FR3099179A1 - METHOD FOR DETERMINING THE EFFECT OF A MUTATION ON THE EXPRESSION OF A GENE OF INTEREST - Google Patents

Abstract

The invention relates to a method for determining the effect of a mutation, in particular a synonymous mutation on the expression of a gene of interest comprising the expression in a cell of a vector comprising a wild-type gene of interest. fused to a first reporter gene and a gene of interest comprising said mutation fused to a second reporter gene, said genes being under the control of a bidirectional promoter. This method is particularly suitable for determining the effect of a mutation previously detected in a patient sample. The invention also relates to the vector used in the method and to the cells expressing said vector.

Description

METHOD FOR DETERMINING THE EFFECT OF A MUTATION ON THE EXPRESSION OF A GENE OF INTEREST

The invention relates to a method for determining the effect of a mutation, in particular a synonymous mutation on the expression of a gene of interest comprising the expression in a cell of a vector comprising a wild-type gene of interest fused to a first reporter gene and a gene of interest comprising said mutation fused to a second reporter gene, said genes being under the control of a bidirectional promoter. This method is particularly suitable for determining the effect of a mutation previously detected in a patient sample. The invention also relates to the vector used in the method and the cells expressing said vector.

The genetic code for translating the information contained in the genome of living cells and for synthesizing proteins is redundant. Thus, most amino acids are encoded by more than one codon. Codons coding for the same amino acid are said to be synonymous. Consequently, a genetic mutation which transforms a codon into another synonymous codon has no effect on the sequence of the synthesized protein.

Synonymous mutations have long been considered to have no consequence on the expression profile and function of the protein. Synonymous mutations have been largely overlooked in human pathology, as these changes have no expected impact on protein sequence. However, very recently, many synonymous mutations have been associated with serious diseases in humans, in particular thanks to genetic screening studies.

Synonymous mutations can lead to molecular, cellular and physiological disturbances and play a role in mRNA splicing (Cartegni L, Chew SL, Krainer AR. Nat Rev Genet. 2002; 3: 285–298), its stability ( Nackley AG, Shabalina SA, Tchivileva IE, et al. Science 2006), its structure (Shen LX, Basilion JP, Stanton VP. Proc Natl Acad Sci USA 1999; 96: 7871–7876) but also in translation and tertiary structure proteins (Kimchi-Sarfaty C, Oh JM, Kim IW, et al. Science 2007).

The study of mutations, especially synonymous mutations and their effects on protein expression and function is necessary to understand the causes of the diseases associated with these mutations.

Currently, mutations, in particular synonymous mutations, are studied by ectopically expressing wild-type and mutated versions of a gene of interest in separate cells, and then measuring the amount of protein produced by immunofluorescence or immunoblot, when antibodies correspondents exist. In this system, the use of distinct cells leads to variability in the expression of wild-type and mutated genes. A precise comparison of the expression profiles of the wild-type and mutated genes, in particular the level of expression and the subcellular localization of the proteins coded by these genes, is therefore not possible.

There thus remains a need to develop a more sensitive method making it possible to precisely compare the expression profiles of the wild-type and mutated genes, making it possible in particular to highlight small differences in expression.

The inventors have developed an experimental system based on the simultaneous expression of a wild-type gene of interest and carrying a mutation under the control of the same bidirectional promoter. Each version of the gene is fused in frame to a different reporter gene to determine the expression profile of each gene. This system is particularly suitable for studying the effect of synonymous mutations.

The present invention thus relates to a method for determining the effect of a mutation, preferably a synonymous mutation, on the expression of a gene of interest in a cell, said method comprising the following steps: i) expression in a cell of a vector comprising a wild-type gene of interest fused to a first reporter gene encoding a first fusion protein and a gene of interest comprising at least one mutation, preferably a synonymous mutation fused to a second reporter gene encoding a second fusion protein, said genes being under the transcriptional control of a bidirectional promoter; ii) detection of the expression of the first and the second fusion protein; iii) comparison of the expression profiles of the first and the second fusion protein. Detection of expression of the first and second fusion protein in ii) may include determining the subcellular localization of the first and second fusion protein. Said step ii) can also comprise the measurement of the level of expression of the first and of the second fusion protein and in this case the step iii) can comprise the comparison of the level of expression of the first and of the second fusion protein, preferably by measuring the ratio of the level of expression of the second fusion protein to the level of expression of the first fusion protein.

In a particular mode, the vector used in the method according to the invention can comprise an inducible bidirectional promoter, preferably inducible by tetracycline or one of its analogs. In another particular mode, the vector used in said method can comprise reporter genes coding for different fluorescent proteins, preferably mCherry and mEGFP. In this case, said method comprises a step ii) of detecting the expression of the first and of the second fusion protein by fluorescence microscopy or by flow cytometry.

Said method according to the invention may further comprise the following normalization steps: iv) expression in another cell of a vector comprising a wild-type gene of interest fused to said first reporter gene coding for a third fusion protein and a second copy of the wild-type gene of interest fused to said second reporter gene encoding a fourth fusion protein, said genes being under the transcriptional control of said bidirectional promoter; v) measuring the level of expression of the third and fourth fusion protein; vi) calculation of the ratio of the level of expression of the fourth fusion protein to the level of expression of the third fusion protein; vii) normalization of the ratio of fusion protein expression levels obtained in iii) by the ratio of fusion protein expression levels obtained in vi).

