Patent Application
20140038239
The invention relates to a meganuclease variant cleaving a DNA target sequence from the human hemoglobin beta gene, to a vector encoding said variant, to a cell, an animal or a plant modified by said vector and to the use of said meganuclease variant and derived products for genome therapy ex vivo (gene cell therapy), and genome engineering.
Hemoglobin (haemoglobin or Hb) molecules are responsible for carrying oxygen via the red blood cells, from the lungs to various parts of the body for use in cellular respiration. Mature haemoglobin (normal adult haemoglobin or Hb A) is a tetrameric protein composed of two types of proteins: two alpha chains and two beta chains. The haemoglobin beta chain is often called beta globin (β-globin). Each protein subunit of hemoglobin carries an iron-containing molecule called heme which is responsible for the oxygen binding. A complete hemoglobin protein is then capable of carrying four oxygen molecules at a time.
The gene which codes for the haemoglobin beta chain (HBB gene or beta globin (β-globin) gene) is located on the short (p) arm of chromosome 11 at position 15.5 (11p15.5). It is composed of 3 exons that are distributed over 1606 base pairs of genomic DNA (Accession number GenBank NC—000011.8 or NT—009237.17). The mRNA (626-bp; accession number GenBank NM—00518.4) is translated into the 147-amino acid sequence of the HBB polypeptide chain [Accession number GenBank NP—00509.1; Online Mendelian Inheritance in Man, OMIM™. Johns Hopkins University, Baltimore, Md. MIM No. 141900 (Nov. 6, 2001)].
Hundreds of alterations have been identified in the HBB gene. Some of these changes result in the production of abnormal forms of hemoglobin, often designated by letters of the alphabet and sometimes also by a name. These abnormal forms of haemoglobin cause sickle cell diseases (sickle cell syndromes or sickle cell anemia syndromes) that include sickle cell anemia (HbSS), hemoglobin SC (HbSC) and hemoglobin SE (HbSE), (Park, K. W., Int. Anesthesiol. Clin., 2004, 42, 77-93; Stuart, M. J. and Nagel R. L., Lancet, 2004, 364, 1343-1360). Other conditions, called beta thalassemias are caused by many genetic mutations that abolish or reduce production of the beta globin subunit of hemoglobin. Hemoglobin sickle-beta thalassemia (HbSBetaThal) is caused when mutations that produce hemoglobin S and beta thalassemia occur together.
Sickle cell diseases are a group of inherited hemoglobin disorders characterized by chronic hemolytic anemia, an increased susceptibility to infections, organ damage, and vascular occlusion causing both acute and chronic pain (Ashley-Koch et al., American Journal of Epidemiology, 2000, 151, 839-845; The metabolic & molecular bases of inherited disease, 8th Ed., Scriver, C. R., c2001: p4571-4636, McGraw-Hill, New York). It is estimated that 70,000 Americans of different ethnic backgrounds have sickle cell disease, and sickle cell syndromes are present in 1 in 400 African Americans. Populations from the Mediterranean basin, the Middle East, and India have also a high frequency of the disease.
The most common beta haemoglobin variant resulting in sickle cell disease is hemoglobin S or HbS which involves the substitution of the glutamic acid at position 6 of the beta globin chain with a valine (E6V).
In sickle cell anemia (HbSS), a common form of sickle cell disease, hemoglobin S or HbS replaces both beta hemoglobin subunits in haemoglobin (Ashley-Koch et al., Am. J. Epidemiol. 2000, 151, 839-845; Sickle cell disease, Sergeant G., Oxford University Press: Oxford, 1985). The mutation causes the abnormal HbS subunits to stick together and form long, rigid molecules. The rigid HbS molecules bend red blood cells into a sickle shape which die prematurely. Therefore the mutation leads to a shortage of red blood cells (anemia). The sickle-shaped cells can also block small blood vessels, causing pain and organ damage.
In other types of sickle cell disease, just one beta hemoglobin subunit is replaced with hemoglobin S. The other beta hemoglobin subunit is replaced with a different abnormal variant, such as hemoglobin C or hemoglobin E. For example, in the hemoglobin SC (HbSC) disease, the beta hemoglobin subunits are replaced by hemoglobin S and hemoglobin C. The severity of this disorder is quite variable, but it can be as severe as sickle cell anemia. Hemoglobin C (HbC) is more common in people of West African descent than in other populations and results from the substitution of the glutamic acid at position 6 by a lysine (E6K). Hemoglobin E (HbE) is a variant of hemoglobin found predominantly in the people of Southeast Asia. The mutated protein results from the substitution of the glutamic acid at position 26 by a lysine (E26K).
Defects in HBB are the cause of beta-thalassemia (Thein S L., Br. J. Haematol., 2004, 124, 264-274). It occurs mostly in Mediterranean and Southeast Asian populations. More than 200 HBB mutations that cause beta thalassemia have been identified. Most of the mutations involve a change in a single nucleotide within the HBB gene, but insertion or deletion has also been reported. The hallmark of beta-thalassemia is an imbalance in globin-chain production in the adult HbA molecule. Absence of beta chain causes beta(0)-thalassemia, while reduced amounts of detectable beta globin causes beta(+)-thalassemia. In the severe forms of beta-thalassemia, the excess alpha globin chains accumulate in the developing erythroid precursors in the marrow. Their deposition leads to a vast increase in erythroid apoptosis that in turn causes ineffective erythropoiesis and severe microcytic hypochromic anemia.
The most common problem for sickle cell patients is the pain episode mainly in the extremities, back, chest, and abdomen (Dwarakanath, G. K. and Warfield C., Hosp. Pract., 1986, 64b; Fields H. L. and Levine J. D., West. J. Med. 1984, 347, 141-143; Vichinski et al., Am. J. Ped. Hematol. Oncol., 1982, 4, 328). The exact cause of the intense pain is unknown. It could be caused by the inflammatory response resulting from the obstruction and sludging of blood flow produced by sickled erythrocytes. Pain episodes are usually managed by the use of analgesic. However, despite the pain, the most serious live threatening complications are bacterial infection and splenic sequestration crisis. Bacterial infection is one of the main causes of death in patients with sickle cell disease (Barrett-Connor, E., Medicine, 1971, 50, 97-112), especially when children are under three years old. As splenic function is decreased or absent in sickle cell anemia, the patients have a high risk of infection within encapsulated organisms such as Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Meningococcus, and others [Powars et al., JAMA, 1981, 245, 1839-1842]. Thus, prophylactic penicillin is always instituted in the young child (Gaston et al., N. Engl. J. Med., 1986, 314, 1593-99). Splenic sequestration crisis is one of the most serious complications and is second cause of death in infants with sickle cell disease. This event usually occurs between the ages of four months and three years. During sequestration episodes sickle cells are trapped in the spleen, causing rapid fall in the hemoglobin level and enlargement of the spleen and others complications. The only treatment today consists of aggressive blood transfusions. Emergent splenectomy is occasionally required (Vichinsky et al., N Engl. J. Med., 1995, 222, 206-213; Haberkern et al., Blood, 1995, 86, 142a).
Daily administration of oral anti-sickling agent like hydroxyurea (Hydrea) is the first effective treatment documented to provide significant benefits in sickle cell disease (Steinberg M H., Scientific World Journal, 2002, 2, 1706-1728; Mehanna A. S., Curr. Med. Chem., 2001, 8, 79-88; Charache et al., N. Engl. J. Med., 1995, 332, 1317-1322). However, the long term benefits of hydroxyurea and toxicities are unknown. Thus, today, blood transfusions are still the best acute treatment for life threatening complications.
A second strategy would be the bone marrow transplantation. It has been used to successfully convert 27 patients in the United States from hemoglobin SS to normal AA or AS (depending on the phenotype of the related marrow donor). However, bone marrow transplantation is still an experimental therapy limited to patients who have enough complications from sickle cell disease to warrant the risk of death (10% mortality) and long-term complications from bone marrow transplantation and who also have a sibling with an identical HLA match (Walters et al., New Engl. J. Med., 1996, 355, 369-376; Walters et al., Biol. Blood Marrow Transplant., 1996, 2, 100-104).
Allogenic hematopoietic stem cell (HSC) transplantation is curative, but this therapeutic option is not available to the majority of patients. The transfer of a regulated β-globin gene in autologous HCSs thus represents a highly attractive alternative treatment (Tisdale J. and Sadelain, M., Semin. Hematol., 2001, 38, 382-392).
HBB could appear to be an excellent candidate for gene therapy: the gene is very small and has been characterized extensively; the disorder is recessively inherited and affects blood cells. However, initial attempt in 1980 failed, largely because of inefficient gene transfer and poor expression of the introduced β-globin genes. Although, much more knowledge was gained about HBB gene expression, there have been no subsequent gene therapy attempts. This is due to the problem of the very tight control of gene expression required into the desired cells. Indeed, the amount of β-globin protein produced must be equal to the amount of α-globin, since the imbalance between β-globin and α-globin chains would result in an α-thalassemia phenotype (Sadelain M. et al., Best. Pract. Res. Clin. Haematol., 2004, 17, 517-534). Nevertheless, ex vivo gene therapy seems to be very attractive, and tremendous achievement has been reached, at least in small animals as mice. Different humanized Sickle Cell Anemia (SCA) mouse models have been obtained by knock-out of the endogenous α- and β-globin genes and knock-in of the human α-, βS- and γ-globin gene (Paszty et al., Science 1997, 278, 876-878; Ryan et al., Science, 1997, 278, 873-876; Chang et al., Proc. Nat. Acad. Sci. USA, 1998, 95, 14886-14890), the sickle cell mice recapitulating the major features found in human sickle cell disease. The sickle cell phenotype could be reversed by integration of a lentivirus-borne human 13-globin gene into bone marrow cells (May et al., Blood, 2002, 99, 1902-1908; Pawliuk et al., Science, 2001, 294, 2368-2371), providing a first step towards true human gene therapy.
Targeted homologous recombination is another alternative that should bypass the problems raised by current approaches. Current gene therapy strategies are based on a complementation approach, wherein randomly inserted but functional extra copy of the gene provide for the function of the mutated endogenous copy. In contrast, homologous recombination should allow for the precise correction of mutations in situ (
Homologous gene targeting strategies have been used to knock out endogenous genes (Capecchi, M. R., Science, 1989, 244, 1288-1292, Smithies, O., Nature Medicine, 2001, 7, 1083-1086) or knock-in exogenous sequences in the chromosome. It can as well be used for gene correction, and in principle, for the correction of mutations linked with monogenic diseases. However, this application is in fact difficult, due to the low efficiency of the process (10−6 to 10−9 of transfected cells). Recently, the gene targeting approach was used to correct the human β-S-globin gene in ES cells from model SCA mice (Chang et al., Proc. Nat. Acad. Sci. USA, 2006, 103, 1036-1040; Wu et al., Blood, 2006, 108, 1183-1188). However, classical selection scheme had to be used, due to the intrinsic low efficiency of the method.
In the last decade, several methods have been developed to enhance this yield. For example, chimeraplasty (de Semir, Nadal et al. 2003) and Small Fragment Homologous Replacement (Goncz, Colosimo et al. 2001; Bruscia, Sangiuolo et al. 2002; Sangiuolo, Bruscia et al. 2002; De Semir and Aran 2003) have both been used to try to correct CFTR mutations with various levels of success.
Another strategy to enhance the efficiency of recombination is to deliver a DNA double-strand break in the targeted locus, using meganucleases. Meganucleases are by definition sequence-specific endonucleases recognizing large sequences (Thierry, A. and B. Dujon, Nucleic Acids Res., 1992, 20, 5625-5631). They can cleave unique sites in living cells, thereby enhancing gene targeting by 1000-fold or more in the vicinity of the cleavage site (Puchta et al., Nucleic Acids Res., 1993, 21, 5034-5040; Rouet et al., Mol. Cell. Biol., 1994, 14, 8096-8106; Choulika et al., Mol. Cell. Biol., 1995, 15, 1968-1973; Puchta et al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 5055-5060; Sargent et al., Mol. Cell. Biol., 1997, 17, 267-277; Cohen-Tannoudji et al., Mol. Cell. Biol., 1998, 18, 1444-1448; Donoho, et al., Mol. Cell. Biol., 1998, 18, 4070-4078; Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101). Such meganucleases could be used to correct mutation responsible for monogenic inherited diseases.
The most accurate way to correct a genetic defect is to use a repair matrix with a non mutated copy of the gene, resulting in a reversion of the mutation. However, the efficiency of gene correction decreases as the distance between the mutation and the DSB grows, with a five-fold decrease by 200 bp of distance. Therefore, a given meganuclease can be used to correct only mutations in the vicinity of its DNA target.
