Detect, localize and quantify vector and therapeutic transgenes using the RNAscope® and BaseScope™ assays

The highly specific and sensitive single-molecule RNA ISH technologies RNAscope® and BaseScope™ are ideal solutions to assess tissue-based gene expression and address questions about therapeutic gene delivery vectors (both viral and non-viral) and transgene expression. With regards to viral vectors such as adeno-associated virus (AAV) and transgene pharmacodynamics and pharmacokinetics, the RNAscope® and BaseScope™ assays are ideal methods to:

  • Visualize vector tissue distribution and transgene expression within morphological context
  • Quantify vector copy number and transgene expression
  • Differentiate transgene from endogenous mRNAs
  • Simultaneously detect vector DNA and transgene RNA
  • Confirm CRISPR/Cas9 gene editing-based therapy with the BaseScope™ assay
  • When detection of protein is not optimal
    • Noncoding RNA (ncRNA), RNA interference (RNAi), and mRNA-based therapies
    • Lack of reliable or available IHC antibodies

Detection methods used in gene therapy studies

Gene therapy is an experimental technique that introduces genetic material into a cell to treat or prevent disease, either by replacing or inactivating a mutated gene or introducing a new gene to fight the disease. Several gene delivery methods are used for this purpose, including viral and non-viral vectors. Adeno-associated virus (AAV) is the most commonly used delivery method due to its gene transfer efficiency, stable transgene expression, broad tropism, and ability to achieve systemic delivery, making it a safe and efficient gene delivery method for non-dividing cells.

However, once administered, confirmation of delivery to and expression in the correct targeted cells, as well as off-targeting effects on other genes, must be accurately assessed. To achieve this, several detection methods have been employed but have limitations:

 

Method

Advantages

Limitations

IHC

  • Detects the transgene protein
  • Provides spatial information
  • Antibody availability
  • Antibody specificity
  • Not quantitative
  • Not ideal for the detection of cells producing a secreted protein
  • Not ideal for the detection of RNAi or ncRNA therapies

Western blot

  • Detects the transgene protein
  • Quantitative
  • No spatial information
  • Antibody availability
  • Not ideal for the detection of cells producing a secreted protein
  • Not ideal for the detection of RNAi or ncRNA therapies

PCR

  • Detects both vector DNA and transgene RNA
  • Quantitative
  • No spatial information
  • No single-cell information

NGS

  • Detects both vector DNA and transgene RNA
  • Quantitative
  • No spatial information

Traditional RNA ISH

  • Detects the transgene RNA
  • Provides spatial information
  • Time consuming
  • Laborious
  • Sensitivity and specificity issues
  • Not quantitative

 

Benefits of RNAscope Assay for gene therapy studies

The highly specific and sensitive single-molecule RNA ISH technologies, RNAscope® and BaseScope™, are ideal solutions to validate correct targeting by gene therapy delivery methods. The advantages of these assays include:

  • Reproducible, consistent assays to validate gene therapies between and within laboratories
  • Robust assays to measure the potency of the vector
  • Quantifiable expression data with spatial information
    • Single-molecule, single-cell resolution
  • Specific probes readily available or can be rapidly designed
    • A single nucleotide difference is sufficient for differential detection
  • Easy to perform assay that can be completed in 1 day

The RNAscope® technology enables visualization and quantification of viral copy number and transgene expression with cell-specific localization in intact, fixed tissue. Some applications of the technology include:

