Wednesday, April 1, 2020

All My Friends Are Dying


My Friend

I’m 39 years old and all my friends are dying.

The medical system is failing us. Poor and wealthy, those with and without insurance. We suffer at the fate of a system that optimizes for revenue instead of alleviating human suffering. Regulatory bodies that care more about protecting pharmaceutical company profits than human lives.

It takes around 10 years for a drug to get approved by the FDA and only 48 drugs were approved in 2019. This isn’t even 48 drugs for unique illnesses that have never had a drug. In fact, a recent study showed that around 65% of FDA submissions are just reformulations or improvements of existing drugs. I mean, there are at least 10 different approved heart-burn drugs that aren’t antacids.

Regulation is killing us, literally. As we don’t get the drugs we need to help us fast enough.
Not only does regulation slow drug development but it allows for high drug prices and little competition. US drug prices are almost four times higher than economically similar countries throughout the world. Pharmaceutical companies make billions, averaging 15%-20% profits when the average for non-pharmaceutical companies is 4%-9%. The government allows this despite over 80% of Americans believing that prescription drug prices should be lower!

I’m not writing to be unbiased because I am not unbiased. It’s no secret that I think that individuals should become more independent of the medical system. I have experimented medically on myself numerous times including a fecal transplant and a few gene therapy experiments. I buy my contacts on the internet from Canada without a prescription. I order blood tests online and draw my own blood. My advocacy for medical freedom, body autonomy and genetic engineering accessibility has brought about a lot of heat. The FDA, the California Medical Board & California Department of Consumer Affairs have investigated me. The state of California has passed a law that specifically targets my company. Why is the system fighting so hard against me? Someone who has never sold a drug, except maybe weed to a friend in high school.

The system is scared and afraid of people functioning on their own. You don’t know enough they say. You’re going to hurt yourself and others. They make trying to help people illegal even when there are no approved treatments.

As I become more well known the number of emails and messages I receive from people suffering from disease and looking for help is becoming insurmountable. I rarely respond anymore. I can’t because I usually end up becoming friends with people and I can’t keep watching my friends die. There was a time in the beginning when I did respond. That’s how I met L and D and how we became friends.

D was diagnosed with Non Small Cell Lung Cancer (NSCLC). She was in her 30s and a never smoker. I quit smoking after I started talking to them. I couldn’t handle the guilt. There are no approved treatments that could cure D’s NSCLC and so she decided to try something radical, something illegal, she wanted to try individualized peptide immunotherapy. It was either that or wait to die.

My PhD in Molecular Biophysics taught me how easy it is to order peptides from companies on the internet. You can have them shipped to your home. If companies won’t ship to you then you create a fake business name, website, sign your emails “Ph.D.” and get a PO Box. You can even get an Employer Identification Number from the IRS without any requirements. Having drugs made for you is within the realm of possibility for anyone even if you have no scientific knowledge. We thought people should know this and so together with L, other PhD scientists, medical doctors and people with cancer we developed a DIY guide on how to order peptide immunotherapies. AS, a friend and medical doctor who helped us with this guide passed away in March 2020.

S was on our cancer email list and we became friends. S also had NSCLC and eventually acquired some illegal immunotherapy peptides. The chances these peptides could help were small but that was better than the nothing that was available. That was better than waiting to die. In Summer 2018, she told me she was going to be in the Bay Area visiting family and she wanted to meet up.

I almost backed out at the last minute because l was scared. But what excuse do you give to a dying person? How do you look them in the eye and refuse them anything? I don’t understand how a government can tell someone dying that they can’t try unapproved treatments because, get this, they may die. Cowards.

The thing I remember most about spending time with S was her smile. The purist smile I have ever seen. S never injected any illegal peptides before she passed away. I don’t know why she wanted to meet. Maybe she just wanted to say goodbye. That’s her hands in this video she created shortly before her death. She passed away in January 2019.

I never met M but he loved to workout and would even do so in his hospital bed. I think it helped him to have something to focus on other than the cancer. We were bros. I remember when he told me he didn’t have long. I told him how much of an inspiration he was to me and that’s the last conversation we had. M passed in December 2018.

The number of friends with cancer I have watched pass away is more than anyone should need to endure.

It’s not just cancer either. K, contacted me because she had muscular dystrophy and could barely move, she couldn’t gain any weight, was frail and close to death. There are human tested gene therapies that have shown promise in Becker muscular dystrophy. For Duchenne muscular dystrophy something as simple as gentamicin sulfate has increased dystrophin levels by as much as 15% when tested in humans. A DIY infusion regimen similar to the study would cost an individual around $600 for the compound. I couldn’t help and she passed in June 2018.

If you have the knowledge, million dollar gene therapies like Glybera can be recreated for under $50k. The patents and regulatory filings provide all the details even down to the DNA sequences used, the dosages needed and administration protocols.

Most any drug can be made by a company in Asia. Just make a post on Alibaba and you will get a quote in less than a week. If you want premade, prepackaged drugs there are many websites that sell from the same manufacturers that are used by US pharmaceutical companies, same packaging and all. You can find these sites pretty easily after searching Reddit for a few minutes.

I hate telling people that the biggest thing between them and a self-administered treatment option is just their own lack of knowledge. That the government makes it so that those like me with knowledge can’t help them order and administer drugs they want to try.

Would you risk going to jail for trying to prevent someone’s death? I feel guilty about that question every day.

According to the government it is ok for an approved medical treatment to kill me. Ok for a self-treatment to kill me. Ok for me to suffer and die without any treatment. If however someone helps me with a treatment, even if the drug has been tested in humans, it is against the law.
People don’t contact me because they are looking for an approved drug. Every single one of them would be willing to try a risky treatment if it meant even a small chance of alleviating their suffering. I have thousands of emails and messages. The husband who has stage 4 cancer, his wife telling me she and her kids can’t live without him. The couple who are drug addicts and would do anything for their daughter to not suffer the same fate. The family that has two kids with muscular dystrophy. The younger brother watching the older deteriorate knowing it won’t be long till he suffers the same fate. Neither child will live past 25.

D passed away in January 2020 and many others die each day without any hope.

See, it’s not money or knowledge that keeps people suffering, it is greedy corporations and the regulatory system that supports them. The system is so set on avoiding blame for someone getting hurt trying something risky that we would rather just let people suffer and die with no chance.

Friday, December 13, 2019

User Interaction Design for Genetic Engineering: Let's Replace Agar with Bagels


Agar plates are a staple of most labs.
They hold moisture well and can have a reasonable distribution of nutrients, chemicals and antibiotics that you may need to grow your organisms.

The problem is for the uninitiated, agar plates aren't intuitive. Without an autoclave, melting all the agar can be a strange process of uncertainty. Streaking out organisms is a mess and the agar is usually so soft most people starting their forays into genetic engineering just destroy the plate.

I have been thinking alot about how to make genetic engineering and lab protocols more intuitive. User interaction design is an important characteristic of making a technology widespread. And let's be honest science is not designed with the user in mind. Have you ever given someone a pipette for the first time and seen them try and use it? lol Fuuuuckkkk you science.

Science is so poorly designed that even PhDs struggle to learn and use new equipment, techniques and protocols. This is compounded by the problem that scientists are notoriously against change. If it works it doesn't need to be changed because generally scientists want to focus all their efforts on doing experiments so they can publish papers. There is no incentive in science to design better ways of doing science.

Running The ODIN, I get to see where people struggle the most when trying to learn genetic engineering and one of the biggest problems is making and using agar plates.

