Biotechnology - The green light saber of the 21th century

Veröffentlicht von: Progress Austria am Mittwoch, Oktober 31, 2018

Autor: DI Martin Stübler, MA, Biotechnologie und Bioökonom.

 

Biotechnology is based on the genetic manipulation of living organisms and bridging the gap between technology and biology. It’s not about creating a healthy and diverse world worth living in. From CRISPER-Cas9 to pharmaceutical-tobacco and from spider-goats to golden-rice the future is going to be very different. Let’s analyze the advantages and benefits as well as the potential threads of such a disruptive technology for the 21th century.

 

What is Biotechnology:

 

There are many good definitions of biotechnology. Merriam-Webster defines it as “the manipulation (as through genetic engineering) of living organisms or their components to produce useful usually commercial products (such as pest resistant crops, new bacterial strains, or novel pharmaceuticals)” [1]


Biotechnology is always based on genetic manipulation. Many tools have been discovered in the last century that enable us to change a given sequence of virtually any organism. Some methods work best for bacteria (e.g. electroporation) others are optimized for cell-culture (eukaryotic cells cultivated in a petri dish) and others are suitable for whole organisms (e.g. gene gun for plants). As the initial definition includes all living beings, let’s use two terms: “micro-biotechnology” and “macro-biotechnology. The micro-biotechnology is the most common form of biotechnology due to easy and safe handling, reproducibility, and elimination of certain ethical concerns.

 

Micro-Biotechnology

 

I like to think of micro-biotechnology as a very small farm of even smaller sheep that produce valuable wool. This microscopic small farm has two big advantages. Firstly, small things don’t have a central nerve system and are therefore not able to experience pain in a way that might lead to ethical concerns. Secondly, the smaller the individual objects are the faster they are able to grow and replicate. It is directly proportional to their surface area. For every biological process the most important parameter is surface area. Surface area becomes even more important the further we go down into the nano world as the most important interactions are defined by surface area and distance. But let’s leave the nano world and raise up one level into the molecular world. The micro-biotechnology, although not directly experienced by us, is still very tangible as we all grew up with microbiological products such as: bread, coffee, cheese, yoghurt, wine, sauerkraut, chocolate and specialties from many other countries such as kombucha kefir, natto, etc. Bridging the gap between microbiological products and micro-biotechnological products we have to introduce genetic manipulation to obtain qualities not achievable by native strains such as new metabolic byproducts. All these applications have in common, that they are using a microbiological organism (bacteria, yeasts or fungi) to transform starting material into products with different nutritional and organoleptic properties. Although this process seems very similar to aging, the outcome is very different and can lead to preservation of food or the production of alcohol. Our ancestors discovered this particularly favorable “aging” already in prehistoric times. With the earliest archaeological evidence of fermentation dating back 13,000 years to residues of a beer with the consistency of gruel, in a cave near Haifa in Israel.

[2]

Beside the ancient food-technological applications modern biotechnology arose from medicinal human needs. In particular the production of insulin from pig-pancreas became too difficult to satisfy the needs of a modern society. The extraction of pig insulin brought many problems such as intolerance for a certain population together with a high variability in product parameters. This lead Eli-Lilly to the idea of taking the human insulin gene and putting it into a well-known fast growing bacteria (Escherichia coli). This event was the breakthrough for modern biotechnology and almost all the insulin production from pig pancreas could be stopped immediately now almost all people with diabetic 1 could be supplied successfully with the method still in use today.

 

Biotechnology vs. Bionic

 

At this point it is important to distinguishes between biotechnology and bionic. The line is not easy to draw but as a rule of thumb whenever a macroscopic static structural, mechanical or dynamic element is copied from nature this is called bionic. Whenever a chemical component, enzymatic pathway or a functional protein is needed this is considered biotechnology. But beforehand it is the job of molecular biology to discover the component and identify the gene sequence. The job of biotechnologists is then to upscale this gene in the host system. This component of interest is in most cases a polypeptide such as a protein used for a therapeutic application. An example for such a protein therapeutic is an antibody. Antibodies of the IgG class are usually composed of four subunits (2 heavy and 2 light peptide-chains). They are produced by B lymphocytes (or B cells).in our body and other mammals and represent a major defense mechanism of our immune system.