In a particular mode, the method according to the invention is characterized in that the mutation of the gene of interest is detected beforehand in a sample from a subject, preferably from a sick subject.

The present invention also relates to a vector comprising two reporter genes under the transcriptional control of a bidirectional promoter, preferably comprising restriction sites allowing the in-phase cloning of genes of interest with reporter genes so as to encode proteins of merger. Preferably, said vector further comprises a wild-type gene of interest fused to the first reporter gene and a gene of interest comprising a mutation fused to the second reporter gene. In another aspect, the invention relates to a cell expressing the vector according to the invention.

Fig. 1

Examples of vectors used in the method according to the invention;

Fig. 2

Correlation of the expression levels per cell of SHH-mEGFP and SHH-mCherry via the method of the invention. A. Microscopic views of human HeLa cells expressing SHH-mEGFP and SHH-mCherry from the vector of the invention. B. Graphic representation of the intensities of each fluorophore per cell. The background noise is subtracted from the signal. The linear relationship between mCherry and mEGFP signals reflects their proportional relationship;

Fig. 3

Robustness of the method according to the invention, inter-experiment variability. Three independent experiments were conducted, and the corresponding normalized ratios calculated and presented graphically. The absence of effect of the SHH c.C522T mutation is maintained between experiments (pvalues=0.1429; 0.0035; 0.0735; non-parametric Wilcoxon t test);

Fig. 4

Colocalization of SHHwt-mEGFP and SHHwt-mCherry. A. Microscopic views of human HeLa cells expressing SHH-mEGFP and SHH-mCherry from the ibis vector. B. Intensity per pixel along the white line in A). C. Superposition of the two SHH-mEGFP and SHH-mCherry curves;

Fig. 5

Highlighting the effect of the SHH c.G552C mutation on the amount of protein produced. The c.G552C mutation of SHH, unlike the c.C522T mutation, leads to a reduction in the amount of the corresponding SHH protein compared to the wt version, with p values less than 10-15 and equal to 7, 7x10-2, respectively (nonparametric Wilcoxon t-test);

Fig. 6

Highlighting the effect of the c.C1206T mutation of SHH on the localization of SHH. A. Microscopic views of human HeLa cells expressing c.C1206T SHH-mEGFP and WT SHH-mCherry from the ibis vector. B. Intensity per pixel along the white line in A). C. Superposition of the two SHH-mEGFP and SHH-mCherry curves. The c.C1206T mutation of SHH (SHH-mEGFP) results in an imperfect overlap with the wild-type version of SHH (SHH-mCherry) compared to the wt/wt situation presented in Figure 2.

The inventors have developed a new vector comprising a wild-type gene of interest fused to a first reporter gene encoding a first fusion protein and a mutated gene of interest fused to a second reporter gene encoding a second fusion protein, said genes being under the control of a bidirectional promoter. The expression of fusion proteins in a cell from this vector as well as their detection and the comparison of their expression profiles make it possible to determine the effect of a mutation on the expression of a gene of interest. By expression profile is meant in particular the expression levels and/or the subcellular localization of the fusion proteins.

The invention thus relates to a vector comprising a wild-type gene of interest fused to a first reporter gene and a gene of interest comprising a mutation fused to a second reporter gene, said genes being under the control of a bidirectional promoter.

By "gene" is meant a nucleic acid molecule encoding a polypeptide. The term gene of interest used in the present application preferably refers to the complementary DNA also called cDNA coding for the product of a gene of interest. The product of a gene can be a peptide or a protein.

The terms "nucleic acid" or "nucleotide sequence" can be used interchangeably to refer to any molecule composed of or comprising nucleotide monomers. A nucleic acid can be an oligonucleotide or a polynucleotide. A nucleotide sequence can be DNA or RNA. A nucleotide sequence can be modified chemically or artificially. The nucleotide sequences can include nucleic acids, peptides, morpholinos, blocked nucleic acids as well as glycol nucleic acids and threose nucleic acids. Each of these sequences differs from natural DNA or RNA by the nature of the backbone. Phosphorothioate nucleotides can be used. Other nucleic acid analogues such as methylphosphonates, phosphoramidates, phosphorodithioatesn N3'P5'-phosphoramidates and oligoribonucleotide phosphorothioates and 2'-0-allyl and 2'-0-methylribonucleotide methylphosphonate analogues can be used in the context of the invention.

The wild-type gene corresponds to the nucleotide sequence, preferably the cDNA of the wild-type gene of interest. By wild gene, we mean the functional gene originally possessed by the species or the most widespread functional form which serves as a reference.

Mutation refers to a change in the nucleotide sequence of the wild-type version of a gene of interest. The mutation can lead to the deletion, insertion or substitution of one or more amino acids of the peptide sequence coded by the gene of interest.

Preferably, the mutation is a synonymous mutation. The synonymous mutation refers to a nucleotide sequence mutation present in the coding region of a gene of interest, for example in an exon of said gene, and which does not modify the translated amino acid sequence.

In the context of the present application, the gene comprising a mutation is called mutated gene.

The term “vector” is understood here to mean a DNA molecule which is indifferently in the form of a single strand or a double strand.

