Any Gene to Any Cell

AAV vectors for gene therapy

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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

Schneider@sirion-biotech.com