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  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N15/09—Recombinant DNA-technology
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
  • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

• the present invention relates to a nucleic acid for the controlled expression of a nucleic acid encoding Cas9 nuclease in an individual.
• the expression of the nucleic acid may be controlled upon consumption of a diet deficient in at least one essential amino acid.
• Genome editing using targetable nucleases is an emerging technology for the precise genome modification of organisms ranging from bacteria to plants and animals, including humans. Its attraction is that it can be used for almost all organisms in which targeted genome modification has not been possible with other kinds of methods.
• ZFNs zinc- finger nucleases
• TALENs transcription-activator like effector nucleases
• meganucleases have enabled the scientific community to generate permanent mutations by introducing double-stranded breaks to activate repair pathways.
• nucleases like ZFN and TALENs
• ZFN and TALENs The capacity of designed nucleases, like ZFN and TALENs, to generate DNA double-stranded breaks at desired positions in the genome has created optimism for therapeutic translation of locus-directed genome engineering.
• these approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.
• CRISPR-Cas9 technology is presently seriously limited by the off-target effect associated with the editing process (i.e. genome editing in unwanted genomic localisation), and by the immunogenicity of the bacterial nuclease Cas9.
• Porteus considers that an "important consideration in determining an appropriate delivery strategy is that genome editing, in contrast to gene-augmentation strategies, is a hit and run approach". Furthermore, Porteus believes that "sustained expression of the nuclease not only is not needed but should be avoided: continued expression of a nuclease increases the probability of deleterious genomic instability and may either compromise the edited cell's fitness or predispose the exposed cell to transformation". Finally, Porteus concludes that "for therapeutic applications that require in vivo editing of cells, the challenge is greater and a solution has not been determined”.
• nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease in an individual comprising:
• regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid, said regulatory polynucleotide being activated in an individual upon consumption of a diet deficient in at least one essential amino acid; and
• nucleic acid encoding a Cas nuclease, which is placed under the control of the said regulatory polynucleotide.
• nucleic acid vector for the controlled expression of a nucleic acid encoding a Cas nuclease, comprising a nucleic acid, as defined herein.
• a still further aspect of the invention relates to a delivery particle comprising a nucleic acid or a nucleic acid vector, as defined herein.
• the invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising (i) a nucleic acid according or a nucleic acid vector or a delivery particle, as defined herein, and (ii) a pharmaceutically acceptable vehicle.
• the invention relates to a host cell comprising the nucleic acid or a nucleic acid vector, as defined herein.
• Another aspect of the invention relates to a pharmaceutical composition, as defined herein, for use as a medicament.
• the invention also relates to a pharmaceutical composition, as defined herein, for use as an active agent for editing the genome into at least one target cell.
• the invention relates to a method for editing the genome into at least one target cell comprising at least the step of administering to an individual in need thereof the pharmaceutical composition, as defined herein.
• the invention relates to a pharmaceutical composition, as defined herein, for use as an active agent for preventing and/or treating a disease.
• An aspect of the invention also relates to a method for preventing and/or treating a disease comprising at least the step of administering to an individual in need thereof the pharmaceutical composition, as defined herein.
• the invention concerns a kit for treating and/or preventing a disease comprising:
• FIGURE 1 Scheme illustrating the GCN2-eIF2a-ATF4 signalling pathway.
• activated GCN2 phosphorylates eIF2a, leading to an up- regulation of the transcription factor ATF4 and its recruitment to AARE sequences to induce target gene expression.
• FIGURE 2 Scheme illustrating the depiction of the AARE-Cas nuclease construct: six copies of the AAREs from Trb3 (black spots) promoter and the Tk minimal promoter compose this construct.
• FIGURE 3 Scheme illustrating the pTrip-2XAARE-NLS-FLAG-CAS9 plasmid.
• pTK indicates the position of the minimal TK promoter;
• 2X AARE indicates the position of the AARE nucleic acids;
• arrow "NLS-FLAG-CAS9” indicates the position of the nucleic acid encoding the Cas9 nuclease;
• arrow "AmpR” stands for the nucleic acid encoding for ampicillin resistance.
• FIGURE 4 Scheme illustrating the pTRIP blast_U6 AAVSl_2xAARE-Cas9- Flag-RFP plasmid.
• the lower panel is in continuity with the upper panel.
• the EcoRl restriction site on the right end of the upper panel refers to the EcoRl restriction site on the left end of the lower panel.
• FIGURE 5 Plots illustrating the Cas9 expression in 293T cells upon induction at TO with either a medium deprived in Leucine (293T-C9 Leu-; plain curve) or a medium comprising tunicamycin (293T-C9 TU; dashed curve).
• Induction is performed at TO and removed at 24 h.
• Expression is monitored 24 h and 48 h after removal of the induction, i.e. at TO+48 h and TO+72 h, respectively.
• the abscissa axis represents the time line (in hours) and the ordinate axis represents the band intensity for Cas9 nuclease, hence is representative of the Cas9 expression.
• the maximum expression of Cas9 is observed after 24 h upon induction, which arbitrarily represents 100% of expression.
• FIGURE 6 Plots illustrating the integration of a donor DNA (Do) at the AAVSl site of the genome of 293T cells.
• 293T cells were transfected with the plasmid 'pTRIP blast_U6 AAVSl_2xAARE-Cas9-flag-RFP' (C9) as well as with the donor plasmid containing a cassette 'AAVSl cut site-GFP-p2a-Puromycin_AAVS 1 cut site' (Do).
