10.4: Biotechnology

Making Human Protein

Proteins can be made only in cells. Until the advent of biotechnology, we extracted proteins from natural sources, e.g., insulin (a protein) was extracted from animal pancreases. Making insulin by biotech came in the nick of time. More people need insulin, and the amount of insulin extracted from animal pancreases is limited by availability of pancreases.

Human proteins can be made by biotechnology, in cell cultures or bacteria (single- cell organisms). The amount isn’t limited as it is when insulin is extracted from pancreases. Activase, the drug for dissolving blood clots (Chap. 7), is made by biotech—the only way this human protein can be made in amounts needed for use as a drug.

The basic method: (1) locate the gene (recipe) for the protein in human DNA, (2) cut out the gene, using special enzymes, (3) insert (splice) the gene into DNA of bacteria (or cell cultures), (4) grow the bacteria to make the proteins (now including the human protein) encoded in their DNA, and (5) extract the human protein that’s been made. Accomplishing all this is easier said than done!**

Bacteria that carry human genes serve as factories for making human proteins. Bacteria multiply fast, so a flask-full can be made quickly. Raw material for this factory is simply the normal nutrients that bacteria (or cell cultures) need.

It’s not only human proteins that are made this way. Calf stomach used to be the source of the enzyme rennin used to make cheese (rennin curdles milk). Rennin made by biotech (inserting the calf gene for rennin into yeast DNA) is a more reliable and cleaner source. It’s been used since 1990 to make most of our cheese.

Besides making specific proteins in large amounts, there are other advantages to making proteins this way. When insulin is made by biotech, it’s human insulin, whereas it’s pig insulin when extracted from pig pancreas. Pig insulin works in humans, but it isn’t exactly the same as human insulin, so some people get an allergic reaction.

Making proteins by biotech avoids hazards such as HIV contamination of blood-clotting factors. When taken from plasma pooled from many donors, an entire batch of blood-clotting factors can be contaminated by just one infected donor. As said earlier, many hemophiliacs were infected with HIV via contaminated blood-clotting factors before the cause of AIDS was known.

^{**For work in developing this technology, Paul Berg was awarded a Nobel Prize in 1980. It’s genetic engineering, but the general term biotechnology is often used instead; genetic engineering sounds ominous to nonscientists.}

Gene Therapy

The first human gene therapy experiment to treat a genetic disease began in 1990. At age 4, Ashanthi De Silva was severely ill with an immuno-deficiency disease caused by a genetic deficiency of a key enzyme. White blood cells taken from her were infected with a retrovirus altered to carry the normal gene she needed into the cells’ DNA. The cells were infused back into her and made the enzyme from its newly inserted gene. She still had to inject some of the enzyme, but the gene therapy enabled her to live on to get a master’s degree, and work to empower rare-disease patient communities. youtube.com/watch?v=IgES04-cSr8.

In 1993, gene therapy, using umbilical cord blood, was used to treat two newborns diagnosed prenatally with another severe immunodeficiency disease.* Gene therapy has been successful in treating others with this disease, but the treatment suffered a setback in 2002 when a child developed a leukemia-like disease after treatment.

In 1989, the defective gene that causes cystic fibrosis was found.† Sticky mucus fills the lungs, making it hard to breathe and causing lung infections and damage. By locating defective genes, scientists can find out what protein is made by a normal version of that gene. Then, by finding out what that protein does, they can begin to figure out the disease.**

In 1991, a normal version of the “cystic fibrosis gene” was inserted into a common-cold virus (altered so it can’t cause a cold). The virus was then dripped into rats’ lungs, causing the lungs to make the desired protein. But when this procedure was tried in human cystic fibrosis patients, it caused unacceptable lung inflammation.

In 1999, gene therapy studies had a major setback when 18-year-old Jesse Gelsinger died 4 days after gene therapy for a disease caused by a defective gene for a liver enzyme. He died from a massive immune response to the common-cold virus that was used to deliver the normal gene for the enzyme to his liver.

In 2017, the FDA (Food and Drug Administration) approved gene-therapy drug (Luxturna) injected into the eye to treat an inherited cause of blindness. In 2019, the FDA approved gene therapy drug (Zolgensma) given intravenously to treat infants with an inherited form of deadly spinal muscular atrophy. Both drugs deliver a normal form of the defective gene using a common-cold virus.

