Patent Grant
9493827
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is herein incorporated by reference in its entirety.
The DNA repair machinery of live cells will seek to repair double strand breaks (DSBs) by homologous recombination (HR) ad integrum or by the more error-prone non-homologous end joining (NHEJ) pathway that essentially religates whatever sequence is available to the open remaining DNA strands. Spontaneous HR is a very rare event in mammalian cells with approximately 1 donor DNA recombination per 106 cells (Cathomen and Joung 2008). This rate increases drastically when cells are exposed to agents that induce DSBs, like e.g. ionizing radiation; also, introduction of DSB by ZFN has been shown to potentiate the likelihood of HR at the cleavage site by a factor of 100-10000 (Durai et al. 2005; Porteus and Carroll 2005), making artificial ZFN a promising tool for genetic manipulation of live cells, including clinical therapy. DSB get repaired quickly, leaving no or only minor nucleotide changes or deletions in the genome, so it has been difficult to obtain data relating to the localization and/or frequency of double-strand breaks occurring in the genome of a cell.
Zinc finger nucleases (ZFNs) are artificial restriction enzymes comprising a zinc finger DNA-binding domain fused to a DNA-cleavage domain. ZFNs are designed to introduce DSBs at virtually any selected genome position. To achieve site-specific genome targeting, ZFNs combine unspecific Fok I endonuclease cleavage domains with arbitrary chosen binding domains of zinc finger proteins (ZFPs). Thereby, ZFN function requires the dimerization of a specific ZFN monomer binding upstream to the target site on the plus strand with a second ZFN-monomer that binds downstream of the target site (Kim et al. 1996; Mani et al. 2005). To reduce cytotoxicity of ZFN, mainly caused by homodimer formation of identical ZFN monomers at off-target sites, a new generation of ZFN has been developed by introducing complementary substitutions of single amino acids in the nuclease domain preventing homodimerization of the Fokl subunits (Miller et al. 2007; Szczepek et al. 2007).
Therapeutic applications of ZFN comprise three main categories of DNA modifications in live cells: i) targeted mutagenesis (gene knock-out), inducing loss of information during NHEJ, ii) gene correction at the target locus by HR involving a homologous donor DNA fragment and iii) targeted integration of an expression cassette by HR into a potentially “safe harbor” sequence whose mutation should be harmless for the targeted cell type. The mode of transfer of ZFN into cells is highly relevant for the success rate and toxicity of specific DNA modification in live cells. It was previously demonstrated that the delivery of ZFN into cells via non-integrating integrase-defective lentiviral vectors (IDLV) has little or no acute cytotoxicity, achieves transfer of the ZFN cDNA into almost every target cell, and has the potential to achieve HR mediated editing of the genome sequence specifically in up to 50% of the transduced cell populations (Lombardo et al. 2007). However, because DSB get repaired quickly, leaving no or only minor nucleotide changes or deletions in the genome, no definitive data has been available neither on the nature nor on the frequency of “off-site” DSB caused by ZFN activity outside of the intended target sequence. This remains an important open issue in using ZFN technology, not least if its application is intended for clinical gene therapy, since the availability of specific genomic editing would minimize or abolish the risk of insertional mutagenesis and oncogenesis observed in preclinical studies and clinical trials with retroviral vectors (Hacein-Bey-Abina et al. 2003; Modlich et al. 2006; Montini et al. 2006; Ott et al. 2006; Hacein-Bey-Abina et al. 2008; Howe et al. 2008).
Thus, means and methods are required for complying with the aforementioned needs. The said technical problem is solved by the embodiments characterized in the claims and herein below.
Accordingly, the present invention relates to a method for determining the in vivo localization of double-strand breaks in a host cell, comprising a) incubating said host cell suspected to comprise DNA double-strand breaks and a linear polynucleotide comprising a known sequence, b) detecting the in vivo insertion sites of said polynucleotide in the genome of said host cell, c) determining the in vivo positions of double-strand breaks, and d) assessing the in vivo localization of double-strand breaks.
The term “DNA double-strand break” or “double-strand break” is understood by the skilled artisan.