A recombinant vector according to the invention is preferably a plasmid vector or an integration vector. In certain embodiments of the invention, the vector is a plasmid. By "plasmid" is meant here a double-stranded circular DNA molecule which has an origin of replication so that it can replicate autonomously in the cell and a selection gene so that it is not lost by the organism through cell multiplication. Many vectors are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form, or integration into the chromosomal material of the host), as well as the nature of the host cell.

Preferably, said vector is an expression vector comprising all the elements necessary for the expression of the genes of interest as defined above. For example, said vector comprises an expression cassette including at least one gene of interest as defined above, under the control of appropriate transcriptional and optionally translational regulatory sequences (promoter, activator, intron, codon d (ATG), stop codon, polyadenylation signal, splice site).

Preferably, the vector is an episomal vector capable of replicating autonomously.

In addition to the coding region, the gene of interest may comprise or be associated with one or more elements which facilitate or increase the expression of said gene, such as activator sequences, response elements, insulators, polyadenylation signals and/or any other functional element.

The genes of interest can be inserted into an expression vector, under the transcriptional control of a bidirectional promoter, to allow the simultaneous expression of the two genes of interest, in particular the wild-type and mutated genes of interest.

By “promoter”, is meant any polynucleotide capable of positively regulating the expression, in a cell, of a nucleotide sequence to which it is linked in an operational manner. A promoter or a promoter sequence is a region of DNA located near a gene and essential for the transcription of DNA into RNA. Promoter sequences are generally located upstream of the transcription start site. Promoter sequences correspond to the region to which RNA polymerase initially binds before starting RNA synthesis.

By “bidirectional promoter”, we mean regulatory regions that can potentially induce the expression of two contiguous genes located in opposite orientations. Preferably, the bidirectional promoter comprises two identical promoters arranged back to back. The fact of using a bidirectional promoter for the two wild-type and mutated genes of interest allows identical transcriptional expression of the two genes of interest and therefore of the two reporter genes to which they are fused.

The bidirectional promoter can be a promoter naturally expressed in humans or a synthetic promoter. Promoters that can be used in the method of the invention include but are not limited to the herpes virus thymidine kinase (TK) gene promoter, CMV promoter, PGK promoter. Preferably the promoter is a CMV promoter.

In a preferred mode, the bidirectional promoter as used in the method of the invention is an inducible bidirectional promoter. The inducible promoter is activated in response to the presence of a particular external compound called an inducer such as antibiotics, hormones or an external condition such as an increase in temperature. The use of an inducible bidirectional promoter makes it possible to simultaneously express the two genes of interest.

In a preferred mode, the inducible promoter is a tetracycline inducible promoter or a derivative thereof. This inducible promoter comprises a minimal promoter operably linked to one or more tetracycline operator(s). Preferably, the bidirectional promoter has a nucleotide sequence SEQ ID NO: 1.

There are two versions of transactivator proteins capable of binding to the tet operator sequence and thus activating downstream gene transcription, one of which can be activated by tetracycline or its derivatives and the other inactivated by these same molecules ( Gossen M. and Bujard H., Proc Natl Acad Sci U S A, 89 (1992) 5547-5551; Gossen M. et al., Science, 268 (1995) 1766-1769). The transcription activator protein comprises two polypeptides, a first which binds to the tet operator sequences and a second which activates transcription. Binding to the tetracycline operator sequences of a transcription activating protein in the presence (tet on system) or absence (tet off system) of tetracycline or one of its analogues allows the activation of the promoter and the transcription of the associated gene.

Tetracycline analogues include, for example, doxycycline, chlorotetracycline and anhydrotetracycline.

The nucleotide sequence encoding the transactivator protein capable of binding to the tet operator sequence may be located on the same vector comprising the tet operator sequence or may be located on a separate vector.

Among the other inducible systems, mention may be made of fusions of VP16 or Gal4 with control protein sequences such as certain nuclear hormone receptors, more or less modified to respond in a specific manner to the presence of hormone analogues.

To be able to distinctly detect the expression of the wild-type gene of interest and of the mutated gene of interest, each of the genes is fused to different reporter genes.

By fusion of two or more genes, we mean the association of two or more genes which code for different proteins or fragments of proteins, the translation of the two fused genes giving a single functional polypeptide. In the context of the invention, the gene of interest is fused in reading phase with the reporter gene to give a fusion protein which makes it possible to detect the protein of interest.

Preferably, the gene of interest is fused to the reporter gene via a "linker", preferably a nucleotide sequence which codes for a peptide sequence which makes it possible to link the different proteins or protein fragments in such a way that the protein adopts a better conformation for protein activity. In particular, the nucleotide sequence codes for a peptide sequence of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 amino acids. In a preferred mode, the linker encodes a peptide sequence consisting of five glycines.

The term "reporter gene" designates a gene whose product (a protein) has a characteristic allowing it to be observed or quantified, for example by detection of a bioluminescence (for example fluorescence) or of an enzymatic activity. Thus, a useful reporter gene in the implementation of the present invention can be chosen from reporter genes coding for a fluorescent protein, and reporter genes coding for an enzyme whose action causes the appearance of a colored product or of an easily characterizable phenotype or any other protein having any other quantifiable activity.

In certain embodiments, a reporter gene according to the invention is chosen from reporter genes encoding an enzyme, such as for example the luciferase gene of Vibrio (Lux), the beta-galactosidase gene (LacZ) or d hydrolase (β-glucoronidase, alkaline phosphatase), the chloramphenicol acetyltransferase (CAT) gene and the beta-lactamase (TEM-1) gene.