• the number of puromycin resistant cells (ordinate axis) are counted upon induction in the presence of tunicamycin (293+Do+C9i Tu), or with a leucine-deprived medium (293+Do+C9i Leu-).
• FIGURE 7 Plots illustrating the integration of a donor DNA (Do) at the AAVSl site of the genome of 293T cells containing one copy of the C9 plasmid (293-C9 cells), similarly as in figure 6.
• 293-C9 cells were transfected with the donor plasmid containing a cassette 'AAVSl cut site-GFP-p2a-Puromycin_AAVS 1 cut site' (Do).
• the number of puromycin resistant cells (ordinate axis) are counted upon induction in the presence of tunicamycin (293_C9+Doi Tu), or with a leucine-deprived medium (293_C9+Doi Leu-).
• the inventors assessed the remarkable features of the nutritional adaptation pathway to a diet deprived of one essential amino acid to achieve a regulatory system ideally suited for gene therapy.
• the inventors found that such a system, based on dietary specific amino acid starvation, does not require the expression of synthetic transcription factors or regulatory proteins nor the administration of pharmacological inducers. It is physiological, non-toxic and is amenable to clinical application.
• This novel nutrition-based regulatory system stands as a physiological approach with the ability to resolve one of the major remaining hurdles in human gene therapy.
• CRISPR Cas nuclease
• WO 2013/068096 disclosed such a controlled expression system for several proteins, and the proof of concept was performed with the expression of the luciferase protein. Chaveroux et al. (Science Signaling, 2015, vol. 8(374), 1-10) took advantage of this system for characterizing the eIF2alpha-ATF4 signalling pathway.
• nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease allows for limiting or avoiding the off-targets, which are usually observed because of a lack of an efficiently controlled expression system (expression "leakage").
• a first aspect of the invention concerns a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease in an individual, comprising:
• regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid, said regulatory polynucleotide being activated in an individual upon consumption of a diet deficient in at least one essential amino acid; and
• the invention also concerns a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease in at least one target cell of an individual, comprising:
• regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid, said regulatory polynucleotide being activated in an individual upon consumption of a diet deficient in at least one essential amino acid; and
• nucleic acid encoding a Cas nuclease, which is placed under the control of the said regulatory polynucleotide.
• controlled expression expression is intended to mean that the expression is induced or turned “on” and shut down or turned “off in a precise manner, with respect to the moment of induction, the duration of induction.
• the Cas nuclease is selected in a group comprising a class I Cas nuclease, a class II Cas nuclease and a class III Cas nuclease.
• a class I Cas nuclease is selected in a group comprising
• a class II Cas nuclease is selected in a group comprising Cas9, Cpfl, Csn2 and Cas4.
• a class III Cas nuclease is selected in a group comprising Cas 10, Csm2 and Cmr5.
• the Cas nuclease is Cas9 nuclease.
• the Cas9 nuclease may originate from a bacterial source, in particular a bacterium selected in a group comprising Acaryochloris marina, Actinomyces naeslundii, Alcanivorax dieselolei, Belliella baltica, Campylobacter jejuni, Corynebacterium diphtheriae, Coriobacterium glomerans, Corynebacterium ulcerans, Desulfomonile tiedjei, Dickeya dadantii, Escherichia coli, Francisella tularensis, Lactobacillus kefiranofaciens, Listeria innocua, Methylobacterium extorquens, Micrococcus luteus, Myxococcus fulvus, Neisseria meningitidis, Pasteurella multocida, Prevotella intermedia, Prochlor
• the Cas9 nuclease may originate from an archaebacterial source, such as e.g. Methanoculleus strengensis.
• Cas9 nuclease disclosed herein encompasses homo logs, paralogs and orthologs and variants of naturally occurring Cas9 nucleases.
• the Cas9 variants may include SpCas9-HFl (Kleinstiver et al; Nature. 2016 Jan 28;529(7587):490-5), fCas9, which is a fusion of catalytically inactive Cas9 to Fokl nuclease (Guilinger et al; Nat. Biotechnol. 2014: 32(6): 577-582), and any rationally engineered Cas9 nucleases with improved specificity as disclosed by Slaymaker et al. (Science. 2016 Jan 1;351(6268):84-8).
• the nucleic acid encoded a Cas9 nuclease and/or vectors encoding a Cas9 nuclease may be commercially available, e.g. from SIGMA-ALDRICH®.
• Cas nucleases may be identified by the means of methods for the directed evolution of proteins Packer and Liu (Nat Rev Genet. 2015 Jul;16(7):379-94).
• the Cas nuclease is a DNA or RNA guided Cas nuclease.
• DNA or RNA guided is intended to mean that in the presence of a guide DNA or RNA, the Cas nuclease is targeted to a nucleic acid, which sequence is complementary with the guide DNA and RNA.
• the expression of a nucleic acid encoding a Cas nuclease may be measured by any suitable method available in the state of the art, including the measure of the mRNA expression, resulting from the transcription of the nucleic acid encoding a Cas nuclease, and/or the measure of the Cas nuclease expression.
• the measure of the Cas nuclease expression may be performed by measuring the expression of the Cas nuclease with anti- antibodies that specifically bind to said Cas nuclease.
• an induced expression may be expressed as a time fold expression as compared to the basal, non- induced expression.
• the induced expression may vary from 2 fold to 10,000 fold, preferably from 4 fold to 500 fold, more preferably from 8 fold to 250 fold, most preferably from 10 fold to 100 fold, as compared to the basal expression.
