A method of testing the safety of a gene therapy vector or a cell modified by the vector

Abstract

A method of assessing the safety of a gene therapy vector comprising: providing a population of genetically modified cells 18 wherein the cells have been transfected with a vector; exposing the genetically edited cells to a carcinogen 20; growing the cells to produce immortal cell lines; and comparing the survival of the test cells with a control 14 to determine if the vector contributes to carcinogenicity. Also claimed is a method of testing the safety of a vector comprising: providing test cells; genetically editing the cells with a vector and exposing cells to a carcinogen or exposing the cells to a carcinogen and modifying the cells with a vector; growing the cells; and comparing the survival of the cells to a control. The cells may be used in immunotherapy. The cells may be CAR-T cells. The cells may be from peripheral blood mononuclear cells or induced pluripotent stem cells (iPSCs). The carcinogen may be ENUA. Also claimed is a method of designing a vector for use in gene therapy by harvesting nucleic acid from the test cells, characterising the modification of the test cells, redesigning the vector, and repeating the method of testing the safety of the vector.

Description

The present invention relates to a method of assessing the safety of a gene therapy vector or a cell modified by the vector for use in gene therapy, for example immunotherapy. In particular, it relates to a method of testing the vector and the safety of CAR-T cells for safe use in cancer immunotherapy. Backg round Gene therapy is a promising approach to deliver therapeutic genes by integration of the vector material carrying the gene permanently into the host genome of cells that require corrective gene augmentation. In this process fully functional genes can be used to treat or correct a genetic disorder where the existing gene in the diseased host is mutated and therefore not generating a functional protein. Gene therapy can deliver genes that provide proteins for normal cell function or for vaccination. Gene therapy can also be used to treat cancer by causing death of target tumour cells. Where immune system cells are engineered to recognise and attack cancer cells, this is known as immunotherapy. CAR-T therapy is a form of immunotherapy used in cancer treatment in which T-cells carrying chimeric antigen receptors (CARs) are provided to a cancer patient. The T-cells are genetically modified to produce the CARs and the CARs enable the T-cells to recognise cancer cells in order to target and destroy them. The process is illustrated schematically in Figure 1. A blood sample 10 is taken from a patient 12. T-cells 14 are isolated or generated from the cells in the blood sample 10, then modified by a vector 16 carrying an expression construct for the CARs thereby generating the CAR-T cells 18. The CAR-T cells are then provided to the cancer patient. Blood cells (PBMC) can be activated to several T-cells lineages and these cells are used to generate T-cells. Alternatively, T-cells, or any other cell intended to be used to target cancer, can be generated following re-programming of any patient cell to an induced pluripotent stem cell (iPSc) and differentiated into the cell of choice for immunotherapy, for example, a T-cell. Markers for cells intended to target cancer are useful for identification. For example, T-cell markers include CD3+, CD4+ and CD8+. CAR-T cells are T-cells that are used to target and kill cancers and are derived from modification of T-cells or any cell intended to be used to kill cancer (to target tumours) often using complex gene transfer vehicles including viruses mainly derived from retroviruses (RV) or from retrotransposons (RP) (Magnani et al. (2020)). CARs are fusion constructs that incorporate an extracellular antigen recognition domain, a transmembrane domain and an intracellular domain including costimulatory and signalling components. An example is lentivirus vectors (LV) that have been engineered to carry CD28 or 4-1BB genes to provide co-stimulation and the chain of the TCR-associated CD3 complex to deliver an activating signal. CAR-T immunotherapy now has a global market with, to date, over 1087 CAR-T clinical trials (ClinicalTrials.gov) and several approved CAR-T products. The RV, such as LV used to create these cells have previously been found associated with clonal dominance or leukaemogenesis following gene therapy for monogenetic disorders. More recently, LV and RP, that can also be used to generate CAR-T, have been found associated with CAR-T cell clonal dominance and persistence due to oncogene activation or gene inactivation, respectively. Of utmost importance is that the modified CAR-T cells, or any immune cell intended for cancer treatment, are efficacious in their action and do not cause side effects. Modified T-cells have been found tumorigenic or have become clonally dominant due to the vector used for therapeutic gene delivery (Howe et al. (2008); Braun etal. (2014); Otteta / . (2006); Miyoshi et al. (1998); Cavazzana-Calvo etal. (2010)). Even though they have been modified to be safe, LV (a subgroup of RV) has also been implicated in clonal dominance (Cesana etal. (2012)). This is due to that fact that these vectors integrate into the chromosomes of the host cell and interact with host cancer genes causing changes in their expression, thereby driving tumorigenesis. Following re-infusion of cells to patients, low-level persistence of immunotherapy cells such as CAR-T cells is known after treatment and re-expansion is rare. However, CAR-T clonal outgrowth has been demonstrated to be associated with LV mediated genotoxicity (Shah etal. (2019)), and recently 2 out of 10 patients treated with a CAR19-carrying transposon developed malignant T-cell lymphomas (Bishop et al. (2021)). As a result of previous findings and concern over potential CAR-T tumorigenesis, the FDA has drafted guidelines for CAR-T safety (]Ttt|3SL6 / wjAmJ[dajjoyZr€^^ documents / considerations-development-chimeric-antigen-receptor-car-t--cell-products). Several strategies have been deployed to understand and even predict gene transfer side effects. However, the lack of reliable "life-like" human-based systems means other models, that use mice and celllines, are limited or untrusted for safety assessment. Examples of safety models include the tumour prone Cdkn2- / - Ifnarl- / -mouse that is predisposed to liver oncogenesis. This is considered sensitive yet biased towards generating false positive tumorigenesis data. The fetal / neonatal mouse model also exposes differences in LVs to induce liver oncogenesis. However, this system is time consuming, costly and oversensitive. The in vitro immortalisation (IVIM) assay uses non-tumour prone or tumour-prone mouse HSc for gene transfer followed by assessment of immortalisation. This model is restricted to murine cells and once again is oversensitive giving little data on genotoxic mechanisms associated with the vector. The serial transformation assay reports retrovirus vector-associated clonal transformation in secondary mouse recipients but also generates false leukaemic positives in the irradiated animals used for cell engraftment. The models developed to understand T-cell tumorigenesis have been reviewed and none are deemed acceptable to accurately support safe product entry to the clinic. Leukaemogenesis is a complex multistage process. T-cells are known to be highly refractory to tumorigenicity with poor remission rates, leaving the field with limited understanding and with clinical case studies revealing complex genetic changes. Hence, there is an urgent need for reliable human-based models of T-cell leukaemia that can predict CAR-T tumorigenesis to replace the current murine and cell models. The present invention seeks to provide an improved method of assessing the safety of a gene therapy vector or a cell modified by the vector. Summary of the Invention According to an aspect of the present invention, there is provided a method of assessing the safety of a gene therapy vector or a cell modified by the vector including: a) providing a population of genetically modified test cells, wherein the test cells have been transfected or infected with a vector carrying exogenous genetic material; b) exposing the population of modified test cells to a carcinogen; c) growing the modified test cells for a period of time sufficient to produce immortal cell lines; d) comparing the survival of the modified test cells with at least one control to determine whether the vector contributes to carcinogenicity. Advantageously all of the cells in the population exposed to the carcinogen have been modified by the vector. Preferably only immune system cells that have been modified are exposed to the carcinogen. For example, modified cells can be isolated using a suitable technique such as FACS. According to another aspect of the present invention, there is provided a method of assessing the safety of a gene therapy vector or a cell modified by the vector including: a) providing a population of test cells to be genetically modified; bi) genetically modifying the population of test cells with a vector carrying exogenous genetic material, selecting modified test cells, and exposing the modified test cells to a carcinogen, or bii) exposing the population of test cells to a carcinogen then modifying the population of test cells with a vector carrying exogenous genetic material; c) growing the modified and carcinogen exposed test cells for a period of time sufficient to produce immortal cell lines; and d) comparing the survival of the modified and carcinogen exposed test cells with at least one control to determine whether the vector contributes to carcinogenicity of the cells. The cells modified by the vector may be for use in gene therapy / immunotherapy. The test cells may be immune system cells and the genetically modified test cells may be for use in immunotherapy. The exogenous genetic material may include an expression cassette that enables immunotherapy. The exogenous genetic material may include chimeric antigen receptors. The test cells may be T cells. The modified test cells may be CAR-T cells. In an embodiment, a) the control is a population of unmodified cells that do not contain a vector carrying exogenous genetic material, and the method includes: b) exposing the population of unmodified control cells to the carcinogen; and c) growing the unmodified control cells for a period of time sufficient to produce immortal cell lines. In an embodiment, a) the control is a