EDITING OF HAEMOGLOBIN GENES

Abstract

The present invention relates to a process for producing a modified nucleic acid, wherein the nucleic acid comprises a mutant haemoglobin B (HBB) gene encoding a mutant Hb-β polypeptide. The process comprises using a base editor, preferably with a gRNA, to edit the mutant HBB gene to change a first (mutant) codon in that gene into a second, non-wild-type codon, wherein the Hb-β polypeptide encoded by that edited HBB gene has a non-wild-type, yet phenotypically-viable, amino acid sequence. The invention also provides a population of isolated haematopoietic stem cells, the stem cells comprising edited HBB genes.

Claims

1. A process for producing a modified nucleic acid molecule, the process comprising the steps: (a) contacting a nucleic acid molecule comprising a mutant HBB gene encoding a mutant Hb-β polypeptide with a base editor, wherein the mutant HBB gene comprises a first non-wild-type codon coding for a first non-wild-type amino acid; and (b) incubating the mutant HBB gene and base editor under conditions such that the base editor is targeted to the nucleotide sequence of the first non-wild-type codon and wherein the base editor edits one or more nucleotides in the first non-wild-type codon to produce a second non-wild-type codon which codes for a second non-wild-type amino acid, thereby producing a modified nucleic acid molecule comprising an edited HBB gene which encodes an edited Hb-β polypeptide, wherein the edited Hb-β polypeptide has a non-wild-type, yet phenotypically-viable, amino acid sequence.

2. A process as claimed in claim 1, wherein: Step (a) comprises contacting a nucleic acid molecule comprising a mutant HBB gene encoding a mutant Hb-β polypeptide with a base editor and a gRNA, wherein the mutant HBB gene comprises a first non-wild-type codon coding for a first non-wild-type amino acid and wherein the gRNA is capable of targeting the base editor to the nucleotide sequence of the first non-wild-type codon of the mutant HBB gene; and Step (b) comprises incubating the mutant HBB gene, base editor and gRNA under conditions such that the gRNA targets the base editor to the nucleotide sequence of the first non-wild-type codon and wherein the base editor edits one or more nucleotides in the first non-wild-type codon to produce a second non-wild-type codon which codes for a second non-wild-type amino acid, thereby producing a modified nucleic acid molecule comprising an edited HBB gene.

3. A process as claimed in claim 1, wherein the HBB gene is a mammalian gene, or a human gene.

4. The process as claimed in claim 1, wherein mutant HBB gene consists of or comprises a nucleotide sequence which encodes an amino acid sequence having 90-99.5%, or 95-99.5% sequence identity to SEQ ID NO: 2.

5. The process as claimed in claim 4, wherein the nucleotide sequence at the codon which corresponds to codon 7 in SEQ ID NO: 1 codes for lysine or valine; and/or the nucleotide sequence at the codon which corresponds to codon 27 in SEQ ID NO: 1 codes for lysine.

6. The process as claimed in claim 5, wherein the nucleotide sequence at the first non-wild-type codon which corresponds to codon 7 in SEQ ID NO: 1 is AAG or GTG; and/or the nucleotide sequence at the first non-wild-type codon which corresponds to codon 27 in SEQ ID NO: 1 is AAG.

7. The process as claimed in wherein the base editor is a programmable nucleic acid binding protein (or an impaired CRISPR-Cas9 mutant, which is capable of being targeted to a target DNA sequence.

8. The process as claimed in claim 7, wherein the base editor is an adenine deaminating editor.

9. The process as claimed in claim 2, wherein: (i) the guide RNA sequence for editing codon 7 is an 18-22 nucleotide guide RNA which is complementary to a nucleotide sequence located in SEQ ID NO: 15, wherein the wild-type complement of codon 7 (CTC) is replaced by CAC; or (ii) the guide RNA sequence for editing codon 27 is an 18-22 nucleotide guide RNA which is located in SEQ ID NO: 16, wherein the wild-type codon 27 (GAG) is replaced by AAG; or (iii) the guide RNA sequence for editing codon 7 is an 18-22 nucleotide guide RNA which is located in SEQ ID NO: 17, wherein the wild-type of codon 7 (GAG) is replaced by AAG.