Agar is not expensive so I'm not looking for something cheaper. If you are still using molecular biology grade agar I applaud you for being a dumb ass. You can purchase 1kg of agar on amazon for $40 or 1kg of "molecular biology grade agar" for $146 both work exactly the same.

What you really need is something that is easy to use, it doesn't shatter into a million pieces if you touch it and is intuitive for most people.

To me alternatives would be something people are familiar with. That we use in our everyday life. I'm just winging it here and not proposing these as alternatives but trying to think differently about science.

I chose a bagel


I wanted to see if I could grow some genetically modified bacteria and propagate the cultures. I know this seems insane and I think I went off the deep end on this one but I think that is sometimes what is required.


First, I wet all the bagel with either water and ampicillin or LB ampicillin (LB is a nutrient media that helps bacteria grow). Using antibiotics like ampicillin is a standard method in genetic engineering to select for the genetic modification. I took some ampicillin resistant GFP engineered bacteria and streaked it on each of the bagel pieces. The fluorescence from GFP makes it easier to track the bacteria through the experiments. Streaking bacteria on a bagel is tough as the surface was soggy and uneven and inoculation loops aren't really made for that. Still I managed to get a little bacteria on.





I put the materials in ziplock type plastic bags. I imagined this would hold the moisture in. I then incubated them at 37C overnight.

The bags did not hold moisture well and the materials seemed to dry out a bit. I think this will be one of the most important things to think about in the design.





Though the light conditions of this image are not comparable to the original taken before the experiment it definitely seems like there is less fluorescence than when I started, I imagine because the bagel dried out. ugh.

Still, its seems like the bacteria survived and so I decided to break off a piece of bagel and use it to inoculate some liquid media and see if I can propagate the culture.


I used a centrifuge to spin down the culture and the bacteria that grew in the culture are fluorescent meaning the bagel inoculation worked.




After 48 hours of having the fluorescent bacteria on the bagel there doesn't appear to be contamination of any kind and the bacteria are still fluorescing.

I don't think bagels are the future of genetic engineering but I don't think agar plates are either.
Most everything that is done in genetic engineering and molecular biology is a complete kludge. The fact that I need like 5 different pieces of equipment to put DNA in bacteria is insane.

Am I really insane for doing this or are the people who continue to do science in such a kludgey way insane?





Tuesday, December 3, 2019

Using My Own Blood Serum For Human Tissue Culture

Growing human cells/tissue in culture is one of the foundations of modern medicine. This typical involves taking human cells from an individual or purchasing them obtained from one of many companies that cell(lol) them.

Human cells have long been assumed difficult and technical to culture but recently I have built out protocols and a class that allows people to culture human cells with minimal equipment and experience in their own kitchen even. I am constantly trying to use the most simplistic and cost effective techniques and materials to lower boundaries in science and thus increase innovation. I want anyone to do experiments with human cells if they want. This adds value for all humans not just Biohackers.

What if all you needed to grow human cells was yourself? Your own blood.

Generally, when growing human or animal cells in culture the liquid media used to grow the cells contains Fetal Bovine Serum (FBS). Fetal Bovine Serum is literally the serum(non-red blood cell part of blood) from fetal cows. After a cow is slaughtered they remove the fetal calf and puncture its heart to extract the serum before they kill it. It is pretty barbaric and doesn't scale well. I mean unless of course we want to farm fetal cows for the sole purpose of extracting their serum?

Why do scientists use FBS instead of serum from other animals? This is not really known. Most people say it is because FBS contains less antibodies than other serums and so is less reactive to the cells in the culture but I am skeptical as to how many animal serums have actually been tested head to head. Scientists do this often where they do something only because other people did it with very vague reasoning why. Scientists tend to be very skeptical of change. In fact, there are serums from adult Chicken, Goat, Horse, Pig, Rabbit and many others. You don't read about these often or at all in the modern scientific literature.

I wondered if fresh serum from my body would work in human tissue culture? I am a trained phlebotomist(true story) so I decided to draw my own blood and use a centrifuge to separate the red blood cells from the serum.



The hardest part is always drawing my own blood. I usually use my median cubital vein on one arm and draw the blood with one hand but thinking about it now I should probably use a vein on my leg so I can use both hands at the same time, oh well, next time? From that blood draw, I obtained around 6mL of my serum. Probably about a third of the blood was serum so if we use the maximum blood donation number of 500mL you can get ~150mL of serum from yourself in one sitting. I should probably say don't try this at home. Drawing your own blood isn't the easiest and you can hurt yourself. Again, I am a trained phlebotomist.

I wanted to grow up some human cells with the serum but I wanted to compare it to FBS and Newborn Calf Serum (NCS), which as the name implies is from newborn calves(I don't know if any calves were hurt in the salvaging of that serum but I assume not). NCS is much less expensive monetarily and much less expensive morally and ethically.

For the experiment I used 3 replicates of HEK 293 cells for each media formulation in a 12 well tissue culture plate and did the experiment twice. I used 10% serum in each case. Above are example pictures. As you can see from the picture that Josiah Juice (my serum) performed as well or better than FBS qualitatively. NCS performed fine though cell growth seemed consistently slightly less than FBS and Josiah Juice. Still NCS seems like a great inexpensive alternative to FBS as NCS costs around 10 times less than FBS! Caveat, these cells were grown up in FBS before being transferred to other serums in the experiment so they are probably under-performing. I imagine that NCS and Josiah Juice cells would do better after the cells acclimatized to the serums on the second or third splitting.

This is pretty fucking cool. Run out of FBS and in a pinch you can use your own serum. But seriously this experiment is more to prove a point.

The biotech and science industry is truly fucked because everyone is doing what everyone else is doing and hoping that somehow their dogged go-getty attitude is somehow going to lead to innovation. What people need to be saying is "Fuck what everyone else has done. Maybe they just did what was readily available to them. I am going to figure shit out instead of copy for a vague reason so I can do my research as fast as possible to try and publish a paper no one cares about."

The explosion of human tissue culture and lab grown meats cannot rely on FBS forever as there is an extremely limited supply that cannot scale. A whole company or industry could be built around supplying a low cost scalable FBS alternative that works just as well or better. Fetal Bovine Serum alone has a market size of $700 million. This will only grow.

Maybe growing human meat using human serum is the answer or maybe that is just my next project.















The nitty gritty...

For working in human cell culture I mostly focus on HEK 293 cells. This is a human embryonic kidney cell line that was modified to be "immortal". These immortalized cells are robust and are great for people to learn cell culture techniques. Obviously, conditions that can be applied to HEK293 cells cannot be applied to _all_ cell lines. However, I think it would also be foolish to think that they can't be applied to many other cell lines.

I culture human cells in a non-CO2 environment. This is because you don't actually need CO2 for tissue culture. The reason people use CO2 is mostly because they use DMEM which contains bicarbonate and CO2 is required to buffer the media. Honestly, I don't get it? Still trying to figure out why people use this contrived method.

I use L15 media. This media is not buffered with bicarbonate so no CO2 is required. In the media, I generally use Ampicillin (100ug/mL), Streptomycin(100ug/mL) and Gentamycin(50ug/mL). This generally prevents most any bacterial contamination that can happen in a non-sterile setup. Yes, that is correct. No sterile hoods or sterile areas are used in my human cell culture. With a little experience and by sterile filtering(0.22uM) the media I rarely experience contamination of cultures.

Briefly, HEK293 cells were grown to confluency in L15 with 10% FBS and the above antibiotics. The cells were washed in PBS and then incubated in a 0.25% Trypsin 0.02% EDTA solution for 5 minutes to removed the adherent cells. The same volume of cells were then added to each well. Each well contained 10% of each serum plus L15 and the above antibiotics. Cells were incubated at 37C with no CO2.