population of genetically modified cells, wherein the cells have been transfected or infected with a vector carrying exogenous genetic material, wherein the vector is known not to contribute to carcinogenicity, and the method includes: b) exposing the population of genetically modified control cells to the carcinogen; and c) growing the genetically modified control cells for a period of time sufficient to produce immortal cell lines. The method may include the step of genetically modifying the population of test cells with a vector carrying exogenous genetic material and selecting only those cells that have been successfully modified. The population of test cells may be obtained from a patient or from a donor. The vector may be a virus. For example, it may be a retrovirus such as a lentivirus. The test cells may be obtained from peripheral blood mononuclear cells. The cells may be generated from iPSCs. The period of time may be at least three weeks or at least four weeks. The period of time may be at least six weeks, seven weeks or eight weeks. The period of time may be at least 10 or 12 weeks. The carcinogen may be a chemical carcinogen, for example it may be ENLIA. The test cells may be CAR-T cells for use in immunotherapy. The test cells such as CAR-T cells may be for use in cancer immunotherapy. According to another aspect of the present invention, there is provided a method of designing a vector for use in gene therapy including carrying out the method of assessing the safety of a gene therapy vector as claimed in any preceding claim, harvesting nucleic acid from surviving test cells modified by a vector determined to contribute to carcinogenicity, carrying out one or more assays to characterise the modification of the test cells by the vector, redesigning the vector on the basis of the results of those assays, and repeating the method of assessing the safety of a gene therapy vector. The assays may include one or more of: (i) an integration site assay to determine vector insertion sites in the host genome; (ii) a gene expression assay to determine changes in gene expression over time; (iii) an RNA transcript assay to identify new transcripts or truncated transcripts over time; (iv) a CpG methylation assay of genes at the integration site; (v) a gene inactivation assay. In some embodiments, at least two assays are carried out, for example, at least two of the above assays. According to another aspect of the present invention, there is provided a method of assessing the safety of a gene therapy vector for use in generating cells for use in immunotherapy including: a) providing a population of genetically modified immune system cells, wherein the immune system cells have been infected or transfected with a vector carrying exogenous genetic material; b) exposing the population of modified immune system cells to a carcinogen; c) growing the modified immune system cells for a period of time sufficient to produce immortal cell lines; d) comparing the survival of the modified cells with a control to determine whether the vector contributes to carcinogenicity. According to another aspect of the present invention, there is provided a method of assessing the safety of CAR-T cells including: 1) providing a population of CAR-T cells; 2) exposing the CAR-T cells to a carcinogen; 3) exposing a negative control population of non-infected T cells to the carcinogen; 4) growing the CART-T cells and the T cells for a period of time sufficient to produce immortal cell lines; 5) comparing the survival of cells in the CAR-T and T cell populations to determine whether the vector contributes to carcinogenicity. According to another aspect of the present invention, there is provided a method of assessing the safety of a gene therapy vector for use in generating cells for use in immunotherapy including: a) providing a population of immune system cells; bi) genetically modifying the population of immune system cells with a vector carrying exogenous genetic material, selecting modified immune system cells, and exposing the modified immune system cells to a carcinogen, or bii) exposing the population of immune system cells to a carcinogen then genetically modifying the population of immune system cells with a vector carrying exogenous genetic material; c) growing the modified and carcinogen exposed immune system cells for a period of time sufficient to produce immortal cell lines; d) comparing the survival of the modified and carcinogen exposed cells with a control to determine whether the vector contributes to carcinogenicity of the cells for use in immunotherapy. According to another aspect of the present invention, there is provided a method of assessing the safety of CAR-T cells including: a) exposing two populations of T cells to a carcinogen, wherein one of the populations is infected with a CAR-T vector and the other is not, wherein infection with the CAR-T vector occurs prior to exposure to the carcinogen or subsequent to exposure to the carcinogen; b) growing the transfected and non-transfected cells for a period of time sufficient to produce immortal cell lines; c) comparing the survival of cells in the transfected and non-transfected cell populations to determine whether the vector contributes to carcinogenicity According to another aspect of the present invention, there is provided a method of treating a patient including carrying out a method as specified above and administering modified cells to the patient, wherein the cells were determined to be modified by a vector that does not contribute to carcinogenicity. In particular the present method provides for assessing the potential for immunotherapy cells such as CAR-T cells to persist, proliferate and become cancerous. List of Figures Embodiments of the present invention are described below, by way of example only, with reference to the accompanying drawings, in which: Figure 1 schematically illustrates the production and use of CAR-T cells in cancer treatment; Figure 2 schematically illustrates a method of an embodiment of the method; Figure 3 is a flow chart illustrating the steps of a method of producing CAR-T cells; Figure 4 schematically illustrates an overview of an embodiment of the method; Figure 5 is a graph showing relative cell numbers over time; Figures 6 to 8 show the results of experiments comparing expansion of cells within a 24-well flat bottom culture plate and a 24-well G-REX® plate; Figure 9 is a graph illustrating T-cell viability after treatment with different carcinogens; Figure 10 shows the results of flow cytometry demonstrating that cells retain their identity following treatments; Figure 11 illustrates that ENDA as a positive control alone generates cell outgrowths; Figure 12 is a series of photographs showing tumorigenic T-cell outgrowths Figure 13 is a schematic diagram of a vector production process; and Figure 14 is a graph showing cancer genes with virus integrations; Figures 15 and 16 are volcano plots showing up- and down-regulated genes; Figures 17 and 18 are dot plots of most enriched signalling pathways; Figure 19 is a dot plot of the most enriched signalling pathways within hallmark gene sets for cancer-specific molecular signatures; and Figure 20 is a heatmap showing the cancer-specific scores across different samples indicating their alignments. Detailed Description Human cells are extremely refractory to pre-malignancy (immortalisation). These cells are also long lived and it can take several years before cancer develops in an individual. Assays to determine the effect of agents that cause pre-malignancy as a pre-requisite to cancer have been developed. These assays usually use strong agents such as carcinogens (for example, chemical carcinogens or radiation) to cause direct changes to the genome or indirect changes such as epigenetic alteration of the genome that can lead to oncogenesis. A number of instances have been reported where gene therapy vectors have been shown associated with cancer development in the clinic and in non-clinical models involving rodents or their cells. As these models are untrusted, the applicant has developed a human model to show RV, LV or adeno-associated virus (AAV) vectors can be associated with genotoxicity. This model measures factors known to be associated with genotoxicity using a range of molecular assays that determine vector interaction that leads to cancer gene expression changes. This model uses human iPSc that are reprogrammed to liver-like cells (HLC) and is suited to a number of treatments but especially those involving gene therapy vector delivery to the liver. The present work explores CAR-T cell outgrowth and / or pre-malignancy prior to their use in immunotherapy. The applicant has developed an assay to determine the potential for tumorigenicity of immune system cells transfected or infected with exogenous genetic material enabling them to be used in immunotherapy. Embodiments of the method are described in relation to CAR-T cells. However, the skilled person will appreciate that the method is applicable to other cells modified for use in immunotherapy, for example, immune system cells such as natural killer cells. Figure 2 schematically illustrates an embodiment of a method according to the invention. A population of genetically modified cells is provided. In the illustrated embodiment of the test (T) a population of CAR-T cells 18 is provided. The CAR-T cells 18, as explained above, are produced by infecting with a viral vector. The CAR-T cells 18 are exposed to a carcinogen 20. Any suitable carcinogen may be used, such as a chemical carcinogen or radiation. The applicant has found ENUA to be particularly effective in the method. Other carcinogens could be used, such as nickel chloride, N-methyl-N-nitrosourea (MNU), benzo(a)pyrene diol epoxide (BPDE), methyl methanesulfonate (MMS), X-rays, and an RV vector carrying the human HTert gene. After 24 hours, the treated cells are then expanded, for example into 6-well plates 22, and grown for at least three to four weeks. Treatment with the carcinogen 20 ensures that even where the vector is safe, the cells do not senesce at around four to six weeks. The cells are then expanded into multi-well plates 24. In practice, 384-well plates would preferably be used. Figure 2 schematically illustrates only 48 wells for clarity, and for illustrative purposes only. The cells are grown in the 384-well plate 24 for a time sufficient to produce immortal cell lines, for example beyond six to eight weeks (for example about 10 weeks) to demonstrate pre-malignancy. After six to eight weeks the number of wells of the multi-well plate 