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WO2025177303A1 - Édition génique médiée par crispr pour induire de l'hémoglobine fœtale - Google Patents

Édition génique médiée par crispr pour induire de l'hémoglobine fœtale

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Publication number
WO2025177303A1
WO2025177303A1 PCT/IN2025/050240 IN2025050240W WO2025177303A1 WO 2025177303 A1 WO2025177303 A1 WO 2025177303A1 IN 2025050240 W IN2025050240 W IN 2025050240W WO 2025177303 A1 WO2025177303 A1 WO 2025177303A1
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WO
WIPO (PCT)
Prior art keywords
gdna
gene editing
editing system
crrna
grna
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Pending
Application number
PCT/IN2025/050240
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English (en)
Inventor
Nupur BHARGAVA
Jitendra VYAS
Aaman BILAKHIA
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Micro Crispr Pvt Ltd
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Micro Crispr Pvt Ltd
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Publication date
Application filed by Micro Crispr Pvt Ltd filed Critical Micro Crispr Pvt Ltd
Publication of WO2025177303A1 publication Critical patent/WO2025177303A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present disclosure relates to a CRISPR mediated gene editing system. More particularly, the present disclosure relates to a gene editing system for fetal hemoglobin induction.
  • a blood monogenic disorder specifically refers to genetic disorders that affect the blood and are caused by mutations in genes involved in the production or function of blood cells.
  • the blood monogenic disorder includes sickle cell anemia (SCD) and betathalassemia.
  • SCD is a monogenic condition that is prevalent and affects millions of people. Over 100,000 persons in the US and roughly 42,000 in Europe are thought to be impacted.
  • Sickle cell anemia the most severe and common form of sickle cell disease (SCD) is an autosomal recessive condition caused by homozygous mutations in which the glutamic acid at position 6 in P-globin is replaced with valine, leading in deoxyhemoglobin polymerization and sickling of red blood cells (RBCs).
  • SCD sickle cell disease
  • the present disclosure relates to a gene editing system to induce expression of fetal hemoglobin (HbF) in hematopoietic stem cells (HSCs).
  • the gene editing system includes a guide RNA (gRNA) extending between a 5'end and a 3' end, and an endonuclease.
  • the gRNA includes at least one Crispr RNA (crRNA) disposed towards the 5' end of the gRNA and a trans-activating Crispr RNA (tracrRNA) disposed towards the 3' end of the gRNA.
  • the crRNA is encoded by at least one of SEQ ID NO. 1 - 6.
  • the crRNA is configured to bind a target strand at a pre-defined locus of a genomic DNA (gDNA).
  • the endonuclease is coupled to the gRNA via the tracrRNA.
  • the endonuclease is configured to introduce a doublestrand break (DSB) in the gDNA.
  • DSB doublestrand break
  • the present disclosure relates to a composition including at least a population of the CD34+ hematopoietic stem cells (CD34+ HSCs) having their respective genomic DNA (gDNA) modified using the gene editing system as described above.
  • the modification is at least one of indel mutations, insertions, deletions, and point mutations in Exon 4 (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCLlla gene (Nucleotide ID: ENST00000642384.1) present on the chromosome 2 of the gDNA.
  • FIG. 1 depicts a gene editing system 100 according to an embodiment of the present disclosure.
  • the system of the present disclosure creates at least one mutation in the gDNA of the HSCs such as indel mutations, insertions, deletions, point mutations, etc.
  • the mutation(s) introduced in the gDNA of the HSCs induces the production of HbF.
  • the HbF in adults, compensates for the abnormal/diseased hemoglobin thereby alleviating symptoms of sickle cell disease,
  • the system of the present disclosure is directed to a pre-determined locus present on one of the chromosomes of human gDNA.
  • the system is directed to at least a portion of the BCLlla gene located on chromosome two and adjacent regions.
  • Fig. 1 depicts an exemplary embodiment of a system 100 of the present disclosure.
  • the system 100 is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based ribonucleoprotein (RNP) complex.
  • the system 100 includes a guide ribonucleic acid (gRNA) 110 and an endonuclease 120.
