Non-viral modification of mesenchymal stem cells

Abstract

Described herein are non-viral methods for transfecting mesenchymal stem cells with nucleic acid constructs expressing one or more functional genes, which, in certain embodiments, may provide high transfection efficiency, high copy number per cell, high cell viability, long-term transient expression, and / or a substantially unaltered pluripotent phenotype. In certain embodiments, such methods may be scalable and / or suitable for large-scale clinical production of modified mesenchymal stem cells. Also described in detail herein are transfected mesenchymal stem cells and mesenchymal stem cell populations, their uses, methods of treating diseases or disorders, such as cancer, using such transfected mesenchymal stem cells, and kits and compositions related thereto.

Description

[Technical field]

[0001] The present invention relates generally to non-viral modification of mesenchymal stem cells. In particular, the present invention relates to non-viral modification of mesenchymal stem cells (MSCs) for therapeutic applications, such as for cancer treatment. [Background technology]

[0002] There are currently >700 clinical trials using mesenchymal stem cells (MSCs) registered in the National Institutes of Health Clinical Trials Database. While MSC-based therapies are considered safe [2], preclinical and clinical data have shown at best moderate efficacy and often ineffectiveness [3-5]. The latest trend to overcome this challenge is the genetic modification of MSCs. In the UK alone, 37% of clinical trials use genetically modified cells, of which 90% use viral carriers for gene delivery to MSCs [6]. Clinical trials are underway with modified MSCs producing cytosine deaminase (CD) for gene-directed enzyme prodrug therapy (GDEPT) [7]. Given the inherent safety and production issues when using viruses [7, 8], highly efficient modification of MSCs using non-viral methods would be desirable but poses considerable challenges.

[0003] Numerous preclinical studies and clinical trials [28-31] have utilized viral vectors as efficient gene delivery vehicles in the engineering of MSCs. While viral gene delivery is highly efficient, there are drawbacks that may include random integration of viral vectors into the host genome, which may interfere with essential gene expression and cellular processes. Even with non-integrating viral vectors, safety risks of viral transduction may arise due to possible presentation of viral antigens on the transduced cells, which may potentially activate immune responses in vivo following transplantation. The production of viral vectors is both labor-intensive and technically demanding, posing challenges for scale-up to increase the number of transgenes. Moreover, it is noteworthy that cells infected with viral vectors typically have low copy numbers (<10 copies / cell). Although viruses have allowed sustained expression of transgenes

[32] , cells infected with viruses typically have low copy numbers (<10 copies / cell) [33, 34]. On the other hand, it has been shown that increased DNA copy numbers can be delivered into individual cells by non-viral methods [35, 36], thus increasing the payload for therapeutic drug delivery, but these methods typically have low transfection efficiencies (often around 0–35%). The production of clinical grade viruses is challenging and typically involves approval as well as the generation of master cell banks of stable producers, resulting in high costs in gene and cell therapeutics [37–39].

[0004] Non-viral methods frequently suffer from shortcomings that prevent clinical application. Non-viral methods, such as cationic polymers, liposomes, electroporation, etc., typically suffer from low efficiency in modifying MSCs at a scale relevant for clinical treatment. Furthermore, non-viral methods, such as electroporation, may have low cell viability that prevents their use at a large scale.

[0005] Transient transfection is a method to rapidly obtain high payloads per cell, avoiding antibiotic selection and weeks of processing time that can cause cellular senescence

[17] and reduce tumor tropism

[18] , as well as safety concerns related to virus-induced MSC transformation

[19] . Although certain non-viral methods have advantages over viral vectors in terms of ease of production, low cost and safety profile

[20] , the lack of widespread adoption for MSC modification is mainly due to low transfection efficiency (0-35%) [21, 22]. Although it is possible to deliver high copy numbers of DNA into cells, transgene expression often remains low. Low expression of transgenes with certain non-viral methods may be due to accumulation of plasmid DNA in non-productive intracellular compartments, resulting in low availability of the plasmid for gene transcription. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Molecular Cloning: A Laboratory Manual (4th edition), 2012, Cold Spring Harbor Laboratory Press [Non-Patent Document 2] "Genes VII", B. Lewin, Oxford University Press (2000) [Non-Patent Document 3] "Molecular Cloning: A Laboratory Manual", Sambrook et al., Cold Spring Harbor Laboratory, 3rd edition (2001) [Non-Patent Document 4] Cold Spring Harb Protoc., July 1, 2016, 2016(7), doi:10.1101 / pdb.prot087163 [Non-Patent Document 5] Oncotarget., October 6, 2017, 8(46):80156~80166

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

[0007] There is a need for additional, alternative and / or improved methods for transfecting MSCs. [Means for solving the problem]

[0008] For many different therapeutic and non-therapeutic applications, stem cells modified to express therapeutic genes or other genes of interest are desirable. Traditionally, in the field of prodrug gene therapy, viral-based genetic modification methods have been the preferred method for modifying stem cells, such as MSCs, in preclinical and clinical studies, because non-viral methods generally provide low transfection efficiency. However, viral-based genetic modification in such applications has inherent safety risks, clinical grade virus can be difficult to produce, and the number of gene copies that can be introduced per cell by viral methods is generally low (often <10 copies per cell). Furthermore, achieving genetic modification of stem cells, such as MSCs, either virally or non-virally, without causing undesirable changes to the phenotype (i.e., pluripotency, immunophenotype, tropism, etc.) of the resulting cells is another challenge facing the field.

[0009] As described in detail herein, the inventors have developed methods of transfecting mesenchymal stem cells with nucleic acid constructs expressing one or more functional genes that are non-viral and may, in certain embodiments, provide high transfection efficiency, high copy number per cell, high cell viability, long-term transient expression, and / or a substantially unaltered pluripotent phenotype. In certain embodiments, such methods may be scalable and / or suitable for large-scale clinical production of modified mesenchymal stem cells. Also described in detail herein are transfected mesenchymal stem cells and mesenchymal stem cell populations, their uses, methods of treating a disease or disorder, such as cancer, using such transfected stem cells, and kits and compositions relating thereto.

[0010] In one embodiment, provided herein are mesenchymal stem cells (MSCs) transfected with a nucleic acid construct expressing one or more functional genes, wherein the MSCs have a pluripotent phenotype that is substantially unaltered by transfection of the nucleic acid construct, and wherein the MSCs are free of viral-based transfection vehicle material.

[0011] In another embodiment, provided herein is a plurality of mesenchymal stem cells (MSCs), wherein at least about 60% of the MSCs are transfected with a nucleic acid construct that expresses one or more functional genes, and wherein the transfected MSCs have a pluripotent phenotype that is substantially unaltered by transfection of the nucleic acid construct, and wherein the MSCs are free of viral-based transfection agent material.

[0012] In another embodiment of the plurality of MSCs, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the MSCs may be transfected with a nucleic acid construct and express one or more functional genes. In further embodiments, the cell viability of the plurality of MSCs may be at least about 70%, at least about 75%, at least about 80%, or at least about 85%.

[0013] In another embodiment of any of the transfected MSCs herein, the MSCs may each be transfected with an average of at least about 1000 copies, at least about 2000 copies, at least about 3000 copies, at least about 4000 copies, at least about 5000 copies, at least about 6000 copies, at least about 7000 copies, at least about 8000 copies, at least about 9000 copies, or at least about 10000 copies of the nucleic acid construct. In another embodiment, one or more functional genes may be transiently expressed in the transfected MSC cells. In another embodiment, the MSCs may be derived from umbilical cord blood, neonatal birth-associated tissue, Wharton's jelly, umbilical cord, cord lining, placenta, or another source of MSC cells. In another embodiment, the MSCs may be adipose tissue-derived MSCs (AT-MSCs), bone marrow-derived MSCs (BM-MSCs), or umbilical cord-derived MSCs (UC-MSCs). In another embodiment, the MSCs may be derived from a human, dog, cat, horse, or another species. In another embodiment, the nucleic acid construct may comprise a CpG-free expression plasmid or another CpG-free expression construct, a scaffold / matrix attachment region (S / MAR), an episomal vector, or an EBNA-1 containing construct.

[0014] In another embodiment of any of the transfected MSCs herein, the MSCs may transiently express one or more functional genes for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, or at least about 17 days following transfection. In another embodiment, the one or more functional genes may include a suicide gene. In another embodiment, the one or more functional genes may include cytosine deaminase (CDy). In another embodiment, the one or more functional genes may include uracil phosphoribosyltransferase (UPRT). In another embodiment, the one or more functional genes may include both CDy and UPRT. In another embodiment, CDy and UPRT may be expressed as a fusion construct. In another embodiment, the one or more functional genes may include a fluorescent protein. In another embodiment, the fluorescent protein may include green fluorescent protein (GFP). In another embodiment, the one or more functional genes may include CDy, UPRT and GFP. In another embodiment, CDy, UPRT and GFP may be expressed as a fusion construct. In another embodiment, the one or more functional genes may include Herpes Simplex Virus Type 1 Thymidine Kinase (HSV-TK) or another thymidine kinase. In another embodiment, the one or more functional genes may include one or more cancer therapy genes or one or more functional genes not related to cancer therapy.

[0015] In another embodiment of any of the transfected MSCs herein, the transfected MSCs may be transfected with a nucleic acid construct using a cationic polymer, a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments, and a second agent capable of stabilizing the microtubule network of the MSCs. In another embodiment, the cationic polymer may comprise linear or branched polyethyleneimine (PEI), poly(amidoamine) PAMAM, or another cationic polymer, or any combination thereof. In another embodiment, the cationic polymer may comprise linear polyethyleneimine (LPEI). In another embodiment, the first agent may comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / cholesteryl hemisuccinate (CHEMS) (DOPE / CHEMS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or another fusogenic lipid, or any combination thereof. In another embodiment, the second agent may comprise a histone deacetylase inhibitor (HDACi), such as a histone deacetylase 6 inhibitor (HDAC6i). In another embodiment, the second agent may comprise SAHA (vorinostat).

[0016] In another embodiment of any of the MSCs herein, the phenotype may include tumor-tropic and / or cancer-tropic properties of the MSCs. In another embodiment, the genetically engineered MSCs of any of the embodiments described herein may be sensitive to treatment with 5-fluorocytosine (5FC) or ganciclovir (GCV). One or more embodiments of the MSCs may a) convert 5FC to 5-fluorouracil (5FU), 5-fluorouridine monophosphate (FUMP) or both, b) convert ganciclovir to ganciclovir monophosphate, or c) a combination of a) and b). In another embodiment, the phenotype may include an immunophenotype in which expression of CD surface markers may be substantially unchanged after transfection.

[0017] In another embodiment of any of the MSCs described herein, the transfected MSCs may be plastic adherent, may express CD105, CD73 and CD90 (>95%), may lack expression of CD45, CD34, CD14 and HLA-DR surface molecules (<2%), may be capable of differentiating in vitro into osteoblasts, adipocytes and chondroblasts, and meet the immunophenotypic criteria defined by the International Society for Cellular Therapy (ISCT). In another embodiment, the transfected MSCs may be undifferentiated.

[0018] In another embodiment of any of the MSCs described herein, the MSCs may be in a cryopreserved state.

[0019] In another embodiment, the MSCs can be used in cancer treatment.In certain embodiments, the cancer can include lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer, or any combination thereof.In another embodiment, the MSCs can be used in combination with 5FC, 5FU, GCV, or any combination thereof.

[0020] In another embodiment, provided herein is a method for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct expressing one or more functional genes, the method comprising exposing the MSCs to a transfection mixture comprising the nucleic acid construct complexed with a cationic polymer, exposing the MSCs to a first agent capable of redirecting endocytosed nucleic acid from an intracellular acidic compartment and a second agent capable of stabilizing the microtubule network of the MSCs, and incubating the MSCs, thereby providing MSCs transfected with the nucleic acid construct.

[0021] In another embodiment of any of the methods described herein, the MSCs may not be centrifuged during exposure to the transfection mixture, during exposure to the first agent and the second agent, during incubation, or any combination thereof. In another embodiment, incubating the MSCs may include gently mixing without centrifugation. In another embodiment, incubating the MSCs may include incubating the MSCs for at least about 2 hours. In another embodiment, incubating the MSCs may include incubating the MSCs for about 2 hours to about 48 hours. In another embodiment, incubating the MSCs may include incubating the MSCs for about 3 hours to about 24 hours, or about 4 hours to about 18 hours.

[0022] In another embodiment of any of the methods described herein, the cationic polymer may comprise a cationic polymer identified to have low cytotoxicity to MSCs. In another embodiment, the cationic polymer may have a size of about 5 kDa to about 200 kDa. In another embodiment, the cationic polymer may comprise linear or branched polyethyleneimine (PEI), poly(amidoamine) PAMAM, or another cationic polymer, or any combination thereof. In another embodiment, the cationic polymer may comprise linear polyethyleneimine (LPEI).

[0023] In another embodiment of any of the methods described herein, the first agent may comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / cholesteryl hemisuccinate (CHEMS) (DOPE / CHEMS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or another fusogenic lipid, or any combination thereof. In another embodiment, the second agent may comprise a histone deacetylase inhibitor (HDACi), such as a histone deacetylase 6 inhibitor (HDAC6i). In another embodiment, the second agent may comprise SAHA (vorinostat).

[0024] In another embodiment of any of the methods described herein, exposing the MSCs to the transfection mixture may include complexing the nucleic acid construct with a cationic polymer to provide a transfection mixture comprising the complexed nucleic acid construct, and adding the transfection mixture to the MSCs. In another embodiment, exposing the MSCs to the transfection mixture may include adding the transfection mixture to the MSCs, and incubating the MSCs with the transfection mixture. In another embodiment, exposing the MSCs to the first and second agents may include replacing the transfection mixture with cell culture medium supplemented with the first and second agents. In another embodiment, exposing the MSCs to the transfection mixture may include removing the culture medium from the MSCs, and replacing the culture medium with the transfection mixture. In another embodiment, exposing the MSCs to the transfection mixture may include incubating the MSCs with the transfection mixture under gentle centrifugation. In another embodiment, the gentle centrifugation may include about 200 g for about 5 minutes.

[0025] In another embodiment of any of the methods described herein, the cell culture medium may comprise complete medium. In another embodiment, the MSCs may be at about 60% confluency and may be seeded about 24 hours prior to exposure to the transfection mixture. In another embodiment, the transfection mixture may comprise the complexed nucleic acid in serum-free DMEM or fresh culture medium.

[0026] In another embodiment of any of the methods described herein, the amount of nucleic acid construct in the transfection mixture to which the MSCs are exposed is greater than or equal to 100 μg / ml of nucleic acid construct per ... 2 In another embodiment, the amount of nucleic acid construct in the transfection mixture to which the MSCs are exposed may be between about 200 ng and about 500 ng per 1.9 cm of surface area. 2In another embodiment, the amount of nucleic acid construct in the transfection mixture to which the MSCs are exposed may be between about 250 ng and about 400 ng per 1.9 cm of surface area. 2 The ratio of cationic polymer to nucleic acid construct may be between about 300ng to about 350ng per 1μg of nucleic acid construct in the transfection mixture. In another embodiment, the ratio of cationic polymer to nucleic acid construct may be between about 1μg to about 30μg of cationic polymer per 1μg of nucleic acid construct in the transfection mixture. In another embodiment, the transfected MSCs may each be transfected with an average of at least about 1000 copies, at least about 2000 copies, at least about 3000 copies, at least about 4000 copies, at least about 5000 copies, at least about 6000 copies, at least about 7000 copies, at least about 8000 copies, at least about 9000 copies, or at least about 10000 copies of the nucleic acid construct. In another embodiment, the nucleic acid construct may comprise a CpG-free expression plasmid or CpG-free expression construct, a scaffold / matrix attachment region (S / MAR), an episomal vector, or an EBNA-1-containing construct.

[0027] In another embodiment of any of the methods described herein, the one or more functional genes may include a suicide gene. In another embodiment, the one or more functional genes may include cytosine deaminase (CDy) and / or thymidine kinase (TK). In another embodiment, the one or more functional genes may include uracil phosphoribosyltransferase (UPRT). In another embodiment, the one or more functional genes may include both CDy and UPRT. In another embodiment, CDy and UPRT may be expressed as a fusion construct. In another embodiment, the one or more functional genes may include a fluorescent protein. In another embodiment, the fluorescent protein may include green fluorescent protein (GFP). In another embodiment, the one or more functional genes may include CDy, UPRT, and GFP. In another embodiment, CDy, UPRT, and GFP may be expressed as a fusion construct. In another embodiment, the one or more functional genes may include herpes simplex virus type 1 thymidine kinase (HSV-TK) or another thymidine kinase. In another embodiment, the one or more functional genes may include one or more cancer therapeutic genes or one or more functional genes not related to cancer therapy. In another embodiment, the one or more functional genes may be transiently expressed in the transfected MSCs. In another embodiment, the MSCs may transiently express the one or more functional genes for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, or at least about 17 days following transfection. In another embodiment, the one or more functional genes may include a fluorescent protein, and the method may further include isolating, selecting, or purifying the transfected MSCs using cell sorting or FACS.

[0028] In another embodiment of any of the methods described herein, the pluripotent phenotype of the transfected MSCs may be substantially unchanged by transfection. For example, and without wishing to be limiting, the pluripotent phenotype may include differentiation potential such that the engineered cells are capable of differentiation into osteogenic, adipogenic, and / or chondrogenic lineages comparable to native MSCs. In another embodiment, the pluripotent phenotype may include tumor-tropic and / or cancer-tropic properties of MSCs. In another embodiment, the pluripotent phenotype may include an immunophenotype in which expression of CD surface markers is substantially unchanged after transfection. In another embodiment, the transfected MSCs may be undifferentiated.

[0029] In another embodiment of any of the methods described herein, the transfected MSCs may be plastic adherent, may express CD105, CD73 and CD90 (>95%), may lack expression of CD45, CD34, CD14 and HLA-DR surface molecules (<2%), may be capable of differentiating in vitro into osteoblasts, adipocytes and chondroblasts, and meet the immunophenotypic criteria defined by the International Society for Cellular Therapy (ISCT).

[0030] In another embodiment of any of the methods described herein, 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%, or at least about 95% of the MSCs are transfected with a nucleic acid construct and may express one or more functional genes. In another embodiment, the cell viability of the transfected MSCs may be at least about 70%, at least about 75%, at least about 80%, or at least about 85%.

[0031] In another embodiment of any of the methods described herein, the method may not include a viral-based transfection agent material.

[0032] In another embodiment of any of the methods described herein, the MSCs may be derived from umbilical cord blood, neonatal birth-related tissue, Wharton's jelly, umbilical cord, umbilical cord lining, placenta, or another MSC cell source. In another embodiment, the MSCs may be adipose tissue-derived MSCs (AT-MSCs), bone marrow-derived MSCs (BM-MSCs), or umbilical cord-derived MSCs (UC-MSCs). In another embodiment, the MSCs may be derived from humans, dogs, cats, horses, or another species.

[0033] In another embodiment of any of the methods described herein, the resulting MSCs may be susceptible to treatment with 5-fluorocytosine (5FC) or ganciclovir (GCV) or both. In another embodiment, the resulting MSCs may a) convert 5FC to 5-fluorouridine (5FU), 5-fluorouridine monophosphate (FUMP) or both, b) convert ganciclovir to ganciclovir monophosphate, or c) a combination of a) and b).

[0034] In another embodiment of any of the methods described herein, the method may include culturing the MSCs in a growth medium, e.g., fresh growth medium, prior to exposing the MSCs to the transfection mixture. In another embodiment, exposing the MSCs to the transfection mixture may include adding the transfection mixture to the MSCs without removing the growth medium from the MSCs, and centrifugation is not performed during the exposing and incubating steps. In another embodiment, exposing the MSCs to the first and second agents may include adding the first and second agents to the MSCs simultaneously with the transfection mixture, sequentially, or in combination with the transfection mixture. In another embodiment, the first and second agents may be added to the MSCs simultaneously with the addition of the transfection mixture to the MSCs, or the first and second agents may be mixed with the transfection mixture and added to the MSCs. In another embodiment, the first and second agents may be added to the MSCs shortly after adding the transfection mixture to the MSCs. In another embodiment, the transfection mixture may not be removed prior to adding the first and second agents to the MSCs. In another embodiment, the exposure period of the MSCs to the transfection mixture may overlap with the exposure period of the MSCs to the first and second agents. In another embodiment, the transfection mixture may not be removed prior to adding the first and second agents to the MSCs.

[0035] In another embodiment of any of the methods described herein, the method may further comprise the step of cryopreserving the transfected mesenchymal stem cells (MSCs) for storage. In another embodiment, the method may further comprise the step of thawing the cryopreserved transfected mesenchymal stem cells in preparation for their use.

[0036] In another embodiment of any of the methods described herein, the transfected MSCs may be defined by any of the embodiments of MSCs described herein. In another embodiment, one or more embodiments of the MSC or MSCs may be produced by any of the methods described herein.

[0037] In another embodiment, provided herein is a MSC or a plurality of MSCs produced by any of the methods described herein.

[0038] In another embodiment, provided herein is the use of any of the MSCs defined herein for treating cancer in a patient in need thereof. In certain embodiments, as non-limiting examples, the cancer may include lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer. In another embodiment, the MSCs may be for use in combination with 5FC, 5FU, GCV, or any combination thereof. In another embodiment, the MSCs may be for use in the manufacture of a medicament for the treatment of cancer. In another embodiment, the MSCs may be for use in combination with 5FC, 5FU, GCV, or any combination thereof.

[0039] In another embodiment, provided herein is a method for treating cancer in a patient in need thereof, which may comprise administering any of the MSCs defined herein to an area proximate to cancer cells of the patient, wherein one or more functional genes in the MSCs may contribute to an anti-cancer effect on the cancer cells.

[0040] In certain embodiments of any of the methods for treating cancer described herein, the cancer may include, for example, lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer.

[0041] In another embodiment of any of the methods for treating cancer described herein, MSCs may be administered simultaneously, sequentially, or in combination with 5FC, 5FU, GCV, or any combination thereof. In another embodiment, the one or more functional genes may include cytosine deaminase (CDy), thymidine kinase (TK), or both. In another embodiment, the one or more functional genes may include uracil phosphoribosyltransferase (UPRT). In another embodiment, the one or more functional genes may include both CDy and UPRT. In another embodiment, CDy and UPRT may be expressed as a fusion construct in MSCs. In another embodiment, the MSCs may transiently express one or more functional genes for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, or at least about 17 days following transfection.

[0042] In another embodiment, any of the methods for treating cancer described herein may further comprise administering 5FC, 5FU, ganciclovir, or any combination thereof to a patient such that the MSCs are exposed to 5FC, 5FU, ganciclovir, or any combination thereof.

[0043] In another embodiment, any of the methods for treating cancer described herein may further comprise the step of producing MSCs by any of the methods defined in any embodiment described herein prior to the step of administering the MSCs.

[0044] In another embodiment, provided herein is a composition comprising the engineered MSCs of any embodiment described herein and at least one of a pharma- ceutically acceptable carrier, diluent, excipient, cell culture medium, or buffer.

[0045] In another embodiment, provided herein is a theranostic agent comprising any of the MSCs of any embodiment described herein.

[0046] In another embodiment, a kit is provided herein for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct that transiently expresses one or more functional genes. In one embodiment, the kit may include MSCs, a nucleic acid construct designed for the transient expression of one or more functional genes, cell culture medium, a cationic polymer, a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments, a second agent capable of stabilizing the microtubule network of MSCs, instructions for carrying out the method described in any embodiment herein, one or more of 5FC, GCV, and / or 5FU. In certain embodiments, the kit may include a cryopreservation buffer or agent, a thawing buffer or agent, or both. In certain non-limiting embodiments, a cryopreservation buffer or solution, such as cryostor10 (Biolife Solutions, USA), may be used. In a further exemplary embodiment, the thawed engineered MSCs may be stored in a hypothermic solution, such as Hypothermosol (Biolife Solutions, USA). As will be apparent to one of skill in the art, other examples may also be used.

[0047] In another embodiment of any of the kits herein, the MSCs may be derived from umbilical cord blood, neonatal birth-related tissue, Wharton's jelly, umbilical cord, umbilical cord lining, placenta, or another MSC cell source. In another embodiment, the MSCs may be adipose tissue-derived MSCs (AT-MSCs), bone marrow-derived MSCs (BM-MSCs), or umbilical cord-derived MSCs (UC-MSCs). In another embodiment, the MSCs may be derived from a human, dog, cat, horse, or another species. In another embodiment, the nucleic acid construct may comprise a CpG-free expression plasmid or another CpG-free expression construct, a scaffold / matrix attachment region (S / MAR), an episomal vector, or an EBNA-1-containing construct. In another embodiment, the cationic polymer may comprise linear or branched polyethyleneimine (PEI), poly(amidoamine) PAMAM, or another cationic polymer, or any combination thereof. In another embodiment, the cationic polymer may comprise linear polyethyleneimine (LPEI). In another embodiment, the first agent may include one or more of DOPC, DPPC, or another fusogenic lipid. In another embodiment, the first agent may include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / cholesteryl hemisuccinate (CHEMS) (DOPE / CHEMS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or another fusogenic lipid, or any combination thereof. In another embodiment, the second agent may include a histone deacetylase inhibitor (HDACi), such as a histone deacetylase 6 inhibitor (HDAC6i). In another embodiment, the second agent may include SAHA (vorinostat). In another embodiment, the one or more functional genes may include a suicide gene. In another embodiment, the one or more functional genes may include a cytosine deaminase (CDy) or a thymidine kinase (TK). In another embodiment, the one or more functional genes may include uracil phosphoribosyltransferase (UPRT). In another embodiment, the one or more functional genes may include both CDy and UPRT. In another embodiment, CDy and UPRT may be expressed as a fusion construct.In another embodiment, the one or more functional genes may include a fluorescent protein. In another embodiment, the fluorescent protein may include green fluorescent protein (GFP). In another embodiment, the one or more functional genes may include CDy, UPRT and GFP. In another embodiment, CDy, UPRT and GFP may be expressed as a fusion construct. In another embodiment, the one or more functional genes may include herpes simplex virus type 1 thymidine kinase (HSV-TK) or another thymidine kinase. In another embodiment, the one or more functional genes may include one or more cancer therapeutic genes or one or more functional genes not related to cancer therapy. In another embodiment, the cationic polymer may include a cationic polymer identified as having low cytotoxicity to MSCs. In another embodiment, the cationic polymer may have a size of about 5 kDa to about 200 kDa. In another embodiment, the ratio of cationic polymer to nucleic acid construct in one or more embodiments of the kit may be about 1 μg to about 30 μg of cationic polymer per μg of nucleic acid construct.

[0048] In another embodiment of any of the kits herein, the kit may be for preparing an MSC-based anti-cancer agent. In another embodiment of any of the kits herein, the kit may include instructions and / or equipment for carrying out any of the methods defined in any one of the embodiments described herein.

[0049] In another embodiment, provided herein is a kit for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct that transiently expresses one or more functional genes, which may include MSCs, a nucleic acid construct designed for the transient expression of one or more functional genes, cell culture medium, a cationic polymer, a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments, a second agent capable of stabilizing the microtubule network of MSCs, instructions for performing any of the methods described herein, one or more of 5FC, GCV, and / or 5FU. In certain embodiments, the kit may include a buffer or agent for cryopreservation, a buffer or agent for thawing, or both, as described above.

[0050] In another embodiment, provided herein is a method for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct expressing one or more functional genes, comprising the steps of culturing the MSCs in a growth medium, adding to the MSCs a transfection mixture comprising the nucleic acid construct complexed with a cationic polymer without removing the growth medium from the MSCs, adding to the MSCs a first agent capable of redirecting endocytosed nucleic acid from an intracellular acidic compartment and a second agent capable of stabilizing the microtubule network of the MSCs, and incubating the MSCs in contact with all of the transfection mixture, the first agent, and the second agent for an incubation period, wherein the first agent and the second agent are added to the MSCs simultaneously with the addition of the transfection mixture, sequentially to the addition of the transfection mixture, or combined with the transfection mixture, and the MSCs are not centrifuged between the addition of the transfection mixture and the end of the incubation period, thereby providing MSCs transfected with the nucleic acid construct.

[0051] In another embodiment of the method for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct expressing one or more functional genes described herein, the incubation period can be at least about 2 hours. In another embodiment, the incubation period can be 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, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours or more.

[0052] In another embodiment, provided herein is a mesenchymal stem cell (MSC) cell or a plurality of MSC cells produced by any of the methods described herein for transfecting MSCs with a nucleic acid construct expressing one or more functional genes. In another embodiment, provided herein is the use of any of the MSC cells or a plurality of MSC cells described herein for treating cancer in a patient in need thereof or for the manufacture of a medicament for the treatment of cancer.

[0053] Described herein is a method for treating cancer in a patient in need thereof.In a particular embodiment of such method, it may comprise administering any of the MSCs defined in one or more embodiments described herein to the area of ​​the patient near cancer cells, wherein one or more functional genes in the MSCs contribute to the anti-cancer effect on the cancer cells.

[0054] In certain embodiments, the cancer may include, for example, lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer.

[0055] In another embodiment, provided herein is a composition comprising any of the MSCs described herein and at least one of a pharma- ceutically acceptable carrier, diluent, excipient, cell culture medium, or buffer.In another embodiment, provided herein is a theranostic agent and / or kit comprising any of the MSCs of any embodiment described herein.