• from 2 fold to 10,000 fold includes 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 75 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1,000 fold, 2,000 fold, 3,000 fold, 4,000 fold, 5,000 fold, 6,000 fold, 7,000 fold, 8,000 fold and 9,000 fold.
• minimal promoter is intended to mean a promoter including all the required elements to properly initiate transcription of a gene of interest positioned downstream.
• minimal promoter and “core promoter” are considered as equivalent expressions.
• the "minimal promoter” includes at least a transcription start site, a binding site for a R A polymerase and a binding site for general transcription factors (TATA box).
• Suitable minimal promoters are known for a skilled artisan.
• a minimal promoter suitable for carrying out the invention may be selected in a group comprising the promoter of the thymidine kinase, the promoter of the ⁇ -globin, the promoter for cytomegalovirus (CMV), the SV40 promoter and the like.
• the individual is a human or a non-human mammal, preferably a human.
• the non-human mammal is selected in a group comprising a pet such as a dog, a cat, a domesticated pig, a rabbit, a ferret, a hamster, a mouse, a rat and the like; a primate such as a chimp, a monkey, and the like; an economically important animal such as cattle, a pig, a rabbit, a horse, a sheep, a goat, a mouse, a rat.
• a pet such as a dog, a cat, a domesticated pig, a rabbit, a ferret, a hamster, a mouse, a rat and the like
• a primate such as a chimp, a monkey, and the like
• an economically important animal such as cattle, a pig, a rabbit, a horse, a sheep, a goat, a mouse, a rat.
• target cell is intended to refer to a cell from the said individual, for which an expression of a Cas nuclease would be beneficial.
• essential amino acid includes histidine (His, H), isoleucine (He, I), leucine (Leu, L), Lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophane (Trp, W) and valine (Val, V).
• At least one essential amino acid is intended to mean 1, 2, 3, 4, 5, 6, 7, 8 or 9 essential amino acid(s).
• a diet deficient in at least one essential amino acid may be administered to an individual for a time period of 5 min to 12 h, which includes 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, 1 h, 1 h 30 min, 2 h, 2 h 30 min, 3 h, 3 h 30 min, 4 h, 4 h 30 min, 5 h, 5 h 30 min, 6 h, 6 h 30 min, 7 h, 7 h 30 min, 8 h, 8 h 30 min, 9 h, 9 h 30 min, 10 h, 10 h 30 min, 11 h, 11 h 30 min.
• a diet deficient in at least one essential amino acid may be administered to an individual once, twice, three times, four times, five times, six times a day, or more.
• the diet deficient in at least one essential amino acid may be administered to an individual once or twice a day.
• the diet deficient in at least one essential amino acid may be administered to an individual early in the morning, e.g. for breakfast, and then the individual may be administered a normal diet for lunch and dinner.
• normal diet is intended to mean a diet that is not deficient in any of the essential amino acids.
• a diet deficient in at least one essential amino acid may be administered to an individual every day, every other day, once a week, twice a week, three times per week.
• a diet deficient in at least one essential amino acid may be administered to an individual for a period of half a day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days 20 days, or more.
• a diet deficient in at least one essential amino acid may be repeated every week, every other week, every month, every month, or more.
• the diet deficient in at least one essential amino acid may be provided by an isoleucine-free, leucine-free and valine-free powdered food product commercially available from NUTRICA METABOLICS®, under the name MILUPA®.
• This diet is adapted to individual having Maple syrup urine disease, which disease appears to affect the branched chain amino acid metabolism.
• a leucine-free, isoleucine-free or valine-free diet may be obtained by mixing the isoleucine-free, leucine-free and valine-free powder with an external source for the 2 remaining amino acids.
• a phenylalanine-free diet may be provided a phenylalanine-free powder, commercially available from MEAD JOHNSON®. This diet is adapted to individual having phenylketonuria.
• the powder is mixed with an adapted a liquid or a semi-solid food that is free of the desired essential amino acid.
• an amino acids starvation can be mimicked by the administration of Halofuginone, or under any other name corresponding to the molecule "4(3H)-Quinazolinone, 7-bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl]-, trans-( ⁇ )-, or commercialized as for example, Halocur, Stenorol, Flavomycin, Lincomix, Stafac.
• amino acid response element (AARE) nucleic acid is selected in a group comprising a nucleic acid of sequence SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4 and SEQ ID No: 5.
• At least one AARE nucleic acid includes at least 2, at least 3, at least 4 and at least 5 AARE nucleic acids.
• the expression “at least one AARE nucleic acid” thus includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 AARE nucleic acids.
• the regulatory polynucleotide comprises at least two amino acids
• the regulatory polynucleotide comprises from one to twenty AARE nucleic acids, preferably from two to ten AARE nucleic acids.
• the regulatory polynucleotide comprises from two to six AARE nucleic acids.
• the regulatory polynucleotide comprises two AARE nucleic acids selected in the group comprising a nucleic acid of sequence SEQ ID NO: 2 and SEQ ID NO: 4.
• the regulatory polynucleotide comprises six AARE nucleic acids of sequence SEQ ID NO: 1.
• the two AARE nucleic acids, or alternatively, the at least two AARE nucleic acids may be identical or distinct.
• the regulatory polynucleotide comprised in the nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease may also be activated upon administration to an individual of halofuginone, tunicamycin, and the like, i.e. compounds which are known to have activating properties of the AARE nucleic acids.
• the invention also concerns a nucleic acid vector for the controlled expression of a nucleic acid encoding a Cas nuclease, comprising a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, as defined herein.
• the nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease according to the invention is incorporated in a vector that is suitable for gene therapy.