10. The process as claimed in wherein the edited HBB gene consists of or comprises: (i) a nucleotide sequence having 90-99.9% nucleotide sequence identity to SEQ ID NO: 1 or a nucleotide sequence encoding an amino acid sequence having 95-99.5% amino acid sequence identity to SEQ ID NO: 2; and wherein (ii) the nucleotide sequence at the codon which corresponds to codon 7 in SEQ ID NO: 1 codes for glycine or alanine; and/or the nucleotide sequence at the codon which corresponds to codon 27 in SEQ ID NO: 1 codes for glycine.

11. The process as claimed in claim 1, wherein: (i) the position of the first non-wild-type codon is codon 7, the wild-type codon at this position is GAG (glutamate), the first non-wild-type (mutant) codon sequence is AAG (lysine), the base editor is an adenine base editor and the second non-wild-type codon is GGG (glycine); or (ii) the position of the first non-wild-type codon is codon 7, the wild-type codon at this position is GAG (glutamate), the first non-wild-type (mutant) codon sequence is GTG (valine), the base editor is an adenine base editor and the second non-wild-type codon is GCG (alanine); or (iii) the position of the first non-wild-type codon is codon 27, the wild-type codon at this position is GAG (glutamate), the first non-wild-type (mutant) codon sequence is AAG (lysine), the base editor is an adenine base editor and the second non-wild-type codon is GGG (glycine).

12. The process as claimed in claim 1, which additionally includes, prior to Step (a), the step of obtaining a sample of haematopoietic stem cells from a subject, or from a human subject, wherein the stem cells comprise nucleic acid molecules comprising mutant HBB genes.

13. The process as claimed in claim 1, which additionally comprises, prior to Step (a), the step of modifying the nucleotide sequences of one or more PAM sites in the vicinity of the first non-wild-type codon in order to increase efficiency of the base-editing process.

14. The process as claimed in claim 1, wherein the process is performed on haematopoietic stem cells which have previously been obtained from a first subject, or from a human subject, wherein the stem cells comprise nucleic acid molecules comprising mutant HBB genes.

15. The process as claimed in claim 14, the process additionally comprises the subsequent step of introducing a population of haematopoietic stem cells comprising modified nucleic acid molecules comprising edited HBB genes, optionally after expansion of the cells, into a second subject, wherein the first and second subjects are the same or related subjects.

16. A population of isolated cells comprising haematopoietic stem cells or progenitor cells comprising edited HBB genes, the edited HBB genes comprising: (i) a nucleotide sequence having 90-99.9% nucleotide sequence identity to SEQ ID NO: 1 or a nucleotide sequence encoding an amino acid sequence having 95-99.5% amino acid sequence identity to SEQ ID NO: 2; and wherein (ii) the nucleotide sequence at the codon which corresponds to codon 7 in SEQ ID NO: 1 codes for glycine or alanine; and/or the nucleotide sequence at the codon which corresponds to codon 27 in SEQ ID NO: 1 codes for glycine.

17. The population of isolated cells as claimed in claim 16, wherein the population of isolated cells comprises at least 20% haematopoietic stem cells or progenitor cells having modified nucleic acid molecules comprising edited HBB genes, or at least 40%, at least 60%, at least 80% or 100% haematopoietic stem cells or progenitor cells having modified nucleic acid molecules comprising edited HBB genes.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0121] FIG. 1. Haemoglobin E is caused by a mutation of codon 27 of the beta globin gene (GAG to AAG). This mutation can be corrected to its canonical sequence using Cas9-ABE7.10 or ABEmax and the guide RNA shown. This enzyme has a 4 bp window and shows processivity meaning that the base will not be corrected simply back to its canonical sequence—it is likely that most cells will be corrected to GGG, which results in the variant haemoglobin Hb Aubenas, which has a normal phenotype. Another variant haemoglobin is also possible but less likely (Hb R27).

