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WO2025122552A1 - Outil de prédiction in silico de sites hors cible de méganucléase - Google Patents

Outil de prédiction in silico de sites hors cible de méganucléase Download PDF

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WO2025122552A1
WO2025122552A1 PCT/US2024/058367 US2024058367W WO2025122552A1 WO 2025122552 A1 WO2025122552 A1 WO 2025122552A1 US 2024058367 W US2024058367 W US 2024058367W WO 2025122552 A1 WO2025122552 A1 WO 2025122552A1
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target
meganuclease
sites
target sites
identified
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Jenny A. Sidrane
Richard Jason LAMONTAGNE
Zhe Zhang
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University of Pennsylvania Penn
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University of Pennsylvania Penn
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
    • G16B35/20—Screening of libraries

Definitions

  • sequence listing file entitled “24-10536PCT_Seq-Listing”, created December 3, 2024, being 11,348 bytes is incorporated by reference herein in its entirety.
  • CRISPR-based genome editing approaches include a guide RNA (gRNA), allowing for prediction of off-target sites based on target site accessibility and the affinity of the gRNA to the target sequence (including both overall nucleotide usage, position-specific nucleotides, and specific mismatch-containing motifs), genome editing by the M2PCSK9 meganuclease is based on a protein-DNA interaction.
  • Substantial protein engineering with a limited ability to model this interaction, is required to change the DNA sequences that this meganuclease recognizes, with some of this previously performed in the optimization of the M1PCSK9 meganuclease which preceded the version utilized in ECUR-506 (Wang et al., 2018). While there are a variety of available in silico prediction tools for CRISPR nucleases, there are no available in silico prediction tools for the M2PCSK9 meganuclease or other meganucleases.
  • the method includes providing a plurality of experimentally identified off-target sites and identifying the best match to the on-target site in each experimentally identified off-target site, and generating a first position weighted matrix (1-PWM) from the best matches.
  • the method further includes identifying the best match to the fPWM in each experimentally identified off-target site, and generating a preliminary position weighted matrix (pPWM) from the best matches.
  • the method further includes performing at least 5 iterations of (b), wherein each iteration comprises finding the best match in each of the experimentally identified off-target sites to the pPWM generated in the iteration prior.
  • the method further includes identifying a final PWM when the pPWM is identical in two or more iterations; and locating the final PWM or a sequence sharing at least 85% identity with the final PWM in the genome, thereby identifying a predicted off-target site in the genome.
  • the nuclease is a meganuclease. In certain embodiments, the meganuclease is the ARCUS meganuclease.
  • FIG. 1 is a venn diagram showing how commonly the predicted motifs from the described in silico prediction tool for the M2PCSK9 meganuclease (ARCUS) occur within the rhesus macaque genome in comparison to the experimentally identified off-target sites from previous studies in rhesus macaques, iPSC-derived human hepatocytes, and human hepatocytes in FRG mice.
  • ARCUS M2PCSK9 meganuclease
  • FIGS. 2A-2C show the results of the experiment described in Example 1. Molecular analysis on the Day 84 liver biopsy samples from each animal were performed to measure transgene copy numbers per diploid genome (FIG. 2A), mRNA expression levels (FIG. 2B), and off-target editing (FIG. 2C).
  • off-target analysis in rhesus macaques is predictive of events that may occur in the human genome through comparison of identified off-target sites within liver samples from a GLP-compliant toxicology study in infant rhesus macaques to studies in human cells (iPSC-derived hepatocytes and chimeric liver-humanized FRG mouse model). This was taken even further through the use of a newly developed in silico prediction tool; out of a total 18,729 unique off-target sites identified in all three studies and by our in silico prediction tool, only two sites identified experimentally were not predicted.
  • M2PCSK9 meganuclease also known as ARCUS.
