NL2029132B1 - Means and methods for assessing genotoxicity - Google Patents
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Abstract
genotoxicity assay for assessing the genotoxic effects of agents on haematopoietic stem and progenitor cells derived from human umbilical cord blood. The present invention provides for means and methods of assessing the genotoxicity of an agent by exposing CD34+ umbilical cord blood cells to the agent and characterizing the CD34+ umbilical cord blood cells by gene sequencing, such as whole genome sequencing.
Description
Means and methods for assessing genotoxicity
INTRODUCTION
An important aspect in the development of novel agents, such as e.g. drugs, pesticides or food additives, is the assessment of the potential of the agent to cause genetic alterations. For some agents a genotoxic effect per se may implicate a safety-risk. For other agents genotoxic effects are related to the effectiveness of an agent in mutating DNA, for example in case of chemotherapeutic agents or radiation.
Amounts or concentrations used of an agent contemplated for its effective use are assessed and amounts or concentrations used to induce possible adverse effects, including unintended or too much of genetic changes are assessed as well.
Ideally, the window of effective use is far removed from when potential adverse effects occur. The use of chemotherapy and antiviral compounds, for example effectively used against cancer cells or viruses, may simultaneously be accompanied by undesirable genotoxic effect in healthy tissue. In such a scenario it is often assessed whether or not an effective amount or concentration of the agent can provide for an acceptable level of genotoxicity.
To test for genotoxic effects of an agent the current state-of-the-art comprises standardized in vitro tests, such as the Bacterial Reverse Mutation Test (Ames test) or mammalian cell line tests in which mutations in selected genes occur (HPRT,
XPRT, TK kinase), and/or chromosomal or micronucleus aberrations (OECD Genetic toxicology Guidance Document Aug 31, 2015). Currently available in vitro tests include single parameter readout (e.g. a gene, a chromosome or a micronucleus) generally focusing on errors induced in a single gene. For example, a commonly used test, the hprt test, detects different genetic events (e.g. base pair substitutions, frameshifts, small deletions and insertions) in the hprt gene located on the X-chromosome.
The current inventors now provide for a highly improved genotoxic assay. The current inventors realized that when assessing genotoxicity, in particular for human use, that the current genotoxicity assays, although useful, are rather restricted, i.e. they take into account only e.g. a particular gene introduced in a (human) cell line.
This means that these genotoxic assays do not focus on the rest of the genome.
Furthermore, the inventors realized that human cell lines, also because they are immortalized, are inherently perhaps more susceptible to genotoxic events and less representative for toxicity in humans. The current inventors realized that ideally by using gene sequencing, of the whole genome or representative thereof, genotoxic events may be highly sensitively detected. The inventors realized that advantageously by using in particular hematopoietic stem cells obtained from cord blood, a population of human cells is provided that allows for detecting such genotoxic events using a sequencing approach. This type of cells has such a low rate of somatic mutations, which highly complicates sequence analysis, that genotoxicity of agents can be efficiently detected therein allowing for a highly convenient and reliable assay that can be completed in only a matter of weeks.
Altogether, the invention provides for highly sensitive in vitro methods for assessing genotoxicity of an agent allowing even detection of very low levels of genotoxicity.
By utilizing CD34+ cells from umbilical cord blood as a means to screen for the genotoxicity of an agent by exposing these cells to said agent and performing further analytic methods on the exposed cells the inventors have now provided for an advantageous highly sensitive and nearly unbiased screening method for genotoxicity. It is highly preferred in accordance with the invention and in the embodiments as described herein that CD34+ umbilical cord blood cells are human
CD34+ umbilical cord blood cells.
Hence, accordingly, in one embodiment, a use of CD34+ umbilical cord blood cells, which are highly preferably human CD34+ umbilical cord blood cells, is provided, wherein said use is in genotoxicity assays. In genotoxic assays of the invention, when these cells are exposed to an agent, genotoxic effect can be easily detected utilizing gene sequencing, e.g. whole genome sequencing. This way, advantageously genotoxic effects on the complete genome can be assessed. This is highly useful for assessing the safety of (potential) drugs or compounds under development. Conversely, such assays may also be used to assess compounds or drugs that are under development to counteract potential genotoxic effects of known agents.
Hence, in another embodiment, an in vitro method is provided for assessing genotoxicity of an agent, said method comprising the steps of: - providing an agent;
- providing CD34+ umbilical cord blood cells; - exposing said CD34+ umbilical cord blood cells to said agent; - assessing genotoxicity of the agent by characterizing said exposed CD34+ umbilical cord blood cells, wherein said characterization comprises gene sequencing of said exposed CD34+ umbilical cord blood cells, preferably whole genome sequencing, to provide for sequence information.
Preferably, the method of the invention includes a comparison with a reference. Accordingly, in a further embodiment, an in vitro method is provided for assessing genotoxicity of an agent, wherein said method comprises the steps of: - providing an agent; - providing CD34+ umbilical cord blood cells; - exposing said CD34+ umbilical cord blood cells to said agent; - exposing in a separate step the provided CD34+ umbilical cord blood cells to a reference agent, or no agent, to provide for a reference; - assessing genotoxicity of the agent by characterizing said exposed CD34+ umbilical cord blood cells and said reference, wherein said characterization comprises gene sequencing of said exposed CD34+ umbilical cord blood cells and said reference, preferably whole genome sequencing, to provide for sequence information and comparing the sequence information of said exposed CD34+ umbilical cord blood cells with the sequence information of said reference.
It is understood that the said reference relates to the medium in which the provided CD34+ umbilical cord blood cells are cultured. In one further embodiment, said reference may have the same medium as the medium in which the agent is provided, but with no agent present therein. In another further embodiment said reference may have the same medium as the medium in which the agent is provided, but not having the agent present, but having a reference agent instead. Such a reference agent may relate to the agent being tested, or may be an agent with a known genotoxic effect or an agent known to not exert a genotoxic effect. It may be highly preferred to not have genotoxicity occurring in the reference as this may more easily allow one to assign DNA changes to be genotoxic effects.
Embodiments of the invention are further described hereinafter with reference to the accompanying drawings:
Fig. 1: Cell viability curves of CD34+ umbilical cord blood cells exposed to ganciclovir (A) and foscarnet (B) treatment. With increasing dose of ganciclovir, the cell viability decreases; allowing determining of the IC50 of exposure to ganciclovir.
Foscarnet does not influence cell viability.
Fig. 2: The y-H2AX mean fluorescence intensity (MFI) of three cord blood samples, each treated with each treatment condition twice (Wicoxon test). Exposure with ganciclovir in the IC50 concentration causes increased staining for y-H2AX, indicating increased levels of double- and/or single-strand breaks.
Fig. 3: The number of single base substitutions of each of the treatment conditions (5uM ganciclovir and/or 200uM foscarnet). Ganciclovir exposure alone enhances the single-base substitution load. Foscarnet did not enhance the single- base substitution load.
Fig. 4: Dose response curves of CD34+ umbilical cord blood cells for each tested cancer treatment (e.g. chemotherapeutic agent (cisplatin (A), maphosphamide (B), doxorubicin (C)) or irradiation (D)). Cell viability is relative to the untreated
HSPCs. Each dot represents a single experimental replicate. With increasing dose of for each tested cancer treatment the cell viability decreases; allowing determining of the IC50’s.
