RAD51 / BRCA2 complex aptamers and uses thereof

Abstract

The invention relates to single-stranded DNA or RNA aptamers and uses thereof. Pharmaceutical compositions and kits comprising such aptamers and uses thereof in the medical field, in particular in the treatment of cancer, are also described.

Description

[0001] TITLE

[0002] RAD51 / BRCA2 COMPLEX APTAMERS AND USES THEREOF

[0003] DESCRIPTION

[0004] FIELD OF THE INVENTION

[0005] The invention relates to single-stranded DNA or RNA aptamers and uses thereof. Pharmaceutical compositions and kits comprising such aptamers and the uses thereof in the medical field, utilizing the principles of Synthetic Lethality, are also described.

[0006] BACKGROUND ART

[0007] Synthetic Lethality (SL) occurs when the combination of two genetic alterations which, individually, are not lethal, causes cell death. This concept is fundamental in oncology research, as it enables the development of treatments capable of selectively eliminating tumor cells, while preserving healthy cells.

[0008] In the context of BRCA2 gene mutations, which interfere with the ability of cells to repair DNA by homologous recombination (HR), SL is exploited through the combined use of drugs such as PARP (poly-ADP-ribose polymerase) inhibitors, including olaparib. BRCA2 is a key protein in the repair of double-stranded DNA breaks, as it facilitates the recruitment of the RAD51 protein, which initiates the search for a homologous sequence for repair. The interaction between BRCA2 and RAD51 is essential for the proper functioning of homologous recombination and for maintaining the integrity of the genome. In tumor cells with BRCA2 mutations, the inability to correctly repair doublestranded DNA breaks makes them particularly dependent on PARP. Usually, PARP repairs single-stranded breaks (SSB), but when it is inhibited, in cells with BRCA2 mutations, where HR does not function correctly, the accumulation of DNA damage becomes lethal.

[0009] By analogy with the alteration of BRCA2 activity induced by mutations of the same protein, the interaction between BRCA2 and RAD51 can be directly blocked. Since this interaction is essential for the recruitment of RAD51 at DNA damage sites and for the initiation of repair by HR, interfering with this interaction in combination with PARP inhibitors induces a lethal effect in tumor cells. Aptamers, short nucleic acid sequences (RNA or DNA) capable of binding specifically to RAD51 , offer a promising solution to inhibit this interaction, destabilizing the BRCA2-RAD51 complex. This alteration affects the ability of tumor cells to repair DNA, increasing the vulnerability thereof to therapies which induce DNA damage, such as chemotherapy and radiotherapy.

[0010] Several approaches to block RAD51 have been investigated, including small molecule inhibitors, antibodies and DNA sequences designed to interfere with the function thereof. However, each of these approaches presents challenges: small molecule inhibitors such as RI-1 bind directly to RAD51 , but the efficacy thereof is limited by the short half life and poor solubility thereof; antibodies provide a viable high specificity alternative, but the development and clinical administration thereof can be complicated; RAD51 targeting aptamers represent a more flexible solution, with the advantage of being engineered for highly specific and targeted binding.

[0011] Some examples of molecules under investigation include Amuvatinib, which reduces RAD51 expression at the mRNA level, and Chicago Sky Blue, which prevents the interaction of RAD51 with ssDNA. However, both have shown clinical limitations related to side effects or stability.

[0012] Specifically altering the interaction between BRCA2 and RAD51 is a crucial step to make tumor cells even more vulnerable. Since this interaction is fundamental for the survival of cells, the inhibition thereof amplifies the effect of PARP inhibitors by altering the repair process of the double-stranded DNA breaks, thereby providing an alternative therapeutic approach within synthetic lethality strategies.

[0013] It is the object of the present invention to develop and provide therapies for treating cancer and SL-connected diseases through new molecules which target the DNA HR repair mechanism, and in particular the RAD51 / BRCA2 complex, offering a new approach to interfere with the formation of the complex and the activity of RAD51 .

[0014] SUMMARY OF THE INVENTION

[0015] For the purpose of the present invention, molecules referred to as aptamers have been developed, short single-stranded oligonucleotides which can adopt specific three- dimensional conformations to selectively interact with the targets thereof, representing a promising strategy.

[0016] Compared to small molecules and antibodies, aptamers offer advantages such as greater solubility, robust stability, and the possibility of reducing side effects.

[0017] Aiming to interfere with the DNA repair mechanism in the HR process, aptamers inhibit the RAD51 / BRCA2 interaction, being, alone or in combination with PARP inhibitors, a valid alternative to current anti-tumor therapies. Within the concept of chemically induced synthetic lethality, the possibility of inhibiting the RAD51 / BRCA2 interaction with aptamers provides a tool with advantages in selectivity, stability, and solubility which are very promising for the development of new anti-tumor therapies.

[0018] This strategy promises to advance precision oncology by offering a targeted therapy with minimized side effects, ushering in a new era in the treatment of cancer and SL- connected diseases.

[0019] Therefore, the present invention focuses on the development of isolated single-stranded DNA or RNA aptamers which aim to target the RAD51 / BRCA2 interaction and prevent the repair of the double-stranded DNA break.

[0020] In a first aspect, the present invention relates to an isolated single-stranded DNA or RNA aptamer, comprising a nucleotide sequence capable of interfering with the RAD51 / BRCA2 complex interface, said nucleotide sequence having two Gs separated by three nucleotides, said nucleotides selected from the group consisting of G, T, U, A, and C, being complementary to amino acids 200-260 of the RAD51 protein and having a GC content of at least 75% of the length thereof, in which the first G of said two G binds histidine 294 of protomer 2 of the RAD51 protein.

[0021] In a second aspect, a pharmaceutical composition is described herein comprising an isolated single-stranded DNA or RNA aptamer according to the present invention, and pharmaceutically acceptable excipients.

[0022] In a third aspect, the invention relates to the use of the isolated single-stranded DNA or RNA aptamer as described herein or of the pharmaceutical composition comprising the isolated single-stranded DNA or RNA aptamer as a medicament.

[0023] In a fourth aspect, the present invention relates to the use of the isolated single-stranded DNA or RNA aptamer in accordance with the invention, or of the pharmaceutical composition comprising the isolated single-stranded DNA or RNA aptamer, in the treatment of cancer.

[0024] In a fifth aspect the invention relates to a pharmaceutical kit comprising: a) isolated single-stranded DNA or RNA aptamer as described herein; and a b) PARP inhibitor, for simultaneous, separate or sequential use.

[0025] In a sixth aspect, the present invention shows a pharmaceutical kit comprising: a) isolated single-stranded DNA or RNA aptamer as shown herein; and a b) PARP inhibitor for simultaneous, separate or sequential use in the treatment of cancer.

[0026] In a seventh aspect, the invention describes a method for treating cancer in a patient affected by such diseases, by using the single-stranded DNA or RNA aptamer as described, or a pharmaceutical composition thereof.

[0027] The dependent claims describe particular embodiments of the invention.

[0028] DESCRIPTION OF THE DRAWINGS

[0029] The invention will now be described in detail with reference to the accompanying figures. Figure 1 : Analysis of the interaction of aptamers with RAD51 . Bio-Layer Interferometry (BLI) analysis showing the binding kinetics of the first six aptamers (Apt_1 -Apt_6 SEQ ID NO:1 -6) and the negative control (rcApt SEQ ID:7) with the RAD51 protein at 25°C. Each curve shows the interaction profile of an individual aptamer, from which it is possible to extrapolate the binding affinities thereof toward RAD51. Circles: experimental points; lines: fitting curves. The curve related to rcApt is reported as raw data, since no binding response was measured.