An alternative, termed “exon knock-in” is featured in
However, although several hundreds of natural meganucleases, also referred to as “homing endonucleases” have been identified (Chevalier, B. S. and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774), the repertoire of cleavable sequences is too limited to address the complexity of the genomes, and there is usually no cleavable site in a chosen gene. For example, there is no cleavage site for a known natural meganuclease in human HBB gene. Theoretically, the making of artificial sequence specific endonucleases with chosen specificities could alleviate this limit. Therefore, the making of meganucleases with tailored specificities is under intense investigation.
Recently, fusion of Zinc-Finger Proteins (ZFPs) with the catalytic domain of the FokI, a class IIS restriction endonuclease, were used to make functional sequence-specific endonucleases (Smith et al., Nucleic Acids Res., 1999, 27, 674-681; Bibikova et al., Mol. Cell. Biol., 2001, 21, 289-297; Bibikova et al., Genetics, 2002, 161, 1169-1175; Bibikova et al., Science, 2003, 300, 764; Porteus, M. H. and D. Baltimore, Science, 2003, 300, 763-; Alwin et al., Mol. Ther., 2005, 12, 610-617; Urnov et al., Nature, 2005, 435, 646-651; Porteus, M. H., Mol. Ther., 2006, 13, 438-446). Such nucleases could recently be used for the engineering of the IL2RG gene in human cells from the lymphoid lineage (Urnov et al., Nature, 2005, 435, 646-651).
The binding specificity of Cys2-His2 type Zinc-Finger Proteins, is easy to manipulate, probably because they represent a simple (specificity driven by essentially four residues per finger), and modular system (Pabo et al., Annu Rev. Biochem., 2001, 70, 313-340; Jamieson et al., Nat. Rev. Drug Discov., 2003, 2, 361-368). Studies from the Pabo laboratories resulted in a large repertoire of novel artificial ZFPs, able to bind most G/ANNG/ANNG/ANN sequences (Rebar, E. J. and C. O. Pabo, Science, 1994, 263, 671-673; Kim, J. S, and C. O. Pabo, Proc. Natl. Acad. Sci. USA, 1998, 95, 2812-2817), Klug (Choo, Y. and A. Klug, Proc. Natl. Acad. Sci. USA, 1994, 91, 11163-11167; Isalan M. and A. Klug, Nat. Biotechnol., 2001, 19, 656-660) and Barbas (Choo, Y. and A. Klug, Proc. Natl. Acad. Sci. USA, 1994, 91, 11163-11167; Isalan M. and A. Klug, Nat. Biotechnol., 2001, 19, 656-660).
Nevertheless, ZFPs might have their limitations, especially for applications requiring a very high level of specificity, such as therapeutic applications. It was recently shown that FokI nuclease activity in fusion acts with either one recognition site or with two sites separated by varied distances via a DNA loop including in the presence of some DNA-binding defective mutants of FokI (Catto et al., Nucleic Acids Res., 2006, 34, 1711-1720). Thus, specificity might be very degenerate, as illustrated by toxicity in mammalian cells and Drosophila (Bibikova et al., Genetics, 2002, 161, 1169-1175; Bibikova et al., Science, 2003, 300, 764-.).
In the wild, meganucleases are essentially represented by homing endonucleases. Homing Endonucleases (HEs) are a widespread family of natural meganucleases including hundreds of proteins families (Chevalier, B. S. and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). These proteins are encoded by mobile genetic elements which propagate by a process called “homing”: the endonuclease cleaves a cognate allele from which the mobile element is absent, thereby stimulating a homologous recombination event that duplicates the mobile DNA into the recipient locus. Given their exceptional cleavage properties in terms of efficacy and specificity, they could represent ideal scaffold to derive novel, highly specific endonucleases.
HEs belong to four major families. The LAGLIDADG family, named after a conserved peptidic motif involved in the catalytic center, is the most widespread and the best characterized group. Seven structures are now available. Whereas most proteins from this family are monomeric and display two LAGLIDADG motifs, a few ones have only one motif, but dimerize to cleave palindromic or pseudo-palindromic target sequences.
Although the LAGLIDADG peptide is the only conserved region among members of the family, these proteins share a very similar architecture (
The making of functional chimeric meganucleases, by fusing the N-terminal I-DmoI domain with an I-CreI monomer (Chevalier et al., Mol. Cell., 2002, 10, 895-905; Epinat et al., Nucleic Acids Res, 2003, 31, 2952-62; International PCT Applications WO 03/078619 and WO 2004/031346) have demonstrasted the plasticity of LAGLIDADG proteins.
Besides, different groups have used a semi-rational approach to locally alter the specificity of the I-CreI (Seligman et al., Genetics, 1997, 147, 1653-1664; Sussman et al., J. Mol. Biol., 2004, 342, 31-41; International PCT Applications WO 2006/097784 and WO 2006/097853; Arnould et al., J. Mol. Biol., 2006, 355, 443-458; Rosen et al., Nucleic Acids Res., 2006, 34, 4791-4800; Smith et al., Nucleic Acids Res., 2006, 34, e149), I-SceI (Doyon et al., J. Am. Chem. Soc., 2006, 128, 2477-2484), PI-SceI (Gimble et al., J. Mol. Biol., 2003, 334, 993-1008) and I-MsoI (Ashworth et al., Nature, 2006, 441, 656-659).
In addition, hundreds of I-CreI derivatives with locally altered specificity were engineered by combining the semi-rational approach and High Throughput Screening:
Two different variants were combined and assembled in a functional heterodimeric endonuclease able to cleave a chimeric target resulting from the fusion of a different half of each variant DNA target sequence (Arnould et al., precited; International PCT Applications WO 2006/097854 and WO 2007/034262), as illustrated on
Furthermore, residues 28 to 40 and 44 to 77 of I-CreI were shown to form two separable functional subdomains, able to bind distinct parts of a homing endonuclease half-site (Smith et al. Nucleic Acids Res., 2006, 34, e149; International PCT Applications WO 2007/049095 and WO 2007/057781).
The combination of mutations from the two subdomains of I-CreI within the same monomer allowed the design of novel chimeric molecules (homodimers) able to cleave a palindromic combined DNA target sequence comprising the nucleotides at positions ±3 to 5 and ±8 to 10 which are bound by each subdomain (Smith et al., Nucleic Acids Res., 2006, 34, e149; International PCT Applications WO 2007/060495 and WO 2007/049156), as illustrated on
The combination of the two former steps allows a larger combinatorial approach, involving four different subdomains. The different subdomains can be modified separately and combined to obtain an entirely redesigned meganuclease variant (heterodimer or single-chain molecule) with chosen specificity, as illustrated on
These variants can be used to cleave genuine chromosomal sequences and have paved the way for novel perspectives in several fields, including gene therapy.
However, even though the base-pairs ±1 and ±2 do not display any contact with the protein, it has been shown that these positions are not devoid of content information (Chevalier et al., J. Mol. Biol., 2003, 329, 253-269), especially for the base-pair ±1 and could be a source of additional substrate specificity (Argast et al., J. Mol. Biol., 1998, 280, 345-353; Jurica et al., Mol. Cell., 1998, 2, 469-476; Chevalier, B. S, and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). In vitro selection of cleavable I-CreI target (Argast et al., precited) randomly mutagenized, revealed the importance of these four base-pairs on protein binding and cleavage activity. It has been suggested that the network of ordered water molecules found in the active site was important for positioning the DNA target (Chevalier et al., Biochemistry, 2004, 43, 14015-14026). In addition, the extensive conformational changes that appear in this region upon I-CreI binding suggest that the four central nucleotides could contribute to the substrate specificity, possibly by sequence dependent conformational preferences (Chevalier et al., 2003, precited).
Thus, it was not clear if mutants identified on 10NNN and 5NNN DNA targets as homodimers cleaving a palindromic sequence with the four central nucleotides being gtac, would allow the design of new endonucleases that would cleave targets containing changes in the four central nucleotides.
The Inventors have identified a series of DNA targets in the human beta globin gene that could be cleaved by I-CreI variants (Table I and
Even though the combined variants were initially identified towards nucleotides 10NNN and 5NNN respectively, and a strong impact of the four central nucleotides of the target on the activity of the engineered meganuclease was observed, functional meganucleases with a profound change in specificity were selected. Furthermore, the activity of the engineered protein could be significantly improved by random mutagenesis and screening, to compare with the activity of the I-CreI protein.
The I-CreI variants which are able to cleave a genomic DNA target from the human HBB gene can be used for genome therapy of sickle cell diseases and beta thalassemia and genome engineering at the beta globin locus (animal models and recombinant cells generation).
For example, the DNA target called HBB6, situated in the coding Exon 1 of HBB (positions 1138 to 1159 of SEQ ID NO: 4;
The meganucleases cleaving HBB8 or HBB5 could be used to correct the E6V mutation. Meganucleases cleaving HBB5 could also be used to correct other mutations at position 6 of the beta globin chain (HbC: E6K). In addition, meganucleases cleaving HBB5 or HBB6 could be used to correct any other amino acid mutation in the vicinity of the cleavage site (E26K). Since the efficiency of gene correction decreases when the distance to the DSB increases (Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101), this strategy would be most efficient with mutations located within 500 bp of the cleavage site. Alternatively, the same meganucleases could be used to knock-in exonic sequences that would restore a functional HBB gene at the HBB locus (
The invention relates to an I-CreI variant wherein at least one of the two I-CreI monomers has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated respectively from positions 26 to 40 and 44 to 77 of I-CreI, and is able to cleave a DNA target sequence from the human beta globin gene.
The cleavage activity of the variant according to the invention may be measured by any well-known, in vitro or in vivo cleavage assay, such as those described in the International PCT Application WO 2004/067736; Epinat et al., Nucleic Acids Res., 2003, 31, 2952-2962; Chames et al., Nucleic Acids Res., 2005, 33, e178 and Arnould et al., J. Mol. Biol., 2006, 355, 443-458. For example, the cleavage activity of the variant of the invention may be measured by a direct repeat recombination assay, in yeast or mammalian cells, using a reporter vector. The reporter vector comprises two truncated, non-functional copies of a reporter gene (direct repeats) and the genomic DNA target sequence within the intervening sequence, cloned in a yeast or a mammalian expression vector. Expression of the variant results in a functional endonuclease which is able to cleave the genomic DNA target sequence. This cleavage induces homologous recombination between the direct repeats, resulting in a functional reporter gene, whose expression can be monitored by appropriate assay.
The admitted numerals of positions of both HBB and meganuclease variants omit counting the first methionine; this explains the discrepancy observed between the usually used numeral annotation and the effective positions observed in the herewith attached sequence listing; more precisely:
as regards HBB numeration, it is in agreement with the numeration given in the OMIM™ database; therefore, the mutations E6K and E26K correspond in the reference sequence (GenBank NP—00509.1 or SEQ ID NO:150 of the herewith attached sequence listing) to mutations in positions 7 and 27 respectively; and
as regards meganuclease numeration, it is in agreement with I-CreI pdb accession code Ig9y.
Amino acid residues in a polypeptide sequence are designated herein according to the one-letter code, in which, for example, Q means Gln or Glutamine residue, R means Arg or Arginine residue and D means Asp or Aspartic acid residue.
Nucleotides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine. For the degenerated nucleotides, r represents g or a (purine nucleotides), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotides), d represents g, a or t, v represents g, a or c, b represents g, t or c, h represents a, t or c, and n represents g, a, t or c.
by “meganuclease”, is intended an endonuclease having a double-stranded DNA target sequence of 12 to 45 bp. Said meganuclease is either a dimeric enzyme, wherein each domain is on a monomer or a monomeric enzyme comprising the two domains on a single polypeptide.
by “meganuclease domain” is intended the region which interacts with one half of the DNA target of a meganuclease and is able to associate with the other domain of the same meganuclease which interacts with the other half of the DNA target to form a functional meganuclease able to cleave said DNA target.
by “meganuclease variant” or “variant” is intented a meganuclease obtained by replacement of at least one residue in the amino acid sequence of the wild-type meganuclease (natural meganuclease) with a different amino acid.
by “functional variant” is intended a variant which is able to cleave a DNA target sequence, preferably said target is a new target which is not cleaved by the parent meganuclease. For example, such variants have amino acid variation at positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target.
by “I-CreI” is intended the wild-type I-CreI having the sequence SWISSPROT P05725 or pdb accession code 1g9y, corresponding respectively to the sequence SEQ ID NO: 1 and SEQ ID NO: 178 in the sequence listing.
by “I-CreI variant with novel specificity” is intended a variant having a pattern of cleaved targets different from that of the parent meganuclease. The terms “novel specificity”, “modified specificity”, “novel cleavage specificity”, “novel substrate specificity” which are equivalent and used indifferently, refer to the specificity of the variant towards the nucleotides of the DNA target sequence.