Fig. 5: The number of single base substitutions (A) and the number of indels (B) per treatment condition (cisplatin (1uM), maphosphamide (10uM), X-ray radiation (2 - 3 Gy (135eV)), doxorubicin (10nM) and negative control). Exposure to IC50 concentrations of each one of cisplatin, maphosphamide and X-ray radiation increases the number of base substitutions and small insertions and deletions.
Fig. 6: Trinucleotide profile of the average induction of mutations per clone of CD34+ clones exposed to maphosphamide. Mutagenic effects caused by maphosphamide are shown by defining the herein displayed corresponding 96 trinucleotide signature. * P < 0.05, Wilcoxon test using bonferroni correction.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.}. The copyright owner has no objection to the facsimile reproduction 5 by anyone of the patent document or patent disclosure, as it appears in the Patent
Office patent file or records, but otherwise reserves all copyright rights whatsoever.
Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. For purposes of the present invention, the following terms are defined below.
As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, a method for administrating a pharmaceutical agent includes the administrating of a plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).
The terms “about” and “approximately”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of 120% or £10%, more preferably £5%, even more preferably £1% and still more preferably £0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
As used herein, the term “and/or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
The terms “genotoxicity”, “genotoxic effect” and “genetic toxicity”, which can used interchangeably, comprise the effect of an agent to induce a change in the DNA sequence as comprised in a cell. It is understood said change usually is not the intended purpose of the agent. In other scenarios, genotoxic effects can be related to the effectiveness of the agent in mutating DNA, for example in case of chemotherapeutic agents or radiation. DNA breaks can occur due to genotoxicity which are subsequently repaired by the cellular DNA repair machinery introducing changes in the DNA. Hence, activation of DNA repair machinery in a cell is a sign of genotoxic effects. Changes to the DNA sequence that can occur due to genotoxicity include insertions, deletions, substitutions (transversions and transitions) and translocations. For example, agents that can have a genotoxic effect are radioactive sources (e.g.as described in the examples), nucleoside analogues (e.g. ganciclovir, remdesivir ), or alkylating agents (e.g. bendamustine or cyclophosphamide). In this context, the term “genotoxic agent” as used herein, refers to a genotoxic substance which may be physical, chemical or biological which can exert a genotoxic effect.
The term “CD34+” as used in the method of the invention refers to the transmembrane giycoprotein (~110 kDa) encoded by the CD34 gene and present in haematopoietic (stem) celis and progenitor cells. Haematopoietic cells expressing *CD34+” are found in bone marrow and in the umbilical cord. CD34+ is a well- established marker for quantification of haematopoietic (stem) cells and progenitor cells. As provided herein CD34+ umbilical cord cells comprise cells isolated from the umbilical cord (blood) by using CD34+ as a marker, for example isolation methods as described hersin, for which numerous means and methods are commercially available.
In a first embodiment, an in vitro method is provided for assessing genotoxicity of an agent, comprising the steps of: - providing an agent; - providing CD34+ umbilical cord blood cells; - exposing said CD34+ umbilical cord blood cells to said agent; - assessing genotoxicity of the agent by characterizing said exposed CD34+ umbilical cord blood cells, wherein said characterization comprises gene sequencing of said exposed CD34+ umbilical cord blood cells, preferably whole genome sequencing, to provide for gene sequence information.
CD34+ umbilical cord blood cells are cells that have been isolated from umbilical cord. Standard means and methods are commercially available to isolate
CD34+ umbilical cord blood cells. For example, as described in the examples herein,
CD34+ umbilical cord blood cells from humans can be easily obtained from human umbilical cord blood by gradient separation (to isolated mononuclear cells, which include CD34+ umbilical cord blood cells) and subsequent positive selection of the
CD34+ cells via magnetic beads conjugated to antibodies directed to human CD34.
An example of a method suitable for selecting and isolating CD34+ umbilical cord blood cells is using a Lymphoprep™ protocol (Stemcell Technologies Inc.) and performing a subsequent positive selection using, for example, a CD34+-UltraPure kit (Miltenyi Biotec).”
It is understood that CD34+ umbilical cord blood cells can be provided freshly prepared. For example, umbilical cord blood obtained from umbilical cord may be frozen and stored until when needed. Alternatively, fractions of blood containing
CD34+ umbilical cord blood cells may be frozen and stored until when needed as well. For example, isolated mononuclear cells may be collected and processed and appropriately stored until when needed. When CD34+ umbilical cord blood cells are needed to be provided in the assay, CD34+ umbilical cord blood cells can be isolated from umbilical cord blood, or fractions thereof, in order to provide for CD34+ umbilical cord blood cells in accordance with the invention. Of course, CD34+ umbilical cord blood cells may be appropriately stored to be used in the methods of the invention as well. In any case, whichever steps one takes to obtain the CD34+ umbilical cord blood cells, these steps highly preferably do not induce differentiation of the cells, as the methods of the invention rely on low grade of somatic mutations of CD34+ umbilical cord blood cells.
CD34+ umbilical cord blood cells can allow for detecting genotoxic events in a highly sensitive manner due to the low rate of somatic mutation background of said cells. In other words, due to their age the DNA of said CD34+ umbilical cord blood cells has been affected only minimally by internal and/or external factors that can lead to DNA-damage such as mutations, indels, single-base substitutions etc. This low background enables that even the lowest levels of genotoxic events caused by an agent to which they are subjected can be detected.
Cells provided in the method of the invention are kept in media suitable for maintaining said cells. For example CD34+ umbilical cord blood cells can be suspended in a media suitable for maintaining hematopoietic stem and progenitor cells in culture. Said media can comprise supplements such as combinations of cytokines (e.g. TPO, SCF, FLT3-ligand, IL-3, IL-6, etc.}, growth factors (e.g. GM-
CSF and/or G-CS), antibiotics (e.g. primocin} and can include serum-free media.
Further supplements may be added to the media to stimulate clonal outgrowth of the said cells if required, for example by supplementing with UM729 and/or
StemRegenin 1. Suitable alternatives to said supplements can be used as well.
Standard media for culturing the cells of interest, e.g. CD34+ umbilical cord cells, are available and known in the art.
The CD34+ umbilical cord blood cells can be suspended in a suitable medium as described hereabove and can be plated in suitable cell culturing dishes or multi- well plates (e.g. 6, 12, 24, 48, 96, 384 wells plates), etc. Depending on the selected cell culturing system (e.g. dish, plate, disk, flask etc.) a suitable cell density can be selected.
In the methods in accordance with the invention an agent is provided. Any agent may be provided to the CD34+ umbilical cord cells as agent to be assessed for its genotoxic effects. In one particular aspect the agent may be suspected of inducing genotoxicity or is known to induce genotoxicity, e.g. commercially available pesticides, chemotherapeutic drugs etc.
As provided herein CD34+ umbilical cord cells ars exposed to said agent.
