HAL Id: hal-02740006
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Development and optimization of a new gene regulation system controlled by nutrition applicable for gene
therapy
Cedric Chaveroux, Alain Bruhat, Valerie Carraro, Julien Averous, Celine Jousse, Anne-Catherine Maurin, Yuki Muranishi, Laurent Parry, Jacques
Mallet, Philippe Ravassard, et al.
To cite this version:
Cedric Chaveroux, Alain Bruhat, Valerie Carraro, Julien Averous, Celine Jousse, et al.. Development and optimization of a new gene regulation system controlled by nutrition applicable for gene therapy. Congrès de la Société Française de Thérapie Cellulaire et Génique (SFTCG), Mar 2014, Toulouse, France. 2014. �hal-02740006�
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Figure 7 : Induction of the AARE-controlled expression of TRAIL in response to an EAA starvation leads to apoptosis in glioblastoma cell line (A) Gli36-Luc cells were infected with lentiviral vectors containing the TRAIL sequence. (B) EAA induction of
AARE-TRAIL system expression leads to apotosis. (C) Kinetic analysis of EAA induction of AARE-AARE-TRAIL system expression. (D) Viability assay of Gli36-Luc cells infected with lentiviral vectors containing AARE-TRAIL or AARE-GFP sequences in leucine-starved Gli36-Luc cells (16 h). (E) TRAIL release measurement (Elisa kit) in culture media, 16 h post leucine starvation. (F) Apotosis induction in parental cells caused by paracrine effect of TRAIL.
Figure 4 : Rapid change in the plasmatic amino acid concentration following the consumption of a complete and an EAA-deficient diet .
(A) Mice were fed either with a control (Ctr) or a leucine-deficient diet (-leu) for 20 to 120 min (B) or refed for 2h on the control diet after a 2h-consumption of the leucine-deficient diet (-leu/+leu).
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Figure 2 : The Gene regulation system containing 2 copies of TRB3 AARE combined to TK minimal promoter provides the highest amino acid inducibility associated to low basal expression. (A) Functional comparison of 3 different AARE sequences.
(B) Functional comparison of 2 different minimal promoters.
Figure 1 : Presentation of the GCN2/eIF2α/ATF4 pathway controlling the AARE-Gene system activity.
GCN2 senses Essential Amino Acids (EAA) deficiency in the blood caused by the consumption of a diet lacking one EAA. Activation of this pathway leads to an increase in the translation of ATF4 transcription factor that functions as a master regulator for transcription of specific target genes playing a crucial role in nutrional stress adaptation.
GCN2 Blood EAA diminution ATF4 Protein synthesis inhibition eIF2α Translational activation Ser 51 phosphorylation AARE Dietary EAA deficiency Transcriptional activation of specific genes ( Trb3…)
ATF4 P
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5 10 R el ati ve L u ci fer ase (fo ld in d u cti o n ) 1 25 15 20 Leucine (µM) 420 140 70 30 0 2XAARE-TK-LUC WT - leu Ctr 40 20 60 80 0 2XAARE-TK-LUC GCN2-/-R el ati ve L u ci fer ase A cti vi tyATF4-/-*
Figure 3 : The AARE-Gene system can be activated by physiological EAA concentrations and requires GCN2 and ATF4
expression. (A) The plasmid construct contains two copies of the TRB3
AARE fused to the minimal promoter thymidine kinase (TK) and the Luciferase (LUC) coding sequence. This transgene is inducible (B) by different concentration leucine (C) via GCN2 (amino acid starvation) and the transcription factor ATF4.
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Development and optimization of a new gene regulation system
controlled by nutrition applicable for gene therapy
Cédric Chaveroux
1
, Alain Bruhat
1
, Valérie Carraro
1
, , Julien Averous
1
, Céline Jousse
1
, Anne-Catherine Maurin
1
,
Yuki Muranishi
1
, Laurent Parry
1
, Jacques Mallet
2
, Philippe Ravassard
2
, Pierre Fafournoux
1
1- Gènes-Nutriments Team, INRA, UMR 1019 Nutrition Humaine, Centre de Clermont-Ferrand-Theix, F-63122 Saint Genès Champanelle, France
2- Biotechnology and Biotherapy Team, Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l’Institut du Cerveau et de la Moelle épinière (CRICM), UMRS 975, Paris, France
INTRODUCTION
Our lab is specialized in nutrition research and mainly in characterizing the molecular mechanisms regulating the cellular adaptation to a nutritional stress. More
specifically, eating a meal devoid of one Essential Amino Acid (EAA) causes a dramatic decrease of the limiting EAA in the blood, which in turn activates the GCN2/
eIF2α/ATF4 signaling pathway (Figure 1). This leads to a translational up-regulation of the transcription factor ATF4 that binds to a particular DNA sequence: the
AARE (Amino Acid Response Element) to initiate a transcription program allowing the stress adaptation.
In gene therapy, the control of transgene expression remains a major concern. Therefore, we have generated an innovative gene regulation system controlled by
nutrition by transfering the AARE in an artificial patented promoter. This system is associated to an EAA-deficient inducing diet developed in our lab.
Generation of a gene regulation system controlled by amino acid availability: the AARE-Gene system.
1
Identification of target tissues and definition of a nutritional protocol dedicated to long term-usage of the AARE-Gene system.