The variant according to the present invention may be an homodimer or an heterodimer. Preferably, both monomers of the heterodimer are mutated at positions 26 to 40 and/or 44 to 77. More preferably, both monomers have different substitutions both at positions 26 to 40 and 44 to 77 of I-CreI.
In a preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 44 to 77 of I-CreI are at positions 44, 68, 70, 75 and/or 77.
In another preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 26 to 40 of I-CreI are at positions 26, 28, 30, 32, 33, 38 and/or 40.
In another preferred embodiment of said variant, it comprises one or more mutations at positions of other amino acid residues that contact the DNA target sequence or interact with the DNA backbone or with the nucleotide bases, directly or via a water molecule; these residues are well-known in the art (Jurica et al., Molecular Cell., 1998, 2, 469-476; Chevalier et al., J. Mol. Biol., 2003, 329, 253-269). In particular, additional substitutions may be introduced at positions contacting the phosphate backbone, for example in the final C-terminal loop (positions 137 to 143; Prieto et al., Nucleic Acids Res., Epub 22 Apr. 2007). Preferably said residues are involved in binding and cleavage of said DNA cleavage site. More preferably, said residues are at positions 138, 139, 142 or 143 of I-CreI. Two residues may be mutated in one variant provided that each mutation is in a different pair of residues chosen from the pair of residues at positions 138 and 139 and the pair of residues at positions 142 and 143. The mutations which are introduced modify the interaction(s) of said amino acid(s) of the final C-terminal loop with the phosphate backbone of the I-CreI site. Preferably, the residue at position 138 or 139 is substituted by an hydrophobic amino acid to avoid the formation of hydrogen bonds with the phosphate backbone of the DNA cleavage site. For example, the residue at position 138 is substituted by an alanine or the residue at position 139 is substituted by a methionine. The residue at position 142 or 143 is advantageously substituted by a small amino acid, for example a glycine, to decrease the size of the side chains of these amino acid residues. More, preferably, said substitution in the final C-terminal loop modifies the specificity of the variant towards the nucleotide at positions ±1 to 2, ±6 to 7 and/or ±11 to 12 of the I-CreI site.
In another preferred embodiment of said variant, it comprises one or more additional mutations that improve the binding and/or the cleavage properties of the variant towards the DNA target sequence from the human beta globin gene.
The additional residues which are mutated may be on the entire I-CreI sequence, and in particular in the C-terminal half of I-CreI (positions 80 to 163). Both I-CreI monomers are advantageously mutated; the mutation(s) in each monomer may be identical or different. For example, the variant comprises one or more additional substitutions at positions: 4, 7, 12, 16, 19, 24, 34, 43, 49, 54, 58, 60, 64, 66, 79, 80, 81, 82, 85, 86, 87, 92, 93, 94, 96, 99, 100, 103, 105, 109, 111, 117, 120, 121, 125, 129, 131, 132, 139, 140, 147, 151, 152, 155, 159, 160, 161, 162 and 163. Said substitutions are advantageously selected from the group consisting of: K4E, K7R, Y12H, F16L, G19S, G19A, 124V, K34R, F43L, T49A, F54L, L58Q, D60N, D60G, V64I, Y66H, S79G, E80G, E80K, I81T, K82R, K82E, H85R, N86S, F87L, Q92R, P93A, F94L, K96R, Q99R, K100R, N103S, V105A, V105I, I109V, Q111H, E117G, D120G, K121R, K121E, V125A, V129A, Q131R, I132V, K139R, T140A, T147A, V151M, L152Q, L155P, K159R, K159E, K160E, S161P, S162P and P163S. Preferably, the variant comprises at least one substitution selected from the group consisting of: G19S, F54L, E80K, F87L, V105A and I132V. More preferably, the variant comprises at least the G19S and E80K substitutions or the F87L and E80K substitutions.
According to a more preferred embodiment of said variant, said additional mutation further impairs the formation of a functional homodimer. More preferably, said mutation is the G19S mutation. The G19S mutation is advantageously introduced in one of the two monomers of an heterodimeric I-CreI variant, so as to obtain a meganuclease having enhanced cleavage activity and enhanced cleavage specificity. In addition, to enhance the cleavage specificity further, the other monomer may carry a distinct mutation that impairs the formation of a functional homodimer or favors the formation of the heterodimer.
In another preferred embodiment of said variant, said substitutions are replacement of the initial amino acids with amino acids selected from the group consisting of: A, D, E, G, H, K, N, P, Q, R, S, T, Y, C, V, L and W.
The variant of the invention may be derived from the wil-type I-CreI (SEQ ID NO: 1 or SEQ ID NO: 178) or an I-CreI scaffold protein having at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity with SEQ ID NO: 178, such as the scaffold SEQ ID NO: 5 (167 amino acids) having the insertion of an alanine at position 2, the D75N substitution, and the insertion of AAD at the C-terminus (positions 164 to 166) of the I-CreI sequence.
In addition, the variants of the invention may include one or more residues inserted at the NH2 terminus and/or COOH terminus of the sequence. For example, a tag (epitope or polyhistidine sequence) is introduced at the NH2 terminus and/or COOH terminus; said tag is useful for the detection and/or the purification of said variant. For example, the variant may have the AAD or ATD sequence inserted at its C-terminus.
The variant may also comprise a nuclear localization signal (NLS); said NLS is useful for the importation of said variant into the cell nucleus. The NLS may be inserted just after the first methionine of the variant or just after an N-terminal tag.
The variant according to the present invention may be an homodimer which is able to cleave a palindromic or pseudo-palindromic DNA target sequence.
Alternatively, said variant is an heterodimer, resulting from the association of a first and a second monomer having different substitutions at positions 26 to 40 and 44 to 77 of I-CreI, said heterodimer being able to cleave a non-palindromic DNA target sequence from the human beta globin gene.
Each monomer (first monomer and second monomer) of the heterodimeric variant according to the present invention may be named with a letter code, after the eleven residues at positions 28, 30, 32, 33, 38, 40, 44, 68 and 70, 75 and 77 and the additional residues which are mutated, as indicated above. For example, KTSHRS/KYSDT+120G or 28K30T32S33H38R40S/44K68Y70S75D77T+120G stands for I-CreI K28, T30, S32, H33, R38, S40/K44, Y68, S70, D75, T77 and G120.
The DNA target sequence which is cleaved by said variant may be in an exon or in an intron of the human beta globin gene.
In another preferred embodiment of said variant, said DNA target sequence is selected from the group consisting of the sequences SEQ ID NO: 6 to 19 (
More preferably, the monomers of the I-CreI variant have at least the following substitutions, respectively for the first and the second I-CreI monomer:
Examples of such variants which cleave the HBB8 target and do not cleave, or cleave with a lower efficiency, the HBB6 target are presented in Tables XVI, XVIII, XX and XXI. For example, the variants presented in Table XVI have the following substitutions:
Preferred variants are those presented in Table XXI:
Examples of such variants which cleave the HBB5 target are presented in
Preferred variants are those presented in Table XII: KNTYQS/ARSER+19S+80K+85R+87L+96R+139R (first monomer) and KSPHRS/KYSDT+81T or KTSHRS/KYSDT+66H+82R+86S+99R+132V+139R+140A (second monomer).
The heterodimeric variant as defined above may have only the amino acid substitutions as indicated above. In this case, the positions which are not indicated are not mutated and thus correspond to the wild-type I-CreI (SEQ ID NO: 1 or 178) or I-CreI N75 scaffold (SEQ ID NO: 5) sequence, respectively. Examples of such heterodimeric I-CreI variants cleaving the HBB DNA targets of Table I (nucleotide sequences SEQ ID NO: 6 to 19) include the variants consisting of a first and a second monomer corresponding to the following pairs of sequences: SEQ ID NO: 123 to 135 (first monomer) and SEQ ID NO: 136 to 148, respectively (second monomer;
Alternatively, the heterodimeric variant may consist of an I-CreI sequence comprising the amino acid substitutions as defined above. In the latter case, the positions which are not indicated may comprise additional mutations, for example one or more additional mutations as defined above.
The invention encompasses I-CreI variants having at least 85% identity, preferably at least 90% identity, more preferably at least 95% (96%, 97%, 98%, 99%) identity with the sequences as defined above, said variant being able to cleave a DNA target from the human beta globin gene.
The heterodimeric variant is advantageously an obligate heterodimer variant having at least one pair of mutations interesting corresponding residues of the first and the second monomers which make an intermolecular interaction between the two I-CreI monomers, wherein the first mutation of said pair(s) is in the first monomer and the second mutation of said pair(s) is in the second monomer and said pair(s) of mutations prevent the formation of functional homodimers from each monomer and allow the formation of a functional heterodimer, able to cleave the genomic DNA target from the human beta globin gene.
To form an obligate heterodimer, the monomers have advantageously at least one of the following pairs of mutations, respectively for the first and the second monomer:
a) the substitution of the glutamic acid at position 8 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the lysine at position 7 with an acidic amino acid, preferably a glutamic acid (second monomer); the first monomer may further comprise the substitution of at least one of the lysine residues at positions 7 and 96, by an arginine,
b) the substitution of the glutamic acid at position 61 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the lysine at position 96 with an acidic amino acid, preferably a glutamic acid (second monomer); the first monomer may further comprise the substitution of at least one of the lysine residues at positions 7 and 96, by an arginine,
c) the substitution of the leucine at position 97 with an aromatic amino acid, preferably a phenylalanine (first monomer) and the substitution of the phenylalanine at position 54 with a small amino acid, preferably a glycine (second monomer); the first monomer may further comprise the substitution of the phenylalanine at position 54 by a tryptophane and the second monomer may further comprise the substitution of the leucine at position 58 or lysine at position 57, by a methionine, and
d) the substitution of the aspartic acid at position 137 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the arginine at position 51 with an acidic amino acid, preferably a glutamic acid (second monomer).
For example, the first monomer may have the mutation D137R and the second monomer, the mutation R51D. The obligate heterodimer meganuclease comprises advantageously, at least two pairs of mutations as defined in a), b) c) or d), above; one of the pairs of mutation is advantageously as defined in c) or d). Preferably, one monomer comprises the substitution of the lysine residues at positions 7 and 96 by an acidic amino acid (aspartic acid (D) or glutamic acid (E)), preferably an aspartic acid (K7E and K96E) and the other monomer comprises the substitution of the glutamic acid residues at positions 8 and 61 by a basic amino acid (arginine (R) or lysine (K); for example, E8K and E61R). More preferably, the obligate heterodimer meganuclease, comprises three pairs of mutations as defined in a), b) and c), above. The obligate heterodimer meganuclease consists advantageously of (i) E8R, E8K or E8H, E61R, E61K or E61H and L97F, L97W or L97Y; (ii) K7R, E8R, E61R, K96R and L97F, or (iii) K7R, E8R, F54W, E61R, K96R and L97F and a second monomer (B) having at least the mutations (iv) K7E or K7D, F54G or F54A and K96D or K96E; (v) K7E, F54G, L58M and K96E, or (vi) K7E, F54G, K57M and K96E. For example, the first monomer may have the mutations K7R, E8R or E8K, E61R, K96R and L97F or K7R, E8R or E8K, F54W, E61R, K96R and L97F and the second monomer, the mutations K7E, F54G, L58M and K96E or K7E, F54G, K57M and K96E. The obligate heterodimer may comprise at least one NLS and/or one tag as defined above; said NLS and/or tag may be in the first and/or the second monomer
The subject-matter of the present invention is also a single-chain chimeric meganuclease (fusion protein) derived from an I-CreI variant as defined above. The single-chain meganuclease may comprise two I-CreI monomers, two I-CreI core domains (positions 6 to 94 of I-CreI) or a combination of both. Preferably, the two monomers/core domains or the combination of both, are connected by a peptidic linker.
The subject-matter of the present invention is also a polynucleotide fragment encoding a variant or a single-chain chimeric meganuclease as defined above; said polynucleotide may encode one monomer of an homodimeric or heterodimeric variant, or two domains/monomers of a single-chain chimeric meganuclease.
The subject-matter of the present invention is also a recombinant vector for the expression of a variant or a single-chain meganuclease according to the invention. The recombinant vector comprises at least one polynucleotide fragment encoding a variant or a single-chain meganuclease, as defined above. In a preferred embodiment, said vector comprises two different polynucleotide fragments, each encoding one of the monomers of an heterodimeric variant.
A vector which can be used in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consist of a chromosomal, non chromosomal, semi-synthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.
Viral vectors include retrovirus, adenovirus, parvovirus (e.g. adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
Preferred vectors include lentiviral vectors, and particularly self inactivacting lentiviral vectors.