This includes exposing the cell culture medium comprising the CD34+ umbilical cord cells. The exposure to said agent can be such that said agent is included in the media in a suitable form, for example as a solid, suspended therein, or dissolved or mixed therein. In one embodiment the CD34+ cells as provided herein are exposed to the agent for at least a sufficient amount of time to ascertain a genotoxic effect of said agent. For example, the agent is added to the medium, or the medium is exchanged for the medium with the agent, and afler a defined exposure period, the medium is exchanged for fresh medium without agent. It is understood that the period of exposure of the cells to said agent is dependent on the subsequent steps in the method, e.g. if the cells are to undergo clonal expansion subsequent lo exposure io said agent. Preferably, when said agent is comprised in the medium, this exposure is for at least 12 hours, at least 24 hours, or at least 48 hours. Preferably said cells arg exposed to said agent for about 72 hours. At most the exposure to said agent is 96 hours, preferably up to (i.e. al most) 72 hours, when the cells are subsequently cionaily expanded. 1 is understood that it is contemplated that there is no maximum exposure of the cells to said agent when the method further comprises direct single cell sequencing. When using single cell sequencing it is contemplated that the cells can be exposed to multiple cycles of a certain agent, for example to mimic a chemotherapy cycle. In case the genes of said cells are sequenced by using singie- cell sequencing then the exposure to said agent is at most § weeks, preferably at mast 4 weeks. When performing single cell sequencing it is preferred fo sequence between about 1 — 5 cells per treatment, preferably about 3 cells per treatment. it is also contemplated herein that the exposure of the CD34+ umbilical cord cells said agent is such that the cells are exposed to a device that briefly, e.g. for a short period of time, exposes the cell culture to sald agent, e.g. wherein said agent includes alpha, beta, gamma or UV irradiation and cells are exposed to an irradiation device, as shown in the examples. Such agents in accordance with the invention, i.e. irradiation, can interact with the CD34+ umbilical cord blood cells and exert potential genotoxic effects. Of course, an agent can also include a source of irradiation, e.g. when an antibody is conjugated with e.g. a radionuclide. in the method of the present invention the genctoxic effect of said agent is assessed by characterizing said exposed CD34+ umbilical cord blood cells. The inventors have surprisingly found that CD34+ umbilical cord blood cells are particularly suitable for characterizing the genotoxic effect of an agent. The inventors have surprisingly found that by using said cells allows for a relatively fast method for assessing genotoxicity, e.g. at most 8 weeks, bul may be performed even quicker,
In one aspect the method according to the invention may be performed by performing the method of the invention directly after the provision of umbilical cord bined, e.g. almost directly taken from the donor of the umbilical cord {blood} and provided in the method of the invention. In other words, the umbilical cord blood may be provided in the method of the invention for separation and isolation of CD34+ cells as provided in the method of the invention without, for example, the further processing and storage of umbilical cord blood.
As said, the inventors have advantageously found that using gene sequencing techniques allows for characterizing the genotoxic effect of an agent, or does not have, on the CD34+ umbilical cord blood cells when exposed to said agent in the methods of this invention. By performing gene sequencing methods gene sequence information can be obtained from the DNA residing in the CD34+ umbilical cord blood cells. Standard means and methods are known and available to perform sequencing and to obtain gene sequence information.
In one embodiment the method in accordance with the invention comprises as gene sequencing method single-cell sequencing. The benefit of utilizing single-cell sequencing in the method of the invention is that the method allows for a relatively quick characterization of the gene sequence information of a CD34+ umbilical cord blood cell, and thus provides a fast way to assess genotoxicity in the methods of the invention.
This way, the time for performing the assay according to the invention., method of the invention wherein single-cell sequencing is used as gene sequencing method is at least 1 weeks, preferably at least 12 days, such as 2 weeks.
In a preferred embodiment the method comprises the assessing of the genotoxicity of an agent by characterizing said exposed CD34+ umbilical cord blood cell by whole genome sequencing, e.g. but not limited to, whole genome bisulfite sequencing, TET-assisted pyridine borane sequencing (TAPS), shotgun sequencing, pairwise-end, and next-generation sequencing, to provide for gene sequencing information, such as representative information on the whole genome sequence of said cells. It is understood that whole genome sequencing provides for a high uniform coverage of sequences across the genome. The skilled person is well aware of whole genome sequencing methods. Gene sequencing information as used herein comprises determining the order of nucleotides making up of a gene, preferably of the whole genome, of said cells. In one preferred embodiment the gene sequencing information comprises indels of the gene or genome. In another preferred embodiment the gene sequencing information comprises single-base substitutions of the gene or genome. The method can comprise that variants are identified from the sequence data. The method may further comprise that high-quality somatic mutations are selected, for example by filtering the variants or clones on variant allele frequency, coverage and/or mapping quality. Additionally the method comprising characterization of said cells may comprise that identified variants are filtered so as to prevent high levels of false-positive indels. Further, the method can comprise filtering of shared mutations between cell clones.
In one aspect of the invention the characterization of the CD34+ umbilical cord derived cells, wherein characterization is by gene sequencing methods comprising that a part, e.g. a single step, a plurality of steps or all steps) of the sequencing of the gene and/or whole genome of said cells is performed by a computer, computer program or any other computational approach and/or method (e.g. deep learning, artificial intelligence etc.). One example thereof is random forest classification performed by a computer({-program). In a further embodiment the in vitro method of the invention is provided wherein said assessment involves comparing said sequence information of agent exposed CD34+ umbilical cord blood cells with sequence information of a reference. It is understood that the said reference relates to the medium in which the provided CD34+ umbilical cord blood cells are cultured. For example, the method wherein an agent is to be compared to a reference comprises that, prior to exposing the provided CD34+ umbilical cord blood cells to an agent, a part of said cells is designated to be exposed to said agent and another part of said cells is designated to be exposed to said reference. Preferably, for comparing the cells exposed to an agent, e.g. the agent that is to be assessed for genotoxic effects, to the cells exposed to a reference, the cells are derived from the same umbilical cord blood, for example from the same donor or from the same sample of umbilical cord blood. It is contemplated that by using the same umbilical cord blood a largely unbiased comparison between the genotoxic effect of an agent and reference can be assessed.
In a further embodiment an in vitro method is provided, wherein said reference is provided by exposing in a separate step the provided CD34+ umbilical cord blood cells to a reference agent and/or no agent and performing thereon the same characterization of the CD34+ umbilical cord blood cells exposed to the agent. It is contemplated that in the method according to the invention said reference may have the same medium as the medium in which the agent is provided, but with no agent present. In another further embodiment said reference may have the same medium as the medium in which the agent is provided, but not having the agent present, but having a reference agent present. Such a reference agent may relate to the agent being tested, or may be an agent with a known genotoxic effect or an agent known to not exert a genotoxic effect.
For example, for the antiviral agent ganciclovir a suitable reference agent may be the antiviral drug, foscarnet. In another non-limiting example a suitable reference agent may be DMSO. In a further non-limiting example a suitable (positive) reference agent for chemotherapeutic drugs may be the chemotherapeutic drug cisplatin, known to have genotoxic effects. It may be highly preferred to at least not have a genotoxic agent, or no reference agent at all, as a reference agent as this most easily allows one to assign DNA-changes to be genotoxic effects of the agent that is to be assessed for genotoxic effects. It may be contemplated to include both a reference inducing genotoxic effects and a reference not inducing genotoxic effects.