The term “determining the in vivo localization of double-strand breaks”, preferably, relates to determining the positions in the genome of a host cell of double-strand breaks occurring in said host cell. It is to be understood that determining the localization of double-strand breaks according to the current specification includes the repair of said double-strand breaks, i.e. the double-strand breaks do no longer exist by the times their position is determined. It is further to be understood that the determination of the in vivo localization of double-strand breaks does not determine the exact position in the genome where the double-strand break occurred. However, the term requires that the position determined is within 10, 25, 50, 100, 250, 500, 1000, 2500, or 5000 nucleotides from the position where the double-strand break occurred for at least 75%, 85%, 90%, 95%, 97%, or 99% of the double-strand breaks examined.
As used herein, the term “incubating” relates to maintaining host cells under controlled conditions favorable for maintenance and/or growth of said host cells, preferably in an incubator. It is, however, also contemplated by the current invention that the host cells are comprised in a tissue or an organism.
The term “host cell” relates to a cell comprising the components required for at least one of the DNA repair systems mediating double strand break repair by homologous recombination ad integrum (HR) or by non-homologous end joining (NHEJ). Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell, even more preferably a human cell, and most preferably the host cell is a K562 cell. Preferably, the host cell is a cell originating from an organism whose genome has been completely sequenced.
The terms “linear polynucleotide comprising a known sequence” or “linear polynucleotide”, preferably, relate to a polynucleotide comprising at least one stretch of nucleotides with a known nucleotide sequence. Preferably, said stretch is at least 18, at least 19, at least 20, at least 25, or at least 50 nucleotides long. Preferably, the nucleotide sequence of the linear polynucleotide is known in its entirety.
It is also contemplated by the current invention that the linear polynucleotide comprises additional sequences. Preferably, said additional sequences code for an integrase-deficient lentivirus, comprising, preferably, an expressible gene for a selectable marker, like, e.g. hygromycin phosphotransferase (Hygromycin B kinase, EC 2.7.1.119) or neomycin-kanamycin phosphotransferase (Kanamycin kinase, EC 2.7.1.95). Preferably, the endonuclease is comprised in said linear polynucleotide in an expressible form.
As used herein, the term “in vivo insertion sites”, preferably, relates to the positions in the genome of a host cell wherein copies of the linear polynucleotide are covalently integrated by means of the cellular HR or the NHEJ systems. It is to be understood that not every DSB induced by an endonuclease and repaired by cellular systems in the presence of a linear polynucleotide will lead to the covalent integration of said linear polynucleotide. However, the term requires that in a given population of host cells used for the determination according to this specification, the number of insertion events is high enough to permit a statistical analysis of the insertion sites. Preferably, at least 50, at least 75, at least 85, at least 90, at least 100, or at least 250 insertion events are analyzed.
The term “in vivo positions of double-strand breaks”, preferably relates to positions in the genome of a host cell where at least one double-strand break occurred.
As used herein, the term “determining the in vivo positions of double-strand breaks” relates to establishing the positions in the genome of a host cell where at least one double-strand break occurred. Preferably, determining the in vivo positions of double-strand breaks comprises the following steps: i) amplifying genomic regions comprising insertion sites. Preferably, amplification is achieved by PCR, more preferably by Linear Amplification Mediated PCR (LAM-PCR, WO/2000/024929), using the information on the known nucleotide sequence comprised in the linear polynucleotide to design specific primers for PCR amplification; ii) Sequencing the amplified polynucleotides obtained in step i); and iii) allocating insertion sites to positions of double-strand breaks.
As used in the current specification, the term “endonuclease” relates to an enzyme hydrolysing phosphodiester bonds within a polynucleotide. Preferably, both strands of DNA are hydrolysed. More preferably, the hydrolysis sites of opposing strands are separated by not more than 100, 50, 25, 20, 15, 10 nucleotides, such that one of the cellular DSB repair systems acts on the ends generated by said hydrolysis. Preferably, the hydrolysis sites are located at a distance of not more than 50, 100, 250, 500, 1000, 2000, 5000, or 10000 nucleotides from the recognition site of the endonuclease. More preferably, the endonuclease is a homing endonuclease or a type I restriction endonuclease. Most preferably, the endonuclease is a Zinc finger endonuclease (ZFN), comprising an endonuclease domain, e.g. the non-specific DNA cleavage domain of the Fokl restriction endonuclease (Kim et al. 1996; Mani et al. 2005), and a DNA binding domain comprising at least one, at least two, or at least three zinc finger domains.
The definitions made above apply mutatis mutandis to the following:
In a further preferred embodiment, the current invention relates to a method for determining the in vivo specificity of an endonuclease, comprising a) incubating a host cell comprising said endonuclease and a linear polynucleotide comprising a known sequence, b) detecting the in vivo insertion sites of said polynucleotide in the genome of said host cell, c) determining the in vivo recognition sites of said endonuclease, and d) assessing the in vivo specificity of said endonuclease.
