Method for screening high target-specific aptamer in multi-tissue co-culture microfluidic device platform

Abstract

The present invention relates to a method for developing personalized targeted anticancer therapy based on aptamers and systemically modeling individuals in a microfluidic device. In one embodiment, the present invention provides a method for developing targeted therapy comprising maintaining targeted cancer cells in co-culture with non-target non-cancer cells using a microfluidic device modularly arranged within a closed system to develop aptamers. In another embodiment, the present invention provides a method for developing targeted therapy comprising maintaining target cells in co-culture with non-target cells using a microfluidic device modularly arranged in a closed system. In this regard, the present invention provides development of aptamers for relevant targets that are in homeostatic balance with fluidic components modulated by co-culture with non-target cells.

Description

TECHNICAL FIELD

[0001] The invention described herein relates to the specificity of aptamers and targeted therapies in the field of personalized medicine. More specifically, it relates to one or more microfluidic devices, methods, assemblies and systems for cell culturing and development of aptamers with higher specificity to desired targets through SELEX technology. In particular, it relates to modeling aggressive cancers for the development of personalized aptamers for patients. BACKGROUND

[0002] Cancers comprise a group of diseases that usually have uncontrolled cell division and the potential to spread to other parts of the body. In particular for the more aggressive disease types, conventional chemotherapy is the main cause of incompatibility for patient survival.

[0003] There are different types of targeted cancer therapies, grouped according to their nature and effect. In general, targeted therapies act by targeting differences that support the survival and growth of cancer cells. The identification of new anticancer compounds can be done through the combination of molecular libraries and ligand evolution processes. In this regard, a technique called SELEX, from Systematic Evolution of Ligands by EXponential enrichment, stands out in the identification and selection of molecules known as aptamers.

[0004] From the Latin Aptus (connection) and the Greek Meros (part), aptamers are composed of synthetic molecules, i.e. not naturally occurring, with specific interactions with target molecules. Strategically, through this interaction, the affinity of a library of nucleic acid combinations is enriched through successive steps of exposure, capture and amplification of the species that best bind to the target of interest. In this regard, aptamers interact with the target (in this case cancer cells) and produce the desired response, capable of altering their functional activity, even positioning, directing, installing and delivering other compounds with therapeutic interest.

[0005] During the traditional SELEX development of aptamers, a negative selection step can be introduced against non-targets, for example, cell types outside the therapeutic purpose. In this case, the combinatorial library is incubated with the negative target with the aim of discarding the molecules that bind to it, to amplify the species that can be called non-ligands. Ideally, negative selection would generate aptamers specific to the target of interest and with no affinity for the negative target. However, it has been proven that the degree of specificity of aptamers is not enough to distinguish cell populations in practice, even after the negative selection step during SELEX.

[0006] As for in vivo variants of SELEX, although specificity is obtained by introducing the aptamer selected in guinea pigs, it is based on the affinity for distorted targets and non-targets of xenogenic animal models.

[0007] On the other hand, the personalization of cancer treatment has become the most successful therapeutic approach. Due to the individuality of the individual and the particular evolution of the pathology of each organism, patients diagnosed with the same type of cancer show different responses to similar treatments. Given the improvement in the specificity of differentiating cell populations, such as cancerous and non-cancerous, the precision of the present innovation consists in developing specific and personalized aptamers for each patient. To this end, the present invention proposes a new screening technique based on microfluidic devices. SUMMARY

[0008] The present invention describes the precision of personalized targeted cancer treatment combining bioengineering and molecular biology techniques. In particular, it is embodied in microfluidic devices that replicate the individual system model of the patient, thus increasing the degree of specificity in identifying personalized therapeutic molecules.

[0009] The present invention provides a method for treating cancer, comprising modeling the patient ex vivo by culturing cancerous and non-cancerous cells of the patient, while taking into account the emergence of systemic properties for the development of personalized targeted therapies.

[0010] In one aspect, the present invention provides a method for modeling the patient systemically according to the location of the cancerous cells, taking into account the configuration and composition of the interactions between different cell populations in the device, allowing the scanning of the desired molecule.

[0011] In addition, it provides a method for screening aptamers with greater specificity and demonstrates the usefulness of the technique in targeting cancer cells with greater precision and personalization levels.

[0012] Taking into account the competition between positive and negative targets of the desired molecule and according to the composition and connection of different cell populations, the present invention also provides a method that favors the balance between the binding affinity and dissociation constant of the desired molecule in the relevant environment.

