Any Gene to Any Cell
AAV vectors for gene therapy
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AAV1 AAV2 AAV5 AAV6 AAV8 AAV9
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WHY WORK WITH SIRION?
OVERCOME MAJOR ROAD BLOCKS IN AAV GENE THERAPY
therapeutic gene
1. Cost-efficiency
through effective
production capabilities
3. Cell/tissue specific gene
expression
2. High level
cell/tissue
tropism4. Less neutralizing events
through immunologic optimization
Invent improved AAV vectors with optimized transduction and
expression properties and low immunogenicity
Next gen AAV
Neutralizing antibodies against WT AAV
capsids (Boutin et al., 2010)
SIRION`S GATEWAY TO FAST CLINICAL VECTORS:
SERVING THE ENTIRE AAV VALUE CHAIN
R&D GMP ready cGMP
Strong project & client commitment
Centralized communication through strategic
partnerships with academics & CDMO
Seamless process transfer
Established manufacturing process from R&D to clinics
Parallel vector development and GMP manufacturing
Long term experience with industrial clients
Engineering
Evolution
Development
USP, DSP
Optimization
Clinics
AAV CAPSID OPTIMIZATION STRATEGIES ENABLE THERAPEUTIC
AAV DEVELOPMENT
Academic network Technology for Discovery ManufacturingDeal & Intellectual
Property
Strategic partnership
with Prof. Grimm of the
University clinic
Heidelberg
world-leading expert in
the field of AAV
engineering
Academic and clinical
development network
Unique AAV capsid
libraries, including
peptide display and
DNA shuffling libraries
Targeted AAV capsid
peptide insertion
Millions of novel variants
In vivo evolution of
capsid libraries with non-
human primates (NHP)
for relevant translational
outcomes
Design & engineering of
transgene cassettes
In-house manufacturing
for pre-clinical studies
Established scalable
production protocols for
GMP ready and cGMP
vectors
Partnership with CDMO
for seamless transition to
clinical development
Risk sharing deal
structure
Exclusive and non-
exclusive IP license with
milestones & royalties
Network & Technology Highlights
LEADING TECHNOLOGY FOR AAV CAPSID OPTIMIZATION
1. Müller et al., Nature Biotech. 2003
2. Waterkamp et al., J Gene Med. 2006
3. Ying et al., Gene Ther. 2010
4. Varadi et al., Gene Ther 2011
Directed Evolution AAV
Proprietary library designs
Library evolution for
AAV1-12 available
Selection in vitro and in
vivo
Peptide insertion libraries Shuffled AAV libraries
Next generation AAVs
Complex shuffled AAV
libraries
Shuffling of AAV1-12
vectors
Deconvolution software
Immunologic optimization
AAV immune evasion
Alanine scanning of VRs
for all AAV serotypes
nAB neutralization assay
for capsid variants
Combinatorial immune
evasion library screening
Preclinical testing of
immune evasion variants
Vector specificity
NGS-guided AAV evolution
NGS guided evolution of
AAVs in vitro and in vivo
Biodistribution of AAV
variants
Barcoded AAV libraries for
complex biodistribution of
multiple AAVs
COMBINED AAV CAPSID & CASSETTE OPTIMIZATION
STRATEGIES FOR HIGHLY POTENT THERAPEUTIC VECTORS
11/21/2018
Bar-coded
AAV library
generation
Sub-library
Primary screening Secondary screening
3-5
selection
rounds
AAV libraries
with unique capsids
Around 50
candidatesNext-
Generation
sequencing
(NGS)
Preclinical
and clinical
development
AA
V o
pti
miz
ati
on
Ca
ss
ett
e o
pti
miz
ati
on
Top candidate
Identification &validation of tissue-specific promotors and enhancers
Tissue-specific
promoters &
enhancers
therapeutic gene
expression driven by
tissue-specific
promoters and
enhancers
Peptide
display
DNA
shuffling
AAV NON-GMP AND GMP MANUFACTURING
Established manufacturing process for all common AAV serotypes - no need for
technology transfer/ process development
Synchronized preclinic and GMP manufacturing processes and quality controls
Centralized communication: Seamless process transfer from development to GMP
partner
Scale up capabilities and high end 2 step purification
Our unique offer
Batch deliveredPurification Timeline
2-Step (FPLC+ Iodixanol
ultracentrifugation)
Standard (1x10E13 VG)
Large (5x10E13 VG)
X-Large (>1x10E14 VG)
2 to 3 weeks
4 to 5 weeks
4 to 5 weeks
Cell Factories
2
8
16
AAV MANUFACTURING FOR PRECLINICS – BE GMP READY
ESTABLISHED GMP AAV VECTOR MANUFACTURING PROCESS
GMP
RAW MATERIALSPRODUCTION
PURIFICATION
F&FQC ANALYTICS
cGMP Master Cell Bank
Expression and packaging
plasmids
HEK293
DNA removalCell factory 10/
HYPERStacks
Primary Capture
Optional:Empty capsid
removal
Clarification
qPCR & ELISA titer
Empty / Full capsid ratio
Endotoxin testing
(Endosafe nexgen PTS,
Charles River)
Measurements of
process/product
related impurities
Polishing and Formulation
Sterile filtration
Fill & Finish Potency assays
Purity check: SDS Silver gel
Integrity of packaged vector
genomes
Sterility test
USP DSP
Cell Lysis
Transient transfection
2 and 3 plasmid system
QUALITY GUARANTEES – NON GMP/ GMP AAV
2-Step purified AAV8 vectors
SDS gel Silver Stain
-VP3
-VP2
-VP1
QC-test Method
Genome Titration qPCR
Capsid Titration ELISA
Purity check SDS PAGE, silver staining
Integrity of packaged vectorgenomes
Agarose gel electrophoresis
Endotoxin Endosafe nexgen PTS, Charles River
Sterility testingTrypton Soja Broth/Sabouraud medium; Oxoid
Mycoplasm testing PCR-based assay
Vector efficiency Transduction
Empty/Full capsid ratio Electron Microscopy
Residual BSA ELISA
Residual Benzonase ELISA
Residual Triton X-100 HPLC
pH, Osmolality, appearance Compendial assays
Residual host cell DNA qPCR
Quality Control (QC) Analytics
VIBALOGICS - OUR PARTNER FOR GMP AAV PRODUCTION
COLLABORATION NETWORK WITH RENOWNED ACADEMIC AND
STRATEGIC PARTNER
AAV peptide insertion library
shuffled AAV library
NGS analysis of top enriched
AAV variants
Barcoded AAV library
Biodistribution analysis with
NGS of barcoded library in
NHP
Project management
Vector engineering
Optimization on transcriptional
side
Production of barcoded AAV
libraries
Process development
GMP-ready vector
manufacturing
QC analytics of vector batches
cGMP manufacturing
Fill & Finish
Prof. Dirk Grimm
Task distribution
SIRION Biotech GmbH
Am Klopferspitz 1985215 MartinsriedGermany
www.sirion-biotech.com
Any Gene to Any Cell
Kathrin Schneider
VP, BD & Sales