A method for inoculating large fractions of central nervous system cells.

Currently, the best inoculation fraction for primate brains is about 2% of the neurons. This is done using injection into the cisterna magna (a small cerebrospinal fluid cavity at the base of the brain) or else intravenous injection. For some diseases this will help. For others, it will not be nearly enough. So, I think about how it might be possible to inoculate most or all of the large brains of humans.

Here’s an interesting paper that suggests a strategy for successfully dosing of neurons in the deep brain. CD898hc single chain antibodies could be made, and their epitope binding region isolated, That binding region could be tested on the surface of a virus capsid to use for targeting to the brain. Would need to test whether a virus capsid would cross the blood brain barrier as well as an antibodies do, but I’d give it decent odds. I think that HSV would be a good vector to work with. AAV is nice, but it’s quite small, and we need a lot more room in the vector if we are going to do base editing.

Here’s a rendering of a rhinovirus that gives some idea of what such an engineered virus might look like. Here, the F’ab region, the functional part of the antibody that has the epitope binding region is attached to the rhinovirus’s cell receptor binding sites. In an engineered virus, those little pigtails would be attached to the virus capsid, and the flattish binding region would be sticking out.

Cold virus with antibody Fabs attached to its binding sites.

Cold virus with antibody Fabs attached to its binding sites.

https://www.rcsb.org/structure/1rvf – Fab complexed with human rhinovirus.
Now, this cold virus is smaller than the Herpese Simplex Virus (HSV) is. Rhinoviruses (cold virus) are about 30 nanometers in diameter. The inner capsid of herpes simplex virus is 125 nanometers, 183 times the volume of the most common gene therapy vectors. So, it’s about 4 times the diameter, of this example I’m using. The most commonly used virus, Adeno-associated virus (AAV), is 22 nanometers, even smaller than a rhinovirus. Adenovirus is 65-80 nanometers in size.

What is a Fab versus whole antibody?

A whole, idealized (because real antibodies flop around) human IgG1 antibody is shown below. The little molecule on the left is what the epitope binding region (hands of the Y) bind to. A and B are roughly the Fab regions. C is the heavy chain. This C region is what the immune system binds to. Almost all antibodies have two protein chains that form the binding region. But that makes it very hard to use molecular biology to express it. So, a special set are used from one set of animals that have single chain epitope binding regions.

A & B – These ends bind to antigens.
C – The Fc chain. Trim21 binds to this.

Below is a model I tweaked to remove the light chain, leaving only the heavy chain part of the antibody. This is a good approximation of what the single chain antibodies are. As much of that binding region as needed can be used. It’s hard to predict exactly how long is optimum. I’d try shortest possible, cut it mid-site, and then at the base of the Y.

Patients with Ebola should be treated with Ghrelin injections, starting early.

Ghrelin stabilizes sepsis and the gut. It works in acute radiation syndrome at 8 gray whole body exposure and up, (LD50 ~4 Gy) where GI syndrome occurs and death follows by septic shock. It works to rescue endotoxic shock.  Ghrelin can rescue patients in septic/multiple organ failure crisis, however, it works best given early.

Human doses of 250-500 micrograms (3.3-6.6 mcg/kg) were well tolerated without achieving a saturation effect.
Ghrelin is not currently available as an FDA approved drug.
To find suppliers search with: CAS No. 304853-26-7, although it should be available from compounding pharmacies, as there are many clinical trials registered.


  1. Kavin G Shah, Rongqian Wu, Asha Jacob, Steven A Blau, Youxin Ji, Weifeng Dong, et al. Human Ghrelin Ameliorates Organ Injury and Improves Survival after Radiation Injury Combined with Severe Sepsis. Molecular Medicine 2009;15(11-12):407-414 http://www.ncbi.nlm.nih.gov/pubmed/19779631.
  2. Lin Chang, Jing Zhao, Jun Yang, Zhaokang Zhang, Junbao Du, Chaoshu Tang. Therapeutic effects of ghrelin on endotoxic shock in rats. European Journal of Pharmacology 2003;473(2-3):171-176 http://www.ncbi.nlm.nih.gov/pubmed/12892835.

I  emailed this to CDC and AAAS as well. It’s very hard to get through to anyone though. One can only hope.

Note: Do NOT give growth hormone for sepsis. Growth hormone has the opposite effect, hastening death of patients in crisis. The reasons why ghrelin, which also causes some stimulated release of growth hormone, works so well but growth hormone alone has the opposite effect are not understood.

