Patent Application
20200264164
The present invention relates to an analytical process for the assessment of the genotoxic potential, also termed mutagenic or transforming activity, of an agent in respect to hematopoietic cells. The agent can be selected from natural and synthetic chemical compounds, and preferably from heterologous expression cassettes, e.g. contained in non-natural viral particles or non-natural viral nucleic acid constructs, e.g. nucleic acid constructs containing a viral element. The heterologous, e.g. viral element can e.g. be a promoter or enhancer region for transgene expression, a viral primer binding site, a viral 5′ region containing a U3 region, and/or a viral packaging signal.
The analytical process is suitable for assessing the genotoxic potential of viral particles for mammalian cells, e.g. for viral particles containing a nucleic acid construct intended for gene therapy. The hematopoietic cells are primary stem cells and/or stem precursor cells of the hematopoietic system, e.g. of human, murine or rat origin. The process is an in vitro process, using the primary stem cells and/or stem precursor cells of the hematopoietic system.
Modlich et al., Blood 108 (8), 2545-2553 (2006) describe that gene transfer to primary murine lineage negative bone marrow cells by adding viral nucleic acid constructs containing a SIN (self-inactivating 3″LTR upon integration into the host genome) nucleic acid construct resulted in lower frequencies of replated cells than an LTR-driven nucleic acid construct after 28 days under cell culture conditions. Higher replating frequencies in the cell culture were detected as cell growth in a limiting dilution series and were found to be indicative of a higher transforming potential of the viral particle. A disadvantage of this method is that the increased cell growth which is determined as an indicator for transformation strongly depends on the myeloid cultivation conditions. The replating phenotype is triggered or accompanied by activation of a very limited number of proto-oncogenes (mainly Mecom). Insertional upregulation of genes of the lymphoid differentiation lineage cannot be detected. A further disadvantage is that the cultivation of cells takes a long time and that the microscopic analysis of cell growth yields no information about the underlying transforming event.
It is an object of the invention to provide an alternative process for analysis of the genotoxic potential of an agent in mammalian hematopoietic cells. Preferably, the alternative process can be performed in a shorter time and/or yields a more detailed picture of the genotoxic potential.
The invention achieves the object by the features of the claims, especially by providing a process for analysis of the genotoxic activity of an agent for hematopoietic cells, the process comprising the steps of adding the agent to hematopoietic stem and progenitor cells in a cell culture under cell culture conditions, cultivating these cells for a pre-determined duration to produce cultivated cells, isolating total RNA, preferably total mRNA, from the cultivated cells and determining the concentration of the RNAs encoding at least one, preferably all of the RNA sequences of Table 1, preferably using the indicated probe sequences. Generally preferred, the RNAs are mRNAs.
and preferably relating the concentrations of the RNAs from cultivated cells to which the agent was added to the concentrations of RNAs determined for cells cultivated under the same cell culture conditions for the same duration in the absence of the agent.
Preferably, the concentration of at least one or all of the RNAs of Table 2 is additionally determined:
The step of relating the concentrations of the mRNAs determined for the cells to which the agent was added with subsequent cultivation to the concentrations of the mRNAs determined for the same type of cells which were cultivated without addition of the agent under the same cell culture conditions results in the determination of differences in the expression levels of these RNAs, which differences in expression levels are characteristic for the genotoxic effect of the agent. As the expression levels of mRNAs have been found to depend on the culture conditions, the cells are cultivated under the same culture conditions and the same duration for each comparison, preferably performed in parallel each time. The cells cultivated under the same cell culture conditions for the same duration in the absence of the agent are also referred to as a negative control or mock.
Preferably, an agent that is known to have a significant genotoxic potential is used under the same culture conditions to provide a positive control, preferably performed each time in parallel to the cells to which the agent was added, and the process comprises the step of comparing the concentrations of the mRNAs determined for the positive control to the concentrations of mRNAs determined for the cells to which the agent to be analysed was added. Generally, the process preferably comprises the comparison of expression levels of the RNAs determined for the cells to which the agent was added to the expression levels of the cellular RNAs determined for the negative control and/or of the positive control, wherein all cells were cultivated under the same cell culture conditions for the same pre-determined duration.
It has been found that the determination of the concentration of these mRNAs allows the differentiation between immortalized cells and non-immortalized cells. Therein, immortalized cells are considered to represent precancerous cells and/or tumour cells, indicating a high genotoxic activity of the agent analysed in the process. Specifically, it has been found that expression levels of the RNAs that are determined for the cells to which the agent was added, which expression levels have differences in comparison to the expression levels of the mRNAs determined for the negative control and/or no or less important differences in comparison to a positive control, are characteristic for the genotoxic activity of the agent. Using a viral particle known to have genotoxic activity as an exemplary agent and murine hematopoietic stem cells and their progenitor cells, significant differences in expression levels of the RNAs were found in comparison to the expression levels determined for the RNAs in a parallel negative control.
