US20190134224A1 - Diet controlled expression of a nucleic acid encoding a pro-apoptotic protein - Google Patents

Diet controlled expression of a nucleic acid encoding a pro-apoptotic protein Download PDF

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US20190134224A1
US20190134224A1 US16/306,832 US201716306832A US2019134224A1 US 20190134224 A1 US20190134224 A1 US 20190134224A1 US 201716306832 A US201716306832 A US 201716306832A US 2019134224 A1 US2019134224 A1 US 2019134224A1
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nucleic acid
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Pierre FAFOURNOUX
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Centre National de la Recherche Scientifique CNRS
Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
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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
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/25—Animals on a special diet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/30—Animal model comprising expression system for selective cell killing, e.g. toxins, enzyme dependent prodrug therapy using ganciclovir
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/30—Vector systems having a special element relevant for transcription being an enhancer not forming part of the promoter region

Definitions

  • the present invention relates to a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein 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.
  • Adoptive T cell therapy initially involves the isolation and ex vivo expansion of tumor specific T cells to achieve greater number of T cells than what could be obtained by vaccination alone.
  • the tumor specific T cells are then infused into patients with cancer in an attempt to give their immune system the ability to overwhelm remaining tumor via T cells recognition of tumor specific antigens, and cancer cells destruction.
  • such a therapy involves manipulating patients' own immune cells to recognize and attack their tumors.
  • TIL tumor infiltrating lymphocytes
  • CAR chimeric antigen receptor
  • CAR technology is administered through the custom-made manufacturing of therapeutic products from each patient's own T-cells.
  • this patient-specific autologous paradigm is a significant limiting factor in the large-scale deployment of CAR technology.
  • Platforms are presently being developed for the production of “off-the-shelf” CAR T-cells from unrelated third-party donor T-cells in the context of allo-transplantations.
  • T cells targeting differentiation antigens
  • T cells targeting differentiation antigens
  • melanocyte differentiation antigens such as MART-1 and gp100
  • vitiligo and uveitis often developed vitiligo and uveitis (Teulings et al., 2014; Pigment Cell Melanoma Res. 2014 November;27(6):1086-96.)
  • T-cell therapy One of the attractions of T-cell therapy is the potential for the transferred cells to persist and expand, thus mediating sustained therapeutic effects. However, any adverse effects will also be similarly sustained, can worsen as the cells proliferate, and be associated with cell proliferation, (Tey, Clin Transl Immunology, 2014 Jun. 20;3(6):e17).
  • the adoptive cell transfer technology needs to incorporate a “suicide” safety component, particularly at the level of the CAR T-cells.
  • a “suicide” safety component particularly at the level of the CAR T-cells.
  • ESC cells have been largely demonstrated in the context of diseases of the hematopoietic system.
  • iPSC induced pluripotent stem cell
  • iPSC-based therapies offer a promising path for patient-specific regenerative medicine.
  • the patient had a series of 18 anti-vascular endothelial growth factor (VEGF) ocular injections for both eyes to cope with the constant recurrence of the disease.
  • VEGF anti-vascular endothelial growth factor
  • the patient experienced no recurrence of neovascularization at the 6 month point and was free from frequent anti-VEGF injections.
  • Her visual acuity was stabilized and there have been no safety related concerns, at least in September 2015, nearly one year after the transplant.
  • HSVtk herpes simplex virus thymidine kinase
  • the viral TK gene product has intrinsic immunogenicity that may cause transduced cells to be rejected by the host immune system in immunocompetent individuals (Berger et al., Blood. 2006 Mar. 15;107(6):2294-302). Additionally, if ganciclovir is used to treat CMV infections in immunocompromised recipients of hemopoietic stem transplants, the use of this suicide gene would result in the unwanted deletion of transduced cells (Bonini et al., Mol Ther. 2007 July;15(7):1248-52).
  • An alternative suicide gene system consists of an inducible caspase 9 (iCasp9) gene together with the small-molecule, bio-inert, chemical induction of dimerization (CID) drug, AP1903.
  • the iCasp9 gene contains the intracellular portion of the human caspase 9 protein, a pro-apoptotic molecule, fused to a drug-binding domain derived from human FK506-binding protein (Straathof et al., Blood. 2005 Jun. 1;105(11):4247-54).
  • Intravenous administration of AP1903 produces cross-linking of the drug-binding domains of this chimeric protein, which in turn dimerizes caspase9 and activates the downstream executioner caspase 3 molecule, resulting in cellular apoptosis.
  • the iCasp9 protein is potentially immunogenic because of the synthetic 20-amino-acid peptide and the hinges between this peptide and the two peptide-moieties are of non-human origin. Although no adverse event was observed in an initial clinical trial, which only included a small cohort of patients there is no guaranty that immunological events will not occurs when a larger number of patients will be enrolled, nor will there even be a guaranty.
