Preparation of polymer / nucleic acid complex solutions

Abstract

The present invention relates to the preparation of solutions of polymer / nucleic acid complexes and the use of such solutions in methods for transfection of cells.

Description

Technical Field

[0001] The present invention relates to the preparation of solutions of polymer / nucleic acid complexes and their use in methods for the transfection of cells.

Background Art

[0002] Transfection is the process of deliberately introducing nucleic acids into eukaryotic cells. There are many situations where it may be desirable or advantageous to introduce various types of exogenous nucleic acids into eukaryotic cells. Commonly used exogenous nucleic acids are plasmid DNA, RNA, siRNA, and oligonucleotides. Once delivered to the cell, the nucleic acids regulate gene expression by driving overexpression or silencing of the gene of interest.

[0003] Gene overexpression is an essential tool in several applications, from understanding the role of the gene of interest (gene research, high-throughput screening) to the production of biologics such as antibodies (protein production) and recombinant viral particles for therapeutic purposes (e.g., virus production for gene and cell therapy).

[0004] Gene silencing is a method used to prevent the expression of a target gene. Gene expression can be partially reduced (gene knockdown) or completely blocked (gene knockout). Because any gene can potentially be targeted, gene silencing is a common technique used to develop gene-based therapies to address single-gene conditions, cancer, and immunotherapy strategies. Transfection has proven to have many uses in numerous different applications. Of particular interest is the transfection of cells with expression vectors, which are widely used in the production of biological agents, including recombinant proteins and viral vectors. For example, a common method of viral vector production involves transfection of primary cells or mammalian / insect cell lines with the vector DNA component, followed by a limited incubation period, and then the recovery of the crude vector from the culture medium and / or cells (Merten, OW. et al., 2014, Pharmaceutical Bioprocessing, 2:183-203). The efficiency of lentiviral vector production is typically influenced by several factors in the “upstream phase,” including [1] the viral serotype / pseudotype used, [2] the composition and size of the transgenic sequence, [3] the medium composition / gas treatment / pH, [4] the transfection reagent / process, [5] the timing of chemical induction and vector recovery, [6] the fragility / viability of cells, [7] the bioreactor shear force, and [8] impurities. Clearly, there are other factors to consider during the “downstream” purification / concentration stages (Merten, OW. et al., 2014, Pharmaceutical Bioprocessing, 2:237-251).

[0005] As the success of viral vector approaches in clinical trials begins to be translated towards regulatory approval and commercialization, attention has been drawn to new bottlenecks in the large-scale production of Good Manufacturing Practice (GMP)-grade vector materials for the manufacture and quality control of pharmaceuticals (Van der Loo JCM, Wright JF., 2016, Human Molecular Genetics, 25(R1):R42-R52). Similar obstacles exist in the production of GMP-grade recombinant proteins.

[0006] A way to overcome this challenge is to find new ways to maximize transfection efficiency during the production of viral vectors or recombinant proteins. Therefore, there is a need in the art to provide alternative improved transfection methods for use in the production of, for example, viral vectors and other biological agents, which can help address known problems associated with the large-scale production of GMP-grade materials such as GMP-grade vector materials.

Summary of the Invention

Means for Solving the Problems

[0007] The inventors have shown that polymer / nucleic acid complexes are stable only for a short time in the presence of salts before having an adverse effect on transfection efficiency. This limits the use of cationic polymers in the large-scale production of biological agents because the optimal incubation period is too short to be practical in large-scale GMP manufacturing.

[0008] The present invention provides an improved method for cell transfection. In this regard, the inventors have surprisingly found that polymer / nucleic acid complexes prepared by mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution (e.g., water), followed by the addition of salt to induce the maturation of the polymer / nucleic acid complex, extend the time for optimal polymer / nucleic acid complex maturation and provide high transfection efficiency. Thus, the time for optimal polymer / nucleic acid complex maturation (i.e., incubation time) can be extended by using an optimal concentration of salt. The inventors have further surprisingly found that the growth of the polymer / nucleic acid complex can be inhibited and the complex stabilized by diluting the polymer / nucleic acid complex solution (therefore diluting the salt), thereby further extending the incubation time. Thus, the method described herein is advantageous because it provides the ability to control the initiation and / or termination of polymer / nucleic acid complex maturation. This is highly advantageous for mass production of GMP-grade materials because it eliminates the time constraint as a problem in the transfection process, i.e., improves the flexibility of the manufacturing process.

[0009] The inventors have surprisingly found that the ability to control the initiation and / or termination of polymer / nucleic acid complex maturation, combined with reduced cytotoxicity, is advantageous because it provides the ability to scale the preparation of polymer / nucleic acid complexes with the number of cells during transfection. This is highly advantageous for the mass production of GMP-grade materials, as it allows the use of higher cell densities for the transfection process (e.g., cell densities achieved using perfusion culture), i.e., improves the capacity and yield of the manufacturing process.

[0010] The present invention relates to a process for preparing solutions of polymer / nucleic acid complexes, and to the use of such solutions for cell transfection in, for example, the production of lentiviral vectors.

[0011] In one embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution. a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes, The salt is not valproate, isobutyrate, or isovalerate.

[0012] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes, The salt is added only before the polymer / nucleic acid complex solution comes into contact with the cells.

[0013] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes, The final salt concentration in step b) is approximately 10 mM to 100 mM.

[0014] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes, The salt has a degree of dissociation of at least 0.95 in a substantially salt-free aqueous solution.

[0015] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) A step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes.

[0016] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) A step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes.

[0017] In a further embodiment, the present invention provides a solution of a polymer / nucleic acid complex obtained or obtainable by the method of the present invention.

[0018] In a further embodiment, the present invention provides a solution comprising a polymer / nucleic acid complex and a salt, wherein the salt concentration is approximately 10 mM to approximately 100 mM.

[0019] In a further embodiment, the present invention provides the use of a solution of the polymer / nucleic acid complex of the present invention for the transfection of nucleic acids into cells.

[0020] In a further embodiment, the present invention provides the use of a solution of the polymer / nucleic acid complex of the present invention in a method for producing retroviral vectors.

[0021] In some embodiments, nucleic acids are selected from DNA, RNA, oligonucleotide molecules, and mixtures thereof.

[0022] In some embodiments, the nucleic acid is DNA, preferably plasmid DNA.

[0023] In some embodiments, the cationic polymer is a polymer-based transfection reagent.

[0024] In some embodiments, the cationic polymer is selected from polyethyleneimine (PEI), dendrimers, DEAE-dextran, polypropyleneimine (PPI), chitosan [poly-(β-1 / 4)-2-amino-2-deoxy-D-glucopyranose], poly-L-lysine (PLL), poly(lactic acid-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), and their derivatives.

[0025] In some embodiments, the cationic polymer is PEI or a derivative thereof, preferably selected from linear PEI, branched PEI, PEGylated PEI, JetPEI, PEIPro®, PEI MAX, and PTG1+.

[0026] In some embodiments, the salt is selected from sodium salts, magnesium salts, potassium salts, calcium salts, phosphates, and mixtures thereof.

[0027] In some embodiments, the salt is phosphate-buffered saline (PBS).

[0028] In some embodiments, the salt is PBS, and PBS is added in step b) to a final concentration of about 0.1X PBS to about 1.0X PBS, preferably to a final concentration of about 0.1X PBS to about 0.3X PBS.

[0029] In some embodiments, the salt is added in step b) to a final concentration of about 10 mM to about 100 mM, preferably in step b) to a final concentration of about 20 mM to about 100 mM.

[0030] In some embodiments, step a) includes mixing a plurality of nucleic acid molecules and a plurality of cationic polymer molecules in a substantially salt-free aqueous solution.

[0031] In some embodiments, the method includes the step of incubating the salt solution produced in step b).

[0032] In some embodiments, the salt solution is incubated for about 1 minute to a maximum of about 2 hours, preferably for about 20 minutes to about 90 minutes.

[0033] In some embodiments, the salt solution is incubated for approximately 60 minutes.

[0034] In some embodiments, the method includes diluting the salt solution prepared in step b) or the salt solution after incubation in step c).

[0035] In some embodiments, the salt is PBS, and the salt solution is diluted to a final concentration of about 0.05X PBS to about 0.15X PBS, preferably to a final concentration of about 0.1X PBS.

[0036] In some embodiments, the nucleic acid functions as a retroviral vector component.

[0037] In some embodiments, the retroviral vector is a replication-deficient retroviral vector.

[0038] In some embodiments, the retroviral vector is a lentiviral vector, preferably HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or Visna.

[0039] In some embodiments, the vector component is a) RNA genome of lentiviral vectors, b) environment or its functional substitute, c) gag-pol or its functional substitute, and / or d) rev or its functional substitute Selected from.

[0040] In some embodiments, the method is e) Depending on the case, a step of culturing mammalian cells, f) A step of transfecting mammalian cells using a solution of a polymer / nucleic acid complex obtained by the method of any one of the preceding claims, g) Depending on the case, a step of introducing a nucleic acid different from the nucleic acid in the polymer / nucleic acid complex solution into mammalian cells, h) Depending on the case, a step of selecting mammalian cells having nucleic acids incorporated into the genome, i) Depending on the case, a step of culturing mammalian cells under conditions in which nucleic acids are expressed, j) Depending on the case, a step of culturing mammalian cells under conditions in which retroviral vectors are produced, k) Depending on the case, the process may include isolating the retroviral vector, It also includes.

[0041] In some embodiments, the method includes the step of culturing mammalian cells.

[0042] In some embodiments, the culture is performed by perfusion culture.

[0043] In some embodiments, the process of culturing mammalian cells by perfusion culture is carried out for approximately 10 to 96 hours.

[0044] In some embodiments, the method further includes the step of inoculating mammalian cells into a cell culture vessel (e.g., a bioreactor) before the step of culturing the mammalian cells.

[0045] In some embodiments, the method further includes a step of performing a rapid medium change after the step of inoculating cells and before the step of transfecting cells. Preferably, the step of culturing mammalian cells includes at least one rapid medium change.

[0046] In some embodiments, the method includes the step of culturing mammalian cells under conditions in which nucleic acids are expressed.

[0047] In some embodiments, the method includes the step of culturing mammalian cells under conditions that produce a retroviral vector.

[0048] In some embodiments, the method includes the step of isolating a retroviral vector.

[0049] In some embodiments, the mammalian cells are HEK293T cells.

[0050] In some embodiments, the mammalian cells are suspension-compatible mammalian cells.

[0051] In some embodiments, the transfection, introduction, and / or culture steps are carried out in a suspension in serum-free medium.

[0052] In some embodiments, the transfection is transient.

[0053] In some embodiments, the transfection, introduction and / or culture steps are carried out in a volume of at least 50 L, preferably in a volume of at least 50 L.

[0054] In some embodiments, nucleic acids different from those in the polymer / nucleic acid complex solution are introduced into mammalian cells by transfection or electroporation.

[0055] In some embodiments, nucleic acids different from the nucleic acids in the polymer / nucleic acid complex solution are those not encoded by the nucleic acids in the polymer / nucleic acid complex solution, such as: a) RNA genome of lentiviral vectors, b) environment or its functional substitute, c) gag-pol or its functional substitute, and / or d) rev or its functional substitute, Encodes any viral vector component selected from the available options.

[0056] In a further aspect, the present invention provides a method for producing a retroviral vector, a) A step of culturing mammalian cells as needed, b) A step of transfecting mammalian cells using a solution of the polymer / nucleic acid complex of the present invention or a solution of the polymer / nucleic acid complex of the present invention, c) Depending on the case, a step of introducing at least one nucleic acid different from the nucleic acid in the polymer / nucleic acid complex solution into mammalian cells, d) Depending on the case, a step of selecting mammalian cells having nucleic acid sequences that encode viral vector components incorporated into their genome, e) A step of culturing mammalian cells under conditions in which a retroviral vector is produced, Includes.

[0057] In some embodiments, the culture is performed by perfusion culture.

[0058] In some embodiments, the process of culturing cells by perfusion culture is carried out for approximately 10 to 96 hours.

[0059] In some embodiments, the method further includes the step of isolating a retroviral vector.

[0060] In some embodiments, the retroviral vector is a replication-deficient retroviral vector.

[0061] In some embodiments, the retroviral vector is a lentiviral vector, preferably HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or Visna.

[0062] In some embodiments, the mammalian cells are HEK293T cells.

[0063] In some embodiments, the mammalian cells are suspension-compatible cells.

[0064] In some embodiments, the method is carried out in a suspension in serum-free medium.

[0065] In some embodiments, steps a), b), c), and / or e) are carried out in a volume of at least 50 L, preferably steps a), b), c), and e) are carried out in a volume of at least 50 L.

[0066] In some embodiments, nucleic acids are introduced into cells by transfection or electroporation in step c).

[0067] In some embodiments, the transfection is transient.

[0068] In some embodiments, at least one nucleic acid optionally introduced into mammalian cells in step b) is not encoded by the nucleic acid in the polymer / nucleic acid complex solution. (i) RNA genome of lentiviral vector, (ii) environment or a functional substitute thereof, (iii) gag-pol or its functional substitute, and / or (iv)rev or its functional substitute, It encodes any lentiviral vector component selected from the group consisting of the following.

[0069] In a further aspect, the present invention provides a method for transfecting mammalian cells, a) A step of culturing mammalian cells in perfusion culture, b) A step of transfecting mammalian cells using a solution of the polymer / nucleic acid complex described herein, Includes.

[0070] In some embodiments, the process of culturing mammalian cells by perfusion culture is carried out for approximately 10 to 96 hours.

[0071] More precisely, mammalian cells are the mammalian cells described herein.

[0072] In some embodiments, the method is carried out in a suspension in serum-free medium.

[0073] In some embodiments, steps a) and / or b) are carried out in a volume of at least 50 L, preferably steps a) and b) are carried out in a volume of at least 50 L.

[0074] In some embodiments, the transfection is transient. [Brief explanation of the drawing]

[0075] [Figure 1] This figure shows the functional titer (TU / mL) results from time-course studies of shaking flasks transfected with PEIPro® transfection mixes incubated at different time intervals. The incubation period of the DNA / PEIPro® complex significantly affects subsequent transfection and lentiviral vector production. The data show that the optimal incubation time for the highest functional titer is 3 minutes, and this decreases rapidly when the incubation time is extended to 15 or 30 minutes. [Figure 2] This figure shows that the dynamics of complex formation can be modified by preparing a transfection mix in water and spiking it with various PBS concentrations. DNA / PEIpro complexes were prepared in a salt-free aqueous solution (H2O), and complex growth was initiated by PBS spiking. (A) When the transfection complex was spiked with 1X PBS, the optimal titer was achieved after 3 minutes of incubation. (B) Adding low concentrations of PBS to the transfection complex allowed for an extension of the incubation time, with 0.2X PBS extending the optimal incubation time to approximately 25 minutes. As an NB-negative control, the complex was prepared in water and then used to transfect cells without PBS spiking. No vectors were constructed to demonstrate that PBS is required to initiate complex growth. [Figure 3]This figure shows that transfection efficiency remained stable for up to 12 hours, and potentially up to 1 week, after dilution of the complex to reduce salt concentration (and stop complex growth). The transfection mix was spiked with 0.2X PBS to initiate complex growth, and then incubated for 30 minutes to reach the optimal complex size. The transfection mix was then diluted with a salt-free aqueous solution (e.g., H2O) to a final PBS concentration of 0.1X. The transfection mix was then stored at 4°C for up to 1 week. A constant amount of the transfection mix was then used to produce GFP lentivirus, and the resulting functional titer was measured. The graph shows that after 12 hours of incubation, the transfection mix was still efficient for producing lentiviral vectors and yielded a functional titer comparable to that of a "fresh" transfection mix. [Figure 4] This figure shows DLS analysis results indicating no complex growth was observed when the DNA:PEIpro complex was prepared in H2O. This correlates with data shown in Figure 2B, which demonstrated that complexes prepared in water and not spiked with PBS did not result in vector production. This suggests that complexes in the 80–86 nm size range are too small to successfully transfect. [Figure 5] DNA / PEIpro complexes were prepared in water, and particle growth was initiated by adding PBS (0.2X). The complexes were incubated for 25 minutes, which corresponds to the optimal time to achieve the highest functional titer (Figure 2B). The complexes were then diluted 2-fold to achieve a final PBS concentration of 0.1X. The complex size was then measured over a further 45 minutes. DLS measurements showed that when the transfection mix was diluted 2-fold, the complex growth stopped, and the size remained between 600 and 800 nm. Correlation of this data with the data shown in Figures 2 and 3 suggests that the optimal complex size for achieving the highest titer is between 600 and 800 nm. [Figure 6]This figure shows that when a transfection mix is ​​spiked with different concentrations of PBS, the rate at which the complex grows to a desirable size for transfection varies depending on the concentration of PBS added. If the PBS concentration is too low (≤0.1X PBS), complex growth does not begin. [Figure 7] This figure shows that any salt can be used to initiate complex growth. The faster the complex growth rate, the lower the salt concentration required to induce complex growth. Graph (A) and Table 2 show the effect of spiking the transfection mix with five different concentrations of NaCl (10mM; 20mM; 50mM; 80mM and 100mM). Only the 80mM and 100mM concentrations initiated significant complex growth. Graph (B) and Table 3 show the effect of spiking the transfection mix with five different concentrations of MgCl2 (10mM; 20mM; 50mM; 80mM; 100mM). [Figure 8] This figure shows the functional titer of therapeutic vectors after production on a 5L bioreactor scale. These results demonstrate that the "aqPEIPro" transfection method is scalable and achieves lentiviral vector titers equivalent to those achieved using cationic lipid-based transfection methods. n=2 for the "aqPEIPro" transfection method and n=4 for the cationic lipid-based transfection method. [Modes for carrying out the invention]

[0076] In vitro method for producing polymer / nucleic acid complex solutions This invention provides an in vitro method for producing a polymer / nucleic acid complex solution. a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) Optionally, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes.

[0077] Cell transfection with exogenous nucleic acids is well known in the art and can be carried out using a variety of viral and nonviral transfection reagents. Transfection overcomes the inherent challenge of introducing negatively charged molecules (e.g., the phosphate backbone of DNA and RNA) into cells with negatively charged membranes. Transfection reagents can be divided into three classes: chemical reagents, cationic lipids, and physical methods.

[0078] Among the chemical reagents used, DEAE-dextran, a cationic polymer, is noteworthy and was one of the first to be used to transfect cultured mammalian cells (Vaheri and Pagano, 1965; McCutchan and Pagano, 1968). Other synthetic cationic polymers, including polybrene (Kawai and Nishizawa, 1984), polyethyleneimine (Boussif et al. 1995), and dendrimers (Haensler and Szoka, 1993; Kukowska-Latallo et al. 1996), have been used to transfer DNA into cells. Calcium phosphate coprecipitation as a transfection method was introduced in the early 1970s (Graham and van der Eb, 1973) and represents another chemical transfection reagent.

[0079] Cationic lipids, such as lipofectamines, represent one of the most common methods for introducing foreign genetic material into cells. The term "liposome" refers to a lipid bilayer that forms colloidal particles in an aqueous medium (Sessa and Weissmann, 1968). Synthetic liposomes were first used to deliver DNA to cells in 1980 (Fraley et al. 1980). The next advance in liposome vehicles was the development of synthetic cationic lipids (Felgner et al. 1987). The cationic head group of lipid compounds associates with negatively charged phosphate on nucleic acids. Liposome-mediated delivery offers advantages such as relatively high gene transfer efficiency, the ability to transfect specific cell types resistant to calcium phosphate or DEAE-dextran, in vitro and in vivo applications, successful delivery of DNA of all sizes from oligonucleotides to yeast artificial chromosomes, RNA delivery, and protein delivery (Kim and Eberwine, 2010; outlined in Stewart et al. 2016). Non-liposomal reagents offer an alternative to liposome-mediated transfection methods. Lipid nanoparticles represent an extension of this transfection method, particularly suitable for the delivery of small molecule drugs in clinical research and therapeutic applications (as outlined in Cullis and Hope, 2017).

[0080] Physical methods for gene transfer were developed in the early 1980s, initially involving direct microinjection into cultured cells or nuclei (Cappechi, 1980). More recently, microinjection has regained popularity in gene editing applications and is being used to deliver CRISPR-Cas9 DNA to the zygote pronucleus to create knockout pigs (Chuang et al. 2017). Electroporation represents another physical method, first reported for gene transfer studies in mouse cells (Wong and Neumann, 1982). This mechanism is based on the use of electrical pulses to perturb the cell membrane and create transient pores that allow nucleic acids to pass into the cell (Shigekawa and Dower, 1988).

[0081] The most widely used nonviral transfection reagents include lipid-based reagents such as lipofectamine and cationic polymers such as PEI. Numerous transfection methods are known in the art (see, for example, Graham et al. (1973), Virology, 52:456; Sambrook et al. (1989), Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986), Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1981), Gene, 13:197). Such techniques can be used to introduce one or more exogenous nucleic acids into suitable host cells.

[0082] As used herein, the terms “transfect” and “transform” refer to the introduction of exogenous nucleic acids (e.g., DNA plasmids) into host cells. Therefore, the terms “transfection” and “transformation” refer to the process of introducing exogenous nucleic acids (e.g., DNA plasmids) into host cells. A cell is “transformed” or “transfected” if the exogenous nucleic acid is introduced inside the cell membrane. Exogenous nucleic acids introduced into host cells may be stably integrated into the host cell’s genome or may be extrachromosomal. Integrated nucleic acids may be maintained within the cell and inherited by progeny cells. Alternatively, exogenous nucleic acids introduced into host cells may be transiently present in the host cell.

[0083] Therefore, as used herein, the term “transfected cell” refers to a cell into which an exogenous nucleic acid has been introduced, or its offspring in which the exogenous nucleic acid is present. Transfected cells can be cultured (i.e., propagated) to transcribe and / or express proteins with the introduced exogenous nucleic acid.

