- Feb 2023
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www.science.org www.science.org
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Quantification of hIDUA-positive cells in DRG (five distinct DRG per animal, n = 3 animals per group), spinal cord (lower motor neurons, two to five distinct sections per animal, n = 3 animals per group), cerebellum, and cortex in NHP (five ×20 magnification fields per region, n = 3 animals per group). Data shown as mean; error bars indicate SD. Wilcoxon test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. See also data file S1.
The difference you see in hIDUA in DRG neurons when miRNA183 is present is incredibly stark. By normalizing the data and showing reductions in sheer percentage, you can really appreciate the effect that the RNA silencing trigger has
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The total number of positive cells per ×20 magnification field was counted manually using the ImageJ or Aperio Image Scope cell counter tool on a minimum of five fields per structure and per animal (exact number of fields or structure counted is noted in the figure legends).
This will help explain the Y-axis you see on some of the scatter plots (e.g., Figure 3, 5). DRG were viewed at 20X magnification and the number of positive cells within the field of view were manually counted.
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At the mRNA level (Fig. 4, bottom row), cytoplasmic ISH signal in transduced DRG neurons was decreased from 42% of area in animals dosed with AAVu68.hIDUA to 7% in animals dosed with AAVhu68.hIDUA-miR183 (Fig. 5A), representing an 83% reduction.
Good validation of their technique demonstrating that mRNA degradation is actually occurring rather than some effect. It is interesting to see the effect that steroids have on promoting increased mRNA transcripts.
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There was no reduction of expression in liver, heart, muscle, or brain cortex with vectors containing the miR183 targets, and expression was enhanced when compared to control GFP vectors in brain cortex (P = 0.03) and heart (P = 0.04) (Fig. 2D and fig. S4).
Very cool to see this play out as the authors alluded to when they described the levels of miRNA183 approximately 1000 times lower in these organs than in DRG. This provides some good evidence to support their hypothesis that miRNA183 may be a viable candidate in targeting AAV transgene expression in DRG.
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Bottom right picture shows immunostaining for the transgene encoded by AAV [green fluorescent protein (GFP) in this case].
A good point of clarification here would be to state if this looking at the same sample set in the previous pictures where other signs of neuron damage are shown. In this bottom right image, I can't quite tell if the same kind of lesions or vacuoles are present as in the other images. In other words, do the previously described symptoms occur where transgene product is present?
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hypereosinophilia
This an excess accumulation of white blood cells call eosinophils. As described by the authors, this is one indication of neuronal degeneration that helps to quantify toxicity.
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Tissues were then frozen in optimum cutting temperature embedding medium and cryosectioned for direct GFP visualization (brain was sectioned at 30 μm, and other tissues at 8 μm). Images were acquired with a Nikon Eclipse Ti-E fluorescence microscope.
I was initially critical of the sample size they described earlier but reading through this specific protocol makes it seem extremely tedious to collect data. Has anyone here performed these types of studies on neurons and/or brain tissue? How tedious does this get?
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The studies were designed to test the hypothesis that AAV-mediated DRG pathology is due to transgene overexpression and to subsequently develop a mitigation strategy.
The authors do a good job making their intent and goals clear throughout the paper. For one reason or another, a lot of papers are ambiguous about their thesis and I think a lot of it has to do with the writing style. The authors just outright state what they are testing, in plain English. And this is published in Science. Note to everyone: keep it simple and unambiguous.
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However, we are beginning to see toxicities that can limit the impact of this technology amid the current explosion of clinical applications of AAV gene therapy.
There's also limitations in the size of transgenes you can effectively use with AAVs, in the order of ~4-5 kb:
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Although these vectors were safe, many gene therapy programs failed in the clinic because of poor transduction.
i.e., these were not good expression vectors because they did not effectively transfer transgenetic material to the host.
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