[0056] These and other features will become more apparent from the following description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0057] [Figure 1]Figure 1 shows the generation of CDy::UPRT_AT-MSCs by one embodiment of the LPEI-based transfection method. The enhancer allows high expression of CDy::UPRT in AT-MSCs. Figure 1A shows that AT-MSCs in 6-well culture vessels were transfected with 1 μg of CDy::UPRT::GFP pDNA complexed with LPEI (1 μg DNA vs. 5 μL LPEI). After centrifugation, the transfection mixture was replaced with fresh medium (with or without TrafEn). After 1 day, representative images were obtained and cells were trypsinized for FACS analysis. Results are shown as mean ± SD (n=4). Untransfected AT-MSCs served as negative control. Significant differences between transfection conditions were calculated using two-tailed Student's t-test. **P<0.01. Figure 1B shows that AT-MSCs (LOT00088) cultured in 24-well vessels were transfected with various amounts of CDy::UPRT expression plasmid using LPEI or Lipofectamine 3000, respectively, followed by centrifugation or the manufacturer's protocol. After 24 h of incubation, cells were fixed with 4% paraformaldehyde and stained for CDy (green) and nuclei (Hoechst stain, blue). Representative images are shown. Bars represent 400 μm. Figure 1C shows that AT-MSCs were transfected with LPEI / CDy::UPRT polyplexes in the presence of TrafEn. One or seven days after modification, cells were lysed for immunoblotting analysis with an antibody targeting CDy. Actin was used as an endogenous control for sample loading. [Diagram 2]2A-2C are bar graphs showing CDy::UPRT expression sensitizing modified AT-MSCs to 5FC and 5FU. FIG. 2A shows CDy::UPRT_AT-MSCs were treated with 5FC 150 μg / mL for the indicated times. Cell viability at each time point was measured by standard MTS assay. At different time points, samples without 5FC treatment served as control. FIG. 2B shows sensitivity to 5FU was compared between unmodified and modified AT-MSCs after 5 days of culture in the presence of 5FU 100 μg / mL. MTS assay was used to measure cell viability after treatment. The condition without 5FU treatment was taken as 100%. Results are shown as mean ± SD (n=4). Significant differences in cell viability between AT-MSCs and CDy::UPRT_AT-MSCs were calculated using two-tailed Student's t-test. **, p<0.005. [Diagram 3] It is shown that CDy::UPRT expression does not affect the standard immunophenotypic profile and differentiation potential. Figure 3A shows AT-MSCs and CDy::UPRT_AT-MSCs labeled with fluorophore-conjugated antibodies and analyzed by flow cytometry according to the manufacturer's instructions. Isotype antibodies were used as respective controls. The histograms show the integrated isotype profile (red), unmodified AT-MSCs (green) and CDy::UPRT_AT-MSCs (blue). Figure 3B shows both cell types cultured for 14 days in medium supplemented for osteogenic differentiation according to the manufacturer's recommendations. At the end of the incubation, cells were stained with Alizarin Red S. Calcium deposits stained with Alizarin Red S are one of the phenotypes suggestive of AT-MSC differentiation. Figure 3C shows unmodified AT-MSCs and CDy::UPRT-expressing AT-MSCs cultured in medium containing components for adipogenic differentiation. After 14 days, cells were stained with Oil Red O. This dye stains oil droplets found within the cells, indicating adipocyte differentiation. Images were acquired at 20× magnification. [Figure 4]It shows that CDy::UPRT expression does not affect the migration ability of AT-MSCs. Figure 4A shows that the migration properties of MSCs were evaluated using a cell invasion assay. First, 200000 or 400000 target cells were plated in 10% FBS-supplemented DMEM in 24-well containers. After 6 hours, the cell culture was washed once with 1x PBS and replaced with serum-free DMEM. CDy::UPRT_AT-MSCs (modified 1 day before the experiment) and non-modified AT-MSCs were loaded onto Matrigel-coated cell inserts. The inserts were transferred to the target cell cultures, respectively. After 24 hours, cell invasion was evaluated by taking fluorescent images of cells stained with Hoechst 33342 under a microscope. The number of migrating AT-MSCs was calculated. The graph shows the average value (n=3) of migrating cells per frame. HEK293T was used as a negative control. Significant differences between 200,000 and 400,000 target cells were calculated using a two-tailed Student's t-test. **, P<0.01. Figure 4B shows images of migrated CDy::UPRT_AT-MSCs stained with Hoechst 33342 taken at 10x magnification. Scale bar represents 400 μm. [Diagram 5]Figure 5 shows the selective cytotoxic anticancer effect of CDy::UPRT_AT-MSC / 5FC mediated against cancer cells in vitro. Figure 5A shows that CDy::UPRT_AT-MSC were co-cultured with U251-MG, MB-MDA231 or MKN1 in 2% FBS supplemented DMEM in the presence or absence of 150 μg / mL 5FC. Therapeutic cells and cancer cell lines were mixed at a ratio of 1 CDy::UPRT_AT-MSC to 5, 10, 50, 100 cancer cells. After 5 days, growth inhibition was assessed spectrophotometrically by standard MTS assay. Growth inhibition efficiency is defined as 100%-(sample / control x 100%). Condition without 5FC treatment served as control. Graph bars represent mean (n=4), +SD. Figure 5B shows bright field images of mixed cultures (1 MSC to 10 cancer cells) taken at the end of the experiment. Scale bar represents 400 μm. Figure 5C shows that the cytotoxic anticancer effect of CDy::UPRT_AT-MSC or AT-MSC against MB-MDA231 was evaluated by indirect coculture. Equal numbers of therapeutic cells and MB-MDA231 were seeded in transwells and 24-well plates, respectively. Cells were cocultured for 4 days in DMEM supplemented with 2% FBS and 5FC 100 μg / mL. Then, transwells were removed and the remaining cells on the culture plates were stained with Hoechst 3222. Fluorescence readouts were acquired with a microplate reader. The efficiency (%) of growth inhibition was defined as 100%-(condition with 5FC / condition without the respective 5FC × 100%). Relative fluorescence units collected from 9 areas of biological triplicates are shown as mean ± SEM. The graph represents the mean + SEM of the results collected from 9 areas of each well. Each image of the remaining cancer cells on the 24-well plate is shown. The scale bar represents 400 μm. [Figure 6]Figure 6 shows the various cytotoxic anticancer effects mediated by CDy::UPRT_AT-MSC / 5FC generated using different transfection methods. AT-MSC (250,000 cells) were transfected with 1 μg of CpG-free CDy::UPRT expression plasmid mediated by LPEI (with or without TrafEn) and Lipofectamine 3000. One day after transfection, CDy::UPRT_AT-MSC were co-cultured with U251-MG, MB-MDA231 or MKN1 in 2% FBS-supplemented DMEM in the presence or absence of 150 μg / mL 5FC. Therapeutic cells and cancer cell lines were mixed in the ratio of 1 CDy::UPRT_AT-MSC to 1 cancer cell (Figure 6A), 5 cancer cells (Figure 6B) or 10 cancer cells (Figure 6C). After 5 days, growth inhibition was evaluated spectrophotometrically by standard MTS assay. The condition without 5FC treatment served as a control. The efficiency of growth inhibition is defined as 100%-(sample / control x 100%). The bars represent the mean (n=4), ±SD. Significant differences between the condition with LPEI+TrafEn and the other methods were calculated using two-tailed Student's t-test. **, P<0.01. [Figure 7]Figure 7 shows that long-term expression allows sustainable anticancer efficiency of CDy::UPRT_AT-MSC. AT-MSC (250,000 cells) were transfected with 1 μg of CpG-free CDy::UPRT expression plasmid in the presence of TrafEn mediated by LPEI. After 1 day (Figure 7A) and 7 days (Figure 7B), modified AT-MSC were harvested and co-cultured with MKN1 and MKN28 cell lines in the presence or absence of 5FC 150 μg / mL at a ratio of 1 MSC to 5 or 10 cancer cells. Growth inhibition was assessed spectrophotometrically by standard MTS assay after 5 days of incubation. Condition without 5FC treatment served as control. Growth inhibition efficiency is defined as 100%-(sample / control x 100%). Bars represent mean values ​​(n=4), ±SD. FIG. 7C shows that cells were lysed for immunoblotting analysis with an antibody targeting CDy 1 and 7 days after modification. Actin was used as an endogenous control for sample loading. Cell lysates of AT-MSCs were collected 1 and 7 days after transfection. Expression of CDy::UPRT was obtained using Western blot analysis. In parallel experiments, modified AT-MSCs were collected 1 (A) or 7 days (B) after transfection. [Figure 8]We show that TrafEn enabled efficient LPEI-based transfection in AT-MSCs. Figure 8A shows that LPEI / pCMV-GFP polyplexes or Lipofectamine 3000 / pCMV-GFP lipoplexes were prepared with various amounts of pDNA. AT-MSCs were transfected with LPEI (1 μg pDNA vs. 10 μL LPEI) or Lipofectamine 3000 according to the centrifugation protocol or manufacturer's instructions, respectively. The fluorescence intensity (RFU) of GFP expression was measured spectrophotometrically (Ex475 / Em509) in nine areas of each biological replicate (n=3). The graph represents the mean value + SEM of RFU. A decrease in cell number was observed with increasing amounts of DNA. Figure 8B shows that AT-MSCs were transfected with LPEI complexed with 200 ng pCMV-GFP in the presence or absence of TrafEn. After 24 hours, cells were trypsinized, pelleted, and resuspended in 1x PBS for flow cytometry analysis. Transfection efficiency was calculated as the percentage of GFP-positive cells normalized to the total cell number quantified by FACS. Bar graphs represent mean ± SD, n=3. Brightfield and fluorescent images were acquired. Representative images are shown. [Figure 9] High transfection efficiency in AT-MSCs isolated from different donors. AT-MSCs were isolated from female donors (LOT00061, Roosterbio) aged 31-45 years. LPEI / pCMV-GFP polyplexes were prepared with various amounts of pDNA at a ratio of 1 μg pDNA to 5 μL LPEI. After 1 day (24 h), representative images were acquired, and then cells were trypsinized, pelleted, and resuspended in 1x PBS for flow cytometry analysis. Transfection efficiency was calculated as the percentage of GFP-positive cells, normalized to the total cell number quantified by FACS. Bar graphs represent the mean ± SD, n=3. Fluorescence images were acquired. Representative images are shown. [Figure 10]Long-term expression of CDy::UPRT::GFP in AT-MSCs. AT-MSCs were transfected with 1.25 μg PEI polyplexes of fused CDy::UPRT::GFP expressing pDNA in the presence of TrafEn. Fluorescent and bright field images were acquired 1, 2, 3, 5, and 8 days after transfection. The fluorescence intensity (RFU) of GFP expression was measured spectrophotometrically (Ex475 / Em509) in 9 areas of the cell culture. The graph represents the mean RFU value + SD of two biological replicates. Significant differences between GFP expression at different days after transfection were calculated using a two-tailed Student's t-test. **P<0.01. [Figure 11] Figure 1 shows adipogenic differentiation of CDy::UPRT::GFP-expressing AT-MSCs. AT-MSCs were transfected with 1.25 μg of fusion CDy::UPRT::GFP-expressing pDNA PEI polyplexes in the presence of TrafEn. 24 hours after transfection, the medium was replaced with adipogenic differentiation medium. After 14 days, cells were stained with Oil Red O. The modified AT-MSCs, as indicated by GFP expression, showed obvious oil droplets, suggesting that the multipotency of AT-MSCs remained unchanged after transfection. [Figure 12]The results show comparable anti-cancer efficiencies of CDy::UPRT_AT-MSC / 5FC and 5FU. Anti-cancer effects were evaluated in U251-MG (Figure 12A), MDA-MB-231 (Figure 12B), and MKN1 (Figure 12C). The therapeutic (anti-cancer) effects of CDy::UPRT_AT-MSC in combination with 5FC were analyzed by co-culture of equal numbers of CDy::UPRT_AT-MSC with cancer cell lines (U251-MG, 2000 cells; MDA-MB-231, and MKN1, 5000 cells). After 1 day, the culture medium was replaced with DMEM supplemented with 2% FBS and various concentrations of 5FC (5, 10, 50, 100 μg / mL). Meanwhile, 4000 cells of U251-MG, 10000 cells of MDA-MB-231, and MKN1 were seeded 24 hours before 5FU treatment. Cell lines were treated with 5FU 5, 10, 50, 100 μg / mL in DMEM supplemented with 2% FBS. After 5 days of incubation, the cytotoxic effect was qualitatively evaluated by a standard MTS assay. Conditions without 5FC and 5FU treatment, set as 100%, served as negative controls. Graphs represent mean values ​​± SD, n=4. [Figure 13] Figure 13 shows the selective growth inhibition effect of CDy::UPRT_AT-MSC / 5FC on cancer cell lines. CDy::UPRT_AT-MSC / 5FC was co-cultured with HS738T (ATCC, CRL-7869), AGS, MKN28, HS746T, NUGC3 and MKN45 (kindly provided by Dr. Yong Wei Peng, National University Cancer Institute, Singapore). Figure 13A shows that the mixed culture was incubated in 2% FBS supplemented DMEM in the presence or absence of 5FC 150 μg / mL. Therapeutic cells and cancer cell lines were mixed at a ratio of 1 CDy::UPRT_AT-MSC to 10 cancer cells. After 5 days, growth inhibition was assessed spectrophotometrically by standard MTS assay. Condition without 5FC treatment served as control. Growth inhibition efficiency is defined as 100%-(sample / control x 100%). Graph bars represent mean values ​​(n=4), ±SD. Figure 13B shows bright field images of mixed cultures taken at the end of the experiment. Scale bar represents 400 μm. [Figure 14]Comparable transfection and anti-cancer efficiencies in stem cells from different origins are shown. Adipose tissue-derived MSCs (AT, Roosterbio), bone marrow-derived MSCs (BM, Roosterbio), and UC-derived MSCs (umbilical cord, ATCC) were transfected in the presence of TrafEn by a centrifugation protocol. 24 hours after transfection, cells were trypsinized and harvested for Western blot analysis (Figure 14A). Cells were lysed for immunoblotting analysis with antibodies targeting CDy and actin. Figure 14B shows that in the same experiment, cells were harvested for co-culture studies with various cancer cell lines at a ratio of 1 MSC to 50 cancer cells. Cells were co-cultured for 5 days in medium containing 100 μg / mL 5FC. At the end of the incubation, the number of remaining cells was assessed spectrophotometrically by measuring the RFU of cells stained with Hoechst 33342 at wavelengths Ex340 / Em488. Conditions with unmodified MSCs served as controls. Percentage of growth inhibition was calculated accordingly. Graphs represent data collected from quadruplicates, mean values ​​± SEM. [Figure 15] Comparable transfection and anticancer efficiencies in various stem cells modified to express HSV-TK are shown. AT-MSC, BM-MSC and UC-MSC were transfected in the presence of TrafEn by centrifugation protocol. 1 μg of pSELECT-zeo-HSV1tk (InvivoGen) was used to transfect 250,000 cells of MSC. 24 hours after transfection, MSC were harvested for co-culture studies with various cancer cell lines at a ratio of 1 MSC to 50 cancer cells. Cells were co-cultured for 5 days in medium containing 100 μg / mL of the prodrug ganciclovir (InvivoGen). At the end of the incubation, the number of remaining cells was assessed spectrophotometrically by measuring the RFU of cells stained with Hoechst 33342 at wavelengths Ex340 / Em488. Conditions with unmodified MSC serve as controls. The percentage of growth inhibition was calculated accordingly. Graphs represent data collected from quadruplicates, mean values ​​± SEM. [Figure 16]Figure 16 shows the time course of CDy::UPRT expression reduction by expression vectors containing CpG islands. AT-MSCs (250,000 cells) were transfected with 1 μg pSELECT-zeo-FcyFur (InvivoGen) in the presence of TrafEn by centrifugation protocol. One, three and seven days after transfection, cells were harvested for Western blot analysis (Figure 16A) and co-culture experiments (Figure 16B). For co-culture experiments, CDy::UPRT modified AT-MSCs were cultured with U251-MG and MDA-MB-231 cells in a ratio of 1 MSC to 1, 5 or 10 cancer cells in DMEM supplemented with 2% FBS and 5FC 100 μg / mL. After 5 days, growth inhibition was assessed spectrophotometrically by standard MTS assay. Conditions without 5FC treatment serve as controls. Percentage of growth inhibition was calculated accordingly. Graphs represent data collected from quadruplicates, mean + SEM. [Figure 17] 1 shows an illustration of an exemplary embodiment of a protocol for MSC transfection. [Figure 18] Figure 1 shows cell viability and transfection efficiency with various amounts of DNA. AT-MSCs were transfected without centrifugation. Genetic modification efficiency and cell viability were measured by flow cytometry analysis. [Figure 19] Figure 1 shows long-term expression of CDy::UPRT in AT-MSCs transfected by the non-centrifugation protocol. Genetic modification efficiency was measured by flow cytometry analysis. [Figure 20] The compatibility of the polymer with different types of MSCs, namely UC-MSCs (FIG. 20A) and BM-MSCs (FIG. 20B), is shown. MSCs were incubated with the transfection mixture for 24 hours without centrifugation. [Figure 21] Figure 1 shows the decrease in cell viability with increasing amounts of DNA and polymer. AT-MSCs were transfected with various polymers without centrifugation. The concentration of linear PEI (<200 kDa) is 1 μg / μL. [Figure 22]Figure 1 shows the decrease in cell viability with increasing amounts of DNA and polymer. UC-MSCs were transfected with various polymers without centrifugation. The concentration of linear PEI (<5 kDa) is 10 μg / μL. [Figure 23] High expression levels per cell by TrafEn method. U2OS cells (Fig. 23A) or AT-MSC cells (Fig. 23B) were infected with lentivirus and incubated for 5 days (left panel of Fig. 23B). In the same experimental set, cells from separate cultures were transfected with PEI in the presence of TrafEn on day 4 (right panel of Fig. 23B). Fluorescence images of infected and transfected cells were taken on day 5. Genetic modification efficiency of lentivirus and TrafEn method for AT-MSC was further measured by flow cytometry analysis. A larger number of cells expressed high levels of GFP in AT-MSC transfected with TrafEn method (right panel of Fig. 23B). [Figure 24] A graph of the number of transfected MSCs obtained in cell vessels of different sizes (FIG. 24A) and the good correlation between MSC number and vessel size (FIG. 24B) are shown. [Diagram 25] 1 shows an illustration of the development of an integrated process for producing large numbers of transfected MSCs using a non-viral transfection method. Factors to consider and / or optimize to achieve the goal are shown. Due to cell diversity, a panel of TrafEn compatible polymers (e.g., PEI) may be screened to obtain an optimal formulation for high transfection efficiency, low cytotoxicity, long-term expression, and scalability of production. [Figure 26] Figure 1 shows cell viability and transfection efficiency at various DNA amounts. Genetic modification efficiency and cell viability were measured by flow cytometry analysis. [Figure 27]It shows that CDy::UPRT expression does not affect the standard immunophenotypic profile and differentiation potential. Figure 27A shows that CDy::UPRT_AT-MSCs were labeled with fluorophore-conjugated antibodies and analyzed by flow cytometry. Isotype is used as a negative control. Figure 27B shows that both cell types were cultured for 14 and 21 days in media supplemented for adipogenic and osteogenic differentiation, respectively. At the end of the incubation, cells were stained with Oil Red O (adipogenesis) or Alizarin Red S (osteogenesis). Oil Red O stains oil droplets found within the cells, suggesting adipogenic differentiation. Calcium deposits stained with Alizarin Red S are one of the phenotypes suggesting AT-MSC differentiation. [Figure 28] See Figure 8A above. Adherent cells were then trypsinized and stained with propidium iodide (PI) and Hoechst 33342 (H33342). Cell viability and total adherent cell number were measured with an NC-3000 cell counter according to the manufacturer's protocol. Untransfected population serves as control. Cell viability (%) represents the percentage of PI negative cells. The percentage of total adherent cell number was calculated relative to the control, which was set as 100%. Data are presented as the mean + SD of experiments performed in biological triplicates. Significant differences between control and transfected samples were calculated using Student's two-tailed t-test. **, p<0.05. [Figure 29] Related to the results shown in Figure 8B above. In the same experiment, the total number of attached cells (left) and cell viability (right) for each condition were measured with a NC-3000 cell counter. Untransfected populations serve as controls. Results are shown as mean ± SD, n=3. [Diagram 30] See Figure 1 above. In the same experiment, the total cell number and cell viability of each condition were measured with a NC-3000 cell counter. The percentage of total adherent cells in the transfected population relative to the control (untransfected) was calculated. Data represent the mean ± SD, n=3. [Diagram 31]Figure 1 shows comparable anti-cancer efficiency of MSCs modified with CD::UPRT and CD::UPRT::GFP. MSCs (200,000 cells) were transfected with 1 μg of CDy::UPRT or CD::UPRT::GFP in the presence of enhancer. One day after transfection, U251-MG cells were co-cultured with CD::UPRT_MSCs in a ratio of 1:1, 1:5 or 1:10 (MSC:cancer cells) in 2% FBS-supplemented DMEM with or without 5FC 100 μg / mL. After 5 days, growth inhibition under treatment conditions was assessed spectrophotometrically by standard MTS assay. Condition without 5FC treatment serves as control set at 0%. Growth inhibition (%) was calculated relative to the control. Data collected from quadruplicates are expressed as mean + SD. [Diagram 32] Antitumor effect of CD::UPRT_AT-MSC in vivo in the presence of 5-fluorouracil (5-FU). To establish subcutaneous tumors, 5x106 temozolomide-resistant U-251MG cells were injected subcutaneously in the dorsal flank region. When tumors reached the target size, 1x106 CD::UPRT_AT-MSC or MSC were injected directly into the subcutaneous tumor. One day later, 5FC 500mg / kg / day was administered daily for 4 consecutive days. The size of the subcutaneous tumor was measured using a digital caliper on the 7th, 11th, and 15th days after MSC administration. The prodrug only group serves as the control group. Tumor volume (mm3) was calculated according to the standard formula V=(W(2)×L) / 2. (A) Box plot shows the distribution of tumor volumes measured from n=5 in each group. The tumor volumes of the treatment groups (CD::UPRT_AT-MSC / 5-FU) showed statistically significant differences (P<0.05) on days 7, 11, and 15. (B) At the end of the experiment, the mice were euthanized. The tumors were extracted and fixed in 4% PFA. Images show tumors extracted from each group (n=5). [Diagram 33]Comparison of expression duration and killing efficiency based on transfection efficiency. AD-MSCs in 24-well culture vessels were transfected with various DNA amounts (200ng-400ng) with or without TrafEn using PEI derivatives (polymer). Two days after transfection, cells were trypsinized for FACS analysis. Both (A) CDUPRTGFP+ (%) and (B) cell viability (%), PI- (%) are shown. Results are shown as mean ± SD (n=3). Untransfected AT-MSCs served as negative control. (C) CD:UPRT:GFP positive cells (%) after harvesting under various transfection conditions, analysis is performed using FACS. (D) Cell viability (%) of co-culture of U251-MG with MSCs transfected under different transfection conditions. Results are shown as mean ± SD (n=6). [Diagram 34] Phenotype of MSCs after transfection. (A) Expression of CD markers (CD90, CD74, CD105, CD14, CD20, CD34 and CD45) on native MSCs (left) and CD:UPRT:GFP MSCs (right). Isotype controls were used as negative controls for FACS analysis. (B) Representative images of Alizarin Red S staining for osteogenic differentiation (top) and Oil Red O staining for adipogenic differentiation (middle) of CD:UPRT:GFP MSCs are shown, as well as an overlay of GFP images and Oil Red O staining (bottom). White arrows point to Oil Red O staining of CD:UPRT:GFP expressing cells after 14 days of differentiation. (C) Fold change in the number of native and modified AD-MSCs that migrated out of fibroblasts and towards U251-MG. Significant differences between the two groups were calculated using unpaired two-tailed Student's t-test. ns indicates not significant, p-value >0.05. [Diagram 35]Figure 2 shows the cytotoxicity of CDUPRTGFP_MSC / 5-FC against TMZR glioma. Transfected AD-MSCs were co-cultured with glioma cell lines (A) U251-MG and U251-MGTMZR40, (B) U87-MG and U87-MGTMZR40, (C) HGCC cell lines, and (D) fibroblasts. Cell viability of the co-cultures was measured after 7 days of incubation with 100 μg / mL 5-FC at different MSC:cancer cells or fibroblasts ratios. Results are shown as mean ± SD (n=6). [Diagram 36] Figure 1 shows the in vivo cytotoxicity of CDUPRTGFP_MSC / 5-FC against U251-MGTMZR40. (A) Tumor volume was measured before and up to 15 days after treatment. (B) Tumor burden was measured after harvesting 15 days after treatment. (C) Percentage change in mouse body weight was measured before and up to 15 days after treatment. Results are shown as mean ± SEM (number of mice at least 6). Significant differences between tumor volume and body weight from native MSC and different numbers of CD:UPRT:GFP_MSC treatments were calculated using Student's unpaired two-tailed t-test. p-value < 0.05 is represented by *, p-value < 0.01 is represented by **, and p-value < 0.005 is represented by ***. ns indicates p-value > 0.05, therefore not significant. [Figure 37] Administration of CD::UPRT::GFP_MSC in combination with 5FC as a treatment for TMZ-resistant glioblastoma (U251-MGTMZR40) is shown. The treatment regimen consisted of intratumoral injection of 1x106 therapeutic or native cells (day 0). One day later, mice were intraperitoneally injected with 500mg 5FC / kg / day once daily for 4 days. Three days after the last dose of 5FC, mice were again injected with engineered stem cells, and this cycle was repeated for the duration of the experiment. After three cycles (50 days after tumor induction or 36 days after the first MSC injection), the experiment was terminated. (A) Measurement of tumor volume at the indicated days after injection of MSC. (B) Images show tumor size 36 days after the first MSC injection. (C) Mouse weight over the course of the experiment. [Figure 38]Treatment data for perianal cancer are shown. The route of administration was intratumoral injection of canine CD::UPRT::GFP_MSC. Latest update (January 2020): No reports of survival or recurrence. [Figure 39] Treatment data for oral melanoma are shown. The route of administration was intratumoral injection of canine CD::UPRT::GFP_MSCs. Latest update (January 2020): Survival. [Diagram 40] The data of thyroid cancer treatment are shown. The route of administration was intratumoral injection of canine CD::UPRT::GFP_MSCs. Latest update (June 2019): Survival. [Diagram 41] Treatment data for soft tissue cancer (cancer ulceration) are shown. Route of administration was intratumoral injection of canine CD::UPRT::GFP_MSC. Latest update (Nov 2018): alive, no recurrence reported. Ultrasound report on Nov 14, 2018: well-defined hypoechoic round mass with dimensions 4x3x2cm in the left anal region. No adhesion to surrounding or deep organs. No metastasis found, especially to sublumbar lymph nodes. Very few 1.5mm very small urinary stones in the bladder, very few in the prostatic urethra. Other organs normal. Complete remission to date. [Diagram 42] Treatment data for nasal tumors are shown. The route of administration was intratumoral injection of canine CD::UPRT::GFP_MSCs. Latest update (January 2020): Survival. [Diagram 43] Treatment data for gastrointestinal cancer. The route of administration was intravenous infusion of canine CD::UPRT::GFP_MSC. Latest update (July 2019): Survival. Ultrasound reports show that the initial proliferation has significantly decreased, despite the fact that there is a second proliferation. [Diagram 44] MSC types from different commercial / collaborative studies were modified with a vector containing the GFP transgene. Graph bars indicate the % of GFP+ population as measured by flow cytometry. [Diagram 45] FIG. 1 shows that MSCs of different origins were engineered to express CD::UPRT::GFP. [Diagram 46]Linearity of scale-up of AD-MSC and UC-MSC on flat-bottom surfaces. (A) Number of live transfected cells plotted against vessel surface area. (B) Representative images of GFP+ (%) from FACS analysis for both AD-MSC and UC-MSC. (C) Percentage of transfection in different culture vessels. [Figure 47] Figure 1 shows the results of testing different microcarriers in AD-MSCs: (A) Description of the microcarriers used (B) The number of viable cells grown on the different microcarriers is plotted against the different days. [Figure 48] Figure 1 shows the expansion of transfection on microcarriers. MSCs were seeded on microcarriers at 1.9 cm2 and transfected with different amounts of DNA plus enhancer. (A) Transfection efficiency, GFP+ (%) and PI- (%) were plotted, (B) representative images were taken at 4x magnification. [Figure 49] Different rates affecting microcarrier scale-up are shown. (A) Transfection efficiency (%) GFP+ (%) and cell viability PI- (%) are shown. Results are shown as mean ± SD (n=3). Untransfected AD-MSCs served as negative control. (B) Representative images of transfected cells were taken at 4x magnification. [Figure 50]Comparison of CD::UPRT::GFP expression and anti-cancer efficiency of AT-MSCs modified by lentivirus or TrafEn-mediated transfection methods. (A) Three days after infection, MSCs were subjected to puromycin 1 μg / mL selection for two weeks. After establishing MSCs stably expressing CD::UPRT::GFP, another set of experiments was set up to generate CD::UPRT::GFP_MSCs by TrafEn-mediated transfection. Two days after transfection, fluorescent images of modified MSCs were acquired. (B) Then, both cultures were harvested for (B) FACS analysis and (C, D) co-culture experiments. The bars in the graph represent the cancer killing efficiency obtained by MTS assay at different ratios of 1 MSC to 1, 5, 50, and 100 cancer cells. Significant differences in the tumor-killing effects of TrafEn-generated CD::UPRT::GFP_MSCs versus lentivirus-generated CD::UPRT::GFP_MSCs were evaluated by two-tailed Student's t-test. **, p-value < 0.005; *, p-value < 0.05. Bright-field images were taken at the end of the co-culture experiments. [Figure 51] 1 shows the results of compassionate use therapy for a 46-year-old patient with renal cell carcinoma. The patient was treated with intratumoral injection of CD::UPRT::GFP expressing MSCs as described herein. [Figure 52] A schematic of a typical centrifugation / spin-based transfection method (top) is shown, compared to an example of a non-centrifugation / spin transfection method (bottom). Data collected on cells treated according to such procedures is also provided (see Example 11). [Diagram 53] 1 shows a schematic of a workflow for cryopreserving modified MSCs (prepared using TrafEn) to allow for long-term storage. As shown, modified MSCs may be cryopreserved and stored. When needed, cells may be removed from storage and prepared for use by thawing in cryogenic liquid. [Figure 54]The results of cell viability (A), expression levels (B) and functional activity (C) of cryopreserved and then thawed modified MSCs are shown in Figure 53. As shown, the modified MSCs maintained high cell viability and expression levels after cryopreservation and storage in cryogenic solution for up to 72 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Described herein are methods for transfecting mesenchymal stem cells with a nucleic acid construct that expresses one or more functional genes. Also described herein are transfected mesenchymal stem cells and mesenchymal stem cell populations, uses thereof, methods of treating a disease or disorder, such as cancer, using such transfected stem cells, and kits and compositions relating thereto. It is understood that the embodiments and examples are provided for illustrative purposes directed to those skilled in the art and are not intended to be limiting in any way.

[0059] For many different therapeutic and non-therapeutic applications, stem cells modified to express a therapeutic gene or another gene of interest are desirable. One example is in the field of prodrug gene therapy, which aims to provide modified stem cells that express an exogenous enzyme that can convert an inactive prodrug into an active therapeutic form at the site where the modified stem cells are introduced into a subject or patient. Traditionally, in the field of prodrug gene therapy, virus-based gene modification approaches have been the preferred approach for modifying stem cells, such as MSCs, in preclinical and clinical studies, because non-viral approaches generally offered low transfection efficiency. Indeed, many preclinical and clinical studies have utilized viral vectors as gene delivery vehicles for stem cell modification. Although viruses can allow sustained expression of transgenes, cells infected with viruses typically have a low payload of transgenes per cell (<10 copies / cell). A higher copy number of transcription units is often desirable, because it can result in higher transgene expression and improve the payload of the cell vehicle in the delivery of therapeutic drugs. The production of clinical grade viruses can be challenging and often involves approval as well as the generation of master cell banks of stable producers, resulting in high costs in gene and cell therapy. Bottlenecks in the manufacture of viral carriers have adversely affected the development and commercialization of cell and gene therapies.

[0060] Although transient transfection has advantages in terms of high payload per cell, which may induce cellular senescence

[40] and / or reduce tumor tropism

[41] , avoids antibiotic selection (and weeks of processing time), and safety concerns related to virus-induced MSC transformation

[42] , the efficiency of non-viral transfection in the field has generally been low. Indeed, although non-viral methods have advantages over viral vectors in terms of ease of production and / or low cost and safety profile

[43] , the lack of widespread adoption in the field of non-viral MSC modification may be due to the low transfection efficiency (0–35%) frequently observed in the field [44, 45]. For example, due to the poor performance of certain chemical-based transfection methods (<5% efficiency)

[46] , human adipose tissue-derived MSCs (AT-MSCs) were engineered by retroviral transduction to express CD::UPRT [47, 48].

[0061] Viral-based genetic modification in such applications has inherent safety risks, clinical grade virus can be difficult to produce, and the number of gene copies that can be introduced per cell by viral methods is generally low (often <10 copies per cell). Furthermore, achieving genetic modification of stem cells, such as MSCs, with high transfection efficiency, either virally or non-virally, without causing undesirable changes to the phenotype (i.e., pluripotency, immunophenotype, tropism, etc.) of the resulting cells, is another challenge facing the field.

[0062] As described in detail herein, the inventors have developed methods of transfecting mesenchymal stem cells with nucleic acid constructs expressing one or more functional genes that are non-viral and may, in certain embodiments, provide high transfection efficiency, high copy number per cell, high cell viability, long-term transient expression, and / or a substantially unaltered pluripotent phenotype. In certain embodiments, such methods may be scalable and / or suitable for large-scale clinical production of modified mesenchymal stem cells. Also described in detail herein are transfected mesenchymal stem cells and mesenchymal stem cell populations, their uses, methods of treating a disease or disorder, such as cancer, using such transfected mesenchymal stem cells, and kits and compositions relating thereto.

[0063] Aspects of the present invention include various embodiments, including but not limited to the following exemplary embodiments.

[0064] Embodiment 1. Mesenchymal stem cells (MSCs) transfected with a nucleic acid construct expressing one or more functional genes, wherein the MSCs have a phenotype in which any one or more of the following phenotypic characteristics are substantially unchanged by transfection of the nucleic acid construct: pluripotency (e.g., differentiation potential), immunophenotype, and / or cancer tropism, and the MSCs do not contain any viral-based transfection agent material.