[0013] Finally, it generates a method that replaces, reduces and improves the use of preclinical development models based on animal experiments, thus allowing the prediction of preliminary results of efficacy, distribution and toxicity.

[0014] Other exemplary aspects and embodiments of the present invention are described in the following sections and appendices. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1The general structure of the microfluidic device according to the application is illustrated and the details of the substrate forming the central chamber with the permeable bottom and the scaffolding function for isolating the desired target are shown.

[0016] Figure 2 The material layers constituting the substrate are illustrated and the details of the central chamber channel for connecting the inlet and outlet are shown.

[0017] Figure 3 An example of a combination of devices for the systemic modeling of a patient from the connection between cell cultures is illustrated and the flow operation in the device controlled by a peristaltic pump is shown.

[0018] Figure 4 The connection of the devices in series and in parallel combination is illustrated. DETAILED DESCRIPTION

[0019] Glossary

[0020] Nucleic acids and oligonucleotides

[0021] Whenever used herein, the term "nucleic acid" or "oligonucleotide" refers to DNA, RNA or any "XNA" molecule, understood as any oligonucleotide with chemical modifications that do not prevent its replication by biochemical methods (such as PCR or sequential chemical synthesis). Examples of these modifications include, but are not limited to: substitution of the hydroxyl group of the nucleotides with halogen or methyl ether groups; use of chiral nucleotides; use of heterogenous nucleic acids, etc.

[0022] Target

[0023] As used herein, the term "target" refers to any entity with a desired objective. To illustrate but not limit the range of possibilities, the target can be: a desired cell population, in which case the target or molecular complex can be located in the cell surface or internal structure; or directly an organic or inorganic molecule, especially a protein, polypeptide, lipid, lipoprotein or glycoprotein.

[0024] Aptamer

[0025] The term "aptamer" refers to a non-naturally occurring nucleic acid folded into a three-dimensional structure from which a specific interaction against a target molecule is obtained. The specific interaction of the aptamer can confer effects such as binding to the target and altering the functional activity, capable of inducing activation, inhibition and promotion of the reactions between the target and other molecules.

[0026] Molecular library

[0027] The "molecular library" referred to throughout this document refers to a group of compounds comprising different numbers of different chemical substances. The library can exhibit different degrees of diversity, such as combinatorial oligonucleotide libraries. The molecular library can also have randomness, in the sense that, generally, almost all the possibilities of three-dimensional configurations given by the degrees of freedom of the molecules are realized by the ligands of the library. The library can be synthesized using various methods known to those skilled in the art, or purchased commercially from third parties.

[0028] Cancer

[0029] For "cancer", the present invention, especially in its scope of application, refers to a group of diseases that have in common uncontrolled cell division and the possibility of forming local solid tumors and metastasizing to other parts of the body. It also refers to tumors, cancer cells and malignant transformed cells, either spontaneous or from environmental factors.

[0030] Cell

[0031] As used herein, "cell" refers to a population of cells, especially but not limited to explants, punctures, biopsies, primary cultures, immortalized or primary cell lines, dissociated, tissue- or even organ-type cell cultures. It also refers especially, but not exclusively, to mammalian cells, which can or can not be derived from a specific patient.

[0032] Non-cancer cell

[0033] As used herein, "non-cancer cell", especially in its scope of application, refers to a cell in a physiological environment and not malignant transformed, which can be derived from explants, punctures, biopsies, primary cultures, immortalized or non-immortalized cell lines, dissociated, tissue- or even organ-type cell cultures. It also refers especially, but not exclusively, to mammalian cells, which can or can not be derived from a specific patient.

[0034] Species

[0035] As used herein, the term "species" refers to the different nucleic acid molecules that constitute the combinatorial library used to develop aptamers.

[0036] Explant

[0037] In the context of this document, "explant" refers to a mammalian tissue cultured in the laboratory, which can be obtained from a specific patient, for whom the present invention can be used to produce anticancer aptamers, by surgical extraction or not. It also refers to any other animal tissue cultured in the laboratory or extracted by surgery.

[0038] Fluid

[0039] "Fluid" is understood as a liquid medium circulating in a microfluidic system, which can include, but is not limited to, buffer solutions, culture media, plasma of human or animal origin.

[0040] insert

[0041] An "insert" in the present invention refers to a semi-permeable membrane used to contain and limit the cell culture without limiting the access to the co-culture of cells by fluid circulation. The present invention comprises a central chamber containing a semi-permeable insert at the bottom, which acts as an anchor or scaffold to culture the desired cells and also to immobilize to display the molecular library. The chamber in the present invention can have different sizes and shapes according to the designed scaffold.