In a particular mode, a reporter gene according to the invention is a reporter gene coding for a fluorescent protein.

Preferably, the first reporter gene codes for a first fluorescent protein, the second reporter gene codes for a second fluorescent protein, and the first and second fluorescent proteins are different. Each of these fluorescent proteins must be suitable for cellular exploration. By "differently fluorescent proteins" is meant proteins which fluoresce in different regions of the optical spectrum, or more specifically proteins which fluoresce at sufficiently different wavelengths that they can be distinguished using a conventional method. for detecting or measuring fluorescence. Thus, the first fluorescent protein can emit in the red (emission wavelength between approximately 605 nm and 780 nm) and the second fluorescent protein can emit in the green (emission wavelength between approximately 510 nm and 570 nm). Alternatively, the first fluorescent protein can emit in blue (wavelength between about 460 nm and 480 nm) and the second fluorescent protein can emit in orange (emission wavelengths between about 585 nm and 605nm). A person skilled in the art knows how to select two different fluorescent proteins to develop a reporter system according to the invention.

The reporter gene which can be used in the context of the present invention is a gene coding for a fluorescent protein belonging to the category of BFPs (such as for example the proteins Sirius, EBFP, SBFP2, EBFP2, Azurite, mKalamal, TagBFP and mBlueberry2) , CFPs (such as for example SCFP3A, mTurquoise, mseCFP, Cerulean, ECFP, CyPet, TagCFP, mTFP1, Midori-ishi Cyan, and mUKG), GFPs (such as for example TurboGFP, AceGFP, Azami Green, ZsGreen, TagGFP2, EGFP , mEGFP, mWasabi, Emerald, Superfolder GFP, Sapphire, T-Sapphire, and SGFP2), YFPs (such as TagYFP, mAmetrine, EYFP, Topaz, SYFP2, Venus, mCitrine, Ypet, PhiYFP, and ZsYellowl), OFPs (such as mBanana, mOrange, Kusabira Orange2, mOrange2, OFP, and TurboRFP), RFPs (such as tdTomato, DsRed2, DsRed, DsRed-Express, TagRFP, TagRFP-T, mTangerine, DsRed-Express2, DsRed-Max , AsRed2, mApple, mStrawberry, mRuby, mRFPl, tdRFP611 and mCherry), or FRFPs (such as eqFP611, tdKeima, mKeima, mRaspberry, tdRFP639, tdKatushka2, mKate, mKate2, mPlum, mNeptune and AQ143).

Preferably, the vector comprises a first reporter gene encoding mEGFP and a second reporter gene encoding mCherry.

In certain embodiments, the vector also comprises restriction sites allowing modification or replacement of the wild-type and/or mutated gene, but optionally also of the promoter, of the first reporter gene, of the second reporter gene. The term "restriction site" designates a particular sequence of nucleotides which is recognized by a restriction enzyme as a cleavage site in the DNA molecule. These restriction sites can, for example, be recognized by enzymes chosen from BamHI, HindIII, KpnI, NdeI, Apal, XbaI, BglII, and EcoRI. Preferably, the restriction sites of the modifiable or replaceable elements such as the wild and mutated genes of interest, promoter, first reporter gene, second reporter gene are all unique and different from each other so that these elements can be modified or replaced specifically and independently of each other. Preferably, the vector comprises at least one restriction site allowing the cloning of a gene of interest in the reading phase with the reporter gene so as to code for a fusion protein.

According to a particular embodiment, the vector can also comprise a selection marker which makes it possible to select the cells expressing this vector. For example, the selection markers can be genes conferring resistance to antibiotics. An antibiotic resistance gene is a gene that produces a protein that makes the cell resistant to antibiotics. Preferably, the antibiotic resistance gene allows selection to be made in prokaryotic cells and is selected, without being limited, from the group consisting of: ampicillin, actinomycin, kanamycin, neomycin, puromycin, gentamycin. In another particular embodiment, the vector of the invention may also comprise a resistance gene making it possible to carry out a selection in eukaryotic cells, such as for example the gene for resistance to hygromycin, blastidicin, geneticin, zeocin , puromycin.

The vector according to the invention is a vector comprising a first reporter gene and a second reporter gene under the transcriptional control of a bidirectional promoter, preferably inducible. In a particular mode, the vector comprises a wild-type gene of interest fused to a first reporter gene and a mutated gene of interest, preferably a gene of interest comprising a synonymous mutation fused to a second reporter gene under the transcriptional control of a bidirectional promoter, preferably inducible. Preferably, the vector comprises restriction sites allowing the cloning in reading phase of the genes of interest upstream of the reporter genes so as to code for fusion proteins. Preferably, the vector according to the invention has a nucleotide sequence SEQ ID NO: 4 or 5, depending on whether or not it comprises the tet transactivator system.

A vector according to the invention can be prepared by any suitable method, the method for obtaining the vector not being a critical or limiting element of the invention.

Methods for preparing such nucleic acid constructs are known in the art and have, for example, been described in several reference books such as Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory: Cold Spring Harbor, and Silhavy, Berman, and Enquist, "Experiments with Gene Fusions", 1984, Cold Spring Harbor Laboratory: Cold Spring Harbor; F.M. Ausubel et al., "Current Protocols in Molecular Biology", 1989, John Wiley & Sons: New York.