[0065] Embodiment 2. A plurality of mesenchymal stem cells (MSCs), at least about 60% of the MSCs being transfected with a nucleic acid construct expressing one or more functional genes, wherein the transfected MSCs have a phenotype in which any one or more of the following phenotypic characteristics are substantially unchanged by transfection of the nucleic acid construct: pluripotency (e.g., differentiation potential), immunophenotype, and / or cancer tropism; and the plurality of mesenchymal stem cells (MSCs) is free of viral-based transfection agent material.

[0066] Embodiment 3. A plurality of MSCs according to embodiment 2, wherein at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the MSCs are transfected with a nucleic acid construct and express one or more functional genes.

[0067] Embodiment 4. A plurality of MSCs according to embodiment 2 or 3, wherein the cell viability of the plurality of MSCs is at least about 70%, at least about 75%, at least about 80%, or at least about 85%.

[0068] Embodiment 5. The MSCs according to any one of embodiments 1 to 4, wherein the transfected MSCs are each transfected with an average of at least about 1000 copies, at least about 2000 copies, at least about 3000 copies, at least about 4000 copies, at least about 5000 copies, at least about 6000 copies, at least about 7000 copies, at least about 8000 copies, at least about 9000 copies, or at least about 10000 copies of the nucleic acid construct.

[0069] Embodiment 6. The MSC according to any one of embodiments 1 to 5, wherein one or more functional genes are transiently expressed in the transfected MSC cells.

[0070] Embodiment 7. The MSCs according to any one of embodiments 1 to 6, which are derived from umbilical cord blood, neonatal birth-related tissue, Wharton's jelly, umbilical cord, umbilical cord lining, placenta, or another MSC cell source.

[0071] Embodiment 8. The MSCs according to any one of embodiments 1 to 7, which are adipose tissue-derived MSCs (AT-MSCs), bone marrow-derived MSCs (BM-MSCs), or umbilical cord-derived MSCs (UC-MSCs).

[0072] Embodiment 9. The MSCs according to any one of embodiments 1 to 8, wherein the nucleic acid construct comprises a CpG-free expression plasmid or another CpG-free expression construct, a scaffold / matrix attachment region (S / MAR), an episomal vector, or an EBNA-1-containing construct.

[0073] Embodiment 10. The MSCs according to any one of embodiments 1 to 9, wherein the transfected MSCs transiently express one or more functional genes for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, or at least about 17 days following transfection.

[0074] Embodiment 11. MSCs according to any one of embodiments 1 to 10, wherein the transfected MSCs are transfected with a nucleic acid construct using a cationic polymer, a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments, and a second agent capable of stabilizing the microtubule network of the MSCs.

[0075] Embodiment 12. The MSCs according to embodiment 11, wherein the cationic polymer comprises linear or branched polyethyleneimine (PEI), poly(amidoamine) PAMAM, or another cationic polymer, or any combination thereof.

[0076] Embodiment 13. The MSCs of embodiment 12, wherein the cationic polymer comprises linear polyethyleneimine (LPEI).

[0077] Embodiment 14. The MSCs according to any one of embodiments 11 to 13, wherein the first agent comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / cholesteryl hemisuccinate (CHEMS) (DOPE / CHEMS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or another fusogenic lipid, or any combination thereof.

[0078] Embodiment 15. The MSCs according to any one of embodiments 11 to 14, wherein the second agent comprises a histone deacetylase inhibitor (HDACi), such as a histone deacetylase 6 inhibitor (HDAC6i).

[0079] Embodiment 16. The MSCs according to any one of embodiments 11 to 15, wherein the second agent comprises SAHA (vorinostat).

[0080] Embodiment 17. The MSCs according to any one of embodiments 1 to 16, wherein the one or more functional genes include a suicide gene.

[0081] Embodiment 18. The MSCs according to any one of embodiments 1 to 17, wherein the one or more functional genes include cytosine deaminase (CDy).

[0082] Embodiment 19. The MSCs according to any one of embodiments 1 to 18, wherein the one or more functional genes include uracil phosphoribosyltransferase (UPRT).

[0083] Embodiment 20. The MSCs according to any one of embodiments 1 to 19, wherein the one or more functional genes include both CDy and UPRT.

[0084] Embodiment 21. The MSCs according to embodiment 20, wherein CDy and UPRT are expressed as a fusion construct.

[0085] Embodiment 22. The MSC according to any one of embodiments 1 to 21, wherein the one or more functional genes include a fluorescent protein.

[0086] Embodiment 23. The MSCs according to embodiment 22, wherein the fluorescent protein comprises green fluorescent protein (GFP).

[0087] Embodiment 24. The MSCs according to embodiment 20, wherein the one or more functional genes include CDy, UPRT and GFP.

[0088] Embodiment 25. The MSCs according to embodiment 24, wherein CDy, UPRT and GFP are expressed as a fusion construct.

[0089] Embodiment 26. The MSCs according to any one of embodiments 1 to 25, wherein the one or more functional genes comprise herpes simplex virus type 1 thymidine kinase (HSV-TK) or another thymidine kinase.

[0090] Embodiment 27. The MSC according to any one of embodiments 1 to 26, wherein the phenotype comprises tumor-tropic and / or cancer-tropic properties of the MSC.

[0091] Embodiment 28. The MSCs according to any one of embodiments 1 to 27, which are sensitive to treatment with 5-fluorocytosine (5FC) or ganciclovir (GCV).

[0092] Embodiment 29. The MSCs according to any one of embodiments 1 to 28, which a) converts 5FC into 5-fluorouridine (5FU), 5-fluorouridine monophosphate (FUMP) or both, b) converts ganciclovir into ganciclovir monophosphate, or c) is a combination of a) and b).

[0093] Embodiment 30. The MSCs according to any one of embodiments 1 to 29, for use in the treatment of cancer, such as lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer.

[0094] Embodiment 31. MSCs for use according to embodiment 30, for use in combination with 5FC, 5FU, GCV, or any combination thereof.

[0095] Embodiment 32. The MSCs according to any one of embodiments 1 to 31, wherein the phenotype comprises an immunophenotype in which expression of CD4 surface markers is substantially unchanged after transfection.

[0096] Embodiment 33. The MSCs according to embodiment 32, wherein the transfected MSCs are plastic-adherent, express CD105, CD73 and CD90 (>95%), lack expression of CD45, CD34, CD14 and HLA-DR surface molecules (<2%), can differentiate in vitro into osteoblasts, adipocytes and chondroblasts, and meet the immunophenotypic criteria defined by the International Society for Cellular Therapy (ISCT).

[0097] Embodiment 34. The MSCs according to any one of embodiments 1 to 33, wherein the transfected MSCs are undifferentiated.

[0098] Embodiment 35. A method for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct expressing one or more functional genes, comprising: exposing the MSCs to a transfection mixture comprising a nucleic acid construct complexed with a cationic polymer; exposing the MSCs to a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments and a second agent capable of stabilizing the microtubule network of the MSCs; and incubating the MSCs; thereby providing MSCs transfected with the nucleic acid construct. A method comprising:

[0099] Embodiment 36 The method of embodiment 35, wherein the MSCs are not centrifuged during exposure to the transfection mixture, during exposure to the first agent and the second agent, during incubation, or any combination thereof.

[0100] Embodiment 37. The method of embodiment 35 or 36, wherein incubating the MSCs comprises gentle mixing without centrifugation.

[0101] Embodiment 38. The method of any one of embodiments 35 to 37, wherein incubating the MSCs comprises incubating the MSCs for at least about 2 hours.

[0102] Embodiment 39. The method of embodiment 38, wherein incubating the MSCs comprises incubating the MSCs for about 2 hours to about 48 hours, or about 3 hours to about 24 hours.

[0103] Embodiment 40. The method of embodiment 39, wherein incubating the MSCs comprises incubating the MSCs for about 4 hours to about 18 hours.

[0104] Embodiment 41. The method of any one of embodiments 35-40, wherein the cationic polymer comprises a cationic polymer identified as having low cytotoxicity to MSCs.

[0105] Embodiment 42. The method of any one of embodiments 35 to 41, wherein exposing the MSCs to a transfection mixture comprises complexing the nucleic acid construct with a cationic polymer to provide a transfection mixture comprising the complexed nucleic acid construct, and adding the transfection mixture to the MSCs.

[0106] Embodiment 43. The method of any one of embodiments 35 to 42, wherein exposing the MSCs to the transfection mixture comprises adding the transfection mixture to the MSCs and incubating the MSCs with the transfection mixture.

[0107] Embodiment 44. The method according to any one of embodiments 35 to 43, wherein exposing the MSCs to the first agent and the second agent comprises replacing the transfection mixture with cell culture medium supplemented with the first agent and the second agent.

[0108] Embodiment 45 The method of embodiment 44, wherein the cell culture medium comprises a complete medium.

[0109] Embodiment 46. The method of any one of embodiments 35-45, wherein the MSCs are about 60% confluent and are seeded about 24 hours prior to exposure to the transfection mixture.

[0110] Embodiment 47. The method of any one of embodiments 35 to 46, wherein the cationic polymer has a size of about 5 kDa to about 200 kDa.

[0111] Embodiment 48. The method of any one of embodiments 35 to 47, wherein the cationic polymer comprises linear or branched polyethyleneimine (PEI), poly(amidoamine) PAMAM, or another cationic polymer, or any combination thereof.

[0112] Embodiment 49. The method of any one of embodiments 35 to 48, wherein the cationic polymer comprises linear polyethyleneimine (LPEI).

[0113] Embodiment 50. The method of any one of embodiments 35 to 49, wherein the first agent comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / cholesteryl hemisuccinate (CHEMS) (DOPE / CHEMS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or another fusogenic lipid, or any combination thereof.

[0114] Embodiment 51. The method of any one of embodiments 35-50, wherein the second agent comprises a histone deacetylase inhibitor (HDACi), such as a histone deacetylase 6 inhibitor (HDAC6i).

[0115] Embodiment 52 The method of embodiment 51, wherein the second agent comprises SAHA (vorinostat).

[0116] Embodiment 53. The method of any one of embodiments 35 to 52, wherein the transfection mixture comprises the complexed nucleic acid construct in serum-free DMEM or in fresh culture medium.

[0117] Embodiment 54. The method of any one of embodiments 35 to 53, wherein exposing the MSCs to the transfection mixture comprises removing culture medium from the MSCs and replacing the culture medium with the transfection mixture.

[0118] Embodiment 55. The method of embodiment 35, wherein exposing the MSCs to the transfection mixture comprises incubating the MSCs with the transfection mixture under gentle centrifugation.

[0119] Embodiment 56. The method of embodiment 55, wherein the gentle centrifugation comprises about 200 g for about 5 minutes.

[0120]

[0046] Embodiment 57. The amount of nucleic acid construct in the transfection mixture to which the MSCs are exposed is greater than or equal to 1000 μg / ml of a surface area of ​​1.9 cm 2 The method of any one of embodiments 35-56, wherein the amount is between about 200 ng and about 500 ng per unit time.

[0121] Embodiment 58. The amount of nucleic acid construct in the transfection mixture to which the MSCs are exposed is greater than or equal to 100 μg / ml of a surface area of ​​1.9 cm 2 58. The method of embodiment 57, wherein the amount is between about 250 ng and about 400 ng per unit time.

[0122] Embodiment 59. The amount of nucleic acid construct in the transfection mixture to which the MSCs are exposed is greater than or equal to 100 μg / ml of a surface area of ​​1.9 cm 2 The method of embodiment 58, wherein the amount is between about 300 ng and about 350 ng per unit time.

[0123] Embodiment 60. The method of any one of embodiments 35 to 59, wherein the ratio of cationic polymer to nucleic acid construct is from about 1 μg to about 30 μg of cationic polymer per μg of nucleic acid construct in the transfection mixture.

[0124] Embodiment 61. The method of any one of embodiments 35 to 60, wherein the one or more functional genes comprises a suicide gene.

[0125] Embodiment 62. The method of any one of embodiments 35 to 61, wherein the one or more functional genes include cytosine deaminase (CDy) and / or thymidine kinase (TK).

[0126] Embodiment 63. The method of any one of embodiments 35 to 63, wherein the one or more functional genes comprises uracil phosphoribosyltransferase (UPRT).

[0127] Embodiment 64. The method of any one of embodiments 35 to 64, wherein the one or more functional genes include both CDy and UPRT.

[0128] Embodiment 65 The method of embodiment 64, wherein CDy and UPRT are expressed as a fusion construct.

[0129] Embodiment 66. The method of any one of embodiments 35 to 66, wherein the one or more functional genes comprises a fluorescent protein.

[0130] Embodiment 67. The method of embodiment 66, wherein the fluorescent protein comprises green fluorescent protein (GFP).

[0131] Embodiment 68. The method of embodiment 64, wherein the one or more functional genes include CDy, UPRT and GFP.

[0132] Embodiment 69 The method of embodiment 68, wherein CDy, UPRT and GFP are expressed as a fusion construct.

[0133] Embodiment 70. The method of any one of embodiments 35 to 69, wherein the one or more functional genes include herpes simplex virus type 1 thymidine kinase (HSV-TK).

[0134] Embodiment 71. The method of any one of embodiments 35 to 70, wherein one or more functional genes are transiently expressed in the transfected MSC cells.

[0135] Embodiment 72. The method of any one of embodiments 35 to 71, wherein the transfected MSCs are each transfected with an average of at least about 1000 copies, at least about 2000 copies, at least about 3000 copies, at least about 4000 copies, at least about 5000 copies, at least about 6000 copies, at least about 7000 copies, at least about 8000 copies, at least about 9000 copies, or at least about 10000 copies of the nucleic acid construct.

[0136] Embodiment 73. The method of any one of embodiments 35 to 72, wherein the phenotype of the transfected MSCs, including any one or more of the pluripotency, immunophenotype, and / or cancer tropism phenotypic characteristics, is substantially unchanged by transfection.

[0137] Embodiment 74. The method of embodiment 73, wherein the phenotype comprises tumor-tropic and / or cancer-tropic properties of MSCs.

[0138] Embodiment 75. The method of embodiment 73 or 74, wherein the phenotype comprises an immunophenotype in which expression of CD40 surface markers is substantially unchanged after transfection.

[0139] Embodiment 76. The method of embodiment 75, wherein the transfected MSCs are plastic-adherent, express CD105, CD73 and CD90 (>95%), lack expression of CD45, CD34, CD14 and HLA-DR surface molecules (<2%), can differentiate in vitro into osteoblasts, adipocytes and chondroblasts, and meet the immunophenotypic criteria defined by the International Society for Cellular Therapy (ISCT).

[0140] Embodiment 77. The method of any one of embodiments 35 to 76, wherein 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%, or at least about 95% of the MSCs are transfected with a nucleic acid construct and express one or more functional genes.

[0141] Embodiment 78. The method of any one of embodiments 35 to 77, wherein the cell viability of the transfected MSCs is at least about 70%, at least about 75%, at least about 80%, or at least about 85%.

[0142] Embodiment 79. The method of any one of embodiments 35 to 78, wherein the transfected MSCs are undifferentiated.

[0143] Embodiment 80. The method of any one of embodiments 35 to 79, which does not include viral-based transfection agent materials.

[0144] Embodiment 81. The method of any one of embodiments 35-80, wherein the MSCs are derived from umbilical cord blood, neonatal birth-related tissue, Wharton's jelly, umbilical cord, umbilical cord lining, placenta, or another source of MSC cells.

[0145] Embodiment 82. The method of any one of embodiments 35 to 81, wherein the MSCs are adipose tissue-derived MSCs (AT-MSCs), bone marrow-derived MSCs (BM-MSCs), or umbilical cord-derived MSCs (UC-MSCs).

[0146] Embodiment 83. The method of any one of embodiments 35 to 82, wherein the nucleic acid construct comprises a CpG-free expression plasmid or another CpG-free expression construct, a scaffold / matrix attachment region (S / MAR), an episomal vector, or an EBNA-1-containing construct.

[0147] Embodiment 84. The method of any one of embodiments 35 to 83, wherein the MSCs transiently express one or more functional genes for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, or at least about 17 days following transfection.

[0148] Embodiment 85. The method of any one of embodiments 35 to 84, wherein the obtained MSCs are sensitive to treatment with 5-fluorocytosine (5FC) or ganciclovir (GCV) or both.

[0149] Embodiment 86. The method of any one of embodiments 35 to 84, wherein the obtained MSCs a) convert 5FC to 5-fluorouracil (5FU), 5-fluorouridine monophosphate (FUMP) or both, b) convert ganciclovir to ganciclovir monophosphate, or c) a combination of a) and b).

[0150] Embodiment 87. The method of any one of embodiments 35 to 86, wherein the one or more functional genes comprise a fluorescent protein, and the method further comprises isolating, selecting or purifying the transfected MSCs using cell sorting or FACS.

[0151] Embodiment 88. The method of any one of embodiments 35 to 87, wherein the transfected MSCs are MSCs as defined in any one of embodiments 1 to 34.

[0152] Embodiment 89. An MSC or a plurality of MSCs produced by the method according to any one of embodiments 35 to 88.

[0153] Embodiment 90. Use of MSCs as defined in any one of embodiments 1 to 34 or 89 for treating cancer, such as lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer, in a patient in need thereof.

[0154] Embodiment 91. The use of embodiment 90, wherein the MSCs are for use in combination with 5FC, 5FU, GCV, or any combination thereof.

[0155] Embodiment 92. Use of MSCs as defined in any one of embodiments 1 to 34 or 89 in the manufacture of a medicament for the treatment of cancer, such as lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer.

[0156] Embodiment 93. The use of embodiment 92, wherein the MSCs are for use in combination with 5FC, 5FU, GCV, or any combination thereof.

[0157] Embodiment 94. A method for treating cancer, e.g., lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer, in a patient in need thereof, comprising: 89. The method of claim 1, further comprising administering MSCs as defined in any one of embodiments 1 to 34 or 89 to a patient in the vicinity of the cancer cells, However, one or more functional genes in the MSCs contribute to an anti-cancer effect on the cancer cells.

[0158] Embodiment 95 The method of embodiment 94, wherein the MSCs are administered simultaneously, sequentially, or in combination with 5FC, 5FU, GCV, or any combination thereof.

[0159] Embodiment 96 The method of embodiment 94 or 95, wherein the one or more functional genes include cytosine deaminase (CDy), thymidine kinase (TK), or both.

[0160] Embodiment 97. The method of any one of embodiments 94 to 96, wherein the one or more functional genes comprises uracil phosphoribosyltransferase (UPRT).

[0161] Embodiment 98. The method of any one of embodiments 94 to 97, wherein the one or more functional genes include both CDy and UPRT.

[0162] Embodiment 99. The method of embodiment 98, wherein CDy and UPRT are expressed as a fusion construct in MSCs.

[0163] Embodiment 100. The method of any one of embodiments 94 to 99, wherein the MSCs transiently express the one or more functional genes for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, or at least about 17 days following transfection.

[0164] Embodiment 101. The method of any one of embodiments 94 to 100, further comprising administering 5FC, 5FU, ganciclovir, or any combination thereof to the patient such that the MSCs are exposed to 5FC, 5FU, ganciclovir, or any combination thereof.

[0165] Embodiment 102. The method according to any one of embodiments 94 to 101, further comprising, prior to the step of administering the MSCs, a step of producing the MSCs by a method defined in any one of embodiments 35 to 88.

[0166] Embodiment 103. A composition comprising the MSCs of any one of embodiments 1 to 34 or 89, and at least one of a pharma- ceutically acceptable carrier, diluent, excipient, cell culture medium, or buffer.

[0167] Embodiment 104. A theranostic agent comprising MSCs according to any one of embodiments 1 to 34 or 89.

[0168] Embodiment 105. A kit for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct that transiently expresses one or more functional genes, comprising: M.S.C. Nucleic acid constructs designed for the transient expression of one or more functional genes; Cell culture medium, Cationic polymers, a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments; A second agent capable of stabilizing the microtubule network of MSCs; Instructions for carrying out the method defined in any one of embodiments 35 to 88. 5FC, GCV and / or 5FU A kit comprising one or more of the following:

[0169] Embodiment 106. The kit of embodiment 105, wherein the MSCs are derived from umbilical cord blood, neonatal birth-related tissue, Wharton's jelly, umbilical cord, umbilical cord lining, placenta, or another source of MSC cells.

[0170] Embodiment 107. The kit of embodiment 105 or 106, wherein the MSCs are adipose tissue-derived MSCs (AT-MSCs), bone marrow-derived MSCs (BM-MSCs), or umbilical cord-derived MSCs (UC-MSCs).

[0171] Embodiment 108. The kit of embodiment 105 or 106, wherein the nucleic acid construct comprises a CpG-free expression plasmid or another CpG-free expression construct, a scaffold / matrix attachment region (S / MAR), an episomal vector, or an EBNA-1-containing construct.

[0172] Embodiment 109. A kit described in any one of embodiments 105 to 108, wherein the cationic polymer comprises linear or branched polyethyleneimine (PEI), poly(amidoamine) PAMAM, or another cationic polymer, or any combination thereof.

[0173] Embodiment 110. The kit of any one of embodiments 105 to 180, wherein the cationic polymer comprises linear polyethyleneimine (LPEI).

[0174] Embodiment 111. The kit of any one of embodiments 105 to 110, wherein the first agent comprises one or more of DOPC, DPPC, or another fusogenic lipid.

[0175] Embodiment 112. The kit of any one of embodiments 105 to 111, wherein the first agent comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / cholesteryl hemisuccinate (CHEMS) (DOPE / CHEMS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or another fusogenic lipid, or any combination thereof.

[0176] Embodiment 113. The kit of any one of embodiments 105 to 112, wherein the second agent comprises a histone deacetylase inhibitor (HDACi), such as a histone deacetylase 6 inhibitor (HDAC6i).

[0177] Embodiment 114 The kit of embodiment 113, wherein the second agent comprises SAHA (vorinostat).

[0178] Embodiment 115. A kit according to any one of embodiments 105 to 114, wherein the one or more functional genes comprises a suicide gene.

[0179] Embodiment 116. The kit according to any one of embodiments 105 to 115, wherein the one or more functional genes include cytosine deaminase (CDy) or thymidine kinase (TK).

[0180] Embodiment 117. The kit of any one of embodiments 105 to 116, wherein the one or more functional genes comprises uracil phosphoribosyltransferase (UPRT).

[0181] Embodiment 118. The kit according to any one of embodiments 105 to 116, wherein the one or more functional genes include both CDy and UPRT.

[0182] Embodiment 119 The kit of embodiment 118, wherein CDy and UPRT are expressed as a fusion construct.

[0183] Embodiment 120. A kit described in any one of embodiments 105 to 119, wherein the one or more functional genes include a fluorescent protein.

[0184] Embodiment 121. The kit of embodiment 120, wherein the fluorescent protein comprises green fluorescent protein (GFP).

[0185] Embodiment 122. The kit of embodiment 118, wherein the one or more functional genes include CDy, UPRT and GFP.

[0186] Embodiment 123 The kit of embodiment 122, wherein CDy, UPRT and GFP are expressed as a fusion construct.

[0187] Embodiment 124. The kit of any one of embodiments 105 to 123, wherein the one or more functional genes comprises herpes simplex virus type 1 thymidine kinase (HSV-TK).

[0188] Embodiment 125. The kit of any one of embodiments 105 to 124, wherein the cationic polymer comprises a cationic polymer identified as having low cytotoxicity to MSCs.

[0189] Embodiment 126. A kit according to any one of embodiments 105 to 125, wherein the cationic polymer has a size of about 5 kDa to about 200 kDa.

[0190] Embodiment 127. A kit according to any one of embodiments 105 to 126, wherein the ratio of cationic polymer to nucleic acid construct in the kit is from about 1 μg to about 30 μg of cationic polymer per 1 μg of nucleic acid construct.

[0191] Embodiment 128. A kit according to any one of embodiments 105 to 124, for preparing an MSC-based anticancer agent.

[0192] Embodiment 129. The kit of embodiment 128, further comprising instructions and / or equipment for carrying out the method defined in any one of embodiments 94 to 102.

[0193] Embodiment 130. The method of any one of embodiments 35-43, 46-53 or 57-88, comprising culturing the MSCs in a growth medium, such as fresh growth medium, prior to exposing the MSCs to the transfection mixture.

[0194] Embodiment 131. The method of embodiment 130, wherein exposing the MSCs to the transfection mixture comprises adding the transfection mixture to the MSCs without removing growth medium from the MSCs, and no centrifugation is performed between the exposing and incubating steps.

[0195] Embodiment 132. The method of embodiment 130 or 131, wherein exposing the MSCs to the first agent and the second agent comprises adding the first agent and the second agent to the MSCs simultaneously with, sequentially with, or in combination with a transfection mixture.

[0196] Embodiment 133. The method of embodiment 132, wherein the first agent and the second agent are added to the MSCs simultaneously with the addition of the transfection mixture to the MSCs, or the first agent and the second agent are mixed with the transfection mixture and added to the MSCs.

[0197] Embodiment 134. The method of embodiment 132, wherein the first agent and the second agent are added to the MSCs shortly after adding the transfection mixture to the MSCs.

[0198] Embodiment 135. The method of any one of embodiments 132 to 134, wherein the transfection mixture is not removed prior to adding the first agent and the second agent to the MSCs.

[0199] Embodiment 136 The method of any one of embodiments 130 to 135, wherein the period of exposure of the MSCs to the transfection mixture overlaps with the period of exposure of the MSCs to the first agent and the second agent.

[0200] Embodiment 137. The method of embodiment 136, wherein the transfection mixture is not removed prior to adding the first agent and the second agent to the MSCs.

[0201] Embodiment 138. A method for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct expressing one or more functional genes, comprising: Culturing the MSCs in a proliferation medium; adding a transfection mixture comprising a nucleic acid construct complexed with a cationic polymer to the MSCs without removing the growth medium from the MSCs; adding to the MSCs a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments and a second agent capable of stabilizing the microtubule network of the MSCs; and incubating the MSCs in contact with all of the transfection mixture, the first agent, and the second agent for an incubation period; wherein the first agent and the second agent are added to the MSCs simultaneously with, sequentially with, or in combination with the transfection mixture; By not centrifuging the MSCs between the addition of the transfection mixture and the end of the incubation period, A method comprising providing MSCs transfected with a nucleic acid construct.

[0202] Embodiment 139. The method of embodiment 138, wherein the incubation period is at least about 2 hours.

[0203] Embodiment 140. The method of embodiment 138, wherein the incubation period is 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, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours or more.

[0204] Embodiment 141. An MSC cell or a plurality of MSC cells produced by the method according to any one of embodiments 130 to 140.

[0205] Embodiment 142. Use of MSCs as defined in embodiment 141 for treating cancer, such as lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer, in a patient in need thereof.

[0206] Embodiment 143. Use of MSCs as defined in embodiment 141 in the manufacture of a medicament for the treatment of cancer, such as lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer.

[0207] Embodiment 144. A method for treating cancer, e.g., lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer, in a patient in need thereof, comprising: Administering MSCs as defined in embodiment 141 to a patient in the vicinity of the cancer cells, A method for treating cancer, wherein one or more functional genes in the MSCs contribute to an anti-cancer effect on the cancer cells.

[0208] Embodiment 145. A composition comprising the MSCs of embodiment 141 and at least one of a pharma- ceutically acceptable carrier, diluent, excipient, cell culture medium or buffer.

[0209] Embodiment 146. A theranostic agent comprising the MSCs described in embodiment 141.

[0210] Embodiment 147. A kit for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct that transiently expresses one or more functional genes, comprising: M.S.C. Nucleic acid constructs designed for the transient expression of one or more functional genes; Cell culture medium, Cationic polymers, a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments; A second agent capable of stabilizing the microtubule network of MSCs; Instructions for carrying out the method defined in any one of embodiments 130 to 140. 5FC, GCV and / or 5FU A kit comprising one or more of the following:

[0211] Transfected mesenchymal stem cells and methods and kits for their production In one embodiment, provided herein are mesenchymal stem cells (MSCs) transfected with a nucleic acid construct expressing one or more functional genes, the MSCs having a pluripotent phenotype that is substantially unaltered by transfection of the nucleic acid construct, wherein the MSCs are free of viral-based transfection agents.

[0212] In another embodiment, provided herein is a plurality of mesenchymal stem cells (MSCs), wherein at least about 60% of the MSCs are transfected with a nucleic acid construct that expresses one or more functional genes, and wherein the transfected MSCs have a pluripotent phenotype that is substantially unaltered by transfection of the nucleic acid construct, and wherein the MSCs are free of viral-based transfection agent material.

[0213] Of course, the MSCs may include any suitable MSCs, such as MSCs derived from umbilical cord blood, neonatal birth-related tissue, Wharton's jelly, umbilical cord, umbilical cord lining, placenta, or another MSC cell source. MSC sources may include human, canine, feline, equine, and others. In certain embodiments, the MSCs may be, for example, one or more of adipose tissue-derived MSCs (AT-MSCs), bone marrow-derived MSCs (BM-MSCs), or umbilical cord-derived MSCs (UC-MSCs).

[0214] In certain embodiments, where modified (transfected) MSCs expressing functional genes are used to treat patients, it is contemplated that the transfected MSCs include MSCs derived from the patient to be treated or from another source or patient. In certain embodiments, the MSCs may be selected for compatibility with the patient to be treated (e.g., to avoid allergic reactions), and the MSCs may or may not originally be derived from the patient to be treated. In certain embodiments, autologous or allogeneic MSCs may be used.

[0215] In certain embodiments, MSCs may be transfected with a nucleic acid construct. Of course, in certain embodiments, MSCs may be transiently transfected with a nucleic acid construct (i.e., the nucleic acid construct may be introduced into a cell site where the one or more functional genes it encodes may be expressed in the cell, but the nucleic acid construct is not integrated into the cell genome) or stably transfected with a nucleic acid construct (i.e., the nucleic acid construct may be integrated into a cell genome where the one or more functional genes it encodes may be expressed in the cell), with or without a selection step (e.g., antibiotic resistance, where such genes are included with the nucleic acid construct).

[0216] In certain embodiments, the MSCs may be transfected using the reagents and / or methods described in detail herein below.

[0217] Of course, the nucleic acid construct may include any suitable nucleic acid sequence suitable for a particular application and suitable for encoding one or more functional genes of interest. In certain embodiments, the nucleic acid construct may generally include any suitable plasmid, expression vector, or other expressible nucleic acid sequence that can result in the production of one or more functional genes / polypeptides encoded by the nucleic acid construct following introduction into a cell. In embodiments where long-term expression is desired, nucleic acid constructs designed for long-term expression and / or to prevent cellular silencing may be used.

[0218] In certain embodiments, the nucleic acid construct may be designed such that the coding region (i.e., the region encoding one or more functional genes of interest) uses codons optimized for expression in a particular organism of interest (e.g., the codons may be optimized for expression in human cells when using human MSCs). In certain embodiments, the nucleic acid construct may be an expressible nucleic acid (i.e., the nucleic acid construct may be designed such that when introduced into or present in any cell, it results in expression of a polypeptide). In certain embodiments, the nucleic acid construct may be DNA or RNA. In certain embodiments, the nucleic acid construct may be a plasmid with appropriate upstream and / or downstream sequences, an expression vector, an mRNA (which in certain embodiments may include sequences appropriate for translation in the cell of interest, e.g., start codons, polyA tails, RBS sequences, and / or others), minicircle DNA, a piece of single-stranded or double-stranded DNA, or others, such that when the nucleic acid construct is introduced into a cell, translation, or transcription and translation, of the nucleic acid construct may occur, providing a polypeptide of the one or more functional genes.