As described herein the method of the invention comprises the provision of gene sequencing information by gene sequencing, preferably by whole genome sequencing or the like. In order to provide for a characterization of said CD34+ umbilical cord blood cells the gene sequencing information can be assessed as to assess any genotoxic effect of the agent, e.g. the agent that is to be assessed for genotoxic effect. Thus, in a further embodiment, accordingly, assessing genotoxicity comprises characterizing the gene sequence information for the presence of one or more of: - structural genomic variations; - genetic mutations, such as substitutions, insertions, and/or deletions; - chromosomal aberrations; - strand asymmetries; - regional mutation frequencies; and - single- and/or double DNA strand breaks.
The method of the invention allows for providing gene sequencing information from agent- or reference-exposed CD34+ umbilical cord cells in various read-outs obtained by a gene sequencing method. Characterization of said one or more CD34+ umbilical cord cells as to assess the genotoxic effect of an agent, may be done by assessing the DNA of said one or more CD34+ cells. The DNA from said CD34+ umbilical cord cells can be assessed for the presence of one or more changes in the
DNA, changes that are associated with genotoxic effects, such as structural genomic variations, genetic mutations, such as substitutions, insertions, and/or deletions, but also transversions and/or transitions, chromosomal aberrations, strand asymmetries, regional mutation frequencies and single- and/or double DNA strand breaks.
It is understood that the exposed or reference human CD34+ umbilical cord blood cells provided gene sequencing require destruction of said cells in order to allow for isolation of the DNA of said cell and subsequent sequencing. Further the
DNA of human CD34+ umbilical cord blood cells used in gene sequencing, or other residual material from the human CD34+ umbilical cord blood cells may not be suitable for further analysis.
In a further embodiment an in vitro method in accordance with the invention is provided, wherein the characterization further comprises one or more of: - assessing micronuclei; - karyotyping; - assessing cell viability; - determining telomere length; - determining mitochondrial DNA copy-number; - assessing cell-cycle interactions; - assessing DNA-repair ; - assessing formation of ROS.
These further characterizations can and preferably are carried out in addition to determining the gene sequencing. Alternatively, it may also be contemplated to characterize genotoxicity using these methods and not perform gene sequencing. It is understood that these genotoxicity characterizations can be representative measures of genotoxicity which can be of interest on their own and thus not necessarily require sequence information. Nevertheless, it is preferred to perform these assays in addition to gene sequencing. If these are assays that require destruction of the cell and/or the DNA that would render the exposed cells not suitable for subsequent gene sequencing methods, it is understood that one may subject part of the exposed cells separate to such further characterization, and subject the other part to gene sequencing. If these assays are compatible with subsequent gene sequencing methods, one may carry out such further characterization first, and subsequently subject the same cells and/or DNA therefrom to gene sequencing.
Examples of such assays are assays to assess the cell viability, such as IC50 assays, MTT assays etc., karyotyping or polymerase chain reaction assays for the determination of mitochondrial DNA copy number. If such further characterization can be carried out after gene sequencing, and/or after processing of a sample for gene sequencing, such may also be contemplated in accordance with the invention.
Examples of such assays are measuring DNA double and/or single strand breaks using an antibody that specifically detects phosphorylated H2AX (yH2AX) protein.
Alternatively, one can also carry out further characterizations on cells that have been subjected to the agent separately (in a separate well or even a separate assay). Of course, in any of these further characterizations, agent exposed human CD34+ umbilical cord blood cells are preferably compared with a reference as described above. In any case, these further characterizations provides for further highly useful information when combined with the assessment of genotoxicity based on gene sequencing. Hence, in a further embodiment, the method in accordance with the invention comprising one of more of said further characterizations, said further characterizations are carried out prior to gene sequencing. In another further embodiment, the method in accordance with the invention comprising one of more of said further characterizations, said further characterizations are carried out separate from gene sequencing.
A highly preferable benefit the method of the invention offers is that a plurality of possible further characterizations of (human) CD34+ umbilical cord cells allows for the assessment of multiple parameters in a single assay combined with gene sequencing.
In a further embodiment an in vitro method is provided, wherein CD34+ umbilical cord blood cells are human CD34+ umbilical cord blood cells. It is contemplated that for assessing the genotoxicity of an agent, for example of chemotherapeutic agents that are commonly used in the treatment of cancers in mammals, particularly in humans, it is highly advantageous for the results derived from the assay that the umbilical cord blood cells provided in the method of the invention are human. One further benefit of the method as provided herein is that it may replace or reduce animal testing and/or may reduce or replace current in vitro and/or in vivo tests. It is contemplated that the use of human CD34+ umbilical cord blood cells, in the method of the invention allows for sufficient assessment of the genotoxicity of an agent from a clinical perspective. Accordingly it is contemplated herein that a benefit of the method of the invention over, for example established methods utilizing different non-human organisms, is that the human umbilical cord blood cells as provided in the method provides results that are considered clinically of higher relevance.
In a further embodiment in the in vitro method of the invention, said CD34+ umbilical cord blood cells, which are preferably human, are isolated from fresh or frozen umbilical cord blood, or part thereof, or are provided fresh or stored until ready to be used. In one embodiment, said CD34+ umbilical cord blood cells are from one donor. The benefit of providing CD34+ umbilical cord blood cells of one donor is that by taking said cells from a single donor said cells display a high degree of sequence identity with little to no somatic mutation background. In contrast, when more than one donor is used, the genetic diversity between donors is usually much greater than the mutations that are introduced in the DNA of the human CD34+ umbilical cord blood cells due to any genotoxic effect. This makes it difficult to assess which sequence variations are caused by genotoxicity and which are derived from genetic diversity between donors. Hence, it is preferred to have CD34+ umbilical cord blood cells from one donor. Nevertheless, it can be contemplated to use different donors, as long as sequence information is provided or is generated from the donor itself and/or its parents such that it can be determined what sequence variation is derived from genotoxicity and which from genetic diversity between donors.
In a further embodiment in the in vitro method in accordance with the invention, said CD34+ umbilical cord blood cells are cultured prior to the step of exposing. It is preferred that the culturing of said CD34+ umbilical cord cells is such that said cells maintain their low somatic mutation background (e.g. approximately — 30 mutations across the whole genome), i.e. by selecting a short time frame. 20 Standard media for culturing the cells of interest, i.e. CD34+ umbilical cord cells, are available and known in the art. Such a step may be in a medium that allows human
CD34+ umbilical cord blood cells to proliferate and recover. It is preferred that said cells are maintained and cultured in said media for at least a number of hours or days, e.g. at least 2 hours, 8 hours, 8 hours, 10 hours, 12 hours, 24 hours, 48 hours or 96 hours, preferably between 12 — 36 hours, more preferably about 24 hours, prior to exposure of said cells to an agent and/or a reference agent. It may be further preferred that cells are maintained and cultured under standard culture conditions of 37°C with 5% CO2. However other suitable standard conditions for incubating said cells can be contemplated. The cells are maintained and cultured prior to the step of exposing to allow said cells to recover from the process of thawing, separating and/or isolating the CD34+ umbilical cord blood cells that has been performed to provide for the CD34+ umbilical cord blood cells. In one aspect of the invention the culturing of the cells in a media is performed at least prior to exposing the cells to an agent or exposing the cells to a reference (e.g. a reference agent or no reference agent).