• the expression "vector that is suitable for gene therapy” is intended to mean that the vector comprises the essential elements for achieving the expression of the nucleic acid encoding a Cas nuclease in a target cell.
• the vector is a viral vector.
• a viral vector is selected in a group comprising an adenoviruse, an adeno-associated virus (AAV), an alphavirus, a herpesvirus, a lentivirus, a non-integrative lentivirus, a retrovirus, vaccinia virus and a bacculovirus.
• AAV adeno-associated virus
• the nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease or the nucleic acid vector, as defined herein may be comprised in a particle, in particular, in association other compounds, such as e.g. with lipids, protein, peptides, or polymers.
• said particle, or “delivery particle” is intended to provide, or “deliver”, the target cells with the nucleic acid encoding a Cas nuclease or the nucleic acid vector comprising the said nucleic acid encoding a Cas nuclease.
• the invention further concerns a delivery particle comprising a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease or a nucleic acid vector, as defined herein.
• the delivery particle may be in the form of a lipoplexe, comprising cationic lipids; a lipid nano-emulsion; a solid lipid nanoparticle; a peptide based particle; a polymer based particle, in particular comprising natural and/or synthetic polymers.
• a polymer based particle may comprise a protein; a peptide; a polysaccharide, in particular chitosan.
• a polymer based particle may comprise a synthetic polymer, in particular, a polyethylene imine (PEI), a dendrimer, a poly (DL- Lactide) (PLA), a poly(DL-Lactide-co-glycoside) (PLGA), a polymethacrylate and a polyphosphoesters.
• PEI polyethylene imine
• PLA poly (DL- Lactide)
• PLA poly(DL-Lactide-co-glycoside)
• PLGA polymethacrylate
• polyphosphoesters a polyphosphoesters
• the delivery particle further comprises at its surface one or more ligands suitable for binding to a target receptor exposed at the membrane of a targeted cell.
• Another aspect of the present invention concerns a pharmaceutical composition
• a pharmaceutical composition comprising (i) a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or a nucleic acid vector or a delivery particle, as defined herein, and (ii) a pharmaceutically acceptable vehicle.
• a pharmaceutical composition comprising (i) a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or a nucleic acid vector or a delivery particle, as defined herein, and (ii) a pharmaceutically acceptable vehicle.
• the formulation of pharmaceutical compositions according to the instant invention is well known to persons skilled in the art.
• a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or a nucleic acid vector or a delivery particle, as defined in the present disclosure may represent the active agent.
• the pharmaceutical composition may comprise a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or a nucleic acid vector or a delivery particle, as defined in the present disclosure, as the only active agent.
• a suitable pharmaceutically acceptable vehicle according to the invention includes any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
• suitable pharmaceutically acceptable vehicles may include, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and a mixture thereof.
• pharmaceutically acceptable vehicles may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the cells.
• auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the cells.
• the pharmaceutical composition may be administered to an individual in need thereof by any route, i.e. by an oral administration, a topical administration or a parenteral administration, e.g., by injection, including a sub-cutaneous administration, a venous administration, an arterial administration, in intra-muscular administration, an intra-ocular administration and an intra-auricular administration.
• a route i.e. by an oral administration, a topical administration or a parenteral administration, e.g., by injection, including a sub-cutaneous administration, a venous administration, an arterial administration, in intra-muscular administration, an intra-ocular administration and an intra-auricular administration.
• the administration of the pharmaceutical composition by injection may be directly performed in the target tissue of interest, in particular in order to avoid spreading of the nucleic acid or the nucleic acid vector comprised in the said pharmaceutical composition.
• the inventors consider that this is particularly important when the brain tissue is target.
• Nucleic acid vector infusions can be conducted with great precision in specific parts of the brain tissue, e.g. by the mean of taking advantage of a magnetic resonance scanner, in particular using frameless stereotactic aiming devices.
• the use of MRI- guidance and new stereotactic aiming devices have now established a strong foundation for neurological gene therapy to become an accepted procedure in interventional neurology.
• an oral formulation according to the invention includes usual excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
• an effective amount of said compound is administered to said individual in need thereof.
• an "effective amount” refers to the amount of said compound that alone stimulates the desired outcome, i.e. alleviates or eradicates the symptoms of the encompassed disease, in particular a genetic disorder.
• nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or a nucleic acid vector or a delivery particle in order to observe the desired outcome.
• the effective amount of the compound to be administered may be determined by a physician or an authorized person skilled in the art and can be suitably adapted within the time course of the treatment.
• the effective amount to be administered may depend upon a variety of parameters, including the material selected for administration, whether the administration is in single or multiple doses, and the individual's parameters including age, physical conditions, size, weight, gender, and the severity of the disease to be treated.
• an effective amount of the active agent may comprise from about 0.001 mg to about 3000 mg, per dosage unit, preferably from about 0.05 mg to about 100 mg, per dosage unit.
• from about 0.001 mg to about 3000 mg includes, from about 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg,
• the active agent may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day.
• an effective amount of the active agent may comprise from about lxlO 5 to about lxlO 15 copies of the nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or the nucleic acid vector or the delivery particle, as defined in the present disclosure, per dosage unit.