Vectors can comprise selectable markers, for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine-guanine phosphoribosyl transferase for eukaryotic cell culture; TRP1 for S. cerevisiae; tetracycline, rifampicin or ampicillin resistance in E. coli.
Preferably said vectors are expression vectors, wherein the sequence(s) encoding the variant/single-chain meganuclease of the invention is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said variant. Therefore, said polynucleotide is comprised in an expression cassette. More particularly, the vector comprises a replication origin, a promoter operatively linked to said encoding polynucleotide, a ribosome-binding site, an RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer. Selection of the promoter will depend upon the cell in which the polypeptide is expressed. Preferably, when said variant is an heterodimer, the two polynucleotides encoding each of the monomers are included in one vector which is able to drive the expression of both polynucleotides, simultaneously. Suitable promoters include tissue specific and/or inducible promoters. Examples of inducible promoters are: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-β-D-thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature. Examples of tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), α-antitrypsin protease, human surfactant (SP) A and B proteins, β-casein and acidic whey protein genes.
According to another advantageous embodiment of said vector, it includes a targeting construct comprising sequences sharing homologies with the region surrounding the genomic DNA cleavage site as defined above.
For instance, said sequence sharing homologies with the regions surrounding the genomic DNA cleavage site of the variant is a fragment of the human beta globin gene comprising positions: 508 to 707, 651 to 850, 701 to 900, 1048 to 1247, 1147 to 1346, 1524 to 1723, 1599 to 1798, 1808 to 2007, 2084 to 2283, 2094 to 2293, 2245 to 2444, 2323 to 2522 and 2523 to 2722 of SEQ ID NO: 4.
Alternatively, the vector coding for an I-CreI variant/single-chain meganuclease and the vector comprising the targeting construct are different vectors.
More preferably, the targeting DNA construct comprises:
a) sequences sharing homologies with the region surrounding the genomic DNA cleavage site as defined above, and
b) a sequence to be introduced, flanked by sequences as in a) or included in sequences as in a).
Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used. Therefore, the targeting DNA construct is preferably from 200 pb to 6000 pb, more preferably from 1000 pb to 2000 pb. Indeed, shared DNA homologies are located in regions flanking upstream and downstream the site of the break and the DNA sequence to be introduced should be located between the two arms. The sequence to be introduced is preferably a sequence which repairs a mutation in the gene of interest (gene correction or recovery of a functional gene), for the purpose of genome therapy. Alternatively, it can be any other sequence used to alter the chromosomal DNA in some specific way including a sequence used to modify a specific sequence, to attenuate or activate the gene of interest, to inactivate or delete the gene of interest or part thereof, to introduce a mutation into a site of interest or to introduce an exogenous gene or part thereof. Such chromosomal DNA alterations are used for genome engineering (animal models/human recombinant cell lines). The targeting construct comprises advantageously a positive selection marker between the two homology arms and eventually a negative selection marker upstream of the first homology arm or downstream of the second homology arm. The marker(s) allow(s) the selection of cells having inserted the sequence of interest by homologous recombination at the target site.
For example,
For correcting the HBB gene, cleavage of the gene occurs in the vicinity of the mutation, preferably, within 500 bp of the mutation (
For example, for correcting the E6V, E6K or E26K mutations in the HBB gene found in sickle cell diseases (HbSS, HbSC and HbSE, respectively), as indicated in
In addition, for correcting the E6V mutation in the HBB gene found in sickle cell anemia (HbSS), as indicated in
Furthermore, for correcting the E26K mutation in the HBB gene found in HbSE, the following variant cleaving the HBB6 DNA target may be used with a targeting construct comprising at least positions 1048 to 1247 of SEQ ID NO: 4 for efficient repair of both the DNA double-strand break and the mutation:
Alternatively, for restoring a functional gene (
The targeting construct comprises the exons downstream of the cleavage site fused in frame (as in the cDNA) and with a polyadenylation site to stop transcription in 3′. The sequence to be introduced (exon knock-in construct) is flanked by introns or exons sequences surrounding the cleavage site, so as to allow the transcription of the engineered gene (exon knock-in gene) into a mRNA able to code for a functional protein (
For making knock-in animals/cells, the targeting DNA construct comprises: a HBB gene fragment which has at least 200 bp of homologous sequence flanking the target site for repairing the cleavage, the sequence of an exogeneous gene of interest, and eventually a selection marker, such as the HPRT gene.
For the insertion of a sequence, DNA homologies are generally located in regions directly upstream and downstream to the site of the break (sequences immediately adjacent to the break; minimal repair matrix). However, when the insertion is associated with a deletion of ORF sequences flanking the cleavage site, shared DNA homologies are located in regions upstream and downstream the region of the deletion.
The modification(s) in the human HBB gene are introduced in human cells, for the purpose of human genome therapy or the making of human recombinant cell lines. However they may also be introduced in humanized cells wherein the endogenous HBB gene has been deleted (knock-out) and a normal or mutated human HBB gene has been introduced anywhere in the genome (transgenic) or specifically at the endogenous HBB locus (knock-in), for the purpose of making animal models of inherited hemoglobin disorders or studying the correction of the mutation by meganuclease-induced homologous recombination. Mice models of inherited hemoglobin disorders are well-known in the art and include the SCA mice, for example (Paszty et al., Science 1997, 278, 876-878; Ryan et al., Science, 1997, 278, 873-876; Chang et al., Proc. Nat. Acad. Sci. USA, 1998, 95, 14886-14890).
The subject-matter of the present invention is also a targeting DNA construct as defined above.
The subject-matter of the present invention is also a composition characterized in that it comprises at least one meganuclease as defined above (variant or single-chain derived chimeric meganuclease) and/or at least one expression vector encoding said meganuclease, as defined above.
In a preferred embodiment of said composition, it comprises a targeting DNA construct, as defined above.
Preferably, said targeting DNA construct is either included in a recombinant vector or it is included in an expression vector comprising the polynucleotide(s) encoding the meganuclease according to the invention.
The subject-matter of the present invention is also the use of at least one meganuclease and/or one expression vector, as defined above, for the preparation of a medicament for preventing, improving or curing a pathological condition caused by a mutation in the beta globin gene as defined above, in an individual in need thereof.
Preferably said pathological condition is selected from the group consisting of: sickle cell diseases (sickle cell anemia, haemoglobin SC, haemoglobin SE) and beta-thalassemias.
The use of the meganuclease may comprise at least the step of (a) inducing in somatic tissue(s) of the donor/individual a double stranded cleavage at a site of interest of the beta globin gene comprising at least one recognition and cleavage site of said meganuclease by contacting said cleavage site with said meganuclease, and (b) introducing into said somatic tissue(s) a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the beta globin gene upon recombination between the targeting DNA and the chromosomal DNA, as defined above. The targeting DNA is introduced into the somatic tissues(s) under conditions appropriate for introduction of the targeting DNA into the site of interest. The targeting construct may comprise sequences for deleting the human beta globin gene and eventually the sequence of an exogenous gene of interest (gene replacement).
According to the present invention, said double-stranded cleavage may be induced, ex vivo by introduction of said meganuclease into somatic cells (hematopietic stem cells) from the diseased individual and then transplantation of the modified cells back into the diseased individual.
The subject-matter of the present invention is also a method for preventing, improving or curing a pathological condition caused by a mutation in the beta-globin gene, in an individual in need thereof, said method comprising at least the step of administering to said individual a composition as defined above, by any means.
The subject-matter of the present invention is further the use of a meganuclease as defined above, one or two polynucleotide(s), preferably included in expression vector(s), for genome engineering of the human beta globin gene for non-therapeutic purposes. The human beta globin gene may be the human endogenous HBB gene (human HBB gene locus; human recombinant cells generation) or a transgene that has been inserted in an animal, for example a mouse (animal models of inherited hemoglobin disorders).
According to an advantageous embodiment of said use, it is for inducing a double-strand break in a site of interest of the beta globin gene comprising a genomic DNA target sequence, thereby inducing a DNA recombination event, a DNA loss or cell death.
According to the invention, said double-strand break is for: repairing a specific sequence in the human beta globin gene, modifying a specific sequence in the human beta globin gene, restoring a functional human beta globin gene in place of a mutated one, attenuating or activating the human beta globin gene, introducing a mutation into a site of interest of the human beta globin gene, introducing an exogenous gene or a part thereof, inactivating or deleting the human beta globin gene or a part thereof, translocating a chromosomal arm, or leaving the DNA unrepaired and degraded.
According to another advantageous embodiment of said use, said variant, polynucleotide(s), vector, are associated with a targeting DNA construct as defined above.
In a first embodiment of the use of the meganuclease according to the present invention, it comprises at least the following steps: 1) introducing a double-strand break at a site of interest of the human beta globin gene comprising at least one recognition and cleavage site of said meganuclease, by contacting said cleavage site with said meganuclease; 2) providing a targeting DNA construct comprising the sequence to be introduced flanked by sequences sharing homologies to the targeted locus. Said meganuclease can be provided directly to the cell or through an expression vector comprising the polynucleotide sequence encoding said meganuclease and suitable for its expression in the used cell. This strategy is used to introduce a DNA sequence at the target site, for example to generate knock-in or transgenic animals, or recombinant human cell lines that can be used for protein production, gene function studies, drug development (drug screening) or as inherited hemoglobin disorders model (study of the disease and of the correction of the mutations by meganuclease-induced homologous recombination).
In a second embodiment of the use of the meganuclease according to the present invention, it comprises at least the following steps: 1) introducing a double-strand break at a site of interest of the human beta globin gene comprising at least one recognition and cleavage site of said meganuclease, by contacting said cleavage site with said meganuclease; 2) maintaining said broken genomic locus under conditions appropriate for homologous recombination with chromosomal DNA sharing homologies to regions surrounding the cleavage site.
In a third embodiment of the use of the meganuclease according to the present invention, it comprises at least the following steps: 1) introducing a double-strand break at a site of interest of the human beta globin gene comprising at least one recognition and cleavage site of said meganuclease, by contacting said cleavage site with said meganuclease; 2) maintaining said broken genomic locus under conditions appropriate for repair of the double-strand break by non-homologous end joining.
The subject-matter of the present invention is also a method for making a modified mouse (knock-in mouse) derived from a humanized mouse comprising a normal/mutated human HBB gene, comprising at least the steps of:
(a) introducing into a pluripotent precursor cell or an embryo of said humanized mouse, a meganuclease, as defined above, so as to induce a double strand cleavage at a site of interest of the human HBB gene comprising a DNA recognition and cleavage site of said meganuclease; and simultaneously or consecutively,
(b) introducing into the mouse precursor cell or embryo of step (a) a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the site of interest upon recombination between the targeting DNA and the chromosomal DNA, so as to generate a genomically modified mouse precursor cell or embryo having repaired the site of interest by homologous recombination,
(c) developing the genomically modified mouse precursor cell or embryo of step (b) into a chimeric mouse, and
(d) deriving a transgenic mouse from the chimeric mouse of step (c).
Preferably, step (c) comprises the introduction of the genomically modified precursor cell generated in step (b) into blastocysts so as to generate chimeric mice.
The subject-matter of the present invention is also a method for making a recombinant human cell, comprising at least the steps of:
(a) introducing into a human cell, a meganuclease, as defined above, so as to into induce a double stranded cleavage at a site of interest of the human globin gene comprising a DNA recognition and cleavage site for said meganuclease; and simultaneously or consecutively,
(b) introducing into the cell of step (a), a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the site of interest upon recombination between the targeting DNA and the chromosomal DNA, so as to generate a recombinant human cell having repaired the site of interest by homologous recombination,
(c) isolating the recombinant human cell of step (b), by any appropriate mean.
The targeting DNA is introduced into the cell under conditions appropriate for introduction of the targeting DNA into the site of interest.
In a preferred embodiment, said targeting DNA construct is inserted in a vector.
The cells which are modified may be any cells of interest. For making knock-in/transgenic mice, the cells are pluripotent precursor cells such as embryo-derived stem (ES) cells, which are well-known in the art. For making recombinant human cell lines, the cells may advantageously be PerC6 (Fallaux et al., Hum. Gene Ther. 9, 1909-1917, 1998) or HEK293 (ATCC # CRL-1573) cells. Said meganuclease can be provided directly to the cell or through an expression vector comprising the polynucleotide sequence encoding said meganuclease and suitable for its expression in the used cell.
For making human recombinant cell lines/transgenic animals expressing an heterologous protein of interest, the targeting DNA comprises a sequence encoding the product of interest (protein or RNA), and eventually a marker gene, flanked by sequences upstream and downstream the cleavage site, as defined above, so as to generate genomically modified cells (human cell) having integrated the exogenous sequence of interest in the human beta globin gene, by homologous recombination.