[0084] As used herein, the term "transfection reagent" refers to a virus or nonviral agent that facilitates nucleic acid-mediated cell transfection.

[0085] As used herein, the terms “polymer / nucleic acid complex,” “polycationic / nucleic acid complex,” and “complex” refer to aggregates of cationic polymers and nucleic acids. Such complexes are also known in the art as polymer-condensed nucleic acids and can be used to transfect a wide range of cells.

[0086] When cationic polymers and nucleic acids are mixed under specific conditions and in the presence of ions, maturation of the polymer / nucleic acid complex occurs due to electrostatic interactions between the cationic polymer and the nucleic acid. Therefore, the present invention can be used with any cationic polymer and any nucleic acid. Furthermore, the present invention can be used for the transfection of any exogenous nucleic acid into cells during the production of any biological agent, such as recombinant protein or lentiviral vector.

[0087] Transfection with cationic polymers involves the condensation of nucleic acids and the subsequent release of exogenous nucleic acids into cells. Therefore, the efficiency of transfection using cationic polymers is related to the nucleic acid binding and dissociation of the polymer.

[0088] Traditionally, transfection complexes (e.g., polymer / nucleic acid complexes) are generated using both the nucleic acid component and the transfection reagent (liposome / polymer) prepared by diluting it with phosphate-buffered saline (PBS), sodium chloride (NaCl), or culture medium. Once the components are mixed, there is typically a "maturation" period for the transfection complex (transfection reagent / nucleic acid complex) related to the physical growth of these complexes to the optimal size for transfection. Beyond this optimal size, complexes that are too small will not deliver sufficient nucleic acid to the cells, and complexes that are too large will hinder endocytosis, thus reducing the efficiency of cell transfection. The dynamic process of transfection complex maturation can be problematic in that the time required to reach the optimal size may be unsuitable for use in large-scale production environments. Often, the time required to physically mix large quantities of DNA and transfection reagents may exceed the optimal time for complex maturation, and the time required to deliver the complex to the production bioreactor must also be considered in the transfection protocol.

[0089] The inventors have shown that polymer / nucleic acid complexes are stable for a short time in the presence of salt before adversely affecting transfection efficiency. This has also been reported by others. For example, manufacturers recommend that the incubation time between the cationic polymer PEIPro® and DNA should not exceed 30 minutes to ensure good transfection efficiency (see Polyplus Transfection PEIPro® DNA Transfection Kit (July 2020): https: / / fnkprddata.blob.core.windows.net / domestic / data / datasheet / PPU / 115-01K.pdf for the virus production protocol CPT115 vL). Because the optimal incubation period is too short to be practically feasible in large-scale GMP production, e.g., on a 200L scale, this limits the use of cationic polymers in the mass production of biological agents, e.g., recombinant proteins or lentiviral vectors.

[0090] To our surprise, the inventors have discovered the following: 1. By mixing the polymer / nucleic acid complex in a substantially salt-free solution (e.g., in water) and then spiking with salt to induce the maturation of the polymer / nucleic acid complex, a solution of the polymer / nucleic acid complex is provided that yields good transfection efficiency, thereby good functional lentiviral vector titer. In other words, by using an optimal concentration of salt, the time for optimal polymer / nucleic acid complex maturation (i.e., incubation time) can be extended. 2. The growth of the polymer / nucleic acid complex can be inhibited, and the complex can be stabilized by diluting the polymer / nucleic acid complex solution (therefore diluting the salt), further extending the incubation time.

[0091] The methods described herein are advantageous because they provide the ability to control both the initiation and termination of polymer / nucleic acid complex maturation. This is highly advantageous at GMP scale because it eliminates the time constraints that are a problem in the transfection process, i.e., improves the flexibility of the manufacturing process.

[0092] The preparation of polymer / nucleic acid complexes in a substantially salt-free aqueous solution (e.g., water) also offers advantages over conventional processes. For example, this allows for the preparation of polymer / nucleic acid complexes at higher concentrations than possible when the complexes are prepared in the presence of salt (e.g., in a medium), and significantly reduces the volume of polymer / nucleic acid complex solution added to the bioreactor. This also increases the total number of cells in the bioreactor during the transfection process, which is expected to increase productivity. Therefore, advantageously, the method of the present invention can be used in conjunction with a perfusion cell culture process prior to the transfection process. The use of perfusion cell culture can advantageously increase the cell density, i.e., the number of cells, in the bioreactor.

[0093] As a further example, the inventors have shown that when mixed in a substantially salt-free aqueous solution (e.g., water), the polymer and nucleic acid do not exhibit complex growth / aggregation, typically forming complexes with a diameter of less than 100 nM. Therefore, both the components and / or mixtures can be efficiently sterilized and filtered before complex formation (i.e., complex maturation) and subsequent addition during the manufacturing process. This adds a very valuable safety step to any manufacturing process, as it ensures the sterility of the complex before it is added to the manufacturing process (e.g., before addition to a bioreactor).

[0094] As a further example, the inventors have also shown that the polymer / nucleic acid complex is stable for several days and actually several weeks once matured in a substantially salt-free aqueous solution (e.g., water) and / or diluted after the complex has matured. This makes it possible to prepare the complex well in advance of the transfection step, significantly reducing the workload associated with this step during manufacturing and improving process flexibility. It also allows for small-scale testing of the transfection efficiency of the complex before use in manufacturing.

[0095] As a further example, the inventors have found that controlling the maturation of polymer / nucleic acid complexes in a substantially salt-free aqueous solution (e.g., water) allows for the preparation of complexes proportional to the number of cells to be transfected (i.e., scaling the concentration and / or volume of the polymer / nucleic acid complex preparation according to the number of cells to be transfected). Furthermore, the lack of toxicity associated with the polymer / nucleic acid complexes of the present invention allows for their use at higher concentrations compared to certain conventional transfection reagents (e.g., lipofectamine). This enables the transfection of a larger total number of cells in the bioreactor, which is expected to increase productivity. This also allows for the preparation of cells for transfection using perfusion culture to increase the total number of cells in the bioreactor for the transfection process.

[0096] Therefore, the methods described herein are not only suitable for use in large-scale GMP manufacturing of materials, but also offer advantages in such manufacturing processes. As used herein, “large-scale” includes standard bioreactor sizes, such as 50L, 200L, 500L, 1000L, and 10,000L.

[0097] The use of transfection enhancers to improve transfection performance and, for example, gene expression, is well known in the art. Examples of transfection enhancers include butyric acid, valproic acid, isobutyric acid, and isovaleric acid, or salts thereof. Salts of butyric acid, valproic acid, isobutyric acid, and isovaleric acid may be any salt, such as sodium or potassium salts (e.g., sodium butyrate). Transfection enhancers are typically added during cell culture after the transfection process. However, such agents may also be added before or during the transfection process, i.e., before or during contact of the transfection mix with the cells.

[0098] As used herein, the term “transfection enhancer” refers to a compound that increases nucleic acid-mediated cell transfection.

[0099] In some embodiments, the salt is not a transfection enhancer. In some embodiments, the salt is not butyrate, valproate, isobutyrate, or isovalerate. Preferably, the salt is not valproate, isobutyrate, or isovalerate. Preferably, the salt is not sodium butyrate.

[0100] In some embodiments, the salt is not added during or after the transfection process, i.e., during or after contact of the transfection mix (e.g., a solution of the polymer / nucleic acid complex) with the cells. In some preferred embodiments, the salt is added only before contacting the solution of the polymer / nucleic acid complex with the cells. Thus, by adding the salt to a mixture of cationic polymer and nucleic acid prepared in a substantially salt-free solution, a solution of the polymer / nucleic acid complex can be obtained that initiates the maturation and growth of the complex and can then be used for transfection.

[0101] In some embodiments, the final concentration of the salt is from about 10 mM to about 500 mM. In some preferred embodiments, the final concentration of the salt is from about 50 mM to about 500 mM. In some embodiments, the salt is PBS, and PBS is added to a final concentration of from about 0.1X PBS to about 3X PBS.

[0102] In some embodiments, the final concentration of the salt is from about 20 mM to about 100 mM. In some embodiments, the salt is PBS, and PBS is added to a final concentration of from about 0.15X PBS to about 0.3X PBS.

[0103] In some embodiments, the salt has a dissociation degree of at least 0.90 (suitably at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99 or 1) in a solution substantially free of salt.

[0104] As used herein, the term "dissociation degree" refers to the proportion of salt molecules that have dissociated into ions in a solution substantially free of salt. The dissociation degree can be calculated using methods known in the art (e.g., Dr Wolfgang Schartl, Basic Physical Chemistry: A Complete Introduction on Bachelor of Science Level (1 (aq) , , (l) , x , , x , , , d , (aq) , y(aq) , y , st , ,

[0106] ,

[0105] Ed. 2014) p. 109, Bookboon, ISBN 978 - 87 - 403 - 0669 - 9).

[0105] The dissociation constant defines the tendency for a substance M x N y to reversibly dissociate (separate) into smaller components M and N in a solution (often aqueous). M x N y(aq) +H2O (l) ⇔xM (aq) +yN (aq)

[0106] The dissociation constant is denoted as K d and is calculated as follows.

number

[0107] The degree of dissociation is the proportion of the original solute molecules that dissociate. This is usually denoted by the Greek letter α. More precisely, the degree of dissociation refers to the amount of solute that dissociates into ions or radicals per mole. For very strong acids and bases, the degree of dissociation is close to 1. Less strong acids and bases have a lower degree of dissociation. A simple relationship exists between this parameter and van't Hoff's factor i. When a solute dissociates into n ions, i = 1 + α(n-1)

[0108] For example, regarding the following dissociations, KCI⇔K + +CI - With n=2, i=1+α (Atkins P. and de Paula J. Physical Chemistry (8th ed. WH Freeman 2006) p.763, ISBN 978-0-7167-8759-4).

[0109] Therefore, in a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution. a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes, The salt is not valproate, isobutyrate, or isovalerate.

[0110] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes, The salt is added only before the polymer / nucleic acid complex solution comes into contact with the cells.

[0111] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes, The final salt concentration in step b) is approximately 10 mM to 100 mM.

[0112] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes, The salt has a degree of dissociation of at least 0.95 in a substantially salt-free aqueous solution.

[0113] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) optionally a step of incubating the salt solution produced in step b), and / or d) A step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes.

[0114] In a further embodiment, the present invention provides an in vitro method for producing a polymer / nucleic acid complex solution, a) A step of mixing nucleic acids and cationic polymers in a substantially salt-free aqueous solution, b) A step of adding salt to the mixture produced in step a) to form a salt solution, c) A step of incubating the salt solution produced in step b), and / or d) Depending on the circumstances, a step of diluting the salt solution produced in step b), or a step of diluting the salt solution after incubation in step c), Includes.

[0115] In some embodiments, step a) includes mixing a plurality of nucleic acid molecules and a plurality of cationic polymer molecules in a substantially salt-free aqueous solution.

[0116] As used herein, the term “substantially salt-free” means that an aqueous solution is salt-free or contains only trace amounts of salt. The salt content of an aqueous solution may be, for example, less than about 10 mM (appropriately less than about 7.5 mM, less than about 5 mM, less than about 2.5 mM, less than about 1 mM, less than about 0.5 mM, or less than about 0.1 mM). A substantially salt-free aqueous solution may contain less than about 1 mM of salt, appropriately less than about 0.5 mM of salt. The salt concentration of a substantially salt-free aqueous solution may be too low to initiate the maturation of polymer / nucleic acid complexes, i.e., the formation of polymer / nucleic acid complexes will not begin in the aqueous solution. The salt concentration of an aqueous solution can be determined using methods known in the art, for example, using a salinity meter. Salt concentrations are generally measured using a direct method using an optical refractometer or determined indirectly by a conductivity meter. Numerous commercially available options are available (https: / / www.pce-instruments.com / english / measuring-instruments / test-meters / salt-meter-kat_40093.htm).

[0117] The following procedure can be used to appropriately determine the solubility of a solid in water. 1) Water 100cm 3 Measure accurately and add to the beaker. 2) Add small amounts of solute until it no longer dissolves. 3) Record the mass of the evaporating dish. 4) Filter the mixture so that undissolved solids remain and the solution is in the evaporating dish. 5) Remove water by heating or evaporation. 6) Weigh the evaporating dish containing the solute and calculate the mass of the dissolved solute (https: / / www.bbc.co.uk / bitesize / guides / z4s48min / revision / 1).

[0118] In some preferred embodiments, step a) includes mixing the nucleic acid and the cationic polymer in a salt-free aqueous solution.

[0119] In some preferred embodiments, the substantially salt-free aqueous solution is water. Therefore, step a) may include mixing the nucleic acid and the cationic polymer in water.

[0120] As used herein, the term “nucleic acid” refers to all forms of nucleic acids, including DNA, RNA, and oligonucleotides. Thus, nucleic acids include genomic DNA, cDNA, spliced ​​or unspliced ​​mRNA, rRNA, tRNA, inhibitory DNA or RNA (e.g., RNAi, microRNA (miRNA), small interfering (si)RNA, short hairpin (sh)RNA, trans-splicing RNA, antisense RNA, or antisense DNA), naturally occurring sequences, synthetic sequences, and intentionally modified or altered sequences (e.g., mutant nucleic acids).

[0121] Therefore, in some embodiments, the nucleic acid is selected from DNA, RNA, oligonucleotide molecules, and mixtures thereof. Preferably, the nucleic acid is DNA, more preferably plasmid DNA.

[0122] In some embodiments, the cationic polymer is a polymer-based transfection reagent. Cationic polymer-based transfection reagents include polyethyleneimine (PEI), dendrimers, DEAE-dextran, polypropyleneimine (PPI), chitosan [poly-(β-1 / 4)-2-amino-2-deoxy-D-glucopyranose], poly-L-lysine (PLL), poly(lactic acid-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), and their derivatives. The derivatives may be chemically modified variants of the cationic polymer, such as PEGylated variants or histidined variants.

[0123] Cationic polymers may be provided in substantially salt-free aqueous solutions such as water, a neutral solution / buffer, or an acidic solution. Cationic polymers can be prepared by dissolving the cationic polymer in an aqueous solvent, a neutral solvent / buffer, or an acidic solvent. Neutral cationic polymer solutions typically have a pH of about 6.0 to about 8.0 (preferably about 6.5 to about 7.5, about 6.8 to about 7.2, or about 7.0 to about 7.2). Acidic cationic polymer solutions typically have a pH of about 0 to about 3.0 (preferably about 0.5 to about 2.0). Examples of neutral solvents / buffers include Tris (Trisma base) and HEPES. Buffer concentrations may range from about 1 mM to about 100 mM (preferably about 2 mM to about 50 mM or about 5 mM to about 20 mM). Examples of acidic solvents include mineral acids (e.g., hydrochloric acid) or organic acids (e.g., glycine hydrochloride solution). Any solvent or buffer can be used to establish or maintain the pH of the cationic polymer solution within an appropriate range without reducing the transfection activity of the cationic polymer. As described herein, the cationic polymer solution may be substantially salt-free.

[0124] PEI is a cationic polymer that offers one of the highest transfection efficiencies for introducing exogenous DNA into mammalian cells, and is therefore one of the most widely used non-viral transfection reagents in the art. PEI can be linear or branched PEI. PEI can have a molecular weight in the range of about 4 kDa to about 160 kDa (appropriately, about 4 kDa to about 160 kDa, about 40 kDa, or about 25 kDa). Linear PEI, branched PEI, and their derivatives are commercially available. Specifically, 40 kDa linear PEI and 25 kDa linear PEI are commercially available. Commercial forms of PEI include linear PEI, branched PEI, PEGylated PEI, JetPEI, PEIPro®, PEI MAX, and PTG1+.

[0125] Therefore, in some preferred embodiments, the cationic polymer is PEI (branched or linear PEI) or a derivative thereof. In some preferred embodiments, the cationic polymer is selected from linear PEI, branched PEI, PEGylated PEI, JetPEI, PEIPro®, PEI MAX, and PTG1+.

[0126] Cationic polymers and nucleic acids can be mixed in solution. Mixing can be carried out in any solution suitable for cationic polymer-based cell transduction. Non-limiting examples of suitable solutions are described herein. The molar or weight ratio of nucleic acids to cationic polymers is not limited, and typical ratios include molar ratios of about 100:1 to about 1:100 (appropriately about 1:1 to about 1:10, about 1:1 to about 1:5, about 1:2 to about 1:4) and weight ratios of about 1:10 to about 5:1 (appropriately about 1:1 to about 5:1). The amounts of nucleic acids and cationic polymers can also be expressed, and are not limited, as the molar ratio of total nitrogen (N) in the cationic polymer to phosphate (P) in the nucleic acid. Typical N:P ratios range from approximately 1:1 to approximately 50:1 (appropriately, approximately 1:1 to approximately 10:1, approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, or approximately 5:1).

[0127] Salts may be suitable for use in cell culture. Those skilled in the art will understand that salts should have low toxicity. Salts suitable for cell culture are well known in the art. Salts may be simple salts or mixtures of simple salts. Salts may be selected from, but are not limited to, sodium (Na) salts, potassium (K) salts, magnesium (Mg) salts, calcium (Ca) salts, phosphate (PO4) salts, or combinations thereof. For example, salts may be NaCl, MgCl2, NaNO3, PBS, or combinations thereof. Those skilled in the art will know that suitable salts may contain a cation of a strong base (e.g., selected from the first and second groups of the periodic table, such as K, Na, Ca, Mg, Ba, etc.) and / or an anion of a strong acid (e.g., Cl - NO3 - SO4 2- You will understand that this includes those that have ).

[0128] In some embodiments, the salt is added to the mixture of cationic polymer and nucleic acid (i.e., in step b)) to a final concentration of about 40 mM to about 500 mM (preferably about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 60 mM to about 350 mM, about 70 mM to about 300 mM, about 75 mM to about 250 mM, or about 80 mM to about 200 mM). In some embodiments, the salt is added to the mixture of cationic polymer and nucleic acid (i.e., in step b)) to a final concentration of about 10 mM to about 100 mM (preferably about 30 mM to about 100 mM or about 50 mM to about 100 mM). In some embodiments, the salt is added to a final concentration of less than about 500 mM (appropriately less than about 450 mM, less than about 400 mM, less than about 350 mM, less than about 300 mM, less than about 250 mM, less than about 200 mM, less than about 150 mM, less than about 100 mM, less than about 80 mM, or less than about 50 mM).

[0129] In some embodiments, the salt is added to the mixture of cationic polymer and nucleic acid (i.e., in step b)) to a final concentration of about 0.01 mM to about 49 mM (preferably about 0.1 mM to about 45 mM, about 5 mM to about 45 mM, about 10 mM to about 40 mM, about 20 mM to about 35 mM). In some embodiments, the salt is added to the mixture of cationic polymer and nucleic acid (i.e., in step b)) to a final concentration of about 27.5 mM. In some embodiments, the salt is added to a final concentration of less than about 49 mM (preferably less than about 45 mM, less than about 40 mM, less than about 35 mM, less than about 32 mM, less than about 30 mM, less than about 25 mM, or less than about 20 mM).

[0130] In some preferred embodiments, the salt is phosphate-buffered saline (PBS). In some embodiments, PBS is added in step b) to a final concentration of about 0.2X PBS to about 10X PBS (appropriately about 0.2X PBS to about 7.5X PBS or about 0.2X PBS to about 5X PBS). In some embodiments, PBS is added in step b) to a final concentration of less than about 10X PBS (appropriately less than about 9X PBS, less than about 8X PBS, less than about 7X PBS, less than about 6X PBS, less than about 5X PBS, or less than about 4X PBS). In some embodiments, PBS is added in step b) to a final concentration of about 0.2X PBS to about 3X PBS (appropriately about 0.2X PBS to about 2.5X PBS, about 0.2X PBS to about 2X PBS, about 0.2X PBS to about 1X PBS or about 0.2X PBS to about 0.5X PBS). In some preferred embodiments, PBS is added to a final concentration of about 0.2X PBS to about 1X PBS. In some embodiments, PBS is added in step b) to a final concentration of less than about 3X PBS (appropriately less than about 2.5X PBS, less than about 2X PBS, less than about 1X PBS, or less than about 0.5X PBS).

[0131] In some preferred embodiments, PBS is added in step b) to a final concentration of about 0.15X PBS to about 0.35X PBS (preferably about 0.20X PBS to about 0.30X PBS). In some embodiments, PBS is added to a final concentration of less than about 0.45X PBS (preferably less than about 0.40X PBS, less than about 0.35X PBS, less than about 0.30X PBS, or less than about 0.25X PBS). In some embodiments, PBS is added in step b) to a final concentration of about 0.2X PBS (preferably about 0.25X PBS, about 0.3X PBS, or about 0.35X PBS).

[0132] A mixture of cationic polymer and nucleic acid can initiate polymer / nucleic acid complex formation in the presence of ions, for example in a salt solution, and be incubated for a desired period to grow the complex to an optimal size for efficient transfection. In some preferred embodiments, the mixture of cationic polymer and nucleic acid is incubated in the presence of salt.

[0133] In some preferred embodiments, the method includes the step of incubating the salt solution produced in step b). The mixture of nucleic acid and cationic polymer (i.e., the salt solution produced in step b)) can be incubated for about 10 seconds to about 4 hours (preferably about 30 seconds to about 3 hours, about 1 minute to about 2 hours, about 5 minutes to about 90 minutes, about 10 minutes to about 75 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to about 45 minutes). The mixture of nucleic acid and cationic polymer (i.e., the salt solution produced in step b)) can be incubated for about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, or about 75 minutes. In some preferred embodiments, the salt solution produced in step b) is incubated for about 20 minutes to about 60 minutes, preferably about 60 minutes. Incubation can be carried out at any temperature suitable for the formation and / or growth of polymer / nucleic acid complexes, such as room temperature.

