FROM LENTIVECTORS TO A GAME CHANGING RNA CARRIER TECHNOLOGY Yann Merlet, Sales engineer Regis Gayon, vectorology & cell engineering manager 06/2017
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FROM LENTIVECTORS TO A GAME CHANGING RNA CARRIER TECHNOLOGY
Yann Merlet, Sales engineerRegis Gayon, vectorology & cell engineering manager
06/2017
www.vectalys.com 2
Vectalys Core Activity
Vectalys Expertise
Activities & Offers
Company overview
01
www.vectalys.com 3
VECTALYS OVERVIEW
Expert in the manufacturing of lentiviral solutions dedicated to hard-to-transfect cells
✓ Privately owned company founded in 2005
✓ Develops, manufactures and commercializes high quality lentiviral toolsproducts dedicated to cell & tissue engineering
DNA DELIVERY RNA DELIVERY
iLVIntegrative lentiviral
vectors
LentiFlashNon-Integrative
lentiviral particles
Stable
expression
Transient
expression
www.vectalys.com
ACTIVITIES & OFFERS
Custom- and premade solutions are dedicated to gene and cell engineering
1. Custom-made lentiviral solutions
2. Premade lentiviral solutions
4
Cell engineering:Cell biosensor
Models engineering
Cell differentiation
Bioproduction
Gene engineering:
Gene expression modulation
Genome editing
Cell engineering:Cell labeling
Cell biosensor
Gene engineering:Genome editing
From 1 ml to 5 ml at 107 TU/ml; From 1 ml to 30 ml at 109 TU/ml
From 20 µl to 5x20 µl at 109 TU/ml
5
VECTALYS INTEGRATED PLATFORM
A comprehensive vectorology platform dedicated to production & analytics
www.vectalys.com
Custom made lentiviral Products Particle Analytics
Premade lentiviral Products Custom Cell & Tissue engineering
www.vectalys.com
VECTALYS COMMITMENTS
Vectalys commits to the success of customers’ projects
▪ Expertise
▪ Human
▪ Ethics
6
11 years of experience in producing high quality of lentiviral particles
Game-changing innovation
Project design & customer-friendly support Training seminars
Confidentiality GMO compliance support IP compliance policy
Over 4000 batches produced for customers and partners programs LentiFlash, a novel non-integrative particle for transient delivery of RNA in delicate cells
www.vectalys.com
IP PORTFOLIO
A portfolio of patents on final product composition & LentiFlash system
7
LentiFlash Technology
Licenses iLV
LentiFlash Applications
Virus-based vector compositions useful for transducing eukaryotic cells
Filed on July 27,2011 USA - Canada - Europe - Israël - China - Singapour - Japan
Production Process
Method for detecting or measuring the impact of a viral vectors compositions on eukaryotic cells and biomarkers used thereof
Filed on July 26, 2012 USA - Canada - Europe - Israël - China - Singapour - Hong-Kong - Japan
Quality Control
Particule rétrovirale comportant au moins deux ARN non viraux encapsidésRetroviral particle comprising at least two non-viral packaged RNA
Filed on May 15, 2015 France - International extension
Particule pour l'encapsidation d'un système d'ingénierie du génomeParticle for genome engineering
Particule virale pour le transfert d'ARNs, notamment dans les cellules impliquées dans la réponse immune
Viral particle for ARNs transfer, in particular in cells involved in the immune response
Filed on May 13, 2016 France
SIN technology
Life Technologies Corp./ Thermofisher
Scientific
DNA Flap technology (cPPT/CTS)
Institut Pasteur
TET inducible promoter
TET Systems
Vectors (Lentiviral Particles)
Oxford Biomedica Ltd
Fluorescent proteins
Clontech, Inc./ Takara Bio US
Fluorescent proteins
Evrogen
Licenses Sequence Of Interest
License LentiFlash
www.vectalys.com 8
A SPECIFIC PROCESS FOR A DEFINED PRODUCT
3 defined products dedicated to specific applications
StartVery high level of transduction onpermissive and “hard-to-transfect”immortalized cell lines
PremiumVery high level of transduction on primary &stem cellsNo induced toxicity or cell damage thanksto the low impurity content
ExpertDirect injection of LV into adult animals forinflammatory, vaccine approaches orLentigenesis
•Titre (TU/ml) : Number of lentiviral functional particles leading to integration of vector DNA
into the host genome.
•PP/TU ratio : Gives a measure of the lentiviral batch quality by discriminating the non
functional physical particles (PP) from the functional particles.
•Specific activity (TU/mg) : Gives the ratio between functional particles and contaminating
proteins in the batch. Specific activity (SA) increases with the purity of the batch.
