Wednesday, October 7, 2020

CRISPR is Dead



Today, the Nobel Prize was awarded for “genome editing” to Emmanuelle Charpentier and Jennifer Doudna. Essentially this was the CRISPR Nobel Prize. If enough of CRISPR has already come so that it is worthy of a Nobel Prize I can’t imagine there is much place to go from here. 


Modifying the genome of organisms has always been of great interest to scientists. Adding or removing genes allows us to understand how things work and allows us to create microorganisms, plants or animals(humans are animals right?) that have traits never seen before. Knowingly modifying the genome of organisms has been done since people understood breeding. Inserting specific DNA elements began in the 1970s but it wasn’t very targeted and was mostly just inserting genes into random places in genomes. It wasn’t until the 1990s that people started to be able to do targeted genome modifications. 


Targeted genome modifications allow highly specific and accurate changes to an organism’s DNA. The co-opting of the cellular mechanism of homologous recombination is the backbone of all modern genome editing technologies including Zinc Finger Nucleases(ZFNs), Transcription activator-like effector nuclease(TALENs) and Clustered regularly Interspaced Short Palindromic Repeats(CRISPR). Both ZFNs and TALENs are mostly synthetic, i.e. they contain a portion that can be engineered to target DNA and a synthetically attached nuclease that will cleave DNA to initiate recombination and gene editing. CRISPR is almost completely natural which makes one wonder how there are so many patents on it’s use, yikes.


CRISPR can modify most any living cell but so can ZFNs, TALENs and other technologies. So why then was CRISPR heralded as the discovery of the century by the MIT Tech Review? Simply, CRISPR is just easier to use because it uses nucleic acid targeting. That makes it cost less and take less time to produce genetic modifications. 


So what has CRISPR made possible? Not much really. Most everything done with CRISPR can be done with one of these other technologies albeit these others are slower and more expensive. CRISPR allows the scaling of genetic engineering so that time and money are much less of a factor. It is the cloud computing of biology at least in my mind.


Despite claims by scientists and pharma companies there is little chance CRISPR will ever be widely used in the clinic to directly treat disease. That is because it suffers from all the same faults as its predecessors and maybe even more so. Gene editing has low efficiency in adult animals(yes humans are animals) no matter the technique used. For instance, if you have a disease that affects the brain you can probably only modify <1% of cells even using the best delivery techniques available. Really, the only way to get rid of genetically inherited diseases using gene editing is by modifying embryos. 


Misleading as it has been CRISPR can’t actually make specific changes to a gene easily in an adult animal. That is because it requires what is called a donor template, basically just a DNA template that cells can use to create the genome modifications. There is no efficient way to use donor templates in an adult animal so all genome edits would need to be gene knock-outs only i.e. you can only use CRISPR to destroy bad genes not modify them to make them good genes. As you can imagine this is very limited in scope when it comes to diseases that can and should be reasonably targeted using genome editing.


To date all human clinical trials involve gene editing technology whether CRISPR or otherwise have failed to show any change in the disease condition. I don’t see this as likely to change. While there might be a disease that can be helped despite the low efficiency of CRISPR chances are that normal gene therapy, that doesn’t edit the genome, will be easier and more successful. There are very few diseases that would require genome editing as opposed to just adding an extra copy of the gene to cells like gene therapy does. It’s not all just conjecture either, just recently in August 2020 the pharma giant Abbvie ended a partnership it had with Editas, one of the major CRISPR players. Apparently, I’m not the only one who sees CRISPR's future in the clinic as limited.


So what applications are left for CRISPR besides contributing to research? Some people are betting on diagnostics, using CRISPR’s ability to target specific sequences of DNA. While this seems reasonable it is unlikely that tried and true methods for DNA detection that use PCR will ever be significantly deplatformed. After that we are scraping the bottom of the bowl of guac.


