With >11 years’ experience in genome editing and stem cell technologies, ASC provides ISO:9001 quality, customizable solution-oriented services for advancing preclinical assay development and drug screening for cell and gene therapy pipelines and bioprocessing/ bioproduction:
- In silico, in vitro and in vivo models for target discovery
- Proof-of-concept studies, IND-enabling preclinical assay development including potency assay, dose ranging, biodistribution and efficacy studies in cell line and animal models for your drug discovery and screening applications
- CMC consultation, titration, biologics analysis, and potency assay development, and more…
Customize each part of your project to fit the stage of your research.
Drug Toxicity and Efficacy Testing
Custom In Vivo Assay
Clinical trials in gene and cell therapies have advanced rapidly in the past decade, reflecting significant advances in scientific innovation with better translation to clinical settings. Assessing the growth in this area of therapeutics, FDA anticipates that by the year 2025 they expect to approve 10-20 gene and cell-based therapy products a year.1 To date, majority of the gene therapies involving delivery to in vivo and ex vivo target cells (Ex. CAR-T), have utilized recombinant viral vectors such as adeno-associated virus (AAV), adenovirus, lentiviruses, vaccinia virus, and retroviruses. As with any therapeutic product developed for use in humans, an initial set of preclinical and non-clinical data aimed at assessing the risks/ safety and benefit/ intended efficacy profile is required. Preclinical (or non-clinical) studies that are conducted in a variety of in vitro (human cell lines) and in vivo (animal) models relevant to the targeted human system or disease, to gather as much safety and efficacy information can maximize the chance of success in human clinical trials.
In particular, gene therapies are highly specific to humans and therefore preclinical testing in human-relevant system is critical. And for virus delivery of gene therapy products, tissue infectivity (viral biodistribution of recombinant viruses) and viral shedding are assessments required by regulatory authorities.2 ASC’s genome editing experts can equip you with clinically relevant cell line or animal models as well as provide you with the technical expertise in designing preclinical assays for your gene therapy programs, such as assessing the right serotype of viral vector, biodistribution, viral shedding, virus titer (and dose determination), potency assays and more.
Examples of Assay Development for a Recombinant Adeno-Associated Virus
Viral biodistribution in mouse tissue
Figure. Standard curve developed to measure viral biodistribution for a recombinant adeno-associated viral (AAV) vector in mouse tissues. Genomic DNA (gDNA) sample from various tissues (heart, kidney, cerebral cortex, testis, lung, spleen, and liver) was extracted from PBS-only injected C57BL/6wild type (WT) mouse tissues, and added to a serial dilution of plasmid containing the gene of interest. Quantitative PCR (qPCR) was performed to generate a standard curve for the biodistribution of the gene of interest. Assay sensitivity: ≤50 copies of plasmid per 1 μg gDNA from mouse tissue. Note: No signal was detected in all negative controls (non-plasmid samples with and without tissue gDNA).
Virus shedding in human fluid samples
Figure. Standard curve developed to measure viral shedding of a recombinant adeno-associated viral (AAV) vector in human body fluid samples. A serial dilution of plasmid containing the gene of interest was spiked in various human body fluid samples (blood, urine, semen and saliva), and the DNA (both viral DNA and gDNA, if possible) was then extracted. Quantitative PCR (qPCR) was performed to generate a standard curve to measure viral shedding. Note: No signal was detected in all negative controls (DNA extracted from normal human body fluid samples without plasmid spiked-in).
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%.
1. U.S. Food & Drug Administration. (2019). Statement from FDA Commissioner Scott Gottlieb, M.D. and Peter Marks, M.D., Ph.D., Director of the Center for Biologics Evaluation and Research on new policies to advance development of safe and effective cell and gene therapies. Retrieved from https://www.fda.gov/news-events/press-announcements/statement-fda-commissioner-scott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics
2. MacLachlan, T. K., McIntyre, M., Mitrophanous, K., Miskin, J., Jolly, D. J., & Cavagnaro, J. A. (2013). Not reinventing the wheel: applying the 3Rs concepts to viral vector gene therapy biodistribution studies. Human Gene Therapy Clinical Development, 24(1), 1-4.