[0134] In the presence of ions, for example, a mixture of cationic polymers and nucleic acids in a salt solution can be diluted with or without incubation of the mixture to stabilize the polymer / nucleic acid complex for storage and subsequent use. In some preferred embodiments, the mixture of cationic polymers and nucleic acids is diluted. In some preferred embodiments, the mixture of cationic polymers and nucleic acids is diluted after the incubation step. Therefore, in some preferred embodiments, the solution of the polymer / nucleic acid complex is diluted after the incubation step.

[0135] In some embodiments, the method includes a step of diluting the salt solution produced in step b) or the salt solution after incubation in step c). In some preferred embodiments, the method includes a step of diluting the salt solution after incubation in step c). The diluent may be a substantially salt-free or salt-free solution, such as water, as described herein.

[0136] In some embodiments, the salt solution is diluted to a final concentration of about 0.05X PBS to about 0.15X PBS, preferably to a final concentration of about 0.10X PBS.

[0137] In some embodiments, the salt solution is diluted to a final concentration of about 5 mM to about 50 mM (preferably about 10 mM to about 40 mM or about 15 mM to about 30 mM). In some embodiments, the salt solution is diluted to a final concentration of less than about 50 mM, less than about 40 mM, less than about 30 mM or less than about 20 mM.

[0138] The polymer / nucleic acid complex solution can be stored at, for example, 4°C after being diluted before use, for example, for transfection.

[0139] Those skilled in the art will understand that the appropriate salt concentration for dilution to initiate or stabilize complex formation varies depending on the ionic strength of the salt used. Selecting an appropriate salt concentration for use in the method of the present invention for specific salts of known ionic strength is within the capabilities of those skilled in the art.

[0140] In some embodiments, the nucleic acid functions as a retroviral vector component.

[0141] In some embodiments, the retroviral vector is a replication-deficient retroviral vector.

[0142] In some embodiments, the retroviral vector is a lentiviral vector, preferably HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or Visna. The lentiviral vector may be a replication-deficient lentiviral vector.

[0143] In some embodiments, the vector component is a) RNA genome of lentiviral vectors, b) environment or its functional substitute, c) gag-pol or its functional substitute, and / or d) rev or its functional substitute Selected from.

[0144] Vector / expression cassette A vector is a tool that enables or facilitates the transfer of an entity from one environment to another. According to the present invention, and as an example, some vectors used in recombinant nucleic acid technology enable entities such as nucleic acid segments (e.g., heterologous DNA segments, e.g., heterologous cDNA segments) to be transferred into target cells and expressed by those target cells. A vector can facilitate the integration of nucleotide sequences encoding viral vector components and the nucleotide sequences encoding viral vector components in order to maintain their expression within target cells.

[0145] A vector may be or may include an expression cassette (also called an expression construct). An expression cassette as described herein includes a region of a transcribable nucleic acid-containing sequence. Therefore, sequences encoding mRNA, tRNA, and rRNA are included within this definition.

[0146] The vector may contain one or more select marker genes (e.g., neomycin resistance genes) and / or traceable marker genes (e.g., genes encoding green fluorescent protein (GFP)). The vector may be used, for example, to infect and / or transduce target cells. The vector may further contain a nucleotide sequence that allows the vector to replicate in the host cell of the problem, for example, a conditionally replicating oncolytic vector.

[0147] The term "cassette" is synonymous with terms such as "conjugate," "construct," and "hybrid," and includes a polynucleotide sequence directly or indirectly conjugated to a promoter. An expression cassette for use in the present invention includes a promoter for the expression of a nucleotide sequence encoding a viral vector component, and optionally a regulator of the nucleotide sequence encoding the viral vector component. Preferably, the cassette includes at least one polynucleotide sequence operably ligated to the promoter.

[0148] The choice of expression cassette, such as plasmid, cosmid, virus, or phage vector, often depends on the host cell into which it is introduced. Expression cassettes can be DNA plasmids (supercoiled, nicked, or linear), minicircle DNA (linear or supercoiled), plasmid DNA containing only the region of interest after removal of the plasmid backbone by restriction enzyme digestion and purification, or DNA produced using an enzyme DNA amplification platform, such as doggybone DNA (dbDNA®) prepared so that the final DNA used is either in a closed ring form or has open breaks (e.g., restriction enzyme digestion).

[0149] Retroviral vector production system and cells A retroviral vector production system (e.g., a lentiviral vector production system) includes a set of nucleotide sequences that encode the components necessary for the production of a retroviral vector (e.g., a lentiviral vector). Therefore, the vector production system includes a set of nucleotide sequences that encode the viral vector components necessary for the construction of retroviral vector particles (e.g., lentiviral vector particles).

[0150] A "viral vector production system," "vector production system," or "production system" should be understood as a system containing the components necessary for lentiviral vector production.

[0151] In one embodiment, the viral vector production system includes Gag and Gag / Pol proteins, as well as nucleotide sequences encoding the Env protein and vector genome sequence. The production system may optionally include nucleotide sequences encoding the Rev protein or its functional substitutions.

[0152] In one embodiment of the present invention, at least one transgene component may be inverted or reversed.

[0153] In one embodiment, at least one transgene component may be inverted or reversed relative to the 5'-3' orientation of the vector genomic RNA. A lentiviral vector genome can be used in which the transgene cassette is inverted, i.e., the transcription unit faces the promoter that drives the vector genomic cassette. Furthermore, if one component of the transgene cassette is reversed and another component is forward, for example, a bidirectional transgene cassette or multiple separate cassettes may be used.

[0154] In one embodiment, the viral vector production system includes a modular nucleic acid construct (modular construct). The modular construct is a DNA expression construct containing two or more nucleic acids used for the production of a lentiviral vector. The modular construct may be a DNA plasmid containing two or more nucleic acids used for the production of a lentiviral vector. The plasmid may be a bacterial plasmid. The nucleic acids may encode, for example, gag-pol, rev, env, or a vector genome. Furthermore, modular constructs designed for the construction of packaging cell lines and producer cell lines may further require encoding transcriptional regulatory proteins (e.g., TetR, CymR) and / or translational repression proteins (e.g., TRAP) and selection markers (e.g., Zeocin®, hygromycin, blastosidine, puromycin, neomycin resistance genes). A modular construct suitable for use in the present invention is described in European Patent No. 3502260, which is incorporated herein by reference in its entirety.

[0155] Since modular constructs for use according to the present invention contain nucleic acid sequences encoding two or more retroviral components in a single construct, the safety profile of these modular constructs has been considered, and further safety features have been directly incorporated into the constructs. These features include the use of insulators for multiple open reading frames of the retroviral vector component, and / or specific orientation and arrangement of retroviral genes in the modular construct. By using these features, it is believed that direct read-through for generating replicable viral particles is prevented.

[0156] Nucleic acid sequences encoding viral vector components may have reverse and / or alternating transcription orientations in a modular construct. Therefore, nucleic acid sequences encoding viral vector components are not presented in the same 5'-to-3' direction, and as a result, viral vector components cannot be produced from the same mRNA molecule. Reverse orientation may mean that at least two coding sequences for different vector components are presented in "head-to-head" and "tail-to-tail" transcription orientations. This can be achieved by providing a coding sequence for one vector component on one strand of the modular construct, e.g., env, and a coding sequence for another vector component on the opposite strand, e.g., rev. Preferably, if coding sequences for three or more vector components are present in a modular construct, at least two of the coding sequences are present in reverse transcription orientation. Therefore, if coding sequences for three or more vector components are present in a modular construct, each component may be oriented such that it is in the opposite 5'-to-3' direction to all adjacent coding sequences of other adjacent vector components; i.e., alternating 5'-to-3' (or transcription) directions may be used for each coding sequence.

[0157] A modular construct for use according to the present invention may include nucleic acid sequences encoding two or more of the following vector components: gag-pol, rev, env, and vector genome. The modular construct may include nucleic acid sequences encoding any combination of the vector components. In one embodiment, the modular construct is as follows: i) RNA genome and rev of a lentiviral vector, or functional substitution thereof, ii) RNA genome and gag-pol of lentiviral vectors, iii) RNA genome and environment of lentiviral vectors, iv) gag-pol and rev, or their functional substitutes, v) gag-pol and env, vi) env and rev, or their functional substitutes, vii) RNA genome of lentiviral vector, rev or its functional substitution, and gag-pol, viii) RNA genome of a lentiviral vector, rev or its functional substitution, and env, ix) RNA genome of lentiviral vector, gag-pol and env, or x) gag-pol, rev, or their functional substitutes, and env, It may contain a nucleic acid sequence that codes for Nucleic acid sequences are reversed and / or alternately oriented.

[0158] In one embodiment, cells for producing a lentiviral vector may contain nucleic acid sequences encoding any one of the combinations i) to x) above, wherein the nucleic acid sequences are located at the same locus and are oriented in opposite and / or alternating directions. The same locus may refer to a single extrachromosomal locus within the cell, e.g., a single plasmid, or a single locus within the cell's genome (i.e., a single insertion site). The cells may be stable or transient cells for producing a retroviral vector, e.g., a lentiviral vector.

[0159] DNA expression constructs may be DNA plasmids (supercoiled, nicked, or linear), minicircle DNA (linear or supercoiled), plasmid DNA containing only the region of interest after removal of the plasmid backbone by restriction enzyme digestion and purification, or DNA produced using an enzyme DNA amplification platform, such as doggybone DNA (dbDNA®) prepared so that the final DNA used is either in a closed ring form or has open breaks (e.g., restriction enzyme digestion).

[0160] In one embodiment, the lentiviral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visnarentivirus.

[0161] A “viral vector-producing cell,” “vector-producing cell,” or “producing cell” should be understood as a cell capable of producing a lentiviral vector or lentiviral vector particle. A lentiviral vector-producing cell may be a “producer cell” or “packaging cell.” One or more DNA constructs of a viral vector system may be stably incorporated into the viral vector-producing cell or maintained episomelly. Alternatively, all DNA components of the viral vector system may be transiently transfected into the viral vector-producing cell. Yet another alternative is that a producing cell stably expressing some of the components may be transiently transfected with the remaining components required for vector production.

[0162] As used herein, the term “packaging cell” refers to a cell that contains the elements necessary for the production of lentiviral vector particles but lacks the vector genome. In some cases, such packaging cells contain one or more expression cassettes capable of expressing viral structural proteins (e.g., gag, gag / pol, and env), typically rev.

[0163] Producer cells / packaging cells can be any suitable cell type. Producer cells are generally mammalian cells, but could also be insect cells, for example.

[0164] As used herein, the terms “producer cell” or “vector-producing / producer cell” refer to a cell containing all the elements necessary for the production of lentiviral vector particles. The producing cell may be a stable producer cell line, or it may be transiently induced, or it may be a stable packaging cell to which the retroviral genome is transiently expressed.

[0165] Vector-producing cells may be cells cultured in vitro, such as tissue culture cell lines. Suitable cell lines include, but are not limited to, mammalian cells such as mouse fibroblast-derived cell lines or human cell lines. Preferably, vector-producing cells are derived from human cell lines.

[0166] Cells and manufacturing methods Solutions of polymer / nucleic acid complexes obtained or obtainable by the in vitro methods described herein, or solutions of polymer / nucleic acid complexes described herein, may be used for cell transfection.

[0167] Therefore, in some embodiments, the in vitro method for producing the polymer / nucleic acid complex solution described herein is e) A step of culturing mammalian cells as needed, f) A step of transfecting mammalian cells using a solution of the polymer / nucleic acid complex obtained by the method described herein, g) Depending on the case, a step of introducing a nucleic acid different from the nucleic acid in the polymer / nucleic acid complex solution into mammalian cells, h) Depending on the case, a step of selecting mammalian cells having nucleic acids incorporated into the genome, i) Depending on the case, a step of culturing mammalian cells under conditions in which nucleic acids are expressed, j) Depending on the case, a step of culturing mammalian cells under conditions in which retroviral vectors are produced, k) Depending on the case, the process may include isolating the retroviral vector, It also includes.

[0168] Ideally, step e) is performed before steps a), b), c) and / or d), for example, before steps a) to d).

[0169] Ideally, step e) is carried out simultaneously with steps a), b), c) and / or d), for example, simultaneously with steps a) to d).

[0170] Ideally, step e) is performed after steps a), b), c) and / or d), for example, after steps a) to d) below.

[0171] In a further embodiment, the present invention provides the use of a solution of the polymer / nucleic acid complex described herein for the transfection of nucleic acids into cells.

[0172] In a further embodiment, the present invention provides the use of a solution of the polymer / nucleic acid complex described herein in a method for producing retroviral vectors.

[0173] In a further aspect, the present invention provides a method for producing a retroviral vector, a) A step of culturing mammalian cells as needed, b) A step of transfecting mammalian cells using a solution of the polymer / nucleic acid complex described herein, c) Depending on the case, a step of introducing at least one nucleic acid different from the nucleic acid in the polymer / nucleic acid complex solution into mammalian cells, d) Depending on the case, a step of selecting mammalian cells having nucleic acid sequences that encode viral vector components incorporated into their genome, e) A step of further culturing mammalian cells under conditions in which a retroviral vector is produced, Includes.

[0174] In a further embodiment, the present invention provides a lentiviral vector produced by any method of the present invention.

[0175] In a further embodiment, the present invention provides a method for generating cells for producing lentiviral vectors, a) A step of culturing mammalian cells as needed, b) A step of transfecting mammalian cells using a solution of the polymer / nucleic acid complex described herein, c) Depending on the case, a step of introducing at least one nucleic acid different from the nucleic acid in the polymer / nucleic acid complex solution into mammalian cells, d) Depending on the case, a step of selecting mammalian cells containing nucleic acid sequences encoding viral vector components, Includes.

[0176] In a further embodiment, the present invention provides a method for generating stable production cells for producing lentiviral vectors, a) A step of culturing mammalian cells as needed, b) A step of transfecting mammalian cells using a solution of the polymer / nucleic acid complex described herein, c) Depending on the case, a step of introducing at least one nucleic acid different from the nucleic acid in the polymer / nucleic acid complex solution into mammalian cells, d) A step of selecting cells containing the nucleic acid that encodes the vector component incorporated into the genome, Includes.

[0177] In a further embodiment, the present invention provides a method for producing transient-producing cells for lentiviral vector production, optionally comprising the steps of: culturing mammalian cells; transfecting the mammalian cells with a polymer / nucleic acid complex solution described herein; and optionally introducing at least one nucleic acid different from the nucleic acid in the polymer / nucleic acid complex solution into the mammalian cells.

[0178] In a further embodiment, the present invention provides cells for producing lentiviral vectors produced by any method of the present invention.

[0179] In a further embodiment, the present invention provides stable production cells for producing lentiviral vectors produced by any method of the present invention.

[0180] In a further embodiment, the present invention provides transient production cells for producing lentiviral vectors produced by any method of the present invention.

[0181] In some embodiments, the nucleic acid of the polymer / nucleic acid complex encodes a retroviral vector component. In some embodiments, the vector component is a) Genome of the viral vector, b) environment or its functional substitute, c) gag-pol or its functional substitute, and / or d) rev or its functional substitute Selected from.

[0182] In some embodiments, the retroviral vector is a replication-deficient retroviral vector.

[0183] In some embodiments, the retroviral vector is a lentiviral vector, preferably HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or Visna.

[0184] In some embodiments, the vector component is a) RNA genome of lentiviral vectors, b) environment or its functional substitute, c) gag-pol or its functional substitute, and / or d) rev or its functional substitute Selected from.

[0185] In some embodiments, the method includes a step of culturing mammalian cells. The step of culturing mammalian cells may be carried out by batch culture, fed-batch culture, concentrated fed-batch culture, or perfusion culture.

[0186] As used herein, the term “batch culture” refers to an operational technique for biotechnology processes, including viral vector production, in which cells are provided with a limited supply of nutrient-containing medium, i.e., cells are cultured in the same medium without the addition of fresh medium and / or supplements, and without the removal of nutrient-depleted medium containing impurities. Therefore, the culture will decrease as nutrients are depleted or other factors, such as the generation of waste or by-products, limit the amount of culture. Typically, the viral vector product is recovered at the end of the process. Batch culture is an upstream processing technique in the production of biomaterials or cell products, such as viral vectors.

[0187] As used herein, the term “fed-batch culture” refers to an operational technique for biotechnology processes, including viral vector production, in which one or more nutrients necessary for cell proliferation and viral vector production (e.g., nutrients that would otherwise be limiting) are supplied to cells in a culture vessel, e.g., a bioreactor, during culture. The viral vector product remains in the culture vessel, e.g., a bioreactor, until the end of the process run in which the viral vector is recovered. One or more nutrients may be supplied continuously or periodically via at least one feed stream without removing a nutrient-deficient medium containing impurities; i.e., one or more nutrients are added to another batch process. The process may be repeated if the cells remain viable at the end of the process run. Fed-batch culture is an upstream processing technique in the production of biomaterials or cell products such as viral vectors.

[0188] As used herein, the term “concentrated fed-batch culture” refers to an operational technique for biotechnology processes, including viral vector production, in which a perfusion culture system, which may be alternating tangential flow (ATF) or tangential flow filtration (TFF), is used in conjunction with an ultrafiltration membrane. In concentrated fed-batch culture, cells are held inside a culture vessel, such as a bioreactor, while nutrient-depleted medium containing impurities is removed and fresh medium is supplied to the cells. This process retains the product in the culture vessel, such as a bioreactor, until the process is complete, as in conventional fed-batch culture, but obtains higher cell and product concentrations in the culture vessel.

[0189] As used herein, the term “perfusion culture” (also known as “continuous perfusion culture” or “perfusion”) refers to an operational technique for biotechnology processes, including viral vector production, in which cells are held inside a culture vessel, such as a bioreactor, while nutrient-depleted medium containing impurities is continuously removed and fresh medium is continuously supplied to the cells. The fresh medium is typically supplied to the cells at the same rate as the nutrient-depleted medium containing impurities is removed. Thus, continuous perfusion involves the continuous removal of small bioreactor working volumes and the introduction of fresh medium into the bioreactor in a volume typically equivalent to the volume of medium removed. Perfusion culture is an upstream continuous processing technique in the production of biomaterials or cell products, such as viral vectors. Viral vector products may remain in the culture vessel, such as a bioreactor, until the end of the process run in which the viral vectors are recovered. For example, this may be the case for intracellularly produced viral vectors such as adenovirus vectors and adeno-associated virus vectors. Alternatively, the viral vector products may be perfused from the culture vessel, such as a bioreactor, and subsequently recovered from the perfusion. For example, this could occur with secreted viral vectors such as lentiviral vectors, when perfusion is performed after viral vector production has begun.

[0190] In one embodiment, the method includes the step of culturing mammalian cells in perfusion culture (e.g., in a perfusion culture system). Continuous perfusion culture is advantageous over batch culture or fed-batch culture in viral vector production because the accumulation of impurities that limit productivity and purity is reduced by the exchange of culture medium.

[0191] The manufacturing process of the present invention is preferably a large-scale process for producing clinical-grade formulations suitable for administration to humans as therapeutic agents. The viral vector compositions described herein are preferably suitable for administration to humans as therapeutic agents.

[0192] The methods according to the present invention as described herein may further include the step of inoculating mammalian cells into a cell culture vessel. Preferably, the step of inoculating mammalian cells into a cell culture vessel is performed before the step of culturing the mammalian cells (for example, culturing mammalian cells in a perfusion culture). “Inoculate” means, for example, introducing cells into the culture medium in the culture vessel.

[0193] Ideally, the process of culturing mammalian cells (for example, culturing mammalian cells in a perfusion culture) should be performed immediately after the process of inoculating mammalian cells into the cell culture vessel.

[0194] In some embodiments, the method further includes a step of performing a rapid medium change after the step of inoculating cells and before the step of transfecting cells, preferably the medium is changed about 20 to about 28 hours after inoculation. Preferably, the medium may be changed about 21 hours (preferably 22, 23, 24, 25, 26, or 27) to about 28 hours after inoculation. Preferably, the medium may be changed about 21 to about 27, 22 to about 26, or 23 to about 25 hours before transfection. Preferably, the medium may be changed about 24 hours before transfection. Preferably, the medium may be changed less than about 21 hours (preferably less than about 22, about 23, about 24, about 25, about 26, about 27, or about 28 hours) after inoculation. Preferably, the step of culturing mammalian cells includes at least one rapid medium change. Cell cultures using rapid medium changes are advantageous over batch or fed-batch cultures in viral vector production for the same reasons as perfusion culture: the accumulation of impurities that limit productivity and purity is reduced by the medium changes.

[0195] As used herein, the term “rapid medium exchange” refers to an operational technique for biotechnology processes, including viral vector production, in which a specified volume of cell culture vessel working volume is removed and a specified volume of fresh medium is introduced into the cell culture vessel in a short discrete time, while the cells are held inside a culture vessel, e.g., a bioreactor as described herein. Thus, the medium, which has been depleted of nutrients by cellular metabolism, as well as impurities and other drugs that may adversely affect viral vector production, such as transfection reagents, are removed, and fresh medium is provided to the cells during the rapid medium exchange. The rapid medium exchange is a sequential process, i.e., the used medium is first removed from the cell culture vessel (e.g., bioreactor), and then the fresh medium is introduced into the cell culture vessel (e.g., bioreactor). The specified volume of fresh medium introduced into the cell culture vessel may be higher, lower, equal to, or equal to the specified volume of cell culture vessel working volume removed. The volume of nutrient-deficient medium containing the removed impurities is typically equal to the volume of fresh medium introduced. A specified working volume of cell culture vessels is typically removed from the vessel, followed by the introduction of a specified volume of fresh medium; that is, the working volume of the cell culture vessel is temporarily reduced during rapid medium exchange before the addition of fresh medium. This differs from conventional perfusion processes where the removal of the working volume of the cell culture vessel and the introduction of fresh medium typically occur simultaneously. Rapid medium exchange is an upstream continuous processing technique in the production of biomaterials or cell products such as viral vectors.