24 containing surviving cell outgrowths resulting from the CAR-T cells 18 is determined. This can be compared with the number of wells of one or more controls (C) containing surviving cell outgrowths. Figure 2 illustrates five different controls (Cl, C2, C3, C4, and C5). The illustrated controls are as follows: Cl) The population of cells is a population of T-cells 14 that have not been genetically modified. It can be seen that treatment with carcinogen 20 results in 10 immortal cell lines forming (as schematically illustrated by the presence of 10 filled in wells). C2) The population of cells is a population of CAR-T cells 18 infected with a vector that is known to be carcinogenic. However, no additional treatment by a carcinogen is performed. Again, 10 immortal cell lines are formed. C3) The population of cells is a population of T-cells 14 that have not been genetically modified. However, no treatment by a carcinogen is performed. All of the cells senesce within four to six weeks and so no immortal cell lines are seen in the multi-well plate 24. C4) The population of cells is a population of CAR-T cells 18 infected with a vector that is known to be carcinogenic. Additional treatment by a carcinogen 20 is performed. This results in an increased frequency of immortal cell lines in the multi-well plate 24 (as schematically illustrated by the presence of 20 filled in wells). C5) The population of cells is a population of CAR-T cells 18 infected with a vector that is known to be safe. Additional treatment by a carcinogen 20 is performed. It can be seen that treatment with carcinogen 20 results in 10 immortal cell lines forming (as schematically illustrated by the presence of 10 filled in wells). The skilled person may wish to include other controls. For example, a population of CAR-T cells 18 infected with a vector that is known to be safe, without carcinogen treatment. This would be expected to result in no immortal cell lines, similar to control C3 above. In preferred embodiments, at least control C2 and / or C5 is included. Turning to the results of the test (T), where the vector is of unknown safety, and the population of CAR-T cells is treated with carcinogen 20, there are two expected outcomes. The first is that there is no increase in immortal cell lines compared to controls having a safe vector or no vector (C5 or C2 as illustrated in Figure 2). This enables a conclusion that the vector is safe for use in gene therapy / immunotherapy (denoted by a "tick" in Figure 2). The second result is that there is an increase in immortal cell lines compared to controls C5 or C2, indicating that the vector is carcinogenic and therefore not safe (denoted by a "cross" in Figure 2). Therefore, if there is a difference in frequency of surviving immortal cells between the test (T) and the control having no vector or a known safe vector (C2, C5), this can be attributed to carcinogenicity arising from modification of the CAR-T cells 18 by the CAR vector 16. It can thus be determined whether the vector contributes to carcinogenicity and therefore whether it is safe or unsafe. Appropriate statistical analysis would be carried out to determine whether any difference in frequency of immortal cell lines derived from CAR-T cells 18 and negative control T-cells 14 is significant would be carried out in a manner known to the skilled person. Immortal cell lines derived from unsafe vectors are then further grown continuously for a period of time until nucleic acid can be obtained, for example for at least four weeks. The nucleic acid from the outgrowths can then be harvested and analysed using the molecular tests described for example in the applicant's earlier patent application publication WO 2019 / 012259, the contents of which are incorporated herein by reference. For example, the previously described molecular methods can be used to identify interactions between the vector and T-cell genome that influence cellular development (oncogenesis) to cancer. The molecular tests can be used to determine vector interactions that cause oncogenesis along with alignment with cancer signatures. On the basis of the assay and the molecular analyses the vector can be modified to make it safe (ie. less interactive with the host genome by not providing activity that contribute to cancer gene activation and cellular outgrowth). After modification, the vector can be re-tested until the vector no longer causes cellular outgrowth. The applicant has thus developed a method of assessing the safety of a gene therapy vector or a cell modified by that vector for use in gene therapy / immunotherapy including: a) providing a population of test cells to be genetically modified; bi) genetically modifying the population of test cells with a vector carrying exogenous genetic material, selecting modified test cells, and exposing the modified test cells to a carcinogen, or bii) exposing the population of test cells to a carcinogen then modifying the population of test cells with a vector carrying exogenous genetic material; c) growing the modified and carcinogen exposed test cells for a period of time sufficient to produce immortal cell lines; and d) comparing the survival of the modified and carcinogen exposed test cells with at least one control to determine whether the vector contributes to carcinogenicity of the cells. In an embodiment, the method involves the following steps: 1) exposing two populations of T-cells to a carcinogen, wherein one of the populations is infected with a CAR-T vector and the other is not, wherein infection with the CAR-T vector occurs prior to exposure to the carcinogen or subsequent to exposure to the carcinogen; 2) growing the infected and non-infected cells for a period of time sufficient to produce immortal cell lines (for a length of time greater than that after which senescence would usually occur, for example, longer than three to four weeks); 5) comparing the survival of cells in the infected and non-infected cell populations to determine whether the vector contributes to carcinogenicity. Human cells are difficult to immortalise, it would therefore be difficult to treat T-cells with, for example, a CAR-LV vector and identify premalignancy in cell culture over a short period of time before the cells die. An advantage of the presently disclosed method is that a population of purified T-cells already infected with a CAR-LV vector (for example) is provided at the outset. This is then treated with a potent carcinogen and the number of immortal outgrowths obtained is compared to those from a control cell culture of T-cells not infected with the vector. The method provides a model sensitive to CAR-T cell outgrowth that can be used to measure the contribution of the vector to cellular outgrowth free from senescence. It can also provide nucleic acid material to analyse for the effects of the vector on the CAR-T cells causing them to outgrow and thus enabling modification of the vector to render it safe. In another embodiment, cells could be treated with a carcinogen and then subsequently infected. However, this does not start with an entire population of infected cells and therefore makes more difficult the procedure to identify the frequency of outgrowing cells associated with vector induced oncogenesis to become immortal over and above those treated only with carcinogen. A preferred embodiment of the method therefore includes the following steps: a) providing a population of genetically modified test cells, wherein the test cells have been transfected or infected with a vector carrying exogenous genetic material; b) exposing the population of modified test cells to a carcinogen; c) growing the modified test cells for a period of time sufficient to produce immortal cell lines; d) comparing the survival of the modified test cells with at least one control to determine whether the vector contributes to carcinogenicity. The method may also include the steps of genetically modifying the population of test cells with a vector carrying exogenous genetic material and selecting only those cells that have been successfully modified. In an embodiment, the method involves the following steps: 1) exposing two populations of T-cells to a carcinogen, wherein one of the populations is infected with a CAR-T or any immunotherapy intended vector and the other is not, wherein infection with the CAR-T vector occurs prior to exposure to the carcinogen or subsequent to exposure to the carcinogen; 2) growing the infected and non-infected cells for a period of time sufficient to produce immortal cell lines (for a length of time greater than that after which senescence would usually occur, for example, longer than three to four weeks); 5) comparing the survival of cells in the infected and non-infected cell populations to determine whether the vector contributes to carcinogenicity. The applicant has used carcinogenic agents and hTERT as +ve controls to generate pre-malignant T-cells from primary PBMC and will use iPSc also for this. The assay can use multi-well plates (such as G-Rex and NaviPlate technology) to aid cellular expansion and detect CAR-T pre malignancy via cellular outgrowth. Starting cultures of over 107 PBMC enable outgrowth at a frequency of approximately 10'4 at present. It is important that the carcinogen is not too potent so that it does not conceal the contribution of the gene therapy vector to oncogenesis. The assay uses minimal carcinogen concentration. Other carcinogens include nickel chloride, benzdiolepoxide (BPDE) at various concentrations, and X-rays of different doses. This means that any potential for CAR LV genotoxicity that increases CAR-T outgrowth frequency can be detected in the multiwall plate. Contributory factors associated with this frequency can be determined using the hlnGetox assay (described in WO 2019 / 012259). The hlnGetox assay was developed with NC3Rs InMutaGene Challenge 21 funding and working with the present assay, will offer a human-based CAR-T tumorigenicity detection platform. Whether or not the identified genetic change that is involved in causing cellular outgrowth away from senescence is due to the vector is identified. In a preferred embodiment, the assay involves the following steps: • Generate T-cells either from patient PBMC or induced pluripotent stem cells • Characterise these cells as T-cells • Expose the cells to a gene therapy vector that is expected to integrate its genome in the T-cell genome to permanently deliver a CAR cassette that produces CAR-T cells • Isolate only infected cells and use these next to uninfected control cells in the model • Expose these cells