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WO2025186392A1 - Édition de génome abca4 thérapeutique pour le traitement d'une maladie stargardt - Google Patents

Édition de génome abca4 thérapeutique pour le traitement d'une maladie stargardt

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Publication number
WO2025186392A1
WO2025186392A1 PCT/EP2025/056149 EP2025056149W WO2025186392A1 WO 2025186392 A1 WO2025186392 A1 WO 2025186392A1 EP 2025056149 W EP2025056149 W EP 2025056149W WO 2025186392 A1 WO2025186392 A1 WO 2025186392A1
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WIPO (PCT)
Prior art keywords
polynucleotide
seq
guide
polynucleotides
abca4
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Pending
Application number
PCT/EP2025/056149
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English (en)
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WO2025186392A8 (fr
Inventor
Alejandro GARANTO IGLESIAS
Robert Wilhelmus Johanna COLLIN
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Stichting Radboud Universitair Medisch Centrum
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Stichting Radboud Universitair Medisch Centrum
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Publication of WO2025186392A1 publication Critical patent/WO2025186392A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the invention relates to the fields of medicine and immunology. In particular, it relates to gene editing systems for the ABCA4 gene.
  • ABCA4 Autosomal recessive mutations in ABCA4 cause Stargardt disease, a progressive disorder characterized by central vision loss and often leading to complete blindness.
  • a typical hallmark of Stargardt disease is the presence of many yellow spots (flecks) distributed throughout the fundus of the patients.
  • the ABCA4 gene is comprised of 50 exons and encodes a protein consisting of 2273 amino acids. This protein is expressed in the outer segments of cone and rod photoreceptor cells and plays an important role in the removal of waste products following phototransduction.
  • variants in ABCA4 can also lead to other subtypes of retinal disease ranging from bull’s eye maculopathy to autosomal recessive cone-rod dystrophy (arCRD; Cremers et al, 1998; Maugeri et al, 2000) and pan-retinal dystrophies (Cremers et al, 1998; Martinez- Mir et al, 1998), depending on the severity of the alleles.
  • arCRD autosomal recessive cone-rod dystrophy
  • pan-retinal dystrophies Cremers et al, 1998; Martinez- Mir et al, 1998), depending on the severity of the alleles.
  • Biallelic ABCA4 variants can be identified in approximately 80% of the cases with STGD1 (Allikmets et al, 1997; Fujinami et al, 2013; Lewis et al, 1999; Maugeri et al, 1999; Rivera et al, 2000; Schulz et al, 2017; Webster et al, 2001 ; Zernant et al, 2011 ; Zernant et al, 2017), and 30% of cases with arCRD (Maugeri et al, 2000), after sequencing coding regions and flanking splice sites.
  • DI deep- intronic
  • the PE alters the mRNA open-reading frame often by introducing a premature stop codon which result in either the degradation of the mRNA or in the synthesis of a truncated non-functional protein.
  • the 49 introns that constitutes ABCA4 there are some which present a higher proportion of causative variants.
  • Such a “hot-spot”’ cluster can for example be found in introns 30 and 36.
  • the invention provides for a composition
  • a gene editing system for the gene editing of an ABCA4 gene comprising a polynucleotide guided gene editing enzyme and at least one of: i) a first set of guide-polynucleotides comprising
  • the guide polynucleotides are encoded by a polynucleotide that is transcribed to provide for the actual guide-polynucleotide.
  • the guide polynucleotides from the first and/or second groups are guide RNAs.
  • the polynucleotide guided gene editing enzyme is encoded by a polynucleotide and/or guide polynucleotides from the first and/or second groups are encoded by or present on a polynucleotide comprised in a vector.
  • the polynucleotide guided gene editing enzyme protein is encoded by a polynucleotide and the guide polynucleotides from the first and/or second groups are each encoded by or each present on another polynucleotide and the polynucleotides are comprised in one vector.
  • the gene editing system is a ribonucleo-protein complex, preferably wherein the guide polynucleotides as defined on claim 1 are encoded by or present on a polynucleotide and the polynucleotide guided gene editing enzyme is present as a protein.
  • the polynucleotide guided gene editing enzyme is a Cas-like or a Cas protein, preferably the Cas protein is a Cas9 protein. In one embodiment, the polynucleotide guided gene editing enzyme comprises a nuclear targeting moiety and/or a cell penetrating peptide (CPP).
  • CPP cell penetrating peptide
  • a first set of guide-polynucleotides comprises or consists of SEQ ID NO: 4 and SEQ ID NO: 10 or SEQ ID NO: 1 and SEQ ID NO: 7;
  • a second set of guide-polynucleotides comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 18 or SEQ ID NO: 13 and SEQ ID NO: 16.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is for ocular administration such as intravitreal administration or subretinal administration.
  • the invention provides for the composition as described herein, for use as a medicament, preferably for use as a medicament for treating an ABCA4 related disease or a condition requiring gene editing of an ABCA4.