  • PCSK9 Proprotein convertase subtilisin kexin 9
  • LDLR low-density lipoprotein receptor
  • VLDLR very low-density lipoprotein receptor
  • LRP1/APOER apolipoprotein E receptor
  • LRP8/APOER2 apolipoprotein receptor 2
  • PCSK9 gene has been targeted for treatment of cholesterol related diseases, it has been demonstrated that the PSCK9 gene locus is a safe harbor for gene targeting for insertion of other, non-PCSK9 transgenes.
  • tools are lacking for prediction of off-target effects of the PCSK9-targeting nucleases that target the PCSK9 gene locus.
  • the “target PCSK9 locus” or “PCSK9 gene locus” is any site in the PCSK9 coding region where insertion of a heterologous transgene is desired.
  • the target PCSK9 locus is in Exon 7 of the PCSK9 coding sequence.
  • the nuclease is a meganuclease that targets PCSK9. Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs), for example, I-Scel. When combined with a nuclease, DNA can be cut at a specific location.
  • the restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ.
  • the nuclease is a member of the LAGLID ADG (SEQ ID NO: 3) family of homing endonucleases.
  • the nuclease is a member of the LCrel family of homing endonucleases which recognizes and cuts a 22 base pair recognition sequence. See, e.g., WO 2009/059195.
  • homing endonucleases are capable of comprehensively redesigning I-Crel and other homing endonucleases to target widely-divergent DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes (WO 2007/047859).
  • the term “homing endonuclease” is synonymous with the term “meganuclease.” See, WO 2018/195449, describing certain PCSK9 meganucleases, which is incorporated herein in its entirety.
  • the PCSK9 meganuclease is that described in WO 2022/232232, sometimes referred to herein as M2PCSK9 or ARCUS.
  • the sequence of the ARCUS meganuclease is reproduced in SEQ ID Nos: 6 and 7.
  • the PCSK9 meganuclease is used in conjunction with a heterologous transgene that does not encode PCSK9 to provide a therapeutic composition for treatment of any number of conditions.
  • a heterologous transgene that does not encode PCSK9 to provide a therapeutic composition for treatment of any number of conditions.
  • One such condition is human ornithine transcarbamylase (OTC) deficiency.
  • OTC human ornithine transcarbamylase
  • an in silico method of predicting off-target sites for a nuclease includes generating a consensus sequence using a position weighted matrix, as further described herein.
  • Each identified site is an “experimentally identified off-target site”.
  • the on-target site is specific for each meganuclease, and can be used as the ARCUS on-target site exemplified herein.
  • the best match at each site is the 22-base subsequence having the highest matching score to the on-target site of the M2PCSK9 meganuclease.
  • the sequences of these matches were used to generate a first position weighted matrix (1-PWM) for the M2PCSK9 motif, as performed previously (Wasserman, W. W., & Sandelin, A. (2004). Applied bioinformatics for the identification of regulatory elements. Nature Reviews Genetics, 5(4), 276-287, which is incorporated herein by reference).
  • a “position weighted matrix” or “PWM” refers to a scoring matrix composed of the log likelihood of each nucleotide in a target sequence. Based on an alignment of all known sites, the total number of observations of each nucleotide is recorded for each position, producing a position frequency matrix (PFM).
  • PFM position frequency matrix
  • position weighted matrices are utilized (e.g., first position weighted matrix, preliminary position weighted matrix), prior to arriving at a final PWM that is used to generate a consensus sequence which is used to predict off-target sites in a particular genome.
  • Multiple iterations are performed to refine the first PWM.
  • 100 iterations were completed to refine the first PWM.
  • local sequences at the 1492 off-target sites are searched to find the best match to the PWM, while the best match at each site is the 22-base subsequence having the highest matching score to the PWM (Wasserman, W. W., & Sandelin, A. (2004)).
  • the best matches are used to generate a preliminary PWM (pPWM) for the next iteration.
  • the pPWM stabilized after ⁇ 10 iterations, generating the final PWM.