[0122] FIG. 2. Sickle cell disease results in conversion of GAG (Glutamate) at position 7 to GTG (Valine). This can be corrected by base editor xCas9-ABE7.10 to GCG (which encodes HbG-Makassar) through editing the Adenine on the opposite strand to Guanine. Alanine at position 7 is described in the literature as HbG-Makassar, which has a normal phenotype. It is likely editing efficiency could be improved by mutating the codon 2 from GTG (valine) to GCG (alanine) which makes a more efficient protospacer active motif (PAM), for the editing of the HbS mutation. Both of these guide RNAs could be used simultaneously.

[0123] FIG. 3. Haemoglobin C (which is the third most important disease causing variant) could also be corrected using base editors to another variant haemoglobin (Hb Lavagna) that has a normal phenotype.

[0124] FIGS. 4A and 4B. Experimental overview of production of the Hb Aubenas variant in wild type CD34.sup.+ human haemopoietic stem and progenitor cells using ABEmax.

[0125] FIG. 5. Creation of over 50% editing of wild type codon 27 from glutamate to glycine (Hb Aubenas).

[0126] FIG. 6. Editing of haemopoietic stem cells from patients with HbE-beta0 thalassaemia.

EXAMPLES

[0127] The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: Production of the Hb Aubenas Variant in Wild Type CD34+ Human Haemopoietic Stem and Progenitor Cells Using ABEmax

[0128] FIG. 4 shows the experimental overview. CD34+ cells were isolated from peripheral blood white cell cones derived from blood donation. These cells were then electroporated with the ABEmax-P2A-GFP plasmid (this was a gift from David Liu Addgene #112101) a separate plasmid to express the guide RNA. GFP positive cells were sorted; cultured for 48h and DNA was extracted and the editing efficiency was assessed by Sanger sequencing.

[0129] FIG. 5 shows that this strategy is capable of creating over 50% editing of wild type codon 27 from glutamate to glycine (Hb Aubenas). Thus it is highly likely that the adjacent adenine will also be base converted to guanine in haemoglobin E, i.e. that AAG will be converted to GAG or GGG.

Example 2: Editing of the HbE Variant in HUDEP Cells

[0130] Human Umbilical cord blood Derived Erythroid Progenitor (HUDEP) cells serve as a good model for human red blood cell production. The HbE mutation was generated in HUDEP cells using spCas9 ribonuclear protein (RNP) and homologous recombination with a single stranded donor template. Cells were sorted into single cell colonies, expanded and genotyped to give a pure population of homozygous cells with the HbE mutation.

[0131] These cells with the HbE mutation were then edited with the ABE 7.10 and the ABEmax base editors using plasmids for the base editors and guide RNAs. A gene editing efficiency of over 80% to Hb Aubenas/WT can be achieved with the ABEmax base editor.

Example 3: Editing of Human CD34+ Haemopoietic Stem and Progenitor Cells from Patients with HbE Related Thalassaemia

[0132] CD34+ cells are isolated from patients with the haemoglobin E mutation using MACS beads (Miltenyi). These cells are edited using the ABE 7.10 and ABEmax base editors using electroporation with a plasmid for the base editor and a plasmid to express the guide RNA.

Example 4: Editing of Human CD34+ Haemopoietic Stem and Progenitor Cells from Patients with Sickle Cell Disease

[0133] CD34+ cells are isolated from patients with the homozygous haemoglobin S mutation using MACS beads (Miltenyi). These cells are edited using the ABE 7.10 and ABEmax base editors using electroporation with a plasmid for the base editor and a plasmid to express the guide RNA.

