Epigenetic reactivation of gamma-globin expression as a novel curative option for Β-hemoglobinopathies

Abstract

Here, the inventors developed a strategy to modify the chromatin status of HSPCs at the HBG promoters to reactivate HbF expression. In particular, the inventors used from 1 to 4 single guide RNA (sgRNA) molecules spanning from the -220 to the -20 region of the HBG promoters together with CRISPR-based epigenome editors, namely dCas9-CBPcore and Tet1-dCas9. The first editor is capable of inserting histone acetylation while the second editor is performing DNA demethylation. Both epigenetic marks are associated with active transcription and are present in fetal erythroid cells expressing HbF. This strategy allows the ex vivo modification of adult HSCs to recreate a fetal-like epigenetic context leading to HbF production. Moreover, it avoids the genotoxicity associated with classical genome editing tools relying on DNA cleavage. Thus, the present invention relates to a method of increasing the expression of gamma globin in a population of eukaryotic cells through epigenome editing.

Description

[0001] EPIGENETIC REACTIVATION OF GAMMA-GLOBIN EXPRESSION AS A NOVEL CURATIVE OPTION FOR B-HEMOGLOBINOPATHIES BACKGROUND OF THE INVENTION

[0002] Sickle cell disease (SCD) and P-thalassemia are genetic diseases caused by mutations in the P-globin locus. In SCD, a point mutation in the HBB gene leads to the formation of the sickle PS-globin chain, which causes the polymerization of sickle hemoglobin (HbS), red blood cell (RBC) sickling, anemia, and organ damage1,2. In P-thalassemia, the partial or total absence of P-globin chains (caused by P+ and po mutations, respectively) leads to the precipitation of noncoupled a-globin chains, apoptosis of erythroid precursors, ineffective erythropoiesis, and anemia3 5. Gene therapy approaches based on the transplantation of autologous, genetically modified hematopoietic stem and progenitor cells (HSPCs) have been investigated as a treatment option for patients lacking a compatible donor for allogeneic HSPC transplantation6. The severity of both SCD and P-thalassemia is lessened by the hereditary persistence of fetal hemoglobin (HbF) in adulthood (HPFH)7. This persistence is due to mutations located 200 to 115 nucleotides upstream of the transcription start sites of the identical HBG1 and HBG2 y-globin promoters. HPFH mutations either generate de novo DNA motifs recognized by transcriptional activators (e.g., KLFI )8 10or disrupt binding sites for transcriptional repressors (e.g., LRF and BCL11A)11. CRISPR-Cas9 nuclease strategies have been exploited to disrupt the LRF and BCL11A repressor binding sites by generating small insertions / deletions (InDeis) that mimic HPFH mutations and reactivate HbF expression12 l5. Recently, base editors, composed of a Cas9 nickase and a deaminase that can either introduce C-to-T or A-to-G point mutations, were developed16and used to introduce HPFH and HPFH-like mutations in the -200 region of the HBG1 / 2 promoters17. This technology, while being more precise and safer than the canonical CRISPR-Cas9 nuclease, can still induce some DNA double-strand breaks (DSB) or generate two single-strand cleavages when targeting the two identical HBG promoters. This can cause the deletion of a 4.9-kb sequence comprising the HBG2 gene and its promoter17. Furthermore, HSCs are highly sensitive to DSBs especially in case of multiple on-target events or concomitant on-target and off-target events18. The Cas9-sgRNA treatment of human HSPCs induces a DNA damage response (DDR) that can lead to apoptosis19,20. In particular, CRISPR-Cas9 can cause p53-dependent cell toxicity and cell cycle arrest, resulting in the selection of cells with a dysfunctional p53 pathway21. Furthermore, the generation of several DSBs can lead to genomic deletions, inversions, translocations, chromosome loss, and chromothripsi s22 25.

[0003] In addition to transcription factors, chromatin-remodelling and modifier complexes (such as the nucleosome remodelling and deacetylase complex) also play a critical role in the y-to-P-globin switch. These complexes include enzymes capable of DNA methylation and demethylation, such as the DNA methyltransferase 3A (DNMT3A) and the ten-eleven translocation methylcytosine dioxygenase 1 (TET1), respectively. Furthermore, histone modifiers including histone acetyltransferases (HATs) and histone deacetylases (HDACs), are implicated in hemoglobin switch. DNMT inhibitors such as decitabine or the HD AC inhibitor vorinostat have been reported to increase y-globin expression26,27. However, the mode of action of these drugs remains uncertain and their non-specific, global effects pose serious safety concerns. Similarly, downregulation of genes encoding components of chromatin-remodelling and modifier complexes was associated to HbF upregulation but, in many cases, this led to impaired cell fitness as these factors play a pleiotropic role on the epigenetic and transcriptional profiles28,29.

[0004] Recently, epigenome editors have been developed and used for precise gene modulation30 32. These tools are composed of a catalytically inactive dead Cas9 (dCas, which is not capable to cleave DNA) fused with effectors that are able to introduce epigenetic modifications such as histone acetylation (e.g. dCas9-P300) or DNA demethylation (e.g. dCas9-Tetl)33,34. In the context of P-hemoglobinopathies, these DNA-cleavage-free technologies have been used to reactivate y globin expression in HEK293T cells33,35 37Therefore, the development of novel, efficacious and safe strategies to modify primary HSPCs through CRISPR-based epigenome editors (and achieve HbF reactivation in their erythroid progeny) is highly desirable.

[0005] SUMMARY OF THE INVENTION

[0006] The present invention is defined by the claims. In particular, the present invention relates to a method of increasing the expression of gamma globin in a population of eukaryotic cells through epigenome editing.

[0007] DETAIL DESCRIPTION OF THE INVENTION

[0008] Here, the inventors developed a strategy to modify the chromatin status of HSPCs and their progeny at the HBG promoters to reactivate HbF expression. In particular, the inventors used from 1 to 4 single guide RNA (sgRNA) molecules spanning from the -220 to the -20 region of the HBG promoters together with CRISPR-based epigenome editors, namely dCas9-CBPcore and Tetl-dCas9. The first editor is capable of inserting histone acetylation while the second editor is performing DNA demethylation. Both epigenetic marks are associated with active transcription and are present in fetal erythroid cells expressing HbF. This strategy allows the ex vivo modification of adult HSCs to recreate a fetal-like epigenetic context leading to HbF production. Moreover, it avoids the genotoxicity associated with classical genome editing tools relying on DNA cleavage.

[0009] Main definitions:

[0010] As used herein, the term “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product”. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Any method known in the art can be used to measure the expression of the gene (e. g. HPLC analysis of protein and RT-qPCR analysis of mRNA.)

[0011] As used herein, the expression "increasing the fetal hemoglobin content” indicates that fetal hemoglobin is at least 5% higher in the eukaryotic cell treated with the epigenome editing platform, than in a comparable, eukaryotic cell, wherein an epigenome editing platform targeting an unrelated locus is present or where no epigenome editing platform is present. In some embodiments, the percentage of fetal hemoglobin expression in the eukaryotic cell is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 1-fold higher, at least 2-fold higher, at least 5-fold higher, at least 10-fold higher, at least 100 fold higher, at least 1000-fold higher, or more than an eukaryotic cell.

[0012] As used herein, the term "gene" refers to a fundamental unit of heredity that encompasses a sequence of nucleotides encoding a specific polypeptide or functional RNA molecule. A gene comprises not only the coding sequence responsible for producing the gene product but also the non-coding sequences that may play roles in regulating gene expression, such as introns and untranslated regions (UTRs). Additionally, a gene includes the promoter region, which is essential for initiating the transcription of the coding sequence.

[0013] As used herein, the term “gamma globin” or “y-globin” has its general meaning in the art and refers to protein that is encoded in human by the HBG1 and HBG2 genes. The HBG1 and HBG2 genes are normally expressed in the fetal liver, spleen and bone marrow. Two y-globin chains together with two a-globin chains constitute fetal hemoglobin (HbF) which is normally replaced by adult hemoglobin (HbA) in the year following birth. The ENSEMBL IDs (i.e. the gene identifier number from the Ensembl Genome Browser database) for HBG1 and HBG2 are ENSG00000213934 and ENSG00000196565 respectively.

[0014] As used herein, the term “promoter” has its general meaning in the art and refers to a nucleic acid sequence which is required for expression of a gene operably linked to the promoter sequence. As used herein, the term HBG1 promoter” refers to the promoter of the HBG1 gene. As used herein, the term “HBG2 promoter” refers to the promoter of the HBG2 gene. HBG1 and HBG2 promoters are identical up to -221 bp upstream the TSSs of these genes.

[0015] As used herein, the term “fusion protein” means a protein created by joining two or more polypeptide sequences together. The fusion polypeptides encompassed in this invention include translation products of a chimeric gene construct that joins the nucleic acid sequences encoding a first polypeptide, e.g., an RNA-binding domain, with the nucleic acid sequence encoding a second polypeptide, e.g., an effector domain, to form a single open-reading frame. In other words, a “fusion protein” is a recombinant protein of two or more proteins which are joined by a peptide bond or via several peptides. The fusion protein may also comprise a peptide linker between the two domains.

[0016] As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

[0017] As used herein, the term “derived from” refers to a process whereby a first component (e.g., a first molecule), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second molecule that is different from the first).

[0018] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

[0019] As used herein, the term “linker” refers to any means, entity or moiety used to join two or more entities. A linker can be a covalent linker or a non-covalent linker. Examples of covalent linkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins or domains to be linked. The linker can also be a non-covalent bond, e.g., an organometallic bond through a metal center such as platinum atom. For covalent linkages, various functionalities can be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea and the like. To provide for linking, the domains can be modified by oxidation, hydroxylation, substitution, reduction etc. to provide a site for coupling. Methods for conjugation are well known by persons skilled in the art and are encompassed for use in the present invention. Linker moieties include, but are not limited to, chemical linker moieties, or for example a peptide linker moiety (a linker sequence). It will be appreciated that modification which do not significantly decrease the function of the RNA-binding domain and effector domain are preferred.

[0020] As used herein, the “linked” as used herein refers to the attachment of two or more entities to form one entity. A conjugate encompasses both peptide-small molecule conjugates as well as peptide-protein / peptide conjugates.

[0021] As used herein, the term “complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base-pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0022] As used herein, the term “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y.

[0023] As used herein, the term “hybridization” or “hybridizing” refers to a process where completely or partially complementary nucleic acid strands come together under specified hybridization conditions to form a double-stranded structure or region in which the two constituent strands are joined by hydrogen bonds. Although hydrogen bonds typically form between adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), other base pairs may form (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0024] As used herein, the term “epigenome editing” refers to a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites (as opposed to whole-genome modifications). Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question. The engineered proteins used for epigenome editing are composed of a DNA binding domain that target specific sequences and an effector domain that modifies epigenomic features. Currently, three major groups of DNA binding proteins have been predominantly used for epigenome editing: Zinc finger proteins, Transcription Activator-Like Effectors (TALEs) and nuclease deficient Cas9 fusions (CRISPR).

[0025] As used herein, the term “epigenome editing enzyme” or “epigenome editor” refers to any engineered molecule designed to alter the epigenomic features at specific genomic sites. These modifications do not change the DNA sequence but influence how genes are expressed by modifying the accessibility of DNA to transcription factors and other DNA-binding proteins. Such enzymes typically consist of a DNA binding domain that targets specific DNA sequences and an effector domain that induces epigenetic changes. Typically, the DNA binding domain is fused to the effector domain.

[0026] As used herein, the term “nuclease” includes an enzyme that induces a break in a nucleic acid sequence, e.g., a single or a double strand break in a double-stranded DNA sequence.

[0027] As used herein, the term “CRISPR / Cas nuclease” has its general meaning in the art and refers to segments of prokaryotic DNA containing clustered regularly interspaced short palindromic repeats (CRISPR) and associated nucleases encoded by Cas genes. In bacteria the CRISPR / Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids). Three types of CRISPR systems have been identified. CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA). The CRISPR / Cas nucleases Cas9 and Cpfl belong to the type II and type V CRISPR / Cas system and have strong endonuclease activity to cut target DNA. Cas9 is guided by a mature crRNA that contains about 20 nucleotides of unique target sequence (called spacer) and a transactivating small RNA (tracrRNA) that also serves as a guide for ribonuclease Ill-aided processing of pre-crRNA. The crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. Cas9 recognizes a trinucleotide (NGG for S. Pyogenes Cas9) protospacer adjacent motif (PAM) to specify the cut site (the 3rdor the 4thnucleotide upstream from PAM).