Batch type Volume
(mL)
Titre
(TU/ml)
PP/TU Specific
activity
(TU/mg )
Start 1-5 >107 <1000 >107
Premium 1-30 >109 <300 >108
Expert 0.5 >109 <300 >5x108
www.vectalys.com 9
PROCESS TRANSFER INTO GMP
Vectalys is engaged in a GMP lentiviral particle production program
Vectalys has experience in small &large-scale batches for research &preclinical development
Customers & partners demand forclinical grade lentiviral vectors
Scalable GMP facilities are identified
Research Development Pre-clinicals Clinicals
Custom Productions Large Scale GMP Production
Consistent production process at all stages
www.vectalys.com 10
LentiFlash technology description
Applications in gene editing
Applications in Immunotherapy
LENTIFLASH TECHNOLOGY
02
www.vectalys.com 11
WHY SWITCH FROM INTEGRATIVE TO NON-INTEGRATIVE TOOLS?
To combine RNA delivery & lentiviral transduction advantages
RNA delivery is a versatile, flexible,and safe means for protein andgene-editing therapies
Lentiviral vectors have the ability toenter 100% of a primary or stem cellpopulation
RNA delivery-mediated by alentiviral particle has the ability totrigger a transient expression inprimary & stem cells
www.vectalys.com 12
CONSTRUCTION OF MS2 & HIV CHIMERAS
HIV provides particle & MS2 the packaging system
MS2
HIV
MCP
RNA
5’LTR RRE cPPT GOI 3’ SIN LTRRSV Pr
GOIPr
MA CA NCp7 p6
RNAMCP
HIV NCP7
+
MCP
HIV NCP7
RNA
MCP
www.vectalys.com 13
FROM Psi- TO MS2-MEDIATING PACKAGING
The LentiFlash transfer plasmid contains no lentiviral sequences
pptrre
CMV
Y
TransgenePromoter
U3 RU5
gag polCMV
VSV GCMV
Psi-mediated packaging MS2-mediated packaging
MA CA NC
TransgenePromoter
gag polCMV
MA CA NC
VSV GCMV
pptrre
CMV
Y
Promoter
U3 RU5
MS2-CP
www.vectalys.com 14
GENE TRANSFER MEDIATED BY LENTIFLASH
The production process is the same as for lentiviral vectors
www.vectalys.com 15
DNA vs. RNA DELIVERY MECHANISMS
Each delivery solution targets specific applications
www.vectalys.com 16
THE RIGHT TOOL FOR THE RIGHT APPLICATION
DNA & RNA deliveries target specific applications
iLV:
Integrative lentiviral vectors
• Stable expression
• DNA delivery
LentiFlash:
Non-Integrative lentiviral particles
• Transient expression
• RNA delivery
Gene overexpression& silencing
Protein & antibodyproduction
Cell labelling & testing
Immunotherapy CAR T-cells
Genome-editing(CRISPR/Cas9)
DC-basedimmunotherapy
Cell differentiation
Cell reprogramming
www.vectalys.com 17
LentiFlash technology description
Applications in gene editing
Applications in Immunotherapy
LENTIFLASH TECHNOLOGY
02
www.vectalys.com 18
IN VITRO CRE-MEDIATED GENE ENGINEERING ASSAY
LentiFlash allows a functional expression of genome engineering enzymes in vitro
LTR U3LTREF1 dsRed LoxLox
+ LentiFlash-CRE
www.vectalys.com 19
IN VIVO CRE EXPRESSION
LentiFlash allows a functional expression of genome engineering
• Local injection of LentiFlash-Cre (107 PP in 5 µL) into the biceps femoris muscle of ROSA26-YFP reporter mice.