  • the gRNA 110 is a polynucleotide sequence including at least one Crispr RNA (crRNA) 110a and a transactivating Crispr RNA (tracrRNA) 110b.
  • the gRNA 110 has a 5' end and a 3'end, and extends therebetween (as shown in Fig. 1).
  • the crRNA 110a is disposed towards the 5' end of the gRNA 110.
  • the crRNA 110a has a pre-defined length ranging from 17 nucleotides to 20 nucleotides.
  • the crRNA 110a forms basepair (or binds) with a pre-defined locus of a genomic deoxyribonucleic acid (gDNA) 10 of, for example, a hematopoietic stem cell(s) (or CD34+ HSCs).
  • Fig. 1 depicts only one of the two strands of the gDNA 10, i.e., a target strand.
  • the complementary strand of the target strand of the gDNA 10 is not depicted in the figures.
  • At least a portion of the target strand of the gDNA 10 binds/interacts and forms base pair with at least a portion of the crRNA 110a (and the gRNA 110).
  • the crRNA 110a forms base-pair with a portion of the BCLlla gene (Nucleotide ID: ENST00000642384.1) on chromosome two or adjacent regions thereof of the gDNA 10.
  • the crRNA 110a base pairs with a portion of the Exon four (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCLlla gene on chromosome two of the gDNA 10.
  • the crRNA 110a is encoded by at least one of SEQ ID No. 1-6.
  • the crRNA 110a encoded by SEQ ID No. 1 binds with at least a portion of a negative strand of chromosome 2 from 60462304 to 60462323, which is part of Exon four of the BCLlla gene of the gDNA 10.
  • the crRNA 110a encoded by SEQ ID No. 2 binds with at least a portion of a positive strand of chromosome 2 from 60462265 to 60462284, which is part of Exon four of the BCLlla gene of the gDNA 10.
  • the crRNA 110a encoded by SEQ ID No. 3 binds with at least a portion of a positive strand of chromosome 2 from 60462273 to 60462292, which is part of Exon four of the BCLlla gene of the gDNA 10.
  • the crRNA 110a encoded by SEQ ID No. 5 binds with at least a portion of a negative strand of chromosome 2 from 60460720 to 60460742, which is part of Exon four of the BCLlla gene of the gDNA 10.
  • the crRNA 110a encoded by SEQ ID No. 6 binds with at least a portion of a negative strand of chromosome 2 from 60460669 to 60460691, which is part of Exon four of the BCLlla gene of the gDNA 10.
  • the tracrRNA 110b is disposed towards the 3'end of the gRNA 110.
  • the tracrRNA 110b forms a binding scaffold for coupling the crRNA 110a (and the gRNA 110) to the endonuclease 120.
  • the endonuclease 120 is coupled to the crRNA 110a (and the gRNA 110) via the tracrRNA 110b.
  • the tracrRNA 110b is encoded by SEQ ID No. 7.
  • the gRNA 110 includes six nucleotides modified with OMe analogs and five nucleotides modified with PS linkages.
  • the six nucleotides modified with the OMe analogs include, from the 5' end to the 3' end, the first and last three nucleotides of the gRNA 110.
  • the five nucleotides modified with the PS linkages include, from the 5' end to the 3' end, the first three and last two nucleotides of the gRNA 110.
  • Chemical modifications of the one or more nucleotides of the gRNA 110 provide stability and protection from exonucleases thereby increasing the half-life of the gRNA 110 in vivo. The chemical modifications also improve editing efficiency of the system 100, reduces risk of innate immune response and reduces off-target activity.
  • the endonuclease 120 of the system 100 is a protein/enzyme that behaves as a nonspecific endonuclease.
  • the endonuclease 120 includes a CRISPR- associated protein 9 (Cas9) (Protein sequence ID No. NP_269215.1) from Streptococcus pyogenes.
  • the endonuclease 120 is high-fidelity Cas9 (HiFi Cas9).
  • the HiFi Cas9 includes the Cas9 protein with a single point mutation, i.e., p.R691A.
  • the tracrRNA 110b includes at least one complementary "direct repeat" sequence region that binds with the endonuclease 120, thereby coupling the endonuclease 120 to the gRNA 110.