The invention also relates to a cell expressing the said vector of the invention.

By "cell" is meant a eukaryotic cell, preferably animal. The invention can be carried out in any cell, in particular mammalian cells. The cells can be cells from primates, including human cells, cells from monkeys, chimpanzees, macaques or baboons. The cells can also be bovine, caprine, ovine, porcine, equine or rabbit cells. The cells can also be rodent cells, in particular mouse, rat, hamster, guinea pig. Examples of mammalian cells therefore include fibroblasts, endothelial cells, epithelial cells, hematopoietic cells. Preferably, the cells are cell lines which represent a homogeneous population of cells, stable after successive mitoses. Preferably, the cell lines are derived from cells taken from an immortalized patient. Mention will be made, by way of non-limiting examples, of 3T3 cells, HeLa cells, U2OS cells, RPE cells, HCT116 cells, HEK293 cells, HEK293T cells, CHO cells or primary fibroblasts.

The vector is expressed in the cell by introducing said vector into the cell. The vector can be introduced into the cell by any method known to those skilled in the art.

The vector can be introduced into the cell by electroporation, conjugation, transfection, transduction, or any other technique known to those skilled in the art. The vector can be associated with a substance allowing it to cross the membrane of the host cells, such as a transporter such as a nanotransporter or a preparation of liposomes, or of cationic polymers, or else introduce it into said host cell using methods physical such as electroporation or microinjection.

Vector-transformed cells can also be used to produce transgenic animals. In another aspect, the invention relates to non-human transgenic animals comprising a cell expressing the vector as described above. The processes for modifying the genetic identity of animals as described in the present application are not likely to cause suffering without substantial medical benefit for humans or animals.

By transgenic animal is meant a non-human animal, preferably a mammal chosen from the group of rodents and in particular mice, rats, hamsters, guinea pigs. Alternatively, the transgenic animal can be chosen from farm animals and in particular pigs, sheep, goats, cattle, equines, in particular horses, lagomorphs, in particular rabbits. The transgenic animal according to the invention can also be chosen from primates, in particular monkeys, such as the macaque, the chimpanzee or the baboon.

To produce transgenic animals, after transformation, the cells are seeded on a feeder layer and/or in an appropriate medium. Cells containing the construct can be detected using a selective medium. To screen for cells expressing the vector, selection genes, such as antibiotic resistance genes, can be inserted into the vector. Positive colonies, ie colonies containing cells expressing the vector, are identified by Southern blot analysis and/or by PCR techniques. The positive cells can then be used to carry out the manipulations on the embryo and in particular the injection of the cells expressing the vector into the blastocysts. As far as the mouse is concerned, the blastocysts are obtained from superovulated females aged 4 to 6 weeks. The cells are trypsinized and the modified cells are injected into the blastocoel of a blastocyst. After injection, the blastocysts are introduced into the uterine horn of pseudo-pregnant females. The females are then allowed to carry to term and the resulting litters are analyzed for the presence of cells possessing the vector.

The vector of the invention is used to determine the effect of a mutation, preferably a synonymous mutation on the expression of a gene of interest. Thus, the invention also relates to a method for determining the effect of a mutation, preferably a synonymous mutation on the expression of a gene of interest in which the vector according to the invention is expressed in a cell. The expression of the two fusion proteins, in particular the level of expression and/or the subcellular localization of the two fusion proteins is then detected and then the expression profiles are compared. The expression of the vector in the cell can be achieved by introducing said vector into the cell. The vector can be introduced into the cell by any method known to those skilled in the art.

The step of detecting the expression of the fusion proteins can correspond to a step of detecting the subcellular localization and/or measuring the level of expression of the fusion proteins coded by the genes of interest fused to the reporter genes of the genetic construct.

In a particular mode, the detection step consists in measuring the level of expression of the first and of the second fusion protein. The detection of the expression of the fusion proteins can be done by detection or measurement of the signal which can correspond for example to the enzymatic activity or the fluorescence of the fusion proteins.

In some embodiments, the first reporter gene encodes a first fluorescent protein and the second reporter gene encodes a second fluorescent protein. The step of detecting the level of expression of the fusion proteins then comprises measuring the intensity of fluorescence emitted by the first fusion protein and measuring the intensity of fluorescence emitted by the second fusion protein. The quantification or measurement of the fluorescence intensity can be carried out by any fluorescence technique known to those skilled in the art, such as fluorescence microscopy, fluorescence spectroscopy, flow cytometry, etc. Thus, for example, the fluorescence measurements can be carried out by fluorescence microscopy by observation between slide and coverslip of the cells. After image acquisition, fluorescence determination can be performed by processing information using software such as PCI or ImageJ (Ramadoss et al, P.N.A.S USA, 2016, 110: 10282-10287). Preferably, for each cell, background noise is removed from the fluorescence intensity measurement.

In another particular mode, the detection step consists in determining the subcellular localization of the fusion proteins coded by the reporter genes fused to the wild-type and mutated genes of interest, in order to highlight a possible cellular localization defect due to the mutation in the gene of interest. In particular, the reporter genes are genes coding for different fluorescent proteins. The detection of fluorescence in the cell can be carried out by any fluorescence technique known to those skilled in the art, in particular by fluorescence microscopy. The various cell organelles can be detected by markers known to those skilled in the art. For example, the nucleus can be marked by DNA labeling using, for example, DAPI.

