Gene Therapy/AAV
Navigating AAV Vector Biodistribution Analysis and Transgene Expression Using RNAscope™ in situ Hybridization Technology
The RNAscope ISH technology is an ideal solution for detecting AAV vector DNA and therapeutic transgene mRNA expression with morphological context, addressing critical questions on tissue biodistribution, cellular tropism, and vector performance in vivo. Screen for transduction efficiency of capsid or vectors designed to escape the immune response.
(1) S12 Nonclinical Biodistribution Considerations for Gene Therapy Products; Guidance for Industry; U.S. Department of Health and Human Services Food and Drug Administration; Center for Drug Evaluation and Research (CDER); Center for Biologics Evaluation and Research (CBER) May 2023; ICH-Safety
Learn How RNAscope Enables Detection of the AAV Vector DNA and Transgene mRNA on the Same Slide
Unlock Answers to Critical Questions for Your AAV-Based Studies.
Elevate Your Studies with RNAscope and Unlock a Deeper Understanding of Your AAV Therapeutic and Transgene Expression.
Morphology-Based Detection: Visualize AAV vector biodistribution and transgene levels at the single-cell level in intact fixed tissue.
Targeted Probes: RNAscope probes can be designed to target unique regions within the promoter of the AAV vector DNA or the transgene mRNA of the viral construct.
Visualize and Quantify: Measure percentage of AAV positive cells for vector and transgene expression.
Codon Optimized Expression Analysis: Distinguish transgene from endogenous sequences, at the single nucleotide level.
Representation of the Gene Therapy Probe Design Strategy
RNAscope probes can be designed to target unique regions within the promotor of the AAV vector DNA or the transgene mRNA of the viral construct.
Why RNAscope?
RNAscope enables tissue-based analysis of viral tropism, transduction efficiency, and persistence over time. Researchers also evaluate dosing, safety and efficacy, and functional markers using RNAscope ISH.
Characterize tissue biodistribution, cellular tropism, and transduction efficiency of your vector at single-cell resolution in conjunction with cell-type specific markers.
Measure abundance of AAV+ cells in target tissues and track vector persistence over time.
Quantify RNA expression of any vector cargo including sequence or codon-optimized human transgenes, CRISPR/Cas9, guide RNAs, or other regulatory non-coding RNAs.
Demonstrate therapeutic efficacy within vector transduced cells and tissues.
Easily Scale from small animal models to non-human primates with the capability to easily distinguish human transgenes from homologous host transcripts.
Recorded Webinar
Julio D Nieves, Associate Director, Imaging, Adverum presents:
2023 ASGCT - ' Using BaseScope Assay for Evaluation of Ocular Biodistribution of Gene Therapy Products' - Webinar
Fig 1. Identification of specific cells transduced by AAV-GFP following subretinal injection using the RNAscope 2.5 HD Duplex assay to simultaneously detect the CB promoter DNA sequence of the AAV vector (green) and the GFP transgene mRNA (red). Staining was observed in almost all of the retinal layers (with the exception of the choroid) in the transduced region, but no staining was observed in the non-transduced naïve region.
Fig 2. BaseScope staining allows visualization and quantification of transgene expression (red arrow) and AAV vector (green arrow) presence in treated liver samples
Products
Probes/Targets
Catalog Probes (Ex: CMV, CBA, WPRE, etc.)
If your gene of interest in not listed in our catalog, ACD can design and manufacture new in situ hybridization probes for you, To order or get more info on Made-to-Order New Targets Probes, please fill in the form below and an ACD Account Executive will contact you.
Made-to-OrderProfessional Assay Services
- Unparalleled expertise in RNAscope and BaseScope ISH
- High capacity and high throughput, delivering more than 10,000 slides per year
- Supporting dozens of gene therapy companies for research studies enabling pre-IND submission
- Team of specialists perform quantitative image analysis using HALO™ Software (Indica Labs)
- Board-certified pathologist review
- Actionable results delivered in weeks rather than months
Resources
Recorded Webinar
Visual detection and quantification of AAV and LV vector biodistribution and transgene expression in preclinical animal models with RNAscope and BaseScope™ in situ hybridization.
Application Note
Application note 'Tissue Biodistribution of AAV-based gene therapy with the RNAscope™ assay'
Flyer
Accelerate Gene Therapy Development with Powerful Visualization Tools for Biodistribution and Functional Analysis
Brochure
Accelerate Gene Therapy Development with RNA and Vector DNA Visualization Tools for Biodistribution and Safety Analysis
Publications
- Assaf, B.T., et al. (2018). Considerations for preclinical safety assessment of adeno-associated virus gene therapy products. Toxicol Pathol. 46(8):1020-1027
- Collaud, F., et al. (2018). Preclinical development of an AAV8-hUGT1A1 vector for the treatment of Crigler-Najjar syndrome. Mol Ther Methods Clin Dev. 12:157-174
- Golebiowski, D., et al. (2017). Direct Intracranial Injection of AAVrh8 Encoding Monkey beta-N-Acetylhexosaminidase Causes Neurotoxicity in the Primate Brain. Hum Gene Ther 28(6): 510-522.
- Kanaan, N. M., et al. (2017). Rationally Engineered AAV Capsids Improve Transduction and Volumetric Spread in the Central Nervous System. Molecular Therapy - Nucleic Acids.
- Borel, F., et al. (2016). Therapeutic rAAVrh10 Mediated SOD1 Silencing in Adult SOD1(G93A) Mice and Nonhuman Primates. Hum Gene Ther 27(1): 19-31.
- Polinski, N. K., et al. (2016). Impact of age and vector construct on striatal and nigral transgene expression. Mol Ther Methods Clin Dev 3: 16082.
- Grabinski, T. M., et al. (2015). A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures. PLoS One 10(3): e0120120.
- Polinski, N. K., et al. (2015). Recombinant adeno-associated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain. Neurobiol Aging 36(2): 1110-1120.
