Slide 1: PCR Methods for the Characterization of AAV-based Drug Product from Titer to Potency
PCR Methods for the Characterization of AAV-based Drug Product from Titer to Potency Pavlina Wolf Gene Therapy Analytical Development 2022
Slide 2: Adeno-Associated Virus (AAV) is an Ideal Vector for Gene Transfer
Adeno-Associated Virus (AAV) is an Ideal Vector for Gene Transfer • AAV is a small (4.7 Kb), single stranded, non-pathogenic DNA virus • Transduces both dividing and non-dividing cells • Long-term transduction following a single administration • Different AAVs exhibit distinct tropism for various ti
Slide 3: rAAV CQAs Emerge as a Function of Vector and Process Design
rAAV CQAs Emerge as a Function of Vector and Process Design Design & Selection Plasmid AAV Cell Line Small Scale Plasmid&AAV Production Purification CQAs Upstream Process Transfection Harvest POTENCY Downstream Process Purification Fill/Finish Formulation Lot Release QC testing 3 CONFIDENTIAL – DO N
Slide 4: PCR Methods in a Typical QC Testing Panel for AAV Lot Release
PCR Methods in a Typical QC Testing Panel for AAV Lot Release VG Titer Transduction Efficiency Transgene Expression Gene Editing Trans-protein expression Trans-protein function 4 CONFIDENTIAL – DO NOT DISTRIBUTE
Slide 5: PCR Methods for Potency Evaluation
PCR Methods for Potency Evaluation VG Titer Transduction Efficiency Transgene Expression Gene Editing Trans-protein expression Trans-protein function 5 CONFIDENTIAL – DO NOT DISTRIBUTE
Slide 6: Two Main Technologies for PCR Quantitation
Two Main Technologies for PCR Quantitation SAMPLE RNA RT DNA PCR qPCR - fluorescence detection ddPCR - direct counting of positive partitions adjusted for probability of co- occupancy with random distribution REPORTER DYE 𝑁𝑁𝑛𝑛𝑛𝑛𝑛𝑛 ln 𝑁𝑁 𝑐𝑐 = − 𝑉𝑉𝑟𝑟𝑟𝑟𝑟𝑟…
Slide 7: Real Time PCR Quantitation
Real Time PCR Quantitation qPCR1 1. Estimates target abundance against a calibration curve Requires standards of known concentration 2. Precision depends on multiple variables from instrument to inhibitors Measure 1.25-1.5-fold differences at best2 Assay precision deteriorates at low copy numb
Slide 8: Droplet Digital PCR Quantitation
Droplet Digital PCR Quantitation ddPCR1 1. Estimates target abundance from positive to total partition ratio2 Requires only known volume. Prior estimate of titer helpful but not essential 2. Precision ≈ # of partitions counts Measure differences as low as <1.2-fold3 Assay precision drops at bo
Slide 9: Case Study: Comparison of VG Titers by qPCR vs ddPCR
Case Study: Comparison of VG Titers by qPCR vs ddPCR 8×10 13 6×10 13 Titer (vg/ml) 4×10 13 2×10 13 0 Lot 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 AAV1 AAV2 AAV3 AAV4 AAV1 AAV2 AAV3 AAV4 Average %CV by qPCR = 17% Average %CV by ddPCR = 11% 9 CONFIDENTIAL – DO NOT DISTRIBUTE
Slide 10: Analytical Criteria for PCR based Method Development
Analytical Criteria for PCR based Method Development SENSITIVITY SPECIFICITY ACCURACY PRECISION LINEARITY • # of copies • Detects target • Differences • Repeatability • Dilutional accurately measured in the assay sequence between Precision and 3-5 non-zero points measured robustness of the • LOD
Slide 11: Acceptance Criteria for PCR Based Titer
Acceptance Criteria for PCR Based Titer SENSITIVITY SPECIFICITY ACCURACY PRECISION LINEARITY • Acceptance • NTC < LOD • QCs spiked • CV ≤ 25% for • qPCR amplification criteria • no in relevant intermediate efficiency between depend on interference in matrix with precision 90-110% assay extracted est
Slide 12: LoB/LoD and LOQ in qPCR
LoB/LoD and LOQ in qPCR Methods to Establish LoD and LoQ LoD Based on 20 Replicate Std Curves • LoB/LoD Calculation per CLSI guideline EP17 LoB = mean blank + 1.645 × σblank LoD = LoB + 1.645 × σlow concentration sample 100 95 • Problem: above equations apply to linear Average Positive (%) respo
Slide 13: Other AAV VG Titer Considerations
Other AAV VG Titer Considerations qPCR vs ddPCR? Titer Considerations • Is ddPCR preferred over qPCR for • Research material typically uses regulatory filings? vendor titer with probe against ITR No specific guidance but ddPCR has region greater precision Overestimated concentration Either ass