After detecting the expression of the two fusion proteins, the expression profiles are compared. By comparison of the expression profiles of the two fusion proteins, is meant in particular the comparison of the expression levels of the fusion proteins and/or the comparison of the subcellular localization of the fusion proteins. The demonstration of differences in the expression profiles of the fusion proteins coded by the reporter genes fused either to the mutated gene or to the wild-type gene, thus makes it possible to characterize the effect of the mutation.

In the particular mode where the expression level of the fusion proteins is measured, the expression profiles can be compared by measuring the ratio between the expression levels of the fusion proteins. In particular, the ratio Ra between the level of expression of the second fusion protein encoded by the mutated gene of interest fused to the second reporter gene and the level of expression of the first fusion protein encoded by the gene of interest wild type fused to the first reporter gene is measured. In the particular case where the reporter gene codes for a fluorescent protein, the ratio Ra corresponds to the ratio between the fluorescence intensity of the second fusion protein coded by the mutated gene of interest fused to the second reporter gene and the intensity of fluorescence of the first fusion protein encoded by the wild-type gene of interest fused to the first reporter gene.

For measuring the expression of the two reporter genes fused to the wild-type and mutated gene of interest, the method according to the invention may also comprise normalization steps. In particular, the normalization comprises the expression in a second cell of said vector as described above comprising not a wild-type gene and a mutated gene but two wild-type genes of interest each fused to said reporter genes. The expression levels of the fusion proteins are measured and then compared. The comparison of the expression levels of the fusion proteins coded by the two reporter genes fused to the two copies of the wild-type gene of interest thus makes it possible to normalize the expression levels of the proteins coded by the same reporter genes fused to the genes of interest wild and mutated. In particular, the ratio R _NORM between the level of expression of the fusion protein coded by the wild-type gene of interest fused to said second reporter gene and the level of expression of the fusion protein coded by the wild-type gene of interest fused to the first reporter gene is measured. In the particular case where the reporter gene encodes a fluorescent protein, the expression level corresponds to the fluorescence intensity. Normalization is performed by dividing the Ra ratio by the R _NORM ratio.

Expression profile abnormalities due to mutations are responsible for many diseases. More than fifty diseases have been characterized as being associated with a synonymous mutation. Examples of diseases associated with synonymous mutations are: androgen insensitivity syndrome, ataxia, cholesterol ester storage disease, chronic granulomatosis, familial colorectal polyposis, hereditary nonpolyposis colorectal cancer, melanomas , Treacher-Collins syndrome, X-linked spinomuscular atrophy of childhood, cystic fibrosis, Hirschsprung's disease, Marfan's syndrome, McArdle's disease, Phenylketonuria, Seckel's syndrome, X-linked hydrocephalus and holoprosencephaly.

Thus, the method according to the invention can be used to determine the effect of a mutation, preferably a synonymous mutation in a gene of interest in a subject. The method according to the invention is thus particularly suitable for identifying in a sick subject the effect of a mutation, preferably a synonym associated with a disease. The identification of the mutation responsible for the disease can thus make it possible to adapt the treatment to the subject. By subject is meant a mammal, preferably a human.

Thus, the invention also relates to a method as described above in which the mutation is detected beforehand in a sample from a subject, preferably from a sick subject.

The sample used may be blood or any other bodily fluid or tissue obtained from an individual. The DNA can be extracted using any method known in the state of the art, such as the methods described in Sambrook et al. (1989). RNA can also be isolated, for example from tissue obtained during a biopsy, using standard methods well known to those skilled in the art, such as phenol-chloroform extraction.

After the nucleic acid extraction and purification steps, PCR amplification using primers can be used to improve signal detection. For example, the isolated RNA can be subjected to reverse transcription and then amplification, such as an RT-PCR reaction using oligonucleotides specific for the mutated site or which allow amplification of the region containing the mutation.

In one embodiment, the mutation can be detected by analysis of the nucleic acid molecule of the gene of interest. The presence or absence of the mutation can be detected by sequencing, amplification and/or hybridization with a specific probe and primers.

Methods for the detection of the mutation are described for example in Molecular Cloning - A Laboratory Manual" Fourth Edition, M.R. Green, J. Sambrook, (Cold Spring Harbor Laboratory, 2012) and Laboratory Protocols for Mutation Detection, Ed. by U. Landegren, Oxford University Press, 1996 and PCR, 2nd edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

The following examples illustrate the invention without limiting its scope.

Examples

1. Construction of the ibis vector

The ibis (induced bifluorescence system) vector allows the inducible and simultaneous expression of two wild-type and mutated versions of genes of interest fused to two fluorophores distinguishable from each other by their fluorescence emission spectra (mEGFP and mCherry ).

The sequences encoding the mEGFP and mCherry fluorophores are placed in divergent orientation and under the control of the same bidirectional promoter inducible by doxycycline. Unique restriction sites are present 5′ of the mCherry (Xho) and mEGFP (SwaI) sequences and allow the insertion of the cDNAs of interest in phase with the mCherry and mEGFP sequences. In addition, a sequence encoding 5 glycine residues is inserted upstream of the sequences of the fluorophores. This highly flexible element of 5 glycines greatly minimizes the risk of disruption of the folding and behavior of proteins of interest by fluorophores.