Droplet digital PCR (ddPCR) is a highly precise method for nucleic acid sequences quantitation, which measures the absolute number of target molecules in a sample, thereby avoiding the dependence of comparison to relative amplification of a known standard sample. In ddPCR, a PCR sample (consists of template DNA, primers, fluorescence-quencher probe, and a PCR master mix) is partitioned into greater than 10,000 droplets. After amplification, the fluorescent droplets containing the target sequence are scored as positive and droplets without fluorescent signal are scored as negative. Poisson statistical analysis of the positive and negative droplets provides absolute quantification of the target sequence.
ASC’s 11+ years’ experience in animal and cell line model genetic engineering and stem cell technology is a powerful resource you can leverage to advance your gene and cell therapy pipeline. Our mission is to equip biotechnology companies with a series of optimized tools for supporting therapeutic target discovery, drug screening, preclinical assay development, preclinical therapeutic development, and bioprocessing/ bioproduction.
Preclinical & Biologics Analysis: From Cell and Animal Modeling to Assay Development
|Cell line models
Cell line characterization
IND-Enabling Studies: We can help you navigate FDA’s increasingly regulated gene and cell therapy requirements from pre-IND, preclinical safety and efficacy requirements all the way to your IND filing. Our multidisciplinary think-tank will work you every step of the way to provide you with scientific consultation for study design development, regulatory compliance, safety and efficacy endpoint determination as well as preclinical custom services to engineer, characterize, validate and test suitable in vitro and in vivo models for target engagement and efficacy assays for your therapeutic candidates.
We can customize each part of your project to fit the stage of your research. Be it cell replacement therapy or adeno-associated virus (AAV) based gene therapy, we can help. Below is a selected list of assays/services that we can help you with:
In vitro and in vivo model generation and evaluation
Potency assay development (DNA/ RNA/ Protein)
Cell viability & Cell-based assays
Consultation for design of experiments
Vector infectivity & copy number testing
Viral TCID50 titer
Titration and immunogenicity assessment
Don’t see a service or assay? Contact us for a free & confidential consultation to discuss your requirements.
Project Workflow: Initiation through Completion
MEET INDUSTRY REGULATORY COMPLIANCE REQUIREMENTS
ISO 9001:2015 Cert # 1100091
ISO 13485:2016 Cert # 1100090
Biosafety Level 2 Laboratory
Studies Performed in a Manner Consistent with Principles of GLP
FDA 21 CFR Part 58
QA Review of Protocols
- Li, R., Baskfield, A., Lin, Y., Beers, J., Zou, J., Liu, C., ... & Zheng, W. (2019). Generation of an induced pluripotent stem cell line (TRNDi003-A) from a Noonan syndrome with multiple lentigines (NSML) patient carrying a p. Q510P mutation in the PTPN11 gene. Stem cell research, 34, 101374.
- Li, R., Pradhan, M., Xu, M., Baskfield, A., Farkhondeh, A., Cheng, Y. S., ... & Rodems, S. (2018). Generation of an induced pluripotent stem cell line (TRNDi002-B) from a patient carrying compound heterozygous p. Q208X and p. G310G mutations in the NGLY1 gene. Stem Cell Research, 101362.
- Poli, M. C., Ebstein, F., Nicholas, S. K., de Guzman, M. M., Forbes, L. R., Chinn, I. K., ... & Coban-Akdemir, Z. H. (2018). Heterozygous Truncating Variants in POMP Escape Nonsense-Mediated Decay and Cause a Unique Immune Dysregulatory Syndrome. The American Journal of Human Genetics, 102, 1-17. https://doi.org/10.1016/j.ajhg.2018.04.010
- Vozdek, R., Long, Y., & Ma, D. K. (2018). The receptor tyrosine kinase HIR-1 coordinates HIF-independent responses to hypoxia and extracellular matrix injury. Sci. Signal., 11(550), eaat0138