Wednesday, August 21, 2019

Does Heating Antibiotics Destroy Them? No

Whether antibiotics are destroyed by heating is probably inconsequential to most people. However, I see scientists and biohackers talk about it way more than you would expect.

Why?

In genetic engineering when scientists modify bacteria or yeast they use antibiotic selection. This means that they give the genetically modified bacteria and yeast antibiotic resistance because it makes it easier to tell which were engineered. Organisms that survive the antibiotics were most likely engineered. This is not always the case, contamination, escapers and natural mutations can give false positives. People doing genetic engineering for their first time experience these issues much more than a seasoned experimenter and so it is important to know what to blame so you can get the experiment correct.


Media is the term used to describe food organisms eat to survive. It basically contains sugars, nitrogen and other macro and micro nutrients. Generally, media is heated to sterilize it, you don't want random bacteria to grow in your media and ruin your experiment.

In a professional lab environment many scientists will use an autoclave which heats to 121C and 15 PSI. While people doing experiments in a more modest setting will use a microwave or an oven which can only go to ~100C before the liquid boils over. In most cases 100C is sufficient to sterilize media. In fact, in many cases not heating and just adding antibiotics is more than enough to sterilize media over the course of a 2-3 day experiment.

When making media scientists wait until after the media cools to add antibiotics. This is good practice. If you can wait 30 minutes no harm is done by adding the antibiotics at a later time. However, this action has led many people to believe that heating antibiotics in any way will destroy them. In fact, it is what I was taught. Only add antibiotics when the media cools to below 50C.

Because I am lazy and always try and do things different than the establishment I started adding antibiotics to my media before it cooled a long time ago and have rarely or never had problems.

I never did a head to head experiment though. I never compared some heated media to non-heated media to media without antibiotics. So I decided to give it a go.


I heated up LB Agar in a microwave and added antibiotics at ~95C.
I used standard working concentrations for bacteria
Kanamycin - 50ug/mL
Ampicillin - 100ug/mL
Chloramphenicol - 35ug/mL
Streptomycin - 100ug/mL
G418 - 200ug/mL

The length of time each antibiotic was at >90C was 5 minutes. The media was allowed to cool at room temperature so the agar plates could solidify.

I also did
LB Agar with no antibiotics
Ampicillin 100ug/mL added at ~50C

I took a tube of DH5a E. coli bacteria and grew to OD 600nm 0.6 in SOC and then plated 10uL - 4 times on each plate. I let all the plates grow overnight for 18 hours at 37C.



As you can see from the plates there is clear growth on the LB Agar plate that had no antibiotics added and there is no growth on the other plates whether the antibiotics were added at >90C or 50C. The antibiotics were not destroyed by heat at least not enough to prevent bacterial growth.

After ~40 hours there is still no growth on any of the plates but the LB Agar without antibiotics plate has some random contaminating strain of bacteria growing



The antibiotics seem to be working fine.

If you don't believe me try the experiment yourself. It is fairly easy to perform.

Caveats
I am not saying "no portion of the antibiotics in the media were destroyed". What I am saying is that it is safe to heat antibiotics and still have enough of them leftover to prevent standard lab bacteria and contaminating bacteria from growing, which is their purpose in this case.

I am not saying this method is the way everyone should make their media. What I am saying is that if you do heat your media with antibiotics in it you are ok and it won't ruin your experiment.

















Wednesday, August 29, 2018

Cultured Meat Will Not Be Realistic Anytime Soon: The Numbers Behind the Hype


Cultured meat, clean meat, synthetic meat, in vitro meat, cellular agriculture, animal free meat, whatever you want to call it, is meat grown in a liquid culture in a bioreactor, a large vat to grow cells. What they do is take muscle cells from an animal and put them in media that has the materials required for growth of the cells. The idea is simple and it is crazy to think that you could just take your own cells grow them and turn them into a meal but you could! One day cannibalism could be the new cool thing. Maybe.
Now cultured meat is said to have advantages over normal meat in that it is technically vegan(no animal is hurt in the process), requires less resources to manufacture and is better for the environment. That sounds great! I mean, who wouldn’t support eating a hamburger that tastes just like a hamburger from a cow except you don’t need to kill the cow and you get to help the environment in the process!
There are a number of start-ups in past few years that have raised a bunch of money on hype. It’s not unreasonable to imagine one day we will all eat cultured meat. The problem is that most people tell you that it is only 2 or 5 years away(Memphis Meats says they will sell cultured meat by 2021) I want to let you know that it is probably much much farther away than that, maybe even 20 years or more.
See, there are few things in our world like food. Human beings constantly eat so the amount of food that needs to be produced to feed everyone is massive. We don’t see it and so are oblivious to the scale and infrastructure required to get us our meat. According to the National Cattlemen’s Beef Association 25 billion pounds of beef is grown, butchered, distributed and sold in the US. Think about that scale, the average semi truck can hold maximum 80,000 lbs. It takes around 390,000 semi trucks packed to capacity to move that meat around. Think about the number of people employed in the beef industry. According to the bureau of labor and statistics around 131,000 people working in Butchering and Meat cutting alone. There are 728,000 cattle ranches in the US! The infrastructure and job training around meat and food is at such a scale it is not even really fathomable. The craziest part is that USDA Choice Beef is sold for the average price of $5.96 / lb! AND people make money off this whole process!
What this tells us is that meat takes more than just cattle and cultured meat takes more than just being able to grow some cells or giving a company tens of millions of dollars.
But why not? It’s not hard to grow cultured meat cells. I mean I have grown up cultured meat in my garage and you probably could too. Just get some freshly butchered meat, sterilize it, and throw it in a petri dish with the proper growth media to feed the cells and some will grow. You can find instructions here on how to isolate and grow mouse myoblasts(A myoblast is a muscle cell. It is what meat is made from).
Ok great but if it is so easy to grow then what’s the problem?
The main problem with cells is that they don’t scale well outside of organisms. It’s easy to grow cattle, you just feed them. In cells, once you try to move from a petri plate to a 1 liter bioreactor to a 100 liter bioreactor to a 1,000 liter bioreactor things become exponentially more difficult each step. Making lots of meat cells is so much harder than making just a little meat cells.

Cultured Meat Needs Antibiotics

Yeah, remember when people told you that these cells are antibiotic and growth horomone free, weeelllll they lied.
Animal cells grown in culture are severely prone to contamination. So much so that most research labs have laminar flow hoods and separate rooms dedicated solely to this purpose. Growing cells can not only get obvious contamination but also contamination that is not detectable by visual inspection or microscopes, the dread of the cell culture world is a teeny tiny bacteria called mycoplasma. Theoretically, you can always just add more antibiotics and anti-fungals. Still this isn’t even mentioning viruses…
From this paper they say you can get about 1gram(g) of myoblast cells per liter(L) of culture media under optimal conditions and the process to grow them takes around 10 days. I have heard that people can get more than 1g / L but not of myoblasts alone. Anne Specht, Ph.D. from the Good food Institute, a pro-cultured meat institute, say that it will take 44 days to grow a single batch at scale. So let’s say 1g / L is an underestimation and 44 days is too long and so overestimate with 10g / L with 10 days. It takes 45L of growth media per pound(lb)(~450g in a lb) of cultured meat. So one hamburger, a quarter pounder(~110g) would need 11L of media and cells! To produce 1000 quarter pounders you would need 11,000 liters of cells!! Imagine trying to keep all the liquid, all the cells, all the air, all the pipes and connections, everything that helps run this bioreactor sterile and clean. Basically, the only way to do this will be with antibiotics. Sterility is going to be an expensive ansd hard learning curve. Bacteria survive even in NASA clean rooms how much more for this?