I have been around CRISPR since near the beginning. The only thing that has remained constant is the hype. Even that has been fading. While it is hard to measure hype Google Trends indicates that for 2020 the topic and search term CRISPR is on track to be the lowest searched since 2016. We now know that CRISPR gene drives don’t really work. No success for CRISPR in the clinic. CRISPR has already been used to edit human embryos. Really, the only thing keeping CRISPR hype alive is probably the MIT Tech Review.

In 2006, RNAi gene silencing was given the Nobel Prize. MIT called it the breakthrough of the decade. I remember everyone being so excited about it! It was the hot topic at conferences and even my graduate school interviews. While RNAi was and always has been a great benefit to researchers its actual application has been extremely limited. It’s taken 13 years from the RNAi Nobel Prize to bring something to the clinic and even then the two drugs have been a bit underwhelming. According to Google Scholar, papers even mentioning RNAi have been on the decline for the past 6 years. The drug approved in the past few years haven't even slowed the decline.


I am sure people will continue to use CRISPR for years to come but I hate to break it to you CRISPR is dead.


 




Thursday, June 25, 2020

Do-It-Yourself: From Scientific Paper to Covid-19 DNA Vaccine

I'll be honest, when the coronavirus pandemic first started I didn't imagine it would last this long. I am constantly planning and looking to the future and it seemed like some temporary nuisance that the media was blowing out of proportion. It was annoying to see people flock to work on coronavirus related things to further their own agenda. Among those were numerous groups of Biohackers who were pretending to solve problems that they couldn't reasonably solve because of lack of access to the SARS-COV2 virus or animal testing models or both. Despite pleas from many many people to get involved in projects I decided to sit the pandemic out and do what I always do - work on shit that would have long-term benefits to the Biohacking and science community. The pandemic kept going. I really didn't imagine it would last this long.

Then in May 2020, an article came out in science magazine where researchers showed that by using a DNA vaccine that codes for the SARS-COV2 spike protein they could create antibodies and protect macaque monkeys from covid-19. Getting good monkey data is basically the best pre-human data you can ever hope to get. Most people only have mouse data. When I see a paper like this the gears in my brain begin to spin because there is a good chance the FDA would approve this for human testing. From the data it is seemingly both safe and effective. I changed my mind. This sounded like a great project and not just because it was coronavirus related. 

This project fits a niche where biohackers have an experimental advantage over academia and industry. With enough knowledge and skill we can perform quick but data laden experiments to show if the same DNA vaccine tested on monkeys would be promising in humans. And instead of taking 2 or 10 years we can have results in two months. Weeks if we wanted to drop an extra $5k. 

For the past month Myself, David Ishee and Dariia Dantseva have been working furiously to develop a step-by-step online class on how you can take a paper with animal data like the one above and use it to develop a research plan to collect usable human testing data. Simultaneously, over the course of the next 2 months we will show you the process we went to recreate the SARS-COV2/covid-19 vaccine from the paper and test it on ourselves. From designing the DNA, testing expression in human cells, ELISAs and more. We will explain how one can do advanced Biomedical research DIY and on a budget.

You ain't fucking going to want to miss this.

With a risk averse scientific and medical system Biohackers are the ones who will push things forward in a reasonable time frame.

Biohackers are needed in society. 

Whether you think I am an idiot for self-experimenting is not the point. The point is that when it comes your turn to suffer and die at the hands of a disease you will hope there is someone like me willing to risk something to try and help.

Biohackers will be trying for you when everyone else has given up.

How do we get more Biohackers then? The problem at the moment is that there are not many people with enough knowledge and enough crazy doing experiments. Science and medicine has become so stratified into a risk averse elitist system because of lack of diversity. I want to change that. I want anyone who has access to an internet connection to have access to the knowledge required to do advanced Biomedical research. I mean, what if we could do this for any drug? Some people with a little ambition, intelligence and crazy could be testing promising drugs in weeks not years. The test pilots of the biological age.

