The Delivery Challenge
Designing the ultimate GABA-A receptor or GABA transporter is only half the battle. The other half is getting the genetic blueprint for these proteins into the neurons of a living brain. The Blood-Brain Barrier (BBB) stands as a formidable fortress, preventing large molecules and most viruses from entering the central nervous system from the bloodstream. Historically, this has necessitated invasive intracranial injections. However, the field of viral vector engineering is rapidly rewriting the rules of engagement.
Enter the AAVs
Adeno-Associated Viruses (AAVs) are the delivery vehicles of choice for modern gene therapy. They are non-pathogenic, trigger minimal immune response, and can provide long-term gene expression in non-dividing cells like neurons. But wild-type AAVs are poor at crossing the BBB. To overcome this, we turn once again to directed evolution. By creating massive libraries of AAV capsids (the protein shell of the virus) and screening them in vivo, researchers have identified variants—such as AAV-PHP.B and AAV-PHP.eB—that can cross the BBB with remarkable efficiency after a simple intravenous injection.
Targeting Specificity: The Right Cell, The Right Time
Once inside the brain, we don't want our engineered proteins expressed everywhere. Expressing a "lazy" GAT-1 transporter in excitatory neurons could be disastrous. Specificity is achieved through the use of **cell-type specific promoters**. For example, placing our engineered gene under the control of the *Dlx5/6* enhancer sequence ensures that it is expressed exclusively in GABAergic interneurons. Alternatively, using the *GFAP* promoter would target expression to astrocytes, suitable for our engineered GAT-3 variants.
Minimizing Off-Target Effects
Safety is paramount. To prevent expression in peripheral organs like the liver (a common sink for systemic AAVs), we incorporate target sites for liver-specific microRNAs (like miR-122) into the viral genome. In liver cells, endogenous miR-122 binds to these sites and degrades the viral RNA, preventing protein production. in the brain, where miR-122 is absent, the therapy remains active. This "detargeting" strategy ensures that our directed evolution masterpieces function only where they are needed: in the neural circuits of the autistic brain.
Excerpt from: Harnessing Directed Evolution Techniques to Target GABA Receptors, Transporters, and GABA Transaminase in ASD by Peter De Ceuster
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