^{*This disease has been called bubble-boy disease—David Vetter, born in 1971, lived his life (12 years) in a sterile, plastic bubble because any infection could be fatal due to his untreatable severe immune deficiency.
†The gene is for a membrane protein that lets chloride ions exit a cell. In cystic fibrosis, chloride ions accumulate inside lung cells, pulling in water from the surrounding mucus, making the mucus thick, hard to cough up, and a favorable site for bacterial infection. White blood cells come in to fight the infection; when the cells die, their DNA (which is thick and viscous) spills into the mucus, thickening it even more. The biotech-made drug pulmozyme was approved in 1993 (the application exceeded 150,000 pages)—the first new treatment in more than 30 years. The inhaled drug is an enzyme that chops up DNA in the lung mucus, thinning it, making breathing easier, reducing lung infections and damage.
**There are huge databases of gene sequences and their proteins, e.g., a certain sequence of bases suggests it’s part of a gene for a membrane protein, just as the phrase add ½ cup oatmeal suggests it’s part of a recipe. Databases are used to find a match to unidentified base sequences, just as we might search databases to find out what books contain the sequence, his knees felt weak as she kissed him.}

Gene Editing

In 2012, a revolutionary DNA editing technique CRISPR-Cas9 was introduced. It’s a game-changer as a fast, simple, and inexpensive way to precisely edit DNA in any organism.* It’s patterned after a defense that bacteria use against viral infections.

Bacteria have odd sections in their DNA called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). Interspersed between these repeats are short segments of DNA that bacteria have taken from invading viruses.

Matching copies of RNA (crispr RNA) are made from these viral DNA segments, just as we make messenger RNA as matching copies of genes in DNA (Figure 10.3). Crispr RNA is cradled in a crispr-associated enzyme (cas9) that cuts DNA. When crispr RNA finds its match in the DNA of an invading virus, it latches onto that segment, and cas9 cuts it out, thereby killing the virus.†

CRISPR-Cas9 is used as a gene-editing tool by replacing the bacterial crispr RNA with a piece of RNA that matches the segment of DNA we want to edit. The RNA homes in on the targeted DNA segment and cas9 cuts it out. This can disable a gene (e.g., to investigate its function), or the segment can be replaced (e.g., to correct a mutation). The possibilities are mind-boggling. Genetic disorders in mice have been cured by editing/repairing genes. Progress is being made for the same in humans, e.g., editing genes to treat sickle cell anemia.

Crops can be made less susceptible to disease and climate change. There’s research into editing DNA of mosquitoes to hamper transmission of malaria and zika virus.**

In 2016, Kathy Niakan, became the first scientist to get regulatory approval to edit genomes of human embryos for research. This allowed her to use CRISPR-Cas9 to selectively disable genes in surplus human embryos donated by fertility treatment patients, to learn their function in the development of healthy embryos. The growth of embryos from a single fertilized cell was limited to 200-300 cells.††

Scientists, ethicists, lawyers, etc., continue to discuss the rapidly expanding use of CRISPRCas9, e.g., to create beagles with twice the normal muscle mass. What about editing DNA in a privately-funded IVF clinic to produce a taller child? CRISPR-Cas9 is both exciting and scary.

^{*Jennifer Doudna and Emmanuelle Charpentier shared a Nobel Prize in 2020 for discovering this technique.
†This is reminiscent of the restriction enzymes that bacteria use to chop up invading viruses. Just as we’ve co-opted restriction enzymes to chop DNA for DNA fingerprinting (Figure 10.4), we’ve co-opted CRISPR as a gene-editing tool.
**Mosquitoes are considered the deadliest creatures on earth; they transmit malaria, zika, yellow fever, chikungunya, dengue, west nile virus, lymphatic filariasis. In 2020, there were about 241 million malaria cases and 627,000 deaths, mostly children.
††In 2018, scientist Jiangku He announced the birth of twin girls whose DNA he had edited as embryos to protect them from HIV infection. He used CRISPR-Cas9 to disable a gene that coded for a membrane protein that lets HIV enter cells. This was shocking, since germline-editing (editing DNA of ova, sperm, or embryo) persists through generations.}

Animal Cloning

Cloning creates a genetic duplicate of an animal. The DNA-containing nucleus of an animal’s cell is removed and inserted in another animal’s ovum that has had its nucleus removed. This means the genes in the resulting embryo are identical to that of the donor.

The 1997 birth of the lamb Dolly was sensational—the first time a mammal had been cloned from an adult cell.* Dolly was cloned from a mammary (breast) cell (and named after Dolly Parton), making her the genetic twin of the ewe who provided the mammary cell. Cloning has practical uses in animal husbandry (e.g., cloning a steer that grows lean and fast on small amounts of feed), medical research, the pharmaceutical industry, etc.

