The older sibling of Project GRFT is Open Insulin. They recently made a quality introductory video about their work which may be viewed here We share the goals of medicine for people, not profit, and valuing open source technology.
Wednesday, June 30, 2021
Thursday, April 22, 2021
Project GRFT in the lab and in the fields - a plan
People have asked the question, “If Griffithsin is such a potent anti-viral, why is it not being manufactured? Why is it not generally available and being used to stop viral outbreaks such as Covid-19?” It’s a very good question. There are a few efforts being made to biomanufacture griffithsin and at least one to get it into clinical trials. In March, 2020, I couldn’t find anything to indicate that it was in any pharma company pipeline. I decided that if no one is working on this, I would put my shoulder to the wheel and do work on its development. First I reached out to researchers around the world who had worked with griffithsin. I reached out to my friends in the biohacking community. There were a few people who responded at first and we started a group of friends to take on a rather enormous project with a goal of making griffithsin available to the world.
The arts of molecular biology and biotechnology demonstrate that
genes from one organism may be placed into another organism which will read the
sequence information and from it make a peptide or polypeptide in the cells. There is a large and thriving industry based
on this. We were searching for the best
method for biohackers with limited resources to use to produce griffithsin on
an epic scale. Most of our experience
with protein expression had been with bacteria so far. E-coli is widely used for this because it
grows rapidly and is relatively inexpensive and easy to work with. Bacteria has certain drawbacks, however. For example bacteria is a prokaryote, lacking
organelles and the protein folding may not work well in eukaryotes such as
animals. On the other end of things,
much pharmaceutical Biomanufacturing uses animal cells for expression. The folding then is correct for animal or
human use. The drawback with animal
cells is that they are difficult to work with, grow slowly, and are extremely
subject to contamination by any number of organisms. Stringent laboratory procedures are needed to
ensure sterility during transfection and the growth cycle. This works well for pharmaceutical companies
with the resources to carry it out, however Biomanufacturing using animal cells
is very expensive to set up and maintain.
Some of the scientists who responded to our inquiries used
plants to express griffithsin with good results. Dr. Yavar Vafaee, Ph.D. had published his
research:
“Heterologous production of recombinant anti-HIV microbicide griffithsin in
transgenic lettuce and tobacco lines” and was very kind in advising and
getting me started in the exciting new direction of plant molecular
biology. We also were fortunate to find Dr. Evangelia
Vamvaka, Ph.D. who had
been working in Dr. Jennifer Doudna’s lab at UCB. She had previously done a great deal of
research with griffithsin and published papers such as “Rice endosperm is cost-effective for the
production of recombinant griffithsin with potent activity against HIV”. After
reading her papers and speaking with her, we decided that her method was the
one that would work the best to meet our biomanufacturing goals as biohackers
with limited resources. Expression in
rice has a number of advantages, such as rice being non-toxic and generally
regarded as safe, and when harvested and dried, it can be stored for long
periods of time without trouble. Dr. Vamvaka’s research shows that the folding
of the protein is correct and that the antiviral properties are present as expressed in rice.
Friday, March 19, 2021
Griffithsin vs Coronaviruses
Griffithsin vs Coronaviruses
On the 18th of March 2021, there are 120,915,219
confirmed cases of SARS-CoV-2 with 2,674,078
confirmed deaths reported (WHO)
UPDATE: As of the 22nd of November 2021, there are now 258,172,735 confirmed cases of SARS-CoV-2 and 5,158,642 confirmed deaths recorded (Johns Hopkins Coronavirus Resource Center)
Please note that SARS is an acronym for severe acute
respiratory syndrome.
The SARS-CoV-2 viral infection may cause extreme damage
to the respiratory system but also affects other organs of the body.
Coronaviruses are single stranded RNA viruses. They are enveloped viruses. The RNA is enclosed inside a protective
spherical coat of protein. These relatively
large virions (virus particles) and have around 74 mushroom shaped protrusions,
the spikes. This gives the virion a look
that some say resembles a crown, which is the origin of the name coronavirus. Some
of the glycoproteins are structural, such as the famous spike protein, and others
are glycosylated non-structural proteins.