  • the endonuclease 120 includes at least two nuclease domains for cleaving each of the two strands of the gDNA 10 and introduce at least one double strand break (DSB) in the gDNA 10.
  • the specificity of the nuclease activity of the endonuclease 120 is governed by the binding of the crRNA 110a with at least a portion of the gDNA 10.
  • the endonuclease 120 includes a HNH domain to cleave the target strand of the gDNA 10 and a RuvC domain to cleave the complementary strand of the gDNA 10.
  • the cleavage of the gDNA 10 by the endonuclease 120 is either close to or at a portion of the target strand where the gRNA 110 base pairs (or binds) with the gDNA 10.
  • the Cas9 endonuclease 120 cleaves the gDNA 10 at a portion of the target strand where the gRNA 110 base pairs (or binds) with the gDNA 10.
  • the system 100 of the present disclosure is described with the examples of Cas9 endonuclease 120, other functionally equivalent endonucleases are within the scope of the teachings of the present disclosure.
  • the system 100 of the present disclosure is introduced within a population of autologous CD34+ HSCs via ex vivo electroporation.
  • the crRNA 110a of the gRNA 110 binds (or forms base pairs) with a portion of the Exon 4 of the BCLlla gene present on the chromosome 2 of the gDNA 10.
  • the endonuclease 120 cleaves the two strands of the gDNA 10 to introduce one DSB.
  • the CD34+ HSCs may repair the DSB of the gDNA 10 via at least one of a non-homologous end joining technique or homology directed repair technique.
  • the repair of the DSB introduces a mutation (one of indel, insertion, deletion) in the BCLlla gene of the gDNA 10 thereby repressing the expression of the BCLlla gene.
  • Repression of the BCLlla gene at least partially removes regulation of expression of the gamma globin genes thereby inducing expression of the gamma globin genes and producing high levels of HbF.
  • the HbF may reduce the symptoms of abnormal hemoglobin.
  • a hematopoietic stem cell having the gDNA 10 with a BCL11A gene, includes at least one of indel mutations, insertions, deletions, and point mutations in Exon 4 (Exons and introns are mapped as per NM_022893.4 and NP_075044.2) of the BCLlla gene (Nucleotide ID: ENST00000642384.1) present on the chromosome 2 of the gDNA 10 introduced by the system 100.
  • composition including a population of the CD34+ HSCs having their gDNA 10 modified using the system 100 is administered to a subject having abnormal hemoglobin.
  • the ability of the gene editing system 100 to modify the autologous HSCs provides accessibility to a larger cohort of patients of sickle cell disease and possibly also transfusiondependent Beta thalassemia as a potential therapeutic alternative.
  • Example 1 Isolation of peripheral blood mononuclear cells (PBMCs)
  • a whole blood sample was drawn from an individual suffering from sickle cell disease (SCD).
  • the whole blood sample was diluted with equal volume of phosphate buffer saline (PBS) to obtain a whole blood mixture.
  • PBS phosphate buffer saline
  • the whole blood mixture was carefully layered on a Ficoll- Paque separation medium in a conical tube.
  • the conical tube was centrifuged at 600 x g for 45 minutes at room temperature with no brake. The tube was recovered from the centrifuge.
  • the tube had three layers, a plasma layer, a Ficoll layer and a PBMC layer disposed therebetween.
  • the PBMC layer was separated and collected into a fresh conical tube.
  • the PBMC layer was diluted with PBS in a ratio of 1:2 and the conical tube was centrifuged at 300 x g for 10 minutes at room temperature to wash the PBMCs. Thereafter, the PBMCs were suspended at 10 7 cells/mL in MACS buffer to obtain a cell suspension.
  • the composition of the MACS buffer was PBS, 0.5% bovine serum albumin (BSA), and 2mM Ethylenediaminetetraacetic acid (EDTA).
  • Example2 CD34+ cell enrichment
  • a MACS column (procured from Miltenyi) was placed in a magnetic field of a MACS separator (procured from Miltenyi). The cell suspension was loaded onto the MACS column. The magnetic field arrested the movement of the MACS microbead bound to CD34+ cells while allowing the other cell types to flow through the MACS column. The MACS column was then washed using 300 pL to 500 pL of MACS buffer to remove any unbound cells. Thereafter, the MACS column was removed from the magnetic field and the flow through/eluent having the CD34+ cells were collected.
