Method for transfecting cells with molecules, composition, kit and application

Abstract

The invention relates to a method, a composition and a kit for transfecting cells with molecules and application. The method comprises the following steps: adding the molecules for transfection into the cells; and modulating the activity of protein kinase C (PKC) in a cell and / or providing a pH-responsive peptide comprising from about 5 to about 20 histidine residues.

Description

[0001] This application is a divisional application of the following parent case:

[0002] Parent case application number: 202080037144.8;

[0003] Parent case application date: March 23, 2020;

[0004] Parent application name: Enhanced transfection. Technical Field

[0005] The present invention relates to the technical field of cell transfection, and in particular to a method, composition, kit and application of transfecting cells with molecules. Background Art

[0006] Gene delivery is a useful tool for studying and manipulating cellular processes as well as for therapeutics. Ex vivo genetic modification of human cells has been shown to significantly enhance their therapeutic potential. In academic and clinical laboratories, the most commonly used nucleic acid delivery vectors are viral vectors. Viral vectors have been shown to be highly efficient. However, viral vectors have the potential to randomly integrate into the host genome, which can interrupt essential gene expression and cellular processes. Due to safety concerns, high cost, and technical difficulties of viral gene delivery, attention has been directed to the development of non-viral gene delivery systems.

[0007] Non-viral gene delivery has relatively high efficiency in many cell lines, but stem cells and post-mitotic cells are known to be resistant to current non-viral delivery methods (0-35% transfection efficiency). One method developed to overcome the low transfection efficiency of non-viral gene delivery is electroporation. Electroporation results in high transfection, but leads to low cell viability after transfection, and scalability is an issue. Alternative methods all require specialized setups, including microinjection, gene guns, sonoporation, lasers, and cell deformation. Currently, achieving high transfection efficiency in difficult-to-transfect cell types using non-viral gene delivery remains unsolved.

[0008] Dixon et al. disclose another recently developed delivery method (PCT Publication No.: WO2015092417, which is incorporated herein by reference). A method for transducing cargo molecules into living cells is disclosed, wherein a delivery molecule comprising a cargo, a glycosaminoglycan (GAG) binding element (which is capable of binding to a GAG on the cell surface) and a protein transduction domain provides efficient transduction of proteins into cells. Modifications to this method are provided in patent application publication No. WO2016207638 (incorporated herein by reference), which discloses a pH-mediated cell delivery vector comprising a cargo or a binding molecule for binding to a cargo, a protein transduction domain, and a GAG binding element capable of binding to a GAG on the cell surface, wherein the GAG ​​binding element is a peptide modified to include one or more histidine residues capable of protonation in an acidic environment. Despite advances in transfection methods, some cell types remain difficult to transfect. Therefore, further improvements are needed.

[0009] To improve transfection of difficult-to-transfect cells, Ho et al. (2016) have demonstrated that chemical inhibition of histone deacetylases (HDACs) can enhance non-viral transfection methods. This suggests that cell activity can be altered to aid the transfection process.

[0010] It is also expected and known that the above transfection methods can transfect molecules other than nucleic acids into cells, such as proteins and nanoparticles. Therefore, there is a need for improved delivery of nucleic acids and other cargoes, such as proteins, peptides and nanoparticles, which would otherwise have difficulty crossing cell membranes. Summary of the invention

[0011] It is an object of the present invention to provide improved transfection efficiency of cells, including cells that are difficult to transfect.

[0012] According to a first aspect of the present invention, there is provided a method for transfecting cells with a molecule, the method comprising the following steps:

[0013] - adding molecules for transfection to said cells; and

[0014] - modulating the activity of protein kinase C (PKC) in said cell and / or providing a pH responsive peptide comprising about 5 to about 20 histidine residues.

[0015] The histidine residues of the pH-responsive peptides can be protonated in the acidic environment of endosomes.

[0016] The present invention unexpectedly found that optionally regulating the PKC activity in the cell with histone deacetylase (HDAC) has a significant effect in significantly enhancing cell transfection, and advantageously transfects cell types that cannot be effectively transfected with gold standard reagents at present. In particular, the present invention has found that the impact of the transfection efficiency regulated by a single HDAC is an efficiency increase in the range of 3-10 times, a single PKC regulation can provide a significantly larger 10-20 times efficiency increase, and HDAC regulation and PKC regulation can provide an unexpected 50-200 times or more efficiency increase together. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 :TPA(T) significantly enhanced the transfection of T2 cells;

[0018] Figure 2 :SAHA(S) significantly enhanced the transfection of RAW264.7 cells;

[0019] Figure 3 :Short-term exposure to SAHA(S) and TPA(T) significantly enhanced the transfection of LCL cells;

[0020] Figure 4 : Transfection of T2 cells;

[0021] Figure 5 : Transfection of LCL cells;

[0022] Figure 6 : Schematic diagram of transfection of rat cerebellum slices;

[0023] Figure 7 : Endosomal escape system of the same peptide;

[0024] Figure 8 : Endosomal escape system on helper peptide;

[0025] Fig. 9 : HDACi activity is at early stages of transfection (during macropinocytosis and endosomal trafficking) rather than at later stages (affecting transcriptional activity);

[0026] Fig.10 : iHMSCs were transfected in the presence of HDAC and PKC inhibition (inhibitors SAHA and GF109203X, GFX);

[0027] Fig.11 : Transfection of LCL in the presence of HDAC and PKC inhibition (inhibitors SAHA and Carvastatin C). DETAILED DESCRIPTION

[0028] PKC regulation

[0029] Modulating the activity of PKC in a cell may include adding an agent to the cell to modulate the activity of PKC in the cell.

[0030] In one embodiment, the regulation of PKC activity in the cell includes the inhibition of PKC activity. Inhibition can include complete (i.e. 100%) inhibition or reduction of activity. In another embodiment, inhibition can include significant or substantial inhibition or reduction of activity. The activity of PKC in the cell can be inhibited by at least 5%. In another embodiment, the activity of PKC in the cell can be inhibited by at least 10%. In another embodiment, the activity of PKC in the cell can be inhibited by at least 20%. In another embodiment, the activity of PKC in the cell can be inhibited by at least 50%. In another embodiment, the activity of PKC in the cell can be inhibited by at least 80%. It will be appreciated by those skilled in the art that the success or level of PKC inhibition can be determined by detecting the phosphorylation state of one or more PKC targets.

[0031] The inhibitor of PKC can inhibit the activity of PKC in the cell. In one embodiment, regulating the activity of PKC in the cell comprises adding a PKC regulator to the cell. The regulator of PKC can be a PKC inhibitor.

[0032] In one embodiment, the regulation of PKC activity in the cell includes the activation of PKC activity. Activation can include significant or substantial activation or increase of activity. The activity of PKC in the cell can increase by at least 5%. In another embodiment, the activity of PKC in the cell can increase by at least 10%. In another embodiment, the activity of PKC in the cell can increase by at least 20%. In another embodiment, the activity of PKC in the cell can increase by at least 50%. In another embodiment, the activity of PKC in the cell can increase by at least 80%. It will be appreciated by those skilled in the art that the success or level of PKC activation can be determined by detecting the phosphorylation state of one or more PKC targets.

[0033] PKC activators can increase the activity of PKC in cells. In one embodiment, the activity of PKC in regulating cells includes adding a PKC regulator to the cells. The regulator of PKC can be a PKC activator. In one embodiment, the regulation of PKC activity in cells includes up-regulating the expression of PKC in cells. Up-regulating expression can be achieved by providing a molecule that can up-regulate PKC. The molecule can be a nucleic acid, such as siRNA. The molecule can be a nucleic acid, such as DNA or RNA, that encodes the PKC for overexpression.

[0034] The modulator of PKC can be selected from the group comprising phorbol 12-myristate 13-acetate (PMA), ingenol 3-angelate (Ingenol 3-angelate, I3A) and bryostatin. In one embodiment, the modulator of PKC comprises phorbol 12-myristate 13-acetate (PMA) (also known as 12-O-tetradecanoylphorbol 13-acetate (TPA)). In another embodiment, the modulator of PKC comprises ingenol 3-angelate (I3A). In another embodiment, the modulator of PKC includes bryostatin. Those skilled in the art will recognize that functional analogs and derivatives of such modulators can also be used as substitutes. In another embodiment, the modulator of PKC can be a gene silencer (such as siRNA) to inhibit the expression of PKC isoforms, thereby reducing its activity.

[0035] In one embodiment, the modulator of PKC can be a PKC inhibitor selected from the group comprising bisindolylmaleimide I (also known as 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide or GFX (GF109203X)), Calphostin C and Go6976 (5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propionitrile); or a combination thereof.

[0036] In one embodiment, the regulation of PKC activity in the cell comprises down-regulating the expression of PKC in the cell. Down-regulating expression can be achieved by providing a molecule that can down-regulate PKC. The molecule can be a nucleic acid, such as an siRNA that can inhibit PKC expression. Those skilled in the art will be familiar with the technology of up-regulating and down-regulating specific proteins (such as PKC) in the cell.

[0037] PKC regulator can be provided at effective concentration and amount to provide required regulation (such as activation). Those skilled in the art will recognize that different PKC regulators may need different concentrations and amounts to effectively regulate PKC. For example, the amount of PKC regulator can be between 10nm and about 10 μM. In another embodiment, the amount of PKC regulator can be between 0.1 and 100 μM. In another embodiment, the amount of PKC regulator can be between 10nm and 1 μM. In another embodiment, the amount of PKC regulator can be between 0.1 and 1 μM.

[0038] The regulation of PKC activity, for example, by adding an agent for regulating PKC activity, can be simultaneously with or after adding a molecule for transfection to the cell. Without being bound by theory, it is understood that PKC regulation is important for the release of the endosomes of the molecule for transfection. Therefore, PKC regulation can be carried out at the appropriate time to enhance the release of the endosomes into the cell.