The term “determining the in vivo specificity” relates to determining the relative number of recognition sites 100% identical with the known recognition site of an endonuclease leading to the insertion of a linear polynucleotide as compared to the total number of insertion sites determined. It is, however, also contemplated by the current specification that for each recognition site identified the similarity to the known recognition site of the endonuclease is determined, so that a consensus sequence can be calculated. A person skilled in the art knows how to obtain an endonuclease with a known recognition site. This is e.g. accomplished by testing the hydrolytic activity of an endonuclease in the presence of various oligo- and/or polynucleotides. Also, e.g. an endonuclease with a known recognition site can be selected, e.g. by phage display. It is, however, also contemplated by the current invention that the known recognition site is e.g. generated by modularly assembling zinc-finger domains of known specificity.
The term “recognition site” relates to sequences in the genome of a host cell bound by the endonuclease of the current specification. Preferably, the binding of a ZFN is mediated by the at least on zinc finger domain interacting with the nucleotides of the recognition site.
As used herein, the term “determining the in vivo recognition sites” relates to establishing the recognition sites bound in vivo by the endonuclease of the current specification. Preferably, determining the in vivo recognition sites comprises the following steps: i) amplifying genomic regions comprising insertion sites. Preferably, amplification is achieved by PCR, more preferably by Linear Amplification Mediated PCR (LAM-PCR, WO/2000/024929 which is herewith incorporated by reference with respect to its entire disclosure content), using the information on the known nucleotide sequence comprised in the linear polynucleotide to design specific primers for PCR amplification; ii) Sequencing the amplified polynucleotides obtained in step i); and iii) allocating insertion sites to recognition sites. Preferably, allocation is achieved by bioinformatic methods, comprising identifying sequences comprising the linear polynucleotide of the current invention, determining genomic sequences adjacent to said linear polynuceotide, aligning said genomic sequences to the genome of the host cell, in silico prediction of potential endonuclease binding sites, and identifying potential binding sites in physical proximity to insertion sites.
In a further preferred embodiment, the current invention relates to a method for obtaining an endonuclease with an altered in vivo specificity, comprising a) providing at least one mutant of an endonuclease with a known recognition sequence, b) determining the in vivo specificity of said mutant of an endonuclease by the method of any one of claims 9 to 13, c) comparing the recognition sites recognized by said at least one mutant endonuclease with the recognition sites recognized by the unmodified endonuclease, and d) obtaining an endonuclease with an altered in vivo specificity.
As used in this specification, the term “altered in vivo specificity”, preferably, relates to an in vivo specificity which is different from the in vivo specificity of an unmodified second endonuclease used for comparison. The alteration is an increase or decrease in the relative number of recognition sites 100% identical with the theoretical recognition site of an endonuclease; e.g. the relative number of recognition sites 100% identical with the theoretical recognition site is increased by at least 5%, 10%, 15%, 25%, or 50%. It is, however, also contemplated that the alteration is a change in the consensus sequence determined by the method of the current invention as described above.
A “mutant of an endonuclease” or “mutant endonuclease” as used herein relates to an endonuclease molecule comprising at least one amino acid exchange and/or at least one insertion and/or at least one deletion of at least one amino acid as compared to the unmodified endonuclease, wherein said mutant endonuclease still has the activity of hydrolyzing DNA. Preferably, said mutant endonuclease is obtained by mutagenizing an expressible gene of an endonuclease with a known recognition site.
As used herein, the term “providing at least one mutant of an endonuclease” relates to making available at least one mutant endonuclease in a host cell. Preferably, said at least one mutant endonuclease is made available by expressing a mutagenized gene for an endonuclease in said host cell.
“Comparing the recognition sites” as used herein relates to comparing the in vivo specificity determined for the mutant endonuclease with the in vivo specificity determined for the unmodified endonuclease. Preferably, the altered in vivo specificity is an increased in vivo specificity, meaning an in vivo specificity wherein an increased relative number of recognition sites is 100% identical with the known recognition site. It is, however, also contemplated by the current specification that an increased in vivo specificity means that the consensus sequence determined by the method of the current invention as described above comprises a lower frequency of alternative nucleotides in at feast one position of the consensus sequence.
