Cell and gene therapy with Cryo-EM | Drug Discovery

Rational design for CAR T-cell receptor

CAR T cell therapy represents a popular approach in the field of cell therapies, harnessing the power of patients' own T cells to identify and eradicate cancer cells. At the core of CAR T-cell therapy lies the Chimeric Antigen Receptor (CAR) receptor, a modular receptor assembled from components derived from immune elements. The antigen-binding domain on the CAR receptor recognizes and binds specific antigens present on the surface of cancer cells, initiating downstream signaling that triggers the destruction of cancer cells. To achieve optimal affinity tuning and specificity, a detailed understanding of the high-resolution 3D protein structure of CAR receptors is important.

CAR (chimeric antigen receptor) on a T cell in complex with CD19 antigen displayed on a tumor cell.

Cryo-EM enables the study of 3D structure of protein complexes at near-atomic resolution. The 3D structure of CAR receptor in complex with cancer antigens, provides insights into their atomic-level interactions and conformational changes. Such details enable researchers to precisely engineer CAR receptors with suitable affinity and specificity, maximizing their therapeutic potential. A study published in Science Immunology showed the application of cryo-EM to solve the molecular structure of CAR receptor bound to CD19, a target antigen for the blood cancers. This information then was used to design several CAR receptors with distinct binding affinity towards CD19 and to generate CAR T cells with unique cancer cell killing capabilities.

Gene therapy involves the delivery of gene editing machinery or a replacement gene to a desired cell/tissue with the help of a suitable delivery vehicle.

Discovery and engineering of gene editor enzymes

Genome editing has become a rapidly growing field for the treatment and prevention of various diseases, as this technology allows for the precise manipulation of a specific genetic sequence inside an organism. Several advanced techniques have emerged, with CRISPR-Cas9 being a prominent example. Recent advancements in cryo-EM technology enables scientists to not only discover new gene editing enzymes but also to perform structure-guided engineering to improve gene editing properties of these enzymes.

There are generally 3 types of tools used:

CRISPR-Cas9 - Comprises clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9). The CRISPR-Cas9 system permits targeted gene cleavage and gene editing in a variety of cells.
Zinc finger nucleases – These were among the first programmable nucleases designed for precise genome editing.
TALENs or Tal effectors – Used for highly precise and efficient gene editing in live cells

Learn more about genome editing tools

Discovery and elucidation of mechanism of action of novel gene editors

Cryo-EM structures reveal gene editors in their native state and elucidates the distinct mechanisms of genome recognition and editing. Structure of SpCas9-ABE8e complex from Lapinaite A et al, Science, 2020.

Structure-guided engineering of gene editors

Cryo-EM structures are used to improve the size, accuracy, speed, and other biochemical properties of existing enzymes or lead to design completely new enzymes. Structure of Cas9 18-20 MM from Bravo J et al, Nature, 2022.

To realize the full therapeutic potential of gene editing systems, it is critical to reduce off-target gene editing, ensure precise editing at the desired target, and developing stable and targetable delivery vehicles. Understanding the structural basis of how these enzymes recognize the target and perform gene editing function will open up new avenues for introducing rational changes to gene editing enzymes to improve their efficiency while avoiding off-targets effects. The use of single particle cryo-EM has been increasingly used to determine the structures of gene editing enzymes. A recent paper by Nakagawa et. al²., used cryo-EM to gain structural insights on CRISPR-Cas12 effectors, a later addition to the CRISPR family but ones that are gaining traction as powerful tool for non-mammalian applications and diagnostics.

Gene delivery vehicle design and characterization

Success of gene therapy is directly correlated with the efficiency of the gene delivery vehicles. These delivery vehicles ensure the targeted delivery of gene editors/gene replacements to specific cells or tissue within the body. Gene delivery vehicles can be generally classified into two categories:

Viral vectors like Adeno-associated viruses (AAV) and Lentiviral-based vectors (LVV)
Non-viral vectors like Liposomes, Lipid nanoparticles (LNPs) and Lipoplexes

Viral vectors are a popular choice for targeted gene delivery. However, there are several challenges associated with these viral vectors such as capsid immunogenicity, limited tropism, and capsid neutralization by human immune system before the viral vector reaches to the target cell/tissue. One of the ways to minimize these side effects are to perform capsid engineering to improve the capsid properties. Capsid engineering is a process that uses high-resolution structure of the viral capsid to introduce precise modifications to improve the capsid for desired properties. Furthermore, cryo-EM can be used for particle characterization enabling the study of the effect of buffer components, excipients, temperature and storage conditions on the integrity and stability of these viral vectors.

Image adapted from Hsu HL et al, Nature Communication, 2020

Structure guided capsid engineering

Capsid engineering involves the deliberate modification of capsid of known AAV serotypes or the engineering of a new type of viral capsid. The goal for capsid engineering is to generate a vector system that can go undetected by the immune system (reduced immunogenicity), can escape neutralization by pre-existing antibodies and is able to target a specific cell or tissue type (tropism). Single particle cryo-EM offers insights into the molecular structure of novel viral capsids and the molecular basis of viral capsid binding to the cell surface receptors or neutralizing antibodies. Once the region of interest is identified, precise structural modification can be made to improve on the desired capsid properties.