  • the invention provides for a use of the composition as described herein for treating an ABCA4-related diseases or a condition requiring gene editing of an ABCA4 gene.
  • the invention provides for a method for gene editing of an ABCA4 gene in a cell, the method comprising contacting the cell with a composition as described herein.
  • the ABCA4-related disease or condition is Stargardt disease.
  • the word "about” or “approximately” when used in association with a numerical value preferably means that the value may be the given value (of 10) more or less 5% of the value.
  • sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.
  • the skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.
  • polynucleotide refers in the context of all embodiments of the present invention to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or mixes or analogs thereof.
  • Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, oligonucleotides and primers.
  • a polynucleotide may comprise one or more modified nucleotides, such as a methylated nucleotide and a nucleotide analogue or nucleotide equivalent wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.
  • Preferred nucleotide analogues and equivalents are described in the section “General definitions”.
  • modifications to the nucleotide structure may be introduced before or after assembly of the polynucleotide.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling compound.
  • a “polynucleotide” can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • a polynucleotide can contain the nucleotide sequence of the full-length cDNA sequence, including the untranslated 5' and 3' sequences, the coding sequences, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • the polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • the polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • Polynucleotides can contain ribonucleosides (adenosine, guanosine, uridine, or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters.
  • Polynucleotides can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine.
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences can be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • ABCA4 Mutations in ABCA4 are amongst the major causes of inherited retinal disease, and in particular lead to a phenotype called Stargardt disease (STGDI), a progressive disorder that affects -1:10,000 individuals worldwide.
  • STGDI Stargardt disease
  • the inventors have identified that a significant amount of ABCA4 mutations affect pre-mRNA splicing of ABCA4. Whilst most of the ABCA4 mutations are spread all over the ABCA4 gene, there are some mutations that tend to cluster, including a series of variants in intron 30 and intron 36 of this gene. From this cluster of variants, several of them are relatively frequent in ABCA4-related diseases.
  • the inventors have developed a genome editing system to remove a substantial part of intron 30 and/or intron 36, without disrupting the gene product. This provides one single approach that can serve for all the variants located in the same intron (as a large part of the intron, harboring pathogenic variants will be removed). This system has been tested in several type of cells, including, patient-derived fibroblasts, as well as iPSC-derived cell models, such as photoreceptor precursor cells.
  • the invention provides for a composition comprising a gene editing system for the gene editing of an ABCA4 gene comprising a polynucleotide guided gene editing enzyme and i) a first set of guide-polynucleotides comprising
  • a guide-polynucleotide according to the present invention comprises at least a guidesequence that is able to hybridize with the target-polynucleotide and is able to direct sequencespecific binding of the gene editing system to the target-polynucleotide to enable gene editing.
  • the guide-polynucleotide is a polynucleotide according to the general definition of a polynucleotide set out here above; a preferred guide-polynucleotide comprises ribonucleotides.
  • the guide polynucleotides from the first and/or second groups are guide RNAs (gRNAs).
  • the gene editing enzyme is encoded by a polynucleotide and/or the guide-polynucleotide is encoded by or present on a polynucleotide.
  • the guide- polynucleotide is encoded by a polynucleotide that is transcribed to provide for the actual guide- polynucleotide.
  • the guide polynucleotide is present in the form of a polynucleotide encoding for said guide-polynucleotide and the guide-polynucleotide is obtained upon transcription of said polynucleotide in a host cell.
  • the polynucleotide guided gene editing enzyme is encoded by a polynucleotide and/or guide polynucleotides from the first and/or second groups are encoded by or present on a polynucleotide comprised in a vector.
  • the polynucleotide guided gene editing enzyme is encoded by a polynucleotide and/or guide polynucleotides from the first and/or second groups are encoded by or present on a polynucleotide comprised in one vector.
  • the whole system is called a “ribonucleoprotein.”
  • the guide-polynucleotides and the polynucleotide guided gene editing enzyme are provided as a as pre-assembled ribonucleoprotein complex.
  • Ribonucleoproteins provided herein can also comprise additional nucleic acids or proteins.
  • the guide polynucleotides from the first and/or second groups are encoded by or present on a polynucleotide and the polynucleotide guided gene editing enzyme is present as a protein.
  • the polynucleotide guided gene editing enzyme is a Cas-like or a Cas protein.
  • a Cas protein or Cas-like protein in the context of all embodiments of the present invention refers to any Cas protein suitable for the purpose of the invention.
  • a Cas protein may comprise enzymatic activity or may not comprise enzymatic activity.
  • Non-limiting examples of Cas proteins include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Casi o, Cas 12a, Csy1 , Csy2, Csy3, Cse1 , Cse2, Csc1 , Csc2 (also known as C2c2), Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx1 S, Csf1 , Csf2, Csf3, Csf4, homologs thereof or modified versions thereof.
