invention, the targeting moiety is directed towards the tumoral marker CSPG4 (Ab-CS126).

In a preferred aspect, the targeting moiety is represented by an antibody or an antibody fragment.

For the purposes of the present invention, the antibody may be selected from the group comprising: be a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a multispecific antibody.

For the purposes of the present invention, an antibody fragment may be selected from the group comprising: Fv, Fab, F(ab’)2, Fab’, dsFv, scFv or sc(Fv)2.

In a preferred embodiment, the targeting moiety is represented by scFv.

In an even preferred embodiment, the targeting moiety is represented by the scFv-9.2.27.

In an aspect of the present invention, the disclosed scFv-9.2.27 comprises the aminoacidic VH sequence of SEQ. ID. No. 3 (see the table below).

In an aspect of the present invention, the disclosed scFv-9.2.27 comprises the VH sequence encoded by the nucleotidic sequence of SEQ. ID. No. 2 (see the table below).

In an aspect of the present invention, the disclosed scFv-9.2.27 comprises the aminoacidic VL sequence of SEQ. ID. No. 5 (see the table below).

In an aspect of the present invention, the disclosed scFv-9.2.27 comprises the VL sequence encoded by the nucleotidic sequence of SEQ. ID. No. 4 (see the table below).

The conjugation of the nanocarrier systems can be carried out according to methods known in the art. For instance, the conjugation of the chitosan nanoparticles can be carried out according to methods known in the art, such as for instance using the EDC chemistry.

According to a third object, the present invention discloses the invention double strand RNA, the invention mature onco-suppressive miR-126-3p sequence and the invention carrier systems for the medical use.

In particular, said medical use is disclosed for the loaded chitosan nanoparticles and by the loaded and targeted chitosan nanoparticles above disclosed.

In an embodiment, the invention double strand RNA, the invention mature onco-suppressive miR-126- 3p sequence, the invention nanocarrier systems, the loaded nanocarrier systems, the loaded and targeted nanocarrier systems, particularly the chitosan nanoparticles and the loaded and targeted chitosan nanoparticles above disclosed are disclosed in the treatment of tumors expressing the tumor marker CSPG4.

In a preferred embodiment, the medical use is disclosed in the treatment of tumors comprising: melanoma, ovarian cancer and lung cancer.

In a preferred aspect, the invention double strand RNA, the invention mature onco-suppressive miR- 126-3p sequence, the invention nanocarrier systems, the loaded nanocarrier systems, the loaded and targeted nanocarrier systems, particularly the chitosan nanoparticles and the loaded and targeted chitosan nanoparticles above disclosed are disclosed for the medical use in the treatment of melanoma.

In a more preferred aspect, the chitosan nanoparticles of the invention are disclosed for the medical use in the treatment of metastatic melanoma resistant to targeted therapy (precision therapy).

With the term “targeted therapy” or “precision therapy” reference is made to the therapeutic treatment directed to the specific target, ie.e BRAFV^600E. In particular, the nanocarrier systems and particularly the chitosan nanoparticles of the invention are disclosed for the medical use in the treatment of metastatic melanoma resistant to the treatment with vemurafenib or dabrafenib.

In a particular aspect, the nanocarrier systems and particularly the chitosan nanoparticles of the invention are disclosed for the medical use in the treatment of metastatic melanoma in combination with

PI3K/AKT inhibitors.

In a preferred aspect, such PI3K/AKT inhibitor is represented by PIK-75.

The hydrochloride salt (CAS 372196-77-5) is here below reported:

The present invention is further disclosed in the following Experimental Section.

Chemical modifications of miR126-3p and stability in human plasma

All the pyrimidines in the sense strand and one of the uridines in the antisense strand of the miR126 of the invention have been modified by adding a methyl group (CH3) at the 2'-0 position of the pentose (2'-0Me). In addition, two 2’-deoxythymidine nucleotide overhangs at their respective 3’-ends have been added to protect this molecule from nuclease cleavage (OMe-miR126) (Figure 1A). The same modifications have been made to negative control sequence (OMe-CTRL) (not shown).