Parkinson’s and gene therapy

In theory, Parkinson’s is a simple disorder.

Red: destructive  Center: normal  Green: normal iron removal

Red: destructive Center: normal Green: normal iron removal

The simple cause of Parkinson’s is lack of, or poor performance from, one of the enzymes to create dopamine. In almost all cases, tyrosine hydroxylase is the problem. Giving people with Parkinson’s L-Dopa allows them to make dopamine using the dopa decarboxylase enzyme. But as you look at this diagram, you see why Parkinson’s can keep getting worse. Look at that the red section on the left. Dopamine producing cells die off due to toxicity produced by the interaction of tyrosine without the tyrosine hydroxylase enzyme. And after those cells die off, motor cells die off. Once that happens, it doesn’t matter if dopamine is restored, and the only thing that will improve Parkinson’s is stem cells, induced pluripotent stem cells (iPSC’s). Gene therapy directed at tyrosine hydroxylase may help, but only in the earliest stages.

Making L-dopa requires iron (Fe) for the tyrosine hydroxylase to work, and human brains build up iron as we age. (Iron build up may by one of the roots of age-related mental decline.)  Eventually, enough free iron and tyrosine is present to react directly with oxygen in the absence of the enzyme. The tyrosine isn’t destroyed, so this reaction can just keep ticking along. Superoxide and charged iron are very reactive and damaging to the cell. If there’s too much, it will kill the cell. And when L-Dopa is given, that turns off whatever tyrosine hydroxylase activity is still going on, which may raise the production of superoxide anion and Fe3+.

Another cause of Parkinson’s can be nutritional. Lack of tyrosine and lack of folic acid can be factors. Supplementing folic acid is a good idea if Parkinson’s runs in your family. Raising tyrosine has worked after symptoms start, but it should be coordinated with your physician. In theory, if your Parkinson’s is genetic, flooding the system with tyrosine might speed up the production of superoxide anion and Fe3+. Taking coenzyme Q10, and vitamins C and E should help, and is a recommended good practice from an early age if Parkinson’s runs in your family.

There are a number of drug treatments and none are perfect. The most effective are mixtures of L-dopa and another drug, such as carbidopa, that stops the conversion of L-dopa into dopamine outside the brain. Entacapone improves transport of L-Dopa into the brain. A good course of therapy will provide these drugs and improve matters. Deprenyl/Eldepryl (seligiline) produces amphetamine and may be associated with higher mortality for currently unknown reasons. Various dopamine agonists stimulate dopamine receptors, but this also results in the receptors becoming less sensitive.

SSRI drugs and ritalin (ADHD treatments) are recommended for Parkinson’s. When the cell death that is caused by Parkinson’s occurs, that affects other neurotransmitters as well. Parkinson’s symptoms are just the most obvious. Patients shouldn’t feel the are required to, but exploring them may be helpful.

Exercise helps people with Parkinson’s. Why is not entirely understood, but probably part of it is stimulation of the motor nerves which probably keeps them in shape. Parkinson’s is improved by passive exercise too, as shown by Parkinson’s patients riding on the back of a tandem bicycle at a faster cadence than they are able to keep by themselves. That effect lasts for several hours, and may slow progress of the disease. Another factor is that fairly high level athletics raises catalase levels in the body, and that would protect against nerve damage from superoxide anion and Fe3+. Last, the DNA of athletic people can be 10-15 years younger by the age of 50 than sedentary people.

It may be the case that when buildup of iron in the brain overwhelms the level of tyrosine hydroxylase to use it, eventually dopamine producing cells begin to die from superoxide.

All of this presents challenges for gene therapy.

  • First, we need to know if the Parkinson’s is due to lack of tyrosine hydroxylase. If it is, gene therapy may help.
  • Second,  we should find out what the iron level is in the substantia nigra. If it’s higher than it should be, a gene therapy may help.
  • In general, a catalase gene therapy should help to minimize brain damage.
  • Some forms of Parkinson’s are caused by mutations in alpha-synuclein and development of Lewy bodies. Anti-synuclein antibodies may be of some help. Around 0.1% of antibodies injected make it across the blood-brain barrier.
  • Another possible treatment for alpha-synuclein mutation is to supply a correct copy by gene therapy.