[0030] Figure 2: FLIM analysis of the Apt1 (SEQ ID NO:1 ) competition assay with RAD51 and BRC4. Phasor plot (phasor plot) from the FLIM analysis which shows the sinusoidal component (s) and the cosinusoidal component (g) on the axes, for the fluorophore Texas Red directly bound to the aptamers Apt1 (SEQ ID NO:1 ) and rcApt (SEQ ID NO:7). The points A-F correspond to different experimental conditions: A= Apt1 alone; B= Apt1 after incubation with RAD51 ; C= Apt1 with RAD51 followed by the addition of BRC4; D = rcApt alone E = rcApt after incubation with RAD51 F = rcApt with RAD51 followed by the addition of BRC4. The results of D, E, F are perfectly superimposed. Figure 3: Effect of Apt1 (SEQ ID NO:1 ) on the localization and formation of foci of RAD51 in response to DNA damage. A: Immunofluorescence images comparing untreated BxPC-3 cells, cells treated with Apt1 and cells treated with rcApt (negative control, SEQ ID NO:7) (left panels), next to images of these sets under DNA stress conditions induced by cisplatin (CPL) (right panels). B: Bar graph quantifying cells positive for the formation of RAD51 foci under physiological conditions (left bars) and under CPL-induced stress (right bars), illustrating the effect of Apt1 in reducing the localization of RAD51 in the nucleus compared to the controls. C: Bar chart showing the levels of nuclear fluorescence of RAD51 under physiological conditions (left bars) and under CPL- induced stress (right bars), highlighting the role of Apt1 in decreasing the nuclear fluorescence of RAD51 , a key factor in the activation of the HR repair pathway for DNA (“ = p-value <0.005).

[0031] Figure 4: Effect of Apt1 (SEQ ID NO:1 ) on the DNA repair mechanism: A: Immunofluorescence images comparing untreated BxPC-3 cells, cells treated with Apt1 and cells treated with rcApt (negative control) B: Bar chart showing the percentage of BxPC-3 cells positive for foci of yH2AX , 96 hours after aptamer transfection. The comparison includes untreated cells (CTRL), cells treated with rcApt (negative control), and cells exposed to Apt1 (*= p-value < 0.05).

[0032] Figure 5: Impact of Apt1 (SEQ ID NO:1 ) on DNA damage in the presence and in the absence of a PARP inhibitor (Olaparib) (SL): Bar chart showing the percentage of BxPC- 3 cells positive for foci of yH2AX72 hours after transfection with the aptamers. The comparison includes on the left the untreated cells (CTRL), the cells treated with Apt1 and rcApt (negative control), and on the right the same conditions combined with treatment with olaparib (**= p-value <0.005), (*= p-value <0.05).

[0033] Figure 6: Effect of increasing concentrations of Apt_1 on the nuclear localization of RAD51 and BRCA2 in response to DNA damage. A. Immunofluorescence images of BxPC-3 cells treated with increasing concentrations of Apt_1 in combination with cisplatin. Nuclei are labeled with DAPI (second column from the left); RAD51 (third column); BRCA2 (fourth column). Bar scale: 10 pm. B. Bar charts showing the quantitative analysis of nuclear fluorescence of RAD51 (light gray) and BRCA2 (dark gray) under DNA damage conditions in the presence of cisplatin and increasing concentrations of Apt_1 . The experiments were performed in biological triplicates and data were collected from about 500 cells per condition. C. Immunofluorescence images of BxPC-3 cells treated with negative control rcApt (top); Bar charts showing the quantitative analysis of nuclear fluorescence of RAD51 (light gray) and BRCA2 (dark gray) under DNA damage conditions in the presence of cisplatin and negative control rcApt (bottom).

[0034] Figure 7: Effect of Apt_1 on cell vitality in BxPC-3 pancreatic carcinoma cells and in 3D spheroids. Both models were treated with increasing concentrations of Apt_1 (SEQ ID NO:1 ) or of the negative control rcApt (50, 100 and 200 nM, corresponding to 0.25, 0.5 and 1 pg / mL), both alone and in combination with 10 pM olaparib (0.1 % DMSO as vehicle control). The quantification of the extent of synthetic lethality and of any potential pharmacological synergy was carried out. (A) Cell vitality in 2D after 72 h of treatment. The results are expressed as mean ± SD (n = 5). Statistical analysis was conducted by one way ANOVA followed by Tukey multiple comparison test. (B) BxPC-3 3D spheroids after 72 h of treatment. (C) Analysis of the synergism between Apt_1 and olaparib.

[0035] Art. 170bis paragraphs 2, 3, and 4 of the Italian Industrial Property Code (CPI)

[0036] As for the provisions of Art. 170-bis, paragraphs 2, 3, and 4 CPI, the following is specified: the provenance of the origin of the biological material used in the present patent application is specified in the application itself, and the genetic manipulation of the aforesaid biological material occurred in compliance with the obligations arising from national and community regulations.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention introduces a new cancer treatment strategy which utilizes DNA aptamers alone or in combination with various already approved drugs. The aptamers of the present invention consist of specific single-stranded DNA sequences for interfering with the RAD51 / BRCA2 interaction which target the RAD51 binding surface. In a first aspect, the present invention relates to an isolated single-stranded DNA or RNA aptamer, comprising a nucleotide sequence capable of interfering with the RAD51 / BRCA2 complex interface, said nucleotide sequence having two Gs separated by three nucleotides, said nucleotides selected from the group consisting of G, T, U, A, and C, being complementary to amino acids 200-260 of the RAD51 protein and having a GC content of at least 75% of the length thereof, in which the first G of said two G binds histidine 294 of protomer 2 of the RAD51 protein.

[0039] The design of the aptamers described herein was carried out by selecting sequences which bind exclusively to the unique features of the portion of the RAD51 protein and have a lower propensity to interact with the remainder of the proteome.

[0040] In particular, these aptamers have been specifically designed to target the binding interface between RAD51 and BRCA2 which consists of 8 BRC repeats of BRCA2, where BRC4 exhibits the highest affinity.

[0041] BRC4 interacts with two distinct pockets of RAD51. RAD51 pocket 1 is formed by the residues Ala157-Met210 and interacts with the FXXA pattern of the BRC4 repeat, while pocket 2 is formed by two alpha helices, consisting of the residues Asn196-Ser214 and Ser239-Gly260, which interact with the LFDE pattern of the BRC4 repeat.

[0042] Pocket 1 promotes the disassembly of RAD51 filaments, while pocket 2 is compatible with the formation of the filaments, an essential step for DNA repair.

[0043] The aptamers were specifically designed to interact with pocket 2 present at the BRCA2 binding interface, which is formed approximately by RAD51 amino acid residues 200- 260, which interact with the BRC4 repeat in BRCA2 so as to inhibit the formation of the RAD51 / BRCA2 complex and simultaneously compromise the functionality of RAD51 .

[0044] For the purposes of the present invention, it is essential that the aptamer contain two guanines (G) separated by three nucleotides (GXXXG), a pattern allowing the interaction with the RAD51 pocket (amino acids 200-260) and comprising the binding interface with BRCA2.

