primary cells and in vivo immunomodulation using ...€¦ · lentiflash-mage a3 t cells harvested...
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Primary cells and in vivo immunomodulation using LentiFlash®, a chimeric RNA delivery technology designed for clinical applications
Christine Duthoit1, Régis Gayon1, Lucille Lamouroux1, Alexandra Iché1, Nicolas Martin1, Florine Samain1, Guillaume Pavlovic2, Tania Sorg2, and Pascale Bouillé1
1Flash Therapeutics – Canal Biotech II. 3 rue des satellites 31400 Toulouse, France; 2Institut Clinique de la Souris, Celphedia, Phenomin, Strasbourg, France. Contact:
Booth #10
Transduction efficiency
LentiFlash® is a cutting-edge technologyfor RNA delivery. It overcomeschallenges posed by DNA delivery asRNA is directly delivered, andtransiently expressed, into thecytoplasm.
Hence, transduced cells are free of viralRNA which is a great advantage fortherapeutic purposes using T cells orHSCs.
It’s also capable of delivering multipleRNA species, such as different codingRNAs and/or Cas9 mRNA + sgRNAs.
Expression duration
Expression duration depends on thehalf-life of the protein encoded by thedelivered RNA.
LENTIFLASH® : A NEW TECHNOLOGY FOR TRANSIENT AND SAFE RNA DELIVERY
MS2-mediated packagingPsi-mediated packaging
Lentiviral vector LentiFlash® particle
MS2 Stem loops
(Prel et al. Mol Ther Methods Clin Dev. 2015)
Close to 100% transduction efficiency Transient and tailored expression
Tailored expression
The dose of LentiFlash® can be tailoredto fit the desired expression level.
Unlike classical integrative lentiviralparticles, LentiFlash® particles cancontain at least 3 different RNA species.
Efficient in vivo recombinase delivery
Local intra-muscular administration of ILV-Cre or LentiFlash®-Cre.
Analyses 2 weeks post injection
DNA
Biodistribution(Cre probe)
RNA
DNA
Efficiency(GFP probe)
RNA
Control ILV LF0.00
0.05
0.10
0.15
0.20
Control ILV LF0.00
0.01
0.02
0.03
0.04
0.05
Control ILV LF0.0
0.1
0.2
0.3
Control ILV LF0.0
0.1
0.2
0.3
0.4
0.5
Data are normalized on Hprt housekeeping gene
mT/mG reporter mice model
Quadriceps histology with GFP immuno-staining shows an efficient local Creactivity, with no excision in other organs.
Collaboration with PHENOMIN-Institut Cliniquede la Souris (ICS)
Increased in vivo efficiency compared to integrative vectors
Higher deletion efficiency with the LentiFlash® than with the integrative lentiviral vector. No residual expression of the Cre recombinase is detected.
DNA and RNA analyse through ddPCRfrom the same sample.
As expected, Cre recombinase is onlydetected with ILV.
GFP reporter is detected at a higher levelafter LentiFlash® injection compared toILV.
LENTIFLASH® : A NEW TECHNOLOGY FOR IMMUNOTHERAPY APPLICATIONS
LentiFlash® delivery of tumoral antigens mediates rejection of progressivetumors
Antigen delivery mediated by LentiFlash®
Dendritic Cells
BoneMarrow
LentiFlash-MAGEA3
Primed DC
1 2
3
4
T cells specificactivation by DC
DC reimplantation
Tumoral mouse cellline expressing
MAGEA3 (Renca)
tumorimplantation
Kinetic follow-up
0
200
400
600
800
1000
1200
1400
0 5 10 15 20 25 30 35
Tum
or
volu
me
(m
m3
)
Days after implantation
Tumor growth into Balb/c mice (Renca model)
RENCA-Mage A3 tumor + DC transduced withLentiFlash-ZsGreen
RENCA-Mage A3 tumor + DC transduced withLentiFlash-Mage A3
T cells harvested from mice transferred with MAGEA3 BMDC respond specifically to MAGEA3 ex vivo
CD25 expression
IFNg secretion
CD8 increase
Mice at the end of in vivo follow-up
Lymph nodecells
LN harvesting
MLR : 1 Renca-MAGEA3 : 1 LNC
Tumoral mouse cellline expressing
MAGEA3 (Renca)
48h co-culture
T cells specificity mediated in vivo by LentiFlash®
Activated T cells were transduced by LentiFlash®
vector (5 pg of p24/cell ) expressing Cas9 and a sgRNAtargeting the human PD1 or CXCR4 genes.
In human primaryT cells with a
sgRNA and Cas9
D1
T cells selection& CD3/CD28
activation
LentiFlash®- CRISPR targeting PD1 or
CXCR4
Flow cytometry
analysis
D0 D7
LENTIFLASH® : A NEW TECHNOLOGY FOR GENE EDITING APPLICATIONS
Human T lymphocytes display high KO efficiencyusing highly purified and concentratedLentiFlash® vectors without affecting viability norproliferation, and preserving the original cellphenotype.
LentiFlash® manages to deliver multiple RNAspecies into all cell types, such as different codingRNA or Cas9 mRNA + sgRNA.
In addition to LentiFlash® transduction efficiency, the KO efficacy depends on sgRNA inherent potency (Yuen et al, NAR 2017), genomic environment, type and state of the targeted cells (activated or not, quiescent or not, cell cycle state).