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JP-7857026-B2 - Compositions and methods for targeting BCL11A

JP7857026B2JP 7857026 B2JP7857026 B2JP 7857026B2JP-7857026-B2

Inventors

  • オークス，ベンジャミン
  • ヒギンズ，ショーン
  • デニー，サラ
  • スタール，ブレット ティ
  • コリン，イザベル
  • アディル，マルーフ
  • アーンズ，コール

Assignees

  • スクライブ・セラピューティクス・インコーポレイテッド

Dates

Publication Date
20260512
Application Date
20211202
Priority Date
20201203

Claims (20)

  1. A system comprising a chimeric CasX variant protein and a guide ribonucleic acid (gRNA) variant, wherein the gRNA variant is a) A scaffold containing the sequence of sequence number 2238 or a sequence having at least 90% sequence identity therewith, and b) A system comprising a targeting sequence complementary to a target nucleic acid sequence containing the B-cell lymphoma/leukemia 11A (BCL11A) gene.
  2. The aforementioned gRNA, a) BCL11A intron, b) BCL11A exon, c) BCL11A intron-exon junction, d) BCL11A regulatory element, and e) intergenetic region, The system according to claim 1, comprising a targeting sequence complementary to a target nucleic acid sequence selected from the group consisting of the following.
  3. The system according to claim 1, wherein the BCL11A gene includes a wild-type sequence.
  4. The system according to claim 1, wherein the gRNA variant is a single-molecule gRNA (sgRNA).
  5. The system according to claim 1, wherein the targeting sequence has a single nucleotide removed from its 3' end, or two, three, four, or five nucleotides removed from its 3 ' end.
  6. The system according to claim 1, wherein the targeting sequence of the gRNA variant is complementary to the sequence of the BCL11A regulatory element, and the regulatory element is selected from the group consisting of the promoter and enhancer regulatory elements of the BCL11A gene.
  7. The system according to claim 6, wherein the targeting sequence of the gRNA variant is complementary to the sequence of an enhancer regulatory element selected from the group consisting of the GATA1 erythrocyte-specific enhancer binding site (GATA1 ) of the BCL11A gene and a sequence located 5 ' to the GATA1 binding site of the BCL11A gene.
  8. The system according to claim 7, wherein the targeting sequence of the gRNA variant includes a sequence that has at least 90%, at least 95%, or at least 100% sequence identity with a sequence selected from the group consisting of UGGAGCCUGUGAAUAAAAGCA (SEQ ID NO: 22), UGCUUUUAUCACAGGCUUCCA (SEQ ID NO: 23), CAGGCUUCCAGGGAAGGUUGUUG (SEQ ID NO: 2949), GAGGCCAAACCCUUCCUUGGA (SEQ ID NO: 2948), AGUGCCAAGCUAACAGUUGCU (SEQ ID NO: 15747), and AUACACUUUGAAGGCUAGUC (SEQ ID NO: 15748 ).
  9. The system according to claim 1, wherein the targeting sequence is ligated to the 3' end of the scaffold of the gRNA variant.
  10. The system according to claim 1, wherein the chimeric CasX variant protein comprises the sequence of SEQ ID NO: 126 or a sequence having at least 90% sequence identity thereto.
  11. The chimeric CasX variant protein further comprises one or more nuclear localization signals (NLS), The one or more NLSs mentioned above, a) at or near the C-terminus of the chimeric CasX variant protein b) at or near the N-terminus of the chimeric CasX variant protein, or c) located at or near the N-terminus and/or near the C-terminus of the chimeric CasX variant protein The system according to claim 1.
  12. The chimeric CasX variant protein can form a ribonucleoprotein complex (RNP) with a gRNA variant, and the RNPs of the chimeric CasX variant protein and the gRNA variant exhibit at least one improved feature compared to the RNP of a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and a reference gRNA containing one of the sequences of SEQ ID NOs: 4-16, wherein the one or more improved features include improved folding of the chimeric CasX variant protein, improved binding affinity to gRNA, improved binding affinity to target DNA, and, in editing target DNA, one or more protospacer adjacent motifs including ATC, CTC, GTC, or TTC. The system according to claim 1, selected from one or more of the following: improved ability to utilize (PAM) sequences, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased nuclease activity, improved cleavage rate of target nucleic acid sequences, increased target strand loading for double-strand breaks, decreased target strand loading for single-strand nicking, decreased off-target breaks, improved binding of non-target DNA strands, improved protein stability, improved protein solubility, improved ribonucleoprotein complex (RNP) formation, higher percentage cleavage-competent RNPs, improved stability of protein:gRNA complexes (RNPs), improved solubility of protein:gRNA complexes, improved protein yield, improved protein expression, and improved fusion characteristics.
  13. The system according to claim 12, wherein if any one of the PAM sequences TTC, ATC, GTC, or CTC is located one nucleotide 5' to the non-target strand of a protospacer having identity with the targeting sequence of the gRNA in the cell assay system, the RNP containing the chimeric CasX variant protein and the gRNA variant exhibits higher editing efficiency and/or binding of the target nucleic acid sequence compared to the editing efficiency and/or binding of the RNP containing the reference CasX protein and reference gRNA in the comparative assay system.
  14. The PAM sequence is TTC, and the targeting sequence of the gRNA variant includes a sequence selected from the group consisting of SEQ ID NOs: 22, 23, 2949, 2948, 15747, and 15748 . The system according to claim 13 .
  15. The system according to claim 13, wherein the increased binding affinity to one or more PAM sequences is at least 1.5 times greater than the binding affinity to any one of the reference CasX proteins of SEQ ID NOs. 1 to 3 for the PAM sequences, and/or the RNPs have a percentage of cleavage-competent RNPs that are at least 5%, at least 10%, at least 15%, or at least 20% higher than the RNPs of the reference CasX protein and the reference gRNAs of SEQ ID NOs. 4 to 16 .
  16. One or more nucleic acids comprising a sequence encoding a chimeric CasX variant protein and a gRNA variant of the system according to any one of claims 1 to 15 .
  17. A nucleic acid comprising a sequence encoding a gRNA variant according to any one of claims 1 to 9 .
  18. A vector comprising a gRNA variant according to any one of claims 1 to 9 , a chimeric CasX variant protein according to any one of claims 11 to 15 , or one or more nucleic acids encoding the chimeric CasX variant protein or encoding or containing the gRNA variant .
  19. The vector according to claim 18, wherein the vector is selected from the group consisting of retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, virus-like particles ( VLPs ), plasmids, minicircles, nanoplasmides, DNA vectors, RNA vectors, lipid nanoparticles, and liposomes.
  20. The vector according to claim 18 , wherein the vector is an AAV vector, and the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, and AAVRh10.