The elements allowing inducibility by doxycycline have already been described (Bornkamm GW et al, NAR, 2005, 33(16): e137) and are carried either on the same plasmid construct in the version of the vector used in the following examples ( Figure 1A, Version 1; SEQ ID NO: 4), or on a separate vector.

In the version of the vector used in the following examples (Version 1), the ibis (between the PacI and NotI sites, Figure 1) and doxycycline inducibility (between the NotI and AscI sites) elements are carried on the episomal plasmid originally described by Bornkamm et al. This vector was modified by eliminating the sequence encoding the mEGFP protein. The sequence encoding the mEGFP protein was inserted on one side of the promoter from which it is separated by a SwaI restriction site and a sequence encoding 5 glycines. The luciferase coding sequence was removed. The sequence encoding the mCherry protein was inserted on the opposite side of the promoter to that of the mEGFP sequence. The sequence encoding the mCherry protein is separated from the promoter by an XhoI restriction site and a sequence encoding 5 glycines. Sequences allowing the selection of bacteria (ampicillin) and vertebrate cells (hygromycin) are also present.

In a second version considered (Figure 1B, Version 2, SEQ ID NO: 5), the elements allowing inducibility by doxycycline are located on a separate plasmid. In this second version, the ibis plasmid is obtained from version 1 by removing the doxycycline inducibility elements by enzymatic digestion (PacI/NotI). The simple closure by ligation of the resulting linearized plasmid makes it possible to obtain the episomal plasmid carrying ibis. Sequences allowing the selection of bacteria (ampicillin) and vertebrate cells (hygromycin) are also present.

The doxycycline inducibility elements obtained in the previous step are inserted into a plasmid such as pCDNA3.1. Sequences allowing the selection of bacteria (kanamycin) and vertebrate cells (G418, geneticin) are also present. This construct is called doxycycline induction element, or DIE.

The SHH wild-type sequence is inserted in phase with the mCherry reporter gene (XhoI site) in all versions of ibis. The sequence carrying the mutations of interest or the wild-type sequence is inserted in frame with the mEGFP reporter gene (SwaI site) for the ibis version to be tested or the normalizing version, respectively. For each gene of interest, there is therefore a version of ibis gene of interest wt-wt and as many mut-wt versions as there are mutations to be tested. These plasmid constructs are obtained by standard DNA cloning techniques.

2. Preparation of cell lines and analysis of fluorescence intensity

Human cell lines are established for each version of ibis. Human HeLa cells are transfected with ibis vectors by lipofection. The transfected cells are then cultured and selected in the presence of hygromycin (100µg/mL). The cells are then cultured to be amplified in a culture medium in the presence of 25 μg/mL of hygromycin.

The cells are seeded and cultured on glass coverslips and the expression induced by treatment with doxycycline (1-5 μg/ml, 20h to 72h) then washed, fixed (4% formaldehyde), stained with DAPI (DNA), mounted between blade and slats then sealed. The fixed cells are then observed and photographed using a fluorescence microscope.

For each cell, the mEGFP (green, I _EGFP ) and mCherry (red; I _mCherry ) fluorescence intensities are measured, reduced by the background noise (I _{EGFP BGD} , I _{mCherry BGD} ) and expressed as a ratio:

Ra= (Ia _EGFP - Ia _{EGFP BGD} )/ (Ia _mCherry - Ia _{mCherry BGD} )

The different measurements obtained by condition (cell line) are expressed in the form of mean and dispersion value. Thus, for two mutations 1 and 2, average ratios Rwt-avg, Rmut1-avg and Rmut2-avg are obtained. These means can be normalized with respect to the wt-wt control condition (ie, divided by Rwt-avg).

An appropriate statistical test (for example variants of the Student's test, unpaired, two-sided and nonparametric) then makes it possible to determine the significance of the differences observed.

3. Effectiveness of the method

The effectiveness of the method according to the invention making it possible to determine the effect of mutations, in particular synonymous, depends on the one hand on a strong correlation of the expression of the reporter genes coding for the proteins EGFP and mCherry in each cell, and on on the other hand the reproducibility of the results obtained between experiments. These two aspects were addressed using the human SonicHedgeHog (SHH) protein.

3.1. D determination of the level of correlation between SHH-EGFP and SHH- mcherry

Human HeLa cells were transfected with an ibis vector comprising two copies of the wild-type SHH gene fused to EGFP and mCherry. The fluorescence intensity of EGFP and mCherry was analyzed by fluorescence microscopy.

The determination of the level of correlation between SHH-EGFP and SHH-mCherry is presented in Figure 2. Analysis by cell of the signals of SHH-mCherry and SHH-mEGFP (Figure 2B) reveals a very strong correlation (R= 0.91 R2=0.82, pvalue<2.2.10 ⁻¹⁶ ) of the expression levels of these two proteins within each cell and satisfies the need for a linear relationship between signals.

In addition, the two proteins SHH-mEGFP and SHH-mCherry are perfectly co-localized at the subcellular level (see also Figures 4 and 6).

3.2. I a reproducibility between experiments

Reproducibility between experiments was determined as follows. The fluorescence intensity ratios being normalized on the mean of the control values, ie SHHwt/wt, a version of SHH containing a mutation, c.C522T, was inserted upstream of the mEGFP sequence and used to establish a cell line corresponding. For each experiment, the normalized ratios on the wt/wt ratios were calculated (Figure 3). Three independent experiments were conducted, and the corresponding normalized ratios calculated and presented graphically. The absence of effect of the SHH c.C522T mutation is maintained between experiments (pvalues=0.1429, 0.0035, 0.0735). Furthermore, the mean and dispersion values are very similar between experiments.