Biotechnological products:

 

The high tech materials of the 20th century steal, glass, plastic and a big array of chemical products changed our life forever. Basically all the materials we are using today haven’t existed 100 years ago. The majority of cloth is made of nylon, polyesters and spandex, containers and boxes are compound materials of plastic-aluminum. All this materials have one thing in common, they are depending on crude oil either as a raw material or as energy source for transformation. Together with oil other elements have become high in demand such as metals and rare soils. From a scientific perspective it was worth the effort, from an economical perspective it was the only choice whereas from an ecological perspective there is a lot left to improve. The 21th century is probably able to overcome many of these problems by technological solutions. Especially modern therapeutically applications will play a crucial role in aging and disease prevention of the 21th century. Most therapeutics are built from the same elements as our body oxygen, carbon, hydrogen nitrogen, calcium and phosphorus together with some trace elements. The big advantage of biological applications is the relative abundance of these elements on our planet. There is no need for life to waist energy for looking for rare soils or special metals such as 20th century humans. The abundance and recyclability of these components makes life on this planet possible. Modern biotechnological production facilities are using the same building blocks to produce high value therapeutically proteins such as antibodies from comparably cheap sources. These sources are defined mixtures of different, sugars, proteins and micronutrients that are used to culture the bacteria in large stainless steel bioreactors. The net value gained from such a fermentation is often 10 000 times the value of the starting material. But this profit margin is usually decreased by the downstream procedure which is separating the active component from the broth. This step is crucial as the product is purified from other components of the fermentation brew. Depending on the purity of the product the price per gram might reach astronomic heights such as for Eculizumab (trade name: Soliris) which costs about US $409,500 for a full therapy [3].

Biotechnological key-technologies for the 21th century need to be cheaper, easier and faster. These technologies are about to raise with the Crisper/Cas9 system at its forefront. Its simplicity and robustness makes it clearly a technology of the 21th century. It was the AAAS's choice for breakthrough of the year in 2015 [4] and is considered one of the most disruptive technologies in 2018 [5] allowing researchers to knock out and rearrange the DNA of any living organism. The simplicity of CRISPR has catalyzed the further development of the scientific technique itself by providing access to genetic manipulation techniques to more people than ever before. Although CRISPER is providing a fantastic tool for the manipulation, the selection process to obtain the clone of interest is still the most labor intensive part.

 

CRISPR is an adaptive immune-system analog in bacteria. Invading pathogens such as bacteriophages (viruses targeting bacteria) are identified and DNA fragments are acquired and transcribed into CRISPR RNAs (crRNAs) to have a set of specifications able to guide cleavage of invading RNA or DNA in the future. This find and remove mechanism can be equipped with different template RNAs (crRNAs) which are not targeting the sequence of a bacteriophage but also a sequence responsible for a hereditary disease. [6]


The CRISPR system works in three stages. In the first stage DNA fragments of invading pathogens are acquired and incorporated into the host. This CRISPR locus are like an internal library spaced between crRNA repeats. In the second stage Cas proteins are expressed which pick up the pre-crRNA and translate them into templates for their work. The fully processed crRNA is composed of two parts. First a spacer sequence responsible for targeting it to the invading genome and second the crRNA repeat sequence, which allows for recognition and binding by Cas proteins. In the last stage, Cas proteins recognize the appropriate target by comparing every sequence they find in the cell to the crRNA. They carry and mediate the cleavage once they find a corresponding match. The guiding sequence is a region of approximately 20-nucleotides similar to a twenty digit bank account number which is unique enough to provide a very high accuracy in identifying the invading genome, thus protecting the host cells from infection. [6]


For the bacterial immune response over a dozen different Cas proteins are available. The Cas 9 protein is so popular due to its simplicity and robustness. It was reengineered by Jennifer Doudna and Emmanuelle Charpentier from a four component system to a two component system making it much easier to use. Since its discovery, Cas9 has been extensively used for genome editing in multiple organisms. Similar to other nucleases, Cas9-mediated genome editing is accomplished by a double strand break (DSB) which triggers DNA repair through intrinsic cellular mechanisms. These repair mechanisms are responsible for the intentional insertion of specific DNA fragments at the site of the initial DSB. [6]

MACRO-Biotechnology

As we have had a look at micro-biotechnological applications so far let’s level up and get from the molecular to the cell level and all the way up to the organism level. When thinking about full macroscopic organisms we can classify them (very simplified) as (i) plants and (ii) animals. At this level we are facing many advantages but also many drawbacks when working with whole organisms. First when looking at the animal-biotechnology we are facing similar ethical questions as for human gene editing. Are we allowed to edit the genome of another species, are we allowed to take out and add genes to make animals more suitable for our needs? Is it ethically okay to produce neon-glowing mice or spider silk producing goats? In my opinion this question is not crucial but “cui bono”. We are basically doing gene editing for all our pets and house animals for more than ten thousand years now resulting in a changed genome, changed set of chromosomes, changed physiology and changed metabolism. Of course we did this primarily with selection tools but the result is still the same. The same holds true for all culture crops, without human input there would be no wheat, rice or corn plants as we know them today. The synergistic history of plants and humans is very well recharged in “The botany of desire” by Michael Pollan showing the deep connection between our needs and how we designed plants to suit us… or the way around, who knows.