[0028] As used herein, the term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3’-5’ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., inek M., Chylinski K., Fonfara I., Hauer M., Doudna I. A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., I. ., McShan W. M., Ajdic D. ., Savic D. ., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., lia H. G., Najar F. Z., Ren Q., ZhuH., SongL., White I, Yuan X., Clifton S. W., RoeB. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by transencoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel ., Charpentier E., Nature 471 :602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” inek M., Chylinski K., Fonfara I., Hauer M., Doudna I. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. therm ophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the term “Cas9” refers to Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC 017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); or Neisseria, meningitidis (NCBI Ref: YP 002342100.1). Typically the Cas9 nuclease comprises the amino acid sequence as set forth in SEQ ID NO: 1

[0029] SEQ ID NO : 1 : Cas9 sequence MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEWD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0030] As used herein, the term “defective CRISPR / Cas nuclease” refers to a CRISPR / Cas nuclease having lost at least one nuclease domain. As used herein, the term “dead Cas9” refers to a CRISPR-associated protein 9 (Cas9) that has been engineered to be catalytically inactive, meaning it no longer has nuclease activity. This modified version of Cas9, also known as dCas9, retains its ability to bind to specific DNA sequences but does not introduce cuts in the DNA. The inactivation is typically achieved through specific point mutations in the nuclease domains of the protein. These mutations prevent the Cas9 from cleaving the DNA strands, thereby allowing it to serve as a programmable DNA-binding protein. The dead Cas9 can then be fused with various effector domains to modulate gene expression or epigenetic states in a targeted manner without causing double-strand breaks in the genome. As used herein, the term “protospacer adjacent motif sequence” or “PAM” refers to an approximately 2-6 base pair DNA sequence that is an important targeting component of a Cas9 nuclease. Typically, the PAM sequence is on either strand, and is downstream in the 5’ to 3’ direction of Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes is 5’-NGG-3’ wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease, e.g., SpCas9, may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence.

[0031] As used herein, the term “protospacer” refers to the sequence (~20 bp) in DNA adjacent to the PAM (protospacer adjacent motif) sequence. The protospacer shares the same sequence as the spacer sequence of the guide RNA. The guide RNA anneals to the complement of the protospacer sequence on the target DNA (specifically, one strand thereof, i.e., the “target strand” versus the “non-target strand” of the target sequence). In order for Cas9 to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand.

[0032] As used herein, the term “guide RNA molecule” generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a Cas9 protein and target the Cas9 protein to a specific location within a target DNA. A guide RNA can comprise two segments: a DNA-targeting guide segment and a protein-binding segment. The DNA-targeting segment comprises a nucleotide sequence that is complementary to (or at least can hybridize to under stringent conditions) a target sequence. The protein-binding segment interacts with a CRISPR protein, such as a Cas9 or Cas9 related polypeptide. These two segments can be located in the same RNA molecule or in two or more separate RNA molecules. When the two segments are in separate RNA molecules, the molecule comprising the DNA-targeting guide segment is sometimes referred to as the CRISPR RNA (crRNA), while the molecule comprising the protein-binding segment is referred to as the trans-activating RNA (tracrRNA). As used herein, the term “sgRNA” has its general meaning in the art and refers to a single guide RNA that is engineered to combine the functionalities of both the crRNA and tracrRNA into a single RNA molecule. As used herein, the term “spacer sequence” refers to a specific sequence of nucleotides within the CRISPR RNA (crRNA) that is complementary to the target DNA sequence. This spacer sequence is integral to guiding the Cas9 nuclease to the precise location in the genome where it will induce a double-strand break. The spacer ensures that the CRISPR / Cas system targets only the desired sequence, thus allowing for highly specific gene editing.

[0033] As used herein, the term “target nucleic acid” or “target” refers to a nucleic acid containing a target nucleic acid sequence. A target nucleic acid may be single-stranded or double-stranded, and often is double-stranded DNA. A “target nucleic acid sequence,” “target sequence” or “target region”, as used herein, means a specific sequence or the complement thereof that one wishes to bind to using the CRISPR system as disclosed herein.

[0034] As used herein, the term “CpG methylation” has its general meaning in the art and refers to the addition of a methyl group to the cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. CpG methylation typically occurs at the 5' position of the cytosine ring, forming 5-methylcytosine. This epigenetic modification is known to play a significant role in regulating gene expression, maintaining genomic stability, and influencing cellular differentiation and development.

[0035] As used herein, the term “histone acetyltransferase” has its general meaning in the art and refers to an enzyme that acetylates conserved lysine residues on histone proteins by transferring an acetyl group from acetyl-CoA to form s-N-acetyl lysine. This modification generally increases gene expression by relaxing chromatin structure, thereby making the DNA more accessible to transcription factors and other DNA-binding proteins. Examples of histone acetyltransferases include p300 and CREB-binding protein (CBP), which play crucial roles in regulating gene expression.

[0036] As used herein, the term “DNA demethylase” refers to an enzyme that removes methyl groups from DNA molecules. This process, known as demethylation, plays a crucial role in regulating gene expression and maintaining cellular identity. By reversing DNA methylation, demethylases enable dynamic and reversible modulation of the epigenetic landscape, allowing for precise control over gene activity in response to various biological signals.

[0037] As used herein, the term “ribonucleoprotein complex,” or “ribonucleoprotein particle” refers to a complex or particle including a nucleoprotein and a ribonucleic acid. A “nucleoprotein” as provided herein refers to a protein capable of binding a nucleic acid (e.g., RNA, DNA). Where the nucleoprotein binds a ribonucleic acid, it is referred to as “ribonucleoprotein.” The interaction between the ribonucleoprotein and the ribonucleic acid may be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).

[0038] As used herein, the term “hematopoietic stem cell” or “HSC” refers to blood cells that have the capacity to self-renew and to differentiate into precursors of blood cells. These precursor cells are immature blood cells that cannot self-renew and must differentiate into mature blood cells. Hematopoietic stem progenitor cells display a number of phenotypes, such as Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA-, Lin- CD34+CD38+IL-3aloCD45RA-, and Lin-CD34+CD38+CD 10+(Daley et al., Focus 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. IIP Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). Within the bone marrow microenvironment, the stem cells self-renew and maintain continuous production of hematopoietic stem cells that give rise to all mature blood cells throughout life. In some embodiments, the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells.

[0039] As used herein, the term "isolated cell" refers to a cell that has been removed from an organism in which it was originally found, or a descendant of such a cell. Optionally the eukaryotic cell has been cultured in vitro, e.g., in the presence of other cells. Optionally the eukaryotic cell is later introduced into a second organism or reintroduced into the organism from which it (or the cell from which it is descended) was isolated. As used herein, the term "isolated population" with respect to an isolated population of cells as used herein refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched.

[0040] As used herein, the expression “substantially pure population of cells” means a population of cells that contains at least 90, 91, 92, 03, 94, 95, 96, 97, 98, or 99% of the desired cell type.

[0041] As used herein, the term "enriching" includes any isolation or sorting process that increases the relative abundance of a desired cell type, or cell types, in a population of cells.

[0042] As used herein, the terms “subject” or “patient” denote a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human, a mouse or a rat. More particularly, the subject according to the invention has or is susceptible to have a 0-hemoglobinopathy (e.g a sickle cell disease or a P-thalassemia). As used herein, the term "P-hemoglobinopathy" has its general meaning in the art and refers to any defect in the structure or function of any hemoglobin of an individual, and includes defects in the primary, secondary, tertiary or quaternary structure of hemoglobin caused by any mutation, such as deletion mutations or substitution mutations in the coding regions of the HBB gene, or mutations in, or deletions of, the promoters or enhancers of such gene that cause a reduction in the amount of hemoglobin produced as compared to a normal or standard condition.

[0043] As used herein, the term "sickle cell disease" has its general meaning in the art and refers to a group of autosomal recessive genetic blood disorders, which results from mutations in a globin gene and which is characterized by red blood cells that assume an abnormal, rigid, sickle shape. They are defined by the presence of pS-globin gene coding for a P-globin chain variant in which glutamic acid is substituted by valine at amino acid position 6 of the peptide: incorporation of the PS-globin in the Hb tetramers (HbS, sickle Hb) leads to Hb polymerization and to a clinical phenotype. The term includes sickle cell anemia (HbSS), sickle-hem oglobin C disease (HbSC), sickle beta-plus- thalassaemia (HbS / p+), or sickle beta-zerothalassaemia (HbS / pO).

[0044] As used herein, the term "P-thalassemia" refers to a hemoglobinopathy that results from an altered ratio of a-globin to P-like globin polypeptide chains resulting in the underproduction of normal hemoglobin tetrameric proteins and the precipitation of free, unpaired a-globin chains.

[0045] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0046] As used herein, the term "therapeutically effective amount" is meant a sufficient amount of population of cells to treat the disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total usage the epigenome editing platform will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, the duration of the treatment, drugs used in combination or coincidental with the population of cells, and like factors well known in the medical arts. In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatmenteffective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. A treatment-effective amount of cells in the composition is dependent on the relative representation of the cells with the desired specificity, on the age and weight of the recipient, and on the severity of the targeted condition. This number of cells can be as low as approximately 103 / kg, preferably 5xl03 / kg; and as high as 107 / kg, preferably 108 / kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. Typically, the minimal dose is 2 millions of cells per kg. Usually 2 to 20 millions of cells are injected in the subject. The desired purity can be achieved by introducing a sorting step. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.

[0047] Methods of the present invention:

[0048] The first object of the present invention relates to a method of increasing fetal hemoglobin content in a eukaryotic cell comprising the step of contacting the eukaryotic cell with an epigenome editing platform that comprises (a) first epigenome editing enzyme wherein the effector domain consists of a histone acetyltransferase, (b) a second epigenome editing enzyme wherein the effector domain consists of a DNA demethylase, (c) a plurality of sgRNAs designed for guiding the epigenome editing enzymes to a target sequences in the region of the HBG1 and / or HBG2 promoter so as to reduce CpG methylation levels, thereby increasing the expression of gamma-globin in said eukaryotic cell.

[0049] Eukaryotic cell:

[0050] In some embodiments, the eukaryotic cell is selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), pluripotent cells (i.e. embryonic stem cells (ES) and induced pluripotent stem cells (iPS)). More preferably the eukaryotic cell is a hematopoietic stem cell.

[0051] In some embodiments, the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells. As used herein, the term “peripheral blood cells” refer to the cellular components of blood, including red blood cells, white blood cells, and platelets, which are found within the circulating pool of blood.

[0052] In some embodiments, the eukaryotic cell is a bone marrow derived stem cell. As used herein the term “bone marrow-derived stem cells” refers to stem cells found in the bone marrow. Stem cells may reside in the bone marrow, either as an adherent stromal cell type that possess pluripotent capabilities, or as cells that express CD34 or CD45 cell-surface protein, which identifies hematopoietic stem cells able to differentiate into blood cells.

[0053] Typically, the eukaryotic cell results from a stem cell mobilization. As used herein, the term “mobilization” or “stem cell mobilization” refers to a process involving the recruitment of stem cells from their tissue or organ of residence to peripheral blood following treatment with a mobilization agent. This process mimics the enhancement of the physiological release of stem cells from tissues or organs in response to stress signals during injury and inflammation. The mechanism of the mobilization process depends on the type of mobilization agent administered. Some mobilization agents act as agonists or antagonists that prevent the attachment of stem cells to cells or tissues of their microenvironment. Other mobilization agents induce the release of proteases that cleave the adhesion molecules or support structures between stem cells and their sites of attachment. As used herein, the term “mobilization agent” refers to a wide range of molecules that act to enhance the mobilization of stem cells from their tissue or organ of residence, e.g., bone marrow (e.g., CD34+ stem cells) and spleen (e.g., Hoxll+ stem cells), into peripheral blood. Mobilization agents include chemotherapeutic drugs, e.g., cyclophosphamide and cisplatin; cytokines, and chemokines, e.g., granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), Fms-related tyrosine kinase 3 (flt-3) ligand, stromal cell-derived factor 1 (SDF-1); agonists of the chemokine (C — C motif) receptor 1 (CCR1), such as chemokine (C — C motif) ligand 3 (CCL3, also known as macrophage inflammatory protein- la (Mip-la)); agonists of the chemokine (C — X — C motif) receptor 1 (CXCR1) and 2 (CXCR2), such as chemokine (C — X — C motif) ligand 2 (CXCL2) (also known as growth-related oncogene protein-P (Gro-P)), and CXCL8 (also known as interleukin-8 (IL-8)); agonists of CXCR4, such as CTCE-02142, and Met-SDF-1,; Very Late Antigen (VLA)-4 inhibitors; antagonists of CXCR4, such as TG-0054, plerixafor (also known as AMD3100), and AMD3465, or any combination of the previous agents. A mobilization agent increases the number of stem cells in peripheral blood, thus allowing for a more accessible source of stem cells.

