Patent Application
20250011844
The present invention relates to a method for determining the level and type of mutation events associated with the use of a targeted genetic modification, such as in the use of designer nucleases/editors, to modify a target site of DNA in a cell population.
Targeted genetic modification, such as designer nuclease/editor technology, is becoming a standard procedure in many laboratories and it has revolutionized not only the basic biology research, but also the diagnostic and the gene therapy field leading to its application in several clinical trials.
Designer nuclease activity evaluation is routinely performed with PCR approaches that amplify a specific region surrounding the cleavage site within the window range of 300 to 700 bp. These methods can be used to evaluate presence of small insertions and deletions (indel) but fail to detect large deletions or mutations that disrupt one primer binding site. Other than the targeted sequence (On-target) editing evaluation, it is necessary to assess the safety of the designer nucleases to verify the quality and quantity of chromosomal aberrations induced at ON- and OFF-target sites by double strand breaks (DSBs). Off-target sites could be at any location in the chromosomal DNA, other than the intended target location, which may cause disruption of essential genes or regulatory sequences.
Different techniques have been used to predict the quality and quantity of OFF-targets using in-silico (COSMID), in-cellula (HTGTS, UDITAS, CAST-seq, GUIDE-seq, IDLV integration, BLISS) or in-vitro (CIRCLE-seq, DIGENOME-seq) methodologies, but with poor resolution and/or accuracy. All these attributes are extremely relevant for gene therapy applications, where a standardised and fully unbiased technique aimed at evaluating the activity of designer nucleases is missing. Furthermore, even rates and quality of therapeutic transgene targeted integration may be over/underestimated or depending by specific cases, impossible to track and therefore surrogated to reporter gene expression constructs (GFP, Luciferase).
In order to tackle these requirements, it is desirable to develop techniques for a quick and unbiased overview of gene editing outcomes after targeted genetic modification, such as designer nuclease treatment, in therapeutically-relevant cells.
According to a first aspect of the present invention, there is provided a method for quantifying mutation events associated with a targeted genetic modification arranged to modify a target site of a targeted-chromosome in a cell population,
The total level of mutagenesis events associated with a targeted genetic modification may be determined by combining the determined mutation events as determined by the one or more analysis strategies A, B, C and D.
Advantageously, the invention provides a method, herein named “MEGA” (Multipurpose Editing Genotoxicity Assessment), which provides a quick and unbiased overview of gene editing outcomes after targeted genetic modification, such as designer nuclease/editor treatment, in therapeutically-relevant cells. This methodology provides an overall single analysis that is more complete, rapid, higher-throughput and consistent than previously available to those assessing the potential genotoxicity of a targeted genetic modification agent or strategy. It takes advantage of digital PCR technology (dPCR) and enables the quantification of double strand breaks (DSBs) in the targeted sites, while discerning the large deletions and the chromosomal aberrations (such as translocations, inversions, unrepaired DSBs). It is also able to quantify the copy number variation of the entire targeted chromosome or the possible loss of 5′ or 3′ chromosome arm portions with respect to the cleavage site. In case of a gene addition approach, where a DNA donor template, either viral or oligonucleotide based, is utilized to knock-in genetic sequences at the ON-target site, this methodology can also detect and quantify the amount of integrated and episomal DNA fragment. Vectors sequences, such as lentiviral or AAV, can be investigated for their stability and integrity before and after transduction in the targeted cell population. This method can make use of few nanograms of genomic DNA (the gDNA amount can be scalable depending on the requested sensitivity) derived from therapeutically relevant primary cells and it complements standard and high-throughput techniques for indel quantification, off-target analysis and chromosomal aberration characterization and quantification.
The Targeted Genetic Modification The targeted genetic modification may derive from targeted nuclease or genome modifiers. The genetic modification may comprise the addition, deletion or substitution of one or more nucleotides in a nucleotide sequence. The genetic mutation may comprise a sequence insertion or deletion, or translocation. The genetic modification may comprise or consist of a single stranded cut (nick), or a double stranded break/cut (DSB). The double stranded cut may be blunt ended, or may leave overhangs, such as sticky ends.
The skilled person will recognise that a “targeted nuclease” may also be referred to as a “designer nuclease” and such terms may be used interchangeably throughout. Targeted nuclease modifications may comprise genetic modifications using the targeted nuclease.
The targeted nuclease may comprise or consist of a RNA-guided endonuclease (RGEN), such as CRISPR/Cas9, Cas-CLOVER, mini-Cas9, or orthologues thereof. In another embodiment, the targeted nuclease may comprise or consist of zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN).
In another embodiment, the targeted genetic modification may comprise or consist of base-editors, prime-editors or targeted transposons.
