Tackling CTE: A Novel Gene Therapy Targeting Tau

Introduction

Tackles, punches, falls, they are all a part of sport. However, what might seem like innocent and harmless events can have devastating consequences. Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative condition caused by repetitive head trauma. It results in cognitive and mood-related symptoms, including memory loss, confusion, and depression. CTE affects a diverse group of people across various sports. One study of 152 brain samples from young athletes who had experienced repetitive head injuries found that 41.4% had CTE (McKee et al., 2013). Yet, current treatments only aim to manage symptoms, rather than prevent or cure the disease itself. It's important to develop therapies that can stop the disease at its root cause so athletes, both young and old, can continue to pursue their passions safely. So, how do we “tackle” this problem?

What Causes CTE?

CTE is caused by the abnormal clumping of the protein tau (McKee et al., 2015). Tau is responsible for maintaining the structural integrity of neurons and allows the transport of molecules along the cell. When tau becomes hyperphosphorylated, it clumps together to form aggregates in the brain, which damage and kill neurons, leading to the neurodegeneration seen in CTE.

PPP3CA is a part of calcineurin, a protein that maintains tau in a normal, healthy form by removing phosphate groups from it (Castiglioni et al., 2024). Studies have shown that in both traumatic brain injury (TBI) and CTE, levels of PPP3CA are reduced. For example, Seo et al., 2017, found that PPP3CA was lower in CTE brain samples at both the gene and protein level when compared to healthy samples. In animal studies, researchers found that decreased levels of PPP3CA in the frontal cortex were correlated with an increased level of hyperphosphorylated tau (Seo et al., 2017)

These findings suggest that the hyperphosphorylation of tau seen following TBI and in CTE may be at least partly due to the reduction in PPP3CA. Therefore, increasing PPP3CA expression in neurons could prevent or reverse tau hyperphosphorylation, potentially stopping the aggregation that results in neurodegeneration. Not only could this approach be used to slow the progression of existing CTE, but if given soon after TBI, it may prevent the development of CTE.

Novel Therapy

To achieve increased expression of PPP3CA, I propose using dCas9-VPR, a technique previously utilised to upregulate gene expression in the brain (Savell et al., 2019). First, a single guide RNA (sgRNA) is designed to target and bind to the region of the PPP3CA gene that controls gene expression by deciding when a gene is switched on. The dCas9-VPR system consists of two parts: the VPR activation domain that binds to the target gene and recruits transcriptional activators to switch on the gene, and an inactive form of the Cas9 enzyme that acts as a scaffold to help hold the VPR domain in place on the gene (Chen and Qi, 2017).

To deliver these components to neurons, they are packaged into split-intein dual AAV vectors (Böhm et al., 2020). AAV vectors are harmless forms of viruses capable of delivering genes into cells. Using dual AAV vectors overcomes the size limit of a single AAV and allows them to cross the blood-brain barrier. The dCas9-VPR protein is split into two halves, one with the N-terminal, i.e. the start of the protein, and one with the end of the protein, the C-terminal. Each half is fused to an intein, a small protein that can join two other protein segments. The IntN intein is fused to the N-terminal half, and IntC is fused to the C-terminal half.

Two AAV vectors are then made:

AAV-N; contains the N-terminal half of dCas9-VPR fused with IntN and a neuron-specific promoter, SYN, to guide the vectors into neurons.

AAV-C; contains the C-terminal half of dCas9-VPR fused with IntC, the same neuron promoter, SYN, and the sgRNA.