For people with significant Parkinson’s the only thing likely to reverse it is iPSC therapy, perhaps with some gene modifications. For instance, gene therapy in iPSC’s, would be a good choice for alpha-synuclein mutation since it is unlikely that this form of Parkinson’s would be caught early enough to intervene effectively with a gene therapy alone. Gene therapy may be able to stall the progress of Parkinson’s, but that’s all that can be expected.

HAART Cocktail Additions

I have speculated that AIDS related dementia, fairly often associated with some kind of aggression, is part of the reproductive evolutionary strategy of HIV.

Unpublished gene chip data. Y scale is number of genes downreg’d. Genes filtered by pathway to remove false responders. Yes, these are gut samples. Even in humans, only 10% of our nervous system is in our head, though we do prize that part very much. There is at least that much in our guts. And note the taste receptors in the gut. What is on our tongues are just a specialized version of what goes all the way down the small intestine. When you eat good food, your gut-brain probably thanks you for it. Probably a good thing our conscious sense of taste doesn’t extend all the way down…

Just saw Dallas Buyers Club last night. It reminded me of when I was working with dying AIDS patients who gave me names of men who had AIDS that were out deliberately trying to infect as many others as possible. One of those patients went through a phase of praying for my early death and little tricks to try to infect me.  There are other diseases that affect behavior such as toxoplasmosis.

HIV hits a lot of gene expressions for neurotransmitters – very early – long before AIDS.  Cannabinoids are downregulated early through glutamate/GAD downregulation, which is why I think HIV+ people should be on Marinol (Dronabinol, tetrahydrocannabinol) or cannabidiol or something like Sativex (GW Pharma).  HIV also has downregulation effect on serotonin. I suspect HIV/AIDS patients would benefit from SSRI/SNRI treatment early.  Some of the behavioral issues physicians see in early stage HIV patients on HAART are probably serotonin downregulation related.

My additions to the HAART cocktail would be:

  • Marinol, Sativex, or medical marijuana preparation.  I do not recommend smoking marijuana for HIV/AIDS. The irritation of the lungs only worsens immune compromise.
  • An SSRI/SNRI drug. (e.g. Prozac, etc.) The usual game of hopscotch trying to find one that the patient likes/tolerates well is required. I suspect early stage may be able to benefit from what would normally be considered subclinical doses for a while.
  • Nortriptyline or Desipramine because these two drugs have been shown to block exit of cytochrome C from cells, which prevents cell death on Huntingtins. Huntingtins genes showed up downregulated, and these drugs may help with prevention/slowing of dementia. They are also pretty benign, unlikely to cause harm. Note that there are interactions between SNRI and tricyclic antidepressants. And I have no data on whether any of the SNRI’s blocks cytochrome C exit or not. So that would lean toward SSRI drugs over SNRI.
  • Xyrem (Gamma hydroxybutyrate (GHB) GHB is available for prescription off-label.  GHB’s a glutamate agonist and improves sleep. Glutamate receptors are downregulated. HIV/AIDS patients often have sleep problems.
  • Baclofen (Gabapentin) might be a reasonable alternative to Xyrem.

New strain of HIV in Africa

Note that the strains in most of Africa are different than in Europe, North and South America. This is consistent with Paul Ewald’s theory that virulent HIV-1 evolved in the bathhouse culture of North America. Also, while subtype C is dominant today in sheer numbers, that has little meaning. What is meaningful here is what it shows about patterns of human sexual interactions.

At 2/3rds of the common strain, 5 year time to progression to AIDS (untreated) from Swedish researchers is significantly less than the average 7-8 years, but I wouldn’t call it extraordinary. Time to AIDS of 2 years is extraordinary. But yeah, it’s worth knowing about.

SIV – Simian immunodeficiency virus. HIV-1 is the most common strain. HIV-2 is a slow progressing strain.

What’s the use of this identification? It should help doctors and patients to make treatment decisions, perhaps help patients keep on HAART. (HIV-1/2 overview)

Of course, in my experience, too many physicians are reluctant to put HIV patients on HAART early, at least in the USA. I’m not sure why that is, perhaps it’s a carryover from the change in attitudes toward prescription of antibiotics. In the case of antibiotics, not prescribing them for every little sniffle is correct. For HIV/AIDS, it’s not.  So patients – shop around. If you have HIV, find a doctor who will be aggressive. But patients, HAART isn’t a picnic in the park, though better than progressing to AIDS – way, way better.