  • any number of iterations can be performed, where the desired outcome is stabilization of the pPWM, i.e., an identical pPWM in two or more consecutive iterations.
  • the final pPWM is identified as the stabilized pPWM.
  • the matching scores of all sites to the PWM had a bimodal distribution with about one third of all sites having scores higher than 0.8 in one peak.
  • the final version of the PWM was used for all follow-up analyses.
  • the consensus sequence was generated from the pPWM using the nucleotide with the highest score at each position.
  • the consensus sequence was TGCCCTGGGGAAAAAAGGGCCA (SEQ ID NO: 5).
  • on-target site refers to the recognition sequence for which a given nuclease has specificity.
  • the recognition site generally occurs only once in a given genome.
  • the meganuclease is the ARCUS meganuclease that targets a region in the PCSK9 locus having the sequence TCCCCTGGGGCAAAGAGGTCCA (SEQ ID NO: 4).
  • off-target activity is affected by the structure of meganucleases and the methods of delivery.
  • experimentally identified off-target site refers to a sequence of a site in the genome that has been observed to have been cleaved by the meganuclease under experimental conditions, as determined using various techniques, such as ITR-seq.
  • predicted off-target site refers to a sequence of a potential site in the genome that may be cleaved by a nuclease, e.g., a meganuclease.
  • the predicted off-target site has the sequence of the consensus sequence as described herein.
  • the predicted off-target site has a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the consensus sequence.
  • the predicted off-target site has a sequence at least 85% identical to SEQ ID NO: 5.
  • the meganuclease is that described in WO 2022/232232, sometimes referred to herein as M2PCSK9 or ARCUS.
  • the meganuclease targets the human beta-2 microglobulin gene, such as those described in WO2017112859A1.
  • the meganuclease targets the human T cell receptor alpha constant region gene, such as those described in WO2017062439A1.
  • the meganuclease targets the human dystrophin gene, such as those described in WO 2011/141820A1.
  • the meganuclease targets the human C-C chemokine receptor type 5 gene (CCR5), such as those described in WO 2014191525A1.
  • the meganuclease targets recognition sequences present in a mutant Rhodopsin P23H allele, such as those described in US 10,758,595.
  • the nuclease is utilized with a donor vector, that provides the coding sequence for a therapeutic transgene.
  • the donor vector contains an expression cassette comprising a nucleic acid sequence encoding a transgene, and regulatory sequences that direct expression of the transgene in the target cell.
  • the transgene encodes a protein that is aberrantly expressed in a liver metabolic disorder or other genetic disorder.
  • the transgene encodes a protein other than PCSK9.
  • proteins include, but are not limited to OTC, low density lipoprotein receptor (LDLr), Factor IX, and Factor VIII.
  • genes which may be delivered via the donor vector include, without limitation, glucose-6-phosphatase, associated with glycogen storage disease or deficiency type 1A (GSD1), phosphoenolpyruvate-carboxykinase (PEPCK), associated with PEPCK deficiency; cyclin-dependent kinase-like 5 (CDKL5), also known as serine/threonine kinase 9 (STK9) associated with seizures and severe neurodevelopmental impairment; galactose- 1 phosphate uridyl transferase, associated with galactosemia; phenylalanine hydroxylase (PAH), associated with phenylketonuria (PKU); gene products associated with Primary Hyperoxaluria Type 1 including Hydroxyacid Oxidase 1 (GO/HAO1), AGXT, branched chain alpha-ketoacid dehydrogenase, including BCKDH, BCKDH-E2, BAKDH- Ela, and B
  • transgenes for delivery include, e.g., those associated with familial hypercholesterolemia (e.g., VLDLr, LDLr, ApoE, see, e.g., WO 2020/132155, WO 2018/152485, WO 2017/100682, which are incorporated herein by reference), muscular dystrophy, cystic fibrosis, and rare or orphan diseases.