  • Cas proteins are known to the person skilled in the art; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.
  • an unmodified Cas protein according to the present invention has DNA cleavage activity, such as e.g. Cas9.
  • Cas protein is a Cas9 protein.
  • Cas protein according is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae.
  • a Cas protein according to the present invention directs cleavage of one or both polynucleotide strands at the location of the target-polynucleotide, such as within the target-polynucleotide and/or within the reverse complement of the target-polynucleotide.
  • At the location of the target-polynucleotide is herein defined as within about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more nucleotides from the first or last nucleotide of a target-polynucleotide; more preferably, within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more nucleotides from the first or last nucleotide of a target-polynucleotide; even more preferably, within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50 nucleotides from the first or last nucleotide of a target-polynucleotide.
  • a Cas protein according to the present invention preferably directs cleavage of one or both polynucleotide strands within about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more nucleotides from the first or last nucleotide of a target-polynucleotide; more preferably, within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more nucleotides from the first or last nucleotide of a target- polynucleotide; even more preferably, within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50 nucleotides from the first or last nucleotide of a target-polynucleotide.
  • the gene editing system as comprised in the composition is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR-associated (Cas) (CRISPR-CRISPR-CRISPR
  • CRISPR system CRISPR-Cas system
  • CRISPR enzyme system CRISPR enzyme system
  • the present invention conveniently provides for a multiplex CRISPR-Cas system.
  • Such multiplex CRISPR-Cas system can conveniently be used for introduction of a donor polynucleotide, deletion of a polynucleotide and polynucleotide library insertion into the genome of a host cell.
  • a multiplex CRISPR-Cas system may refer to the use of one of more Cas proteins, one of more guide- polynucleotides and/or one or more donor polynucleotides.
  • CRISPR-Cas complex refers in the context of all embodiments of the present invention to a complex comprising a guide-polynucleotide hybridized to a target-polynucleotide and complexed with a Cas protein.
  • a non-mutated Cas protein such as but not limited to the Cas9 protein of Streptococcus pyogenes
  • the formation of the CRISPR-Cas complex results in cleavage of one or both polynucleotide strands in or near (e.g. within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target-polynucleotide.
  • a target-polynucleotide according to the present invention (defined below herein) is associated with a PAM sequence (defined below herein) and the PAM sequence is preferably immediately downstream (3') of the target-polynucleotide; the formation of the CRISPR-Cas complex typically results in cleavage of one or both polynucleotide strands 3 base pairs upstream (5') of the PAM sequence.
  • a target-polynucleotide according to the present invention is preferably associated with a protospacer adjacent motif (PAM), which is a short polynucleotide recognized by the CRISPR-Cas complex.
  • PAM protospacer adjacent motif
  • the target-polynucleotide and PAM are linked wherein the PAM is preferably immediately downstream (3') of the target-polynucleotide.
  • the exact sequence and length of the PAM may vary, e.g. different Cas proteins and nucleases of similar function may require different PAM sequences.
  • a preferred PAM according to the present invention is a polynucleotide of 2 to 8 nucleotides in length.
  • a preferred PAM is selected from the group consisting of 5'-XGG-3', 5'- XGGXG-3', 5'-XXAGAAW-3', 5'-XXXXGATT-3', 5'-XXAGAA-3', 5'-XAAAAC-3', wherein X can be any nucleotide or analog thereof, preferably any nucleotide; and W is A or T.
  • a more preferred PAM is 5'-XGG-3' such as preferably 5'-AGG-3'.
  • the PAM is preferably matched with the Cas protein.
  • the most widely used CAS/CRISPR system is derived from S.
  • a preferred PAM for a Neisseria meningitidis Cas protein is 5'-XXXXGATT-3'; a preferred PAM for a Streptococcus thermophilus Cas protein is 5'-XXAGAA-3'; a preferred PAM for a Treponema denticola is 5'-XAAAAC-3'.
  • a preferred PAM matches the Cas protein used.
  • a Cas protein according to the present invention may be engineered to match a different PAM than the native PAM matching the wild-type Cas protein. As such, the CRISPR-Cas system according to the present invention may be used for customized specific targeting.
  • the PAM site is recognized by the CRISPR-system nuclease.
  • the guide-polynucleotide preferably also comprises a sequence that has a specific secondary structure and allows binding of the Cas protein to the guide-polynucleotide.
  • sequence is known in the art as tracr RNA, tracr sequence, tracr scaffold or guide-polynucleotide structural component, these terms are used interchangeably herein; wherein the tracr is the abbreviation for transactivating CRISPR; tracrRNA thus means transactivating CRISPR RNA.
  • the tracrRNA in the original CRISPR-Cas system is the endogenous bacterial RNA that links the crRNA (guide-sequence) to the Cas nuclease, being able to bind any crRNA.