[0039] The regulation of PKC activity (such as inhibition) can be temporary.For example, regulation can occur enough time to help transfection process.The regulation of PKC activity (such as activation) can last 4 hours or shorter time.The regulation of PKC activity (such as activation) can be carried out after starting transfection.The regulation of PKC activity (such as activation) can be up to 3 hours after starting transfection.In one embodiment, the regulation of PKC activity (such as activation) can be carried out in the time between about 1 hour to about 3 hours after starting transfection.In another embodiment, the regulation of PKC activity (such as activation) can be carried out in the time between about 12 to about 24 hours after starting transfection.In another embodiment, the regulation of PKC activity (such as activation) can be carried out in the time between about 0 to about 24 hours after starting transfection.

[0040] HDAC Regulation

[0041] The method for transfecting cells with molecules may also include regulating histone deacetylase (HDAC) in the cell. In one embodiment, the regulation of HDAC activity in the cell includes the inhibition of HDAC activity. The regulation of HDAC in the cell (such as inhibition) can be carried out simultaneously with the regulation (such as activation) of PKC activity in the cell. In one embodiment, HDAC is suppressed and PKC is activated. In another embodiment, HDAC is suppressed and PKC is suppressed. In another embodiment, HDAC is activated and PKC is activated. In another embodiment, HDAC is activated and PKC is suppressed.

[0042] Advantageously, several HDAC inhibitors (HDACi) with more specific or broader specificity have demonstrated a certain level of transfection-promoting activity in a variety of cell lines without toxicity.

[0043] HDAC inhibition can include complete (i.e. 100%) inhibition or reduction of HDAC activity. In another embodiment, inhibition can include significant or substantial inhibition or reduction of HDAC activity. The activity of HDAC in the cell can be suppressed by at least 5%. In another embodiment, the activity of HDAC in the cell can be suppressed by at least 10%. In another embodiment, the activity of HDAC in the cell can be suppressed by at least 20%. In another embodiment, the activity of HDAC in the cell can be suppressed by at least 50%. In another embodiment, the activity of HDAC in the cell can be suppressed by at least 80%. It will be appreciated by those skilled in the art that the success or level of HDAC inhibition can be determined by assay, such as histone deacetylase assay (e.g., histone deacetylase assay kit provided by Sigma-Aldrich / Merck, fluorometry or its equivalent assay and kit).

[0044] Inhibition of HDAC may comprise the use of an HDAC inhibitor. Thus, in one embodiment, the method of transfecting a cell with a molecule may further comprise adding an HDAC inhibitor during or prior to transfection with the molecule.

[0045] HDAC inhibitors can be selected from the group comprising suberoylanilide hydroxamic acid (SAHA), panobinostat, trichostatin A (TSA), tubastatin A (tubastatin A) and valproic acid. In one embodiment, HDAC inhibitors include suberoylanilide hydroxamic acid (SAHA). In another embodiment, HDAC inhibitors include panobinostat, trichostatin A (TSA). In another embodiment, HDAC inhibitors include tubastatin A. In another embodiment, HDAC inhibitors include valproic acid. Those skilled in the art will recognize that functional analogs and derivatives of such inhibitors can also be used as substitutes. In another embodiment, HDAC inhibitors can be gene silencers (such as siRNA) to inhibit the expression of HDAC, thereby reducing its activity.

[0046] In one embodiment, the regulation of HDAC activity in the cell includes enhancing HDAC activity, for example by providing an HDAC enhancer. The HDAC enhancer may include N-acetylthiourea. Additionally or alternatively, an HDAC inhibitor may be inhibited to enhance HDAC activity. For example, the HDAC inhibitor TSA may be inhibited. The TSA inhibitor may include ISTA1.

[0047] In one embodiment, the regulation of HDAC activity in the cell includes the expression of HDAC in the cell being raised. Up-regulating expression can be achieved by providing a molecule that can raise HDAC. The molecule can be a nucleic acid, such as siRNA. The molecule can be a nucleic acid (such as DNA or RNA) encoding an HDAC for expression (such as overexpression). The molecule that enhances HDAC activity can include a vector encoding HDAC and an optional promoter that drives expression. Those skilled in the art will be familiar with the technology of raising and lowering specific proteins (such as HDAC) in cells.

[0048] Combinations of PKC and HDAC inhibition may include the use of PMA / TPA in combination with one or more of panobinostat, TSA, or SAHA.

[0049] In one embodiment, wherein the cell to be transfected is a T-cell or a B-cell or a hybrid thereof, the PKC modulator may comprise PMA / TPA. In one embodiment, wherein the cell to be transfected is a 2 hybrid human T / B cell, the PKC modulator may comprise PMA / TPA.

[0050] In one embodiment, wherein the cells to be transfected are macrophages, the PKC regulator may include PMA / TPA, which may be further used with the HDAC inhibitor SAHA. In one embodiment, wherein the cells to be transfected are lymphoblastoid cell line cells (LCL), the PKC regulator may include PMA / TPA, which may be further used with the HDAC inhibitor SAHA. LCL cells may be lymphoblastoid human B cells.

[0051] HDAC regulators can be provided in effective concentrations and amounts to provide the desired HDAC activity inhibition. Those skilled in the art will recognize that different HDAC inhibitors may require different concentrations and amounts to effectively inhibit HDAC. For example, the amount of the HDAC inhibitor can be between 10nM and about 100 μM. In another embodiment, the amount of the HDAC regulator can be between 0.1 and 100 μM. In another embodiment, the amount of the HDAC regulator can be between 0.1 and 10 μM. In another embodiment, the amount of the HDAC regulator can be between 1 and 10 μM.

[0052] HDAC inhibition can be temporary. For example, inhibition can occur for a sufficient time to help the transfection process. HDAC inhibition can last for 4 hours or less. HDAC inhibition can be after starting transfection. HDAC inhibition can be up to 6 hours after starting transfection. In one embodiment, HDAC inhibition can be the time between about 1 hour to about 6 hours after starting transfection.

[0053] In one embodiment, HDAC inhibition and PKC modulation (such as inhibition) can occur substantially simultaneously with each other.

[0054] Molecules used for transfection

[0055] The molecule used for transfection may not itself modulate the activity of protein kinase C (PKC) in the cell. The molecule used for transfection may not comprise a protein kinase C (PKC) modulator or may consist of a protein kinase C (PKC) modulator, such as an inhibitor or activator.

[0056] The molecule used for transfection can include nucleic acids, such as nucleic acid vectors. The molecule used for transfection can include oligonucleotides. The molecule used for transfection can include any one of the groups selected from siRNA, modified messenger RNA (mRNA), microRNA, DNA, PNA, LNA or its construct. In one embodiment, the molecule used for transfection is DNA. Nucleic acids encoding or non-coding proteins or peptides, such as DNA. Nucleic acids, such as DNA, can contain one or more gene sequences and / or one or more regulatory sequences.

[0057] In another embodiment, the molecule used for transfection may include a protein. The molecule used for transfection may include a peptide. The molecule used for transfection may include a protein related to physiology or metabolism. The molecule used for transfection may include an intracellular protein. The molecule used for transfection may include a signaling protein, which is a protein involved in a signaling pathway. The molecule used for transfection may include a protein involved in cell expression or metabolic regulation. The molecule used for transfection may include a protein involved in cell division. The molecule used for transfection may include a protein involved in cell differentiation (such as stem cell differentiation). The molecule used for transfection may include a protein required for inducing pluripotent stem cells. The molecule used for transfection may include a protein involved in cardiac cell differentiation. The molecule used for transfection may include a marker, such as a protein marker. The molecule used for transfection may include a bacterial or bacterial-derived protein. The molecule used for transfection may include a mammalian or mammalian-derived protein. The molecule used for transfection may be any peptide, polypeptide or protein. The molecule used for transfection may include a research, diagnostic or therapeutic molecule. The molecule used for transfection may include a transcriptional regulator, i.e., a member of signal generation. Molecules used for transfection may comprise enzymes or their substrates, proteases, modulators of enzyme activity, perturbimers and peptide aptamers, antibodies, modulators of protein-protein interactions, growth factors or differentiation factors.

[0058] The molecule used for transfection can be a precursor protein. For example, an excision domain can be provided in the delivery molecule, and the delivery molecule is arranged to be cut when entering the cell or after entering the cell. The molecule used for transfection can be a protein arranged to be post-translationally modified in the cell. Once entering the cell, the molecule used for transfection can be arranged to have functionality. For example, the molecule used for transfection may not have function until after transfection into the cell.

[0059] The molecule used for transfection may include any intracellular molecule. The molecule used for transfection may include any protein or molecule with intracellular function (mode of action), intracellular receptor, intracellular ligand or intracellular substrate. The molecule used for transfection may include proteins or molecules that are naturally / normally internalized into the cell. The molecule used for transfection may include proteins that are intended to be delivered or displayed on the cell surface, such as cell surface receptors. The molecule used for transfection may be selected from any one of the groups comprising therapeutic molecules; drugs; prodrugs; functional proteins or peptides, such as enzymes or transcription factors; microproteins or peptides; and toxins; or their encoding nucleic acids.

[0060] In one embodiment, the molecule used for transfection may comprise a transcription factor or a nucleic acid encoding a transcription factor. Additionally or alternatively, the molecule used for transfection may comprise a growth factor or a nucleic acid encoding a growth factor.

[0061] The molecules used for transfection can be selected from any one of the group comprising toxins, hormone transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD, myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF4, C / EBP, SP1, CCAAT-box binding protein, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS-binding protein, STAT, GATA-box binding protein, such as GATA-3, transcription factors such as HIF1a and RUNT, forkhead family of winged helix proteins, carbamoyl synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetacetate hydrolase (fumarylacetacetate hydrolase), arginase, ... hydrolase), phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor VIII, factor IX, cystathionine β-synthase, branched-chain ketoacid decarboxylase, isovaleryl-coA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, β-glucosidase, pyruvate carboxylate, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, H protein, T protein, cystic fibrosis transmembrane regulator (CFTR) sequence, dystrophin cDNA sequence, Oct-3 / 4 (Pou5f1), Sox2, c-Myc, Klf4, RPE65 Nanog and SoxB1; or fragments thereof, and / or combinations thereof.