[0219] Suitable expression vector technologies for overexpressing or introducing into cells specific functional genes / polypeptides are known in the art (see, for example, Molecular Cloning: A Laboratory Manual (4th ed.), 2012, Cold Spring Harbor Laboratory Press). As known to those skilled in the art, nucleotide sequences for expressing specific polypeptides may code for or include features as described in "Genes VII", B. Lewin, Oxford University Press (2000) or "Molecular Cloning: A Laboratory Manual", Sambrook et al., Cold Spring Harbor Laboratory, 3rd ed. (2001). The nucleotide sequence encoding the specific functional gene / polypeptide of interest may be incorporated into a suitable vector, for example a commercially available vector. The vector may be individually constructed or modified using standard molecular biology techniques, for example as outlined in Sambrook et al. (Cold Spring Harbor Laboratory, 3rd ed. (2001)). Those skilled in the art will recognize that the vector may include nucleotide sequences encoding desired elements that may be operably linked to the nucleotide sequence encoding the functional gene / polypeptide. Nucleotide sequences encoding such desired elements may include a transcriptional promoter (e.g., a constitutive or inducible promoter), a transcriptional enhancer, a transcriptional terminator, and / or an origin of replication. Selection of an appropriate vector may depend on several factors, including, but not limited to, the size of the nucleic acid to be incorporated into the vector, the type of transcriptional and translational regulatory elements desired, the desired level of expression, the desired copy number, whether chromosomal integration is desired, the type of selection process (if any) desired, or the host cell or host range to be transformed.

[0220] It should be understood that the nucleic acid construct may encode one or more functional genes. The one or more functional genes may generally include any suitable functional genes encoding one or more functional RNAs, peptides, polypeptides, or proteins of interest. It should be understood that the one or more functional genes are typically selected to suit the particular application to which the modified mesenchymal stem cells are applied. As an example where the modified MSCs are used in a prodrug gene therapy approach, the one or more functional genes may include an enzyme capable of converting an inactive or low activity prodrug into an active form, such that exposure of the modified MSCs to the prodrug results in the formation of an active drug to treat surrounding cells and / or tissues. In certain embodiments, the one or more functional genes may include, for example, a suicide gene that may convert the prodrug into an active form that harms both the modified MSCs and surrounding diseased cells. In another embodiment, the one or more functional genes may include one or more cancer therapeutic genes, or one or more functional genes that are not related to cancer treatment and may, for example, have another therapeutic or non-therapeutic function.

[0221] Given the teachings herein, numerous examples of prodrug gene therapy systems, including both suitable prodrugs and corresponding functional / suicide genes, will be known to those of skill in the art. Some examples of functional genes that can be used, and their corresponding prodrugs (destinations), are provided in Table 1 below.

[0222] [Table 1]

[0223] Further examples of functional genes that can be used may include any suitable functional gene that produces a nucleic acid or polypeptide product that may be useful in the treatment of a disease or disorder of interest. Of course, a wide variety of nucleic acids, peptides, polypeptides and proteins with therapeutic activity are known to those skilled in the art and may be included in the nucleic acid constructs described herein. By way of example, genes that have been introduced into MSCs for cancer treatment in the field and that may be incorporated into the constructs and methods described herein may include:

[0224] [Table 2]

[0225] Further examples of functional genes that can be used may include genes for cancer therapy (Table 3) and / or genes for use in further therapeutic applications (Table 4).

[0226] [Table 3]

[0227] [Table 4]

[0228] The inherent tumor tropism of MSCs [9, 10] suggests that they can be used as cellular vehicles to deliver anticancer drugs specifically to tumors and their metastatic sites. Numerous GDEPT clinical trials with MSCs have shown promising results that may warrant further development into phase II clinical trials [7-11]. Such an approach may facilitate enzymatic localized and / or controlled conversion of non-toxic prodrugs in the vicinity of target cells. A “bystander effect” may enhance cytotoxicity against target cells [7]. The anticancer potential of specific CD4+-producing MSCs has been validated in a wide range of solid tumors, including gastric cancer [12-14], breast cancer [15, 16], and glioblastoma [17-19]. Preclinical studies have shown that cytosine deaminase / 5-fluorocytosine (CD4+ / 5FC) expression is highly robust, and as low as 4% of CD4+-positive cells in the tumor mass is sufficient for tumor eradication [20-22]. An advancement in the CD / 5FC system was the inclusion of uracil phosphoribosyltransferase (UPRT), a pyrimidine salvage enzyme that bypasses the rate-limiting enzymes dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT) to directly convert 5FU to 5-fluorouridine monophosphate (FUMP) [23-26]. CD::UPRT / 5FC can enhance the conversion of 5FC to its active metabolites by 30- to 1500-fold compared to CD / 5FC and 5FU [24, 27].

[0229] In certain embodiments, the one or more functional genes may include a suicide gene. By way of example, in certain embodiments, the one or more functional genes may include cytosine deaminase (CDy), uracil phosphoribosyltransferase (UPRT), or both. In certain embodiments, the one or more functional genes may include cytosine deaminase (CDy), uracil phosphoribosyltransferase (UPRT), herpes simplex virus type 1 thymidine kinase (HSV-TK), or another thymidine kinase, or any combination thereof. In certain embodiments, the one or more functional genes may be expressed separately or as a fusion construct. In certain embodiments, the nucleic acid construct may include two or more functional genes, or the nucleic acid construct may be provided as a mixture of two or more separate nucleic acid constructs, for example, each of which expresses a different functional gene of interest.

[0230] In certain embodiments, the one or more functional genes may include a fluorescent protein or another marker or tag. In certain embodiments, the fluorescent protein may be for use in identifying and / or evaluating successfully transfected MSCs. In certain embodiments, the fluorescent protein may be for use in separating, isolating, selecting or purifying transfected MSCs from non-transfected MSCs or other cells. In certain embodiments, the fluorescent protein may enable, for example, FACS-based cell sorting to quantify, purify or isolate transfected MSCs. In certain embodiments, the one or more functional genes may include, for example, green fluorescent protein (GFP).

[0231] In certain embodiments, the one or more functional genes of the nucleic acid construct may include CDy and UPRT, which may or may not be expressed as a fusion construct. In certain embodiments, the one or more functional genes may further include a fluorescent protein, such as green fluorescent protein (GFP), which may or may not be expressed as a fusion construct.

[0232] In certain embodiments, the one or more functional genes of the nucleic acid construct may comprise a selection gene, e.g. an antibiotic resistance gene, for selecting transfected or stably transfected MSCs.

[0233] While the exact copy number in any transfected MSC may vary somewhat, in certain embodiments it is contemplated that the transfected MSCs may each be transfected with, on average, at least about 1000 copies, at least about 2000 copies, at least about 3000 copies, at least about 4000 copies, at least about 5000 copies, at least about 6000 copies, at least about 7000 copies, at least about 8000 copies, at least about 9000 copies, or at least about 10000 copies of the nucleic acid construct.

[0234] In the case where a plurality or population of MSCs are transfected, in certain embodiments, it is contemplated that 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%, or at least about 95% of the MSCs may be transfected with a nucleic acid construct and may express one or more functional genes following transfection. Thus, in certain embodiments where a plurality or population of MSCs are transfected, the transfection efficiency may be any value between about 60% and about 100%, including any value rounded to the nearest tenth or any subrange therebetween. In certain embodiments, the transfection efficiency may be 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%, or at least about 95%. In certain embodiments, the transfection efficiency may be calculated as the % of cells expressing one or more functional genes of interest.

[0235] In further embodiments, it is contemplated that the cell viability of the MSCs may be at least about 70%, at least about 75%, at least about 80%, or at least about 85%. In certain embodiments, cell viability may be measured using any suitable methodology generally known to one of skill in the art in view of the teachings herein, such as the propidium iodide assay described in Cold Spring Harb Protoc., July 1, 2016, 2016(7), doi:10.1101 / pdb.prot087163.

[0236] In certain embodiments, the transfected MSCs may have a pluripotent phenotype that is substantially unchanged by transfection with a nucleic acid construct. Of course, the transfected MSCs may be transfected with a nucleic acid construct, and one or more functional genes encoded by the nucleic acid construct may be expressed in the transfected MSCs. However, in many applications, it is desirable that the phenotype of the transfected MSCs is otherwise substantially unchanged compared to the MSCs before transfection. By way of example, the MSCs have a pluripotent phenotype that may be desirable for certain applications of the transfected MSCs. Thus, in certain embodiments, the phenotype of the transfected MSCs is pluripotent and has not been further differentiated compared to the MSCs before transfection.

[0237] In certain embodiments, the pluripotent phenotype of the transfected MSCs, which may be substantially unchanged compared to the pluripotent phenotype of the MSCs prior to transfection, may comprise an immunophenotype in which expression of CD surface markers by the MSCs is substantially unchanged following transfection. By way of example, in certain embodiments, the transfected MSCs may be plastic adherent, may express CD105, CD73 and CD90 (>95%), may lack expression of CD45, CD34, CD14 and HLA-DR surface molecules (<2%), and may be capable of differentiating in vitro into osteoblasts, adipocytes and chondroblasts, for which they meet the immunophenotypic criteria defined by the International Society for Cellular Therapy (ISCT) (see Cytotherapy, 2006, 8(4):315-7, and https: / / www.celltherapysociety.org / news / 390154 / FDA-Grand-Rounds-cites-ISCTs-minimal-criteria-for-defining-MSCs.htm, which are incorporated herein by reference in their entireties). To this end, in certain embodiments, an acceptable percentage for identifying a positive marker (i.e., for a CD marker to be considered expressed) is that at least about 95% of the cells of the population express the marker after transfection, and an acceptable percentage for expressing a negative marker (i.e., for a CD marker to be considered not expressed) is that after transfection, the population lacks expression of the specific marker in at least 98% of the cells of the population. For example, in certain embodiments, the cells may lack expression of the HLA-DR marker after transfection just as unmodified MSCs lack expression of the HLA-DR marker, suggesting that the phenotype has not been substantially altered by the transfection, and that the MSC quality and phenotype have not been adversely altered by the transfection.

[0238] Generally, in certain embodiments, an immunophenotypic marker or another phenotypic marker may be considered to be unchanged by transfection if the expression profile of the relevant marker in a transfected cell is substantially unchanged (i.e., less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% change) compared to the expression profile of the same cell prior to transfection, or of a native cell (i.e., an unmodified / untreated control cell), or an equivalent or comparable cell that has not been transfected. In certain embodiments, an immunophenotypic marker or another phenotypic marker may be considered to be unchanged by transfection if the expression profile of the relevant marker in the transfected MSC cells is substantially unchanged (i.e., less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% change) compared to the expression profile of native MSC cells (i.e., unmodified / untreated MSC cells).

[0239] Of course, the transfected MSCs described herein may express one or more functional genes following transfection. Thus, in such embodiments, the transfected cells may be expressing one or more functional genes following transfection and may differ from comparable untransfected MSCs in that respect. As such, reference herein to a pluripotent phenotype that is substantially unchanged by transfection may reflect that one or more phenotypic characteristics (including but not limited to pluripotent characteristics) other than the expression of functional genes may be substantially unchanged in the MSCs following transfection. Examples of phenotypic characteristics that are substantially unchanged following transfection are described in detail herein and may include, for example, any one or more of pluripotent characteristics, immunophenotypic characteristics, cancer tropism characteristics, and / or other phenotypic characteristics.

[0240] In certain embodiments, the pluripotent phenotype of MSCs that is substantially unchanged by transfection may include tumor tropism and / or cancer tropism properties of MSCs, particularly when the transfected MSCs are used to treat cancer. In certain embodiments, the tumor tropism and / or cancer tropism properties of MSCs may be measured by a cell invasion assay, which is described in more detail in Example 1 below. In certain embodiments, the tumor tropism and / or cancer tropism properties of the transfected MSCs may be considered to be unchanged if there is no substantial loss of the tumor tropism and / or cancer tropism properties after transfection (i.e., the tumor tropism and / or cancer tropism properties may be substantially the same or may be enhanced after transfection).

[0241] In certain embodiments, transfected MSCs may not include virus-based transfection mediator materials.It should be understood that the virus-free transfection method for preparing transfected MSCs described herein is provided in detail below.Therefore, in certain embodiments, transfected MSCs described herein may not include (i.e., may not contain) virus-based transfection mediator materials, which may include, for example, phage proteins and / or nucleic acids, viral membrane components, viral nucleic acids, and / or viral proteins, typically found in virus-based gene or nucleic acid delivery techniques.

[0242] In certain embodiments, it is contemplated that the transfected MSCs described herein may be transfected with a nucleic acid construct and may express one or more functional genes for a period of time appropriate for the patient treated with the transfected MSCs to benefit. It has been found herein that the currently developed method provides transfected MSCs, including transiently transfected MSCs, that express one or more functional genes for an extended period of time. Thus, in certain embodiments, the transfected MSCs may transiently express one or more functional genes for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, or at least about 17 days following transfection.

[0243] In certain embodiments, provided herein is a population of MSC cells or a composition comprising a plurality of MSC cells, wherein there is about 90% or more cells expressing MSC markers, about 80% or more cells viability as measured by a standard viability test, about 70% or more MSCs testing positive for the transgene as tested by flow cytometry, or any combination thereof.Preferably, for the population of MSC cells or a composition comprising a plurality of MSC cells, there is about 90% or more cells expressing MSC markers, about 80% or more cells viability as measured by a standard viability test, and about 70% or more MSCs testing positive for the transgene as tested by flow cytometry.

[0244] In certain embodiments, it is contemplated that the methods described herein may be used to provide transfected MSCs that express one or more functional genes for a long period of time, even if the transfection is transient. In certain embodiments, it is contemplated that if long-term expression is desired, the nucleic acid construct may be designed to provide long-term transient expression of one or more functional genes. By way of example, in certain embodiments, it has been found herein that long-term expression of one or more functional genes can be achieved when the nucleic acid construct comprises a CpG-free expression plasmid. Based on this discovery, the skilled artisan will recognize various options for increasing the duration of transient expression in light of the teachings herein. In certain embodiments, it is contemplated that the nucleic acid construct may comprise a CpG-free expression plasmid or another CpG-free expression construct, a scaffold / matrix attachment region (S / MAR), an episomal vector, or an EBNA-1-containing construct. Further examples of features related to long-term expression can be found in Molecular Therapy, 2006, 14(5):613-626; J Biol Chem, 2000, 275(39):30408-16; Nucleic acids research, 2014, 42(7):e53-e53; and DOI: https: / / doi.org / 10.1016 / j.ymthe.2006.03.026, each of which is incorporated herein by reference in its entirety. In certain embodiments, some or all of the features may be implemented in the design of the nucleic acid constructs described herein. In certain embodiments, some or all of the features may be implemented as modules that can be added to the nucleic acid constructs described herein. For example, in certain CD::UPRP:GFP constructs described and used herein, CpG-free and S / MAR features / modules were used in the nucleic acid constructs.

[0245] In certain embodiments, transfected MSCs may be produced by any of the methods described herein. By way of example, in certain embodiments, transfected MSCs may be transfected with a nucleic acid construct using a cationic polymer, a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments, and a second agent capable of stabilizing the microtubule network of MSCs. Further description of such methods and components is provided below. By way of example, in certain embodiments, the cationic polymer may comprise linear or branched polyethyleneimine (PEI), the first agent may comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / cholesteryl hemisuccinate (CHEMS) (DOPE / CHEMS), and / or the second agent may comprise a histone deacetylase inhibitor (HDACi), such as SAHA (vorinostat).

[0246] In certain embodiments, such as where the functional genes include one or more of cytosine deaminase (CDy), uracil phosphoribosyltransferase (UPRT), or herpes simplex virus type 1 thymidine kinase (HSV-TK), the MSCs may be sensitive to treatment with 5-fluorocytosine (5FC) or ganciclovir (GCV). In certain embodiments, the transfected MSCs may convert a) 5FC to 5-fluorouridine (5FU), 5-fluorouridine monophosphate (FUMP), or both, b) ganciclovir to ganciclovir monophosphate, or c) a combination of a) and b). In certain embodiments, the MSCs may be for use in cancer treatment. In certain embodiments, the transfected MSCs may be for use in combination with 5FC, 5FU, GCV, or any combination thereof.

[0247] In certain embodiments, the transfected MSCs may be substantially undifferentiated.

[0248] In yet another embodiment, a method for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct expressing one or more functional genes, comprising: exposing the MSCs to a transfection mixture comprising a nucleic acid construct complexed with a cationic polymer; exposing the MSCs to a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments and a second agent capable of stabilizing the microtubule network of the MSCs; and incubating the MSCs; thereby providing MSCs transfected with the nucleic acid construct. A method is provided herein comprising:

[0249] Examples of suitable MSCs, nucleic acid constructs and functional genes have already been described in detail herein.By way of example, in certain embodiments, one or more functional genes may include suicide genes; cytosine deaminase (CDy) and / or thymidine kinase (TK); uracil phosphoribosyltransferase (UPRT); both CDy and UPRT, whether or not provided as fusion constructs; fluorescent proteins, such as green fluorescent protein (GFP); CDy, UPRT and GFP, whether or not provided as fusion constructs; herpes simplex virus type 1 thymidine kinase (HSV-TK); or any combination thereof.

[0250] In certain embodiments, the MSCs may be derived from umbilical cord blood, neonatal birth-related tissue, Wharton's jelly, umbilical cord, umbilical cord lining, placenta, or another MSC cell source. In certain embodiments, the MSCs may be adipose tissue-derived MSCs (AT-MSCs), bone marrow-derived MSCs (BM-MSCs), or umbilical cord-derived MSCs (UC-MSCs). In other embodiments, the MSCs may be derived from humans, dogs, cats, horses, or another species.

[0251] In certain embodiments, the nucleic acid construct may comprise a CpG-free expression plasmid or another CpG-free expression construct, a scaffold / matrix attachment region (S / MAR), an episomal vector, or an EBNA-1 containing construct.

[0252] In certain embodiments, the cationic polymer may comprise any suitable cationic polymer or polycationic polymer or partial cationic polymer capable of complexing with the nucleic acid construct and delivering the nucleic acid construct into the MSC upon exposure to the MSC. In certain embodiments, the cationic polymer may be selected from polyethyleneimine, polycationic amphiphiles, DEAE dextran, cationic polymers, derivatives thereof, or any combination thereof. In certain embodiments, the cationic polymer may comprise a cationic polymer such as a dendrimer, branched polyethyleneimine (BPEI), linear polyethyleneimine (LPEI), poly(amidoamine) (PAMAM), XtremeGENE HP®, or any combination thereof. In certain embodiments, the cationic polymer may comprise LPEI. In certain embodiments, the cationic polymer may be, for example, a homopolymer, copolymer, or block copolymer. In certain embodiments, the cationic polymer may have a size of about 5 kDa to about 200 kDa. In certain embodiments, the cationic polymer may have a size of about 5 kDa or less. In certain embodiments, the cationic polymer may have a size of about 200 kDa or more. In certain embodiments, the cationic polymer may comprise linear or branched polyethyleneimine (PEI), poly(amidoamine) PAMAM, or another cationic polymer, or any combination thereof. In certain embodiments, the cationic polymer may comprise linear polyethyleneimine (LPEI).

[0253] In certain embodiments, the amount of nucleic acid construct in the transfection mixture to which the MSCs are exposed is greater than or equal to 100 μg / ml for a surface area of ​​1.9 cm 2 In certain embodiments, the amount of nucleic acid construct in the transfection mixture to which the MSCs are exposed can be between about 200 ng and about 500 ng per 1.9 cm of surface area.2 In certain embodiments, the amount of nucleic acid construct in the transfection mixture to which the MSCs are exposed can be between about 250 ng and about 400 ng per 1.9 cm of surface area. 2 In certain embodiments, the amount of nucleic acid construct to which the MSCs are exposed may be between about 300 ng and about 350 ng per 1.9 cm of surface area. 2 The amount of iodine per milligram may be any value between about 200 ng and about 500 ng rounded to the nearest tenth, or any subrange therebetween.

[0254] In certain embodiments of any of the foregoing methods, the ratio of cationic polymer to nucleic acid construct can be from about 1 μg to about 30 μg of cationic polymer per μg of nucleic acid construct in the transfection mixture, or any value therebetween rounded to the nearest tenth, or any subrange therebetween.

[0255] In certain embodiments, the N / P of the cationic polymer and the nucleic acid may be in the range of about 5 to about 100, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 109, 102, 104, 105, 106 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 or 100, or any value therebetween rounded to the first decimal place or any subrange therebetween.

[0256] In certain embodiments, the transfection mixture may include a complexation buffer, cell culture medium or cell buffer, or any combination thereof.

[0257] In certain embodiments, the first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments may comprise any suitable agent capable of directing genetic material away from non-productive acidic compartments of the cell. In another embodiment, the first agent may comprise a lipid, a peptide fusogenic agent, or a combination thereof. In certain embodiments, the first agent may comprise DOPE, CHEMS, DPPC, or DOPC, or any combination thereof. In certain embodiments, the first agent may comprise hemagglutinin (HA2 peptide), influenza-derived fusogenic peptide diINF-7, the T domain of diphtheria toxin, a polycationic peptide, such as polylysine and / or polyarginine, or any combination thereof. In certain embodiments, the first agent may comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / cholesteryl hemisuccinate (CHEMS) (DOPE / CHEMS). Various ratios of DOPE:CHEMS can be used, for example, in certain embodiments, ratios between about 9:1 and about 1:9, such as about 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, may be used. In certain embodiments, a mixture of fusogenic and helper lipids may be used, such as a mixture of DOPE and CHEMS. In certain embodiments, three lipids may be used. For example, in certain embodiments, a mixture of DPPC:DOPE:CHEMS may be used in various ratios.

[0258] In certain embodiments, the second agent capable of stabilizing the microtubule network of MSCs may comprise any suitable agent that stabilizes microtubules or its network. In certain embodiments, the second agent may be capable of enhancing tubulin acetylation. In certain embodiments, the second agent may be selected from histone deacetylase inhibitors (HDACi), such as histone deacetylase 6 inhibitors (HDAC6i), tubulin binding agents (TBAs), and siRNAs that can directly or indirectly affect the stability of the microtubule network. In certain embodiments, the HDACi may comprise tubastatin A, belinostat, bufexamac, panobinostat, PCI-24781, SAHA (vorinostat), scripteid, trichostatin A, valproic acid, B2, salermide, sirtinol, or any combination thereof. In certain embodiments, the second agent may comprise histone deacetylase inhibitors (HDACi), such as SAHA (vorinostat).

[0259] In certain embodiments, the first and second agents together may form TrafEn™, an abbreviation for trafficking enhancer, which directs genetic material, or a complex containing genetic material, into a productive pathway for efficient transfection.

[0260] Examples of suitable first agents capable of redirecting endocytosed nucleic acids from intracellular acidic compartments and suitable second agents capable of stabilizing the microtubule network of MSCs, and TrafEN™, are described in detail in International Publication No. WO 2014 / 070111, entitled A Novel Reagent for Gene-Drug Therapeutics, and in Ho YK et al., Enhanced Non-Viral Gene Delivery by Coordinated Endosomal Release and Inhibition of β-Tubulin Deactylase, Nucleic Acids Research, 2017, 45(6):e38, both of which are incorporated by reference in their entireties.

[0261] In certain embodiments of the methods, the MSCs are not centrifuged during exposure to the transfection mixture, during exposure to the first agent and the second agent, during incubation, or any combination thereof. In certain embodiments of the methods, incubating the MSCs may optionally include gentle mixing without centrifugation.

[0262] In certain embodiments, centrifugation may be used to promote rapid attachment of polymer-complexed DNA to cells. In that case, the transfection mixture may be removed immediately or shortly after centrifugation to minimize toxicity of free cationic polymer (not substantially spun down by centrifugation) to the cells. Therefore, in certain embodiments, centrifugation may be used to transfect cells, especially when using smaller scales that can be easily centrifugated. By way of example, the centrifugation method may include, for example, adding the transfection mixture to the cells, centrifuging to attach the nucleic acid complexes onto the cells, removing the transfection mixture to remove free polymer from contact with the cells, and replacing with fresh medium that may contain TrafEn or to which TrafEn may be added.

[0263] In certain embodiments, it may be desirable to operate on a larger scale, for example in livestock and / or human therapeutic indications. Centrifugation may be undesirable at such a larger scale. Centrifugation may also be undesirable in other cases, for example in applications where centrifugation equipment is unavailable, inconvenient, and / or expensive. As described in detail herein, it has been found that in certain embodiments, centrifugation can be omitted. When centrifugation is omitted, in certain embodiments, the incubation time may be extended, for example, from about 2 hours to about 24 hours, to allow sufficient contact of the cells with the polymer-complexed DNA. The presence of free polymer during this incubation time may be toxic, depending on the cells and polymer used. Therefore, in certain embodiments, the selection of the cationic polymer may be appropriately adjusted for the particular cell type and incubation time to reduce or avoid toxicity to the cells, as described in detail herein.

[0264] As described in detail herein, the methods described herein, which may or may not use centrifugation, can provide high transfection efficiency (e.g., >about 70%).

[0265] In embodiments where centrifugation is omitted, the method may be more easily scalable, for example to accommodate large production scales for preclinical and / or clinical testing. However, the inventors have found that when centrifugation during transfection is omitted, the incubation time during transfection can be extended to achieve high transfection efficiency. Thus, in certain embodiments where centrifugation is omitted, incubating the MSCs may include incubating the MSCs for at least about 2 hours. In certain embodiments, incubating the MSCs may include incubating the MSCs for about 2 hours to about 24 hours, or about 4 hours to about 18 hours, or any value between 2 hours and 24 hours, rounded to the nearest tenth, or any subrange therebetween.

[0266] The inventors further confirmed that in embodiments where centrifugation is omitted and / or incubation time is extended, the selection of cationic polymer may be adjusted appropriately, since certain cationic polymers may cause toxicity, which may be undesirable, especially when incubation time is extended. Furthermore, different types of MSCs (i.e., various types, origins, cell lines, and growth conditions) may exhibit different resistance to cationic polymers and / or extended incubation periods. Therefore, in certain embodiments, the transfection method may be adapted for the individual MSCs used. Example 2 below provides some examples where DNA amount / conditions and cationic polymer selection was made to avoid toxicity and obtain high transfection efficiency during transfection of certain MSCs. Therefore, in certain embodiments of the methods described herein, the cationic polymer may comprise a cationic polymer identified to have low cytotoxicity to MSCs of a particular application. In certain embodiments, the cationic polymer may be selected, for example, by size and / or charge number. In certain embodiments, a larger certain cationic polymer may be preferred, for example, for some cells, but may be somewhat toxic to other cells. In some cases, smaller and / or less charged cationic polymers may typically be less toxic but may exhibit lower transfection efficiency and / or transfection rate in certain cell lines. In certain embodiments, for example, TrafEn may be used to increase transfection efficiency.

[0267] In certain embodiments, the buoyant density of the medium, which may vary between cell types, may be considered when selecting the cationic polymer and / or polymer-DNA complex, as this may have an effect on the rate of attachment of the polymer-DNA complex on the cell. In certain embodiments, the complex and / or medium may be selected to promote good attachment on the cell, and / or the cationic polymer may be selected such that the free cationic polymer is non-toxic or has low toxicity to the particular cell.

[0268] In certain embodiments, it is contemplated that cationic polymers may be screened to identify those that provide suitable transfection efficiency and / or cell viability for the particular MSCs of interest, since transfection efficiency and / or cell viability are two noteworthy features identified herein to measure the level of compatibility of the cationic polymer with a particular MSC type / donor for providing efficient transfection without centrifugation. In certain embodiments, the cationic polymer may be selected to not cause significant or deleterious levels of cytotoxicity during, for example, an incubation period of at least about 2 hours or at least about 4 hours. If the cationic polymer is non-toxic to the cells, the incubation period may be continued for a longer period. In certain embodiments, toxicity may be assessed by any suitable method, for example, a propidium iodide assay. In certain embodiments, cell viability (or cell viability target) may be about 70% or more after transfection.

[0269] In certain embodiments of any of the above methods, exposing the MSCs to a transfection mixture may include complexing the nucleic acid construct with a cationic polymer to provide a transfection mixture comprising the complexed nucleic acid construct, and adding the transfection mixture to the MSCs. In other words, exposing the MSCs to a transfection mixture preferably includes pre-complexing or mixing the nucleic acid construct with the cationic polymer prior to adding the MSCs.

[0270] In another embodiment of any of the above methods, exposing the MSCs to the transfection mixture may comprise adding the transfection mixture to the MSCs and incubating the MSCs with the transfection mixture.

[0271] In another embodiment of any of the above methods, exposing the MSCs to the first agent and the second agent may include adding the first agent and the second agent simultaneously with or shortly after adding or exposing the MSCs to the transfection mixture, which in certain embodiments may be performed where centrifugation is omitted.

[0272] In another embodiment of any of the above methods, exposing the MSCs to the first and second agents may include adding the first and second agents simultaneously with the transfection mixture in exposing the MSCs to the transfection mixture, or may include adding the first and second agents to MSCs that have already been contacted with the transfection mixture (i.e., the transfection mixture does not have to be removed prior to adding the first and second agents). In certain embodiments, the step may be performed where centrifugation is omitted.

[0273] In yet another embodiment of any of the above methods, exposing the MSCs to the first and second agents may include replacing the transfection mixture with cell culture medium supplemented with the first and second agents. In certain embodiments, the step may be performed using centrifugation to promote rapid attachment of the polymer-complexed DNA to the cells so as to reduce toxicity of free polymer to the cells. In certain embodiments, the cell culture medium may include complete medium.

[0274] In certain embodiments of any of the above methods, the MSCs may be about 60% confluent and may be seeded about 24 hours prior to exposure to the transfection mixture.

[0275] In certain embodiments of any of the above methods, the transfection mixture may comprise the complexed nucleic acid in serum-free DMEM or in fresh culture medium.

[0276] In further embodiments of any of the above methods, exposing the MSCs to the transfection mixture may include adding the transfection mixture to the cells without removing the culture / growth medium from the cells prior to adding the transfection mixture (which may or may not further include fresh culture medium), which in certain embodiments may be performed where centrifugation is omitted.

[0277] In certain embodiments of the above method, the step of exposing the MSCs to the transfection mixture comprises: Optionally, replacing the culture / growth medium in which the cells are cultured with fresh culture / growth medium; and adding the transfection mixture to the cells (which may or may not further contain fresh culture medium, or which may be added simultaneously or sequentially with fresh culture medium), without removing the culture / growth medium (if present) from the cells prior to adding the transfection mixture may include:

[0278] It is therefore contemplated that in certain embodiments the cell culture / growth medium may be replaced or refreshed prior to adding the complexed nucleic acid construct to the cells in order to provide fresh culture medium prior to transfection, which in certain embodiments may be done where centrifugation is omitted.

[0279] In further embodiments of any of the above methods, exposing the MSCs to the transfection mixture may include removing culture medium from the MSCs and replacing the culture medium with the transfection mixture, which in certain embodiments may be performed using centrifugation to promote rapid attachment of the polymer-complexed DNA to the cells.

[0280] In certain embodiments, it is contemplated that exposing MSCs to the transfection mixture may include incubating MSCs with the transfection mixture under gentle centrifugation. For example, it is contemplated that centrifugation may be performed in certain embodiments when the method is performed on a small scale and / or a suitable centrifugation device is available. Gentle centrifugation may be performed to avoid toxicity of free polymer after adding the polymer-nucleic acid construct. In certain embodiments, the method may include adding the transfection mixture to the cell culture, gentle centrifugation (e.g., about 5 minutes) to attach the polymer-nucleic acid construct complex to the cells, and removing the transfection mixture (containing the free polymer, which is relatively small and does not substantially spin down by centrifugation). In certain embodiments, gentle centrifugation may include about 200 g for about 5 minutes.

[0281] In certain embodiments of the methods described herein, for example, transfection may be performed in flat-bottom vessels in which the amount of reagent is increased, matched to the cell density, and the amount of DNA is increased according to the surface area of ​​the cell culture vessel.

[0282] In certain embodiments of the methods described herein, MSCs may be cultured on microcarriers (e.g., microbeads) and therefore may be suspended during transfection, optionally with shaking or other agitation. In certain embodiments, the microcarriers may comprise microbeads. In certain embodiments, the microcarriers may comprise type 1 porcine collagen coated microcarriers. In certain embodiments, the microcarriers may comprise Cytodex®3. In certain embodiments using microcarriers, transfection may be performed at elevated cell density under shaking, which may allow for large-scale production. Furthermore, the rpm during shaking can be adjusted depending on the type of vessel and the density / number of microcarriers used.