  • familial hypercholesterolemia e.g., VLDLr, LDLr, ApoE
  • WO 2020/132155 e.g., WO 2018/152485, WO 2017/100682, which are incorporated herein by reference
  • muscular dystrophy e.g., cystic fibrosis, and rare or orphan diseases.
  • Examples of such rare disease may include spinal muscular atrophy (SMA), Huntingdon’s Disease, Rett Syndrome (e.g., methyl-CpG-binding protein 2 (MeCP2); UniProtKB - P51608), Amyotrophic Lateral Sclerosis (ALS), Duchenne Type Muscular dystrophy, Friedrichs Ataxia (e.g., frataxin), progranulin (PRGN) (associated with non- Alzheimer’s cerebral degenerations, including, frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA) and semantic dementia), among others.
  • SMA spinal muscular atrophy
  • Huntingdon’s Disease e.g., methyl-CpG-binding protein 2 (MeCP2); UniProtKB - P51608)
  • ALS Amyotrophic Lateral Sclerosis
  • Duchenne Type Muscular dystrophy e.g., frataxin
  • PRGN progranulin
  • FTD frontotemporal dementia
  • PNFA progressive
  • genes include, carbamoyl synthetase I, ornithine transcarbamylase (OTC), arginosuccinate synthetase, arginosuccinate lyase (ASL) for treatment of arginosuccinate lyase deficiency, arginase, fumaryl acetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin, rhesus alpha- fetoprotein (AFP), rhesus chorionic gonadotrophin (CG), glucose-6-phosphatase, plasma protease Cl inhibitor (SERPING1) associated with hereditary angioedema, porphobilinogen deaminase, cystathione beta-synthase associated with homocystinuria, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl
  • Still other useful gene products include enzymes such as may be useful in enzyme replacement therapy, which is useful in a variety of conditions resulting from deficient activity of enzyme.
  • enzymes that contain mannose-6-phosphate may be utilized in therapies for lysosomal storage diseases (e.g., a suitable gene includes that encoding P-glucuronidase (GUSB)).
  • suitable transgene for delivery may include human frataxin delivered in an AAV vector as described, e.g., PCT/US20/66167, December 18, 2020, US Provisional Patent Application No. 62/950,834, filed December 19, 2019, and US Provisional Application No. 63/136,059 filed on January 11, 2021 which are incorporated herein by reference.
  • transgene for delivery may include human acid-a-glucosidase (GAA) delivered in an AAV vector as described, e.g., PCT/US20/30493, April 30, 2020, now published as WO2020/223362A1, PCT7US20/30484, April 20, 2020, now published as WO 2020/223356 Al, US Provisional Patent Application No. 62/840,911, filed April 30, 2019, US Provisional Application No. 62.913,401, filed October 10, 2019, US Provisional Patent Application No. 63/024,941, filed May 14, 2020, and US Provisional Patent Application No. 63/109,677, filed November 4, 2020 which are incorporated herein by reference.
  • GAA human acid-a-glucosidase
  • transgene for delivery may include human a-L-iduronidase (IDUA) delivered in an AAV vector as described, e.g., PCT/US2014/025509, March 13, 2014, now published as WO 2014/151341, and US Provisional Patent Application No. 61/788,724, filed March 15, 2013 which are incorporated herein by reference.
  • IDUA human a-L-iduronidase
  • Other useful therapeutic products include those expressed in muscle, including heart muscle.
  • Other useful therapeutic products encoded by the transgene include hormones and growth and differentiation factors including, without limitation, insulin, glucagon, glucagon- like peptide 1 (GLP-1), growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF- I and IGF-II), any one of the
  • transgenes useful herein include those for treating mucopolysaccharidosis type I-VII (IDUA, IDS, GNA, HGSNAT, NAGLU, SGSH, GALNS, GLB1, ARSB, GUSB).