[0062] The growth factor may include adrenomedullin (AM); angiopoietin (Ang); autotaxin; bone morphogenetic protein (BMP); ciliary neurotrophic factor (CNTF); leukemia inhibitory factor (LIF); interleukin-6 (IL-6); colony-stimulating factor (CSF); factor); macrophage colony stimulating factor (M-CSF); granulocyte colony stimulating factor (G-CSF); granulocyte macrophage colony stimulating factor (GM-CSF); epidermal growth factor (EGF); ephrin; erythropoietin (EPO); fibroblast growth factor (FGF); glial cell line-derived neurotrophic factor (GDNF); neurotrophic factor neurturin; neurotrophic factor persephin; artemin; growth differentiation factor-9 (GDF9); hepatocyte growth factor (HGF); hepatoma-derived growth factor (HDGF); insulin; insulin-like growth factor; interleukins; keratinocyte growth factor (KGF); migration stimulating factor (MSF); macrophage stimulating protein (MSP), also known as hepatocyte growth factor , myostatin (GDF-8), neuregulin, neurotrophin, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin, placental growth factor (PGF), platelet-derived growth factor (PDGF), renal enzyme (RNLS), anti-apoptotic survival factor, T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor, transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and Wnt group of growth factors, or a combination thereof, and / or nucleic acids encoding such growth factors.

[0063] The nucleic acid to be transfected may upregulate or be capable of upregulating a growth factor in a cell. In one embodiment, the nucleic acid to be transfected upregulates or is capable of upregulating the expression of BMP2 and / or VEGF in a cell.

[0064] The nucleic acid to be transfected may encode a transcription factor. The nucleic acid to be transfected may encode BMP2 and / or VEGF.

[0065] In one embodiment, the molecule used for transfection may comprise a protein complex, such as a viral particle or a virus-like particle (VLP). The viral particle or virus-like particle (VLP) may also comprise a nucleic acid.

[0066] In one embodiment, the molecule used for transfection can include nanoparticles, such as metal nanoparticles or polymer nanoparticles. The nanoparticles can be rods, such as metal rods. The nanoparticles can be porous. The molecule used for transfection can include nanostructures. The molecule used for transfection can include superparamagnetic iron oxide nanoparticles (SPION). The molecule used for transfection can include non-small molecules.

[0067] The molecules used for transfection can include non-covalently bound complexes, such as protein-protein complexes, protein-mRNA complexes, protein-noncoding RNA complexes, protein-lipid complexes and protein-small molecule complexes. Examples of such complexes are RNA-induced silencing complexes (RISC) and spliceosomes.

[0068] The molecules used for transfection can have a molecular weight of at least 1KDa. The molecules used for transfection can have a molecular weight of at least 5KDa. The molecules used for transfection can have a molecular weight of at least 10KDa. The molecules used for transfection can have a molecular weight of at least 20KDa. The molecules used for transfection can have a molecular weight of 400KDa or less. The molecules used for transfection can have a molecular weight of 300KDa or less. The molecules used for transfection can have a molecular weight between about 0.5KDa and about 400kDa. The molecules used for transfection can have a molecular weight between about 1KDa and about 400kDa. The molecules used for transfection can have a molecular weight between about 0.5KDa and about 200kDa. The molecules used for transfection can have a molecular weight between about 1KDa and about 200kDa. The molecules used for transfection can have a molecular weight between about 2KDa and about 300kDa. The molecules used for transfection can have a molecular weight between about 20KDa and about 300kDa. Molecules used for transfection may have a molecular weight between about 20 kDa and about 100 kDa.

[0069] When the molecule used for transfection comprises amino acids, the length of the molecule used for transfection can be between about 20 and about 30,000 amino acids. The length of the molecule used for transfection can be between about 20 and about 10,000 amino acids. The length of the molecule used for transfection can be between about 20 and about 5,000 amino acids. The length of the molecule used for transfection can be between about 20 and about 1,000 amino acids. The length of the molecule used for transfection can be at least about 20 amino acids. The length of the molecule used for transfection can be at least about 100 amino acids.

[0070] In one embodiment, the molecule for transfection is combined with the transfection delivery molecule, compounded, embedded in the transfection delivery molecule or connected to the transfection delivery molecule. In one embodiment, the molecule for transfection is the cargo of the transfection delivery molecule. The molecule for transfection (i.e. cargo) can be capable of being combined with the transfection delivery molecule, such as ion or covalent binding. The molecule for transfection (i.e. cargo) can include an element for combining the transfection delivery molecule. The molecule for transfection can include biotin, or streptavidin. The molecule for transfection can be biotinylated. The molecule for transfection can include an affinity tag, which can be combined with a complementary affinity tag on the transfection delivery molecule.

[0071] The molecule for transfection may be a fusion peptide comprising the molecule for transfection and a transfection delivery molecule.

[0072] The terms "molecule for transfection" and "cargo" are used interchangeably herein.

[0073] Transfection delivery molecules

[0074] The transfection delivery molecule can be selected from the group comprising nucleic acids, peptides, proteins, viral particles, virus-like particles, non-viral molecules, synthetic polymers and glycosaminoglycan (GAG) binding enhanced transduction (GET)-cargo molecules. In one embodiment, the transfection delivery molecule comprises a glycosaminoglycan (GAG) binding enhanced transduction (GET)-cargo molecule (referred to herein as a "GET system" or "GET molecule").

[0075] The advantageous transfection efficiency of the GET system is described in detail in PCT Publication No.: WO2015092417. GET molecules used together with PKC modifiers, and optionally further with HDACi, advantageously produce significantly higher transfection levels than gold standard transfection techniques such as the Lipofectamine product (from Life Technologies).

[0076] In one embodiment, the transfection delivery molecule may be a glycosaminoglycan (GAG) binding enhanced transduction (GET) delivery molecule comprising:

[0077] -goods

[0078] -GAG binding element, capable of binding to GAGs on the cell surface; and

[0079] -Protein transduction domain.

[0080] In another embodiment, the transfection delivery molecule can be a glycosaminoglycan (GAG) binding enhanced transduction (GET) delivery molecule comprising:

[0081] - a cargo binding molecule for binding a cargo, and optionally wherein the cargo is bound to said cargo binding molecule;

[0082] -GAG binding element, capable of binding to GAGs on the cell surface; and

[0083] -Protein transduction domain.

[0084] The GAG ​​binding element may be different from the protein transduction domain. The GAG ​​binding element may be different from the protein transduction domain in structure and / or sequence. Relative to the protein transduction domain, the GAG ​​binding element may be specific for different cell surface molecules. The GAG ​​binding element may be more specific for cell surface molecules than the protein transduction domain. In one embodiment, the GAG ​​binding element is not a protein transduction domain as described herein. As described herein, in one embodiment, the protein transduction domain is not a specific GAG binding element. Those skilled in the art will understand that the protein transduction domain may or may not bind to GAGs on the cell surface, but such binding is not specific or preferential to GAGs. Relative to the non-specific binding of GAGs by the protein transduction domain, the GAG ​​binding element may preferentially bind to GAGs.

[0085] The GAG ​​binding element may be a heparan sulfate glycosaminoglycan (HS-GAG) binding element, which is capable of binding to HS-GAG on the surface of a cell.

[0086] Heparan sulfate glycosaminoglycans (HS-GAGs) are proteoglycans in which two or three HS chains are tightly attached to cell surface or extracellular matrix proteins. It is in this form that HS binds to a variety of protein ligands and regulates a variety of biological activities, including developmental processes, angiogenesis, blood coagulation, and tumor metastasis. Heparan sulfate is a member of the glycosaminoglycan family and is structurally closely related to heparin. Both consist of repeating disaccharide units that are variably sulfated. The most common disaccharide unit in heparan sulfate consists of glucuronic acid (GlcA) linked to N-acetylglucosamine (GlcNAc), typically accounting for around 50% of the total disaccharide units.

[0087] The GAG ​​binding element may have a specific affinity for GAG. The HS-GAG binding element may have a specific affinity for HS-GAG. The HS-GAG binding element may comprise a heparin binding domain (HBD) or a variant thereof. The heparin binding domain variant may comprise a truncated heparin binding domain, or an extended heparin binding domain. The GAG ​​binding element may comprise any protein, peptide or molecule that specifically or preferentially binds to GAG. The HS-GAG binding element may comprise any protein, peptide or molecule that specifically or preferentially binds to HS-GAG.

[0088] The HS-GAG binding element may comprise at least a portion of the heparin binding domain of heparin binding EGF-like growth factor (HB-EGF). The heparin binding domain may comprise P21 of HB-EGF. The heparin binding domain may comprise a truncation, extension or functional variant of P21.

[0089] The HS-GAG binding element may comprise the heparin binding domain of fibroblast growth factor or a functional variant thereof.

[0090] The HS-GAG binding element may be selected from any one of the group including FGF, antithrombin such as ATIII, VEGF, BMP, Wnt, Shh EGF and PDGF; or variants thereof. The HS-GAG binding element may comprise any one of FGF2, FGF7 or PDGF. The HS-GAG binding element may comprise one or more heparin binding sulfate domains (e.g., domains A, B or C) of any FGF protein. The HS-GAG binding element may comprise FGF4. The HS-GAG binding element can comprise FGF1 HBD A (heparan sulfate binding domain A (the first HBD domain of FGF1)), FGF2 HBD A (heparan sulfate binding domain A), FGF4 HBD A (heparan sulfate binding domain A), FGF1 HBD C (heparan sulfate binding domain C), FGF2 HBD B (heparan sulfate binding domain B), FGF2 HBD C (heparan sulfate binding domain C), FGF4 HBD C (heparan sulfate binding domain C), FGF7 HBD B (heparan sulfate binding domain B), FGF7 HBD C (heparan sulfate binding domain C), antithrombin such as ATIII, VEGF or PDGF, or variants thereof.