  • a guide-polynucleotide structural component may be comprised of a single polynucleotide molecule or may be comprised of two or more molecules hybridized to each other; or two or more molecules which associate with Cas protein or other nucleases of similar function. Such components of a guide-polynucleotide structure may be referred to as a tracr sequence and a tracr-mate sequence.
  • the guide-polynucleotide preferably also comprises a tracr sequence and/or a tracr-mate sequence.
  • the polynucleotide guided gene editing enzyme comprises a nuclear targeting moiety and/or a cell penetrating peptide (CPP).
  • Means of introducing the gene editing enzyme into cells can be done by using nucleartargeting moiety and/or cell-penetrating peptides (CPP).
  • CCPP cell-penetrating peptides
  • a cell-penetrating peptide is linked to the CRISPR enzymes.
  • the CRISPR enzymes and/or RNA guides are coupled to one or more CPPs to transport them inside cells effectively. Examples of CPP are described in WO2016073433, incorporated by reference herein.
  • the first set of guide-polynucleotides comprises or consists of SEQ ID NO: 4 and SEQ ID NO: 10 or SEQ ID NO: 1 and SEQ ID NO: 7;
  • the second set of guide-polynucleotides comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 18 or SEQ ID NO: 13 and SEQ ID NO: 18.
  • the composition of the invention further comprises a pharmaceutically acceptable excipient.
  • a pharmaceutical composition may comprise any pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant, solubilizer and/or diluent.
  • Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer and/or diluent may for instance be found in Remington, 2000.
  • a preferred route of administration is through ocular administration such as intravitreal or subretinal administration.
  • the administration is through injection of an aqueous solution or specially adapted formulation for intraocular administration.
  • EP2425 814 discloses an oil in water emulsion especially adapted for intraocular (intravitreal) administration of peptide or nucleic acid drugs. This emulsion is less dense than the vitreous fluid, so that the emulsion floats on top of the vitreous, avoiding that the injected drug impairs vision
  • the gene editing systems described herein, or components thereof, nucleic acid molecules thereof, or nucleic acid molecules encoding or providing components thereof, can be delivered by various delivery systems such as vectors, e.g., plasmids, viral delivery vectors, such as adeno- associated viruses (AAV), lentiviruses, adenoviruses, and other viral vectors, or combinations thereof.
  • vectors e.g., plasmids
  • viral delivery vectors such as adeno- associated viruses (AAV), lentiviruses, adenoviruses, and other viral vectors, or combinations thereof.
  • AAV adeno- associated viruses
  • the invention provides for a viral vector expressing
  • a polynucleotide guided gene editing enzyme preferably a Cas-like or a Cas protein for the gene editing of an ABCA4 gene and at least one of: i) a first set of guide-polynucleotides comprising
  • the guide polynucleotide comprises at least one of SEQ ID NO: 1 or SEQ ID NO: 4,
  • the guide polynucleotide comprises at least one of SEQ ID NO 7 or SEQ ID NO 10; and ii) a second set of guide-polynucleotides comprising
  • the guide polynucleotide comprises SEQ ID NO: 13 and
  • the guide polynucleotide comprises at least one of SEQ ID NO: 16 or SEQ ID NO: 18.
  • the viral vector recombinant adeno-associated virus (rAAV) vector may be used for delivery.
  • AAV vectors can be based on one or more of several capsid types, including AAV1 , AV2, AAV5, AAV6, AAV8, AAV8.2.
  • Exemplary AAV vectors and techniques that may be used to produce rAAV particles are known in the art (see, e.g., Aponte-Ubillus et al. (2016) Appl. Microbiol. Biotechnol. 102(3): 1045-54; Zhong et al. (2012) J. Genet. Syndr. Gene Ther. S1 : 008; West et al. (1987) Virology 160: 38-47 (1987); Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-60); U.S. Pat. Nos. 4,797,368 and 5,173,414; and International Publication Nos. WO 2015/054653 and WO 93/24641 , each of which is incorporated by reference).
  • Nonviral delivery vectors encompassing lipid, polymer, peptide, and inorganic nanoparticle-based delivery systems, and nanoclews have firmly established nonviral delivery methodologies as a feasible substitute for viral vectors.
  • peptides and peptide-like materials combine the capability to extensively engineer properties facilitates the delivery of RNP (ribonucleoprotein) entities, irrespective of their size and molecular weight.
  • Peptide- mediated Cas9-RNP delivery can be accomplished through either covalent conjugation to the Cas9 protein or noncovalent ionic interactions with the negatively charged Cas9 RNP.
  • the gene editing systems described herein, or components thereof, nucleic acid molecules thereof, or nucleic acid molecules encoding or providing components thereof can be carried or delivered by non-viral delivery or carriers such as nonviral delivery vectors, encompassing lipid, polymer, peptide, and inorganic nanoparticle-based delivery systems, and nanoclews.
  • the RNP (which in one embodiment of the invention comprises Cas9 and at least one of the gRNAs as described herein) is conjugated with a peptide to form a nanocomplex.