In another embodiment, the targeted genetic modification may be a viral vector integration into the DNA, such as genomic DNA.
In one embodiment the targeted nucleic acid is DNA. The targeted DNA may be genomic DNA. Alternatively, the targeted DNA may be mitochondrial DNA.
The targeted nucleic acid may comprise a target/recognition sequence of between 8 and 40 nucleotides. The skilled person will recognise that the length of target sequence required may depend on the targeted genetic modification technology used. For example, CRISPR/Cas9 may recognise a sequence of about 20 nucleotides.
In one embodiment, the targeted nucleic acid is part of a nucleic acid molecule/strand that acts as a template for the dPCR reaction.
The “target cleavage/editing site” may refer to the specific site of cleavage of the targeted genetic modification, for example a nick or double-stranded break for insertion, deletion or substitution of nucleotide residues.
The target cleavage/editing site may be in a gene or regulatory sequence. The target cleavage/editing site may be in gene region Xp11 or Xq22 for the WAS or BTK genes, respectively.
The two pairs of primers may be designed to obtain the same amplicon length, for example about 60-120 nucleotides in length. In a preferred embodiment, the first region of DNA may be between 60 and 120 nucleotides in length. In another embodiment, the first region of DNA may be between 40 and 2000 nucleotides in length.
In a preferred embodiment, the distance between the first and second regions of DNA to be amplified in the reference control assay may be about 150-250 nucleotides. In another embodiment, the distance between the first and second regions of DNA to be amplified in the reference control assay may be about 0-10 kb in length.
In a preferred embodiment, the second region of DNA may be between 60 and 120 nucleotides in length. In another embodiment, the second region of DNA may be between 40 and 2000 nucleotides in length.
In one embodiment, the reference control analysis is used to quantify the genomic or DNA integrity. The target first and second regions of this analysis strategy may be located on a chromosome different to that of the chromosome targeted for the targeted genetic modification, such as nuclease cleavage. This may avoid variations in the reference control analysis strategy, such as from potential chromosomal aberrations that may occur in the targeted chromosome, i.e., LOH.
The relative level (e.g., ratio) of amplified first and second regions of DNA in the non-target chromosome may be determined by measuring the quantity of dPCR droplets having combined first and second labelled probe detections relative to the quantity of dPCR droplets having first-only or second-only labelled probe detections. The genomic integrity/fragmentation may be calculated by the ratios among the single positive droplets with the double positive droplets.
The values obtained from the non-targeted chromosome in the modified cell population may be normalised against the values obtained in the unmodified cell population as a control.
The two pairs of primers may be designed to obtain the same amplicon length, for example about 60-120 nucleotides in length. In a preferred embodiment, the 5′ region of DNA that is 5′ to the target cleavage/editing site in the targeted chromosome may be between 60 and 120 nucleotides in length. In another embodiment, the 5′ region of DNA that is 5′ to the target cleavage/editing site in the targeted chromosome may be between 40 and 2000 nucleotides in length.
The distance between the 5′ and 3′ regions of DNA to be amplified in the flanking analysis may be at least 40 nucleotides. In a preferred embodiment, the distance between the 5′ and 3′ regions of DNA to be amplified in the flanking analysis may be about 40-400 nucleotides. In another embodiment, the distance between the 5′ and 3′ regions of DNA to be amplified in the flanking analysis may be about 40-200 nucleotides. In another embodiment, the distance between the 5′ and 3′ regions of DNA to be amplified in the flanking analysis may be about 0-10 kb.
The 5′ region of DNA that is 5′ to the target cleavage/editing site in the targeted chromosome may be at least 20 nucleotides from the target cleavage/editing site. The 5′ region of DNA that is 5′ to the target cleavage/editing site in the targeted chromosome may be within 200 nucleotides of the target cleavage/editing site. In another embodiment, the 5′ region of DNA that is 5′ to the target cleavage/editing site in the targeted chromosome may be distanced between 20 and 200 nucleotides from the target cleavage/editing site. In another embodiment, the 5′ region of DNA that is 5′ to the target cleavage/editing site in the targeted chromosome may be distanced 0-10 kb from the target cleavage/editing site.
In a preferred embodiment, the 3′ region of DNA that is 3′ to the target cleavage/editing site in the targeted chromosome may be between 60 and 120 nucleotides in length. In another embodiment, the 3′ region of DNA that is 3′ to the target cleavage/editing site in the targeted chromosome may be between 40 and 2000 nucleotides in length.
