Cryo-EM in Gene Delivery

Leveraging the power of cryo-EM in gene therapy and delivery

Gene therapy is a strategy which attempts to modify a patient’s genes through the delivery of genetic material designed to carry out a specific gene addition or edit. Gene addition typically involves delivery of a gene to the patient’s cells, often to compensate for the loss of activity by the patient’s protein variant. Gene editing leverages a number of techniques to achieve:

• Gene insertion – creates targeted breaks in DNA with instructions to repair the breaks by adding in genetic material with correct functioning.
• Gene inactivation – Creates targeted breaks in DNA without repair instructions with the goal of turning off a defective gene.
• Gene correction – Creates targeted breaks in DNA with instructions to repair defective regions of a gene without fully replacing the original genetic code.

Types of Gene Editors

A wide array of nucleases are being explored for clinical development, which requires a deep understanding of their native cellular function, regulation, and the impact of any protein engineering activities undertaken to modulate the activity and specificity of the encoded proteins.

Structural biology has long provided critical insights into the structure-function relationships of various gene editing tool proteins. For example, crystallographic analysis of the I-OnuI meganuclease and multiple reengineered variants demonstrated the feasibility of introducing a large number of mutations into an editor to allow for recognition of multiple unique genomic targets sites without alteration of the protein scaffold.⁽¹⁾

Zinc finger nucleases and transcription activator-like effector nucleases (TALENS) have also been studied by crystallography. In both classes, DNA recognition is specified by the editor protein itself, meaning that extensive protein engineering (and structure validation) is needed to modulate the recognition and activity behaviors of the editor.  

More recently, CRISPR-Cas9 and related systems have received tremendous attention as gene editing tools, as they are able to accurately and efficiently target and cleave any segment of double-stranded DNA based solely on the sequence of the loaded guide RNA. Engineering of the CRISPR-like editor, either through modification of its sequence or through the insertion of domains with complementary functionality, further expands the functional range of this editor class. Strategies for Cas9 engineering were recently reviewed by the team at Beam Therapeutics.⁽²⁾

Structural biology and the structure-function relationship of gene editors

While crystallography has been successfully used to study CRISPR systems, for example the high-resolution characterization of Type III-A effector subunits⁽³⁾, cryo-EM has been instrumental in uncovering the mechanisms of CRISPR-Cas9, especially for fully assembled complexes. The method continues to be an essential tool in characterizing engineered variants as tools for gene therapy. One example was the elucidation of the high-resolution structure of Cas9 fused to a natural transfer RNA deaminase that was evolved to give an adenine base editor that works on DNA.⁽⁴⁾ The structure provides a detailed blueprint for ongoing protein engineering of similar base editors to increase their versatility and controllability for use in patients.

In recent years, advancements in cryo-EM methods have made high resolution structure determination more routine, supporting rapid, cost-effective delivery of structures. Analogous to Structure-Based Drug Design (SBDD) for small molecule therapeutics, robust and reproducible workflows now combine with an ever-expanding toolkit for automated data processing to ensure ongoing structure support for gene editing efforts.

Leveraging cryo-EM in the delivery of gene therapies 

Regardless of the gene therapy approach, effector molecules must make it into the cells of the patient. In vivo delivery systems can be either viral (e.g., Adeno-associated virus (AAV), lentivirus, or more) or non-viral (e.g., lipid nanoparticles (LNPS) or virus-like particles). Each delivery vehicle needs to be developed with an eye to cell/tissue tropism, specificity, avoidance of host immune responses, and the consistency of cargo loading.

Cryo-EM in viral vector development: High resolution structure determination of viruses is a relatively straightforward application of cryo-EM when viral vectors exhibit minimal pleiomorphism and high symmetry. Resolutions better than 2.0 Å (and even better), can be quickly and reliable achieved, allowing for:

• Structural characterization of the parent virus, delineating important features of surface decorations, loops and capsid protein conformations that determine which receptors the virus binds to in human cells.
• Structures of parent and/or engineered virus variants in complex with antibodies to better define engineering strategies that would minimize clearance by host immune responses.
• Structural comparisons of virus variants to better understand behavioral differences at a structure-function level.

Furthermore, cryo-EM imaging can provide important insights at lower resolution and with much higher throughput, including details of general morphology, aggregation, size distribution, circularity, lamellarity, and membrane thickness.

Of critical importance to the delivery of gene therapies is an assessment of the packaging capability of a given virus and development process. Cryo-EM workflows allow for quantitative assessment of empty versus full capsids and provide important details on partially loaded, and “other” particles. The very low sample volumes required make this method applicable at the earliest stages of vector selection and development. This information-rich method offers significant advantages over methods like analytical ultracentrifugation (AUC) where material requirements and throughput are limiting. Similarly, these characteristics can be reported for non-replicating virus-like particles.

Cryo-EM for non-viral delivery vehicles: Lipid nanoparticles are self-assembling, dynamic structures capable of carrying nucleic acids, like siRNA, mRNA and DNA, as well as small molecule therapeutics. Knowledge of particle morphology is of tremendous interest for its impact on biological activity, biodistribution, and toxicity, but morphology can be challenging to characterize due to the variation in attributes typically observed in these particle formulations. Cryo-EM imaging workflows are well suited for such samples, offering a direct visualization of the particles in a sample in near-native states, and avoids artifacts that may be introduced in negative stain (NS) TEM workflows. Reliable size and morphology information can be returned, along with details on small particle populations that are rarely detected in light scattering methods.⁽⁵⁾

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