[0045] In particular, the first guanine of the GXXXG pattern inserts at the interface between protomer 1 and protomer 2 (the fundamental protein subunits which repeat to form the RAD51 filament), at histidine 294 of protomer 2. If a purine is not present at that position, the aptamer is not capable of establishing the TT-TT interaction with histidine. Furthermore, if there is an adenine instead of guanine, the stabilizing contribution resulting from the electrostatic interaction between the partial negative charges of the guanine oxygen and the nitrogen atoms of the histidine side chain would also be absent. These sequences are further characterized by a greater percentage of G compared to C, which is mainly present between the interaction sites and absent in the reverse complement sequences, which do not interact with RAD51 .

[0046] In an embodiment, the aptamer composition has a GC content greater than 90% of the total length.

[0047] The aptamers were designed so as to have maximum affinity toward this specific binding interface, thus minimizing the potential off-target effects on RAD51 .

[0048] The aptamers described herein are distinguished not only by specificity and precise targeting of the RAD51 / BRCA2 interface, but also by the advantageous reduced length thereof.

[0049] In an embodiment, the sequences of the present invention have a length between 6 and 21 nucleotides, preferably between 9 and 18 nucleotides, more preferably between 12 and 16 nucleotides, even more preferably of 15 nucleotides.

[0050] With a length between 6 and 21 nucleotides, these aptamers provide greater stability, reduce the risk of non specific interactions and improve tissue penetration. These features make it a more potent and efficient therapeutic agent, capable of overcoming the drawbacks associated with preceding RAD51 inhibitors.

[0051] In a further embodiment the aptamer interferes with the interface of the RAD51 / BRCA2 complex with a binding affinity with Kd values in the range from 100 nM to 2500 pM.

[0052] For the purposes of the present invention, single-stranded DNA or RNA aptamers are represented in the attached sequence listing.

[0053] Table 1 reports the sequences of the single-stranded DNA aptamers:

[0054] The single-stranded DNA aptamers (SEQ ID NO: 1 - SEQ ID NO: 29) can be converted to single-stranded RNA aptamers by replacing T with U, where necessary, without compromising functionality and efficacy.

[0055] Preferably the isolated single-stranded DNA or RNA aptamer in accordance with the invention has a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, more preferably the sequence is SEQ ID NO: 1.

[0056] In a second aspect, a pharmaceutical composition is described herein comprising an isolated single-stranded DNA or RNA aptamer according to the present invention, and pharmaceutically acceptable excipients.

[0057] The pharmaceutical composition comprising an isolated single-stranded DNA or RNA aptamer according to the present invention can be variously formulated, for example it can be administered in the form of tablets, capsules, granules, effervescent granules, orodispersible granules, beads, drops, sticks, syrups, and sachets or as an injectable solution.

[0058] Preferably the pharmaceutical composition is in the form of nanoparticles, liposomes or viral vectors such as adenovirus.

[0059] The aptamers described in the present invention introduce an innovative approach to target the RAD51 / BRCA2 interaction and hinder the repair of the double-stranded DNA break. They offer advantages compared to previously developed compounds, such as:

[0060] - High solubility: nucleic acid molecules have higher solubility than the heterocyclic compounds, which is essential for formulation and administration.

[0061] - High specificity and affinity: it is possible to control both specificity and affinity through rational, data-driven design, enabling precise targeting for therapeutic and diagnostic applications.

[0062] - Facilitated entry into cells: using various vectors, aptamers can penetrate into cells without solubility issues.

[0063] - Greater stability: aptamers are stable for more than 2 days within the cell and chemical modifications could provide further stability in a complex biological context.

[0064] - Improved imaging: when conjugated with fluorophores, aptamers enable targeted imaging, providing clearer diagnostic results.

[0065] - Versatile targeting: aptamers can be conjugated with a wide range of molecules, expanding utility in different applications.

[0066] - Cost-effective synthesis: the synthesis of aptamers by chemical synthesis offers a cost-effective alternative to the production of recombinants in bioreactors.

[0067] - Reversible binding: permits the controlled release of conjugated molecules under specific conditions.

[0068] - Customizable for multiplexing: permits the simultaneous use for detection and multiplex treating, targeting multiple pathways or markers.

[0069] In a third aspect, the invention relates to the use of the isolated single-stranded DNA or RNA aptamer as described herein or of the pharmaceutical composition comprising the isolated single-stranded DNA or RNA aptamer as a medicament.

[0070] In a fourth aspect, the present invention relates to the use of the isolated single-stranded DNA or RNA aptamer in accordance with the invention, or of the pharmaceutical composition comprising the isolated single-stranded DNA or RNA aptamer, in treating cancer or in diseases where the induction of synthetic lethality is necessary. Non- exhaustive examples of pathologies where the induction of synthetic lethality improves the prognosis are tumors lacking mutations in the BRCA1 or BRCA2 genes or in the RAD51 gene.

[0071] These aptamers not only represent a leap forward in cancer therapy, but also exemplify the evolution of therapeutic strategies, promising greater efficacy and specificity in interrupting the interactions between pathological proteins.

[0072] In a preferred embodiment, the isolated single-stranded DNA or RNA aptamer described herein or the pharmaceutical composition comprising the isolated single-stranded DNA or RNA aptamer are used in the treatment of pancreatic carcinoma, prostate cancer, breast cancer, ovarian carcinoma, uterine tumor, stomach cancer, colon carcinoma, retinoblastoma, melanoma, and thyroid carcinoma.

[0073] In a fifth aspect the invention relates to a pharmaceutical kit comprising: a) isolated single-stranded DNA or RNA aptamer as described herein; and a b) PARP inhibitor, for simultaneous, separate or sequential use.

[0074] In a sixth aspect, the present invention shows a pharmaceutical kit comprising: a) isolated single-stranded DNA or RNA aptamer as shown herein; and a b) PARP inhibitor, for simultaneous, separate or sequential use in the treatment of cancer.

[0075] The aptamers described herein have demonstrated an effective impairment of the DNA repair mechanisms in cells, interfering with the activity of the RAD51 / BRCA2 complex. The effect thereof could be further enhanced and extended to other DNA repair mechanisms when they are applied with other compounds, enhancing the efficacy thereof.

[0076] The aptamers of the present invention can be used alone or in combination with other anticancer drugs already in clinical use, for example in diseases connected to synthetic lethality.

[0077] The aptamers specific for the RAD51 / BRCA2 complex described herein are particularly relevant in the context of SL.

[0078] In the case of BRCA2, SL occurs when a mutation thereof blocks homologous recombination (HR) and simultaneously the repair of single-stranded breaks (SSB) is inhibited. This effect can also be obtained by targeting the protein essential for HR, RAD51.

[0079] The aptamers described in this invention, by blocking the interaction between RAD51 and BRCA2, make cells particularly vulnerable to the accumulation of DNA damage when PARP is inhibited, which controls SSB repair.

[0080] This strategy offers a new and precise method to target tumor cells, potentially widening the use of PARP inhibitors to a wider population of patients, including those without BRCA mutations.

[0081] In a preferred embodiment, the use of the pharmaceutical kit of the present invention is directed to the treatment of pancreatic carcinoma, prostate cancer, breast tumor, ovarian carcinoma, uterine tumor, stomach cancer, colon carcinoma, retinoblastoma, melanoma, and thyroid carcinoma.

