PRIORITY

This application corresponds to the U.S. National Phase of International Application No. PCT/EP2019/075101, filed Sep. 19, 2018, which, in turn, claims priority to European Patent Application No. 18196438.8 filed Sep. 25, 2018, the contents of which are incorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 17, 2021, is named LNK_226 US_SEQ_LIST.txt and is 12,232 bytes in size.

BACKGROUND OF THE INVENTION

Genome editing describes the targeted modification of the genomes of any kind of cell type of interest with so-called “designer nucleases”.

Several designer nucleases are known, which are also designated as “programmable nucleases” or “engineered nucleases”. Examples thereof are zinc-finger nuclease (ZFNs), transcriptional activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) which may be derived from the clustered regularly interspaced repeat (CRISPR/Cas) prokaryotic adaptive immunity system. Such means are important and widely used for genome editing not only in cultured cells but also in whole organisms. Designer nucleases have an origin in nature but are artificially modified in order to act in an intended manner.

Genome editing has broad applications and has been successfully employed to genetically modify prokaryotic and eukaryotic microorganisms, crops, livestock, model organisms for research, cell lines for drug screening, and various cell types or organs for therapeutic applications. For most of these applications, the specificity of the employed designer nuclease is a key parameter to ensure maintenance of genome integrity of the edited cell type.

In the context of clinical applications in humans, relevant cell types that have been edited with designer nucleases include hematopoietic stem cells, B and T cells, epidermal stem cells, pluripotent stem cells, liver cells, muscle cells, and retinal cells. Relevant disease targets include (but are not limited to) hereditary disorders, in particular hereditary disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, infectious diseases, or cancer.

Before employing genome editing in transplantable cell types ex vivo or before applying gene editing tools in vivo directly in the patient, the designer nucleases need to be carefully evaluated with respect to activity and specificity. Specificity of engineered endonucleases is the key for any clinical translation of gene editing in order to maintain genome integrity and to reduce the risk of inducing oncogenic mutations. The consequence of designer nuclease induced mutagenesis at so-called off-target sites and/or the resulting chromosomal aberrations are often referred to as genotoxicity that eventually could lead to cancer.

The term “on-target site” is used in the present application to designate a site at which a DNA double strand break is intended to be introduced by using “designer nucleases”. Such intended site of action is usually designated as “on-target site”.

The designer nucleases have a certain sequence specificity and thus work at such “on-target site”. The designer nucleases may, however, also work at so-called “off-target sites” which show a certain degree of sequence homology to the “on-target site”. The term “off-target site” as used herein refers to a site where the designer nucleases have activity and which usually have a sequence that is not identical to the target sequence of the designer sequence. An “off-target site” relates to a sequence other than an “on-target site” that is cleaved by the designer nucleases. The fact that designer nucleases have an activity even at sites different from the on-target site may be due to phenomena that can be caused by various reasons. The downside of designer nucleases cleaving at off-target sites is that this can result in undesired side effects like mutations, deletions, sequence inversions and other disturbances in the genome which should be avoided.

In general, designer nuclease induced off-target activity can lead to short insertion/deletion (indel) mutations, large chromosomal deletions, chromosomal inversions, as well as chromosomal translocations. On the molecular level, off-target activity occurs when the DNA binding moieties of designer nucleases bind to sequences in the genome that share homology to the actual target site. Much effort has been invested in increasing the safety of genome-editing tools in the past decade, leading to better designer nucleases with much higher specificity.

Nonetheless, a thorough preclinical assessment of designer nuclease specificity is a clearly stated requirement by the regulatory bodies, such as the Paul Ehrlich Institute in Germany or the U.S. Food and Drug Administration (FDA). There is a need for applied diagnostic methods which are highly sensitive and allow skilled persons not only to measure off-target mutagenesis but also chromosomal aberrations and/or any other unexpected genomic modifications with high sensitivity.

To assess the genotoxic risk associated with the application of designer nucleases such as CRISPR-Cas nucleases, several methods have been developed to determine either off-target activity of designer nucleases or designer nuclease induced chromosomal aberrations. In principle, these methods can be subdivided into computer-based prediction algorithms (in silico methods), in vitro test methods and cell-based methods. All of these methods rely on next generation sequencing (NGS) and are typically employed in a two-step process: A ‘screening assay’ is first used to identify all potentially possible off-target sites in the genome of interest. A subsequent ‘confirmatory assay’ is used to sequence the potential off-target sites defined in the screening test in the genome of the gene edited cells.

In silico prediction algorithms are based on well-defined parameters, including similarity to the target sequence (Lee et al. (2016), Mol Ther 24, 475-487). They represent a fast and relatively cheap ‘screening assay’, but more often than not, those algorithms miss critical off-target sites. In contrast to the in silico analysis, experimental methods allow for the identification of off-targets independently of predetermined parameters and are consequently less biased. However, experimental methods are more laborious and more expensive. In addition, they are subject to technical limitations and some of them lack sensitivity.