First class will be live-streamed Sunday June 28th at 11AM PT at https://youtube.com/josiahzayner/

Saturday, April 25, 2020

Why We Shouldn't Be Forced To Wear Masks


It has become glaringly obvious in the past few months that science has let us down. I don’t blame them. The situation the world is in is complicated because we don’t have enough information on what we should do and how we should act. We look to our “experts” for help but generally they are probably only slightly more insightful than yourself.

In Oakland, CA where I live there is a mandatory order to wear masks when in public. This is despite as recently as March 31, 2020 the World Health Organization(WHO) has suggested people not wear masks to help prevent the spread of coronavirus. On April 6th, 2020 they released interim guidance suggesting that people in the community should wear masks, though they are not effective alone. The CDC suggests we should wear masks or rather cloth face coverings and save masks for medical professionals. So what gives? Should we or should we not wear masks?

There have been a number of scientific studies in a number of different live settings that have shown that face masks alone don’t reduce influenza like illness spread in a statistically significant way. A meta study of 15 of these studies also agrees. However, a recent study done in a lab environment disagrees. When there is conflicting information from scientific papers and organizations how do we decide who is correct and who is incorrect?

This is one of the major problems with science today, science is never wrong. You can almost always find a paper that agrees with the point you are trying to make. Glyphosate is a probable carcinogen according to the International Agency for Research on Cancer (IARC) but according to the WHO and European Union it isn’t. Do you believe the WHO over the IARC? Why? Because one seems more reputable? So the one with the best reputation wins? Is that what science comes down to for you?
Who do we believe and who do we trust?

This happens often in science, conflicting information. None of the studies are technically wrong and so it is up to the bias of the reviewer/interpreter/observer to determine what evidence they want to agree with. If we even have evidence enough to disagree.

Models and data analysis are an interpretation of data that is guided by a human. We are left with a simple choice: either believe it was modeled correctly or don’t. How can you say it wasn’t modelled correctly if you don’t even have information on how it was modeled?

In March, The Imperial College in London released a report that said up to 2.2 million people in the US might die from the coronavirus. The CDC ran models that suggested anywhere from 200,000 to 1.7 million might die. These numbers caused mass hysteria and the lockdown of most states. Despite the fact that 200,000-2.2 million is almost about as big a range as you can get in this prediction. Fortunately, we will most likely underperform and have nearer to 100,000 deaths or less in the US(the current number of coronavirus related deaths on April 24 stands at 51,000 with over 26,000 from New York and New Jersey alone, who may either be overcounting or undercounting depending on who you ask). It is unknown whether any of the models took into account preventative measures and I imagine the lockdown has decreased the number of coronavirus related deaths but by how many? In this scenario could the models ever be wrong? If all the deaths stopped after 4,000, like in China, would there be anyone who would say these models were wrong or would they instead point to preventative measures that helped save us from the predictions of the models?

The models can never be wrong. This makes it so the science can never be wrong. I have seen it time and time again. Where scientists _decide_ some scientific publication is wrong and look at it under the most intense scrutiny that they fail to give similarly to papers that support their argument and a _consensus_ is reached. Do we have great evidence that covid-19 is causing a really high death rate? Not really. Testing has been extremely underdone or needs to be tripled to reopen according to the NYT. Why then are scientists trying so hard to dismiss studies that suggest the number of infected people is much higher than by what testing so far in the US has been done? They really don’t know if these studies are incorrect as evidence for a high death rate is based on an extreme lack of testing. Still, they have decided a low death rate is incorrect and so that is what we are to believe.

The powers that be from on high have decided that this science is correct and this science is incorrect. The problem is that there is no empirical way to decide whether one piece of science is more correct than a piece that disagrees. It requires humans to judge and humans are fallible and prone to bias. Here is where modern science breaks down. It is more about social and political posturing to achieve the most number of people to support your argument than it is of empiricism. If you disagree with the consensus you are automatically wrong. I mean, consensuses have never been wrong before amirite(see sarcasm).