In a further embodiment said CD34+ umbilical cord blood cell clones are selected and expanded after the exposure to said agent. It is preferred that (a part of) the CD34+ umbilical cord cells are sorted, selected and clonally expanded to the exposure of said cells to the agent or reference (e.g. a reference agent or no reference agent). Preferably, clonal expansion is such that sufficient cells are obtained suitable for whole genome sequencing. Preferably, multiple clones may be selected. This way, genotoxic events that have occurred in one or multiple cells can be advantageously determined. Prior to sorting, CD34+ umbilical cord blood cells having been exposed to an agent or reference preferably are washed with suitable washing buffer and preferably are stained for sorting. Sorting of the clones can be performed by utilizing one or more antibodies suitable, such as CD34-BV421 (Biolegend, clone 581 1:20), CD38-PE (clone HIT2, 1:50), CD45RA-PerCP-Cy5.5 (clone HI30, 1:20), CD11c-FITC (clone 3.9, 1:20), CD16-FITC (clone 3G8, 1:20),
CD90 APC (5E10, 1:200) and/or Lineage cocktail FITC (CD3/CD14/CD19/CD20/CD58, clones UCHT1, HCD14, 3G8, HIB19, 2H7, HCD586, respectively, 1:20). It is preferred that for sorting the CD34+ umbilical cord blood cell clones at least a CD34-specific antibody is used. A skilled person is able to determine and utilize suitable ways for sorting CD34+ umbilical cord blood cells after exposure of said cells to an agent or reference. In one aspect of the invention the human CD34+ umbilical cord blood cell clones are obtained after an IC50 assay, i.e. viable cells that remained after the IC50 assay. Clonal expansion comprises culturing the cells for a period of time, for example a few days or weeks, e.g. at least 3 days, 1 week, 2 weeks, preferably for about 3 — 6 weeks. In one non-limiting example clones are expanded until confluent for at least 70% in a cell culturing system, e.g. in a 384-well plate (Greiner). Suitable media for the clonal expansion of the CD34+ umbilical cord cells as used herein comprises a media suitable for supporting hematopoietic stem and progenitor cells in culture such as described extensively herein. It is preferred that said media comprises supplemented UM729 and SR-1.
Accordingly, in a further embodiment, a method for assessing genotoxicity of an agent is provided, comprising the steps of:
- providing an agent; - providing CD34+ umbilical cord blood cells, which cells have been allowed to recover, - exposing said CD34+ umbilical cord blood cells to said agent; - exposing in a separate step the provided CD34+ umbilical cord blood cells to a reference agent, or no agent, to provide for a reference; o wherein said exposure steps are for up to 72 hours; - subsequently, assessing genotoxicity of the agent by characterizing said exposed CD34+ umbilical cord blood cells and said reference, wherein said characterization comprises gene sequencing of said exposed CD34+ umbilical cord blood cells and said reference, preferably whole genome sequencing, to provide for sequence information and comparing the sequence information of said exposed CD34+ umbilical cord blood cells with the sequence information of said reference.
In yet a further embodiment, a method for assessing genotoxicity of an agent is provided, comprising the steps of: - providing an agent; - providing CD34+ umbilical cord blood cells, which cells have been allowed to recover, - exposing said CD34+ umbilical cord blood cells to said agent; - exposing in a separate step the provided CD34+ umbilical cord blood cells to a reference agent, or no agent, to provide for a reference; o wherein said exposure steps are for up to 72 hours; - subsequently, select cell clones, after the exposure step and allowing the cell clones to expand, for example for about 4-6 weeks; - assessing genotoxicity of the agent by characterizing said selected clones of said exposed CD34+ umbilical cord blood cells and said reference, wherein said characterization comprises gene sequencing of said exposed
CD34+ umbilical cord blood cells and said reference, preferably whole genome sequencing, to provide for sequence information and comparing the sequence information of said exposed CD34+ umbilical cord blood cells with the sequence information of said reference.
When selecting cell clones it is preferred that at least 2 cell clones, preferably at least 2 or more cell clones, for example 3 cell clones, are selected.
In another aspect of the invention said CD34+ umbilical cord blood cells are exposed to a defined amount and/or concentration of said agent. Said agent can be known or suspecied of exhibiting a genotoxic and/or cytotoxic effect. in another aspect said cells are exposed ic an amount or concentration of said agent that is known or suspecied {o exhibit a genotoxic effect.
In a further embodiment the in vitro method of the invention is provided, wherein the exposure to said agent comprises the exposure of the cells to one or more titration series of the agent. It is preferred to expose the cells to one or more titration series of the agent for evaluating the genotoxic effect of a plurality of concentrations and/or amounts of the agents used in the method of the invention.
When performing a titration series of one or a plurality of agents it is preferred that each titration concentration to be tested is provided on a separate well, dish or other culturing system comprising a part of the CD34+ umbilical cord cells as provided herein. Suitable titration series are dependent on the agent provided in the method.
A skilled person is able to determine suitable titration series for testing the agent in the method as provided herein.
In a further embodiment, the titration series which is part of the characterization step comprises an IC50 assay. In an IC50 assay the cell viability is assessed, i.e. IC50 is the concentration under the culture conditions selected of which it is determined that 50% of the cells exposed to the agent remain viable.
Preferably, and in a further embodiment, the said viable cells obtained after determining said IC50 are subjected to gene sequencing. It is highly advantageous, when in the methods of the invention different agents are to be compared, cells obtained after the IC50 assay may be selected from a particular concentration range resulting in a defined cell survival range. For example, when at a certain concentration, or concentration range, 40 — 80 % of the cells remain viable, these cells may be selected for subsequent gene sequencing. Performing an IC50 assay is beneficial for use in the method of the invention, because different agents shown to have similar effect on the cell (e.g. 40-60% of cells exposed to the agent survive), at least with regard to cytotoxicity, can be compared with regard to genotoxicity.
Consequently, performing an IC50 assay prior to selecting cells for gene sequencing,
allows for an advantageous comparison between different agents and/or references of the genotoxic effects. Further, performing an IC50 assay allows for determining dose responses of agents at different concentrations. For example, some chemotherapeutic agents such as 5-fluoruracil, are known to exhibit a mutagenic effect at a low concentration, whereas a higher concentration has almost no mutagenic effect. When a genotoxic event results in a DNA change, and is not subsequently repaired by the DNA machinery, the DNA change is retained in the genome in subsequent cell divisions. Such a genotoxic event may also be referred to as a mutagenic event. Hence, it is understood that the methods and uses as described herein for assessing genotoxicity may also be referred to as methods and uses for assessing mutagenicity instead. It is understood that the terms mutagenic and genotoxic are terms that may be used interchangeably in the field.