[0091] The HS-GAG binding element can be selected from any one of the group including hepatocyte growth factor, interleukin, morphogen, HS-GAG binding enzyme, Wnt / Wingless, endostatin, viral protein, such as foot-and-mouth disease virus protein, annexin V, lipoprotein lipase; or its HS-GAG binding fragment. The HS-GAG binding element can comprise any protein, peptide or molecule capable of specifically binding to HS-GAG.

[0092] Those skilled in the art will appreciate that "variants" include functional variants, which may have some sequence differences from known, reported, published or claimed sequences, but the variants can still bind to HS-GAG. Conservative amino acid substitutions are also included in the meaning of "variants".

[0093] The HS-GAG binding element may comprise the amino acid sequence KRKKKGKGLGKKRDPCLRKYK (P21, SEQ ID NO.1). The HS-GAG binding element may comprise a sequence having at least 80% identity to SEQ ID NO.1. The HS-GAG binding element may comprise a sequence having at least 90% identity to SEQ ID NO.1. The HS-GAG binding element may comprise a sequence having at least 95% identity to SEQ ID NO.1. The HS-GAG binding element may comprise a sequence having at least 98% identity to SEQ ID NO.1. The HS-GAG binding element may comprise a sequence having at least 99% identity to SEQ ID NO.1.

[0094] The HS-GAG binding element may comprise the amino acid sequence GRPRESGKKRKRKRLKPT (PDGF, SEQ ID NO.3). The HS-GAG binding element may comprise a sequence having at least 80% identity to SEQ ID NO.3. The HS-GAG binding element may comprise a sequence having at least 90% identity to SEQ ID NO.3. The HS-GAG binding element may comprise a sequence having at least 95% identity to SEQ ID NO.3. The HS-GAG binding element may comprise a sequence having at least 98% identity to SEQ ID NO.3. The HS-GAG binding element may comprise a sequence having at least 99% identity to SEQ ID NO.3.

[0095] The HS-GAG binding element may comprise the amino acid sequence TYASAKWTHNGGEMFVALNQ ((FGF7, HBD B) SEQ ID NO.5). The HS-GAG binding element may comprise a sequence having at least 80% identity to SEQ ID NO.5. The HS-GAG binding element may comprise a sequence having at least 90% identity to SEQ ID NO.5. The HS-GAG binding element may comprise a sequence having at least 95% identity to SEQ ID NO.5. The HS-GAG binding element may comprise a sequence having at least 98% identity to SEQ ID NO.5. The HS-GAG binding element may comprise a sequence having at least 99% identity to SEQ ID NO.5.

[0096] The HS-GAG binding element may comprise the amino acid sequence TYRSRKYTSWYVALKR ((FGF2, HBD B) SEQ ID NO.7). The HS-GAG binding element may comprise a sequence having at least 80% identity to SEQ ID NO.7. The HS-GAG binding element may comprise a sequence having at least 90% identity to SEQ ID NO.7. The HS-GAG binding element may comprise a sequence having at least 95% identity to SEQ ID NO.7. The HS-GAG binding element may comprise a sequence having at least 98% identity to SEQ ID NO.7. The HS-GAG binding element may comprise a sequence having at least 99% identity to SEQ ID NO.7.

[0097] Sequence identity can be determined using standard BLAST alignment parameters (provided by http: / / www.ncbi.nlm.nih.gov / ).

[0098] The GAG ​​binding element may comprise a GAG binding antibody, or a variant or fragment thereof. The HS-GAG binding element may comprise a HS-GAG binding antibody, or a variant or fragment thereof. The antibody fragment may be an antibody variable domain, scFv, diabody, FAb, Dab, F(ab)'2, a heavy and light chain dimer, or a single chain structure. The antibody variant may comprise a protein scaffold comprising a CDR, an antibody mimetic, or a DARPin.

[0099] The GAG ​​or HS-GAG binding element may comprise a nanobody (a single domain antigen binding fragment derived from a heavy chain antibody, devoid of light chains and naturally occurring in Camelidae).

[0100] A single domain antibody may comprise a VHH fragment comprising CDR1, CDR2 and CDR3, wherein

[0101] CDR1 may comprise or consist of the amino acid sequence of GFTVSSNE or GFFAFSSYA;

[0102] CDR2 may comprise or consist of the amino acid sequence of ISGGST or IGTGDT; and

[0103] CDR3 may comprise or consist of the amino acid sequence of GRRLKD or SLRMNGWRAHQ.

[0104] A single domain antibody may comprise a VHH fragment comprising CDR1, CDR2 and CDR3, wherein

[0105] CDR1 may comprise or consist of the amino acid sequence of GFTVSSNE;

[0106] CDR2 may comprise or consist of the amino acid sequence of ISGGST; and

[0107] CDR3 may comprise or consist of the amino acid sequence of GRRLKD.

[0108] Alternatively, CDR3 may comprise the amino acid sequence GMRPRL, HAPLRNTRTNT, GSRSSR, GRTVGRN, GKVKLPN, SGRKGRMR, SLRMNGWRAHQ or RRYALDY.

[0109] A single domain antibody may comprise a VHH fragment comprising CDR1, CDR2 and CDR3, wherein

[0110] CDR1 may comprise or consist of the amino acid sequence of GFAFSSYA;

[0111] CDR2 may comprise or consist of the amino acid sequence of IGTTGDT; and

[0112] CDR3 may comprise or consist of the amino acid sequence of SLRMNGWRAHQ.

[0113] Alternatively, CDR3 may comprise the amino acid sequence LKQQGIS, AMTQKKPRKLSL, HAPLRNTRTNT, GMRPRL, RRYALDY or SGRKYFRARDMN.

[0114] The HS-GAG binding element may comprise anti-HS scFv antibodies AO4B08, AO4B05, AO4F12, RB4CB9, RB4CD12, RB4EA12 or RB4EG12 (as described by Jenniskens et al. (2000. The Journal of Neuroscience, 20(11):4099–4111) and Smits et al. (2006. METHODS INENZYMOLOGY, VOL. 416, pp.61-87), which are incorporated herein by reference); or a fragment thereof. The HS-GAG binding element may comprise AO4B08. The HS-GAG binding element may comprise CDR1, CDR2 and CDR3 of AO4B08, AO4B05, AO4F12, RB4CB9, RB4CD12, RB4EA12 or RB4EG12. The HS-GAG binding element may comprise CDR1, CDR2 and CDR3 of AO4B08.

[0115] The HS-GAG binding element may comprise HS3A8, LKIV69, EW3D10, EW4G2, NS4F5, RB4EA12, HS4E4, or HS4C3 (as described in Wijnhoven et al. (2008) Glycoconj J 7 25:177–185) and Smits et al. (2006. METHODS IN ENZYMOLOGY, VOL. 416, pp. 61-87, incorporated herein by reference). The HS-GAG binding element may comprise HS4E4 or HS4C3. The HS-GAG binding element may comprise CDR1, CDR2, and CDR3 of HS3A8, LKIV69, EW3D10, EW4G2, NS4F5, RB4EA12, HS4E4, or HS4C3. The HS-GAG binding element may comprise CDR1, CDR2 and CDR3 of HS4E4 or HS4C3.

[0116] The HS-GAG binding element may comprise SEQ ID NO: 15 or 17 (AO4B08). The HS-GAG binding element may comprise SEQ ID NO: 11 or 13 (HS4C3). The HS-GAG binding element may comprise an antibody or antibody fragment, a heavy chain and / or a light chain. The HS-GAG binding element may comprise an antibody or antibody fragment, a heavy chain comprising HCDR1, HCDR2 and HCDR3 chains and / or a light chain comprising LCDR1, LCDR2 and LCDR3.

[0117] In one embodiment of the invention, the protein transduction domain is not a GAG. In another embodiment, the protein transduction domain is different from a GAG.

[0118] The protein transduction domain may be hydrophilic or amphiphilic. The protein transduction domain may comprise a majority of hydrophilic amino acid residues. The protein transduction domain may comprise a majority of arginine and / or lysine amino acid residues. The protein transduction domain may comprise a periodic sequence having a repeated amino acid sequence motif. The protein transduction domain may comprise an osmotic protein, a TAT, such as HIV-derived TAT, a MAP or a transporter, pVec or pep-1.

[0119] When referring to "a majority" of the residues, those skilled in the art will understand that this includes greater than 50% of the residues. A majority may be 55%, 60%, 70%, 80%, 90% or 95% of the residues.

[0120] The protein transduction domain may be selected from any one of the group comprising:

[0121] Penetratin or Antenapedia PTD RQIKWFQNRRMKWKK;

[0122] HIV transactivator protein (TAT) YGRKKRRQRRR;

[0123] Synembryn B(SynB)1RGGRLSYSRRRFSTSTGR;

[0124] SynB3 RRLSYSRRRF;

[0125] PTD-4PIRRRKKLRRLK;

[0126] PTD-5RRQRRTSKLMKR;

[0127] Chicken house virus (FHV) coat - (35-49)RRRRNRTRRNRRRVR;

[0128] Brome mosaic virus (BMV) Gag-(7-25)KMTRAQRRAAARRNRWTAR;

[0129] Human T-cell lymphotrophic virus (HTLV)-II Rex-(4-16)TRRQRTRRARRNR;

[0130] D-Tat GRKKRRQRRRPPQ;

[0131] R9-Tat GRRRRRRRRRPPQ;

[0132] transporterGWTLNSAGYLLGKINLKALAALAKKIL chimera;

[0133] microtubule-associated protein (MAP) KLALKLALKLALALKLA;

[0134] Streptavidin binding peptide (SBP) MGLGLHLLVLAAALQGAWSQPKKKRKV;

[0135] Folate binding protein (FBP) GALFLGWLGAAGSTMGAWSQPKKKRKV;

[0136] Human 3-methyladenine-DNA glycosylase (MPG)

[0137] ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya;

[0138] 10MPG - Nuclear Localization Sequence (NLS)

[0139] ac - GALFLGFLGAAGSTMGAWSQPKSKRKV - cya;

[0140] Pep - 1ac - KETWWETWWTEWSQPKKKRKV - cya; and

[0141] Pep - 2ac - KETWFETWFTEWSQPKKKRKV - cya; or

[0142] Polyarginines such as RxN (4 < N < 17) chimeras, polylysines such as KxN

[0143] (4 < N < 17) chimeras, (RAca)6R, (RAbu)6R, (RG)6R, (RM)6R, (RT)6R, (RS)6R,

[0144] R10, (RA)6R, R7 and R8.