The step of relating the concentrations of the mRNAs determined for the hematopoietic cells to which the agent was added to the concentrations of mRNAs determined for the hematopoietic cells cultivated under the same cell culture conditions for the same duration in the absence of the agent preferably is by calculating the ratio of the specific mRNA concentrations. Generally, the concentration ratio can be replaced by the ratio of measurement values measured for the mRNA concentrations, e.g. by the ratio of fluorescence measurement values. Preferably, only the mRNAs are considered as having an expression level which significantly differs from the expression determined for the negative control. For example, a significant difference can be defined for a difference of at least a log2 of the concentration ratio bigger than +1 or smaller than −1. This exemplary cut-off for expression levels which compared to the negative control differ at least by such a factor can be used in order to limit the RNAs to those which have an important difference in expression level.
Preferably, for agents containing a nucleic acid, the copy number of this nucleic acid in relation to the genome of the cells is determined. In this manner, the influence of the copy number can be taken into account in the determination of the genotoxic risk of an agent. For agents not containing a nucleic acid, the concentration of the agent should be measured in order to take into account the concentration effect, for example by dividing the concentration of RNAs determined in the cells with agent by the concentration of the added agent.
The culture conditions comprise the composition of the cultivation medium, cultivation temperature, the composition of the gas atmosphere, and the movement of the cultivation medium, e.g. by shaking, stirring, or static incubation.
A preferred medium for the hematopoietic stem cells and their progenitor cells used in the process contains growth-promoting cytokines, especially the following cytokines: stem cell factor (SCF), preferably in addition interleukin-3 (IL-3), Flt3-ligand, interleukin-11 (IL-11), and more preferably additionally interleukin-7 (IL-7), and further optionally additionally interleukin-6 (IL-6), interleukin-15 (IL-15), thrombopoietin (TPO) and further optionally additionally at least one notch ligand (DL1 or DL4).
An advantage of the process of the invention is that it is not restricted to measure presence of cells that proliferate during a long period of cultivation as e.g. in the process of Modlich (2006), as it has been found that the RNA isolated in the process according to the invention does not necessarily contain, and preferably not contain relevant levels of the mRNA Lmo2, whereas in cells treated in the process of Modlich (2006) all cells that finally proliferated, when analysed by the process of the invention were found to express Lmo2. Therefore, the process of the invention has the advantage of identifying Lmo2, which is an indicator for precedence of adverse events in gene therapy, e.g. an indicator associated with the development of leukemia by gene therapy. As a further advantage, the process is not dependent on the expression of Lmo2 by the cells to be analysed.
Following addition of the agent to the cells in cell culture and prior to cultivating the cells, the process can comprise the step of incubating the cells in the presence of the agent and removing the agent, e.g. by exchanging the culture medium of the cells in the cell culture for fresh medium without the agent, e.g. following a prior incubation period of 1 to 3 d.
Preferably, the agent is a non-natural nucleic acid construct, optionally packaged as a viral particle. The non-natural nucleic acid construct can e.g. contain a non-natural or a natural nucleic acid sequence having the ability to integrate into genomic DNA, e.g. at least a sequence having homology to the cell, a viral primer binding site, a viral 5′ region containing a U3 region, and/or a viral packaging signal promoter sequence.
When the agent comprises a nucleic acid construct, e.g. the agent is a viral particle, the process preferably comprises the step of performing a quantitative analysis of the nucleic acid construct in total DNA obtained during the pre-determined duration from an aliquot of the cultivated cells for determination of the average copy number of the agent in the mammalian cell. Preferably, the quantitative analysis of the nucleic acid construct of the agent in total DNA obtained from the cells is made by quantitative PCR (polymerase chain reaction) using a primer pair specific for the nucleic acid construct, and preferably also a primer pair specific for a genomic DNA section of the cells, in a separate or in the same PCR. The step of collecting DNA from the cells for the quantitative analysis of the nucleic acid construct can e.g. be made at 2 to 8 d, preferably 4 to 8 d, more preferably 4 d following the addition of the agent to mammalian cells in a cell culture, in order to avoid detection of viral nucleic acids from episomes or from transiently infected cells.