In another example, determining the 1C50 allows for assessing the concentration of an agent as provided herein that reduces cell viability such that insufficient cells survive to allow for gene sequencing.
Accordingly, in yet another further embodiment, a method for assessing genotoxicity of an agent is provided, comprising the steps of: - providing an agent; - providing CD34+ umbilical cord blood cells, which cells have been allowed to recover, - exposing said CD34+ umbilical cord blood cells to said agent, wherein said exposure comprises performing an IC50 assay; - exposing in a separate step the provided CD34+ umbilical cord blood cells to a reference agent, or no agent, to provide for a reference; co wherein said exposure steps are for up to 72 hours; - determine cell viability after said exposure to determine IC50 - subsequently, select cell clones, wherein said clones are preferably selected from a concentration range resulting in about 40% - 60% cell survival after the exposure step and allowing the cell clones to expand, for example for about 4-6 weeks; - assessing genotoxicity of the agent by characterizing said selected clones of said exposed CD34+ umbilical cord blood cells and said reference, wherein said characterization comprises gene sequencing of said exposed
CD34+ umbilical cord blood cells and said reference, preferably whole genome sequencing, to provide for sequence information and comparing the sequence information of said exposed CD34+ umbilical cord blood cells with the sequence information of said reference.
When more than 60% survival of said CD34+ cells is achieved a different concentration of said agent is selected and tested. When nearly 100% (e.g. 100%, 99%, 98%, 97%, 95%) of said CD34+ cells survive then the IC50 of a second more or less comparable agent is assessed, and, for the agent that resulted in a cell viability of nearly 100%, a 100x higher concentration that the C50 of those second comparable agents.
In a further embodiment in the in vitro method of the invention, the agent is an antiviral agent, chemotherapeutic agent, a biochemical agent, a pesticide, or a food additive. In methods in accordance with the invention, as described and embodied herein, said agents that can be tested can comprise any agents. It may be preferred to select antiviral agents, chemotherapeutic agents, biochemical agents, pesticides and/or food additives. These types of agents are in particular subjected to genotoxicity assays. Genotoxic effect that can be exerted by such agents include undesirable genotoxic effects, for example the induction of an undesirable and clinically unacceptable amount of changes to the DNA or to the cell (e.g. change in telomere length). The methods of the invention therefore allow assessing the safety of an agent, e.g. the risk and or extent of exhibiting undesirable genotoxic effects, of an agent as provided herein.
By means of the method of the invention a meaningful assessment can be made to the (undesirable) genotoxic effect such an agent may have on a subject, preferably of a human, who is exposed to said agent, for example, but not limited, by administering, topically applying, inhaling and/or being in any manner directly exposed to said agent. Such assessment is not necessarily restricted to CD34+ umbilical cord blood cells, as CD34+ umbilical cord blood cells as used in methods of the invention can be regarded a model system of an organism, such as human.
Hence, any results thereof may be regarded as an indication of effects in human.
As for some agents, the effect is to exert also comprises genotoxic effects, the methods of the invention can also be used to select for effective agents. For example, chemotherapeutic agents (e.g. cisplatin) have genotoxic effects both on cancer cells and/or healthy cells. The therapeutic dose is selected such that the genotoxic effect allows to differentiate between cancer cells and non-diseased cells.
Nonetheless, potential chemotherapeutic agents, or other agents with desired genotoxic properties, may be selected utilizing the methods of the invention. Agents that may be of interest to test in methods in accordance of the invention also include
CRISPR/Cas, TALENs and Zinc fingers and the like (Ferrari, et al., Nat Rev Genet 22, 216-234 (2021)). Such agents, are designed to introduce specific DNA changes.
As it is important to assess potential genotoxic effects, it may in particularly useful for these agents to be tested in methods in accordance with the invention, as such agents may have undesired off-target effects, i.e. induce changes outside of the intended change.
Also, advantageously, the method allows for the assessment of genotoxic effects of one or more agents. When it is desirable to include a plurality (e.g. 2, 3, 4, 5, 10, 25, 50, 100, 1.000, 10.000, ..., ...., etc.) of agents in the assessment for genotoxic effect of said agents the CD34+ umbilical cord blood cells as provided herein can be allocated to (e.g. culturing of said cells in) a plurality of cell culturing systems, e.g. multiple dishes or wells etc., thus allowing for the exposure of a plurality of agents. As such, the method of the invention is considered to be highly suitable for high-throughput screening of the genotoxic effect of a plurality of agents.
It is preferred that, when exposing CD34+ umbilical cord blood cells in a plurality of culturing systems to a plurality of agents as provided herein, that said CD34+ cells are derived from the same umbilical cord (blood), and thus preferably stem from one donor of said umbilical cord (blood). It is also preferred that in case different agents are compared, for each agent IC50 is determined and cells (in case of single cell sequencing) or expanded clones, obtained from concentrations resulting in similar viability for each agent are subjected to gene sequencing. it is also understood that not only single agents can be screened utilizing any of the methods of the Invention, but combinations of agents can be tested as well.
This way, optimal combinations of agents from a genotoxic effect can be selected, and combinations exerting undesirable genotoxic effects can be discarded.
In certain aspects of the invention said agent is a chemotherapeutic agent or radiation, e.g. ionizing radiation, suspected of inducing genotoxicity in a cancer cell, e.g. a mutated cell, and/or in a healthy cell. it is contemplated herein that induction of genotoxicity in a cancer cell, and thus the effectiveness {g.g. the therapeutic gffectivensss) of a chemotherapeutic agent or radiation, can be closely associated with undesirable genctoxicity in healthy, non-cancerous, cells. Therefore the precise detection of said genotoxic effect of an agent is of high importance for such drugs.
The methods of the invention therefore allow to assess the efficacy of an agent as well as safety aspects of an agent.
In another embodiment, the means and methods in accordance with the invention also allow to assess and screen for compounds for reducing genotoxicity.
For example, such compounds may be tested in means and methods wherein in the step of exposing the CD34+ umbilical cord blood cells to the agent, such a compound is included. When compared with the effect of not including the said compound, it can be assessed whether or not genotoxic effects of the agent were reduced. It is also understood that of course in scenarios where the agent is to have a genotoxic effect, the assay can also be used to determine whether or not certain compounds may diminish activity of a compound or improve it.
In yet another embodiment, provided herein are further uses of the CD34+ umbilical cord blood cells, for example in methods according to the invention. In an embodiment of the invention a use of isolated CD34+ umbilical cord blood cells in a genotoxicity assay is provided.
In a further embodiment the invention relates to a use of isolated CD34+ umbilical cord blood cells in a genotoxicity assay wherein said genotoxic assay is for assessing genotoxicity of an agent or for assessing the capacity of a compound to reduce the genotoxicity of an agent. For example, a potential assay could be adding antioxidants to a certain genotoxic treatment to counteract induced (secondary) oxidative stress-induced mutagenesis. Hence, in another further embodiment, a method in accordance with the invention is provided assessing the capacity of a compound to reduce the genotoxicity of an agent, wherein said method comprises the steps of: - providing an agent known to cause genotoxicity; - providing a compound; - providing CD34+ umbilical cord blood cells; - exposing said CD34+ umbilical cord blood cells to said agent;
- exposing in a separate step CD34+ umbilical cord blood cells to said agent in the presence of said compound; - exposing in a separate step the provided CD34+ umbilical cord blood cells to a reference agent, or no agent, to provide for a reference; - assessing any potential reduction induced by the compound in genotoxicity of the agent by characterizing said exposed CD34+ umbilical cord blood cells in the presence and absence of said compound, and said reference, wherein said characterization comprises gene sequencing, preferably whole genome sequencing, to provide for sequence information and comparing the sequence information of said exposed CD34+ umbilical cord blood cells with and without the compound and said agent with the sequence information of said reference.