Cultured Meat is Not Vegan

To grow the cells at more than a snail’s pace you need to use FBS. FBS is Fetal Bovine Serum, literally, the filtered non-red/white blood cell part of blood from fetal cows. The reason fetal serum is used is because it contains all the growth factors that are needed and fetal cows don’t have all the immunological parts that would cause them to kill or inhibit the cells you are trying to grow. Theoretically, you don’t need to use FBS you could possible use Newborn Calf Serum(NCS) which is the serum from newborn cows, much easier and less expensive to obtain but it doesn’t work as well and is still not vegan. NCS is also more immunogenic and so is less likely to work well. Adult serum is not usually used because it has lots of immunological factors that make it difficult for cells to grow but maybe we can eventually figure out an easy way get adult serum to work and harvest it from humans? Would that be vegan?
*Disclosure I donated plasma in college for money when I was a poor undergrad so it’s not an unreasonable idea
Are there good cheap vegan alternatives to FBS that work as well? There are plenty of non-serum based alternatives but none that work well enough to make people switch over.

Structuring those Infras

When you think about meat we tend to think about the animal and the grocery store because that is all we really know and see. The biggest problem with cultured meat is not whether you can make a meatball or a hamburger, that’s the easy part. The hard part is how to scale it to be able to produce enough to sell to consumers and make a profit.
In order to get your meat to the store first, you have the farmer or cattlehand. The average feedlot(let’s call it a farm) has 40 head of cattle. There are around 728,000 beef farms and ranches or about 15,000 per state if they were divided equally. This produces 25 billion pounds of beef. Say a cultured meat company aims to take 1% of the market or 250 million pounds of beef. At traditional prices($5.96 / lb) that beef is worth about $1.5 billion.
Let’s just say for argument sake that a company like Memphis Meats really only needs to make 1 million pounds of cultured meat a year. Just a note, 1 million pounds a year is tiny, Impossible Foods, a plant based meat start-up just started scaling up to 1 million pounds a month and they aren’t even selling in grocery stores. Ok, still, how would a cultured meat company do that? You need to start out by building a warehouse filled with giant vats to grow cells called bioreactors. Now, most bioreactors are around 2000L or smaller. For reference, a chinese company that grows massive amounts of cells for other purposes, WuXi, created a 14 tank 2000L system for $150 million. Now, this warehouse has a max output of 28,000L, if they run flawlessly at 10g / L of cells and a new culture every 10 days you could make around 23,000 lbs of meat a year. Cattle take around three years to grow to full weight. So cultured meat is definitely more time efficient. Still, even if you could make 10x more cultured meat cells or 100g / L it would still only be 230,000 lbs of meat a year. That is still not alot though. Ok, so, what if every liter produced 1 kg or 2.2lbs of meat even though this is a theoretically impossibility because that is (1kg)1000g of cells per (1L)1000g of water, i.e. there is no water left only cells, but let’s just see how the math turns out.
Every 10 days you would make around 62,000 lbs of cultured meat and you can do that 36 times a year so you have a total of 2.2 million lbs of cultured meat a year. Also, understand that the animal serum industry only produces around 700,000 L of FBS media to grow the cells each year and to run the bioreactors that much would require over 1,000,000 L! So even if you could produce an impossible number of cultured meat, you would need an unyet accomplished amount of media and all to grow only 2.2 million lbs of cultured meat, which is less than 0.01% of the 25 billion lb beef market. Even using theoretical impossibilities and a state of the art bioreactor system one could only make less than 0.01% of the current market in a year. Are you skeptical yet?
It gets worse though, at consumer prices reasonably comparable to cattle beef say $10 / lb, that 2.2 million lbs of cultured meat would only be $22 million a year. If the media to grow the cells only cost $22 / L you would still make no profit. Unfortunately, it costs a lot more!

The Cost of Making Cultured Meat

FBS is the media used to grow cultured meat cells and it is expensive. You are talking about $1000 for one liter. Yes, one liter. Maybe that price can be driven down by greater demand but fetal cows aren’t exactly the easiest thing to come by. So that 45L / lb of cells is going to cost you $45k. There are definitely bulk discounts so I am sure the price is lower but go back and compare that with the $5.69 / lb of USDA choice beef charged to the consumer. Even if you get a 75% discount at bulk a full pound of cultured meat would cost $11k!!! This is also without replacing the media. Generally during cell culture the media will be replaced multiple times to help the cells keep growing at a good rate.
It is true that there are other ways to grow cells that don’t use FBS. Memphis Meats claims that they can get the price down to $2400 / lb, which seems great when previous prices were in the hundreds of thousands of dollars. But this number is ridiculously high even if it is true. To produce that 0.01% market share of 2.2 million lbs of cultured meat would cost around $5 billion. While at market rate that 2.2 million lbs would only produce around $22 million in revenue.
But let’s theorize again. Cells can grow in many many different types of media. In reality, all you need to grow these cells is some carbon source, a nitrogen source and the proper pH. Newborn Calf Serum(NCS), cost about 20% of FBS at $174 / liter and while that seem like a great deal it doesn’t work as well so that 10g / L of cells is probably much lower but let’s say it is not. So 45L x $174 is about $7.8k / lb using NCS. Even if you can make 100g of cultured meat per liter you are still talking $750 / lb for just the growth media alone.
But hey start-ups just wing it sometimes so let’s say you just use something like Gatorade with a cheap nitrogen source. Six gallons(22L) worth of gatorade mix costs $18 on Amazon. So using something as cheap as Gatorade mix would still cost you $36 / lb at 10g of cultured meat per liter. Of course this is without paying anyone, purifying, packing, processing and shipping. So a bottom number for cultured meat is probably above $36 / lb. Anne Specht, Ph.D. of the Good Food Institute calculated that unless significant advances happen the lowest you can recreate culture media at scale is for $377 / L. So it’s going to be ridiculously hard to make cultured meat at a comparable price but even if someone does they can’t just sell it. They still need to deal with the FDA.

FDA

The FDA will probably regulate cultured meat. What exactly does this mean? At the moment no one knows. Does it mean a lengthy process in order to get the product approved for safety or does it mean just simple tests to show that the product is not contaminated? To me this seems to be almost as scary as the scaling problem. The FDA is notoriously lengthy to work with and so you can imagine approving the use of cultured meats possibly taking years.

The Future of Meat is Plant Based

Let’s be honest. In order to create a food product that can scale it needs to fit inside the existing ecosystem of infrastructure. The best bet for something like this would be a plant/fungal based replacement. I have no connection to Impossible Foods but have eaten their plant based burger a few times. Their burger is composed of plant based products and then uses a plant hemoglobin(blood) mimic called leghemoglobin. This leghemoglobin is produced in yeast using genetic engineering so it can be made at scale. It is purified from yeast and added to the plant based burger to give it a “meaty” taste. Their “fake” meat is no different than countless others on the market and so can be processed and made in mostly the same besides one ingredient. The Impossible burgers tastes pretty burger like. The texture is not completely perfect and generally the restaurants that serve it will sear the burger to give it some texture.
Selling a new food is all about scaling and Impossible Foods knew that. The next big food start-ups will also know that. Make a staple with mostly plants/fungus say pork, chicken, bacon? And add something completely new to it like a little bit of cultured fat cells. Not too much that it costs alot but enough that it is a unique product that people will want to try. This makes it so the infrastructure investment is minimal and once you create the product your path to market is clear.
Finally, let me take a minute to apologize. I’m not saying that cultured meat is a bad thing. I am not saying don’t invest in it or try to do it. If no money is ever invested in cultured meat then it will never become realized. My biggest problem is that people either can’t do basic maths or don’t care to and so make wild exaggerations about how cultured meat is so much more amazing than cattle beef when it really is just a more expensive, antibiotic laden replacement that can’t scale well.

