The applications of biotechnology · AP® Biology AP Biology

Gene therapy and vaccines based on nucleic acids

Children affected by severe combined immunodeficiency were born with no defenses and had to live in sterile bubbles, until it became possible to introduce a working copy of the ADA gene into their own lymphocytes and return them to their bodies cured. How can we use nucleic acids not just to study a disease, but as a direct medicine inside human cells?

Two ways of using genetic material as a medicine

To correct a heritable disease caused by a faulty gene, a vector (often a modified virus, unable to cause disease) introduces a working copy of that gene into the cells.

To train the immune system against a virus, an mRNA vaccine introduces only the instructions for making one protein of the pathogen; the cells read it, make the protein temporarily and the immune system learns to recognize it, without the mRNA ever being integrated into the patient's DNA.

Why are attenuated viruses used as gene therapy vectors rather than injecting naked DNA directly into the tissues?

Show answer

Because viruses have very efficient natural mechanisms for crossing the cell membrane and carrying the genetic material to the nucleus, preventing nucleases from destroying it.

Following an mRNA vaccine into the cell

Why can't naked mRNA simply be injected, and what happens once it gets inside a cell?

Barriers: mRNA is a large, hydrophilic molecule whose phosphate backbone is negatively charged. The hydrophobic core of the phospholipid bilayer and the negatively charged outer surface of the membrane repel it, so it cannot diffuse across. In addition, RNases in blood and tissues degrade free RNA within minutes.

Solution: the mRNA is packed inside lipid nanoparticles. The lipid shell protects it from RNases, hides its charge, and lets the particle fuse with the membrane or enter by endocytosis, which releases the mRNA into the cytosol.

In the cytosol, ribosomes read the codons and translate the mRNA into the pathogen protein (for example, a viral surface protein). The mRNA is then broken down, so the protein is made only for a short time. The immune system detects the protein and builds memory against it.

Your turn: a classmate claims that "mRNA vaccines can permanently alter your DNA." Refute it using where the mRNA is and what it would need.

The mRNA stays in the cytosol, while the DNA is protected inside the nucleus. Cells have no enzyme that copies this mRNA into DNA and inserts it into a chromosome (that would need reverse transcriptase and integrase, as in retroviruses). The mRNA is also degraded within hours to days. So it is not gene therapy and it does not change the genome.

Practice★☆☆

Name two applications of biotechnology in healthcare and briefly explain what each consists of.

Solve

Practice★☆☆

What is gene therapy? Give an example of a disease targeted by this strategy and explain its underlying principle.

Solve

Practice★★★

During the development of an mRNA vaccine against an enveloped pathogenic virus, the research team must overcome several physiological and cellular obstacles: a) If free…

During the development of an mRNA vaccine against an enveloped pathogenic virus, the research team must overcome several physiological and cellular obstacles:

a) If free mRNA were injected directly into the bloodstream, it would be degraded within minutes and would be unable to cross the plasma membrane. Justify what biological barriers (molecular and electrostatic) prevent its entry, and explain how encapsulation in lipid nanoparticles solves this problem.
b) Once in the host cell cytosol, describe which organelle translates the mRNA and refute the erroneous claim that "mRNA vaccines can permanently alter the patient's genome," providing two biological arguments based on the flow of genetic information and cellular compartmentalization.
c) Compare the safety of this vaccine with a traditional attenuated virus vaccine in an individual with severe immunodeficiency: predict what risk of reversion or pathogenic replication the attenuated vaccine poses that does not exist at all with the mRNA vaccine, and justify your reasoning.

Solve

Production of recombinant drugs: the case of insulin

From pig pancreases to recombinant bacteria

Until the FDA approved the first recombinant human insulin in 1982, the insulin used to treat diabetes was extracted from the pancreases of pigs and cattle: an expensive process, limited by the number of animals and carrying a risk of allergic reactions because the protein is not exactly human.

Today the human insulin gene, introduced into bacteria (or yeast) by molecular cloning, makes it possible to produce identical human insulin in bioreactors, in practically unlimited quantities and at a much lower cost per unit.