  • AA amino acids
  • GCN2 protein kinase which phosphorylates the ⁇ subunit of eukaryotic initiation factor 2 (eIF2 ⁇ ) on serine 51, leading to up-regulation of the translation of the activating transcription factor (ATF4).
  • eIF2 ⁇ eukaryotic initiation factor 2
  • ATF4 activates transcription of specific target genes through binding to the Amino Acid Response Element (AARE).
  • AARE Amino Acid Response Element
  • the patent application WO 2013/068096 disclosed an expression cassette including a gene of interest under the control of an inducible promoter, which includes at least one AARE regulatory sequence and a minimal promoter.
  • the present invention relates to a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein in an individual, comprising:
  • the present invention relates to a nucleic acid vector for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, comprising a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, according to instant invention.
  • the invention in another aspect, relates to a delivery particle comprising a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein or a nucleic acid vector, as defined in the instant invention.
  • a still further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising (i) a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, or a nucleic acid vector, or a delivery particle as defined herein, and (ii) a pharmaceutically acceptable vehicle.
  • the present invention relates to a host cell comprising the nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein or a nucleic acid vector, as defined herein.
  • the present invention relates to a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, as defined herein, for use as a medicament.
  • the present invention relates to a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, as defined herein, for use as an active agent for inducing apoptosis into at least one target cell.
  • the present invention relates to a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, as defined herein, for use as an active agent for treating and/or preventing a tumor.
  • the present invention relates to a method for inducing apoptosis into at least one target cell comprising at least the step of administering to an individual in need thereof of the nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein or the nucleic acid vector, as defined herein.
  • the present invention relates to a method for treating and/or preventing a tumor comprising at least the step of administering to an individual in need thereof of the nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein or the nucleic acid vector, as defined herein.
  • kits for treating and/or preventing a tumor comprising:
  • FIG. 1 Scheme illustrating the GCN2-eIF2 ⁇ -ATF4 signalling pathway.
  • activated GCN2 phosphorylates eIF2 ⁇ , leading to an up-regulation of the transcription factor ATF4 and its recruitment to AARE sequences to induce target gene expression.
  • FIG. 2 Scheme illustrating the depiction of the AARE-gene construct: two copies of the AAREs (grey boxes) from Trb3 promoter and the Tk minimal promoter compose this construct.
  • FIG. 4 Plot illustrating the measurement of luciferase expression in vivo.
  • Transgenic mice were used in which the AARE-TK-luciferase construct was stably integrated. Following a 16 h-fasting period, transgenic mice were fed on a control diet (Ctrl) or on a diet devoid of leucine (-Leu). Bioluminescence imaging was performed for 3 to 12 hours after the beginning of the meal. Signal intensity as a result of photon detection is graded from red (highest number of photons) to blue (lowest intensity) (not shown).
  • ROI Regions Of Interest
  • FIG. 6 Scheme illustrating the strategy for on/off expression regulation of a transgene via the AARE-Driven Expression system. Pulses of diets swapping the lacking amino acid (isoleucine, phenylalanine or lysine) were performed. AARE-Luc transgenic mice were subjected to three starvation sequences each including sequentially: a 12-hours fasting (white shape, Fa), a 6-hours feeding period on an EAA-deficient diet (gray shapes, —I; —F; —K) and a 30-hours of recovery period with a control diet (gray shape, C). A quarter circle represents 6 hours. Black arrows indicate time points for bioluminescence acquisition. Signal intensity as a result of photon detection is graded from red (highest number of photons) to blue (lowest intensity) (not shown).
  • FIG. 8 Scheme illustrating the nutritional protocols for a 24-day utilization of the AARE-Driven Expression system. Mice were subjected to a repetition of four times the nutritional cycle presented above (-EAA group). A control group (Ctrl group) was fed on a control diet only (see FIG. 6 for the references).
  • FIG. 9 Plot illustrating the body weight, expressed in percent of the initial value, observed during the nutritional protocols described in FIG. 8 above. Ctrl (open squares) and -EAA (closed squares) groups are depicted. No significant difference was observed between the 2 groups.
  • FIG. 10 Plot illustrating the lean mass, expressed in percent of the initial value, observed during the nutritional protocols described in FIG. 8 above. Ctrl (open squares) and -EAA (closed squares) groups are depicted. No significant difference was observed between the 2 groups.
  • FIG. 11 Plot illustrating the fat mass, expressed in percent of the initial value, observed during the nutritional protocols described in FIG. 8 above. Ctrl (open squares) and -EAA (closed squares) groups are depicted. No significant difference was observed between the 2 groups.