Friday, October 13, 2017

The First Attempt At Human CRISPR Gene Editing

The first attempt at human CRISPR gene editing did not occur in a hospital or University or in a clinical trial by some $100 million funded company. Instead, it happened in small cramped room in San Francisco in front of 30 or so people who squeezed in to listen to a talk about how biohackers are making genetic and cellular modification accessible.


This is the first time in the history of the Earth that humans are no longer slaves to the genetics they are born with. As I write this, the FDA is in the process of approving the first human gene therapy treatment. Still it's too slow for me, clinical trials have been going on since before 2008. I want to accelerate that. I want people to have a choice about their genetics. 

To push it forward I did a CRISPR experiment on myself.

How did I do this? CRISPR is some complicated new technology that is really hard! Well, at least that is what the press and media and maybe even some Scientists say. Unfortunately, it's not true. It just took one piece of DNA that contains the Cas9 protein and a guideRNA(gRNA) targeted to the exon 1 of the Myostatin gene and I injected my muscle with it. This DNA then enters some of my cells and both the Cas9 protein and the gRNA would be made by my cells and this molecular complex would target my myostatin gene and cut it. This would lead to Non-Homologous End Joining(NHEJ) and effectively some of the myostatin copies would not work. When myostatin is not working to stop muscle growth, muscles grow.  

Myostatin knock-out dog on left, normal dog on right.
Image from
https://www.technologyreview.com/s/542616/first-gene-edited-dogs-reported-in-china/

I won't look like that dog though. The DNA only entered the area of injection in my forearm. However, the point of this experiment is not whether the CRISPR gene editing changed my muscles(too early to tell). Or that the efficiency was 100% and better than anything available. The point is that we are on the cusp of  humanity changing. This is the first of many people who will change their genomes. This will happen for medical reason, for science, athletics or maybe just because people wanted to or were bored.

I am sure many people will try and dismiss this as unscientific or how clinical trials will need to be run. The problem is that old world is dead. It died long ago and biohackers are creating a new one in its place. This is a world where the only important outcome is the one intended by the user. The FDA is meant to allow the creation of mass market drugs that companies can profit off of. It was never meant to regulate the genetics of individuals.

What could be more of a human right than to be able to decide what genes create you? 

About The Experiment

The one thing about a technology is that it takes infrastructure to grow. If everyone needs to program an app to use an app you would have very few people using apps and that is the state of lots of biotech and all of genetic engineering at the moment.

Fortunately, because of the hype, CRISPR has reached the place of infrastructure threshold because of all the companies that have been getting investments. What has happened is that lots of companies have been building up supporting technology to sell to the companies with lots of money. This means that the technology becomes more accessible to everyone. 

This is even the case for things like using CRISPR on humans. So why aren't people doing it or trying it? Are they scared? Don't want to? Or just don't understand how easy it is? Seriously, it is so easy that someone with little or no biotech knowledge could order DNA that contains Cas9 and a gRNA(a complete CRISPR system) and use it on themselves if they wished for under $400.

I'll be honest here. CRISPR modification is a bit overboard. In 99% of cases CRISPR modification is probably the wrong thing to do. Transient transfection has virtually no hazards and can lead to the same outcome. Still CRISPR is a fascinating technology. It blows my mind that in 2017 the technology to modify your genome is so easy to use. So I set about to use it.

One of the most common genes to target for proof of concept CRISPR or genetic engineering experiments is myostatin. Myostatin inhibits muscle growth so when you knock-out the gene it makes things have more muscle and become physically stronger. Examples(https://www.nature.com/articles/srep25029). A knock-out generally just refers to a mutation that in some way causes the gene to not be made properly. In some genetic engineering methods you remove most of the gene by literally having it cut-out and replaced, this is where the term knock-out stems from. In CRISPR a knock-out from NHEJ often occurs from missense mutations that shift the whole DNA coding of the protein to make gibberish and so the gene doesn't work.

What about doing it on adults? There have been a few CRISPR experiments on non-embryos such as https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4883596/ but most experiments with CRISPR are on embryos because then you can edit every cell easily as there are one or very few. This doesn't mean it doesn't work on adults just that you are working with percentages of cells rather than all the cells in the organism. 

Methods of getting DNA into cells can be as simple as putting the DNA in water/PBS and injecting it. The efficiency of this however is pretty low and is probably in the < 26% range of DNA getting into a cell. Still with billions of DNA molecules per injection this can be a decent number of cells. This technique of injecting DNA with PBS has been used in Phase I clinical trials(https://sci-hub.cc/http://online.liebertpub.com/doi/pdf/10.1089/hum.2004.15.1065). 

So has myostatin ever been edited or changed in an adult? There have been experiments on using DNA containing follistatin(follistatin is a myostatin inhibitor which stops myostatin from working instead of messing with the gene) and this has shown muscle growth in mice and macaques(non-human primates (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852878/). It can take months to see a difference in muscle growth though. Still this is not CRISPR.

CRISPR generally has lower efficiency than just the efficiency of getting the DNA into the cells because once in the cell the editing needs to take place and in the case of NHEJ a mutation needs to be made. So in order to make my injection more robust I mixed the DNA with PEI(polyethylenimine). This molecule has long been known to get DNA into cells more efficiently when injected or mixed with cells in culture (http://www.pnas.org/content/92/16/7297.short). 

The question of whether gene engineering will occur by injecting DNA with Cas9 and gRNA is mostly a non-question. There is an extremely high probability that the CRISPR DNA entered my cells and that some were edited. The biggest question is how many cells were edited and were there enough to have any physical effect? Even after the experiment I don't know. As only time and more experiments will tell.

A big question people have is, "What about off-target effects?"
According to a recent battle of the CRISPR off-target effects. Even using a gRNA that has predicted off-target sites in two mice led to 12 Single Nucleotide changes and 16 InDels(insertions or deletions) total among them.

In comparison in a human cell there is estimated to be anywhere from ~40,000 to ~150,000 DNA lesions caused by oxidative DNA damage at any given time. Cells also undergo ~50 double strand breaks per cell per cell cycle 

This is not to say that increasing double strand breaks(DNA being cut) is in any way helpful but more that human cells are very robust. Especially when the double strand breaks from CRISPR are targeted and are non-random meaning that through proper choice they chance that the can lead to a mutation that causes cancer or similar is even smaller than naturally occurring breaks.

To this day there has been no known occurrence of CRISPR causing spontaneous cancers to form.

I think it is ok to be fearful. But as a Scientist I think it is also a goal to look at fear and see if it is reasonably backed by numbers and understanding and compare that to the risk you are willing to take. You have about 5-10 trillion non-red blood cells in your body undergoing at least 50 double strand breaks every few days (not counting red blood cells because they make up a large portion of the bodies total cells and are boring). I can't imagine a few billion extra targeted double strand breaks would add much to risk. 10 billion extra double strand breaks(200 million cells at 50 double strand breaks from CRISPR(overestimate)) would amount to 0.0001% of the total going on in the body in the period of ~ a week. Over the course of a lifetime it would be infinitesimally small to be exposed to one CRISPR editing dose.