• from about lxlO 5 to about lxlO 15 copies includes 2xl0 5 , 3xl0 5 , 4xl0 5 , 5xl0 5 , 6xl0 5 , 7xl0 5 , 8xl0 5 , 9xl0 5 , lxlO 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 , 5xl0 6 , 6xl0 6 , 7xl0 6 , 8xl0 6 , 9xl0 6 , lxlO 7 , 2xl0 7 , 3xl0 7 , 4xl0 7 , 5xl0 7 , 6xl0 7 , 7xl0 7 , 8xl0 7 , 9xl0 7 , lxlO 8 , 2xl0 8 , 3xl0 8 , 4xl0 8 , 5xl0 8 , 6xl0 8 , 6xl0 8 , 7x
• the invention concerns a host cell comprising the nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease or a nucleic acid vector, as defined herein.
• the target cell and/or the host cell may be selected among a prokaryotic cell or an eukaryotic cell.
• a "prokaryotic cell” encompasses a bacterial cell and an archaebacterial cell.
• the target cell and/or the host cell is a eukaryotic cell.
• a "eukaryotic cell” encompasses a yeast, an algae cell, a plant cell, an animal cell, preferably a mammal cell and more preferably a human cell.
• the eukaryotic cell is a mammal cell, preferably a human cell.
• a target cell and/or a host cell may encompass, without limitation, a cell of the central nervous system, an epithelial cell, a muscular cell, an embryonic cell, a germ cell, a stem cell, a progenitor cell, a hematopoietic stem cell, a hematopoietic progenitor cell, an induced Pluripotent Stem Cell (iPSC).
• a cell of the central nervous system an epithelial cell, a muscular cell, an embryonic cell, a germ cell, a stem cell, a progenitor cell, a hematopoietic stem cell, a hematopoietic progenitor cell, an induced Pluripotent Stem Cell (iPSC).
• iPSC induced Pluripotent Stem Cell
• the target cell and/or the host cell is not a stem cell, a progenitor cell, a germinal cell or an embryonic cell.
• the target cell and/or the host cell may belong to a tissue selected in a group comprising a muscle tissue, a nervous tissue, a connective tissue, and an epithelial tissue.
• the target cell and/or the host cell may belong to an organ selected in a group comprising a bladder, a bone, a brain, a breast, a central nervous system, a cervix, a colon, an endometrium, a kidney, a larynx, a liver, a lung, an oesophagus, an ovarian, a pancreas, a pleura, a prostate, a rectum, a retina, a salivary gland, a skin, a small intestine, a soft tissue, a stomach, a testis, a thyroid, an uterus, a vagina. • Uses
• Another aspect of the invention concerns a pharmaceutical composition
• a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or a nucleic acid vector, or a delivery particle, as defined herein, and a pharmaceutically acceptable vehicle, for use as a medicament.
• the invention also relates to the use of a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, as defined herein, for the preparation or the manufacture of a medicament.
• the invention concerns a pharmaceutical composition
• a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or a nucleic acid vector, or a delivery particle, as defined herein, and a pharmaceutically acceptable vehicle, for use as an active agent for editing the genome into at least one target cell.
• Another aspect of the invention further relates to the use of a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, as defined herein, as an active agent for editing the genome into at least one target cell.
• the edition of the genome may be performed in vivo, in vitro or ex vivo.
• the edition of the genome may be performed as in Komor et al. Nature; 2016 Apr 20;533(7603):420-4.
• the target cell has at least a genetic mutation.
• the genetic mutation is present in a gene selected in a group comprising MTTP; CNGB3; SLC39A4; TRMU; ACOX1; ADA; ABCD1 ; SAMHD1; MAN2B1; HBA; ATRX; COL4A3; COL4A4; COL4A5; ALMS1; SLC12A6; ASL; CYP19A1; SLC35A3; ASNS; AGA; TTPA; ATM; SACS; BBS10; BBSl ; BBS2, BBS 12; CIITA; BSND; GP1BA; HSD3B2; ACAT1; GPR56; BTD; BLM; ASPA; CPS1; CPT1A; CPT2; RAB23; RJVIRP; SLC6A8; GAMT; CYP27A1; NDRG1; PRPS1; GJB1 ; VPS 13 A; CH
• G6PC SLC37A4; GAA; AGL; GBE1; PYGM; PFKM; BCS1L; HFE2; TFR2; ALDOB;
• TECPR2 HPS1; HPS3; HMGCL; HLCS; CBS; MTHFR; MTRR; HYLS1; SLC25A15;
• EDA EDA; ALPL; GNE; MED 17; IVD; TMEM216; RGPRIP1L; LAM A3; LAMB3; LAMC2; GALC; TGM1; CEP290; RDH12; RPE65; LCA5; CRB1; LRPPRC; GLE1; EIF2B5;
• CAPN3 DYSF; SGCG; SGCA; SGCB; FKRP; DLD; STAR; LPL; HADHA; SLC7A7;
• GRHPR HOGA1; SEPSECS; ABCB11; PCCA; PCCB; CTSK; PDHA1; PDHB; PTS; ATP6V1B1; EYS; CERKL; FAM161A; DHDDS; PEX7; AGPS; ESC02; SLC17A5;
• ACADVL FKTN; ATP7B; LIP A; RSI; IL2RG; PEX1 ; PEX2; PEX6 and PEX10.
• the present invention concerns a pharmaceutical composition
• a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or a nucleic acid vector, or a delivery particle, as defined herein, and a pharmaceutically acceptable vehicle, for use as an active agent for treating and/or preventing a disease.
• the disease is selected in a group comprising a genetic disorder, an infectious disease and a cancer.
• the disease is a genetic disorder.