The pre-determined duration of cultivating the cells following the step of adding the agent to the cells in culture can e.g. amount to 10 to 15 days, and this duration can optionally be divided into a first and a second partial duration, wherein the first partial duration of cultivating the cells is followed by a step of diluting the cells and then by cultivating the cells for the second partial duration, e.g. by re-plating the cells at lower cell number and cultivating for a second partial duration. In this embodiment, the step of diluting the cells for a second partial duration of incubation can increase the selection pressure on the cells and therefore result in more pronounced differences in the translation of mRNA species. Preferably, the first partial duration can be 2 to 10 d, preferably 6 to 8 d, and the second partial duration that follows the dilution of the cells for further cultivation can be 5 to 12 d, e.g. 5 to 7 d.
Optionally, the pre-determined duration of cultivating the cells, optionally the first partial duration can be 2 to 6 d, preferably 3 to 4 d. Such duration can have the advantage of determining concentrations of mRNAs, which preferably are mRNAs, characteristic for general toxicity of agents, whereas longer durations, especially durations of 8 to 15 d, have been found to result in specific differences in expression levels of the mRNAs regarding genotoxicity. This shows a specific advantage of the process of the invention in that it generally results in the identification of genotoxic activity of an agent at higher sensitivity and identification but also the identification of a general toxicity of an agent that prevents or reduces proliferation of the cells.
A further advantage of the process of the invention is that it does not require the replating step needed for microscopic analysis of transformed or immortalized cells and hence can use shorter pre-determined durations of cultivating cells as e.g. used in the process of Modlich et al. (2006), which uses an expansion plus limiting dilution of the murine hematopoietic stem and progenitor cells in culture for 4 weeks following transduction of cells.
The primary hematopoietic stem cells and/or stem progenitor cells obtained freshly or used after a freeze-thaw-cycle, which are used in the process of the invention, e.g. of murine origin, can be cultivated under cell culture conditions prior to adding the agent, adding nothing or an inactive compound for the negative control, and adding a known genotoxic agent as a positive control, respectively, to separate cells in culture at the same prior incubation period. This cultivation period prior to adding the agent can e.g. last for 1 to 3 d, e.g. 2 d for freshly obtained primary cells.
The step of determining the concentration of the mRNAs can be by reverse transcription followed by quantitative PCR, preferably by hybridisation of the mRNA, preferably the total RNA isolated from the cultivated cells, to nucleic acid probes which are immobilized on a substrate, e.g. arranged on a substrate as an array of probes. Arrays of nucleic acid probes immobilized on a substrate are generally known as microarrays which are suitable for quantification of hybridizing mRNAs. Optionally, concentration of mRNAs can be quantified by next generation sequencing of the reverse transcribed RNAs.
In an optional embodiment, the process contains the evaluation of the mRNAs which are considered as deregulated. Therein, the mRNAs are considered deregulated for which subsequent to normalization, preferably Quantil normalization (e.g. performed using the R-package limma linear models for microarrays), for the concentration ratio with the negative control a loge of bigger than +1 or smaller than −1 is found.
Preferably additionally, a normalized enrichment score (NES) is calculated for each of the mRNAs considered deregulated. The NES is calculated as described by Subramanian et al., Proc Natl Acad Sci USA (2005) Oct. 25; 102(43):15545-50. In short, the NES is calculated by the steps of generating a numerical matrix of the protein encoding genes, creating a running-sum statistic with a maximum deviation from zero (Enrichment Score), determination of a nominal P value (Estimation of Significance Level) and adjusting the Enrichment Score for Multiple Hypothesis Testing (Normalized Enrichment Score). Therein, an NES above 2.0 indicates a high probability that the agent has transformed the cells, an NES below 1.2 indicates a low probability that the agent has genotoxic potential, and an intermediate NES, i.e. between 1.2 and 2.0 indicates that the agent has potential genotoxic activity.
Preferably, in a subsequent step databanks are searched for the mRNAs considered deregulated, the databanks containing RNAs, which preferably are mRNAs, which have been found associated with diseases, e.g. relevant in diseases, e.g. in tumour patients, e.g. in AML, ALL or MDS patients, or in mouse models of such a disease. A hit in each databank for an RNA considered deregulated adds weight to this RNA as the overlap percentage for a hit in the databank. The result of the addition of the weight, e.g. the percentage of overlap to the at least one databank, e.g. to the log2 ratio for each mRNA, yields a numerical value reflecting the genotoxic potential.
It was found that this calculation of a value reflecting the genotoxic potential for viral particles which are currently considered as having a low genotoxic potential yields values close to zero, and that this calculation for known mutagenic viral particles yields values which are significantly bigger.