If you still operate under the idea that science delivers us truth and facts I beg you to reconsider your position and understand that beneath the peer review, the data collection, the choice to publish what experiments and what to leave out, is a whole pool of bias and misinformation. Scientists are just as biased as everyone else, maybe more so, because they have their reputation on the line. But we put them up in the heavens and pretend that they are presenting the truth to the rest of us. When they have no more access to the truth than anyone else.


Science might be doing us more harm than good but it depends on who you ask.

Wednesday, April 15, 2020

Scientists Can't Save Us If There Aren't Many Scientists

Two Normal Scientists

As a former academic scientist it is hard for me to comprehend how many scientists there actually are. When I was in graduate school I was surrounded by scientists. My friends were scientists, my colleagues, the friends of my friends were. I had lunch with scientists, got hammered out of my gourd with scientists and had sex with scientists. The only people I interacted with that weren’t scientists were the bartenders at The Cove Lounge and my family.

With the ‘rona pandemic scaring the shit out of people we all hope the scientists are there to save us. Ya’ know like some Outbreak bullshit. Last minute, save your lover. Where is Dustin Hoffman when we need him? There are so many articles about scientists and companies doing their part that it seems like everyone in the world is working to save us. I have long pondered the question of how many scientists there actually are. If we were to conjure up all biomedical scientists and pay them to work on the ‘rona exclusively how many would that be? I imagine alot but only 2% of the US(~6 million people) has a PhD in anything. That number seems low to me. I guess I know too many PhDs and it skews my estimate. When I actually started to figure out how many scientists there are it was even more shocking. 

Trying to quantify the number of scientists that are working to save us from disease is tough as there really is no data on it. Instead, what we can do is use the data that is available to indirectly infer how many scientists there are out there working in biomedical research.

According to the NSF there are around 200,000 employed PhD biological scientists in the US. If we try and narrow down the number to those engaged in biomedical research the number drops closer to 50,000-100,000. Understand, scientists aren’t just PhDs. PhDs are actually the smallest group of scientists. There are about twice as many people receiving Master’s degree so if we assume similar employment rates for Master’s degrees as we do with PhDs that adds another ~200,000 people to the biological researcher workforce. For bachelor’s degrees ~2 million have been received in Biological sciences since 1990. It is difficult to find the numbers of people with Bachelor’s degrees who are employed in biomedical research. If we use Indeed.com, a website which includes many or most job postings, and look for entry level job openings in biomedical research in the US the number is somewhere around 6,000. If we can assume most jobs stay on the market for 3 months that is about 24,000 entry level jobs a year. If we assume the number of jobs available is proportional to the number of people who obtained degrees (which isn’t always true but is our best bet) we have around 300,000 - 400,000 total employees with Bachelor's degrees. If we add up those numbers we have a total of around 450,000 - 700,000 active biomedical research scientists in the US. 

That number is just so much smaller than I imagined. The crazy thing is that many of these people are teachers, managers or work in non-research roles so even 450,000 is probably a high estimate. But the world is a pretty big place and the US is small in the whole scheme of things. Right? I mean...

Finding data on how many biomedical scientists are in every country is near impossible but fortunately (unfortunately?) most countries contribute so little to the total that we can just ignore them. According to the NSF, China awards around twice as many science and engineering bachelor’s degrees as the US but only 85% as many PhDs. The Euro8(Germany, United Kingdom, France, Spain, Italy, Portugal, Sweden, and Romania) awards around the same number of bachelor’s degrees as the US while also awarding about 1.5x more PhDs. If we can extrapolate the job numbers from the US we are looking at around 800,000 bachelor’s + 200,000 masters + 85,000 PhDs and around 1.1 million biomedical researchers in China, 400,000 bachelor’s + 300,000 master’s + 150,000 PhDs and around 850,000 in Euro8. I know it seems weird to just choose these countries but from the data the number of biomedical researchers in other countries not mentioned do not significantly contribute to the worldwide total from all the numbers I can find.