  • the peptide is an amphipathic peptide.
  • the peptide is an amphipathic peptide that is conjugated to a fatty acid moiety.
  • the fatty acid moiety is a monounsaturated such as for example oleic acid.
  • the composition, nanocomplex or vector according to the invention is for use as a medicament.
  • the medicament is for use in treating, the preventing, or delaying an ABCA4 related disease or a condition requiring gene editing of an ABCA4.
  • treatment is understood to include the prevention and/or delay of the ABCA4-related disease or condition.
  • An individual which may be treated using the composition or vector according to the invention may already have been diagnosed as having an ABCA4-related disease or condition.
  • an individual which may be treated using the composition or vector according to the invention according to the invention may not have yet been diagnosed as having a ABCA4- related disease or condition but may be an individual having an increased risk of developing a ABCA4-related disease or condition in the future given his or her genetic background.
  • a preferred individual is a human being.
  • the ABCA4-related disease or condition preferably is Stargardt disease.
  • the invention further provides for the use the composition for the gene editing of an ABCA gene according to the invention, the nanocomplex according to the invention a vector according to the invention or a composition according to the invention for treating an ABCA4-related disease or condition requiring gene editing of ABCA4.
  • Each feature of all medical use embodiment herein has earlier been defined herein and is preferably such feature as earlier defined herein.
  • the invention further provides for, a method of treatment of an ABCA4-related disease or condition requiring gene editing of ABCA4, comprising said method comprising contacting a cell of said individual with an composition, nanocomplex or vector according to the invention.
  • a method of treatment of an ABCA4-related disease or condition requiring gene editing of ABCA4 comprising said method comprising contacting a cell of said individual with an composition, nanocomplex or vector according to the invention.
  • the invention further provides for a method for a method for gene editing of an ABCA4 gene in a cell, said method comprising contacting the cell, preferably a retina cell, with an composition according to the invention, the nanocomplex according to the invention or the vector according to the invention.
  • the features of this aspect are preferably those defined earlier herein.
  • Contacting the cell with the composition or vector according to the invention may be performed by any method known by the person skilled in the art. Use of the methods for delivery of the several aspects of the invention as described earlier herein is included. Contacting may be directly or indirectly and may be in vivo, ex vivo or in vitro.
  • gRNA30-1 (SEQ ID NO: 10); gRNA30-2 (SEQ ID NO: 4); gRNA30-3 (SEQ ID NO: 1); gRNA30-4 (SEQ ID NO: 7); gRNA30-5 (SEQ ID NO: 8); gRNA30-6 (SEQ ID NO: 2); gRNA36-1 (SEQ ID NO: 13); gRNA36-2 (SEQ ID NO: 18); gRNA36-3 (SEQ ID NO: 16); gRNA36-4 (SEQ ID NO: 17); gRNA36-5 (SEQ ID NO: 14).
  • Figure 2 Analysis of the PIR efficacy of the RPNC-mediated editing in fibroblast at DNA level either in intron 30 (A) or intron 36 (B).
  • A) On top, representative electrophoresis gel of the amplification of the intron 30 by PCR in both control and patient fibroblast cell lines.
  • Edited band 1 (E1) indicates the expected edited band after treating with gRNAs 30-1 (SEQ ID NO:4) and 30-2 (SEQ ID NO:10) while the edited band 2 (E2) represents the expected band after the editing with gRNAs 30-3 (SEQ ID NO:1) and 30-4 (SEQ ID NO:7).
  • the 5'UTR region of RPE65 was amplified as loading control.
  • the edited band 1 (E4) represented the expected band after editing employing gRNAs 36-1 (SEQ ID NO:13) and 36-2 (SEQ ID NO:18), while edited band 2 (E3) pointed the expected edited band after gRNAs 36-1 (SEQ ID NO:13) and 36-3 (SEQ ID NO:16) mediated editing.
  • the 5'UTR region of RPE65 was amplified as loading control.
  • MQ indicates the negative control of the PCR; NT means Non-treated cells; P means cells treated with the peptide but without Cas9 or gRNAs (negative control of the transfection).
  • CHX indicates if cell were treated with (+) or without (-) cycloheximide 24 hours prior harvesting. Statistical significance with respect to the untreated condition (NT +) is indicated as * p ⁇ 0.05 or ** p ⁇ 0.01 using one-way ANOVA after Bonferroni correction.
  • Figure 3 Analysis of the effectiveness of the lipopeptide-mediated editing in photoreceptor precursor cells at DNA.
  • the full-length (FL) amplicon and the expected edited bands (E1 or E4) after the treatment with the selected gRNAs were indicated on the right edge of the gel.
  • the 5'UTR region of RPE65 was amplified as loading control.
  • a graph chart representing the overall result indicating the percentage of the full-length transcript (FL) and of the edited band (E) per each condition. Each bar is represented by mean ⁇ SD (n 2).