The databanks contain genes which when deregulated can have a genotoxic effect at least of viral particles. The databanks, e.g. at least one, are selected from the following ones:
The invention is now described by way of examples with reference to the figure, which shows in
Murine primary hematopoietic stem precursor cells that were isolated, cryopreserved, thawed, cultivated for 3 d in serum-free medium, preferably in StemSpan medium containing SCF, Flt3-L, IL-3, IL-11, IL-7 and expanded in IMDM medium (same cytokines) for 2 d at 37° C., 5% CO2 atmosphere in 24-well cell culture dishes. At day 0, the agent, a positive control and medium as a negative control were added to separate dishes of the cultivated cells. The exemplary agent suspected of having a genotoxic activity was a viral particle containing a nucleic acid construct coding for the IL-2 receptor common gamma chain under an elongation factor 1 alpha short promoter, designated LVEFS. The positive control was a viral particle containing a nucleic acid construct with intact LTR regions (promoter/enhancer regions) from the spleen focus forming virus, designated RVSF.
The cells with added agent, positive control and negative control, respectively, were incubated under cell culture conditions for 4 days, then an aliquot of each cell culture was removed for extraction of genomic DNA, from which the copy number of the viral nucleic acid sequence was determined. In detail, the primers of Table 3 were used to detect the viral elements indicated.
The cultivated cells were expanded in IMDM medium containing cytokines SCF (100 ng/ml), IL-3 (20 ng/ml), Flt-3L (100 ng/ml), IL-11 (100 ng/ml), and IL-7 (100 ng/ml) for further 4 days, then collected and re-plated at a 1:10 dilution in fresh medium of this composition and cultivated for another 7 d. Alternatively, the medium in addition contained OP9-DL1 or DL4 at a concentration of 5 μg/ml. Then total RNA was collected from the cells using RNAzol and the extraction kit from Zymo Research.
The RNA from each culture was analysed for the concentration of the following RNAs of Table 1: AFAP1L1, ALS2CL, ARX, BEX6, CCDC102A, DCTD, EHD3, GM805, GUCY1A3, MEX3A, MYCT1, NAIP1, NOLC1, PA2G4, PDGFRB, PHLDA2, SLA2, SLC12A2, SOX14, SOX4, SPNS2, TBXA2R, TC1649157, TIE1, TMEM176A, TMEM176B, TOX, ZFPM1. Additionally, the following RNAs of Table 2 were analysed: AJUBA, BHLHA15, CP, ENSMUST00000103558, GLRP1, GRTP1, LCK, NAP002592-003, PIGA, PRMT7, SLC35F2, TGM2, TIMP3, ZFP184, AKAP13, ASB2, ATP9A, CCR7, CPLX2, DUSP2, ELOVL6, EPOR, FGF3, IGF1R, ITK, JAKMIP1, KLRB1F, LMO2, MEF2C, MIDI, MND1, MYB, MYC, PIM2, PRDM16, RAMP1, RASGRP2, SEMA7A, SH3KBP1, SLC43A1, SMAD3, ST3GAL6, TNFSF13B, VARS. For analysis of the RNAs, hybridisation to immobilized specific oligonucleotides contained on a chip, followed by optical detection using laser irradiation of the fluorescent dye Cyanine 3 were used.
The ratio of the mRNA concentrations were determined as the quotient of the fluorescence signal intensity determined for the cells to which the agent was added to the signal intensity determined in the negative control for each mRNA species.
The differentiation between immortalized cells and non-immortalized cells was verified by performing the conventional limiting dilution protocol of the Modlich (2006) assay in parallel. Differences between immortalized and non-immortalized cells can be assessed by the measurement of the mRNA species of Table 1. For determination of the numerical value discriminating the genotoxic potential, a normalized enrichment score (NES) was calculated for the significantly expressed mRNAs using the GSEA method. The exemplary agent LVEFS had a NES of −1.4 which was significantly different from the positive control RVSF with an NES of 2.62.
For determination of the genotoxic potential according to the database RTCGD, the significantly increased concentrations of RNA species of Table 1 and of the additional RNAs of Table 2 that were determined in the cells to which the exemplary agent LVEFS or RVSF was added, were analysed for overlaps with the database. The sample LVEFS had a mean overlap of 0 out of 44 and the RVSF 17 out of 44. This corresponds to a p-value <0.001 and is regarded highly significant.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15202623.3 | Dec 2015 | EP | regional |
| 16185408.8 | Aug 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/081351 | 12/16/2016 | WO | 00 |
