Acoustic methods for transduction and transfection

Abstract

The present invention relates to an acoustophoretic method for introducing an exogenous nucleic acid (3) into a cell (2), comprising the step of continuously sweeping the frequency of a sound wave from a first frequency f1 to a second frequency f2 using a sweep time in the range of 1 ms to 100 ms, where f2 is higher than f1. The present invention also relates to a method for transducing a cell (2), to a cell obtained by the method of the present invention, and to an acoustophoretic device (1) for introducing an exogenous nucleic acid (3) into a cell (2), comprising a chamber (11), at least two inlets (121, 122), at least two outlets (131, 132), and at least one sound wave generator (14) configured to continuously sweep the frequency of a sound wave from a first frequency f1 to a second frequency f2 using a sweep time in the range of 1 ms to 100 ms, where f2 is higher than f1.

Description

[Technical field]

[0001] The present invention relates to a method and device for introducing exogenous nucleic acids into cells using acoustophoresis. In particular, the present invention relates to a method and device for the transduction or transfection of cells using acoustophoresis. The present invention also relates to modified cells obtained by said method. [Background technology]

[0002] Cellular transfection and transduction constitute important tools for understanding cell biology and therapeutic modulation of gene expression. A significant number of techniques, classified as viral (transduction) and non-viral (transfection), have been developed to introduce nucleic acids into target cells. Transduction techniques are highly effective methods for gene delivery; however, they are plagued by immune responses and insertional mutagenesis. Due to concerns about the risks of using viral vectors, transfection techniques have gradually gained attention, but the achievable transfection efficiency is not as good as transduction, and transduction is significantly cheaper than transfection.

[0003] Viral vectors such as retroviruses and adenoviruses have been shown to be efficient for transduction, however, these viral vectors are difficult to deliver and therefore expensive, and traditional transduction methods exhibit high multiplicities of infection, i.e., the viral vectors are used in large excess compared to the cells.

[0004] Non-viral techniques such as electroporation, microinjection, and lipofection have been reported. Acoustic-induced transfection, also known as sonoporation, has been developed using acoustic cavitation of microbubbles to enhance the delivery of nucleic acids. Such ultrasound-mediated transfection, which essentially relies on acoustic cavitation, has a significant drawback because cell membranes can be severely disrupted and subsequently cause lethal damage. Thus, cell viability is extremely low in conventional acoustic-induced transfection.

[0005] Furthermore, current transduction and transfection methods are not easily scalable to the large numbers of cells required in clinical protocols, are relatively costly, have low gene transfer efficiencies, and cannot be scaled up for commercialization.

[0006] Thus, there is a need for a method of gene transfer by transfection or transduction that has one or more of the following advantages: high gene transfer efficiency, low cost, low multiplicity of infection, exhibits high cell viability, and is easily scalable by allowing treatment of large numbers of cells in a short amount of time.

[0007] Applicant has discovered that this need can be met by a non-invasive acoustophoretic method for introducing exogenous nucleic acid into cells, which involves forcibly confining the cells and the exogenous nucleic acid to a defined volume by deflecting the cells into a suspension containing the nucleic acid.

[0008] In this regard, as disclosed in International Patent Publication No. 2021 / 074385, the applicant has developed an acoustophoresis device implemented for introducing exogenous nucleic acids into cells.

[0009] Although such acoustophoretic devices provide sufficient satisfaction, their implementation, known for introducing foreign nucleic acids into cells, may under certain circumstances cause fluid flow in the acoustic chamber when the fluid absorbs the acoustic energy transmitted by the sound wave generator, also known as acoustic streaming. When acoustic streaming is created, it results in a circulatory motion or vortex that can cause agitation in the fluid mixture. This phenomenon is nonlinear and disrupts the laminar flow in the chamber. Such parasitic fluid flow in the chamber may be detrimental to concentrating cells at the desired height of the chamber.

[0010] The present invention aims to improve the efficiency of methods for introducing exogenous nucleic acids into cells by better controlling the concentration of cells at a desired height of the chamber. Summary of the Invention

[0011] The present invention provides a method for introducing an exogenous nucleic acid into a cell, comprising the steps of: i. a chamber configured to be associated with at least one acoustic wave generator for generating acoustic waves within the chamber; at least one acoustic wave generator configured to generate acoustic waves in the chamber; at least two inlets in fluid communication with the chamber, including a first inlet and at least one second inlet; at least two outlets in fluid communication with the chamber, the outlets including a first outlet and at least one second outlet; Providing an acoustophoretic device comprising: ii. injecting a suspension containing exogenous nucleic acid into the chamber at a first inlet and injecting a suspension of cells into the chamber at at least one second inlet; iii. applying an acoustic field by generating acoustic waves in a chamber; iv. sequentially sweeping the frequency of the sound wave from a first frequency f1 to a second frequency f2 using a sweep time in the range of 1 ms to 100 ms, where f2 is higher than f1, such that the sound wave sequentially presents all frequencies in the range [f1, f2] from the first frequency f1 to the second frequency f2 during the sweep time; v. collecting the suspension containing the cells modified with the exogenous nucleic acid at the first outlet; The present invention relates to a method comprising the steps of:

[0012] Sequentially sweeping the frequency of the acoustic wave from a first frequency f1 to a second frequency f2 avoids secondary fluid flows within the chamber that are detrimental to the concentration of cells in the center of the chamber. In particular, this sweeping step avoids acoustic streaming within the chamber.

[0013] Acoustic waves are generated to create an acoustic pressure node in the chamber where cells congregate. A first frequency f1 and a second frequency f2 are selected such that a sweep in the range [f1, f2] results in oscillations around the acoustic pressure node.

[0014] In one embodiment, the nucleic acid is carried by a viral vector or macrovesicle. In one embodiment, the first frequency f1 and the second frequency f2 are each in the range of 0.1 MHz to 4 MHz. In one embodiment, the method further comprises a further step after step (iv) of sweeping the acoustic frequency from the second frequency f2 to a third frequency f3 using a sweep time in the range of 0.5 ms to 10 ms, where f2 is above f3. In one embodiment, the first frequency f1 is equal to the third frequency f3. In one embodiment, the power applied to the acoustic generator is in the range of 0.2 W to 50 W. In one embodiment, the generated acoustic waves are volumetric acoustic waves, surface acoustic waves, standing surface acoustic waves, multidimensional acoustic waves, or standing acoustic waves. In one embodiment, the multiplicity of infection is in the range of 0.5 to 30. In one embodiment, the cell is selected from a Chinese hamster ovary (CHO) cell, an NSO hybridoma cell, a baby hamster kidney (BHK) cell, a human cell, a regulatory T cell, a helper T cell, a cytotoxic T cell, a memory T cell, an effector T cell, a gamma delta T cell, a Jurkat T cell, a CAR-T cell, a B cell, or a NK cell, a peripheral blood mononuclear cell (PBMC), a HEK293T, algae, a plant cell, a tumor infiltrating lymphocyte (TIL), or a bacterium.

[0015] The present invention also provides a method for transducing a cell, comprising the steps of: i a chamber configured to be associated with at least one acoustic wave generator for generating acoustic waves within the chamber; at least one acoustic wave generator configured to generate acoustic waves in the chamber; at least two inlets in fluid communication with the chamber, including a first inlet and at least one second inlet; at least two outlets in fluid communication with the chamber, the outlets including a first outlet and at least one second outlet; Providing an acoustophoretic device comprising: ii. injecting a suspension of viral vectors containing nucleic acid into the chamber at a first inlet and injecting a suspension of cells into the chamber at at least one second inlet; iii. applying an acoustic field by generating acoustic waves in a chamber; iv. sequentially sweeping the frequency of the sound wave from a first frequency f1 to a second frequency f2, where f2 is higher than f1, using a sweep time in the range of 1 ms to 100 ms, such that the sound wave sequentially presents all frequencies in the range [f1, f2] from the first frequency f1 to the second frequency f2 during the sweep time; v. collecting the suspension containing the nucleic acid modified cells at the first outlet; The present invention relates to a method comprising the steps of:

[0016] In one embodiment, the first frequency f1 and the second frequency f2 are in the range of 0.1 MHz to 4 MHz, respectively. In one embodiment, the method further comprises a further step after step (iv) of sweeping the acoustic frequency from the second frequency f2 to the third frequency f3 using a sweep time in the range of 0.5 ms to 10 ms, where f2 is higher than f3. In one embodiment, the cell is selected from Chinese Hamster Ovary (CHO) cells, NSO hybridoma cells, Baby Hamster Kidney (BHK) cells, human cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, effector T cells, γδ T cells, Jurkat T cells, CAR-T cells, B cells, or NK cells, peripheral blood mononuclear cells (PBMCs), algae, plant cells, HEK293T, tumor infiltrating lymphocytes (TILs), or bacteria.

[0017] The present invention also relates to a cell obtained by the method according to the invention.

[0018] The present invention also provides an acoustophoresis apparatus for introducing an exogenous nucleic acid into a cell, comprising: a chamber configured to associate with at least one acoustic wave generator for generating acoustic waves within the chamber extending along a longitudinal axis; at least one acoustic wave generator configured to generate acoustic waves in the chamber; at least two inlets in fluid communication with the chamber, said inlets including a first inlet for introducing a suspension containing exogenous nucleic acid into the chamber and at least one second inlet for introducing a suspension of cells into the chamber; at least two outlets in fluid communication with the chamber, the outlets including a first outlet for collecting a suspension containing the cells modified with the exogenous nucleic acid and at least one second outlet; Including, at least one sound wave generator is configured to sequentially sweep the frequency of the sound wave from a first frequency f1 to a second frequency f2 using a sweep time in the range of 1 ms to 100 ms, where f2 is higher than f1, and the sound wave sequentially presents all frequencies in the range [f1, f2] from the first frequency f1 to the second frequency f2 during the sweep time; This relates to a sonophoresis device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition In the present invention, the following terms have the following meanings.

