Team:UCL/Project/Chemotaxis

INSERTING THE CIRCUIT

Ways to get GEM cells into the brain

It is all very well having designed a genetic circuit to combat a disease, but unless it can be feasibly transfected into the correct cells, or unless one can easily implant the transfected cells in sufficient quantity, in the correct locales and without significant bodily damage, that circuit is practically useless. Therefore, we have considered some methods of getting our genetically engineered microglial (GEM) cells into the brain that could plausibly be seen in future clinical practice.

In Vivo: Lipid-Peptide-DNA Nanocomplexes

There is active research interest in performing gene therapy in the brain. One method of gene delivery involves using synthetic nanoparticle formulations to transfect cells in vivo. Nanocomplexes are constructed from lipids and receptor-targeting peptides, which self-assemble around DNA structures intended for transfection, such as recombinant plasmids. They are capable of efficient targeted transfection (Hart 2010 ) but must be inserted into the brain via micro-neurosurgery as they are far too big to pass through the blood-brain barrier.

These lipid-peptide-DNA (LPD) nancomplexes have been shown to be able to engage cell surface receptors (or if the lipids are cationic, anionic moieties such as proteoglycans) and activate endocytosis; the cell internalises the LPD nanocomplex. It is taken to the endosome for degradation, but as the LPD nanocomplex disassembles its lipids fuse with the lipid bilayer of the endosome and destabilise it to form pores through which the peptide-DNA (PD) complex can escape. The peptide tag defends the DNA from marauding nuceleases until the DNA can be taken up by the nucleus, for example during division (Hart 2010 ). Non dividing cells, such as neurons, can be targeted by engaging cytoplasmic importins with the PD. Without this feature, the transfection can be targeted just to dividing brain cells, such as microglia and tumorous brain cells (glioblastoma)(Hart 2010 ).

Writer et al. found that LPD nanocomplexes were effectively taken up by microglial cells when they were injected into the brains of rats. This is because the injection causes damage to the brain tissue in the needle's path and the microglia are attracted to this damage. As immune cells, it is their function to respond to such damage. Since the LPD nanocomplexes are foreign particles, the microglia ingest them and become transfected (Writer et al. 2012). This suggests that non-protein targeted LPD nanocomplexes (or microglia specific targeted LPD nanocomplexes) are effective at microglial transfection. The stability of the transfection was not gauged in this study, and more research is, of course, required.

LPD nanocomplexes can be gadolinium-labelled to allow them to show up in an MRI scan (Writer et al. 2012). This would allow us to estimate the degree of transfection and the spread of genetic information from the injection site in vivo. The dispersal of the LPD nanocomplexes is significant, because it is a major factor in determining the efficacy of a single dose. Maximal dispersal minimises the number to injection sites required, and therefore the amount of damage the brain must sustain from repeated micro-neurosurgery. Dispersal in the brain could be maximised by using anionic, as opposed to cationic, lipids in the LPD nanocomplex as this negates electrostatic 'friction' with proteoglycans on cell surfaces.

Stem Cell Derived

Embryonic stem cells, or even induced pluripotent stem (iPS) cells, could be differentiated into human microglia capable of acting as a genetic circuit chassis or the vehicle for forms of gene therapy. Protocols for generating embryonic stem cell-derived microglia (ESdM) (Tsuchiya et al. 2005) have been developed, and genetically engineered ESdM have been used in research as a gene therapy vehicle that helps treat an autoimmune disease by expressing neurotrophin 3 (Beutner et al. 2012). This Nature paper by Beutner et al. concluded that genetically engineered EDsM represented a new way to treat inflammatory conditions and repair cellular damage in the brain, which is, essentially, what our circuit is designed to do on a more diverse and ambitious scale. Engineered ESdM can be transplanted intravenously (in blood vessels around the brain) and then engraft into the central nervous system (Beutner et al. 2012). Alternatively, intelligently genetically engineered ESdM and iPS derived microglia they could be inserted into the brain using microsurgery, as already discussed.