Lipopolysaccharide, the “killer app” of plasmid gene therapy

 

I’m kidding about the “killer app” of course.

LPS

LPS – Section A, on the left is the poly-saccharide part made of sugar molecules. B, on the right, is the lipid, or oil part. Rendered using Rasmol.

3fxi_bio_r_500

TLR4 receptor together with MD-2, which form the fully capable receptor. You can see little LPS molecules in there, lower left. RCSB 3FXI

Diagram of TLR4 operation.

Lipopolysaccharide (LPS), also known as endotoxin, kills. LPS is the broken down cell walls of bacteria.LPS triggers signalling of toll-like receptor 4 (TLR4).  TLR4 is one of a pair of large protein molecules that manage to recognize this pattern in bacteria. If you had no TLR4, you would be in serious trouble, because you couldn’t respond to the presence of infecting bacteria. So animals evolved the TLR4 receptor so we can detect their presence, and every animal has it.

The problem with LPS is that it causes septic shock. Injection with purified LPS is the lab model for sepsis. In animals, the LD50 is around 6 milligrams per kilogram of body weight.

So here is where it gets interesting. Because the most reliable way to make plasmids is to do it in E. coli bacteria. (Oh, dear, oh dear.)

A cluster of Escherichia coli bacteria magnified 10,000 times. (Wikipedia commons) You can see the cell walls of these bacteria. The lipid tails of LPS point inward, toward the center of the bacteria. The visible surface is saccharide (linked sugars).

Thousands of plasmids can be made by each little E. coli bacteria. We culture the bacteria, then break open their little cells. That releases the plasmids that are floating around inside of the bacteria. This mess is centrifuged, and the clear liquid that contains the plasmids is drawn off. So far, so good. But a fair amount of the LPS is also floating in the water along with the plasmids. It’s enough that you can kill a 25 gram lab mouse with one little injection. The amount of LPS varies, but it’s usually at least 1.5 milligrams per milliliter of water, and can be 10 to 50 milligrams. (One milliliter of water = 1 gram of water. There are 1,000 milliliters in a gram.)

A 25 gram mouse only needs 150 micrograms (0.15 milligrams) to get an LD50 dose. Rats are a little better. A lab rat weighs about 500 grams, so it needs 20 times as much, or 3 milligrams. But the injections for rats are also larger, so you will usually kill your rats with uncleaned native preparations.

There are two ways to clean the LPS out. You can use ultra-centrifugation in cesium chloride gradient, but it doesn’t always work. You can also use a methods that has the DNA of the plasmids stick to a surface and wash away the LPS. This loses quite a bit of DNA.

And there are kits for it that are supposed to let you clean LPS on the lab bench – easy-peasy. Just one problem with that. I couldn’t make them work well enough. And nobody I know has done it either. A professor said that it nearly killed his lab mice when he injected them. So, yes, those kits work. Without it, his mice would have croaked. Instead, they just got very sick. They work, but not well enough.

Seriously, I tried to make those kits work for almost a whole year. When I couldn’t do it, I tried creating new cleaning protocols of my own. For instance, I mixed the plasmid preparations (we call them preps) with oil. Then I let them separate, drew off the oil and repeated it. That also worked – some. It was incremental, and removed about 7% of the LPS with each iteration. To remove 99.5% of LPS in the prep, it would take 75 iterations – in theory. But I lost too much water each time. After 10 iterations I had half the fluid I had to begin with, and I had lost a lot of DNA along with it.

It may be possible to further protect against LPS by adding LALF (derived from Limulus polyphemus amebocytes ) to the preparation. But I haven’t tried that method yet. It’s partial. It might work if you combined the clean prep kit with it. Emphasis on might. It should generate an immune response to the LALF protein though, so it may only work the first time you inject.

But even if LPS doesn’t make your animal sick it is highly activating to the immune system. For gene therapy, you don’t want that. In gene therapy, you want the immune system to forget about what just came in, “Nothing to see here. Move along. Move along.” For gene therapy, you do anything you can to evade the immune system’s notice.

And that is why I send my stuff out to companies that specialize in producing clean preps. They either use the ultra-centrifuge method, or they use the method that adheres DNA to a surface and washes away the LPS. When they are done, they test it using Limulus amebocyte lysate (LAL) to certify the LPS level. You get certified material and you won’t kill your mice. You won’t make them sick. You will minimize immune system activation. It isn’t cheap at $10,000 or so, but it’s the way to go once you get past cell culture.

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