[0054] Epigenome editing enzyme:

[0055] In some embodiments, the epigenome editing enzyme consists of a DNA binding domain that is fused to an effector domain.

[0056] DNA binding domain:

[0057] In some embodiments, the DNA binding domain is selected from the group consisting of Zinc finger proteins, Transcription Activator-Like Effectors (TALEs) and nucleases.

[0058] In some embodiments, the DNA binding domain consists of a nuclease, preferably a CRISPR / Cas nuclease, even more preferably a defective CRISPR / Cas nuclease. Thus, in some embodiments, the DNA binding domain consists of a defective CRISPR / Cas nuclease. The sequence recognition mechanism is the same as for the non-defective CRISPR / Cas nuclease. Typically, the defective CRISPR / Cas nuclease of the invention comprises at least one RNA binding domain. The RNA binding domain interacts with a guide RNA molecule as defined hereinafter. However, the defective CRISPR / Cas nuclease of the invention is a modified version with no nuclease activity. Accordingly, the defective CRISPR / Cas nuclease specifically recognizes the guide RNA molecule and thus guides the epigenome editing enzyme to its target DNA sequence.

[0059] In some embodiments, the defective CRISPR / Cas nuclease can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. In some embodiments, the nuclease domains of the protein can be modified, deleted, or inactivated. In some embodiments, the protein can be truncated to remove domains that are not essential for the function of the protein. In some embodiments, the protein is truncated or modified to optimize the activity of the RNA binding domain.

[0060] In some embodiments, the CRISPR / Cas nuclease consists of a mutant CRISPR / Cas nuclease i.e. a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. In some embodiments, the mutant has the RNA-guided DNA binding activity, but lacks one or both of its nuclease active sites. In some embodiments, the mutant comprises an amino acid sequence having at least 50% of identity with the wild type amino acid sequence of the CRISPR / Cas nuclease. Various CRISPR / Cas nucleases can be used in this invention. Non-limiting examples of suitable CRISPR / CRISPR / Cas nucleases include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966. See e.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entireties.

[0061] In some embodiments, the CRISPR / Cas nuclease is derived from a type II CRISPR- Cas system. In some embodiments, the CRISPR / Cas nuclease is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polar omonas naphthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalter omonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina, inter alia.

[0062] Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvCl subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H841 A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5): 1173-83 (2013).

[0063] In some embodiments, the Cas9 variants having mutations other than D10A or H840A are used, which e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at DIO and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvCl subdomain). In some embodiments, the Cas9 variant is a dead Cas9.

[0064] Effector domain:

[0065] In some embodiments, the effector domain consists of a effector domain. More particularly, the effector domain consists of a histone acetyltransferase or a DNA demethylase.

[0066] In some embodiments, the effector domain consists of a histone acetyltransferase. Histone acetyltransferases (HATs) are a group of enzymes that play a crucial role in regulating gene expression by modifying chromatin structure. These enzymes achieve this by transferring acetyl groups from acetyl -CoA to the lysine residues on histone proteins, which leads to a more relaxed chromatin structure and facilitates transcriptional activation. In some embodiments, the histone acetyltransferase is selected from the group consisting of p300 protein, CREB binding protein (CBP; an analog of p300), GCN5, or PCAF, or fragment thereof. One of the well- studied HATs is the CREB-binding protein (CBP), a versatile coactivator that interacts with numerous transcription factors and regulates various cellular processes, including cell growth, differentiation, and apoptosis. CBP functions as a histone acetyltransferase by acetylating histones H3 and H4, thereby promoting the assembly of transcriptional machinery at gene promoters and enhancers. The ability of CBP to acetylate non-histone proteins, such as transcription factors and signaling molecules, further underscores its pivotal role in gene regulation and cellular homeostasis. The broad substrate specificity and multifaceted functions of CBP make it an indispensable component of the epigenetic landscape, influencing gene expression patterns in response to diverse physiological and environmental cues. In the context of epigenome editing, the incorporation of CBP as the effector domain within a CRISPR / Cas9-based system offers a powerful tool for targeted gene activation. By fusing CBP to a defective Cas9 (dCas9) that retains its DNA-binding capability but lacks nuclease activity, researchers can direct the histone acetyltransferase activity of CBP to specific genomic loci. This targeted acetylation can modulate chromatin accessibility and enhance transcriptional activation of desired genes. In some embodiments, the effector domain is a fragment of CBP, in particular, the core domain of CBP (aa 1084-1701) as described in Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer-promoter interactions. Wang K, Escobar M, Li J, Mahata B, Goell J, Shah S, Cluck M, Hilton IB. Nucleic Acids Res. 2022 Aug 12;50(14):7842-7855. In some embodiments, the effector domain consists of a DNA demethylase. In some embodiments, the effector domain that is capable to remove DNA methylation is selected among ten-eleven translocation (TET) dioxygenases, specifically, TET1, TET2, and TET3. TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) in an Fe (II) / a-ketoglutarate-dependent manner. Notably, 5fC and 5caC can be excised by thymine-DNA glycosylase (TDG), and the modified site returns to the unmethylated status through base excision repair (BER). Therefore, these enzymes regulate active turnover of DNA methylation. In some embodiments, the effector domain is ten-eleven translocation methylcytosine dioxygenase 1 (TET1). TET1 has a pivotal role in the process of active DNA demethylation, a critical mechanism involved in regulating gene expression and maintaining cellular identity. By oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), TET1 alters the epigenetic landscape, thereby influencing chromatin structure and accessibility. This activity of TET 1 can be harnessed in epigenome editing to precisely modulate gene expression patterns. Employing TET1 as an effector domain within a CRISPR / Cas9-based system presents a powerful approach to reprogram specific genomic loci. By fusing TET1 to a defective CRISPR / Cas9 (dCas9) that retains its DNA-binding capability but lacks nuclease activity, researchers can direct the demethylase activity of TET1 to targeted genomic regions. This fusion allows for the site-specific removal of methyl groups from DNA, leading to the activation or repression of gene expression depending on the context of the target site.

[0067] In some embodiments, the effector domain is fused to the N-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the effector domain is fused to the C-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the defective CRISPR / Cas nuclease and the effector domain are fused via a linker. Suitable linker motifs and linker configurations will be apparent to those of skill in the art. In some embodiments, suitable linker motifs and configurations include those described in Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-69, the entire contents of which are incorporated herein by reference.

[0068] In some embodiments, the fusion protein may comprise additional features. Other exemplary features that may be present are localization sequences, such as nuclear localization sequences (NLS), cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable localization signal sequences and sequences of protein tags are provided herein, and include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable features will be apparent to those of skill in the art.

[0069] sgRNA molecules:

[0070] In particular, the sgRNAs include a region that is complementary and capable of hybridization to a pre-selected target site of interest in the promoter of HBG1 or HBG2. In some embodiment, this guide sequence can comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the region of base pairing between the guide sequence and the corresponding target site sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length. In some embodiments, the guide sequence is about 17-20 nucleotides in length, such as 20 nucleotides. Typically, a software program is used to identify candidate CRISPR target sequences on both strands of the DNA nucleic acid molecule containing the HBG genes based on desired guide sequence length and a CRISPR motif sequence (PAM) for a specified CRISPR enzyme. One requirement for selecting a suitable target nucleic acid is that it has a 3 ' PAM site / sequence. Each target sequence and its corresponding PAM site / sequence are referred herein as a Cas-targeted site. Type II CRISPR system, one of the most well characterized systems, needs only Cas 9 protein and a guide RNA complementary to a target sequence to affect target cleavage. For example, target sites for Cas9 from S. pyogenes, with PAM sequences NGG, may be identified by searching for 5'-Nx-NGG-3' both on the input sequence and on the reversecomplement of the input. Since multiple occurrences in the genome of the DNA target site may lead to nonspecific genome editing, after identifying all potential sites, the program filters out sequences based on the number of times they appear in the relevant reference genome. For those CRISPR enzymes for which sequence specificity is determined by a “seed” sequence, such as the 11-12 bp 5' from the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Thus, to avoid editing at additional genomic loci, results are filtered based on the number of occurrences of the seed:PAM sequence in the relevant genome. The user may be allowed to choose the length of the seed sequence. The user may also be allowed to specify the number of occurrences of the seed:PAM sequence in a genome for purposes of passing the filter. The default is to screen for unique sequences. Filtration level is altered by changing both the length of the seed sequence and the number of occurrences of the sequence in the genome. The program may in addition or alternatively provide the sequence of a guide sequence complementary to the reported target sequence(s) by providing the reverse complement of the identified target sequence(s). Further details of methods and algorithms to optimize sequence selection can be found in U.S. application Ser. No. 61 / 836,080; incorporated herein by reference.

[0071] Typically, the sgRNA comprises a spacer sequence selected from Table 1. In some embodiments, the epigenome editing platform of the present invention comprises the g-28, g-115, g-197, g-211 sgRNAs wherein the g-28 sgRNA comprises the space sequence as set forth in SEQ ID NO:6, the g-115 sgRNA comprises the spacer sequence as set forth in SEQ ID NO:5, the g-197 sgRNA comprises the spacer sequence as set forth in SEQ ID NO:3 and the g-211 sgRNA comprise the spacer sequence as set forth in SEQ ID NO:2.

[0072] The sgRNA of the present invention can be made by various methods known in the art including cell-based expression, in vitro transcription, and chemical synthesis. The ability to chemically synthesize relatively long RNAs (as long as 200 mers or more) using TC-RNA chemistry (see, e.g., U.S. Pat. No. 8,202,983) allows one to produce RNAs with special features that outperform those enabled by the basic four ribonucleotides (A, C, G and U). In particular, the RNA molecule of the present invention can be made with recombinant technology using a host cell system or an in vitro translation-transcription system known in the art. Details of such systems and technology can be found in e.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entireties.

[0073] In some embodiments, the sgRNA may include one or more modifications. Such modifications may include inclusion of at least one non-naturally occurring nucleotide, or a modified nucleotide, or analogs thereof. Modified nucleotides may be modified at the ribose, phosphate, and / or base moiety. Modified nucleotides may include 2’-O-methyl analogs, 2’-deoxy analogs, or 2’ -fluoro analogs. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. The use of locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine.

[0074] Vectors:

[0075] In some embodiments, the different components of the epigenome editing platforms of the present invention are provided to the eukaryotic cell through expression from one or more expression vectors. For example, the nucleic acids encoding the sgRNA or epigenome editing enzymes can be cloned into one or more vectors for introducing them into the eukaryotic cell. The vectors are typically prokaryotic vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the sgRNA or the epigenome editing enzymes herein disclosed. Preferably, the nucleic acids are isolated and / or purified. Thus, the present invention provides recombinant constructs or vectors having sequences encoding one or more of the sgRNAs or epigenome editing enzymes described above. Examples of the constructs include a vector, such as a plasmid or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In some embodiments, the construct further includes regulatory sequences. A “regulatory sequence” includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as inducible regulatory sequences. The design of the expression vector can depend on such factors as the choice of the eukaryotic cell to be transformed, transfected, or infected, the desired expression level, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with eukaryotic hosts are also described in e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press). The vector can be capable of autonomous replication or integration into a host DNA. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as tetracycline or ampicillin resistance in E. coli. Any of the procedures known in the art for introducing foreign nucleotide sequences into host cells may be used. Examples include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell.

[0076] In some embodiments, the different components of the epigenome editing platform of the present invention are provided to the population of cells through the use of an RNA-encoded system. For instance, the editing system may be provided to the population of cells through the use of a chemically modified mRNA-encoded adenine or cytidine base editor together with modified guide RNA as described in Jiang, T., Henderson, J.M., Coote, K. et al. Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979 (2020). In particular, engineered RNA-encoded editing enzymes (e.g. ABE) system are prepared by introducing various chemical modifications to both mRNA that encoded the epigenome editing enzyme and guide RNA. In particular said modifications consist in uridine depleted mRNAs modified with 5-methoxyuridine: synonymous codons may be introduced to deplete uridines as much as possible without altering the coding sequence and replaced all the remaining uridines with 5-methoxyuridine. Said optimized base editing system exhibits higher editing efficiency at some genomic sites compared to DNA-encoded system. It is also possible to encapsulate the modified mRNA and guide RNA into lipid nanoparticle (LNP) for allowing lipid nanoparticle (LNP)-mediated delivery.