Description

Cross-reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63/1200233,885, filed on 3 December 2020, the contents of which are incorporated herein by reference in their entirety. Advocacy by Reference of Sequence Listing This application includes a sequence listing in ASCII format submitted via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy was created on 1 December 2021, named SCRB_030_01WO_SeqList_ST25.txt, and has a file size of 8.78 MB. Fetal hemoglobin (similarly hemoglobin F, HbF, or α2γ2) is the primary oxygen carrier protein in the human fetus. HbF has a different composition from adult hemoglobin, allowing it to bind more strongly to oxygen and enabling the developing fetus to reabsorb oxygen from the mother's bloodstream. HbF is a tetramer of two adult α-globin polypeptides and two fetal β-like γ-globin polypeptides. During pregnancy, the replicated γ-globin gene constitutes the main gene transcribed in the β-globin cluster. After birth, γ-globin is replaced by adult β-globin. This process is called the "fetal switch" and involves the expression of BCL11A (a regulator of HbF silencing) (Sankara, V.G., et al. Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A. Science 322(5909):1839-1842(2008), Liu, N., et al. Direct Promoter Repression by BCL11A Controls the Fetal to...). Adult Hemoglobin Switch. Cell 173(2):430 (2018). In healthy adults, the composition of hemoglobin is hemoglobin A (approximately 97%), hemoglobin A2 (2.2–3.5%), and hemoglobin F (<1%) (Thomas, Can and Lumb, A.B. Physiology of hemoglobin. Continuing Education in Anaesthesia Critical Care & Pain. 12(5):251–256 (2012)). Hemoglobin disorders are, in most cases, hereditary monogenic disorders inherited as autosomal codominant traits. Common hemoglobin disorders include sickle cell anemia, as well as alpha-thalassemia and beta-thalassemia. Hemoglobin disorders are most common in people from Africa, the Mediterranean region, and Southeast Asia. Most hemoglobin disorders, including sickle cell anemia, are simply structural abnormalities of the globin protein itself. Sickle cell anemia arises from a point mutation in the β-globin structural gene HBB, resulting in the production of abnormal hemoglobin (HbS) and a decrease in the blood's oxygen-carrying capacity. Thalassemia, in contrast, usually results in insufficient production of normal globin protein, often leading to a deficiency or absence of adult hemoglobin (HbA) through mutations in regulatory genes. In β-thalassemia, β-globin is deficient, and increased γ-globin expression reduces the α-globin-β-globin imbalance underlying the pathophysiology of anemia in this condition (Liu, N., et al. Direct Promote Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch. Cell 173(2):430 (2018)). Both sickle cell disease and thalassemia can cause anemia. B-cell lymphoma/leukemia 11A (BCL11A) is a protein encoded by the BCL11A gene in humans. During hematopoietic cell differentiation, this gene is downregulated and has been found to play a role in suppressing fetal hemoglobin production. BCL11A is the major repressor protein for hemoglobin F production by binding to the gene encoding the γ subunit in its promoter region (Sankara VG, et al. Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 322:1839 (2008)). Increased gamma globin reduces the clinical severity of beta-abnormal hemoglobin disorders, sickle cell disease, and beta-thalassemia, which are caused by mutations or decreased expression of beta-globin. Therefore, gene editing of BCL11A to increase gamma globin expression beyond the remaining approximately 1% of fetal hemoglobin has been proposed as an attractive therapeutic strategy in adults with abnormal hemoglobin disorders (Smith, E.C., et al. Strict in vivo specification of the Bcl11a hemoglobin enhancer. Blood 128(19):2338(2016)). The emergence of CRISPR/Cas systems and the programmable nature of these minimal systems has facilitated their use as versatile technologies for genomic manipulation and engineering. To date, the use of CRISPR/Cas systems for the treatment of hemoglobin disorders has been limited to ex vivo cell editing followed by transplantation into subjects suffering from the underlying hemoglobin disorder. Therefore, in subjects with these diseases, there is a need for compositions and methods to modulate BCL11A to reduce direct in vivo suppression of the γ-globin gene promoter. To address this need, compositions and methods for targeting the BCL11A gene are provided herein. This disclosure relates to a composition of a modified class 2, type V CRISPR protein and a guide nucleic acid used to modify a target nucleic acid containing the BCL11A gene in cells. The class 2, type V CRISPR protein and guide nucleic acid are modified to passively enter target cells. The