The 3′ region of DNA that is 3′ to the target cleavage/editing site in the targeted chromosome may be at least 20 nucleotides from the target cleavage/editing site. The 3′ region of DNA that is 3′ to the target cleavage/editing site in the targeted chromosome may be within 200 nucleotides of the target cleavage/editing site. In another embodiment, the 3′ region of DNA that is 3′ to the target cleavage/editing site in the targeted chromosome may be distanced between 20 and 200 nucleotides from the target cleavage/editing site. In another embodiment, the 3′ region of DNA that is 3′ to the target cleavage/editing site in the targeted chromosome may be distanced 0-10 kb from the target cleavage/editing site.
The relative level of amplified 5′ and 3′ regions of DNA in the targeted chromosome may be determined by measuring the quantity of dPCR droplets having combined third and fourth (5′ and 3′) labelled probe detections relative to the quantity of dPCR droplets having third(5′)-only or fourth(3′)-only labelled probe detections.
The linkage percent (i.e. between the probed sequences, which is represented by the double positive droplets) may be normalized by the genomic integrity derived in the reference control analysis and may represent gross chromosomal aberrations. The copy number variation between the flanking analysis and the reference control analysis may represent large deletions.
The values obtained from the non-targeted chromosome in the modified cell population may be normalised against the values obtained in the unmodified cell population as a control.
In one embodiment, the values in the targeted chromosome is normalised against the values in the reference control of the non-targeted chromosome, and normalised against the relative values obtained from the unmodified cell population control.
The same approach can be multiplexed with assays, carrying different dyes, designed over the vector of interest to study its integrity before and after transduction.
In one embodiment, the fifth primer pair to amplify a region of DNA that includes the target cleavage/editing site in the targeted chromosome, may comprise a forward primer that is 5′ to the target cleavage/editing site and a reverse primer that is 3′ to the target cleavage/editing site.
The “fifth labelled probe” may otherwise be termed an “on-target probe”. The fifth labelled probe arranged to hybridise with the point of cleavage at the target in the amplified DNA that has been modified by the targeted genetic modification may be arranged to span the cleavage/edited site. It is understood that the sequence of the target cleavage/editing site of the modified nucleic acid will be the desired/designed sequence after the successful modification by the targeted genetic modification. The fifth labelled probe may be capable of spanning the entire genetic modification region. Alternatively, the fifth labelled probe may span between an unmodified/non-targeted region of the nucleic acid and a modified region comprising the genetic modification, for example in cases where the genetic modification is an insertion that is longer than the probe. The fifth labelled probe may hybridise to a region that spans between juxtaposed sequences of the original/unmodified sequence and an insert sequence, or juxtaposed sequences of the original/unmodified sequence having a deletion therebetween. The juxtaposed sequences at the target cleavage/editing site following genetic modification, or nucleotide substitutions at the target cleavage/editing site, may form a new sequence that would be unique for targeting by the probe.
In one embodiment, the fifth labelled probe may comprise a minor groove binding domain (MGB), for example to increase the sensitivity towards mutations.
The “sixth labelled probe” may otherwise be termed a “distal-target probe”. In a preferred embodiment, the sixth labelled probe arranged to hybridise with the amplified DNA at a site that is not the target cleavage/editing site may be targeted at a region that is at least 20 nucleotides far from the cleavage site. In another embodiment, the sixth labelled probe arranged to hybridise with the amplified DNA at a site that is not the target cleavage/editing site may be targeted at a region that is 0-10 kb far from the cleavage site.
The relative level of on-target versus distal-target probe hybridisations in the targeted chromosome may be determined by measuring the quantity of dPCR droplets having combined fifth and sixth (on-target and distal-target) labelled probe detections (indicating the presence of unmodified sequence) relative to the quantity of dPCR droplets having sixth(distal-target)-only labelled probe detections (indicating the presence of genetic modification or unknown genetic modification).
The values obtained from the non-targeted chromosome in the modified cell population may be normalised against the values obtained in the unmodified cell population as a control. The double positive (two probe) copy number difference from the reference control analysis and the unmodified control sample may be used to determine the absolute amount of small indels, large deletions and chromosomal aberrations together, including a targeted integration from a desired genetic modification.
In a preferred embodiment, the targeted sub-telomeric regions arranged to be amplified by the sixth primer pair and a seventh primer pair may be within 20 megabases of the respective telomere end. In another embodiment, the targeted sub-telomeric regions arranged to be amplified by the sixth primer pair and a seventh primer pair may be within 0-100 Mb of the respective telomere end. It is preferable to design the amplification regions on unique sequence regions as far as possible from the target cleavage/editing site to ensure that all potential mutations detected within the targeted chromosome are LoH.
The amplified regions of sub-telomeric regions arranged to be amplified by the sixth primer pair and a seventh primer pair may be preferentially between about 60 and 120 nucleotides in length.