The sequence of interest may be any gene coding for a certain protein/peptide of interest, included but not limited to: reporter genes, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, disease causing gene products and toxins. The sequence may also encode an RNA molecule of interest including for example a siRNA.
The expression of the exogenous sequence may be driven, either by the endogenous human beta globin promoter or by an heterologous promoter, preferably a ubiquitous or tissue specific promoter, either constitutive or inducible, as defined above. In addition, the expression of the sequence of interest may be conditional; the expression may be induced by a site-specific recombinase (Cre, FLP . . . ).
Thus, the sequence of interest is inserted in an appropriate cassette that may comprise an heterologous promoter operatively linked to said gene of interest and one or more functional sequences including but mot limited to (selectable) marker genes, recombinase recognition sites, polyadenylation signals, splice acceptor sequences, introns, tags for protein detection and enhancers.
For making animal models of inherited-hemoglobin disorders, the targeting DNA comprises the correct/mutated human HBB gene sequence, flanked by sequences upstream and downstream the cleavage site, so as to generate animals having corrected the mutation in the HBB gene or animals having inserted a mutated HBB gene (sickle globin gene, for example).
The meganuclease can be used either as a polypeptide or as a polynucleotide construct encoding said polypeptide. It is introduced into mouse cells, by any convenient means well-known to those in the art, which are appropriate for the particular cell type, alone or in association with either at least an appropriate vehicle or carrier and/or with the targeting DNA.
According to an advantageous embodiment of the uses according to the invention, the meganuclease (polypeptide) is associated with:
According to another advantageous embodiment of the uses according to the invention, the meganuclease (polynucleotide encoding said meganuclease) and/or the targeting DNA is inserted in a vector. Vectors comprising targeting DNA and/or nucleic acid encoding a meganuclease can be introduced into a cell by a variety of methods (e.g., injection, direct uptake, projectile bombardment, liposomes, electroporation). Meganucleases can be stably or transiently expressed into cells using expression vectors. Techniques of expression in eukaryotic cells are well known to those in the art. (See Current Protocols in Human Genetics: Chapter 12 “Vectors For Gene Therapy” & Chapter 13 “Delivery Systems for Gene Therapy”). Optionally, it may be preferable to incorporate a nuclear localization signal into the recombinant protein to be sure that it is expressed within the nucleus.
Once in a cell, the meganuclease and if present, the vector comprising targeting DNA and/or nucleic acid encoding a meganuclease are imported or translocated by the cell from the cytoplasm to the site of action in the nucleus.
For purposes of therapy, the meganucleases and a pharmaceutically acceptable excipient are administered in a therapeutically effective amount. Such a combination is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of the recipient. In the present context, an agent is physiologically significant if its presence results in a decrease in the severity of one or more symptoms of the targeted disease and in a genome correction of the lesion or abnormality.
In one embodiment of the uses according to the present invention, the meganuclease is substantially non-immunogenic, i.e., engender little or no adverse immunological response. A variety of methods for ameliorating or eliminating deleterious immunological reactions of this sort can be used in accordance with the invention. In a preferred embodiment, the meganuclease is substantially free of N-formyl methionine. Another way to avoid unwanted immunological reactions is to conjugate meganucleases to polyethylene glycol (“PEG”) or polypropylene glycol (“PPG”) (preferably of 500 to 20,000 daltons average molecular weight (MW)). Conjugation with PEG or PPG, as described by Davis et al. (U.S. Pat. No. 4,179,337) for example, can provide non-immunogenic, physiologically active, water soluble endonuclease conjugates with anti-viral activity. Similar methods also using a polyethylene-polypropylene glycol copolymer are described in Saifer et al. (U.S. Pat. No. 5,006,333).
The invention also concerns a prokaryotic or eukaryotic host cell which is modified by a polynucleotide or a vector as defined above, preferably an expression vector.
The invention also concerns a non-human transgenic animal or a transgenic plant, characterized in that all or part of their cells are modified by a polynucleotide or a vector as defined above.
As used herein, a cell refers to a prokaryotic cell, such as a bacterial cell, or an eukaryotic cell, such as an animal, plant or yeast cell.
The subject-matter of the present invention is also the use of at least one meganuclease variant, as defined above, as a scaffold for making other meganucleases. For example a third round of mutagenesis and selection/screening can be performed on said variants, for the purpose of making novel, third generation meganucleases.
The different uses of the meganuclease and the methods of using said meganuclease according to the present invention include the use of the I-CreI variant, the single-chain chimeric meganuclease derived from said variant, the polynucleotide(s), vector, cell, transgenic plant or non-human transgenic mammal encoding said variant or single-chain chimeric meganuclease, as defined above.
The I-CreI variant according to the invention may be obtained by a method for engineering I-CreI variants able to cleave a genomic DNA target sequence from the human beta globin gene, comprising at least the steps of:
(a) constructing a first series of I-CreI variants having at least one substitution in a first functional subdomain of the LAGLIDADG core domain situated from positions 26 to 40 of I-CreI,
(b) constructing a second series of I-CreI variants having at least one substitution in a second functional subdomain of the LAGLIDADG core domain situated from positions 44 to 77 of I-CreI,
(c) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet at positions −10 to −8 of the I-CreI site has been replaced with the nucleotide triplet which is present at positions −10 to −8 of said genomic target and (ii) the nucleotide triplet at positions +8 to +10 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions −10 to −8 of said genomic target,
(d) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet at positions −5 to −3 of the I-CreI site has been replaced with the nucleotide triplet which is present at positions −5 to −3 of said genomic target and (ii) the nucleotide triplet at positions +3 to +5 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions −5 to −3 of said genomic target,
(e) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet at positions +8 to +10 of the I-CreI site has been replaced with the nucleotide triplet which is present at positions +8 to +10 of said genomic target and (ii) the nucleotide triplet at positions −10 to −8 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions +8 to +10 of said genomic target,
(f) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-CreI site wherein (i) the nucleotide triplet at positions +3 to +5 of the I-CreI site has been replaced with the nucleotide triplet which is present at positions +3 to +5 of said genomic target and (ii) the nucleotide triplet at positions −5 to −3 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present at positions +3 to +5 of said genomic target,
(g) combining in a single variant, the mutation(s) at positions 26 to 40 and 44 to 77 of two variants from step (c) and step (d), to obtain a novel homodimeric I-CreI variant which cleaves a sequence wherein (i) the nucleotide triplet at positions −10 to −8 is identical to the nucleotide triplet which is present at positions −10 to −8 of said genomic target, (ii) the nucleotide triplet at positions +8 to +10 is identical to the reverse complementary sequence of the nucleotide triplet which is present at positions −10 to −8 of said genomic target, (iii) the nucleotide triplet at positions −5 to −3 is identical to the nucleotide triplet which is present at positions −5 to −3 of said genomic target and (iv) the nucleotide triplet at positions +3 to +5 is identical to the reverse complementary sequence of the nucleotide triplet which is present at positions −5 to −3 of said genomic target, and/or
(h) combining in a single variant, the mutation(s) at positions 26 to 40 and 44 to 77 of two variants from step (e) and step (f), to obtain a novel homodimeric I-CreI variant which cleaves a sequence wherein (i) the nucleotide triplet at positions +3 to +5 is identical to the nucleotide triplet which is present at positions +3 to +5 of said genomic target, (ii) the nucleotide triplet at positions −5 to −3 is identical to the reverse complementary sequence of the nucleotide triplet which is present at positions +3 to +5 of said genomic target, (iii) the nucleotide triplet at positions +8 to +10 of the I-CreI site has been replaced with the nucleotide triplet which is present at positions +8 to +10 of said genomic target and (iv) the nucleotide triplet at positions −10 to −8 is identical to the reverse complementary sequence of the nucleotide triplet at positions +8 to +10 of said genomic target,
(i) combining the variants obtained in steps (g) and (h) to form heterodimers, and
(j) selecting and/or screening the heterodimers from step (i) which are able to cleave said genomic DNA target from the human beta globin gene.
One of the step(s) (c), (d), (e) or (f) may be omitted. For example, if step (c) is omitted, step (d) is performed with a mutant I-CreI site wherein both nucleotide triplets at positions −10 to −8 and −5 to −3 have been replaced with the nucleotide triplets which are present at positions −10 to −8 and −5 to −3, respectively of said genomic target, and the nucleotide triplets at positions +3 to +5 and +8 to +10 have been replaced with the reverse complementary sequence of the nucleotide triplets which are present at positions −5 to −3 and −10 to −8, respectively of said genomic target.
Steps (a), (b), (g), (h) and (i) may further comprise the introduction of additional mutations at other positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target, at positions which improve the binding and/or cleavage properties of the mutants, or at positions which either prevent or impair the formation of functional homodimers or favor the formation of the heterodimer, as defined above.
The additional mutations may be introduced by site-directed mutagenesis and/or random mutagenesis on a variant or on a pool of variants, according to standard mutagenesis methods which are well-known in the art, for example by using PCR. Site-directed mutagenesis may be advantageously performed by amplifying overlapping fragments comprising the mutated position(s), as defined above, according to well-known overlapping PCR techniques. In addition, multiple site-directed mutagenesis, as described in
In particular, random mutations may be introduced on the whole variant or in a part of the variant, in particular the C-terminal half of the variant (positions 80 to 163) to improve the binding and/or cleavage properties of the mutants towards the DNA target from the gene of interest. Site-directed mutagenesis at positions which improve the binding and/or cleavage properties of the mutants, for example at positions 19, 54, 80, 87, 105 and/or 132, may also be combined with random-mutagenesis. The mutagenesis may be performed by generating random/site-directedmutagenesis libraries on a pool of variants, according to standard mutagenesis methods which are well-known in the art and commercially available.
Preferably, the mutagenesis is performed on one monomer of the heterodimer formed in step (i) or obtained in step (j), advantageously on a pool of monomers, preferably on both monomers of the heterodimer of step (i) or (j).
Preferably, at least two rounds of selection/screening are performed according to the process illustrated by
The cleavage activity of the improved meganuclease obtainable by the method according to the present invention may be measured by a direct repeat recombination assay, in yeast or mammalian cells, using a reporter vector, by comparison with that of the initial meganuclease. The reporter vector comprises two truncated, non-functional copies of a reporter gene (direct repeats) and the genomic DNA target sequence which is cleaved by the initial meganuclease, within the intervening sequence, cloned in a yeast or in a mammalian expression vector. Expression of the meganuclease results in cleavage of the genomic DNA target sequence. This cleavage induces homologous recombination between the direct repeats, resulting in a functional reporter gene (LacZ, for example), whose expression can be monitored by appropriate assay. A stronger signal is observed with the improved meganuclease, as compared to the initial meganuclease. Alternatively, the activity of the improved meganuclease towards its genomic DNA target can be compared to that of I-CreI towards the I-CreI site, at the same genomic locus, using a chromosomal assay in mammalian cells (Arnould et al., J. Mol. Biol., 2007, 371, 49-65).
The (intramolecular) combination of mutations in steps (g) and (h) may be performed by amplifying overlapping fragments comprising each of the two subdomains, according to well-known overlapping PCR techniques.
The (intermolecular) combination of the variants in step (i) is performed by co-expressing one variant from step (g) with one variant from step (h), so as to allow the formation of heterodimers. For example, host cells may be modified by one or two recombinant expression vector(s) encoding said variant(s). The cells are then cultured under conditions allowing the expression of the variant(s), so that heterodimers are formed in the host cells, as described previously in the International PCT Application WO 2006/097854 and Arnould et al., J. Mol. Biol., 2006, 355, 443-458.
The selection and/or screening in steps (c), (d), (e), (f) and/or (j) may be performed by using a cleavage assay in vitro or in vivo, as described in the International PCT Application WO 2004/067736, Arnould et al., J. Mol. Biol., 2006, 355, 443-458, Epinat et al., Nucleic Acids Res., 2003, 31, 2952-2962 and Chames et al., Nucleic Acids Res., 2005, 33, e178.
According to another advantageous embodiment of said method, steps (c), (d), (e), (f) and/or (j) are performed in vivo, under conditions where the double-strand break in the mutated DNA target sequence which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination-mediated repair of said DNA double-strand break.
The subject matter of the present invention is also an I-CreI variant having mutations at positions 26 to 40 and/or 44 to 77 of I-CreI that is useful for engineering the variants able to cleave a DNA target from the human beta globin gene, according to the present invention. In particular, the invention encompasses the I-CreI variants as defined in step (c) to (f) of the method for engineering I-CreI variants, as defined above, including the variants of Tables II, III, VI, VIII, X, XIII, XIV, XVII and XIX. The invention encompasses also the I-CreI variants as defined in step (g) and (h) of the method for engineering I-CreI variants, as defined above, including the variants of the sequence SEQ ID NO: 38 to 76, 80 to 122, 163, 223 to 230 (combined variants of Tables II, III, VI, XIII, XIV and XVII).