  • FIG. 12 Plot illustrating the muscles' tissue weight at the end of the nutritional protocols depicted in FIG. 8 . Mice were sacrificed and three skeletal muscles (soleus, gastrocnemius and tibialis) were collected and weighted. Data are expressed in percent of control group. No significant difference was observed between the 2 groups.
  • FIG. 14 Plot illustrating the relative luciferase activity measured from the livers of the mice treated as described in FIG. 13 . Livers have been collected and imaged for bioluminescence and then the corresponding protein homogenates were assayed for luciferase activity determination.
  • FIG. 15 Plot illustrating the intra-pancreatic delivery and induction of the AARE-Luc following lentiviral transduction.
  • Administration of lentiviral particles containing the AARE-TK-Luc (LV-AARE-Luc) was performed into the pancreas of wild type mice.
  • mice were challenged with the nutritional protocol (-Ile diet following O/N starvation).
  • Light emission was quantified using ROIs covering the pancreatic area. Signal intensity as a result of photon detection is graded from red (highest number of photons) to blue (lowest intensity) (not shown).
  • FIG. 16 Plot illustrating the relative luciferase activity measured from the pancreas of the mice treated as described in FIG. 15 .
  • the pancreas have been collected and imaged for bioluminescence and the corresponding protein homogenates were assayed for luciferase activity determination.
  • FIG. 17 Plot illustrating the intra-hippocampus delivery and induction of the AARE-Luc following lentiviral transduction.
  • Stereotaxic administration of lentiviral particles containing AARE-Luc constructs was performed into the hippocampus of wild type mice. Two constructs were used: the AARE-TK-LUC construct (LV-AARE-Luc), injected in the left part of the hippocampus, and a control TK-LUC construct (LV-Luc) where AARE sequences were removed, infused in the right part.
  • LV-AARE-Luc the AARE-TK-LUC construct
  • mice were challenged with the nutritional protocol (-Ile diet).
  • FIG. 18 Plot illustrating the tumor growth inhibition by TRAIL expression using the AARE-Driven Expression system.
  • Gli36-luc cells (2 ⁇ 10 6 ) were implanted subcutaneously in nude mice.
  • mice were subjected to the nutritional protocol described in FIG. 8 : one group fed a control diet (Ctrl; open squares) and a second group fed with an alternation of EAA-deficient diets (-EAA; closed squares).
  • Tumor growth was monitored by bioluminescence imaging on the first day after Gli36-luc implantation (T0) and subsequently every week.
  • FIG. 19 Plot illustrating the tumors weight analysis from mice treated as in FIG. 18 .
  • FIG. 20 Scheme illustrating the nutritional protocol for controlling the TRAIL-dependent apoptosis.
  • tumors from the Ctrl group fed a control diet (Ctrl/Ctrl) were excised at day 21.
  • a control diet (Ctrl/Ctrl)
  • -EAA/-Ile isoleucine
  • FIG. 21 Results of the analysis of the proteins homogenates from the excised tumors of mice treated as in FIG. 20 . Proteins homogenates were analysed by western blot for detection of TRAIL (short and long exposures), cleaved-PARP, and ATF4 levels. Actin levels are shown as a loading control. Quantification of the western blot reveals that the level of TRAIL under EAA-diet was estimated to be over hundred fold more intense than the faint signal obtained under control conditions.
  • 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 and provides a “safety switch” for therapy approaches based on a “suicide strategy”.
  • a key part in the experimental data below concerns the pro-apoptotic protein TRAIL.
  • the corresponding set of experiments were not only carried out to address issues dealing with cancer, but also as a model system to address the issues of background activities and the dynamics of the system.
  • a gene regulatory system should be regulated over a wide dose range of the inducer, within a safe dose of the vector, and exhibit a low level of background expression.
  • the AARE/suicide gene system is ideally suited to act as a suicide safety device, resulting in no possible immune response.
  • a suicide safety device is not necessary for the function of CAR T-cells, nor for the maturation/differentiation of stem/iPS cells.
  • the presence of such a safety device would represent an indispensable safeguard in these corresponding therapies.
  • the AARE-based safety devices are perfectly secure by nature.
  • An unforeseen expression of the suicide gene which would destroy the CAR T-cells (or the iPSC-derived cells), would not arm the patient, in considering that he could easily receive another infusion of therapeutic “off-the-shelf” CAR T-cells.
  • AARE-based suicide systems considerably broaden the field and development of safety switches.
  • the whole family of caspases can be considered.
  • AARE suicide systems do not necessarily rely on protein of non-human origin to perform the suicide task.