To verify the functional efficiency of OMe-miR126 sequence, it has been evaluated its ability to inhibit the expression of PI3KR2 (p850) subunit of PI3K, a key molecule in PI3K/AKT signalling pathway, reported as a direct target of miR126-3p (Guo C, 2008; Felli N, 2013). Specifically, A375M-VR cell line was transfected with OMe-miR126 or relative negative control whereas commercial miR126 sequence, Dharm-miR126-3p (Invitrogen) and Dharm-CTRL have been used as reference (Felli N, 2013). Immunoblotting analysis showed a comparable reduction of p850 levels (50-40%) (Figure 1 B, left panel). A time-course evaluation has shown that the inhibitory effect of 0Me-miR126 is still very strong 72 hours after transfection (Figure 1 B, right panel). Another important issue to be addressed before choosing the sequence for in vivo experiments, was the RNA molecules stability in plasma. To this end, OMe-miR126 and Dharm-miR126 sequences in PBS containing 50% human plasma at 37°C have been compared. The results showed that OMe-miR126 degradation was slower than Dharm-miR126. Specifically, the time course reported in Figure 1 C shows that, starting from the same quantity of OMe-miR126 and Dharma-miR126 oligos, no reduction and a reduction to one-third is observed, respectively, after 6 hours of incubation. Furthermore, a complete degradation of Dharm-miR126 was observed after 24 hours whereas OMe-miR126 was still clearly visible (~80 % of the initial quantity). To verify the lifetime of OMe-miR126, the incubation has been extended to 48 hours and observed its complete disappearance (not shown). Based on the evidence of efficacy in reducing target expression and stability in plasma, efforts have been focused on OMe-miR126 in further experiments. A transient transfection of A375M- VR with this oligo has been performed and a significant cell growth inhibition after OMe-miR126 overexpression (Figure 1 D) has been observed.

The results described so far show that the chemical changes introduced in miR126-3p canonical sequence to obtain OMe-miR126 does not change its functionality and make it much more stable in the presence of plasma enzymes.

OMe-miR126 sequence in combination with BRAF inhibitors

It has been evaluated the cell growth inhibitory effect of OMe-miR126 sequence on metastatic melanoma cell line (A375M) and its derived dabrafenib resistant sub-line (A375M-DR), generated by exposing parental cells to increasing concentrations of the drug.

As shown in Figure 2A, the ICso values of vemurafenib (704.7 nM), dabrafenib (41 nM) and PIK-75 (73,5 nM) were reduced by half when the treatments were performed in combination with OMe-miR126 (329.6 nM, 22.3 nM and 40.3 nM respectively), Dharm-miR126 was used as positive reference, since reproduce a sequence for which the synergy effect had already been demonstrated (Pedini F, 2019). Also in drugs combination (vemurafenib + PIK-75 or dabrafenib + PIK-75), ICso values were reduced in association with OMe-miR126 (see Figure 2A upper panel).

Since our aim was to evaluate a therapeutic strategy to treat metastatic melanoma resistant to the standard therapy with BRAF inhibitors, the inhibitory effect of OMe-miR126 and its potential synergistic role with PIK-75 on A375M-DR have been evaluated. As reported in Figure 2B, the ICso of PIK-75 decreased from 153.3 nM to 120.5 nM. To calculate the potential synergistic effect, the Excess Over Bliss (EOB) score (Liu Q et al. 2018) has been used. This parameter is used to describe the phenomenon where the combined effects of two or more drugs are greater than the sum of their individual effects (EOB>0 synergism, EOB=0 indipendent, EOB<0 antagonism). The values obtained indicated that OMe-miR126 is synergistic with single drug (vemurafenib, dabrafenib or PIK-75) or with their combination (vemurafenib + PIK-75 or dabrafenib + PIK-75) (Figure 3).

The same experimental design was replicated using another melanoma cell line, SKMEL28, and its dabrafenib- resistant counterpart, SKMEL28-DR with similar results (data not shown).

The results described demonstrate that OMe-miR126 synergizes with BRAF and PI3K inhibitors and their combination in inhibiting the growth of both sensitive and resistant metastatic melanoma cell lines.