3.3. M highlights defects in cell localization

Finally, the ability of the method to highlight cellular localization defects of proteins of interest depends on the perfect colocalization of the mCherry and mEGFP versions. This crucial property of the system is illustrated in Figure 4 in which images of cells expressing SHHwt-mEGFP and SHHwt-mCherry indicate the superposition of the signals and, therefore, their perfect colocalization. In more detail: after induction by doxycycline of the expression of the fusion proteins, the cells are seeded and cultured on glass slides and the expression induced by treatment with doxycycline (1-5 μg/ml) then fixed ( 4% formaldehyde, stained with DAPI (DNA), mounted between slides and coverslips and sealed. The fixed cells are then observed and photographed using a fluorescence microscope (epifluorescence or confocal microscopy).

4. Method and practical illustration by highlighting the effect of mutations of SHH on the amount of SHH produced

In order to validate the capacity of this system to highlight the effect of mutation on the quantity of protein produced, two SHH mutations, one frequent in the general population and without effect (c.C522T), the other found in patients with SHH deficiency -holoprosencephaly, c.C552G- were used as negative and positive control, respectively.

For each version of ibis (SHHwt/wt, SHHc.C522T/wt, SHHc.C552G/wt) human cells in culture are transfected and selected in the presence of hygromycin (100 µg/ml, 8-12 days typically). After selection the cells are ready to use.

The cells are cultured on glass slides and the expression induced by treatment with doxycycline (1-5 μg/ml, 20h to 72h) then fixed (4% formaldehyde), stained with DAPI (DNA), mounted and observed under a microscope. fluorescence, used to photograph cells under the different conditions as illustrated in Figure 2. The fluorescence intensity ratios are then calculated for each cell, then normalized on the average of the SHHwt/wt cell ratios for each of the three independent experiments performed (Figure 5).

5. Method and practical illustration by highlighting the effect of SHH mutations on the localization of SHH

In order to validate the ability of this system to highlight the effect of mutation on the localization of a protein of interest, the wild-type version of SHH and a version carrying the c.C1206T mutation were used.

For each version of ibis (SHHwt/wt, SHH c.C1206T/wt) human cells in culture are transfected and selected in the presence of hygromycin (100 μg/ml, typically 8-12 days). After selection the cells are ready for use.
The c.C1206T mutation of SHH (SHH-mEGFP) leads to imperfect overlap with the wild-type version of SHH (SHH-mCherry) compared to the wt/wt situation presented in Figure 2. This result reveals the deleterious effect of the c .1206 of SHH on the localization of SHH and which underlies the pathogenicity of this mutation.

Claims (10)

Method for determining the effect of a mutation on the expression of a gene of interest in a cell, the said method comprising the following steps:
i) expression in a cell of a vector comprising a wild-type gene of interest fused to a first reporter gene encoding a first fusion protein and a gene of interest comprising at least one mutation fused to a second reporter gene encoding a second fusion protein, said genes being under the transcriptional control of a bidirectional promoter,
ii) detection of the expression of the first and of the second fusion protein,
iii) comparison of the expression profiles of the first and the second fusion protein. Method according to claim 1 characterized in that the mutation is a synonymous mutation. Method according to Claim 1 or 2, characterized in that step ii) comprises determining the subcellular localization of the first and of the second fusion protein. Method according to any one of Claims 1 to 3, characterized in that step ii) comprises measuring the level of expression of the first and of the second fusion protein and step iii) comprises comparing the level of expression of the first and second fusion protein, preferably by measuring the ratio of the level of expression of the second fusion protein to the level of expression of the first fusion protein. Method according to any one of Claims 1 to 4, in which the bidirectional promoter is an inducible promoter, preferably inducible by tetracycline or one of its analogues. A method according to any of claims 1 to 5 wherein the reporter genes encode different fluorescent proteins, preferably mCherry and mEGFP and preferably wherein said expression of the first and second fusion protein is detected by fluorescence microscopy or flow cytometry. A method according to any of claims 4 to 6 further comprising the steps of:
iv) expression in another cell of a vector comprising a wild-type gene of interest fused to said first reporter gene coding for a third fusion protein and a second copy of the wild-type gene of interest fused to said second reporter gene coding for a fourth protein fusion, said genes being under the transcriptional control of said bidirectional promoter,
v) measurement of the level of expression of the third and of the fourth fusion protein,
vi) calculating the ratio of the level of expression of the fourth fusion protein to the level of expression of the third fusion protein,
vii) normalization of the ratio of fusion protein expression levels obtained in iii) by the ratio of fusion protein expression levels obtained in vi). Method according to any one of Claims 1 to 7, characterized in that the mutation of the gene of interest is detected beforehand in a sample from a subject, preferably from a sick subject. Vector comprising two reporter genes under the transcriptional control of a bidirectional promoter, preferably comprising restriction sites allowing the cloning in reading phase of genes of interest with reporter genes so as to code for fusion proteins, characterized in that it further comprises a wild-type gene of interest fused to the first reporter gene and a gene of interest comprising a mutation fused to the second reporter gene. A cell expressing the vector according to claim 9.