Single-chain chimeric meganucleases able to cleave a DNA target from the gene of interest are derived from the variants according to the invention by methods well-known in the art (Epinat et al., Nucleic Acids Res., 2003, 31, 2952-62; Chevalier et al., Mol. Cell., 2002, 10, 895-905; Steuer et al., Chembiochem., 2004, 5, 206-13; International PCT Applications WO 03/078619 and WO 2004/031346). Any of such methods, may be applied for constructing single-chain chimeric meganucleases derived from the variants as defined in the present invention.
The polynucleotide sequence(s) encoding the variant as defined in the present invention may be prepared by any method known by the man skilled in the art. For example, they are amplified from a cDNA template, by polymerase chain reaction with specific primers. Preferably the codons of said cDNA are chosen to favour the expression of said protein in the desired expression system.
The recombinant vector comprising said polynucleotides may be obtained and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques.
The I-CreI variant or single-chain derivative as defined in the present the invention are produced by expressing the polypeptide(s) as defined above; preferably said polypeptide(s) are expressed or co-expressed (in the case of the variant only) in a host cell or a transgenic animal/plant modified by one expression vector or two expression vectors (in the case of the variant only), under conditions suitable for the expression or co-expression of the polypeptide(s), and the variant or single-chain derivative is recovered from the host cell culture or from the transgenic animal/plant.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, which refers to examples illustrating the I-CreI meganuclease variants and their uses according to the invention to Table XXII which summarizes the different sequences, described in the present invention, as well as to the appended drawings in which:
28K30N32T33Y38Q40S44A68R70S75E77R80K96R (KNTYQS/ARSER/80K96R)+KTSHRS/KYSDT (panel A) or
28K30N32T33Y38Q40S44A68R70S75E77R80K96R (KNTYQS/ARSER/80K96R)+KNSRRS/KYSDT (panel B). H10, H11, H12 are positive controls of different strength.
It should be clearly understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they in no way constitute a limitation.
This example shows that I-CreI mutants can cut the HBB5.3 DNA target sequence derived from the right part of the HBB5 target in a palindromic form (
The method for producing meganuclease variants and the assays based on cleavage-induced recombination in mammal or yeast cells, which are used for screening variants with altered specificity are described in the International PCT Application WO 2004/067736; Epinat et al., Nucleic Acids Res., 2003, 31, 2952-2962; Chames et al., Nucleic Acids Res., 2005, 33, e178, and Arnould et al., J. Mol. Biol., 2006, 355, 443-458. These assays result in a functional LacZ reporter gene which can be monitored by standard methods.
The target was cloned as follow: oligonucleotide corresponding to the target sequence flanked by gateway cloning sequence was ordered from Proligo: 5′ tggcatacaagttttggtctcctgtacaggagaccaacaatcgtctgtca 3′ (SEQ ID NO: 33). Double-stranded target DNA, generated by PCR amplification of the single stranded oligonucleotide, was cloned using the Gateway protocol (INVITROGEN) into yeast reporter vector (pCLS1055,
I-CreI mutants cleaving 10GGT_P or 5CCT_P were identified as described previously in Smith et al, Nucleic Acids Res., 2006, 34, e149; International PCT Applications WO 2007/049095, WO 2007/057781, and Arnould et al., J. Mol. Biol., 2006, 355, 443-458; International PCT Applications WO 2006/097784, WO 2006/097853, respectively for the 10GGT_P or 5CCT_P targets. In order to generate I-CreI derived coding sequence containing mutations from both series, separate overlapping PCR reactions were carried out that amplify the 5′ end (aa positions 1-43) or the 3′ end (positions 39-167) of the I-CreI coding sequence. For both the 5′ and 3′ end, PCR amplification is carried out using Gall OF (5′-gcaactttagtgctgacacatacagg-3′; SEQ ID NO: 34) or Gal10R (5′-acaaccttgattggagacttgacc-3′; SEQ ID NO: 35) primers specific to the vector (pCLS0542,
Screening was performed as described previously (Arnould et al., J. Mol. Biol., 2006, 355, 443-458). Mating was performed using a colony gridder (QpixII, Genetix). Mutants were gridded on nylon filters covering YPD plates, using a low gridding density (about 4 spots/cm2). A second gridding process was performed on the same filters to spot a second layer consisting of different reporter-harboring yeast strains for each target. Membranes were placed on solid agar YPD rich medium, and incubated at 30° C. for one night, to allow mating. Next, filters were transferred to synthetic medium, lacking leucine and tryptophan, with galactose (1%) as a carbon source, and incubated for five days at 37° C., to select for diploids carrying the expression and target vectors. After 5 days, filters were placed on solid agarose medium with 0.02% X-Gal in 0.5 M sodium phosphate buffer, pH 7.0, 0.1% SDS, 6% dimethyl formamide (DMF), 7 mM β-mercaptoethanol, 1% agarose, and incubated at 37° C., to monitor β-galactosidase activity. Results were analyzed by scanning and quantification was performed using appropriate software.
To recover the mutant expressing plasmids, yeast DNA was extracted using standard protocols and used to transform E. coli. Sequence of mutant ORF were then performed on the plasmids by MILLEGEN SA. Alternatively, ORFs were amplified from yeast DNA by PCR (Akada et al., Biotechniques, 2000, 28, 668-670), and sequence was performed directly on PCR product by MILLEGEN SA.
I-CreI combinatorial mutants were constructed by associating mutations at positions 44, 68, 70, 75 and 77 with the 28, 30, 32, 33, 38 and 40 mutations on the I-CreI N75 or D75 scaffold, resulting in a library of complexity 1935. Examples of combinations are displayed on Table II. This library was transformed into yeast and 3456 clones (1.8 times the diversity) were screened for cleavage against HBB 5.3 DNA target (tggtctcctgt_P; SEQ ID NO: 25). 366 positives clones were found, which after sequencing and validation by secondary screening turned out to correspond to 176 different novel endonucleases (see Table II). The 20 novel endonucleases presented in Table II correspond to the sequences SEQ ID NO: 38 to 57 in the sequence listing. Examples of positives mutants cutting the HBB 5.3 target are shown in
Throughout the text and figures of the examples, combinatorial mutants sequences are named with an eleven letter code, after residues at positions 28, 30, 32, 33, 38, 40, 44, 68 and 70, 75 and 77. For example, KCSRQS/KASDI stands for I-CreI K28, C30, S32, R33, Q38, S40, K44, A68, S70, D75 and 177 (I-CreI 28K30C32S33R38Q40S44K68A70S75D77I). Parental mutants are named with a six letter code, after residues at positions 28, 30, 32, 33, 38 and 40 or a five letter code, after residues at positions 44, 68, 70, 75 and 77. For example, KCSRQS stands for I-CreI K28, C30, S32, R33, Q38 and S40K, and KASDI stands for I-CreI K44, A68, S70, D75 and I77.
This example, shows that I-CreI variants can cleave the HBB5.4 DNA target sequence derived from the left part of the HBB5 target in a palindromic form (
Both sets of proteins are mutated at position 70. However, two separable functional subdomains exist (Smith et al, Nucleic Acids Res., 2006, 34, e149 and International PCT Applications WO 2007/060495 and WO 2007/049156). That implies that this position has little impact on the specificity in base 10 to 8 of the target. Therefore, to check whether combined mutants could cleave the HBB5.4 target, mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving 5AGG_P (caaaacagggt_P; SEQ ID NO: 23) were combined with the 28, 30, 32, 33, 38, 40 mutations from proteins cleaving 10AAG_P (caagacgtcgt_P; SEQ ID NO: 21).
See example 1 except that 25 ng of the PCR product and 75 ng of vector DNA (pCLS 1107,
I-CreI combinatorial mutants were constructed by associating mutations at positions 44, 68, 70, 75 and 77 with the 28, 30, 32, 33, 38 and 40 mutations on the I-CreI N75 or D75 scaffold, resulting in a library of complexity 920. Examples of combinatorial mutants are displayed on Table III. This library was transformed into yeast and 1728 clones (1.9 times the diversity) were screened for cleavage against HBB5.4 DNA target (caagacagggt_P; SEQ ID NO: 26). 25 positives clones were found, which after sequencing and validation by secondary screening turned out to be correspond to 19 different novel endonucleases (see Table III). The 19 novel endonucleases presented in Table III correspond to the sequences SEQ ID NO: 58 to 76 in the sequence listing. Examples of positives mutants cutting the HBB5.4 target are shown in
I-CreI mutants able to cleave each of the palindromic HBB5 derived targets (HBB5.3 and HBB5.4) were identified in examples 1 and 2. Pairs of such mutants (one cutting HBB5.3 and one cutting HBB5.4) were co-expressed in yeast. Upon co-expression, there should be three active molecular species, two homodimers, and one heterodimer. It was assayed whether the heterodimers that should be formed cut the HBB5.2 and HBB5 targets.
a) Cloning of Mutants in Yeast Expression Vector Marked with KanR
To co-express two I-CreI mutants in yeast, mutants cutting the HBB5.4 sequence were subcloned in a a yeast expression vector marked with a kanamycin resistance gene (pCLS1107,
Yeast strain expressing a mutant cutting the HBB5.3 target in pCLS0542 expression vector was transformed with DNA coding for a mutant cutting the HBB5.4 target in pCLS1107 expression vector. Transformants were selected on −L Glu+G418 medium.
Mating was performed using a colony gridder (QpixII, Genetix).
Mutants were gridded on nylon filters covering YPD plates, using a low gridding density (about 4 spots/cm2). A second gridding process was performed on the same filters to spot a second layer consisting of different reporter-harbouring yeast strains for each target. Membranes were placed on solid agar YPD rich medium, and incubated at 30° C. for one night, to allow mating. Next, filters were transferred to synthetic medium, lacking leucine and tryptophan, adding G418, with galactose (1%) as a carbon source, and incubated for five days at 37° C., to select for diploids carrying the expression and target vectors. After 5 days, filters were placed on solid agarose medium with 0.02% X-Gal in 0.5 M sodium phosphate buffer, pH 7.0, 0.1% SDS, 6% dimethyl formamide (DMF), 7 mM β-mercaptoethanol, 1% agarose, and incubated at 37° C., to monitor β-galactosidase activity. Results were analyzed by scanning and quantification was performed using appropriate software.
Co-expression of mutants cleaving the HBB5.3 (8 mutants) and HBB5.4 (10 mutants) sequences resulted in efficient cleavage of the HBB5.2 target in almost all cases (
I-CreI mutants able to cleave the non palindromic HBB5.2 target were identified by assembly of mutants cleaving the palindromic HBB5.3 and HBB 5.4 target. However, none of these combinations was able to cleave HBB5, which differs from HBB5.2 only by 3 bp at positions −2, +1 and +2 (
Therefore, protein combinations cleaving HBB5.2 were mutagenized, and mutants cleaving HBB5 efficiently were screened. According to the structure of the I-CreI protein bound to its target, there is no contact between the 4 central base pairs (positions −2 to 2) and the I-CreI protein (Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier B. S. and Stoddard B. L., Nucleic Acids Res., 2001, 29, 3757-3754; Chevalier et al., J. Mol. Biol., 2003, 329, 253-269). Thus, it is difficult to rationally choose a set of positions to mutagenize, and mutagenesis was done on the whole protein. Random mutagenesis results in high complexity libraries, and the complexity of the variants libraries to be tested was limited by mutagenizing only one of the two components of the heterodimers cleaving HBB 5.2.
Thus, proteins cleaving HBB 5.4 were mutagenized, and it was tested whether they could efficiently cleave HBB 5 when coexpressed with proteins cleaving HBB 5.3.
