A Platform Approach to Functional Potency Assay Development for Gene Therapy Products

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A PLATFORM APPROACH TO FUNCTIONAL POTENCY ASSAY DEVELOPMENT FOR GENE THERAPY PRODUCTS

November 20, 2019 Karen Doucette, Director of Operations Gene Therapy Analytical Development Summit

Renaissance Boston Waterfront Hotel

TABLE OF CONTENTS

• Streamlining development of functional potency assays

• Case studies for diverse product types with custom and complex MOA

• Bridging the gap between R&D and cGMP for commercially viable potency assays

Multiple delivery systems:

 Plasmid DNA  Viral vectors  Bacterial vectors  Human gene editing  Patient-derived cellular gene therapy

Selection based on:

 Tissue tropism  Packaging capacity  Immunogenicity

Image: CBER, US FDA Mechanism of action depends on: https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/what-gene-therapy

 In vivo or ex vivo  Vector or non-vector  Viral vector or non-viral vector

Potency is defined as “the specific ability or capacity of the product…to effect a given result.”

Validation Parameters:

 Accuracy  Precision (Repeatability, Intermediate Precision)  Specificity  Linearity and Range  System Suitability  Robustness

Multi-Step Process Based on Central Dogma of Molecular Biology 1. Gene is delivered to cell

2. Gene is turned on

Potency assay must reflect expression and activity 3. Gene encodes for protein

4. Protein is functional

Infection Transcription Translation Activity

Protein functions Vector/ • Antibody DNA mRNA Protein Function • Enzyme Plasmid • Messenger • Structure • Transport • Storage Gene Cell Nucleus Cytoplasm Body • Channels • Binding • Other

Multi-Step Process Based on Central Dogma of Molecular Biology 1. Gene is delivered to cell

DOE for Transduction Efficiency 2. Gene is turned on

MOI Time Points Enhancers 3. Gene encodes for protein

1x104 24 hr Sodium butyrate 4. Protein is functional 1x105 48 hr Neuraminidase 72 hr Etoposide Infection None

Virus: AAV2-CMV-GFP Cells: HEK293 cells (P48)

Vector/ No AAV-GFP 4 MOI 1x105 DNA mRNA Protein Function MOI 1x10

Plasmid

FSC Gene Cell Nucleus Cytoplasm Body MFI119 MFI605 GFP

Design: • AAV9-GFP under ubiquitous promoter • 6 cell lines selected for screening based on literature for potential be permissibility to AAV9 • 2 MOIs • With and without enhancer (neuraminidase) Transduction Efficiency Screening

Conclusions: 120 • 2 cell lines were transducible without enhancer (#2 100 and #6) • Enhancer increased transduction in 5 of 6 cell lines 80 (green bars) 60 without enhancer • Cell line #6 had the highest transduction efficiency, 40 and comparable efficiency with and without enhancer, with enhancer 20

so this cell line was selected %of expressing cells GFP 0 1 2 3 4 5 6 Cell Line #

Multi-Step Process Based on Central Dogma of Molecular Biology 1. Gene is delivered to cell

2. Gene is turned on

3. Gene encodes for protein

4. Protein is functional

Infection Transcription

Vector/ Ubiquitous Tissue-Specific DNA mRNA Protein Function Plasmid Promoters Promoters (TSP) CMV Liver CßA Retina EF1a Brain Gene Cell Nucleus Cytoplasm Body SV40 Others

Design: • AAV5-XXX-eGFP • Cell line from target tissue MOI 1E5 / CMV • Ubiquitous promoter (top panels) and tissue-specific promoter (TSP, bottom panel) • 2 MOIs • With and without enhancers (sodium butyrate and etoposide) MOI 1E6 / CMV

Conclusions: • Capsid is permissible to the cell line MOI 1E6 / TSP • Enhancers increased transduction with ubiquitous promoter • Cell line does not contain sufficient endogenous transcription factors for TSP

Multi-Step Process Based on Central Dogma of Molecular Biology 1. Gene is delivered to cell

2. Gene is turned on

3. Gene encodes for protein

4. Protein is functional

Infection Transcription Translation

Specificity from endogenous protein: Vector/ DNA mRNA Protein Function Plasmid  Subtract background from NTC  Knock out endogenous with CRISPR  Knock down endogenous with LV-shRNA Gene Cell Nucleus Cytoplasm Body

ABCG2/BCRP Knockdown

VC  Cell line with endogenous expression  Lentiviral shRNA constructs with 5 different sequences  Western blot and RT-qPCR to confirm reduced expression  Stable knockdown cell line used for FDA-recommended functional activity assays

Multi-Step Process Based on Central Dogma of Molecular Biology 1. Gene is delivered to cell

2. Gene is turned on

3. Gene encodes for protein

4. Protein is functional

Infection Transcription Translation Activity

Protein Functions Vector/ DNA mRNA Protein Function • Antibody Plasmid • Enzyme • Messenger • Structure • Transport Gene Cell Nucleus Cytoplasm Body • Storage • Channels • Binding • Other

Enzyme Transporter Transcription Ligand Interaction Factor X X X X

E X Y Promoter

Y Y Y Y

LC-MS/MS for LC-MS/MS for ELISA or cell-based PLA assay for X binding formation of accumulation of ddPCR for mRNA bioassay for binding of X to Y with no known product Y substrate X expression of Y causing downstream Y downstream mechanism e.g. RPE65 ABCA4 훃-catenin VEGF ??

Therapeutic Gene

Start functional potency assay development as early as possible

Phase 1/2 Phase 3

Start bridging the gap from R&D to cGMP once assay feasibility is established

Potency Feasibility/ Development/ Qualification Spec Setting Validation Assay POC Optimization

4-6 months 4-6 months 3-6 months 3-6 months 3-6 months

Maintain a development narrative to justify assay conditions based on product knowledge

R&D BRIDGE GMP

R&D scientists with different skill sets Establish assay-specific time and Multiple GMP analysts must be trained in Analysts for POC (cell culture, molecular material requirements for training new multiple lab techniques for a single assay biology, bioassay, analytical) analysts on custom assays

Explore commercial sources and GMP R&D grade, may come from academic grade material during assay GMP grade supplied by approved vendors Reagents or non-GMP sources with no incoming optimization, gather data to set specs and part of reagent management system reagent specifications for critical reagents QA begin vendor qualifications, and Vendors May not be approved by QA explore alternatives if vendor does not Approved by QA meet quality requirements Begin IOPQ process, and plan for May not be validated or have Validated (IOPQ) with inter- and intra-lab Equipment feasibility and cost of establishing redundancy redundancy redundancy of customized equipment Pursue validation package if available, Validated (i.e., meet Part 11/ Annex 11 data Software May not be validated establish compliant work-around integrity requirements for unique logins, audit procedures if not trail, etc.) Protocol driven and recorded in lab Transferred to SOP and forms, with system Processes Process map, involve QC lab early notebooks and sample suitability criteria established

Using traditionally R&D assays for GMP release

 Involve the QC lab early Business Process Modeling and Notation (BPMN)

 Prepare QA to qualify R&D vendors and Example: What the task of splitting a cell culture flask looks like in BPMN validate R&D equipment & software Each Task of the assay/SOP can be mapped in a similar fashion  Process Map to streamline transfer to GMP

Robust

Reproducible

Compliant R&D protocol step of “Split flask” = a 4-6 step process in a method SOP

Meet our experts here at the conference

Visit any of our locations Boston – Philadelphia - San Diego

https://www.absorption.com/kc/potency-assay-guide-2019/

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