The copy number variation of one of the two 5′ and 3′ sub-telomeric amplicons and the reference control assay estimates the loss of heterozygosity in the targeted chromosome.
The eighth primer pair is designed to recognise specifically the donor DNA sequence.
The ninth primer pair to amplify a region of the genomic DNA, may amplify a region that is close to the target cleavage/editing site. In a preferred embodiment, the ninth primer pair to amplify a region of the genomic DNA, may amplify a region that is outside a homology region utilized in the donor DNA and/or at least 50 bp from the cleavage site. In one embodiment, the ninth primer pair to amplify a region of the genomic DNA, may amplify a region that is outside a homology region utilized in the donor DNA and/or 0-10 kb from the cleavage site.
The relative level of amplified donor and genomic regions of DNA in the modified cell population may be determined by measuring the quantity of dPCR droplets having combined ninth and tenth (donor and genomic) labelled probe detections (indicating they are linked and there has been a donor integration) relative to the quantity of dPCR droplets having ninth(donor)-only or tenth(3′ genomic)-only labelled probe detections (indicating no linkage and no site specific integration—the donor remains episomal).
The linkage percent (i.e., where the linked probed sequences are represented by double positive droplets) may represent the amount of targeted integration. Single positive droplets derived by the donor specific probe may represent the amount of donor DNA integrated in the genomic DNA and/or in an episomal state. The linkage percent (i.e., represented by double positive droplets) may be normalized by the genomic integrity derived in the reference control assay. Additionally, or alternatively, the copy number ratio determined between the KI-OT assay and the reference control assay may represent the amount of donor DNA not integrated in the targeted locus. In one embodiment, the values obtained from the non-targeted chromosome in the modified cell population may be normalised against the values obtained in the unmodified cell population as a control.
Knock-In Analysis (in-Out Strategy)
In one embodiment, a further analysis is provided as follows:
The knock-in analysis may be conducted on the modified cell population. The copy number ratio may be determined against a control amplicon of the same amplicon size. The control amplicon may be distanced away from the cleavage site or may be on a non-targeted chromosome.
Combinations of the analysis strategies on the treated cell population may be conducted in parallel or conducted sequentially. Two or more, or all, of the analysis strategies on the treated cell population may be carried out in separate dPCR reactions (e.g., may not share reagents). In another embodiment, two or more analysis strategies may be provided in the same dPCR reaction (e.g., may share reagents). Where combinations of analysis strategies are conducted in the same dPCR reaction, the labelled probes may be distinguishable between the different analysis strategies, for example by fluorescing at different wavelengths.
Where two or more, or all the analysis strategies are conducted, the nucleic acid from the modified cell population may be from the same population/culture and/or the same targeted genetic modification. In particular, portions of the modified nucleic acid may be distributed into two or more analysis strategies to be run in parallel or sequentially.
The primers described herein may be any suitable length for priming a dPCR reaction. In one embodiment, the primers are at least 8 nucleotides in length. In another embodiment, the primers are about 8-40 nucleotides in length. In another embodiment, the primers are about 10-40 nucleotides in length. In another embodiment, the primers are about 8-30 nucleotides in length. In another embodiment, the primers are about 10-30 nucleotides in length. In another embodiment, the primers are about 15-25 nucleotides in length.
Where combinations of analysis strategies are conducted in the same dPCR reaction, the primer pairs may have substantially similar or compatible melting and annealing temperatures, for example the melting and annealing temperatures (Tm) of a primer pair for each analysis strategy may be within 5° C. or preferably within 3° C. of the respective melting and annealing temperatures of the primer pair of another analysis strategy.
The primers may comprise oligonucleotide, such as DNA, or nucleotide analogues thereof. Nucleotide analogues may comprise LNA (locked nucleic acid), PNA (peptide nucleic acid), PMO (phosphorodiamidate morpholino oligomer) or combinations thereof.
The primer pairs may be selected from any of the primer pairs provided in Tables 1-5 herein, or combinations thereof. The skilled person may select appropriate primer pairs and combinations in accordance with the analysis strategies being conducted.
In one embodiment, the label of the labelled probes is a fluorescent label. The labelled probes may be 5′ labelled with a fluorescent dye, such as a fluorescein. In one embodiment, the labelled probes may be labelled with fluorescein, such as fluorescein amidite (FAM) or 2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC). The fluorescein amidite may be 6-FAM.
The skilled person will recognise that any suitable fluorescent dyes may be used to label the probes herein, for example labelled probes may be selected from fluorescein amidite (FAM), TET, 2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC), hexachloro-fluorescein (HEX), and Cy3.5, or fluorescent dyes providing substantially similar wavelength emissions.