[0020] "Longitudinal axis of a chamber" refers to a line connecting the set of centers of the cross-sections of the chamber. The longitudinal axis of a chamber may be straight or curved and may be contained in a plane that may be a plane of symmetry for some or all of the cross-section of the chamber.

[0021] "Multiplicity of infection" (MOI) refers to the ratio of agent (e.g., phage or more generally virus, bacteria) to target (e.g., cell) for infection. In the present invention, multiplicity of infection refers to the number of viral vector particles per cell present in the chamber of the acoustophoresis apparatus.

[0022] "Naked nucleic acid" refers to free nucleic acid (also referred to herein as a plasmid), i.e., nucleic acid that is not contained or carried in a viral vector or microvesicle.

[0023] "Nucleic acid" refers to DNA or RNA.

[0024] "Transduction" refers to the reaction consisting of the transfer of genetic material (eg, nucleic acid such as DNA or RNA) from a donor to a recipient (eg, a cell) via a viral vector.

[0025] "Transfection" refers to a reaction consisting of the introduction of nucleic acid (eg, DNA or RNA) into a eukaryotic cell.

[0026] Detailed Description The present invention relates to a method for introducing exogenous nucleic acid into a cell.

[0027] The method comprises: i. a chamber configured to be associated with at least one acoustic wave generator for generating acoustic waves within the chamber; at least one acoustic wave generator configured to generate acoustic waves in the chamber; at least two inlets in fluid communication with the chamber, including a first inlet and at least one second inlet; at least two outlets in fluid communication with the chamber, the outlets including a first outlet and at least one second outlet; Providing an acoustophoretic device comprising: ii. injecting a suspension containing exogenous nucleic acid into the chamber at a first inlet and injecting a cell suspension into the chamber at at least one second inlet; iii. applying an acoustic field by generating acoustic waves in a chamber; iv. sequentially sweeping the frequency of the sound wave from a first frequency f1 to a second frequency f2 using a sweep time in the range of 1 ms to 100 ms, where f2 is higher than f1, such that the sound wave sequentially presents all frequencies in the range [f1, f2] from the first frequency f1 to the second frequency f2 during the sweep time; v. collecting the suspension containing the cells modified with the exogenous nucleic acid at the first outlet; Includes.

[0028] The method of the present invention uses an acoustophoretic device (described herein below) that includes a sound wave generator(s) for transfection or transduction of cells. The acoustophoretic device is used to bring cells into close proximity with foreign nucleic acid (DNA or DNA or both), i.e. by confining the cells and foreign nucleic acid in the same fluid layer. This increases the probability of contact between the cells and the foreign nucleic acid, thus facilitating transfer of the nucleic acid into the cells. This confinement of the cells with the foreign nucleic acid is advantageously performed using a non-invasive and low-pressure method to enhance transduction or transfection.

[0029] A suspension containing the exogenous nucleic acid and a suspension containing the cells are injected at separate inlets and flow through the chamber with laminar flow, resulting in at least two layers of fluid flowing through said chamber without mixing.

[0030] The chamber extends along a longitudinal axis and comprises two opposing walls extending along the longitudinal axis (herein referred to as longitudinal walls), the two opposing walls being perpendicular to said longitudinal axis (herein referred to as transverse walls), the longitudinal walls comprising an entry wall, i.e. the wall of the chamber coupled to the sound generator, and an opposing wall. The first inlet is preferably located equidistant from these two longitudinal walls, such that the suspension containing the exogenous nucleic acid injected at said first inlet forms a central layer of flow in the chamber.

[0031] Acoustophoresis is used for the manipulation of particles in a fluid using sound waves. The application of an acoustic field in a chamber by generating sound waves using a sound wave generator induces localized regions of high and low pressure that are created in the chamber, in particular an acoustic pressure node is created in the center of the chamber (depending on the selected frequency at which the sound wave generator operates), a focusing surface is located on the acoustic pressure node, and an acoustic radiation force (ARF) is created in the chamber. This force exerted on the particles is also dependent on their size, the larger the particles, the greater the force exerted. The magnitude of the force depends on the density of the particles and their compressibility with respect to the fluid medium, and increases with the volume of the particles. This results in the movement of the particles present in the chamber according to their size.

[0032] In the method of the present invention, cells with micrometer size undergo much larger ARF than foreign nucleic acid with nanometer size. Thus, only cells move due to their micrometer size by applying acoustic field, whereas foreign nucleic acid is not affected by the application of acoustic field due to its small nanometer size. This allows only cell deflection in the chamber. Cells are confined to a small fluid volume where nucleic acid is already present, without contact or pressure, and with continuous flow to transduce or transfect them. Advantageously, dilution of foreign nucleic acid in a larger fluid volume is avoided, thereby reducing the chance of nucleic acid / cell encounter.

[0033] Upon application of the acoustic field in the chamber, the ARF typically pushes the cells toward the central pressure node with a force equivalent to 100 times gravity, a force greater than the combined action of fluid drag and gravity, so that the cells are deflected from their initial suspension into the central layer of fluid, i.e., the suspension containing the exogenous nucleic acid. This forced confinement of the cells and the exogenous nucleic acid in the same fluid layer greatly facilitates the transfer of the exogenous nucleic acid into the cells, resulting in modified cells.

[0034] Thus, the cell-containing suspension is modified with exogenous nucleic acid and then flows to a first outlet and the cell-depleted suspension flows to at least one second outlet for collection.

[0035] The present invention relies on the spontaneous bias of cells into a suspension containing exogenous nucleic acid in a continuous flow suspension, which is particularly advantageous since it allows for better transfer of said exogenous nucleic acid into cells, e.g., enhanced transduction efficiency when the exogenous nucleic acid is contained in a viral vector, or enhanced transfection efficiency in the case of naked exogenous nucleic acid.

[0036] In the methods of the invention, both the cells and the exogenous nucleic acid flow through the chamber with a continuous fluid stream (same or different flow rates), so that the methods of the invention do not rely on gravitational settling to collect the modified cells, i.e., the cells do not form clusters that settle from the acoustic field to the bottom of the chamber. Advantageously, the same chamber can be used to treat small or large volumes of cells.

[0037] The continuous sweeping of the frequency of the acoustic wave from the first frequency f1 to the second frequency f2 avoids secondary fluid flow in the chamber, which is detrimental to the concentration of cells in the center of the chamber. In particular, this sweeping step avoids acoustic streaming in the chamber, which refers to the fluid flow that occurs in the acoustic chamber when the fluid absorbs the acoustic energy transmitted by the sound wave generator. When acoustic streaming is created, it results in a circulating motion or vortex that can cause agitation in the fluid mixture. This phenomenon is nonlinear and disrupts the laminar flow in the chamber. In the method of the present invention, the frequency is preferably about 1 MHz, and acoustic streaming is essentially considered to be a frequency below 1 MHz. The circulating motion in the chamber resulting from acoustic streaming preferentially disrupts the smallest biological objects, i.e., foreign nucleic acid, disrupts the laminar flow of the fluid layer, and dilutes the foreign nucleic acid in the chamber. This leads to poor proximity between cells and foreign nucleic acid. Therefore, the continuous frequency sweep is important to avoid the occurrence of this phenomenon.

[0038] Advantageously, the method of the present invention has a limited number of steps and is easy to perform, which also exhibits reduced costs, increased transduction or transfection efficiency, high cell viability, and low MOI.

[0039] In other words, the method of the present invention is a method for enhancing the introduction of exogenous nucleic acid into cells.

[0040] Protein expression is measured, for example by Western blot or immunostaining, to confirm successful introduction of the foreign nucleic acid into the cells.

[0041] In one embodiment, in step (iv), the frequency of the sound wave is swept continuously from a first frequency f1 to a second frequency f2 using a sweep time in the range of 1 ms to 100 ms, i.e. the frequency of the sound wave varies continuously from the first frequency f1 to the second frequency f2 such that all frequencies in the range [f1-f2] are swept.

[0042] In one embodiment, the first frequency f1 and the second frequency f2 are each in the range of 0.1 MHz to 4 MHz, preferably 0.5 MHz to 2 MHz, more preferably 0.8 MHz to 1.5 MHz, and even more preferably 0.9 MHz to 1.1 MHz.

[0043] Low frequencies, for example around 1 MHz, are particularly suitable for large cells. The thickness of the channel is inversely proportional to the frequency, and low frequencies allow for thicker channels and therefore higher throughput. For example, for cells with a diameter larger than 20 μm, frequencies less than or equal to 1 MHz show good deflection efficiency.

[0044] In a preferred configuration of this embodiment, the first frequency f1 is in the range of 0.5 MHz to 1.5 MHz, more preferably 0.8 MHz to 1.2 MHz, and even more preferably 0.9 MHz to 1 MHz.

[0045] In another preferred configuration of this embodiment, the second frequency f2 is in the range of 0.9 MHz to 2 MHz, more preferably 1 MHz to 1.5 MHz, and even more preferably 1.05 MHz to 1.2 MHz.

[0046] In another preferred configuration of this embodiment, the range [f1-f2] is the resonant frequency f res is concentrating on.

[0047] In one embodiment, step (iv) is performed 100 to 1000 times per second, preferably 250 to 750 times per second, more preferably 400 to 600 times per second. Advantageously, repeating step (iv) reduces the risk of acoustic streaming.

[0048] In a preferred configuration of this embodiment, step (iv) is performed approximately 500 times per second.

[0049] In one embodiment, the method further comprises a further step (called step (iv')) after step (iv) of continuously sweeping the frequency of the sound wave from a second frequency f2 to a third frequency f3 using a sweep time in the range of 0.5 ms to 10 ms, where f2 is higher than f3, and the sound wave continuously presents all frequencies in the range [f2, f3] from the second frequency f2 to the third frequency f3 during the sweep time. Thus, the frequency of the sound wave continuously varies from the second frequency f2 to the third frequency f3 such that all frequencies in the range [f2-f3] are swept.