Slide 14: Regulatory Guidance for Potency Testing of GT Products
Regulatory Guidance for Potency Testing of GT Products What is Potency? Guidance Documents • Potency is defined as “the specific ability or • FDA-2008-D-0520: Guidance for Industry: capacity of the product, as indicated by Potency Tests for Cellular and Gene appropriate laboratory tests or by Therap
Slide 15: Potency of AAV Drug Product
Potency of AAV Drug Product • A gene therapy vector relies on 2 main biological activities for its potency 1. the ability to transfer a genetic sequence to a cell 2. the biological effect of the expressed genetic sequence cells in vitro Biological Assays tissue Platforms to Measure Potency in vivo N
Slide 16: EDIT-103 for Autosomal Dominant Retinitis Pigmentosa (adRP)
EDIT-103 for Autosomal Dominant Retinitis Pigmentosa (adRP) • An inherited autosomal dominant retinal disease leading to blindness in later life • Symptoms: o Decreased night vision (nyctalopia) o Loss of peripheral vision (tunnel vision), and eventually significant decline in central vision • No ap
Slide 17: EDIT-103: Dual AAV-Based Knockout and Replace Therapeutic Strategy
EDIT-103: Dual AAV-Based “Knockout and Replace” Therapeutic Strategy • Agnostic to any RHO mutation – thus will knockout any dominant gain-of-function rhodopsin mutant • Step 1: Both mutant and normal endogenous RHO will be knocked out in the treated area • Step 2: Exogenous normal RHO (resistant to
Slide 18: Dual Vector Strategy Adds Complexity to Potency Assay Design
Dual Vector Strategy Adds Complexity to Potency Assay Design • EDIT-103 delivers two transgenes Vector 1 • Expression of Cas9 endonuclease RHO x AAV5 #1 promoter SaCas9 Vector 2 • Expression of gRNA • Expression of coRHO replacement at mRNA RT-ddPCR readout • coRHO expression-driven restoration to
Slide 19: In-Vitro Potency Assay Platform Strategy
In-Vitro Potency Assay Platform Strategy SUITABLE CELL LINE MEASURABLE FEATURES Transgene Target expression editing CONFIDENTIAL – DO NOT DISTRIBUTE
Slide 20: In-Vitro Potency Assay Platform Selection
In-Vitro Potency Assay Platform Selection In-Vitro Platform AAV5 Transducibility • HEK293T cells • contain but don’t express endogenous RHO target • AAV5 transducible • Additional cell line engineering required to enable the expression of transgenes under RHO promoter • Cell culture conditions optim
Slide 21: ddPCR Readouts for mRNA Expression and Target Editing
ddPCR Readouts for mRNA Expression and Target Editing RT-ddPCR for coRHO mRNA expression Drop-off ddPCR for RHO gene editing Drop-off Reference Primers/Probe Primers/Probe FAM FAM HEX Reference Gene Cut Site HEX HEX Target Gene xxx INDEL 21 CONFIDENTIAL – DO NOT DISTRIBUTE
Slide 22: Protein Replacement Potency Assay
Protein Replacement Potency Assay Expression Response Next Steps in Development 120 • Assay will be transferred to AD/QC for (Normalized to Reference) 100 method optimization / validation Establish Relative Potency GOI Expression 80 Optimize key assay steps 60 Establish system suitability crit
Slide 23: Editing Potency Assay
Editing Potency Assay Editing Response Next Steps in Development 120 • Assay will be transferred to AD/QC for (Normalized to Reference) method optimization / validation 100 Establish Relative Potency 80 Optimize key assay steps %Editing 60 Establish system suitability criteria 40 • Assay accep
Slide 24: Summary
Summary VG Titer Assay Development Potency Assay Development • Don’t rely on ITR based vendor • Start potency assay development titers early! • Develop target gene specific assays Technically challenging Slow TAT on assays • Test primers and probes in silico PCR based assays to prove editing a
Slide 25: Acknowledgements
Acknowledgements Mariacarmela Allocca Lily Maxham Racheal D’Souza Mark S. Shearman Pallavi Gambhire Cristina Silvescu Georgia Giannoukos Adrian Timmers Lily Li Khushboo Undavia Zoe Liu Jennifer Wendelken Eugenio Marco Kate Zhang * Alphabetical order according to last names 25 CONFIDENTIAL – DO NOT D