[0082] In a seventh aspect, the invention describes a method for treating cancer by using the single-stranded DNA or RNA aptamer as described or a pharmaceutical composition thereof.

[0083] Embodiments of the single-stranded DNA aptamers of the present invention are given below by way of illustration.

[0084] EXAMPLES

[0085] The examples set forth herein describe the development of single-stranded DNA (ssDNA) aptamers having RAD51 as target with high specificity and affinity.

[0086] In particular, these aptamers interfere with the RAD51 / BRCA2 interaction, essential for homologous recombination (HR), a DNA repair mechanism on which tumor cells heavily rely due to the genomic instability thereof when targeted by anti-tumor treatments.

[0087] In brief, the aptamers exemplified below were designed by predicting protein-nucleic acid interaction propensities and were validated by bio layer interferometry (BLI) to confirm the binding affinity thereof for RAD51 .

[0088] Fluorescence lifetime imaging microscopy (FLIM) suggests a competitive mechanism of the aptamers at the BRCA2 binding site, preventing the recruitment and function of RAD51.

[0089] In-cell assays have demonstrated that these aptamers specifically reduce the nuclear transport of RAD51 and the formation of foci, effectively compromising the HR pathway. Using the phosphorylated variant of the histone yH2AX as a marker of double-stranded breaks, it was verified that the aptamers of the present invention significantly compromise the DNA repair mechanism.

[0090] Example 1 : Assessment of the interaction force of the aptamers with the RAD51 protein The predicted binding propensities between the aptamers Apt_1 -Apt_6 (SEQ ID NO:1 - 6) and RAD51 , including the reverse complementary sequence of Apt1 , here referred to as rcApt (SEQ ID NO:7), as a negative control were validated. The binding affinity of the aptamers was confirmed through the determination of the dissociation constants (Kds) thereof by BLI, in which a stronger interaction is indicated by a lower Kd.

[0091] The highest interaction propensity scores, respectively 12.47 and 12.44, were assigned to Apt_1 (SEQ ID NO:1 ) and Apt_2 (SEQ ID NO:2), which exhibited the lowest Kds, respectively 1 16 ± 14 nM and 138.0 ± 4 nM.

[0092] Kds of 188.0 ± 13 nM and 413 ± 1 1 nM were determined for Apt_3 (SEQ ID NO:3) and Apt_4 (SEQ ID NO:4), both with an interaction propensity score of about 10. Kds of 936 ± 39 nM and greater than 2 pM were exhibited by Apt_5 (SEQ ID NO:5) and Apt_6 (SEQ ID NO:6), both with interaction scores below 9.

[0093] The rcApt negative control (SEQ ID NO:7) showed no binding, as expected.

[0094] The results of these analyses, as shown in table 2, indicate that the DNA sequences of Apt_1 -Apt_6, predicted to strongly interact with the specific area of RAD51 , showed a trend in which a higher propensity score correlates with a lower Kd (Figure 1 ).

[0095] Table 2. List of the sequences of the preferred aptamers, predicted interaction force, and experimental validation of the affinities. Example 2: Competition of Apt1 on the RAD51 / BRCA2 interaction

[0096] FLIM is a powerful technique for the exploration of interactions and molecular dynamics within biological systems. This method facilitates the identification of specific interactions between a protein and a molecule labeled with a fluorophore.

[0097] In the specific case, potential interactions with other molecules on the fluorescence lifetime of a fluorophore were analyzed.

[0098] In this experiment, it was sought to confirm the ability of the aptamers to bind the interface between RAD51 and BRCA2, since the aptamer design was optimized for this portion of the protein.

[0099] Instead of full length BRCA2 (384 kDa), a smaller conventionally used peptide, designated BRC4, a 4 kDa peptide derived from one of the eight BRC repeats of BRCA2 and extensively characterized for the specific interaction thereof with RAD51 and for the ability thereof to mimic the regulatory effect which BRCA2 exerts on RAD51 during DNA HR repair was selected. In particular, between the eight repeats of BRCA2, BRC4 exhibits the greater affinity for RAD51 .

[0100] As a proof of concept, the best-performing aptamer, Apt_1 (SEQ ID NO:1 ), was evaluated using this technique. As a control, the reverse and complementary sequence of Apt_1 , rcApt (SEQ ID NO:7), which does not bind RAD51 , was included. In order to detect the behavior of the aptamers, Apt_1 and rcApt were conjugated with Texas Red fluorophore at the 5' end.

[0101] The FLIM experiments were performed under different conditions: A) Apt_1 alone (unbound); B) Apt_1 incubated with RAD51 ; C) Apt_1 with RAD51 followed by the addition of BRC4; D) rcApt alone; E) rcApt after incubation with RAD51 ; and F) rcApt with RAD51 followed by the addition of BRC4.

[0102] The results of D), E) and F) are perfectly superimposable.

[0103] The fluorescence lifetime decay of 1 pM Apt_1 was analyzed before (A) and after (B) the addition of 5 pM RAD51 , revealing an increase in the slow component (T slow) from 5.01 ± 0.1 ns to 5.32 ± 0.2 ns (Table 3). This increase in T slow confirms an interaction between RAD51 and Apt_1. The introduction of BRC4 into this system (C) led to a reduction of T slow to 5.09 ns, suggesting a partial displacement of Apt_1 from the binding site thereof by BRC4, thereby reintroducing the aptamer into solution. The observed intermediate lifetime of 5.09 ± 0.1 ns supports the premise that Apt_1 was both in solution and partially bound to RAD51 , reinforcing the hypothesis that Apt_1 specifically targets the RAD51 -BRCA2 interaction site.

[0104] A parallel experiment with rcApt consistently showed a T slow of 5.01 ± 0.1 ns under all tested conditions: alone D), with RAD51 E) and with BRC4 F). This uniformity in the decay time between these conditions confirms that the negative control rcApt interacts with neither RAD51 nor BRC4.

[0105] Furthermore, the decomposition of the fluorescence signal into percentages of "slow" and "fast" components revealed the presence of multiple fluorescence states, each of which influences the fluorescence lifetime differently. The fast component, which indicates a short lifetime, suggests a rapid return to the ground state, while the slow component, which indicates a longer lifetime, suggests a slower return.

[0106] The increase in the slow component of Apt_1 from 0.71 to 0.82 upon addition of RAD51 , and the subsequent return thereof to 0.71 upon addition of BRC4, supports the idea that BRC4 competes with Apt_1 for the binding site of RAD51 (Table 3).

[0107] Using the phasor approach for fluorescence decay analysis, the sine component (s) and the cosine component (g) were plotted, enabling visual differentiation of the molecular environments that influence our aptamer-fluorophore conjugates (Figure 2). A clear shift of the phasor plot from condition A) to B) indicates binding of Apt_1 with RAD51 , while the addition of BRC4 shifts the plot to condition C), suggesting competition of BRC4 with Apt_1 for the binding site of RAD51 . The experiment with rcApt showed no changes under any condition, highlighting the specificity of the interaction of Apt_1 with RAD51 . These results validate the specific interaction of Apt_1 with RAD51 and clearly suggest that Apt_1 binds to the same interaction area on RAD51 as BRC4, underscoring the potential of the aptamers of the present invention as therapeutic agents.

