OriginALS

Description

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a devastating neurodegenerative disease characterized by the progressive degeneration of motor neurons in the brain and spinal cord¹. The disease is fatal, with an average survival rate of only 3–5 years after diagnosis². The exact cause of ALS is still unknown. Several genetic factors have been identified and are currently being investigated further. No specific environmental factor has been definitively shown to cause ALS³. ALS can be classified as familial or sporadic, depending on whether or not there is a family history of the disease. Familial ALS (FALS) accounts for 10% of all ALS cases, while Sporadic ALS (SALS) accounts for the other 90%⁴ Although SALS and FALS appear identical clinically and pathologically, the genetics of FALS are better understood. Therefore, the medications developed thus far are mainly based on the research conducted on FALS rather than SALS.

A.L.S Pathophysiology – Reactive Gliosis

The defining feature of ALS is the death of motor neurons in the motor cortex, brainstem and the spinal cord². It is not fully understood why neurons die in ALS, but this neurodegeneration is thought to involve many different cellular and molecular processes⁵.

Recent evidence suggests that in both SALS and FALS, non-neuronal cells, astrocytes and microglia, directly contribute to motor neuronal damage and cell death. Under disease or injury conditions, astrocytes change their normal morphology and function, gaining a new neurotoxic function- rapidly killing motor neurons^6-7. These so called "reactive astrocytes" change their gene expression relative to quiescent astrocytes. Two such distinguishing genetic markers are Steap4 and Timp1 genes, expressed exclusively in reactive astrocytes^8-11.

A recent study has shown that activated microglia cells are required to induce reactive astrocytes, by secreting cytokines such as IL-1α and TNF-α¹². The synthesis of both these cytokines is mediated by the NF-kB transcription factor¹³. NF-kB activation in microglia causes motor neurons death in vitro as well as in vivo. Heterozygous inhibition of NF-kB in microglia substantially delayed disease progression in ALS mice model¹⁴.

The process where microglia secrete an excess of cytokines and induce reactive astrocytes is an inflammatory process called reactive gliosis. Reactive gliosis has been identified as a common process to ALS and other neurodegenerative diseases, such as Huntington’s and Parkinson’s disease¹².

Motivation

To date, there is no cure for ALS. Available medications focus on treating symptoms and providing supportive care, with the goal of improving quality of life and prolonging patient survival⁵. Currently there are two main medications sold in the market for treating ALS: Riluzole and Edaravone.

Riluzole has been found to modestly prolong survival by about 2-3 months¹⁵, although the mechanism of its action is poorly understood. Edaravone has been shown to slow the decline in only a small group of patients who meet very specific criteria¹⁶. To date, there is no evidence that either Riluzole or Edaravone are significantly effective in prolonging survival in most cases of ALS (FALS and SALS alike)¹⁷.

OriginALS Goal

Our objective, as the BGU-IGEM team, is to develop a therapeutic system that will ultimately prolong survival of ALS patients via novel genetic engineering techniques.

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A Two Dimensional Approach

We divide our project into two approaches: 1) identify and specifically target reactive astrocytes (and not normal resting astrocytes) by their distinguishing markers and prevent their toxic activities by promoting an intrinsic apoptotic signal 2) inhibit the secretion of toxic pro-inflammatory cytokines by microglia cells.

The Astrocyte Pathway:
In the astrocyte pathway, we aim to identify and eliminate only reactive astrocytes. In order to identify the reactive astrocytes, we will use the promoters of the distinguishing markers, Steap4 and Timp1. The Timp1 and Steap4 genes have high levels of expression specifically in reactive astrocyte when compared to other cells in the central nervous system (CNS)¹⁸.

These two promoters will control the expression of dCas9 and its accompanying gRNA¹⁹. Each component in our CRISPR system is placed on a different plasmid and have a different promoter: the dCas9 protein and the transcription factors will be expressed under the pTimp1 promoter, while the gRNA will be expressed under the pSteap4 promoter, with ribozyme flanked RNA.

Only if both plasmids will enter the target cell and these promoters will be activated, the dCas9 and the gRNA will join together, to target a synthetic promoter that express an exogenous Caspase3. In order to add further specificity, we plan to deliver these plasmids using the Adeno-Associated Virus-9 (AAV9)²⁰. This modified virus targets and delivers its contents specifically to astrocyte cells²¹. The activation of caspase 3 by the dCas9 system will eventually lead to apoptotic death of the reactive astrocytes²².

The Microglia Pathway:
In the microglia pathway, we will knockout the IKKβ gene in microglia cells, using the CRISPR-Cas9 system²³ delivered by AAV6²⁰ virus that targets microglia cells²⁴. The IKKβ gene is transcribed and translated into a kinase which has a role as an activator upstream to the NFkB complex assembly. Therefore, the knockout will ensure a decrease in the neuro-inflammation transducing classical NFkB signal²⁵.

Aiming for specific microglia expression, we chose to combine the pF4/80 promoter, which is a known macrophage marker²⁶ with AAV6 viral vector. The pF4/80 promoter will be responsible for the expression of the Cas9 protein, while the U6 promoter, which is a non-specific promoter, is responsible for the expression of the IKKβ guide RNA sequence. Our CRISPR Cas9 system will target and knock out the IKKβ gene, resulting in inhibition of inflammatory cytokines synthesis and thus reduce the formation of new reactive astrocytes in the system.

Summary

To summarize, in our two-dimensional approach, we will target reactive astrocytes and microglia cells.
The goal of the astrocyte pathway is to remove the harmful activity of existing reactive astrocytes. This will be achieved by inducing apoptosis in the existing reactive astrocytes. All this without damaging the normal resting astrocytes.
In the microglia pathway we want to prevent the formation of new reactive astrocytes. We plan to achieve our goal by reducing the secretion of cytokines from activated microglia.

This novel combined approach is aimed to break the cycle of unregulated reactive gliosis, and hopefully will slow down the progression rate of both familial and sporadic ALS. By combining the removal of toxic cells with the prevention of the formation of new ones, the number of toxic astrocytes can be significantly reduced, thus slowing down the progression rate of ALS.

Future Applications

As reactive astrocytes and microglia are associated not only with ALS but other neurodegenerative afflictions included are Huntington's, Alzheimer's, Parkinson’s disease and brain injuries¹², our approach may be suitable to these "reactive gliosis" afflictions by developing treatments based on our novel method to target the reactive astrocytes and microglia

Figure 5: The presence of reactive astrocytes in various neurodegenerative diseases.<br/>Liddelow, Shane A., et al. "Neurotoxic reactive astrocytes are induced by activated microglia."<br/>Nature (2017)

References

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