Green particles of an unidentified virus
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CryoEM and the Application to Gene Therapy

History Of Electron Microscopy (EM)

Viruses have been identified as the causative agents of diseases in animals and plants since the early 1900s and, as early as 1940, the transmission electron microscope (TEM) provided scientists with their first images of these viruses. Improvements to this technology include negative staining methods developed around 1960, when scientists could begin to elucidate 2D and 3D structural models of larger viruses.

Two landmark Nobel Prizes were awarded for new discoveries in TEM; the first was for crystallographic electron microscopy (A. Klug, 1982) and the second for cryo-electron microscopy, or cryoEM (J. Dubochet, J. Frank, and R. Henderson, 2017), paving the way for the amazing ultra-high 3Å resolution. To put this in perspective, modest-sized proteins like albumin can be structurally imaged and modeled molecularly using cryoEM. The smallest viruses (parvoviruses) are 200 Å in diameter, which means they are 70-fold larger than the lower limit of resolution of cryoEM, and as such, exceptional detail can be visualized. High-resolution structures of this nature were at one time only possible using x-ray crystallography, but without crystal-induced structural artifacts and no need for fixative or staining techniques, the rapid sample freezing technique allows us to see the viruses as they exist in nature.

Why is EM Important?

Viewing virus samples at such high resolution allow a fine morphological analysis of virus capsid integrity as well as the genetic payload. While this level of magnification may seem like a lot, the techniques are now being applied to gene therapy vectors to visualize critical quality attributes relevant to how they’re manufactured. For example, many factors can have significant impacts on gene packaging efficiency (i.e., genome size, vector quality, packaging cells, transfection reagents, etc.), leading to lot variability in the resulting therapeutic vector.

The critical quality attribute most in need of measuring is the empty/full/partially filled capsid ratio. Combined with genome titering, this largely affects the dosing accuracy between different lots of manufactured vector, as only the properly loaded capsids are therapeutically potent.

The Ideal Approach

Several orthogonal methods can help elucidate the vector loading status, and it is useful to employ multiple methods like analytical ultracentrifugation (AUC) or capillary isoelectric focusing (cIEF) to monitor the manufacturing process. It should be noted that both AUC and cIEF lack visual confirmation on vector quality, as the data is not a visual picture of the product, but simply infers proper quality attributes.

With CryoEM, the direct imaging of the vector can be used to verify critical quality attributes seen in other methods and provide photographic evidence of the conclusion. It can show other informative attributes as well, such as ruptured vector and aggregation. Both of these qualities may influence the product’s final formulation.

How We Can Help

Charles River has recently added support for CryoEM and integrated this service as part of our Biophysical Laboratory Services in Shrewsbury, MA. CryoEM can now be combined with AUC and other orthogonal methods to look at empty, full, and/or partial aggregation events as well as other morphological characteristics that may result in response to your manufacturing process.

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