[0042] ligand

[0043] The term "ligand" used in the present invention refers to any chemical substance that has a specific three-dimensional structure capable of binding to other chemical substances. Examples of ligands in the context of the present invention are, but not limited to: oligonucleotides, peptides and proteins.

[0044] molecule

[0045] In this context, the term "molecule" represents a group of atoms, which can be the same or different, arranged by covalent bonds. It is used as a reference to the species of composition of matter that can be transformed by chemical reactions to produce new chemical species.

[0046] microfluidics

[0047] When used to describe the present invention, "microfluidics" and its grammatical derivatives refer to the technology well known to those skilled in the art that uses devices and tools to precisely control the flow path of fluids, the proportions of solution components and their combinations in a coordinated and planned manner, related or not to any process or physical and chemical analysis, working at picometer, nanometer, micrometer or millimeter scale.

[0048] non-target or negative target

[0049] The term "non-target" or "negative target" as used herein refers to the opposite description of "target", i.e. not intended to establish an interspecies interaction description. The negative target is used to remove ligand substances with undesirable functions that interact with substances other than the target.

[0050] appropriate period

[0051] The term "appropriate period" and its linguistic variants, as used herein, refers to any specific interval of time necessary to achieve the objectives of the process. As described herein, these time periods can be known or can be determined as needed to obtain satisfactory results in certain specific applications within the scope of the present invention.

[0052] SELEX

[0053] The term SELEX is derived from the acronym of Systematic Evolution of Ligands by Exponentialenrichment, used to refer to the technique involving in vitro evolution experiments in combinatorial libraries.

[0054] substrate

[0055] A "substrate" herein refers to the material that constitutes the microfluidic device, which can be, but is not limited to, liquid crystal polymer, polydimethylsiloxane, polystyrene, low temperature co-fired ceramic, photopolymer additive manufacturing, 3D printing, etc.

[0056] method

[0057] The present invention as presented herein is embodied in microfluidic devices, systems, assemblies and methods that serve as a platform for the development of aptamers, improving the degree of molecular specificity to the desired target. In one aspect, one or more methods are provided as a tool to improve the screening of specific aptamers for relevant applications in biological complex environments. In another aspect, the method is particularly applicable, but not limited to, the iterative process of screening compounds, such as the SELEX technique for aptamer development.

[0058] According to the present invention as presented herein, it is provided that:

[0059] a) a substrate forming a channel connecting an inlet and an outlet;

[0060] b) a substrate forming a chamber with a permeable bottom and a scaffold function for cell culture;

[0061] c) a combination of substrates forming a microfluidic device unit;

[0062] d) organism modeling by modularly arranged combinations of device units in a closed system;

[0063] e) a device for guiding the circulation flow of fluids according to the desired target of positive and negative to be targeted;

[0064] f) a platform for maintaining a modular arrangement of temperature, pressure and pH values compatible with physiology;

[0065] g) a platform allowing the exposure of a library of molecules in a complex biological environment.

[0066] In the present invention, as Figure 1As shown, the device's substrate is composed of layers of different materials that remain connected through different physical and chemical treatments. When using different materials such as glass, organic silicone-based organic polymers (PDMS), photo-cured resins, co-fired ceramic devices (LTCC) or polystyrene (PS), they are bonded by exposing each layer of material to plasma, followed by immediate contact, pressing each layer against each other for a period of time. In another technical aspect, when built using photo-cured resins through 3D printing technology, light in the wide ultraviolet (UV) spectrum is used to polymerize the device. When produced through 3D printing using photo-cured resins, the device can even have its channels coated with a hydrophobic solution. Regarding the microfluidic channels, when printed with photo-cured resins, they are treated with isopropyl alcohol to keep the surface well flattened. When built using LTCC layers, the channels are washed with different concentrations of EDTA.

[0067] To minimize fluid leakage from the device during operation, especially in the connection between the insert and the device, another improvement in the normal operation of the device includes the use of a sealing ring or silicone polymer with a diameter of 10 mm and a thickness of 1.7 mm, correctly inserted into the support groove. The dimensions of the sealing ring are not limited to the aforementioned dimensions, but can vary according to the possibilities within the scope of the present invention described herein, mainly depending on the technical specifications of the insert, which can vary. The insert groove is made only of 3D printed photo-cured resin and includes a recess for accommodating the sealing ring, in order to connect the device and the insert.