[0077] In some embodiments, the different components of the epigenome editing platform of the present invention are provided to the population of cells through the use of ribonucleoprotein (RNP) complexes. For instance, the epigenome editing enzymes can be precomplexed with one or more sgRNAs to form a ribonucleoprotein (RNP) complex. The RNP complex can thus be introduced into the eukaryotic cell. Introduction of the RNP complex can be timed. The cell can be synchronized with other cells at Gl, S, and / or M phases of the cell cycle. RNP delivery avoids many of the pitfalls associated with mRNA, DNA, or viral delivery. Typically, the RNP complex is produced simply by mixing the proteins (i.e. the epigenome editing enzyme) and one or more sgRNAs in an appropriate buffer. This mixture is incubated for 5-10 min at room temperature before electroporation. Electroporation is a delivery technique in which an electrical field is applied to one or more cells in order to increase the permeability of the cell membrane. In some embodiments, the editing efficiency can be improved by adding a transfection enhancer oligonucleotide.

[0078] Population of edited cells:

[0079] A further object of the present invention relates to a population of eukaryotic cells that are edited according to the method of the present invention.

[0080] Methods of treatment:

[0081] A further object of the present invention relates to a method for increasing fetal hemoglobin levels in a subject in need thereof, the method comprising transplanting a therapeutically effective amount of the population of eukaryotic cells obtained by the editing method as above described.

[0082] In some embodiments, the population of cell is autologous to the subject, meaning the population of cells is derived from the same subject.

[0083] In some embodiments, the subject has been diagnosed with a hemoglobinopathy. The method of the present invention is thus particularly suitable for the treatment of P-hemoglobinopathies.

[0084] In some embodiments, the P-hemoglobinopathy is a sickle cell disease. In some embodiments, the hemoglobinopathy is a P-thalassemia.

[0085] In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatmenteffective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. A treatment-effective amount of cells in the composition is dependent on the relative representation of the cells with the desired specificity, on the age and weight of the recipient, and on the severity of the targeted condition. These amount of cells can be as low as approximately 103 / kg, preferably 5xl03 / kg; and as high as 107 / kg, preferably 108 / kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. Typically, the minimal dose is 2 million of cells per kg. Usually 2 to 20 million of cells are injected in the subject. The desired purity can be achieved by introducing a sorting step. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.

[0086] Kits

[0087] This invention further provides kits containing reagents for performing the abovedescribed methods, including all component of the epigenome editing platform as disclosed herein. To that end, one or more of the reaction components, e.g., sgRNAs, and nucleic acid molecules encoding for the epigenome editing enzymes for the methods disclosed herein can be supplied in the form of a kit for use. In some embodiments, the kit comprises one or more epigenome editing enzymes and one or more sgRNAs. In some embodiments, the kit can include one or more other reaction components. In some embodiments, an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate. Examples of additional components of the kits include, but are not limited to, one or more host cells, one or more reagents for introducing foreign nucleotide sequences into host cells, one or more reagents (e.g., probes or PCR primers) for detecting expression of the guide RNA or epigenome editing enzymes or verifying the target nucleic acid's status, and buffers or culture media for the reactions. The kit may also include one or more of the following components: supports, terminating, modifying or digestion reagents, osmolytes, and an apparatus for detection. The components used can be provided in a variety of forms. For example, the components (e.g., enzymes, RNAs, probes and / or primers) can be suspended in an aqueous solution or as a freeze-dried or lyophilized powder, pellet, or bead. In the latter case, the components, when reconstituted, form a complete mixture of components for use in an assay. The kits of the invention can be provided at any suitable temperature. For example, for storage of kits containing protein components or complexes thereof in a liquid, it is preferred that they are provided and maintained below 0° C., preferably at or below -20° C., or otherwise in a frozen state. The kits can also include packaging materials for holding the container or combination of containers. Typical packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, micro-particles and the like) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like). The kits may further include instructions recorded in a tangible form for use of the components.

[0088] FIGURES

[0089] Figure 1. Epigenetic status of the HBG promoters in adult and fetal erythroid cells. (A) ChlP-seq analysis of HBG1 / 2 promoters (highlighted). Epigenetic modifications include H3K27 acetylation (H3K27ac), H3K4 trimethylation (H3K4me3), H3K9 acetylation (H3K9ac), H3K27 trimethylation (H3K27me3), H3K9 trimethylation (H3K9me3) in adult (A) and fetal erythroblasts (F). (B) Percentage of P-like globin mRNA (HBG and HBB) expression measured by RT-qPCR in adult erythroblasts at day 6 of the erythroid differentiation (Adult 1), adult erythroblasts at day 13 of the erythroid differentiation (Adult 2), edited adult erythroblasts harboring the disrupted LRF binding site (HPFH 1), edited adult erythroblasts harboring the KLF1 binding site (HPFH 2), and fetal erythroblasts (Fetal) at day 6 (circle) or day 13 (triangle) of erythroid differentiation. Of note, for 2 of the 3 fetal liver donors, we collected cells at day 13 since at day 6 the differentiation towards the erythroid lineage was delayed compared to the first donor and a relatively large fraction of the cells was not committed towards the erythroid lineage. P-like globin expression was normalized to HBA. (C) Schematic representation of the HBG gene on chromosome 11. Boxes indicate exons. The sequence corresponding to the LRF binding site, before (GGGCCCCTTCCCCAC (SEQ ID NO: 17) - CCCGGGGAAGGGGTG (SEQ ID NO: 18) and after base editing (GGGCCCCCCCCCCAC (SEQ ID NO: 19) -CCCGGGGGGGGGGTG (SEQ ID NO:20) - GGGCCCCTCCCCCAC (SEQ ID NO:21) -CCCGGGGTGGGGGTG (SEQ ID NO: 22)), is reported. (D) Average methylation of CpGs withing the HBG promoters by bisulfite sequencing. (E) Methylation analysis of individual CpGs. All data are expressed as mean ± SD from 4 biologically independent experiments (3 adult donors for Adult 1 and HPFH 1, 3 adult donors for Adult 2 and HPFH2, 1 fetal donor at day 6, 2 fetal donors at day 13). Asterisks indicate level of statistical significance; **P<0.01; ***p<0.001; ****P<0.0001; no asterisk = not significant (Unpaired t-test).

[0090] Figure 2. Epigenetic reactivation of HBG in the HEK293T cell line. (A) Schematic representation of the HBG1 / 2 gene. Black boxes depict exons. Lines represent sgRNAs (named after the distance in nucleotides of the cleavage site from the TSS). Pins represent the CpGs.

[0091] (B, C) Relative expression of HBG genes normalised to GAPDH expression measured by RT-qPCR. Cells were transfected with plasmids expressing the editors alone or together with plasmids expressing the different sgRNAs and analysed 3 days after transfection. Data are expressed as means ± SD from 3 independent experiments. (D) Time course analysis of HBG expression at day 3, 6, 9, 13, 17 after transfection. Data are expressed as means ± SEM from 2 independent experiments. (E) Methylation analysis of CpGs within the HBG promoters by bisulfite sequencing in untreated HEK293T. Data are expressed as means ± SD from 3 independent experiments.

[0092] Figure 3. Erythroid differentiation of HSPCs is not impacted by epigenome editing. Frequency of (A) CD36+, (B) CD71+ and (C) CD235A+ cells at day 13 and 19 of erythroid differentiation, as measured by flow cytometry analysis in control and edited samples. Control cells (TE) were electroporated with TE buffer. (D) Frequency of enucleated cells at day 13, 16, and 19 of erythroid differentiation as measured by flow cytometry analysis of DRAQ5 nuclear staining. (E) Frequency of CD49d+, BAND3+ and CD49d+ / BAND3+ in 7AAD- / CD235+ cells at day 13 and 19 of erythroid differentiation as measured by flow cytometry. (F) Average methylation of CpGs withing the HBG promoters by bisulfite sequencing at day 13 of erythroid differentiation. All data are expressed as means ± SD from 3 independent experiments. Each symbol represents a different donor. (G) Western blot analysis of Cas9 expression in HSPCs from 2 healthy individuals (left panel) or in the K562 cell line (right panel). Cells treated with epigenome editors were collected at an early time point (12 or 24 hours) and at a later time point (Day 6 after electroporation). As positive control we electroporated K562 cells with plasmids expressing Cas9 or CBP-dCas9 enzymes and collect them after 48 hours. Actin was used as a loading control, a-globin was used in HSPCs and HSPC-derived erythroblast as a differentiation control. (H) Representative gating strategy for population analysis on live, single erythroid cells at day 19 of the differentiation in unstained (top panel), CD235a-mono-stained (middle panel) and stained cells (low panel) in order to determine HbF expression via flow cytometry. Within the HbF-negative population, two distinct cell populations are observed, likely due to differences in autofluorescence between enucleated and nucleated cells. (I) Percentage of HbF-positive cells measured by flow cytometry at day 6 of erythroid differentiation from cells as in G (2 donors HD HSPCs). (J) Expression of P-, Gy-, and Ay- globin chains measured by RP-HPLC in RBCs. P-like-globin expression was normalized to a-globin. The a- / non-a-globin ratio is reported on top of the graph. All data are expressed as means ± SD from 3 independent experiments. Each symbol represents a different donor. Asterisks indicate level of statistical significance; *P<0.05; **P<0.01; ***P<0.001; no asterisk = not significant (Unpaired t-test). Figure 4. HbF reactivation in the erythroid progeny of SCD HSPCs treated with epigenome editors. (A) Scheme of the experimental procedure used for experiments in nonmobilized or bone marrow-derived SCD HSPCs. (B) Methylation analysis of CpGs within the HBG promoters by bisulfite sequencing. Cells electroporated with sgRNAs and mRNA expressing the editors were analyzed at day 13 of the erythroid differentiation. (C) Percentage of P-like globins (HBG and HBB) expression normalized to HBA expression measured by RT-qPCR at day 13 of the erythroid differentiation. (D) Fold-change increase of HBG primary transcripts normalized to HBA expression measured by RT-qPCR. (E) Cas9 expression normalized to HBA expression measured by RT-qPCR at day 13 of erythroid differentiation. HSPCs (n = 2) electroporated with TE buffer or editors collected and analyzed by RT-qPCR 3 days after transfection served as positive controls. (F) Histogram plot of HbF-positive RBCs measured by flow cytometry at day 20 of the erythroid differentiation. The two peaks in the HbF negative population might derive from the different autofluorescence of enucleated and nucleated cell populations. (G) Percentage of HbF-positive RBCs measured by flow cytometry.

[0093] (H) HbF and HbS levels measured by CE-HPLC in RBCs at day 19 of the erythroid differentiation. The percentage of each Hb type was calculated over the total Hb tetramers. (I) Frequency of sickle red blood cells measured 3 hours after 02 deprivation (normalized to the TE controls). (J) Representative pictures of red blood cells at 20% 02 and after 3 hours at 0% 02. All data are expressed as means ± SD from 3 independent experiments. Each symbol represents a different donor. Asterisks indicate level of statistical significance; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; no asterisk = not significant (Unpaired t-test).

[0094] Figure 5. Progenitor counts and HbF reactivation in BFU-Es after epigenome editing. (A) Scheme of the experimental procedure used for experiments in bone marrow-derived SCD HSPCs. (B) Colony-forming cell frequency in control and edited samples. (C) Methylation analysis of CpGs within the HBG promoters by bisulfite sequencing in pools of BFU-E colonies (>25 colonies obtained in 3 independent experiments). (D) Percentage of P-like globins (HBG and HBB) expression normalized to HBA expression measured by RT-qPCR. (E) Fold-change increase of HBG primary transcripts normalized to HBA expression measured by RT-qPCR. (F) Cas9 expression normalized to HBA expression measured by RT-qPCR. HSPCs (n = 2) electroporated with TE buffer or editors collected and analyzed by RT-qPCR 3 days after transfection served as positive controls (same data as in Figure 3e). (G) HbF and HbS levels measured by CE-HPLC in BFU-Es. The percentage of each Hb type was calculated over the total Hb tetramers. All data are expressed as means ± SD from 3 independent experiments. Each symbol represents a different donor. Asterisks indicate level of statistical significance; *P<0.05; no asterisk = not significant (Unpaired t-test).