[0145] The protein transduction domain may comprise polyarginine or polylysine. The protein transduction domain may comprise arginine and lysine repeats. The protein transduction domain may comprise arginine residues, such as consecutive arginine residues. The protein transduction domain may consist essentially of arginine residues. The protein transduction domain may comprise arginine repeats, such as 4 - 20 arginine residues. The protein transduction domain may comprise 8 arginine residues. The protein transduction domain may comprise from about 6 to about 12 arginine residues. The protein transduction domain may comprise from about 7 to about 9 arginine residues.

[0146] The protein transduction domain may comprise from about 4 to about 12 amino acid residues. The protein transduction domain may comprise from about 6 to about 12 amino acid residues. The protein transduction domain may comprise from about 7 to about 9 amino acid residues. The protein transduction domain may comprise at least about 4 amino acid residues. The protein transduction domain may comprise at least about 6 amino acid residues.

[0147] The protein transduction domain may comprise lysine residues, such as consecutive lysine residues. The protein transduction domain may consist essentially of lysine residues. The protein transduction domain may comprise lysine repeats, such as 4 - 20 lysine residues. The protein transduction domain may comprise 8 lysine residues. The protein transduction domain may comprise from about 4 to about 12 lysine residues. The protein transduction domain may comprise from about 6 to about 12 lysine residues. The protein transduction domain may comprise from about 7 to about 9 lysine residues.

[0148] The protein transduction domain may comprise Q and R residues, such as consecutive QR repeat residues. The protein transduction domain may consist essentially of Q and R residues. The protein transduction domain may comprise QR repeats, such as 4-20 QR repeat residues. The protein transduction domain may comprise 8 QR repeat residues. The protein transduction domain may comprise about 6 to about 12 QR repeat residues. The protein transduction domain may comprise about 7 to about 9 QR repeat residues.

[0149] In one embodiment, the cargo is conjugated to a cargo-binding molecule. The cargo can be conjugated to the cargo-binding molecule during manufacture, after manufacture, before use, or during use of the delivery molecule.

[0150] The cargo binding molecule can be a carrier of the cargo molecule. A single cargo binding molecule can bind and carry multiple cargo molecules. The cargo binding molecule can protect the cargo before internalization into the cell. The cargo binding molecule may be able to bind biotin on a biotinylated cargo. The cargo binding molecule may be able to bind nucleic acid-based cargo. The cargo binding molecule may be able to bind peptide-based cargo. The cargo binding molecule may be able to bind antibody cargo, or a fragment or mimetic thereof. The cargo binding molecule may be able to bind nanoparticle cargo, such as metal or polymer nanoparticles. The cargo binding molecule may be functionally inactive in the cell, but may carry or bind active cargo. The cargo binding molecule may include a chemical linker molecule. The cargo binding molecule may include an affinity tag. The cargo binding molecule may include a peptide or protein. The cargo binding molecule may include mSA2 (monomeric streptavidin 2). The cargo binding molecule may include a nucleic acid-interactive peptide, such as LK15. The cargo binding molecule may include an antibody binding molecule, such as an IgG binding protein. The IgG binding protein may include Staphylococcus aureus IgG binding protein SpAB. Those skilled in the art will appreciate that any suitable pair or group of molecules can be used for the cargo and cargo-binding molecules, provided there is sufficient binding or affinity between them.

[0151] The bond or interaction between the cargo and the cargo-binding molecule can be reversible or degradable, for example in an intracellular environment.

[0152] The GAG ​​binding element and the protein transduction domain can be combined with the cargo and / or cargo binding molecule by direct chemical conjugation or through a connector molecule. The GAG ​​binding element and the protein transduction domain can be combined with the cargo by direct chemical conjugation or through a connector molecule. The GAG ​​binding element and the protein transduction domain can be combined with the cargo binding molecule by direct chemical conjugation or through a connector molecule. The GAG ​​binding element, the protein transduction domain and the cargo can be a single fusion molecule (for example, it can be encoded and transcribed as a single peptide molecule). The GAG ​​binding element, the protein transduction domain and the cargo binding molecule can be a single fusion molecule (for example, it can be encoded and transcribed as a single peptide molecule). The protein transduction domain and the GAG ​​binding element can be located on the side of the cargo binding molecule and / or the cargo.

[0153] The length of the delivery molecule may be about 10 to about 30,000 amino acids. The length of the delivery molecule may be about 20 to about 30,000 amino acids. The length of the delivery molecule may be about 30 to about 30,000 amino acids. The length of the delivery molecule may be about 40 to about 30,000 amino acids. The length of the delivery molecule may be about 10 to about 10,000 amino acids. The length of the delivery molecule may be about 20 to about 10,000 amino acids. The length of the delivery molecule may be about 40 to about 10,000 amino acids. The length of the delivery molecule may be about 10 to about 3000 amino acids. The length of the delivery molecule may be about 20 to about 3000 amino acids. The length of the delivery molecule may be about 40 to about 3000 amino acids. The length of the delivery molecule may be about 10 to about 1000 amino acids. The length of the delivery molecule may be about 20 to about 1000 amino acids. The length of the delivery molecule may be about 40 to about 1000 amino acids. The length of the delivery molecule may be about 40 to about 500 amino acids. The length of the delivery molecule may be about 10 to about 500 amino acids. The length of the delivery molecule may be about 20 to about 500 amino acids. The length of the delivery molecule may be about 100 to about 3000 amino acids. The length of the delivery molecule may be at least about 100 amino acids.

[0154] The delivery molecule can be a single fusion molecule. The cargo, HS-GAG binding element and protein transduction domain can be fused together. The HS-GAG binding element and protein transduction domain can be located on the side of the cargo. The cargo, HS-GAG binding element and protein transduction domain can be connected together by one or more linker molecules.

[0155] The delivery molecule may have a molecular weight of at least 1KDa. The delivery molecule may have a molecular weight of at least 5KDa. The delivery molecule may have a molecular weight of at least 10KDa. The delivery molecule may have a molecular weight of at least 20KDa. The delivery molecule may have a molecular weight of 400KDa or less. The delivery molecule may have a molecular weight of 300KDa or less. The delivery molecule may have a molecular weight of about 0.5KDa to about 400kDa. The delivery molecule may have a molecular weight of about 1KDa to about 400kDa. The delivery molecule may have a molecular weight of about 0.5KDa to about 200kDa. The delivery molecule may have a molecular weight of about 1KDa to about 200kDa. The delivery molecule may have a molecular weight of about 2KDa to about 300kDa. The delivery molecule may have a molecular weight of about 20KDa to about 300kDa. The delivery molecule may have a molecular weight of about 20KDa to about 300kDa. The delivery molecule may have a molecular weight of about 20KDa to about 100kDa.

[0156] The cargo may be capable of binding (such as ionically or covalently) to the protein transduction domain and / or the GAG ​​binding element.

[0157] In one embodiment, the transfection delivery molecule includes a molecule for delivery (cargo). In particular, the molecule for delivery (ie, cargo) and the transfection delivery molecule may be referred to as a "transfection delivery molecule".

[0158] Cells / Cell Populations

[0159] The cell may be a mammalian cell, such as a human cell. The cell may be a cancer cell. The cell may be a stem cell. The cell may be a mutant cell. The cell may include a cell population. The cell population may be a mixed population of cell types. The cell may be a mesenchymal stem cell, such as an iHMSC. The cell may be an embryonic stem cell. The cell may be a pluripotent stem cell, such as a human pluripotent stem cell (hPSC).

[0160] In one embodiment, the cell or cell population used for transfection is a blood cell, such as LCL or RAW246.7. In one embodiment, the cell or cell population used for transfection is a microglia cell, such as BV2.

[0161] In one embodiment, the cell or cell population used for transfection can be selected from the group consisting of peripheral blood mononuclear cells (PBMC), hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), induced pluripotent stem cells (IPSC), human embryonic stem cells (HESC), sperm, oocytes, skeletal muscle cells, brain cells and lung cells, or a combination thereof.

[0162] Lung cells may include alveolar cells. Brain cells may include neurons and / or glial cells. Skeletal muscle cells may include myocytes and / or myoblasts.

[0163] Those skilled in the art will recognize that the number of cells to be transfected can vary depending on the application and whether the transfection is in vivo or in vitro. In one embodiment, such as in vitro, the number of cells to be transfected includes at least 1000 cells, 5000 cells, 1x103 cells, 5x103 cells, or 8x103 cells, or more. In another embodiment, such as in vivo, the number of cells to be transfected includes at least 1000 cells, 5000 cells, 1x103 cells, 5x103 cells, 8x103 cells, 1x104 cells, 1x105 cells, 1x106 cells, 1x107 cells, or 1x108 cells, or more.

[0164] The method may further comprise centrifuging the delivery molecule together with the cells to be transfected, for example, to facilitate their contact and increase transfection efficiency.

[0165] Other methods detailed

[0166] The method can be performed in vitro or in vivo. In one embodiment, where the method is performed in vivo, the cells to be transfected can be in a subject. The subject can be a mammal, such as a human.

[0167] In one embodiment, wherein the method is performed in vitro, the cells to be transfected can be isolated from a subject. The cells can be a maintained cell line, or a maintained cell culture extracted from a subject.

[0168] In one embodiment, wherein the method is implemented in vivo, PKC regulators and / or molecules for transfection can be administered to a subject. In one embodiment, HDAC inhibition is further provided, and HDAC inhibitors can be administered to a subject. PKC regulators and / or molecules for transfection can be provided in a pharmaceutically acceptable excipient. HDAC inhibitors can be provided in a pharmaceutically acceptable excipient. Administration can be systemic and / or directly carried out on the original position cells to be transfected.