  • the expression of a suicide transgene must irremediably be followed by the execution of corresponding cell, in which the suicide gene is endogenously expressed, allowing no time for any immune reaction.
  • the list of suicide genes could include “toxins” from various origins, provided that the gene products are not secreted and that their expressions are intimately associated with the killing of cells.
  • a first aspect of the invention concerns a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein in an individual, comprising:
  • pro-apoptotic protein is intended to mean a protein that physiologically participates to programmed cellular death, or apoptosis, upon reception by the cell of a suitable signal.
  • 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 expression of a nucleic acid encoding a pro-apoptotic protein 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 pro-apoptotic protein, and/or the measure of the pro-apoptotic protein expression.
  • the measure of the pro-apoptotic protein expression may be performed by measuring the expression of the pro-apoptotic protein with anti-antibodies that specifically bind to said pro-apoptotic protein.
  • 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 RNA 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.
  • essential amino acid includes histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), Lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophane (Trp, W) and valine (Val, V).
  • the expression “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.
  • 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.
  • 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 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.
  • 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, 20 AARE nucleic acids.
  • the regulatory polynucleotide comprises at least two AARE nucleic acids.
  • the regulatory polynucleotide comprises from one to twenty AARE nucleic acids, preferably from one 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 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 pro-apoptotic protein 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 pro-apoptotic protein may be selected in a group comprising Apoptosis-inducing factor 1 (AIF, AIFM1), Adenylate kinase isoenzyme 2 (AK2), Annexin A1 (Annexin-1, Annexin I, Lipocortin I, Calpactin II, Chromobindin-9, p35, Phospholipase A2 inhibitory protein, ANXA1, ANX1, LPC1, ANNEXIN-1, LIPOCORTIN I), Apoptotic protease-activating factor 1 (APAF1, CED4), Nucleolar protein 3 (Myp, Nop30, ARC, NOL3), Cell death regulator Aven (AVEN, PDCD12), Bcl2 antagonist of cell death (Bcl-2-binding component 6, Bcl-XL/Bcl-2-associated death promoter, Bcl-2-like 8 protein, BAD, BCL2L8, BBC2), Bcl-2 homologous antagonist
  • the pro-apoptotic protein is selected in a group comprising TRAIL, FAS receptor, FAS-associated protein, FADD, caspase 1, caspase 3, caspase 7, caspase 8 and caspase 9.
  • the pro-apoptotic protein is TRAIL.
  • the invention also concerns a nucleic acid vector for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, comprising a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, as defined herein.
  • the nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein 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 pro-apoptotic protein in a target cell.
  • the vector is a viral vector.
  • a viral vector is selected in a group comprising an adenovirus, an adeno-associated virus, an alphavirus, a herpesvirus, a lentivirus, a non-integrative lentivirus, a retrovirus and a vaccinia virus.
  • the invention further concerns a delivery particle comprising a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein or a nucleic acid vector, as defined herein.
  • the delivery particle may be in the form of a lipoplex, 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 pro-apoptotic protein, or a nucleic acid vector or a delivery particle, as defined herein, and (ii) a pharmaceutically acceptable vehicle.
  • compositions according to the instant invention are well known to persons skilled in the art.
  • a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, 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 pro-apoptotic protein, 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.
  • 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 cancer.
  • nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, 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, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100
  • 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 1 ⁇ 10 5 to about 1 ⁇ 10 15 copies of the nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, or the nucleic acid vector or the delivery particle, as defined in the present disclosure, per dosage unit.
  • from about 1 ⁇ 10 5 to about 1 ⁇ 10 15 copies includes 2 ⁇ 10 5 , 3 ⁇ 10 5 , 4 ⁇ 10 5 , 5 ⁇ 10 5 , 6 ⁇ 10 5 , 7 ⁇ 10 5 , 8 ⁇ 10 5 , 9 ⁇ 10 5 , 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 8 ,
  • the invention concerns a host cell comprising the nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein or a nucleic acid vector, as defined herein.
  • the host cell is a eukaryotic cell.
  • a “eukaryotic cell” encompasses 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 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 host cell is not a stem cell, a progenitor cell, a germinal cell or an embryonic cell.
  • 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 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.
  • 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 host cell according to the instant invention may be a cancer cell, in particular a cancer cell selected in a group comprising a leukaemia cell, a carcinoma cell, a sarcoma cell, a lymphoma cell, a craniopharyngioma cell, a bastoma cell, a melanoma cell, a glioma cell and a mesothelioma cell.
  • the cancer cell is selected in a group comprising an esthesioneuro-blastoma cell, a glioblastoma cell, a hepatoblastoma cell, a medulloblastoma cell, a nephroblastoma cell, a neuroblastoma cell, a pancreatoblastoma cell, a pleuropulmonary blastoma cell, a retinoblastoma cell.