[0050] A preferred configuration of this embodiment sweeps all frequencies in a defined range, ie, [f1-f2], then [f2-f3], successively, back and forth over a predefined sweep time.

[0051] According to one embodiment, the third frequency f3 is in the range of 0.1 MHz to 4 MHz, preferably 0.5 MHz to 2 MHz, more preferably 0.8 MHz to 1.5 MHz, even more preferably 0.9 MHz to 1.1 MHz.

[0052] According to one embodiment, the first frequency f1 is equal to the third frequency f3. In this embodiment, a further step (iv') corresponds to a return sweep or frequency sweep using a sweep time the same as or different from the sweep time in step (iv).

[0053] According to one embodiment, the sweep time of the further step (iv') is equal to or different from the sweep time of step (iv). In a preferred configuration of this embodiment, the sweep time of the further step (iv') is equal to the sweep time of step (iv).

[0054] According to one embodiment, the sweep rate of steps (iv) and (iv') is in the range of 1 kHz to 10 kHz, preferably 5 kHz to 10 kHz. Advantageously, a small sweep rate allows a more effective acoustic streaming suppression.

[0055] According to one embodiment, the residence time of the cells in the chamber is in the range of 1 second to 60 seconds, preferably 5 seconds to 30 seconds, more preferably 5 seconds to 15 seconds. The method of the present invention is advantageously faster than conventional transduction methods, where the transfer of nucleic acid into cells can take from 10 minutes to several hours. Thus, in the same time as conventional transduction methods, e.g. 10 minutes, a larger number of cells are modified using the method of the present invention. For example, the method of the present invention allows treating (i.e. transducing or transfecting) 50,000 to 150,000 cells per minute.

[0056] According to one embodiment, the nucleic acid is carried in a viral vector or a microvesicle, in this embodiment, the method of the invention is a transduction method.

[0057] In a specific configuration of this embodiment, the viral vector is selected from retroviruses, such as adenoviruses, paramyxoviruses, or lentiviruses. Retroviruses are characterized by their ability to reverse transcribe their RNA genome into cDNA copies, which are then stably integrated into the genome of host cells. In this embodiment, the method for introducing foreign nucleic acid into cells is a method for transducing cells, in which the viral vector carries nucleic acid into cells by entering the cell itself or by inserting nucleic acid into the cell.

[0058] Examples of retroviruses include simple and complex retroviruses (e.g., lentiviruses), which contain two copies of positive-stranded RNA with associated viral reverse transcriptase (RT) located within an inner core along with structural and enzymatic proteins including nucleocapsid (NC), capsid (CA), integrase (IN), and protease (PR), which is surrounded by an outer protein layer including matrix (MA) protein and encapsulated in interspersed envelope glycoproteins (ENV).

[0059] Lentivirus and retrovirus gene delivery vectors utilize retrovirus replication to provide stable integration of desired nucleic acid sequences.Transfection of foreign nucleic acid only results in transient transgene expression, but the activity of viral integrase in retrovirus and lentivirus-based systems allows stable integration of foreign transgenes, which are then inherited and continuously expressed during repeated cell divisions.The important characteristic of both lentivirus and retrovirus vectors is that they produce replication-deficient or self-inactivating particles.This allows delivery of desired sequences without continuing viral replication in target cells.

[0060] According to another embodiment, the nucleic acids are naked nucleic acids (also referred to herein as plasmids), i.e. they are not contained or carried by a viral vector. In this embodiment, the method for introducing an exogenous nucleic acid into a cell is a method for transfection of the cell, where the naked nucleic acid enters the cell.

[0061] According to one embodiment, the cell is selected from a Chinese hamster ovary (CHO) cell, an NSO hybridoma cell, a baby hamster kidney (BHK) cell, a human cell, a regulatory T cell, a helper T cell, a cytotoxic T cell, a memory T cell, an effector T cell, a gamma delta T cell, a Jurkat T cell, a CAR-T cell, a B cell, or a NK cell, a peripheral blood mononuclear cell (PBMC), a HEK293T, an algae, a plant cell, a tumor infiltrating lymphocyte (TIL), or a bacterium.

[0062] According to one embodiment, the cells and the nucleic acid are suspended in the same fluid medium, in particular in a buffer. In a preferred configuration of this embodiment, the medium is Roswell Park Memorial Institute medium (RPMI) containing 10% FBS fetal bovine serum and 1% penicillin / streptomycin.

[0063] The buffer is selected for its acoustic and fluid qualities, and its compatibility with cells and nucleic acids. The buffer containing the nucleic acid is designed to remain in the focal plane of the chamber, for example by adding a solute or colloidal suspension to reduce turbulence, or by diluting it by adding a solvent such as water. The buffer is also designed to reduce the chaotic exchange of chemicals and particle types with the suspension.

[0064] According to one embodiment, the buffer can be, but is not limited to, CPD (Citrate-Phosphate-Dextrose), SSP+, RPMI medium, LB (Lysogeny Broth), DMSO (Dimethyl Sulfoxide), methylcellulose, HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid), PBS (Phosphate Buffered Saline), CMRL medium (containing phenol red), DMEM (Dulbecco's Modified Eagle's Medium), or a mixture thereof.

[0065] According to one embodiment, the buffer is configured to avoid accidentally modifying the cells that must be deflected, for example by avoiding chemical compounds that are undesirable for the cells, by reducing the osmotic forces applied to the cells in the buffer, and / or by maintaining a suitable pH range for the cells.

[0066] According to one embodiment, the multiplicity of infection is in the range of 0.5 to 30, preferably 0.5 to 20, more preferably 0.5 to 5, even more preferably 0.7 to 1. Methods for transduction at low multiplicity of infection are particularly advantageous since fewer viral vectors are used with high transfection efficiency. As mentioned, viral vectors can be difficult to provide and are expensive commodities. It is particularly interesting to avoid wasting viral vectors by bringing them closer to the number of cells. For example, compared to typical transduction, the methods of the invention show at least a 20% increase in transduction efficiency using 10-fold fewer viral vectors.

[0067] In a preferred configuration of this embodiment, the multiplicity of infection is 1, ie, one viral vector is used per cell.

[0068] According to one embodiment, the number of cells treated by the method of the invention is in the range of 50 000 cells / min to 150 000 cells / min. In a particular configuration of this embodiment, the number of cells treated by the method of the invention is in the range of 50 000 cells / min to 150 000 cells / min at an injection rate of 0.5 ml / min for the suspension of cells. Thus, the method of the invention is easily scalable to treat large numbers of cells as required by clinical protocols.

[0069] According to one embodiment, the suspension of cells and / or the suspension containing the exogenous nucleic acid further comprises an additive, such as polybrominated hexadimethrine, a polypeptide consisting of three functional domains (C domain, H domain, and CS-1 site) derived from human fibronectin protein, lipofectin, lipofectamine, or a cationic peptide, such as protamine. The first additive is used to increase the efficiency of transduction of certain cells by retroviruses (and lentiviruses) in cell culture, and is typically used in an amount of 10 pg / ml or less. The second additive enhances the efficiency of retrovirus- and lentivirus-mediated transduction in hematopoietic cells, including hematopoietic stem cells and terminally differentiated cells, such as primary T cells and macrophages.

[0070] According to one embodiment, the generated acoustic waves are volumetric acoustic waves, surface acoustic waves, standing surface acoustic waves, multi-dimensional acoustic waves, or standing acoustic waves.

[0071] According to one embodiment, only one sound pressure node is created within the chamber upon application of the sound field, preferably said sound pressure node is located in the center of the chamber.

[0072] In a particular configuration of this embodiment, the sound pressure node is located at mid-height (e / 2, which is the thickness of the chamber).

[0073] According to one embodiment, the power applied to the acoustic wave generator is in the range of 0.2W-50W, preferably 0.2W-25W, more preferably 0.5W-10W, even more preferably 1W-4W.

[0074] In a preferred configuration of this embodiment, the power applied to the acoustic generator is 2W.

[0075] According to one embodiment, the infusion and collection rates are in the range of 0.1 ml / min to 100 ml / min, both rates depending on the species involved (cells, viral vectors).

[0076] In certain configurations of this embodiment, the collection rate is in the range of 0.1 ml / min to 10 ml / min, more preferably 0.5 ml / min to 5 ml / min, and most preferably 0.5 ml / min to 1.5 ml / min.

[0077] In a particular configuration of this embodiment, the injection rate of the suspension of cells and the injection rate of the suspension containing exogenous nucleic acid are in the range of 0.1 ml / min to 100 ml / min. Preferably, the injection rate of the suspension of cells and the injection rate of the suspension containing exogenous nucleic acid are in the range of 0.1 ml / min to 10 ml / min, more preferably 0.5 ml / min to 5 ml / min, and most preferably 0.5 ml / min to 1 ml / min.

[0078] In a particular configuration of this embodiment, the acoustophoresis device includes two inlets (a first inlet and a second inlet), and the injection rates of the suspension containing the cells and the suspension containing the exogenous nucleic acid are equal.

[0079] In another configuration of this embodiment, the acoustophoresis device comprises three inlets, a first central inlet and two second peripheral inlets, and the injection rate of the suspension containing exogenous nucleic acid is higher than the injection rate at the two second peripheral inlets. In particular, the ratio between the injection rate of the suspension containing exogenous nucleic acid and the injection rate at each of the two second peripheral inlets is in the range of 1.2 to 2, preferably 1.6 to 1.9, and more preferably corresponds to 1.8.