Example 5: Editing of Haemopoietic Stem Cells from Patients with HbE-Beta0 Thalassaemia

[0134] Patient-derived CD34+ cells were incubated with two plasmids, the base editor containing plasmid ABEmax and a plasmid containing the gRNA and green fluorescent protein (GFP). The cells were electroporated and cultured for 24 h. They were subsequently sorted for GFP positive cells. These cells were cultured for another few days prior to harvesting for DNA. The HBB gene was amplified using PCR and the sequence obtained using high throughput sequencing.

[0135] The results are shown in FIG. 6. This shows that the thalassaemic allele was unedited, but that the beta E allele was converted to the sequence for Hb Aubenas in 49.6% of alleles and WT in 36.5%. 3.9% were converted to a previously-undescribed Hb variant.

REFERENCES

[0136] BLACKWELL, R. Q., OEMIJATI, S., PRIBADI, W., WENG, M. I. & LUI, C. S. 1970. Hemoglobin-G Makassar—Beta6 Glu-!Ala. Biochimica Et Biophysica Acta, 214, 396-+. [0137] GAUDELLI, N. M., KOMOR, A. C., REES, H. A., PACKER, M. S., BADRAN, A. H., BRYSON, D. I. & LIU, D. R. 2017. Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage. Nature, 551, 464-+. [0138] HU, J. H., MILLER, S. M., GEURTS, M. H., TANG, W. X., CHEN, L. W., SUN, N., ZEINA, C. M., GAO, X., REES, H. A., LIN, Z. & LIU, D. R. 2018. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 556, 57-+. [0139] JINEK, M., CHYLINSKI, K., FONFARA, I., HAUER, M., DOUDNA, J. A. & CHARPENTIER, E. 2012. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337, 816-821. [0140] KOBLAN, L. W., DOMAN, J. L., WILSON, C., LEVY, J. M., TAY, T., NEWBY, G. A., MAIANTI, J. P., RAGURAM, A. & LIU, D. R. 2018. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol. [0141] KOMOR, A. C., KIM, Y. B., PACKER, M. S., ZURIS, J. A. & LIU, D. R. 2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533, 420-4. [0142] KOMOR, A. C., ZHAO, K. T., PACKER, M. S., GAUDELLI, N. M., WATERBURY, A. L., KOBLAN, L. W., KIM, Y. B., BADRAN, A. H. & LIU, D. R. 2017. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv, 3, eaao4774. [0143] KURITA, R., SUDA, N., SUDO, K., MIHARADA, K., HIROYAMA, T., MIYOSHI, H., TANI, K. & NAKAMURA, Y. 2013. Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells. PLoS One, 8, e59890. [0144] LACAN, P., FRANCINA, A., PROME, D., DELAUNAY, J., GALACTEROS, F. & WAJCMAN, H. 1996. Hb Aubenas [beta 26(B8)Glu->Gly]: A new variant normally synthesized, affecting the same codon as in Hb E. Hemoglobin, 20, 113-124. [0145] METTANANDA, S., FISHER, C. A., HAY, D., BADAT, M., QUEK, L., CLARK, K., HUBLITZ, P., DOWNES, D., KERRY, J., GOSDEN, M., TELENIUS, J., SLOANE-STANLEY, J. A., FAUSTINO, P., COELHO, A., DOONDEEA, J., USUKHBAYAR, B., SOPP, P., SHARPE, J. A., HUGHES, J. R., VYAS, P., GIBBONS, R. J. & HIGGS, D. R. 2017. Editing an alpha-globin enhancer in primary human hematopoietic stem cells as a treatment for beta-thalassemia. Nat Commun, 8, 424. [0146] NISHIDA, K., ARAZOE, T., YACHIE, N., BANNO, S., KAKIMOTO, M., TABATA, M., MOCHIZUKI, M., MIYABE, A., ARAKI, M., HARA, K. Y., SHIMATANI, Z. & KONDO, A. 2016. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, 353. [0147] OLD, J., HARTEVELD, C. L., TRAEGER-SYNODINOS, J., PETROU, M., ANGASTINIOTIS, M. & GALANELLO, R. 2012. In: ND (ed.) Prevention of Thalassaemias and Other Haemoglobin Disorders: Volume 2: Laboratory Protocols. Nicosia, Cyprus. [0148] TRAKARNSANGA, K., GRIFFITHS, R. E., WILSON, M. C., BLAIR, A., SATCHWELL, T. J., MEINDERS, M., COGAN, N., KUPZIG, S., KURITA, R., NAKAMURA, Y., TOYE, A. M., ANSTEE, D. J. & FRAYNE, J. 2017. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nat Commun, 8, 14750. [0149] VIPRAKASIT, V., WIRIYASATEINKUL, A., SATTAYASEVANA, B., MILES, K. L. & LAOSOMBAT, V. 2002. Hb G-Makassar [beta 6(A3)Glu->Ala; codon 6 (GAG->GCG)]: Molecular characterization, clinical, and hematological effects. Hemoglobin, 26, 245-253.