  • CD34+ cells were resuspended at 10 7 cells/mL in a HSPC culture medium (procured from Stem cell technologies) to obtain a CD34+ cell culture.
  • CD34+ cells were separated and labelled with anti-human mice CD34-PE conjugated antibodies (procured from BD Biosciences) and analyzed using flow cytometry (BD Accuri C6 Plus procured from BD Biosciences) and microscopy (CKX53SF procured from Olympus) to assess the purity and viability of the CD34+ cell population in the CD34+ cell culture.
  • flow cytometry BD Accuri C6 Plus procured from BD Biosciences
  • CKX53SF procured from Olympus
  • the system 100 of the present disclosure was introduced within the CD34+ cells obtained from Example 2 above via ex vivo electroporation (by following the DZ100 method in a Lonza 4D-Nucleofector apparatus). Thereafter, the CD34+ cells were cultured at 37°C, under 5% CO2 and analyzed after periodic intervals of incubation.
  • Fig. 4 depicts the CD34+ cells, 18 hours after nucleofection.
  • Fig. 4a depicts the CD34+ cells, 10 days after nucleofection/differentiation.
  • Group WT represents a wild-type CD34+ cell population that were not being modified with the system 100 of the present disclosure.
  • Group A represents the population of CD34+ cells being modified with the system 100 having crRNA 110a encoded by SEQ ID No. 1.
  • Group B represents the population of CD34+ cells being modified with the system 100 having crRNA 110a encoded by SEQ ID No. 2.
  • Group C represents the population of CD34+ cells being modified with the system 100 having crRNA 110a encoded by SEQ ID No. 3.
  • Group D represents the population of CD34+ cells being modified with the system 100 having crRNA 110a encoded by SEQ ID No. 4.
  • the gDNA 10 was isolated using DNeasy Blood and Tissue kit (procured from Qiagen, #69506) from all the groups of CD34+ cells obtained in Example 3 above.
  • the DNeasy Blood and Tissue kit includes a lysis buffer (buffer AL), a DNeasy mini spin column, wash buffer 1 (buffer AW1), wash buffer 2 (buffer AW2), and elution buffer (buffer AE).
  • Buffer AL lysis buffer
  • Buffer AW1 wash buffer 1
  • buffer AW2 wash buffer 2
  • AE elution buffer
  • lysis buffer buffer AL
  • 200 pL of the lysis buffer buffer AL
  • 200 pL of 96-100% ethanol was added to the micro-centrifuge tube and mixed thoroughly by vortexing the micro-centrifuge tube.
  • the contents of the micro-centrifuge tube were loaded onto the DNeasy mini spin column placed in a 2 mL collection tube.
  • the DNeasy mini spin column was centrifuged at more than 6,000 x g for 1 min. The eluent/flow-through and the collection tube was discarded. The DNeasy mini spin column was then placed in a new 2mL collection tube.
  • 500 pL of the wash buffer 1 (buffer AW1) was added to the DNeasy mini spin column and centrifuged at more than 6,000 x g for 1 min. The eluent/flow-through and the collection tube was discarded. The DNeasy mini spin column was then placed in a new 2mL collection tube. 500 pL of the wash buffer 2 (buffer AW2) was added to the DNeasy mini spin column and centrifuged at more than 20,000 x g for 3 min. The eluent/flow-through and the collection tube was discarded. The DNeasy mini spin column was then placed in a new micro-centrifuge tube.
  • the gDNA 10 of all the groups were loaded in the wells of an agarose gel along with a lkb standard DNA ladder (procured from New England Biolabs).
  • the pMAX GFP was loaded in one of the wells of the agarose gel as a positive control.
  • the agarose gel was run and the then viewed under UV light as shown in Fig. 5.
  • Example 5 T7 endonuclease assay
  • the gDNA 10 isolated in Example 4 above was subjected to PCR amplification to amplify a portion of Exon 4 of the BCL11A gene.
  • the primers used in the amplification reaction includes a forward primer encoded by SEQ ID No. 8 and a reverse primer encoded by SEQ ID No. 9.