  • Full-length (FL) transcript and edited (E1) band were indicated at the right side of the gel.
  • the 5'UTR region of RPE65 was amplified as loading control.
  • a graph bar representing the average result in which each bar is represented by mean ⁇ SD (n 2).
  • MQ indicates the negative control of the PCR;
  • NT Nontreated cells;
  • P+C means cells treated with Lipopeptide and Cas9 ribonucleoprotein, but not with gRNAs;
  • Pept means cells treated only with lipopeptide and Cas9 means cells treated only with Cas9 RNP.
  • CHX indicates if cell were treated with (+) or without (-) cycloheximide 24 hours prior harvesting.
  • gRNA30-1 SEQ ID NO: 10
  • gRNA30-2 SEQ ID NO: 4
  • gRNA36-1 SEQ ID NO: 13
  • gRNA36-2 SEQ ID NO: 18
  • FIG. 4 Analysis of the effect of the genome editing of intron 30 ofABCA4 gene in photoreceptor precursor cells (PPCs) at the RNA level.
  • ACTB was amplified as loading control MQ indicates the negative control of the PCR;
  • NT means Nontreated cells.
  • CHX indicates if cell were treated with (+) or without (-) cycloheximide 24 h prior to harvesting.
  • * p ⁇ 0.05 Ordinary One-way ANOVA after Bonferroni correction).
  • FIG. 5 Overview of PacBio data on intron 30 ofABCA4 (genome editing target) using control and patient-derived PPCs transfected with RPNCs with gRNAs 30-1 and 30-2
  • A) Graph bars of the percentage of reads in the predicted edited region by employing the “.BAM” (left) or “.CRAM” (right) files. In all cases the edited samples present a 30% reduction of the reads, corresponding to deleted reads after Cas9-RNP editing.
  • Figure 6 Analysis of the effectiveness of the lipopeptide-mediated editing in retinal organoids derived from photoreceptor precursor.
  • Administration of the selected gRNA was either via the medium or via the medium and injection to identify penetrance and the level of editing achieved.
  • MQ indicates the negative control of the PCR;
  • NT indicates non-treated cells.
  • gRNA design and selection gRNA sequences were designed employing two different web tools: CRISPOR (http://crispor.tefor.net/) and CHOPCHOP (https://chopchop.cbu.uib.no/), using intron 30 and intron 36 of ABCA4 gene as a target sequence. The most promising sequences from the output were selected based on ranking and considering the predicted efficacy and off-targets.
  • gRNAs were selected (Table 2) and amplified from the ABCA4 gene template (primers SEQ ID NO: 19-41) to be further cloned in pairs into pX458 (Addgene plasmid # 48138; http://n2t.net/addgene:48138; RRID:Addgene_48138) for initial evaluation in HEK293T cells.
  • pX458 Additional plasmid # 48138; http://n2t.net/addgene:48138; RRID:Addgene_48138
  • gRNAs were combined into the pX458 based on their upstream or downstream position from the hot-spot cluster. The best combinations of gRNAs were subsequently ordered as crRNA for further evaluation using the Cas9 RNP system.
  • a total of 400,000 cells of HEK293T were transfected with the pX458 vector (Addgene, #48138) in which the designed gRNAs were introduced after the U6 promoter. Double-gRNAs pX548 constructs (Addgene, #48138).
  • the transfection was done using FuGENE®-HD (Promega, Madison, Wl, USA) following a 3:1 FuGENE®-HD Transfection Reagent:DNA ratio.
  • the FuGENE®- HD/DNA mixture was delivered via an Opti-MEM reduced serum medium (Gibco, Waltham, MA, USA). Samples were harvested 48 h after transfection to proceed with DNA analysis.
  • Photoreceptor precursor cell (PPC) differentiation and characterization iPSC lines were previously characterized elsewhere.
  • the differentiation from induced pluripotent stem cells (iPSC) to photoreceptor precursor cells was conducted by adapting the protocols already published by Gonzalez-Cordero et al. in 2017 and Capowsky et al. in 2019.
  • iPSCs were seeded as single cells and maintained E8F until becoming confluent. At that point, cells were grown in E6 medium for 2 days.
  • NIM Neural Induction Medium
  • BMP4 R&D Systems, Minneapolis, MN, USA
  • All PPC conditions were harvested by scraping the cells after washing the wells with 1x PBS. Experiments were performed in two independent differentiation batches. In order to detect the PE, cells were treated with CHX 24 hours before harvesting them.
  • RNA from day 0 and day 30 of differentiation was employed. In total 1 microgram of RNA was used to synthetize cDNA by using Superscript VILO Master Mix (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's instructions. Then, a quantitative PCR (qPCR) analysis was setup with the GoTaq Real-Time Quantitative PCR Master kit (Promega, Madison, Wl). The reactions were processed in triplicates and run using the Applied Biosystem Quantstudio 5 Digital system.