Where a pair of probes is used in an analysis strategy, the probes may be labelled differently, such that they are distinguishable. For example, a pair of probes in an analysis strategy may comprise two sets of oligonucleotides, where the two sets are labelled with different fluorescent dyes. In an embodiment where combinations of probes and/or analysis strategies are provided in a single dPCR reaction, each probe type may be labelled differently, such that they are distinguishable according to their excitation wavelength.
The labelled probes described herein may be any suitable length for specifically hybridising to a substantially complementary target sequence. In one embodiment, the labelled probes are at least 6, 7, 8, 9 or 10 nucleotides in length. In another embodiment, the labelled probes are about 8-40 nucleotides in length. In another embodiment, the labelled probes are about 10-40 nucleotides in length. In another embodiment, the labelled probes are about 8-30 nucleotides in length. In another embodiment, the labelled probes are about 10-30 nucleotides in length. In another embodiment, the labelled probes are about 15-25 nucleotides in length.
Where two or more labelled probes are used in an assay, the two or more labelled probes may have substantially similar or compatible annealing temperatures to their respective target sequences, for example the annealing temperatures may be within 10° C. or preferably within 3° C. of each other. In another embodiment, the annealing temperatures may be within 10% or preferably within 5% of each other.
Where combinations of analysis strategies are conducted in the same dPCR reaction, the labelled probes may have substantially similar or compatible annealing temperatures to their respective target sequences, for example the annealing temperatures of labelled probes for each assay may be within 10% or preferably within 5% of the respective annealing temperatures of the labelled probes of another analysis strategy. In another embodiment, the annealing temperatures may be within 10° C. or preferably within 3° C. of each other.
The Tm of the labelled probes may be about 5-15° C. higher than the primers. The labelled probes may further comprise a Minor Groove Binding (MGB) domain, for example to increase the annealing temperature and improve the positive signal intensity over the background and the sensitivity towards mutations.
The labelled probes may comprise oligonucleotide, such as DNA, or nucleotide analogues thereof. Nucleotide analogues may comprise LNA, PNA, PMO or combinations thereof.
The labelled probes may be selected from any of the labelled probes provided in Tables 1-5 herein, or combinations thereof. The skilled person may select appropriate probe combinations in accordance with the analysis strategy being conducted.
The dPCR Labelled Probe Detections in Droplets
dPCR droplets may be individually measured and quantified for labelled probe detections, for example by a dPCR droplet reader, which scans each droplet for the labels of successfully hybridised probes, such as scanning for fluorescent wavelengths emitted by the probe labels. dPCR droplets may be sorted and counted using an adapted FACS method to sort and count droplets instead of cells, for example with a (FADS fluorescent activated droplet sorter) device specific for the droplets.
The cell population may comprise cells that are prokaryotic or eukaryotic. Preferably the cells are eukaryote cells. In one embodiment the cells are mammalian cells, such as human cells.
The cells may be stem cells, such as iPSCs or ESCs. The cells may be germline or somatic cells. In one embodiment the cells may be immune cells, such as lymphocytes (T cells, B cells or natural killer (NK) cells), neutrophils, and monocytes/macrophages. The cells may comprise a mixed population of cell types.
In one embodiment, the cells may be associated with a disease or condition, such as cancer cells or infected cells. In one embodiment, the cells may be associated with a mutation or infection causing a disease or condition.
The modified cell population and unmodified cell population may be of the same cell type and/or source. In particular, the modified cell population and unmodified cell population may be substantially identical, other than the modified cell population has been treated with a targeted genetic modification.
The cell number may be sufficient for a treatment. In one embodiment, at least 1000 cells are provided. Preferably at least 50,000 cells are provided.
The DNA of the unmodified- and modified cell population may be extracted from the cells such that it is suitable as a template for the dPCR. Preferably, the genomic DNA is extracted from the cells of the unmodified- and modified cell population.
The skilled person will be familiar with various genomic DNA extraction techniques that may be used. In a preferred embodiment, the genomic DNA is extracted with a technique that maintains a high degree of genomic integrity with an average fragment length size >15 kb. In one embodiment, the genomic DNA is extracted by the salting-out method, for example as described by Miller et al (Nucleic Acids Research, 1988, 16, 1215.), which is herein incorporated by reference. Suitable high molecular weight extraction methods may be used by the skilled person, for example by glass-bead precipitation, such as provided by the Monarch® HMW DNA Extraction Kit (New England Biolabs Inc.).
DNA extraction methods can damage the DNA creating fragments of all sizes. The method of the invention advantageously takes the genomic integrity into account and can further use an extraction technique providing a more intact genome, thereby increasing the accuracy of the determination.