[0196] The culture vessel may be a bioreactor, wave bag, roller bottle, or flask. Preferably, the culture vessel is a bioreactor.

[0197] In one embodiment, the suspension culture may be in a culture vessel such as a bioreactor. The bioreactor may have a capacity (e.g., working capacity) of, for example, about 0.1 liters to about 1000 liters, or about 0.1 liters to about 500 liters.

[0198] For example, the capacity such as the working capacity can be about 0.1 liter to about 0.25 liter, about 0.5 liter to about 250 liters, about 1 liter to about 200 liters, about 5 to about 180 liters, about 10 to about 150 liters, about 15 to about 100 liters, about 20 to about 80 liters, or about 30 to about 50 liters.

[0199] In one embodiment, the capacity such as the working capacity can be about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000 or 2000 liters.

[0200] In one embodiment, the working capacity can be about 2 liters to about 100 liters.

[0201] In one embodiment, the working capacity can be about 5 liters.

[0202] In one embodiment, the working capacity can be about 50 liters.

[0203] In one embodiment, the working capacity can be about 200 liters.

[0204] In one embodiment, the working capacity can be about 1000 liters.

[0205] In some embodiments, the method includes culturing mammalian cells under conditions in which nucleic acid is expressed.

[0206] In some embodiments, the method includes culturing mammalian cells under conditions in which a retroviral vector is produced.

[0207] In some embodiments, the method includes isolating a retroviral vector.

[0208] In some embodiments, the mammalian cells are HEK293T cells.

[0209] In some embodiments, the mammalian cells are suspension-compatible mammalian cells.

[0210] As used herein, the term “suspension-compatible cells” refers to cells that typically grow in an adhesion manner suited to suspension growth. Methods for adapting cells to suspension are known in the art. For example, adaptation to suspension growth can be achieved by continuous serum reduction during cell passage. This yields suspension-compatible cells that grow in serum-free medium, i.e., in the absence of serum such as fetal bovine serum (FBS).

[0211] Therefore, in some embodiments, the transfection, introduction, and / or culture steps are carried out in a suspension in serum-free medium.

[0212] The method of the present invention can be carried out on adherent cells cultured in a suspension system. For example, adherent cells can be cultured on microcarriers in a suspension system.

[0213] In some embodiments, the transfection is transient.

[0214] The polymer / nucleic acid complex methods and solutions described herein are suitable for use in large-scale production of materials. Non-limiting examples of large-capacity culture vessels suitable for use in the present invention include spinner flasks (maximum capacity approximately 36 L), wave bags (maximum capacity approximately 100 L), and bioreactors (maximum capacity approximately 10,000 L).

[0215] In some embodiments, the transfection, introduction and / or culture steps are carried out in a volume of at least 50 L (preferably at least 60 L, at least 75 L, at least 100 L, or at least 200 L), preferably in a volume of at least 50 L (preferably at least 60 L, at least 75 L, at least 100 L, or at least 200 L).

[0216] In some embodiments, nucleic acids different from those in the polymer / nucleic acid complex solution are introduced into mammalian cells by transfection or electroporation.

[0217] In some embodiments, nucleic acids different from the nucleic acids in the polymer / nucleic acid complex solution are those not encoded by the nucleic acids in the polymer / nucleic acid complex solution, such as: a) RNA genome of lentiviral vectors, b) environment or its functional substitute, c) gag-pol or its functional substitute, and / or d) rev or its functional substitute, Encodes any viral vector component selected from the available options.

[0218] In some embodiments, the method further includes a step of repeating steps (i), (j), and / or (k). In some embodiments, the method further includes a step of repeating steps (i), (j), and (k). In some embodiments, the method further includes a step of repeating steps (i) and (k). In some embodiments, the method further includes a step of repeating steps (j) and (k). Steps (i), (j), and / or (k) may be repeated multiple times.

[0219] As described above, perfusion culture is advantageous over batch culture or fed-batch culture in viral vector production because the accumulation of impurities that limit productivity and purity is reduced by medium exchange. This makes it possible to achieve higher cell densities. Therefore, the length of the perfusion step can be varied according to the target cell density for transfection. Appropriately, cells can be cultured continuously in the perfusion cell culture system for a long period of time. Appropriately, cells can be cultured continuously in the perfusion cell culture system from inoculation to the transfection step, or immediately before the transfection step.

[0220] In some embodiments, cells are cultured in a perfusion culture system for approximately 0.5 hours to approximately 168 hours (preferably approximately 0.5 hours to approximately 156 hours, approximately 0.5 hours to approximately 144 hours, approximately 0.5 hours to approximately 132 hours, approximately 0.5 hours to approximately 120 hours, approximately 0.5 hours to approximately 108 hours, approximately 0.5 hours to approximately) prior to the step of transfecting mammalian cells with the polymer / nucleic acid complex solution described herein. The cells are cultured for 96 hours, approximately 0.5 hours to approximately 84 hours, approximately 0.5 hours to approximately 72 hours, approximately 0.5 hours to approximately 60 hours, approximately 0.5 hours to approximately 48 hours, approximately 0.5 hours to approximately 36 hours, approximately 0.5 hours to approximately 32 hours, approximately 0.5 hours to approximately 28 hours, approximately 0.5 hours to approximately 24 hours, approximately 0.5 hours to approximately 22 hours, approximately 0.5 hours to approximately 20 hours, approximately 0.5 hours to approximately 18 hours, or approximately 0.5 hours to approximately 16 hours. Preferably, the cells are cultured in a perfusion culture system for approximately 0.5 hours to approximately 24 hours, approximately 0.5 hours to approximately 22 hours, approximately 0.5 hours to approximately 20 hours, or approximately 0.5 hours to approximately 18 hours, for example, approximately 0.5 hours to approximately 20 hours.

[0221] In some embodiments, the cells are cultured in a perfusion culture system for about 1 hour to about 168 hours (preferably about 1 hour to about 156 hours, about 1 hour to about 144 hours, about 1 hour to about 132 hours, about 1 hour to about 120 hours, about 1 hour to about 108 hours, about 1 hour to about 96 hours, about 1 hour to about 84 hours, about 1 hour to about 72 hours, about 1 hour to about 60 hours, about 1 hour to about 48 hours, about 1 hour to about 36 hours, about 1 hour to about 32 hours, about 1 hour to about 28 hours, about 1 hour to about 24 hours, about 1 hour to about 22 hours, about 1 hour to about 20 hours, about 1 hour to about 18 hours, or about 1 hour to about 16 hours) prior to the step of transfecting mammalian cells with a solution of the polymer / nucleic acid complex described herein. Preferably, the cells are cultured in a perfusion culture system for about 1 to 24 hours, about 1 to 22 hours, about 1 to 20 hours, or about 1 to 18 hours, for example, about 1 to 20 hours.

[0222] In some embodiments, cells are cultured in a perfusion culture system for about 12 to about 168 hours (preferably about 12 to about 156 hours, about 12 to about 144 hours, about 12 to about 132 hours, about 12 to about 120 hours, about 12 to about 108 hours, about 12 to about 96 hours, about 12 to about 84 hours, about 12 to about 72 hours, about 12 to about 60 hours, about 12 to about 48 hours, about 12 to about 36 hours, about 12 to about 32 hours, about 12 to about 28 hours, about 12 to about 24 hours, about 12 to about 22 hours, about 12 to about 20 hours, about 12 to about 18 hours, or about 12 to about 16 hours) prior to the step of transfecting mammalian cells with a solution of the polymer / nucleic acid complex described herein. Preferably, the cells are cultured in a perfusion culture system for about 12 to 24 hours, about 12 to 22 hours, about 12 to 20 hours, or about 12 to 18 hours, for example, about 12 to 20 hours.

[0223] In some embodiments, the transfection step is performed immediately after the step of culturing mammalian cells in perfusion culture.

[0224] In some embodiments, the transfection step is performed about 0.5 hours to about 24 hours (preferably about 0.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours or about 24 hours, preferably about 1 hour) after the step of culturing mammalian cells in perfusion culture.

[0225] In some embodiments, the transfection step is performed after the step of culturing mammalian cells in perfusion culture for at least about 0.5 hours to at least about 24 hours (appropriately, at least about 0.5 hours, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, or at least about 24 hours, preferably at least about 1 hour).

[0226] In some embodiments, perfusion culture is performed by removing approximately 50% to approximately 1000% (appropriately approximately 50% to approximately 900%, approximately 50% to approximately 800%, approximately 50% to approximately 700%, approximately 50% to approximately 600%, approximately 50% to approximately 500%, approximately 50% to approximately 400%, approximately 50% to approximately 300%, approximately 50% to approximately 250%, approximately 50% to approximately 200%, approximately 50% to approximately 150%, approximately 50% to approximately 100%, approximately 100%, approximately 150%, approximately 200%, approximately 300%, approximately 400%, approximately 500%, approximately 600%, approximately 700%, approximately 800%, approximately 900%, or approximately 1000%) of the suspension cell culture volume and introducing an appropriate volume of fresh medium into the suspension cell culture for the entire duration of the perfusion culture. Therefore, in some embodiments, perfusion culture is performed at approximately 50% to approximately 1000% (appropriately, approximately 50% to approximately 900%, approximately 50% to approximately 800%, approximately 50% to approximately 700%, approximately 50% to approximately 600%, approximately 50% to approximately 500%, approximately 50% to approximately 400%, approximately 50% to approximately 300%, approximately 50% to approximately 250%, approximately 50% to approximately 200%, approximately 50% to approximately 150%, and approximately 50%). This is done by removing approximately 100%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% of the working volume of the cell culture vessel, such as a bioreactor, and introducing an appropriate volume of fresh medium into the cell culture vessel, such as a bioreactor. Preferably, this volume is removed over the entire duration of the perfusion culture. The volume of fresh medium introduced into the cell culture vessel may be greater than, less than, equal to, or equal to the working volume of the cell culture vessel removed during the perfusion culture. In a preferred embodiment, the volume of fresh medium introduced into the suspended cell culture is equal to the volume of the suspended cell culture removed during the perfusion culture.

[0227] Suitably, a volume of the suspension cell culture of about 50% to about 1000% (suitably, about 50% to about 900%, about 50% to about 800%, about 50% to about 700%, about 50% to about 600%, about 50% to about 500%, about 50% to about 400%, about 50% to about 300%, about 50% to about 250%, about 50% to about 200%, about 50% to about 150%, about 50% to 100%, 100%, 150%, 200%) can be removed, and the volume of fresh medium introduced into the suspension cell culture can be equivalent to the volume of the suspension cell culture removed.

[0228] The suspension cell culture volume removed in the perfusion culture process can be at least about 50% (suitably, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900% or at least about 1000%) of the suspension cell culture volume.

[0229] The suspension cell culture volume removed in the perfusion culture process can be less than about 50% (suitably, less than about 100%, less than about 150%, less than about 200%, less than about 250%, less than about 300%, less than about 400%, less than about 500%, less than about 600%, less than about 700%, less than about 800%, less than about 900% or less than about 1000%) of the suspension cell culture volume.

[0230] In a preferred embodiment, about 100% to about 200% (suitably about 150%) of the suspension cell culture volume is removed in the perfusion culture process, and the volume of fresh medium introduced into the suspension cell culture can be equivalent to the volume of the suspension cell culture removed.

[0231] In some embodiments, perfusion culture is performed using flow rates of approximately 50% to 500% of the daily suspension cell culture volume (appropriately, approximately 50% to 450%, 50% to 400%, 50% to 350%, 50% to 300%, 50% to 250%, 50% to 200%, 50% to 150%, 50% to 100%, 100%, 150%, and 200%). Therefore, in some embodiments, perfusion culture is performed using a flow rate of about 50% to about 500% per day of the cell culture vessel working volume, for example, the bioreactor working volume (appropriately, about 50% to about 450%, about 50% to about 400%, about 50% to about 350%, about 50% to about 300%, about 50% to about 250%, about 50% to about 200%, about 50% to about 150%, about 50% to about 100%, about 100%, about 150%, about 200%). Preferably, perfusion culture is performed using a flow rate of about 50% to about 200% per day of the suspension cell culture volume (appropriately, about 50% to about 150% or about 50% to 100%). Preferably, perfusion culture is performed using a flow rate of about 200%, about 150%, or about 100% per day of the suspension cell culture volume. Preferably, perfusion culture is performed using a flow rate of approximately 150% of the daily suspension cell culture volume.

[0232] As described above, the use of rapid medium changes is advantageous in viral vector production compared to batch culture or fed-batch culture because the accumulation of impurities that limit productivity and purity is reduced by the medium changes. This makes it possible to achieve higher cell densities.

[0233] In some embodiments, rapid medium changes are completed within approximately 0.1 to 8 hours (ideally, approximately 0.2 to 8 hours, 0.3 to 8 hours, 0.4 to 8 hours, 0.5 to 8 hours, 1 to 8 hours, 1.5 to 8 hours, 2 to 8 hours, 2.5 to 8 hours, 3 to 8 hours, 4 to 8 hours, and 5 to 8 hours).

[0234] Rapid medium changes can be completed within approximately 0.1 to 5 hours (ideally within approximately 0.2 to 5 hours, 0.3 to 5 hours, 0.4 to 5 hours, 0.5 to 5 hours, 1 to 5 hours, 1.5 to 5 hours, 2 to 5 hours, 2.5 to 5 hours, 3 to 5 hours, and 4 to 5 hours). Rapid medium changes can be completed within approximately 0.1 to 3 hours (ideally within approximately 0.2 to 3 hours, 0.3 to 3 hours, 0.4 to 3 hours, 0.5 to 3 hours, 1 to 3 hours, 1.5 to 3 hours, 2 to 3 hours, and 2.5 to 3 hours).

[0235] Rapid culture medium changes can be completed in less than approximately 0.5 hours (ideally less than approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, and 8 hours).

[0236] In some embodiments, rapid medium exchange is performed by removing approximately 50% to 99% of the volume of the suspended cell culture and introducing an appropriate volume of fresh medium into the suspended cell culture. Thus, in some embodiments, rapid medium exchange is performed by removing approximately 50% to 99% of the working volume of the cell culture vessel, e.g., the working volume of a bioreactor, and introducing an appropriate volume of fresh medium into the cell culture vessel, e.g., a bioreactor. The volume of fresh medium introduced into the cell culture vessel may be greater than, less than, equal to, or equal to the working volume of the cell culture vessel removed during rapid medium exchange. In preferred embodiments, the volume of fresh medium introduced into the suspended cell culture is equal to the volume of the suspended cell culture removed during rapid medium exchange.

[0237] Appropriately, approximately 55% to 99% (appropriately, approximately 60% to 99%, 65% to 99%, 70% to 99%, 75% to 99%, 80% to 99%, 85% to 99%, and 90% to 99%) of the suspension cell culture volume may be removed during rapid medium exchange p, and the volume of fresh medium introduced into the suspension cell culture may be equivalent to the volume of suspension cell culture volume removed.

[0238] Appropriately, approximately 55% to 95% (appropriately, approximately 60% to 90%, 65% to 85%, 70% to 80%, 70% to 85%, and 75% to 85%) of the suspension cell culture volume may be removed during rapid medium exchange, and the volume of fresh medium introduced into the suspension cell culture may be equivalent to the volume of suspension cell culture volume removed.

[0239] In a preferred embodiment, approximately 70% to approximately 85% (preferably approximately 75% or approximately 80%) of the volume of the suspended cell culture is removed, and the volume of fresh medium introduced into the suspended cell culture may be equivalent to the volume of the suspended cell culture removed.

[0240] The volume of the cell culture suspension removed may be at least about 50% (appropriately at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%).

[0241] The volume of suspension cell culture removed may be less than approximately 55%, less than approximately 60%, less than approximately 65%, less than approximately 70%, less than approximately 75%, less than approximately 80%, less than approximately 85%, less than approximately 90%, less than approximately 95%, or less than approximately 99%.

[0242] In some embodiments, rapid medium changes are performed 0 to about 24 hours before transfection (appropriately about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 to 24 hours). In some embodiments, a rapid medium change is performed 0 to about 3 hours before transfection (preferably about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2 or 2.5 to about 3 hours). A rapid medium change may be performed 0 to about 2 hours before transfection (preferably about 0.1, 0.2, 0.3, 0.4, 0.5, 1 or 1.5 to about 2 hours). Rapid medium changes should be performed before transfection at approximately 0.1 hours, 0.2 hours, 0.3 hours, 0.4 hours, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, and 7 hours. It may be performed in less than approximately 7.5 hours, less than approximately 8 hours, less than approximately 8.5 hours, less than approximately 9 hours, less than approximately 9.5 hours, less than approximately 10 hours, less than approximately 11 hours, less than approximately 12 hours, less than approximately 13 hours, less than approximately 14 hours, less than approximately 15 hours, less than approximately 16 hours, less than approximately 17 hours, less than approximately 18 hours, less than approximately 19 hours, less than approximately 20 hours, less than approximately 21 hours, less than approximately 22 hours, less than approximately 23 hours, or less than approximately 24 hours. In a preferred embodiment, rapid medium exchange is performed about 1 hour before transfection.

[0243] In some embodiments, cells may be in the exponential growth phase during perfusion culture. After the perfusion culture step, the cells grow to approximately 1 x 10⁻⁶. 5 cells / mL ~ approx. 1x10 9 Cells / mL (Appropriately, about 2 x 10⁶) 5 cells / mL ~ approx. 5x10 8 cells / mL, approximately 3x10 5 cells / mL ~ approx. 1x10 8cells / mL, approximately 4x10 5 cells / mL ~ approx. 5x10 7 cells / mL, approximately 5x10 5 cells / mL ~ approx. 1x10 7 cells / mL, approximately 6x10 5 cells / mL ~ approx. 5x10 6 cells / mL, approximately 7x10 5 cells / mL ~ approx. 1x10 6 The final cell density may be approximately 1 x 10⁶ cells / mL. After the perfusion culture process, the cells will be approximately 1 x 10⁶ cells / mL. 5 cells / mL ~ approx. 5x10 7 Cells / mL (Appropriately, about 2 x 10⁶) 5 cells / mL ~ approx. 1x10 7 cells / mL, approximately 3x10 5 cells / mL ~ approx. 5x10 6 cells / mL, approximately 4x10 5 cells / mL ~ approx. 1x10 6 cells / mL, approximately 5x10 5 cells / mL ~ approx. 1x10 6 The final cell density may be (cells / mL). The cell number may increase by at least about 25% (appropriately, at least about 50%, at least about 70%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%) during the perfusion culture process. The cell count may increase by approximately 25% (appropriately, approximately 50%, 70%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000%) during the perfusion culture process. The cell count may increase by approximately 0.5 times (appropriately, approximately 1 time, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, or 25 times) during the perfusion culture process.

[0244] The polymer / nucleic acid complex solution may be in contact with cells (i.e., incubated) for transfection for an unspecified period. For example, the time may range from about 20 minutes to about 24 hours (appropriately, about 30 minutes to about 20 hours, about 1 hour to about 16 hours, about 2 hours to about 12 hours, or about 3 hours to about 8 hours). To reduce the cytotoxic effect of the cationic polymer on cells, the culture medium may be replaced with fresh medium after transfection.

[0245] Cells may be in the exponential growth phase before or at the time of contact with the polymer / nucleic acid complex solution. Cells may be in the exponential growth phase before or at the time of contact with the polymer / nucleic acid complex solution, approximately 1 x 10⁻¹⁶ cells. 5 cells / mL ~ approx. 1x10 8 Cells / mL (Appropriately, about 2 x 10⁶) 5 cells / mL ~ approx. 5x10 6 cells / mL, approximately 3x10 5 cells / mL ~ approx. 4x10 6 cells / mL, approximately 4x10 5 cells / mL ~ approx. 3x10 6 cells / mL, approximately 5x10 5 cells / mL ~ approx. 2x10 6 cells / mL, approximately 6x10 5 cells / mL ~ approx. 1x10 6 It may have a cell density of (cells / mL).

[0246] In some embodiments, the polymer / nucleic acid complex:cell ratio used in the transfection process is 1 x 10⁻¹⁰ 6 Approximately 0.1 μg of DNA per cell (ideally, 1 x 10⁶ 6 Approximately 0.2 μg of DNA per cell, 1 x 10⁻⁶ 6 Approximately 0.3 μg of DNA per cell, 1 x 10⁻⁶ 6 Approximately 0.4 μg of DNA per cell, 1 x 10⁶ 6 Approximately 0.5 μg of DNA per cell, 1 x 10⁶ 6 Approximately 0.6 μg of DNA per cell, 1 x 10⁻⁶ 6 Approximately 0.7 μg of DNA per cell, 1 x 10⁻⁶ 6 Approximately 0.8 μg of DNA per cell, 1 x 10⁻⁶ 6Approximately 0.9 μg of DNA per cell, 1 x 10⁶ 6 Approximately 1 μg of DNA per cell, 1 x 10 6 1.5 μg of DNA or 1 x 10⁶ per cell 6 The amount of DNA per cell is 2 μg. Preferably, the polymer / nucleic acid complex:cell ratio used in the transfection step is 1 x 10⁻¹⁶. 6 Each cell contains approximately 0.6 μg of DNA.

[0247] In some embodiments, the polymer / nucleic acid complex:cell ratio used in the transfection process is 1 x 10⁻¹⁰ 6 Approximately 0.3 μg of DNA per cell (ideally, 1 x 10⁶ 6 Approximately 0.6 μg of DNA per cell, 1 x 10⁻⁶ 6 Approximately 1 μg of DNA per cell, 1 x 10 6 1.5 μg of DNA or 1 x 10⁶ per cell 6 The amount of DNA per cell is 2 μg. Preferably, the polymer / nucleic acid complex:cell ratio used in the transfection step is 1 x 10⁻¹⁶. 6 Each cell contains approximately 0.6 μg of DNA.