[0283] Another approach using flat-bottom vessels and microcarriers follows similar steps, for example: step 1 → exposing MSCs to a transfection mixture containing a nucleic acid construct complexed with a cationic polymer, step 2 → exposing MSCs to a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments and a second agent capable of stabilizing the microtubule network of MSCs, and step 3 → incubating MSCs to provide MSCs transfected with the nucleic acid construct. Surface area cm 2 The number of cells added per vessel and the cell culture volume used are adapted for each vessel. In microcarrier applications, the shaking speed may be adjusted to prevent the microcarriers from clumping.

[0284] In certain embodiments of the methods described herein, incubating the MSCs during transfection may comprise a rotating bioreactor type agitation (e.g., a rotating Erlenmeyer flask), a wave bioreactor, a rotating wall bioreactor, a stirred tank bioreactor, or a shaker type agitation for at least a portion of the incubation time (see FIG. 52 for further examples).

[0285] In certain embodiments of any of the above methods, the one or more functional genes may be transiently expressed in the transfected MSCs, stably expressed in the transfected MSCs, or a combination thereof.

[0286] In certain embodiments of any of the foregoing methods, the transfected MSCs may each be transfected with an average of at least about 1000 copies, at least about 2000 copies, at least about 3000 copies, at least about 4000 copies, at least about 5000 copies, at least about 6000 copies, at least about 7000 copies, at least about 8000 copies, at least about 9000 copies, or at least about 10000 copies of the nucleic acid construct.

[0287] In certain embodiments of any of the above methods, the pluripotent phenotype of the transfected MSCs may be substantially unchanged by transfection. In certain embodiments, the pluripotent phenotype may include tumor-tropic and / or cancer-tropic properties of the MSCs. In certain embodiments, the pluripotent phenotype may include an immunophenotype in which the expression of CD surface markers may be substantially unchanged after transfection. In certain embodiments, the transfected MSCs may be plastic-adherent, express CD105, CD73 and CD90 (>95%), lack expression of CD45, CD34, CD14 and HLA-DR surface molecules (<2%), and may differentiate in vitro into osteoblasts, adipocytes and chondroblasts, fulfilling the immunophenotypic criteria defined by the International Society for Cellular Therapy (ISCT). The phenotype of the transfected cells is described in detail herein above and in the Examples below.

[0288] In certain embodiments of any of the foregoing methods, 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%, or at least about 95% of the MSCs are transfected with a nucleic acid construct and may express one or more functional genes.

[0289] In certain embodiments of any of the foregoing methods, the cell viability of the transfected MSCs may be at least about 70%, at least about 75%, at least about 80%, or at least about 85%.

[0290] In certain embodiments of any of the above methods, the transfected MSCs may be undifferentiated.

[0291] In certain embodiments of any of the foregoing methods, the method may not include a viral-based transfection agent material.

[0292] In certain embodiments of any of the foregoing methods, the MSCs may transiently express the one or more functional genes for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, or at least about 17 days following transfection.

[0293] In certain embodiments, the resulting MSCs may be sensitive to treatment with 5-fluorocytosine (5FC) or ganciclovir (GCV) or both. In certain embodiments, the resulting MSCs may a) convert 5FC to 5-fluorouridine (5FU), 5-fluorouridine monophosphate (FUMP) or both, b) convert ganciclovir to ganciclovir monophosphate, or c) a combination of a) and b).

[0294] In certain embodiments, the functional gene or genes may include a fluorescent protein, and the method may further include isolating, selecting, or purifying the transfected MSCs using cell sorting or FACS. This step may be performed, for example, when a particularly high purity is desired. Of course, such isolation, selection, or purification may be optional, since it is intended that, for example, in clinical applications, a population that is about ≧70% positive for the therapeutic gene may be obtained without further steps of isolation, selection, or purification, in certain embodiments as described herein above, which may be acceptable.

[0295] In yet another embodiment of the above method, there is provided a method for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct expressing one or more functional genes, comprising: Culturing the MSCs in a proliferation medium; adding a transfection mixture comprising a nucleic acid construct complexed with a cationic polymer to the MSCs without removing the growth medium from the MSCs; adding to the MSCs a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments and a second agent capable of stabilizing the microtubule network of the MSCs; and incubating the MSCs in contact with all of the transfection mixture, the first agent, and the second agent for an incubation period; wherein the first agent and the second agent are added to the MSCs simultaneously with, sequentially with, or in combination with the transfection mixture; Methods are provided herein that provide for MSCs transfected with a nucleic acid construct by not centrifuging the MSCs between the addition of the transfection mixture and the end of the incubation period.

[0296] In certain embodiments, culturing MSCs in growth medium may include providing the cells with fresh growth medium (i.e., replacing spent or partially spent growth medium with fresh growth medium) or adding fresh growth medium to spent or partially spent growth medium.

[0297] In certain embodiments, the incubation period may be at least about 2 hours.

[0298] In certain embodiments, the incubation period can be 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, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours.

[0299] In certain embodiments of any of the foregoing methods, the method may produce any of the transfected MSCs described herein.

[0300] In yet another embodiment, provided herein is a MSC or a plurality of MSCs produced by any of the methods described herein.

[0301] In another embodiment, provided herein is a composition comprising any of the MSCs described herein and at least one of a pharma- ceutically acceptable carrier, diluent, excipient, cell culture medium, or buffer.

[0302] In certain embodiments, the pharma- ceutically acceptable carrier, diluent, excipient, cell culture medium or buffer may include, for example, any suitable PBS buffer, cryopreservation medium, matrigel, or hydrogel. In certain embodiments, provided herein is a composition comprising a suspension of MSCs as described herein in PBS or another buffer or cell culture medium. In another embodiment, provided herein is a composition comprising MSCs as described herein frozen with cryopreservation medium.

[0303] In yet another embodiment, provided herein is a theranostic agent comprising any of the MSCs described herein. By way of example, in certain embodiments, the theranostic agent may comprise MSCs expressing both a therapeutic or suicide gene and a fluorescent protein. The MSCs may have cancer- and / or tumor-tropic properties and may be used to indicate the location of cancer or tumor cells using fluorescence, at which point of fluorescence (where the suicide gene is used) a prodrug may be added, for example, to provide an anti-cancer or anti-tumor effect.

[0304] In yet another embodiment, a kit for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct that transiently expresses one or more functional genes, comprising: M.S.C. Nucleic acid constructs designed for the transient expression of one or more functional genes; Cell culture medium, Cationic polymers, a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments; A second agent capable of stabilizing the microtubule network of MSCs; instructions for carrying out the methods described herein; 5FC, GCV and / or 5FU Provided herein are kits comprising one or more of:

[0305] In certain embodiments, the MSCs may be any MSCs described herein. In certain embodiments, the MSCs may be derived from umbilical cord blood, neonatal birth-related tissue, Wharton's jelly, umbilical cord, umbilical cord lining, placenta, or another MSC cell source. In certain embodiments, the MSCs may be adipose tissue-derived MSCs (AT-MSCs), bone marrow-derived MSCs (BM-MSCs), or umbilical cord-derived MSCs (UC-MSCs). In other embodiments, the MSCs may be derived from humans, dogs, cats, horses, or another species.

[0306] In certain embodiments, the nucleic acid construct may be any nucleic acid construct described herein and the one or more functional genes may be any one or more functional genes described herein. In certain embodiments, the nucleic acid construct may comprise a CpG-free expression plasmid or another CpG-free expression construct, a scaffold / matrix attachment region (S / MAR), an episomal vector, or an EBNA-1-containing construct.

[0307] In certain embodiments, the cationic polymer may comprise any cationic polymer described herein. In certain embodiments, the cationic polymer may comprise linear or branched polyethyleneimine (PEI), poly(amidoamine) PAMAM, or another cationic polymer. In certain embodiments, the cationic polymer may comprise linear polyethyleneimine (LPEI). In certain embodiments, the cationic polymer may comprise a cationic polymer identified as having low cytotoxicity to MSCs. In certain embodiments, the cationic polymer may have a size of about 5 kDa to about 200 kDa.

[0308] In certain embodiments, the first agent may include any suitable first agent described herein. In certain embodiments, the first agent may include one or more of DOPC, DPPC, or another fusogenic lipid. In certain embodiments, the first agent may include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) / cholesteryl hemisuccinate (CHEMS) (DOPE / CHEMS), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or another fusogenic lipid, or any combination thereof.

[0309] In certain embodiments, the second agent may include any suitable second agent described herein. In certain embodiments, the second agent may include a histone deacetylase inhibitor (HDACi), such as SAHA (vorinostat).

[0310] In certain embodiments, the one or more functional genes may include a suicide gene; cytosine deaminase (CDy); thymidine kinase (TK); uracil phosphoribosyltransferase (UPRT); both CDy and UPRT, which may or may not be provided as a fusion construct; a fluorescent protein, such as green fluorescent protein (GFP); CDy, UPRT and GFP, which may or may not be provided as a fusion construct; herpes simplex virus type 1 thymidine kinase (HSV-TK); or any combination thereof.

[0311] In certain embodiments, the ratio of cationic polymer to nucleic acid construct in the kit may be from about 1 μg to about 30 μg of cationic polymer per 1 μg of nucleic acid construct.

[0312] In certain embodiments, the kit may be for preparing an MSC-based anti-cancer drug. In certain embodiments, the kit may further include instructions and / or devices for carrying out the methods for treating cancer described herein. In certain embodiments, a syringe or another suitable injection device may be provided for intratumoral or intravenous or subcutaneous injection or infusion of MSC. In certain embodiments, the MSC may be embedded in a biomaterial, such as gelfoam, for administration.

[0313] In certain embodiments, methods are provided herein for scalable, non-viral genetic modification of mesenchymal stem cells (MSCs) for cancer therapy. In certain embodiments, the methods described herein may include transfecting MSCs with one or more suicide genes in the presence of a formulation of transfection enhancer (TrafEn). In certain embodiments, such methods may include using a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments and a second agent capable of stabilizing the microtubule network of MSCs. In certain embodiments, high efficiency modification in terms of number and expression of modified cells may provide for the generation of highly effective MSCs expressing therapeutic genes, such as the suicide gene cytosine deaminase (CD). In certain embodiments, the modified MSCs may be administered to a patient with a tumor or cancer. In certain embodiments, the therapeutic gene expressed by the MSCs may convert a prodrug into a toxic drug that shrinks or eliminates the tumor mass. In certain embodiments, the methods described herein may be used in the manufacture of a medicament for treating cancer and / or another indication. Further described herein are methods and kits for delivering genetic material into cells. In certain embodiments, MSCs may be modified with generally any suitable cancer targeting therapeutic gene and / or generally any other suitable therapeutic gene for treating generally any other suitable disease and / or disorder.

[0314] Uses and methods of treating diseases or disorders, such as cancer, using transfected mesenchymal stem cells - Patents.com Provided herein are transfected MSCs that may express one or more functional genes, as well as methods and kits for preparing transfected MSCs, as described in detail herein. In certain embodiments, the one or more functional genes may include one or more therapeutically active genes, for example, producing one or more therapeutically active RNAs, peptides, polypeptides or proteins. Thus, the MSCs described herein can be understood to be generally intended for use in the treatment, prevention or amelioration of any disease or disorder for which the one or more functional genes are therapeutically active. The following discussion is primarily related to the treatment of cancer, but the skilled artisan will recognize, in light of the teachings herein, that a variety of other diseases or disorders are also contemplated herein.

[0315] In one embodiment, provided herein is the use of any of the MSCs described herein to kill cancer cells.

[0316] In one embodiment, provided herein is a use of any of the MSCs described herein for treating cancer in a patient in need thereof.

[0317] In certain embodiments, the cancer may include any one or more of various types of solid tumors (e.g., thyroid cancer, sarcoma, lymphoma, squamous cell carcinoma, etc.). Because MSCs can exhibit significant tropism, it is contemplated that the site of the cancer may generally be anywhere and the site does not present a significant issue. Furthermore, in certain embodiments, it is contemplated that the MSCs and treatments described herein may be tailored to individual cancers, and that the MSC prodrug strategies described herein are generally cancer type independent.

[0318] In certain embodiments, the cancer may include, for example, lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer, or any combination thereof.

[0319] In certain embodiments, the patient may include a vertebrate, a mammal, or a human.

[0320] In certain embodiments, the MSCs may be for use in combination (either simultaneously, sequentially, or in admixture) with one or more additional or therapeutic agents that are active against the disease or disorder being treated, for example, one or more anti-cancer agents where the disease or disorder is cancer.

[0321] In certain embodiments, the MSCs may be intended for use in combination with one or more corresponding prodrugs (either simultaneously, sequentially, or in admixture), especially when the one or more functional genes expressed by the transfected MSCs include suicide genes or express enzymes that convert the prodrugs into their active forms. By way of example, in certain embodiments, the one or more functional genes may include cytosine deaminase (CDy); thymidine kinase (TK); uracil phosphoribosyltransferase (UPRT); both CDy and UPRT, which may or may not be provided as a fusion construct; herpes simplex virus type 1 thymidine kinase (HSV-TK); or any combination thereof, and may be intended for use in combination with 5FC, 5FU, GCV, or any combination thereof.

[0322] In another embodiment, provided herein is the use of any of the MSCs described herein in the manufacture of a medicament for the treatment of cancer.In certain embodiments, the MSCs may be for use in combination with 5FC, 5FU, GCV, or any combination thereof.

[0323] In certain embodiments, the MSCs described herein may be for administration to a patient via generally any suitable technique appropriate for the patient and / or disease being treated. By way of example, it is contemplated that in certain embodiments, the MSCs may be administered systemically (e.g., intravenous injection) or locally (e.g., local injection or local implantation) to a patient. In certain embodiments, the MSCs may be administered intravenously, e.g., as described in Oncotarget., Oct. 6, 2017, 8(46):80156-80166, or intracranially, e.g., as described in Clin Cancer Res., Jun. 15, 2017, 23(12):2951-2960, each of which is incorporated herein by reference in its entirety. In certain embodiments, the MSCs described herein may be for administration to a patient via intraportal, intraperitoneal, intravenous, intratumoral, subcutaneous, intracranial injection or infusion, or embedded in a hydrogel or gelfoam for administration or implantation to a patient.

[0324] In yet another embodiment, there is provided a method for treating cancer in a patient in need thereof, comprising: administering MSCs as described herein to a patient in an area adjacent to the cancer cells; However, methods are provided herein in which one or more functional genes in MSCs contribute to an anti-cancer effect on cancer cells.

[0325] In certain embodiments, the patient may include a vertebrate, a mammal, or a human.

[0326] In certain embodiments, the cancer may include, for example, lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer, or any combination thereof.

[0327] In certain embodiments, the MSCs described herein may be administered to a patient via generally any suitable technique appropriate for the patient and / or disease being treated. By way of example, it is contemplated that in certain embodiments, the MSCs may be administered systemically (e.g., intravenous injection) or locally (e.g., local injection, intratumoral injection, subcutaneous injection, or implantation or infusion) to a patient. In certain embodiments, the MSCs may be administered intravenously, e.g., as described in Oncotarget., Oct. 6, 2017, 8(46):80156-80166, or intracranially, e.g., as described in Clin Cancer Res., Jun. 15, 2017, 23(12):2951-2960, each of which is incorporated herein by reference in its entirety. In certain embodiments, the MSCs described herein may be for administration to a patient via intravenous, intratumoral, subcutaneous, intracranial injection or infusion, or embedded in a hydrogel or gelfoam for administration or implantation to a patient.

[0328] Of course, in certain embodiments, MSCs may be administered to a patient such that cells associated with a disease or disorder (e.g., cancer or tumor cells) are in contact with or in suitable proximity to the MSCs, such that one or more functional genes expressed by the MSCs may exert a therapeutic effect on the cells associated with the disease or disorder (either directly or indirectly via (e.g.) prodrug conversion). In certain embodiments, MSCs may have tropism properties for cells associated with a disease or disorder that aid in the placement of the MSCs in suitable proximity to the cells associated with the disease or disorder. In certain embodiments, MSCs may be administered in direct contact with the cells associated with the disease or disorder or within a proximal range of about 1-3 cm from the cells associated with the disease or disorder. In certain embodiments, direct contact of MSCs with disease cells, e.g., cancer cells, may be unnecessary, as prodrug therapy may provide a bystander effect.

[0329] In certain embodiments, MSCs may be administered in combination with one or more corresponding prodrugs (either simultaneously, sequentially, or in admixture), especially when the one or more functional genes expressed by the transfected MSCs include suicide genes or express enzymes that convert prodrugs into their active forms. By way of example, in certain embodiments, the one or more functional genes may include cytosine deaminase (CDy); thymidine kinase (TK); uracil phosphoribosyltransferase (UPRT); both CDy and UPRT, which may or may not be provided as a fusion construct; herpes simplex virus type 1 thymidine kinase (HSV-TK); or any combination thereof, and may be for use in combination with 5FC, 5FU, GCV, or any combination thereof.

[0330] In certain embodiments, one or more functional genes of the MSCs and the prodrug may be designed such that conversion of the prodrug to an active form may target or kill the MSCs in addition to surrounding cells (e.g., cancer cells or tumor cells) associated with the disease or disorder. Therefore, in certain embodiments where the MSCs are to be used in combination with another agent, e.g., a prodrug, it may be desirable to keep the MSCs and the prodrug separate from each other until the MSCs are first contacted with the appropriate cells of the patient, so that the MSCs are not killed or inactivated before they can provide a therapeutic effect. In certain embodiments, it may take at least some time for the MSCs to migrate directional toward the tumor, so that in certain embodiments, it is contemplated to delay administration of the prodrug until the MSCs are in close proximity to the tumor, for example.

[0331] In certain embodiments, the MSCs may transiently express one or more functional genes for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, or at least about 17 days following transfection, following administration to a patient, or both.

[0332] In certain embodiments of the methods described herein, the methods may further include administering a prodrug, e.g., 5FC, 5FU, ganciclovir, or any combination thereof, to the patient such that the MSCs are exposed to the prodrug, e.g., 5FC, 5FU, ganciclovir, or any combination thereof, and may convert the prodrug to an active form.

[0333] In certain embodiments of any of the foregoing methods, the method may further comprise the step of producing the transfected MSCs using any of the production methods described herein prior to the step of administering the MSCs.

[0334] In certain embodiments, MSCs may be expanded and / or cultured prior to transfection, for example for large-scale MSC engineering, in certain embodiments such expansion may be desirable. EXAMPLES

[0335] Example 1 Non-viral modification of mesenchymal stem cells for cancer therapy. An efficient non-viral process for engineering mesenchymal stem cells for gene-directed enzyme prodrug cancer therapy Modification of mesenchymal stem cells (MSCs) for prodrug gene therapy was performed by viral and non-viral gene delivery systems. Due to the low efficiency (0-50%) of conventional transfection methods, viral methods were widely used in preclinical and clinical studies. The embodiments described herein demonstrate polyethylenimine (PEI)-based modification of human adipose tissue-derived MSCs (AT-MSCs) with >90% efficiency in the presence of a first agent, e.g., a fusogenic lipid, and one or more of a second agent, e.g., a histone deacetylase 6 inhibitor (HDAC6i). The cellular phenotype of MSCs remained unchanged after modification, a desirable feature for clinical application of MSCs. Equipped with this method, the anticancer effect of modified MSCs producing fusogenic yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) was investigated in glioma, breast and gastric cancer cell lines. Due to the efficient conversion of 5-fluorocytosine, CDy::UPRT_AT-MSCs showed significant cytotoxic effects against human gastric cancer, breast cancer, and glioblastoma in vitro. More than 80% inhibition was observed in gastric MKN1 cell line when directly co-cultured with 1% therapeutic CDy::UPRT_AT-MSC / 5FC. Long-term expression was possible up to 7 days after transfection using a CpG-free expression plasmid. CDy::UPRT_AT-MSCs harvested 7 days after transfection showed efficient inhibition of 85% and 95% in gastric MKN1 and MKN28 cell lines, respectively. This result suggests that the method described here may provide an alternative process for MSC-based prodrug therapy without using viral vectors.

[0336] Accumulating evidence on the inherent tumor tropism of MSCs has opened up an emerging platform to utilize MSCs as cellular vehicles to deliver anticancer drugs, especially to tumors and their metastatic sites [9, 10]. Recently, numerous GDEPT clinical trials with MSCs have shown promising results that warrant further development into phase II clinical trials [7-11]. The therapeutic approach allows for enzymatic localized and controlled conversion of non-toxic prodrugs in the vicinity of target cells. The “bystander effect” enhances cytotoxicity against target cells [7]. The anticancer potential of CD4+-producing MSCs has been validated in a wide range of solid cancers, including gastric cancer [12-14], breast cancer [15, 16], and glioblastoma [17-19]. Preclinical studies have shown that cytosine deaminase / 5-fluorocytosine (CD4+ / 5FC) expression is highly robust, and as low as 4% of CD4+ positive cells in the tumor mass is sufficient for complete eradication of the tumor [20-22]. A significant advance in the CD / 5FC system was the inclusion of the pyrimidine salvage enzyme uracil phosphoribosyltransferase (UPRT), which bypasses the rate-limiting enzymes dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT) to directly convert 5FU to 5-fluorouridine monophosphate (FUMP) [23-26]. CD::UPRT / 5FC enhances the conversion of 5FC to its active metabolites by 30-1500-fold compared to CD / 5FC and 5FU [24, 27].

[0337] Transient transfection may have a high payload per cell, may induce cellular senescence

[40] , and may reduce tumor tropism

[41] , and avoids antibiotic selection and weeks of processing work, as well as safety concerns related to virus-induced MSC transformation

[42] . Although non-viral methods have advantages over viral vectors in terms of ease of production, low cost, and safety profile

[43] , their lack of widespread adoption for MSC modification is mainly due to the low transfection efficiency (0–35%) typically encountered [44, 45]. For example, due to the poor performance of chemical-based transfection methods (<5% efficiency)

[46] , human adipose tissue-derived MSCs (AT-MSCs) were engineered by retroviral transduction to express CD::UPRT [47, 48].

[0338] In this study, we demonstrated that it was possible to modify AT-MSCs and another MSC source with high efficiency using a cationic polymer in combination with TrafEn, enabling the development of therapeutic MSCs that produce CDy::UPRT without the need to use viruses or establish stable cell lines.

[0339] result An efficient non-viral LPEI-based transfection method for engineering AT-MSCs To evaluate the transfection efficiency of LPEI and Lipofectamine 3000 (L3K), AT-MSCs (age range 18-30 years) were transfected with a plasmid encoding a GFP reporter gene in 24-well tissue culture vessels. Although the number of transfected cells was higher when LPEI was used, the number of adherent cells was lower than when L3K was used (Figure 8A). Although cell viability remained high after transfection, the number of adherent cells was significantly reduced after LPEI-mediated transfection compared to the untransfected control. The number of adherent cells further decreased with the use of increasing amounts of pDNA (Figure 28), consistent with previous observations (Swiech et al., BMC Biotechnology, 11(114), 2011; McCall et al., Frontiers in Moleuclar Neuroscience, 5, (2012); Ho et al., Bioscience Reports, 38, 2018; Madeira et al., Journal of Biotechnology, 151, 130-136, 2011). Attempts to achieve increased adherent cell numbers by transfecting AT-MSCs with 200 ng of pDNA while using smaller amounts of polymer only resulted in a significant decrease in transfection efficiency (Figure 8B).

[0340] We then investigated the use of enhancer()35 for increased transfection with small amounts of pDNA (200 ng) and various DNA:polymer ratios (Figure 8b and Figure 29). More than 80% of AT-MSC cells were transfected (Figure 8B and Figure 29) with adherent cell numbers and viability (Figure 29) comparable to untransfected controls. We then extended the study to include another AT-MSC isolated from another donor (age range 31-45 years). Using the same protocol, transfection efficiency was as high as 90% of the cells transfected (Figure 9).

[0341] To establish a reliable protocol for genetic modification of AT-MSCs, transfection with LPEI and Lipofectamine 3000 was tested and compared with different DNA amounts. To check the efficiency of different parameters, AT-MSCs (age range 18-30 years) were transfected with a plasmid encoding a GFP reporter gene in 24-well tissue culture vessels (Figure 8A). Obviously, AT-MSCs were less easily transfected with Lipofectamine 3000. While LPEI outperformed Lipofectamine 3000 in terms of transfection efficiency, a significant drop in cell number was observed, suggesting a potential cell load.

[0342] However, attempts to transfect AT-MSCs with small amounts of pDNA and polymer to reduce cell load substantially reduced transfection efficiency (Figure 8B). Our previous work, which was dedicated to elucidating the intracellular trafficking pathways of polyplexes, led to the development of a TrafEn-based method

[54] . TrafEn can include histone deacetylase inhibitors (HDACi) and fusogenic lipids. In the study herein, we investigated the potential of this reagent in enabling efficient transfection with small amounts of pDNA and polymer. More than 80% of AT-MSC cells were transfected without cell load (Figure 8B). We then extended the study to include another AT-MSC isolated from another donor (age 31-45 years). Using the same protocol, we achieved a transfection efficiency close to 90% (Figure 9).

[0343] Human adipose tissue-derived mesenchymal stem cells (AT-MSCs, RoosterBio) were isolated from a female donor (LOT00088, age 18-30 years). AT-MSCs were maintained in hMSC High Performance Basal Medium (RoosterBio). Breast cancer cell line MDA-MB-231 (HTB-26, ATCC) and primary human dermal fibroblasts (ATCC, PCS-201-012) were cultured and maintained according to the manufacturer's instructions. Glioma cell line U-251MG was kindly provided by Paula Lam (Duke NUS Medical School). U-251MG cell line was cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS, Biowest). Gastric cancer cell lines MKN1 and MKN28 were kindly provided by Dr. Yong Wei Peng (National University Cancer Institute, Singapore). Gastric cancer cell lines were cultured in RPMI (Roswell Park Memorial Institute medium, Thermo Scientific) supplemented with 10% FBS. Cells were maintained at 37°C in a humidified atmosphere and 5% CO2.

[0344] Characterization of theranostic CDy::UPRT_AT-MSCs and identification of the functionality of CDy::UPRT_AT-MSCs To generate AT-MSCs expressing fused cytosine deaminase and uracil phosphoribosyltransferase (CDy::UPRT_AT-MSC), AT-MSCs were transfected with LPEI following the centrifugation protocol. Based on GFP analysis of AT-MSCs transfected with CDy::UPRT::GFP in the presence of TrafEn, a transfection efficiency close to 80 ± 2.3% was achievable (Figure 1A). As shown by flow cytometry analysis, the majority of transfected cells expressed high levels of GFP in the presence of TrafEn. Although increasing the amount of DNA improved the transfection efficiency of LPEI and Lipofectamine 3000, the performance was still insufficient (Figure 1B). These data suggest that TrafEn is required to promote intracellular trafficking of polyplexes in AT-MSCs

[49] . It was confirmed that CDy::UPRT expression was maintained for at least 7 days after transfection (FIG. 1C; FIG. 10).

[0345] Based on immunocytostaining analysis, transfection efficiency was significantly improved at small amounts of pDNA (200 ng) in the presence of the enhancer. In the absence of the enhancer, increasing the amount of pDNA slightly increased the transfection efficiency of LPEI and Lipofectamine 3000 (Figure 1). Extending this observation, we constructed a fusion gene encoding cytosine deaminase, uracil phosphoribosyltransferase, and green fluorescent protein (CDy::UPRT:GFP) for direct visualization and quantification. In the presence of the enhancer, transfection efficiency was significantly increased (about 80%) compared to the use of LPEI alone (about 40%, Figure 1), without significant changes in viability (Figure 30). Notably, there was no significant difference in the anticancer efficiency of AT-MSCs modified with CDy::UPRT:GFP or CDy::UPRT (Figure 31). Taken together, the results indicate a significant improvement in transfection of AT-MSCs by the enhancer, which likely shares a similar mechanism with the promotion of intracellular trafficking of pDNA in BM-MSCs (Ho et al., Nucleic Acids Research, 45(38), 2017).

[0346] The transgene of interest was introduced into AT-MSCs at passages 3-5. For each well (6-well plate format), LPEI (PEI MAX, Polyscience) 5mg / mL was added to pDNA in serum-free DMEM at different ratios of pDNA to PEIMAX. The mixture was incubated at room temperature for 15 minutes in a total volume of 100μL. The pDNA:LPEI ratio was calculated according to the amount of pDNA (μg):volume of LPEI 1mg / mL (μl). Then, to prepare the transfection mixture, the LPEI / pDNA complex was added (1:20) to serum-free DMEM medium. The culture medium was removed and replaced with the transfection mixture, followed by gentle centrifugation at 200g for 5 minutes. After centrifugation, the transfection mixture was removed and replaced with complete medium supplemented or not with TrafEn. TrafEn consists of DOPE / CHEMS (Polar Avanti Lipid) and Vorinostat (SAHA, Bio Vision). Cells were incubated for 24 hours before analysis.

[0347] Flow cytometry, Western blot, and immunocytostaining were performed as previously described

[49] . Briefly, flow cytometry: The percentage of fluorescent positive cells was quantified by an Attune NxT flow cytometer system (ThermoFisher Scientific), and raw data was analyzed using Invitrogen Attune NxT software (ThermoFisher Scientific). Imaging: Cell images were captured on an EVOS FL cell imaging system (ThermoFisher Scientific) equipped with three fluorescent cubes to examine DAPI (Ex357 / Em447), GFP (Ex470 / Em510) fluorescence. Western blot: Samples were analyzed by immunoblotting using sheep anti-CDy (PA185365, ThermoFisher Scientific) and monoclonal anti-β-actin (A2228, Sigma-Aldrich), respectively. Immunocytostaining: Samples were labeled with sheep anti-CDy and Alexa Fluor 488 donkey anti-sheep fluorescent secondary antibodies (A11015, ThermoFisher Scientific). Image acquisition was performed using an EVOS FL Cell Imaging System. All images were taken with the same optical settings.

[0348] Expression of CDy::UPRT sensitized AT-MSCs to the prodrug 5FC (exposure of CDy::UPRT-modified AT-MSCs to 5FC decreased cell viability over time) (Figure 2A), indicating the functionality of the phosphoribosyltransferase domain of the CDy transgene. Furthermore, CDy::UPRT_AT-MSCs showed increased susceptibility to the suicide effect in the presence of the active cytotoxic drug 5FU (Figure 2B). This effect was likely due to the activity of the UPRT transgene, which catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate

[25] . This is consistent with observations in other studies [19, 55].

[0349] The phenotypic properties of AT-MSCs are not affected by the LPEI-based transfection method. For future clinical applications, it is desirable to ensure that the quality of AT-MSCs remains unchanged after genetic modification. To exclude the possibility that high transfection efficiency in AT-MSCs was achieved at the expense of their quality, their characteristics were evaluated according to the main criteria defined by the International Society for Cellular Therapy (ISCT)

[51] . To investigate the possibility that high transfection may indeed modify the phenotype of AT-MSCs, immunophenotyping of CDy::UPRT_AT-MSCs was performed by standard FACS analysis using markers defined by the International Society for Cellular Therapy (ISCT)

[51] . The immunophenotype of CDy::UPRT_AT-MSCs was analyzed by standard FACS analysis for potential changes in the expression of surface markers, relative to unmodified AT-MSCs. CDy::UPRT_AT-MSCs showed an identical profile compared to unmodified AT-MSCs. Both cell types were found to be positive for CD90, CD73, and CD105, and negative for CD14, CD20, CD34, CD45, and HLA-DR (Figure 3A, Figure 27B). Expression of CDy::UPRT did not affect the differentiation potential of AT-MSCs toward osteogenic (Figure 3B, Figure 27A) and adipogenic (Figure 3C, Figure 27A) lineages. Notably, the presence of oil droplets in CDy::UPRT::GFP-expressing AT-MSCs provides direct evidence for the differentiation potential of AT-MSCs after transfection (Figure 11). The oil droplets suggested that the potential to differentiate toward the adipogenic lineage was not affected by transfection and transgene expression (Figure 11). In a separate study, chondrogenic differentiation was also not affected after transfection using this method (not shown).