[0080] The present invention also provides a method for transducing a cell, comprising the steps of: i. a chamber configured to be associated with at least one acoustic wave generator for generating acoustic waves within the chamber; at least one acoustic wave generator configured to generate acoustic waves in the chamber; at least two inlets in fluid communication with the chamber, including a first inlet and at least one second inlet; at least two outlets in fluid communication with the chamber, the outlets including a first outlet and at least one second outlet; Providing an acoustophoretic device comprising: ii. injecting a suspension of viral vectors containing nucleic acid into the chamber at a first inlet and injecting a suspension of cells into the chamber at at least one second inlet; iii. applying an acoustic field by generating acoustic waves in a chamber; iv. sequentially sweeping the frequency of the sound wave from a first frequency f1 to a second frequency f2, where f2 is higher than f1, using a sweep time in the range of 1 ms to 100 ms, such that the sound wave sequentially presents all frequencies in the range [f1, f2] from the first frequency f1 to the second frequency f2 during the sweep time; v. collecting the suspension containing the nucleic acid modified cells at the first outlet; The present invention relates to a method comprising the steps of:

[0081] All the preceding embodiments and advantages apply to this transduction method.

[0082] This transduction method uses an acoustophoretic device, described hereinafter, to effect cell transduction.

[0083] According to one embodiment, the first frequency f1 and the second frequency f2 are each in the range of 0.1 MHz to 4 MHz.

[0084] According to one embodiment, the method further comprises a further step after step (iv) of sweeping the acoustic frequency from a second frequency f2 to a third frequency f3 using a sweep time in the range of 0.5 ms to 10 ms, called step (iv'), where f2 is above f3.

[0085] In one embodiment, the cell is selected from a Chinese hamster ovary (CHO) cell, an NSO hybridoma cell, a baby hamster kidney (BHK) cell, a human cell, a regulatory T cell, a helper T cell, a cytotoxic T cell, a memory T cell, an effector T cell, a gamma delta T cell, a Jurkat T cell, a CAR-T cell, a B cell, or a NK cell, a peripheral blood mononuclear cell (PBMC), algae, a plant cell, HEK293T, a tumor infiltrating lymphocyte (TIL), or a bacterium.

[0086] The present invention also provides a method for transfecting a cell, comprising the steps of: i. a chamber configured to be associated with at least one acoustic wave generator for generating acoustic waves within the chamber; at least one acoustic wave generator configured to generate acoustic waves in the chamber; at least two inlets in fluid communication with the chamber, including a first inlet and at least one second inlet; at least two outlets in fluid communication with the chamber, the outlets including a first outlet and at least one second outlet; Providing an acoustophoretic device comprising: ii. injecting a suspension of plasmids into the chamber at a first inlet and injecting a suspension of cells into the chamber at at least one second inlet; iii. applying an acoustic field by generating acoustic waves in a chamber; iv. sequentially sweeping the frequency of the sound wave from a first frequency f1 to a second frequency f2, where f2 is higher than f1, using a sweep time in the range of 1 ms to 100 ms, such that the sound wave sequentially presents all frequencies in the range [f1, f2] from the first frequency f1 to the second frequency f2 during the sweep time; v. collecting the suspension containing the plasmid-modified cells at the first outlet; The present invention relates to a method comprising the steps of:

[0087] All the preceding embodiments and advantages apply to this transduction method.

[0088] This transfection method uses an acoustophoretic device, described hereinafter, to effect the transfection of cells.

[0089] Traditional transfection methods create holes in the cell's plasma membrane to allow nucleic acids to enter the cell. Typically, cells are subjected to electroporation, in which the cells are exposed to an electric field to increase the permeability of the cell membrane, or sonoporation, in which the cells are exposed to ultrasound to induce pore formation in the cell membrane.

[0090] The present invention also relates to a cell obtained by any one of the methods of the invention.

[0091] The present invention also provides an acoustophoresis apparatus for introducing an exogenous nucleic acid into a cell, comprising: a chamber configured to be associated with at least one acoustic wave generator for generating acoustic waves within the chamber extending along a longitudinal axis; at least one acoustic wave generator configured to generate acoustic waves in the chamber; at least two inlets in fluid communication with the chamber, said inlets including a first inlet for introducing a suspension containing exogenous nucleic acid into the chamber and at least one second inlet for introducing a suspension of cells into the chamber; at least two outlets in fluid communication with the chamber, the outlets including a first outlet for collecting a suspension containing the cells modified with the exogenous nucleic acid and at least one second outlet; Including, at least one sound wave generator is configured to sequentially sweep the frequency of the sound wave from a first frequency f1 to a second frequency f2, f2 being higher than f1, using a sweep time in the range of 1 ms to 100 ms, such that the sound wave sequentially presents all frequencies in the range [f1, f2] from the first frequency f1 to the second frequency f2 during the sweep time; This relates to a sonophoresis device.

[0092] The acoustophoretic device is configured to implement any of the methods of the present invention.

[0093] According to one embodiment, the at least two inlets are located at one end of the chamber and the at least two outlets are located at the other end along the longitudinal axis of the chamber.

[0094] According to one embodiment, the first inlet and the first outlet are aligned along a focusing plane created at the mid-height of the chamber upon application of the acoustic field, said first inlet and said first outlet being hereinafter referred to as the "central inlet" and the "central outlet", respectively.

[0095] According to one embodiment, the at least one second inlet and the at least one second outlet are not aligned along a focusing plane created at the mid-height of the chamber upon application of the acoustic field, said at least one second inlet and said at least one second outlet being hereinafter referred to as the "peripheral inlet" and the "peripheral outlet", respectively.

[0096] According to one embodiment, an acoustophoretic device includes two inlets, a first inlet and a second inlet, and two outlets, a first outlet and a second outlet, where the first inlet and the first outlet are aligned along a focusing plane and are referred to as the "central inlet" and the "central outlet", respectively, and the second inlet and the second outlet are not aligned along a focusing plane and are referred to as the "peripheral inlet" and the "peripheral outlet", respectively.

[0097] According to one embodiment, the acoustophoretic device includes three inlets, a first central inlet and two second peripheral inlets, and three outlets, a first central outlet and two second peripheral outlets. The first inlet and the first outlet are aligned along a focusing plane and are referred to as "central inlet" and "central outlet", respectively, and the second inlet and the second outlet are not aligned along a focusing plane and are referred to as "peripheral inlet" and "peripheral outlet", respectively. Furthermore, the second inlets are each located on either side of the longitudinal axis of the chamber, and the second outlets are each located on either side of the longitudinal axis of the chamber. In other words, the second outlets are located opposite each other along the longitudinal axis, and the second outlets are located opposite each other along the longitudinal axis.

[0098] The sound wave generator is configured to emit sound waves, said sound waves having a defined amplitude and a defined frequency, said sound wave generator being located between the first inlet and the first outlet. The sound waves allow the cells to move from their initial suspension into the suspension containing the exogenous nucleic acid, resulting in an enhanced transduction or transfection response by confining the cells to the same fluid layer as the exogenous nucleic acid.

[0099] According to one embodiment, the acoustic wave generator is configured to generate bulk acoustic waves (BAW), i.e. volumetric acoustic waves that propagate in three dimensions, i.e. acoustic waves that propagate between the acoustic wave generator and the chamber through the bulk of the coupling material. Generating bulk acoustic waves in the chamber advantageously allows for the treatment (i.e. transduction or transfection) of large volumes of cell suspensions, and allows for efficient and easy temporary coupling between the acoustic wave generator and the chamber.

[0100] At least one acoustic wave generator does not generate a surface acoustic wave (SAW), a standing surface acoustic wave (SSAW), or a traveling surface acoustic wave (TSAW). A surface acoustic wave (SAW) refers to an acoustic wave that propagates along a surface between the acoustic wave generator and the chamber. A surface acoustic wave (SSAW) refers to a SAW that oscillates in time but whose peaks do not propagate in space. A traveling surface acoustic wave (TSAW) refers to a SAW whose peaks propagate in space. The use of SSAW limits the primary coupling capability between the acoustic wave generator and the chamber and the volume that can be processed, making it a poor candidate for the production of suspensions that are susceptible to contamination. The use of traveling waves (e.g., TSAW) renders the device of the present invention ineffective.

[0101] According to one embodiment, the acoustic wave generator is a piezoelectric transducer, which may be made of ceramics, such as lead zirconate, titanate, potassium niobate, or sodium tungstate; crystals, such as quartz, tourmaline, or gallium phosphate; III-V or II-VI semiconductors, such as gallium nitride or zinc oxide; polymers, such as polyvinylidene fluoride, polyvinylidene chloride, or polyimide; or mixtures thereof.

[0102] In a particular configuration of this embodiment, the transducer is a longitudinal resonator, which allows for efficient and easy implementation of a transient coupling between the transducer and the chamber.

[0103] In one embodiment, the acoustic wave generator is configured to emit acoustic waves at a frequency in the range of 0.1 MHz to 4 MHz, preferably 0.5 MHz to 2 MHz, more preferably 0.8 MHz to 1.5 MHz, even more preferably 0.9 MHz to 1.1 MHz. The frequency of the generated waves is selected depending on the particles moving in the suspension, in particular said frequency is selected based on the size of the particles to be deflected.

[0104] In one embodiment, the acoustic generator is external to the chamber.

[0105] In one embodiment, the acoustic wave generator generates an acoustic force field across the thickness of the chamber rather than the width, this embodiment is particularly advantageous as it allows for the formation of a fluid layer along the thickness of the chamber.

[0106] In one embodiment, the acoustic waves have an angle of incidence relative to the longitudinal axis of the chamber in the range of 85° to 95°, for example 89° to 91°.

[0107] In one embodiment, the acoustic waves have an angle of incidence relative to the longitudinal axis of the chamber of substantially 90°.

[0108] According to one embodiment, the acoustic generator is longitudinally coupled to a wall of the chamber, in particular to a longitudinal wall, in other words, the acoustic generator extends along the same longitudinal axis as the chamber.