[0108] Table 3. The table shows the fastest (T fast) and slowest (T slow) components of the fluorescence decay time, the sine component (s), the cosine component (g) and the values of the fast and slow components obtained from fluorescence lifetime imaging microscopy (FLIM). These measurements relate to the Texas Red fluorophore conjugated to the aptamers Apt1 and rcApt, with detailed variations subsequent to the addition of RAD51 and BRC4. The observed change in the T values for Apt1 in the presence of RAD51 confirms the existence of a binding between the two. On the other hand, the T values for the control molecule, rcApt, remain unchanged, confirming the absence of interaction with RAD51 .

[0109] Example 3: Effect of aptamers on the sub-cellular localization of RAD51 and on the formation of foci of RAD51

[0110] The impact of the DNA aptamer Apt_1 (SEQ ID NO:1 ) on the sub-cellular localization of RAD51 and on the formation of foci of RAD51 , crucial for the HR pathway in DNA repair, was analyzed.

[0111] Under induced stress conditions, Apt_1 considerably reduced the nuclear transport of RAD51 and the formation of nuclear foci, thereby promoting cell death.

[0112] The assessment focused on two main aspects: the sub-cellular distribution of RAD51 and the formation of local response hubs at DNA damage sites (foci).

[0113] Under physiological conditions, RAD51 is uniformly distributed between the cytoplasm and the nucleus, a balance which is altered during cellular stress, as in the case of Double-Stranded Breaks (DSB), as a result of which RAD51 is transported into the nucleus to facilitate repair through HR.

[0114] Using immunofluorescence microscopy, the effect of the aptamer Apt_1 and of a negative control (rcApt) on the distribution of RAD51 in BxPC-3 pancreatic carcinoma cells treated with cisplatin to induce DNA damage was assessed.

[0115] The analysis demonstrated that untreated cells maintain a basal level of nuclear RAD51 , which increases significantly with treatment with cisplatin, indicating activation of the HR pathway (Figure 3A and 3C). However, the cells to which Apt_1 was administered exhibited nuclear levels of RAD51 comparable to those of untreated cells, suggesting an effect of Apt_1 on the nuclear recruitment of RAD51 , which thus limits the capacity of RAD51 in DNA repair. This effect is specific to Apt_1 , since the cells treated with rcApt did not exhibit significant changes compared to treatment with cisplatin alone.

[0116] Furthermore, the formation of nuclear foci of RAD51 , a marker of active HR repair, was significantly reduced in cells treated with Apt_1 compared to those treated with cisplatin alone or with rcApt, reinforcing the potential of Apt_1 to interfere with the HR pathway. Quantitative confirmation was provided by Imaged analysis, which showed a decrease in the number of cells positive for foci of RAD51 following treatment with Apt_1 , suggesting a direct impact on the efficacy of HR-mediated repair (Figure 3B).

[0117] These results provide strong evidence of the capacity of Apt_1 to modulate the process of DNA repair in tumor cells, specifically inhibiting the nuclear transport of RAD51 and the formation of foci of RAD51 . Targeting this crucial interaction, the aptamers described herein provide a new approach to cancer therapy, improving the efficacy of existing treatments by sensitizing cancer cells to DNA damage, highlighting the potential thereof as promising candidates for precision oncology.

[0118] These examples strikingly and convincingly demonstrate the efficacy of these aptamers in targeting RAD51 by binding directly to the RAD51 / BRCA2 interaction region, thereby hindering the translocation of RAD51 into the nucleus.

[0119] Without access to the nucleus, RAD51 is not capable of carrying out the vital role thereof in DNA repair. This impairment of DNA repair mechanisms, on which cancer cells heavily rely, suggests that cancer treatments could be significantly more effective when Apt_1 is used in combination with targeted anti-tumor drugs. By inhibiting a key pathway which cancer cells use to repair damaged DNA, Apt_1 could increase the susceptibility of these cells to therapeutic interventions.

[0120] Example 4: Capability of aptamers to interfere with DNA repair

[0121] The impact of Apt_1 (SEQ ID NO:1 ) on cellular DNA damage response and repair processes was explored, with particular focus on HR, using the phosphorylated variant of the histone yH2AX as a marker of DSB, a critical indicator of cellular DNA damage and repair dynamics (Figure 4).

[0122] Nuclear yH2AX puncta (also referred to as foci), indicative of double-stranded breaks (DSB), were monitored to assess the influence of Apt_1 on the DNA repair pathway.

[0123] Under physiological conditions, cells exhibit a baseline level of DSBs, which are efficiently repaired through natural repair mechanisms. However, observations over a period of 96 hours after aptamer transfection revealed a significant increase in DSB in cells treated with Apt_1 .

[0124] In particular, in the presence of Apt_1 , the foci-positive cells are double those present both in the untreated controls (CTRL) and in the conditions treated with the negative control rcApt (Figure 4). This increase in DSB demonstrates that Apt_1 disrupted the normal repair process, likely targeting and inhibiting the HR pathway, essential for the accurate repair of DSB.

[0125] These results provide compelling evidence that Apt_1 acts to compromise the DNA repair mechanism mediated by HR.

[0126] Furthermore, the set of these data highlights the potential of aptamers as therapeutic agents to target tumor cells, which rely heavily on HR to survive in the face of DNA damage.

[0127] Example 5: Effect of the aptamers on the accumulation of DNA damage in the context of synthetic lethality

[0128] At this point, the effect of the aptamers of the present invention was explored in the context of diseases associated with synthetic lethality (SL), a strategy to obtain selective death of tumor cells while sparing healthy cells, targeting critical survival pathways.

[0129] In particular, olaparib, a PARP inhibitor (PARPi) used in tumors in which BRCA1 or BRCA2 are mutated, resulting in HR deficiency, has demonstrated great potential (Figure 5). By combining olaparib with an HR inhibitor, the aim was to sensitize tumor cells to the treatment, independently of the genetic mutation status. This strategy could improve the efficacy of the treatment in various types of cancer, by acting on shared DNA repair pathways.

[0130] After confirmation of the efficacy of Apt_1 (SEQ ID NO:1 ) in combination with olaparib, we explored SL. Apt_1 disrupts the DNA repair process, potentially increasing cell death in combination therapies.

[0131] By fluorescence microscopy, the levels of yH2AX, which indicate DNA double-stranded breaks (DSB), were monitored. Cells treated with olaparib and Apt_1 showed a significant increase in foci of yH2AX compared to controls, indicating greater DNA damage (Figure 5).

[0132] This result underscores the potential of Apt_1 as a therapeutic agent by exploiting the vulnerabilities of tumor cells in DNA repair mechanisms.

[0133] Example 6: Effect of increasing concentrations of Apt 1 on the nuclear localization of RAD51 and BRCA2 in response to DNA damage. Under physiological conditions, RAD51 is mainly localized in the cytoplasm. In order to participate in DNA repair by HR, RAD51 must be translocated into the nucleus and recruited to sites of DNA damage by BRCA2. In response to double-stranded breaks (DSB), RAD51 and BRCA2 accumulate in the nucleus forming characteristic nuclear foci, markers of regions of active repair.

[0134] In order to evaluate the effect of Apt_1 on the BRCA2 mediated nuclear recruitment of RAD51 , the subcellular localization of these proteins was analyzed under stress conditions. DNA damage was induced by means of cisplatin, a platinum-based chemotherapeutic agent which causes inter- and intra-strand crosslinks by covalently binding to DNA bases. Such adducts distort the structure of DNA and are repaired primarily through the HR pathway.