Recombination is fundamental to HIV replication. HIV crosses over while reproducing 2-3 times for every copy made. Combined with the very high mutation rate that means virtually every virus is some sort of mutant, this forms the basis for HIV’s evolutionary survival strategy. HIV will evolve to fill any niche it can find. So if there is a population that vectors it from person to person, it will evolve to the maximum virulence that it can and still keep transmitting successfully. 


I split off the HAART cocktail stuff into a separate post.

HIV/anti-viral – puzzle from the press

DRACO is supposed to be something from MIT that will fight all viruses.  Watch out for DRACO – MIT Scientist Develops Vaccine To “Cure All Viral Diseases”

…HIV or hepatitis, emerging viruses such as avian or swine influenza, and highly lethal viruses such as Ebola or smallpox…

As part of the PANACEA (for Pharmacological Augmentation of Nonspecific Anti-pathogen Cellular Enzymes and Activities) project, researchers from MIT Lincoln Laboratory have developed and demonstrated a novel broad-spectrum antiviral approach, called DRACO (for Double-stranded RNA [dsRNA] Activated Caspase Oligomerizer). DRACO selectively induces apoptosis, or cell suicide, in cells containing any viral dsRNA, rapidly killing infected cells without harming uninfected cells. As a result, DRACO should be effective against virtually all viruses, rapidly terminating a viral infection while minimizing the impact on the patient.

Like many such notices in the pop-sci press, it is hard to figure, and the headline is overwrought, as is the article.

  • HIV is a single stranded RNA virus, not a double, and has no dsRNA stage. So as described, this couldn’t work against them.
  • Smallpox is a DNA virus, so this wouldn’t work.

Ebola, hepatitis viruses and influenza viruses could be susceptible to this method.

My guess is that they might have a small molecule that ramps up Rig-1.

Is this worthwhile? Certainly, as an anti-viral for a limited subset of viruses.

What is it? I’m not sure. I’d guess it’s a small molecule, but don’t hold me to that.

A new human antibody to treat AIDS

Technology park is advertising a new human antibody to treat AIDS. The claim is that this human can neutralize 98% of HIV isolates around the world. They are trying to drum up interest from pharma – or somebody.

I am guessing that this offering is from Baltimore’s lab. (Antibody-based protection against HIV infection by vectored immunoprophylaxis.) but there could be plenty of others by now.

Bottom line: This mAB should do a little worse than any single anti-viral drug. That is not to say it shouldn’t be available! It absolutely should, because it will be a late-term treatment for AIDS when a the HIV virus in a patient has escaped control by current anti-virals. But looking forward, what the developers of such therapies should be looking for is not a single magic bullet mAb. They should be looking for several mAbs that together neutralize all known isolates of HIV. They should be looking for a HAART made of mAbs. A HAART made of mABs (say 5-15 mABs to different epitopes) would be extremely valuable in the developed world because it would not have the unpleasant side-effects that make HIV/AIDS patients take vacations from their drugs. If we can get HIV patients to stay on their drugs, then those patients won’t spread HIV/AIDS. And if we have a mAb HAART used prophylactically by high-risk people, that will get us one step closer to eliminating HIV/AIDS.

For patients, the superior way to deliver mAbs that neutralize HIV is by gene therapy because it lasts longer and is much cheaper.

So, let’s analyze this single-mAb for HIV/AIDS.
Business problem: This product needs a white-knight. I’ve interacted with the VC/Pharma funding system and it is very narrow. They want to field products that are used daily, and this does come close enough there. But, it’s not a pill, which is the holy-grail of pharma. Small molecules are way easier to make and deliver. Also, this product will interfere with existing pharma products that are very lucrative. HAART revenue is about the same as a mAb, but manufacturing and channel costs are much lower than for mAbs.

Biology analysis:

Their product should work for a subset of HIV/AIDS patients for a while. But 2% isolate escape is actually pretty high. You see, HIV is a quasi-species. It mutates like crazy. One of my first projects in grad school was to look at the range of mutations in HIV. I worked out the HIV mutation tree within a host, and the range of quasi-species of HIV is far greater than the range of quasi-species that transmit between hosts. Many, if not the majority of those mutants may be non-viable, particularly outside the host. But the infective power of large quantities in the bloodstream of what would be a non-transmissible variant external to the host well may be significant within that host. (Lost that cite at the moment, but it’s a Nature paper showing apparent transfer from T-cell to T-cell by membrane contact.) If 2% of the quasi-species variants escape control by this antibody intra-host, the virus will evolve around it. Period. 98% should guarantee escape from control. I would expect that the timeline plot of numbers of patients HIV escaping control by this mAb would resemble an F distribution. i.e. front-loaded with a long tail. I would guess (just a guesstimate now) that the center of that distribution would be around 1.5-2 years.

