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JP-7856581-B2 - Base editing approaches for the treatment of β-hemoglobin disorders

JP7856581B2JP 7856581 B2JP7856581 B2JP 7856581B2JP-7856581-B2

Inventors

  • ミッチョ，アンナリタ
  • アントニウ，パナヨティス
  • カヴァッツァーナ，マリナ

Assignees

  • アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル
  • ユニヴェルシテ・パリ・シテ
  • アシスタンス ピュブリク－オピトー ドゥ パリ
  • フォンダシオン・イマジネ

Dates

Publication Date
20260511
Application Date
20210512
Priority Date
20200513

Claims (8)

  1. A method for increasing the fetal hemoglobin content in isolated eukaryotic cells, comprising the steps of: contacting isolated eukaryotic cells with a gene editing platform comprising (a) at least one base editing enzyme and (b) at least one guide RNA molecule for guiding the base editing enzyme to at least one target sequence in the HBG1 or HBG2 promoter, thereby editing the promoter, and subsequently increasing the expression of gamma globulin in the eukaryotic cells; The gene editing platform introduces a -198T>C mutation into the HBG1 or HBG2 promoter, thereby enabling the KLF1 activator to bind to the promoter ; or the gene editing is used to edit the -200 region within the HBG1 or HBG2 promoter, thereby disrupting the binding site to the LRF repressor , and The base editing enzyme and the corresponding guide RNA molecule are as follows: The base editing enzyme is ABEmax, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 49; The base editing enzyme is CBE-NRCH, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; The base editing enzyme is CBE-SpG, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; The base editing enzyme is CBE-SpRY, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; and The base editing enzyme is CBE-SpRY, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 53; A method selected from the group consisting of the following .
  2. The method according to claim 1, wherein the gene editing platform introduces at least one mutation selected from the group consisting of -201C>T, -200C>T, -197C>T, -196C>T, -195C>T, and -194C>T into the HBG1 or HBG2 promoter, thereby disrupting the binding site to the LRF suppressor.
  3. The method according to claim 1, wherein the eukaryotic cells are selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), and pluripotent cells (i.e., embryonic stem cells (ES) and induced pluripotent stem cells (iPS)).
  4. The method according to claim 1, wherein multiple guide RNA molecules are designed to target multiple sequences within the HBG1 or HBG2 promoter.
  5. The method according to claim 1, wherein multiple base editing enzymes, along with multiple guide RNA molecules, are designed to target multiple sequences within the HBG1 or HBG2 promoter.
  6. A pharmaceutical composition for increasing fetal hemoglobin levels in subjects requiring it, comprising a therapeutically effective amount of a population of eukaryotic cells obtained by the method described in any one of claims 1 to 5 .
  7. The method according to claim 1, wherein the eukaryotic cells are derived from a subject diagnosed with a hemoglobin disorder such as sickle cell disease or β-thalassemia.
  8. A kit for increasing the fetal hemoglobin content in eukaryotic cells, comprising a gene editing platform comprising (a) at least one base editing enzyme and (b) at least one guide RNA molecule for guiding the base editing enzyme to at least one target sequence within the HBG1 or HBG2 promoter, The gene editing platform introduces a -198T>C mutation into the HBG1 or HBG2 promoter, thereby enabling the KLF1 activator to bind to the promoter ; or the gene editing is used to edit the -200 region within the HBG1 or HBG2 promoter, thereby disrupting the binding site to the LRF repressor , and The base editing enzyme and the corresponding guide RNA molecule are as follows: The base editing enzyme is ABEmax, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 49; The base editing enzyme is CBE-NRCH, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; The base editing enzyme is CBE-SpG, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; The base editing enzyme is CBE-SpRY, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; and The base editing enzyme is CBE-SpRY, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 53; A kit selected from the group consisting of the following .