Conversely, the capacity of a compound to enhance genotoxicity of an agent can likewise be determined.
In a further and different embodiment the invention relates to a use of isolated
CD34+ umbilical cord blood cells in the method of the invention as broadly described and embodied herein.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention.
Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.
All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.
Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect,
description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.
It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.
It will be understood that all details, embodiments and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments and preferences for all aspect separately.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art.
Example 1: assessing genotoxicity of antiviral agents on CD34+ umbilical cord blood cells
Human CD34+ differentiation
CD34+ cells were separated and isolated from human umbilical cord blood by lymphoprep gradient separation and subsequent positive selection using the CD34+-
UltraPure kit (Miltenyi Biotec) according to manufacturer's instructions.
Cells were incubated overnight at 37°C, 5% 02 and 5% CO2.
Incubation with agents
After overnight incubation cells were treated with increasing concentrations of the following antiviral compounds: ganciclovir (Sigma Aldrich), foscarnet sodium (Sigma Aldrich), a combination of the two compounds or DMSO as vehicle control.
Cells were incubated for 24 hours, after which DNA damage as assessed by y-
H2AX-staining and by WGS of clonally expanded cells.
Cell viability
While ganciclovir caused dose-dependent cell death at micromolar concentrations, foscarnet did not induce cell death at any of the tested concentrations (Figure 1A+B).
Flow cytometry
For y-H2AX-staining, 100,000-200,000 CD34+ cells were resuspended in permeabilization buffer containing 0.5% saponin, 0.5% BSA, 10mM HEPES, 140mM
NaCl, 2.5mM CaCl2 in water, pH 7.4, sterile filtered. Anti-yH2A.X (Ser139) FITC (Merk) or Mouse IgG isotype antibody (X) were added to samples and cells were incubated for 20 min on ice. After staining, cells were washed with 0.1% saponin in
PBS and resuspended in FACS buffer (1x PBS, 2-5% FBS, 2mM EDTA, 2mM NaN3) prior to flow cytometric analysis.
After drugs incubation, cells were harvested and washed with PBS. 100.000-200.000
CD34+ cells were resuspended in ice-cold fixative solution (2.5% formaldehyde and 0.93% methanol in sterile filtered PBS), incubated for 20 min at 4°C and transferred to a 96 well plate. Fixed samples were washed twice with PBS. Next, cells were resuspended in permeabilization buffer containing 0.5% saponin, 0.5% BSA, 10mM
HEPES, 140mM NaCl, 2.5mM CaCl2 in water, pH 7.4, sterile filtered. Anti-yH2A.X (Ser139) FITC (Merk) or Mouse IgG isotype antibody (X) were added to samples and cells were incubated for 20 min on ice. After staining, cells were washed with 0.1% saponin in PBS and resuspended in FACS buffer (1x PBS, 2-5% FBS, 2mM EDTA, 2mM NaN3) prior to flow cytometric analysis on a Beckman Coulter CytoFLEX S.
Statistical analysis
A Wilcoxon test was also used to compare y-H2AX levels in in vitro treated cord blood cells. P values were Benjamini & Hochberg (FDR) corrected for multiple testing (R stats::p.adjust, option ‘method = “fdr”) (Figure 2).
Whole genome sequencing
For analysis of single-cell mutagenesis caused by antiviral treatment, CD34+ cells were sorted as single cells into flat-bottom 384-well plates (Greiner), using the same antibody mix and sorting strategy as for bone marrow and peripheral blood
HSPCs. Cells were clonally expanded for 4-6 weeks, after which DNA was isolated (QlAamp DNA micro kit, Qiagen) and sent for whole genome sequencing.
DNA was isolated from the clonally expanded HSPCs using the DNeasy DNA
MicroKit (Qiagen), according to the manufacturer's instructions. Libraries for Illumina sequencing were generated from 20-50 ng of genomic DNA using standard protocols (IIlumina). Samples were sequenced to 15-30x base coverage (2 x 150 bp) on an lumina NovaSeq 6000 system. This enables us to reach a sensitivity that would not have been picked up with a conventional assay. Sequence reads were mapped against the human reference genome (GRCh38) using the Burrows-Wheeler Aligner v0.7.5a mapping tool with settings ‘bwa mem —c 100 —-M’ (Li et al., 2009). Sequence reads were marked for duplicates using Sambamba v0.6.8. Realignment was performed using the Genome Analysis Toolkit (GATK) version 3.8-1-0(Depristo et al., 2011)..
Structural variants
Structural variant calling was done with the GRIDSS-purple-linx pipeline of the
Hartwig Medical Foundation(Cameron et al.). All resulting structural variants were checked by hand in the IGV (Thorvaldsdottir et al., 2013) and false positive results were excluded. SVs could only be inspected of patients for which an MSC normal control was available.
Results
Both ganciclovir and the combination treatment caused substantial DNA damage, visualized by y-H2AX staining, while foscarnet exposure alone did not cause considerable cell death.
HSPCs exposed to ganciclovir or to the combination therapy showed increased numbers of single base substitutions as compared to HSPCs exposed to foscarnet alone or untreated clones (Figure 3).
Conclusion
Our experimental setup enabled us to detect increased levels of single base substitutions in CD34+ umbilical cord blood cells after exposure to ganciclovir.
Example 2: assessing genotoxicity of chemotherapeutic agents or radiation on
CD34+ umbilical cord blood cells
Human CD34+ differentiation
CD34+ cells were separated and isolated from human umbilical cord blood by lymphoprep gradient separation and subsequent positive selection using the CD34+-
UltraPure kit (Miltenyi Biotec) according to manufacturer's instructions.
Cells were incubated overnight at 37°C, 5% O2 and 5% CO2.
Incubation with agents
After overnight incubation cells were either exposed to 2 or 3 Gray X-ray (135eV) irradiation using a CellRad irradiation device (Precision X-ray) or were treated with increasing concentrations of any one of the following chemotherapeutic agents maphosphamide (Niomech - IIT GmbH), cisplatin (Accord), doxorubicin (Accord), cytarabine (Hospira) and incubated for 72 hours.
Sorting of clones
Cells were single cell sorted using a Sony SH800S flow cytometer in transparent polystyrene 384-well plates (Greiner), containing 75 uL of HSPC culture medium per well consisting supplemented with UM729 and SR-1. After plating, the 384-well plates were wrapped in polyethylene wrap and placed in an incubator for 37°C, ambient O2 for 4 weeks. Clones were harvested between 4-6 weeks after sorting when >70% confluent.