[0350] To investigate the phenotype of CDy::UPRT-producing AT-MSCs, cells were labeled with an MSC phenotyping kit (Miltenyi Biotech) consisting of CD73, CD90, CD105, CD14, CD20, CD34, CD45, and HLA-DR antibodies according to the manufacturer's instructions. The expression of markers was then analyzed by FACS. A high-quality MSC population consists of >95% CD90, CD105, and CD73 positive cells. The population expressing CD14, CD20, CD34, CD45, and HLA-DR may be less than 1%

[51] . The multipotency of AT-MSCs was confirmed by their ability to differentiate into osteogenic and adipogenic lineages [52, 53]. Differentiation of AT-MSCs was induced by StemPro™ Osteogenic Differentiation Kit and StemPro™ Adipogenic Differentiation Kit (ThermoFisher Scientific). Unmodified AT-MSCs were used as controls. The phenotype and differentiation potential of CDy::UPRT producing AT-MSCs are not significantly different from unmodified AT-MSCs.

[0351] CDy::UPRT_AT-MSCs maintain their targeting for cancer cell lines in vitro The chemotactic response of AT-MSCs toward cytokines released by cancer cells is desirable for effective targeting of tumor cells

[10] . Therefore, it is desirable that genetic modification does not change the tropism of AT-MSCs toward cancer cells. Here, we used an invasion assay to examine the potential effect of LPEI-based transfection on the tumor tropism of AT-MSCs. We examined the directional migration of AT-MSCs through the extracellular matrix in the presence of cancer cells. The directional migration of AT-MSCs through the extracellular matrix indicates the tropism of AT-MSCs toward cancer cells. Invasion of AT-MSCs through the extracellular matrix was significantly induced by MDA-231-MB, U251-MG, and MKN1, but not by HEK293T (Figure 4A). This observation is consistent with other studies that utilized HEK293T as a non-cancer cell line control [56, 57]. The comparable migration numbers of AT-MSCs and CDy::UPRT_AT-MSCs suggest that the tumor homing ability was not affected by LPEI-mediated transfection and CDy::UPRT expression in AT-MSCs. To further confirm that AT-MSC migration depends on specific chemokines secreted by cancer cells, we hypothesized that a higher number of cancer cells would result in an increase in migrating AT-MSCs. Indeed, a significantly higher number of CDy::UPRT_AT-MSCs migrated toward U-251MG and MKN1. On the other hand, a moderate increase in migrated AT-MSCs was found in the condition with MDA-MB-231 cell line (Figure 4). The number of CDy::UPRT_AT-MSCs that invaded through the extracellular matrix depended on the number of cancer cells, with a higher number of cells migrating toward U-251MG and MKN1 and a lower number of cells migrating toward MDA-MB-231 cell line (Figure 4).

[0352] For each treatment, quadruplicates of AT-MSC, MKN1, MKN45, MDA-MB-231 (10,000 cells per well) and U-251MG (4000 cells per well) were plated in 96-well plates. After 24 hours, the culture medium was replaced with medium containing various concentrations of 5-fluorocytosine (5-FC, InvivoGen) or 5-fluorouracil (5FC, InvivoGen). After 1-5 days, the plates were subjected to the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). The colorimetric readout was measured spectrophotometrically at 490 nm. Results were expressed as the percentage of cell viability relative to cells in the condition without 5-FC or 5-FU (set at 100%).

[0353] An exemplary method for cell invasion assay is as follows: The tumor tropism of AT-MSC was measured using BD Biocoat™ Matrigel invasion chamber (BD Biosciences). Cancer cell lines or HEK293T cells were loaded into the lower well of a 24-well plate. After 24 hours, unmodified AT-MSC and CDy::UPRT-producing AT-MSC in serum-free DMEM were added onto the invasion chamber. The lower well was washed with 1×PBS, poured with serum-free DMEM, and assembled for invasion assay. After 24 hours of incubation, the non-invaded cells and Matrigel were removed from the inside of the insert. The invaded cells were stained with Hoechst 33342 (ThermoFisher Scientific) and photographed by an imaging system. The number of cells in three frames was counted.

[0354] CDy::UPRT_AT-MSC / 5FC-mediated cytotoxicity in vitro Proof of the cytotoxic effect of CDy::UPRT_AT-MSC on target cells is key to employing LPEI-based transfection / TrafEn in generating theranostic MSCs for prodrug cancer therapy. The action of cytosine deaminase / 5FC in growth inhibition is commonly evaluated by MTS assay. First, the anticancer efficiency of CDy::UPRT_AT-MSC / 5FC and 5FU was compared in glioma, breast and gastric cancer cell lines (Figure 12). The comparable anticancer effect of CDy::UPRT_AT-MSC / 5FC and 5FU suggests a high efficiency of converting 5FC into a cytotoxic drug. At a 1:1 ratio of CDy::UPRT_AT-MSC to cancer cells, the anticancer effect was comparable to the direct pharmacological effect of 5FU. To further investigate the therapeutic potential of CDy::UPRT_AT-MSC / 5FC, cells were co-cultured with target cancer cells at various ratios of MSC to cancer cells (Figure 5A). Even at a 1:50 co-culture ratio of CDy::UPRT_AT-MSC / 5FC with U251-MG, MDA-MB-231 and MKN1, approximately 57%, 69% and 89% growth inhibition could be observed, respectively. This ratio of mixed culture represents 2% therapeutic cells in the cancer cells. When 10% therapeutic cells were used, more than 86% growth inhibition was obtained in all cancer cells. It is noteworthy that 85% growth inhibition was observed in the MKN1 population with only 1% therapeutic cells. No growth inhibition was observed in the co-culture without 5FC, suggesting the lack of anti-cancer properties of AT-MSC (Figure 5B).

[0355] An exemplary method for the direct co-culture method is as follows: Quadruplicates of gastric and breast cancer cell lines (5000 cells) and U-251MG (2000 cells) were plated in a 96-well plate. After 5 hours, increasing numbers of either unmodified AT-MSCs or CDy::UPRT-producing AT-MSCs were added to the cancer cell cultures at ratios of 1 AT-MSC to 1, 5, 10, 50, and 100 cancer cells. After 1 day, the culture medium was replaced with 2% FBS-supplemented DMEM with or without 5-FC (0-150 μg / mL). After 5 days, cell viability was measured by proliferation assay. The condition without 5-FC was set as 100%.

[0356] Considering the situation when therapeutic cells do not directly contact with cancer cells in vivo, indirect co-culture experiments were used to obtain the cytotoxic effect of CDy::UPRT_AT-MSC / 5FC. Four days after exposure of MDA-MB-231 to CDy::UPRT_AT-MSC / 5FC, nearly 90% growth inhibition was observed (Figure 5C). In the absence of cell-cell adhesion, the anti-cancer efficiency of CDy::UPRT_AT-MSC / 5FC compares well with the direct co-culture model. Taken together, these data suggest that potential cytotoxic anti-cancer effects can be exerted when therapeutic cells are in contact with or in close proximity to target cells. Next, the study was extended to compare the sensitivity of normal mixed gastric cells (Hs738-nontransformed human fetal gastrointestinal cells) with five gastric cancer cell lines. CDy::UPRT_AT-MSC / 5FC selectively exerted cytotoxic anticancer effects against gastric cancer cell lines (Figure 13), suggesting specific targeting of the therapeutic cells / 5FC to cancer cells, but not to normal cells.

[0357] An exemplary method for the indirect co-culture method is as follows: MB-MDA-231 cells were plated in 24-well plates (5×10 per well). 4 Cells). AT-MSC or CDy::UPRT_AT-MSC (5 × 10 per well 4 Cells) were plated on transwells (Corning, C05 / 3422). After 6 hours of incubation, inserts containing therapeutic cells were transferred into wells containing MB-MDA-231 with or without 5FC. Cytotoxic effects were evaluated after 4 days of incubation. Transwells were removed and the culture medium was replaced with 1×PBS containing Hoechst 3222 1 μg / mL. Stained cells were analyzed using a Synergy H1 microplate reader at excitation and emission wavelengths of 358 nm and 461 nm, respectively. RFU was recorded in 9 areas of the cell culture with a gain setting of 80.

[0358] LPEI / TrafEn enhancer generates highly potent CDy::UPRT_AT-MSC We hypothesized that high expression of suicide genes may be important in the process of generating therapeutic AT-MSCs. We then compared the efficacy of therapeutic cells treated with different protocols tested by transfecting AT-MSCs with pCMV-GFP (Figure 8), or a CpG-free plasmid encoding CDy::UPRT (Figure 1). As expected, the anti-cancer efficiency of therapeutic cells prepared with different protocols significantly depended on the transfection efficiency of each protocol (Figure 6). The anti-cancer efficiency of CDy::UPRT_AT-MSCs generated in the presence of TrafEn (enhancer) significantly exceeds that of the alternative methods, especially in MB-MDA-231 and U251-MG. These two cell lines showed lower sensitivity to the toxicity of 5FU (Figure 12), and higher concentrations of 5FU were required for their growth inhibition. Complete growth inhibition was observed in all cancer cell lines co-cultured with CDy::UPRT_AT-MSCs generated in the presence of TrafEn at a ratio of 1 MSC to 10 cancer cells. It is noteworthy that the method described herein can be applied to MSCs obtained from various origins (Figure 14) and to other suicide genes, such as Herpes Simplex Virus Type 1 Thymidine Kinase (HSV-TK) (Figure 15). Transfection protocols using enhancers generated modified MSCs with similar efficacy regardless of the cell source (adipose-derived, bone marrow-derived or umbilical cord-derived MSCs, Figure 14). Furthermore, MSCs were successfully transfected with other suicide genes, such as Herpes Simplex Virus Type 1 Thymidine Kinase (HSV-TK) (Figure 15).

[0359] Long-term expression of CDy::UPRT in AT-MSCs by transient transfection Based on evidence of in vivo biodistribution of MSCs, it is expected that MSCs will require 1–4 days, depending on the route of administration and the site of the tumor, to disseminate among residual tumors and home to distant foci of tumors [29, 58, 59]. To ensure continuous expression of the suicide gene, viral transduction followed by antibiotic selection has been employed in the preparation of modified MSCs for prodrug therapy [19, 29, 60]. Continuous expression of CDy::UPRT was demonstrated up to 7 days after transfection (Figure 1C). To verify that modified AT-MSCs remained functional within a time frame appropriate for substantial anticancer effects, the cancer killing efficiency of modified MSCs harvested from days 1 and 7 after transfection was measured in parallel studies. Comparable growth inhibition was obtained with CDy::UPRT_AT-MSCs harvested at days 1 and 7 after transfection (Figure 7).

[0360] In this study, we intentionally chose a CpG-free plasmid because enhanced and prolonged expression was observed with CpG-free plasmids [61, 62]. In addition, the plasmid backbone used in this study contains a matrix attachment region (MAR) to improve the stability of gene expression

[63] . Indeed, when an expression plasmid lacking these two features was used, the expression of CDy::UPRT was severely reduced at 3 days after transfection. As expected, the anticancer efficiency of modified AT-MSCs harvested at 7 days after transfection was 400-fold lower than that of their counterparts harvested at 1 day after transfection (Figure 16). Although AT-MSCs modified in the presence of TrafEn showed significant anticancer efficacy compared to the alternative methods, the efficiency of growth inhibition decreased over time, suggesting that the bottleneck lies in the events following successful gene delivery. It is noteworthy that TrafEn enabled excellent anticancer effects despite the time-dependent decrease in the functionality of modified AT-MSCs.

[0361] Indeed, transient transfection allowed long-term expression of CDy::UPRT in AT-MSCs. To investigate the persistence of transgene expression and function in modified AT-MSCs, the anticancer efficiency of modified MSCs harvested from 1 and 7 days after transfection was examined. Clearly, the expression of the transgene CDy::UPRT was significant over a period of 7 days after transfection (Figure 7C), consistent with the observations using CD::UPRT::GFP (Figure 10). Comparable growth inhibition of cancer cells was observed by CDy::UPRT_AT-MSCs harvested 1 day (Figure 7A) or 7 days (Figure 7B) after transfection.

[0362] Administration of MSCs The method of administration of MSCs may depend on the type of cancer and / or the specific application. Described herein is the development of an upstream process for generating therapeutic MSCs for suicide prodrug therapy. Modified MSCs may be administered intravenously, for example, in adenocarcinoma treatment (TREAT-ME Phase 1 Study, Oncotarget., Oct. 6, 2017; 8(46):80156-80166). In certain embodiments, intracranial administration may be performed, for example, in patients with recurrent high-grade gliomas, intracranial administration of CD-NSCs was performed during tumor resection or biopsy (Clin Cancer Res., Jun. 15, 2017; 23(12):2951-2960).

[0363] Prodrug / gene combinations. This study further concerns a cytosine deaminase / uracil phosphoribosyltransferase fusion gene and 5-flucytosine system. A thymidine kinase / ganciclovir system was also tested. Again, the TrafEn method outperformed other non-viral methods in anti-cancer efficiency (Figure 14B). The method described here can be used as a platform for non-viral modification of various MSC types for suicide gene / pro-drug systems known in the art, for example, as listed in Table 3 of J Clin Invest., May 1, 2000, 105(9):1161-1167, which is incorporated herein by reference in its entirety.

[0364] Comparison of non-viral transfection with viral transfection The reagent formulation, TrafEn (described in PCT / SG2013 / 000464), allows for productive expression of transgenes with high copies of intracellular plasmid DNA, resulting in the generation of MSCs with high therapeutic payloads in this study. A notable advantage of the TrafEn system over viral methods is the early onset of transgene expression (Figure 23A) and significantly higher expression per cell (Figure 23B). This feature allows for a shortened cell preparation step and a higher payload of suicide genes per cell, potentially lowering production costs and the number of MSCs to be used for therapy.

[0365] Generation of stable cell lines. The generation of stable cell lines strives to obtain a large number of transfected cells. Antibiotic selection is very laborious (2-3 weeks) and can potentially compromise MSC quality

[88] , cause cell senescence

[88] , reduce tumor tropism

[89] , as well as raise safety concerns regarding virus-induced MSC transformation

[90] . Without antibiotic selection, low transfection efficiency may not be sufficient for clinical applications. Given the effective high transfection using the process developed and described herein, there is no longer a need to generate stable cell lines. With therapeutic genes subcloned into cpg-free vectors, high expression of CDUPRT over a 7-day period was as effective in eliminating cancer cells as expression 1 day after transfection (Figure 7A, Figure 7B). While selection is intended to be compatible with the transfection methods described herein, the transfected MSCs herein were not selected because the efficiency was >80%. It is contemplated that in certain embodiments, the generation of stable cell lines will not be necessary for therapy.

[0366] CD::UPRT_AT-MSC-Mediated Tumor Growth Inhibition In Vivo To examine this approach in vivo, CD::UPRT_AT-MSCs were injected directly into subcutaneous (sc) tumors. Considerable research with rather conflicting results has been noted regarding the use of unmodified MSCs for experimental cancer therapy (Christodoulou et al., Stem Cell Res Ther, 9(336), 2018). To ensure that the anti-cancer effect was due to the expression of CD::UPRT, a cell control group (MSC+5FC) was used in addition to a pro-drug control group (5FC). In this study, MSC+5FC did not exert any significant tumor-promoting or anti-tumor effects. A significant inhibition of tumor growth was observed in the treated group (Figure 32). With one cycle of treatment, an average of 45% reduction in tumor size was observed in the treated group 3 days after the last 5FC administration (day 7 in Figure 32A). Overall tumor size in the treated group was significantly smaller than the pro-drug and cell control groups (Figure 32B).

[0367] The results provide in vivo evidence of GDEPT effects in a subcutaneous mouse model - the glioblastoma cell line U251MG.

[0368] Consideration The therapeutic effect of MSC-mediated GDEPT has been demonstrated in various preclinical [9, 20, 21] and clinical trials [7, 9]. Due to the lack of efficacy of alternative non-viral based methods, viruses are used for genetic modification of MSCs. This study shows the use of a virus-free formulation and protocol to generate therapeutic AT-MSCs with high payload for prodrug cancer therapy. The beneficial results of the efficient transfection method were established in the substantial functional improvement in terms of anticancer efficacy of CDy::UPRT producing AT-MSCs. The method described herein does not alter the quality and characteristics of AT-MSCs and is applicable to various types of MSCs and therapeutic genes.

[0369] In the development process of MSC-based prodrug gene therapy, gene delivery methods play a crucial role. To engineer MSCs towards GDEPT, viral vectors are routinely used in preclinical studies and clinical trials [28-31]. Retroviruses are frequently used in the generation of CDy-expressing AT-MSCs [19, 47, 48, 60, 64]. Briefly, AT-MSCs were transduced with retrovirus-containing medium three times on three consecutive days. The cells were further expanded in the presence of antibiotic G418 for 10 days before use. An alternative method is the lentiviral gene delivery system. Efficiency up to 80% was reported with single transduction of AT-MSCs at an MOI of 150 [65, 66]. Unfortunately, polybrene can potentially inhibit MSC proliferation

[67] .

[0370] We report a facile PEI-based transfection method that is fast, simple, economical, and does not require antibiotic selection or multiple transfections compared to viral gene delivery systems. Since each cell can be transfected with several thousand copies of DNA [35, 36], the majority of cells were found to express high levels of the gene of interest (Figure 1, Figure 8B), which may explain the high efficacy of CDy::UPRT_AT-MSC. Indeed, CDy::UPRT_AT-MSC generated with PEI + enhancer showed anticancer efficiency comparable to 5FU, suggesting efficient conversion of the prodrug 5FC to a toxic drug (Figure 13). Complete inhibition of MD-MBA-231, U251-MG, MKN45, and MKN1 cell lines could be achieved using 10% therapeutic AT-MSC generated by PEI-based transfection in the presence of TrafEn without further purification or antibiotic selection after transfection (Figure 6C). On the other hand, the low transfection efficiency of PEI alone and Lipofectamine 3000 in AT-MSCs (Figure 1) resulted in low anti-cancer efficiency, especially in MD-MBA-231 and U251-MG cell lines (Figure 6).

[0371] The phenotype and properties of therapeutic AT-MSCs prepared by the method reported herein compare favorably with data presented in other studies in which AT-MSCs were retrovirally modified to express CDy or CDy::UPRT for prodrug cancer therapy [19, 47, 48, 60, 64]. The phenotypic markers, differentiation potential, and tumor tropism of CD::UPRT_AT-MSCs were evaluated against unmodified AT-MSCs, confirming that our method does not affect the quality of AT-MSCs (Figures 3, 4), a desirable feature for theranostic applications of modified AT-MSCs

[51] . Consistent with other reports [60, 68, 69], CDy::UPRT_AT-MSCs exerted cytotoxicity in indirect coculture experiments (Figure 5C), further confirming that cell-cell adhesion is not required for the bystander effect. It is also noteworthy that therapeutic MSCs are sensitive to the CDy::UPRT / 5FC system (Figure 2), thus preventing the long-term survival of therapeutic cells and fulfilling the requirement of cell vectors

[69] . At a ratio of 1 therapeutic MSC to 10 cancer cells, Kucerova et al. showed a remarkable 40% growth inhibition of MDA-MB-231 in the presence of CDy_AT-MSCs generated by retroviral transduction

[47] . Interestingly, modification of AT-MSCs with CDy::UPRT did not improve the anticancer effect

[16] , leading to the approach of a combinatorial prodrug therapy of CDy::UPRT / 5FC and HSV-TK / GCV. Surprisingly, at 10% therapeutic cells, we observed nearly 91% growth inhibition of MDA-MB-231 (Figure 5A), which may be due to the higher expression level of suicide genes per cell.

[0372] In a clinical trial of CDy expressing neural stem cells, recurrent glioma patients were given an 11-day treatment regimen

[70] . In in vivo animal studies, the duration of treatment ranged from 6 days [28, 29, 47] to 23 days

[29] depending on the cancer type. Often, modified MSCs were given weekly for a period of 3 weeks. In this study, we successfully demonstrated long-term expression of CDy::UPRT in AT-MSCs transfected with PEI+TrafEn without antibiotic selection for up to 7 days (Figure 1, Figure 7). Expression of the suicide gene in transiently transfected AT-MSCs could be sustained throughout the required duration of the treatment regimen

[70] . This endorses the adoption of non-viral gene delivery systems in the development of stem cell-based prodrug therapy. Apparently, AT-MSCs modified with CDy::UPRT::GFP showed anticancer efficiency comparable to CDy::UPRT (data not shown). Instead of antibiotic selection, which can potentially affect MSC quality

[40] , GFP tagging may be utilized for flow cytometric isolation of CD::UPRT-positive AT-MSCs, further streamlining the workflow for theranostic cell generation.

[0373] The accelerating number of gene and cell therapy clinical trials suggests a thrilling era that promises the emergence of treatment paradigms for diseases that previously lacked therapeutic options. This trend has resulted in a tremendous demand for clinical-grade virus manufacturing capabilities. The lack of virus production has been a bottleneck for the advancement and commercialization of cell and gene therapy

[71] . This study describes a useful tool for polymer-based ex vivo MSC modification that can be highly scalable and economical. The proposed workflow circumvents the limitations in viral vector supply and facilitates MSC modification without compromising the quality and anticancer efficacy of theranostic MSCs. Having an efficient, non-viral-based gene modification workflow, it is noticeable that the method can be useful in achieving further therapeutic effects via comfortable co-transfection of multiple anticancer genes, which may significantly broaden the perspective of therapeutic strategies for cancer treatment.

[0374] Conflicting reports on the role of MSCs in tumor inhibition and growth have recently emerged. This discrepancy appears to be mainly due to technical differences and inherent biological heterogeneity. Nevertheless, genetically modified MSCs are believed to provide a more suitable strategy for cancer treatment, because genetically modified MSCs are typically safer and more efficient than unstable and heterogeneous natural MSCs.

[0375] This study achieved effective modification of AT-MSCs with high efficiency without the use of viruses for the generation of theranostic AT-MSCs for prodrug cancer therapy. Approximately half of the cell population was transfected with a commercially available polymer (PEI-max), and the efficiency was significantly improved in the presence of enhancers with low toxicity (Figure 1a). This modification process did not require purification or antibiotic selection due to high expression of >70% of CD4+-expressing MSC cells, consistent with release testing for human clinical trials. Attempts to develop novel cationic polymers and lipids to modify MSCs have previously met with limited success due to low transfection efficiency or high cytotoxicity. Recently, poly(β-amino-ester) (PBAE) polymer constructs were reported to transfect MSCs with high efficiency and low toxicity. Although the cells were successfully modified, the migration ability was significantly affected.

[0376] In order to use AT-MSCs as therapeutic targeted drug delivery vehicles, it is desirable that the process used to modify AT-MSCs does not significantly alter their phenotypic characteristics and behavior, including their multipotency and migratory and invasive capabilities. The lack of significant differences in the expression of phenotypic markers and differentiation potential of modified and natural AT-MSCs (Figure 3) is an important criterion for theranostic applications of modified AT-MSCs. Intrinsic tumor tropism is a key feature for the homing / migratory properties of MSCs as cellular vehicles for delivering therapeutics. Despite the high degree of transgene overexpression, the migratory ability of modified cells was comparable to that of natural MSCs in the presence of cancer cells in vitro (Figure 4).

[0377] To improve the efficacy and safety of conventional cancer chemotherapy, a number of GDEPT systems have been utilized in cancer treatment. Among the enzyme / prodrug systems tested in recent studies, CD::UPRT was the most effective and was used with stem cells in clinical trials. In the present study, CD::UPRT modified cells efficiently inhibited the proliferation of MD-MBA-231, U251-MG, MKN45, and MKN1 cell lines by only 10% of the therapeutic AT-MSCs. It is noteworthy that MDA-MB-231 proliferation was inhibited by only about 90% at a ratio of 1 therapeutic MSC to 10 cancer cells (Figure 5A). In a similar ratio, Kucerova et al. demonstrated only 40% proliferation inhibition of the same cell type when using AT-MSCs modified by retroviral transduction (Kucerova et al., J Gene Med, 10, 1071-1082, 2008). However, another study reported a reduction of approximately 60% in cell numbers in co-cultures of MDA-MB-231 with virally transduced CDy::UPRT_MSCs at a ratio of 1 MSC to 4 cancer cells (Kucerova et al., Stem Cell Research, 8, 247-258, 2012). Intratumoral administration of CD::UPRT_AT-MSCs / 5FC demonstrated that MSCs modified by non-viral methods can exert a fairly high anti-cancer effect (Figure 32). In comparable study designs, Kwon et al. (Kwon et al., Clinical and Experimental Otorhinolaryngology, 6, 176-183, 2013) and Nouri et al. (Nouri et al., Journal of Controlled Release: Official Journal of the Controlled Release Society, 200, 179-187, 2015) reported inhibition of tumor growth, but not regression, with one and six doses of CD_MSC / 5FC treatment cycles, respectively. Without wishing to be bound by theory, it is believed that modifications using the process developed herein may result in increased payload, resulting in more effective killing of cancer cells.

[0378] MSC-mediated CD / 5FC treatment has been proposed as a strategy to overcome the systemic toxicity of 5-FU (You et al., Journal of Gastroenterology and Hepatology, 24, 1393-1400, 2009; Kwon et al., Clinical and Experimental Otorhinolaryngology, 6, 176-183). No significant changes in patient weight or other direct side effects were observed throughout the in vivo studies herein (data not shown), as shown in other studies (You et al., Journal of Gastroenterology and Hepatology, 24, 1393-1400, 2009; Kwon et al., Clinical and Experimental Otorhinolaryngology, 6, 176-183). Due to the mitigation of systemic toxicity, repeated injections of CD-MSCs may be possible to enhance anticancer activity. Moreover, it is noteworthy that therapeutic MSCs are sensitive to the CDy::UPRT / 5FC system (Figure 2), thus limiting the survival of therapeutic cells and fulfilling a key requirement of the “hit-and-run” strategy, without leaving any trace of cellular mediators (Mohr et al., Cancer Letters, 414, 239-249, 2018).

[0379] Depending on the route of administration and the site of the tumor, 1-4 days may be adequate for MSCs to biodistribute to residual tumors and home to distant foci of tumors (Aboody et al., Sci Transl Med, 5, 184ra159, 2013). In a recent clinical trial for advanced gastrointestinal cancer, patients received three treatment cycles with modified MSCs followed by a prodrug 48-72 hours later (von Elnem et al., Int J cancer, 2019). In parallel, a prodrug (5FC) was administered 4 days after administration of modified neural stem cells, and the modified cells were functional throughout the entire 7-day course of 5-FC (Portnow, Clinical Cancer Research: An Official Journal of the Americanl Association for Cancer Research, 23, 2951-2960, 2017). Thus, the transiently transfected AT-MSCs with long-term expression of CD::UPRT herein (see FIG. 7) are believed to be effective for the duration of the treatment regimen.

[0380] A slow spin process was used to reduce toxicity due to prolonged exposure to polyplexes (Ho et al., Nucleic Acids Research, 45(e38), 2017; Boussif et al., Gene Ther, 3, 1074-1080, 1996). In this study, we describe an in vitro, non-viral process with high efficacy and high cell viability using cationic polymers to engineer theranostic AT-MSCs for GDEPT. We showed that the phenotypic characteristics and migratory capacity of the modified cells were comparable to native MSCs, despite high overexpression of the transgene. These cells were highly efficient in inhibiting the proliferation of cancer cells in vitro. Thus, this process for modifying AT-MSCs could provide an effective and safer alternative to viral transduction for stem cell-based cancer therapy and could be useful for a wide range of applications.

[0381] Mesenchymal stem cells (MSCs) have emerged as promising vehicles for gene-directed enzyme prodrug therapy (GDEPT). Therapeutic efficacy can be improved using enhanced MSCs preconditioned with, for example, cytokines and / or growth factors, abiotic conditions, pharmaceuticals, and / or genetically modified and / or reprogrammed. The tumor-tropic properties of MSCs suggest that this vehicle provides an effective targeted therapy for tumors and metastatic diseases. A key step in the modification of MSCs is the delivery of genes with high efficiency and low cytotoxicity. Due to the low efficiency of conventional transfection methods, viral methods have been used to transduce MSCs in preclinical and clinical studies. The results herein show efficient transfection (>80%) of human adipose tissue-derived MSCs (AT-MSCs) using economical polyethylenimine in the presence of fusogenic lipids and histone deacetylase 6 inhibitors. Notably, the cell phenotype of MSCs remained unchanged after modification. AT-MSCs modified with the fusion transgene yeast cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT) showed potent cytotoxic effects against glioma, breast and gastric cancer cells in vitro. The efficiency of elimination of gastric cell lines was effective even using AT-MSCs 7 days after transfection, suggesting sustained expression and function of the therapeutic gene. Furthermore, significant regression of sc tumors was achieved by direct injection of a single dose of therapeutic MSCs. An efficient modification process for MSC-based prodrug therapy is provided herein as an alternative to the use of viral vectors.

[0382] In this study, a highly efficient modification process of AT-MSCs using cationic polymers in combination with enhancers is developed and described, enabling CDy::UPRT-producing theranostic MSCs without the need to use viruses or establish stable cell lines. Furthermore, this MSC modification process is donor-independent and can be used in a wide range of applications.

[0383] Methods and Materials cell culture Human adipose tissue-derived mesenchymal stem cells (AT-MSCs, RoosterBio) were isolated from a female donor (LOT00088, age 18-30 years). AT-MSCs were maintained in hMSC High Performance Basal Medium (RoosterBio). Breast cancer cell line MDA-MB-231 (HTB-26, ATCC) and primary human dermal fibroblasts (ATCC, PCS-201-012) were cultured and maintained according to the manufacturer's instructions. Glioma cell line U-251MG was kindly provided by Paula Lam (Duke NUS Medical School). U-251MG cell line was cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS, Biowest). Gastric cancer cell lines MKN1 and MKN28 were kindly provided by Dr. Yong Wei Peng (National University Cancer Institute, Singapore). Gastric cancer cell lines were cultured in RPMI (Roswell Park Memorial Institute medium, Thermo Scientific) supplemented with 10% FBS. Cells were maintained at 37°C in a humidified atmosphere and 5% CO2.

[0384] Construction of cpg-free expression plasmid containing CD::UPRT Plasmid DNA (pDNA) expressing fused cytosine deaminase and uracil phosphoribosyltransferase (4265 bp pSELECT-zeo-FcyFur (https: / / www.invivogen.com / pselect-zeo-fcyfur)) was purchased from InvivoGen. Construction of CpG-free expression plasmids of CD::UPRT was performed by cross-wrapping in vitro assembly (CLIVA) cloning technique

[50] . Briefly, Lucia in pCpGfree-Lucia plasmid (InvivoGen) was replaced with CD::UPRT using pSELECT-zeo-FcyFur as a template in polymerase chain reaction (PCR) (https: / / www.invivogen.com / pcpgfree). All pDNAs were propagated in Escherichia coli DH5α GT115 strain (InvivoGen) under selection with the antibiotic zeocin as indicated. Plasmids were purified using the EZNA endo-free plasmid maxi kit according to the manufacturer's instructions (Omega Bio-tek).

[0385] Transfection procedure Common centrifugation methods: The transgene of interest is introduced into AT-MSCs at passages 3-5. For each well (6-well plate format), 5 mg / mL LPEI (PEI MAX, Polyscience) was added to pDNA in serum-free DMEM at different ratios of pDNA to PEIMAX. In certain embodiments, the N / P ratio may range from 5 to 100 depending on the polymer selected. The mixture was incubated at room temperature for 15 min in a total volume of 100 μL. The pDNA:LPEI ratio was calculated according to the amount of pDNA (μg):volume of 1 mg / mL LPEI (μl). The LPEI / pDNA complex was then added (1:20) to serum-free DMEM medium to prepare the transfection mixture. The culture medium was removed and replaced with the transfection mixture, followed by gentle centrifugation at 200 g for 5 min. After centrifugation, the transfection mixture was removed and replaced with complete medium supplemented or not with TrafEn. TrafEn consists of DOPE / CHEMS (Polar Avanti Lipid) and vorinostat (SAHA, Bio Vision) in a ratio of 9:2, with SAHA at 1.25 μM. Cells were incubated for 24 hours before analysis.

[0386] The above protocols are generally exemplary / representative of the methods used in the experiments shown in Figures 1-16, 20 and 23 and may be subject to variation and modification depending on the particular experiment.