I would not expect this mAb to do better than any single anti-viral that is normally effective against HIV. We know that HIV evolves around single drugs eventually, and that those drugs start with 100% coverage of all known isolates. That evolution problem is why we have HAART to treat HIV/AIDS now. We use multiple drugs, typically three or more different anti-virals so that to escape control, a virus will need 3 major mutations simultaneously. Since for all practical purposes, that does not happen, as long as HAART is maintained, it usually keept HIV/AIDS in check. Typically, when a patient has escape variants when on HAART, it means that they fudged on their drug regimen. They stopped taking one or two, or perhaps all of their drugs, and did so more than once. Why do patients do this? Because those anti-virals make a person feel lousy and look unattractive. For young men who have lived to be attractive, this is not easy.

The half-life of a full IgG antibody in the human body is roughly 40 days, unless it is being used up a a rapid pace. I would expect this to require dosing at least once a week, and probably twice. But it would be worth studying how mAb titers behave over time when injected into a patient with HIV/AIDS. It may well be possible to back off dosing rate or quantity after a while. (Note that the half-life of synthetic antibodies like micro-bodies/nano-bodies is much lower because they get eliminated by the kidneys.)

The discussion in the advertisement about 25 million AIDS orphans is misleading. Yes, there are millions of AIDS orphans that continue to destabilize third world nations. These orphans are putty in the hands of warlords. Yes, this is important. But this therapy requires clean, sterile needles. It requires a cold chain (refrigeration) to keep it potent. We can’t even vaccinate against measles in Africa effectively for those reasons. (Needles get re-used afterward by injecting drug users and spread hepatitis and HIV.) The combination of problems makes measles vaccine efficacy in most of Africa about 50%. So this therapy is not practical to deliver to the AIDS patients who matter most.

Then the cost of manufacturing and delivering an injectable mAb medication such as this is high. As reference points, Humira (adalimumab) costs $831 per shot every 2 weeks – $21,606 per year. Enbrel (etanercept) costs $20,000 per year. And those are mAbs delivered to a large market.  We can barely afford to try to deliver vaccines that cost less than a dollar a dose. Implying that this mAb could plausibly be paid for in those regions is for all practical purposes a lie. The cost is prohibitive. Completely prohibitive.

That said, thg offering looks like a marketer wrote it up. The marketer is doing his or her best to rep the product, but does not have the in-depth understanding of it that the source lab has.

Lipopolysaccharide and humans

LPS is bad stuff. But for humans, LPS is far worse.

We humans, it turns out, are unique in a very specific way. Immune system receptors for various things are called toll-like receptors (TLRs). There are quite a few of them, TLRs 1-13.

In most animals, each TLR is paired with a Siglec. Siglecs aren’t numbered for the TLR that they work with. But TLR4, the one for LPS, is normally controlled by Siglec 13.

Each TLR turns the immune system on. The Siglecs damp the TLR response if it gets too high. They don’t work 100%. If they did, no animal would die of sepsis. But they make several thousand-fold difference.

We humans don’t have a working copy of Siglec-13. We haven’t had it for a long, long time, because Neanderthals didn’t have them either. We split with Neanderthals around 1 million years ago. That’s 83,000 grandma’s in the past.

The LPS LD50 for a 25 gram mouse is 150 micrograms. That is 150 millionths of a gram. Not very much, but it works out to around 6 milligrams per kilogram. (1 kg = 2.2 pounds).

If humans had working Siglec-13, we would have an LD50 of around 420 milligrams, based on 6 milligrams per kilogram. But instead, the LD50 for humans is… 300 micrograms.

That’s right. It takes just double the dose of LPS to kill a 25 gram mouse as it does to kill a 70,000 gram man.

That’s why, for injection into a human being, it is even more important to have ultra-clean plasmid preps. And the price for a prep for a human clinical trial reflects the level of cleanliness. Instead of $10,000 or so, human clinical trial material costs around $700,000.

Yes, some of that is “what the traffic will bear.” Some of that is regulatory cost. But it’s also because you just can’t make a mistake.