In a further preferred embodiment, the current invention relates to a kit for determining the in vivo specificity of an endonuclease, comprising a polynucleotide providing a linear form in a host cell and a manual.
The term “kit” as used herein refers to a collection of the aforementioned compounds, means or reagents of the present invention which may or may not be packaged together. The components of the kit may be comprised by separate vials (i.e. as a kit of separate parts) or provided in a single vial. Moreover, it is to be understood that the kit of the present invention is to be used for practising the methods referred to herein above. It is, preferably, envisaged that all components are provided in a ready-to-use manner for practising the methods referred to above. Further, the kit preferably contains instructions for carrying out the said methods. The instructions can be provided by a user's manual in paper- or electronic form. For example, the manual may comprise instructions for interpreting the results obtained when carrying out the aforementioned methods using the kit of the present invention.
All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.
The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.
To analyze whether IDLV are captured into preexisting DSB similar to what has been described for AAV derived vectors (Miller et al. 2004) and therefore can serve as a stable genetic marker for the temporary DSB, we introduced multiple DSB by gamma-irradiation in K562 cells and determined the frequency of integrated vector forms. K562 cells transduced with a GFP expressing IDLV prior to irradiation (48 hours after transduction) showed a frequency of GFP positive cells of 82.5% on day 5 after transduction, whereas non-irradiated cells revealed 68.7% GFP positive cells. 20 days after IDLV transduction 13.5% of the gamma-irradiated cells were still GFP positive, whereas only 6.2% of the non-irradiated cells showed GFP expression. These levels sustained for the whole observation period (35 days), an observation well in line with an increase in the frequency of IDLV integrations that are not diluted out by cell division (
We analyzed the residual integration pattern of an IDLV carrying the D64V mutation in the viral integrase in transduced K562 cells (Donoru). Lentivirus integration sites (IS) of the IDLV in the cellular genome were studied by LAM-PCR (Schmidt et al. 2007) and nrLAM-PCR (Gabriel et al. 2009). Analysis of more than 100 IS from IDLVn transduced K562 cells (as well as ˜500 IDLV IS obtained from other cell lines showed a close to random integration profile with no obvious preference of insertion into gene coding regions or other genomic structures of the human genome, contrary to what has been described for ICLV (
To examine whether the integration pattern of IDLV changes after ZFN treatment, we analyzed IS in K562 cells infected with IDLV expressing ZFN either targeting Exon 3 of the CCR5 gene locus or Exon 5 of the IL2RG gene locus. These cells were co-infected with a non-integrating donor vector harboring a GFP expression cassette under control of the human PGK promoter, flanked by regions homologous to the respective target site (CCR5wt/hc or IL2RG1/hi, respectively). We had previously shown the targeted integration of the PGK-GFP expression cassette by HR in up to 50% in these cells (Lombardo et al. 2007).
We identified IDLV integrations by unbiased nrLAM and/or LAM-PCR optimized for accessing a large portion of the human genome by using the enzymes HpyCH4V, MseI, Tsp509I or MspI.
Deep sequencing of the amplified IDLV-genome junctions revealed 282 unique lentivirus LTR insertions in CCR5wt/hc samples. These IS were distributed throughout the genome (
Additionally, we analyzed the same ZFP fused to the recently described obligate heterodimeric Fokl nuclease domain (Miller et al. 2007). In cells treated with this advanced ZFN architecture, we detected 85 IDLV integrations in samples coinfected with the CCR5 homologous donor vector (CCR5muF/hc). Of those, 32 IS (37.6%) were located closer than 1.9 kb distance to the ZFN target site in the CCR5 gene, most of them (24 IS) within a 60 bp window surrounding the target site.
Insertion site analysis of cells transduced only with the IDLV donor vector harboring homologous sequences to the CCR5 target site (IDLVhc) in absence of the CCR5 targeting ZFN revealed 66 IS, 4 (6.1%) of them located within the CCR5 gene. These IS were located between 388 bp upstream and 988 bp downstream of the target locus, most likely representing spontaneous homologous recombination events (
In the case of the IL2RG1/hi approach, LAM PCR revealed 111 different genomic IDLV insertion loci, of which 13 (11.7%) mapped to the IL2RG locus, at most 1.1 kb apart from the ZFN target locus. Eight of these 13 IS were located within 16 bp distance to the ZFN target site (
To analyze the proportion of ZFN induced off-target DSB more precisely, we sought to eliminate the possibility of HR competing with NHEJ. We repeated and expanded our analyses in K562 cells coinfected with CCR5- or IL2RG specific ZFN-expressing IDLV and a donor IDLV without any homology regions to either target site. Due to the lack of homology between donor IDLV and target locus, integration of the GFP expression cassette could not be attributed to HR. Therefore IDLV should be captured sequence independent into any DSB through NHEJ. (nr)LAM-PCR analysis of CCR5wt/n, samples showed 95 IS from which 29 (30.5%) mapped to the CCR5 locus. 26 of these IS were located closer than 55 bp apart from the ZFN target site.