Random mutagenesis libraries were created on a pool of chosen mutants by PCR using Mn2+ or by a two-step PCR process using dNTP derivatives 8-oxo-dGTP and dPTP as described in the protocol from JENA BIOSCIENCE GmbH for the JBS dNTP-Mutagenesis kit. Primers used are preATGCreFor (5′-gcataaattactatacttctatagacacgcaaacacaaatacacagcggccttgccacc-3′; SEQ ID NO: 77) and ICreIpostRev (5′-ggctcgaggagctcgtctagaggatcgctcgagttatcagtcggccgc-3′; SEQ ID NO: 78). Approximately 25 ng of the PCR product and 75 ng of vector DNA (pCLS 1107,
The yeast strain FYBL2-7B (MAT a, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202) containing the HBB5 target in the yeast reporter vector (pCLS1055,
Four mutants cleaving HBB5.4 (KGSYQS/RYSET, KGSYQS/ARSER, KGSYQS/TRSER, KNTYQS/ARSER according to the nomenclature of Table IV) were pooled, randomly mutagenized and transformed into yeast. 2280 transformed clones were then mated with a yeast strain that contains (i) the HBB5 target in a reporter plasmid (ii) an expression plasmid containing a mutant that cleaves the HBB5.3 target (KTSHRS/KYSDT or KNSRRS/KYSDT according to nomenclature of Table IV). After mating with this two yeast strains, one positive clone was found to cleave the HBB5 target. In a control experiment, this positive clone was not found to trigger cleavage of HBB5 without co-expression of the KTSHRS/KYSDT or KNSRRS/KYSDT I-CreI mutants. In conclusion, this positive clone contained proteins able to form heterodimers with KTSHRS/KYSDT or KNSRRS/KYSDT leading to cleavage activity for the HBB5 target. The yeast screening assay leading to the identification of the positive clone is shown in
I-CreI mutants cleaving the palindromic HBB5.3 target were identified and tested by co-expression with mutants cleaving the palindromic HBB5.4 target for their ability to cleave the non palindromic target HBB5 (Example 3). Nevertheless none of the heterodimeric combinations tested was able to cut the HBB5 sequence. In order to obtain a cleavage of this target, I-CreI mutants cleaving the HBB5.3 sequence were randomly mutagenized or site directed mutagenized and new variants cleaving HBB5 with high efficacy, when co-expressed with mutant cleaving the HBB5.4 target, were screened.
Six amino-acid substitutions which enhance the activity of I-CreI derivatives were individually introduced into the coding sequence of proteins cleaving HBB5.3. These mutations correspond to the replacement of Glycine 19 with Serine (G195), Phenylalanine 54 with Leucine (F54L), Glutamic Acid 80 with Lysine (E80K), Phenylalanine 87 with Leucine (F87L), Valine 105 with Alanine (V105A) and Isoleucine 132 with Valine (I132V). The resulting mutagenized proteins were then tested for their ability to induce efficient cleavage of the HBB5 target, upon coexpression with a mutant cleaving HBB5.4.
Same protocol as in example 4 except that 25 ng of the PCR product and 75 ng of vector DNA (pCLS0542,
Libraries were created by PCR on a pool of initial mutants cleaving HBB5.3. As an example, to introduce the G195 substitution into the coding sequences of the mutants, two separate overlapping PCR reactions were carried out that amplify the 5′ end (residues 1-24) or the 3′ end (residues 14-167) of the I-CreI N75 coding sequence. For both the 5′ and 3′ end, PCR amplification is carried out using a primer with homology to the vector (Gal10F 5′-gcaactttagtgctgacacatacagg-3′ (SEQ ID NO: 34) or Gal10R 5′-acaaccttgattggagacttgacc-3′ (SEQ ID NO: 35)) and a primer specific to the I-CreI coding sequence for amino acids 14-24 that contains the substitution mutation G195 (G19SF 5′-gccggctttgtggactctgacggtagcatcatc-3′ (SEQ ID NO: 151) or G19SR 5′-gatgatgctaccgtcagagtccacaaagccggc-3′ (SEQ ID NO: 152)). The resulting PCR products contain 33 bp of homology with each other. The PCR fragments were purified. Finally, approximately 25 ng of each of the two overlapping PCR fragments and 75 ng of vector DNA (pCLS0542,
The same strategy was used with the following pair of oligonucleotides to create the other libraries containing the F54L, E80K, F87L, V105A and I132V substitutions, respectively:
The yeast strain FYBL2-7B (MATα, ura3Δ851, trp1Δ63, leu2Δ1, lys2A202) containing the HBB 5 target in the yeast reporter vector (pCLS1055,
Libraries obtained by random mutagenesis and by site directed mutagenesis were constructed on a pool of fourteen initial I-CreI mutants cleaving HBB5.3 target (Table VI). 2232 and 372 transformed clones for each library were then mated with a yeast strain that (i) contains the HBB5 target in a reporter plasmid (ii) expresses the mutant 28K30N32T33Y38Q40S44A68R70S75E77R+80K+96R also called KNTYQS/ARSER+80K+96R, an optimized variant cleaving the HBB5.4 target already described in example 4.
The identification of some positives mutants harbouring the G19S mutation after site directed mutagenesis and cutting efficiently the HBB 5 target are shown in
As an example, the sequences of 14 I-CreI optimized mutants cleaving the HBB5.3 target when forming heterodimer with the mutant KNTYQS/ARSER+80K+96R cleaving HBB5.4 are compiled in Table VII. These optimized variants correspond to the sequences SEQ ID NO: 164 to I77.
As described in example 4, just one I-CreI optimized mutant called KNTYQS/ARSER+80K+96R cleaving the palindromic HBB5.4 target was already identified from the heterodimeric yeast screening of a library obtained by random mutagenesis and tested by co-expression with mutants cleaving the palindromic HBB5.3 target. In order to increase the number of the I-CreI cutters for the HBB5 sequence, another set of libraries corresponding to site-directed mutagenesis of a pool of mutants cleaving the HBB5.4 target by the insertion of the six amino-acid substitutions G19S, F54L, E80K, F87L, V105A and I132V were constructed.
The resulting proteins were tested for their ability to induce efficient cleavage of the HBB5 target.
Same protocol as in example 5 except that 25 ng of each of the two overlapping PCR fragments and 75 ng of vector DNA (pCLS 1107,
The yeast strain FYBL2-7B (MAT a, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202) containing the HBB 5 target in the yeast reporter vector (pCLS1055,
Libraries constructed by site directed mutagenesis were derived from a pool of twelve I-CreI mutants corresponding to eleven initial mutants and one optimized mutant called KNTYQS/ARSER+80K+96R cleaving HBB5.4 target (Table VIII). 372 transformed clones for each library were then mated with a yeast strain that (i) contains the HBB5 target in a reporter plasmid (ii) expresses the mutant 28K30T32S33R38H40S/44K68S70S75N77I also called KTSRHS/KSSNI, an initial variant cleaving the HBB5.3 target already described in example 3.
Some positives optimized mutants harbouring the G19S mutation after site directed mutagenesis and cutting efficiently the HBB 5 target are shown in
The results of the experiments of mutagenesis of mutants targeting the palindromic targets HBB5.3 and HBB5.4 lead to the identification of optimized mutants cleaving more efficiently the sequence HBB5 when tested in heterodimeric co-expression yeast screening assay (see examples 5 and 6). Moreover, as shown in example 6 (Table IX), the sequences of some optimized mutants (KNTYQS/ARSER+80K+87L, KNTYQS/ARSER+19S+80K), indicate that the association of the substitutions E80K with F87L and G19S with E80K within the same mutant can lead to an increase of it's efficiency of cleavage of the HBB5 target. In order to obtain a large panel of I-CreI mutants with a maximal efficacy of cleavage of the HBB5 target, new libraries of random and multiple site-directed mutagenesis (
Same protocol as in example 4. For the libraries derived from the pool of the mutants targeting HBB5.3 or HBB5.4 sequences, 25 ng of the PCR product and 75 ng of vector DNA pCLS0542 linearized by digestion with NcoI and EagI (
Same protocol as in example 5 except that to obtain a multiple insertion of the G19S, F54L, E80K, V105A and I132V substitutions into the coding sequences of the mutants cleaving HBB5.3 and HBB5.4 targets, six groups of separate overlapping PCR reactions were carried out that amplify internal fragments of the I-CreI N75 coding sequence containing the sequences between the different mutations. As an example, for the multiple site directed mutagenesis for the insertion of the mutations G19S and F54L, PCR amplification is carried out using primers specific to the I-CreI coding sequence for amino acids 14-24 that contains or not the substitution G19S (G19SF 5′-gccggctttgtggactctgacggtagcatcatc-3′ (SEQ ID NO: 151) and G19 wtF 5′-gccggctttgtggacggtgacggtagcatcatc-3′ (SEQ ID NO: 187)) with primers specific to the I-CreI coding sequence for amino acids 49-59 that contains or not the substitution F54L (F54LR 5′-cactagtttgtccagcagccaacggcgctgggt-3′ (SEQ ID NO: 154) and F54 wtR 5′-cactagtttgtccagaaaccaacggcgctgggt-3′ (SEQ ID NO: 188), leading to the generation of four fragments of PCR with different ends.
The same strategy was used with the following groups of oligonucleotides to create the other internal fragments:
The resulting overlapping PCR products contain 15 bp of homology with each other. The PCR fragments corresponding to each internal region were then purified pooled and I-CreI coding sequences containing or not the mutations G19S, F54L, E80K, V105A and I132V were generated by PCR assembly. Finally, approximately 25 ng of each assembled PCR fragment obtained with the mutants cleaving HBB5.3 and HBB5.4 targets and 75 ng of vector DNA pCLS0542 (
The yeast strain FYBL2-7B (MAT a, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202) containing the HBB 5 target in the yeast reporter vector (pCLS1055,
Libraries of random mutagenesis or multiple site directed mutagenesis were derived from a pool of eight and six I-CreI mutants corresponding to optimized mutants cleaving the targets HBB5.3 and HBB5.4 (Table X). 2232 or 2790 transformed clones for each kind of library were obtained. The clones corresponding to the libraries derived from the HBB5.3 mutants were mated with a yeast strain that (i) contains the HBB5 target in a reporter plasmid (ii) expresses the mutant 28K30N32S33Y38Q40S/44A68R70S75E77R+24V+43L+80K+87L+96R also called KNSYQS/ARSER+24V+43L+80K+87L+96R, a refined variant cleaving the HBB5.4 target already described in example 6. The clones corresponding to the libraries derived from the HBB5.4 mutants were mated with a yeast strain that (i) contains the HBB5 target in a reporter plasmid (ii) expresses the mutant 28K30S32S33H38R40S/44K68Y70S75D77T also called KSSHRS/KYSDT, an optimized variant cleaving the HBB5.3 target already described in example 5.
Examples of positives mutants cutting efficiently the HBB5 target identified with the two kinds of mutagenesis are shown in the
Sequencing of such positive clones allowed the identification of 98 and 45 novel optimized mutants leading to efficient cleavage of HBB5 target in yeast heterodimeric screen assay. The sequences of some I-CreI optimized mutants cleaving the HBB5.3 and HBB5.4 targets are compiled in Table XI.
Several I-CreI refined mutants able to efficiently cleave the HBB 5 target in yeast when forming heterodimers were identified in example 7. In order to validate and characterize the heterodimer displaying the maximal efficacy to cleave the HBB 5 sequence in CHO cells, the efficiency of combinations of mutants able to cut the HBB 5 target were tested using an extrachromosomal assay in mammalian cells.
1) Materials and methods
The target of interest was cloned as follows: oligonucleotide corresponding to the target sequence flanked by gateway cloning sequence was ordered from PROLIGO. Double-stranded target DNA, generated by PCR amplification of the single stranded oligonucleotide, was cloned using the Gateway protocol (INVITROGEN) into CHO reporter vector (pCLS1058,
The ORF of I-CreI optimized mutants cleaving the HBB5.3 and HBB5.4 targets identified in example 7 were re-cloned in pCLS1069 (
CHO cells were transfected with Polyfect® transfection reagent according to the supplier's protocol (QIAGEN). 72 hours after transfection, culture medium was removed and 150 μA of lysis/revelation buffer for β-galactosidase liquid assay was added (typically 1 liter of buffer contained: 100 ml of lysis buffer (Tris-HCl 10 mM pH7.5, NaCl 150 mM, Triton X100 0.1%, BSA 0.1 mg/ml, protease inhibitors), 10 ml of Mg 100× buffer (MgCl2 100 mM, β-mercaptoethanol 35%), 110 ml ONPG 8 mg/ml and 780 ml of sodium phosphate 0.1M pH7.5). After incubation at 37° C., OD was measured at 420 nm. The entire process was performed on an automated Velocity11 BioCel platform. Per assay, 150 ng of target vector was cotransfected with 12.5 ng of each HBB5 mutant expression vector (12.5 ng of vector encoding the mutant cleaving the palindromic HBB5.3 target and 12.5 ng of vector encoding the mutant cleaving palindromic HBB5.4 target).
Mutants described in Table XI were first re-cloned in the mammalian expression vector pCLS1069 (
The
This example shows that I-CreI mutants can cut the HBB8.3 DNA target sequence derived from the right part of the HBB8 target in a palindromic form (
Both sets of proteins are mutated at position 70. However, two separable functional subdomains exist (Smith et al, Nucleic Acids Res., 2006, 34, e149 and International PCT Applications WO 2007/057781 and WO 2007/049095). That implies that this position has little impact on the specificity in base 10 to 8 of the target. Therefore, to check whether combined mutants could cleave the HBB8.3 target, mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving 5TCT_P (caaaactagt_P) were combined with the 28, 30, 32, 33, 38 and 40 mutations from proteins cleaving 10AGA_P (cagaacgtcgt_P).