Synthesis and analysis of single chain variable Fragment 9.2.27 (scFv-9.2.27)

Chitosan nanoparticles have been used for OMe-miR126 direct delivering within metastatic melanoma cells. In particular, chitosan based nanoparticles (CSs) have been conjugated with a scFv designed with the variable heavy chain (VH), and the variable light chain (VL) of the murine mAb 9.2.27 specific for the melanoma marker CSPG4. The scFv-9.2.27 was produced and purified according to previous publish data (Flego et al., 2021 ; Ascione et al. 2019). The purified scFv-9.2.27 were detected in a Western-Blot experiment with HRP- conjugated anti-6His mAb (Figure 4A). To evaluate the binding capacity of scFv-9.2.27 towards its target antigen, an immunofluorescence assay was performed by flow cytometry on A375M melanoma and 293FT cell lines, expressing high and low levels of surface antigen CSPG4, respectively. The results obtained showed that this antibody can selectively interact with CSPG4 membran receptor (Figure 4B). To evaluate a direct toxic effect of the scFv-9.2.27 protein, in vitro treatments of A375M-DR have been performed with increasing quantity of this soluble protein. As shown in Figure 4C, no toxic effect was observed up to 1 pg/ml.

The data described above, demonstrates that scFv-9.2.27 is able to effectively and specifically recognize the melanoma specific marker CSPG4. Furthermore, it has no effect on the growth of A375M-DR up to a concentration of 1 pg/ml.

Synthesis and characterization of nanoparticles

Synthesis of antibody-conjugated nanoparticles to direct OMe-miR126 delivering

CSs preparation, OMe-miR126 entrapping within CSs to obtain CS@miR126s and eventually scFv- 9.2.27@CSs conjugation to obtain the complex scFv-9.2.27@CSs@126s (hereafter related as Ab-CS126s), were performed as reported in Figure 5A (left panel). In brief, chitosan nanoparticles were prepared by polyelectrolyte complexation method, mixing two solutions of oppositely charged polymers, in a 1 :1 v/v ratio. Net charge and size of polyplexes are mainly determined by the N/P ratio and by increasing the N/P ratio from 5 to 10 (at fixed CS deacetylation degree) and a lower molecular weight was required to achieve efficient complexes stabilization. 1 mg/mL chitosan solution was prepared by dissolving the polymer in 0,1 M aqueous solution of acetic acid (pH=4). Then, 80 pg of oligonucleotide were diluted with an appropriate volume of sterile double-distilled water to obtain an N/P ratio of 10. The nucleic acid solution was added drop-wise to 500 pL of the chitosan solution under magnetic stirring. The mixture was kept under constant agitation for 15 minutes, left at RT for 30 minutes to allow the spontaneous formation of nanoparticles and then stored for 24 h at 4°C before use.

Entrapment efficiency of loaded nucleic acid

The amount of unbound nucleic acid was determined by an UV-Vis spectrophotometer. Samples were centrifuged at 14000 rpm and 4°C, for 30 minutes, to facilitate the precipitation of polyplexes and supernatants were analyzed spectrophotometrically. An entrapment efficiency of 70 % ±9.3 was achieved according to the following formula,

E%= (Al- Af)/AI x 100

Ai = initial amount of nucleic acid added to the solution (pg)

Af = final amount of nucleic acid in the supernatant (pg)

EDC (1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide)/NHS(N-Hydroxysuccinimide) chemistry was used to conjugate chitosan nanoparticles with the antibody fragment. 500 pg of chitosan NPs were precipitated by centrifugation (14000 rpm, 30 minutes, 4°C). The pellet was suspended in 500 pl of PBS (pH=7.4). Subsequently, 50 pg of antibody were diluted in an equal volume of MES buffer (pH=6). EDC and NHS in 2:5 molar ratio, were added to the reaction mixture and kept under magnetic stirring for 15 minutes. After adjusting the pH to 7, the NPs solution was added, incubating the reaction mixture at room temperature for 2 hours under continuous magnetic stirring. The solution was then transferred into a dialysis membrane and finally placed in a beaker containing sterile distilled water. Dialysis was allowed for 5 days. The amount of bound antibody was evaluated by means of spectrophotometric measurements. The total amount of bound antibody was higher than 80 % with Ab/CS weight ratio of 1 :10 (Figure 5A, right panel).

Characterization of nanoparticles

First, the dimensional distribution and polydispersion index (Pdl) of CS126s and Ab-CS126 were measured with DLS (Figure 5B). A very slight difference between the dimensional values has been observed, due to the small size of the antibody used. This is in fact a protein fragment, with a molecular weight of ~30 kDa and a relatively low steric envelope. The average Pdl for all nanoparticle samples was 0,2. The low Pdl indicates the good quality of the systems.