  • Another aspect of the invention concerns a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, as defined herein, 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 pro-apoptotic protein, as defined herein, for the preparation or the manufacture of a medicament.
  • the invention concerns a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, as defined herein, for use as an active agent for inducing apoptosis 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 pro-apoptotic protein, as defined herein, as an active agent for inducing apoptosis into at least one target cell.
  • the induction of apoptosis may be performed in vivo, in vitro or ex vivo.
  • the target cell is a tumor cell.
  • the tumor cell is selected in a group comprising a cell from 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),
  • the present invention concerns a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, as defined herein, for use as an active agent for treating and/or preventing a tumor.
  • the tumor is selected in a 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), a small mela,
  • 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 invention concerns a nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, as defined herein, for use as an active agent for adoptive cell transfer.
  • the methods disclosed herein may be achieved in vitro, in vivo or ex vivo.
  • Another aspect of the present invention concerns a method for inducing apoptosis into at least one target cell comprising at least the step of administering to an individual in need thereof of the nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein or the nucleic acid vector, as defined herein.
  • the invention concerns a method for treating and/or preventing a tumor comprising at least the step of administering to an individual in need thereof of the nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein or the nucleic acid vector, 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 (Ile, 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.
  • nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein or the nucleic acid vector may be formulated as a pharmaceutical composition, as described above.
  • 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.
  • 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.
  • 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 cancer.
  • nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, 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.
  • the invention concerns a kit for treating and/or preventing a tumor comprising:
  • the anti-tumor compound may be selected by a skilled in the art from the compounds commonly employed in chemotherapy.
  • the anti-tumor 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.
  • mice maintenance and all experiments have been approved by our institutional-animal care and use committee, in conformance with French and European Union laws (permission to experiment on mice B63-150, local alle committee CEMEA CE10-13, animal facilities agreement C6334514, GMO agreement 4756CA-I).
  • C57Bl/6 transgenic mice expressing the luciferase gene under the control of AARE were engineered in our lab as described in Chaveroux et al. (Science signaling; 2015, 8(374):rs5).
  • Fisher rats and BalB/C and C57Bl/6 mice were housed in an animal facility at INRA.
  • Nude mice were purchased from Janvier labs (SM-NU-6S-M).
  • SM-NU-6S-M Janvier labs
  • six 6-week-old males per group were used. Animals were excluded from the study if they died during the period of time between intratumoral or tissular delivery of lentivirus and feeding protocols.
  • Examiners were not blinded with respect to diet administration and treatments. Animals had ad libitum access to food and water at all times, unless otherwise indicated.
  • Pancreatic injections were conducted as follow: 6-week-old males C57Bl/6 mice were anesthetized with isoflurane and a laparotomy through midline incision was made. The lentiviral vector solution containing 2 ⁇ 10 7 particles was administrated into the splenic vein. Ten days after injection, 16-h fasted mice were fed a control or a diet devoid of isoleucine for 6 hours. At the end of the experiment, whole body and excised pancreas bioluminescence were measured by using a bioluminescence imaging system (IVIS spectrum, PerkinElmer).
  • IVIS spectrum Bioluminescence imaging system
  • lentiviral preparation in 2- ⁇ L volume was injected unilaterally into the dentate gyrus region of the hippocampus. The preparation was injected with a speed of 0.5 ⁇ L/min over a period of 4 min by using a Hamilton 5- ⁇ L syringe and a 27 G syringe.
  • Hydrodynamic injections were prepared by diluting 50 ⁇ g of pGL3-2XAARE TRB3-Tk-LUC plasmid in a saline physiological buffer corresponding to 10% body volume of the mouse and were administrated over a 5-sec period in to the tail vein of 6-week-old males BalB/C mice (6 animals per group). Twenty-four hours after injection, 16-h fasted mice were fed a control or a diet devoid of isoleucine for 6 hours. At the end of the experiment, whole body and excised livers bioluminescence were measured by using a bioluminescence imaging system (IVIS spectrum, PerkinElmer).
  • IVIS spectrum Bioluminescence imaging system
  • Tumors were obtained by injection of 2 ⁇ 10 6 Gli36-luc cells suspended in 200 ⁇ L of DMEM into the left flank region. After a 1-week period for xenografts implantation, mice were ranked according to light emission of the tumors and then distributed into the experimental groups to insure equal average tumors volumes between the groups. Tumors were then injected with 10 9 lentiviral particles and subjected to the indicated nutritional conditions. After a 1-week period for xenografts implantation, tumors were injected with 10 9 lentiviral particles and subjected to the indicated nutritional conditions. The sizes of the resultant tumors were measured weekly with a bioluminescence imaging system (IVIS spectrum, Perkin-Elmer). At endpoint, mice were sacrificed and the tumors were harvested surgically, weighted, photographed and flash-frozen for subsequent protein analysis.