[0068] In the present invention, the microfluidic device comprises a central chamber, the bottom of which contains a semi-permeable insert with a restriction or support function for the cultivation of the desired cells. In addition, in the case of exposure of a molecular library for aptamer selection, the insert is also used to perform the target immobilization. The chamber in the present invention can have different sizes and shapes, and according to the design of the insert support, the diameter ranges from 4 to 120 mm, and the depth ranges from 3 to 30 m, or according to the standard size specifications of the cell culture wells. In the present invention, the insert supports the anchoring of the semi-permeable insert to accommodate the cell culture, which can be made of polyethylene terephthalate, polycarbonate or other bioinert materials, and the insert's pores can vary between 1 and 8 μΜ.

[0069] As Figure 2The cross section formed between the channel and the central chamber, in order to direct the device fluid through the semi-permeable insert housed in the bottom into the chamber, ensures a continuous flow of the channel fluid to fill the chamber. Depending on the specific needs of the desired cell culture, different channel diameters can be used to achieve the required flow rate and local pressure, and the device is able to adapt sufficiently to these changes while maintaining the same flow rate by locally changing the caliber of the channel before each chamber, whose internal circular section can vary from 0.4 to 1.5 mm 2 are not equal.

[0070] The platform comprises devices connected by silicone tubes fixed by protrusions on the surface of the devices, where the outlet opening of one device is connected to the inlet opening of at least one other device and a peristaltic pump. As Figure 3 As shown, these devices are modularly arranged in a closed system, with the aid of a pump that directs the circulation flow, thus making the fluid continuously pushed in circulation between the devices. The target cells confined within the central chamber of the devices are co-cultured with other non-target cells, i.e. the devices remain connected in a closed system, exchanging signal molecules through the circulation fluid, that is, in addition to the fluid composition, the culture is exposed to signal molecules secreted by other cells that regulate the fluid medium in itself or in the system. The fluids used in the microfluidic devices can be, but are not limited to, phosphate saline solution, Hanks balanced salt solution, HEPES and the like buffer solutions; cell culture media such as MEM, DMEM, F-12, RPMI, adjusted to pH 7. The fluid can consist of a defined medium supplemented with growth factors or supplemented with fetal bovine serum or patient serum concentrate. The microfluidic system of the present invention comprises a pump with peristaltic movement to control the flow through the devices, which can vary continuously between 0.5 mL / min and 3 mL / min or in a programmed cycle of 5 to 180 min.

[0071] As Figure 4As shown, the devices in which the cells of interest are cultured are arranged in series and / or parallel combinations with the negative targets according to the in situ localization of the positive targets in the organism. Different combinations can be used to refer to the disposition of the cells of interest in the blood circulation flow in the individual. Broadly speaking, the method of the present invention provides for modeling the patient as an external closed system, in which the devices are arranged in different combinations for each specific individual, without the need to manufacture a specific new device. By way of example, but not limited to this example, for the development of a targeted therapy for pancreatic tumors, the flow of the device must pass through a device 1 with lung cell culture, then split into two parallel devices, one device 3 containing a culture of pancreatic cancer cells and the other device 4 containing a culture of cells surrounding the tumor or non-malignantly transformed pancreatic cells. The fluid is collected and directed to device 5 containing a culture of liver cells, then to device 6 containing a culture of bone marrow. Finally, the fluid is directed to a peristaltic pump and again to device 1.

[0072] For all the ways in which the devices can be arranged to organize the positive and negative targets, the cell cultures of interest must be limited to mobilize the fluid components, including molecules from the combinatorial library, for aptamer selection and ligand binding material recovery. For platform operation, the fluid is at a temperature of 37°C, in a humid atmosphere and 5% CO2 during the circulation through the arrangement of devices. After a sufficient time to equilibrate the binding and dissociation rates of the fluid components that circulate through the targets and establish a steady-state balance between the negative and positive targets, the nucleic acid combinatorial library is introduced to screen aptamers by the reference technique SELEX. The application of the library of molecules to screen aptamers by SELEX through the present invention can be exemplified by the library of nucleic acids. The nucleic acid library can be produced by sequential chemical synthesis and / or enzymatic reactions, consisting of about 10 9 to 10 15 Such oligonucleotides contain 70 to 100 nucleotides and have a central random region flanked by conserved regions for primer hybridization and polymerase chain reaction amplification.