[0169] The method of transfecting cells with molecules can be a non-viral transfection method. A person skilled in the art will understand that "non-viral transfection method" includes any transfection method in which the molecule used for transfection is not of viral origin.

[0170] Advantageously, non-viral transfection methods allow for shorter durations of transgene expression, are capable of delivering DNA of flexible sizes, are less expensive, easier to prepare, and produce little or no immune response in vivo compared to viral transfection methods.

[0171] In vitro transfection can be performed in cell growth medium, buffer, or saline.

[0172] pH-mediated endosomal escape

[0173] In one embodiment, a transfection delivery molecule may be used in combination with a pH responsive peptide comprising about 5 to 20 histidine residues, such as for delivery.

[0174] Histidine exhibits considerable buffering capacity and thus becomes protonated at the low pH of late endosomes or lysosomes. There is a "proton sponge" hypothesis describing that unprotonated residues such as the imidazole ring of histidine can absorb protons as they are pumped into the endosome / lysosome, which causes more protons to be pumped in, leading to an increased influx of Cl ions and water. The combination of osmotic swelling due to repulsion between protonated amine groups and swelling of positively charged residues in the cargo leads to rupture of the endosomal / lysosomal membrane and subsequent release of its contents into the cytoplasm.

[0175] Increased proton transport into late endosomes and lysosomes occurs via the ATP-driven V-ATPase. This pump is able to continuously transport protons, as long as sufficient ATP is available in the cytoplasm, and the goal of V-ATPase activity is to maintain a proton gradient across the vesicle membrane. GET peptides may buffer lysosomes, however, the V-ATPase pump maintains most vesicles acidic by increasing the influx of protons. Due to the "proton sponge" hypothesis, no changes in pH were observed even with the increased proton influx.

[0176] In one embodiment, a pH responsive peptide comprising about 5 to 20 histidine residues can be added to a transfection delivery molecule, such as a GAG peptide as described herein. In particular, the pH responsive peptide can be combined with or compounded with a transfection delivery molecule, such as a GAG peptide as described herein. The pH responsive peptide can be covalently bound to a transfection delivery molecule, such as a GAG peptide as described herein, for example in the form of a fusion peptide.

[0177] In another embodiment, the pH responsive peptide may be an auxiliary peptide provided with the transfection delivery molecule. In one embodiment, the pH responsive peptide may be contained within a nanoparticle that is released in an endosomal vesicle.

[0178] In another embodiment, the pH responsive peptide may replace the PTD (protein transduction domain / CPP) of a transfection delivery molecule, such as the GAG ​​peptides described herein.

[0179] In one embodiment, the pH responsive peptide comprises 5-20 histidine residues and a nucleic acid interaction sequence or consists of it. In another embodiment, the pH responsive peptide comprises 5-12 histidine residues and a nucleic acid interaction sequence or consists of it. In another embodiment, the pH responsive peptide comprises 8-20 histidine residues and a nucleic acid interaction sequence or consists of it. In another embodiment, the pH responsive peptide comprises 5-15 histidine residues and a nucleic acid interaction sequence or consists of it. In another embodiment, the pH responsive peptide comprises 8-15 histidine residues and a nucleic acid interaction sequence or consists of it. In another embodiment, the pH responsive peptide comprises 8-12 histidine residues and a nucleic acid interaction sequence or consists of it. In another embodiment, the pH responsive peptide comprises 9-11 histidine residues and a nucleic acid interaction sequence or consists of it. In another embodiment, the pH responsive peptide comprises 10 histidine residues and a nucleic acid interaction sequence or consists of it.

[0180] The nucleic acid interaction sequence is capable of binding to nucleic acid, preferably non-specific binding to nucleic acid, for example, by its charge binding. The nucleic acid interaction sequence may be amphiphilic and therefore also has an endosomal escape function. The length of the nucleic acid interaction sequence may be between 5 and 30 residues. The length of the nucleic acid interaction sequence may not exceed 25, 30 or 40 residues. The nucleic acid interaction sequence may contain or consist of a plurality of K and / or L residues. The nucleic acid interaction sequence may contain or consist of 10-25 K and / or L residues. The nucleic acid interaction sequence may contain or consist of 15 K and / or L residues. The K and L residues may alternate, or the pH responsive peptide may contain repeating units of KLL and / or KLLL. The nucleic acid interaction sequence may contain KLLKLLLKLLLKLLK (SEQ ID NO: 19) or a variant thereof. The variant may have at least 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID NO: 19.

[0181] In one embodiment, the pH responsive peptide comprises or consists of the sequence KLLKLLLKLLLKLLKHHHHHHHHHH (referred to herein as "LK15-10H") (SEQ ID NO: 20), or a variant thereof. The variant may have at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 20. In one embodiment, the pH responsive peptide comprises or consists of the sequence KLLKLLLKLLLKLLK (H5-20) (SEQ ID NO: 21).

[0182] In one embodiment, wherein the pH responsive peptide is combined with a transfection delivery molecule (such as a GAG peptide), the pH responsive peptide can replace the protein transduction domain of the transfection delivery molecule (such as a GAG peptide). In another embodiment, wherein the pH responsive peptide is combined with a transfection delivery molecule (such as a GAG peptide), in addition to the protein transduction domain of the transfection delivery molecule (such as a GAG peptide), the pH responsive peptide can also be provided.

[0183] In one embodiment, the transfection delivery molecule having a pH responsive peptide comprises or consists of the following sequence:

[0184] FLHTYRSRKYTSWYVALKRKLLKLLLKLLLKLLKHHHHHHHHHH (SEQ ID NO: 22) (FGF2B-LK15-10H) (also referred to herein as "44").

[0185] In one embodiment, the transfection delivery molecule having a pH responsive peptide comprises or consists of the following sequence:

[0186] TYRSRKYTSWYVALKRKLLKLLLKLLLKLLKHHHHHHHHHHRRRRRRRRR (SEQ ID NO: 23) (FGF2B-LK15-10H-8R) (also referred to herein as "FLHR").

[0187] In one embodiment, the transfection delivery molecule having a pH responsive peptide comprises or consists of the following sequence:

[0188] TYRSRKYTSWYVALKRKLLKLLLKLLLKLLKRRRRRRRRHHHHHHHHHH (SEQ ID NO:24) (FGF2B-LK15-8R-10H) (also referred to herein as "FLRH").

[0189] In one embodiment, the transfection delivery molecule having a pH responsive peptide comprises or consists of the following sequence:

[0190] TYRSRKYTSWYVALKRRRRRRRRRRKLLKLLLKLLLKLLKHHHHHHHHHH (SEQ ID NO: 25) (FGF2B-8R-LK15-10H) (also referred to herein as "FRLH")

[0191] Other aspects

[0192] According to another aspect of the present invention, there is provided a composition comprising:

[0193] - a modulator of PKC and / or a pH responsive peptide comprising about 5 to 20 histidine residues; and

[0194] -Molecules used for transfection.

[0195] Preferably, 5-20 histidine residues of the pH responsive peptide are capable of being protonated in the acidic environment of endosomes.

[0196] In one embodiment of the composition, the PKC modulator may be a PKC inhibitor. The composition may also include an HDAC inhibitor. The composition may be a pharmaceutically acceptable composition.

[0197] According to another aspect of the present invention, there is provided a kit comprising:

[0198] - a modulator of PKC and / or a pH responsive peptide comprising about 5 to 20 histidine residues; and

[0199] -Molecules used for transfection.

[0200] In one embodiment of the kit, the PKC modulator may be a PKC inhibitor.

[0201] The kit may also comprise an HDAC inhibitor.The components of the kit may be provided individually or as a combination.

[0202] The kit may also include centrifuge tubes, and optionally a centrifuge.

[0203] The molecules used for transfection may be in lyophilized form.The kit may further comprise a reconstitution solution, such as a buffer.

[0204] According to another aspect of the present invention, there is provided a method for enhancing cell transfection using a transfection molecule, the method comprising the following steps:

[0205] - adding molecules for transfection to the cells; and

[0206] - modulating the activity of protein kinase C (PKC) in cells and / or providing a pH-responsive peptide comprising about 5 to 20 histidine residues to enhance endosomal release in cells.

[0207] pH-responsive peptides can be protonated in endosomes after internalization.

[0208] The molecule used for transfection may not itself modulate the activity of PKC in the cell.The molecule used for transfection may not comprise or consist of a PKC modulator (eg, an inhibitor or activator).

[0209] According to another aspect of the present invention, there is provided use of a PKC regulator and / or a pH-responsive peptide comprising about 5 to 20 histidine residues for improving cell transfection efficiency.

[0210] Alternatively, HDAC regulators can be used in combination with PKC regulators to improve cell transfection efficiency. In particular, this use can be used in combination with HDAC inhibitors. GET-mediated transfection can be used.

[0211] The method of the present invention can be used in gene therapy.

[0212] According to another aspect of the present invention, there is provided a gene therapy method comprising administering to a subject:

[0213] (a) a PKC modulator and / or providing a pH responsive peptide comprising about 5 to 20 histidine residues; and

[0214] (b) A molecule for transfection, wherein the molecule for transfection comprises a nucleic acid.

[0215] The gene therapy method may further comprise administering an HDAC inhibitor.

[0216] According to another aspect of the present invention, there is provided a method of treating a subject, comprising administering to the subject:

[0217] (a) a PKC modulator and / or providing a pH responsive peptide comprising about 5 to 20 histidine residues; and

[0218] (b) A molecule for transfection, wherein the molecule for transfection comprises a therapeutically effective molecule.

[0219] The therapeutically effective molecule may be a nucleic acid, protein or peptide as described herein. The therapeutically effective molecule may be a nucleic acid encoding a gene sequence and / or a regulatory sequence (eg, as described herein).

[0220] According to another aspect of the present invention, there is provided a method of treating a subject, comprising administering to the subject a cell that has been transformed according to the method of the present invention.

[0221] Cells that have been transformed according to the methods of the present invention may contain molecules used for transfection within the cells.