[0387] Common non-centrifugation methods: Cell Preparation: Cell Expansion- Culture medium was removed from the refrigerator and allowed to warm at room temperature. MSC vials (500,000 cells / vial) were removed from the liquid nitrogen dewar and immediately thawed in a 37°C water bath by vigorous agitation. Note: In certain embodiments, MSCs may be obtained from different origins / species / donors. The wells of the vials were sprayed with 70% alcohol before transferring to the safety cabinet. The cells were aseptically transferred to a 15 mL centrifuge tube. 4 mL of culture medium was slowly (dropwise) added to the cells. Centrifuge at 200×g for 5 minutes. The supernatant was carefully removed without disturbing the cell pellet. The cells were resuspended in 10 mL of culture medium. Mix well and seed the cells in a 1× T75 vessel. Cell growth was monitored using a microscope to ensure that the culture reached >80% confluence before harvesting. After 2 days the cells were ready to use.

[0388] Cell Preparation: Cell Growth- To harvest the cells, the vessel was transferred into a safety cabinet and the waste medium was removed. The medium was removed and rinsed once with 3 ml of 1x PBS. The wash was aspirated. 2 mL of Accutase was added to the flask and incubated at 37°C (3-5 min). Gently tapped to remove any remaining cells from the flask surface. 4 mL of fresh culture medium was added to quench the Accutase activity. The cell suspension was transferred to a 15 mL centrifuge tube. Centrifugation was performed at 200 x g for 5 min. The supernatant was aspirated and the cells were resuspended in 10 mL of fresh medium. Mix well and transfer 0.1 mL of cells to a microcentrifuge tube for cell counting. The cell count was 0.1-1 x 10 6 The cell suspension may be subcultured, frozen, or used for generation of CD::UPRT expressing MSCs. For subculture, a concentration of 5000-7000 cells / cm is required. 2 (cell culture surface). Replenish with fresh medium as appropriate. Cells may be subcultured up to passage 6. For cryopreservation, centrifuge cells at 200 x g for 5 minutes. Aspirate the supernatant and resuspend cells at 1 million cells / mL in KBM Banker 2. Aliquot 500 μL of suspended cells into each vial.

[0389] Generation of CD::UPRT expressing MSCs: Cell seeding - Optimal confluency of MSCs is around 60%. Cells were seeded 24 hours prior to transfection. Note: TrafEn™ Reagent is stable in the presence of serum and antibiotics. Standard culture medium can be used throughout the entire experiment. Recommended cell numbers to seed at least 2x T175 24 hours prior to transfection to prepare 10 million cells:

[0390] [Table 5]

[0391] * The cell number may vary due to different proliferation rates of MSCs.

[0392] Generation of CD::UPRT-expressing MSCs: DNA transfection protocol Preparation of reagents: Fusogenic lipid, 1st drug: supplied as 1x working stock. HDACi, 2nd drug: HDACi is dissolved in DMSO. Aliquot and store diluted solutions at -20°C.

[0393] Step 1: Complex formation Dilute 9-50 μg DNA in 1500 μL complexation buffer. Vortex for 5 seconds to mix. Add cationic polymer to diluted DNA. Vortex for 5 seconds to mix. * 1.5-30 µg polymer to 1 µg DNA. Incubate the transfection mixture at room temperature for 15 min.

[0394] Step 2: Preparation of TrafEn mixture Mix 0.2-1 mg of the first drug and 0.2-1 mg of the second drug during the incubation of the transfection mixture. Mix immediately by pipetting. Do not vortex. Incubate at room temperature for 10-20 minutes.

[0395] Step 3: Transfection Add 2500 μL of fresh culture medium to the transfection reagent / DNA mixture. Add 4200 μL of the transfection reagent / DNA mixture dropwise into the culture vessel. Do not remove the growth medium from the cells before adding the transfection reagent / DNA.

[0396] Add the TrafEn mixture dropwise to the culture vessel.

[0397] Mix by gently rocking the culture vessel back and forth and side to side.

[0398] The culture vessel is returned to the incubator.

[0399] Cells were used 24 hours after transfection.

[0400] Generation of CD::UPRT-expressing MSCs: Cell harvesting - To harvest the cells, transfer the vessel into a safety cabinet and remove the waste medium. Remove the medium and rinse once with 10 mL of 1x PBS. Aspirate the wash. Add 5 mL of Accutase to the flask and incubate at 37°C (3-5 min). Gently tap to remove residual cells from the flask surface. Add 10 mL of fresh culture medium to quench Accutase activity. Transfer the cell suspension to a 15 mL centrifuge tube. Mix well and transfer 0.1 mL of cells to a microcentrifuge tube for cell counting - total cell count for each flask can be approximately 5 million. Centrifuge at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells in 10 mL of 1x PBS. Centrifuge at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells in 10 mL of 1x PBS. Centrifuge at 200 x g for 5 min. Aspirate the supernatant. Resuspend the cells in the remaining PBS. The cells are ready for use.

[0401] The above protocols are generally exemplary / representative of the methods used in the experiments shown in Figures 17-19, 21-22, 24, and 26-27, and may be modified and adapted depending on the particular experiment. The experiments shown in the identified figures typically involve a 1.9 to 75 cm 2The experiment was carried out in a culture vessel.

[0402] In certain embodiments, non-centrifugation methods, such as those described above, may be suitable for large-scale MSC modification, e.g., for cells with a surface area of ​​about 175 cm. 2 Large scale operations of the invention may be amenable to such non-centrifugal methods.

[0403] Expression analysis Flow cytometry, Western blot and immunocytostaining were performed as previously described

[49] .

[0404] Flow cytometry: The percentage of fluorescent positive cells was quantified by an Attune NxT flow cytometer system (ThermoFisher Scientific), and raw data was analyzed using Invitrogen Attune NxT software (ThermoFisher Scientific).

[0405] Imaging: Cell images were captured with an EVOS FL Cell Imaging System (ThermoFisher Scientific) equipped with three fluorescence cubes for examining DAPI (Ex357 / Em447), GFP (Ex470 / Em510) fluorescence.

[0406] Western blot: Samples were analyzed by immunoblotting using sheep anti-CDy (PA185365, ThermoFisher Scientific) and monoclonal anti-β-actin (A2228, Sigma-Aldrich), respectively.

[0407] Immunocytostaining: Samples were labeled with sheep anti-CDy and Alexa Fluor 488 donkey anti-sheep fluorescent secondary antibodies (A11015, ThermoFisher Scientific). Image acquisition was performed using an EVOS FL Cell Imaging System. All images were taken with the same optical settings.

[0408] Characterization and differentiation potential of CDy::UPRT-producing AT-MSCs To investigate the phenotype of CDy::UPRT-producing AT-MSCs, cells were labeled with an MSC phenotyping kit (Miltenyi Biotech) consisting of CD73, CD90, CD105, CD14, CD20, CD34, CD45, and HLA-DR antibodies according to the manufacturer's instructions. The expression of markers was then analyzed by FACS. A high-quality MSC population consists of >95% CD90, CD105, and CD73 positive cells. The population expressing CD14, CD20, CD34, CD45, and HLA-DR may be less than 1%

[51] . The multipotency of AT-MSCs was confirmed by their ability to differentiate into osteogenic and adipogenic lineages [52, 53]. Differentiation of AT-MSCs was induced by StemPro™ Osteogenic Differentiation Kit and StemPro™ Adipogenic Differentiation Kit (ThermoFisher Scientific). Unmodified AT-MSCs were used as controls. The phenotype and differentiation potential of CDy::UPRT producing AT-MSCs should not differ significantly from unmodified AT-MSCs.

[0409] Cell viability assay For each treatment, quadruplicates of AT-MSC, MKN1, MKN45, MDA-MB-231 (10,000 cells per well) and U-251MG (4000 cells per well) were plated in 96-well plates. After 24 hours, the culture medium was replaced with medium containing various concentrations of 5-fluorocytosine (5-FC, InvivoGen) or 5-fluorouracil (5FC, InvivoGen). After 1-5 days, the plates were subjected to the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). The colorimetric readout was measured spectrophotometrically at 490 nm. Results were expressed as the percentage of cell viability relative to cells in the condition without 5-FC or 5-FU (set at 100%).

[0410] In vitro drug susceptibility For each treatment, quadruplicates of AT-MSC, MKN1, MKN45, MDA-MB-231 (10,000 cells per well) and U-251MG (5000 cells per well) were plated in 96-well plates. After 24 hours, the culture medium was replaced with medium containing various concentrations of 5-fluorocytosine (5-FC, InvivoGen) or 5-fluorouracil (5FC, InvivoGen). After 1-5 days, the plates were subjected to the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). The colorimetric readout was measured spectrophotometrically at 490 nm. Results were expressed as the percentage of cell viability relative to cells in the condition without 5-FC or 5-FU (set at 100%).

[0411] In vitro anti-cancer effect of CDy::UPRT-producing AT-MSCs Direct co-culture: quadruplicates of gastric and breast cancer cell lines (5000 cells) and U-251MG (2000 cells) were plated in 96-well plates. After 5 hours, increasing numbers of either unmodified AT-MSCs or CDy::UPRT-producing AT-MSCs were added to the cancer cell cultures at ratios of 1 AT-MSC to 1, 5, 10, 50, and 100 cancer cells. After 1 day, the culture medium was replaced with 2% FBS-supplemented DMEM with or without 5FC (0-150 μg / mL). After 5 days, cell viability was measured by a proliferation assay (commercial assay - https: / / www.promega.sg / products / cell-health-assays / cell-viability-and-cytotoxicity-assays / celltiter-96-non_radioactive-cell-proliferation-assay-_mtt_ / ?catNum=G4000). The condition without 5-FC was set as 100%.

[0412] Indirect co-culture: MB-MDA-231 cells were plated in 24-well plates (5 × 10 4Cells). AT-MSC or CDy::UPRT_AT-MSC (5 × 10 per well 4 Cells) were plated on transwells (Corning, C05 / 3422). After 6 hours of culture, inserts containing therapeutic cells were transferred into wells containing MB-MDA-231 cell line with or without 5FC. Cytotoxic effects were evaluated after 4 days of incubation. Transwells were removed and culture medium was replaced with 1×PBS containing Hoechst 3222 1 μg / mL. Stained cells were analyzed using a Synergy H1 microplate reader at excitation and emission wavelengths of 358 nm and 461 nm, respectively. RFU was recorded in 9 areas of the cell culture with a gain setting of 80. Growth inhibition after treatment is calculated relative to the control (co-culture of non-transfected AT-MSC cells with MB-MDA-231 cells).

[0413] In vivo anti-cancer effect of CDy::UPRT-producing AT-MSCs Female nude mice aged 5 to 6 weeks were purchased from InVivos and used for in vivo studies following an IACUC approved protocol (R18-1383). Mice were anesthetized by isoflurane inhalation and 5 × 10 6 Temozolomide-resistant U-251MG cells were injected sc into the dorsal lateral region (one tumor per mouse). Tumor growth was checked with digital calipers. Tumors grew to an average volume of 80–200 mm. 3 Treatment was initiated when the mice reached a size of 1×10. All mice were randomly distributed into three groups, each containing 5 mice. The prodrug control group was injected daily with the prodrug. The cell control group was injected daily with 1×10 6 In addition to intratumoral injections of 1×10 MSCs, the prodrug was injected daily. 6In addition to intratumoral injection of CD::UPRT_AT-MSC, prodrugs were injected daily. Modified or unmodified MSCs were administered intratumorally (single dose) on day 0. One day later, mice were administered ip with 500 mg / kg 5FC for 4 consecutive days. Tumor size and body weight were measured before cell injection (day 0) and on days 7, 11, and 15 after MSC administration.

[0414] Cell invasion assay The tumor tropism of AT-MSC was measured using BD Biocoat™ Matrigel invasion chamber (BD Biosciences). Cancer cell lines or HEK293T cells were loaded into the lower well of a 24-well plate. After 24 hours, unmodified AT-MSC and CDy::UPRT-producing AT-MSC in serum-free DMEM were added onto the invasion chamber. The lower well was washed with 1×PBS, poured with serum-free DMEM, and assembled for the invasion assay. After 24 hours of incubation, the non-invaded cells and Matrigel were removed from the inside of the insert. The invaded cells were stained with Hoechst 33342 (ThermoFisher Scientific) and photographed by an imaging system. The number of cells in three frames was counted.

[0415] statistical analysis When using Student's t-test, unpaired two-tailed tests were used and changes in readouts were assumed to follow a normal distribution.

[0416] Abbreviation MSC: mesenchymal stem cells, PEI: polyethyleneimine, HDAC6i: histone deacetylase 6 inhibitor, CD::UPRT: fusion protein of yeast cytosine deaminase::uracil phosphoribosyltransferase, CD: cytosine deaminase, GDEPT: gene-directed enzyme prodrug therapy, UPRT: uracil phosphoribosyltransferase, FUMP: 5-fluorouridine monophosphate, 5FC: 5-fluorocytosine, 5FU: 5-fluorouracil, DPD: dihydropyrimidine dehydrogenase, OPRT: orotate phosphoribosyltransferase, GFP: green fluorescent protein, AT-MSC: human adipose tissue-derived MSC, DMEM: Dulbecco's modified Eagle's medium, FBS: fetal bovine serum, pDNA: plasmid DNA, MOI: multiplicity of infection, HSV-TK: herpes simplex virus type 1 thymidine kinase.

[0417] Example 2 A scalable method for highly efficient transfection of MSCs Methods that omit centrifugation may improve scalability. However, it may result in extended incubation times, which can be a challenge if the cationic polymer is not appropriately selected. Process development to optimize the protocol for each MSC donor / type may be desirable. In this study, we identified key steps that enable the generation of clinically useful modified MSCs for therapeutic purposes (Figure 17). Examples of desirable features of the protocol are listed in Table 5.

[0418] [Table 6]

[0419] Scalability An important aspect for clinical application of MSC prodrug gene delivery is the production scalability of the gene delivery method. Previous reports on TrafEn to enhance centrifugation-based polymer transfection had limited scalability. This study describes the addition of polyplexes and TrafEn directly to any culture vessel, without the need for centrifugation. The particular difference with this development is the duration of exposure to polymer and TrafEn. In this study, the appropriate formulation of polymer and TrafEn can be incubated with cells without the need for centrifugation. It may provide a means to scale up production, since the need to centrifuge large containers is neither necessary nor convenient (Figure 24A-B). It is noteworthy that the transfection efficiency remained unchanged regardless of the surface area of ​​the cell culture, suggesting the feasibility of transferring the enhancing effect of TrafEn to preclinical and clinical scale.

[0420] To scale up the production of these highly transfected MSCs, it is desirable to achieve compatibility of cationic polymers with TrafEn. In an exemplary process development (Figure 17), various linear (LPEI) and branched (BPEI) polymers with molecular weights (MW) ranging from <4 kDa to 200 kDa were evaluated for high transfection efficiency and low cytotoxicity without the need for centrifugation. Depending on the MSC type, donor and culture conditions, the most appropriate composition can be selected and then enhanced using TrafEn (e.g. Figure 20). Thus, a key step is the identification of a compatible polymer that can be enhanced with TrafEn.

[0421] In certain embodiments, the polymer type, polymer structure (linear, branched), and / or polymer size may be selected or adapted for individual cell types. As an example, the results in FIG. 20 suggest that MSCs in FIG. 20A prefer large polymers and MSCs in FIG. 20B prefer small polymers. In certain embodiments, the MSCs may comprise UC-MSCs and the polymer may comprise a polymer greater than about 50 kDa, between about 50 kDa and about 200 kDa, or greater than about 200 kDa. In certain embodiments, the MSCs may comprise BM-MSCs and the polymer may comprise a polymer less than about 50 kDa, between about 50 kDa and about 5 kDa, or less than about 5 kDa. In certain embodiments, the polymer may comprise LPEI.

[0422] Low toxicity and high efficiency With reference to Figure 26, for successful use of MSCs in prodrug therapy, cell health is desirable to ensure functionality of MSCs during treatment. At DNA amounts below 350ng, maximum transfection efficiency (>90%) was achieved without significant loss of cell viability as shown by propidium iodide exclusion assay (Figure 26). This data suggests the robustness of the embodiment of the workflow to modify MSCs with high efficiency without introducing significant cytotoxicity to MSC cultures.

[0423] DNA optimization (1 in Figure 17) DNA content Large amounts of DNA can result in cytotoxicity. The DNA range used for MSC transfection was 1.9 cm. 2 The results show that the amount of DNA ranges from 100 to 500 ng per surface area of ​​100 μg / mL. As shown in Figure 18, increasing the amount of DNA was not beneficial due to high cytotoxicity.

[0424] DNA vector design The use of appropriate DNA vector design can prolong transgene expression. The goal of stable cell line generation is to obtain a large number of transfected cells. Antibiotic selection is very labor-intensive (2-3 weeks) and can potentially compromise MSC quality

[17] , induce cell senescence

[17] , reduce tumor tropism

[18] , and raise safety concerns regarding virus-induced MSC transformation

[19] . Without antibiotic selection, the low transfection efficiency of conventional methods may not be sufficient for clinical applications.

[0425] Given the effective high transfection using the process developed and described herein, the generation of stable cell lines may not be necessary. With therapeutic genes subcloned into CpG-free vectors, high expression of CDy::UPRT over a 7-day period was relatively effective in eliminating cancer cells compared to expression 1 day after transfection (Figure 7). Long-term expression was observed using a transfection protocol that did not include centrifugation (Figure 19). Expression of CDy::UPRT significantly decreased over time, resulting in a reduced anticancer effect of modified MSCs (Figure 16).

[0426] In addition to removing one or more CpG islands from the plasmid construct, the method was compatible with the use of other plasmids known in the art, such as those listed in Jackson DA et al., Designing Nonviral Vectors for Efficient Gene Transfer and Long-Term Gene Expression, Molecular Therapy, 14:613-26, which is incorporated herein by reference in its entirety. Suitable vectors may include those vectors shown to provide long-term expression, such as scaffold / matrix attachment region (S / MAR), episomal vectors, and EBNA-1 containing vectors.

[0427] Polymer optimization (Figure 17-2) In an embodiment of the invention, a suitable polymer can be incubated with cells without the need for centrifugation. This can provide a means to scale up production, since the need to centrifuge large containers is not necessary or convenient. Extension of exposure and incubation time of cells and transfection mixture beyond 20 minutes can introduce cytotoxicity. Certain polymers can be cytotoxic under certain conditions. For example, Ho et al., Enhanced transfection of a macromolecular lignin-based DNA complex with low cellular toxicity, Biosci. Rep. (2018) 38:1-9, faced toxicity with lignin-PGEA-PEGMA.

[0428] To ensure higher quality and higher transfection efficiency, the selection of a compatible polymer with the cell type may be desirable. Various linear (LPEI) and branched (BPEI) polymers with molecular weights (MW) ranging from 2 kDa to 200 kDa were evaluated for high transfection efficiency and low cytotoxicity without the need for centrifugation. Depending on the MSC type, donor and culture conditions, the most appropriate composition was selected and then enhanced using TrafEn. For example, Figure 19 shows that LPEIs of <200 kDa and <5 kDa were compatible with umbilical cord MSCs (UC-MSCs) and BM-MSCs, respectively.

[0429] In addition to polymer compatibility, optimization of the amount of polymer can enhance cell viability. Cytotoxicity is highly related to the amount of polymer and DNA, with increasing amounts resulting in increased toxicity, as indicated by a lower % of propidium iodide (PI) negative cells. When the commercial polymer content and concentration were unknown (e.g., reagents such as Turbofect, Polyfect, and Transficient), a decrease in cell viability was observed with increasing amounts of polymer. For PEI-based polymers, amounts of polymer ranging from 1 μg to 30 μg relative to 1 μg of DNA were tested (Figures 20, 21).

[0430] This study yielded an exemplary method for highly efficient transfection of MSCs, which may include the following steps.

[0431] Cell preparation Cell expansion: Remove the culture medium from the refrigerator and allow to warm at room temperature. Remove the MSC vials (500,000 cells / vial) from the liquid nitrogen dewar and immediately thaw in a 37°C water bath by vigorous agitation. MSCs can be obtained from different origins / species / donors. Spray the wells of the vials with 70% alcohol before transferring to a safety cabinet. Aseptically transfer the cells to a 15 mL centrifuge tube. Slowly (dropwise) add 4 mL of culture medium to the cells. Centrifuge at 200 x g for 5 min. Carefully remove the supernatant without disturbing the cell pellet. Resuspend the cells in 10 mL of culture medium. Mix well and seed the cells into 1 x T75 vessels. Observe cell growth using a microscope and ensure the culture reaches >80% confluence before harvesting. After 2 days the cells are ready to use.

[0432] Cell expansion: To harvest the cells, transfer the vessel into a safety cabinet and remove the spent medium. Remove the medium and rinse once with 3 mL of 1x PBS. Aspirate the wash. Add 2 mL of Accutase to the flask and incubate at 37°C (3-5 min). Gently tap to remove any remaining cells from the flask surface. Add 4 mL of fresh culture medium to quench the Accutase activity. Transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 200 x g for 5 min. Aspirate the supernatant and resuspend the cells in 10 mL of fresh medium. Mix well and transfer 0.1 mL of cells to a microcentrifuge tube for cell counting. Cell numbers should be in the range of 0.1-1 x 106 cells / mL. The cell suspension can be used for subculture, freezing, or for generation of CD::UPRT expressing MSCs. For subculture, 5000-7000 cells / cm 2 (cell culture surface). Replenish with fresh medium as appropriate. Cells can be subcultured up to 6 passages. For cryopreservation, centrifuge cells at 200 x g for 5 minutes. Aspirate the supernatant and resuspend cells at 1 million cells / mL in KBM Banker 2. Aliquot 500 μL of suspended cells into each vial.

[0433] Generation of CD::UPRT-expressing MSCs Cell seeding: The optimal confluence of MSCs is about 60%. Cells are seeded 24 hours before transfection. Note: TrafEn™ Reagent is stable in the presence of serum and antibiotics. Standard culture medium can be used throughout the experiment.

[0434] DNA Transfection Protocol Preparation of reagents: Fusogenic lipid, 1st drug: supplied as 1x working stock. HDACi, 2nd drug: HDACi is dissolved in DMSO. Aliquot and store diluted solutions at -20°C.

[0435] Step 1: Complex formation Dilute 9-50 μg DNA in 1500 μL complexation buffer. Vortex for 5 seconds to mix. Add cationic polymer to diluted DNA. Vortex for 5 seconds to mix. * 1.5-30 µg polymer to 1 µg DNA. Incubate the transfection mixture at room temperature for 15 min.

[0436] Step 2: Preparation of TrafEn mixture Mix 0.2-1 mg of the first drug and 0.2-1 mg of the second drug during the incubation of the transfection mixture. Mix immediately by pipetting. Do not vortex! Incubate at room temperature for 10-20 minutes.

[0437] Step 3: Transfection Add 2500 μL of fresh culture medium to the transfection reagent / DNA mixture. Add 4200 μL of the transfection reagent / DNA mixture dropwise to the culture vessel. Do not remove the growth medium from the cells before adding the transfection reagent / DNA. Add the TrafEn mixture dropwise to the culture vessel. Gently rock the culture vessel back and forth and side to side to mix. Return the culture vessel to the incubator. 24 hours after transfection, the cells are ready to use.

[0438] Cell harvest: To harvest cells, transfer vessels into a safety cabinet and remove waste medium. Remove medium and rinse once with 10 mL 1x PBS. Aspirate wash. Add 5 mL Accutase to flask and incubate at 37°C (3-5 min). Gently tap to remove residual cells from flask surface. Add 10 mL fresh culture medium to quench Accutase activity. Transfer cell suspension to a 15 mL centrifuge tube. Mix well and transfer 0.1 mL of cells to a microcentrifuge tube for cell counting - total cell count for each flask should be approximately 5 million. Centrifuge at 200 x g for 5 min. Aspirate supernatant and resuspend cells in 10 mL 1x PBS. Centrifuge at 200 x g for 5 min. Aspirate supernatant and resuspend cells in 10 mL 1x PBS. Centrifuge at 200 x g for 5 min. Aspirate supernatant. The cells are resuspended in the remaining PBS and are ready to use.

[0439] Example 3 Development of transfection methods and processes for any of a variety of MSC cell types Process optimization of one or more steps may be desirable to empirically identify conditions for TrafEn to enhance MSC transfection, as shown in Figure 17. We have found that the method provides >70% efficiency in expression duration, scalability, and increased quality of modified MSCs after modification.

[0440] Different MSC types can be efficiently transfected. Various types of MSCs have been used for cancer treatment (Table 2). The method was validated in various MSC types (bone marrow, adipose-derived, umbilical cord) for prodrug gene therapy (Figure 14B). The embodiments of the method described herein can be used for any of the MSC types, for example, adipose MSCs, BM MSCs (e.g., Roosterbio), UC MSCs (e.g., ATCC, cell applications), umbilical cord lining MSCs (e.g., cell research corporation).

[0441] Process development for any of the various MSCs (Figure 25) Cellular diversity With reference to FIG. 14B, we show the comparable transfection and anti-cancer efficiencies in stem cells from different origins. Adipose tissue-derived MSCs (AT, Roosterbio), bone marrow-derived MSCs (BM, Roosterbio), and UC-derived MSCs (umbilical cord, ATCC) were transfected in the presence of TrafEn by a centrifugation protocol. 24 hours after transfection, cells were trypsinized and harvested for Western blot analysis (FIG. 14A). Cells were lysed for immunoblotting analysis with antibodies targeting CDy and actin. In the same experiment, cells were harvested for co-culture studies with various cancer cell lines at a ratio of 1 MSC to 50 cancer cells (FIG. 14B). Cells were co-cultured for 5 days in medium containing 100 μg / mL 5FC. At the end of the incubation, the number of remaining cells was assessed spectrophotometrically by measuring the RFU of cells stained with Hoechst 33342 at wavelengths Ex340 / Em488. Conditions with unmodified MSCs served as controls. Percentage of growth inhibition was calculated accordingly. Graphs represent data collected from quadruplicates, mean + SEM.

[0442] Selection of TrafEn In certain embodiments, the selection of TrafEn may be based on [1] transfection efficiency, [2] cell viability, or both.

[0443] In certain embodiments, TrafEn-compatible transfection reagents may be selected / optimized by screening a library of polymers, which may include commercially available polymers, e.g., Turbofect (ThermoScientific), Jetprime (Poplyplus transfection). PEI is one example of a polymer identified herein.

[0444] Selection of Nucleic Acid Constructs The design and amount of DNA may be selected and optimized based on a particular application. In certain embodiments, the DNA design may be based on [1] duration of expression, [2] transfection efficiency, or both. The amount of DNA may be optimized based on [1] transfection efficiency, cell viability, or both.

[0445] Selection of cationic polymer The cationic polymer may be selected and optimized based on the particular application, in certain embodiments, by screening a library of available polymers ranging in size from about 4 kDa to about 200 kDa.

[0446] The polymer ratio may be selected and optimized based on the particular application, in certain embodiments, the polymer ratio may be selected and optimized by testing within a range of about 5-100 of N / P.

[0447] In certain embodiments, the type and ratio of polymers may be selected based on a balance between transfection efficiency and cell viability.

[0448] In certain embodiments, selection and / or optimization may be performed for a particular gene-based application, and may include polymer screening, DNA amount screening, and polymer / DNA ratio screening. The results of the screening may determine the protocol / workflow based on the results of [1] transfection efficiency >70% and [2] cell viability >70%.

[0449] optimization Culture conditions Cell growth (seeding): Optimal confluency of MSCs may be around 60%. MSCs may be seeded 24 hours prior to transfection. Note: TrafEn™ Reagent is stable in the presence of serum and antibiotics. Standard culture medium can be used throughout the entire experiment.

[0450] Cell health: After transfection, cell viability is preferably >70% as defined by propidium iodide assay. Cell mass is preferably not significantly sacrificed in terms of differentiation potential and phenotypic markers.

[0451] Cell density: Cell density may typically range from about 60-90% in certain embodiments. For example, transfection may be tested and optimized at densities of about 60%, about 70%, about 80%, or about 90%.

[0452] Passage number: In certain embodiments, efficiency can be consistent for MSCs from passages 1-25, as long as the MSCs are not senescent.

[0453] Optimization of DNA transfection: Complexation: For example, dilute 9-50 μg of DNA in 1500 μL of complexation buffer. Vortex for 5 seconds to mix. Add cationic polymer to the diluted DNA. Vortex for 5 seconds to mix. * 1.5-30 μg polymer to 1 μg DNA. Incubate the transfection mixture at room temperature for 15 minutes. Optimization can be based on DNA amount. Approximately 1.9 cm 2 For a surface area of ​​about 100 to about 500 ng of DNA, amounts of DNA greater than about 500 ng may, in certain embodiments, be toxic to stem cells of certain embodiments.

[0454] Preparation of TrafEn mixture: During incubation of the transfection mixture, mix, for example, 0.2-1 mg of the first and second agents. Of course, in certain embodiments, the amount and / or ratio of the first and second agents may be varied based on the cell type used. For example, in certain embodiments, the ratio of fusogenic lipids to helper lipids may be varied. The second agent may include, for example, an HDACi that targets HDAC6. The first and second agents may include those described in WO 2014 / 070111, which is incorporated herein by reference in its entirety.

[0455] Transfection medium: For example, 2500 μL of fresh culture medium is added to the transfection reagent / DNA mixture. The transfection medium may be used for the preparation of DNA-polymer complexes. For example, DNA and polymer may be added to the transfection medium and incubated for about 10 to about 45 minutes. After incubation, the transfection mixture may be added directly to the cell culture.

[0456] Transgene vector design: Transgene vectors can vary. Generally, a transgene vector can include a promoter and a transgene. For long-term expression, codon optimization to remove CpG islands, addition of modules such as S / MAR, and / or promoter optimization may be used.

[0457] Incubation period: In certain embodiments, the incubation may be at about 37 degrees Celsius for, for example, about 2 to about 48 hours, or about 2 to about 24 hours.

[0458] The range of various conditions including DNA amount, polymer, cell density and TrafEn formulation may be determined accordingly and adapted to the MSC source. After process optimization, the product may include a specific workflow / protocol and optimized reagents (plasmid DNA, polymer, TrafEn) that can be formulated in kit form in certain embodiments. Such an approach provides process robustness and / or facilitates good reproducibility of transfection results in different laboratories and settings.

[0459] Example 4 Additional results for non-spin protocols The anticancer efficacy of CD::UPRT_MSCs was examined (in vitro and in vivo data) using a temozolomide-resistant glioblastoma model. The MSC source was adipose tissue-derived MSCs (AT-MSCs) from Roosterbio.

[0460] Consistent with the release criteria for the use of virally modified MSCs for cancer therapy (EU Clinical Trial Registration Number: 2012-003741-15), comparable levels of cell modification (>75%) and cell viability (>80%) by any non-viral gene delivery strategy are highly desirable. Using TrafEn-mediated transfection, the protocol showed a transfection efficiency of >90% with a cell viability of >80% (Figures 33A and 33B, note that modified MSCs were prepared according to the non-centrifugation protocol described above, although both centrifugation and non-centrifugation protocols can be used). This exceeded the specified threshold of 80% cell viability based on the TREATME-1 protocol recently used in clinical trials (Niess, 2015; von Elnem, 2019). It was achieved 2 days after transfection without the need for antibiotic selection. Furthermore, the results showed a correlation of transfection efficiency with the cancer cell killing ability of the modified MSCs (Figure 33C and Figure 33D), confirming that TrafEn can be used to generate economical, off-the-shelf style allogeneic MSC-GDEPT.

[0461] After obtaining a large number of CD:UPRT:GFP_MSCs, the properties of the modified stem cells were evaluated. According to the properties defined by ISCT (Dominici, Cytotherapy 8, 315-317, 2006), modified MSCs should maintain CD marker expression and differentiation potential. Regarding the characterization of CD markers, similar to native MSCs, more than 95% of CD:UPRT:GFP_MSCs expressed CD73, CD90 and CD105, and less than 2% of cells expressed CD14, CD20, CD34 and CD45 (Figure 34A). After induction, CD:UPRT:GFP expressing cells were able to differentiate into both osteogenic and adipogenic lineages (Figure 34B), properties similar to native MSCs. After modification of AT-MSCs, no significant differences in tumor tropism were observed between native and transfected AT-MSCs (Figure 34C). AT-MSCs were found to migrate specifically to cancer cell lines in preference to non-cancerous cells, i.e., fibroblasts. This data suggests that TrafEn-mediated transfection does not affect the MSC phenotype as a cellular vector for tumor targeting.