[0095] Figure 6. Transcriptome and DNA methylome profiles of epigenome-edited HSPCs. (A) Percentage of P-like globins (HBG and HBB) expression normalized to HBA expression measured by RT-qPCR. Analysis performed 3 days after electroporation (3 donors SCD HSPCs). (B) Percentage of HbF-positive cells measured by flow cytometry at day 6 of erythroid differentiation (3 donors SCD HSPCs). (C) Venn diagram reporting the distribution of upregulated genes between the different conditions. (D) In silico predicted off-target sites for each of the sgRNA. Off-targets were classified as intergenic or intragenic. The list of off-target genes has been cross-referenced with the list of DEGs. The predicted off-target genes were either not expressed or not dysregulated. (E) CpG methylation profiles in a magnified view of a ±l-kb region centered on the HBG2 (above) and HBG1 (below) promoter region. Individual dots indicate the average methylation of each CpG. Linear regression was applied to the smoothed curve. (F) Volcano plots from RNA-seq analyses showing differential gene expression between controls and cells treated with Tetl-4xsgRNAs. RNA-seq was performed 72 h after electroporation in a healthy donor. The horizontal dashed line indicates the threshold on the false discovery rate (FDR <0.05), and the vertical dashed lines correspond to the threshold on log2FC> 1 or < -1. Upregulated genes are indicated in red and downregulated genes in blue. Genes in grey are not differentially expressed according to the applied thresholds. We observed 163 upregulated and 98 downregulated genes (log2FC> 1 or < -1; FDR < 0.05). (G) Expression levels of LINE and SINE elements, in control and edited conditions. Expression values are reported as counts per million (cpm). (H) DNA methylation levels (%) of control and Tetl-edited conditions in the same elements analyzed in G.

[0096] Figure 7. Transcriptome and DNA methylome profiles of epigenome-edited HSPCs. (A) Volcano plots showing differential gene expression between cells treated with CBP+Tetl alone (left), Tetl-4xsgRNAs (middle) and CBP+Tetl-4xsgRNAs (right) and control cells. RNA-seq was performed 72 h after electroporation in 3 different donors with SCD. The horizontal dashed line indicates the threshold on the false discovery rate (FDR < 0.05), and the vertical dashed lines correspond to the threshold on log2FC > 1 or < -1. Upregulated genes are indicated in red and downregulated genes are in blue. Genes in grey are not differentially expressed. (B) Gene set enrichment analyses on Hallmark gene sets from MSigDB were performed on up-regulated DEG. The enriched Hallmark gene sets are reported. We reported the number of up-regulated genes belonging to each gene set. HM, hallmark. (C) CpG methylation profiles in a ±25-kb genomic region centered on the TSS of HBG1. Individual dots indicate the average methylation of each CpG. Linear regression was applied to the smoothed curve (bottom panel) representing the percentage of methylation levels across the 50-kb locus encompassing HBG1 and HBG2. (D) Correlation between log2FC in gene expression of the top 10 up- and down-regulated genes and their variation in DNA methylation levels. We calculated the average DNA methylation in a ±25-kb genomic region centered on the TSS of DEGs in control vs treated cells.

[0097] Figure 8. Engraftment and multi-lineage differentiation of epigenome-edited HSPCs and HbF expression in their erythroid progeny. (A) Scheme of the experimental procedure used for experiments of HD HSPC xenotransplantation. Epigenome editor mRNAs and sgRNAs were co-transfected in HD HSPCs. Control and edited cells were xenotransplanted into NBSGW immunodeficient mice 1 day after transfection. Mice were euthanized 16 weeks after transplantation and their hematopoietic tissues and organs were collected and analyzed.

[0098] (B) Engraftment of human cells in NBSGW mice transplanted with control (mock-transfected) or treated HSPCs 16 weeks post-transplantation (n = 3 ctr group, n= 6 treated group). Chimerism is calculated as the percentage of human CD45+ cells in the total murine and human CD45+ cell population in BM, spleen, thymus, and peripheral blood. (C) Frequency of human T (CD3) and B (CD19) lymphoid, myeloid (CDllb, CD14, and CD15), erythroid (CD235, CD36 and CD71) cells and HSPCs (CD34) in BM and (D) spleen, of mice transplanted with control and edited HSPCs, 16 weeks after the transplantation (n = 3 ctr group, n= 6 treated group). (E) Percentage of P-like globin (HBG and HBB mRNAs normalized to HBA expression and (F) fold-change increase of HBG primary transcripts normalized to HBA expression measured by RT-qPCR in BM cells sorted for CD235a. (G) Percentage of HbF-positive cells measured by flow cytometry in BM-derived human CD235a-positive cells. (H) HbF and HbA levels measured by CE-HPLC in human CD235a+ BM cells. The percentage of each Hb type was calculated over the total Hb tetramers. All data are expressed as mean ± SD. Each point represents an individual mouse; in the treated group squares indicate samples treated with Tetl and circles depict samples treated with CBP+ Tetl. Asterisks indicate level of statistical significance; *P<0.05; no asterisk = not significant (Unpaired t-test).

[0099] Figure 9. Edited HSPCs prior to engraftment in NBSGW immunodeficient mice. (A) Methylation analysis of CpGs within the HBG promoters by bisulfite sequencing in HSPCs and early erythroid cells 6 days after electroporation. (B) Percentage of HbF-positive cells measured by flow cytometry in early erythroid populations. (C) Colony forming cell frequency in control and edited samples 14 days after electroporation. (D) HbF and HbS levels measured by CE-HPLC in BFU-E colonies. The percentage of each Hb type was calculated over the total Hb tetramers. (E) Expression of P-,Gy-, andAy- globin chains measured by RP-HPLC in BFU-E colonies. P-like-globin expression was normalized to a-globin. The a- / non-a-globin ratio is reported on top of the graph. (F) Average methylation of CpGs withing the HBG promoters by bisulfite sequencing in single BFU-E colonies differentiated from control and epigenome edited HSPCs. Data are expressed as means ± SD. (G) Percentage of P-like globin mRNA (HBG and HBB) expression measured by RT-qPCR in single BFU-E colonies differentiated from control and epigenome edited HSPCs. P-like globin expression was normalized to HBA. Data are expressed as means ± SD. Asterisks indicate level of statistical significance; **P<0.01; ****P<0.0001; no asterisk = not significant (Unpaired t-test).

[0100] Figure 10. HBG reactivation by Tetlvl in differentiated HUDEP-2 cells. (A) Schematic representation of epigenetic editors. Tetl and Tetlv2 plasmids were modifies to contain the T7 promoter sequence for mRNA in vitro production. (B) Methylation analysis of CpGs within the HBG promoters by bisulfite sequencing. Cells electroporated with sgRNAs and mRNA expressing the editors were analyzed after 8 days of differentiation. In some samples, Sanger sequencing was unsuccessful (empty cell). (C) Percentage of P-like globins (HBG and HBB) expression normalized to HBA expression measured by RT-qPCR after 8 days of differentiation. (D) HbF levels measured by flow cytometry after 8 days of differentiation.

[0101] (E) HbF and HbA levels measured by CE-HPLC. The percentage of each Hb type was calculated over the total Hb tetramers. (F) Expression of P-,Gy-, andAy- globin chains measured by RP-HPLC. P-like-globin expression was normalized to a-globin. All data are means ± SD from 2-4 independent experiments.

[0102] EXAMPLE:

[0103] Results:

[0104] Different epigenetic marks characterize the HBG promoters in adult versus fetal primary erythroblasts.

[0105] To provide a comprehensive characterization of the epigenetic landscape associated with the HBG promoters, we reanalysed publicly available ChlP-sequencing data comparing HSPC-derived erythroblasts from adult and fetal human samples38. The levels of histone marks typically associated with active transcription, such as histone 3 lysine 27 acetylation (H3K27ac), histone 3 lysine 4 trimethylation (H3K4me3) and histone 3 lysine 9 acetylation (H3K9ac), were high in fetal erythroblasts, while these modifications were absent in adult erythroblasts which do not express y-globin (Figure 1A). On the contrary, repressive histone modifications (H3K27me3 and H3K9me3) were absent in both adult and fetal cells (Figure 1A). These data confirmed the close correlation between activating histone marks, the activity of the HBG promoters and y-globin expression. We then investigated DNA methylation in erythroid cells derived from adult or fetal liver HSPCs (expressing mainly HBB and HBG, respectively; Figure IB), or from HSPCs treated with base editors to generate HPFH and HPFH-like mutations disrupting the binding site for the LRF transcriptional repressor (HPFH1) or creating a binding site for the KLF1 transcriptional activator (HPFH2) in the HBG promoters17(Figure 1C). These edited fetal-like cells showed increased levels of y-globin compared to the control cells (up to -70% of the total P-like globin mRNAs) (Figure IB).

[0106] Adult cells expressing low levels of HbF displayed a general hypermethylation of all CpGs within the HBG promoters. On the contrary, edited cells and fetal erythroblasts (characterized by high HbF expression) showed substantially reduced CpG methylation levels (Figure ID and E). These results show a positive correlation between HBG gene activity and DNA demethylation. Furthermore, these data suggest the CpG methylation is a highly dynamic process that can be modulated to achieve HBG gene activation.

[0107] Targeted histone acetylation induces HBG activation in HEK293T.

[0108] Activation of the y-globin promoters in the erythroid progeny of primary healthy donor HSPCs was reported by using a zinc finger DNA binding domain coupled with the transcriptional activator VP64 (ggl-VP64)39and in cell lines using conventional CRISPRa tools (transcriptional activators such as Cas9-VP64, Cas9-VPR) or with CRISPR-based epigenome editors containing a lysine acetyl-transferase domain (P300 or CBP) able to perform histone acetylation (Cas9-P300, Cas9-CBP)33,35 37Although significant y-globin reactivation was achieved, these strategies were not able to sustain HBG expression over time. Previous studies demonstrated that, at least in the context of a repressive epigenome editing system, simultaneous modulation of DNA methylation and histone modifications is crucial for ensuring the persistence of epigenetic modifications31. Thus, we combined deposition of histone acetylation (via dCas9-CBP) with DNA demethylation (using Tetl-dCas9) to recreate an epigenetic landscape similar to that observed in fetal cells at the HBG promoters and achieve stable HBG activation.

[0109] First, plasmids coding for the epigenome editor dCas9-CBP core (CBP) and the sgRNAs targeting the HBG promoters (g-28, g-115, g-165, g-197, g-21 l)12>35(Figure 2A) were co-transfected in the HEK293T cell line. Three days after transfection, we evaluated HBG expression by RT-qPCR. Control cells transfected with either the dCas9 or CBP plasmid alone, as well as those transfected with dCas9 and four sgRNAs, showed negligent HBG expression. Conversely, CBP strongly increased y-globin transcription with all the sgRNAs, reaching >103-fold increase in HBG expression (compared to dCas9 only), except for g -165 which led to low gene activation (Figure 2B-2C) An even higher activation (up to 104-fold change) was observed using four sgRNAs, with the best combination being g -28, -115, -197, -211 (referred hereafter as 4x sgRNA) (Figure 2B-2C). Next, we tested the long-term efficacy of our strategy by maintaining the transfected cells in culture for 17 days and analysing HBG expression at different time points. In addition to CBP, we tested Tetl-dCas9 (Tetl), which is capable of removing DNA methylation. Although high-level of HBG expression was initially observed by RT-qPCR in cells treated with either CBP alone or in combination with Tetl (together with the optimal sgRNA combination), HBG reactivation was not sustained over time, eventually returning to baseline levels at the final time point (Figure 2D). Moreover, Tetl alone was not able to reactivate HBG expression neither 3 days post-transfection nor at later timepoints (Figure 2D). To investigate the failed HBG reactivation observed with Tetl, we analyzed the methylation status of the CpGs in the HBG promoters in the HEK293T cell line. Although this cell line does not express y-globin, all the CpGs were poorly methylated, suggesting that DNA methylation does not play a role in regulating HBG expression in HEK293T cells (Figure 2E).

[0110] Overall, these results showed that CBP-mediated histone acetylation is a robust approach to induce HBG gene transcription in HEK293T and the combination of four sgRNAs results in a more potent gene activation compared to the individual sgRNAs. Nevertheless, CBP is not sufficient to obtain a long-term activation in this cell model. Furthermore, HEK293T are not the appropriate cellular model to evaluate Tetl-dCas9 effect on reactivation of y-globin genes and to provide insights on the stability of gene activation obtained when combining DNA and histone epigenetic modifications.