[0222] According to another aspect of the present invention, a PKC modulator and / or a pH-responsive peptide comprising about 5 to 20 histidine residues is provided for use as a drug in combination with a molecule for transfection.

[0223] The use can be used for gene therapy. In another embodiment, the use can be used for treating or preventing a disease. In another embodiment, the use can be used for tissue repair or replacement. The tissue can be soft tissue or bone tissue.

[0224] The treatment or prevention may be for the treatment or prevention of a monogenic disease. The monogenic disease may be selected from any one of the group consisting of sickle cell disease, cystic fibrosis, polycystic kidney disease, Tay-Sachs disease, alpha 1-antitrypsin deficiency, and primary ciliary dyskinesia.

[0225] Treatment or prevention can be used to treat or prevent diseases or conditions that can be treated or prevented by overexpression of growth factors. Treatment or prevention can be used to treat or prevent diseases or conditions, including wound healing, tissue repair, bone repair, diabetic ulcers, neurodegeneration (e.g., Alzheimer's disease or Parkinson's disease), or osteoarthritis.

[0226] definition

[0227] A person skilled in the art may understand "modulator" to describe an agonist, inverse agonist or antagonist, ie a modulator can increase or decrease the activity of its target.

[0228] One skilled in the art may understand "inhibitor" to describe an inverse agonist or antagonist, ie an inhibitor reduces or stops the activity of its target.

[0229] The term "temporary regulation", including "temporary inhibition" or "temporary activation" should be understood as the regulation of activity in the cell is not permanent. For example, upon deactivation or absence of the regulator, the original cell activity can be restored. In contrast, permanent regulation can include genetic regulation, such as knocking out a gene by mutation.

[0230] The terms "transfection" and "transduction" are used interchangeably herein. They are understood to refer to the process of introducing molecules such as nucleic acids or proteins into cells.

[0231] The terms "transfected", "transduced" or "transformed" in relation to cells are understood to mean that the cells have internalized the molecules used for transfection. The molecules used for transfection can be in the cytoplasm and / or the nucleus, i.e., not sequestered in endosomes. Successful transfection can be measured by reporter gene expression (such as GFP or other fluorescent proteins and flow cytometry). Those skilled in the art will appreciate that the method may not require determination of transfection rate or efficiency each time or for the practice of the method. The term "gene therapy" is understood to mean the introduction of genetic material, such as genes and / or regulatory elements, into one or more cells of a subject to provide a therapeutic benefit or prevent a condition or disease.

[0232] Those skilled in the art will appreciate that optional features of one embodiment or aspect of the invention may be applicable to other embodiments or aspects of the invention where appropriate.

[0233] Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

[0234] Figure 1 -TPA (T) significantly enhances transfection of T2 cells. Experiments performed on 20,000 T2 hybrid human T / B cells, measured 24 hours after transfection of 1 μg pCMV-Gluc pDNA using GET. T = TPA (μM), S = SAHA (μM). TPA appears to be the main effect on T2 cell transfection efficiency.

[0235] Figure 2 - SAHA (S) significantly enhances transfection of RAW264.7 cells. Experiments performed on 20,000 RAW264.7 mouse macrophages, measured 24 hours after transfection of 1 μg pCMV-Gluc pDNA using GET. T = TPA (μM), S = SAHA (μM).

[0236] Figure 3 - Short exposure to SAHA (S) and TPA (T) significantly enhances transfection of LCL cells. Experiments performed on 20,000 LCL lymphoblastoid human B cells, measured 24 hours after transfection of 1 μg pCMV-Gluc pDNA using GET. T = TPA (μM), S = SAHA (μM). TPA was shown to have the greatest effect on transfection of LCL cells, and SAHA enhanced TPA. Without these modulators, there was virtually no transfection.

[0237] Figure 4 - Transfection of T2 cells. Experiments performed on 20,000 T2 hybrid human T / B cells, measured 24 hours after transfection of pCMV-Gluc pDNA with GET. 1X = 1 μg pCMV-Gluc pDNA with GET. 0.2X = 0.2 μg pCMV-Glu cpDNA with GET. T = TPA (μM), S = SAHA (μM).

[0238] Figure 5 - Transfection of LCL cells. Experiments performed on 20,000 LCL lymphoblastoid human B cells, measured 24 hours after transfection of pCMV-Gluc pDNA with GET. 1X = 1 μg pCMV-Gluc pDNA with GET. 0.2X = 0.2 μg pCMV-Gluc pDNA with GET. T = TPA (μM), S = SAHA (μM).

[0239] Figure 6 -Schematic diagram of transfection of rat cerebellar slices.

[0240] Experiments were performed on postnatal day 8 (P8) pups. Cerebellum was dissected from the brain in ice-cold preparation medium using a stereomicroscope. 350 μm thick sections were made in sagittal direction using a McIlwain tissue chopper or vibratome. Microwell cell culture inserts (0.25 ml per well, 50 μl on top) were used. Transfection was initiated on days 3-5 in vitro (D3-5) by adding the transfectant (100 μl) to the sections in the top well.

[0241] Figure 7 - Endosomal escape system of the same peptide. Human skin fibroblasts were transfected with pCMV-gluc and FLR, FLH and FRLH with decreasing peptide:pDNA charge ratios (5:1, 4:1, 3:1, 2:1, 1:1). Gluc was measured on day 1 post-transfection, with 1 ug pDNA transfected into 2x105 cells in 12-well plates. Bars are SD. N=3.

[0242] Figure 8 - Endosomal escape system on helper peptide. Human skin fibroblasts were transfected with pCMV-gluc and FLR with or without increasing ratios of FLH and FRLH (5:1, 4:1, 3:1, 2:1, 1:1 of FLR, helper peptide composition to pDNA to 5:1 peptide charge). Gluc was measured on day 1 post-transfection, 1 ug pDNA was transfected into 2x105 cells in 12-well plates. Bars are SD. N=3.

[0243] Fig. 9 -HDACi activity is early in transfection (during macropinocytosis and endosomal transport) rather than late (affecting transcriptional activity). Human immortalized MSCs were transfected by rapid transfection (5 min) using pCMV-gluc and FLR:FLH (1:1). Transfection was removed within the first 6 hours of uptake / transfection and exposed to SAHA (1-100uM, S1-100) for 1 hour. Gluc was measured on day 1 post-transfection, and 1ug pDNA was transfected into 12-well plates with 2x105 cells. Bars are SD. N=3.

[0244] Fig.10 - iHMSCs were transfected in the presence of HDAC and PKC inhibition (inhibitors SAHA and GF109203X, GFX). GFX0 = 0 μM GF109203X, GFX0.1 = 0.1 μM GF109203X, GFX1 = 1 μM GF109203X, S0 = 0 μM SAHA, S1 = 1 μM SAHA, S10 = 10 μM SAHA.

[0245] Fig.11 - Transfection of LCL in the presence of HDAC and PKC inhibition (inhibitors SAHA and Carvstatin C). CPC0 = 0 μM Carvstatin C, CPC0.1 = 0.1 μM Carvstatin C, S0 = 0 μM SAHA, S1 = 1 μM SAHA, S10 = 10 μM SAHA.

[0246] Materials used

[0247] pCMV-Gluc is a mammalian expression vector that expresses Gaussia luciferase under the control of the CMV promoter. It was obtained from New England Biolabs (NEB) https: / / international.neb.com / products / n8081-pcmv-gluc-2-control-plasmid#Product%20Information.

[0248] pGM (pGM206) is a CpG-free version of the DNA vector expressing Gaussia luciferase (pG4-hCEFI-soGluc) (http: / / spiral.imperial.ac.uk:8080 / bitstream / 10044 / 1 / 19179 / 2 / Biomaterials_32_10_2011.pdf).

[0249] One skilled in the art will recognize that any suitable control vector may be used to demonstrate transfection efficiency.

[0250] Example sequences for transfection delivery molecules

[0251] Examples of HS-GAG binding sequences

[0252] P21 amino acid sequence

[0253] (SEQ ID NO.1)

[0254] KRKKKGKGLGKKRDPCLRKYK

[0255] P21 nucleotide sequence (including methionine / ATG):

[0256] (SEQ ID NO:2)

[0257] aagcgcaagaagaagggcaaaggcctgggcaagaagcgcgatccgtgcctgcgcaagtataag

[0258] PDGF (194-211) amino acid sequence:

[0259] (SEQ ID NO.3)

[0260] GRPRESGKKRKRKRLKPT

[0261] PDGF (194-211) nucleotide sequence:

[0262] (SEQ ID NO:4)

[0263] ggccgcccgcgcgaaagcggcaaaaaacgcaaacgcaaacgcctgaaaccgacc

[0264] FGF7B amino acid sequence:

[0265] (SEQ ID NO.5).

[0266] TYASAKWTHNGGEMFV AL NQ

[0267] FGF7B nucleotide sequence:

[0268] (SEQ ID NO:6)

[0269] Acctatgcgagcgcgaaatggacccataacggcggcgaaatgtttgtggcgctgaaccag

[0270] FGF2 HBD B (247-262) amino acid sequence:

[0271] (SEQ ID NO.7).

[0272] TYRSRKYTSWYVALKR

[0273] FGF2 HBD B (247-262) nucleotide sequence:

[0274] (SEQ ID NO:8)

[0275] acctatcgcagccgcaaatataccagctggtatgtggcgctgaaacgc

[0276] Nucleotide encoding 8R protein transduction domain sequence:

[0277] (SEQ ID NO:9)

[0278] CGA AGA CGC AGG AGA CGT CGA AGG

[0279] Example of delivery molecule nucleotide sequence (P21-cargo-8R):

[0280] (SEQ ID NO: 10)

[0281] aagcgcaagaagaagggcaaaggcctgggcaagaagcgcgatccgtgcctgcgcaagtataagNcgaagacgcaggagacgtcgaagg

[0282] N = cargo nucleic acid sequences of varying length (ie, the number of nucleotide residues may vary), or other molecular entities.