Which fundamental molecular property makes it possible for a bacterium to decode a human gene and make exactly the same insulin?

Show answer

The universality of the genetic code: the same mRNA codons code for the same amino acids in both bacteria and human cells.

Practice★★☆

The diagram summarizes, in four numbered stages, how human insulin is produced today. Explain what happens in each stage and account for the advantages this method…

The diagram summarizes, in four numbered stages, how human insulin is produced today. Explain what happens in each stage and account for the advantages this method offers compared to extracting insulin from pig or cattle pancreases.

Solve

GM crops

Every growing season, the corn borer caterpillar tunnels into corn stalks and ruins entire harvests unless tons of chemical insecticides are sprayed repeatedly, harming other insects and the soil. Is it possible to give the plant itself permanent biological defences by incorporating the defensive abilities of another living thing?

Practice★★☆

Bt corn incorporates a gene from the bacterium Bacillus thuringiensis that produces an insecticidal toxin. State two advantages of this GM crop and two risks or…

Bt corn incorporates a gene from the bacterium Bacillus thuringiensis that produces an insecticidal toxin. State two advantages of this GM crop and two risks or criticisms raised against it.

Solve

Bioremediation and water treatment

Two scales of the same idea

In a wastewater treatment plant, bacteria and other microorganisms break down the dissolved organic matter before the water is returned to the river, in tanks where the available oxygen is controlled to favour their activity.

In a hydrocarbon spill at sea, bacteria that can use oil as a source of carbon and energy are employed, breaking it down into simpler compounds. To work effectively they need oxygen, nutrients (nitrogen, phosphorus) and a suitable temperature; this is why fertilizers are sometimes added to the affected area to speed up the process.

Why does adding fertilizers containing nitrogen and phosphorus speed up the biological removal of an oil slick on the coast?

Show answer

Because hydrocarbons provide plenty of carbon but lack other essential bioelements; the fertilizer removes that limiting factor and allows the degrading bacteria to multiply.

Energy flows through, matter is recycled

In a coastal ecosystem, producers fix 10,000 kJ of energy per square meter per year. How much reaches the top consumers, and what happens to the matter?

Only about 10% of the energy passes to the next trophic level; the rest is lost mainly as heat from cellular respiration. Producers: 10,000 kJ. Primary consumers: 1,000 kJ. Secondary consumers: 100 kJ. Tertiary consumers: 10 kJ.

Energy enters as sunlight and leaves as heat, so it flows in one direction and must be supplied continuously. Chemical elements such as carbon, nitrogen and phosphorus are not lost: decomposers (bacteria and fungi) break down dead organisms and wastes and return these elements to the soil and water, where producers take them up again.

When people disturb this cycle, there are consequences. Fertilizer runoff from the Midwest that reaches the Gulf of Mexico causes algal blooms. When the algae die, decomposers use up the oxygen and create a "dead zone." Bioremediation uses the same decomposer metabolism, but on purpose.

Practice★★★

A biomedical consortium proposes applying CRISPR/Cas9 technology to viable human zygotes to correct an autosomal dominant mutation responsible for Huntington's disease…

A biomedical consortium proposes applying CRISPR/Cas9 technology to viable human zygotes to correct an autosomal dominant mutation responsible for Huntington's disease prior to embryo implantation.

a) Describe the two essential molecular components of the CRISPR/Cas9 system that must be microinjected into the zygote, and explain how they act in coordination to recognize and accurately cut the target DNA sequence.
b) In experimental trials, two major technical problems are detected: off-target cuts and genetic mosaicism (some cells of the blastocyst carry the edit while others retain the original unedited allele). Justify at the molecular and embryonic developmental level why mosaicism arises, and predict what health consequences it would entail for the resulting individual.
c) Distinguish precisely between somatic gene editing and germline gene editing. Scientifically justify why germline modification in embryos faces far greater ethical and legal opposition in the international community than gene therapy applied to the somatic cells of an adult patient.