  • qPCR quantitative PCR
  • the expression levels of eight markers (OCT3/4, OTX2, CRX, RECOVERIN, PAX6, NRL, OPN1SW, ABCA4 and RPE65) and a housekeeping gene (GUSB) were studied to evaluate the success of the differentiation.
  • Each sample was normalized against the expression of the housekeeping gene and compared with iPSC (day 0) using the 2“ (AACt) method (using primers SEQ ID NO: 42-59).
  • gRNAs were delivered as crRNA-tcrRNA complex (IDT, Coralville, IA, USA). To do that, gRNAs sequences were synthetized as crRNA and diluted in duplex buffer to a final concentration of 200 pM. Then, the crRNA:tracrRNA duplex was assembled by mixing the two RNA oligos in 1 :1 equimolar concentration together with duplex buffer to reach a final duplex concentration of 50 pM. For the peptide-mediated delivery of the Cas9:crRNA:tracrRNA (RNP) complex of each gRNA was first diluted to a final concentration of 2.5 pM in nuclease-free water.
  • RNP Cas9:crRNA:tracrRNA
  • Cas9 RNP (Sigma, San Luis, MO, USA) stock was diluted in the reconstruction solution to have a final concentration of 1530 nM. For cellular delivery, it was diluted 2.5 times in OptiMEM (Sigma) and mixed together with the crRNA:tracrRNA complex at 2.5 pM (per gRNA) for 15 minutes at room temperature to form RNP.
  • ribonucleoprotein (RNP) ratios were equal to Cas9 (25nM)/gRNA1 (25nM) and gRNA2 (25nM) at a 1 :2 proportion.
  • Peptide stock was diluted in nuclease-free water with 1 % of acetic acid to enhance solubility, subsequently dilutions were prepared with Opti-MEM (Gibco).
  • the peptide was prepared to have a final concentration of 6 pM (around 1 :2:150).
  • the peptide was then thoroughly mixed with the gRNAs-Cas9 RNP solution and incubated together for a duration of 20 minutes at room temperature. This process resulted in the creation of RPNC for PIR with determined molar ratios.
  • these nanocomplexes were supplemented to the cells for experimentation. Samples for RNA analysis were treated with CHX 24 hours before harvesting.
  • the optimal concentration of peptide was determined by a dose-response experiment that was conducted using increasing concentrations of the peptide (data not shown).
  • Fibroblast cells were subjected to RNP nanocomplex for PIR delivery using concentrations ranging from 1 pM (1 :2:25) to 12 pM (1 :2:300), while PPCs were treated with concentrations ranging from 3 pM (1 :2:75) to 30 pM (1 :2:750).
  • PPCs RPNCs were delivered to day 20-22 PPCs, and samples were collected on day 30 or 31 .
  • RPNCs were delivered the day after seeding 200,000 cells in a 6 well-plate. After RPNC delivery, fibroblasts were subsequently trans-differentiated to boost the expression of ABCA4.
  • fibroblasts were trans-differentiated during 10 days using an adapted from Seko et al. 2018.
  • the culture medium was replaced by DMEM/ Ham's F-12 Nutrient Mix medium (1 :1) (Thermo Fisher Scientific) supplemented with 1 % of FCS and 0.2 % primocin.
  • medium Prior to each use, medium was supplemented with 40 ng/ml bFGF, 20 ng/ml EGF, and 1x of fibronectin and B27. The medium was changed every other day until the end of the trans-differentiation.
  • DNA isolation was conducted by using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Then, a total of 40 ng of the obtained DNA was employed for the PCR to amplify intron 30 or intron 36. The 5’ UTR of RPE65 was employed as loading control. PCRs were conducted using Ampli TaqGold 360 mix (Applied Biosystems; Waltham, MA, USA) mixed with the corresponding primers (as included in the sequence listing SEQ ID NO: 60-65) a concentration of 10 pM.
  • the PCR program included a denaturation step of 94 °C for 10 min, followed by 35 cycles of melting (94 °C for 30 s), annealing (58 °C for 30 s), and extension (72 °C for 2 min and 30 sec) steps, with a final elongation step of 72 °C for 5 min. Finally, the semi-quantification of the different transcripts was performed using Imaged software. 80 The information regarding the whole genome sequencing (WGS) and PacBio analysis is explained Supplemental Material.
  • RNA isolation was isolated using the Nucleospin RNA kit (Machery Nagel Duren, Germany) according to the manufacturer’s instructions. Total RNA concentrations were measured using Nanodrop 2000 and 1 pg of total RNA was used for cDNA synthesis using VILO superscript IV cDNA synthesis kit (Thermo Fisher Scientific) following the manufacturer’s recommendations. Next, a total of 50 ng of cDNA was used as a template to amplify the pseudoexon of intron 30. As loading control, ACTB was also amplified. A complete list of primers (as included in the sequence listing SEQ ID NO: 66 -73).