  • IVIS spectrum Bioluminescence imaging system
  • Plasma amino acids were purified, i.e., 100 ⁇ L of plasma was added to 30 ⁇ L of sulfosalicylic acid solution (1 mol/L in ethanol with 0.5 mol/L thiodiglycol) which was previously evaporated.
  • sulfosalicylic acid solution 1 mol/L in ethanol with 0.5 mol/L thiodiglycol
  • norleucine as an internal standard to evaluate sample treatment efficiency, which was then used to correct the raw values.
  • Amino acid concentrations were determined using an L8900 amino acid analyzer (ScienceTec, Courtaboeuf, France) with BTC 2410 resin (Hitachi Chemical).
  • Fat and lean masses were determined by placing restrained individual mice into the mouse EchoMRI-100 instrument (Echo Medical Systems LLC). For muscle wasting assessment, after 24 days of experiment, the gastrocnemius, soleus and tibialis anterior hind limb skeletal muscles were weighted. Results from mice fed EAA-deficient diets were reported to the control group data and expressed as a percentage.
  • DMEM F12 Dulbecco's modified Eagle's medium F12
  • DMEM F12 Base 10% fetal bovine serum.
  • DMEM F12 Base DMEM F12 Base
  • Gli36-Luc cells were a gift of Dr. Shah K. (Harvard Medical School, Boston, Mass.).
  • GCN2 ⁇ / ⁇ and PERK ⁇ / ⁇ MEFs were given by Drs D. Ron and H. Harding (Institute of Metabolic Science, Cambridge, UK).
  • PKR ⁇ / ⁇ MEFs were from Dr John C Bell (Ottawa health research institute, Canada). KO MEFs were validated by PCR and Western Blot analyses and Gli36-Luc cells by luciferase assays. All the cell lines were mycoplasma free. Gli36-luc cells were transduced with either the LV-AARE-eGFP or LV-AARE-TRAIL vectors by using an MOI of 10, in the presence of polybrene (5 ⁇ g/mL). 48 hours after infections, cells were transferred to 10 cm dishes and maintained for experimental purposes.
  • 2XAARE TRB3-Tk-LUC, 2XAARE CHOP-Tk-LUC and 2XAARE ATF3-Tk-LUC plasmids were constructed by inserting SacI-XhoI double-stranded oligonucleotides containing two copies of the different AARE sequence into the pcDNA3-TK-Luc plasmid.
  • the 2XAARE TRB3- ⁇ -globin-LUC construct was obtained by replacing the TK minimal promoter sequence flanked by the Xho1 and HindIII restriction sites with a double-stranded sequence corresponding to the various AARE sequences.
  • 2XAARE TRB3-Tk-eGFP and 2XAARE TRB3-Tk-TRAIL lentiviruses were obtained by synthesizing eGFP and the human TRAIL cDNA sequences (GeneCust) flanked by Nco1 and Xba1 restriction sites. DNA cassettes were then inserted in an HIV-INS vector containing two copies of the AAREs from the Trib3 gene.
  • 2XAARE TRB3-Tk-eGFP and 2XAARE TRB3-Tk-TRAIL lentiviruses were produced in the Vectorology facility (ICM, Paris).
  • the viability of the cells was measured with the XTT cell viability kit (Cell signaling tech., 9095). Apoptosis was evaluated by flow cytometry analysis with the ANXAS/PE/7-AAD Apoptosis detection kit (BD Biosciences, 559763) following the manufacturer's instructions. Regarding ELISA assays, 16 h after treatment the media were collected and used for TRAIL protein determination with the Human TRAIL/TNFSF10 Quantikine ELISA kit (R&D systems, DTRL00).
  • Trb3, Chop and Atf3 genes are up-regulated following activation of the GCN2-eIF2 ⁇ -ATF4 pathway, and implicates the recruitment of ATF4 to specific AAREs to induce their expression ( FIG. 1 ).
  • AAREs are present as a single copy of the core sequence within the Chop and Atf3 promoter, or as a repetition of three copies in the Trb3 promoter.
  • eIF2 ⁇ can be phosphorylated in mammalian tissues by three other kinases: PKR (activated by dsRNA and cytokines), PERK (activated by endoplasmic reticulum stress), or HRI (activated by heme deficiency). HRI is expressed in an erythroid cell-specific manner; therefore the attention was focused on the role of the ubiquitously expressed kinases GCN2, PKR and PERK.
  • FIG. 3 shows that the blood leucine level was increased during the post-prandial period after consumption of a control meal. In sharp contrast, leucinemia dramatically decreased as early as 30 min after consumption of a diet devoid of leucine.