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Figure 5 : The reversible AARE-Gene system is induced the most in response to diets lacking Isoleucine, Valine or Threonine and can be used for transgene delivery in the liver, pancreas or the brain (A) Process for generation of the AARE-LUC mice strain. (B)
Inducibility test of the AARE-Gene system in response to a diet devoid of one Essential Amino Acid (EAA) or one Non-Essential Amino Acid (NEAA) in the AARE-LUC transgenic mice. (C) Identification of tissues sensitive to EAA deficiency. (D) Demonstration of the reversibility of transgene expression following the consumption of a complete diet
B
Ctr -Leu -Lys -Thr -Val -IleuDorsal Ventral
-Ala -His -Phe -Met -Trp
1.2 1.0 0.8 0.6 0.4 0.2 Radiance (p/sec/cm2/sr) Color scale Min = 1.52e6 Max = 1.38e8 X108 6h Dorsal Ventral -Ileu 6h +Ctr 6h -Ileu 6h -Ileu 6h +Ctr 24h -Ileu 6h +Ctr 48h -Ileu 6h +Ctr 72h Ctr 1.2 1.0 0.8 0.6 0.4 0.2 Radiance (p/sec/cm2/sr) Color scale Min = 1.52e6 Max = 1.38e8 X108 insertion infection Oocytes (1 cell) LUCIFERASE
Homozygous AARE-LUC mice strain Lentivirus production N LUCIFERASE AARE Transgène LUCIFERASE AARE Transgène 2XAARE LUCIFERASE 10 20 To tal fl u x (fo ld in d u cti o n -AA/ C tr ) 1 60 40 50 30
-Leu -Lys -Thr -Val -Ileu
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Ventral Dorsal-His -Phe -Met -Trp -Ala
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EAA NEAA Duodenum Kidney Spleen Muscle (gastroc.) Lung Heart Ctr -Ileu -Lys -Thr Ctr -Ileu -Lys -Thr Liver Pancreas BAT WAT Brain Re la ti ve lu cif er as e act ivi ty (fo ld in d u ct io n /Ct r) 1 5 2 3 4 6 7 -Ileu -Lys -Thr Re la ti ve lu cif er as e act ivi ty (f o ld in d u ct io n /Ct r) 1 4 3 2 -Ileu -Lys -Thr Duodenum Kidney Spleen Muscle (gastroc.) Lung Heart Ctr -Ileu -Lys -Thr Ctr -Ileu -Lys -Thr Liver Pancreas BAT WAT Brain Re la ti ve lu cif er as e act ivi ty (fo ld in d u ct io n /Ct r) 1 5 2 3 4 6 7 -Ileu -Lys -Thr Re la ti ve lu cif er as e act ivi ty (f o ld in d u ct io n /Ct r) 1 4 3 2 -Ileu -Lys -ThrD
Figure 6 : Rotation of several diets lacking one EAA (Ileu, Phe, Lys) doesn’t affect neither (A) body weight nor (B) fat and (C) lean mass. For the
nutritional protocol (top left panel), a quarter circle represents a period of 6 h. Black arrows indicate time points for body parameters measurements.
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C
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Figure 6 : Validation of the AARE-Gene system functionality after tissue injection in vivo. (A) Lentiviral vectors were injected into the hippocampus of the left
hemisphere of rats. (B) Two weeks after injection the animals were fasted overnight and then fed either a control (Ctr) or threonine-deficient diet (-Thr). Six hours after the beginning of the meal, the animals were sacrificed, brains dissected and luciferase activity was measured.
R el ati ve L u ci fe ra se ac ti vi ty 300 200 150 100 50 0 250 350 400 Ctr - Thr
Constructs: AARE-TK-Luc TK-Luc Left Hippocampus (injected) AARE-TK-Luc AARE-TK-Luc Left hemisphere without hippocampus 450 500 Right hippocampus (non-injected) Lentivirus production insertion Injection in the hippocampus LUCIFERASE AARE Transgène LUCIFERASE AARE Transgène 2XAARE LUCIFERASE
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Demonstration of functional proofs on concept by controlling transgene expression through the AARE-Gene system .
Lentivirus production insertion Human glioblastoma cells infection LUCIFERASE AARE Transgène LUCIFERASE AARE Transgène 2XAARE TRAIL
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CONCLUSION:
Our lab has develop a new gene regulation system controlled by nutrition displaying multiple advantages : (1) use of endogenous molecular
mechanisms meaning no requirement of pharmacological inducers and expression of regulatory proteins, (2) no toxicity of an EAA limitation in a short time (3)
possibility to target the brain (no blood-brain barrier restriction) and other tissues (pancreas, liver…), (4) system reversibility allowing transgene deliveries by pulses,
avoiding development of therapy resistance following a prolonged exposure to the therapeutic protein.
We are looking for collaborations and partners interested in developing and testing this AARE-Gene system for gene therapy
applications in different pathological models.
Contact: pierre.fafournoux@clermont.inra.fr, alain.bruhat@clermont.inra.fr, cedric.chaveroux@clermont.inra.fr
Relative Luciferase Activity
0 10 20 30 40 - leu Ctr 50 60 2X AARE Trb3 LUCIFERASE
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2X AARE Chop LUCIFERASE*
2X AARE Atf3 LUCIFERASE*
Relative Luciferase Activity
0 20 40 60 100 - leu Ctr 120 140 2X AARE Trb3 LUCIFERASE