to chemical carcinogens and passage these cells to a tissue culture vessel that enables outgrowth of immortal CAR-T cells from single cells as outgrowing clones that may be counted • Expand immortal CAR-T clones to a number that can be used to isolate enough nucleic acid material for molecular examination of the cause of pre-malignancy contributed by the gene therapy vector Embodiments of the invention provide a method that involves the following: • Use of T-cells that have been subjected to a gene therapy vector in order to generate CAR-T cells in a safety model. • Isolation of these cells and exposure of these to chemical carcinogens that provide genetic changes to these cells that may complement changes associated with the gene therapy vector in causing cells to become immortal or pre-malignant. • Detection of outgrowing CAR-T cells that are free from senescence • Analysis of the cells for the contribution by the vector to oncogenesis Bioinformatics and omic analysis of the data that supports vector involvement in oncogenesis can be used to design novel gene therapy vectors that have reduced contribution to supporting oncogenesis. The methods disclosed in the present application provide several advantages. Currently, T-cells are obtained from patient PBMC cells or other patient cells or T-cells derived from any iPSc (these can be commercially available) and require conversion to CAR-T before re infusion to the patient requiring cancer treatment. These cells are currently not screened for pre-malignancy and therefore present a risk of becoming cancerous. The present methods enable the CAR-T vector (such as CAR-LV) to be investigated for its contribution to premalignancy. Although this would take longer than the time required for the cells to be useful for cancer treatment, the protocol provides a measure of vector safety. If T-cells are generated from iPSc they can be considered 'off the shelf' for cancer treatment without delay having been tested for no outgrowth using the assay. These cells might benefit from gene editing so as not to be subjected to host immune responses. The procedure, therefore uses either PBMC or iPSc derived T-cells or other cells intended for immunotherapy. However, the assay will be useful to pre-screen LV vectors that do or do not cause CAR-T immortality and could therefore be used for modifying these vectors to prevent any vector contributing to pre-malignancy to generate safer CAR-T cells derived or not derived from a patient for immediate immunotherapy. The alternative iPSc / CAR-T approach will therefore produce 'off the shelf' screened CAR-T safe for immediate immunotherapy. In embodiments that use iPSc cells, these can be used to generate CAR-T cells whilst also testing the CAR-T vector (for example, CAR-LV) for its contribution to causing cancer. An additional advantage is that once safe CAR-T / CAR-LV vectors have been identified that generate CAR-T that do not outgrow due to the vector, the CAR-T cells or their originating iPSc cells can be gene edited to prevent them recognising or being recognised by the host as foreign. This modification, therefore, generates 'off the shelf' CAR-T cells that can be used to treat any patient cancer. The methods disclosed herein may be used to test the contribution by any gene therapy vector towards cellular pre-malignancy. It may be useful to predict safety of CAR-T or any T-cell or non-T-cells for gene therapy in patients. EXAMPLES Example 1 - Generation of T-cells from iPSc T cells can be generated from induced pluriopotent stem cells and infected with a CAR vector, then isolated by FACS to obtain a population of 100% vector infected cells that are then subjected to ENUA (carcinogen) treatment. T cells can also be isolated using markers: CD4+, CD8+ and CD3+. Protocol: iPSc (3.5xl06) are used for each well of an aggrewell 6-well plate. Counted cells are centrifuged at 300xg for 5 minutes. The supernatant is aspirated and cells resuspended in 2.5ml of EB formation medium (StemCell Technologies) to obtain a final concentration of 1.4xl06 cell / ml (3.5xl06 cells / well). Pre-treatment of Aggrewell 6 well plate: Day 0. Aggrewell™ 400 plates are firstly rinsed with anti-adherence rinsing solution, then 2.5ml of warm EB formation Medium is added to each well before 2.5 ml of iPSc cell suspension (500 cells / microwell in a total volume of 5ml) is added and mixed with the medium The plate is then centrifuged at lOOxg for 3 minutes and incubated at 37°C and 5% CO2. Day 2. Cell medium is changed for fresh EB medium A, slowly touching the side of the well and then cells are incubated at 37°C for 24hrs Day 3. The medium is changed again using EB medium B again, slowly touching the side of the well and left to incubate at 37°C for 24hrs Day 5 Warm DMEM / F-12 with 15 mM HEPES and EB Medium B and to each AggreWell™ 400 plate. Place a 37pm reversible strainer on top of a 50 mL conical tube. Note: Ensure the arrow on the strainer is pointing upwards. Use a new strainer and a new tube for each AggreWell™400 well to be harvested. To dislodge EBs from the microwell, firmly pipette medium up and down around the surface of the well and transfer the EB suspension to the strainer. Note: Aggregates will remain on the strainer; any unincorporated single cells will flow through. Rinse the entire surface of the well with DMEM / F-12 to collect any remaining EBs. Pass rinse over the strainer. Repeat step the previous step until all EBs have been removed from the well. One or two repeats should be sufficient to dislodge all EBs. Examine the well under a microscope to ensure that all EBs have been removed. Discard flowthrough. Flip the strainer and place on top of a fresh 50 mL conical tube. Wash with 2.5 mL of EB Medium B to collect the EBs into the tube. Using a serological pipette, mix EBs to evenly distribute them in the suspension. Using a serological pipette, add 2.5 mL of EB suspension into one well of a fresh non-tissue culturetreated 6-well plate. Move the 6-well plate in several quick, short, back-and-forth and side-to-side motions to distribute the EBs across the surface of the wells. Note: Even distribution of EBs is important to avoid aggregation. If EBs aggregate into a few large clumps, differentiation may be affected. 4. Incubate at 37°C and 5% CO2 for 2 days. Day 7. Warm EB Medium B to room temperature. Using a 5 mL serological pipette, gently add 2.5 mL of EB Medium B to each well. Move the plate in several quick, short, back-and-forth and side-to-side motions to distribute the EBs across the surface of the wells. Place the 6-well plate in a 37°C incubator at 37°C and 5% CO2 for 3 days. Day 10. Warm EB Medium B to room temperature and using a 5 mL serological pipette, slowly remove half (2.5 mL) of medium from each well. Them add 2.5 mL of fresh EB Medium B. d. Incubate at 37°C and 5% CO2 for 2 days. Day 12. Add DMEM / F-12 with 15 mM HEPES to Collagenase Type II to prepare a 2500 U / mL Collagenase II Solution. Gently pipette EBs and cells up and down in the wells to ensure all EBs are in suspension. Transfer the suspension from one well to a 15 mL conical tube. Centrifuge the EB suspension at 300xg for 5-10 minutes. Carefully aspirate the supernatant and add 1 mL of Collagenase II Solution to the pellet from one well. Gently pipette up and down to resuspend. Incubate at 37°C for 20 minutes. Add 3 mLTrypLE™ Express to the suspension. Gently pipette up and down to mix. Incubate at 37°C for 20 minutes. Gently pipette the suspension up and down to break up any remaining clumps. Add 6 mL of DMEM / F-12 with 15 mM HEPES. Centrifuge at 300xg for 5 -10 minutes. Remove and discard the supernatant. Optional: Pass the suspension through a 37 pm strainer to remove clumps and obtain a single-cell suspension. Resuspend the cell pellet in EasySep™ Buffer (Catalog #20144), RoboSep™ Buffer (Catalog #20104), or PBS containing 2% fetal bovine serum and 1 mM EDTA. Medium should be free of Ca++ and Mg + + . Isolate CD34+ cells using EasySep™ Human CD34 Positive Selection Kit II. Perform the CD34+ isolation using the protocol optimized for ES or iPS cell cultures; reduce the number of separations in the magnet to 2 to increase yield while retaining a sufficient CD34+ purity for further culture. Flow Cytometry: For phenotype assessment of hematopoietic progenitor cells by flow cytometry, use the following fluorochrome-conjugated antibodies: • Anti-Human CD34 Antibody, Clone 581 (Catalog #60013) • Anti-Human CD34 Antibody, Clone 8G12 (Catalog #60121) The protocol above is summarised in Table 1 below Table 1 - Protocol for generation of haematopoietic stem cells Day 0 Seeding in AggreWell Plate Day 1 - Day 2 EB medium A Mesoderm formation Day 3 EB medium A Day 4 - Transfer to a 6 well plate ■w r- Day 5 POPPING OUT Day 6 - Day 7 EB medium B Day 8 - Day 9 - Day 10 EB medium B Day 11 - Hematopoietic specification Day 12 DMEM / F-12 with 15 mM HEPES to Collagenase Type II Harvest and dissociate EBs, isolate CD34 Proceed to differentiation to T cells using the STEMdiffTM T cell kits 5 provided by STECELL Technologies to generate T cells. Example 2 - Protocol to Generate CAR-T Cells Overview Cell populations (PBMCs and CD4 / CD8 T-cellsJ are isolated from peripheral blood and activated with TransAct™ activation reagent for 24-48 hours. Activated cells are transduced with lentiviral vectors and expanded before harvest. The steps are shown in the flow chart of Figure 3. PBMC isolation 15mL of Histopaque 1077 (Sigma, #10771) is added to Leukosep tubes (Greiner Bio-one, #227290) at room temperature (RT) and the tubes are centrifuged at 1000g for 1 minute, in accordance with the manufacturer's instructions. Whole peripheral blood isolated from healthy donors are diluted 1:1 with Dulbecco's PBS (GIBCO, # 14190-094) and added to each Leukosep tube, with a maximum of 33mL of diluted blood added to each tube. Leukosep tubes are then centrifuged at 800g for 15 minutes at RT with no / slow brake to ensure that buffy coat layers are not disturbed. The cloudy mononuclear layer, visible in the mid-upper part of the tube is removed using a Pasteur pipette (Fisher, #10652842) and transferred into 50mL centrifuge tubes (Corning, #430290). Up to three buffy coat layers from the same donor are transferred into a single 50mL centrifuge tube, in order to ensure sufficient removal of remaining Histopaque and plasma from the cell pellets. Cell pellets are washed three times - tubes are topped up to the 50mL mark with PBS, centrifuged at 400g for 10 minutes, supernatant is removed and cell pellets flicked to aid re-suspension of cell pellets. Prior to centrifugation on the