Description

Field of invention: This invention relates to medicine, particularly to the field of hematology. Background of the invention: Beta-hemoglobin disorders (beta-thalassemia and sickle cell disease (SCD)) are monogenic disorders caused by mutations in the beta-globin gene locus that affect the synthesis or structure of adult hemoglobin (Hb). Beta-thalassemia is caused by mutations in the beta-globin gene (HBB) locus that reduce (β + ) or eliminate ( β0 ) the production of beta-globin chains in the adult hemoglobin (HbA) tetramer, leading to the deposition of unbound α-globin chains, death of erythroid cells, and severe anemia (1) . In SCD, A>T mutations in the HBB gene cause the substitution of glutamic acid at position 6 of the beta-globin chain with valine ( βS ), which is the cause of deoxygenation-induced polymerization of sickle cell hemoglobin (HbS). These initial events induce sickle-shaped red blood cells (RBCs), hemolysis, vascular occlusive crisis, and multiple organ failure, often accompanied by a severely shortened life expectancy (2) . The only definitive treatment for β-hemoglobin disorders is allogeneic hematopoietic stem cell (HSC) transplantation from an HLA-compatible donor, an option applicable to less than 30% of patients (3) . Gene therapy approaches based on the transplantation of the patient's own genetically modified HSCs are being studied as a treatment option for patients without compatible donors (4) . Genome editing technologies based on direct gene modification have been used to develop therapeutic approaches for β-hemoglobin disorders. These approaches use designer nucleases such as the CRISPR/Cas9 system that induce double-strand breaks (DSBs) in DNA via a single guide RNA (gRNA) complementary to a specific genomic target (4) . The clinical history of β-hemoglobin disorders shows that the severity of both β-thalassemia and SCD is mitigated by the synthesis of fetal γ-globin in adulthood, which is typically associated with gene mutations (deletions or point mutations) within the HBB cluster, also known as mutations in hereditary hyperfetal hemoglobinemia (HPFH) (5) . Fetal hemoglobin (HbF) compensates for the deficiency of adult hemoglobin in β-thalassemia, and γ-globin exerts a potent anti-sickling effect in SCD by replacing mutants of the sickle β-chain (4) . In particular, mutations in the two identical promoters of the γ-globin genes (HBG1 and HBG2) either generate de novo DNA motifs recognized by transcription activators (TAL1, KLF1 and GATA1) (6, 7, 8) or disrupt the binding sites of transcription repressors (LRF and BCL11A) (9) . As a potential treatment for both β-thalassemia and SCD, several genome editing strategies have been developed that aim to reactivate fetal gamma globin expression in the patient's hematopoietic stem/progenitor cells (HSPCs) by disrupting cis-regulatory sequences through deletions or insertions that mimic mutations of hereditary hyperfetal hemoglobinemia in the HSPCs (10) . CRISPR/Cas9 disruption of the LRF and BCL11A repressor binding sites within the gamma globin promoter efficiently reactivates fetal hemoglobin expression and remits the SCD RBC phenotype (1) . However, most deletions disrupting repressor binding sites are caused by microhomology-mediated end joining (MMEJ), which may not be effective in the quiescent HSC fraction of the HSPC population that is regrowing over a long period of time, as the HSPC population is mostly composed of dividing progenitor cells (11, 12) . It is noteworthy that HSCs are highly sensitive to DNA DSBs (13) , especially when multiple on-target events are present, or when on-target and off-target events occur simultaneously. Even when using highly specific gRNAs, treatment of human HSPCs with Cas9/gRNA can induce a DNA damage response, leading to apoptosis (14) . CRISPR/Cas9 can induce P53-dependent cytotoxicity and cell cycle arrest, resulting in negative selection of cells with a functional P53 pathway (15) . Furthermore, the occurrence of several on-target DSBs, simultaneous on-target and off-target DSBs, or even a single on-target DSB carries the risk of deletions, inversions, and translocations (16) . Therefore, the development of novel, effective, and safe therapeutic strategies for β-hemoglobin disorders based on precise base editing is preferred over DNA repair induced by DSBs. In recent years, cytosine and adenine base editing enzymes (CBE and ABE) based on the CRISPR system have been shown to be able to cause pinpoint changes in DNA with little to no DSB (17) . The basic components of base editing enzymes are non-catalytic Cas9 nucleases and deaminases; these ultimately result in the conversion of C-G to T-A or A-T to G-C (for CBE and ABE, respectively) (18) . Base editing approaches enable precise DNA repair with virtually no DSB, thus eliminating the risk of DSB-induced apoptosis, translocation, and major DNA insertion or deletion. Furthermore, base editing has lower levels of off-target activity than