The experimental procedure is as described in example 1.
I-CreI combinatorial mutants were constructed by associating mutations at positions 44, 68, 70, 75 and 77 with the 28, 30, 33, 38 and 40 mutations on the I-CreI scaffold, resulting in a library of complexity 1600. This library was transformed into yeast and 2880 clones (1, 8 times the diversity) were screened for cleavage against the HBB8.3 DNA target (cagacttctgt_P; SEQ ID NO: 31). 264 positives clones were found, which after sequencing and validation by secondary screening turned out to be correspond to 126 different novel endonucleases (see Table XIII). The 24 novel endonucleases presented in Table XIII correspond to the sequences SEQ ID NO: 80 to 103. Examples of positives mutants cutting the HBB8.3 target are shown in
This example shows that I-CreI variants can cleave the HBB8.4 DNA target sequence derived from the leftpart of the HBB8 target in a palindromic form (
Both sets of proteins are mutated at position 70. However, two separable functional subdomains exist (Smith et al, Nucleic Acids Res., 2006, 34, e149 and International PCT Applications WO 2007/049095 and WO 2007/057781). That implies that this position has little impact on the specificity in base 10 to 8 of the target. Therefore, to check whether combined mutants could cleave the HBB 8.4 target, mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving 5CCT_P (caaaaccctgt_P) were combined with the 28, 33, 38 and 40 mutations from proteins cleaving 10TGA_P (ctgaacgtcgt_P).
The experimental procedure is as described in example 2.
I-CreI combined mutants were constructed by associating mutations at positions 44, 68, 70, 75 and 77 with the 28, 30, 32, 33, 38 and 40 mutations on the I-CreI scaffold (Table XIV), resulting in a library of complexity 1720. This library was transformed into yeast and 3168 clones (1, 8 times the diversity) were screened for cleavage against the HBB8.4. 141 positives clones were found, which after sequencing and validation by secondary screening turned out to be correspond to 93 different novel endonucleases (see Table XIV). The 19 novel endonucleases presented in Table XIV correspond to the sequences SEQ ID NO: 104 to 122. Examples of positives mutants cutting the HBB8.4 target are shown in
We have previously identified I-CreI mutants able to cleave each of the palindromic HBB 8 derived targets (HBB8.3 and HBB8.4, see examples 9 and 10). Pairs of such mutants in yeast (one cutting HBB 8.3 and one cutting HBB 8.4) were co-expressed in yeast. Upon coexpression, there should be three active molecular species, two homodimers, and one heterodimer. It was tested whether the heterodimers cut the HBB8.2 and HBB8 targets.
The experimental procedure is as described in example 3.
Co-expression of mutants cleaving the HBB 8.3 (11 mutants) and HBB 8.4 (15 mutants) sequences resulted in efficient cleavage of the HBB 8.2 target in almost all cases (
As is evident from the above, the invention is not at all limited to those of its embodiments, implementations and applications which have just been described more explicitly; it encompasses, on the contrary, all the variants which may occur to the specialist in this field, without departing from the framework or the scope of the present invention.
I-CreI mutants able to cleave the non palindromic HBB8 target were identified by co-expression of mutants cleaving the palindromic HBB8.3 and mutants cleaving the palindromic HBB8.4 target (Example 11). To increase the number and efficacy of I-CreI mutants able to cleave the non palindromic HBB8 target, mutants cleaving the palindromic HBB8.3 target were site directed mutagenized and new variants cleaving HBB8 with high efficacy, when co-expressed with mutants cleaving the HBB 8.4 target, were screened.
Six amino-acid substitutions which enhance the activity of I-CreI derivatives were individually introduced into the coding sequence of proteins cleaving HBB8.3. These mutations correspond to the replacement of Glycine 19 with Serine (G195), Phenylalanine 54 with Leucine (F54L), Glutamic Acid 80 with Lysine (E80K), Phenylalanine 87 with Leucine (F87L), Valine 105 with Alanine (V105A) and Isoleucine 132 with Valine (I132V). The resulting mutagenized proteins were then tested for their ability to induce efficient cleavage of the HBB8 target, upon coexpression with a mutant cleaving HBB8.4.
The experimental procedure is as described in example 5.
Libraries containing the six amino-acids substitutions (Glycine19 with Serine, Phenylalanine 54 with Leucine, Glutamic Acid 80 with Lysine, Phenylalanine 87 with Leucine, Valine 105 with Alanine and Isoleucine 132 with Valine) were constructed on a pool of seventeen initial I-CreI mutants cleaving HBB8.3 target (Table XVII). 372 transformed clones for each library were then mated with a yeast strain that (i) contains the HBB8 target in a reporter plasmid (ii) expresses the mutant 28K30N32T33C38Q40544D68A70575K77R also called KNTCQS/DASKR, a variant cleaving the HBB8.4 target described in example 11.
After mating with this yeast strain, 186 new I-CreI clones were found to cleave the HBB8 target more efficiently than the initial mutants when forming heterodimers with a mutant cleaving the HBB8.4 target. These 186 mutants (72 clones with G19S mutation, 60 clones with F54L mutation, 21 clones with E80K mutation, 5 clones with F87L mutation, 9 clones with V105A mutation and 19 clones with I132V mutation) were then rearranged (wells A1 to H9 of the rearranged plate) and submitted to a validation screen conducted exactly in the same conditions as the first one (
The sequence of 25 I-CreI optimized mutants cleaving the HBB8.3 target when forming heterodimer with the mutant KNTCQS/DASKR cleaving HBB8.4 are listed in Table XVIII (SEQ ID NO: 231 to 255). Some of these I-CreI mutants are expected mutants due to the site-directed mutagenesis, but also contain unexpected mutations probably due to the PCR reaction and micro-recombination between mutants of the pool used for the libraries construction.
In order to test the specificity of cleavage of the HBB8 target, the 93 optimized mutants with the simple mutations F54L, F87L, V105A and I132V cleaving the HBB8.3 target were mated with a yeast strain that contains the HBB8.5 or the HBB6.5 target (
I-CreI mutants able to cleave the non palindromic HBB8 target were identified by co-expression of mutants cleaving the palindromic HBB8.3 and mutants cleaving the palindromic HBB8.4 target (Example 11). To increase the number and efficacy of I-CreI mutants able to cleave the non palindromic HBB8 target, mutants cleaving the palindromic HBB8.4 target were also site directed mutagenized and new variants cleaving HBB8 with high efficacy, when co-expressed with mutants cleaving the HBB 8.3 target, were screened.
The six mutations (G19S, F54L, E80K, F87L, V105A and I132V) described in example 12 were individually introduced into the coding sequence of proteins cleaving HBB8.4, and the resulting proteins were tested for their ability to induce efficient cleavage of the HBB8 target, upon co-expression with a mutant cleaving HBB8.3.
Site-directed mutagenesis libraries were created by PCR on a pool of initial mutants cleaving HBB8.4 as described in example 5. The PCR fragments were purified. Approximately 25 ng of each of the two overlapping PCR fragments and 75 ng of vector DNA (pCLS1107,
Libraries containing the six amino-acids substitutions were constructed on a pool of fourteen initial I-CreI mutants cleaving HBB8.4 target (Table XIX). 372 transformed clones for each library were then mated with a yeast strain that (i) contains the HBB8 target in a reporter plasmid (ii) expresses the mutant 28K30T32S33H38R40S44K68Y70S75N77Y also called KTSHRS/KYSNY, a variant cleaving the HBB8.3 target described in example 11.
After mating with this yeast strain, 166 new I-CreI clones were found to cleave the HBB8 target more efficiently than the initial mutants when forming heterodimers with a mutant cleaving the HBB8.3 target. These 166 mutants (93 clones with G19S mutation, 6 clones with F54L mutation, 12 clones with E80K mutation, 17 clones with F87L mutation, 23 clones with V105A mutation and 15 clones with I132V mutation) were then rearranged and submitted to a validation screen conducted exactly in the same conditions as the first one (
Some examples of 20 I-CreI optimized mutants cleaving the HBB8.4 target when forming heterodimer with the mutant KTSHRS/KYSNY cleaving HBB8.3 are listed in Table XX. Some of these I-CreI mutants are expected mutants due to the site-directed mutagenesis, but also contain unexpected mutations probably due to the PCR reaction and micro-recombination between mutants of the pool used for the libraries construction.
In order to test the specificity of cleavage of the HBB8 target, the 93 optimized mutants with the simple mutations F54L, E80K, F87L, V105A and I132V cleaving the HBB8.4 target were mated with a yeast strain that contains the HBB8.6 or the HBB6.6 target (
In examples 12 and 13, we have identified I-CreI refined mutants able to efficiently cleave the HBB8 target in yeast. In this example, we tested the ability of combinations of the mutants cleaving the HBB8.3 and HBB8.4 sequences to cut the HBB8 target using an extrachromosomal and a chromosomal reporter assay in CHO mammalian cells.
Protocol of cloning of HBB8 derived targets in a vector for CHO screen, re-cloning of meganucleases and extrachromosomal assay in mammalian cells is as described in example 8.
CHO cell lines harbouring the reporter system (
Comparison of the cleavage activity of heterodimers of I-CreI improved mutants against the two targets HBB8 and HBB6 was first monitored using the extrachromosomal assay in mammalian CHO cells. As it can be seen in Table XXI and
The activity of one of the most efficient HBB8 heterodimer containing the two I-CreI optimized mutants HBB8.3_H3 and HBB8.4_A4 was also tested using a chromosomal assay in a CHO cell line containing either the HBB8 or the HBB6 targets. This chromosomal assay has been extensively described in a recent publication (Arnould et al., J Mol Biol, 2007, 371, 49-65). Briefly, a CHO cell line carrying a single copy transgene was first created. The transgene contains a human EF1α promoter upstream an I-SceI cleavage site (
The two cell lines were co-transfected with the repair matrix and various amounts of the vectors expressing the meganucleases. The frequency of repair of the LacZ gene increased from a maximum of 3.9×10−3 with the cell line harboring the HBB6 target, to a maximum of 8.8×10−2 with the cell line harboring the HBB8 target (
These finding confirm that in a chromosomal context the HBB8 target with the mutation responsible of the Sickle Cell Anemia is also more efficiently cleaved than HBB6 target derived from the wild type locus. Such result is in agreement with the results obtained with the extrachromosomal assay implying that I-CreI optimized mutants may be used for genomic correction of a mutation responsible of a genetic disease.
I-CreI refined mutants able to efficiently cleave the HBB5 and HBB8/6 targets in yeast and in mammalian cells (CHO K1 cells) have been identified in examples 7, 8, 12, 13 and 14. It was therefore useful to construct the molecular tools to test the efficiency of the HBB meganucleases to correct the mutation responsible of the Sickle cell Anemia. For that purpose, a Knock-in matrix (KI) was designed to analyse the induction of homologous recombination by the heterodimeric combinations of I-CreI refined mutants at the HBB locus of the lymphoblastoid human SC-1 (ATCC_CRL—8756) cell line.
1) Materials and methods
The Knock-in matrix consists on an hygromycin resistance coding sequence (CDS) cloned between two human HBB homology arms [LH HBB of 1730 bp from 4032375 to 4034105 and RH HBB of 2136 bp from 4034106 to 4036242 in the genomic contig NT009237.17. Position 4034105 is within the HBB5 target (position 11 of the HBB5 target) and corresponds to position 1247 on SEQ ID NO: 4 (
Human lymphoblastoid SC-1 cell line (ATCC_CRL—8756) is cultivated in complete RPMI 1640 medium supplemented with 20% fetal calf serum, penicillin, streptomycin, fungizon, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate. After transfection, selection is performed using Hygromycin in complete medium. After ten days of selection with Hygromycin, negative selection to Ganciclovir is applied to select clones that are resistant to Hygromycin and Ganciclovir. Such clones are amplified and genomic DNA extracted.
Knock-in events may be detected by PCR analysis on genomic DNA using the pair of primers located respectively in the human HBB sequence upstream of the LH HBB left homology arm and in the hygromycin CDS, to obtain a KI specific PCR amplification. The sequencing of the PCR amplified fragments would allow to determine the efficiency of the correction of the Sickle Cell Anemia mutation.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCTIB2007/003169 | Jul 2007 | IB | international |
| PCTIB2008/002524 | Jun 2008 | IB | international |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12670571 | May 2010 | US |
| Child | 13909771 | US |