These envelope and spike glycoproteins play a role in the infection of
cells by attaching to a host cell and acting as a doorway for endocytosis or
the taking in of the now naked RNA by the receptors of the cell (the envelope
does not enter the cell). Once inside the host cell, in the cytoplasm, the RNA can
instruct the ribosomes to replicate more of the virus.
Remembering that GRFT is a lectin, with the ability to
bind carbohydrates, we can now start to see how the antiviral action of GRFT is
to attach to the glycoproteins, such as the spike protein and block the viral
entry into the host cell.
3 deadly coronaviruses,
SARS, MERS, and Covid-19
SARS coronavirus first appeared in humans in 2002 with 8096
reported cases.
MERS (Middle East respiratory syndrome) had an outbreak
in 2012 with 2260 reported cases.
Covid-19 first appeared in 2019
SARS and MERS show a higher percentage of fatalities
than Covid-19 with MERS being the most deadly.
Covid-19 is more easily transmissible than SARS or
MERS.
SARS, MERS and Covid-19 were infecting animals,
probably bats, at first and then zoonotic transmission occurred, the jump from
animal to human. Besides bats, with
MERS, camels were a reservoir of the virus and humans were infected from camels
(Please don’t touch a sick camel, or at least wash your hands after).
Once a coronavirus made the jump, human to human
transmission spread it quickly.
Is it
probable that GRFT can prevent infection by coronaviruses?
We had been doing research related to Griffithsin for a
few years and in March 2020 when the covid-19 pandemic was sweeping across the
world, we searched through our library of papers and found one, published in
the March 2010 issue of Journal of Virology:
Broad-Spectrum In
Vitro Activity and In Vivo Efficacy of the Antiviral Protein Griffithsin against
Emerging Viruses of the Family Coronaviridae. (O'Keefe BR,
Giomarelli B, Barnard DL, Shenoy SR, Chan PK, McMahon JB, Palmer KE, Barnett
BW, Meyerholz DK, Wohlford-Lenane CL, McCray PB Jr. Broad-spectrum in vitro
activity and in vivo efficacy of the antiviral protein griffithsin against
emerging viruses of the family Coronaviridae. J Virol. 2010 Mar;84(5):2511-21.
doi: 10.1128/JVI.02322-09. Epub 2009 Dec 23. Erratum in: J Virol. 2010
May;84(10):5456. PMID: 20032190; PMCID: PMC2820936)
This excellent paper shows the results of many
experiments. 4 strains of SARS were
tested individually in vitro and griffithsin showed remarkable antiviral
action, preventing the viral cytopathic cell death from the viral infection. GRFT bound to the Coronavirus spike protein.
Experiments were also successfully performed on other coronaviruses that don’t
infect humans but are found in animals. GRFT was also shown to be of very low toxicity
to the host cells.
In vivo experiments were performed on mouse models using a
mouse-adapted SARS virus.
Control groups of mice got nothing at all (sham) or only GRFT
(control) but the important groups observed were the mice getting SARS virus
alone and the group getting both SARS and doses of GRFT. The griffithsin was applied into the tiny
mouse nostrils, because that is a good place to administer it when the virus
attacks the respiratory system. Picture
for a moment, scientists in lab coats pipetting into mouse nostrils! This may have caused the mice some discomfort
but it also saved them from dying from SARS.
The mouse group that got only the virus sickened, losing weight, and
then 70% of the group died, while 30% recovered.
The group of lab mice that got the SARS virus and also doses
of GRFT up their tiny noses didn’t lose weight and had 100% survival.