  • the hepatic mRNA encoding Trb3 is significantly induced one hour following the beginning of a leucine-free meal.
  • the delay to obtain measurable bioluminescence is due to the necessity to accumulate enough luciferase.
  • this experiment provided a clear indication that the AARE-Driven Expression System functions in vivo as well.
  • Gene therapy may require long-term expression of the transgene.
  • long-term EAA deprivation is not physiologically relevant
  • the ability of maintaining a long-term expression of the transgene was tested by taking advantage of the flexibility the AARE-Driven Expression System.
  • pulses of amino acid deprived diet swapping each of the lacking EAA were performed.
  • AARE-driven luciferase mice were subjected for 6 days to a nutritional cycle as described in FIG. 6 .
  • Monitoring abdominal bioluminescence was performed every day ( FIG. 7 ). The quantification of bioluminescence clearly showed that transgene expression returned to a near-basal level 24 h after the dietary challenges. Re-induction followed similar kinetics with maximal levels depending on the lacking AA.
  • Feeding cycles of an EAA-deficient diet have no effects on protein metabolism, provided that the amino acid differs from one cycle to the other.
  • Body weight, percentage of lean and fat mass and weight of muscle was monitored in animals that have been subjected for 24 days to the above cycling nutritional protocol (4 nutritional cycles as shown FIG. 8 ). As illustrated in FIG. 9-12 , none of these physiological parameters was modified relative to control.
  • pancreatic tissues were transduced with a lentiviral vector carrying luciferase driven by AAREs.
  • a clear induction of luminescence was observed in the pancreas in response to the inducer diet, which was confirmed by measuring luciferase activity in protein extracts ( FIGS. 15 and 16 ). No signal was detected in other parts of the mice.
  • lentiviral injection of the AARE-Driven Luc expression system was performed in the hippocampus of rats, a cerebral region that is easily accessible by stereotaxic injection. The left hippocampus received lentiviral particles containing the AARE-Luc sequence, whereas the right received particles without AARE sequences.
  • luciferase activity was measured in brain extracts. As shown in FIG. 17 , brain extracts of the left hippocampus containing the vector harboring the AARE-Driven Luc expression system exhibited increased luciferase activity, whereas the right hippocampus and the non-injected area of the left hippocampus expressed a background activity.
  • the AARE-Driven Expression System can be pharmacologically induced in liver and in pancreas following intra-peritoneal injection of halofuginone, a pharmacological activator of GCN2 that mimics amino-acid starvation by inhibiting prolyl-tRNA synthetase 18 (not shown).
  • Pharmacological activation of GCN2 could provide an alternative to the nutritional protocol.
  • a careful assessment of the specificity, pharmacokinetics and potential adverse effects of halo fuginone is being awaited.
  • TRAIL was chosen as a gene of interest.
  • TRAIL is a secreted cytokine that acts in a paracrine manner by binding to specific death receptors to initiate apoptosis.
  • TRAIL has been intensely investigated, particularly in glioblastoma cells. It has a short biological half-life (30 min) and is rapidly cleared from the body after systemic administration. However, prolonged exposure of normal human cells to TRAIL could still be toxic.
  • the long-term “nutrition-based” protocol may represent a suitable solution to regulation of TRAIL expression.
  • Human Gli36-luciferase cells were used as a model for glioblastoma cells. These cells, which constitutively expressed luciferase, were transduced with the AARE-Driven TRAIL Expression System. First, it was ascertained that TRAIL protein expression was induced in response to leucine starvation, secreted into the culture medium, had a paracrine effect on surrounding cells and triggered apoptosis (not shown).
  • TRAIL expression prevented tumor growth with no apparent toxicity at a gross level (-EAA group), whereas similar animals fed on a control diet showed no inhibition (Ctrl group) ( FIGS. 18 and 19 ).
  • TRAIL protein was detected solely in tumors from the -EAA group, after 6 h of the -Ile diet period. As expected, the nutritional protocol by itself had no effects on tumor growth (not shown).
  • this nutrition-based regulatory system takes advantage of the adaptive GCN2-eIF2 ⁇ -ATF4 signal transduction pathway that senses amino acid deficiency. This pathway, which is conserved from yeast to human, is triggered when omnivorous animals are confronted with food restricted in one EAA, a situation that may occur frequently in the wild if only a single plant protein source is accessible.
  • the activation of the GCN2-eIF2 ⁇ -ATF4 pathway occurs exclusively in response to the consumption of an EAA unbalanced diet and is engaged in no other human nutritional conditions.
  • Physiological conditions such as fasting, do not affect the amino acids blood concentration to such an extent that it would trigger the GCN2 pathway.