The dPCR
In one embodiment, extracted DNA from the cell population may be digested into smaller fragments, for example by restriction enzyme digestion. This may facilitate distribution of the DNA into the droplets of the dPCR. Preferably, the restriction enzyme cleavage sites are not located in or close (e.g. 2 bp or less) to the assays of interest to prevent interference of the amplification and analysis.
Distribution of the nucleic acid may be facilitated by dilution of the nucleic acid. Therefore, in one embodiment, the nucleic acid solution may be diluted to be in the correct range of quantification. For example, Poisson distribution may be used for the absolute quantification calculation.
The amplification reagent for dPCR DNA amplification may otherwise be termed a “master mix” or “amplification mix”. The skilled person will understand that an amplification mix may comprise all the reagents necessary for droplet generation and PCR amplification of the DNA. Such components may comprise reaction buffer, polymerase, and dNTPs. A DNA polymerisation reporter molecule, such as a DNA-binding dye (e.g., Evagreen™) may also be provided in the amplification mix, for example to allow monitoring of the amplification reaction using a real-time PCR. The DNA-binding dye may be constructed of two monomeric DNA-binding dyes linked by a flexible spacer. In the absence of DNA, the dimeric dye can assume a looped conformation that is inactive in DNA binding. When DNA is available, the looped conformation can shift via an equilibrium to a random conformation that is capable of binding to DNA to emit fluorescence.
The amplification reagents may be divided equally between the dPCR droplets.
The skilled person will be able to provide suitable conditions for the amplification reaction to occur, including suitable temperature and incubation times.
About 20-30 ng of the nucleic acid, such as human gDNA, may be provided for distribution within the droplets. In another embodiment, 10-100 ng of nucleic acid, such as gDNA, may be provided for distribution within the droplets (e.g., for diploid human genomic DNA). In another embodiment, 25-100 ng of nucleic acid, such as gDNA, may be provided for distribution within the droplets.
Alternatively, about 10-20 ng of the nucleic acid, such as human gDNA, may be provided for distribution within the droplets. In another embodiment, 5-50 ng of nucleic acid, such as gDNA, may be provided for distribution within the droplets (e.g., for diploid human genomic DNA).
The amount of DNA may be sufficient to result in no more than 20% of positive droplets for the analysis strategy of interest. This advantageously avoids formation of double positive droplets by chance and can reduce the impact of the normalization for genomic integrity.
The final concentration of primers may be about 1 μM. In one embodiment, the final concentration of primers may be about 0.2-1 μM.
The final concentration of labelled probes may be about 250 nM. In one embodiment, the final concentration of labelled probes may be about 50-500 nM.
The dPCR may be conducted with at least 1000 droplets. Preferably at least 10000 droplets per analysis is used. The droplets' size and volume may be consistent/standardised (i.e., substantially equal) within the population of droplets.
The dPCR droplet preparation, reaction and processing may be conducted by a suitable dPCR system and a reader, such as the QX200™ Droplet Reader/QuantaSoft™ Analysis Pro Software (Bio-Rad Laboratories), Naica® system—Multiplex Crystal Digital PCR™/Crystal Miner Software (STILLA technologies), or QIAcuity Digital PCR System/QIAcuity Software Suite.
According to another aspect of the present invention, there is provided the use of the method of the invention herein for screening potential targeted genetic modification agents, such as designer nucleases, for therapeutic use.
The skilled person will recognise that the method of the invention may be adapted to determine the genomic or DNA integrity of any nucleic acid, such as a vector, or a viral genomic nucleic acid (e.g., viral DNA).
The targeted genetic modification may be in any nucleic acid type. Therefore, the term “targeted-chromosome in a cell population” may be substituted with “targeted nucleic acid in a nucleic acid population”. For example, the “cell population” may be substituted herein with a “virus population”, and the “targeted-chromosome” and “non-targeted chromosome” may be a “targeted viral genome” and non-targeted viral genome” respectively. In a further example, the “cell population” may be substituted herein with a “microbial population”, and the “targeted-chromosome” and “non-targeted chromosome” may be a “targeted genome” and non-targeted genome” respectively.
According to another aspect of the present invention, there is provided a method for quantifying mutation events associated with a targeted genetic modification arranged to modify a target site of a nucleic acid, such as DNA or RNA,
The total level of mutagenic events associated with a targeted nucleic acid modification may be determined by combining the determined mutation events as determined by the one or more analysis strategies 1, 2, and 3.
The nucleic acid may be DNA, such as vector DNA. In another embodiment, the nucleic acid may be a viral genome, such as viral genomic DNA. In another embodiment the nucleic acid may be bacterial DNA.
