Potency assays are essential to validate the biological function of your cell and gene therapy biotherapeutics and to establish a quality product that is efficacious and safe. ASC’s multidisciplinary expertise in genetic engineering, cell-based assays and therapeutics (including stem cell technology), and adeno-associated virus (AAV)-based gene therapy, can design and develop preclinical assays specific to your early-stage biotherapeutics to enable critical go-no-decisions and regulatory compliant data for IND-filing: assays to determine mechanism of actions at a DNA/ RNA/ protein levels, tissue-specificity and infectivity of AAV serotypes, activity assays to determine gene expression and reduction of disease markers, in vivo assays for determining cell replacement function, and more.
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Example of Pharmacodynamic (PD) Study to Evaluate CRISPR/Cas9 Genome Editing Efficiency of a Recombinant Adeno-Associated Viral Vector
In Vitro Indel detection using droplet digital PCR (ddPCR) following CRISPR-SaCas9 genome editing at a specific genetic locus of choice in HEK293T cells
Figure. HEK293T cells in 12-well cell-culture plates were co-transfected with 0.5µg CMV-SaCas9 plasmid and 1µg U6-GuideRNA plasmid (1:2 ratio) using Lipofectamine3000. Indel detection at the locus was done by ddPCR using NHEJ genome editing detection drop-off assay (Bio-Rad) 48 hours post-transfection. The assay included one set of primers and two probes annealing the amplicon (FAM-labeled Reference probe and HEX-labeled Drop-off probe). Indel (blue, 37.6%) and unedited (orange) amplicon droplet populations are indicated. Gray droplets population includes droplet with no target sequence.
In Vivo knock-in detection in tissue samples at a specific genetic locus in a Humanized mouse model using ddPCR following CRISPR-SaCas9 genome editing
Figure. Knock-in abundance following CRISPR-SaCas9 genome editing at a specific genetic locus of choice in C57BL/6 WT mouse. Combined delivery of two recombinant AAV viruses containing the gene of interest and SaCas9-GuideRNA was applied at a total dose of 1.8E+11VG/Kg. To determine the knock-in abundance, two ddPCR primer-probe assays were used: the first assay includes a primer set that amplifies the target locus only following insertion (targeting the junction of insertion), and a FAM-labeled probe. The second assay, including a HEX-labeled probe, targets a reference region and is used to count all genome copies. KI abundance is calculated by the ratio of FAM-positive droplets (KI amplicons) to all HEX positive droplets (Reference amplicons). Gray droplets population includes droplet with no target sequence. KI% detected here is 10%.
Potency of a drug or biologic (cell and gene therapy; CGT) is the specific ability of capacity of a product in terms of the concentration or amount required to produce a defined effect. This is measured by appropriate laboratory tests specific to the mechanism of action of the drug. For biotherapeutic products, it means a valid biologic assay or a matrix of assays to determine the claimed biological activity of the drug. With respect to gene therapy products, each product has a unique mechanism of action and therefore unique potency. Unlike pharmaceutical drug candidates, developing assays to determine potency of cell and gene therapy products by quantitative measurement of its capacity to elicit a defined biological effect is very complex and not fully defined. Potency assay should be quantitative and expressed in units of activity calibrated against an international/ national/ in-house reference standard(s).
FDA guidance recommends that potency assays include in vitro and/or in vivo tests as well as full characterization of the biological processes early in the drug development phase in order to assure identity, quality, purity, strength (potency), and stability of the product. In fact, the more the product is characterized early-on in the development through sensitive and precise preclinical assays, the better the understanding of the safety and efficacy of the product. Some of the challenges for developing potency assays tests for CGT products include: the inherent variability of starting materials in case of cell-based therapies, limited lot size and materials for testing, limited stability, lack of appropriate reference standards, interactions between multiple active ingredients (vectors containing multiple genes), complex MoA (Ex. infection, integration, and expression of transgene), and in vivo fate of the product (Ex. migration from site of administration, cellular differentiation, viral infection and transgene expression). Such challenges require stepwise analysis of the investigational product, and incremental development of potency assays.
Applied StemCell has a unique blend expertise to provide custom services for designing preclinical potency assays for early-stage cell and gene therapy products, that are compliant with FDA guidance and for enabling IND filing.
For example, in the case of gene therapy products using adeno-associated viral (AAV) vector-based delivery, we offer in vitro and in vivo tests to assay potency of the drug for its specific mechanisms of action at DNA, RNA and protein levels using ddPCR, qPCR, western blots, ELISA, immunohistochemistry, and more. Potency assay matrix would also include cell line and animal model selection/ development specific to gene therapy drug and specific promoter, viral transduciblity and infectivity, tissue-selectivity of AAV serotype(s), dose-ranging studies, activity assays (enzymatic activity), assay of relative potency against a reference material, mitigation of common impurities during sample processing, and more. We will develop and optimize assays specific to your investigational drug to ensure specificity, validity, and reproducibility.