[0073] After a period of exposure of the nucleic acid library, the target cell culture is collected to extract the ligand molecules. The adhered ligands can be highlighted by procedures that change the three-dimensional structure of the ligand, such as temperature (70-95°C), high concentrations of salts (such as KCl, NaCl, MgCl2, etc.), change in pH by treatment with acidic or basic solutions, denaturing agents such as urea, ethidium bromide, phenol-chloroform method, etc. or by chemical bond competition (with high concentrations of imidazole, glutathione or monoclonal antibodies of known targets). The recovered molecules are amplified by polymerase chain reaction and recombined in single-stranded form, and then they can be subjected to subsequent selection cycles or to the identification of the aptamer. When some cycles are performed, more copies of ligands that show higher affinity tend to be enriched by binding competition, and in the proposed application 3 to 8 cycles are required to obtain the aptamer. With the present application, the method embodied by the platform means a new technical effect for the SELEX technique, providing specificity for the use of aptamers in biological complexity and related environments, such as, but not limited to, personalized targeted cancer therapy. On the contrary, the application of the SELEX technique to corresponding positive and negative targets that are disconnected from the steady-state equilibrium results in an unsatisfactory level of specificity for therapeutic applications.

[0074] According to the present application, the specificity of the developed aptamer mimics the degree obtained by the SELEX technique in vivo in the animal guinea pig, with the advantage of simultaneously providing a target and an environment derived from the patient himself for therapeutic purposes.

[0075] The invention described herein provides a method for screening aptamers with high specificity by target cells:

[0076] a) on the device substrate, a support is fixed in a central chamber, which contains inserts with the appropriate porosity for limiting the cell type of interest;

[0077] b) by coupling the projections of the substrate with connectors and silicone tubes, the device is modularly arranged in a closed system by a combination of series and / or parallel connections;

[0078] c) the channel and chamber system of the device is filled with fluid at 37°C;

[0079] d) the cell culture of interest is contained in the respective central chambers of the device;

[0080] e) the fluid in the system is circulated for a time that can vary between 10 and 72 hours;

[0081] f) the molecular combinatorial library is injected into the system for circulation for 10 to 90 minutes;

[0082] g) the fluid circulating in the system is discarded and replaced at 37°C;

[0083] h) Collecting the target cell culture restricted in the device central chamber;

[0084] i) Extracting molecules from the combinatorial library bound to the target cells;

[0085] j) Amplification and recombination of the molecule set extracted from the target cells;

[0086] k) Identifying candidate molecules for aptamers.

[0087] Industrial applicability

[0088] In clinical applications, when explant samples from cancer patients are co-cultured and arrayed into targets and non-targets, the method can collect enough specificity necessary to develop personalized aptamers for the patient. Aptamers developed by the present invention against cancer cells of a given patient can be used in targeted anti-cancer therapy for that patient, for example, as carriers for radiotherapy, chemotherapy, immunotherapy, and gene therapy.

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Claims

1. A method for screening aptamers with high target specificity in a microfluidic device platform used for co-culture of cell cultures, wherein, The method is performed within an array of microfluidic devices forming a closed system for establishing a steady-state equilibrium between target and non-target cells; wherein each microfluidic device comprises a substrate having a central chamber, an inlet, and an outlet, and the method includes the following steps: (a) In the central cavity of each microfluidic device substrate, a support containing inserts, wherein the inserts are semi-permeable inserts; (b) By using a combination of series and / or parallel connections, silicone tubes are used to connect the inlet and outlet of different microfluidic device substrates, and the devices are modularly arranged into a closed system. (c) Channel and chamber systems of equipment filled with fluid at 37°C; (d) The cell cultures of interest are contained in the various central chambers of the apparatus; (e) Allow the fluid in the system to circulate for a sufficiently long time; (f) Inject the molecular combinatorial library into the system and circulate it for a period of time; (g) Discard and replace the circulating fluid in the system at 37°C; (h) Collect the target cell culture confined in the central chamber of the device; (i) Extracting molecules from a library of combinatorial molecules that bind to target cells; (j) Amplification and recombination of molecular groups extracted from target cells; (k) Identify candidate molecules for aptamers; Wherein, the cell culture of interest described in step (d) includes cancerous target cells and non-cancer non-target cells; Step (f) is performed after a steady-state equilibrium has been established between the target cells and non-target cells, as well as the fluid components flowing through the closed system.

2. The method according to claim 1, wherein, This method is based on developing targeted therapy vectors using aptamers developed for specific patients, and has applications in the delivery of drugs such as chemotherapy, radiotherapy, immunotherapy, and anticancer gene therapy.

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