CN-122003501-A - Compositions and methods for treating hemoglobin disorders

Abstract

Provided are gRNA molecules and nucleic acids, complexes, compositions, cells, and kits thereof. Also provided are methods of increasing HbF expression levels in progeny erythrocytes of human cd34+ HSPCs by genome editing, which methods are useful for treating hemoglobinopathies, such as β -thalassemia and sickle cell disease.

Inventors

• QU SU
• ZHAO YUEMENG
• Tian Xingxue
• ZHAO XIAOPING

Assignees

• 上海天泽云泰生物医药股份有限公司

Dates

Publication Date

20260508

Application Date

20240927

Priority Date

20230928

Claims (20)

1. A guide RNA (gRNA) molecule comprising a targeting domain complementary to a target sequence within an HBG1 promoter region located between chr11:5,248,269 and 5,249,857 of human genome hg19 or an HBG2 promoter region located between chr11:5,253,188-5,254,781 of human genome hg 19.
2. The gRNA molecule of claim 1, the target sequence is located between about 210bp upstream to about 100bp upstream of the transcription start sites of the HBG1 and HBG 2.
3. The gRNA molecule of any one of claims 1-2, comprising a tracr sequence and a crRNA sequence.
4. The gRNA molecule of any one of claims 1-3, which is a two-component guide RNA molecule.
5. The gRNA molecule of any one of claims 1-3, which is a single-component guide RNA molecule (sgRNA).
6. The gRNA molecule of any one of claims 1-5, wherein (1) contacting a CRISPR/Cas system comprising the gRNA molecule with the target sequence, or (2) introducing a CRISPR/Cas system comprising the gRNA molecule into a cell in which a gene comprising the target sequence is located, a base insertion or deletion (indel) capable of being formed at or near the target sequence.
7. The gRNA molecule of claim 6, the CRISPR/Cas system comprising a Cas12b nuclease and/or a functionally active fragment thereof.
8. The gRNA molecule of claim 6, the CRISPR/Cas system comprising a Cas12i nuclease and/or a functionally active fragment thereof.
9. A gRNA molecule of claim 3, the tracr sequence comprising the nucleotide sequence set forth in SEQ ID No. 60.
10. The gRNA molecule of claim 3, wherein the crRNA sequence comprises a nucleotide sequence selected from any one of SEQ ID NOs 61-66.
11. The gRNA molecule of any one of claims 1-10, comprising a nucleotide sequence selected from any one of SEQ ID NOs 12-59 and 67-78.
12. A nucleic acid molecule encoding the gRNA molecule of any one of claims 1-11.
13. A vector comprising the nucleic acid molecule of claim 12.
14. The vector of claim 13, selected from the group consisting of lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes Simplex Virus (HSV) vectors, plasmids, microloops, nanoplasmms, and RNA vectors.
15. A Ribonucleoprotein (RNP) complex comprising the gRNA molecule of any one of claims 1-11 and a nuclease of a CRISPR/Cas system.
16. The RNP complex of claim 15, the nuclease is a class II V-type Cas enzyme.
17. The RNP complex of claim 16, the nuclease is a Cas12b nuclease.
18. The RNP complex of claim 17, the Cas12b nuclease comprises a variant thereof, an ortholog thereof, and/or a functionally active fragment thereof.
19. The RNP complex of claim 17 or 18, the Cas12b nuclease is a Cas12b nuclease (AaCas 12 b) from Alicyclobacillus acidiphilus.
20. The RNP complex of claim 19, the AaCas b nuclease comprises one or more of the following mutations relative to the wild-type AaCas b nuclease: (1) Replacing one or more amino acid residues in the wild-type AaCas b nuclease that interact with a pre-spacer adjacent motif (PAM) with positively charged amino acid residues; (2) Substitution of one or more amino acid residues in the wild-type AaCas b nuclease involved in opening a DNA duplex with an amino acid residue having an aromatic ring, and/or (3) Substitution of one or more amino acid residues in the RuvC domain of the wild-type AaCas b nuclease that interact with a single-stranded DNA substrate with positively charged amino acid residues or hydrophobic amino acid residues, Wherein the amino acid sequence of the wild-type AaCas b nuclease is shown as SEQ ID NO. 91.