[0283] Two versions of the nanobody variant of each ScFv antibody were made; one with the same sequence as the ScFv vHH domain (framework domain 1-CDR1-framework domain 2-CDR2-framework domain 3-CDR3-IgA hinge domain / framework domain 4), and one with the CDR1, 2, and 3 domains grafted to a common vHH domain sequence. Both versions had comparable activity, and the grafted version was created to demonstrate that simply grafting the CDR domains onto a common antibody was also effective.

[0284] The following are the sequences of HS4C3 and AO4BO8 ScFv vHH and grafted vHH:

[0285] HS4C3 ScFv vHH

[0286] (SEQ ID NO:11)

[0287] EVQLVESGGGLVQPRGSLRLSCAASGFTVSSNEMSWIRQAPGKGLEWVSSISGGSTYYADSRKGRFTISRDNSKNTLYLQMNNLRAEGTAAYYCGRRLKDPSTPPTPSPSTPPTPSPS

[0288] CDR1 GFTVSSNE

[0289] CDR2-ISGGST

[0290] CDR3 GRRLKD

[0291] HS4C3 ScFv vHH nucleotide sequence (SEQ ID NO: 12)

[0292] gaagtgcagctggtggaaagcggcggcggcctggtgcagccgcgcggcagcctgcgcctgagctgcgcggcgagcggctttaccgtgagcagcaacgaaatgagctggattcgccaggcgccgggcaaaggcctggaatgggtgagcagcattagcggcggcagcacctattatgcggatagccgcaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtatctgcagatgaacaacctgcgcgcggaaggcaccgcggcgtattattgcggccgccgcctgaaagatccgagcaccccgccgaccccgagcccgagcaccccgccgaccccgagcccgagc

[0293] HS4C3-grafted vHH

[0294] (SEQ ID NO:13)

[0295] QVQLVESGGGSVQAGGSLRLSCTASGFTVSSNELGWFRQAPGQERWAVAAISGGSTYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCGRRLKDWGQGTQVTVSSPSTPPTPSPSTPPTPSPS

[0296] CDR1 GFTVSSNE

[0297] CDR2 ISGGST

[0298] CDR3 GRRLKD

[0299] Nucleotides of HS4C3-grafted vHH

[0300] (SEQ ID NO:14)

[0301] caggtgcagctggtggaaagcggcggcggcagcgtgcaggcgggcggcagcctgcgcctgagctgcaccgcgagcggctttaccgtgagcagcaacgaactgggctggtttcgccaggcgccgggccaggaacgctgggcggtggcggcgattagcggcggcagcacctattatgcggatagcgtgaaaggccgctttaccattagccgcgataacgcgaaaaacaccgtgaccctgcagatgaacaacctgaaaccggaagataccgcgatttattattgcggccgccgcctgaaagattggggccagggcacccaggtgaccgtgagcagcccgagcaccccgccgaccccgagcccgagcaccccgccgaccccgagcccgagc

[0302] AO4B08 ScFv vHH

[0303] (SEQ ID NO:15)

[0304] EDQLVESGGGLVQPGGSLRPSCAASGFAFSSYALHWVRRAPGKGLEWVSAIGTGGDTYYADSVMGR FTISRDNAKKSLYLHMNSLIAEDMAVYYCSLRMNGWRAHQPSTPPTPSPSTPPTPSPS

[0305] CDR1 GFAFSSYA

[0306] CDR2 IGTGGDT

[0307] CDR3 SLRMNGWRAHQ

[0308] AO4B08 ScFv vHH nucleotide sequence

[0309] (SEQ ID NO:16)

[0310] gaagatcagctggtggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcccgagctgcgcggcgagcggctttgcgtttagcagctatgcgctgcattgggtgcgccgcgcgccgggcaaaggcctggaatgggtgagcgcgattggcaccggcggcgatacctattatgcggatagcgtgatgggccgctttaccattagccgcgataacgcgaaaaaaagcctgtatctgcatatgaacagcctgattgcggaagatatggcggtgtattattgcagcctgcgcatgaacggctggcgcgcgcatcagccgagcaccccgccgaccccgagcccgagcaccccgccgaccccgagcccgagcaccccgccgaccccgagcccgagc

[0311] AO4B08-grafted vHH

[0312] (SEQ ID NO:17)

[0313] QVQLVESGGGSVQAGGSLRLSCTASGFAFSSYALGWFRQAPGQERWAVAAIGTGGDTYYADSVKGR FTISRDNAKNTVTLQMNNLKPEDTAIYYCSLRMNGWRAHQWGQGTQVTVSSPSTPPTPSPSTPPTPSPS

[0314] CDR1 GFAFSSYA

[0315] CDR2 IGTGDT

[0316] CDR3 SLRMNGWRAHQ

[0317] Nucleotide sequence of AO4B08-grafted vHH

[0318] (SEQ ID NO:18)

[0319] caggtgcagctggtggaaagcggcggcggcagcgtgcaggcgggcggcagcctgcgcctgagctgcaccgcgagcggctttgcgtttagcagctatgcgctgggctggtttcgccaggcgccgggccaggaacgctgggcggtggcggcgattggcaccggcggcgatacctattatgcggatagcgtgaaaggccgctttaccattagccgcgataacgcgaaaaacaccgtgaccctgcagatgaacaacctgaaaccggaagataccgcgatttattattgcagcctgcgcatgaacggctggcgcgcgcatcagtggggccagggcacccaggtgaccgtgagcagcccgagcaccccgccgaccccgagcccgagcaccccgccgaccccgagcccgagc

Claims

1. A method for transfecting cells with a molecule, characterized in that The method comprises the following steps: adding a molecule for transfection to the cells; and The activity of protein kinase C (PKC) in the cell is modulated and / or a pH responsive peptide comprising about 5 to about 20 histidine residues is provided. 2 . The method according to claim 1 , wherein the modulation of the PKC activity in the cell comprises activation of the PKC activity.

3. The method according to claim 1 or 2, wherein the regulation of PKC activity is provided by adding a PKC regulator selected from the group consisting of phorbol 12-myristate 13-acetate (PMA), ingenol 3-angelate (I3A) and bryostatin; or its functional analogs and derivatives; or the PKC regulator is a gene silencer siRNA configured to inhibit the expression of PKC.

4. The method according to any one of the preceding claims, wherein the modulation of PKC activity is at a time between about 1 hour and about 3 hours after initiation of transfection.

5. The method according to any one of the preceding claims, further comprising regulating histone deacetylase (HDAC) in the cell.

6. The method of claim 5, wherein the modulation of HDAC activity is provided by adding an HDAC inhibitor selected from the group consisting of suberoylanilide hydroxamic acid (SAHA), panobinostat, trichostatin A (TSA), tubastatin A and valproic acid; or functional analogs and derivatives thereof; or the HDAC modulator is a gene silencer siRNA configured to inhibit the expression of HDAC.

7. The method according to any one of the preceding claims, wherein PMA / TPA activation of PKC activity is provided in combination with one or more of the HDAC inhibitors panobinostat, TSA or SAHA.

8. The method according to any one of the preceding claims, wherein the molecule used for transfection comprises or consists of a nucleic acid, a protein, a peptide or a nanoparticle.

9. The method of claim 8, wherein the nucleic acid is a DNA encoding one or more gene sequences and / or one or more regulatory sequences.

10. The method according to any one of the preceding claims, wherein the molecule used for transfection comprises a transcription factor or a nucleic acid encoding a transcription factor.

11. The method according to any one of the preceding claims, wherein the molecule used for transfection comprises a growth factor or a nucleic acid encoding a growth factor.

12. The method according to any one of the preceding claims, wherein the molecule for transfection is bound to, complexed with, embedded within or linked to a transfection delivery molecule.

13. The method of claim 12, wherein the transfection delivery molecule is selected from the group comprising nucleic acids, peptides, proteins, viral particles, virus-like particles, non-viral molecules, synthetic polymers and glycosaminoglycan (GAG) binding enhanced transduction (GET)-cargo molecules, or pH-mediated variants thereof.

14. The method according to any one of the preceding claims, wherein the cell or cell population for transfection is selected from the group comprising peripheral blood mononuclear cells (PBMC), hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), induced pluripotent stem cells (IPSC), human embryonic stem cells (HESC), sperm, oocytes, skeletal muscle cells, brain cells and lung cells or a combination thereof.

15. The method according to any one of the preceding claims, wherein the pH responsive peptide further comprises a sequence of 10 to 25 K and / or L residues.

16. The method according to any one of the preceding claims, wherein the pH responsive peptide comprises or consists of the sequence KLLKLLLKLLLKLLK(H5-20).

17. A composition, characterized in that Include: A modulator of PKC and / or a pH responsive peptide comprising about 5 to about 20 histidine residues; and Molecules used for transfection.

18. The composition of claim 17, further comprising an HDAC modulator.

19. A kit, characterized in that: Include: A modulator of PKC and / or a pH responsive peptide comprising about 5 to about 20 histidine residues; and Molecules used for transfection.

20. The kit according to claim 19, further comprising an HDAC modulator.

21. A use of a composition, characterized in that: Used to improve cell transfection efficiency; The composition comprises a modulator of PKC and / or a pH responsive peptide comprising about 5 to about 20 histidine residues, optionally in combination with an HDAC modulator for improving cell transfection efficiency.

22. A use of a composition, characterized in that: For use in the preparation of gene therapy drugs; The composition comprises, (a) a modulator of PKC and / or a pH responsive peptide comprising about 5 to about 20 histidine residues; and (b) A molecule for transfection, wherein the molecule for transfection comprises a nucleic acid.

23. An application of a composition, characterized in that: For the preparation of medicines; The composition comprises, (a) a modulator of PKC and / or a pH responsive peptide comprising about 5 to about 20 histidine residues; and (b) A molecule for transfection, wherein the molecule for transfection comprises a therapeutically effective molecule.

24. A use of cells, characterized in that: For the preparation of medicines; The cells are transfected with molecules using the method of any one of claims 1 to 16.

25. A use of a composition, characterized in that: for use in combination with molecules for transfection to prepare drugs; The composition comprises a modulator of PKC and / or a pH responsive peptide comprising about 5 to about 20 histidine residues.

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