Chitosan nanoparticles are generally characterized by a positive surface charge, due to the presence of amino groups positively charged at acidic pH. The positive charge is also optimal for the internalization of nanovectors in cells, as cellular membranes exhibit a negative charge (Dowaidar M, 2018; Faust JJ, 2014). As shown in Figure 5B, the average (^-potential value was +37,8 ± 4,3 mV for CS126s and it was +33,3 ± 1 ,2 mV for Ab- CS126s. CS-126s and Ab-CS126s have been characterized for their size distribution by NanoSight analysis, confirming the DLS data and revealing an average concentration of CS-126s and Ab-CS126s preparation corresponding to 2,2x10¹¹ particles/ml and 1 ,7x10¹⁰ particles/ml, respectively (Figure 5C and data not shown). The experimental data above described demonstrates that the used conditions for synthesizing nanoparticles are able to efficiently entrap OMe-miR126 molecules and to show on theirs surfaces the Ab molecules useful for melanoma cell targeting.

Physicochemical characterization of CS126 and Ab-CS126 nanoparticles

The chitosan nanoparticles morphology has been studied by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). From SEM/TEM analyses, polyplexes appeared as highly aggregated globular nanostructures (Figure 6A). After nanoparticles conjugation with antibody fragment, nanoparticles have a greater dispersion capacity in an aqueous environment, showing a spherical shape. Since SEM/TEM analyses are carried out on dried samples, micrographs showed usually NPs dimensions lower than those obtained from analysis in aqueous solution. Under these conditions, the nanoparticles are strongly hydrated and for this reason dimensions detected by DLS are larger. For better characterization of our nanocarriers, the quantity of scFv-9.2.27 bound to Ab-CS126 has been evaluated by WES analysis (Figure 6B). A direct comparison with the positive control (soluble scFv-9.2.27) allows to estimate an amount of 250 pg of scFv-9.2.27 /6.8X10⁷ Ab-CS126. As the last step, before performing functional experiments, the stability of CS126 in human plasma has been evaluated. Figure 6C shows that the CS nanoparticles extend the stability of OMe-miR126 after the 48 hours of incubation at which time-point it has been observed a complete disappearance of the sequence, as indicated by gel electrophoresis.

The data reported demonstrates that both CS-126s and Ab-CS126s have spherical morphological features. The scFv-9.2.27 is conjugated to the Ab-CS126s and the entrapped OMe-miR126 has improved stability in a 50% serum medium compared to the free oligo.

In vitro internalization of CS126 and Ab-CS126

To assess the internalization efficiency of CS126 and Ab-CS126, melanoma cell line A375M-DR has been incubated with increasing amounts of particles conjugated with a FITC molecule. As reported in Figure 7A (left panel), immunofluorescence microscopy indicates substantial internalization of the FITC signal in both CS126 and Ab-CS126 cell incubation. However, while CS126 showed a dispersed distribution with free aggregate and peripheral staining of the cells, AbCS126 stain diffusely the cells suggesting the internalization of the complex. Flow cytometric analysis showed that after 20 minutes of incubation with 1 ,6 X 10⁸ particles, approximately 100% of the cells were FITC positive (Figure 7A, right panel). To evaluate the real ability to transfer OMe-miR-126 within cells, the amount of OMe-miR-126 entrapped within the CS126 and Ab-CS126 has been measured by qRT-PCR, obtaining 1 .2x10 ⁸ and 1.7x10’¹⁰ pMole/nanoparticles respectively (Figure 7B). Then, the amount of OMe-miR-126 after A375M-DR incubation with the same number of CS126 and Ab- CS126 nanoparticles has been evaluated by qRT-PCR, and it has been found a ~56-fold and ~7.3-fold increase for CS126 and Ab-CS126, respectively (Figure 7C). Considering the different amount of miR126- molecules entrapped in CS126 compared with Ab-CS126, it is concluded that Ab-CS126s have an uptake efficiency 9.5 times higher than CS126 on the acceptor A375M-DR melanoma (Figure 7D). The same experiments were performed on SKMEL28 cell line (data not shown).