• the genetic disorder is selected in a non-limiting group comprising Abetalipoproteinemia; Achromatopsia; Acrodermatitis Enteropathica; Acute Infantile Liver Failure; Acyl-CoA Oxidase I Deficiency; Adenosine Deaminase Deficiency; Adreno leukodystrophy, X-Linked; Aicardi-Goutieres Syndrome; Alpha- Mannosidosis; Alpha-Thalassemia; Alpha-Thalassemia Mental Retardation Syndrome; Alport Syndrome; Alstrom Syndrome; Andermann Syndrome; Argininosuccinic Aciduria; Aromatase Deficiency; Arthrogryposis, Mental Retardation, and Seizures; Asparagine Synthetase Deficiency; Aspartylglycosaminuria; Ataxia With Isolated Vitamin E Deficiency; Ataxia-Telangiectasia; Autosomal Recessive Spastic Ataxia of
• the disease is an infectious disease.
• the infectious disease selected in a non-limiting group comprising Anaplasmosis; Anthrax; Babesiosis; Botulism; Brucellosis; Burkholderia mallei infection (glanders); Burkholderia pseudomallei infection (melioidosis); Campylobacteriosis; Carbapenem-resistant Enterobacteriaceae infection (CRE); Chancroid; Chikungunya infection; Chlamydia infection; Ciguatera; Clostridium difficile infection; Clostridium perfringens infection (Epsilon Toxin); Coccidioidomycosis fungal infection (Valley fever); Creutzfeldt- Jacob Disease, transmissible spongioform (CJD); Cryptosporidiosis; Cyclosporiasis; Dengue Fever; Diphtheria; E.
• Anaplasmosis Anaplasmosis
• Anthrax Bacesiosis
• Botulism Brucellosis
• Coli infection Eastern Equine Encephalitis (EEE); Ebola Hemorrhagic Fever (Ebola); Ehrlichiosis; Arboviral or parainfectious encephalitis; Non-polio enterovirus infection; D68 enterovirus infection, (EV-D68); Giardiasis; Gonococcal infection (Gonorrhea); Granuloma inguinale; Type B Haemophilus Influenza disease, (Hib or H-flu); Hantavirus pulmonary syndrome (HPS); Hemolytic uremic syndrome (HUS); Hepatitis A (Hep A); Hepatitis B (Hep B); Hepatitis C (Hep C); Hepatitis D (Hep D); Hepatitis E (Hep E); Herpes; Herpes zoster, zoster VZV (Shingles); Histoplasmosis; Human Immunodeficiency Virus/ AIDS (HIV/ AIDS); Human Papillomarivus (
• the disease is a cancer.
• the cancer is selected in a non-limiting group comprising a bladder cancer, a bone cancer, a brain cancer, a breast cancer, a cancer of the central nervous system, a cancer of the cervix, a cancer of the upper aero digestive tract, a colorectal cancer, an endometrial cancer, a germ cell cancer, a glioblastoma, a Hodgkin lymphoma, a kidney cancer, a laryngeal cancer, a leukaemia, a liver cancer, a lung cancer, a myeloma, a nephroblastoma (Wilms tumor), a neuroblastoma, a non-Hodgkin lymphoma, an oesophageal cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a pleural cancer, a prostate cancer, a retinoblastoma, a skin cancer (including a melanoma),
• ex vivo manipulations and/or therapy may be encompassed within the scope of the instant invention, which would include stem cells and progenitor cells, hematopoietic stem and progenitor cells, induced Pluripotent Stem Cell (iPSC), and adult cells from different species.
• iPSC induced Pluripotent Stem Cell
• nucleic acids and the nucleic acid vectors encompassed by the instant invention may be employed to engineer animal or plant models, e.g. animal models for preclinical studies, bearing in mind the fundamental ethical principles.
• Another aspect of the present invention concerns a method for editing the genome into at least one target cell comprising at least the step of administering to an individual in need thereof the pharmaceutical composition, as defined herein.
• the invention concerns a method for preventing and/or treating a disease comprising at least the step of administering to an individual in need thereof the pharmaceutical composition, as defined herein.
• the methods above further comprise a step of providing the individual with a diet deficient in at least one essential amino acid, in particular an amino acid selected in a group comprising histidine (His, H), isoleucine (He, I), leucine (Leu, L), Lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophane (Trp, W) and valine (Val, V).
• the methods above alternatively comprise a step of administering a compound known to activate the AARE nucleic acid comprised in the regulatory polynucleotide, in particular a compound selected in a group comprising halofuginone, tunicamycin, and the like.
• the disease is selected in a group comprising a genetic disorder, an infectious disease and a cancer.
• the pharmaceutical composition may be administered to an individual in need thereof by any route, i.e. by an oral administration, a topical administration or a parenteral administration, e.g., by injection, including a sub-cutaneous administration, a venous administration, an arterial administration, in intra-muscular administration, an intra-ocular administration, and an intra-auricular administration.
• routes i.e. by an oral administration, a topical administration or a parenteral administration, e.g., by injection, including a sub-cutaneous administration, a venous administration, an arterial administration, in intra-muscular administration, an intra-ocular administration, and an intra-auricular administration.
• Other modes of administration employ pulmonary formulations, suppositories, and transdermal applications.
• an oral formulation according to the invention includes usual excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
• an effective amount of said compound is administered to said individual in need thereof.
• an "effective amount” refers to the amount of said compound that alone stimulates the desired outcome, i.e. alleviates or eradicates the symptoms of the encompassed disease, in particular a genetic disorder.
• nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, or a nucleic acid vector or a delivery particle in order to observe the desired outcome, comprised in a pharmaceutical composition, as defined herein.
• the effective amount of the compound to be administered may be determined by a physician or an authorized person skilled in the art and can be suitably adapted within the time course of the treatment.