Cryo-EM assisted capsid engineering

Discovery and characterization of novel viral vectors

High-resolution capsid structure provides a platform for capsid engineering. Structural details can also be used to identify new features for novel viral vector identification, regulatory approval as well as for Intellectual property (IP) protection. Hsu et.al³, published research detailing a novel capsid which shared sequence similarity with AAV2, one of three serotypes approved for commercial use in patients. Their 2.5 Å resolution structure identified critical amino acid residues that contributed to the characteristics of their novel vector that they obtained from clinical samples.

Density map of new AAV capsid. Figure from Hsu et. al. 2020 under a creative commons license.

Viral capsid-receptor interaction

Mapping of receptor interaction sites help facilitate AAV engineering with enhanced or unique targetability. Furthermore, capturing these small differences on how different AAV serotypes interact with cell surface receptors and utilizing them to train artificial intelligence/machine learning algorithms will help generate diverse AAV capsids with unique specificity and targetability.

Zhang R et al used single particle cryo-EM to understand the molecular basis cell surface receptor binding of three AAV serotypes (AAV1, AAV2 and AAV5).

Image adapted from Zhang R et al, Nature Communications, 2019, illustrating the use of cryo-EM to understand receptor binding of three AAV serotypes.

Capsid Immunogenicity optimization

Cryo-EM structures provide insights into AAV neutralization mechanisms and to rational design of escape variants with minimal disruption to cell tropism and gene expression. Smith JK and Agbandje-McKenna M, used cryo-EM to identify neutralizing antibody (Nab) hotspots on the vector surface and combined this information to perform site directed mutagenesis to eliminate these hotspots to create viral vectors with the ability to avoid preexisting immunity against gene delivery vectors.

Image adapted from Smith JK and Agbandje-McKenna M, PLOS Pathogens, 2018, illustrating the use of cryo-EM to visualize areas of antibody binding to improve the efficacy of viral vectors for gene therapy.

Viral vector characterization using cryo-EM image analysis

It is well established that an imbalance in the ratio of genome containing AAV particles to the empty AAV particles in a gene therapy formulation affects the therapeutic efficacy. Furthermore, it is important to characterize that capsid engineering efforts did not negatively impact viral capsid properties such as capsid stability. Production process and storage conditions can also negatively affect the stability, integrity, and infectivity of virus particles. Therefore, comprehensive viral vector characterization is essential to ensures efficacy and safety of gene therapy products.

Determination of empty-full AAV capsid ratio

The ratio of empty to full viral particles is a critical factor that affects the overall efficacy of AAV based gene therapy. An imbalanced ratio can reduce the therapeutic payload delivered to target cells, compromising the desired therapeutic effect. Accurate characterization of the empty-to-full ratio allows researchers and developers to optimize vector production and ensure maximum efficiency in delivering the therapeutic cargo.

Regulatory agencies around the world such as US-FDA and EU-EMA are asking to report accurate quantification of the ratio of empty vs. full particle in gene therapy formulations.

Revealing particle morphology

The structural integrity and morphology of viral vectors play a vital role in their ability to effectively deliver therapeutic genes.
Aggregated and broken viral particles are considered as impurities and their accurate estimation is required by regulatory agencies. Such information can also be utilized in the process optimization stage to optimize the viral vector production process.

Decoding surface features

The surface properties of viral vectors determine their interactions with target cells, host immune responses, and potential risks associated with off-target effects. Cryo-EM provides the unique capability to capture detailed images of the viral vector's surface structure, allowing for the identification of critical features such as smooth capsid verse capsid with a spiky surface in case of Lentivirus based viral vectors. This high-resolution information enables scientists to fine-tune vector design and enhance their specificity, reducing potential adverse effects and improving overall therapeutic efficacy.

Cryo-EM projection images of Env− HIV-1 mature VLPs and SIV virions from cryo-electron tomography. Image adapted from Ni T et al., Structure Biology, 2021 and Zanetti G et al., PLOS Pathogens, 2006.

Once of the biggest advantage that cryo-EM offers is the accurate characterization of multiple critical quality attributes (CQA) just from a single data set. In the table below, several quality attributes that are needed to characterize both Adeno-associated viruses (AAVs) and Lentiviral vectors (LVVs) to ensure the optimal formulation is being developed.

CQA for Adeno-associated viruses	CQA for Lentiviral-based vectors	Cryo-EM characterization
Capsid content: Empty/Genome containing	Virion vs. vesicles
Morphology	Virion size
Broken capsids	Smooth vs. spikey particles
Particle aggregation	Spike density
Particle size	Transduction efficiency (or transduction inhibitory particles)
Impurities	Core morphology
	Impurities

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