The data above reported indicate that both CS126 and Ab-CS126 nanoparticles improve the internalization efficiency.

Evaluation of CSFITCs and Ab-CSFITCs stability: in vitro and in vivo experiments

In vivo stability kinetics of CSFITC and Ab-CSFITC has been evaluated by analysis of mice plasma withdrawn at different times after injection of fluorescent particles and it has been observed a rapid disappearance of fluorescent particles that was under the limit of detection after 60 minutes with both CSFITC or Ab-CSFITC (Figure 8A, upper panel). Conversely, in in vitro experiments, CSFITC and Ab-CSFITC maintain their fluorescence until six days of incubation in plasma, with more stability showed by Ab-CS FITC (Figure 8A, bottom panel), suggesting that the disappearance of in vivo fluorescence is due to distribution/elimination of the particles and not to degradation. To clarify this important aspect, the main organs of CSFITC and Ab- CSFITC injected mice have been analyzed by immunofluorescence microscopy. As shown in Figure 8B a strong FITC signal is present in lung, liver, kidney and spleen. As a final step before starting in vivo treatments, the potential toxic effect of CS126 and Ab-CS126 has been evaluated after three weeks of repeated administration to mice. No toxic effect was observed, as indicated by the maintenance of normal weight and health of the treated mice (data not shown).

The above reported data finally indicate that CSFITC and Ab-CSFITC, when in vivo administered, do not undergo degradation mediated by plasma enzymes and distribute in the animal's organs including liver and lungs, which are extremely relevant as frequent localization of melanoma metastases.

In vivo experiments

As it is not possible to produce metastases from melanoma cells resistant to dabrafenib treatment (A375M- DR) through intravenous injection, a reliable animal model has been developed that allows to measure the spread of metastatic tumors over time. This has been obtained by injecting A375M-DR-LucGFP cells into the spleens of 5-week-old female severely immunodeficient NSG mice, using a dose of 5x10⁴ cells in 200 pL of PBS (Figure 9A). To avoid a predominant growth of tumors in the injection site, spleen was removed after 30 minutes. The progression of metastases was monitored by bioluminescence imaging of the inoculated cells, using the I VIS optical imaging system, on a weekly basis. About two weeks after inoculation, when the signal was barely detectable, the mice were randomly allocated to one of six treatment arms:

1 ) Control Group (nine mice); 2) PIK-75 (six mice); 3) Ab-CS126 (six mice); 4) PIK-75 + CS126 (four mice); 5) PIK-75 + Ab-CS (-) (five mice); 6) PIK-75 + Ab-CS126 (eight mice). All the treatments group received dabrafenib intraperitoneally three times a week, PIK-75 was given daily, and Ab-CS126s were given intravenously three times a week.

The statistical analysis, which was based on the comparison of the slopes of the tumor growth curves for each group, showed a significant inhibition in the group treated with PIK-75 + Ab-CS126 compared to the other groups (Figure 9B). After the animals were sacrified, the organ analysis confirmed a lower tumor growth in the liver and a very strong reduction of lung metastases formation. Figure 9C shows representative images for each group.

Overall, these findings indicate that Ab-CS126 in combination with PI3K/AKT inhibitors could be a promising treatment option for patients who have become resistant to conventional therapy.

Apoptotis Detection in Liver and Lung mice cryopreserved tissue sections

To assess the effectiveness of combination PIK-75 + Ab-CS126, a Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay has been performed on liver and lung sections of NSG-treated mice and compared the results with those from the control group. The control groups consisted of tissues obtained from untreated healthy animals and mice treated with the PIK-75 + Ab-CS126 combination but not injected with A375M-DR cells. As shown in Figure 10, there is a strong positive signal in both liver and lung sections of the NSG-treated mice, and no apoptotic signals were observed in the control mice. It is interesting to note that the lungs of mice treated with PIK-75 + Ab-CS126 showed no visible metastases on In vivo Imaging System (I VIS) (Figure 9C, right panel), but were strongly positive for TUNEL staining that indicates apoptosis. This suggests that most of the cells that invaded the lung underwent death after treatment with PIK-75 + Ab- CS126. To verify the actual presence of the nanoparticles in mouse tissue, staining has been performed using an antibody capable of recognizing scFv-9.2.27, as shown in Figure 10B, the kidney section of a mouse treated with anti-scFv-9.2.27 was positive for the staining.

