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Design

Our team spent over six months designing our project to create a concept which would be applicable not only for the iGEM competition, but also would be viable for professional applications.

The plan

Our hypothesis states that removing reactive astrocytes from the central nervous system (CNS) will slow down the rate of neuron degeneration under reactive gliosis conditions. The proposed therapeutic approach has two main objectives: remove existing reactive astrocytes while simultaneously preventing the creation of new reactive astrocytes. We wish to achieve these objectives by inducing apoptosis in reactive astrocytes and inhibiting the secretion of pro-inflammatory cytokines by microglia.

We decided to inhibit cytokine production through inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway¹.

We chose to implement this inhibition using the CRISPR-Cas9 system, as this revolutionary tool allows targeted gene editing and the flexibility to test targets simply by adding different gRNAs.

The second system aims to remove reactive astrocytes from the CNS. We decided to achieve this by activating apoptosis, as programmed cell death will prevent further damage to the CNS. We chose to induce apoptosis by expressing Caspase-3, whose activation plays a central role in apoptosis. Here we deliberated between activating an endogenic Caspase 3 promoter or inserting an exogenous Caspase 3 under a promoter of our choice.

This version reaches its active conformation without activation through cleavage while the endogenic Caspase3 must undergoes proteolysis in order to activate apoptosis, thus we therefore considered it less controllable and reliable². Additionally, using reversed-caspase-3 (rev-caspase-3) gives us complete control over the characteristics of the promoter and affects the effector stage of apoptosis.

The rev-caspase-3 expression will be induced by deactivated Cas9, this time CRISPR system will be used to induce gene expression rather than for gene splicing.

A critical aspect of our entire system is specificity, in order to prevent damage to healthy astrocytes and to improve the treatment efficiency. We decided to increase specificity by constructing a three level hierarchy: specific delivery to our target cells, a two-plasmid system, reactive astrocytes specific promoters.

It's All About Specificity

In order to select specific promoters, we turned to the literature and examined RNA sequencing and microarray datasets. We found that the F4/80 is expressed specifically in macrophage cells. With specific delivery to the CNS, this promoter would target the microglia, which are the macrophage representatives in the CNS. Reactive astrocytes change their expression profile significantly when compared to resting astrocytes³. Based on this, we selected pSteap4 and pTimp1, as these genes are highly expressed in reactive astrocytes and expressed in negligible amounts in resting astrocytes and other brain cells such as neurons and microglia^4-7.

The 2015 BGU-iGEM team, Boomerang, has developed a system to specifically target cancer cells. In this system, they split the CRISPRcas9 and its gRNA onto two separate plasmids, meaning that only if both these plasmids enter the target cell and only if both these plasmids are expressed will the system be activated. We decided to build on this previous ingenious strategy and implement a two-plasmid system in our project as well. This technique will increase the probability of the expression of our constructs in our target cells, and decrease chances of off-target activity, thus increasing the safety of any potential therapy derived from this project.

Most prominently, we wished to include a specific delivery system which could be viable and acceptable for administration to patients. After much deliberation we chose to use Adeno-associated virus types 6 and 9, which directly target microglia and astrocytes, respectively. For more info on AAV6 and AAV9 specific delivery and why they are viable for a human medication in the future, visit our safety page. At this point we had chosen all the components necessary for our two-dimensional therapeutic approach.

The Model

Before commencing with the technical design of our plasmids, we had to decide on a suitable model to work with. This model needed to allow us to stimulate ALS disease conditions that will be also practical based on our available facilities and time constraints. When considering our long-term goals, we examined the notions of both zebra fish and mouse models. We chose to base our design on the mouse models.

Next, we considered primary mouse cells versos cell lines. Although primary cells model ALS conditions more accurately than cell-lines, it is very laborious and, requires expertise that we do not have and it is very time consuming and delicate. Furthermore, although our project focuses on ALS, the therapy we propose is very general as it targets downstream processes common to all cases of ALS (rather than a specific model such as SOD1) and in fact applicable to any condition which exhibits reactive gliosis.

Therefore, murine CNS cell-lines were our choice of models, as they are less time consuming and can be used to model more general reactive gliosis conditions.

Microglia Pathway Plasmids

The microglia destined plasmids were designed based on a pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA as a backbone⁸. This backbone expresses Staphylococcus aureus CRISPRcass9 (SaCas9) conjugated under the cytomegalovirus (CMV) promoter, a U6 promoter with a location for gRNA insertion and the SaCas9 scaffold, inverted terminal repeats (ITR) for the AAV 2/6 hybrid, bacterial and mammalian origins of replication (Ori), and Ampicillin resistance⁸.

We chose to work with the SaCas9 as it is smaller compared to streptococcus pyogenes Cas9 (SpCas9), making it easier to pack in the target viral vectors.

We designed 5 guide RNAs (gRNAs) complementary to exons 2, 4, 5 of the IKBKB gene with the purpose of causing a deleterious mutation in the IKBKB gene. We inserted the gRNA sequences under the U6 promoter (a strong RNA polymerase III promoter). To confer specificity to our system, we cloned the murine pF4/80 promoter and inserted it instead of the CMV promoter. Next, we added an enhanced green fluorescent protein (EGFP) sequence under a T2A self-cleaving peptide, to allow the EGFP expression in conjugation with the SaCas9, therefore providing a selection method for successful insertion in our cells. The name of the plasmid is pX601 F4/80 EGFP gRNA (AAV-dCas9 plasmid)