[0248] In the method of the present invention, the vector component may include the gag, env, rev, and / or RNA genome of a viral vector. The nucleotide sequences encoding the vector component can be introduced into cells simultaneously or sequentially in any order.

[0249] In some embodiments of the methods and uses of the present invention, suitable producing cells or cells for producing lentiviral vectors are cells capable of producing viral vectors or viral vector particles when cultured under suitable conditions. Therefore, the cells typically contain nucleotide sequences encoding vector components, which may include gag, env, rev, and the RNA genome of the lentiviral vector. Suitable cell lines include, but are not limited to, mammalian cells such as mouse fibroblast-derived cell lines or human cell lines. They are generally mammalian cells, including human cells such as HEK293T, HEK293, CAP, CAP-T, or CHO cells, but may also be insect cells such as SF9 cells. Preferably, the vector-producing cells are derived from human cell lines. Therefore, such suitable producing cells can be used in any of the methods or uses of the present invention.

[0250] Methods for introducing nucleotide sequences into cells are well known in the art and have been previously described. Therefore, the introduction of nucleotide sequences encoding vector components, including gag, env, rev, and RNA genomes of lentiviral vectors, into cells using conventional techniques in molecular biology and cell biology is within the scope of the skills of those skilled in the art.

[0251] Stable producing cells may be packaging or producer cells. To produce producer cells from packaging cells, a vector genomic DNA construct can be introduced stably or transiently. Packaging / producer cells can be produced by transduction of a retroviral vector expressing one of the vector components, namely the genome, gag-pol component, and envelope, into a suitable cell line, as described in International Publication No. 2004 / 022761.

[0252] Alternatively, nucleotide sequences can be transfected into cells, and then integration into the producing cell genome occurs rarely and randomly. Transfection methods using solutions of polymer / nucleic acid complexes described herein can be carried out using methods well known in the art. For example, a stable transfection process may use constructs engineered to facilitate concatemerization. Those skilled in the art will be aware of methods to facilitate the integration of nucleotide sequences into producing cells. For example, linearizing a nucleic acid construct may be helpful if it is naturally circular. Less random integration methodologies may include nucleic acid constructs containing shared regions homologous to endogenous chromosomes of mammalian host cells to induce integration into selected sites within the endogenous genome. Furthermore, if recombination sites are present on the construct, these can be used for targeted recombination. For example, a nucleic acid construct may contain loxP sites that enable targeted integration when combined with Cre recombinase (i.e., using a Cre / lox system derived from a P1 bacteriophage). Alternatively, or in addition, the recombination site is an att site (e.g., from a lambda phage) that enables site-specific integration in the presence of lambda integrase. This makes it possible to target lentiviral genes to loci in the host cell genome, enabling high and / or stable expression.

[0253] Other methods of targeted recombination are well known in the art. For example, targeted recombination at selected chromosomal loci can be promoted by using methods that induce targeted breaks in genomic DNA. These methods often involve the use of methods or systems to induce double-strand breaks (DSBs), e.g., nicks in the endogenous genome, to induce repair of the breaks by physiological mechanisms such as non-homologous end joining (NHEJ). Breaks can be caused by the use of specific nucleases such as engineered zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the use of a CRISPR / Cas9 system with an engineered crRNA / tracr RNA ("single guide RNA") to guide specific breaks, and / or the use of Argonaut system-based nucleases (e.g., from T. thermophilus).

[0254] Packaging / producer cell lines can be used that can be prepared by incorporating nucleotide sequences using lentiviral transduction alone, nucleic acid transfection alone, or a combination of both.

[0255] Methods for producing retroviral vectors from cells, particularly for processing retroviral vectors, are described in International Publication No. 2009 / 153563.

[0256] In one embodiment, the producing cells may contain an RNA-binding protein (e.g., tryptophan RNA-binding attenuation protein, TRAP) and / or a Tet repressor (TetR) protein or an alternative regulatory protein (e.g., CymR).

[0257] The production of lentiviral vectors from producing cells may be by transfection, by production from stable cell lines which may include an induction step (e.g., doxycycline induction), or by a combination of both. The transfection method can be carried out using methods well known in the art, examples of which have been previously described.

[0258] Cells, packaging cell lines, or producer cell lines transiently transfected with components encoding lentiviral vectors are cultured to increase cell number, virus number, and / or viral titer. Cell culture is performed to enable the cells to metabolize and / or grow and / or divide and / or produce the viral vector of the present invention. This can be achieved by methods well known to those skilled in the art, including, for example, providing nutrients to the cells in a suitable culture medium. Methods may include surface-adherent growth, growth in suspension, or a combination thereof. Culture can be performed in tissue culture flasks, tissue culture multiwell plates, dishes, roller bottles, wave bags, or bioreactors, for example, using batch, fed-batch, or continuous systems. In order to achieve large-scale production of viral vectors by cell culture, it is preferable in the art to have cells that can grow in suspension. The optimal conditions for culturing cells are well known (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (1973), and RIFreshney, Culture of animal cells: A manual of basic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

[0259] Preferably, the cells are first "bulked up" in a tissue culture flask or bioreactor, and then grown in a multi-layer culture vessel or large bioreactor (over 50 L) to produce the vector-producing cells of the present invention.

[0260] Preferably, cells are grown in an adhesive manner to produce the vector-producing cells of the present invention.

[0261] Preferably, cells are grown in suspension mode to produce the vector-producing cells of the present invention.

[0262] Polymer / nucleic acid complex solution In a further embodiment, the present invention provides solutions of polymer / nucleic acid complexes obtained or obtainable by any of the in vitro methods described herein.

[0263] Accordingly, in further embodiments, the present invention provides a solution comprising a polymer / nucleic acid complex and a salt, wherein the salt concentration is approximately 10 mM to approximately 500 mM. In some embodiments, the salt concentration is approximately 50 mM to approximately 500 mM.

[0264] In further embodiments, the present invention provides a solution comprising a polymer / nucleic acid complex and a salt, wherein the salt concentration is approximately 0.1 mM to approximately 49 mM. In some embodiments, the salt concentration is approximately 10 mM to approximately 49 mM.

[0265] In some embodiments, the salt is PBS, and the PBS concentration is approximately 0.15X PBS to approximately 3X PBS.

[0266] In some embodiments, the concentration of PBS is less than approximately 0.3X PBS.

[0267] Methods for transfecting mammalian cells In a further aspect, the present invention provides a method for transfecting mammalian cells, a) A process of culturing mammalian cells, b) A step of transfecting mammalian cells using a solution of the polymer / nucleic acid complex described herein, Includes.

[0268] In some embodiments, the process of culturing mammalian cells is carried out by perfusion culture.

[0269] Therefore, in a further aspect, the present invention provides a method for transfecting mammalian cells, a) A step of culturing mammalian cells in perfusion culture, b) A step of transfecting mammalian cells using a solution of the polymer / nucleic acid complex described herein, Includes.

[0270] More precisely, mammalian cells are the mammalian cells described herein.

[0271] In some embodiments, the method is carried out in a suspension in serum-free medium.

[0272] In some embodiments, steps a) and / or b) are carried out in a volume of at least 50 L, preferably steps a) and b) are carried out in a volume of at least 50 L.

[0273] In some embodiments, the transfection is transient.

[0274] The steps of culturing mammalian cells (e.g., in perfusion culture) and transfecting mammalian cells are carried out as described herein.

[0275] Retroviral vectors and lentiviral vectors Viral vectors can also be called vectors, vector virions, or vector particles.

[0276] Retroviral vectors can originate from or be derived from any suitable retrovirus. Numerous different retroviruses have been identified. Examples include mouse leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney's mouse leukemia virus (MoMLV), FBR mouse osteosarcoma virus (FBR MSV), Moloney's mouse sarcoma virus (Mo-MSV), Abelson's mouse leukemia virus (A-MLV), avian myelocytoplasmosis virus-29 (MC29), and avian erythroblastosis virus (AEV). A detailed list of retroviruses can be found in Coffin et al. (1997) “Retroviruses”, Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763.

[0277] Retroviruses can be broadly classified into two categories: "simple" and "complex." Retroviruses can be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are lentiviruses and spumaviruses. A review of these retroviruses is presented in Coffin et al (1997).

[0278] Lentiviruses are part of a larger group of retroviruses. A detailed list of lentiviruses can be found in Coffin et al. (1997) “Retroviruses”, Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763. Briefly, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human autoimmune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototype “slow virus” Visna-Maedivirus (VMV), as well as related canine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), Maedivisna virus (MVV), and bovine immunodeficiency virus (BIV). In one embodiment, the lentiviral vector is derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV, or visnarentivirus.

[0279] Lentiviridae distinguish lentiviruses from retroviruses in that they have the ability to infect both dividing and non-dividing cells (Lewis et al (1992) EMBO J 11(8):3053-3058 and Lewis and Emerman (1994) J Virol 68(1):510-516). In contrast, other retroviruses such as MLV cannot infect non-dividing cells or slowly dividing cells, such as the cells that make up muscle, brain, lung, and liver tissue.

[0280] A lentiviral vector, as used herein, is a vector comprising at least one component portion that can be derived from a retrovirus. Preferably, the component portion is involved in the biological mechanism by which the vector infects or transduces target cells and expresses NOI.

[0281] Retroviral vectors can be used to replicate NOIs in vitro in compatible target cells. Accordingly, this specification describes a method for producing the protein in vitro by introducing the vector of the present invention into compatible target cells in vitro and growing the target cells under conditions that result in NOI expression. The protein and NOI can be recovered from the target cells by methods well known in the art. Suitable target cells include mammalian cell lines and other eukaryotic cell lines.

[0282] In some embodiments, the vector may have an "insulator" gene sequence that acts as a barrier, blocking the interaction between the promoter and the enhancer and reducing read-through from adjacent genes.

[0283] In one embodiment, insulators are present between one or more retroviral nucleic acid sequences to prevent promoter interference and read-through from adjacent genes. When insulators are present in the vector between one or more retroviral nucleic acid sequences, each of these insulator genes may be positioned as an individual expression unit.

[0284] The basic structure of retroviral and lentiviral genomes shares many common features, such as packaging signals that enable genome packaging, primer binding sites, integration sites that allow integration into the target cell genome, and 5'LTR and 3'LTR located between or within the gag / pol and env genes (which are polypeptides necessary for the assembly of viral particles) that encode the packaging components. Lentiviruses have further features, such as the rev gene and RRE sequence in HIV, which enable the efficient transport of the RNA transcript of the integrated provirus from the nucleus to the cytoplasm of infected target cells.

[0285] In proviruses, these genes are flanked at both ends by a region called a long-terminal repeat (LTR). LTRs are responsible for provirus integration and transcription. LTRs also function as enhancer promoter sequences, allowing them to regulate the expression of viral genes.

[0286] The LTR itself is an identical sequence that can be divided into three elements called U3, R, and U5. U3 originates from a sequence specific to the 3' end of the RNA. R originates from a sequence repeated at both ends of the RNA, and U5 originates from a sequence specific to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.

[0287] In the typical retroviral vectors described herein, at least a portion of one or more protein-coding regions essential for replication can be removed from the virus; for example, gag / pol and env may be absent or non-functional. This results in a defect in viral vector replication.

[0288] Lentiviral vectors may originate from either primate lentiviruses (e.g., HIV-1) or non-primate lentiviruses (e.g., EIAV).

[0289] Generally speaking, a typical retroviral vector production system involves the isolation of the viral genome from essential viral packaging functions. These components are typically supplied to the producing cells on a separate DNA expression cassette (or known as a plasmid, expression plasmid, DNA construct, or expression construct).

[0290] The vector genome contains an NOI. The vector genome typically requires a packaging signal (ψ), an internal expression cassette containing an NOI, (occasionally) a post-transcription element (PRE), typically a central polyprint lactucle (cppt), 3'-ppu, and a self-inactivating (SIN)LTR. The R-U5 region is necessary for the correct polyadenylation of both the vector genomic RNA and NOI mRNA, as well as the reverse transcription process. The vector genome may optionally contain an open reading frame that enables vector production in the absence of rev, as described in International Publication No. 2003 / 064665.

[0291] The packaging functions include the gag / pol and env genes. These are necessary for the production of vector particles by the producing cells. By providing these functions in trans to the genome, the production of replication-deficient viral vectors is facilitated.

[0292] Gamma retroviral vector production systems are typically three-component systems requiring genome, gag / pol, and env expression constructs. HIV-1 lentiviral vector production systems may further require the provision of an accessory gene, rev, and that the vector genome contain a rev response element (RRE). EIAV-based lentiviral vectors do not require rev to be provided in trans if an open reading frame (ORF) is present in the genome (see International Publication No. 2003 / 064665).

[0293] Typically, both "external" promoters (driving the vector genome cassette) and "internal" promoters (driving the NOI cassette), encoded within the vector genome cassette, are potent eukaryotic or viral promoters, as are those that drive other vector system components. Examples of such promoters include CMV, EF1α, PGK, CAG, TK, SV40, and ubiquitin promoters. Potent "synthetic" promoters (e.g., the JeT promoter), such as those generated by DNA libraries, can also be used to drive transcription. Alternatively, tissue-specific promoters, such as rhodopsin (Rho), rhodopsin kinase (RhoK), cone-rod homeobox-containing gene (CRX), neuroretinal-specific leucine zipper protein (NRL), vitiligo macular dystrophy 2 (VMD2), tyrosine hydroxylase, neuron-specific neuron-specific enolase (NSE) promoter, astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, human α1-antitrypsin (hAAT) promoter, phosphoenolpyruvate carboxykinase (PEPCK), and liver fatty acid-binding protein. Transcription can be driven using the following promoters: quality promoter, Flt-1 promoter, INF-β promoter, Mb promoter, SP-B promoter, SYN1 promoter, WASP promoter, SV40 / hAlb promoter, SV40 / CD43, SV40 / CD45, NSE / RU5' promoter, ICAM-2 promoter, GPIIb promoter, GFAP promoter, fibronectin promoter, endoglin promoter, elastase-1 promoter, desmin promoter, CD68 promoter, CD14 promoter, and B29 promoter.

[0294] The production of retroviral vectors involves either transient cotransfection of the producing cells with these DNA components, or the use of a stable producing cell line in which all components are stably integrated into the producing cell genome (e.g., Stewart HJ, Fong-Wong L, Strickland I, Chipchase D, Kelleher M, Stevenson L, Thoree V, McCarthy J, Ralph GS, Mitrophanous KA and Radcliffe PA. (2011). Hum Gene Ther. Mar;22(3):357-69). Another approach is to use stable packaging cells (in which the packaging components are stably integrated) and then transiently transfect them with a vector genome plasmid as needed (e.g., Stewart, HJ, MALeroux-Carlucci, CJSion, KA Mitrophanous and PA Radcliffe (2009). Gene Ther. Jun;16(6):805-14). It is also possible to create incomplete alternative packaging cell lines (in which only one or two packaging components are stably incorporated into the cell line) and transiently transfect the missing component to produce a vector. Producing cells may also express regulatory proteins such as members of the tet repressor (TetR) protein family of transcription factors (e.g., T-Rex, Tet-On, and Tet-Off), members of the kumate-inducible switch system family of transcription factors (e.g., kumate repressor (CymR) proteins), or RNA-binding proteins (e.g., TRAP-tryptophan-activated RNA-binding protein).

[0295] In one embodiment of the present invention, the viral vector is derived from EIAV. EIAV has the simplest genomic structure of a lentivirus and is particularly preferred for use in the present invention. In addition to the gag / pol and env genes, EIAV encodes three other genes: tat, rev, and S2. tat acts as a transcriptional activator of the viral LTR (Derse and Newbold (1993) Virology 194(2):530-536 and Maury et al (1994) Virology 200(2):632-642), and rev regulates and modulates viral gene expression via the rev response element (RRE) (Martarano et al. (1994) J Virol 68(5):3102-3111). The mechanisms of action of these two proteins are thought to be broadly similar to similar mechanisms in primate viruses (Martarano et al. (1994) J Virol 68(5):3102-3111). The function of S2 is unknown. Furthermore, the EIAV protein Ttm has been identified, which is encoded by the first exon of tat spliced ​​into the env coding sequence at the initiation of the transmembrane protein. In an alternative embodiment of the present invention, the viral vector is derived from HIV, which does not encode S2, but unlike EIAV, encodes vif, vpr, vpu, and nef.

[0296] The term "recombinant retroviral vector or lentiviral vector" (RRV) refers to a vector containing sufficient retroviral genetic information to enable the packaging of an RNA genome into a viral particle that can transduce target cells in the presence of a packaging component. Transduction of target cells may involve reverse transcription and integration into the target cell genome. RRVs carry nonviral coding sequences that are delivered to target cells by the vector. RRVs cannot replicate independently and cannot produce infectious retroviral particles within target cells. Typically, RRVs lack functional gag / pol and / or env genes and / or other genes essential for replication.

[0297] Preferably, the RRV vector of the present invention has the smallest viral genome.

[0298] As used herein, the term “minimal viral genome” means that a viral vector has been engineered to remove non-essential elements while retaining essential elements that provide the necessary functions for infecting, transducing, and delivering NOI to target cells. Further details of this strategy can be found in International Publication No. 1998 / 17815 and International Publication No. 99 / 32646. Minimal EIAV vectors lack tat, the S2 gene, and optionally rev, neither of which are supplied in trans in the production system. Minimal HIV vectors lack vif, vpr, vpu, tat, and nef.

[0299] The expression plasmid used to produce the vector genome in the producing cell may contain transcriptional regulatory sequences operably linked to the retroviral genome to direct the transcription of the genome in the producing / packaging cell. All third-generation lentiviral vectors have a deletion in the 5'U3 enhancer-promoter region, and the transcription of the vector genomic RNA is driven by a different viral promoter, such as a heterologous promoter like the CMV promoter, as described later. This feature enables vector generation independently of tat. Some lentiviral vector genomes require additional sequences for efficient virus production. For example, in the case of HIV in particular, this may include RRE sequences. However, the need for RREs for the (separate) GagPol cassette (and the dependence on the rev provided in trans) can be reduced or eliminated by codon optimization of the GagPol ORF. Further details of this strategy can be found in International Publication No. 2001 / 79518.

[0300] Other sequences that perform the same function as the rev / RRE system are also known. For example, a functional analogue of the rev / RRE system is found in Mason Pfizer monkey virus. This is known as a constitutive transport element (CTE) and contains an RRE-type sequence in the genome that is thought to interact with factors in infected cells. Cellular factors can be considered rev analogues. Therefore, CTEs can be used as an alternative to the rev / RRE system. Any other functional equivalents of the Rev protein that are known or will become available may be relevant to the present invention. For example, it is known that the Rex protein of HTLV-I can functionally substitute for the Rev protein of HIV-1. Rev and RRE may not be present in the vector for use in the method of the present invention, or may be non-functional, and alternative rev and RRE, or functionally equivalent systems, may exist.

[0301] As used herein, the term “functional substitute” means a protein or sequence that has an alternative sequence that performs the same function as another protein or sequence. The term “functional substitute” is used herein interchangeably with “functional equivalent” and “functional analogue” for the same meaning.

[0302] SIN Vector The viral vectors described herein may be used in a self-inactivating (SIN) configuration in which the viral enhancer and promoter sequences are deleted. SIN vectors can be constructed and transduced into non-dividing target cells in vivo, ex vivo, or in vitro with similar efficacy to that of non-SIN vectors. Transcriptional inactivation of the long terminal repeat (LTR) in SIN proviruses should prevent vRNA recruitment and further reduce the likelihood of replication-capable virus formation. This should also allow for regulated gene expression from the internal promoter by eliminating the cis-effect of the LTR.

[0303] As an example, self-inactivating retroviral vector systems are constructed by deleting transcriptional enhancers or enhancers and promoters within the U3 region of the 3'LTR. After a series of vector reverse transcriptions and incorporations, these changes are copied to both the 5'LTR and 3'LTR, producing transcriptionally inactive "proviruses." However, any promoters within the LTR in such vectors remain transcriptionally active. This strategy is used to eliminate the influence of enhancers and promoters in the viral LTR on transcription from genes located within it. Such effects include increased or suppressed transcription. This strategy can also be used to eliminate downstream transcription from the 3'LTR to genomic DNA. This is of particular concern in human gene therapy, where preventing accidental activation of endogenous oncogenes is crucial (Yu et al., (1986) PNAS 83:3194-98; Marty et al., (1990) Biochimie 72:885-7; Naviaux et al., (1996) J. Virol. 70:5701-5; Iwakuma et al., (1999) Virol. 261:120-32; Deglon et al., (2000) Human Gene Therapy 11:179-90). SIN lentiviral vectors are described in U.S. Patent Nos. 6,924,123 and 7,056,699.

[0304] Replication-deficient lentiviral vector In the genome of a replication-deficient lentiviral vector, the gag / pol and / or env sequences may be mutated and / or non-functional.

[0305] In typical lentiviral vectors described herein, at least a portion of the coding region of one or more proteins essential for viral replication can be removed from the vector. This results in a loss of viral vector replication. A portion of the viral genome can also be replaced with an NOI to create a vector containing an NOI that can be transduced into non-dividing target cells and / or its genome can be incorporated into the target cell genome.

[0306] In one embodiment, the lentiviral vector is a non-embedded vector as described in International Publication No. 2006 / 010834 and International Publication No. 2007 / 071994.

[0307] In further embodiments, the vector has the ability to deliver sequences that do not contain or lack viral RNA. In further embodiments, heterologous binding domains (heterologous to gag) located on the delivered RNA and homologous binding domains on Gag or GagPol can be used to ensure packaging of the delivered RNA. Both of these vectors are described in International Publication No. 2007 / 072056.

