Hey, remember a while back when I mentioned how scientists used evolution (random mutation and natural selection) in the lab to ‘perfect’ gene therapy vectors?

Theyve just one it again! In a BIG way!

Molecular Evolution of Adeno-associated Virus for Enhanced Glial Gene Delivery.

These folks were looking for a way to get man-made viruses to deliver therapeutic genes to infected/defective cells. Now, you dont want these viruses to infect EVERY cell of a sick person, necessarily, you want to target the specific, sick cells. A golden target for these folks is glial cells– as their malfunctions have been connected to basically every neurodegenerative disease you can think of– Lou Gehrigs Disease, Parkinsons, Huntingtons, MS, hell, even migraines.

A barrier to any putative gene therapy for these diseases has been the fact we dont have adeno associated viral vectors that infect glial cells. In the lab, you can dump a LOT of virus on astrocytes, and some of the serotypes will infect them, but that just doesnt work in vivo.

Why not let evolution make you an adeno associated virus that likes to infect glial cells, and only glial cells?

STEP 1: MUTATION
The paper I talked about before? They randomized their viral vectors by shuffling bits of the structural protein around. It was a random as they could get it.

This paper, they made things even MOAR random! “… random mutagenesis, DNA shuffling, AAV peptide display, and a new semi-random loop replacement method…” AHHH! Thats some mighty fine random!

STEP 2: NATURAL SELECTION
They put this super-random mixture of viruses on primary astrocytes from human donors. Some variants wont get in at all. Some will get in kinda, and some will kick ass.

The ones that get in kinda/great will produce lots of babby viruses.

Take the babby viruses, put them on new astrocytes.

Take THEIR babby viruses, put them on new astrocytes.

Koerbe et al only had to passage their babby viruses 4-5 times to get a population of viruses that LOVED astrocytes!!

STEP 3: DOES IT WORK IRL?
Koerbe took their astrocyte-specific viruses and gave them a gene to deliver, GFP. So when they injected these viruses into rats, they could see where the viruses went by looking for green glowing cells. Result? Lots of green in the rat brains (even though human cells were used for selection).

Bonus: You dont really need to know how this is working for it to work. As in, we dont need to figure out each and every step in adeno associated viruses life cycle and selection pressures at each stage and intelligently design viral vectors that can avoid those challenges. We can let evolution do all the work for us, and we can figure out the details later!

Importantly, while a lack of detailed mechanistic knowledge of specific gene delivery barriers in this and other cases can preclude rational design strategies to improve vector properties, a broader “black box” approach to engineer efficient AAV vectors through random diversification and high-throughput selection can still succeed.

You know, like, when you had to do group projects in elementary school? And whoever was the ‘smartest’ in the group ended up doing all the work, but everyone else got a slice of the A? Evolution is smarter than us. Lets let it do all the work, and we can take credit for the new therapies it figures out.

In summary, by employing directed evolution with a diverse array of novel AAV libraries including a new peptide loop replacement library, we engineered novel AAV vectors capable of highly efficient delivery to astrocytes in vitro and importantly to astrocytes and Müller glia in vivo….
… modulating mutant superoxide dismutase-1 expression within astrocytes may reduce neuronal cell death in amyotrophic lateral sclerosis, or therapeutic gene delivery may prevent the accumulation of amyloid beta-plaques in Alzheimer’s disease. Furthermore, because Müller cells span the entire retina and surround every class of neuron present within this tissue, transduction of Müller cells would permit spread of neurotrophic factors throughout all layers of this tissue to significantly augment existing therapies for retinitis pigmentosa, age-related macular degeneration and neovascularization.