So I decided to go for it. I ordered the DNA online. There are many companies and websites that do this and so I will not mention the name of the company because I don't want to make any problems for myself or others.

Anyone can order DNA and can find a gRNA as long as they know the name of a gene(try it out with MSTN or myostatin). You can get a little more technical like I did and decide where you want to target and type of Cas9 used and other things but generally even if you had no idea really what you were doing you could come out on top.

Instead of having someone replicate the DNA to make lots for the injection(you can pay companies <$200) I grew up the bacteria that contained the CRISPR DNA myself and purified it. I basically did a double purification to make sure it was pretty pure(endotoxin free!). I used gel electrophoresis to assess the quality further. I then prepared 40ug of DNA with PEI in 350uL and it was ready for injection. 

The DNA sequence for the DNA I used can be found at: https://drive.google.com/open?id=0B_R75gIJvkFUMXBLb01VbUpjME0

After the injection there has been no swelling and only mild tenderness from plunging the needle into my muscle. The tenderness went away within two days of the injection. I am measuring both relaxed and flexed muscle circumference around the forearm where the site of the injection was.

The way to test to see how many cells were modified would require some "deep sequencing" i.e. I would need to do a muscle tissue biopsy and would need to sequence the myostatin gene in thousands of my muscle cells. I have contacted some companies for quotes.

Though this experiment was on me. I don't want it to be only about me. I want it to be about how this technology is inexpensive and easy to use. I want to it to be about helping people use this technology to better their lives and so I wrote a guide that shows people the steps of how easy it is to make your own CRISPR system.

The guide I wrote is meant to be general. Not meant to be in depth and weigh down people with details as I think genetic engineering shouldn't only be for people who can program DNA from scratch. Especially with the state of modern technology you don't need to and shouldn't need to know how to create gRNAs from scratch or their orientation &c. &c.

For people interested The CRISPR DNA is also listed on The ODIN but less for making money off of it(we are selling it dirt cheap) and more for making it accessible. Understand, the DNA we sell is not injectable. Once people have the DNA they can replicate it indefinitely in bacteria and grow up as much as they want and share it. It requires groups of people to make Scientific progress and effect societal change. I can't be the only one doing this stuff!

One of the main reasons I did this is to push the field of genetic engineering forward. Everyone wants to just talk about this stuff. They ask should we do it? I say, how can we not do it when we have a technology that can help people. 


This is the second time I have genetically modified myself. You can hear about the first successful attempt at https://www.youtube.com/watch?v=DCzZvb6wqi8


Monday, October 2, 2017

DIY Guide to Creating a Peptide Immunotherapy Cancer Vaccine

I received an email over a year ago from Lars. His wife Dyanne, a never smoker, had developed Non-Small Cell Lung Cancer. He was reaching out to me to see if CRISPR would be a viable way to try and treat his wife. He lamented at how the medical system and academic scientists he contacted had given up on her to die. Eventually, we decided that CRISPR was probably not the best thing to try and they started down the path of immunotherapy instead. What we found was that a patient could use cutting edge peptide immunotherapy for a few hundred dollars or euros by ordering their own peptides directly from synthesis companies. We started out by helping to coach people through the process of ordering and working with peptides and as the demand increased we decided to do something more.

See the fucked up thing about the world is not the scammers peddling fake cancer treatments, it's the medical doctors peddling infinite wisdom but refusing to do anything, much less everything that they can to help someone who is dying. What lengths would you be willing to goto to save your loved one or partner and does not every human deserve that same dignity and respect? Maybe you say this is irresponsible as if letting someone die without the right to do whatever they want with their body is totally fine.



See, so many people are scared of what might happen if people all over start trying things like this but ya' know what I am scared of? I am scared of people just being left to die because people are to worried about saving their own career and their own paycheck than saving a human life.


So after much thought and deliberation myself, Lars and a few other caregivers and individuals with cancer decided to make a guide based on things people had been trying to some success. Maybe a first of its kind guide(with hopefully many more to come) on how to treat someone who has NSCLC using peptide immunotherapy.
https://goo.gl/omorJA
We are not advertising this as a cure-all or even cure. We are putting this out there as a means of help and hope because one of the saddest things to realize in life is that world doesn't have your back. Sometimes in your hardest moments you need to be your own hope. But you can and that's the amazing thing. It's possible that out there in the scientific literature or through self-experimentation you can help yourself and possibly others.

As I write this there are people dying of cancer and other diseases. Maybe close to home, maybe far away. But if they don't inspire you to learn more, work harder and try harder. What the fuck are we doing Science for?

Wednesday, February 15, 2017

How to Genetically Engineer a Human in Your Garage - Part III - The first round of experiments


Whenever I start to work on something I have trouble sleeping. The problem is that I am always working on something so I always have trouble sleeping. It's not worry or anxiety, it's hard to describe it without saying that it is just excitement. Not like happy excited but it is an anticipation. When I am working on an experiment, to me it's not even really Science, I am building a world. I am taking this world that exists as a dream in my mind and creating it in the physical world. I stay up at night imagining how to do that.

Starting the experiment to genetically modify myself was scary and daunting. The world I created was so intricate I didn't know if I could possibly share it with anyone or even succeed at creating it.

It is one of the issues I always run into and one thing I have a hard time communicating to journalists. They want access to this sacred part of my life but treat me like a paycheck. They want their sound bite and I try and give it to them but how would I explain my motives? When they were many and complex. To create something beautiful, to test out new technology to try and develop something that others could use, for medical use and on and on The reasons were so arrayed, I had been thinking about it for months. How do you explain a months worth of thoughts to a person who wants to condense it down to one sentence? Even those you are closest to?

And then you start to think about the experiment. How I read countless papers, researched DNA plasmids, viruses and virus serotypes, mammalian expression elements, chemicals to use and try, DNA vaccines, experiments on mice, and you ask me how am I going to do it? Is it safe? How do I explain the reason for every choice, is it possible unless you read the papers I read and thought through everything like I did?

I didn't want to do it alone. I don't want to do it alone. I want to share it but I want to share it without the soundbites.



There are 3 main parts to topical or sub-cutaneous genetic modification
1. Hair removal
2. Skin Stripping
3. DNA application



Hair removal
Not as complicated as it sounds or maybe more complicated than it sounds? First you need some depilatory cream. Yeah, WTF is that shit?I remember sitting there reading this paper on topical DNA transfection in mice and they are talking about depilatory cream and how it can help transfection efficiency and I was wondering how I could get my hands on this stuff. I come to find out that it is Nair, well you could have just said that.

*Sidenote - Though this example is kind of a joke one of my issues with Science is that even a trained a Ph.D.(myself) oftentimes has trouble deciphering papers. The information you really want to know like DNA sequences and techniques is often hidden and obscured in papers. Someone might say "Well those papers are not about the sequence and that is just too much information so whatever". Until recently I was trying to piece together a DNA sequence from a paper about DNA sequences and they weren't in the paper!!!! How does one even publish a paper about new synthetic promoters and not have the exact promoter sequences used in the paper?!!!! I could have pieced it back together from the clues but I figured I would email the author instead and they responded that the sequences were patented and so I would need a license and they didn't give them to me!!!! WTF! Standing on the shoulders on giants? More like standing on the shoulders of patents and paywalls so no one can advance Science!

Nair works as you would expect it. Easy to use on the arm for hair removal.