LF-CRE ILV-CRE
P. Ravassard Hopital Pitié-Salpétrière
LoxLox PFY
LoxLox YFP
+CRE
www.vectalys.com 20
HUMAN T CELLS & hHSC TRANSDUCTION BY LENTIFLASH
The right tool for an efficient transient expression in primary cells
www.vectalys.com 21
LENTIFLASH & CANCER IMMUNOTHERAPIES
CRISPR/Cas9 delivery-mediated by LentiFlash particles leads to efficient PD1 knockout in activated primary T cells
D1 D3
T cells selection
& CD3/CD28
activation
T1FACS
analysis
D0 D7
T2
✓ Human activated T cells were transduced by a range ofLentiFlash-CRISPR (Cas9 + gRNA), once (T1) or twice (T2)
✓ Lentiflash delivering Cas9 is used as control of PD1 editing
✓ PD1 expression is analyzed by FACS 4 days after 2nd
transduction
www.vectalys.com 22
LENTIFLASH & ANTIVIRAL STRATEGIES
CRISPR/Cas9 delivery-mediated by LentiFlash particles leads to efficient CXCR4 knockout in activated primary T cells
D1 D3
T cells selection
& CD3/CD28
activation
T1FACS
analysis
D0 D7
T2
✓ Human activated T cells were transduced by a range ofLentiFlash-CRISPR (Cas9 + gRNA), once (T1) or twice (T2)
✓ Lentiflash delivering Cas9 is used as control of CXCR4editing
✓ CXCR4 expression is analyzed by FACS 4 days after 2nd
transduction
www.vectalys.com 23
LentiFlash technology description
Applications in gene editing
Applications in Immunotherapy
LENTIFLASH TECHNOLOGY
02
www.vectalys.com
DENDRITIC-BASED VACCINES
Antigen delivery mediated by LentiFlash inhibits tumoral development in vivo
24
Dendritic Cells
(CD11c+)
Bone Marrow LENTIFLASH-
MAGEA3 Primed DC
1 2
3 4
CD8+ and CD4+ specific Activation
(DC presentation of TAA via MHC I and II)
Tumor growth
Positive Animals
Intradermic
reimplantation of DC
Tumoral mouse cell
line expressing MAGE
A3 (Renca)
tumor
implantationKinetic
follow-up
3
0
100
200
300
400
500
0 2 4 6 8 10 12 14 16 18
Tum
or
volu
me
(m
m3
)
Days after implantation
Tumor growth into Balb/c mice
RENCA-Mage A3 cells + Dendritic cells transduced with RLP-ZsGreen
RENCA-Mage A3 cells + Dendritic cells transduced with RLP-Mage A3
0
25
50
75
100
0 2 4 6 8 10 12 14 16 18
% p
ost
ive
An
imal
s
Days after implantation
Positive animals for tumor growth
RENCA-Mage A3 cells + Dendritic cells transduced with RLP-ZsGreen
RENCA-Mage A3 cells + Dendritic cells transduced with RLP-Mage A3
www.vectalys.com 25
LENTIFLASH PARTICLE PROPERTIES
LentiFlash is a versatile and efficient cell transfer system for RNA delivery
1. The lentiviral production platform is convenient for LentiFlashproduction
2. The LentiFlash particles leads to efficient delivery in primary & stemcells both in vitro and in vivo
3. RNAs delivered by LentiFlash are rapidly bioavailable, leading to highand short-term expression of the transferred messenger
4. The LentiFlash particle contains an increased ratio of RNA to particlecontent compared to the LV parental particle
www.vectalys.com 26
FLASHCELL COMPANY
03
www.vectalys.com 27
A NEW COMPANY NAMED FLASHCELL
Vectalys has set up a spin-off named FlashCell to carry out the future therapeutic applications
▪ With its lentiviral-based bioproduction and non-integrative RNA delivery capabilities,
Vectalys has developed LentiFlash technology with broad potential therapeutic
applications from gene editing to cell-based immunotherapy
▪ Vectalys has licensed LentiFlash technology to a new company FlashCell for therapeutic
purposes including:
• proprietary therapeutic programs,
• partnerships and licensing agreements with pharmaceutical and biotechnological
companies for the utilization, further development and global commercialization of the
non-integrative RNA delivery system resulting from the initial discovery and the
development programs efforts.
www.vectalys.com 28
A LEGITIME MOVE TO THERAPEUTIC APPLICATIONS
FlashCell is part of the Vectalys’s history
VECTALYS
Lentiviral tools manufacturing
Integrative Lentiviral vectors
DNA delivery
Non-integrative LentiFlash
RNA delivery
MANUFACTURING PLATFORM from R&D to GMP GRADES
High quality lentiviral manufacturing platform
from basic research to clinical applications
FlashCellTherapeutic unit
DEVELOPMENT OF LENTIFLASH TOOLS FOR THERAPEUTIC
APPLICATIONS
In different fields: antiviral strategies, Cancer
immunotherapies, Neuromuscular & Skin disorders
www.vectalys.com
A LEGITIME MOVE TO THERAPEUTIC APPLICATIONS
FlashCell is part of the Vectalys’ history
VECTALYS
Lentiviral tools manufacturing
Integrative Lentiviral vectors
DNA delivery
Non-integrative LentiFlash
RNA delivery
MANUFACTURING PLATFORM from R&D to GMP GRADES
High quality lentiviral manufacturing platform
from basic research to clinical applications
FlashCellTherapeutic unit
DEVELOPMENT OF LENTIFLASH TOOLS FOR THERAPEUTIC
APPLICATIONS
In different fields: antiviral strategies, Cancer
immunotherapies, Neuromuscular & Skin disorders
THANK YOUwww.vectalys.com
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