  • IDUA mucopolysaccharidosis type I-VII
  • IDS mucopolysaccharidosis type I-VII
  • GNA mucopolysaccharidosis type I-VII
  • HGSNAT HGSNAT
  • NAGLU NAGLU
  • SGSH GALNS
  • GLB1 ARSB
  • GUSB GUSB
  • Exemplary sequences for useful for treating MPSI can be found in WO 2019/010335, which is incorporated herein by reference.
  • Exemplary sequences for useful for treating MPSII can be found in WO 2019/060662, which is incorporated herein by reference.
  • Exemplary sequences for useful for treating MPSIIIa can be found in WO
  • a can mean one or more than one.
  • a cell can mean a single cell or a multiplicity of cells.
  • the term “specificity” means the ability of a nuclease to recognize and cleave double-stranded DNA molecules only at a particular sequence of base pairs referred to as the recognition sequence, or only at a particular set of recognition sequences.
  • the set of recognition sequences will share certain conserved positions or sequence motifs, but may be degenerate at one or more positions.
  • a highly-specific nuclease is capable of cleaving only one or a very few recognition sequences. Specificity can be determined by any method known in the art.
  • exogenous nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the position in which it exists in a chromosome, or host cell.
  • An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same expression cassette or host cell, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements.
  • heterologous when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid.
  • the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene.
  • the term “host cell” may refer to the packaging cell line in which a vector (e.g., a recombinant AAV) is produced from a production plasmid.
  • the term “host cell” may refer to any target cell in which expression of the transgene is desired.
  • a “host cell,” refers to a prokaryotic or eukaryotic cell that contains a exogenous or heterologous nucleic acid sequence that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • the term “host cell” refers to cultures of cells of various mammalian species for in vitro assessment of the compositions described herein.
  • the term “host cell” refers to the cells employed to generate and package the viral vector or recombinant virus. Still in other embodiment, the term “host cell” is intended to reference the target cells of the subject being treated in vivo for the diseases or conditions as described herein. In certain embodiments, the term “host cell” is a liver cell or hepatocyte.
  • a “subject” is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or gorilla.
  • a patient refers to a human.
  • a veterinary subject refers to a non-human mammal. In certain embodiments, the subject does not have a defect in their PCSK9 gene.
  • a “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
  • the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be “gutless” - containing only the gene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication.
  • sequence identity “percent sequence identity” or “percent identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g. of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
  • percent sequence identity may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof.
  • a fragment is at least about 8 amino acids in length and may be up to about 700 amino acids. Examples of suitable fragments are described herein.
  • the term “substantial homology” or “substantial similarity,” when referring to amino acids or fragments thereof, indicates that, when optimally aligned with appropriate amino acid insertions or deletions with another amino acid (or its complementary strand), there is amino acid sequence identity in at least about 95 to 99% of the aligned sequences.
  • the homology is over full-length sequence, or a protein thereof, e.g., a cap protein, a rep protein, or a fragment thereof which is at least 8 amino acids, or more desirably, at least 15 amino acids in length. Examples of suitable fragments are described herein.
  • highly conserved is meant at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity. Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art.
  • aligned sequences or alignments refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.
  • AAV alignments are performed using the published AAV9 sequences as a reference point. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs.
  • Such programs include, “Clustal Omega”, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using FastaTM, a program in GCG Version 6.1. FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.
  • percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • Multiple sequence alignment programs are also available for amino acid sequences, e.g., the “Clustal Omega”, “Clustal X”, “MAP”,
  • the term “about” refers to a variant of ⁇ 10% from the reference integer and values therebetween.
  • “about” 40 base pairs includes ⁇ 4 (i.e., 36 - 44, which includes the integers 36, 37, 38, 39, 40, 41, 42, 43, 44).
  • ⁇ 4 i.e., 36 - 44, which includes the integers 36, 37, 38, 39, 40, 41, 42, 43, 44.
  • the term “about” is inclusive of all values within the range including both the integer and fractions.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder associated with high mortality.