[0111] Removal of DNA methylation and deposition of histone acetylation reactivates HbF and corrects the sickling cell phenotype in the erythroid progeny of HSPCs We then reasoned that additional studies using primary cells carrying hypermethylated y-globin promoters were needed to assess the long-term effects of DNA demethylation and histone acetylation on y-globin expression. To this aim, we delivered the editing reagents as RNA in HSPCs obtained from patients with SCD. Cells were electroporated with CBP and Tetl editors alone or in combination with 4x sgRNA. The dCas9 with no effector but with 4x sgRNA was used as an additional control. After electroporation, cells were differentiated towards the erythroid lineage (Figure 4A). No significant differences were observed in the expression of early and late erythroid markers and in the enucleation rate between control and treated cells, showing no impact of the editing procedure on erythroid differentiation (Figure 3A-3E). While control primary erythroblasts showed highly methylated CpGs, within the HBG promoters, epigenome editors caused a general demethylation with Tetl and CBP+Tetl being more effective than CBP alone (Figure 4B and 3F). DNA methylation levels correlated with y-globin mRNAs expression (measured by RT-qPCR) accounting for up to 40% of the total P-like globin transcripts in Tetl- and CBP+Tetl -treated samples (Figure 4C). The analysis of primary transcripts confirmed an active transcription of HBG genes in treated cells (Figure 4D) even in the absence of detectable epigenome editor expression, suggesting durable HBG gene activation (Figure 4E and 3G). Flow cytometry revealed 80% of HbF-positive RBCs in all the edited samples (Figure 4F-4G and 3H-3I). HbF reactivation was also observed by cation-exchange HPLC (CE-HPLC) with 30% of HbF tetramers in samples treated with CBP and >40% HbF in Tetl- and CBP+Tetl -treated cells. Concomitantly HbS levels were substantially reduced (Figure 4H). These results were confirmed by reversed-phase HPLC (RP-HPLC) analysis of individual globin chains (Figure 3 J). Finally, to evaluate the correction of the sickling phenotype, RBCs were incubated under hypoxic conditions to induce HbS polymerization. After 3 hours, we observed 40% reduction in the frequency of sickle cells in CBP-treated samples and 50% in the Tetl and CBP+Tetl conditions, reaching levels similar to those observed in asymptomatic carriers (Figure 4I-4J).

[0112] Overall, these findings demonstrate that DNA methylation plays a direct role in HbF silencing, which can be reverted using epigenetic modifiers. This might represent a safe and efficacious strategy for P-hemoglobinopathies, such as SCD.

[0113] Persistence of HbF expression after treatment with epigenome editors.

[0114] To assess the safety of the approach and to measure HbF production in cells that underwent a higher number of cell divisions compared to erythroblasts in liquid culture, HSPCs were plated in a semi-solid medium that allows the clonal growth and differentiation of erythroid and granulocyte / monocyte progenitors (Figure 5A). The frequency of erythroid (BFU-E) colonies and granulocyte / monocyte (CFU-GM) colonies were similar in all the treated conditions and comparable to the controls, demonstrating no impact of epigenome editing on hematopoietic progenitors (Figure 5B). As observed in liquid cultures, pools of BFU-E colonies treated with editors and sgRNAs presented a general hypomethylation in all the CpGs of the HBGH2 genes (Figure 5C). These results correlated with the increase of y-globin transcripts in treated samples (even when epigenome editors are no longer expressed), with Tetl and CBP+Tetl being more potent than CBP alone (Figure 5D-5F). Finally, all the treated cells showed HbF reactivation (as measured by CE-HPLC), which was more pronounced in Tetl and CBP+Tetl samples (Figure 5G).

[0115] Specificity profile of epigenome editors targeting HBG

[0116] To evaluate the occurrence of unintended outcomes at the RNA level (e.g., due to off-target activity of epigenome editors), we analyzed the transcriptome of control and edited HSPCs derived from 3 different SCD donors 72 hours after electroporation. RT-qPCR analysis showed increased HBG expression in HSPCs treated with CBP+Tetl -4xsgRNAs, which was less pronounced in Tetl-4xsgRNAs samples, suggesting that HBG reactivation after Tetl-mediated DNA demethylation alone necessitates other factors to be expressed upon erythroid differentiation (Figure 6A). Accordingly, erythroid populations derived from edited HSPCs showed similar frequencies of HbF-expressing cells (Figure 6B). We then compared by RNA sequencing (RNA-seq) mock-electroporated HSPCs and HSPCs treated with CBP+Tetl alone, Tetl-4xsgRNAs or CBP+Tetl -4xsgRNAs. Cells that received only editor mRNAs showed 14 upregulated genes and no downregulated genes (log2 fold change (log2FC)> l or <-l, respectively; false discovery rate (FDR) <0.05). In HSPCs electroporated with Tetl or CBP+Tetl and the 4x sgRNAs, we observed 101 and 113 differentially expressed genes (DEGs; 81 and 80 upregulated, 20 and 33 downregulated), respectively (Figure 7A and Dt). Most of the upregulated genes were in common between the different conditions (Figure 6C) and were involved in RNA sensing and response to viruses (e.g., IFI44L, IFI44, IFI6, IRF7, IRF9, OAS1-3). In fact, a functional enrichment analysis focusing on MSigDB Hallmark gene sets confirmed an enrichment in genes associated with interferon alpha and gamma pathways (Figure 7B). Accordingly, the FC of DEGs increased with the amount of electroporated RNA (Figure 7A). Importantly, we did not observe dysregulation of genes involved in HbF regulation and DNA damage response or in silico predicted sgRNA-dependent off-target genes (Figure 6D and 7A, and Data not shown). Finally, we observed no changes in the expression profile of transposable elements (such as LINE and SINE, which can be sensitive to DNA demethylation), in the different edited conditions compared to the controls (Figure 6G and Data not shown).

[0117] To further assess the specificity of our strategy, we analyzed the native DNA methylome using direct genomic DNA sequencing with Oxford Nanopore Technology in healthy donor (HD)-derived HSPCs treated with Tetl-4xsgRNAs. Focusing on a 50-kb region centered on the HBGH2 genes, we detected a decrease in 5-mC restricted to the HBG promoter regions in treated cells compared to controls (Figure 6E and 7C). Specifically, we quantified a 38% DNA methylation decrease at the HBGH2 promoters, while only -7% in the surrounding regions. In parallel, 5-hmC levels were increased, confirming that this mark is positively correlated with y-globin expression.

[0118] We then examined the variation in DNA methylation levels in a ±25-kb region surrounding the transcription start site (TSS) of each DEG identified via RNA-seq (Figure 6F and Data not shown). Our data revealed only a modest decrease in the average DNA methylation (0 to -7%) in treated cells, observed across both upregulated and down-regulated genes (Figure 7D and Data not shown). In treated cells, we analyzed -17,000 genes with unchanged expression levels and identified a subset of genes (400 with reduced DNA methylation and 64 with increased methylation) that exhibited changes in DNA methylation, which were not associated with gene dysregulation (Data not shown). Finally, in silico predicted sgRNA-dependent off-targets showed a modest change in DNA methylation (from -7% to +10%) that did not influence gene expression (Data not shown). Finally, we found no differences in DNA methylation levels at repetitive elements in control vs treated cells (Figure 6H and Data not shown). Altogether, these results show a modest transcriptional response related to cellular sensing to RNA and highlight the specificity of our DNA DSB-free strategy.

[0119] Epigenome editing in repopulating hematopoietic stem cells

[0120] To evaluate the capability of edited HSPCs to engraft and differentiate in vivo and test the long-term effects of epigenome editors, we transplanted human HSPCs edited with Tetl or CBP+Tetl into immunodeficient NBSGW mice (Figure 8A). The HBG promoters were hypermethylated in control input cells, while a diminished methylation was detected in treated samples (Figure 9A). Erythroid cells differentiated in vitro from edited HSPCs showed high levels of HbF expression (Figure 9B). Moreover, similar progenitor frequencies were observed in the different conditions, whereas only edited BFU-E presented an increased production of HbF and y-globin chains (Figure 9C-9E). A clonal analysis revealed that DNA methylation was significantly reduced in Tetl- and Tetl+CBP-treated BFU-E and this was accompanied by a substantial increase m HBG expression (Figure 9F-9G).

[0121] Sixteen weeks after transplantation, we measured the frequency of human CD45+ cells and the proportion of the different human lineages in the hematopoietic tissues. We observed no differences between control and treated animals in the chimerism (measured as percentage of human CD45+ cells on the total human and murine hematopoietic CD45+ cells) in the bone marrow (BM), spleen, thymus and blood, and in the frequency of lymphoid, myeloid and erythroid cells or CD34+ HSPCs (Figure 8B-8D). These results indicate that epigenome editing does not affect engraftment and differentiation potential of HSCs.

[0122] Importantly, CD235a+ erythroid cells sorted from BM of animals treated with epigenome editors showed increased HBG mRNA and primary transcripts (Figure 8E-8F). Finally, we observed a higher percentage of HbF+ cells by flow cytometry (Figure 8G) and an increase in HbF production in edited cells compared to the control group (Figure 8H).

[0123] Further optimization of the strategy in HUDEP-2 cells

[0124] Previous results showed efficient epigenetic reactivation of HBG in HSPC-derived erythroblasts treated with Tetl combined with 4 sgRNAs mapping to the HBG promoters. However, HSPC showed dysregulation of genes involved in RNA sensing and inflammation. To increase HBG reactivation and limit gene dysregulation, we tested a more efficient version of the Tetl editor and reduce the number of sgRNAs. Nunez et al. developed a Tetl-dCas9 editor enzyme with a linker (80 amino acids, aa) between Tetl and dCas9 that is longer compared to previous version of this editor (16 aa-long linkers)40. Moreover, while the previous editor contains a murine Tetl domain, the version developed by Nunez et al. incorporates a human TET1 domain. This editor showed increased gene activation in HEK293T cells. Therefore, we adapted this editor for mRNA in vitro production by adding the T7 promoter (referred from here after as Tetlv2) and tested it in the HUDEP-2 erythroid cell line (Figure 10A). HUDEP-2 cells were electroporated with mRNA encoding Tetl or Tetlv2 together with sgRNAs targeting the HBG promoters. In addition to our benchmark of 4 sgRNAs (g-28, g-115, g-197, g-211; 4x sgRNA), we tested individual sgRNAs or a combination of the two sgRNAs recognizing the 5’ and 3’ ends of the HBG promoter region (g-28 and g-211; 2x sgRNA). The cells were expanded for 6 days after the electroporation and then terminally differentiated for 8 days to obtain late-stage erythroblasts that were analysed for DNA methylation, globin expression and hemoglobin production.

[0125] DNA methylation in late-stage erythroblasts was high (76-88%) in the control groups and it decreased to less than 50% in the conditions with Tetl or Tetlv2 and the combination of the same 4 sgRNAs. Individual sgRNAs in combination with Tetlv2 resulted to be less efficient in demethylating, whereas the combination of g-28 and g-211 allowed to reach a demethylation level similar to Tetl or Tetlv2 with 4 sgRNA (Figure 10B). RT-qPCR showed a similar increase of HBG transcripts in HUDEP-2 treated with Tetl or Tetlv2 and 4 or 2 sgRNAs. Cells treated with individual sgRNAs showed lower HBG reactivation. These results were confirmed by flow cytometry and HPLC analyses with the combination of 2x sgRNA reaching levels of HbF production comparable to those obtained with 4x sgRNA (Figure 10C-10F).

[0126] In conclusion, the new optimized Tetlv2 perform as well as the original Tetl enzyme previously used, but the combination of only two sgRNAs seems sufficient to achieve robust levels of HBG activation and HbF production. Notably, the epigenetic modification introduced were still observed 14 days after the treatment (6 days of expansion + 8 days of differentiation), suggesting a durable effect of the epigenetic editing.

[0127] Material & methods:

[0128] Plasmids

[0129] Plasmids used in this study include dCas9 (Addgene, #100091), Tetl-dCas9 (Addgene, #136650), dCas9-CBPcore (Addgene, #179543) and TETv4 (Addgene #167983). The plasmids used as template for mRNA in vitro transcription were obtained by substituting the coding sequence of pCMV-T7-ABE8e-nSpRY-P2A-EGFP (Addgene, #185912) with the desired editor through Gibson assembly cloning (NEB) following manufacturer’s recommendation. All the sgRNA were cloned into the pMA128 (provided by M. Amendola, Genethon, France).

[0130] sgRNA design

[0131] To generate the plasmids expressing the sgRNAs recognizing the specific protospacer (Table 1), oligonucleotides were annealed, and the duplexes were ligated into the Bbsl-digested MA128 plasmid. For RNA-mediated base editing we used chemically modified synthetic sgRNAs harboring 2'-O-methyl analogs and 3'-phosphorothioate nonhydrolyzable linkages at the first three 5' and 3' nucleotides (Synthego).