I was working on a plant-biotechnological project during grad-school producing a tobacco plant expressing a monoclonal antibody to treat HIV. The general idea was to grow the necessary medicine directly in the countries of need (SSA, Sub Saharan Africa) and supply the local communities not only with the seeds to regrow the plants as often as needed but also with simplified purification methods able to extract the life-saving antibodies from the toxic plants. The tobacco plant has many advantages such as easy cultivation, fast growing and every plant produces several thousand seeds. The nicotine present in all plant parts ensures the plant isn’t consumed as a food source. Every therapeutic application is tightly linked to a defined dose of the active ingredient (in this case the antibody) and the time-release-profile of this active ingredient. The proper estimation of necessary antigen concentration in the plant source is the main difficulty in getting FDA approval of whole plant drugs. Some products have already entered pre-clinical testing such as tomatoes, corn, carrots, potatoes and algae for direct treatment or disease prevention of Cholera, Coronaviruses, Ebola, Yellow fever, Dengue fever and H5N1 viruses [7]. Certain factors remain a challenge for edible vaccines but the advantage of producing vaccines on a local decentralized basis without the need of a cold chain makes edible vaccines for immunization an undisputed goal for next generations.

The United nations have started the campaign “Zero hunger” to eradicate world hunger by 2030 [8]. This desirable goal should be of global interest and many nations are contributing enormous amounts of resources to achieve it. But very often it is not the lack of caloric energy intake that is causing reduced immune function, blindness or death in developing countries but rather the scarcity of key nutrients. Clinical and subclinical vitamin A deficiency remains the biggest problem, affecting 250 million schoolchildren worldwide [9]. Recently, food-based interventions to increase the availability of vitamin A–rich foods and their consumption have been suggested as a realistic and sustainable alternative to overcome vitamin A deficiency in developing countries. One well known example is “Golden Rice” a south East Asian rice variety (Oryza sativa) which was genetically modified to express a temperature stable Vitamin A precursor (β-carotine) which is converted to Vitamin A in the human body after oral consumption. Genetically engineered ‘‘Golden Rice’’ (because of the color) contains up to 35 µg β-carotene per gram of rice. The corresponding vitamin A levels that can be achieved after consuming this bio-fortified rice were determined by Tang et al and showed a significant improvement in rice-consuming populations that commonly exhibit low vitamin A status. The bioavailability is the key factor here and corresponding vitamin A levels can only be determine by blood samples. In a study by Tang et al demonstrated that 100 g uncooked rice (130-200g cooked rice) provides 500–800 µg retinol (vitamin A) this represents about 80%-100% of the estimated daily dietary requirement. Which shows, that β-Carotene derived from Golden Rice is effectively converted to vitamin A in humans.

Golden rice is an excellent examples of a biotechnological application with a positive direct impact on the developing countries of the Middle East but it is based on many patents. Syngenta Seeds AG was able to achieve patent wavers for all patents included for humanitarian use. The Golden Rice Humanitarian Board gained the right to sublicense the technology to breeding institutions in developing countries, free of charge. And families are able to use the technology for free without exceeding an annual income of $10.000. The farmers are allowed to reuse the harvested grain as seed for the following season without charge or penalty. Despite all this golden rice is still experiencing public resistance especially in developed countries. These countries are ironically not experiencing major retinol (vitamin A) deficiencies and are therefore probably more likely to reject a new technology which is not suiting their needs.

Biotechnology as an integral part of our everyday life will gain more importance in the 21th century than it has ever before with direct applications in gene-therapy, vaccines, food production and the still unmentioned topic of CO2 compensation or personal protection by a bullet proof skin produced by “spider goats” (goats producing spider silk after genetic manipulation) at Utah State University [11].

Many steps have been accomplished, but public awareness of “gene editing” as a neutral tool is probably the key aspect and course-setting for all future developments. Critical thinking and constant questioning should not only be qualities of the new generations but of a developed society as a whole. Everybody can contribute to this development by bringing this topic to discussion and taking opposing arguments into account while keeping an open ear and an open mind.

Sources:

[1] https://www.merriam-webster.com/dictionary/biotechnology

[2] https://www.bbc.com/news/world-middle-east-45534133

[3] https://en.wikipedia.org/wiki/Eculizumab

[4] https://www.sciencemag.org/news/2015/12/and-science-s-2015-breakthrough-year

[5] https://disruptionhub.com/2018-disruptive-trends/

[6] https://www.annualreviews.org/doi/pdf/10.1146/annurev-biochem-060815-014607

[7] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492011/pdf/vaccines-05-00014.pdf

[8] https://www.un.org/sustainabledevelopment/wp-content/uploads/2018/09/Goal-2.pdf

[9] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682994/pdf/ajcn8961776.pdf

[10] http://www.goldenrice.org/Content1-Who/who4_IP.php

[11] https://www.theguardian.com/science/2012/jan/14/synthetic-biology-spider-goat-genetics

 



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