AAV vectors for gene therapy. Any Gene to Any Cell

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1 AAV vectors for gene therapy Any Gene to Any Cell

2 % Population WHY WORK WITH SIRION? OVERCOME MAJOR ROAD BLOCKS IN AAV GENE THERAPY Invent improved AAV vectors with optimized transduction and expression properties and low immunogenicity 1. Cost-efficiency through effective production capabilities Next gen AAV therapeutic gene 3. Cell/tissue specific gene expression 2. High level cell/tissue tropism 4. Less neutralizing events through immunologic optimization AAV1 AAV2 AAV5 AAV6 AAV8 AAV9 Neutralizing antibodies against WT AAV capsids (Boutin et al., 2010)

3 SIRION`S GATEWAY TO FAST CLINICAL VECTORS: SERVING THE ENTIRE AAV VALUE CHAIN Engineering Evolution Development USP, DSP Optimization Clinics 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

4 AAV CAPSID OPTIMIZATION STRATEGIES ENABLE THERAPEUTIC AAV DEVELOPMENT Network & Technology Highlights Academic network Technology for Discovery Manufacturing Deal & 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 nonhuman 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 nonexclusive IP license with milestones & royalties

5 LEADING TECHNOLOGY FOR AAV CAPSID OPTIMIZATION Peptide insertion libraries Shuffled AAV libraries Immunologic optimization Vector specificity Directed Evolution AAV Proprietary library designs Library evolution for AAV1-12 available Selection in vitro and in vivo Next generation AAVs Complex shuffled AAV libraries Shuffling of AAV1-12 vectors Deconvolution software 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 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 1. Müller et al., Nature Biotech Waterkamp et al., J Gene Med Ying et al., Gene Ther Varadi et al., Gene Ther 2011

6 Cassette optimization AAV optimization COMBINED AAV CAPSID & CASSETTE OPTIMIZATION STRATEGIES FOR HIGHLY POTENT THERAPEUTIC VECTORS Primary screening Secondary screening AAV libraries with unique capsids Peptide display DNA shuffling 3-5 selection rounds Bar-coded AAV library generation Around 50 candidates Next- Generation sequencing (NGS) Sub-library Top candidate Identification &validation of tissue-specific promotors and enhancers 11/21/2018 Tissue-specific promoters & enhancers therapeutic gene expression driven by tissue-specific promoters and enhancers Preclinical and clinical development

7 AAV NON-GMP AND GMP MANUFACTURING Our unique offer 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

8 AAV MANUFACTURING FOR PRECLINICS BE GMP READY Purification Batch delivered Cell Factories Timeline Standard (1x10E13 VG) 2 2 to 3 weeks 2-Step (FPLC+ Iodixanol ultracentrifugation) Large (5x10E13 VG) 8 4 to 5 weeks X-Large (>1x10E14 VG) 16 4 to 5 weeks

9 ESTABLISHED GMP AAV VECTOR MANUFACTURING PROCESS USP DSP GMP RAW MATERIALS PRODUCTION PURIFICATION F&F QC ANALYTICS cgmp Master Cell Bank HEK293 Cell Lysis qpcr & ELISA titer Expression and packaging plasmids Cell factory 10/ HYPERStacks Transient transfection 2 and 3 plasmid system DNA removal Clarification Primary Capture Optional:Empty capsid removal Empty / Full capsid ratio Integrity of packaged vector genomes Endotoxin testing (Endosafe nexgen PTS, Charles River) Polishing and Formulation Sterile filtration Measurements of process/product related impurities Fill & Finish Potency assays Purity check: SDS Silver gel Sterility test

10 QUALITY GUARANTEES NON GMP/ GMP AAV Quality Control (QC) Analytics 2-Step purified AAV8 vectors QC-test Genome Titration Method qpcr SDS gel Silver Stain -VP1 -VP2 -VP3 Capsid Titration Purity check Integrity of packaged vector genomes Endotoxin Sterility testing Mycoplasm testing Vector efficiency Empty/Full capsid ratio Residual BSA Residual Benzonase Residual Triton X-100 ph, Osmolality, appearance Residual host cell DNA ELISA SDS PAGE, silver staining Agarose gel electrophoresis Endosafe nexgen PTS, Charles River Trypton Soja Broth/Sabouraud medium; Oxoid PCR-based assay Transduction Electron Microscopy ELISA ELISA HPLC Compendial assays qpcr

11 VIBALOGICS - OUR PARTNER FOR GMP AAV PRODUCTION

12 COLLABORATION NETWORK WITH RENOWNED ACADEMIC AND STRATEGIC PARTNER Task distribution Prof. Dirk Grimm 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

13 Kathrin Schneider VP, BD & Sales SIRION Biotech GmbH Am Klopferspitz Martinsried Germany Any Gene to Any Cell