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Analytical Platforms For Supporting Biodistribution Studies For AAV Gene Therapy

 Analytical Consideration For Supporting Biodistribution Studies For AAV Gene Therapy

The biodistribution studies should be designed to answer the following questions

  • Profiling and quantity of the DNA/RNA of the gene therapy products in the different tissues
  • Expression and profiling of the target RNA gene or protein products in different tissues
Profiling and quantity of the DNA/RNA of the gene therapy products in the different tissues

Profiling and quantification of the DNA/RNA of the gene therapy products and biofluids are the primary object of the BD studies. These data provides the selective tropism of the AAV WT, serotypes and AAV variants to the different tissues. It has become even more important as there are increased efforts to identify novel AAVs with improved selective tropism and low immunogenicity. Both screening of natural variants and capsid engineering are extensively employed.  

Currently, real-time quantitative polymerase chain reaction (qPCR) is considered the ‘gold standard’ for measurement of specific DNA (or, with a reverse transcription step, RNA as well) presence in tissues/biofluids. Digital droplet PCR is an emerging platform for these measurement. Quantification of nucleic acid sequences is important for assessing the relative amount of genetic material from a GT product and determining the kinetics of its accumulation or decay. The quantitative methods should be sufficiently sensitive with acceptable reproducibility. 

Expression and profiling of the target RNA gene or protein products in different tissues

In addition of tissue specific tropism of AAV vectors, the tissue specific promoters are engineered to drive the selective expression of the target tissues. For example, albumin promoter or ApoE promoter are used to drive expression in the liver. In these cases, quantitation of the target RNA and proteins provide addition information of the selective expression. It helps identify differential expression of genes in intended vs unintended tissues and evaluate the safety risk and benefit. This also help tease out the correlation of presence of DNA/RNA of gene therapy with the gene expression.

The methods for detection/quantitation of  RNA/proteins include: enzyme-linked immunosorbent assay (ELISA); immunohistochemistry (IHC); western blot; in situ hybridisation (ISH); digital PCR; flow cytometry; various in vivo and ex vivo imaging techniques; and other evolving technologies. 


The details on quantitation methods and case studies are provided in the link below

  • Real Time PCR
  • Digital Droplet PCR
  • Enzyme Linked Immunosorbent Assay 
  • Western Blot 
  • In-Situ Hybridization
  • Flow Cytometry  


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