First phase 1 clinical study of GRFT for prevention of HIV

 Researchers at Albert Einstein College of Medicine completed phase one clinical study of a griffithsin topical.  This is designed to be applied as a gel to the vagina before sex to inhibit HIV infection.  Phase one trials are done are to see what effect the pharmaceutical has on healthy humans.   The results of this study are very promising,  This is a huge step forward, and we look forward to the next phases of clinical trials.

Here's a link to the paper:

 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0261775 

Meanwhile our group is looking into what the optimal pH of a buffer for adhesion of DNA to W and AU microparticles would be.   We would like to hear from anyone who has done particle bombardment on the subject of what buffers worked best.

important considerations when preparing projectiles

 Basically when coating particles we need to pay careful attention to:

1. pH: This is most important!
2. Timing of incubation
3. Temperature of incubation
4. Salt concentration
5. Size of particles
6. Freshness of Spermidine (0.1M)
7. 2.5 M CaCl2 is filter sterilized and aliquots prepared ready to use.
8. Purity and concentration of DNA
9. Keeping particles suspended in the buffer solution while adding the spermidine and CaCL2
10. We have seen problems with particles sticking to the 1.5 Ml centrifuge tubes. DNA Lo-Bind tubes should work better.

Preparing particles - theory

 

What is happening when we coat either Au or W particles?   

Adsorption

Adsorption is where a layer of molecules cling to a surface.

1.       There is the adsorbate (i.e. DNA plasmid)

2.       There is the adsorbent (i.e. W)

What attracts the adsorbate and the adsorbent to each other so that they are held together in Adsorption?

DNA has a – charge.  W has + charge.  Under the right conditions, a bond is formed.  If the DNA is in solution with the W and the bond forms, the DNA will precipitate and adsorption occurs.  The DNA is not present in the solution anymore, it is now coating the W microparticle.

That’s very beautiful and simple to understand but as may be expected the devil’s in the details. 

preparing rice seed to grow embryos and calluses

 Here's Hector in the lab as he prepares rice seed to grow on media plates

https://youtu.be/Qdc9-8sgymg

note: the husked seeds were washed with bleach to sterilize them

Hector's work on project GRFT

Introducing Mr. Hector Vera who is one of our talented, dedicated and hard working volunteers at project GRFT.  Hector speaks from outside a laboratory in Oakland, California where our gene guns are now being built and tested.  

Open Insulin - changing the paradigm

 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.   

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.  

Our group, Project GRFT formed, started meeting and working.   Our goal is to grow transgenic rice containing the polypeptide griffithsin and to make this rice available worldwide for research.        To be continued....