  • OTC deficiency adeno-associated virus (AAV) neonatal gene therapy would only provide shortterm therapeutic effects as the non-integrated genome gets lost during hepatocyte proliferation.
  • Nuclease-mediated, site-specific integration of an OTC mini gene cassette in a safe harbor in the genome would provide long-term therapeutic benefits to patients with OTC deficiency.
  • rhesus macaques were used in non-GLP-compliant POC pharmacology studies.
  • the M2PCSK9 meganuclease targets a 22- bp sequence present in the human and rhesus macaque PCSK9 gene.
  • rhesus macaques can be used to evaluate on-target editing (pharmacology) and safety/toxicology.
  • newborn and infant rhesus macaques have similar anatomical and physiological features as human infants and will allow for the use of the intended clinical ROA (IV).
  • PCSK9 levels were followed in all newborn animals including the donor-only control animals over time.
  • PCSK9 levels on Day 0 varied between the newborns.
  • Nine animals showed a trend of reduced PCSK9 levels post vector administration including one donor-only control animal, while the while the remaining five animals showed persistent or transient elevation of PCSK9 levels post dosing.
  • a liver biopsy via laparotomy was performed. Transduction efficiencies of hOTC in liver were evaluated by dual ISH with hOTC- and M2PCSK9-specific probes to detect transgene mRNA, and by OTC immunofluorescence to detect human OTC protein, followed by quantification on scanned slides.
  • the three animals 21-111, 21-113, and 21- 122) with pre-existing anti-AAVrh79 binding antibodies at the time of dosing did show any OTC-positive hepatocytes by both methods.
  • the two donor-only control animals showed low level ( ⁇ 1%) of hOTC transduction.
  • liver biopsy samples from each animal were performed to measure transgene copy numbers per diploid genome, mRNA expression levels, on-target editing, and off-target editing (FIG. 2A-2C). Consistent with the transduction efficiency analyses, the two animals (21-157 and 21-175) in Group 6 had the highest hOTC vector GC (FIG. 2A), hOTC mRNA (FIG. 2B), and on-target indel% (FIG. 2C).
  • the M2PCSK9 vector GC in animals were 2-fold to 7-fold lower than the hOTC vector GC, while M2PCSK9 mRNA levels were 23-fold and 765-fold lower than the hOTC mRNA levels (FIG. 2A and 2B).
  • Off-target activity evaluated by ITR-seq identified 2 to 40 potential off-targets in the Day 84 liver biopsy samples in this study. Off-target editing will be further characterized by amplicon-seq on the potential off-target sites.
  • CRISPR-based genome editing approaches include a guide RNA (gRNA), allowing for prediction of off-target sites based on target site accessibility and the affinity of the gRNA to the target sequence (including both overall nucleotide usage, position-specific nucleotides, and specific mismatch-containing motifs), genome editing by the M2PCSK9 meganuclease is based on a protein-DNA interaction.
  • gRNA guide RNA
  • Substantial protein engineering with a limited ability to model this interaction, is required to change the DNA sequences that this meganuclease recognizes, with some of this previously performed in the optimization of the M1PCSK9 meganuclease which preceded the version utilized in ECUR-506 (Wang et al., 2018).
  • the matching scores of all sites to the PWM had a bimodal distribution with about one third of all sites having scores higher than 0.8 in one peak.
  • the final version of the PWM was used for all follow-up analyses.
  • This novel in silico prediction tool was then used to determine whether there were overlapping sites between different data sets, including those identified in the liver samples obtained in the GLP-compliant toxicology study in infant NHPs administered with ECUR- 506 (Non-clinical Study 2020-007) and those identified in the human genome in human hepatocytes derived from iPSCs and a chimeric liver-humanized mouse model. While the prediction of off-target sites in the human genome could be validated based on a number of overlapping off-target sites identified, the inherent differences between proteimDNA and gRNA:DNA interactions mean additional caveats to in silico prediction for meganucleases need to be considered when interpreting these data compared to similar predictive methods for CRISPR nucleases.