[0308] Vector titer Those skilled in the art will understand that there are numerous different methods for determining the titer of a viral vector. The titer is often expressed as transduction units / mL (TU / mL). The titer can be increased by increasing the number of infectious particles and by increasing the specific activity of the vector preparation.

[0309] NOI and polynucleotides The polynucleotides of the present invention may include DNA or RNA. They may be single-stranded or double-stranded. It will be understood by those skilled in the art that numerous different polynucleotides may encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, it will be understood that, using routine techniques, nucleotide substitutions can be made that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention in order to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is expressed.

[0310] Polynucleotides can be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or lifespan of the polynucleotides of the present invention.

[0311] Polynucleotides, such as DNA polynucleotides, can be prepared recombinantly, synthetically, or by any means available to those skilled in the art. They can also be cloned by standard techniques.

[0312] Longer polynucleotides will generally be produced using recombinant methods, for example, polymerase chain reaction (PCR) cloning techniques. This involves producing a primer pair (e.g., about 15–30 nucleotides) adjacent to the target sequence to be cloned; contacting the primers with mRNA or cDNA obtained from animal or human cells; performing PCR under conditions that result in amplification of the desired region; isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel); and recovering the amplified DNA. The primers may be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0313] Common retrovirus vector elements Promoter and enhancer NOI and polynucleotide expression can be controlled using transcriptional regulatory elements or translational repression elements, including regulatory sequences such as promoters, enhancers, and other expression regulatory signals (e.g., the tet repressor (TetR) system), or transgene repression in vector-producing cell lines (TRIPs) or other regulators of NOI described herein.

[0314] Functional prokaryotic promoters and promoters can be used in eukaryotic cells. Tissue-specific or stimulus-specific promoters may be used. Chimeric promoters containing sequence elements from two or more different promoters may also be used.

[0315] Appropriate promotion sequences are strong promoters, including those derived from the genomes of viruses such as polyomavirus, adenovirus, fowlpox virus, bovine papillomavirus, arowanasarcoma virus, cytomegalovirus (CMV), retroviruses, and simian virus 40 (SV40), or those derived from heterologous mammalian promoters such as actin promoter, EF1α, CAG, TK, SV40, ubiquitin, PGK, or ribosomal protein promoter. Alternatively, tissue-specific promoters, such as rhodopsin (Rho), rhodopsin kinase (RhoK), cone-rod homeobox-containing gene (CRX), neuroretinal-specific leucine zipper protein (NRL), vitiligo macular dystrophy 2 (VMD2), tyrosine hydroxylase, neuron-specific neuron-specific enolase (NSE) promoter, astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, human α1-antitrypsin (hAAT) promoter, phosphoenolpyruvate carboxykinase (PEPCK), and liver fatty acid-binding protein. Transcription can be driven using the following promoters: quality promoter, Flt-1 promoter, INF-β promoter, Mb promoter, SP-B promoter, SYN1 promoter, WASP promoter, SV40 / hAlb promoter, SV40 / CD43, SV40 / CD45, NSE / RU5' promoter, ICAM-2 promoter, GPIIb promoter, GFAP promoter, fibronectin promoter, endoglin promoter, elastase-1 promoter, desmin promoter, CD68 promoter, CD14 promoter, and B29 promoter.

[0316] NOI transcription can be further increased by inserting enhancer sequences into the vector. While the enhancers are relatively orientation and position independent, eukaryotic virus-derived enhancers such as the SV40 enhancer and the CMV initial promoter enhancer may be used. The enhancers can be spliced ​​into the vector at a 5' or 3' position relative to the promoter, but are preferably located 5' from the promoter.

[0317] The promoter may further include features to ensure or increase expression in appropriate target cells. For example, features may be conserved regions, such as Pribnow boxes or TATA boxes. The promoter may include other sequences that affect (e.g., maintain, enhance, or decrease) the expression level of a nucleotide sequence. Suitable other sequences include Sh1-introns or ADH introns. Other sequences may include inductive elements such as temperature, chemical, light, or stress-inducible elements. There may also be suitable elements to enhance transcription or translation.

[0318] Regulators of NOI A complex factor in the generation of retroviral packaging / producer cell lines and retroviral vector production is that the constitutive expression of certain retroviral vector components and NOIs is cytotoxic, leading to the death of cells expressing these components and thus preventing vector production. Therefore, the expression of these components (e.g., gag-pol and envelope proteins, e.g., VSV-G) can be regulated. The expression of other non-cytotoxic vector components, e.g., rev, can also be regulated to minimize the metabolic burden on cells. Thus, the modular constructs or nucleotide sequences encoding vector components and / or cells described herein may include cytotoxic and / or non-cytotoxic vector components associated with at least one regulatory element. As used herein, the term “regulatory element” refers to any element that can be increased or decreased in relation to the expression of a relevant gene or protein. Regulatory elements include gene switch systems, transcriptional regulatory elements, and translational repression elements.

[0319] Numerous prokaryotic regulatory systems have been adapted to generate gene switches in mammalian cells. Many retroviral packaging and producer cell lines are regulated using gene switch systems (e.g., tetracycline and kmeate-inducible switch systems), and thus can turn on the expression of one or more retroviral vector components during vector production. Gene switch systems include those involving transcription factor (TetR) proteins (e.g., T-Rex, Tet-On, and Tet-Off), those involving transcription factor kmeate-inducible switch systems (e.g., CymR proteins), and those involving RNA-binding proteins (e.g., TRAP).

[0320] One such tetracycline-inducible system is the tetracycline repressor (TetR) system based on the T-REx® system. For example, in such a system, the tetracycline operator (TetO2) is positioned such that its first nucleotide is 10 bp from the 3' end of the last nucleotide of the TATATAA element of the human cytomegalovirus major pre-early promoter (hCMVp), and then TetR can act as a repressor on its own (Yao F, Svensjo T, Winkler T, Lu M, Eriksson C, Eriksson E., 1998, Hum Gene Ther; 9: 1939-1950). In such a system, NOI expression can be controlled by a CMV promoter in which two copies of the TetO2 sequence are inserted in tandem. The TetR homodimer, in the absence of an inducer (tetracycline or its analog doxycycline [dox]), binds to the TetO2 sequence and physically blocks transcription from the upstream CMV promoter. In the presence of the inducer, it binds to the TetR homodimer, causing an allosteric change that prevents it from binding to the TetO2 sequence, resulting in gene expression. The TetR gene can be codon-optimized because it has been found to improve translational efficiency, leading to tighter control of TetO2 regulatory gene expression.

[0321] The TRIP system, described in International Publication No. 2015 / 092440, provides an alternative method for suppressing NOI expression in producing cells during vector production. TRAP-binding sequence (e.g., TRAP-tbs) interactions form the basis of transgene protein suppression systems for retroviral vector production, particularly when it is desirable that constitutive and / or strong promoters, including tissue-specific promoters, drive the transgene, especially when the expression of the transgene protein in producing cells results in a decrease in vector titer and / or induces an immune response in vivo due to viral vector delivery of the transgene-derived protein (Maunder et al, Nat Commun. (2017) Mar27;8).

[0322] In short, the TRAP-tbs interaction forms a translation block and represses the translation of the transgene protein (Maunder et al, Nat Commun. (2017) Mar27;8). The translation block is effective only in producing cells and therefore does not interfere with DNA or RNA-based vector systems. The TRiP system can repress translation when the transgene protein is expressed from constitutive and / or potent promoters, including tissue-specific promoters derived from one or more cistronic mRNAs. Unregulated expression of the transgene protein has been shown to reduce the vector titer and affect the quality of the vector product. Repression of the transgene protein for both transient and stable PaCL / PCL vector production systems is beneficial to producing cells to prevent a decrease in vector titer, where toxicity or molecular weight issues may lead to cellular stress, the transgene protein may induce an immune response in vivo due to viral vector delivery of the transgene-derived protein, the use of gene-edited transgenes may result in on / off targeted effects, and the transgene protein may affect the elimination of the vector and / or envelope glycoprotein.

[0323] Envelope and pseudotype formation In one preferred embodiment, the lentiviral vectors described herein are pseudotyped. In this regard, pseudotyped vectors can offer one or more advantages. For example, the env gene product of an HIV-based vector restricts these vectors to infecting only cells that express a protein called CD4. However, if the env gene in these vectors is replaced with an env sequence derived from another enveloped virus, they may have a broader infection spectrum (Verma and Somia (1997) Nature 389(6648):239-242). As an example, the researchers pseudotyped an HIV-based vector having a glycoprotein derived from VSV (Verma and Somia (1997) Nature 389(6648):239-242).

[0324] In another embodiment, the Env protein may be a modified Env protein, such as a mutant Env protein or an engineered Env protein. Modifications may be made or selected to introduce targeting ability, reduce toxicity, or for other purposes (Valsesia-Wittman et al 1996 J Virol 70:2056-64; Nilson et al (1996) Gene Ther 3(4):280-286; and Fielding et al (1998) Blood 91(5):1802-1809 and references cited therein).

[0325] The vector can be pseudotyped with any selected molecule.

[0326] As used herein, "env" means the endogenous lentiviral envelope or heterologous envelope as described herein.

[0327] VSV-G The envelope glycoprotein (G) of the rhabdovirus vesicular stomatitis virus (VSV) is an envelope protein that has been shown to be able to pseudotype certain enveloped viruses and viral vector virions.

[0328] The ability of pseudotyped MoMLV-based retroviral vectors in the absence of retroviral envelope proteins was first demonstrated by Emi et al. (1991) Journal of Virology 65:1202-1207. International Publication No. 1994 / 294440 teaches that retroviral vectors can be successfully pseudotyped with VSV-G. These pseudotyped VSV-G vectors can be used to transduce a wide range of mammalian cells. More recently, Abe et al. (1998) J Virol 72(8) 6356-6361 teaches that non-infectious retroviral particles can be made infectious by the addition of VSV-G.

[0329] Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-7 successfully pseudotyped the retrovirus MLV containing VSV-G, resulting in a vector with a modified host range compared to its native MLV. VSV-G pseudotyped vectors have been shown to infect not only mammalian cells but also cell lines derived from fish, reptiles, and insects (Burns et al. (1993) ibid.). They have also been shown to be more efficient than conventional bidirectional envelopes for various cell lines (Yee et al., (1994) Proc. Natl. Acad. Sci. USA 91:9564-9568, Emi et al. (1991) Journal of Virology 65:1202-1207). Because the VSV-G protein's cytoplasmic tail can interact with the retroviral core, it can be used to pseudotype specific retroviruses.

[0330] Providing non-retroviral pseudotyping envelopes such as the VSV-G protein offers the advantage of enriching vector particles to high titers without losing infectivity (Akkina et al. (1996) J. Virol. 70:2581-5). Retroviral envelope proteins, likely consisting of two non-covalently linked subunits, are clearly unable to withstand the shear forces during ultracentrifugation. Interactions between subunits can be disrupted by centrifugation. In comparison, the VSV glycoprotein is composed of a single unit. Therefore, pseudotyping of the VSV-G protein can offer potential advantages both in efficient target cell infection / transduction and during the manufacturing process.

[0331] International Publication No. 2000 / 52188 describes the construction of pseudotyped retroviral vectors from stable producer cell lines containing vesicular stomatitis virus-G protein (VSV-G) as a membrane-bound viral envelope protein, and provides the gene sequence of the VSV-G protein.

[0332] Ross River virus The Ross River virus envelope has been used to pseudotype non-primate lentiviral vectors (FIV), primarily transducing the liver after systemic administration (Kang et al., 2002, J. Virol., 76:9378-9388). Efficiency was reported to be 20 times greater than that obtained with VSV-G pseudotyped vectors, and it caused less cytotoxicity, as measured by serum levels of liver enzymes suggestive of hepatotoxicity.

[0333] Baculovirus GP64 The baculovirus GP64 protein has been shown to be a viable alternative to VSV-G for the large-scale production of high-titer viruses required for clinical and commercial applications (Kumar M, Bradow BP, Zimmerberg J (2003) Hum Gene Ther. 14(1):67-77). Compared to VSV-G pseudotyped vectors, GP64 pseudotyped vectors have similar broad tropism and similar intrinsic titers. Since GP64 expression does not kill cells, HEK293T cell lines constitutively expressing GP64 can be constructed.

[0334] Alternative envelope Other envelopes that provide a reasonable titer when used with pseudotyped EIAV include Mocola, rabies, Ebola, and LCMV (lymphocytic choriomeningitis virus). Intravenous injection of lentiviruses pseudotyped with 4070A into mice resulted in maximum gene expression in the liver.

[0335] Packaging sequence When used in the context of this invention, the terms “packaging signal,” interchangeably referred to as “packaging sequence” or “psi,” are used to refer to a non-coding sequence necessary for capsid formation of the retroviral RNA chain during viral particle formation. In HIV-1, this sequence maps to a locus extending from upstream of the major splice donor site (SD) to at least the gag start codon (which may include part or all of the 5' sequence to nucleotide 688 of gag). In EIAV, the packaging signal includes the R region to the 5' coding region of Gag.

[0336] As used herein, the terms “elongation packaging signal” or “elongation packaging sequence” refer to the use of sequences around the psi sequence that further elongates within the gag gene. Including these additional packaging sequences can increase the efficiency of vector RNA insertion into the viral particle.

[0337] The determinants of feline immunodeficiency virus (FIV) RNA capsid formation have been shown to be distinct and discontinuous, comprising one region (R-U5) at the 5' end of the genomic mRNA and the other region mapped within 311nt proximal to the gag (Kaye et al., J Virol. Oct;69(10):6588-92(1995).

[0338] Internal ribosome entry site (IRES) Insertion of IRES elements enables the expression of multiple coding regions from a single promoter (Adam et al (above); Koo et al (1992) Virology 186:669-675; Chen et al 1993 J. Virol 67:2142-2148). IRES elements were first found at the untranslated 5' end of picornaviruses, facilitating cap-independent translation of viral proteins (Jang et al (1990) Enzyme 44:292-309). When located between open reading frames of RNA, IRES elements enable efficient translation of downstream open reading frames by facilitating ribosome entry at the IRES element, followed by downstream translation initiation.

[0339] A review article on IRES was presented by Mountford and Smith (TIG May 1995 vol. 11, No. 5: 179-184). Numerous different IRES sequences are known, including those derived from encephalomyocarditis virus (EMCV) (Ghattas, IR, et al., Mol. Cell. Biol., 11:5848-5859 (1991); BiP protein [Macejak and Sarnow, Nature 353:91 (1991)]; the Antennapedia gene (exons d and e) of Drosophila [Oh, et al., Genes & Development, 6:1643-1653 (1992)] and the Antennapedia gene of poliovirus (PV) [Pelletier and Sonenberg, Nature 334:320-325 (1988); see also Mountford and Smith, TIG11, 179-184 (1985)].

[0340] IRES elements derived from PV, EMCV, and porcine varicella-zoster virus have long been used in retroviral vectors (Coffin et al., see above).

[0341] The term "IRES" includes any sequence or combination of sequences that function as an IRES or improve the function of an IRES. IRESs may be of viral origin (e.g., EMCV IRES, PV IRES, or FMDV 2A-like sequences) or cellular origin (e.g., FGF2 IRES, NRF IRES, Notch 2 IRES, or EIF4 IRES).

[0342] In order for the IRES to initiate translation of each polynucleotide, the IRES should be placed between or before polynucleotides in the module construct.

[0343] Nucleotide sequences used for developing stable cell lines require the addition of selection markers to select cells in which stable integration occurs. These selection markers can be expressed as a single transcription unit within the nucleotide sequence, or it may be preferable to initiate translation of the selection marker with a polycistronic message using an IRES element (Adam et al. 1991 J. Virol. 65, 4985).

[0344] Gene orientation and insulators It is well known that nucleic acids are directional, and this ultimately affects mechanisms such as transcription and replication in cells. Therefore, genes, when part of the same nucleic acid construct, can have relative orientations to each other.

[0345] In certain embodiments of the present invention, at least two nucleic acid sequences present at the same locus in a cell or construct may be oriented in opposite and / or alternating directions. In other words, in certain embodiments of the present invention at this particular locus, a pair of consecutive genes do not have the same orientation. This may help prevent both transcriptional and translational read-through when the regions are expressed within the same physical location in the host cell.

[0346] Having alternating orientations benefits retroviral vector production when the nucleic acids required for vector production are based on the same gene locus within the cell. This can also improve the safety of the resulting construct by preventing the creation of replicable retroviral vectors.

[0347] When nucleic acid sequences are in reverse and / or alternating orientations, the use of an insulator can prevent inappropriate expression or silencing of NOIs from their surrounding genes.

[0348] The term "insulator" refers to a class of DNA sequence elements that, when bound to an insulator-binding protein, have the ability to protect a gene from surrounding regulatory signals. Two types of insulators exist: enhancer-blocking and chromatin barrier. When an insulator is located between a promoter and an enhancer, its enhancer-blocking function shields the promoter from the enhancer's transcriptional promotion (Geyer and Corces 1992; Kellum and Schedl 1992). Chromatin barrier insulators function by preventing the progression of nearby condensed chromatin, resulting in transcriptionally active chromatin regions replacing transcriptionally inactive chromatin regions, thus silencing gene expression. Insulators that inhibit heterochromatin diffusion, and therefore gene silencing, prevent this process by recruiting enzymes involved in histone modification (Yang J, Corces VG. 2011; 110: 43-76; Huang, Li et al. 2007; Dhillon, Raab et al. 2009). Insulators can possess one or both of these functions, and chicken β-globin insulator (cHS4) is one such example. This insulator is the most widely studied vertebrate insulator, is very G+C rich, and possesses both enhancer-blocking and heterochromatic barrier functions (Chung JH, Whitely M, Felsenfeld G. Cell. 1993; 74: 505-514). Other such insulators with enhancer-blocking functions include, but are not limited to, the following: Human β-globin insulator 5 (HS5), human β-globin insulator 1 (HS1), and chicken β-globin insulator (cHS3) (Farrell CM1, West AG, Felsenfeld G., Mol Cell Biol. 2002 Jun;22(11):3820-31; J Ellis et al. EMBO J. 1996 Feb1;15(3):562-568).In addition to reducing undesirable distal interactions, insulators also help prevent promoter interference between adjacent retroviral nucleic acid sequences (i.e., when a promoter from one transcription unit impairs the expression of an adjacent transcription unit). When insulators are used between each retroviral vector nucleic acid sequence, the reduction in direct read-through helps prevent the formation of replicable retroviral vector particles.

[0349] Insulators may be present between each of the retroviral nucleic acid sequences. In one embodiment, the use of insulators prevents promoter-enhancer interactions from one NOI expression cassette interacting with another NOI expression cassette in the nucleotide sequence encoding the vector component.

[0350] An insulator may be present between the vector genome and the gag-pol sequence. This thus limits the possibility of producing replicable retroviral vectors and "wild-type"-like RNA transcripts, improving the construct's safety profile. The use of insulator elements to improve the expression of stably incorporated multigene vectors is described in Moriarity et al, Nucleic Acids Res. 2013 Apr;41(8):e92.

[0351] Vector titer Those skilled in the art will understand that there are numerous different methods for determining the titer of a lentiviral vector. The titer is often expressed as transduction units / mL (TU / mL). The titer can be increased by increasing the number of vector particles and by increasing the specific activity of the vector preparation.

[0352] therapeutic use The lentiviral vectors described herein, or cells or tissues transduced with the lentiviral vectors described herein, may be used in medicine.

[0353] Furthermore, the lentiviral vectors described herein, the cells produced by the present invention, or cells or tissues transduced with the lentiviral vectors described herein may be used in the preparation of pharmaceuticals for delivering the desired nucleotides to the target sites where they are needed. Such use of the lentiviral vectors or transduced cells of the present invention may be for therapeutic or diagnostic purposes, as described above.

[0354] Therefore, cells transduced by the lentiviral vector described herein are provided.

[0355] "Cells transduced by viral vector particles" are understood as cells into which nucleic acids carried by viral vector particles have been transferred, particularly as target cells.

[0356] In a preferred embodiment, the nucleotide of interest provides a therapeutic effect.

[0357] "Target cells" should be understood as cells that are desirable to express NOI. NOI can be introduced into target cells using the viral vector of the present invention. Delivery to target cells can be performed in vivo, ex vivo, or in vitro.

[0358] NOIs may have therapeutic or diagnostic applications. Suitable NOIs include, but are not limited to, sequences encoding enzymes, cofactors, cytokines, chemokines, hormones, antibodies, antioxidant molecules, engineered immunoglobulin-like molecules, single-chain antibodies, fusion proteins, immune costimulatory molecules, immunomodulatory molecules, chimeric antigen receptors, transdomain-negative variants of target proteins, toxins, conditional toxins, antigens, transcription factors, structural proteins, reporter proteins, intracellular localization signals, tumor suppressor proteins, growth factors, membrane proteins, receptors, vascular active proteins and peptides, antiviral proteins and ribozymes, and their derivatives (such as derivatives with associated reporter groups). NOIs may also encode microRNAs. While we do not wish to be constrained by theory, it is thought that microRNA processing is inhibited by TRAP.

[0359] In one embodiment, NOI may be useful in the treatment of neurodegenerative disorders.

[0360] In another embodiment, NOI may be useful in treating Parkinson's disease.

[0361] In another embodiment, the NOI may encode one or more enzymes involved in dopamine synthesis. For example, the enzymes may be one or more of the following: tyrosine hydroxylase, GTP-cyclohydrolase I, and / or aromatic amino acid dopa decarboxylase. Sequences for all three genes are available (GenBank® accession numbers X05290, U19523, and M76180, respectively).

[0362] In another embodiment, the NOI may encode vesicular monoamine transporter 2 (VMAT2). In an alternative embodiment, the viral genome may include an NOI encoding aromatic amino acid dopa decarboxylase and an NOI encoding VMAT2. Such a genome may be used in the treatment of Parkinson's disease, particularly in conjunction with peripheral administration of L-DOPA.