At present, several experimental ‘screening assays’ are employed to determine off-target sites and are likely sensitive enough to be considered for preclinical evaluation of designer nuclease specificity, like for example:a) EP 3 219 810 (whole genome sequencing)b) Guide-Seq (Tsai et al. (2015), Nat Biotechnol 33, 187-197),c) BLISS (Yan et al. (2017), Nat Commun 8, 15058),d) Digenome-Seq (Kim et al. (2015), Nat Methods 12, 237-243), ande) Circle-Seq (Tsai et al. (2017), Nat Methods 14, 607-614).

Guide-Seq is a cell-based method that introduces short double-stranded oligodeoxynucleotides (dsODN) into the cell in addition to the designer nuclease. Once the designer nuclease cuts the genome, the short dsODN is integrated by the cellular DNA repair machinery into the resulting DNA double-strand breaks, and can then serve as a starting point for high-throughput sequencing. This method works well but only in certain human cell lines whose genome can differ considerably from the genome of the patient.

BLISS detects actual DNA double strand breaks in cells by means of an in vitro oligo ligation to the available DNA ends. The ligated DNA is in vitro transcribed and the library sequenced by high-throughput sequencing. Digenome-Seq and Circle-Seq are in vitro methods, which are based on the cleavage of the whole genome or circularized genome fragments with CRISPR-Cas.

For Digenome-Seq, whole-genome sequencing is performed on the in vitro cleaved genomes, which will yield sequence reads with the same 5′ ends at cleavage sites that can then be computationally identified. To reach the necessary coverage and therefore sufficient sensitivity, Digenome-Seq must be performed on high-throughput sequencing machines, such as the Illumine HiSeq line. In consequence, the application of Digenome-Seq is rather expensive.

In Circle-Seq sequencing adapters are ligated to the cleaved 5′ ends, which then can be used for NGS to identify the off-target sites. However, Circle-Seq may suffer from potential biases resulting from the need to circularize genomic DNA and requires large amounts of input DNA.

Hence, Circle-Seq cannot be performed if only limited amount of sample, e.g. a biopsy, is available. In all cases, these experimentally determined off-target sites must be validated in the patient's cells using NGS-based ‘confirmatory assays’, such as multiplexed targeted amplicon sequencing, to establish an actual specificity profile of the nucleases in the clinically relevant target cells.

Importantly, the above-described methods allow researchers to predict off-target sites which are cleaved by the designer nuclease of choice, but none of them enable an assessment of gross chromosomal aberrations induced by programmable nucleases, such as recently described (Kosicki et al. (2018), Nat Biotechnol 36, 765-771).

Two further described methods, high-throughput genome-wide translocation sequencing (HTGTS) and uni-directional targeted sequencing methodology (UDiTaS) can identify translocations or other chromosomal aberrations induced by designer nucleases. HTGTS (WO 2016/081798) and UDiTaS (WO 2018/129368) disclose methods relating to the detection of non-specific DNA double-strand breaks in the genome. These two methods also allowed for the identification of translocation events but the described bioinformatic analyses as well as the biased genomic fragmentation (use of Tn5 tagmentation for UDiTaS, restriction enzymes for HTGTS) considerably limit sensitivity of these approaches. HTGTS does neither state a lower limit of detection (LLoD) nor sensitivity. UDiTaS's LLoD is indicated as 0.1%.

All known methods cannot identify chromosomal rearrangements that are not related to off-target activity of a designer nuclease. In particular, these methods cannot identify homology-mediated chromosomal rearrangements that are triggered by on-target activity of a designer nuclease.

HTGTS and UDiTaS are not quantitative with regard to unknown translocation events.

HTGTS and UDiTaS were not shown to work on genomic DNA harvested from a gene-edited, clinically relevant cell type, i.e. hematopoietic stem cells.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method that can identify genomic modifications, including gross chromosomal aberrations, and that is (i) highly sensitive, (ii) highly specific, (iii) quantitative, (iv) able to detect previously undescribed types of chromosomal rearrangements, and (v) performed directly on genomic DNA isolated from the clinically relevant cell type. The method is designated herein as CAST-Seq (chromosomal aberration analysis by single targeted linker-mediated PCR).

CAST-Seq is based on single targeted linker-mediated PCR (LM-PCR) and uses decoy primers to enhance the signal-to-noise ratio. This method allows to identify off-target sites and to detect genomic modifications derived from both on- and off-target activity of designer nucleases, including large deletions, inversions and translocations with unmatched sensitivity. Importantly, because CAST-Seq's high sensitivity, the assay can be performed with less than 1 μg of genomic DNA as input. CAST-Seq can therefore be applied directly to any clinically relevant human cell type of choice, including ex vivo gene edited cells before transplantation or cells derived from a biopsy of gene edited organs. This unique setup and the fact that CAST-Seq is performed directly in the gene edited cell type or tissue of interest, CAST-Seq may make an NGS-based confirmation assay redundant by straightforwardly uniting the ‘screening test’ with the ‘confirmatory test’. CAST-Seq may therefore substantially improve the process by detecting chromosomal aberrations at “on-target sites” and “off-target-sites”.