[0109] According to one embodiment, the acoustic generator is temporarily coupled to the chamber. Temporarily coupled means releasably coupled. This embodiment allows the use of a non-disposable acoustic generator and a disposable chamber, thereby still ensuring sterility of the chamber, which is a fundamental point in the handling of biological objects. The acoustic generator is not glued to the chamber, for example using epoxy resin.

[0110] In a preferred configuration of this embodiment, the temporary coupling is provided by using a coupling layer between the wall of the chamber and the acoustic generator. This advantageously prevents the presence of air bubbles between the chamber and the acoustic generator and ensures improved contact between the chamber and the acoustic generator. The coupling layer allows for a much more efficient transfer of acoustic energy between the acoustic generator and the chamber.

[0111] According to one embodiment, said coupling layer is made from a thermoplastic elastomer, a thermoplastic polyurethane or a silicone.

[0112] According to one embodiment, said coupling layer is made from an oil or a mixture containing an oil.

[0113] In particular, examples of coupling layers include, but are not limited to, liquids or gels, such as water, hydrogels, vegetable oils, mineral oils, greases, polymers, derivatives thereof, or mixtures thereof. The coupling elements should be as thin and homogenous as possible, i.e., free of any bubbles or microbubbles within the coupling elements or on the contact surfaces.

[0114] In one embodiment, the chamber extends along a longitudinal direction and has a cross-section having a width measured along a first transverse axis and a thickness measured along a second transverse axis perpendicular to the first transverse axis, the width being greater than or equal to the thickness, and the chamber has a first wall and a second wall along the second transverse axis.

[0115] According to one embodiment, the chamber has a rectangular shape, a cylindrical shape, or other shape. In a preferred configuration of this embodiment, the chamber is a rectangular parallelepiped characterized by a length (along the longitudinal axis A1), a width, and a thickness, which makes it easier to apply sound waves to the parallelepiped without disturbance than any other shape. The chamber may also be referred to herein as a "channel."

[0116] According to one embodiment, the chamber is not a microfluidic channel, and thus the device of the present invention is not a microfluidic device. The use of non-microfluidic channels allows to significantly and easily increase the flow rate in the chamber, thus reducing the processing time for a given volume while keeping the shear rate low enough so that the particles are not damaged during the process, and thus allowing the device to be used to provide cell therapy treatments.

[0117] According to one embodiment, the chamber has a width of 1 mm to 50 mm, preferably 5 mm to 20 mm, more preferably 5 mm to 15 mm, the width being the diameter if the chamber is cylindrical.

[0118] According to one embodiment, the chamber has a thickness in the range of 150 μm to 2 mm, preferably 300 μm to 780 μm.

[0119] According to one embodiment, the chamber has a length in the range of 5 cm to 20 cm, preferably 5 cm to 15 cm.

[0120] In one embodiment, the acoustic wave has a wavelength λ and the thickness of the chamber is substantially a multiple of λ / 4.

[0121] According to one embodiment, the chamber includes an inner wall made from a material that has an acoustic impedance greater than the acoustic impedance of a fluid flowing through said chamber.

[0122] In a particular configuration of this embodiment, the chamber or the inner wall of said chamber is made of a material selected from organic or inorganic polymers; metals; gels, such as hydrogels; glasses, such as quartz glass, borosilicate glass, crystals, such as silicon; ceramics, such as silicon carbide; resins; derivatives thereof or mixtures thereof. The material of the chamber or its inner walls should be easy to sterilize after use.

[0123] Examples of organic polymers include, but are not limited to, poly(methyl methacrylate) (PMMA), polyurethane, silicone, polyethylene, polymethylpentene, polystyrene, polycarbonate, polydimethylsiloxane, medical grade polymers, or disposable plastics.

[0124] Examples of metals include, but are not limited to, steel or stainless steel. Advantageously, stainless steel allows for good transmission of acoustic energy to the fluid in the chamber. Additionally, steel walls may act as reflectors in the device.

[0125] Advantageously, PMMA has an acoustic impedance two times higher than fluids, and stainless steel has an acoustic impedance 40 times higher than fluids. Furthermore, PMMA is advantageously compatible with all cells and is optically transparent, i.e., allowing one to see the interior of the chamber, and thus the fluids therein.

[0126] The device of the present invention does not include a reflector configured to reflect sound waves in the chamber, it uses the layer of free air outside the chamber as a potential reflector.

[0127] According to one embodiment, the chamber is sterile.

[0128] According to one embodiment, the device is disposable. The device is normally in contact with fluids that may become contaminated. Being disposable ensures good hygiene, regulatory compliance, and saves time. In this embodiment, the system is replaced after use.

[0129] According to another embodiment, the device and all its parts are sanitizable, in which embodiment, the device can be sanitized using a bath wash, a sanitizing wipe, or any other means known to those skilled in the art.

[0130] In one embodiment, the device is a portable device, in which the dimensions of the device and the connections between the parts of the device allow for easy transportation of the device.

[0131] According to one embodiment, the device further comprises at least one pump means connected to the chambers. The pump means is configured to manage the flow of fluids in the device, i.e. in the chambers. In particular, the pump means is capable of applying a controlled and constant flow rate at each inlet and / or chamber of the device, advantageously allowing the maintenance of sterility of the chambers. In this embodiment, the pump means is a mechanical pump, preferably the pump means is a peristaltic pump. In a particular configuration, each inlet is connected to one pump means.

[0132] According to one embodiment, the acoustophoresis device further comprises a flow rate sensor configured to measure and / or control the flow rate in the chamber, which allows for checking and adjusting the flow rate so that the flow of fluid in the chamber is as smooth as possible in accordance with Poiseuille flow.

[0133] In a particular configuration of this embodiment, the flow sensor is a non-contact flow sensor, i.e., the sensor does not come into contact with any fluid in the chamber. This is particularly advantageous as non-contact flow rate measurement / monitoring ensures sterility of the devices and fluids herein.

[0134] In one embodiment, the acoustophoretic device further comprises a pressure sensor.

[0135] In one embodiment, the acoustophoresis device further comprises a concentration sensor for measuring the concentration of cells and / or nucleic acids in the chamber.

[0136] In one embodiment, the device further includes an electronic controller configured to monitor the flow rate of the fluid in the chamber based on data collected from the pressure sensor, the concentration sensor, and / or the flow rate sensor.

[0137] In one embodiment, the device further comprises a temperature sensor and a temperature control system configured to monitor and control the temperature of the fluid flowing in the chamber. In particular, the temperature control system is configured to ensure that said temperature remains below a threshold value. This embodiment is particularly advantageous because it ensures that the chamber does not heat up during activation of the sound wave generator (which results in heat), and thus the fluid in the chamber does not heat up. This protects the cells and nucleic acids in the chamber from degradation due to excessive heat or unexpected activation.

[0138] In a particular configuration of this embodiment, said threshold value is 35°C, preferably 30°C, more preferably 25°C, and even more preferably 20°C.

[0139] In a particular configuration of this embodiment, the temperature control system is a cooling system, such as, for example, a Peltier cooling system or a water cooling system.

[0140] In one embodiment, the acoustophoretic device includes multiple acoustic wave generators, preferably the device includes 2, 3, or 4 acoustic wave generators. This embodiment is advantageous because it allows for good force distribution along the chamber. The acoustic wave generators are preferably aligned along the longitudinal axis (A1) of the chamber.

[0141] In a particular configuration of this embodiment, the acoustic waves generated by the acoustic wave generators may be the same or different.

[0142] The use of multiple acoustic generators is advantageous when the fluid is flowing at high velocities. In the first instance, the fluid velocity increases as the time of flight under the acoustic generator decreases. This may require multiple acoustic generators to be used to achieve focusing.

[0143] When multiple acoustic wave generators are used, at least one of them may generate acoustic waves along the width of the chamber.

[0144] In one embodiment, the device includes multiple chambers, each one connected to an acoustic wave generator. The chambers are aligned in parallel. In this embodiment, several volumes may be simultaneously treated with acoustic waves in the chambers before being collected, ensuring that a large amount of cells can be treated in a short time.

[0145] In one embodiment, fluid communication between different parts of the device includes any means known to those skilled in the art, such as flexible manifolds or tubing and clamps or valves, etc. Their representation in the drawings is not representative of their dimensions and positions. [Brief description of the drawings]

[0146] [Figure 1] FIG. 1 is a schematic diagram of the application of an acoustic field to cells within a chamber. [Figure 2A] FIG. 2A is a schematic diagram of an acoustophoresis device 1 according to a first embodiment of the present invention. [Figure 2B] FIG. 2B is a schematic diagram of the acoustophoresis apparatus 1 illustrated in FIG. 2A in which cells 2 and exogenous nucleic acid flow within a chamber 11. [Figure 3A] FIG. 3A is a schematic diagram of an acoustophoresis device 1 according to a second embodiment of the present invention. [Figure 3B] FIG. 3B is a schematic diagram of the acoustophoresis apparatus 1 illustrated in FIG. 3A in which cells 2 and exogenous nucleic acid flow within chamber 11. [Figure 4A] FIG. 4A is a schematic diagram of an acoustophoresis device 1 according to a third embodiment of the present invention. [Figure 4B] FIG. 4B is a schematic diagram of the acoustophoresis device 1 according to the fourth embodiment of the present invention. [Figure 5A] FIG. 5A shows a sweep of the acoustic frequency from a first frequency of 0.9 MHz to a second frequency of 1.1 MHz using a sweep time of 1 ms. [Figure 5B] FIG. 5B is an expanded view of FIG. 5A, representing only the first 0.2 ms sweep, to more easily visualize the effect of the frequency sweep. [Figure 6] Figure 6 is a statistical analysis of transduction efficiency by flow cytometry showing the percentage of GFP positive cells in different conditions. A: control sample, cells without acoustic in a tissue culture well plate without virus, B: cells without acoustic in a tissue culture well plate with viral vector, C: cells in the flow channel with acoustic without virus, D: cells collected from the top side outlet 132 of the flow channel with acoustic power of 1 W in the presence of viral vector, E: cells collected from the outlet 131 of the flow channel with acoustic power of 1 W in the presence of viral vector, F: cells collected from the bottom side outlet 132 of the flow channel with acoustic power of 1 W and viral vector, G: cells collected from the top side outlet 132 of the flow channel with acoustic power of 2 W and viral vector, H: cells collected from the outlet 131 of the flow channel with acoustic power of 2 W and viral vector, I: cells collected from the bottom side outlet 132 of the flow channel with acoustic power of 2 W and viral vector. n=3 independent biological experiments. The percentage of transduction efficiency of samples from the central outlet (E and H) compared to the control value (B) has been quantified by performing a T-test; P values ​​≥ 0.05 correspond to ns, 0.01–0.05 to *, and 0.001–0.01 to **.