Insertion site analysis of the samples treated with CCR5 specific ZFP fused to the mutated obligate heterodimeric Fokl nuclease domain revealed 290 IS in samples coinfected with the nonhomologous donor vector (CCR5muF/n). 71 (24.5%) of these IS were not exceeding 3.1 kb to the ZFN target site, whereas 52 IS were positioned within a 60 bp window surrounding the target site.
For the IL2RG targeting ZFN, we compared two different sets of zinc finger proteins. By (nr)LAM-PCR analysis we detected 208 IS in the IL2RG1/n setting, from which 17 IS (8.2%) mapped to the target locus. From the IL2RG2/n setting we retrieved 248 IS, from which 21 (8.5%) mapped to the target locus. These “on-target” IS were located at most at 585 bp distance to the site were the ZFN induced DSB is expected.
As a reminder, none of the IS obtained from the samples treated with the nonhomologous donor IDLV (IDLVn) alone was located in the vicinity to the CCR5 or IL2RG target site.
Out of the 377 unique IS retrieved from CCR5wt/hc and CCR5wt/n samples, on top of the 88 IS located within the CCR5 gene 80 additional IS were located in 13 different chromosomal regions, each of these loci carrying more than one integrated IDLV (
In the samples transduced with the obligate heterodimeric Fokl ZFN (CCR5muF/hc and CCR5muF/n), 49 of the 375 IS identified by LAM-PCR have been found to be located in 7 other chromosomal loci separate from the CCR5 ZFN target site, each of them harboring at leastmore than 2 closely related IS (
For the IL2RG1/hi and IL2RG1/n samples, we could identify 38 out of 318 IS in 15 genomic loci outside the target region, which itself harbored 30 IS. Each of these loci carried two to four IDLV insertions (Table 3). As integrations into such small regions are very unlikely to occur by chance, genomic loci which carry more than 1 IS in a very close proximity to each other may represent potential off-target hotspots for the respective ZFN.
Out of the 248 IS for the IL2RG2/n setting 32 IS were detected in 15 chromosomal loci, which harbored more than one IDLV integrant in close proximity. Three of these loci (FAM133B, SLC31A1 and SEC16A) harbored integrations in IL2RG1/n and IL2RG2/n samples and SEC16a had also an integration event in the IL2RG1/hi transduced cells (Table 3).
We hypothesized that if ZFN target fidelity was substantial, off-target restriction of the ZFN should most likely affect the sequences of the genome most homologous to the original target motif. An in silico search of the human genome for possible off-target sites of ZFN action on the basis of sequence similarity to the intended target site of the ZFN heterodimer allowed to rank sequences most likely to be subject to cleavage by ZFN. As ZFN dimerization is most effective if the binding sites of the ZFN monomers are separated by 5-6 nucleotides, we searched for off-target binding sites allowing spacing of ZFN monomers between 0-10 nucleotides. In addition to the intended heterodimerization of two different ZEN monomers, we also searched for genomic sites supporting the formation of homodimers from each of the two ZFN monomers. The human genome does indeed contain numerous of these theoretical in silico off-target loci, which show only few mismatches to either target site. However, partial sequence homology is found to be more abundant for the CCR5 target site. Table 1 lists all genomic sites with partial homology to the ZFN target sites with at most two (CCR5) or three (IL2RG) mismatching nucleotides. From this top list with only minor differences to the target site, IDLV insertions have been detected in the ABLIM2 gene (4 IS) and in the CCR2/FLJ78302 gene (41 IS), both previously described known off-target sites of the CCR5 specific ZFN (Perez et al. 2008). These off-site motifs show 96 or 92% sequence similarity to the CCR5 target site, respectively. In case of the IL2RG specific ZFN, 2 IS had been detectable by LAM-PCR in the KIAA0528 gene (88% sequence homology to the target site). To determine the accuracy of our in silico modeling, we compared our data with the real IS identified by LAM PCR. Strikingly, we indeed found 150 IS in 66 loci where an integration event of the IDLV occurred within a 150 bp distance to a partially homologous ZFN target site, with more than 70.8% sequence similarity to the original CCR5 target site (Table 2). In cells treated with the IL2RG specific ZFN, 45 genomic loci with partial sequence homology to the target site carried 63 IDLV integrants in total (Table 3).
