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Written by Qiu Yichen - Wang Sizhen, Fang Yiyi edited - Xia Ye
Epilepsy is characterized by spontaneous and intermittent seizures, and overactive brain circuits are an important pathogenesis
behind epilepsy.
Nearly 1% of the global population is currently affected by epilepsy [1].
In the process of traditional drug treatment or surgical treatment, the drug can spread to the entire brain, but it cannot target the treatment of overactive abnormal brain areas [1], and surgical treatment is only suitable for a small proportion of patients with drug-resistant epilepsy [1], so there are still one-third of epilepsy patients who need to endure the severe effects of seizures such as seizures
。
Different from the traditional way of correcting erroneous gene sequences, new gene therapies greatly broaden the scope of application by carrying functional genes to achieve therapeutic effects, and can also be applied to non-hereditary neurological diseases and effectively control the process of diseases
.
Previous studies have shown that marker genes for hyperactive neurons can be detected during the pathogenesis of many neurological diseases, and the mechanism of seizures is that overactive neurons lead to abnormally activated brain circuits that produce convulsions [2].
In recent years, gene therapies targeting epilepsy have shown that regulating hyperactive brain regions can effectively control spontaneous seizures [3-6], but these therapies are not effective in distinguishing between "healthy" and "healthy" hyperactive" neurons
.
This means that 'healthy' neurons may also be over-controlled in activity, negatively
affecting normal brain activity.
November 3, 2022, Kullmann and Kullmann of the Institute of Epilepsy, School of Neurology Research, University College London Lignani's team published a title titled "On-demand cell-autonomous gene therapy for brain circuit disorders" in the journal Science Research papers
.
Researchers have developed a new gene therapy that can autonomously identify overactive cells, so that the functional genes carried can be expressed only in these recognized cells and not expressed in normally active cells
.
After successfully regulating overactive cells, the gene therapy voluntarily stops expression, forming a closed loop 'close – loop'
.
This gene therapy does not require additional drugs or optoelectronic instruments to assist in control, and it can complete the targeted treatment of overactive brain areas in itself, which has certain enlightenment for targeted therapy
.
Dr.
Qiu Yichen is the first author, Professor Dimitri Kullmann (FRS FMedSci MAE) and Gabriele Lignani The assistant professor is the co-corresponding author
.
The research team first screened a series of genes that respond to and express neuronal stimulation, including early immediate genes, such as CFOS, Arc and egr1
.
In addition to the promoters of these natural genes, the authors also tested some synthetic promoters (such as ESARE).
NPAS4-derived）
。 The commonality of these promoters is to guide gene expression by sensing intracellular calcium signals, hence the name activity-dependent promoter
.
In the previous literature, this activity-dependent promoter has been widely used to label active neurons [7].
Using adeno-associated virus (AAV) as a vector, the authors designed to test these promoters by linking them to potassium channels in different combinations
.
Since potassium channels can reduce nerve cell activity by modulating ion balance, the "switched-on" state can be entered when the designed combination is successfully initiated for expression, that is, the promoter is activated, mediating the high expression of downstream potassium channels, thereby weakening neuronal excitability (Figure).
1）
。
In neurons cultured in vitro, the authors tested two different potassium channels, KCNA1 (Kv1.
1) and KCNJ2 ( Kir2.
1）
。 First in PTX (picrotoxin, a GABA receptor blocker)-activated neurons, the authors confirmed that promoter activation levels increase in tandem with changes in neuronal activity and in the presence of neurons Peak after 6 h (Figure 1).
Figure 1: Activity-dependent gene therapy senses hyperactive neurons and enhances the expression
of carrier genes.
When neuronal cellsWhen not overactive, gene therapy automatically stops working
.
(Image source: Qiu, etal.
, Science, 2022)
Next author in PTX Different promoters and a combination of two different potassium channels were tested in the activated neurons to screen the group with the best effect
.
The authors found that the cfos-KCNA1 combination had significantly lower neuronal electrical activity parameters compared to the control group (Figure 2
。 Other promoter and potassium combinations have not achieved such a significant effect
.
Figure 2The cfos-KCNA1 combination inhibits neuronal hyperactivity
.
(Source: Qiu, etal.
, Science, 2022)
Subsequently, the authors further observed the different combinations in Whether PTZ (Pentylenetetrazol) is effective in mouse models of induced epilepsy (Figure 3).
The authors first injected AAV-loaded promoter in combination with potassium channels in the hippocampal brain of mice, and then induced convulsions by PTZ injection to induce its expression
in overactive cells.
The authors found that the cfos-KCNA1 combination performed the most again
.
The results of electrophysiological experiments showed that the action potential frequency of neurons expressing the combination was significantly reduced, and the current threshold was significantly increased
.
In addition, by immunofluorescence staining, the authors confirmed that the combination was mainly expressed in excitatory neurons, while the expression levels in GABA-positive suppressor neurons were extremely low
.
Therefore, the authors identified cfos-KCNA1 as the best combination
.
To test whether the cfos-KCNA1 combination can switch on when neuronal activity is abnormal, and switch off after neuronal activity returns to normal under the action of potassium ions After injecting AAV9 cfos-KCNA1 in the hippocampus on both sides, the authors performed multiple acute induction
of PTZ in the injected mice.
The authors hypothesize that the first PTZ-induced tics will switch-on the gene therapy, increasing the expression of KCNA1, the potassium channel, and inhibiting or reducing the symptoms
of tics in subsequent induction experiments.
After 24 h, the authors performed PTZ induction for a second time and observed a reduction in the severity of tics in mice injected with cfos-KCNA1.