[0363] In another embodiment, the NOI may encode a therapeutic protein or a combination of therapeutic proteins.

[0364] In another embodiment, NOI may encode one or more proteins selected from the group consisting of glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary body neurotrophic factor (CNTF), neurotrophin-3 (NT-3), acid fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), insulin growth factor-2, VEGF-A, VEGF-B, VEGF-C / VEGF-2, VEGF-D, VEGF-E, PDGF-A, PDGF-B, PDFG-A, and heterodimers and homodimers of PDFG-B.

[0365] In another embodiment, the NOI may encode one or more anti-angiogenic proteins selected from the group consisting of angiostatin, endostatin, platelet factor 4, pigment epithelial-derived factor (PEDF), placental growth factor, restin, interferon-α, interferon-inducible protein, gro-beta and tubedown-1, interleukin (IL)-1, IL-12, retinoic acid, anti-VEGF antibodies or their fragments / variants, e.g., aflibercept, thrombospondin, VEGF receptor proteins, e.g., those described in U.S. Patent No. 5,952,199 and U.S. Patent No. 6,100,071, and anti-VEGF receptor antibodies.

[0366] In another embodiment, the NOI may encode an anti-inflammatory protein, antibody, or protein or antibody fragment / variant selected from NF-κB inhibitors, IL-1β inhibitors, TGFβ inhibitors, IL-6 inhibitors, IL-23 inhibitors, IL-18 inhibitors, tumor necrosis factor α and tumor necrosis factor β, lymphotoxin α and lymphotoxin β, LIGHT inhibitors, α-synuclein inhibitors, tau inhibitors, β-amyloid inhibitors, and IL-17 inhibitors.

[0367] In another embodiment, the NOI may encode a cystic fibrosis membrane conductance regulator (CFTR).

[0368] In another embodiment, the NOI may encode a protein that is normally expressed in ophthalmic cells.

[0369] In another embodiment, the NOI may encode a protein that is normally expressed in photoreceptor cells and / or retinal pigment epithelial cells.

[0370] In another embodiment, the NOI may encode a protein selected from the group including RPE65, aryl hydrocarbon interaction receptor protein-like 1 (AIPL1), CRB1, lecithin retinal acetyltransferase (LRAT), photoreceptor-specific homeobox (CRX), retinal guanylate cyclise (GUCY2D), RPGR interaction protein 1 (RPGRIP1), LCA2, LCA3, LCA5, dystrophin, PRPH2, CNTF, ABCR / ABCA4, EMP1, TIMP3, MERTK, ELOVL4, MYO7A, USH2A, VMD2, RLBP1, COX-2, FPR, harmonin, Rab escort protein 1, CNGB2, CNGA3, CEP290, RPGR, RS1, RP1, PRELP, glutathione pathway enzymes, and optisin.

[0371] In other embodiments, the NOI may encode human coagulation factor VIII or factor IX.

[0372] In other embodiments, NOI is phenylalanine hydroxylase (PAH), methylmalonyl-CoA mutase, propionyl-CoA carboxylase, isovaleryl-CoA dehydrogenase, branched-chain keto acid dehydrogenase complex, glutaryl-CoA dehydrogenase, acetyl-CoA carboxylase, propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, carbamoyl-phosphate synthase ammonia, ornithine transcarbamylase, glucosylceramidase β, α-galactosidase A, glucosylceramidase It may encode one or more metabolically involved proteins selected from the group including β, cystinosine, glucosamine (N-acetyl)-6-sulfatase, N-acetyl-α-glucosaminidase, N-sulfoglucosamine sulfohydrolase, galactosamine-6-sulfatase, arylsulfatase A, cytochrome B-245β, ABCD1, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase, arginase 1, alaning's lycoxylate aminotransferase, ATP-binding cassette, and subfamily B members.

[0373] In other embodiments, the NOI may encode a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In one embodiment, the CAR is an anti-5T4 CAR. In other embodiments, NOI may encode B cell maturation antigen (BCMA), CD19, CD22, CD20, CD47, CD138, CD30, CD33, CD123, CD70, prostate-specific membrane antigen (PSMA), Lewis Y antigen (LeY), tyrosine-protein kinase transmembrane receptor (ROR1), mucin 1, cell surface binding (Muc1), epithelial cell adhesion molecule (EpCAM), endothelial growth factor receptor (EGFR), insulin, protein tyrosine phosphatase, non-receptor type 22, interleukin 2 receptor α, interferon induced by helicase C domain 1, human epidermal growth factor receptor (HER2), glypican 3 (GPC3), disiaroganglioside (GD2), mesothelin, or vesicular endothelial growth factor receptor 2 (VEGFR2).

[0374] In other embodiments, the NOI may encode a chimeric antigen receptor (CAR) for an NKG2D ligand selected from the group including ULBP1, 2 and 3, H60, Rae-1a, b, g, d, MICA, and MICB.

[0375] In further embodiments, NOI may include SGSH, SUMF1, GAA, common gamma chain (CD132), adenosine deaminase, WAS protein, globin, α-galactosidase A, δ-aminolevulinic acid (ALA) synthase, δ-aminolevulinic acid dehydratase (ALAD), hydroxymethylbilan (HMB) synthase, uroporphyrinogen (URO) synthase, uroporphyrinogen (URO) decarboxylase, coproporphyrinogen (COPRO) oxidase, and This can encode rotoporphyrinogen (PROTO) oxidase, ferrochelatase, α-L-iduronidase, iduronate sulfatase, heparan sulfamidase, N-acetylglucosaminidase, heparin-α-glucosaminide N-acetyltransferase, 3N-acetylglucosamine 6-sulfatase, galactose-6-sulfate sulfatase, β-galactosidase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, and hyaluronidase.

[0376] In addition to NOIs, vectors may also contain or encode siRNA, shRNA, or regulatory shRNA. (Dickins et al. (2005) Nature Genetics 37:1289-1295, Silva et al. (2005) Nature Genetics 37:1281-1288).

[0377] Indications The retroviral vectors and AAV vectors according to the present invention can be used to deliver one or more NOIs useful for the treatment of disorders listed in International Publication No. 1998 / 05635, International Publication No. 1998 / 07859, and International Publication No. 1998 / 09985. The nucleotide of interest may be DNA or RNA. Examples of such diseases are given below.

[0378] Impairment of response to cytokines and cell proliferation / differentiation activity; immunosuppressant or immunostimulatory activity (e.g., for treating immunodeficiency including infection by human immunodeficiency virus, for regulating lymphocyte proliferation; for treating cancer and many autoimmune diseases, and for preventing transplant rejection, or for inducing tumor immunity); regulation of hematopoiesis (e.g., for treating myeloid or lymphoid diseases); promotion of bone, cartilage, tendon, ligament and nerve tissue growth (e.g., for treating wound healing, burns, ulcers and periodontal disease and neurodegeneration); inhibition or activation of follicle-stimulating hormone (regulation of fertility); chemotactic / chemotactic activity ( For example, to mobilize specific cell types to the site of injury or infection; hemostatic and thrombolytic activity (for example, to treat hemophilia and stroke); anti-inflammatory activity (for example, to treat septic shock or Crohn's disease); macrophage inhibitory activity and / or T cell inhibitory activity, and therefore anti-inflammatory activity; anti-immune activity (i.e., inhibitory effect on cellular and / or humoral immune responses, including non-inflammatory responses); inhibition of the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as upregulation of fas receptor expression in T cells.

[0379] Malignant disorders including cancer, leukemia, growth, invasion and spread of benign and malignant tumors, angiogenesis, metastasis, ascites and malignant pleural effusion.

[0380] Autoimmune diseases, including arthritis, such as rheumatoid arthritis, hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen disorders, and other diseases.

[0381] Vascular diseases including arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome, cardiovascular effects, peripheral vascular disease, migraine and aspirin-dependent antithrombosis, stroke, cerebral ischemia, ischemic heart disease, or other diseases.

[0382] Diseases of the gastrointestinal tract, including peptic ulcers, ulcerative colitis, Crohn's disease, and other related conditions.

[0383] Liver diseases, including hepatic fibrosis and cirrhosis.

[0384] Hereditary metabolic disorders, including phenylketonuria (PKU), Wilson's disease, organic acidemia, urea cycle disorders, cholestasis, and other diseases.

[0385] Diseases of the kidneys and urinary tract, including thyroiditis or other glandular disorders, glomerulonephritis or other diseases.

[0386] Disorders of the ear, nose, and throat, including otitis or other ear, nose, and throat disorders, dermatitis or other skin conditions.

[0387] Dental and oral disorders, including periodontal disease, periodontitis, gingivitis, or other dental / oral diseases.

[0388] Orchitis or epididymitis - Testicular diseases including orchitis, infertility, testicular trauma, or other testicular disorders.

[0389] Gynecological diseases including placental dysfunction, placental insufficiency, recurrent miscarriage, eclampsia, pre-eclampsia, endometriosis, and other gynecological disorders.

[0390] Ophthalmic disorders, such as Leber congenital amaurosis (LCA) including LCA10, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveitis, optic neuritis, glaucoma (including open-angle glaucoma and juvenile congenital glaucoma), intraocular inflammation, such as retinitis or cystoid macular edema, sympathetic ophthalmitis, scleritis, retinitis pigmentosa, macular degeneration including age-related macular degeneration (AMD), and juvenile macular degeneration including Best's disease, Best's disease, Best vitiligo macular degeneration, Stargardt disease, Usher syndrome, Doin honeycomb retinal dystrophy, and Saw Bie's macular dystrophy, juvenile retinoschisis, Chondrod's dystrophy, corneal dystrophy, Fuchs' dystrophy, Leber congenital amaurosis, Leber hereditary optic neuropathy (LHON), Addey syndrome, Oguchi's disease, degenerative Fondus disease, ocular inflammation caused by ocular trauma or infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, such as excessive scarring after glaucoma filtration surgery, ocular graft rejection, and other ocular diseases such as diabetic macular edema, retinal vein occlusion, RLBP1-associated retinal dystrophy, choroidemia, and achromopsia.

[0391] Parkinson's disease, complications and / or side effects of treatment for Parkinson's disease, AIDS-related dementia, combined HIV-associated encephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease and other degenerative diseases, CNS conditions or disorders, stroke, post-polio syndrome, psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Fabry disease, Gaucher disease, cystinosis, Pompe disease, metachromatic leukodystrophy Fee, Wiscott-Aldrich syndrome, adrenoleukodystrophy, beta-thalassemia, sickle cell disease, Guillaime-Barré syndrome, Sydenham chorea, myasthenia gravis, pseudotumor brain, Down syndrome, Huntington's disease, CNS compression or trauma or infection of the CNS, muscular atrophy and dystrophy, diseases, conditions or disorders of the central and peripheral nervous system, neurological and neurodegenerative disorders including motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy, and spinal cord and detachment injuries.

[0392] Cystic fibrosis, mucopolysaccharidosis, mucopolysaccharidosis including, for example, Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome C, Sanfilippo syndrome D, Hunter syndrome, Hurler-Scheie syndrome, Morquio syndrome, ADA-SCID, X-linked SCID, X-linked chronic granulomatous disease, porphyria, hemophilia A, hemophilia B, post-traumatic inflammation, bleeding, coagulation and acute phase reactions, cachexia, anorexia, acute infection, septic shock, infectious diseases, diabetes, complications or side effects of surgery, complications and / or side effects of bone marrow transplantation or other transplantation, complications and / or side effects of gene therapy due to infection by a virus carrier or AIDS, for example, other diseases or conditions of tissues and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue, etc., for the prevention and / or treatment of graft rejection in the case of natural or artificial cell transplantation, to suppress or inhibit humoral and / or cellular immune responses.

[0393] siRNA, microRNA, and shRNA In certain other embodiments, NOIs include microRNAs. MicroRNAs are a very large group of small RNAs produced naturally in organisms, at least some of which regulate the expression of target genes. The forming members of the microRNA family are let-7 and lin-4. The let-7 gene encodes a small, highly conserved RNA species that regulates the expression of endogenous protein-coding genes during insect development. The active RNA species is initially transcribed as a precursor of about 70 nt, which is processed after transcription into a mature form of about 21 nt. Both let-7 and lin-4 are transcribed as hairpin RNA precursors, which are processed into their mature forms by the Dicer enzyme.

[0394] In addition to NOIs, vectors may also contain or encode siRNA, shRNA, or regulatory shRNA (Dickins et al. (2005) Nature Genetics 37:1289-1295, Silva et al. (2005) Nature Genetics 37:1281-1288).

[0395] Post-transcriptional silencing (PTGS), mediated by double-stranded RNA (dsRNA), is a conserved cellular defense mechanism for regulating the expression of foreign genes. Random incorporation of elements such as transposons or viruses is thought to trigger the expression of dsRNAs that activate sequence-specific degradation of homologous single-stranded mRNA or viral genomic RNA. This silencing effect is known as RNA interference (RNAi) (Ralph et al. (2005) Nature Medicine 11:429-433). The RNAi mechanism involves processing long dsRNAs into double helix sequences of approximately 21-25 nucleotides (nt)RNA. These products are called small interfering RNAs or silencing RNAs (siRNAs), which are sequence-specific mediators of mRNA degradation. In differentiated mammalian cells, dsRNAs longer than 30 bp have been found to activate the interferon response, leading to a shutdown of protein synthesis and nonspecific mRNA degradation (Stark et al., Annu Rev Biochem 67:227-64 (1998)). However, this response can be avoided by using 21 nt siRNA double helix, which allows for the analysis of gene function in cultured mammalian cells (Elbashir et al., EMBO J. Dec3;20(23):6877-88 (2001), Hutvagner et al., Science. Aug3, 293(5531):834-8. Eupub Jul12 (2001)).

[0396] Pharmaceutical composition This disclosure provides pharmaceutical compositions comprising cells or tissue transduced with the lentiviral vector described herein or the viral vector described herein, in combination with a pharmaceutically acceptable carrier, diluent, or excipient.

[0397] This disclosure provides a pharmaceutical composition for treating an individual by gene therapy, comprising a therapeutically effective amount of a lentiviral vector. The pharmaceutical composition may be for use in humans or animals.

[0398] The composition may contain pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The selection of pharmaceutically acceptable carriers, excipients, or diluents can be made in relation to the intended route of administration and standard pharmaceutical practices. In addition to carriers, excipients, or diluents, the pharmaceutical composition may also contain any suitable binders, lubricants, suspending agents, coatings, solubilizers, and other carriers that can assist or increase the entry of the vector into the target site (e.g., a lipid delivery system).

[0399] Where appropriate, the composition may be administered by any one or more inhalations; in the form of suppositories or pessaries; topically in the form of lotions, solutions, creams, ointments or powders; by the use of skin patches; orally in the form of tablets containing excipients such as starch or lactose; or alone or mixed with excipients in the form of capsules or eggs; or by any one or more in the form of elixirs, solutions or suspensions containing flavorings or colorings; or parenterally, for example, by intracavitary, intravenous, intramuscular, intracranial, intraocular, intraperitoneal or subcutaneous injection. In the case of parenteral administration, the composition may be best used in the form of a sterile aqueous solution that may contain other substances, such as salts or monosaccharides sufficient to make the solution isotonic with blood. In the case of buccal or sublingual administration, the composition may be administered in the form of tablets or lozenges that can be formulated in a conventional manner.

[0400] The lentiviral vectors described herein may also be used ex vivo to transduce target cells or tissues before transferring them to a patient who requires them. Examples of such cells may be autologous T cells, and examples of such tissues may be donor corneas.

[0401] Mutants, derivatives, analogs, homologs, and fragments In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses the use of their variants, derivatives, analogues, homologs, and fragments.

[0402] In the context of the present invention, a variant of any given sequence is a sequence in which a specific sequence of residues (whether amino acid residues or nucleic acid residues) is modified so that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. Variant sequences can be obtained by adding, deleting, substituting, modifying, replacing, and / or muting at least one residue present in naturally occurring proteins.

[0403] As used herein with respect to the proteins or polypeptides of the present invention, the term “derivative” includes any substitution, mutation, modification, substitution, deletion and / or addition of one (or more) amino acid residues to or from a sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0404] As used herein with respect to polypeptides or polynucleotides, the term “analog” includes any mimic, i.e., a chemical compound having at least one of the endogenous functions of the polypeptide or polynucleotide it mimics.

[0405] Typically, amino acid substitutions, such as 1, 2, or 3 to 10 or 20 substitutions, may be made, as long as the modified sequence retains the desired activity or capability. Amino acid substitutions may include the use of analogues that do not exist in nature.

[0406] The proteins used in this invention may also undergo silent changes, including deletions, insertions, or substitutions of amino acid residues, resulting in functionally equivalent proteins. Intentional amino acid substitutions may be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues, as long as the endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged head groups having similar hydrophilic values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0407] Conservative substitutions can be performed, for example, according to the following table. Amino acids in the same block in the second column, preferably in the same line in the third column, may be substituted for each other.

[0408] [Table 1]

[0409] The term "homolog" refers to an entity that has a certain degree of homology to the wild-type amino acid sequence and wild-type nucleotide sequence. The term "homology" can be considered synonymous with "identity."

[0410] In this context, homologous sequences are interpreted as including amino acid sequences that are at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the target sequence, preferably at least 95%, 97%, or 99% identical. Typically, homology includes the same active site, etc., as the target amino acid sequence. While homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, homology is preferably expressed in terms of sequence identity.

[0411] In this context, homologous sequences are interpreted as including nucleotide sequences that are at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the target sequence, and preferably at least 95%, 97%, 98%, or 99% identical. While homology can also be considered in terms of similarity, in the context of the present invention, homology is preferred to be expressed in terms of sequence identity.

[0412] Homology comparisons can be performed visually, or more generally, with the help of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

[0413] Homology percentages can be calculated over consecutive sequences, that is, by aligning one sequence with the other and directly comparing each amino acid in one sequence with its corresponding amino acid in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignment is performed over only a relatively small number of residues.

[0414] While this is a very simple and consistent method, it fails to take into account, for example, that in otherwise identical sequence pairs, a single insertion or deletion in a nucleotide sequence may cause subsequent codons to fall out of alignment, and therefore, when the overall alignment is performed, it can result in a significant decrease in the homology percentage. As a result, most sequence comparison methods are designed to produce an optimal alignment that takes possible insertions and deletions into account without excessively disadvantageing the overall homology score. This is achieved by inserting "gaps" into the sequence alignment in an attempt to maximize local homology.

[0415] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, thereby ensuring that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible achieves a higher score than a sequence with many gaps, reflecting a higher relevance between the two sequences being compared. "Affine gap cost" typically imposes a relatively high cost for the presence of gaps and a smaller penalty for each subsequent residual within a gap. This is the most commonly used gap scoring system. A high gap penalty naturally produces an optimized alignment with fewer gaps. Most alignment programs allow for correction of the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

[0416] Therefore, calculating maximum percentage homology requires first generating an optimal alignment, taking gap penalties into account. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12:387). Other software capable of performing sequence comparisons includes, but is not limited to, the BLAST package (see Ausubel et al. (1999) ibid. - Ch.18), FASTA (Atschul et al. (1990) J.Mol.Biol.403-410), and the GENEWORKS comparison tools suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, the GCG Bestfit program is preferred. Another tool called BLAST2 sequencing is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett (1999) 174(2):247-50; FEMS Microbiol Lett (1999) 177(1):187-8).

[0417] While the final homology percentage can be measured in terms of identity, the alignment process itself is not typically based on all-or-nothing pair comparisons. Instead, a scaled similarity score matrix is ​​commonly used, which assigns a score to each pair comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is ​​the BLOSUM62 matrix, which is the default matrix for the BLAST program suite. The GCG Wisconsin program generally uses either public default values ​​or custom symbol comparison tables, if provided (see the user manual for further details). In some applications, it is preferable to use the public default values ​​of the GCG package, or a default matrix such as BLOSUM62 in the case of other software.

[0418] Once the software has generated the optimal alignment, it is possible to calculate the homology percentage, preferably the sequence identity percentage. The software typically does this as part of the sequence comparison and generates a numerical result.

[0419] A "fragment" is also a variant, and this term typically refers to a selected region of a polypeptide or polynucleotide of interest, either functionally or, for example, in an assay. Therefore, a "fragment" refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

[0420] Such mutants can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. If an insertion is made, synthetic DNA encoding the insertion can be produced along with 5' and 3' flanking regions corresponding to naturally occurring sequences on either side of the insertion site. The flanking regions contain convenient restriction sites corresponding to locations in the naturally occurring sequence, so that the sequence is cleaved by an appropriate enzyme and the synthetic DNA is linked to the cleavage. The DNA is then expressed according to the present invention to produce the encoded protein. These methods are merely examples of the many standard techniques known in the art for manipulating DNA sequences, and other known techniques can also be used.

[0421] All variants, fragments, or homologs of the regulatory protein suitable for use in cells and / or modular constructs of the present invention retain the ability to bind to the homologous binding site of NOI such that NOI translation is suppressed or prevented in viral vector-producing cells.

[0422] All mutant fragments or homologs of the binding site retain the ability to bind to the homologous RNA-binding protein such that NOI translation is suppressed or prevented in viral vector-producing cells.

[0423] Codon optimization The polynucleotides used in this invention (including the NOI and / or components of the vector production system) can be codon-optimized. Codon optimization has been previously described in International Publication No. 1999 / 41397 and International Publication No. 2001 / 79518. Different cells use certain codons differently. This codon bias corresponds to a bias in the relative abundance of a particular tRNA in a cell type. It is possible to increase expression by altering the codons in the sequence to match the relative abundance of the corresponding tRNA. Similarly, it is possible to decrease expression by deliberately selecting codons for which the corresponding tRNA is known to be rare in a particular cell type. Thus, further degrees of translational control are available.

