A combinatorial gene therapy for focal epilepsy based on coordinated expression of multiple GABA-A receptor subunits
Abstract:
In spite of the many available antiepileptic drugs (AEDs) and of other therapeutic approaches (surgery, brain stimulation, ketogenic diet, etc.), about one-third of epilepsy patients do not get control of their seizures. The search for conceptually new therapeutic approaches is therefore a priority, and gene therapy is often a consideration. In fact, drug-resistant focal epilepsies represent an accessible target for gene therapy, because it may be sufficient to inject the vector directly in the brain parenchyma and express the therapeutic gene(s) in the relatively restricted brain area that generates focal seizures. Attempts in this direction have been made with genes that can modify cell (or circuit) function and control hyperexcitability, such as some channels, neurotransmitters, or receptors. These strategies have shown some but not totally satisfactory success in pre-clinical models. This may be due to a number of factors; first, epilepsy occurs because of a multitude of changes in neural cells and neuronal circuits and, therefore, using a single gene approach is unlikely to be fully effective; second, there may be homeostatic changes occurring following gene therapy administration that, in time, attenuate the desired effects; third, expression of non-human proteins entails the risk of immune reactions.
We plan to use a conceptually new combinatorial gene therapy based on lentiviral vectors (LV), in the frame of an innovative chemogenetic approach. Current chemogenetic approaches use Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), i.e. modified receptors that no longer respond to the endogenous ligand but to an otherwise inert drug. A problem with this approach is that the mutant receptor is a potentially immunogenic exogenous protein. In addition, the use of a new drug (the DREADD ligand) requires in depth assessment of its safety. The alternative approach that we propose is to overexpress endogenous receptors that mediate the response to clinically used AEDs, as these receptors will not cause immune reactions. We will focus on GABA-A receptors highly sensitive to benzodiazepines and barbiturates, that contain the alpha1, beta3 and gamma2 subunits. We will design vectors expressing a combination of these subunits under control of a promoter (CaMKII) that is specifically active in excitatory neurons. Thus, these vectors are expected to increase endogenous GABA-mediated inhibition on excitatory neurons, and to increase their responsiveness to benzodiazepines and/or barbiturates in the injected epileptogenic region, which implicates relatively lower drug concentrations in other regions of the brain and, therefore, lower risk of side effects.
We plan to use a conceptually new combinatorial gene therapy based on lentiviral vectors (LV), in the frame of an innovative chemogenetic approach. Current chemogenetic approaches use Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), i.e. modified receptors that no longer respond to the endogenous ligand but to an otherwise inert drug. A problem with this approach is that the mutant receptor is a potentially immunogenic exogenous protein. In addition, the use of a new drug (the DREADD ligand) requires in depth assessment of its safety. The alternative approach that we propose is to overexpress endogenous receptors that mediate the response to clinically used AEDs, as these receptors will not cause immune reactions. We will focus on GABA-A receptors highly sensitive to benzodiazepines and barbiturates, that contain the alpha1, beta3 and gamma2 subunits. We will design vectors expressing a combination of these subunits under control of a promoter (CaMKII) that is specifically active in excitatory neurons. Thus, these vectors are expected to increase endogenous GABA-mediated inhibition on excitatory neurons, and to increase their responsiveness to benzodiazepines and/or barbiturates in the injected epileptogenic region, which implicates relatively lower drug concentrations in other regions of the brain and, therefore, lower risk of side effects.
Dettagli progetto:
Referente scientifico: Simonato Michele
Fonte di finanziamento: Bando PRIN 2022
Data di avvio: 18/10/2023
Data di fine: 18/10/2025
Contributo MUR: 126.416 €
Cofinanziamento UniFe: 30.127 €
Partner:
- Università degli Studi di FERRARA (capofila)
- Università degli Studi di ROMA "La Sapienza"