  • the blood concentration of all amino acids would be maintained through a compensation mechanism involving increased proteolysis in liver and then in muscle.
  • the GCN2-eIF2 ⁇ -ATF4 pathway has been found in numerous tissues such as liver, pancreas and in different parts of the brain, revealing thereby the large potential applicability of the AARE-driven expression system to numerous diseases. It is unique and exhibits no inherent toxicity, as it is not based on an exogenous ligand-inducible system relying on non-human/viral transcriptions factors/regulatory proteins, which could generate immune responses. Moreover, it obviates the need of pharmacological inducers, which could generate toxic effects, especially for long-term treatments.
  • the nutrient-based regulatory system offers a robust and flexible means to precisely tune the expression of a desired gene since the inducer diets are composed of free amino acids that are easily absorbed, thereby leading to a rapid EAA blood drop kinetics and triggering of therapeutic transgene.
  • the level of expression of a given transgene will depend upon the specific EAA lacking in the diet.
  • a diet composed of free amino acid taken by a patient, in the morning, is not anticipated to be a challenge for the patients.
  • ready-to-eat diet package would be similar to taking food complements.
  • a medical formula of leucine free diet available on the market and validated in Maple syrup urine disease (MSUD), could be readily used in conjunction with the AARE system.
  • novel formula tailored to the individual needs and tastes of the patient can be developed. Following the induction period, patients could after a few hours resume eating normal diet. When required, sustained expression of the transgene can be obtained by alternating diets, every day or every few days.
  • GDNF glial cell line-derived neurotrophic factor
  • the dynamics of a given regulatory system is a key feature in addressing the pharmacological properties of a given medication supplied through gene transfer.
  • a gene regulatory system should be regulated over a wide dose range of the inducer, within a safe dose of the vector, and exhibit a low level of background expression.
  • the study of the pro-apoptotic TRAIL cytokine described in the Results section provided a direct and quantitative comparison between transgene expression and the resulting physiological/clinical effect.
  • the level of TRAIL expressed in the induced tumor was, in the absence of dietary isoleucine, estimated to be over hundred fold more intense than the faint signal obtained under control conditions.
  • TRAIL expression prevented tumor growth with no apparent toxicity at the gross level, whereas animals fed on a control diet showed no tumor inhibition.
  • Such a high ratio of induction associated with the nutritional protocol offers great flexibility in adapting transgene expression levels to optimal therapeutic levels, and to abrogate the background activity when required.
  • the AARE system may, in particular situations, act as an endogenously controlled system in view of the fact that the eIF2 ⁇ -ATF4 signalling pathway is part of one of the three branches of the ER stress response.
  • AARE-dependent transcription can become internally activated in situations such as certain tumors or certain brain areas of patients suffering from Alzheimer's or Parkinson's disease.
  • the expression of a therapeutic gene could be triggered endogenously, without resorting to an EAA-deficient diet.
  • the therapeutic factor is a secreted protein with paracrine effect
  • the therapeutic effect may be obtained by injection of the vector into neighbouring healthy tissue followed by dietary induction.
  • the nutrition-based regulatory system was shown to be effective in the context of a vector backbone, such as that of the lentiviral vector which transduces many types of cells efficiently and is among the most promising viral vectors currently used in gene therapy trials.
  • This finding calls for the testing of other vectors for their capacities to express the AARE system, which may further exemplify the flexibility and potential of the nutrition-regulated system.
  • the AARE system highlights a new concept in the field of gene therapy, that synthetic diets can enable a tight and robust temporal control of therapeutic transgene expression, thereby unlocking a frequent hurdle in the translation of gene therapy protocols to clinical fruition.
  • Thymidine kinase (Tk) minimal promoter Regulatory polypeptide comprising the Thymidine kinase (Tk) minimal promoter and six copies of the AARE nucleic acid sequence from the TRIB3 gene: SEQ ID NO: 6 GGTACCGATTAGCTCCGGTTTGCATCACCCGGACCGGGGGATTAGCTCCG GTTTGCATCACCCGGACCGGGGGATTAGCTCCGGTTTGCATCACCCGGAC CGGGGGCCGGGCGCGCGTGCTAGCGATTAGCTCCGGTTTGCATCACCCGGAC CGGGGGATTAGCTCCGGTTTGCATCACCCGGACCGGGGGGGATTAGCTCCGG TTTGCATCACCCGGACCGGGGACTCGAGGTCCACTTCGCATATTAAGGTG ACGCGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCGCTTAACAG CGTCAACAGCGTGCCGC cDNA of TRAIL protein: SEQ ID NO: 7 ATGGCTATGATGGAGGTCCAGGGGACCCAG

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