[0424] Many viruses, including retroviruses, utilize a large number of rare codons, which can be modified to correspond to commonly used mammalian codons, thereby achieving increased expression of a target gene, such as NOI, or packaging components in mammalian-producing cells. Codon frequency tables are publicly known in the art for mammalian cells and various other organisms.

[0425] Codon optimization of viral vector components offers numerous other advantages. The alteration of their sequences ensures that the nucleotide sequences encoding the packaging components of the viral particles, necessary for assembly in producer / packaging cells, have the RNA instability sequences (INS) removed. Simultaneously, the amino acid sequence coding sequences of the packaging components are maintained so that the viral component encoded by the sequence remains the same, or at least sufficiently similar, without compromising the functionality of the packaging component. In lentiviral vectors, codon optimization also overcomes Rev / RRE requirements for export, making the optimized sequence Rev-independent. Codon optimization also reduces homologous recombination between different constructs within the vector system (e.g., between overlapping regions in the gag-pol and env open reading frames). Therefore, the overall effect of codon optimization is a significant increase in viral titer and improved safety.

[0426] In one embodiment, only codons related to INS are codon-optimized. However, in a much more preferred practical embodiment, sequences are codon-optimized in their entirety, with a few exceptions, for example, sequences containing the frameshift region of gag-pol (see below).

[0427] The lentiviral vector gag-pol gene contains two overlapping reading frames encoding the gag-pol protein. The expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosome "slipping" during translation. This slippage is thought to be caused, at least partially, by ribosomal arrest RNA secondary structures. Such secondary structures are located downstream of the frameshift site of the gag-pol gene. In the case of HIV, the overlapping region extends from nucleotide 1222 (nucleotide 1 is A in gag ATG) downstream of the start of gag to the end of gag (nt 1503). As a result, a 281 bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon-optimized. Retaining this fragment allows for more efficient expression of the Gag-Pol protein. In the case of EIAV, the start of the overlap is interpreted as nt 1262 (nucleotide 1 is A in gag ATG), and the end of the overlap is interpreted as nt 1461. To ensure that frameshift regions and gag-pol duplication are preserved, the wild-type sequence is retained at nt 1156-1465.

[0428] Derivatives from optimal codon usage frequencies may be created, for example, to adapt to favorable restriction sites, and conservative amino acid changes may be introduced into the Gag-Pol protein.

[0429] In one embodiment, codon optimization is based on a low-expression mammalian gene. The third base, and possibly the second and third bases, can be modified.

[0430] It will be understood that, due to the degenerate nature of the genetic code, a large number of gag-pol sequences can be achieved by those skilled in the art. Furthermore, many retroviral variants have been described that can be used as starting points for generating codon-optimized gag-pol sequences. Lentiviral genomes can be highly diverse. For example, there are many quasi-species of HIV-1 that are still functional. This is also true for EIAV. These variants can be used to enhance specific parts of the transduction process. Examples of HIV-1 variants can be found in the HIV Database, operated by Los Alamos National Security, LLC, at http: / / hiv-web.lanl.gov. Details of EIAV clones can be found in the National Center for Biotechnology Information (NCBI) database, located at http: / / www.ncbi.nlm.nih.gov.

[0431] The codon-optimized gag-pol sequence strategy can be used for any retrovirus. This applies to all lentiviruses, including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1, and HIV-2. Furthermore, this method can be used to increase the expression of genes derived from HTLV-1, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERV), MLV, and other retroviruses.

[0432] Codon optimization can make gag-pol expression Rev-independent. However, to enable the use of anti-rev or RRE factors in lentiviral vectors, it would be necessary to make the viral vector production system completely Rev / RRE-independent. Therefore, the genome also needs to be modified. This is achieved by optimizing the vector genome components. Advantageously, these modifications also result in the production of a safer system where all the additional proteins are absent in both producer and transduction cells.

[0433] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing embodiments of this disclosure. Numerical ranges include the number defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in 5' to 3' orientation, and amino acid sequences are written from left to right in amino to carboxyl orientation.

[0434] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, is also specifically disclosed unless the context explicitly indicates otherwise. Each smaller range between any stated value or intervening value within the stated range and any other stated value or intervening value within that stated range is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and subject to any specifically excluded limits within the stated range, each range of either limit, neither limit, or both limits included in the smaller range is also included in this disclosure. If the stated range includes one or both limits, the range excluding one or both of the limits that they include is also included in this disclosure.

[0435] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise explicitly indicated by the context.

[0436] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and are comprehensive or open-ended, and do not exclude additional unlisted components, elements, or process steps. The terms “comprising,” “comprises,” and “comprised of” also include “consisting of.”

[0437] The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing in this specification should be construed as an acknowledgment that such publications constitute prior art to the attached claims.

[0438] The present invention will now be further illustrated by examples, which are intended to help those skilled in the art to carry out the invention and are not intended in any way to limit the scope of the invention. [Examples]

[0439] Materials and methods Preparation of PEIPro / DNA complex The standard method for preparing PEIpro is as follows: 1. Prepare the "PEI mix". Add PEIPro to the culture medium in a ratio of approximately 1:20. After addition is complete, mix briefly by swirling. 2. Prepare the "DNA mix". Add the DNA plasmid to the culture medium in a ratio of approximately 1:20. 3. Combine the PEI and DNA mix. Then, add the PEI mixture to the DNA mixture in a 1:1 ratio, either by pouring or pumping. Next, incubate the transfection mixture at room temperature to allow complex formation.

[0440] The aqueous PEIpro method is as follows: 1. Prepare the "PEI mix". Add PEIPro to water in a ratio of approximately 1:20. After addition is complete, mix briefly by swirling. Neither the addition time nor the subsequent incubation time is time-dependent. 2. Prepare the "DNA mix." Add the DNA plasmid to water in a ratio of approximately 1:20. Neither the addition time nor the subsequent incubation time is time-dependent. 3. Combine the PEI and DNA mix. Then, add the PEI mixture to the DNA mixture in a 1:1 ratio by pouring or pumping. After completion, give the mixture a quick swirl. Neither the addition time nor the incubation time after addition is time-dependent. 4. Spike the PBS. Add 10X PBS in a ratio of approximately 1:50 of 10X PBS to the final transfection volume so that the final concentration of PBS equals 0.2X. Briefly swirl the mixture immediately after addition and start the timer. Incubate the mixture at room temperature to allow complex formation.

[0441] Transfection may be performed after the above steps. For the bioreactor experiment, the transfection mix was added to the bioreactor via a hand pump after a 30-minute incubation. The incubation period varied in the shaking flask test where time-course experiments were performed.

[0442] Shaking flask experimental conditions To investigate the effect of transfection complex incubation time on GFP vector production using DNA / PEIPro complexes prepared in culture medium, a single transfection mix was prepared as described above and left at room temperature for up to 60 minutes after complex formation. At predetermined intervals, a fixed amount of approximately 2 mL was taken from the bulk transfection mix and used to transfect individual E125 Erlenmeyer flasks seeded with HEK293T cells. The final volume in the shaking flask was 40 mL, resulting in a transfection volume ratio of 1:20.

[0443] Example 1 - When the transfection mix is ​​prepared in culture medium, the optimal incubation time for achieving the highest titer is too short to be applicable to GMP manufacturing. The data demonstrate that the incubation period of the DNA / PEIPro® complex significantly affects subsequent transfection and lentiviral vector production (Figure 1). The data show that the optimal incubation time for the highest titer is approximately 3 minutes, and this decreases rapidly when the incubation time is extended to 15 or 30 minutes. This represents a significant problem with developing a PEIPro® process for large-scale GMP manufacturing purposes. Compared to a standard lipofectamine 2000CD-based process that allows for a robust process with a maximum complex stability of 6 hours as the minimum time limit, the requirement that the complex must be prepared and added to the bioreactor within 5 minutes imposes a significant time limit on the process itself, making it impractical for large-scale GMP production.

[0444] Example 2 - The kinetics of complex formation can be modified by preparing the transfection mix in water and varying the PBS concentration used to initiate DNA / PEIPro® complex formation. The transfection complex was prepared using a GFP vector as described above under the subheading "Preparation of PEIPro / DNA Complex". The bulk transfection mix was prepared in water and divided into three methods. A proportion of the transfection complex was spiked with a volume of 10X PBS so that the final concentration of PBS was equal to 1X. A portion of the transfection mix was spiked with 10X PBS so that the final concentration was equal to 0.2X. The remaining transfection mix was left untouched and not spiked with PBS. The two PBS-spiked preparations were then incubated at room temperature for up to 2 hours. At predetermined intervals, a constant volume of approximately 2 mL was taken from the bulk transfection mix and used to transfect individual E125 Erlenmeyer flasks seeded with HEK293T cells. The final volume in the shaking flask was 40 mL, so the transfection volume ratio was 1:20.

[0445] Nine shaking flasks were transfected with a 1X spike transfection mix at the following time intervals: 1 min; 2 min; 3 min; 4 min; 5 min; 6 min; 7 min; 15 min; 30 min; 60 min; and 120 min. Twelve additional shaking flasks were transfected with a 0.2X PBS transfection mix at 1 min; 2 min; 3 min; 4 min; 6 min; 10 min; 15 min; 20 min; 25 min; 30 min; and 40 min.

[0446] At the end of the GFP vector production process, samples were taken from each shaking flask and functional titer was determined using a transduction assay based on flow cytometry. The results show that the kinetics of complex growth can be modified by varying the PBS concentration (Figure 2). Figure 2A shows that when the transfection complex was spiked with 1X PBS, the optimal titer was achieved after 3 minutes of incubation. Figure 2B shows that spiking the transfection complex with a lower concentration of PBS can extend the incubation time. Spike with 0.2X PBS extended the optimal incubation time to approximately 25 minutes. As a negative control, the complex was prepared in water and then used to transfect cells without PBS spikes. No vectors were produced that showed PBS was required to initiate complex growth.

[0447] Example 3 - Transfection efficiency is stable for up to 12 hours, and potentially up to 1 week, after diluting the transfection mixture to reduce salt concentration (and after stopping complex growth). The transfection mix was prepared as described above, spiked with 0.2X PBS to initiate complex growth, and then incubated for 30 minutes to reach the optimal size of the complex. The transfection mix was then diluted with a salt-free aqueous solution (i.e., H2O) to a final PBS concentration of 0.1X. The transfection mix was then stored at 4°C for up to one week. To test the stability of the diluted PEIPro® / DNA complex, the titer of the functional lentiviral vector achieved using the complex was evaluated after various incubation times following dilution. GFP lentivirus was then produced using a fixed amount of the transfection mix, and the resulting functional titer was measured. The results demonstrate that the transfection efficiency of the diluted complex was maintained over time (Figure 3) and that it was stable at 4°C for at least one week. In particular, the graph shows that after 12 hours of incubation, the transfection mix was still efficient at generating lentiviral vectors and yielded comparable functional titers compared to the “fresh” transfection mix.

[0448] Example 4 - Dynamic light scattering analysis showing the size over time of DNA:PEIpro complexes prepared in H2O (Zetasizer Nanometer ZS, Malvern Instrument, Malvern, UK). DNA / PEIpro complexes were prepared in H2O as described above, and then samples of the transfection mixture were taken at 2-minute intervals over 60 minutes and analyzed using a Zetasizer to measure the complex size. Figure 4 shows that the complex size did not increase over 60 minutes. This correlates with the data shown in Figure 2B, which demonstrated that complexes composed of water alone did not result in vector production. This data, along with DLS measurements, suggests that complexes in the 80–86 nm size range are too small to successfully transfect.

[0449] Example 5 - Diluting the transfection mix 2-fold stops growth at the optimal particle size. The DNA / PEIpro complex was prepared in H2O as described above, and particle growth was initiated by adding PBS to a final concentration of 0.2X. The complex was incubated at room temperature for 25 minutes, which corresponded to the optimal time for achieving the highest functional titer (see Figure 2B). The complex was then diluted 2-fold to a PBS concentration of 0.1X. The complex size was then measured over time for up to 45 minutes. DLS measurements showed that when the transfection mix was diluted 2-fold, the complex growth stopped, and the size remained between 600 and 800 nm (Figure 5). Correlation of this data with the functional data generated in Examples 2 and 3 above suggests that the optimal complex size for achieving the highest titer is between 600 and 800 nm.

[0450] Example 6 - DLS analysis shows that different concentrations of PBS affect the kinetics of complex growth. DNA / PEIpro complexes were prepared in water as described above, and particle growth was initiated by adding three different concentrations of PBS (0.1X; 0.15X and 0.2X), followed by incubation at room temperature. Samples were then taken every 2 minutes and analyzed using a Zetasizer (Zetasizer Nanometer ZS, Malvern Instrument, Malvern, UK). The results show that when the transfection mix is ​​spiked with different concentrations of PBS, the rate at which the complex grows to the optimal size for transfection differs depending on the concentration of PBS added. If the PBS concentration is too low (<0.15X PBS), it is not sufficiently concentrated to initiate complex growth (Figure 6). Table 1 shows the effect of different PBS concentrations on complex growth in relation to the time it takes to reach the optimal size.

[0451] [Table 2]

[0452] Example 7 - Any salt can be used to initiate compound propagation. DNA / PEIpro complexes were prepared in H2O and then spiked with different concentrations of NaCl or MgCl2. Figure 7A and Table 2 show the effects of spiking the transfection mix with four different concentrations of NaCl (10mM; 20mM; 50mM; 80mM and 100mM). Only the 80mM and 100mM concentrations initiated significant complex growth. Figure 7B and Table 3 show the effects of spiking the transfection mix with four different concentrations of MgCl2 (10mM; 20mM; 50mM; 80mM; 100mM). Because MgCl2 has a higher ionic strength, complex growth initiated at lower concentrations, and the time it took to reach the optimal complex size was faster than with NaCl.

[0453] [Table 3]

[0454] [Table 4]

[0455] The functional potency obtained on a 5L scale using Example 8-aqPEIPro transfection is favorable compared to that obtained when therapeutic vector products are produced using cationic lipid-based transfection methods. Six 5L bioreactors were used in this study. Four bioreactors were transiently transfected using a cationic lipid-based transfection method, and the other two bioreactors were transfected using the described aqPEIPro® method. Both bioreactors were transiently transfected with an HIV-1 vector genome encoding 5T4-CAR. This therapeutic vector is intended for genetic modification of T cells that can target and kill tumor cells expressing the antigen 5T4 (Owens, LG; Sheard, VE et al J Immunother. 2018 Apr; 41(3): 130-140).

[0456] Samples were taken at the end of the manufacturing process, and functional titers were determined using transduction assays and flow cytometry.

[0457] The functional potency values ​​obtained on a 5L scale in this study were favorable compared to those obtained when the product was produced using a cationic lipid-based transfection method, and the data appeared more consistent (n=2). The average crude yield obtained using the aqueous PEIPro method was 3.5375 x 10⁻¹⁰, compared to the cationic lipid-based transfection method (n=4). 5 TU / mL (standard deviation=+ / -1.8x10 5 Compared to TU / mL, 4.82.25x10 5 TU / mL (standard deviation=+ / -6.8x10 4 This indicates that a titer of TU / mL was obtained. This suggests that the aqPEIPro transfection method works as well as cationic lipid-based transfection methods and is scalable to 5L (Figure 8).

[0458] All publications referenced in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described form for carrying out the invention, which will be apparent to those skilled in the art in molecular biology or related fields, are intended to be within the scope of the following claims.

Claims

1. 1. An in vitro method for producing a solution of polymer / nucleic acid complexes, comprising: a) mixing the nucleic acid and the cationic polymer in a substantially salt-free aqueous solution; b) adding salt to the mixture produced in step a) to form a salt solution; and c) optionally incubating the salt solution produced in step b); an in vitro method for preparing said solution of polymer / nucleic acid complexes comprising:

2. i) the nucleic acid is selected from DNA, RNA, an oligonucleotide molecule, and mixtures thereof; ii) the nucleic acid is DNA, preferably plasmid DNA; iii) the cationic polymer is a polymer-based transfection reagent; iv) the cationic polymer is selected from polyethyleneimine (PEI), dendrimers, DEAE-dextran, polypropyleneimine (PPI), chitosan [poly-(β-1 / 4)-2-amino-2-deoxy-D-glucopyranose], poly-L-lysine (PLL), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), and derivatives thereof; v) the cationic polymer is PEI or a derivative thereof, preferably selected from linear PEI, branched PEI, PEGylated PEI, JetPEI, PEIPro®, PEI MAX and PTG1+; vi) the salts are selected from sodium salts, magnesium salts, potassium salts, calcium salts, phosphate salts and mixtures thereof; and / or vii) The method of claim 1, wherein the salt is phosphate buffered saline (PBS).

3. i) the salt is PBS, and PBS is added in step b) to a final concentration of about 0.1X PBS to about 1.0X PBS, preferably the PBS is added to a final concentration of about 0.1X PBS to about 0.3X PBS; or ii) the salt is added in step b) to a final concentration of about 10 mM to about 100 mM, preferably the salt is added in step b) to a final concentration of about 20 mM to about 100 mM.

4. incubating the salt solution produced in step b), Depending on the situation i) incubating the salt solution for about 1 minute up to about 2 hours; ii) incubating the salt solution for about 20 minutes to about 90 minutes; or iii) incubating the salt solution for about 60 minutes; The method according to any one of claims 1 to 3.

5. 5. The method of any one of claims 1 to 4, wherein the salt is PBS and the salt solution is diluted to a final concentration of about 0.05X PBS to about 0.15X PBS, preferably, the salt solution is diluted to a final concentration of about 0.1X PBS.

6. the nucleic acid encodes a retroviral vector component; The method of any one of claims 1 to 5, optionally wherein the retroviral vector is a replication-defective retroviral vector.

7. The method of claim 6, wherein said retroviral vector is a lentiviral vector, preferably said lentiviral vector is HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or Visna.

8. The vector component comprises: i. the RNA genome of the lentiviral vector; ii. env or a functional substitute thereof; iii. gag-pol or a functional substitute thereof; and / or iv.rev or its functional substitute The method of claim 7, wherein the compound is selected from the group consisting of

9. e) optionally culturing mammalian cells in perfusion culture; f) transfecting mammalian cells with a solution of polymer / nucleic acid complexes obtained by the method according to any one of claims 1 to 8; g) optionally introducing into said mammalian cells a nucleic acid different from the nucleic acid of the solution of polymer / nucleic acid complexes; h) optionally selecting mammalian cells having said nucleic acid integrated into their genome; i) optionally culturing said mammalian cells under conditions whereby said nucleic acid is expressed; j) optionally culturing said mammalian cells under conditions whereby said retroviral vector is produced; k) optionally isolating said retroviral vector; The method of any one of claims 1 to 8, further comprising:

10. i. The method comprises culturing mammalian cells in perfusion culture, Optionally, said step of culturing the mammalian cells in perfusion culture is carried out for about 10 hours to about 96 hours; ii. culturing said mammalian cells under conditions whereby said nucleic acid is expressed; iii. culturing said mammalian cells under conditions in which said retroviral vector is produced; and / or iv. The method of claim 9, further comprising the step of isolating said retroviral vector. Claim 11: i. The mammalian cell is a HEK293T cell; ii. the mammalian cells are suspension adapted mammalian cells; iii. the transfection, introduction and / or culture steps are performed in suspension in serum-free medium; iv. the transfection is a transient transfection; v. the transfection, introduction and / or culture steps are carried out in a volume of at least 50 L, preferably the transfection, introduction and culture steps are carried out in a volume of at least 50 L; vi. A nucleic acid different from the nucleic acid of the solution of the polymer / nucleic acid complex is introduced into the mammalian cell by transfection or electroporation; and / or vii. A nucleic acid different from the nucleic acid of the solution of the polymer / nucleic acid complex is not encoded by the nucleic acid of the solution of the polymer / nucleic acid complex, i) the RNA genome of the lentiviral vector; ii) env or a functional surrogate thereof; iii) gag-pol or a functional substitute thereof; and / or iv) rev or a functional substitute thereof; The method of claim 9 or 10, wherein the viral vector component encodes any viral vector component selected from the group consisting of:

12. A solution of polymer / nucleic acid complexes obtained or obtainable by the method according to any one of claims 1 to 11, Optionally, a solution of said polymer / nucleic acid complex obtained or obtainable by a method according to any one of claims 6 to 8.

13. 13. Use of a solution of polymer / nucleic acid complexes according to claim 12 for transfection of said nucleic acid into cells.

14. a) culturing mammalian cells in perfusion culture; b) transfecting said mammalian cells with a solution of a polymer / nucleic acid complex according to any one of claims 6 to 8 or with a solution of a polymer / nucleic acid complex according to claim 12; 1. A method for transfecting a mammalian cell, comprising: Depending on the situation i. the step of culturing the mammalian cells in perfusion culture is carried out for about 10 hours to about 96 hours; ii. the mammalian cell is a HEK293T cell; iii. the mammalian cells are suspension adapted cells; iv. performed in suspension in serum-free medium; v. step a) and / or step b) are carried out in a volume of at least 50 L, preferably step a) and step b) are carried out in a volume of at least 50 L; vi. The transfection is a transient transfection; The method.

15. Use of a cationic polymer in an in vitro method for controlling the initiation and / or termination of maturation of a polymer / nucleic acid complex, said method comprising: a) mixing a nucleic acid and a cationic polymer in a substantially salt-free aqueous solution; b) adding salt to the mixture produced in step a) to form a salt solution; and c) incubating the salt solution produced in step b); The use comprising:

16. A method according to any one of claims 1 to 11, comprising diluting the salt solution produced in step b) or diluting the salt solution after incubation in step c).

17. The use according to claim 15, wherein the method comprises a step of diluting the salt solution produced in step b) or a step of diluting the salt solution after incubation in step c).

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