  • Visualization of vector tissue distribution and transgene expression with morphological context in any animal model
  • Quantification of vector copy number, transgene expression and AAV+ cell number in target and non-target tissues
  • Differentiation of transgene from endogenous mRNAs
  • Simultaneous detection of vector DNA and transgene RNA
  • Confirmation of CRISPR/Cas9 gene editing-based therapy with the BaseScope™ assay
  • Obtaining visual information about penetration of AAV from the vasculature into the target tissue 
  • Quantification of cell-specific transgene RNA expression over time to assess expression maintenance and transduced cell clonality
    • Distinguish uniform expression vs. clonal populations with heterogeneous expression
    • Examine stability of transgene over time
  • Visualization of the vector ITR with spatial resolution
  • Detection of off-targeting effects by validating targeting of correct gene in the correct tissue or cell type
  • When detection of protein is not optimal
    • Noncoding RNA (ncRNA), RNA interference (RNAi) and mRNA-based therapies
    • Lack of reliable or available IHC antibodies
    • Identification of the producing cell when targeting a secreted protein
    • Targeted gene contains many splice variants/isoforms, making antibody specificity a concern


AAV transgene structure and the RNAscope® or BaseScope™ probe design
 


Schematic of AAV transgene detection in a cell with the RNAscope® or BaseScope™ assay

 

The RNAscope® assay in publications on gene therapy:

 

  • Detection of rAAV2/5 DNA in the rat striatum:
    Neuroscience research uses gene therapy approaches to deliver therapeutic agents or to generate animal models. Grabinski et al. injected rAAVs into the striatum of rats and used RNAscope® ISH to detect ectopic viral DNA in free-floating brain sections. Robust rAAV DNA ISH signal was detected in the injected striatum but absent in the uninjected hemisphere. This study is the first demonstration of a method for identifying neurons transduced with rAAVs using RNAscope® ISH for the viral genome particles. 
    A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures
     
  • Impact of age and vector construct on viral-mediated gene transfer in rat brain:
    Gene therapy using viral vectors has shown great promise in preclinical models of Parkinson's disease but clinical trial success remains elusive. Polinski et al. found that advanced age impacts the efficacy of multiple viral vectors in the brain, including AAV and lentivirus. Dual RNAscope® ISH-IHC was used to visualize the cellular location of viral particles and demonstrate that in both the young and aged brain, viral genomes overwhelmingly colocalized with NeuN neurons, but were rarely observed in astrocytes. Also, contrary to other reports, there was no increase in S100β astrocytes in the aged striatum compared to young striatum. RNAscope® ISH also showed that different viral vectors result in lower GFP expression in different regions of the aged brain.
    Impact of age and vector construct on striatal and nigral transgene expression
    Recombinant
    adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain
     
  • Silencing of the Huntington’s disease gene with RNAi using AAV9-mediated delivery of microRNA:
    Many studies have examined the ability of RNA interference (RNAi) via AAV to downregulate the Huntington’s disease gene Htt in transgenic mouse models of Huntington’s disease but few silencing studies have been done in knock-in mouse models such as the Q140/Q140 mouse. Keeler et al. injected an AAV9 construct expressing an artificial micro RNA against mouse Htt (miRHtt) into the striatum of Q140/Q140 mice. Investigation of striatal Htt at the cellular level by RNAscope® ISH revealed ~50% lower Htt mRNA expression in AAV9-GFP-miRHtt-injected striatum due to a partial reduction in the number of copies of mutant Htt mRNAs per cell.
    Cellular analysis of silencing the Huntington’s disease gene using AAV9 mediated delivery of artificial microRNA into the striatum of Q140/Q140 mice
     
  • Delivery of the secreted protein EPO to the liver via mRNA-nanoparticle therapy:
    Non-viral delivery systems for therapeutic use have predominantly focused on plasmid DNA, siRNA, miRNA, and antisense oligonucleotides. DeRosa et al. explored the delivery of exogenous mRNA therapy, which provides transient production of therapeutic proteins without the need for nuclear delivery or risk of insertional mutagenesis. Lipid nanoparticles were used to deliver hEPO and hFIX mRNA by IV injection. RNAscope® ISH confirmed delivery of hEPO and hFIX mRNA to hepatocytes and showed that, following mRNA delivery, there was increased EPO mRNA, serum protein, and hematocrit, demonstrating that the exogenous mRNA-derived protein maintains normal activity similar to endogenous protein.
    Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system
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