One way to get a steady and large source of a particular human protein is to insert the human gene into a lactating animal, such that the protein is excreted in the milk. One then milks the animal and extracts the human protein from the milk. (It isn’t sold to drink!) The stumbling block has been that not only is it very hard to get the gene inserted this way, but the gene isn’t passed on to offspring (it isn’t in the animal’s ova). In contrast, cloning provides a way to reproduce an animal in which the gene has been successfully inserted.

^{*In becoming liver cells, heart cells, etc., certain genes are turned on or off in an embryonic stem cell’s DNA, e.g., brain cells don’t make the digestive enzyme lactase because the gene to make it is turned off—permanently it was thought.}

Plant Genetics

In crossbreeding, genes mix “by chance.” It takes many crosses and a long time to get what you want. Biotech is faster and more precise.

Plant genes can be edited, e.g., by CRISPR-Cas9. A single gene can be transferred to get what you want in one shot. The gene even can be from another species, e.g., a gene that protects fish against freezing put into a plant to prevent frost damage.

Altered bacteria (like the use of altered retroviruses in gene therapy) can be used to insert genes. Agrobacterium tumefaciens normally causes disease by inserting its genes into plant DNA. Plant geneticists can remove the disease-causing genes from this bacterium (disarm it), and then use the bacteria as a vehicle to transfer a desired gene into plants.

One such gene is for a protein in the coat of a virus that infects and damages crops. It was noticed that crops infected with a mild strain of a virus weren’t susceptible to infection with a more damaging strain of the virus (like the cowpox infection in dairy maids conferring immunity to smallpox; see Chap. 7). It was found that one of the virus’s proteins, alone, confers the same protection (the basis of some human vaccines).

Scientists could then cut out the gene for this viral protein, insert it into disarmed Agrobacterium tumefaciens, and let this bacterium do what it does naturally—insert the gene into plant DNA. This genetically altered plant can now make this viral protein for its own protection—it’s “vaccinated” against the plant disease. (When we eat a plant with this “foreign protein,” it’s digested into its component amino acids.)

Agricultural biotech can have a huge impact on health in developing countries where food is scarce and the nutritional quality of the staple diet is inadequate. Vitamin A deficiency causes blindness, much suffering, and millions of deaths each year. Rice, a dietary staple for half the world’s population, lacks beta-carotene (which our bodies can convert to vitamin A). Golden Rice was created by inserting genes to enable rice to produce beta-carotene, giving it its golden color.

Creating Golden Rice was a huge scientific endeavor. Beta-carotene isn’t a protein, so it wasn’t a matter of inserting a single gene. Biosynthesis required several genes for the needed enzymes (proteins) that had to work in sequence—a breakthrough in biotech. It was also a humanitarian endeavor, the seeds to be offered free to poor farmers in developing countries.

The 8-year project was headed by Ingo Potrykus and Peter Beyer and funded by the Rockefeller Foundation, culminating in the year 2000 by publication in the journal Science and a Time magazine cover featuring Potrykus.

For the next 20 years, Golden Rice wasn’t allowed to be grown commercially. Potrykus retired in 1999 at age 65 from his professorship at the Institute of Plant Sciences of the Swiss Federation of Technology, but continued to work to make Golden Rice available. In 2013, he met with the Pope, who gave Golden Rice his personal blessing.

In 2016, more than 100 Nobel laureates signed a letter asking Greenpeace to end its opposition to Golden Rice and to GMOs (genetically modified organisms) in general. In July 2021, the Philippines became the first country to approve the commercial planting of Golden Rice.

In 2015, the Innate™ potato passed muster as being as safe and nutritious as regular potatoes. It’s Innate in that the inserted genes come from other varieties of potatoes. The potato is genetically modified to produce less acrylamide (a carcinogen to rodents and possibly humans) when deepfried and to prevent black spots from forming when raw potatoes are bruised (lots of potatoes are discarded because of bruising). In response to anti-GMO voices, McDonald’s announced it won’t use this potato.*

^{*The Flavr Savr tomato has a similar story. The gene for the enzyme that causes rapid deterioration was knocked out, so the tomatoes last longer after picking and can be picked when ripe and tasty. Campbell Soup Co. helped finance its development but, in response to anti-GMO voices, announced in 1993 that they wouldn’t use it in their products.}