[0147] Exemplary embodiments of the present invention The application of a sound field to cells 2 in a chamber between a transmitting wall 111 and an opposing wall 112 of the chamber is shown in Figure 1. The transmitting wall 11 and the opposing wall are longitudinal walls of the chamber, i.e. both walls extend along the longitudinal axis of the chamber. The transmitting wall is coupled to a sound wave generator 14 and the opposing wall may act as a reflector. As shown in Figure 1, the application of a sound field to cells 2 in the chamber induces the movement of cells 2 and allows deflecting and concentrating the cells 2 at the nodes of the sound field.

[0148] In this example, ultrasound waves are generated in a chamber between a reflector (here the facing wall 112) and a transmitting wall 111 associated with the sound generator 14. This allows the creation of an acoustic pressure node in the centre of the chamber (depending on the selected frequency at which the sound generator 14 operates), a focusing surface 15 is located on the acoustic pressure node and acoustic radiation forces (ARF) are generated in the chamber. The ARF pushes the cells 2 towards the pressure node with a force equivalent to up to 100 times gravity. The cells 2 suspended in fluid may then migrate to the acoustic pressure node and remain trapped at the focusing surface 15 thereafter.

[0149] This is particularly advantageous as it makes it possible to localize the cell 2 within the fluid at a particular location in the chamber, in particular the fluid at the focusing surface 15, without using any mechanical force, pressure or invasive techniques that may damage the cell 2.

[0150] In the first embodiment shown in FIGS. 2A and 2B, the acoustophoretic device 1 includes: a chamber 11 associated with an acoustic wave generator 14 for generating acoustic waves within the chamber 11, the chamber extending along a longitudinal axis A1; an acoustic wave generator 14 longitudinally aligned with the longitudinal axis A1 of the chamber 11; two inlets in fluid communication with the chamber 11, including a first inlet 121 for introducing a suspension containing exogenous nucleic acid 3 into the chamber 1 and a second inlet 122 for introducing a suspension containing cells 2 into the chamber 1; two outlets in fluid communication with the chamber 1, including a first outlet 131 and a second outlet 132 for collecting a suspension containing cells modified with an exogenous nucleic acid; Includes.

[0151] The chamber extends along a longitudinal axis A1, has a first transverse axis A2 and a second transverse axis A3 perpendicular to the first transverse axis A2, a width greater than or equal to a thickness, and the chamber has first and second walls along the second transverse axis. The length of the chamber is measured along the longitudinal axis A1, the width is measured along the first transverse axis A2, and the thickness is measured along the second transverse axis A3. The chamber includes a transmission wall 111 coupled to the acoustic wave generator 14 and an opposing wall 112 opposing the transmission wall.

[0152] The chamber 11 may be divided into three main zones: an injection zone including inlets 121, 122, an active zone where sound waves are generated, and an exit zone including outlets 131, 132. The active zone is designed to optimize acoustic efficiency in a controlled manner through the selection of materials, layer thicknesses, and width of the chamber 11. The length of the active zone is in the range of 1 cm to 20 cm, preferably 1 cm to 10 cm, more preferably 1 cm to 5 cm.

[0153] In this embodiment, the acoustophoresis device 1 comprises two inlets 121, 122 and two outlets 131, 132, the longitudinal axes of the first inlet 121 and the first outlet 131 are aligned with the longitudinal axis A1 of the chamber 11 and with the focusing surface 15 located in the centre of the chamber 11, i.e. at approximately the mid-height of said chamber 11. The longitudinal axes of the second inlet 122 and the second outlet 132 are aligned, said axis being perpendicular to the longitudinal axis A1 of the chamber 11, i.e. aligned with the first transverse axis A2.

[0154] A suspension of exogenous nucleic acid 3 is injected into the first inlet 121 in a laminar flow, and a suspension of cells 2 is injected into the second inlet 122 in a laminar flow, thus forming two fluid layers that flow in the chamber 11 without turbulence. Upon application of the acoustic field generated by the acoustic generator 14, the cells 2 are deflected from their initial suspension into the central layer of fluid, i.e., the suspension containing the exogenous nucleic acid 3, as the ARF pushes the cells 2 towards the focusing surface 15 with a force greater than the combined action of the fluid drag and gravity. Due to their respective magnitudes, only the cells 2 are deflected, while the flow of exogenous nucleic acid remains undisturbed. Once the cells 2 and the exogenous nucleic acid 3 are collected in the same fluid layer, the exogenous nucleic acid 3 is transferred to the cells 2, resulting in modified cells. The modified cells are collected at the first outlet 131.

[0155] This embodiment is particularly advantageous since this forced confinement of the cells 2 and the foreign nucleic acid 3 in the same fluid layer greatly facilitates the transfer of said foreign nucleic acid 3 into the cells 2 .

[0156] In a second embodiment shown in FIGS. 3A and 3B, the acoustophoretic device 1 includes: a chamber 11 associated with an acoustic wave generator 14 for generating acoustic waves within the chamber 11, said chamber extending along a longitudinal axis A1; an acoustic wave generator 14 longitudinally aligned with the longitudinal axis A1 of the chamber 11; three inlets in fluid communication with the chamber 11, including a first inlet 121 for introducing a suspension containing exogenous nucleic acid 3 into the chamber 1 and two second inlets 122 for introducing a suspension containing cells 2 into the chamber 1; three outlets in fluid communication with the chamber 1, including a first outlet 131 and two second outlets 132 for collecting a suspension containing cells modified with an exogenous nucleic acid; Includes.

[0157] The chamber extends along a longitudinal axis A1, has a first transverse axis A2 and a second transverse axis A3 perpendicular to the first transverse axis A2, a width greater than or equal to a thickness, and the chamber has first and second walls along the second transverse axis. The length of the chamber is measured along the longitudinal axis A1, the width is measured along the first transverse axis A2, and the thickness is measured along the second transverse axis A3. The chamber includes a transmitting wall 111 coupled to the acoustic generator, and an opposing wall 112 facing the transmitting wall.

[0158] In this embodiment, the acoustophoresis device 1 comprises three inlets 121, 122 and three outlets 131, 132. The longitudinal axes of the first inlet 121 and the first outlet 131 are aligned with the longitudinal axis A1 of the chamber 11 and with the focusing plane 15 located in the center of the chamber 11, i.e. at approximately the mid-height of the chamber 11. The longitudinal axes of the second inlet 122 and the second outlet 132 are aligned. Said axes are perpendicular to the longitudinal axis A1 of the chamber 11, i.e. aligned with the first short axis A2. Furthermore, said second inlets 122 are located on either side of the longitudinal axis A1 of the chamber, respectively, and said second outlets 132 are located on either side of the longitudinal axis A1 of the chamber, respectively. In other words, the second inlets 122 are positioned opposite one another along the longitudinal axis A1, and the second outlets 132 are positioned opposite one another along the longitudinal axis A1.

[0159] A suspension of exogenous nucleic acid 3 is injected into the first inlet 121 in a laminar flow, and a suspension of two cells 2, preferably the same suspension, is injected into the second inlet 122 in a laminar flow, thus forming three fluid layers that flow in the chamber 11 without turbulence. Upon application of the acoustic field generated by the acoustic generator 14, the cells 2 are deflected from their initial suspension into the central layer of fluid, i.e. the suspension containing the exogenous nucleic acid 3, as the ARF pushes the cells 2 towards the focusing surface 15 with a force greater than the combined action of the fluid drag and gravity. Due to their respective sizes, only the cells 2 are deflected, while the flow of the exogenous nucleic acid remains undisturbed. Once the cells 2 and the exogenous nucleic acid 3 are gathered in the same fluid layer, the exogenous nucleic acid 3 is transferred into the cells 2, resulting in modified cells. The modified cells are collected at the first outlet 131.

[0160] This embodiment is particularly advantageous since this forced confinement of cells 2 and foreign nucleic acid 3 in the same fluid layer greatly facilitates the transfer of said foreign nucleic acid 3 into cells 2. Furthermore, having two inlets for the injection of a suspension of cells 2 increases the number of cells 2 that can be modified with foreign nucleic acid.

[0161] In the third embodiment shown in FIG. 4A, the acoustophoretic device 1 is identical to the acoustophoretic device 1 according to the first embodiment illustrated in FIG. 2A, but includes a plurality of acoustic wave generators 14 coupled to the transmitting wall of the chamber 11, said acoustic wave generators 14 being longitudinally aligned with the longitudinal axis A1 of the chamber 11.

[0162] This embodiment is particularly advantageous since it increases the active area of ​​the chamber, i.e. the area where the sound waves are generated in the chamber 1. This also allows for a good force distribution along the chamber 1.