TABLE-US-00003 SEQUENCES SEQ ID NO: 1 Genomic DNA sequence of the wild-type human HBB gene (excluding 5′UTR) ATGGTGCATCTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGG CAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGTTGGTAT CAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGA GACAGAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTAT TGGTCTATTTTCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAG AGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGG CAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTGCTCGGTGCCTTTAGTG ATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACACTGAGT GAGCTGCACTGTGACAAGCTGCACGTGGATCCTGAGAACTTCAGGGTGAG TCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTT CATGTCATAGGAAGGGGATAAGTAACAGGGTACAGTTTAGAATGGGAAAC AGACGAATGATTGCATCAGTGTGGAAGTCTCAGGATCGTTTTAGTTTCTT TTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCT TTTTTTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATT GTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAA AAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGC TTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATT GATACATAATCATTATACATATTTATGGGTTAAAGTGTAATGTTTTAATA TGTGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAA AAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATA CTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGC CTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCA ATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGAT GTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTG CTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAG GCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCT GGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCA CCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAAT GCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCT ATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATT ATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTT CATTGC SEQ ID NO: 2 Amino acid sequence of the wild-type human HBB polypeptide. MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLS TPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVD PENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH SEQ ID NOs: 3 and 4 gRNA genomic target: TGGTAAGGCCCTGGGCAGGT RNA sequence: UGGUAAGGCCCUGGGCAGGU SEQ ID NOs: 5 and 6 gRNA genomic target: TTCTCCACAGGAGTCAGATG RNA sequence: UUCUCCACAGGAGUCAGAUG SEQ ID NOs: 7 and 8 gRNA genomic target: TTCTCCACAGGAGTCAGGTG RNA sequence: UUCUCCACAGGAGUCAGGUG SEQ ID NOs: 9 and 10 gRNA genomic target: AGATGCACCATGGTGTCTGT RNA sequence: AGAUGCACCAUGGUGUCUGU SEQ ID NOs: 11 and 12 gRNA genomic target: AGGTGCACCATGGTGTCTGT RNA sequence: AGGUGCACCAUGGUGUCUGU SEQ ID NOs: 13 and 14 gRNA genomic target: TCCTAAGGAGAAGTCTGCCG RNA sequence: UCCUAAGGAGAAGUCUGCCG SEQ ID NO: 15 5′-end of genomic DNA sequence of the wild-type human HBB gene covering potential gRNAs for editing HbS: GACTTCTCCACAGGAGTCAGATGCACCAT SEQ ID NO: 16 Genomic DNA sequence of the wild-type human HBB gene covering potential gRNAs for editing HbE: GAAGGTGGTAAGGCCCTGGGCAGGTTGGT SEQ ID NO: 17 Genomic DNA sequence of the wild-type human HBB gene covering potential gRNAs for editing HbC: CTGACTCCTAAGGAGAAGTCTGCCGTTAC