It was shown that the gene therapy successfully controlled the activity of overactive neurons and attenuated seizures
.
Two weeks later, the authors performed a third PTZ induction and speculated that because the mouse brains were not overactive, the gene therapy would switch-off and no longer work
.
The authors observed no significant difference in seizure severity between mice injected with cfos-KCNA1 and control groups in the third PTZ-induction experiment.
This set of experiments proved that when there is neuronal hyperactivity, the cfos-KCNA1 combination can be switch-on and reduce the severity of tics; When there are no hyperactive neurons in the brain, the combination switch-off provides an effective proof
for the closed-loop model.
Figure 3 cfos-KCNA1 inhibits hyperactive neurons and attenuates seizures in PTZ-induced experiments (Image source: Qiu, etal.
, Science, 2022)
Next, the authors asked whether cfos-KCNA1 gene therapy could achieve the purpose of reducing the frequency of epilepsy in a chronic epilepsy model
。 The authors tested the combination in a KA (kainic acid)-induced mouse model of chronic epilepsy and used EcoG to record data
on brain wave activity.
The authors found that the mice treated with AAV cfos-KCNA1 had a nearly 80 percent reduction in epilepsy frequency compared to the control group, with some mice no longer experiencing seizures.
This aroused the author's curiosity, how did the combination reduce the frequency of seizures? The authors found that in addition to generalized seizures, another feature of epilepsy, spikes, may be an important controlling factor
.
The authors found that the spike wave phenomenon was sufficient to initiate CFOS activity
.
By studying the brainwave atlas data, the data showed that the spike wave activity of mice in the treatment group gradually decreased, in addition to the power spectrum, coastline</Parameters such as B33 > The authors also found that these spikes were sufficient to induce the expression of the cfos-KCNA1 combination and control the activity of
neurons.
This may explain why the gene therapy can reduce the frequency of epilepsy in mice and even make seizures
no more.
The authors then conducted a series of memory and behavioral tests, and the treated mice showed no negative effects
.
Figure 4 AAV cfos-KCNA1 reduced the frequency of epilepsy in a mouse model of chronic epilepsy, and spike was sufficient to induce the gene therapy and effectively control neuronal activity, which has achieved the purpose of
reducing epilepsy.
(Source: Qiu, etal.
, Science, 2022)
Finally, the authors are also assembling human brains The gene therapy was tested in a model of human cortical assembloids (hCAs) (Figure 5).
In this experiment, the authors used an individual that fused cortical spheroids and inhibitory subpallial spheroids The human brain assembles and carries out a series of important expression gene markers, proving that the two have been well integrated into one
.
Compared with human brain organoids, human brain assemblies integrate a variety of cell bodies to better simulate the process of brain maturation [8].
The authors used potassium chloride (KCl) stimulation to simulate high-intensity neuronal activity (epileptiform activity, high potassium) in human brain assemblies expressing the gene therapy model) to induce the expression
of causative therapy.
4 h later, the authors added KCl for a second time to induce high-intensity neuronal activity and observed that the overall neuronal activity of the cfos-KCNA1 expression group was significantly lower than that of the control group
.
This shows the good expression and effect of this gene therapy in humanoid brain models, which is highly consistent
with the results of mouse experiments.
Figure 5 In the humanoid brain model, the authors also observed the expression of cfos-KCN1, and the overall activity of the expression group was significantly reduced
.
(Source: Qiu, etal.
, Science, 2022)
Article conclusion and discussion, inspiration and prospects
This paper proposes a new gene therapy idea
based on the regulation of neuronal autonomous activity.
This activity-dependent gene therapy, which can effectively distinguish between 'healthy' and 'hyperactive' neurons, by using an activity-dependent promoter (cfos-promoter) to control the expression
of potassium ions (KCNA1).
The combination has achieved good results
in mouse models of chronic epilepsy and human stem cell-induced humanoid brain models.
Compared with its previous gene therapy ideas, this gene therapy uses a simple design to achieve the purpose of 'close-loop', getting rid of the dependence of the instrument, and selecting to express cfos and potassium channel KCNA1 by brain activity , and therefore more targeted
.
At the same time, there are still some unsolved mysteries
about how the specific treatment of this gene therapy works.
For example, how it works in the brain network, where its opening and hanging thresholds are, how long potassium ions are expressed on the cell surface, and why the rest of the promoters and KCNK2 potassium channels tested by the authors did not achieve the same effect
.
At the same time, in the clinic, when the frequency of some epilepsy patients is low, can the gene therapy still have a role
?
Finally, the authors propose that since neuronal overactivity in addition to epilepsy is also associated with other neurological system diseases, the therapy could theoretically be generalized to more neurological diseases, such as Parkinson's and migraines
.
For example, adjust the expression of promoters and ion channels to adapt to other disease characteristics to achieve the best therapeutic effect
.
Original link: www.
science.
org/doi/10.
1126/science.
abq6656
First author: Dr.
Yichen Qiu (left); Corresponding authors: Assistant Professor Gabriele Lignani (middle), Professor Dimitri Kullmann (right).
(Photo courtesy of Kullmann and Lignani team, School of Neurology Research/Institute of Epilepsy, University College London).
Welcome to scan the code to join Logical Neuroscience Literature Study 2
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[9] The Neuron-Niu Jianqin/Yi Chen Ju team discovered the coupling mechanism between the differentiation of oligodendrocytes and the formation of foot processes in developing oligodendrocyte cells
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Recommended high-quality scientific research training courses【1】Symposium on patch clamp and optogenetics and calcium imaging technology (January 7-8, 2023, Tencent Meeting)【2】The 10th NIR Training Camp (Online: 2022.11.
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