[0132] Table 1. List of sgRNA spacer sequences.

[0133]

[0134] mRNA in vitro transcription

[0135] 10 pg of editors-expressing plasmids were digested overnight with 20 Units of a restriction enzyme that cleaves once after the stop codon. The linearized plasmids were purified using the PCR purification kit (QIAGEN) and were eluted in 30 pl of DNase / RNase-free water.

[0136] 1 pg of linearized plasmid was used as template for the in vitro transcription (ivt) reaction (MEGAscript, Ambion). The ivt protocol was modified as follows. The GTP nucleotide solution was used at a final concentration of 3.0 mM instead of 7.5 mM and the anti -reverse cap analog N7-Methyl-3'-O-Methyl-Guanosine-5'-Triphosphate-5'-Guanosine (ARCA, Trilink) was used at a final concentration of 12.0 mM resulting in a final ratio of Cap:GTP of 4:1 that allows efficient capping of the mRNA. The incubation time for the ivt reaction was reduced to 30 minutes. The polyadenylation step was performed following manufacturer's guidelines (Poly-A tailing kit, Ambion). mRNA was precipitated using lithium chloride and resuspended in TE buffer in a final volume that allowed to achieve a concentration of >1 pg / pl. The mRNA quality was evaluated using Tapestation 2200 (Agilent).

[0137] Cell line culture and transfection

[0138] HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of fetal bovine serum (Lonza) and 1% penicillin / streptomycin (Gibco). For transfections, 1.5xl05cells / condition were seeded 24h before in a 24-well plate and then transfected with polyethylenimine (PEI) with epigenome editor-expressing plasmids and sgRNA-containing plasmid in a ratio 3:1 with a total of 1 ug DNA transfected. In the combinations with less sgRNAs, the MA128 plasmid was used to reach 1 ug of total DNA per transfection. 24h after transfection the medium was changed. Cells were collected 72h after transfection (or at indicated time points for time course experiment) for qRT-PCR analysis.

[0139] HUDEP-2 cells were cultured in expansion medium containing StemSpan SFEM (Stem Cell Technologies) supplemented with 1 pg / mL of doxycycline (Sigma), IxlO'6M of dexamethasone (Sigma), 100 ng / mL of human stem cell factor (hSCF, Peprotech), 3 UI / mL of EPO (EPREX 10000U from Necker pharmacy), 1% penicillin and streptomycin, and L-glutamine. The cells were expanded at a concentration of 100,000 cells / mL. HUDEP-2 were differentiated in Iscove's Modified Dulbecco’s Medium (IMDM) supplemented with 5% AB human serum, 3 U / mL of EPO, 10 pg / mL of insulin, 330 ug / mL of holo-transferrin, 100 ng / mL of hSCF, 2 U / mL of heparin, 1 pg / mL of doxycycline, 1% Pen / Strep and 1% L-glutamine. The cell concentration varied during the differentiation: 200,000 cells / mL at day 1-2; 350,000 cells / mL at day 2-3; 500,000 cells / mL at day 4-7 and up to 1,000,000 cells / mL after day 8.

[0140] HSPC purification and culture

[0141] Adult human non-mobilized peripheral blood CD34+ HSPCs from SCD patients and fetal human CD34+ HSPCs from fetal liver of a healthy donor were obtained from the “Hopital Necker-Enfants malades” Hospital (Paris, France). Written informed consent was obtained from all adult subjects. All experiments were performed in accordance with the Declaration of Helsinki. The study was approved by the regional investigational review board (reference: DC 2022-5364, CPP Ile-de-France II “Hopital Necker-Enfants malades”). HSPCs were purified by immunomagnetic selection with MACS columns (Miltenyi Biotec) after immunostaining with the CD34 MicroBead Kit (Miltenyi Biotec). 48 hours before transfection, CD34+cells were thawed and cultured at a concentration of 5xl05cells / ml in StemSpan (STEMCELL Technologies) supplemented with penicillin / streptomycin (Gibco), 250 nM StemRegenin (STEMCELL Technologies), 38 nM UM171 (STEMCELL Technologies), and the following recombinant human cytokines (PeproTech): human stem cell factor (SCF) (300 ng / ml), Flt-3L (300 ng / ml), thrombopoietin (TPO) (100 ng / ml), and interleukin-3 (IL-3) (60 ng / ml).

[0142] RNA transfection

[0143] 0.5xl05to 2xl05CD34+HSPCs or 2xl05HUDEP-2 cells per condition were transfected with 2 pg of each enzyme encoding mRNA, and a mix of synthetic sgRNAs (2 ug each). The P3 Primary Cell 4D-Nucleofector X Kit S (Lonza) was used with the CA137 program (Nucleofector 4D). Cells transfected with TE buffer or with the enzyme-encoding mRNA only served as negative controls.

[0144] Colony-forming cell (CFC) assay

[0145] CD34+HSPCs were plated at a concentration of 1000 cells / mL in a methylcellulose-based medium (GFH4435, Stem Cell Technologies) under conditions supporting erythroid (BFU-E) and granulocyte / monocyte (CFU-GM) differentiation. BFU-E and CFU-GM colonies were counted after 14 days. Colonies were randomly picked and collected as bulk populations (containing at least 25 colonies) to evaluate hemoglobin production by HPLC.

[0146] HSPC differentiation Transfected HSPCs were differentiated into mature RBCs using a three-phase erythroid differentiation protocol. During the first phase (day 0 to day 6), cells were cultured in a basal erythroid DOUAY medium supplemented with 100 ng / ml recombinant human SCF (PeproTech), 5 ng / ml recombinant human IL-3 (PeproTech), 3 lU / ml EPO Eprex (Janssen-Cilag) and 10-6 M hydrocortisone (Sigma). During the second phase (day 6 to day 9), cells were co-cultured with MS-5 stromal cells in the basal erythroid DOUAY medium supplemented with 3 lU / ml EPO Eprex (Janssen-Cilag). During the third phase (day 9 to day 13), cells were co-cultured with stromal MS-5 cells in a basal erythroid DOUAY medium without cytokines. During the last phase (day 13 to day 20), heat-inactivated human AB serum (10%) was added to the basal erythroid DOUAY medium. Erythroid differentiation was monitored by flow cytometry analysis of CD36, CD71, GPA, BAND3, and CD49d erythroid surface markers and analysis of enucleated cells using the DRAQ5 double-stranded DNA dye.

[0147] 7AAD was used to identify live cells.

[0148] Flow cytometry analysis

[0149] HSPC-derived erythroid cells and HUDEP-2 cells were fixed with 0.05% cold glutaraldehyde and permeabilized with 0.1% TRITON X-100. After fixation and permeabilization, cells were stained with an antibody recognizing GPA erythroid surface marker (1 / 100 PE-Cy7-conjugated anti-GPA antibody, 563666, BD Pharmingen) and an antibody recognizing HbF (1 / 5 FITC-conjugated anti-HbF antibody, clone 2D12552829 BD). Flow cytometry analysis of CD36, CD71, GPA, BAND3 and CD49d erythroid surface markers was performed using a V450-conjugated anti-CD36 antibody (1 / 20 561535, BD Horizon), a FITC-conjugated anti-CD71 antibody (1 / 50 555536, BD Pharmingen), a PE-Cy7-conjugated anti-GPA antibody (1 / 100 563666, BD Pharmingen), a PE-conjugated anti-BAND3 antibody (1 / 50 9439, IBGRL) and an APC-conjugated anti-CD49d antibody (1 / 20 559881, BD). Flow cytometry analysis of enucleated or viable cells was performed using double-stranded DNA dyes DRAQ5 (65-0880-96, Invitrogen) and 7AAD (559925, BD), respectively. All analyses were performed using the Novocyte Flow Cytometry system (Agilent). Data were analysed using the FlowJo (BD Biosciences) software.

[0150] Western blot

[0151] HSPCs, HSPC-derived erythroblasts and K562 cells were harvested after electroporation at indicated time points (12 hours, day 1, day 2, day 6). Cells were lysed with RIPA buffer (Thermo Fisher Scientific) supplemented with cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche) and incubated on ice for 30 minutes. Lysates were sonicated using the Sonics Vibra-Cell VCX750 (2 cycles of 10 seconds, 9 seconds ON and 1 second OFF, amplitude 50%). Following sonication, samples were centrifuged at 13,000 x g for 12 min at 4°C and the soluble fractions were collected. Proteins were quantified with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) following manufacturer instructions. At least 30 pg of total protein were mixed with SDS loading buffer, heated and loaded on NuPAGE™ Bis-Tris Mini Protein Gels, 4-12% (Thermo Fisher Scientific) using NuPAGE™ MOPS SDS Running Buffer (Thermo Fisher Scientific). Proteins were transferred to a PVDF membrane via wet transfer in buffer containing 10% Tris / Glycine, 0.5% SDS and 10% ethanol. Membranes were blocked with 3% BSA in PBS-Tween (PBS + 0.1% Tween-20) and incubated with antibodies specifically recognizing Cas9 (Diagenode, Cl 5200216), actin (Millipore MAB1501) and a-globin (Santa Cruz 31110) at 4°C overnight. Membranes were washed at least 3 times PBS-Tween and then incubated with HRP-conjugated secondary antibodies (Santa Cruz). Protein bands were visualized using a ChemiDoc XRS imaging system (BioRad).

[0152] RP-HPLC analysis of globin chains

[0153] RP-HPLC analysis was performed using a NexeraX2 SIL-30AC chromatograph and the LC Solution software (Shimadzu). A 250 x 4.6 mm, 3.6 pm Aeris Widepore column (Phenomenex) was used to separate globin chains by HPLC. Samples were eluted with a gradient mixture of solution A (water / acetonitrile / trifluoroacetic acid, 95:5:0.1) and solution B (water / acetonitrile / trifluoroacetic acid, 5:95:0.1). The absorbance was measured at 220 nm.

[0154] CE-HPLC analysis of hemoglobin tetramers

[0155] Cation-exchange HPLC analysis was performed using a NexeraX2 SIL-30AC chromatograph and the LC Solution software (Shimadzu). A 2 cation-exchange column (PolyCAT A, PolyLC, Columbia, MD) was used to separate hemoglobin tetramers by HPLC. Samples were eluted with a gradient mixture of solution A (20 mM bis Tris, 2 mM KCN, pH = 6.5) and solution B (20 mM bis Tris, 2mM KCN, 250 mM NaCl, pH = 6.8). The absorbance was measured at 415 nm.

[0156] Sickling assay

[0157] HSPC-derived mature RBCs obtained at the end of the erythroid differentiation, were incubated under gradual hypoxic conditions (20% O2 for 20 min; 10% O2 for 20 min; 5% O2 for 20 min; 0% O2 for 60-120 min) and a time course analysis of sickling was performed in real-time by video microscopy. Images were captured every 20 min using a Spinning disk confocal microscope (Zeiss) and a 40x objective. Throughout the time course, images were captured and then processed with ImageJ to determine the percentage of non-sickle RBCs per field of acquisition in the total RBC population. Approximately 500 cells were counted per condition.

[0158] DNA and RNA extraction

[0159] Genomic DNA was extracted using PURE LINK Genomic DNA Mini kit (LifeTechnol ogies), or Quick-DNA / RNA Miniprep (ZYMO Research) following manufacturer’ s instructions. Total RNA from HEK293T was extracted using the RNeasy Micro kit (QIAGEn) following manufacturer’s instructions. RNA from erythroid cells was extracted using Quick-DNA / RNA Miniprep (ZYMO Research) and treated with DNase (DNase I kit; Invitrogen) according to manufacturer’s instructions.

[0160] cDNA synthesis and RT-qPCR

[0161] Synthesis of cDNA from HEK293T samples was performed using the High-capacity cDNA reverse transcription Kit (ThermoFisher Scientific). Mature transcripts from HSPC-derived erythroblast were reverse-transcribed using SuperScript First-Strand Synthesis System for RT-qPCR (Invitrogen) with oligo (dT) primers. RT-qPCR was performed using primers listed in Table 2, the iTaq universal SYBR Green master mix (Biorad), and the CFX384 Touch Real-Time PCR Detection System (Biorad).

[0162] Table 2. List of primers used for RT-qPCR.