The term “digital PCR (dPCR)” refers to a method for performing digital PCR that is based on water-oil emulsion droplet or physical partitioning technology. A sample of nucleic acid with a master mix of reagents is spread into physical partitions or fractionated into thousands of droplets (e.g., 20,000 droplets) with the aim of the droplets/partitions only having a single targeted nucleic acid molecule. A simultaneous PCR amplification reaction is carried out on the targeted template nucleic acid molecules present in the individual droplets. The term “digital PCR (dPCR) may be used interchangeably with “digital droplet PCR (ddPCR)”.
Genotoxicity describes the property of an agent able to alter the genetic function within a cell causing unwanted mutations/effects, which may lead to functional impairment or disease development (e.g., cancer, therapy impairment, differentiation impairment).
Reference herein to an “insertion” is understood to mean a genetic modification that involves a sequence of nucleic acid being inserted into the sequence of another nucleic acid.
Reference herein to a “deletion” is understood to mean a genetic modification that involves a sequence of nucleic acid being removed, or otherwise termed “deleted”.
Reference herein to an “indel” is understood to mean a genetic modification that may be an insertion or deletion.
“Translocation” refers to a type of chromosomal abnormality in which a chromosome and a portion of it recombines to a different chromosome.
“Loss of heterozygosity (LOH)” is understood to be a gross chromosomal event that results in loss of chromosomal regions.
“Homologous recombination” is understood to be a type of genetic recombination in which genetic information is exchanged between two similar or identical nucleic acid molecules.
Reference herein to “donor DNA” or “DNA donor” is understood to mean a sequence of DNA that has been provided from a non-chromosomal sequence, such as synthetic DNA, or a vector.
The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention.
Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.
The primers and probes listed herein may be used, as an example, in the method and/or compositions of the invention.
Optimal primer and probe design should follow standard qPCR/dPCR rules particularly those regarding specificity, secondary structures, % GC content, primer dimers among multiplexed assays, melting temperatures (Tm), and the presence of single nucleotide polymorphisms (SNPs). All assays should return a robust signal using the same PCR program parameters with a clear separation between the negative and positive populations. Probes running in multiplex should have a distinct fluorophore compatible with the relative device recommendations.
The Tm of the primers should be within the same temperature range, while the probes should be 5-15 Tm degrees higher than the primers. Difficult sequences may also require a probe with the Minor Groove Binding (MGB) or locked nucleic acid (LNA) modification domain to increase the annealing temperature and improve the positive signal intensity over the background.
Two pairs of primers should be designed to obtain the same amplicon length of ca. 60-120 bp. The distance between the two assays should be of ca. 150-250 bp. The target of this assay should ideally be located on a chromosome different to that of the chromosome targeted for nuclease cleavage. (
One pair of primers should be designed surrounding the cleavage point with two probes designed to target two loci in the region amplified by the primers. One of them should be placed directly across the cleavage site (cleavage probe) whilst the other probe should be placed at least 20 or 25 bp distant from it (distal probe).
Two pairs of primers should be designed 5′ and 3′ of the cleavage site leaving a space of at least 20 bp from the cleavage site. The distance between the 5′ assay forward primer and the 3′ reverse primer should be as short as possible.
Two assays should be designed in sub-telomeric regions of the targeted chromosome 5′ and 3′ of the cleavage site and ideally 2-10 Mb distant from the chromosomal ends.
One assay should be designed upstream and adjacent to the donor homology regions (HA), while the other assay is located inside the donor cassette in a specific sequence (DN). In the case of a donor introducing a single mismatched nucleotide, a further probe specificity test is advised (MGB probes would better distinguish a single nucleotide polymorphism). The distance between the assays should as short as possible.
Alternative 5th Targeted Integration “in/Out” Strategy Assay:
The In/Out targeted integration strategy requires a pair of primers and one probe that are specific for the integrated cassette in the targeted locus. One primer should be designed inside the donor cassette and the other outside the donor cassette. In case of short donor cassettes, the probe can be the only specific element recognising the donor sequence.
The distance between the primers should be as short as possible. A control assay should be designed far from the cleavage site or preferentially on another allele and it should match the In/Out amplicon length for a more precise quantification.
dPCR Reaction Protocol
Around 10-100 ng gDNA (30-50 ng range for diploid human genomic DNA) per well. This amount typically generates the required number of droplets. The final concentration of primers and probes is 1 μM and 250 nM, respectively.
All assays should be performed in duplicate or in triplicate to average the final readout and to possibly remove wells that resulted in a technical failure.