• the effective amount to be administered may depend upon a variety of parameters, including the material selected for administration, whether the administration is in single or multiple doses, and the individual's parameters including age, physical conditions, size, weight, gender, and the severity of the disorder to be treated.
• Another aspect of the invention also relates to a method for editing the genome into at least one target cell comprising the steps of:
• nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease, as disclosed herein;
• a donor nucleic acid comprising a nucleic acid intended to replace the target genomic nucleic acid
• the Cas nuclease Upon induction of the Cas nuclease, the Cas nuclease will promote single strand or double strand break(s) in the genomic target nucleic acid, with the assistance of the guide DNA or RNA, and on the donor nucleic acid. Subsequently, the nucleic acid from the donor nucleic acid may be integrated in the genome in the place of the genomic target nucleic acid.
• the induction of the expression of the Cas nuclease may be performed by providing to the target cell a medium that is deficient in at least one essential amino acid or a medium that comprises halofuginone and/or tunicamycin.
• the target nucleic acid has a genetic mutation.
• the invention concerns a kit for treating and/or preventing a disease comprising:
• the disease is selected in a group comprising a genetic disorder, an infectious disease and a cancer.
• the expression "pharmaceutically active compound” is intended to mean a compound having a benefit towards the prevention and/or the treatment of a given disease.
• the pharmaceutically active compound is an antimicrobial compound, which may be suitably selected by a skilled in the art from the compounds commonly employed to combat an infectious disease, in particular, a bacterial, a fungal or a viral infection.
• the antimicrobial compound is an antibiotic selected in a group comprising a penicillin, in particular penicillin and amoxicillin; a carbapenem, in particular imipenem; a cephalosporin, in particular cephalexin; an aminoglycoside, in particular gentamicin and tobramycin; a tetracycline, in particular tetracycline and doxycycline; a macrolide, in particular erythromycin and clarithromycin; a quinolone, in particular ciprofloxacin and levofloxacin; and a sulphonamide, in particular sulfamethizole and sulfamethoxazole.
• a penicillin in particular penicillin and amoxicillin
• a carbapenem in particular imipenem
• a cephalosporin in particular cephalexin
• an aminoglycoside in particular gentamicin and tobramycin
• a tetracycline in particular tetra
• the antimicrobial compound is an antiviral agent selected in a non-limiting group comprising a neuraminidase inhibitor; a nucleoside analogue of guanine; a nucleoside analogue of thymidine; a nucleotide reverse transcriptase inhibitor; and a protease inhibitor.
• the pharmaceutically active compound is an anti-cancer compound, which may be suitably selected by a skilled in the art from the compounds commonly employed in chemotherapy.
• the anti-cancer compound may be selected in a group comprising an alkylating agent, a purine analogue, a pyrimidine analogue, an anthracycline, bleomycin, mytomycin, an inhibitor of topo-isomerase 1, an inhibitor of topo-isomerase 2, a taxan, a monoclonal antibody, a cytokine, an inhibitor of a protein kinase, and the like.
• Figure 1 illustrates the GCN2-eIF2a-ATF4 signaling pathway.
• activated GCN2 phosphorylates eIF2a, leading to an up-regulation of the transcription factor ATF4 and its recruitment to AARE sequences to induce target gene expression.
• Figure 2 illustrates the overall strategy for constructing a nucleic acid encoding a Cas nuclease under the regulation of Tk minimal promoter and six copies of the AARE nucleic acid from Trb3 (black spots).
• a cellular model derived from HEK 293T cells bearing a single copy of GFP transgene is used (293T GFP cell line).
• This cell line is co-transduced with 2 different lentiviral vectors.
• the first one expresses a FLAG tagged version of CAS9 (Shen et al Cell Res. 2013 Apr 2. doi: 10.1038/cr.2013.46; SEQ ID NO: 8) placed under the control of the 2X AARE-TK regulation promoter (SEQ ID NO: 6 and SEQ ID NO: 7).
• the second vector expresses a guide RNA specifically targeting the GFP reporter gene (gRNA GFP ) under the control of the U6 promoter a RNA polymerase III promoter (Ma, H et al. Mol Ther Nucleic Acids 2014 doi: 10.1038/mtna.2014.12).
• Both lentiviral vectors have been constructed in the pTRIP lentiviral backbone (Zennou et al, 2000; Cell 101, 173-185).
• nucleic acid of the pTrip-2XAARE-NLS-FLAG-CAS9 plasmid (SEQ ID NO: 9) is represented in Figure 3.
• the transduced cells are amplified in culture. In absence of induction the transduced cells are amplified in culture. In absence of induction the transduced cells are amplified in culture. In absence of induction the transduced cells are amplified in culture. In absence of induction the transduced cells are amplified in culture. In absence of induction the transduced cells are amplified in culture. In absence of induction the transduced cells are amplified in culture. In absence of induction the
• 2XAARE-CAS9 cells and the EFla-CAS9 cells are lysed and the expression of CAS9 is monitored by quantitative RT PCR and the amount of CAS9 protein expression followed by Western blot detection of the FLAG tag. Under such condition only the ubiquitously expressed CA9 is detected and quantified.
• the transduced cells are placed in culture in a specific medium depleted in either Leu or Thr.
• the induction of CAS9 expression in the 2XAARE-CAS9 cells is monitored with time both at the level of mRNA and protein. The optimal treatment period is thus determined.