An estimate of the worldwide total number of individuals actively employed in biomedicine in any capacity is  around 3 million.

Being hands-on in science is essential to being able to plan out research experiments and perform research. When you aren’t using equipment you lose your touch and sense of intuition for what is possible and on what time scale. There is a phrase in science that people use for someone who knows their way around a lab and can make things work. Scientists say that the person has “got hands” or has “good hands”.

Most scientists you have heard of or revered don't have good hands. Nobel Prize researchers generally didn't do the actual experiments that lead to the Nobel Prize. People say it is the process of science and that everyone moves away from doing lab work but it really doesn’t make much sense. Scientists at the prime of their career and ability, generally quit doing actual hands-on science. 

In an average research lab of 10 people there are around 3 people who participate in a support role and not active research. These positions would be the lab Principal Investigator(PI), the lab manager, administrative roles and roles like dishwasher. If we subtract that 30% we are at around 2.1 million biomedical non-support role researchers worldwide. At MIT, the typical lab is under 10 people and I imagine that number is even less elsewhere. I have worked in labs with one or two others and those other two were in support roles and not active in research. These numbers are probably an underestimate.

Our final worldwide total is around 2.1 million biomedical non-support role researchers who can do hands-on work. 

Around 50% of US research funding is for applied research which is considered research that can contribute directly to a product or outcome. I know, I know but basic science research will contribute to helping us _eventually_. While I don’t necessarily share that sentiment and my own published research is evidence of that fact, I am talking about research that can contribute now. Like, if say, a virus was spreading. From the NSF we know that 25% of researchers work at for profit institutions and let’s just say for profit institutions are generally doing applied research. Drug companies got to make drugs amirite? That leaves 50% of the other 75% as those doing applied research that can contribute directly to a biomedical product or outcome now. If we use these numbers to extrapolate to our global number

This leaves us with around 1.3 million people worldwide doing applied biomedical non-support role hands-on research.

If around 7% of those are PhD researchers, as from the initial numbers above, that is only 91,000 PhDs doing applied biomedical non-support role hands-on research worldwide!!!! 

The lack of experienced scientists doing hands-on research hurts us all. One of the goals of my company, The ODIN, is to train up people who can do hands-on scientific research. In the past year we have trained nearly 2,000 people to do so. The problem isn’t schools not letting in enough people, it is the simple problem of marketing. Convincing people that they are capable and then training them.

The World Health Organization(WHO) has a system that provides codes and nomenclature for over 33,000 diseases. If no one researched anything in biomedical science but diseases that would be less than 3 PhD scientists per disease in the world. 

Here is the problem, when new threats come around like CoVid-19 the number of scientists with actual knowledge and skill to be able to work towards developing a treatment is tiny. If you imagine that vaccine development for viruses is a subset of a field in virology and then factor in familiarity with coronaviruses you are down to very few people. In 2016, coronaviruses were listed by the World Health Organization as in urgent need of R&D. Using Pubmed, I found 12 scientific publications in 2017 that mention (coronavirus or CoV) and vaccine in the title, 6 in 2018 and 16 in 2019

Despite multiple previous coronavirus pandemics why wasn’t there more research on coronavirus vaccines done? 

It's not funding. The NIH alone invests $41 Billion in medical research a year. The top 20 Pharma companies invest around $100 billion. ChinaEurope and Japan add near another $100 billion. Bringing the total Biotech R&D investment per year to somewhere near a quarter of a trillion dollars.

Honestly, I imagine it is because there just aren’t enough scientists to go around. 

We should be afraid and not because of CoVid-19. There are so many diseases that humans suffer and die from on a daily basis that have no treatment or cure. That shit needs to change and change fast. We need more scientists that can do hands-on research because what we have just ain't cutting it.






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.