Skin Stripping

Your actual live skin cells are hidden under layers of dead skin, the stratum corneum.
https://training.seer.cancer.gov/images/melanoma/skin.jpg

You can imagine that if you are trying to genetically engineer cells they need to be living and alive so that they can take the DNA you put in them and turn it into protein(jellyfish green fluorescent protein in my case!). What this means is that you need to remove the stratum corneum. Now this next part is no joke. Scientific papers indicate that the best methods are either tape stripping or tooth brush scrubbing and I quote:

"The shaved dorsal skin was then either stripped by applying Blenderm tape (3M Health Care, St. Paul, MN) five times and/or brushed using a tooth brush with rounded firm nylon bristles. The number of brushing strokes varied from 50 to 200."

I still haven't tried the tooth brush method because I just can't find it in my heart to scrub my skin 200 times with a tooth brush. Instead, I have mostly stuck with the tape stripping method. It does hurt. Some tapes work better than others. I usually use duct tape or packaging tape. I usually stop when putting alcohol on it burns.




DNA Application

Now this is a complicated one that I still haven't figured out because there are so many different things to try and so many different variables. Topical application experiments of DNA have been extremely limited as far as I can find. You can use water, or some ions or salt or Tris or DMSO or ...... you get it. Do you cover it or evaporate it or inject it, electroporate it? I think alot of times with experiments it is easy to become caught up in what is the best way to do something instead of just doing it. Obviously, doing this experiment with no knowledge or preparation would be foolish but I have learned that I will never be 100% ready. I don't know what DNA application method is best and over the course of my experiments so far I don't know if I have found out yet either.

Normally, I would just apply it to my skin and then evaporate it using a heat gun of sometype. This seemed to work the best and fastest and wouldn't hurt the integrity of the DNA I imagine. I also tried out just putting tape over the drop so it was just in constant contact with my skin.



I had a plan but I expected that from the beginning things wouldn't goto plan but I adapt well. hahaha.

Visualization

Initially I hoped that I would be able to see the genetic engineering in my skin by looking for fluorescence by exciting the protein and then looking for it using a visual filter. I can say that my first experiments doing this most likely didn't work. They could have worked but it didn't seem like I would ever be able to definitively tell if the cells were making the jellyfish GFP.



The first round of experiments didn't work or were inconclusive so I decided to try new things. I decided to try and use a virus and I decided that I might need to try new analysis methods that would require a skin sample!


Monday, January 30, 2017

How to Genetically Engineer a Human in Your Garage - Part II - DNA got what I need




Ohhh babbbyyy, you, you got what I need


To embark on an experiment to start genetically modifying myself I needed to obtain DNA and not just any DNA, some that was specifically encoded to work with humans. This is because not all DNA works with every organism. In fact most organism have their own unique codes that allow and don't allow certain DNA to work with them. Kind of like a security system for the cell. Like the CRISPR kits we sell won't work on humans without modification, they only work on bacteria.

The purpose of most DNA used for genetic engineering is to make proteins1 The targets I wanted to start with were Green Fluorescent Protein(GFP) and Red Fluorescent Protein(RFP). These are genes from Jellyfishes. I figured that in a best case scenario I would be able to actually see my cells glow and fluoresce if they were engineered. Worst case scenario is that I would need to extract the mRNA to look for the presence of these new genes in my cells but they would be easy to find if works because they are not endogenous(natural) to human beings.

Anyways, good thing for me there has been a bunch of work on modifying human cells with fluorescent proteins in petri dishes or cell culture as it is called. What this means is that there is already a bunch of DNA in existence that was made to work with human cells I just needed to find it and obtain it. Which can be more difficult than you think.

I am not going to out any companies specifically in this post because they may or may not want to know they are or have sent their DNA to me for the purpose of human genetic engineering. I am sure you can ask around or do research online and find companies that will sell to you.

I set about obtaining pieces of plasmid DNA2,3.
And scary but not scary I wanted to obtain a virus4 that could put this jellyfish DNA into my cells.

One of the most difficult things about being a Biohacker is that companies sometimes won't sell things to you. Not because there are any laws regarding the sales but because they want to control who is able to do Science. Fuck that shit.


How to Obtain DNA
This is a best practices in how to obtain things that companies try and keep from individuals because they think we are stupid. Some cases may take much less and some cases much more.


  1. Start a business. What this basically means is register a domain name and go through the process of setting up an email address so you can send emails from that domain. It legitimizes you 100x more than a gmail.com email address and the whole thing costs like $12 on godaddy.

  2. Get a Ph.D. j/k j/k you can always just add Ph.D. after your name in an email. No one checks or can check. There is no database of Ph.D.s

  3. Have a FedEx account #. I am pretty sure these are free and giving someone a FedEx account number has gotten me around the "We don't ship to home addresses" a few times.

  4. Social Engineer them. It is blatant discrimination that companies won't sell materials because they think us people outside of academia or industry are too incompetent to use them properly or might hurt ourselves. And don't try and tell me it is for safety when I can purchase a gun, knife or car sooo much easier and those can outright kill people. Shit bleach and kitchen cleaner are more hazardous than any chemical in my lab and I can purchase those in a grocery store. Seriously, things like DNA are unduly controlled and we, at The ODIN, hope to do something about it in 2017. 

One of the companies I used is one of the largest suppliers of plasmids. They actually will ship to most anyone and most any address as far as I can tell. They have even encouraged me to lie to them to bypass their own review system. #FunFact


Ok, so what is the difference between using plasmid DNA and a virus?

Current, technology makes using a virus cost and skill prohibitive. Though I could purchase the virus for $500 or so it came in a limited supply. The benefit however is that it should work out of the box. I would not need to do much to make it ready. Plasmids are inexpensive, they don't work as well in terms of human engineering as viruses do but once you have some you can replicate as much as you want indefinitely. You can give it to friends to replicate and use and they are easy to modify for new uses.

I wanted to explore multiple avenues with my goal being to eventually focus on plasmid DNA because I want to work with technology that is cheap and accessible.

At first I began working with plasmid DNA. Though it is somewhat easy to work with it requires that one know how to do bacterial cell culture, plasmid purification and maybe even ethanol precipitation. You need a centrifuge and lab grade pipettes and probably an incubator and shaker. While most of this stuff will only run someone a few hundred dollars it is still a hindrance. However, I started a document on best practices for plasmid purification for human genetic modification. What one can do however is send the plasmid DNA or bacteria with plasmid DNA to a company and have them just purify you up a bunch. Depending on how much you need this can run from $100-$400+
Making and purifying your own viruses while not challenging from a technical or safety sense it is challenging from an equipment and resources sense. One would need to be able to do human or mammalian cell culture and transfection and have materials to purify the virus. Doable but these techniques are far outside the scope of a garage lab at the moment. Maybe not so much in the near future though?

The DNA in the virus came prepackaged and ready to go and despite what people might think, AAV is considered Biosafety containment level 1 by most places(which is the least strict and is on the same level as Sodium Chloride or Agar i.e. it is safe).

Obvi, after I received my DNA it was time for the experiments to begin! 


Genetic engineering in humans usually occurs as intramuscular injections or systemic injections(i.e. into the blood stream. Both of these ways seemed silly as they made it difficult to perform any testing to see if the experiment worked. Muscle biopsy on myself? YIKES! I decided on topical skin and subcutaneous(under the skin) delivery of DNA! The crazy thing is that there hasn't been as much scientific literature and research as I expected on this topic. I did collect a sampling of papers however that I found interesting and you can find them here: https://drive.google.com/open?id=0B_R75gIJvkFUMkhrbUdZRzhscGc


July 5th 2016 was the first day I started experimenting on myself.