• the promoter 2xAARE contains 6 binding sequences for the transcription factor ATF4, which is rapidly induced in conditions of essential amino acids (EAA) starvation, or other cellular stress such as the stress induced to the endoplasmic reticulum by Tunicamycin (Tu).
• EAA essential amino acids
• Tunicamycin Tunicamycin
• the Cas9 gene of Streptococcus pyogenes (spCas9) fused to a flag tag, an autocatalytic P2A peptide and the red fluorescent protein (RFP) was cloned under the control of the 2xAARE enhancer containing 4 binding sites for ATF4 and the minimal promoter of thymidine kinase gene (TKm) derived from the Herpes simplex virus (HSV; Figure 4).
• This HIV-derived lentiviral vector allows stable expression of the resistance gene Blasticidin for selection of integration events of the vector.
• a second cassette contains the U6 promoter and the guide RNA AAVS1 (SEQ ID NO: 10) and the CRISPR- associated RNA scaffold, allowing cut below the ATG of the human gene PPP1R12C.
• a third cassette of expression contains the gene spCas9-flag-RFP under control of the promoter 2xAARE-TKm.
• This plasmid was used to produce lentiviral particles according to standard protocol of co-transfection of the vector plasmid, + a plasmid encoding the VSV envelop (pVSV), + a plasmid encoding the HIV Rev gene (pRev), + a plasmid encoding the Gag and Pol genes of HIV (p8.9), in 293T cells. After 48 h, cells supernatants were harvested, ultra-centrifuged for concentration and stored at -80°C until use. Vector stocks were tittered with real time quantitative PCR to measure viral RNA copies of genomes/ml (Saeed et al; Mol Ther Nucleic Acids. 2014 Dec 2;3:e213).
• 293T cells were transduced with 100 vRNAc per cell and then selected with blasticidin at 2 ⁇ g/ml (Sigma-Aldrich®). Non-transduced 293T cells all died, while transduced cells grew normally indicating that they all contained at least one copy of the vector pTRIP blast_U6 AAVS 1 2xAARE-Cas9-fiag-RFP.
• Proteins concentration were measured using Bradford test and 30 ⁇ g of lysate mixed with loading buffer and beta-mercaptoethanol and heated at 95°C for 5 minutes, was loaded on a denaturing SDS 10% polyacrylamide gel. Proteins were separated with electrophoresis. Migration was monitored with colored ladder.
• Proteins on the gel were transferred to a nitrocellulose membrane through semi-dry transfer and membrane was used for immunoblot using an anti-FLAG M2 MAB (Sigma-Aldrich®), then detected with a secondary anti-mouse antibody coupled to HRP. Peroxydase activity was revealed with Chemiluminescent HRP substrate (Luminata crescendo -Millipore®) and pictured with a chemiluminence detector Fusion FX7 (Vilber®).
• the density of the detected bands of SPCas9-Flag-RFP of 193 kDa was quantified with the software of the Fusion FX7 detector (Vilber®).
• the level of beta-actin in each sample was also measured in the same way but using an anti-beta actin primary antibody.
• the density of the bands corresponding to Cas9-flag-RFP was normalized with the density of beta-actin.
• Cas9-induced double strand breaks are repaired by the cell machinery and produces insertions and deletions (indels) at the site of cutting, thus re-hybridization of PCR bands obtained from a population of cells containing a mixture of different indels produces DNA fragments containing mismatches. These are cut by the T7 nuclease releasing smaller bands of DNA.
• the system for the controlled expression of Cas9, on a mode ON/OFF, as disclosed herein provides a tool for safely editing the genome. Indeed, in the absence of induction, the absence of any detectable leakage of the expression system provides a safety feature for preventing any unwanted genome editing.
• the rapid expression may be shut down as soon as the removal of induction is effective.
• 293T cells were transfected (Ca 2+ phosphate method) with the plasmid 'pTRIP blast_U6 AAVSl_2xAARE-Cas9-fiag-RFP' as well as with the donor plasmid containing a cassette 'AAVSl cut site-GFP-p2a-Puromycin_AAVS 1 cut site', accepting cuts with Cas9 + gRNA AAVSl on 5' and 3' of the GFP-p2a- Puromycin gene.
• the chosen AAVSl site targeted by the guide R A in the genome of 293T cells includes the ATG start codon of the gene PPP1R12C. When targeted, it will allow insertion of the released GFP-p2a-Puromycin cassette in the place of the exon 1 of PPP1R12C and subsequently expression by the PPP1R12C promoter. In this case, recombinant cells express GFP and become resistant to puromycin allowing their selection and to count the clones corresponding to integration events.
• the donor construct gives resistant clones to puromycine only when both plasmids 'pTRIP blast_U6 AAVSl_2xAARE-Cas9-flag-RFP' (C9) and donor 'pAAVSl cut site-GFP-p2a-Puromycin_AAVS 1 cut site' (Do) are provided together with 2xAARE induction (i) either with a Leucine deprived medium (i Leu-) or a Tunicamycin- containing medium (i Tu), but not with complete medium (ni). This indicates that Cas9 activity for targeted integration requires induction of the promoter 2xAARE.
• this system may be turned on and turned off very precisely, this system offers a safe tool for providing genome editing and hence gene therapy in individuals in need thereof.
• AARE sequence from the TRIB3 gene SEP ID NO: 1
• AARE sequence from the CHOP gene SEP ID NO: 2
• AARE sequence from the ATF3 gene SEP ID NP: 4 agcgttgcatcacccc
• AARE sequence from the SNAT2 gene SEP ID NO: 5
• Thymidine kinase minimal promoter nucleic acid SEP ID NO: 6

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