final wash step, a 20pL sample of cell solution is removed and counted using the NC-250™ NucleoCounter® (ChemoMetec) as per manufacturer's instructions. Depending on the experimental set up, isolated PBMCs are either progressed to CD4+ and CD8+ T-cell isolation. Red blood cell (RBC) lysis is performed when RBC contamination remains subsequent to gradient density centrifugation and a PBMC population is required, therefore the remaining RBCs would not be removed through CD4 / CD8 T-cell isolation. Sufficient numbers of PBMCs are transferred into a 50mL centrifuge tube and centrifuged at 300xg for 10 minutes before pouring off supernatant. lOx RBC Lysis Solution (Miltenyi Biotec, #130-094-183) is diluted with room temperature (RT) tissue-culture grade water to generate a lx RBC lysis solution. Cell pellets are resuspended in 25mL of lx RBC lysis solution and mixed well before incubating at RT for 10 minutes. The cell solution is centrifuged at 300xg for 10 minutes and the supernatant is discarded before cell pellets were resuspended in an appropriate volume of TexMACS™ media (Miltenyi Biotec, #130-097-196). CD4 / CD8 Isolation Subsequent to the isolation of PBMCs from peripheral blood, the cell solution is further enriched to isolate for either CD4+ or CD8+ T-cells alone, or a mixed population of CD4+ and CD8+ T-cells. Cell pellets are resuspended with 80pL of cold AutoMACS® buffer (Miltenyi Biotec, #130-091-221) and 20pL of FcR blocking reagent (Miltenyi Biotec, #130-059-901) for every 107 cells before being mixed and incubated at RT for 5 minutes. At this point, the appropriate microbeads are added depending on the T-cell population required: - CD4+ T-cell isolation: 20pL of CD4 microbeads (Miltenyi Biotec, #130-045-101) for every 107 cells - CD8+ T-cell isolation: 20pLof CD8 microbeads (Miltenyi Biotec, #130-045-201) for every 107 cells - CD4+ and CD8+ T-cell isolation: 20pL of CD4 microbeads for every 107 cells and 20pL of CD8 microbeads for every 107 cells The samples are mixed well and then incubated for 15 minutes at 4°C, at which point the AutoMACS® Pro-Separator (Miltenyi Biotec) can be set up with the appropriate buffers, according to the manufacturer's instructions. Subsequent to the incubation, the cells are washed with 1mL of cold AutoMACS® buffer for every 107 cells centrifuging at 300xg for 10 minutes. Supernatants are poured off and cell pellets were resuspended in 500pL of AutoMACS® buffer for every 108 total cells. At this point, the samples are transferred to the AutoMACS® proseparator and the "PosselS" protocol is used for a sensitive positive selection of the magnetically labelled T-cells. Within this protocol, the sample is loaded into the AutoMACS® proseparator and sequentially run through two columns sat within magnets which are turned on for cell separation. Non-magnetically labelled cells are eluted as the negative fraction, with the columns being washed thoroughly with AutoMACS® buffer. Once non-magnetically labelled cells were eluted, the magnets turn off and the magnetically labelled cells were flushed out of the columns as the positive fraction. The positive fractions are then washed twice by adding up to 15mL of PBS, centrifuging at 300xg for 10 minutes and discarding the supernatant. Samples are resuspended in an appropriate volume of TexMACS™ media and counted on the NC-250™ NucleoCounter® Cell samples are then either cryopreserved for later use or activated and transduced with vector. Activation of PBMC cells Isolated PBMCs are resuspended in TexMACS™ media, supplemented with 100 International Units (IU) per ml IL-2 (Sigma, #SRP3085) and a 1:100 dilution of TransAct™ T-cell activation reagent (Miltenyi Biotec, #130-109-104 / 130-111-160), at a density of IxlO6 cells per mL lxlO6 cells are plated into each well of a 24-well flat bottom cell culture plate (Greiner Bio-One, #662160) and incubated in a humidified incubator at 37°C with 5% CO2 for two days. T-cells are harvested from flat bottom cell culture plates, re-counted and resuspended to a density of lxlO6 cells / mL in TexMACS™ media supplemented with 100 lU / mL IL-2, and re-plated into flat bottom cell culture plates adding lxlO6 cells per well. The appropriate number of wells are transduced with the appropriate lentiviral vectors with the addition of a spinnoculation step where cells are centrifuged for 2 hours at 32°C before being incubated at 37°C with 5% CO2 for two days. T-cells CD4 / CD8 T-cells are resuspended in TexMACS™ media, supplemented with the cytokine cocktail of choice (either lOOIU / mL IL-2 or a combination of lOng / mL IL-7 (Miltenyi Biotec, #130-095-362) and lOng / mL IL-15 (Miltenyi Biotec, #130-095-764)) and a 1:100 dilution of TransAct™ T-cell activation reagent, at a density of lxlO6 cells per mL. T-cells are plated either directly into 24-well G-REX® plates (Wilson-Wolf, #80192M) and 6-well G-REX® plates (Wilson-Wolf, #80240M), adding lxlO6 cells or 4xl06 cells per well respectively prior to transduction, or seeded into 24-well flat bottom culture plates at a density of lxlO6 cells / mL for 24 hours prior to harvest and counting, following the protocol described in section Error! Reference source not found., and then seeded into 24-well and 6-well G-REX® plates, adding lxlO6 cells or 4xl06 cells per well respectively prior to transduction. Infection of T-cells The required volume of lentiviral vector to transduce known numbers of T-cells at a specific multiplicity of infection (MOI). The appropriate volume of lentiviral vector is removed from the -80°C storage location and thawed on ice for 10 minutes. Vials of lentiviral vectors are pooled together where appropriate and the required volume of lentiviral vector for the required MOI is added into each well of the culture plate. Cells are incubated at 37°C with 5% CO2 within a humidified incubator for 48 hours. Cell Expansion Cell expansion was carried out using standard 24-well flat bottom plates or G-REX plates. Standard 24-well flat bottom culture plates: Subsequent to transduction of T-cells, T-cells cultured within standard 24-well flat bottom culture plates must be split to a density of between 7.5xl05 - IxlO6 cells / mL every 2-3 days to ensure sufficient nutrient availability for expansion. T-cells are split by the addition of media to bring the cell density down to the recommended density, with half of the cell solution transferred into a new well if required. The appropriate volume of cytokine is added to each well subsequent to cell splitting to ensure the appropriate final concentration of cytokine is maintained throughout the culture procedure. G-REX® plates: After 48 hours of transduction, T-cells are transferred into G-REX® plates with either IxlO6 cells transferred into each well of a 24-well G-REX® plate or 4xl06 cells transferred into each well of a 6-well G-REX® plate, depending on the number of T-cells transduced. The appropriate volume of TexMACS™ media as per manufacturer's instruction and cytokines are added to each well with sufficient volume of the appropriate cytokines added to achieve 100 lU / mL IL-2 or lOng / mL of IL-7 and IL-15. Cells are incubated at 37°C with 5% CO2 within a humidified incubator for 72 hours. After 72 hours, the appropriate volume of either fresh IL-2 or IL-7 / IL-15 is added to each well to ensure a final concentration of 100 ILI / mL or lOng / mL respectively. Cells are incubated at 37°C with 5% CO2 within a humidified incubator for 48 hours. After a further 48 hours (at day 7 post-transduction), a media change is performed. G- REX® plates sre carefully removed from the incubator and a strippette is used to remove 6.5mL of TexMACS™ media from each well of 24-well G-REX® plates, or 30mL of TexMACS™ media from each well of 6-well G-REX® plates. Care must be taken to ensure that the strippette does not disturb the T-cells settled at the bottom of each well. The appropriate volume of warm TexMACS™ media supplemented with either 100 lU / mL IL-2 or lOng / mL IL-7 / IL-15 is added into each well of the G-REX® plates. Cells are incubated at 37°C with 5% CO2 within a humidified incubator for 48 hours. After 48 hours (Day 9 posttransduction), the appropriate volume of IL-2 (100 lU / mL) or IL-7 / IL-15 (lOng / mL) is added to each well. Cells are incubated at 37°C with 5% CO2 within a humidified incubator for 72 hours. After a further 72 hours (Day 12 post-transduction), T-cells are harvested from G-REX® plates by mixing with a strippette and transferring the cell solution into the appropriately sized centrifuge tube. Duplicate wells are pooled together. At this point, samples of cell solution are removed for counting on the NC-250™ NucleoCounter® and transduction efficiency analysis. Example 3 - Overview of the Assay With reference to Figure 4, commercially obtained PBMC were activated (day 0) and counted then placed in G-Rex plate for treatment with chemical carcinogens and / or transduced with MLV carrying the hTERT gene (day 1). Cells were seeded onto NaviPlates (day 20) for detection of outgrowths. ENUA was found to provide measurable numbers of outgrowths. ENUA +ve control treated cells versus untreated T-cells will be infected with a CAR-LV to determine the effect of the vector on outgrowth or to determine CAR-T tumorigenicity. Outgrowths are counted by plate reader - Agnostic assay. Clones and bulk cultures will be expanded for hlnGetox analysis. Figure 5 shows the trend in cell numbers over time. After seeding, treated and control T-cells, CAR-T cells contract in numbers due to cell death. Outgrowths begin to grow and pre-malignant cells last beyond six weeks. Example 4 - Comparison of Cell Expansion within 24-well Flat Bottom Culture Plate and 24-well G-Rex® Plate Cell populations were cultured in 24-well G-Rex® plates for 10 days (transferred to G-REX® on day 3 post isolation) with average fold expansion reaching 140 fold. The results are shown in Figures 6 to 8. Comparison of the fold expansion over the culture period demonstrated significantly better fold expansion of PBMCs in G-REX® plates. Error bars display Standard Error of the Mean (SEM). n=4 for 24-well flat bottom plates. n=ll for 24-well G-REX® plates. A two-tailed paired T test was performed to analyse significance. *** indicates that P <0.001. PBMCs were cultured in either 24 well standard flat bottom plates or 24 well G-REX plates 24 well flat bottom plate - N=4 24 well G-REX plate -N = ll .Error bars display SEM. Example 5 - Treatment of Cells with Agents to Determine the Effects of CAR. LV on CAR-T Outgrowth Example agent 1: Exposure to carcinogen To infected CAR-T cells grown in G-REX wells, a range of