Gene Therapy/AAV
Adeno-associated virus (AAV) has proven to be an effective gene therapy delivery vehicle. The primary considerations in gene therapy preclinical in vivo animal studies are vector biodistribution, cell-specific uptake and transgene expression, as well as safety against germ line transmission. While assays of extracted RNA and DNA from bulk tissue are simple, these provide only an average value for an entire complex tissue and lack cellular resolution.
RNA and DNA in situ hybridization using RNAscope™ and BaseScope™ assays combine the quantitative, molecular measurement, like PCR, with single-cell resolution in the context of intact tissue morphology. Moreover, RNAscope enables simultaneous detection of AAV vector DNA and transgene mRNA expression, and it can be multiplexed with cell markers. It is a simple and reliable method for analysis of AAV in routine FFPE tissues.
- AAV vector DNA detection and quantification in intact fixed tissue
- Probes designed to AAV promoter regions and anti-sense strand from AAV DNA detection
- Detection of therapeutic human- and codon-optimized transgene RNA without cross-hybridization to endogenous transcripts
- Morphology-based AAV vector biodistribution and transgene mRNA expression analysis in intact animal model tissues
- Visual and quantifiable assays to measure percentage of cells positive for vector and transgene expression
- Multiplexed analysis of cell-specific AAV and transgene mRNA with cell marker probes, e.g., neuronal, astrocyte, glia, hepatocytes, Kupfer cell markers
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-OrderPharma 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.
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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.
Application Note
Application note "Detection of immune cell checkpoint and functional markers in the tumor microenvironment by the RNA in situ hybridization RNAscope® assay'
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