Description

Compositions and methods for treating hemoglobin disorders Technical Field The application relates to the field of biological medicine, in particular to a molecule for treating hemoglobin diseases, a composition containing the molecule, a method and application thereof. Background Hemoglobinopathies are a very heterogeneous class of hereditary anaemia, and can include alpha-hemoglobinopathies and beta-hemoglobinopathies, as well as abnormal hemoglobin quantification, based on the lack of globin chain classification. Beta-thalassemia is a heterogeneous autosomal recessive genetic anemia characterized by reduced or absent beta-globin chain synthesis resulting in a disease associated with hemoglobin a (HbA). Excessive type a hemoglobin chains can lead to dyserythropoiesis, erythrocyte precursor intramedullary apoptosis, and hemolytic anemia. Beta-thalassemias are clinically divided into two types, depending on the severity of the symptoms, heavy beta-thalassemias (or β0 in which case the production of the beta-globin chain ceases due to mutation) and medium beta-thalassemias (or β+ in which case the production of the beta-globin chain decreases). Severe beta thalassemia is a serious medical condition requiring routine blood transfusion. Sickle Cell Disease (SCD) is also a hereditary blood disease caused by defects in β -globin chain production, which includes sickle cell anemia, sickle hemoglobin C disease (HbSC), sickle β+ thalassemia (HbS/β+) and sickle β 0 thalassemia (HbS/β0). Studies have shown that all forms of beta-hemoglobinopathy are caused by mutations in the beta-globin structural gene (HBB). Beta-thalassemia is caused by mutations in more than 200 different beta-globin genes. SCD is caused by an a to T point mutation in the sixth codon, which results in E6V substitution in defective β -globin (β s). The human β -globin locus consists of five genes located in the short region of chromosome 11 and is responsible for producing the β -globin chain of HAb. The locus comprises delta, gamma-A (HBG 1), gamma-G (HBG 2) and epsilon globin in addition to the beta-globin gene. A16 kb long Locus Control Region (LCR) located upstream of the HBB locus to 40-60kb is thought to regulate the differential expression of the beta-globin-like gene throughout development. In the late neonatal period, the gamma-A and gamma-G expression of the fetus is inhibited, and the beta-globin gene of the adult starts to express, which is called gamma-to-beta globin expression conversion. Fetal hemoglobin (HbF) Hereditary Persistence (HPFH) is a benign lesion, and a large amount of HbF is still produced after adulthood. Heterozygote patients with HPFH and β -hemoglobinopathy have mild clinical symptoms, while homozygous patients with these diseases have severe symptoms. HPFH is typically caused by mutations in the β -globin gene cluster or the γ -globin promoter region that either create new binding sites for the red blood cell activator or disrupt the binding sites for the inhibitor. The point mutations at the proximal promoters of HBG1 and HBG2 are divided into two distinct groups, approximately 115bp and 200bp upstream of the Transcription Start Site (TSS) of the replicated gamma-globin gene, respectively. The inhibitor that plays a major role in gamma-globin gene silencing is BCL11A, which binds to-115 bp. Recently, ZBTB7A, also known as LRF or FBI-1, was identified as the second major inhibitor of binding to the site at-200 bp. These findings indicate that introducing artificial HFPH-like mutations into the promoter region to reactivate gamma-globin gene expression is a therapeutic strategy with application prospects for beta-hemoglobinopathies. In China, although improvement in clinical treatment reduced mortality in infants, treatment of most patients with β -hemoglobin is still dominated by supportive treatment. Current treatments aim to alleviate symptoms and treat complications, including regular blood transfusion, inhibition of erythropoiesis, iron removal, and pain relief. Human Leukocyte Antigen (HLA) identical siblings Bone Marrow Transplantation (BMT) or HLA matched donor umbilical Cord Blood Transplantation (CBT) is another viable treatment, but at risk of Graft Versus Host Disease (GVHD). However, allogeneic BMT is only suitable for a small fraction of patients with β -hemoglobin, as most patients cannot find a perfectly matched non-related donor. The novel therapeutic approach using autologous transgenic HSPCs can avoid the need for perfectly matched bone marrow or cord blood donors, thereby avoiding the risk of GVHD after transplantation. Whole genome association studies have shown that some Single Nucleotide Polymorphisms (SNPs) located on the erythrocyte-specific enhancer at the BCL11A locus are associated with downregulation of BCL11A expression, sustained adult gamma-globin expression, and clinical manifestations of mild beta-hemoglobinopathies. Gene therapy with corrected beta-globin is also considered a possi