The genomic DNA needs to be mixed in the master mix and only then, aliquoted in the several tubes that will receive the different combinations of primers and probes. This will decrease the pipetting variability among the different assays (with 3 colour and the internal control assay this would not be strictly required).
An untreated (UT) sample should be also analysed alongside edited samples to normalize the inter-assay data fluctuations. The genomic DNA should possibly be from the same donor to control for person-to-person variations and/or other variabilities.
To avoid formation of double positive droplets by chance and to reduce the impact of the normalization for genomic integrity, the amount of sample DNA should result in no more than 20% of positive droplets for the assays of interest.
Quantifying the mutation events in accordance with the invention herein may be determined and analysed according to the following mathematical principles.
The quantifications given by the digital droplet polymerase chain reaction (dPCR) machine (copies/ul) may be normalised twice for all the assays. Three colour dPCR, carrying the house-keeping internal control in multiplex, allows a more precise normalization. For two colour dPCR, the control assay may be carried out in another well.
This double normalization allows for the removal of the inter- and intra-assay variabilities caused by different factors such as variable quantities of reagents or assay lengths.
Loss of heterozygosity (LOH) can be calculated utilizing the Norm2 ratios of the relative assays designed in the sub-telomeric p and q regions:
LoH%=(Norm2−1)*100
This parameter will indicate the gain or the loss of copies of the two chromosomal portions studied. This parameter may be plotted separately from the other parameters since an allele with a LOH can still retain the edited sequence.
Small indels can be calculated from the Indels assays formed by the “cut” and “distal” probe.
The “Small indels %” parameter should be negative indicating the loss of copies respect to the distal probe. When plotting, the absolute value may be provided.
When this assay is utilized, and considering the UT control sample and a control assay, it is possible to also quantify other chromosomal mutations % (accounting mainly for large deletions, insertions and translocations) data:
The “Small indels/o” and “Mutations/o” parameters should be negative indicating the loss of copies relative to the controls. When plotting the absolute value may be provided. Those parameters are already normalised for the amount of alleles, in the case of a X assay in XY context no other normalizations may be required and the values will reflect the percentage copies with respect to the original allelic number.
When this assay is utilized per se, and not in the MEGA context, it can provide relative information such as the relative percentage of small indels:
Calculate base distance from Forward primer 5′ base of Channel 1(ch1) assay to the Forward primer 5′ base of channel2 (ch2) assay=DistAssay1.
Calculate base distance from Reverse primer 5′ base of Channel 1(ch1) assay to the Reverse primer 5′ base of channel2 (ch2) assay=DistAssay2.
Calculate the number of single positive droplets (Dch#) for the genomic integrity assays obtained from the dPCR and calculate the following ratio with all the relative positive droplets (Dch#all):
This value may be utilized in the Flanking assays and the Homologous Recombination (HR) assays to account for the genomic integrity by multiplying this value to the assays distance.
Depending on the sequence complexity and the strategy utilized for the targeted integration, it is possible to design two different strategies to calculate the targeted insertion.
In/out strategy. No possibility to check for the episomal/randomly integrated donor DNA. No need for normalizations (in/out design with independent control assay of the same length). Normalization is required if the control and reference chromosomes amounts are not equal (e.g., X assayed in XY context).
Donor KI-OT strategy. Needs genomic integrity normalization (Donor KI-OT design). Report the average distance between the two HR assays as similarly calculated for the genomic integrity assay. Here we describe the assay designed on the donor as DN and the one outside the homology arm as HA.
In “OutDroplets”, the number of droplets that are on average more likely to be single positive because of the genomic fragmentation rather than double positive can be estimated. The OutDroplets number can be used to normalize the single positive droplets (nDDN) from the DN assay that is supposed to be read as double positive.
The linkage between the DN assay and the HA assay can be calculated as described in Regan J F et al. (PlosOne 2013. doi:10.1371/journal.pone.0118270) (which is herein incorporated by reference), with some modifications described herein.
This Parameter will represent the category of the “other aberrations” representing mutations such as translocations, open ends, inversions, chromothripsis repairs.
In this case two main factors can be considered:
This % linkage calculation can be adopted to check vector integrity of any kind. The two assays will be designed within the vector sequence at the two extremities.
The percent of episomal, off targets driven and random integrated donor DNA (% Episomal/OT) is calculated from the single positive droplets normalized by the genomic integrity and the amount of the control assay.
3rd assay. Large deletions and chromosomal aberrations
This evaluation will discern the large deletions from the two sides in relation to the cleavage site.
Those mutations will remove biases derived from the targeted integration or other mutations.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2115834.0 | Nov 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2022/052772 | 11/3/2022 | WO |