[0462] Treatment of glioblastoma multiforme (GBM) is a major challenge and unmet need due to the high rate of recurrent GBM with temozolomide (TMZ) resistance. GDEPT could potentially be offered as a second-line therapy for TMZ non-responders. Herein, we verified whether the non-viral CD:UPRT:GFP_MSC / 5-FC system is effective against temozolomide (TMZ)-resistant glioma cell lines. Both parental and TMZ-resistant U251-MG cell lines were sensitive to their modified MSCs plus 5-FC (Figure 35A). Cell viability of the co-culture increased in an MSC dose-dependent manner. Higher cell viability was observed with decreasing MSC-to-cancer ratio, suggesting decreased killing. Similar observations were observed in U87-MG and U87-MGTMZR40 (Figure 35B). We next verified whether it is true for glioma cell lines derived from HGCC patients, previously reported to be TMZ-resistant. Similarly, efficient killing of HGCC glioma cell lines is observed (Figure 35C). To confirm the toxicity of this system to non-cancerous cells, we examine the cytotoxicity of CD:UPRT:GFP_MSC / 5-FC in fibroblast cultures. Interestingly, fibroblasts were less sensitive, 47.78±2.93%, even at a 1:1 ratio of MSC to fibroblasts (Figure 35D).

[0463] Next, TMZ-resistant cells, U251-MG TMZR40 The in vivo cytotoxic effect of prodrug therapy was investigated in a subcutaneous mouse model using CD:UPRT:GFP / MSC. Mice showed a significant reduction in tumor volume with a single intratumoral administration of cells. The difference between the modified cell treatment group and the native cell control group was significant as early as 7 days after treatment and was maintained over a 15-day period (Figure 36A). Tumors were harvested 15 days after treatment and a significant difference in tumor burden was observed between native and modified MSC (Figure 36B). Interestingly, the increased dose (0.5×10) with modified cells reduced tumor volume. 6 vs 1×10 6) showed similar changes in tumor volume (Figure 36A, grey and light blue circles, and Figure 36B). An important finding of this study was that there appeared to be no significant systemic toxicity, as measured by changes in mouse body weight (Figure 36C). After completion of three cycles of treatment, long-term tumor suppression was observed (Figure 37).

[0464] Example 5 Large Animal Study- Multi-Site Investigational Study A large animal study was conducted. The primary objectives achieved in this study were: 1) No significant side effects 2) Reduction in tumor size 3) Improved quality of life 4) Life extension.

[0465] Modified MSCs were prepared by expansion to 20-30 million cells in a 500 cm2 culture vessel following the non-centrifugation protocol described above.

[0466] Animal enrollment was not related to tumor type or age of the dog. Inclusion criteria included animals with biopsy data. Exclusion criteria included patients with tumors that could not be sampled or with systemic illness (e.g., significant fever, immunosuppression, organ failure). The anticancer effect of GDEPT was demonstrated in dogs and cats presenting with various cancers, including lymphoma (lymphadenopathy), thyroid cancer, melanoma, perianal cancer, soft tissue cancer, nasal cancer, gastrointestinal cancer, and lymphoma (blood-borne). In all cases, blood tests were performed before and after each cycle, with no significant changes in BUN and ALKP values. Overall, the clinical picture suggests the clinical usefulness and excellent safety profile of CD::UPRT_MSC / 5FC described herein.

[0467] The data provided herein are representative cases of each cancer type. Figure 38 shows perianal cancer data, Figure 39 shows oral melanoma data, Figure 40 shows thyroid cancer data, Figure 41 shows soft tissue sarcoma (cancer ulceration) data, Figure 42 shows nasal tumor data, and Figure 43 shows gastrointestinal cancer data.

[0468] FIG. 38 shows treatment data for perianal cancer. Route of administration was intratumoral injection of canine CD::UPRT::GFP_MSC. Latest update (January 2020): Survival, no recurrence reported. FIG. 39 shows treatment data for oral melanoma. Route of administration was intratumoral injection of canine CD::UPRT::GFP_MSC. Latest update (January 2020): Survival. FIG. 40 shows treatment data for thyroid cancer. Route of administration was intratumoral injection of canine CD::UPRT::GFP_MSC. Latest update (June 2019): Survival. FIG. 41 shows treatment data for soft tissue sarcoma (cancer ulceration). Route of administration was intratumoral injection of canine CD::UPRT::GFP_MSC. Latest update (November 2018): Survival, no recurrence reported. Ultrasound report on November 14, 2018 showed the presence of a well-defined hypoechoic round mass with dimensions of 4 x 3 x 2 cm in the left anal region. No adhesion to surrounding or deep organs. No metastasis was found, especially to the sublumbar lymph nodes. Only a few tiny urinary stones of 1.5 mm were present in the bladder and only a few in the prostatic urethra. Other organs are normal. Complete remission to date. Figure 42 shows the treatment data of the nasal tumor. The route of administration was intratumoral injection of canine CD::UPRT::GFP_MSC. Latest update (January 2020): Survival. Figure 43 shows the treatment data of the gastrointestinal cancer. The route of administration was intravenous infusion of canine CD::UPRT::GFP_MSC. Latest update (July 2019): Survival. From the ultrasound report, the first proliferation has significantly decreased, despite the fact that there is a second proliferation. Details of this study are presented below with the following treatment plan, with the exception that modified human MSCs were used in the feline patients. All canine patients were treated with umbilical cord lining MSCs or adipose tissue-derived MSCs extracted from canine donors.

[0469] 1.1 Rationale for dose, schedule and route of administration of CD::UPRT::GFP_MSC The dose tested in animal studies was 10 x 10 6 ~40×10 6 The doses ranged from 5 to 20 × 10 modified MSCs and were well tolerated at all doses. 6A dose of therapeutic cells was safely administered by direct intratumoral injection. 6 Doses of 10 × 10 were safely administered by direct intravenous injection. Patients with both superficial and internal masses were administered a maximum of 10 × 10 6 (intratumoral administration) and 20x10 6 The patients were treated with a combination of therapeutic MSCs (administered intravenously). The maximum tolerated dose has not been identified in the current research studies.

[0470] The study will use a maximum volume of 1 mL and 10 mL for intratumoral and intravenous administration, respectively.

[0471] 1.2 Rationale for the dose, schedule, and route of administration of 5FC The 5FC doses tested ranged from 20 mg / kg / day to 50 mg / kg / day, and all doses were well tolerated. The study used doses of 35 to 50 mg / kg / day over a 4-day course of oral 5FC, which was repeated for each cycle of treatment. Information on preparation of oral 5FC was provided for patient owners.

[0472] 1.3 Study duration and follow-up An attempt was made to follow up treated patients. All patients enrolled in the study were followed for survival.

[0473] 1.4 Study Completion Criteria The clinical trial sponsor and patient owners reserve the right to terminate the study at any time.

[0474] 1.5 Treatment Schedule and Follow-Up Plan The duration of each of the three cycles of treatment is 21 days.

[0475] 1.6 Treatment Guide Because patients may have solid tumors of various sizes at various locations (internal or superficial), the investigator may select any of the doses of modified MSCs in Table 1. When selecting a treatment, the investigator should consider the patient's medical records regarding tumor size and location.

[0476] [Table 7]

[0477] 1.7 Procedure: Intratumoral injection 1. Carefully wipe the surface of the body with disinfectant for 10 seconds and allow to air dry.

[0478] 2. Approximately 0.5 mL of therapeutic cells will be withdrawn by the physician using a syringe. An Ultra-fine II short needle (30G insulin syringe) will be used. Note: In the case of tumor masses containing a large amount of pus, remove as much pus as possible before starting the injection of therapeutic cells.

[0479] 3. Aspirate the entire contents of the Eppendorf tube into the syringe.

[0480] 4. For superficial tumors, administer therapeutic cells intratumorally using multiple injections. Whenever possible, inject perpendicular to the tumor mass to a depth of 0.8 cm. Inject the entire contents of the tube as follows: approximately 100 μL for up to 5 injections (0.5 mL total). Slowly inject the appropriate volume of vector over approximately 10 seconds and allow to sit for 20-25 seconds before removing the needle. Slowly remove the needle and repeat the injection, taking care to distribute the injection throughout the tumor mass.

[0481] 1.8 Procedure: Intravenous Administration Intravenous administration is performed at a flow rate of 10 mL over 30 min. Therapeutic cells are prepared in a total volume of 5-10 mL.

[0482] 1.9 Oral administration of 5FC 5FC is administered orally in capsule form twice daily.

[0483] Procedures and evaluation during testing 2.2. Physical Examination The physical examination will include cardiac and pulmonary auscultation, abdominal examination, and lymph node palpation. Examination of additional systems will be performed as clinically indicated.

[0484] 2.3. Vital Signs Temperatures and weights are recorded at each visit.

[0485] 2.4 Routine Laboratory Evaluation A CBC with differential and platelet count and a chemistry panel (electrolytes, BUN, creatinine, estimated glomerular filtration rate [at screening], total bilirubin, alkaline phosphatase, ALT and AST, LDH, and uric acid) will be performed.

[0486] 2.5. Monitoring for Potential Adverse Effects Throughout each study, owners should be sent a questionnaire regarding signs of adverse effects, e.g., patients will be monitored for hair loss, rash, nausea, vomiting, diarrhea, and loss of appetite.

[0487] Example 6 The TrafEn transfection method is independent of the MSC source In this example, the effect of TrafEn on increased transfection is shown in the following MSC sources: human: umbilical cord-derived, umbilical cord endometrium-derived, adipose tissue-derived and bone marrow-derived MSCs. The results are shown in Figure 44. Different commercial MSC types were modified with a vector containing the GFP transgene. The bars represent the % of the GFP+ population as measured by flow cytometry. Figure 45 shows the results for canine umbilical cord endometrium-derived and adipose tissue-derived MSCs. MSCs of different origins were effectively modified to express CD::UPRT::GFP.

[0488] This study was performed using the non-centrifugation protocol described above.

[0489] As shown in Figure 44, the effect of TrafEn was measured in different MSC types. (A) UC-MSC (Cell Applications), AD-MSC and BM-MSC (Roosterbio) were transfected with pMAXGFP in the presence or absence of TrafEn using a non-centrifugation protocol. Fluorescence images were acquired one day after transfection. Representative images are shown. (B) Umbilical cord lining (Cell Research Corp), fetal tissue (BTI collaborator), umbilical cord (Cell Applications), dog fat (iVET Animal Hospital), human fat and bone marrow (Roosterbio) were transfected with pMAXGFP in the presence of TrafEn using a non-centrifugation protocol. Two days after transfection, cells were harvested for FACS analysis. The graph shows the percentage of cells that expressed GFP.

[0490] Figure 45 shows results from human umbilical cord lining (Cell Research Corp) and human adipose tissue (Hayandra). MSCs were transfected with CD::UPRT::GFP expression vector in the presence of TrafEn using a non-centrifugation protocol. Brightfield and fluorescent images were acquired two days after transfection. Cells were then harvested for FACS analysis. FACS profiles show different expression levels in the two types.

[0491] Example 7 Scaled-up generation of engineered MSC cells-flat bottom and microbead culture systems In this example, scale-up options for generating engineered MSC cells were examined. Two approaches were developed, the first being a flat-bottom system and the second being a microbead culture system.

[0492] In the flat-bottom transfection system, a method for transfection using a compatible cationic polymer and enhancer (e.g., TrafEn) was developed to efficiently transfect MSCs. The scalability of this method for producing modified cells was examined by increasing the surface area of ​​the flask. T175 flasks (area 175 cm) were used. 2 ) was found to be highly correlated with the number of AD-MSCs and UC-MSCs, with a coefficient of determination R2 close to 1 (Figure 46A). The results obtained show that close to 3 million GFP+ cells can be obtained using a T175 flask. Furthermore, the transfection efficiency remained unchanged at over 90% with increasing culture vessel size (Figure 46B). Linearity was established and the transfection efficiency was measured using a Corning® CellSTACK® 10 stack chamber (6,360 cm 2 ) is approximately 1.1 x 10, which is sufficient for the treatment of one human patient. 8 To validate these findings, transfections were performed in a Corning® CellSTACK® 1 chamber (Figure 46C). Clearly, upscaling of transfections is feasible to generate sufficient engineered cells for, for example, preclinical or clinical studies.

[0493] This study was performed using the non-centrifugation protocol described above.

[0494] Figure 46 shows the linearity of scale-up of AD-MSC and UC-MSC on flat-bottom surfaces. AD-MSC and UC-MSC were transfected with CD::UPRT::GFP expression vector in the presence of TrafEn using a non-centrifugation method. Cells were harvested 2 days after transfection without medium change. (A) The number of live transfected cells was plotted against the vessel surface area. (B) Representative images of GFP+ (%) from FACS analysis for both AD-MSC and UC-MSC. (C) Canine umbilical cord endometrial MSCs (Cell Research Corp) were plated at 15000-20000 cells / cm2 in different vessels. After 1 day, cells were transfected with CD:UPRT::GFP polyplexes in the presence of TrafEn. After 24 hours of incubation, cells were harvested. The total number of collected cells was measured with an NC-3000 automated cell counter. GFP expression was analyzed by flow cytometry. The graph shows the % of GFP positive cells in different cell numbers harvested from the culture vessel.

[0495] Since MSCs are adhesive to surfaces, growth in bioreactors / shaker flasks may utilize microcarriers for attachment. In the microcarrier-based (e.g., microbead-based) transfection system described in this example, several microcarriers were examined to identify their suitability for MSC growth. The microcarriers presented different characteristics summarized, such as their diameter, density, coating, and charge (Figure 47A). To identify the suitability of different microcarriers for MSC growth, tests were performed by measuring cell viability. Growth on uncoated microcarriers Cytodex® 1 and P PLUS 102-L resulted in the lowest cell numbers (Figure 47B), and with Cytodex® 1, there were no viable MSCs, as in the control sample without microcarriers. MSCs grew well on Cytodex® 3, a type 1 porcine collagen-coated microcarrier, resulting in the highest cell numbers on day 5. Therefore, Cytodex® 3 was selected for further testing. The number of transfected cells correlated linearly with the total surface area of ​​the microcarriers (Figure 48). Interestingly, adapting MSCs to suspension resulted in a decrease in GFP+ expression (%) with increasing cell numbers. Since flask shaking speed can affect cell aggregation that may physically impede exposure to polyplexes, after seeding, the agitation speed of MSCs was increased to improve cell dispersion. When the shaking speed was increased to 70 rpm, more cells were transfected (>90% of cells expressed GFP). Interestingly, the increase in speed was associated with a decrease in GFP expression (%) with lower cell numbers (1x10 6 ~3×10 6 ) reduced cell viability, possibly due to increased shear stress. This was alleviated by increasing the cell number at seeding (4 × 10 6 cells) (Figure 49). In this study, approximately 4 × 10 6 GFP+ cells were cultured in a 125 mL Erlenmeyer flask, approximately 150 cm 2 This result was obtained from a 50 mL volume on 1.25 L microcarriers. 2A 4 L Erlenmeyer flask containing an area of ​​100 μg / ml will allow for a cell density of approximately 1 x 10 8 It showed the potential to ascend to cells.

[0496] This study was performed using the non-centrifugation protocol described above with an additional shaking step, further details of which are provided below.

[0497] Microcarrier Culture Protocol AD-MSCs (RoosterBio) were cultured in various microcarriers, namely C-GEN 102, Pro-F 102, P Plus 102-L (Thermofisher), Cytodex® 1 microcarrier beads, Cytodex® 3 microcarrier beads (GE Healthcare's Life Sciences) and Synthemax® II microcarriers (Corning) according to the manufacturer's instructions. Briefly, the microcarriers were hydrated in PBS (20 mg / mL) and then sterilized using an autoclave at 121°C for 30 min. 1.9 cm 2 A microcarrier surface area of ​​1000 μg / well was used in a 24-well plate. Prior to culturing AD-MSCs, the microcarriers were equilibrated in complete medium at 37° C. for 1 h prior to use. AD-MSCs were then cultured and seeded on the microcarriers at agitation speeds of 50 rpm or 70 rpm for proliferation and transfection studies.

[0498] Figure 47 shows the results of using different microcarriers in AD-MSCs. (A) Description of the microcarriers used. (B) The number of viable cells grown on the different microcarriers is plotted for different days.

[0499] Figure 48 shows the results of expansion from microcarriers on plates to flasks. Human AD-MSCs were plated onto microcarriers according to total surface area. Larger surface areas are obtained with increasing numbers of microcarriers in culture. Cells were cultured in 1000 x cm 3 20~40x10 3Cells were plated on microcarriers. One day later, cells were transfected with pMAXGFP polyplexes in the presence of TrafEn. One day after transfection, cells were harvested for cell counting and FACS analysis. (A) The number of live transfected cells was plotted against the vessel surface area. (B) Representative images of transfected cells were taken at 4x magnification.

[0500] For microcarrier transfection, AD-MSCs were cultured on 1.9 cm of Cytodex® 3 microcarriers in 24-well non-adherent plates. 2 Different seeding densities were seeded on Cytodex®3 at an agitation speed of 50 rpm 24 h prior to transfection. Similar to flat-bottom transfection, polymer-DNA complexes were added to the cell cultures in a drop-wise manner after 15 min of incubation. Similarly, transfection enhancer was supplemented to the complete medium prior to the addition of polyplexes. For microcarrier (large scale) transfection, AD-MSCs were seeded on Cytodex®3 at appropriately optimized cell densities for various surface areas (20,000-40,000 / cm2). 2 ) in 125 mL Erlenmeyer flasks at agitation speeds of 50 rpm or 70 rpm for transfection. The agitation speed remains constant throughout incubation and production of modified MSCs.

[0501] Example 8 Non-viral modified MSCs show greater anti-cancer efficacy than lentiviral modified MSCs This example aims to compare the cancer killing efficiency of CD::UPRT::GFP_MSCs generated by lentivirus and TrafEn-mediated transfection. To this end, CD::UPRT::GFP stably expressing MSCs were generated by lentivirus infection followed by antibiotic selection. Fluorescence image and flow cytometry analysis (Figure 50A, Figure 50B) suggest that the overall expression level of the transgene is significantly lower in the transduced MSCs (stable expressing CD::UPRT::GFP). Notably, 19% and 25.7% of the TrafEn-modified population expressed medium (blue) and high (orange) levels of CD::UPRT::GFP, respectively (Figure 50B). In the transduced MSC population, <20% of the population expressed medium levels of CD::UPRT::GFP. Thus, the transduced MSCs exerted a lower cancer killing efficiency, especially at the ratio of 1 MSC to 50 and 100 cancer cells.

[0502] Figure 50 shows the results of comparing CD::UPRT::GFP expression and anti-cancer efficiency of AT-MSCs modified by lentivirus or TrafEn-mediated transfection. (A) Three days after infection, MSCs were subjected to puromycin 1 μg / mL selection for two weeks. After establishing MSCs stably expressing CD::UPRT::GFP, another set of experiments was set up to generate CD::UPRT::GFP_MSCs by TrafEn-mediated transfection. Two days after transfection, fluorescent images of modified MSCs were acquired. (B) Then, both cultures were harvested and subjected to (B) FACS analysis and (C, D) co-culture test. The bars in the graph represent the cancer killing efficiency at different ratios of 1 MSC to 1, 5, 50, and 100 cancer cells obtained by MTS assay. The significant difference in the tumor-killing effect of CD::UPRT::GFP_MSCs generated by the TrafEn method compared to CD::UPRT::GFP_MSCs generated by lentivirus was evaluated by two-tailed Student's t-test. ** , p-value < 0.005; * , p-value < 0.05. Bright-field images were taken at the end of the co-culture experiments.

[0503] method: Non-viral MSCs: Prepared using the non-centrifugation protocol described above.

[0504] Lentivirus: MSCs were infected with a lentiviral vector carrying CD::UPRT::GFP. MSCs were transduced with lentivirus at MOI 5 in the presence of 8 μg / mL polybrene. One day after infection, the culture medium was replaced with fresh medium containing 1 μg / mL puromycin for one week. Cells were harvested for FACS analysis and co-culture studies.

[0505] Expression profile. Using flow cytometry analysis, the expression profiles of MSCs modified with CD::UPRT::GFP_lentivirus and CD::UPRT::GFP_TrafEn are different. The three markers in the FACS profile show high (orange), medium (blue) and low (currant) levels of expression. Higher expression suggests a higher payload of CD::UPRT::GFP. MSCs modified with the TrafEn method (non-centrifugation) results in the generation of a population with high expression of CD::UPRT::GFP (19%), whereas lentivirus modified MSCs do not. Higher payload may result in higher cancer killing efficiency.

[0506] Cancer killing efficiency Treatment of cells with TrafEn-modified MSCs resulted in significantly higher cancer-killing efficiency than lentivirus-modified MSCs.

[0507] Example 9 Case study: Feline lymphoma, intravenous injection of CD::UPRT::GFP expressing human adipose tissue-derived MSCs Therapeutic cells were prepared as follows: human adipose tissue-derived MSCs were transfected and cryopreserved. On the day of administration, frozen CD::UPRT::GFP_MSCs were thawed and formulated in cryo for intravenous administration. With the understanding that lymphoma potentially involves bone marrow without obvious measurable mass, it was decided that an appropriate sign of improvement would be PCV (Packed Cell Value). During the treatment period, the number of transfusions required by the patient decreased from 3 times per week to 0 or 1 time per week. An increase in PCV values ​​suggests that anemia is less severe. Additionally, the following observations were reported by the owner: 1) More active 2) Restoration of previous habits such as greeting the owner at the door and interacting with the owner 3) Improves appetite.

[0508] [Table 8]

[0509] The treatment plan for this study is as described in the canine treatment protocol outlined above. MSCs will be modified in the presence of TrafEn using a non-centrifugation method as described above.

[0510] Example 10 Compassionate use: Intratumoral injection of CD::UPRT::GFP expressing MSCs in a 46-year-old patient with recurrent cell carcinoma A 46-year-old patient with renal cell carcinoma was administered compassionate use therapy. The patient was treated with intratumoral injection of CD::UPRT::GFP expressing MSCs as described herein. The results are shown in Figure 51.

[0511] Adipose tissue MSCs were extracted from patient tissue obtained by liposuction. After expansion of MSCs, a cell bank is created in Hayandra Peduli's GMP facility. The transfection protocol was optimized. Six million MSCs were transfected in T225 flasks in the presence of TrafEn, with a maximum transfection efficiency of 75%. The TrafEn protocol and reagents are transferred to the GMP facility to generate CD::UPRT::GFP_MSCs for compassionate use.

[0512] Modified MSCs were harvested on the day of treatment and prepared in 2 mL Plasmalyte. 20-50 million cells were injected intratumorally at 20 sites at the periphery of the tumor mass. One day after MSC administration, 5FC was administered to the patient via oral administration for a total of 2000 mg 5FC / day (4 x 5FC pills 500 mg per day). 5FC was administered for 4 days.

[0513] The administration cycle of MSC and 5FC was repeated every week. Photographs of the tumor mass and data on pain levels one week after each treatment cycle are shown.

[0514] Example 11 Centrifugation vs. non-centrifugation FIG. 52 provides an illustration of a typical centrifugation / spin-based transfection method (top) compared to an example of a non-centrifugation / spin transfection method (bottom).

[0515] In centrifugation / spin-based transfection, cells are seeded in a vessel and DNA / polymer complexes are added. In the depicted example, a spin step is performed for approximately 5-10 minutes as shown. The medium is then removed and enhancer (i.e., TrafEn) is added. Data collected from adipose tissue-derived MSCs so treated are also provided.

[0516] For comparison, the non-centrifugation / spin transfection method is shown in the lower diagram of Figure 52. Several options for this method are depicted. In one embodiment, cells are seeded in a vessel, DNA / polymer complexes are added, and an enhancer (e.g., TrafEn) is also added, followed by incubation for at least about 24 hours (now with non-spin / centrifugation). The medium is then removed and transfected MSCs are fed. Data is shown for adipose tissue-derived MSCs subjected to such treatment.

[0517] Another embodiment of the non-centrifugation / spin transfection method is shown in Figure 52 (bottom). In this embodiment, cells are seeded (upper graph / image - on flat bottom; lower image - on microbeads) and enhancer (e.g. TrafEn) is added as well as DNA / polymer complexes. Incubation is performed with shaking (e.g., other options such as bioreactor types including spin flasks, wave bioreactor systems, rotating wall bioreactor designs, and stirred tank bioreactor designs are also depicted) for at least about 24 hours. Then, harvesting is performed by spinning (e.g., about 300g for 3 minutes in the depicted example), adding 1x PBS, spinning again (e.g., about 300g for 3 minutes in the depicted example), adding trypsin, shaking at 100 rpm, and quenching by adding medium. Filtration is performed with washing (1x PBS in the depicted example). Spinning (e.g., about 300g for 3 minutes in the depicted example) thus obtaining a cell pellet for downstream analysis. Data are presented for cells so treated, providing a comparison of polymer only versus polymer plus TrafEn.

[0518] Example 12 Cryopreserved modified MSCs (generated using TrafEn) are viable and functional Figure 53 provides an illustration of a workflow for cryopreserving modified MSCs (prepared using TrafEn) to allow for long-term storage. As shown, modified MSCs may be cryopreserved and stored. When needed, the cells can be removed from storage and prepared for use by thawing in cryogenic liquid.

[0519] Figure 54 shows the results of cell viability, expression levels and functional activity of modified MSCs that were cryopreserved and then thawed as shown above in Figure 53. As shown, the modified MSCs maintained high cell viability and expression levels after cryopreservation, thawing and storage in cryogenic liquid for up to 72 hours.

[0520] Cells were thawed in hypothermosol and stored for up to 3 days at 4° C. Cell viability and % CD::UPRT::GFP+ cells were measured daily.

[0521] One or more exemplary embodiments have been described by way of example, and those skilled in the art will appreciate that numerous variations and modifications can be made without departing from the scope of the invention, as defined in the claims.

[0522] [References] TIFF0007644017000009.tif179170TIFF0007644017000010.tif212170TIFF0007644017000011.tif212170TIFF0007644017000012.tif212170 TIFF0007644017000013.tif212170TIFF0007644017000014.tif212170TIFF0007644017000015.tif212170TIFF0007644017000016.tif212170 TIFF0007644017000017.tif212170TIFF0007644017000018.tif212170TIFF0007644017000019.tif212170TIFF0007644017000020.tif212170 TIFF0007644017000021.tif212170TIFF0007644017000022.tif212170TIFF0007644017000023.tif212170TIFF0007644017000024.tif212170

[0523] All references cited herein, and elsewhere in the description, are hereby incorporated by reference in their respective entireties.

Claims

1. 1. A method for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct expressing one or more functional genes, comprising: exposing the MSCs to a transfection mixture comprising a nucleic acid construct complexed with a cationic polymer; exposing the MSCs to a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments and a second agent capable of stabilizing the microtubule network of the MSCs; and incubating the MSCs; thereby providing MSCs transfected with the nucleic acid construct. Including, the MSCs are not centrifuged during exposure to the transfection mixture, during exposure to the first agent and the second agent, during incubation, or during a combination thereof; The first agent comprises a fusogenic lipid, the fusogenic lipid being selected from the group consisting of DOPE, CHEMS, DPPC, DOPC, and combinations thereof, and the second agent is selected from a histone deacetylase inhibitor (HDACi); method.

2. 2. The method of claim 1, wherein incubating the MSCs comprises incubating the MSCs for between 2 hours and 48 hours.

3. 3. The method of claim 1 or 2, wherein exposing the MSCs to the first agent and the second agent comprises replacing the transfection mixture with cell culture medium supplemented with the first agent and the second agent.

4. 4. The method of claim 1, wherein the one or more functional genes comprises a suicide gene.

5. 5. The method of claim 1, wherein the one or more functional genes comprise cytosine deaminase (CDy), uracil phosphoribosyltransferase (UPRT), or both.

6. 6. The method of any one of claims 1 to 5, wherein the transfected MSCs are each transfected with an average copy number of at least 1000 copies, at least 2000 copies, at least 3000 copies, at least 4000 copies, at least 5000 copies, at least 6000 copies, at least 7000 copies, at least 8000 copies, at least 9000 copies, or at least 10000 copies of the nucleic acid construct.

7. The method of any one of claims 1 to 6, wherein the pluripotent phenotype of the transfected MSCs is substantially unchanged by transfection.

8. The method of claim 7, wherein the pluripotent phenotype comprises tumor-tropic and / or cancer-tropic properties of MSCs.

9. 9. The method of claim 7 or 8, wherein the pluripotent phenotype comprises an immunophenotype in which expression of CD surface markers is substantially unchanged after transfection, and the transfected MSCs are plastic adherent, express CD105, CD73 and CD90 (>95%), lack expression of CD45, CD34, CD14 and HLA-DR surface molecules (<2%), can differentiate in vitro into osteoblasts, adipocytes and chondroblasts, and fulfill the immunophenotypic criteria defined by the International Society for Cellular Therapy (ISCT).

10. 10. The method of claim 1, wherein exposing the MSCs to the transfection mixture comprises adding the transfection mixture to the MSCs without removing growth medium from the MSCs, and no centrifugation is performed between the exposing and incubating steps.

11. 11. The method of any one of claims 1, 2, and 4 to 10, wherein exposing the MSCs to the first agent and the second agent comprises adding the first agent and the second agent to the MSCs simultaneously with the transfection mixture, sequentially with the transfection mixture, or in combination with the transfection mixture.

12. 12. The method of any one of claims 1, 2, and 4 to 11, wherein the exposure period of the MSCs to the transfection mixture overlaps with the exposure period of the MSCs to the first agent and the second agent, and the transfection mixture is not removed prior to adding the first agent and the second agent to the MSCs.

13. 1. A method for transfecting mesenchymal stem cells (MSCs) with a nucleic acid construct expressing one or more functional genes, comprising: Culturing the MSCs in a proliferation medium; adding a transfection mixture comprising a nucleic acid construct complexed with a cationic polymer to the MSCs without removing the growth medium from the MSCs; adding to the MSCs a first agent capable of redirecting endocytosed nucleic acids from intracellular acidic compartments and a second agent capable of stabilizing the microtubule network of the MSCs; and incubating the MSCs while in contact with all of the transfection mixture, the first agent, and the second agent for an incubation period; thereby providing MSCs transfected with the nucleic acid construct. Including, the first agent and the second agent are added to the MSCs simultaneously with the addition of the transfection mixture, sequentially with the addition of the transfection mixture, or in combination with the transfection mixture; Between the addition of the transfection mixture and the end of the incubation period, the MSCs were not centrifuged. The first agent comprises a fusogenic lipid, the fusogenic lipid being selected from the group consisting of DOPE, CHEMS, DPPC, DOPC, and combinations thereof, and the second agent is selected from a histone deacetylase inhibitor (HDACi); method.

14. 14. The method of any one of claims 1 to 13, wherein the MSCs are transfected with a nucleic acid construct expressing one or more functional genes, the MSCs have a pluripotent phenotype that is substantially unchanged by transfection of the nucleic acid construct, and the MSCs are free of viral-based transfection agent material.

15. 15. The method of any one of claims 1 to 14, wherein the transfected MSCs transiently express one or more functional genes for at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, or at least 17 days following transfection.

16. 16. The method of any one of claims 1 to 15, wherein the transfected MSCs are for use in the treatment of cancer, wherein the cancer is lymphoma, clear cell carcinoma, glioblastoma, temozolomide-resistant glioblastoma, perianal cancer, oral melanoma, thyroid cancer, soft tissue cancer, cancer ulceration, nasal tumor, or gastrointestinal cancer.

17. 17. The method of any one of claims 1 to 16, wherein the MSCs are for use in combination with 5-fluorocytosine (5FC), 5-fluorouracil (5FU), ganciclovir (GCV), or a combination thereof.

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