These results strongly support that metastatic melanoma cells in both liver and lung are reached and killed by the treatment with modified miR126 incapsulated in CS nanoparticles bearing anti-CPSG4 antibody.

Application to tumor expressing CSPG4

Preliminary data (see Figure 1 1 indicate elevated levels of the membrane marker CSPG4 in the ovarian (A2780 and SK-OV3) and lung (H1975 and HCC827) tumor cell lines. Furthermore, the lipofectamine-mediated transduction of the OMe-miR126 sequence into SK-OV3 line has shown a synergistic role of this sequence with inhibitors of the PI3K/AKT pathway (PIK-75 and BKM120), consistent with previous observations in melanoma (see Figure 12).

Liposome as carrier systems

LPNs in the range 100-150 nm can be formulated by microfluidic reactor through a multiple lipidic composition able to complex the nucleic acids a provide an hydrated layer improving colloidal stability and protein adsorption. Available technologies allow the incorporation of the invention miRNA (OMe-miR126) and the functionalization with anti-CSPG4 scFv antibodies into properly designed liposomal particles for the use of a liposomal vector in the therapy of CPSG4-expressing cancers with OMe-miR126.

Claims

1. A double strand RNA comprising a mature onco-suppressive miRNA sequence represented by miR- 126-3p having the sense strand sequence corresponding to SEQ. ID. No. 6 and by the anti-sense strand sequence corresponding to SEQ ID. No. 7: modified in that all the pyrimidine residues of the sense strand and the fourth uridine residue in the antisense strand are modified by O-methylation at the 2’-O-pentose (2’-0Me).

2. The double strand RNA according to the preceding claim, wherein said sense strand and said antisense strand comprise two 2’-deoxythymidine residues at each 3’-terminal.

3. The double strand RNA according to the preceding claim 1 or 2, which is delivered through a suitable nanocarrier system.

4. The double strand RNA according to the preceding claim, wherein said nanocarrier system is represented by chitosan nanoparticles or liposomes.

5. The double strand RNA according to claim 3 or 4, wherein said nanocarrier system is loaded with said double strand RNA.

6. The double strand RNA according to any one of claims 3 to 5, wherein said nanocarrier system is linked to a targeting moiety.

7. The double strand RNA according to any one of claims 3 to 5, wherein said nanocarrier system is covalently linked to a targeting moiety.

8. The double strand RNA according to any one of claims 6 or 7, wherein said targeting moiety is represented by an antibody or an antibody fragment.

9. The double strand RNA according to the preceding claim, wherein said antibody is selected from the group comprising: a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a multispecific antibody.

10. The double strand RNA according to claim 8 or 9, wherein said antibody fragment is selected from the group comprising: Fv, Fab, F(ab’)2, Fab’, dsFv, scFv or sc(Fv)2.

11. The double strand RNA according to any one of claims 8 to 10, wherein said targeting moiety is represented by scFv.

12. The double strand RNA according to any one of claims 6 to 11 , wherein said targeting moiety is represented by scFv-9.2.27 having the aminoacidic VH sequence of SEQ. ID. No. 3 and the aminoacidic VL sequence of SEQ. ID. No. 5.

13. The double strand RNA according to any one of the preceding claims for the medical use.

14. The double strand RNA for the medical use according to claim 13 in the treatment of tumors expressing the tumor marker CSPG4.

15. The double strand RNA for the medical use according to the preceding claim, wherein said tumors expressing the tumor marker CSPG4 comprise: melanoma, ovarian cancer and lung cancer.

16. The double strand RNA according to the preceding claim for the medical use in the treatment of melanoma resistant to targeted therapy.

17. The double strand RNA for the medical use according to the preceding claim 15 or 16, wherein melanoma is melanoma resistant to the treatment with vemurafenib or dabrafenib.

18. The double strand RNA for the medical use according to any one of the preceding claims 14 to 17 for the medical use in the treatment of tumor in combination with PI3K/AKT inhibitors.

19. The double strand RNA for the medical use according to the preceding claim for the medical use in the treatment of metastatic melanoma in combination with PI3K/AKT inhibitors.

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