[0163]

[0164] DNA methylation analysis

[0165] Genomic DNA was treated with bisulfite using EZ DNA Methylation-GoldTM kit following manufacturer's instructions (ZYMO research). After bisulfite conversion, DNA was PCR-amplified using the PyroMark PCR kit (QIAGEN) (Table 3), then purified using the QIAquick PCR Purification kit (QIAGEN). The PCR products were cloned in pCR4-TOPO vector following the TOPO™ TA Cloning™ kit for sequencing protocol. The ligation products were transformed into E. Coli One Shot™ TOP 10 competent bacteria (ThermoFisher Scientific). Plasmid extracted from single bacterial colonies were sequenced by Sanger sequencing and the results were analysed using the Quantification Tools for Methylation Analysis (QUMA) tool. Alternatively, bisulfite converted DNA was first PCR-amplified using the PyroMark PCR kit (QIAGEN) and primers containing specific DNA stretches (MR3 for forward and MR4 for reverse primers; Table 3). Amplicons were then purified using Ampure XP beads (Beckman Coulter) and used as template for a second PCR using the Phusion High-Fidelity polymerase (NEB) with primers containing Unique Dual Index (UDI) Illumina-compatible barcodes annealing to MR3 and MR4 sequences (Table 3). The amplicons were pooled equimolarly, purified using the High Pure PCR product purification kit (Sigma-Aldrich), and sequenced using Illumina NovaSeq 6000 system (paired-end sequencing; 2*150-bp) to obtain a minimum of 100.000 reads per amplicon. NGS data were analysed using CRISPResso241, measuring the percentage of converted / unconverted cytidines in CpGs nucleotides. Sequenced CpGs were aligned to hg38 reference genome, and their position are reported in Table 4.

[0166] Table 4. Genomic position of HBG promoters CpG

[0167]

[0168]

[0169] In silico predicted off-targets

[0170] The sgRNA-dependent off-target sites were predicted in silico using COSMID42. The following settings were used for each sgRNA: PAM suffix: NGG; number of allowed mismatched with no InDeis: 3; number of allowed mismatched with 1-base Del: 2; number of allowed mismatched with 1-base Ins: 2.

[0171] Whole transcriptome sequencing

[0172] Total RNA was isolated using the Quick-DNA / RNA Miniprep Kit (Zymo) including a DNAse treatment step. RNA quality was assessed by capillary electrophoresis using High Sensitivity RNA reagents with the Fragment Analyzer (Agilent Technologies) and the RNA concentration was measured by using both Xpose spectrophometry (Trinean) and Fragment Analyzer (Agilent Technologies) capillary electrophoresis. RNA-seq libraries were prepared starting from 100 ng of total RNA using the RNA Library Prep Kits with Polaris Depletion (Watchmaker) as recommended by the manufacturer to deplete rRNA and globin mRNAs. qPCR according to the Illumina qPCR Quantification Protocol Guide was performed to quantify the RNA-seq libraries. An equimolar pool of the final indexed RNA-Seq libraries was prepared and sequenced using the Illumina NovaSeq X system (paired-end sequencing; 2*100-bp, 50 million reads per library).

[0173] Read quality was assessed using FastQC [version 0.11.9; https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ]. Adapter sequences and low-quality bases (Q < 20) were trimmed from raw reads with BBDuk [version 38.92; https: / / sourceforge.net / projects / bbmap / ]; moreover, the first 10 nucleotides were forcetrimmed for low quality. Reads shorter than 35 bp post-trimming were discarded. Trimmed reads were aligned to the human reference genome (hg38) using STAR [version 2.7.9a]43. Raw gene counts were generated in R-4.1.1 using the featureCounts function of the Rsubread package [version 2.8.1]44and the GENCODE 44 basic gene annotation for hg38 reference genome. Raw gene counts were normalized to counts per million mapped reads (CPM) and to fragments per kilobase of exon per million mapped reads (FPKM) using the edgeR R package [version 3.36.0]45; only genes with a CPM greater than 1 in at least 3 samples were retained for differential analysis. Differential gene expression analysis was performed using the glmQLFTest function (using donor as a blocking variable for SCD samples) or the exactTest function (for HD samples) of the edgeR R package. Genes with FDR < 0.05 and absolute log2 fold change > 1 were defined as differentially expressed. Functional enrichment analysis on MsigDB Hallmark gene sets was performed using the enricher function of the clusterProfiler R package [version 4.12.5]46. To investigate the expression profile of transposable elements, we used the TEtranscripts package (ADD REF). Firstly, trimmed reads were aligned to the human reference genome (hg38) using STAR [version 2.7.9a]43, setting winAnchorMultimapNmax to 200 and outFilterMultimapNmax to 100, as recommended by TEtranscripts authors. The raw count matrix for transposable elements was obtained by the TEcount function of TEtranscripts package, using the hg38 GTF files for TE annotation provided in the TEtranscripts web site (https: / / www.mghlab.org / software / tetranscripts). Raw counts for transposable elements were collapsed by families and groups and normalized to counts per million mapped reads (CPM) using the edgeR R package [version 3.36.0]45within R-4.1.1. Differential gene expression analysis was performed using the glmQLFTest function (using donor as a blocking variable for SCD samples) or the exactTest function (for HD samples) of the edgeR R package.

[0174] Nanopore-based genomic DNA sequencing and combined methylation / hydroxymethylation analysis

[0175] Genomic DNA (0.8 pg) was fragmented using a Covaris g-TUBE to achieve an average fragment size of 8-9 kb. The DNA was brought to a final volume of 50 pL and centrifuged at 5,500 x g for 1 minute. The tube was then inverted, and the centrifugation was repeated for an additional 1 minute. The sheared DNA fragments were processed using the SQK-LSK114 library preparation kit protocol from Oxford Nanopore Technologies. This included end-repair, A-tailing of the DNA, and adapter ligation. Libraries were individually sequenced on a PromethlON P2i device, using one flow cell (FLO-PRO114M) per sample to achieve an average coverage of 3 OX. Raw Pod5 data were basecalled using MinKNOW to detect 5mCpG and 5hmCpG modifications. The resulting reads were aligned to the CHM13-T2T and GRCh38.pl4 annotated human genome assemblies using Minimap2, generating BAM files. Methylation and hydroxymethylation data were extracted and visualized using Modbamtools and Methylartist, enabling both global analyses and locus-specific investigations. For the analysis of repetitive elements, the BED files containing the locations of SINE and LINE transposable elements were retrieved from RepeatMasker.org. The analysis was then performed using Bedtools for the intersection of regions of interest and Modkit for the annotation and quantification of associated epigenetic modifications. Xenotransplantation of HSPCs in NBSGW mice

[0176] NOD.Cg-KitW-41JTyr +PrkdcscidI12rgtmlWjl / ThomJ (NBSGW) mice were housed in a pathogen-free facility. Control or edited mobilized CD34+ cells (3 * 105 cells per mouse) were transplanted into non-irradiated NBSGW male or female mice of 6-9 weeks of age via retro-orbital sinus injection. NBSGW mice were conditioned with busulfan (Sigma- Aldrich) injected intraperitoneally (15 mg / kg body weight) 24 hours before transplantation. Sixteen weeks after transplantation, NBSGW primary recipients were euthanized. Cells were harvested from bone marrow (BM), thymus, spleen, and blood, and stained with antibodies against the following murine and human surface markers: murine CD45 (1 / 50 mCD45-VioBlue; Miltenyi Biotec), human CD45 (1 / 50 hCD45-APCvio770; Miltenyi Biotec), human CD3 (1 / 50 CD3-APC; Miltenyi Biotec), human CD14 (1 / 50 CD14- PECy7; BD Biosciences), human CD15 (1 / 50 CD15-PE; Miltenyi Biotec), human CDllb (1 / 100 CDllb-APC; Miltenyi Biotec), human CD19 (1 / 100 CD19-BV510; BD Biosciences), human CD235a (1 / 50 CD235a-PE; BD Biosciences), human CD71 (1 / 10 CD71-APC; BD Biosciences), CD36 (1 / 50 CD36-FITC; BD Bio- sciences), and CD34 (1 / 100 CD34-PE-Vio770; Miltenyi Biotec). Cells were analyzed by flow cytometry using the Novocyte analyzer (Agilent) and the FlowJo software (BD Biosciences). Human BM CD45+ cells were sorted by immunomagnetic selection with CD45 MicroBeads (Miltenyi Biotec) and subjected to immunostaining with biotinylated antibodies that recognized the following surface markers: CD3 (dilution 1 / 25, clone HIT3a; BD), CD19 (dilution 1 / 25, clone HIB19; BD), B220 (dilution 1 / 50, clone RA3-6B2; BD), Teri 19 (dilution 1 / 50, clone TER-119; BD), and mCD117 (clone 2B8; BD). BM cells were washed and incubated with 20 pL of Anti-Biotin beads (Miltenyi Biotec). After washing, the cells were magnetically purified using an LS column (Miltenyi Biotec) according to the manufacturer’s instructions. Cells from the negative fraction were immuno-stained with the following antibodies: CD235a-PE (dilution 1 / 5000, BD) and hCD45-BV510 (dilution 1 / 100, BD). The hCD451ow / - / CD235ahigh cells were sorted using the MA900 cell sorter (Sony Biotechnology, San Jose, CA) and subjected to DNA methylation, RT-qPCR, RP-HPLC and CE-HPLC analyses. Flow cytometry analysis for HbF was performed as previously described on cells from the negative fraction. All experiments and procedures were performed in compliance with the French Ministry of Agriculture’s regulations on animal experiments and were approved by the regional Animal Care and Use Committee (APAFIS#2019061312202425_v4). Mice were housed in a temperature- (20°C-22°C) and humidity (40%-50%)-controlled environment with 12:12 hour light-dark cycle and fed ad libitum a standard diet. ChlP-seq data source

[0177] We analyzed publicly available H3K4me3, H3K27ac, H3K9ac, H3K27me3 and H3K9me3 ChlP-seq datasets in human primary fetal, adult and HEK293T47’48. For the histone modifications in human primary fetal and adult, we downloaded the hgl8-mapped read bed files from Gene Expression Omnibus (GSM908038, GSM908039, GSM908050, GSM908051, GSM908048, GSM908049, GSM908042, GSM908043, GSM908040, GSM908041) and converted them to the hgl9 build of the human genome using the UCSC liftOver tool. We used MACS213 to build signal tracks and call peaks. For HEK293T dataset, we downloaded the hgl9-mapped read bed files from Gene Expression Omnibus (GSM4301071, GSM4301076, GSM4301082, GSM4301086).

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Claims

CLAIMS:1 A method of increasing fetal hemoglobin content in a eukaryotic cell comprising the step of contacting the eukaryotic cell with an epigenome editing platform that comprises (a) first epigenome editing enzyme wherein the effector domain consists of a histone acetyltransferase, (b) a second epigenome editing enzyme wherein the effector domain consists of a DNA demethylase, (c) a plurality of sgRNAs designed for guiding the epigenome editing enzymes to a target sequences in the region of the HBG1 and / or HBG2 promoter so as to reduce CpG methylation levels, thereby increasing the expression of gamma-globin in said eukaryotic cell.2 The method according to claim 1 wherein the eukaryotic cell is selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), pluripotent cells (i.e. embryonic stem cells (ES) and induced pluripotent stem cells (iPS)).3 The method according to claim 1 or 2 wherein the DNA binding domain of the epigenome editing enzymes consists of a defective CRISPR / Cas nuclease.4 The method according to claim 3 wherein the defective CRISPR / Cas nuclease is a a dead Cas9.5 The method according to any one of claims 1 to 4 wherein the effector domain of the first epigenome editing enzyme is a CREB binding protein (CBP) or a fragment thereof such as the core domain of CBP.6 The method according to any one of claims 1 to 4 wherein the effector domain of the second epigenome editing enzyme is TET1.7 The method according to any one of claims 1 to 6 wherein the sgRNA comprises a spacer sequence selected from Table 1.8 The method according to any one of claims 1 to 7 wherein the epigenome editing platform comprises the g-28, g-115, g-197, g-211 sgRNAs wherein the g-28 sgRNA comprises the space sequence as set forth in SEQ ID NO: 6, the g-115 sgRNA comprises the spacer sequence as set forth in SEQ ID NO:5, the g-197 sgRNA comprises the52spacer sequence as set forth in SEQ ID NO:3 and the g-211 sgRNA comprise the spacer sequence as set forth in SEQ ID NO:2.9 A population of eukaryotic cells that are obtainable by the method according to any one of claims 1 to 8.10 A method for increasing fetal hemoglobin levels in a subj ect in need thereof, the method comprising transplanting a therapeutically effective amount of the population of eukaryotic cells according to claim 9.11 The method according to claim 10 wherein the subject suffers from a P- hemoglobinopathy such as sickle cell disease or P-thalassemia.53

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