[0135] A commercial SPAD fluorescence microscope was used to evaluate the localization of RAD51 and BRCA2 under conditions of DNA damage, in the presence or absence of increasing concentrations of Apt_1 (Figure 6). Under untreated (physiological) conditions, RAD51 showed a mainly cytoplasmic localization, while BRCA2 was diffusely distributed between cytoplasm and nucleus (Figure 6A and B, “Untreated” panels). After treatment with cisplatin, both proteins accumulated in the nucleus (the Figure 6A and B, “Cisplatin” panels), consistent with the role thereof in HR repair.

[0136] However co-treatment with Apt_1 compromised such a nuclear relocalization in a concentration-dependent manner (Figure 6A and B). At 25 nM Apt_1 no effects were observed; at 50 nM Apt_1 only slight and non-significant changes were detected. Conversely, 100 nM and 200 nM Apt_1 markedly reduced the nuclear accumulation of RAD51 and prevented the nuclear enrichment of BRCA2, restoring a distribution like that of unstressed physiological conditions (Figure 6A and B).

[0137] Pearson correlation analysis confirmed the statistical significance of the effect at 100 nM and 200 nM Apt1 (Table 4). No impact was observed with the reverse-sequence control rcApt (Figure 6C, Table 4).

[0138] These results demonstrate that higher concentrations of Apt_1 significantly compromise the nuclear accumulation of RAD51 and the recruitment of BRCA2, potentially interfering with HR mediated DNA repair.

[0139] Table 4. Pearson correlation coefficients (± standard error) representing the correlation between the subcellular localization of RAD51 and BRCA2 under DNA damage conditions, with or without increasing concentrations of Apt_1 .

[0140] Example 7: Effect of increasing concentrations of Apt 1 on cell viability and on synthetic lethality in combination with olaparib.

[0141] Cell viability assays were conducted in BxPC-3 pancreatic carcinoma cells exposed to increasing concentrations of Apt_1 (SEQ ID NO:1 ) and of the negative control rcApt (50, 100, and 200 nM, corresponding to 0.25, 0.5, and 1 pg / mL), either alone or in combination with 10 pM olaparib (0,1 % DMSO as vehicle control).

[0142] In order to quantify the extent of synthetic lethality and any pharmacological synergy, the Chou and Talalay model was adapted, particularly suited to experimental designs based on fixed concentrations of one of the agents.

[0143] As shown in Figure 7, rcApt did not exhibit significant effects on cell viability, either alone or in combination with olaparib. Conversely, Apt_1 induced a dose-dependent reduction of cell viability and significantly potentiated olaparib mediated cell death. The pharmacological interaction indices (i. index < 0.8) confirmed a synergistic effect for Apt_1 at 100 and 200 nM, demonstrating the presence of a chemically induced synthetic lethality (Figure 7A).

[0144] In order to evaluate the therapeutic potential under more physiological conditions, the combinatorial effect of Apt_1 with olaparib was also examined in BxPC-3 3D spheroids, which more faithfully recapitulate tumor architecture from the standpoint of cell-cell and cell-matrix interactions of the in vivo microenvironment. Consistent with the 2D data, rcApt had no significant effects, while Apt_1 showed a dose-dependent increase in cell death, amplified by treating in combination with olaparib. In this system, the synergistic effect (i. index < 0.8) was detected at 200 nM Apt_1 , probably in relation to differences in penetration and cellular dynamics in the three-dimensional model (Figure 7B). It is further noted that, in non-tumorigenic cell lines (MCF-10A), no dose-dependent effect on cell death is observed.

[0145] Furthermore, the assessment of the volume of the spheroids revealed that Apt_1 (200 nM) and olaparib (10 pM) significantly reduce tumor growth, with a further enhanced effect when administered in combination. Analyses using a fluorescent assay with calcein acetoxymethyl ester (Calcein-AM) and propidium iodide (PI), which enable distinguishing viable cells (green fluorescence) from dead cells, permeable to PI (red fluorescence), have confirmed an increase in cell death following the combined treatment (Figure 7C).

[0146] Overall, these results demonstrate that Apt_1 , in combination with olaparib, induces chemically induced synthetic lethality in 2D and 3D models, with a dose-dependent synergistic effect. The consistency of the data obtained in the two experimental systems strengthens the therapeutic potential of Apt_1 and provides a solid basis for future preclinical studies aimed at dose optimization and at pharmacological development in the field of pancreatic carcinoma therapy.

[0147] Materials and methods

[0148] BIOLAYER INTERFEROMETRY (BLI)

[0149] BLI was employed for determining the dissociation constants (Kds) of the aptamers with RAD51. The experiments were conducted using the Octet Red instrument (ForteBio, Inc., Menlo Park, CA) at 25 °C. All the steps of the assay were performed in the binding buffer, a solution of 50 mM Tris HCI buffer at pH 8, containing 200 mM KCI, 0.25 mM EDTA, 1 mM DTT, 10% glycerol and 0.01 % Tween-20. Streptavidin-coated biosensors were selected to allow loading the biotinylated DNA aptamers at a concentration of 2 pg / mL. The protocol to generate the binding curves between the aptamers and RAD51 was set as follows: Baseline of 180 seconds; aptamer loading step on the sensors of 300 seconds; washing step of 120 seconds; association step of 600 seconds with increasing protein concentrations (from 15 nM to 15 pM, based on binding strength); dissociation step of 600 seconds. The Kd values were obtained by fitting the response intensity (wavelength shift at the time of binding) to the protein concentration at the stationary state. The experiments were performed in triplicate.

[0150] FLUORESCENCE AND LIFETIME IMAGING MICROSCOPY (FLIM)

[0151] FLIM images were acquired using the digital frequency-domain (DFD) FLIMbox (ISS inc., Champaign, IL), coupled to an A1 RMP multiphoton microscope (Nikon, Japan), calibrated with a 10 pM solution of coumarin 153 in methanol (t= 4.3 ns). The samples were excited via 2-photon excitation with a Chameleon Ultrall Ti:Sapphire laser (Coherent inc., Saxonburg, PA) tuned to 850 nm through a 60x oil immersion objective (NA=1.45). Fluorescence-emitted photons were filtered with a 525 / 50 nm BP filter for coumarin 153 and 605 / 70 nm for aptamers labeled with Texas red. Analyses were performed with the ISS VistaVision multi-image phasor analysis software, where the sine (s) and cosine (g) coordinates are defined as gFh = mF,h cos(cpF,h) sFh = mF,h sin(cpF,h)

[0152] Where m is the modulation, <p is the acquired phase of the h-th pixel in the FLIM image, and F is the laser repetition frequency.

[0153] HANDLING CELL CULTURES

[0154] BxPC-3 pancreatic carcinoma cells were cultured in RPM1 1640 medium (Thermofisher, 1 1875093), supplemented with 2 mM L-glutamine, 100 mg / l penicillin, 100 mg / l streptomycin and 10% fetal bovine serum (FBS). The cells were maintained at 37°C in a 5% CO2 atmosphere and were divided when they reached confluence. For microscopic analysis, the cells were seeded at a concentration of 150000 cells / mL on 24-well plates with coverslips. After one day, the complete medium was replaced with FBS-free medium for 18h-24h, to synchronize the cells at a similar growth step. New complete medium was then introduced and, after a 2 hour recovery period, the cells were treated with Lipofectamine 2000 (Thermofisher, 11668019) with or without aptamers at 0.5 pg / mL, following the manufacturer's instructions. After transfection, the cells were allowed to recover overnight before further treatments.