Footnotes

1. Proteins are awesome. They are little molecular machines inside cells that let cells do things. Read about them!

2. Plasmid DNA is small circular DNA that is separately from the host cells genomic DNA(compared to genomic DNA. Genomic DNA is the DNA that is transferred from a parent cell. The standard set of genes each cell comes with). Plasmid DNA is circular so that it can be replicated easy by organisms such as bacteria. This allows researchers to grow bacteria and extract the plasmid to use for other purposes.

3. Here is a link to the DNA sequences of the plasmids and virus I used and am using: https://drive.google.com/open?id=0B_R75gIJvkFUMkhrbUdZRzhscGc

4. The virus in question is a non-replicative(it doesn't replicate and reproduce) Adeno Associated Virus(AAV). It has been used a bunch on humans and shown to be super safe. Fortunately, I was actually also able to obtain AAV1 which is efficacious across a number of tissues including keratinocytes(which make skin).


Wednesday, January 25, 2017

How to Genetically Engineer a Human in Your Garage - Part I

My name is Josiah Paul Zayner. If my name sounds "religious" that's because it is, Josiah was a King of Judah in the Hebrew.Christian Bible, Paul was an apostle of Jesus. I have three brothers Zachariah(older) and Micah and Jedidiah(both younger).

I don't remember a time when my Mom and my Biological father were together. He was severely abusive. We were poor. We lived on a farm. It wasn't a huge farm like people might imagine but a small animal farm in rural Indiana. We collected eggs from our chickens to eat and drank dehydrated milk. I have never met anyone else who drank dehydrated milk. I don't remember much of my childhood because of the trauma from abuse. I only see small flashes like a nightmare one can still feel emotionally but barely recall what exactly happened. This includes the now infamous family story of discharging the the gun my Mom had in the car door for protection. My Mom's second husband was also abusive. I ran away from home twice. Living with computer hacker friends in Oklahoma and Texas after high school.

My Mom is an amazing human. I would not be where I am today without her and her willingness to fight for her family.

Me(on the right side) and My Brothers circa 1987

Growing up I was never particularly what one would consider smart or gifted. I have just always liked to build and create things. I was mostly an introvert and would often get lost in my head, I still do. In high school I was mostly a jock but navigated through most groups. It was in high school that I found out about computers. I found out that if I wanted to learn something I could just read and teach myself. 

I made a discovery today.  I found a computer.  Wait a second, this is
cool.  It does what I want it to.  If it makes a mistake, it's because I
screwed it up.  Not because it doesn't like me...
                Or feels threatened by me...
                Or thinks I'm a smart ass...
                Or doesn't like teaching and shouldn't be here...
        Damn kid.  All he does is play games.  They're all alike.
My high school teachers made fun of me because I would always be reading in class. "What are you going to be a book reviewer one day?" and then the class would laugh and I would go back to my reading. I was almost expelled from High School twice. The first time I punched a kid a bunch and he ended up in the hospital. They handcuffed me in class. I was arrested and suspended. The second was for writing a program called "HackTheSchool.exe" and putting it on the shared network drive so anyone could access it. The program was a joke, it didn't actually hack the school but teachers back then didn't have enough knowledge to read the code and be able to tell the difference. In lieu of expulsion I was suspended and banned from ever using a computer in the school that was connected to a network or the internet. They had a special computer in my programming class that was unplugged from any network just for me.

Me on the computer at home circa 1999 

I eventually became a "computer hacker" and was a member of Legions of the Undeground(LoU) a hacking group. While that story is long and for another time it is the foundation of why I started my company The ODIN and have invested heavily in Biohackers. During that time in the 1990s and early 2000s I saw kids like me who were awful at school, came from broken homes, who could code circles around and out technical 99% of the world. All of us were self-taught. Little know fact is that you can still find a piece of software I wrote back then on The ODIN website and people still download it. Nowadays all of these people have great jobs at tech companies and most have still never finished college.

When I was 19, I was able to obtain a job at Motorola doing programming and networking. I worked there at the end of the dotcom bubble and then during the burst. I was laid off with tens or hundreds of thousands of other people. The one thing I took away from Motorola came from Dennis Tsai, he told me "People can take away your job but they can never take away what is up here (pointing at his head)." I could have taken another Tech Job but instead decided to goto school and learn. Fill my head with information that no one could ever take away.

Because I did so poorly in high school, I started back at a community college for around 1.5 years before transferring. I ended up majoring in Plant Biology and obtaining a BA from Southern Illinois University and though I did much better than I did in high school I just wasn't good at standardized education. Still there was a minor bit of meritocracy left back then and my Master's Degree at Appalachian(la-chun) State led to being accepted to a Ph.D. program at the University of Chicago.
I owe a huge debt to Ece Karatan, Tobin Sosnick and Tao Pan who taught me in Graduate School. Without them I wouldn't be who and where I am. I also think they owe me an apology because it has taken me years to unteach myself the critical nature I was driven into. I was taught to question everything and be constantly vigilant and critical. While this taught me to be logical and rigorous I think it stole my creativity, it stole my ability to dream and believe. Graduate school was a beautiful and extremely painful experience. It's hard for me to recommend graduate school, I think it really depends on the individual.

After graduate school I was awarded a fellowship working as a Synthetic Biologist at NASA engineering bacteria to break down and recycle materials for long-term space travel or when we colonize other planets. That was an interesting life experience but if you really want to read about that you can check out previous blog posts Here, Here and Here. Let's just say I don't work well in the system. I don't do well with rules that don't make much sense or people who are more interested in improving their position than contributing. Fuck the system.

The two guys named Bob who ran the NASA Fellowship program and Me




My last day at NASA and no one is around because no one is ever around.
Jan. 5th 2016


2015 culminated with me leaving my fellowship at NASA because I was fed up with the system, with everything. Outside I saw so many people hungering to be genetic designers and at NASA people hired to do the same siting on their asses. I began to work at The ODIN full-time. 

My goal at The ODIN has been to give people access to resources and knowledge so that they can Biohack. I have seen what hackers can do and usually because of their different path in life have unique insights into problems and tend to be more creative than trained and pedigreed Scientists. 

If a poor kid like me can end up at NASA and now running their own company but never quite be at the top of their class, never scoring well on tests no matter how much effort I put in, something is wrong with the school system. I can program 3 or 4 languages pretty fluently, can do electronics and basic circuit design and have a Ph.D. in Biophysics but even in graduate school no matter how much I studied I would always end up with lower grades than my peers. The education system is broken. The social system is broken. Or maybe they are all work fine and it is just me who is broken.

That's why I fight.

I fight for access for everyone. But I fight fiercely for the broken among us. Those who need hope.

You deserve better.

In 2016, I began to fight hard. I explored microbiome modification for health and privacy and did a thorough Scientific analysis that you can read Here, Here and Here. I found that it was possible to successfully transplant a gut microbiome and it alleviated alot of suffering in my life. It hurt and was hard.

In Sept. I ran the first conference for Biohackers(http://biohacktheplanet.com), which I worked my ass off for and poured so much into. It was amazing and turned out so much better than I could have ever hoped.

But still there is so much more to be done. The system is fucked up and so many people who hunger for knowledge and the ability to help others can't.

I didn't grow up wanting to be a Scientist or Biohacker or Genetic Designer. I was too poor to dream of that possibility. But, I did grow up wanting to help people and this has lead me to a project that will be a culmination of my life's work so far, Genetically Modifying humans. Genetically modifying myself.

This is me. I am a Genetic Designer.


Sorry, this post wasn't about the actual human genetic engineering experiment but I feel I need to give my background to tell my story.