Whole genome sequencing
Cells were sorted in flat-bottom 384-well plates (Greiner) filled with 75 yL of
HSPC culture medium per well supplemented with UM729 and SR-1. Clones were harvested between 4-6 weeks after sorting when >70% confluent. DNA was isolated using a QlAamp DNA Micro kit (Qiagen) according to manufacturers' instructions.
Libraries were generated using 50 ng genomic DNA using the Illumina TruSeq Nano kit (Illumina). The library was sequenced on Novaseq 6000 sequencers at a depth of 15- 30x base coverage. Reads were mapped to the human reference GRCh38 genome using the Burrows- Wheeler Aligner v0.7.5a "BWA-MEM -c 100 -M".
Duplicate sequencing reads were marked using Sambamba v0.6.8.
Variants in all cord-blood derived HSPCs were called using GATK
Haplotypecaller version 4.1.3.0 using default settings. Variants were filtered using
GATK 4.1.3.0 using the following filter settings for SBS: --filter-expression 'QD < 2.0' -filter-expression 'MQ < 40.0" --filter- expression 'FS > 60.0" --filter-expression 'HaplotypeScore > 13.0' --filter-expression 'MQRankSum < -12.5' --filter-expression 'ReadPosRankSum < -8.0' -filter-expression 'MQO >= 4 && ((MQO / (1.0 * DP)) > 0.1)' --filter-expression 'DP < 5' --filter-expression 'QUAL < 30' -- filter-expression 'QUAL >= 30.0 && QUAL < 50.0’ --filter-expression 'SOR > 4.0' -filter-name 'SNP_LowQualityDepth' --filter-name 'SNP_MappingQuality' --filter-name 'SNP_StrandBias' - -filter-name 'SNP_HaplotypeScoreHigh' --filter-name 'SNP_MQRankSumLow' --filter-name 'SNP_ReadPosRankSumLow' --filter-name 'SNP_HardToValidate' --filter-name 'SNP_LowCoverage' --filter-name 'SNP_VeryLowQual' --filter-name 'SNP_LowQual' --filter- name 'SNP_SOR' -cluster 3 -window 10". The following settings were used to filter all other variants: filter_criteria = "--filter-expression 'QD < 2.0' --filter-expression 'ReadPosRankSum < -20.0' --filter- expression 'FS > 200.0" --filter-name 'INDEL_LowQualityDepth' --filter-name 'INDEL_ReadPosRankSumLow’ --filter-name 'INDEL_StrandBias".
Somatic mutation filtering
After variant calling, we used an in-house developed filtering script SMuRF to select only high-quality somatic mutations. For all variants, the position in the original alignment file (. bam) was assessed and a pileup for each variant position was performed to obtain the VAF for each variant. For clones sequenced at 30x depth, we filtered all variants with the following settings: variant allele frequency (VAF) 20.3, base coverage of 210, MQ quality of 60 or higher. For all cord blood clones sequenced at 15x depth, we filtered for a VAF of = 0.15 and a coverage of 25, MQ quality 60 or higher.
In addition, all variants were filtered using a blacklist based on variants present in sequencing data from bulk bone marrow MSCs obtained from non-related healthy individuals. To prevent high levels of false-positive indels, a blacklist consisting of 15x Novaseq data based on fetal clones was used to filter recurring indels as sequencing artefacts. Finally, in all cord blood clones shared mutations between clones were filtered.
Structural variant calling
Structural variants were determined using GRIDSS, integrating purity, ploidy, structural variants and copy number using the GRIDSS-PURPLE-LINX pipeline. All structural variants obtained from the LINX output were inspected using Integrated
Genome Viewer (IGV, version 2.8.2) and false positive structural variants were removed.
Results
Cell viability
We used a treatment regimen that caused 40% - 60% cytotoxicity in the cells (see Figures 4 A — D).
Whole genome sequencing
The additive mutational effect of chemotherapy exposure can be easily detected in these cells, because of the low background mutation load, as these cells harboured at birth on average 30.8 unique single base substitutions and 4.5 indels genome-wide (95% confidence intervals are 23.8 — 37.9 and 3.0 — 6.0, respectively).
We exposed CD34+ cord blood cells to maphosphamide (10uM) and doxorubicin (10 nM). We also exposed CD34+ cord blood cells with cisplatin and ionizing radiation detect increased levels of single base substitutions, indels and larger structural variation after exposure to cisplatin as well as ionizing radiation or cisplatin.
We assessed the number of single base substitutions (Fig. 5A) and indels (Fig. 5B) in chemotherapy-exposed CD34+ cord blood cells (* P < 0.05, Wilcoxon test using bonferroni correction) and assessed mutagenic effects by defining the corresponding 96 trinucleotide signatures (Fig. 6).
Conclusion
Our experimental setup enabled us to detect increased levels of single base substitutions, indels and larger structural variation in CD34+ umbilical cord blood cells after exposure to cisplatin as well as ionizing radiation.
In addition, we found mutagenic effects for maphosphamide exposure and experimentally defined the corresponding 96 trinucleotide signatures.
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| NL2029132A NL2029132B1 (en) | 2021-09-06 | 2021-09-06 | Means and methods for assessing genotoxicity |
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| WO (1) | WO2023033652A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070259370A1 (en) * | 2002-11-06 | 2007-11-08 | The Government Of The Usa As Represented By The Secretary Of The Dept Of Health And Human Services | Detection of mutational frequency and related methods |
| WO2019160998A1 (en) * | 2018-02-13 | 2019-08-22 | Twinstrand Biosciences, Inc. | Methods and reagents for detecting and assessing genotoxicity |
| WO2021022046A1 (en) * | 2019-07-31 | 2021-02-04 | Bioskryb, Inc. | Genetic mutational analysis |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070259370A1 (en) * | 2002-11-06 | 2007-11-08 | The Government Of The Usa As Represented By The Secretary Of The Dept Of Health And Human Services | Detection of mutational frequency and related methods |
| WO2019160998A1 (en) * | 2018-02-13 | 2019-08-22 | Twinstrand Biosciences, Inc. | Methods and reagents for detecting and assessing genotoxicity |
| WO2021022046A1 (en) * | 2019-07-31 | 2021-02-04 | Bioskryb, Inc. | Genetic mutational analysis |
Non-Patent Citations (3)
| Title |
|---|
| FERRARI ET AL., NAT REV GENET, vol. 22, 2021, pages 216 - 234 |
| GHADERI M ET AL: "A comparison of DNA damage induced by aflatoxin B1 in hepatocyte-like cells, their progenitor mesenchymal stem cells and CD34^+ cells isolated from umbilical cord blood", MUTATION RESEARCH. GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS, ELSEVIER, AMSTERDAM, NL, vol. 719, no. 1-2, 3 February 2011 (2011-02-03), pages 14 - 20, XP027575901, ISSN: 1383-5718, [retrieved on 20100929] * |
| JAICHANDRAN SIVALINGAM ET AL: "Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells", MOLECULAR THERAPY, vol. 24, no. 3, 1 March 2016 (2016-03-01), US, pages 607 - 619, XP055394819, ISSN: 1525-0016, DOI: 10.1038/mt.2015.223 * |
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