Important points made that stand out:
“Due to the proven threat from
SARS-CoV infections and the possibility of
future zoonotic
transmission of coronaviruses, efforts have
been initiated to
identify agents that could either reduce
infection or suppress
the deleterious cytokine response to SARS-CoV
infection”
“The broad range of Coronaviridae species
sensitive to GRFT is a significant attribute for this antiviral protein, as
this group of viruses appears to be capable of continuing zoonotic evolution
and transfer to human hosts”
10 years later, a novel Coronavirus made the
leap from animal to human hosts, there was an outbreak, and the world was
unprepared to stop it. As we all know it became a raging global pandemic.
In 2016, an article was published in the
journal Antiviral Research about in vitro studies done using GRFT to stop MERS
from infecting cells:
Middle East respiratory
syndrome coronavirus infection is inhibited by griffithsin.
Jean
K. Millet, Karin Seron, Rachael N. Labitt, Adeline Danneels, Kenneth E. Palmer,
Gary R. Whittaker, Jean Dubuisson, Sandrine Belouzard
Again, GRFT was shown to bind to the spike
protein and prevent infection, this time by the MERS coronavirus.
Again, scientists urged more development of
griffithsin:
“In conclusion, griffithsin has a low
cytotoxicity, likely interacts with any coronavirus spike proteins because of
their highly glycosylated nature and is able to hamper coronavirus spike
protein functions. Griffithsin should be considered as an interesting drug
candidate to develop for the treatment and/or prevention of current but also
future emerging coronavirus infections.”
3 years later, in Wuhan, China, a novel
coronavirus started infecting humans and as humans we were unprepared and
defenseless.
In a 2020 letter to the editor of Virilogica
Sinica, we find confirmation that the entry inhibiting antiviral GRFT also
works on the SARS Cov-2
Yanxing Cai, Wei Xu, Chenjian Gu, Xia Cai, Di
Qu, Lu Lu, Youhua Xie & Shibo Jiang
One interesting development reported in this
letter regards the pan-coronavirus fusion inhibitor targeting the HR1 domain of
human coronavirus spike, EK1 working synergistically with GRFT.
We could easily see from the research that has
been done that GRFT has the potential to prevent a coronavirus outbreak from
becoming a pandemic. It may be too late
to do anything about the present one that we are facing. Vaccines have been developed, tested, produced
and deployed in one year which is a huge accomplishment. It’s
not a matter of if but a matter of when the next coronavirus makes the leap
from animal to human. We were not
prepared for the covid-19 onslaught. A
small but dedicated group of scientists and biohackers, decided to start to prepare
for the next outbreak without delay.
To be continued….
Tuesday, March 9, 2021
Project GRFT: The story so far
In this and the posts to follow, we will introduce project GRFT, what this project is doing, and why it's important. GRFT = Griffithsin. The first part of our story starts here.
**********************************************************************************************************************************************************************
Seaweed
In the
1800’s a widow, Mrs. Amelia Griffiths (1768-1858), roamed the seacoasts of
Devon, Cornwall and Dorset with her wicker basket in hand, passionately collecting
and cataloging the marine algae commonly known as seaweed. In honor of Mrs. Griffiths and her
contributions to the science of phycology, the Swedish marine botanist Carl Adolph
Agardh named a genus of red marine algae “Griffithsia”.
Griffithsia setacea from
Griffiths’ books. © 2014 Royal Albert Memorial Museum & Art Gallery, Exeter
City Council
Griffithsia
are eukaryotic organisms of the Rhodophylum (Rhodo, from the ancient Greek,
Rhodon, Rose and phylum which in the taxonomy stack is below kingdom and above
class). Griffithsia are found in the oceans of the world. They dance gracefully in
Neptune’s realm off the Coast of New Zealand.
Viruses
In the late
1800’s, tobacco plantations in the Ukraine and Bessarabia (now Moldova) were,
like many other plantations worldwide, infected with a disease that caused
spots on the leaves causing a mottled appearance or mosaic pattern. This was called mosaic disease because the
patterns sometimes resembled mosaics.
The disease could and did destroy entire tobacco crops. Scientists in Russia and Europe started an
inquiry into the cause of the disease.