[0163] In the fourth embodiment shown in FIG. 4B, the acoustophoretic device 1 is identical to the acoustophoretic device 1 according to the first embodiment illustrated in FIG. 3A, but includes a plurality of acoustic wave generators 14 coupled to the transmitting wall of the chamber 11, said acoustic wave generators 14 being longitudinally aligned with the longitudinal axis (A1) of the chamber 11.

[0164] This embodiment is particularly advantageous since it lengthens the active area of ​​the chamber 1, i.e. the area where the sound waves are generated in the chamber 1. This also allows for a good force distribution along the chamber 1.

[0165] Working Example The invention is further illustrated by the following examples.

[0166] Example 1: Transduction efficiency by frequency sweep material and method A suspension containing Jurkat E6 cells, RPMI medium containing 10% FBS and 1% penicillin / streptomycin is prepared at a number of 100000 cells / ml. The cells were used in RPMI medium containing 10% FBS and 1% penicillin / streptomycin.

[0167] Prepare a suspension of nucleic acid-containing viral vectors, including VLP-VSV(GFP), (Virus-Like Particle-Vesicular Stomatitis Virus (Green Fluorescent Protein)) as viral vector at a concentration of 2 μl / ml in RPMI medium containing 10% FBS and 1% penicillin / streptomycin.

[0168] Therefore, the MOI is less than 1.

[0169] The transduction of cells is carried out as follows: i. a chamber associated with a sound wave generator for generating sound waves within the chamber; three inlets in fluid communication with the chamber, including a first central inlet for introducing a suspension containing exogenous nucleic acid into the chamber, and two second peripheral inlets for introducing a suspension of cells into the chamber; three outlets in fluid communication with the chamber, including a first central outlet for collecting a suspension containing cells modified with exogenous nucleic acid, and two second peripheral outlets; The present invention provides an acoustophoretic device comprising: ii. A suspension of viral vectors is injected at a first central inlet of the chamber and simultaneously a suspension of cells is injected at two second peripheral inlets of the chamber. iii. Applying an acoustic field to generate sound waves within the chamber. iv. performing a frequency sweep of the sound wave, said frequency sweep having two parts defining a sweep cycle: A first part with a start frequency f1 of 0.9 MHz and an end frequency f2 of 1.1 MHz using a sweep time of 1 ms (see Figures 5A and 5B for illustration with different frequencies); The second part called "return sweep" with a start frequency f2 of 1.1 MHz and an end frequency f1 of 0.9 MHz using a sweep time of 1 ms. Including, Therefore, the total sweep time for one sweep cycle including part 1 and part 2 is 2 ms. The signal is continuous wave, ie, the sweep cycle is repeated continuously approximately 500 times per second. v. A sample containing the nucleic acid modified cells is collected at a first central outlet.

[0170] The transduction parameters for each study are detailed in Table I below. [Table 1]

[0171] Table I shows the experimental protocols for both static (sample 1) and continuous flow acoustic conditions (samples 2-4). The static conditions represent conditions where cells and viruses are incubated in a cell culture dish, while the continuous flow acoustic conditions represent conditions where cells and viruses are injected into the channel of the acoustophoresis device and exposed to acoustic waves. The acoustic exposure time represents the time the cells are exposed to acoustic waves (approximately the time it takes for the cells to pass through the active area of ​​the channel at a flow rate of 0.5 ml / min).

[0172] The first sample corresponds to static conditions (without sonication) using 24-well tissue culture plates for transduction. After 72 hours of incubation of the virus and cells, the transduction efficiency was quantified. A control experiment was performed under the same conditions without the virus.

[0173] Sample 2 is a control experiment for the continuous flow acoustic conditions: cells alone were injected into the channels of the acoustophoresis device and exposed to acoustic waves.

[0174] Sample 3 was obtained under continuous flow acoustic conditions with cells and viruses injected into the inlets 122 and 121, respectively, of the channels of the acoustophoresis device, as shown in FIG. 3A, and then exposed to 1 W acoustic waves to increase their proximity.

[0175] Sample 4 was obtained under continuous flow acoustic conditions with cells and viruses injected into the inlets 122 and 121 of the channels of the acoustophoresis device, respectively, as shown in Figure 3A, and then exposed to 2 W acoustic waves to increase their proximity.

[0176] All samples were collected from the outlet of the channel, placed into 24-well cell culture plates, and incubated in a 37° C., 5% CO 2 incubator for 72 h, after which the transduction efficiency was quantified by flow cytometry.

[0177] Samples (1-4) were then collected and placed in cytometry tubes. The tubes were analyzed in a hemocytometer (FACS CantoII). After the first passage, a solution of propidium iodide (PI): 10 μg / ml per sample was added, followed by cytometry analysis, and dead cells in each sample were measured and later excluded in the statistical quantification. Transduction efficiency was quantified using a gate of viable single cells that were positive for green fluorescence (GFP). result [Table 2] TIFF2025511164000004.tif69132

[0178] Table II shows the experimental conditions and transduction efficiency results for samples obtained under static or continuous-flow acoustic conditions at acoustic powers between 0 W and 2 W, with the experiments repeated three times. Static conditions (labeled "static" in Table II) refer to conditions where cells and viruses were incubated in cell culture dishes, while continuous-flow acoustic conditions (labeled "continuous flow" in Table II) refer to conditions where cells and viruses were injected into the channels of an acoustophoresis device and exposed to acoustic waves.

[0179] In each set of experiments (1–4), the experimental conditions in Table I were reproduced, resulting in four samples per experiment: one sample from static conditions (-1), one control sample in continuous flow acoustic conditions (no virus, -2), and two samples from continuous flow acoustic conditions (-3 and -4).

[0180] In static experiments, i.e., experiments performed under static conditions, cells and viruses were added to a 24-well cell culture plate without exposure to acoustics. For continuous flow acoustics, cells and viruses were injected into the inlet 122 referenced in Figure 3A. The control experiment (listed as "(Control)" in Table II) refers to an experiment performed with only cells without virus.

[0181] In experiments (1-3, 1-4), (2-3, 2-4), and (3-3, 3-4), cells were injected into each of the side inlets 122 mentioned in FIG. 3A at a flow rate of 0.25 ml / min. Thus, the total cell flow rate was 0.5 ml / min. Viruses conjugated with green fluorescent protein (GFP) were injected into the central inlet 121 at the same flow rate of 0.5 ml / min. This ensures that there is no disruption of laminar flow as the cell and virus fluid layers flow through the channel, and that the fluid layers are parallel and stable. Both the virus and cells are exposed to acoustic waves with a power of 1 W / 2 W for about 30 seconds. The frequency sweep is performed as detailed herein above. The force generated by phonophoresis moves the cells towards the center of the channel and deflects them towards the virus layer, thus increasing the cell / virus proximity and transduction occurs. Samples were then collected from all outlets and the transduction efficiency was analyzed by flow cytometry. As the majority of the virus was present at outlet 131, the samples collected at this outlet were used as the outcome for the acoustic experiment. However, suspensions collected at the other outlets were analyzed. A constant MOI=0.7 was used in all three experiments using the same VLP-VSV(GFP) virus. The percentage of cell viability indicates the percentage of live / viable cells using propidium iodide (PI) solution staining measured by flow cytometry in all samples. The percentage of transduction efficiency corresponds to the viable cells that were positive for GPF.

[0182] From Table II, it is clear that continuous flow acoustic conditions with an acoustic power of 1 W significantly improves transduction efficiency and cell viability.

[0183] Example 2: Comparative Example Between Transduction with and without Frequency Sweeping material and method Stationary Condition Each test was performed with a control run and a static run, neither of which was exposed to sound waves. In the control run, the virus was added to the cells in a petri dish and washed immediately after. In the static run, the virus was added to the cells in a tissue culture petri dish and then washed after 90 minutes. In both the control and static runs, an MOI of 25 was used. In these tests, baculovirus is used to transduce green fluorescent protein (GFP) into JurkatT cells.

[0184] Table III below shows the transduction efficiency results of four replicate experiments. [Table 3]

[0185] From Table III it is clear that the longer the virus and cells remain in the Petri dish the greater the transduction efficiency.

[0186] Continuous Flow Conditions - with Frequency Sweep (Method of the Invention) Experiment 3 from Example 1 was reproduced using RPMI medium containing Jurkat E6 cells as the cell suspension and a lentivirus gene conjugated with green fluorescent protein (GFP) as the virus suspension.

[0187] The acoustic channel of the acoustophoresis device is a two-part molded PMMA (poly(methyl methacrylate)) channel bonded with chloroform and epoxy glue. The geometry is identical to FIG. 3A. The distance between walls 111 and 112 is 750 μm and the width of the channel is 10 mm. The flow rate of the virus suspension at inlet 121 is 0.5 ml / min and the flow rate of the cell suspension at each inlet 122 is 0.25 ml / min (so the flow rate of the cell suspension is 0.5 ml / min), so the total flow rate of the suspension entering the channel is 1 ml / min.

[0188] A frequency sweep of a sound wave is performed, said frequency sweep having two parts that define a sweep cycle: A first part with a start frequency f1 of 0.9 MHz and an end frequency f2 of 1.1 MHz using a sweep time of 1 ms (see Figures 5A and 5B for illustration with different frequencies); The second part called "return sweep" with a start frequency f2 of 1.1 MHz and an end frequency f1 of 0.9 MHz using a sweep time of 1 ms. Including, Therefore, the total sweep time for one sweep cycle including part 1 and part 2 is 2 ms. The signal is continuous wave, ie, the sweep cycle is repeated continuously approximately 500 times per second.

[0189] Continuous flow condition - no frequency sweep RPMI medium containing Jurkat E6 cells and a lentivirus gene conjugated to green fluorescent protein (GFP) were used as the cell suspension and virus suspension, respectively.