[0155] IMMUNOFLUORESCENCE SAMPLE PREPARATION FOR THE INVESTIGATION OF THE BEHAVIOR OF RAD51

[0156] On the day following transfection, half of the sample cells were treated with cisplatin 50 pM for 2 hours to induce DNA damage, while the other half was left untreated as control. After cisplatin treatment, the cells were rinsed twice with phosphate-buffered saline (PBS) and then fixed for 10 minutes at ambient temperature with a 4% paraformaldehyde solution prepared in PBS. After three PBS washings, the cells were permeabilized with Triton-X 100 at 0.1 % in PBS for 5 min, followed by another three PBS washings.

[0157] The cells were treated with 5% BSA prepared in PBS for 30 minutes as a blocking step. The cells were then exposed to a primary antibody to detect the RAD51 protein using rabbit anti-RAD51 antibody (Bio Academia, 70-001 ) prepared with 5% bovine serum albumin (BSA) in PBS at a 1 :1000 dilution and incubated for 1 hour at room temperature. Next, an anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Thermofisher, A-1 1008) was added at a 1 :1000 dilution and incubated for a further hour at room temperature.

[0158] After three washings with PBS, the cells were treated for 5 minutes with a 0.5 pg / mL solution of 4',6-diamidino-2-phenylindole (DAPI) prepared in PBS. After a further three rinses in PBS, the coverslips were positioned face down on glass slides using ProLong™ Diamond Antifade Mountant mounting liquid (Thermofisher, P36965).

[0159] IMMUNOFLUORESCENCE SAMPLE PREPARATION FOR THE INVESTIGATION OF RAD51 AND BRCA2 COLOCALIZATION

[0160] For the fluorescence analysis of RAD51 and BRCA2, the slides containing fixed cells were examined using the Nikon NSPARC confocal microscope, equipped with a Plan Apo Lambda S 40x / 1 .25 NA silicone immersion objective. The images were acquired with a projection zoom set to 1 Airy Unit (AU), a constant pixel size equal to 70 nm, and a dwell time of 0.4 nsec.

[0161] DAPI excitation was carried out with a 405 nm laser, with power transmission set to 4%, and the fluorescence was collected within the 430-463 nm interval. Alexa Fluor® 488 was excited with a 488 nm laser (power 4%), with fluorescence detection within the 503- 545 nm interval. Alexa Fluor® 563 was excited with a 561 nm laser (power 10%), with fluorescence detection within the 582-618 nm interval.

[0162] For each experimental condition, at least eight representative images were collected. Quantitative analysis of the fluorescence intensity and of the formation of foci was conducted on a minimum of 200 nuclei per sample.

[0163] IMMUNOFLUORESCENCE SAMPLE PREPARATION FOR THE INVESTIGATION OF THE EFFECT OF DNA DAMAGE IN THE CONTEXT OF SYNTHETIC LETHALITY

[0164] In order to evaluate the effect of the aptamers on physiological DSB, the cells were analyzed three days after transfection, evaluating those positive for foci of yH2AX . In order to determine the impact of the aptamers on SL, one day after transfection of the aptamers olaparib was added at a concentration of 50 pM. The formation of foci of yH2AX was analyzed four days after transfection.

[0165] In both cases the cells were rinsed twice with PBS and then fixed for 10 minutes at room temperature with a 4% paraformaldehyde solution prepared in PBS. After three PBS washings, the cells were permeabilized with Triton-X 100 at 0.1 % in PBS for 5 min, followed by another three PBS washings. The cells were treated with 5% BSA prepared in PBS for 30 minutes as a blocking step. Next, a primary antibody against phosphohistone H2AX (Ser139), clone JBW301 (Merck / Sigma Aldrich, 05-636), at a 1 :1000 dilution in BSA / PBS at 5% was used and incubated for 1 hour at room temperature. The cells were then treated with anti-mouse secondary antibody conjugated with Alexa Fluor 647 (Thermofisher, A-21236) at a 1 :1000 dilution and incubated for a further hour at ambient temperature.

[0166] After three washings with PBS, the cells were treated for 5 minutes with a 0.5 pg / mL solution of 4',6-diamidino-2-phenylindole (DAPI) prepared in PBS. After another three rinses in PBS, the coverslips were positioned face down on glass slides using the mounting liquid ProLong™ Diamond Antifade Mountant (Thermofisher, P36965). IMMUNOFLUORESCENCE IMAGE ACQUISITION

[0167] The slides containing the fixed cells were examined with the Nikon A1 R confocal microscope, using the 60x objective with 4 channel detectors and by the Nikon NIS- Elements Advanced Research software version 5.30.02 (64 bit). The fluorescence patterns were determined by tracing a line on the selected cells and evaluating the fluorescence intensity of the three fluorophores (DAPI, RAD51 and TexasRed) pixel by pixel. At least 10 images were examined per condition. In order to calculate the fluorescence intensity and the number of foci, at least 600 cells were used per sample.

[0168] IMMUNOFLUORESCENCE IMAGE ANALYSIS

[0169] The internal macro was executed using Imaged software for counting foci of RAD51 with and without cisplatin and in the absence / presence of Apt1 or rcAptl labeled with a fluorophore, to evaluate the impact of the aptamers on the DSB repair mechanism. In order to measure the intensity of the nuclear fluorescence of RAD51 , the nuclear region was identified using the nuclear stain DAPI. In order to segment the nuclei, a gaussian filter was applied to the images to smooth them and reduce noise. The image threshold was adjusted (Otsu method) to differentiate the signal of interest from the background. Finally, a watershed algorithm was applied to the binary image to separate nuclei which could be touching. The particle analysis parameters were set as follows: size range from 1 to infinity; circularity between 0.5 and 1 . Once identified in the nuclear region and added to the manager of the region of interest (ROI), the nuclear fluorescence was quantified in the green channel, corresponding to RAD51 . The fluorescence data were normalized based on the nuclear area.

[0170] In order to estimate the fluorescence intensity ratio between the cytoplasmic and nuclear RAD51 signal, a macro for Imaged was designed with the following operational pipeline. The program was configured to measure the area and the mode of the fluorescence intensity of ROI which represent nuclear and cytoplasmic areas in the images. A gaussian blur with Sigma 1 was applied to the DAPI channel to facilitate segmentation of the nuclear region with greater confidence. The image was segmented using the Otsu criterion, an automatic and robust threshold parameter. Holes in the binary images were filled with the Imaged algorithm and were used to create a region of interest.

[0171] The second channel - the RAD51 signal - was processed in an analogous manner. A gaussian blur with sigma 0.5 was applied and the image was segmented using the MinError function; this made it possible to obtain a binary image of the entire cell by virtue of the RAD51 distribution. The binary image of the nucleus was subtracted from that of the entire cells to obtain the image of the cytoplasmic area. Finally, the intensity and the area of the two regions of interest, the cytoplasmic one and the nuclear one, were measured. The ratio between the nuclear signal and the cytoplasmic signal was used to discriminate between control cells and treated cells.