  • PCR mixtures also contained 1x PCR buffer with MgCb (Roche, Manheim, Germany), 1x Q-solution (QIAGEN), 2.5 mM of MgCb, 2 pM of dNTPs, 0.2 mM of each primers and 0.5 U Taq DNA Polymerase (Roche).
  • PCR products were resolved by electrophoresis and all the transcripts were verified by Sanger. Semiquantitative analysis was conducted using Imaged software. 80
  • Example 1 Design and test ofgRNAs in HEK293T showed high efficiency in editing both intron 30 and intron 36.
  • intron 30 and intron 36 Based on the fact that there are two introns in which a significant number of DI variants are found (intron 30 and intron 36), we hypothesized that the partial removal of the corresponding intron can be a therapeutic strategy to target multiple DI variants simultaneously.
  • Different guide-RNA (gRNA) sequences were designed along intron 30 (upstream and downstream of the hot-spot cluster). The idea was to combine two gRNAs (one of each region) to excise the hot-spot cluster by removing part intron sequence. In total, 12 gRNAs were designed for intron 30 (Table 2). The same approach was followed also in intron 36, for which a total of 6 gRNAs were designed to be able to remove the hot-spot cluster also present in this intron (Table 2).
  • gRNAs sequences were cloned in the plasmid pX458, which contains the CRISPR-Cas9 machinery allowing the gRNAs to cut within the target sequence. Subsequently, gRNAs were tested in pairs in HEK293T cells to assess their genome editing capacity (Figure 1). Although all tested pairs were capable of genome editing, for the next experiments the best performing cells were selected. For intron 30, the pair 30 1 +2 (SEQ ID NO:4 and SEQ ID NO: 10 resp.) and 30 3+4 (SEQ ID NO:1 and SEQ ID NO: 7 resp.) were the ones selected since both presented the best editing efficacies.
  • pair 30 1 +2 was able to remove a bigger part of intron 30, covering a larger number of variants ( Figure 1 ; left).
  • pairs 36 1 +2 SEQ ID NO:13 and SEQ ID NO: 18 resp.
  • 1 +3 SEQ ID NO:13 and SEQ ID NO: 16 resp.
  • Genome editing was achieved in control and patient-derived fibroblast cells.
  • gRNAs were delivered together with the Cas9 protein as ribonucleoprotein (RNP) to further validate the findings on HEK293T cells.
  • RNP ribonucleoprotein
  • fibroblast cells derived from both a control individual and a STGD1 patient carrying the variant c.4539+2001 G>A in a heterozygous manner.
  • gRNA was delivered together with Cas9 and a modified LAH5 (LAH5 as described in Oktem, et al, Pharmaceutics 15.10 (2023): 2500) this complex will be referred to as an RNPC complex herein.
  • RPNC were delivered to both control and patient cells.
  • RPNC delivery was performed and was followed by a 10-day transdifferentiation protocol to boost the expression levels of ABCA4 mRNA and PE.
  • ABCA4 pathogenic variant present on the patient line is located in intron 30, only the gRNAs against that region were tested in that cell line allowing DNA and RNA analysis of the genome editing effect.
  • the pair for intron 30 and the pair of intron 36 were tested in the control fibroblast line to evaluate the genome editing capacity at the DNA level.
  • iPSC-derived photoreceptor precursor cells are a heterogeneous culture of retinal-like cells that have been shown to have increased expression levels of ABCA4.7.
  • two iPSC- derived PPC cultures were again employed: patient carrying the c.4539+2001 G>A variant in heterozygous manner derived from the fibroblast cells used in previous section, and its corresponding isogenic control iPSCs.
  • the previously identified as the most efficacious pairs of gRNAs for both introns were evaluated (gRNA pair 30 1 +2 and 36 1 +2).
  • Example 4 On- and off-target analysis revealed no off-target effects and the correct cleavage of intron 30.
  • Example 5 Example 5: Genome editing in Retinal Organoids
  • the mixture was delivered either to the organoids as 200 pl of total medium or as 100 pl in addition to 1 pl of the transfection mixture being injected in the center of the organoid using a Hamilton needle. If we compared the organoids injected they show higher levels of editing compared with those in which the transfection mixture was delivered only in the medium. To better compared all four replicates, the same samples were all amplified in the same reaction and resolved in a gel next to each other to avoid differences during the PCR reaction (figure 6). In general, the second condition with injection provided higher levels of editing. We hypothesize that this is because cells are targeted both from the inside and the outside, increasing the amount of cells exposed to the transfection mixture.

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Abstract

La présente invention concerne le domaine de la médecine. En particulier, l'invention concerne un système d'édition de gènes qui peut être utilisé dans le traitement, la prévention et/ou le retard de maladies associées à ABCA4 telles que la maladie de Stargardt.
PCT/EP2025/056149 2024-03-06 2025-03-06 Édition de génome abca4 thérapeutique pour le traitement d'une maladie stargardt Pending WO2025186392A1 (fr)

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