carcinogens are added individually and cells are then characterised for cell surface markers for CD4+, CD8+ or CD3+ using FLOW cytometry. Carcinogens are used to cause genome changes that result in CAR-T outgrowth via avoidance of senescence (untreated T-cells usually senesce between 3-5 weeks post culture). Carcinogen examples include ENUA, MNU and nickel chloride and are incubated with the infected CAR-T cells for between 30 minutes to 24 hours. Exposed cells are given 24 hours to recover before their viabilities are determined. Cells are then transferred to 96 well plates at a range of dilutions to achieve single cell derived outgrowths observed by a fraction of the 96 well plate generating expanding cells in individual wells. Clones suspected to be outgrowths are then transferred to G-REX® plates for expansion to isolate nucleic acids for assay. Example agent 2: Exposure to hTERT vector hTERT expression is known to support cellular immortalisation. Expression of hTERT has been shown to efficiently cause cells to avoid senescence and is, therefore an ideal agent to be used as a positive control for T-cell outgrowth. Retrovirus vectors (RV) or exogenous hTERT (encoding the catalytic subunit of human telomerase) can be transferred to T-cells for this purpose. Cells activated from PBMC or iPSc grown in G-REX wells are infected with RV carrying the hTERT cDNA and given 24 hours for gene expression and further characterised for cell surface markers for CD4+, CD8+ or CD3+ using FLOW cytometry and their viabilities determined. Cells are then transferred to 96 well plates at a range of dilutions to achieve single cell derived outgrowths 5 observed by a fraction of the 96 well plate generating expanding cells in individual wells. Clones suspected to be outgrowths are then transferred to back to G-REX plates for expansion to isolate nucleic acids for assay. Treatments for a typical experiment to determine CAR-T outgrowth are 10 summarised in Table 2 below. Table 2 - Treatments for a typical experiment to determine CAR-T outq rowth T cell treatments Outgrowth frequency None -ve control None GFP LV infection -ve control None CD19 LV infection experimental None hTERT RV infection 4-ve control Extended lifespan ENUA Carcinogen 4-ve control lxlO'4 GFP+carcinogen 4-ve control lxlO'4 CD19+carcinogen Experimental plus +ve control Pending hTERT-i-carcinogen -i-ve control Extended lifespan T cells are exposed to treatments to determine cell outgrowth frequency. Cell infected with CAR-LV are used to identify the frequency of outgrowth associated with the vector and positive controls are used to show CAR-T outgrowth is achieved. These results show that T-cells can be immortalised using chemical carcinogens or a positive control vector (hTERT), but not using the CD19 LV vector alone or a negative control GFP LV. Example 6 - Comparison of Different Carcinogens Flow cytometry was carried out on treated cells to identify the percentage of cell expression CD3+, CD4+ and CD8+ T cell markers. The results are shown in Table 3. Table 3 - T-cell flow cytometry of treated cells %CD3+ %CD4+ %CD8+ No carcinogen 99.2 % 67.7 % 22.3 % GFPLV 98.9 % 85.6 % 11.8% CD19 LV 99.8 % 92.1% 1.91 % hTERT 79.0 % 79.4 % 6.99 % ENU 98.8 % 85.0 % 12.6 % GFP+ENUA 99.9 % 97.9 % 1.77 % CD19+ENUA 77.0 % 66.0 % 14.1% hTERT+ENUA 71.4 % 69.1% 12.8 % CAR6 (d 8) 89.8 % 49.5 % 40.8 % Following activation, and 4-ve control treatments ENUA was identified as the most effective carcinogen providing detectable pre-malignant outgrowths past 6 weeks. Flow cytometery showed percentage of CD4 and CD8 +ve out of CD3+ with and without +ve and -ve control treatments. GFPLV = -ve control LV, CD19LV = CAR carrying vector, hTERT= MLV carrying hTERT 4-ve control vector, ENUA = powerful chemical carcinogen 4-ve control. These data show that cells treated with carcinogens retain their T-cell markers, confirming that they remain T-cells after treatment. Figure 9 illustrates T-cell viability after treatment. Cells were counted following activation, treatment and expansion in G-Rex® plates. GFPLV = -ve control LV, CD19LV = CAR carrying vector, hTERT= MLV carrying hTERT 4-ve control vector, ENUA = powerful chemical carcinogen 4- ve control. Flow cytometry also revealed that the cells retain their identity following treatments as determined by CD4-i-ve and CD8-i-ve markers (Figure 10). These results demonstrate that the cells still have the characteristics and markers of T-cells. Example 7 - Expansion of Cells in NaviPlates To identify outgrowths of T-cells following treatment with ENUA and expansion in G-Rex® plates, cells were seeded onto NaviPlates at different densities. NaviPlates with 384 well formats were used. Outgrowths that could be counted were identified at densities of 100, 1000 or above cells / well. At present clonal outgrowth can be detect down to 100 cell / well in a 340 well set up. Example 8 - Generation of Outgrowths by ENUA Cells were treated with carcinogens and grown in Naviplates over time. In most cases, cells become senescent after 3-4 weeks. However certain wells showed cells growing in wells past 6 weeks indicating they were immortal. As shown in Figure 11, treatment with ENUA by itself provided a sensitive detection of tumorigenic clones. To find and optimise positive controls that generated outgrowths that could be detected by NaviPlates after treatment, ENUA, MNU, X-ray and nickel chloride and a RV carrying hTERT, carcinogens were tested. ENUA / hTERT and ENUA alone were found to provide viable colonies that remained proliferative and expanded more than 6 weeks in culture. Non-treatment resulted in colonies that do not survive past 3-4 weeks. Following treatments, colonies of outgrowths were identified. Cells would appear initially as proliferating colonies, then in most cases die at approximately 3-4 weeks post treatment. ENUA or ENUA / hTERT treatments generated colonies that proliferate past the 6-8 week time points (shown by the white arrows in Figure 12). These colonies could be isolated and expanded in Phase II for hlnGetox analysis to identify any association of the vector with outgrowth if increased colony frequency occurs. Example 9 - Optimisation of Vector Production This process is illustrated schematically in Figure 13. Dayl: Seed T175 flasks with 3xl07 HEK cells. Incubate overnight at 37°C and 5% CO2. Day 2 am: Tranfect HEK cells with plasmids plus Jet-PRIME transfection reagent. Day 2 pm: Media change 4-6 hours post-transfection. Day 3: Image to confirm transfection. Day 4: Harvest virus and concentrate via sucrose cushion at 4 °C. Day 5: Resuspend viral pellets, aliquot at store aliquots at -80 °C. Set up titre determination assay. Day 7: Analyse HEK transduction efficiency via flow cytometry. Calculate biological titre. Vectors are produced via transient transfection of HEK 293 cells (Figure 13) or from the pBabeh MLV PA317 cell line. LV transfection was carried out using PEI, FugeneR6 or Genejuice reagents. Transfection was followed by media changes and ultracentrifugation then re-suspension of virus pellets and infection of fresh HEK cells. Cells were passaged and media removed to test for replication competent lentivirus (RCL). MLV vector generated from PA317 cells can be titrated by p24 gag assay. LV.VSVg.hPGK.CD19-41BB-CD3z.IZW = CAR vector was produced by transient transfection of 293T cells. The vector LV SIN / GFP vector which carries the GFP reporter gene acted as the -ve control LV (GFP LV). The PA317 cell line continuously producing pBabehTERT was kindly provided by Dr Yasu Takeuchi UCL and produced by plating these cells in culture, allowing them to become confluent, changing their medium for DMEM 10%FCS and harvesting the vector after 24 hours. Example 10 - Molecular Analysis of Vector Integration Sites Materials and Methods Nucleic acid isolation: DNA and RNA samples were isolated from transduced samples according using DNeasy Blood &Tissue Kit and RNeasy Mini Kit (Qiagen, Manchester) respectively, according to manufacturer's instructions. RNA was treated with DNAase I to remove contaminants, according to manufacturer's instructions (Qiagen). Nucleic acid concentration and purity was analysed using NanoDrop™ 2000c spectrophotometer (ThermoFisher Scientific, Hemel Hempstead). Nucleic acid integrity analysis: DNA samples were initially analysed for vector presence. Therefore, a vector specific primer pair was designed and used in a standard PCR with lOng DNA. Products were applied to 2% gel electrophoresis to visualize PCR amplicons and the detection of expected bands. LV insertion site analysis: Viral vector integration site analysis was performed using Sharing-Extension Primer Tag Selection / Linear-Mediated Polymerase Chain Reaction (S-EPTS / LM-PCR), which is a shearing DNA based integration site analysis method, followed by next-generation sequencing. Raw sequencing data were trimmed based on quality (Phred) and filtered for containing both molecular barcodes at full identity. Remaining reads were further analysed using GENE-IS. Briefly, sequences were further trimmed and only sequences containing the expected vector-specific stretch were considered for following steps. First, sequences were aligned to the human genome (UCSC assembly release number hg38) by Burrows-Wheeler Aligner (BWA) MEM algorithm. Potential integration sites were then mapped with BLAST at a minimum alignment identity percentage of 95%. Adjacent genes and other features were annotated according to RefSeq database. For each detected integration site, the relative sequence count compared to all sequences was calculated. Biologically relevant IS clusters, called common integration sites (CIS), were analysed using a graphs based approach. Any IS detected was considered as node that contained the IS locus. If the distance of two nodes was less than 50kb, the nodes were connected and resulting nodes sets considered as CIS. A list of over 700 well-defined cancer genes was compiled from the Cancer Gene Census database (https: / / cancer.sanqer.ac.uk / census). Cancer gene data was obtained from Ensembl human genes (http: / / www.ensembl.org / biomart / martview / : version GRCh38.plO). Relative frequencies of integration sites, that were detected within a lOOkb window of a TSS of a cancer-related gene, were analysed. Results The results of the EPTS / LM-PCR molecular analysis of LV infected cells are shown in Table 4. Table 4 - Integration of vector in cancer genes pHV LV IS in Cancer genes isolated from infected cells HDLBP MIR4801 ATP13A4 LOC102467213 SPDYC EIF2AK4 LINC01090 UBAP2L JRKL-AS1 BRPF3 TEX36-AS1 PDCD10 POU3F2 LINC01568 CPE POM121L12 