[0172] In order to assess the increase in the presence of foci of yH2AX within nuclei due to DNA damage, an Imaged macro was developed, which establishes a comprehensive operating protocol for automated image analysis in a specific directory. The image parameters, such as the directory path, file format, and channels designated for segmentation and analysis, were configured at the beginning. A reference image, in particular one of cells treated with cisplatin, was selected to adjust the threshold parameters essential for the segmentation of the signal of interest. After this initial configuration, a gaussian blur with a sigma value of 1 was applied to the DAPI channel to improve the accuracy of the segmentation of nuclear regions. Segmentation was performed using Otsu’s criterion, an automated and robust thresholding method. The holes within the binary images were filled using the Imaged algorithm, thereby facilitating the delineation of individual nuclei as regions of interest (ROI) through the Analyze Particles function. The parameters were set to include items with a size of at least 5000 pixels, a circularity range of 0.5-1 .00 and to exclude the items at the periphery of the image.

[0173] For the analysis of the yH2AX signal, a gaussian blur with a sigma of 0.5 was applied, followed by segmentation using threshold values determined by the reference image. This process generated a binary image of foci of yH2AX. Each nuclear ROI containing the foci was quantified by calculating the average value of the pixels within the yH2AX binary image; an average greater than one was indicative of a positive ROI. The proportion of positive nuclei to the total number of nuclei was used to distinguish between control cells and treated cells, as outlined in the study. From the detailed description and the Examples given above, the advantages achieved by the single-stranded DNA aptamers of the present invention are apparent.

[0174] CELL VIABILITY ASSESSMENT

[0175] The cell viability of 2D cultures following treatment with the aptamers was assessed by MTT cell assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. BxPC-3 cells were seeded at a concentration of 7500 cells / well in standard 96 well plates, allowed to adhere for 24h and treated with the aptamers Apt1 or rcApt (50, 100 or 200 nM), alone or in combination with olaparib (10 pM) for 72h. At the end of the treatment, a sterile solution of MTT (5 mg / mL) dissolved in PBS 1 X was added to each well, at the final concentration of 0.5 mg / mL. The plates were further incubated at 37°C for 4h to allow the conversion of MTT into water-insoluble formazan crystals. The crystals were further solubilized by adding to each well a 1 :1 volume of a 10% SDS solution, 0.01 M HCI. The following day, the absorbance of each well at the wavelengths of 570 nm and 690 nm was read by a microplate reader. The same type of assay was adapted and employed for the assessment of cell viability also in 3D spheroids. BxPC-3 cells were seeded at a concentration of 30000 cells / well in 96-well plates with the bottom coated with 1 % sterile agarose in 1 X PBS. The spheroids were allowed to grow for 96h, then treated for 72h and subjected to MTT assay.

[0176] CALCULATION OF THE PHARMACOLOGICAL INTERACTION INDEX

[0177] The assessment of the antineoplastic effect of the combination of aptamers and olaparib on pancreatic adenocarcinoma cell cultures was obtained by calculating the pharmacological interaction index (i. index) according to the adaptation of the method developed by Chou and Talalay obtained by applying the following formula (i. index < 0.8 indicates synergism; 0.8 < i. index < 1.2 indicates an additive effect; i. index > 1.2: indicates antagonism): i. index = (Viability % [aptamer+olaparib]) / (Viability % [aptamer] Viability % [olaparib])

[0178] ASSESSMENT OF CELL DEATH BY MEANS OF VITAL DYES AND SPHEROIDAL VOLUME The effect of the aptamer treatment on cell death in 3D spheroids was assessed by staining with fluorescent vital dyes. 3D spheroids of BxPC-3 cells were obtained in 96 well plates and treated with Apt1 or rcApt (50, 100 or 200 nM), alone or in combination with olaparib (10 pM). Upon completion of the treatment, calcein acetoxymethyl ester (Calcein-AM) and propidium iodide (PI) were added to each well at a concentration of

[0179] 2.5 and 3.75 pM respectively, then the plates were incubated at 37°C for 45 min. Upon completion of the incubation, fluorescence microscopy images were acquired by a Leica DMI6000 B inverted microscope, equipped with a Leica DFC360 FX camera and filters for FITC, TRITC, and DAPL In order to determine the effect of the treatments on cell death, the fluorescent signal of Calcein-AM (Excitation = 485 nm, Emission = 530 nm) and PI (Excitation = 535 nm, Emission = 620 nm) was measured by a Tecan Spark® microplate reader and the results were expressed as the PI / Calcein-AM ratio. For the assessment of the spheroid volume over the treatment time, visible light microscopy images (Leica DM16000 B inverted microscope, Leica DFC360 FX camera) were acquired every 24h post treatment and processed with Imaged software using the

[0180] SA_NJ macro. The spheroid volume was calculated using the formula:

[0181] Spheroid Volume (V) = 0.5 X (Length) X (Width)2.

Claims

CLAIMS1 . An isolated single-stranded DNA or RNA aptamer comprising a nucleotide sequence capable of interfering with the RAD51 / BRCA2 complex interface, said nucleotide sequence: having two Gs separated by three nucleotides, said nucleotides selected from the group consisting of G, T, U, A, and C; being complementary to amino acids 200-260 of the RAD51 protein; and having a GC content of at least 75% of the length thereof, wherein the first G of said two Gs binds histidine 294 of protomer 2 of the protein RAD51 .

2. The isolated single-stranded DNA or RNA aptamer according to claim 1 , wherein said nucleotide sequence has a length in the range from 6 to 21 nucleotides, preferably in the range from 9 to 18, more preferably in the range from 12 to 16 nucleotides, most preferably 15 nucleotides.

3. The isolated single-stranded DNA or RNA aptamer according to any one of claims 1 or 2, wherein said aptamer interferes with the RAD51 / BRCA2 complex interface with a binding affinity with Kd values in the range from 100 nM to 2500 pM.

4. The isolated single-stranded DNA or RNA aptamer according to any one of claims 1 to 3, wherein said sequence is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

5. The isolated single-stranded DNA or RNA aptamer according to any one of claims 1 to 4, wherein said sequence is SEQ ID NO: 1 .

6. A pharmaceutical composition comprising an isolated single-stranded DNA or RNA aptamer according to any one of claims 1 to 5 and pharmaceutically acceptable excipients.

7. An isolated single-stranded DNA or RNA aptamer according to any one of claims 1 to 5 or a pharmaceutical composition comprising an isolated single-stranded DNA or RNA aptamer according to claim 6, for use as a medicament.

8. An isolated single-stranded DNA or RNA aptamer according to any one of claims 1 to 5 or a pharmaceutical composition comprising an isolated single-stranded DNA or RNA aptamer according to claim 6, for use in the treatment of cancer.

9. The isolated single-stranded DNA or RNA aptamer according to any one of claims 1 to 5 or the pharmaceutical composition comprising an isolated single-stranded DNA or RNA aptamer according to claim 6, for use according to claim 8, wherein said cancer is selected from the group consisting of pancreatic carcinoma, prostate cancer, breast cancer, ovarian carcinoma, uterine tumor, stomach cancer, colon carcinoma, retinoblastoma, melanoma, and thyroid carcinoma.

10. A pharmaceutical kit comprising: a) an isolated single-stranded DNA or RNA aptamer according to any one of claims 1 to 5; and a b) PARP inhibitor, for simultaneous, separate or sequential use.1 1 . A pharmaceutical kit comprising: a) an isolated single-stranded DNA or RNA aptamer according to any one of claims 1 to 5; and a b) PARP inhibitor, for simultaneous, separate or sequential use in the treatment of cancer.

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