[0190] An acoustic chamber was used, which contained an ultrasonic transducer and a reflector. The virus and cells were placed in the acoustic chamber to form a closed environment. The recirculation loop drew fluid from one end of the acoustic chamber, and then recirculated the fluid into the acoustic chamber through the other end. The acoustic chamber had a volume of about 1 ml, and the recirculation loop had a volume of about 3 ml. The transduction test was performed using the acoustic chamber. The acoustic power was 1 W. result [Table 4]

[0191] The experiments performed with frequency sweeping used 10 times less virus (MOI) for effective transduction compared to the experiments performed without frequency sweeping. The method of the invention is therefore particularly suitable for therapeutic applications with CAR-T cells. The range of cell concentrations used in the experiments performed with frequency sweeping resulting in cell treatment rates of 50,000-150,000 cells / min allows the experiments to be scaled up to treat a larger number of cells and virus particles compared to the experiments performed without frequency sweeping. The method of the invention treats 1.5-4.5 times more cells per minute and the increase in transduction is 30% higher in this case (an increase of 2.6 versus 2.0).

[0192] Example 3 material and method Experiment 3 from Example 1 was reproduced using RPMI medium containing Jurkat E6 cells as the cell suspension and a lentiviral gene conjugated with green fluorescent protein (GFP) as the virus suspension.

[0193] The acoustic channel of the acoustophoresis device is a two-part molded PMMA (poly(methyl methacrylate)) channel bonded with chloroform and epoxy glue. The geometry is identical to FIG. 3A. The distance between walls 111 and 112 is 750 μm and the width of the channel is 10 mm. The flow rate of the virus suspension at inlet 121 is 0.5 ml / min and the flow rate of the cell suspension at each inlet 122 is 0.25 ml / min (so the flow rate of the cell suspension is 0.5 ml / min), so the total flow rate of the suspension entering the channel is 1 ml / min.

[0194] A frequency sweep of a sound wave is performed, said frequency sweep having two parts that define a sweep cycle: A first part with a start frequency f1 of 0.9 MHz and an end frequency f2 of 1.1 MHz using a sweep time of 1 ms (see Figures 5A and 5B for illustration with different frequencies); The second part called "return sweep" with a start frequency f2 of 1.1 MHz and an end frequency f1 of 0.9 MHz using a sweep time of 1 ms. Including, Therefore, the total sweep time for one sweep cycle including part 1 and part 2 is 2 ms. The signal is continuous wave, ie, the sweep cycle is repeated continuously approximately 500 times per second.

[0195] result All three replicates of the acoustic experiment provided reproducible results in terms of enhanced transduction at 1 W acoustic power.

[0196] FIG. 6 represents the transduction efficiency results of all three experiments shown in Table III under the experimental conditions described in Table II. The transduction efficiency percentage is a quantitative measurement of transduction efficiency by flow cytometry showing the percentage of live T cells that are GFP positive: A: control sample without acoustics in static conditions in tissue culture well plates containing only cells and no virus; B: cells without acoustics in static conditions in tissue well plates containing cells and viral vectors. The encounter of cells and viruses increases the transduction efficiency by 2.5 times compared to the condition of the control state, from an average efficiency of 35.5% to an average efficiency of 68.3% when 1 W acoustic power is applied to the acoustic channel shown in column E. The increase in acoustic power increases the transduction efficiency from 35.5% to 56.67% shown in column H. The expression of GFP was tested in cells collected from the outlet 132 to verify that there is less virus at these outlets, as shown in columns D, F of FIG. 6 when applying 1 W, and G and I when applying 2 W. The increase in transduction efficiency is not as high as outlet 131, and there is less cell concentration at both outlets 132 compared to outlet 131. This proves that the cells injected at inlet 122 do indeed migrate to the central layer of the channel and mix with the virus more easily than without ultrasound. [Explanation of symbols]

[0197] 1-Acoustophoresis Apparatus 11-Chamber 111-Transmission Wall 112-Facing Wall 121-First Entrance 122-Second Entrance 131-1st Exit 132-Second Exit 14-Sound Generator 15-Focusing plane 2-cell 3-Foreign nucleic acid A1-Longitudinal axis A2-First short axis A3-Second short axis

Claims

1. A method for introducing an exogenous nucleic acid (3) into a cell (2), comprising the steps of: i. a chamber (11) configured to be associated with at least one acoustic wave generator (14) for generating acoustic waves within the chamber (11); at least one acoustic wave generator (14) configured to generate acoustic waves in said chamber (11); at least two inlets (121, 122) in fluid communication with said chamber (11), including a first inlet (121) and at least one second inlet (122); at least two outlets (131, 132) in fluid communication with said chamber (11), including a first outlet (131) and at least one second outlet (132); Providing an acoustophoretic device (1), comprising: ii. injecting a suspension containing exogenous nucleic acid (3) into said chamber (11) through said first inlet (121) and injecting a suspension of cells (2) into said chamber (11) through said at least one second inlet (122); iii. applying an acoustic field by generating acoustic waves in said chamber (11); iv. Using a sweep time ranging from 1 ms to 100 ms, change the frequency of the sound wave to a first frequency f １ to the second frequency f ２ continuously sweeping up to f ２ GA １ and the acoustic wave has a first frequency f during the sweep time. １ to the second frequency f ２ Range up to [f １ , f ２ ] in succession; v. collecting the suspension containing the cells (2) modified with the exogenous nucleic acid (3) at said first outlet (131); Including, method.

2. The method of claim 1, wherein the nucleic acid (3) is carried in a viral vector or a microvesicle.

3. The method of claim 2, wherein the multiplicity of infection is in the range of 0.5 to 30.

4. The method of claim 1, wherein the nucleic acid (3) is a naked nucleic acid.

5. First frequency f １ and a second frequency f ２ The method according to any one of claims 1 to 4, wherein each of the frequencies is in the range of 0.1 MHz to 4 MHz.

6. The second frequency f is scanned using a sweep time in the range of 0.5 ms to 10 ms. ２ to the third frequency f ３ A further step after step (iv) of sweeping the sonic frequency up to f ２ GA ３ The method according to any one of claims 1 to 5, further comprising the steps:

7. The first frequency f １ is the third frequency f ３ The method of claim 6, wherein the ?

8. The method according to any one of the preceding claims, wherein the power applied to the acoustic wave generator (14) is in the range of 0.2W to 50W.

9. The method according to any one of claims 1 to 8, wherein the cell (2) is selected from Chinese hamster ovary (CHO) cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, human cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, effector T cells, γδ T cells, Jurkat T cells, CAR-T cells, B cells, or NK cells, peripheral blood mononuclear cells (PBMCs), HEK293T, algae, plant cells, tumor infiltrating lymphocytes (TILs), or bacteria.

10. A method for transducing a cell (2), comprising the steps of: i. a chamber (11) configured to be associated with at least one acoustic wave generator (14) for generating acoustic waves within the chamber (11); at least one acoustic wave generator (14) configured to generate acoustic waves in said chamber (11); at least two inlets (121, 122) in fluid communication with said chamber (11), including a first inlet (121) and at least one second inlet (122); at least two outlets (131, 132) in fluid communication with said chamber (11), including a first outlet (131) and at least one second outlet (132); Providing an acoustophoretic device (1), comprising: ii. injecting a suspension of a viral vector containing a nucleic acid (3) into said chamber (11) at said first inlet (121) and injecting a suspension of cells (2) into said chamber (11) at said at least one second inlet (122); iii. applying an acoustic field by generating acoustic waves in said chamber (11); iv. Using a sweep time ranging from 1 ms to 100 ms, change the frequency of the sound wave to a first frequency f １ to the second frequency f ２ continuously sweeping up to f ２ GA １ and the acoustic wave has a first frequency f during the sweep time. １ to the second frequency f ２ Range up to [f １ , f ２ ] in succession; v. collecting the suspension containing the cells (2) modified with the nucleic acid (3) at said first outlet (131); Including, method.

11. First frequency f １ and a second frequency f ２ 11. The method of claim 10, wherein each of is in the range of 0.1 MHz to 4 MHz.

12. The second frequency f is scanned using a sweep time in the range of 0.5 ms to 10 ms. ２ to the third frequency f ３ A further step after step (iv) of sweeping the sonic frequency up to f ２ GA ３ The method according to claim 10 or 11, further comprising the step of:

13. The method according to any one of claims 10 to 12, wherein the cell (2) is selected from Chinese hamster ovary (CHO) cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, human cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, effector T cells, γδ T cells, Jurkat T cells, CAR-T cells, B cells, or NK cells, peripheral blood mononuclear cells (PBMCs), algae, plant cells, HEK293T, tumor infiltrating lymphocytes (TILs), or bacteria.

14. A cell obtainable by the method according to any one of claims 1 to 9.

15. An acoustophoresis device (1) for introducing a foreign nucleic acid (3) into a cell (2), comprising: a chamber (11) configured to be associated with at least one acoustic wave generator (14) for generating acoustic waves within the chamber (11) extending along a longitudinal axis; at least one acoustic wave generator (14) configured to generate acoustic waves in said chamber (11); at least two inlets (121, 122) in fluid communication with said chamber (11), said inlets (121, 122) including a first inlet (121) for introducing a suspension containing an exogenous nucleic acid (3) into said chamber (11) and at least one second inlet (122) for introducing a suspension of cells (2) into said chamber (11); at least two outlets (131, 132) in fluid communication with said chamber (11), said outlets (131, 132) including a first outlet (131) for collecting a suspension containing cells (2) modified with an exogenous nucleic acid (3) and at least one second outlet (132); Including, The at least one acoustic wave generator (14) uses a sweep time in the range of 1 ms to 100 ms to change the frequency of the acoustic wave to a first frequency f １ to the second frequency f ２ and configured to continuously sweep up to f ２ GA １ and the acoustic wave has a first frequency f during the sweep time. １ to the second frequency f ２ Range up to [f １ , f ２ ] in succession, Sonic phoresis apparatus (1).

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