  • the amplicons obtained above from the Group WT was separately added to the amplicons obtained from the Groups A, C, and D in a ratio of 1:1 by weight of the respective amplicons to obtain respective amplicon mixtures.
  • the respective amplicon mixtures of Groups, A, C, D and the amplicons of the Group WT were kept inside the thermal cycler, and the thermal cycler was programed for 1 cycle of denaturation at 95°C for 5 mins, and then gradually cooled to 25°C - 37°C to obtain a heteroduplex DNA (hDNA) of the respective groups.
  • the hDNA forms only if there were any mis-match in base pair formation between the amplicons of the Groups A, C, D and the amplicons of Group WT due to in/del mutation(s) in the amplicons.
  • a T7 endonuclease mixture was prepared by adding 10 U of T7 endonuclease I (procured from New England Biolabs, #M0302L) in 2 pl of IX reaction buffer (NEBBufferTM 2 procured from New England Biolabs). 200ng of the hDNA obtained above for respective groups were separately added to the T7 endonuclease mixture and incubated at 37°C for 15-30 minutes. During incubation, the T7 endonuclease I enzyme cleaves all the mis-matched base- pairs in the hDNA. The activity of the T7 endonuclease I was stopped by heating the T7 endonuclease mixture at 80°C for 5 minutes.

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Abstract

La présente invention concerne un système d'édition génique permettant d'induire l'expression de l'hémoglobine fœtale (HbF) dans les cellules souches hématopoïétiques (CSH). Le système d'édition génique inclut un ARN guide (ARNg 110) se déployant entre une extrémité 5' et une extrémité 3', et une endonucléase. L'ARNg comprend au moins un ARN Crispr (ARNcr) situé vers l'extrémité 5' de l'ARNg et un ARN Crispr trans-activateur (ARNtracr) situé vers l'extrémité 3' de l'ARNg. L'ARNcr est codé par au moins l'une de SEQ ID NO 1 à 6. L'ARNcr est conçu pour lier un brin cible à un locus prédéfini d'un ADN génomique (ADNg). L'endonucléase (120) est couplée à l'ARNg via l'ARNtracr (110b). L'endonucléase est conçue pour introduire une cassure double brin (DSB) dans l'ADNg.
PCT/IN2025/050240 2024-02-19 2025-02-18 Édition génique médiée par crispr pour induire de l'hémoglobine fœtale Pending WO2025177303A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3280803A1 (fr) * 2015-04-06 2018-02-14 The Board Of Trustees Of The Leland Stanford Junior University Arn guides chimiquement modifiés pour la régulation génétique médiée par crispr/cas
RU2018127636A (ru) * 2015-12-28 2020-02-03 Новартис Аг Композиции и способы лечения гемоглобинопатий
US20220047637A1 (en) * 2018-11-29 2022-02-17 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
CN114364799A (zh) * 2019-08-28 2022-04-15 甘李药业股份有限公司 编辑造血干/祖细胞中bcl11a基因的方法
EP3371306B1 (fr) * 2015-11-04 2023-01-04 Crispr Therapeutics AG Matériels et méthodes pour le traitement d'hémoglobinopathies

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3280803A1 (fr) * 2015-04-06 2018-02-14 The Board Of Trustees Of The Leland Stanford Junior University Arn guides chimiquement modifiés pour la régulation génétique médiée par crispr/cas
EP3371306B1 (fr) * 2015-11-04 2023-01-04 Crispr Therapeutics AG Matériels et méthodes pour le traitement d'hémoglobinopathies
RU2018127636A (ru) * 2015-12-28 2020-02-03 Новартис Аг Композиции и способы лечения гемоглобинопатий
US20220047637A1 (en) * 2018-11-29 2022-02-17 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
CN114364799A (zh) * 2019-08-28 2022-04-15 甘李药业股份有限公司 编辑造血干/祖细胞中bcl11a基因的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BAUER, DANIEL E. ET AL.: "An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level.", SCIENCE, vol. 342, no. 6155, 2013, pages 253 - 257, XP093087302, DOI: 10.1126/science.1243745 *

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