CPNE4 XPO5 These data show that LV integrates into cancer genes. Further 5 investigations may include using total RNA from isolated clones to reveal any changes in the expression of the genes with LV insertions. Figure 14 shows results from clones after infection where cancer genes with virus integrations are determined by EPTS / LM-PCR and with altered expression using q-RT PCR. Example 11 - Differential analysis of Gene Expression Cancer genes identified by EPTS LM PCR are then used for analysis of their expression changes compared to uninfected cells. Materials and Methods Cloning iPSC: Prior to single cell cloning, iPSCs were transduced with lentiviral vectors as previously described. On day of SCC complete mTeSRl (StemCell Technology) medium with conditioned medium at a ratio of 1:1 was prepared. The medium was supplemented with 10 pM Y-27,632 (Calbiochem) to enhance the cell survival. Transduced positive GFP cells were washed with PBS (Sigma-Aldrich) once and gently dissociated using Gentle Cell Dissociation reagent (StemCell Technology) for 15 minutes. The single cells were resuspended in mTeSRl and Y-27632 and counted by haemocytometer. To make the final 2 cells / ml, 4.8 pl of cell solution was transferred to 12 ml of complete / conditioned medium and 500 pl from the cell suspension was dispensed per well of a 24-well plate. This was to ensure the plate was seeded at a density of 1 cell / well. Following seeding, the cells were undisturbed for 7 to 10 days. After 7 days, the plate was scanned for colonies. The cells from each colony were expanded and harvested for DNA / RNA extraction. 33 (pHR LV) and 7 (pHV LV) clones were isolated from infected samples respectively. qRT-PCR: RNA was extracted from 3D hepatospheres using RNAeasy Mini RNA Extraction Kit (Qiagen) according to manufacturer's instructions. RNA quantity and quality were evaluated using NanodropTM200c. Following this step, cDNA was amplified using the RT2 First Strand Kit (Qiagen) following the manufacturer's instruction. qPCR was performed with TaqMan Fast Advance Mastermix and primer pairs, using a Roche LightCycler 480 real-time PCR system. Gene expression was normalised to housekeeping gene; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed as relative expression over 3D hepatospheres on day 0 of differentiation as control sample. qPCR was conducted in triplicate and data was analysed using Roche LightCycler 480 software. Results Table 5 below shows identification of LV integration sites (IS) in cancer genes (CG) in iPSc by EPTS / LM-PCR after infection. Shown are IS in oncogenes or proto-oncogenes after infection. Table 5 - Integration of pHV LV vector in cancer genes with unregulated expression in isolated clones Clone number Gene name 1 FMR1NB, FRAS1 2 DNAH7, OR52N4 3 DAZAP1, TMEM 232 4 CTNNA3, WDFY2, BASP1P1 5 BASP1P1, MGAT4C, MAPK10, MGF14, TMEM189 6 DANCR, CTC1 7 CTC1, CTC2, DANCR, PCGF3 8 ATRNL1, IP6K1, ANGPT1, ZMYM4, VBP1, FUT9, MIR4431, NLGN4X 9 IRAKI, RPS6KA4, FRYL, LEF1, IL26 10 AXIN1, LINC01571, MED13L, SOX8, THTPA, CASC6, CSK, RBFOX3, VPS13C, ANKRD11 11 LIN01249, PLCG1, ADAM21P1, LINC01249, MGAT4C, MARS 5 Table 5 shows results from clones after infection where cancer genes with virus integrations are determined by EPTS / LM-PCR and with altered expression using q-RT PCR. Single cell IPSC clones isolated from bulk infected LV populations and expanded for DNA and RNA extraction. Isolated DNA was subjected to EPTS / LM-PCR IS analysis of genes in proximity to IS (lOOkbp 5' and 3' window). Primer sets particular to each IS was designed and generated for q-RTPCR analysis on RNA from each clone to quantify gene expression for each gene compared to uninfected iPSc. Genes are listed that were found upregulated associated with each vector with significance of p<0.05. Dicr'i icc inn Lf I S3) Im U 33 IU11 These data show that following insertion, compared to the expression of cancer genes in uninfected cells, LV alters the expression of gene close to the site of insertion. Figures 15 to 18 show differential analysis of gene expression between LV infected iPSC versus uninfected iPSC controls. Figures 15 and 16 are volcano plots showing up- (LogFC>0; right) and down-regulated (LogFC<0; left) genes in pHR (Figure 15) or pHV-infected (Figure 16) iPSc compared with uninfected iPSc. Statistical significance is indicated in different colours. Figures 17 and 18 are dot plots of most enriched signalling pathways within GO term biological process gene sets for pHR (Figure 17) or pHV infected (Figure 18) iPSC. Mean Log2 fold changes are indicated by colour gradients. The Benjamini-Hochberg adjusted p values are indicated by dot sizes (control uninfected = CTRL). Example 12 - Genotoxicity Assessed Using Cancer-Specific Signature Scores Bioinformatic analysis pipeline: An analytical pipeline in R (v,4.0.2) was established. Raw data was curated into feature-sample matrices and streamlined into ExpressionSet objects using BioBase (v.2.50.0). The subsequent analysis mainly included differential analysis, gene set enrichment analysis, signature score assessment, and weighted gene co-expression network analysis. Differential Analysis: limma (v3.46.0) was used for differential analysis on count matrices. First, we normalised and conducted Iog2-transformation on count matrices. Next, design matrices were constructed using phenotypic data and fitted to the processed matrices. Following empirical Bayes moderation, differentially expressed genes (DEGs) were identified and those with an absolute Iog2 fold-change greater than 1 and BH-adjusted p-values less than 0.01 were retained. Lastly, these DEGs were visualised using EnhancedVolcano (v. 1.8.0). Gene Set Enrichment Analysis (GSEA): clusterprofiler (v3.18.1) was used for GSEA. First, gene expression levels were ranked from highest to lowest by comparing transcriptomic samples from study groups against controls. Next, Hallmark gene sets or GO terms from MSigDB (v.7.5.1) were used to perform GSEA, resulting in BH-adjusted p values, ratio of genes from each set, and other attributes. Lastly, treeplot or cnetplot were used to visualise the results. Signature Score Analysis: Molecular signatures for specific cancers were defined using TCGA RNA-seq datasets. Differential analysis criteria for these signature genes were set at Iog2 fold-change >2 and BH-adjusted p-values <0.01. The scaled mean expression levels of these genes were then determined and visualised using pheatmap (vl.0.12). Weighted Gene Co-expression Network Analysis (WGCNA): WGCNA (v.1.70-3) was used for the analysis. Normalised and transposed count matrices were used at the outset and network topology analysis were performed, resulting in an optimal soft threshold power. Topological overlap matrix was constructed using the blockwiseModules command and traits associated with each module were determined by calculating hypothetical central genes. Lastly, potential key drivers in selected modules were identified by using the intramodularConnectivity command. To infer the probability of carcinogenesis induced by pHR or pHV lentiviruses in iPSC or HLC, we first defined cancer-specific signatures by performing differential analysis of genes highly expressed in each cancer type compared with each normal tissue. Through pathway analysis hallmark gene sets (Figure 19), we found that these signatures are associated with pathways regarding nucleic acid synthesis / metabolism, active transcription, cell proliferation, as E2F Targets and G2M Checkpoint are enriched regardless of cancer types. We utilised these signatures to score transcriptomes of iPSC or HLC infected with pHR or pHV lentiviruses on Day 3 or Day 30 and compared with respective controls (Figure 20). In general, infected iPSC are characterised by their higher cancer scores than infected HLC. This might be due to the nature of iPSC that are rapidly proliferating than differentiated cells. On Day 3, pHR-infected iPSC or HLC have higher cancer scores than pHV-infected iPSC or HLC. Figures 19 and 20 show evaluation of the potential carcinogenic impact of pHR or pHV lentiviral infection. Figure 19 represents dot plots of most enriched signalling pathways within Hallmark gene sets for cancerspecific molecular signatures. Expression percentages of gene sets are indicated by dot sizes. The Benjamini-Hochberg adjusted p values are indicated by colour gradients. Figure 20 shows a heatmap illustrating the cancer-specific scores for both iPSCs and HLCs under various conditions, including different days post-infection and vector subtype. Notably, iPSCs generally exhibit higher cancer propensity scores than HLCs, potentially due to their higher proliferation. At Day 3 postinfection, pHR infected cells demonstrate elevated cancer-specific scores relative to pHV infected cells, consistent with earlier observations. By Day 30, however, pHV infected iPSCs exhibit higher scores than their pHR infected counterparts, suggestive of carcinogenesis. All optional and preferred features and modifications of the described embodiments and dependent claims are usable in all aspects of the invention taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another. It will be appreciated by those skilled in the art that changes could be made to the embodiments and examples described above without departing from the broad inventive concept. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the claims. References Bishop, D.C., et al., Development of CAR T-cell lymphoma in 2 of 10 patients effectively treated with piggyBac-modified CD19 CAR T cells. Blood, 2021. 138(16): p. 1504-1509. Braun, C.J., et al., Gene therapy for Wiskott-Aldrich syndrome—longterm efficacy and genotoxicity. Sci Transl Med, 2014. 6(227): p. <d— / I Cl O J ■ Cavazzana-Calvo, M., et al., Transfusion independence and HMGA2 activation alter gene therapy of human beta-thalassaemia. Nature, 2010. 467(7313): p. 318-22. Cesana, D., et al., Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations. J Clin Invest, 2012. 122(5): p. 1667-76. Howe, S.J., et al., Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest, 2008. 118(9): p. 3143-50. Magnani, C.F., et al., Transposon-Based CAR T Cells in Acute Leukemias: Where are We Going? Cells, 2020. 9(6). Miyoshi, H., et al., Development of a self-inactivating lentivirus vector. J Virol, 1998. 72(10): p. 8150-7. Ott, M.G., et al., Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med, 2006. 12(4): p. 401-9. Shah, N.N., et al., Clonal expansion of CAR T cells harboring lentivector integration in the CBL gene following anti-CD22 CAR T-cell therapy. Blood Adv, 2019. 3(15): p. 2317-2322.