To quantify the ZFN activity at the most likely off-site loci, we sequenced 15 genomic loci per ZFN target system by pyrosequencing after exposure to the different ZFN described above. These Loci were chosen based on the presence of a partial homologous target site in the vicinity of a identified IS or for the reason that more than 1 IS has been detected in this locus. After high-throughput sequencing of the amplified loci the obtained sequences have been analysed for signs of NHEJ, namely small insertions or deletions at these loci.
To determine the sequence binding specificity of the different ZFP, we aligned the 11 most probable binding sites of the identified off target Loci. This comparative analysis of off-site sequence homologies allows to rapidly optimize zinc finger motifs (
γ-Irradiation of IDLV Transduced K562 Cells
1×104 K562 cells were seeded into a 12-well plate and transduced with a GFP expressing LV 24 h later with 0.6 μg HIV-1 gag p24. Cells have been γ-irradiated with 2.5 Gy 48 h after transduction. GFP expressing cells were counted by FACS for further 34 days.
Insertion Site Analysis by LAM-PCR.
To identify insertion sites of the IDLV LAM-PCR was performed as previously described using the enzymes Tsp509I, MseI, HpyCH4V and MspI (Schmidt et al. 2007). In brief, genomic DNA from transduced cell samples was preamplified by linear PCR using LTR-specific biotinylated primers. PCR products were captured on solid phase by magnetic beads (Dynabeads). After synthesis of the second strand, restriction digest of ds DNA and ligation of a linker cassette two additional rounds of exponential nested PCRs were performed. The resulting amplicons were sequenced using the Roche/454 platform and sequences obtained were aligned to the human genome via BLAT (BLAST like alignment tool) (Kent 2002)
Pyrosequencing Using the 454 Platform (Roche).
PSR amplicons were prepared as suggested by the manufacturer. An additional PCR (‘Fusionprimer-PCR’) with fusionprimers containing individual barcode sequences of 6 bases was carried out. 40 ng of purified LAM-PCR products served as template for the fusionprimer PCR reaction. PCR conditions: Initial denaturation 2 minutes at 95° C.; followed by 12 cycles at 95° C. for 45 s, 60° C. for 45 s and 72° C. for 60s. Final elongation was 5 minutes at 72° C. 15 μl of the PCR-products were analysed on a 2% agarose gel. DNA concentration was measured with the ND-1000 Spectrophotometer (Thermo Scientific).
IS Data Analysis of IDLV Transduced
LAM-PCR amplicon sequences have been identified through sequence alignment using BI2Seq (Altschul et al. 1990) and the Smith-Waterman algorithm (Smith and Waterman 1981; Gotoh 1982). After trimming, the sequences were aligned to the human genome using the assembly from UCSC (RefSeq genes and RepeatMasker; Alignment March 2006). IS were considered as valid if a LTR-genome junction was present and the flanking genomic region showed a unique sequence match of at least of 95% after alignment to the human genome using BLAT (Kent 2002)
In Silico Prediction of Off-Target Loci
In order to identify sequence parts homologous to the ZFN motives, the human genome was scanned for all possible 3mers contained in the ZFN motives. All matches were extended to full motive length depending on the location of the 3mer within the motive. Between the two ZFN cassettes a possible spacer from 0 to 10 nucleotides was considered. Consent was displayed in capital letters while mismatches with the ZFN motive were reported in lowercase. The information about the homologs was directly linked to the IS for further analyses.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2011/050380 | 1/13/2011 | WO | 00 | 9/7/2012 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2011/086118 | 7/21/2011 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6514706 | Von Kalle et al. | Feb 2003 | B1 |
| 20070134796 | Holmes et al. | Jun 2007 | A1 |
| 20080131962 | Miller | Jun 2008 | A1 |
| 20080159996 | Ando et al. | Jul 2008 | A1 |
| 20090111119 | Doyon et al. | Apr 2009 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 0024929 | May 2000 | WO |
| Entry |
|---|
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| Number | Date | Country | |
|---|---|---|---|
| 20130143204 A1 | Jun 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61295135 | Jan 2010 | US |