Was it a mold or a bacteria? Was
it something else? Conducting
experiments, they found that the agent which infected the plants passed through
filters with a small pore size that would have stopped bacterial cells. It could not be seen with the microscopes in
use at the time. It would not grow on
prepared media on which bacteria or fungi would thrive. So this was something as yet undiscovered. Dmitry I. Ivanovsky and Martinus W. Beijerinck
started the groundwork for what would become Virology, the study of viruses.
What is a
virus? First we should look at what a
virus is not. Animal, vegetable, or
mineral it is not. Fungus or bacteria it
is not. Viruses can't be considered
cells or independently living organisms. Perhaps viruses are best imagined as
being exquisite alien machines running a program to inject nucleotides into living cells causing the cellular mechanism
to produce more of the virus. Viruses
are made of nucleotides often dressed up in a protein coat or capsid.
Not all
viruses cause health problems. Some we
find useful for a wide variety of applications including Biomanufacturing by
the expression of of recombinant protein in organisms. Viruses considered pathogenic to humans only
number about 100 - 200 varieties. These disease causing viruses, however,
represent a major health concern and are a significant cause of mortality and
suffering worldwide.
Prevention of viral outbreaks
Prevention
of the spread of pathogenic viruses may be accomplished in several ways, for
example:
1.
Physical
distancing, physical barriers.
These
keep the virus particles, known as virions, away from cells by methods such as
masking and wearing other PPE, also by air filtration.
2. Vaccines
Vaccination
stimulates the immune response with the body producing antibodies against the
particular virus. Vaccination usually
only requires 1-3 doses of the vaccine and may provide immunization for a
lifetime.
Antivirals
Antivirals prevent the virus from hijacking cells in the host and replicating. The antiviral works typically by blocking entry into the cell by the virus. Antivirals need to be taken whenever there is danger of the transmission of a virus. Antivirals generally work against pathogenic viruses when administered before or at the first stages of infection.
Antiviral lectins
A lectin
may be defined as a carbohydrate binding protein.
Lectins are
found in everywhere in nature including in the food we eat.
An example
of an antiviral lectin is BanLec or banana lectin. Found in the bananas Musa acuminata and Musa balbisiana, BanLec has been shown to be effective
against HIV.
Other
examples of effective antiviral lectins found in nature are cyanovirin-N,
scytovirin, and microvirin.
Griffithsin
Of all the known antiviral lectins
found in nature, perhaps griffithsin, the red seaweed protein, is the shining
star. Besides being free of significant toxicity, griffithsin is the most
powerful of any of the lectins at inhibiting enveloped viruses from entering
cells.
Griffithsin,
or GRFT is a 121 amino acid, 12.7 kDa protein.
Wild type
GRFT:
Please take
a look at position 31 above. In wild
GRFT, this is a non-standard amino acid and in recombinant GRFT this is
generally replaced by substituting alanine (the red A).
The yellow
arrows designate the secondary B-sheet structures of the protein.
GRFT is a
dimer and has 3 triangular prismatic blades in the Beta sheets. These separately have been shown to have some
antiviral properties, however together in griffithsin’s domain swapped dimer they
are more powerful, just as two hands can grab and hold with more strength than
one hand. This dimeric structure has six
carbohydrate binding sites that work together.
An
interactive 3D model of the GRFT protein structure may be found here, scroll down to "Structure" and play with it.
Future episodes to follow!
Monday, February 8, 2021
work in progress
Thanh's gene gun rapid fire magazine design for our hardware working group.
Please help support the project:
(new link)
https://charity.gofundme.com/o/en/campaign/help-us-build-a-gene-gun
Thursday, December 3, 2020
We come bearing arms for life.
Have you ever dreamed of a gun that can save lives instead of destroying lives?
The guns we make are guns are for saving lives.
That's what we do.
Will you help to make it real?
Any donation is appreciated.
https://charity.gofundme.com/o/en/campaign/help-us-build-a-gene-gun
😷
Wednesday, October 31, 2018
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