  • off-target sites identified across the GLP- compliant toxicology study in rhesus macaques with ECUR-506 and those identified with the M2PCSK9 meganuclease in human cells (iPSC-derived human hepatocytes and FRG mice), we also added in off-target sites predicted to occur based on our newly developed inhouse prediction tool.
  • CRISPR-based genome editing approaches include a gRNA allowing for prediction of off-target sites based on target site accessibility due to genomic DNA structure and epigenetic modifications, and the affinity of the gRNA to the target sequence (including both overall nucleotide usage, position-specific nucleotides, and motifs with some nucleases allowing for up to 3 bp mismatches), genome editing by the M2PCSK9 meganuclease is based on a protein-DNA interaction.
  • off-target sites looked for motifs of 22 bp or less that commonly occur in identified off-targets (the M2PCSK9 meganuclease has a 22-nucleotide target site within the PCSK9 gene).
  • the resulting 17,165 predicted off-target sites were then compared to identified potential M2PCSK9 meganuclease off-target sites compiled from the totality of unique off-target sites identified across three different studies using different methods, including sites identified in the rhesus genome in previous studies with the M2PCSK9 meganuclease, identified off-target sites in the human genome in iPSC-derived hepatocytes, and sites identified in the chimeric liver-humanized FRG mouse model, as further described below:
  • identified off-target sites in macaque liver samples are located predominantly in the intragenic regions of the genome and, therefore, would not affect expression of endogenous genes.
  • the majority of potential off-target sites identified by GUIDE-seq on M2PCSK9-transduced human iPSC cells or by ITR-seq on the humanized FRG mice liver samples also reside in intronic, non-coding regions of the human genome.
  • the data presented here demonstrate sustained and therapeutically beneficial editing in non-human primate liver with an engineered meganuclease and provide a data set on in vivo genome editing efficacy, immune toxicity, and safety.
  • potential for off-target editing has been thoroughly assessed in NHPs, which provide a relevant model for translation to events that may occur in patients. While we cannot predict whether patient-specific single nucleotide polymorphisms throughout the genome could increase off-target editing at a particular site, we do not see areas of specific concern.

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Abstract

L'invention concerne un procédé in silico pour fournir <i /> un site hors cible prédit d'une nucléase ayant un site sur cible dans un génome. Le procédé comprend la génération d'une séquence consensus, telle que décrite ici. Dans certains modes de réalisation, la nucléase est une méganucléase qui cible PCSK9. Dans certains modes de réalisation, la nucléase est ARCUS.
PCT/US2024/058367 2023-12-04 2024-12-04 Outil de prédiction in silico de sites hors cible de méganucléase Pending WO2025122552A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040031072A1 (en) * 1999-05-06 2004-02-12 La Rosa Thomas J. Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement
WO2017017673A2 (fr) * 2015-07-28 2017-02-02 Yeda Research And Development Co. Ltd. Protéines stables et procédés pour leur conception
US20190295689A1 (en) * 2014-01-27 2019-09-26 Georgia Tech Research Corporation Methods and systems for identifying crispr/cas off-target sites
US11680254B2 (en) * 2017-04-21 2023-06-20 Precision Biosciences, Inc. Engineered meganucleases specific for recognition sequences in the PCSK9 gene

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040031072A1 (en) * 1999-05-06 2004-02-12 La Rosa Thomas J. Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement
US20190295689A1 (en) * 2014-01-27 2019-09-26 Georgia Tech Research Corporation Methods and systems for identifying crispr/cas off-target sites
WO2017017673A2 (fr) * 2015-07-28 2017-02-02 Yeda Research And Development Co. Ltd. Protéines stables et procédés pour leur conception
US11680254B2 (en) * 2017-04-21 2023-06-20 Precision Biosciences, Inc. Engineered meganucleases specific for recognition sequences in the PCSK9 gene

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