Claims

1. A method of assessing the safety of a gene therapy vector or a cell modified by the vector including:a) providing a population of genetically modified test cells, wherein the test cells have been transfected or infected with a vector carrying exogenous genetic material;b) exposing the population of modified test cells to a carcinogen;c) growing the modified test cells for a period of time sufficient to produce immortal cell lines;d) comparing the survival of the modified test cells with at least one control to determine whether the vector contributes to carcinogenicity.

2. A method of assessing the safety of a gene therapy vector or a cell modified by the vector including:a) providing a population of test cells to be genetically modified;bi) genetically modifying the population of test cells with a vector carrying exogenous genetic material, selecting modified test cells, and exposing the modified test cells to a carcinogen, orbii) exposing the population of test cells to a carcinogen then modifying the population of test cells with a vector carrying exogenous genetic material;c) growing the modified and carcinogen exposed test cells for a period of time sufficient to produce immortal cell lines; andd) comparing the survival of the modified and carcinogen exposed test cells with at least one control to determine whether the vector contributes to carcinogenicity of the cells.

3. A method as claimed in claim 1 or 2, wherein the test cells are immune system cells and the genetically modified test cells are for use in immunotherapy.

4. A method as claimed in claim 1, 2 or 3, wherein the exogenous genetic material includes chimeric antigen receptors.

5. A method as claimed in any preceding claim, wherein the test cells are T cells.

6. A method as claimed in claim 5, wherein the modified test cells are CAR-T cells.

7. A method as claimed in any preceding claim wherein:a) the control is a population of unmodified cells that do not contain a vector carrying exogenous genetic material,wherein the method includes:b) exposing the population of unmodified control cells to the carcinogen; andc) growing the unmodified control cells for a period of time sufficient to produce immortal cell lines.

8. A method as claimed in any preceding claim wherein:a) the control is a population of genetically modified cells, wherein the cells have been transfected or infected with a vector carrying exogenous genetic material, wherein the vector is known not to contribute to carcinogenicity,wherein the method includes:b) exposing the population of genetically modified control cells to the carcinogen; andc) growing the genetically modified control cells for a period of time sufficient to produce immortal cell lines.

9. A method as claimed in any preceding claim including the step of genetically modifying the population of test cells with a vector carrying exogenous genetic material and selecting only those cells that have been successfully modified.

10. A method as claimed in any preceding claim, wherein the population of test cells is obtained from a patient.

11. A method as claimed in any preceding claim, wherein the population of test cells is obtained from a donor.

12. A method as claimed in any preceding claim, wherein the vector is a virus.

13. A method as claimed in any preceding claim, wherein the vector is a retrovirus.14 A method as claimed in any preceding claim, wherein the vector is a lentivirus.

15. A method as claimed in any preceding claim, wherein the test cells are obtained from peripheral blood mononuclear cells.

16. A method as claimed in any preceding claim, wherein the cells are generated from iPSCs.

17. A method as claimed in any preceding claim, wherein the period of time is at least three weeks or at least four weeks.

18. A method as claimed in any preceding claim, wherein the period of time is at least six weeks.

19. A method as claimed in any preceding claim, wherein the period of time is at least eight weeks.

20. A method as claimed in any preceding claim, wherein the period of time is at least 12 weeks.

21. A method as claimed in any preceding claim, wherein the carcinogen is a chemical carcinogen.

22. A method as claimed in any preceding claim, wherein the carcinogen is ENUA.

23. A method as claimed in any preceding claim, wherein the test cells are CAR-T cells for use in immunotherapy.

24. A method of designing a vector for use in gene therapy including carrying out the method of assessing the safety of a gene therapy vector as claimed in any preceding claim, harvesting nucleic acid from surviving test cells modified by a vector determined to contribute to carcinogenicity, carrying out one or more assays to characterise themodification of the test cells by the vector, redesigning the vector on the basis of the results of those assays, and repeating the method of assessing the safety of a gene therapy vector.5 25. A method as claimed in claim 24, wherein the assays include oneor more of:(i) an integration site assay to determine vector insertion sites in the host genome;(ii) a gene expression assay to determine changes in gene expression 10 over time;(iii) an R.NA transcript assay to identify new transcripts or truncated transcripts over time;(iv) a CpG methylation assay of genes at the integration site;(v) a gene inactivation assay.15

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