Well, technically, you can make a mistake and use things like Enbrel, steroids and rapamycin to save the life of a test subject. And anyone doing human clinical trials better have those on hand, along with a well defined protocol for following patients just in case. It should never happen, but that’s always true.

Lipopolysaccharide, the “killer app” of plasmid gene therapy


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


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.


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.

Transfection methods – Which is best?

Several basic types of transfection: gene gun (effective on almost anything), electroporation (oldie but goodie), liposomes, polymer complexes, and viral particles, with or without reverse transcriptase.

Cell culture transfection

Liposomes are sometimes problematic, because they kill cells, but these days there are kits from Sigma-Aldrich and others that are quite good. Polymer complexes like Turbofect from Thermofisher also work well, distinguished from liposomes in that they cling to the DNA, and work just about as well with large DNA strands as with short plasmids of a few thousand base pairs. But nobody really cares about that, you just want your DNA to get into the cells so you can move on. Usually, in cell culture, those are just fine.

There are electroporation systems that work in cell culture for both adherent and non-adherent cells. It typically takes a little more handling, and if you don’t set equipment right, you’ll smoke your little cells. But I’ve found electroporation to work fine, even when things are a bit off. Cells are pretty tough if you keep them cold, etc.

The nice thing about gene guns is that you can use them easily right on a plate. Bang, it’s done. They don’t do so well with suspended cells, because the particles won’t penetrate much into water. But usually you can get around that by spinning and draining cells, or culturing them on plates the way it’s usually done when creating monoclonal antibodies. Sterile technique is a lot trickier then though. You’ll need to take great care with your facility, use UV lamps and wipedowns all the time.

Not so many people use viral particles for cell culture, and that’s something I don’t have experience with, so I won’t say any more about it.

In vivo transfection

All of the above work in-vivo also, although you have to prep a bit differently. For instance, to use a gene gun with a plasmid into muscle tissue, you’ll have to open the skin and lay bare a enough muscle. You’ll need to keep the distance correctly, and may want to protect non-inoculation areas from getting transfected. Aluminum foil will do fine surrounding the area if you care.

I like electroporation into muscle also. This is done with a needle surrounded by fine tines that surround the needle in the muscle. The needle becomes negative terminal and the tines around it positive, to drive DNA into cells. This method has been shown to work well, at least in large animals.

In addition to these methods, I like using microbeads of 2-5 microns composed of PEG, PLG or something like that, with your DNA embedded into it. That’s an optimum size for uptake into cells. The drawback is that this can generate an immune inflammatory response – mild, but that needs to be compensated for. (And Butterfly Sciences has some vehicle formulations for that.)

I think that injection into mouse muscle is difficult. Their muscles are so small, and syringes are large enough, that I think it’s pretty easy to screw up the injection. So I’d prefer not to work with anything smaller than a rat.

Special notes about viral particles

Viral gene therapy is more widely known than bare DNA plasmid therapy. In this method, a recombinant virus is engineered containing a gene of interest. Adenoviruses, adeno-associated virus and lentiviruses are commonly used. Viral vectors can be made specific to a tissue type by careful engineering of the virus’s attachment moities.

This method has several drawbacks:

  • The doses required can cause death through a sepsis-like syndrome, although animals are less likely to suffer this consequence because their immune systems are more robust than humans. Jesse Gelsinger died this way. If you get into this kind of trouble, try a shot of Enbrel.
  • There is some risk of cancer. It may be low, but it is there. AAV has a low rate of integration into chromosomes, very low. But it’s not zero.
  • With retrovirus deliver of genes the risk of cancer is significant. Around half of all the children given gene therapy for SCID died as a result.
  • Viral delivery of genes is irreversible. Viruses disseminate in the body, and cannot be removed surgically. In theory, in some limited cases, like synovial fluid, the viral vector should not get outside the synovium. But what about injection errors? Those happen. With plasmids, there is a small section of muscle where the transfection is virtually entirely localized.
  • If the viral vector is accidentally injected into the wrong person, you have a serious problem. Of course, that shouldn’t happen, but when thinking about products that will be sent out all over, things happen. If it’s an animal product, someone might use it on a human. Medical errors happen all the time, from amputation of the wrong leg, to administering the wrong dose of medication. Count on it happening sooner or later.

Both viruses and bare DNA (plasmids) are workable. I prefer the plasmid system because it’s more forgiving. Transfection efficiency is not as good as viral delivery, but it’s good enough.