Solve

Forensic DNA fingerprinting and the ethical debate on embryo editing

At the scene of a burglary in Houston, investigators who upload profiles to the national CODIS database find that, all that remains is a tiny speck of dried saliva on the rim of a glass, invisible to the naked eye and containing barely a few picograms of biological material. How can that almost undetectable amount of DNA become irrefutable evidence that singles out one suspect among billions of people?

From a minimal sample to an identification

From a drop of blood, a hair with its root or traces of saliva, PCR amplifies several specific STRs of the genome until there is enough DNA to analyze them.

The number of repeats at each STR analyzed varies so much between people that the combination of several of them is, in practice, unique to each individual (except identical twins). The amplified fragments are separated by size using gel (or capillary) electrophoresis: DNA is negatively charged, so it moves toward the positive pole, and shorter fragments travel farther. Comparing that pattern with that of a suspect, a victim or an alleged parent allows forensic identifications and paternity tests with extremely high reliability. The FBI's CODIS database in the U.S. compares profiles at 20 core STR loci.

Why can't two monozygotic twins be told apart by a standard forensic analysis of STR repeats?

Show answer

Because they come from the fertilization of a single zygote that later divided, so they share exactly the same DNA sequence and the same number of repeats at all loci.

Practice★★★

In a forensic investigation, a minute bloodstain is found on a victim's clothing (Sample). The police interrogate two suspects (S1 and S2). In addition, in a parallel…

In a forensic investigation, a minute bloodstain is found on a victim's clothing (Sample). The police interrogate two suspects (S1 and S2). In addition, in a parallel civil case, a woman (M) claims paternity of her child (C) from S1. The lab analyzes three short tandem repeat (STR) loci:

Individual / Sample	Locus 1	Locus 2	Locus 3
Forensic sample	12, 14	9, 11	15, 15
Suspect 1 (S1)	12, 14	9, 11	15, 15
Suspect 2 (S2)	12, 12	9, 11	15, 17
Mother (M)	10, 14	7, 9	14, 15
Child (C)	12, 14	9, 11	15, 16

a) Interpret the forensic results: can either of the suspects be ruled out? Does this match allow one to state with absolute $100\ \%$ certainty that the non-excluded suspect is the perpetrator of the crime? Justify your answer considering statistical probability and the need to analyze more loci.
b) Analyze the paternity test data: deduce the obligate paternal alleles that the child (C) must have received by comparing their genotype with that of their mother (M). Based on these, determine with reasoned arguments whether S1 can be the biological father of C.
c) Explain at the chromosomal and molecular level what it means for an individual to exhibit two different values at an STR locus versus only one (as in Locus 3 of S1), and what analytical technique allows these alleles to be distinguished after PCR amplification.

Solve

Solving a case that combines biotechnology and immunity

To solve a new situation, identify the molecular modification, the cell involved, its function and one limitation. An application is not explained just by naming a technique.

Designing a reasoned response

T lymphocytes are modified so that they express a receptor that recognizes a molecule on certain tumor cells. How are genetic engineering and immunity related?

A DNA construct carrying the receptor gene can be introduced into the lymphocytes. Restriction enzymes and ligase make it possible to assemble recombinant DNA; targeted editing with CRISPR uses a guide RNA and a nuclease to act on a specific sequence. These are different operations.

Cytotoxic T lymphocytes destroy target cells through cellular mechanisms. If the target also appears in healthy tissues, these may be damaged: specificity, efficacy and safety must be evaluated, rather than claiming that recognizing a tumor antigen removes all risk.

A trial of efficacy needs a suitable comparison, evaluation criteria and control of variables. A difference observed without a comparison does not by itself demonstrate the effect of the treatment.

Practice★☆☆

What is gene therapy? Give an example of a disease targeted by this strategy and explain its underlying principle.

Solve

Let's practice! 2 medium exercises in this section

★★☆ Bt corn incorporates a gene from the bacterium Bacillus thuringiensis that produces an…★★☆ The diagram summarizes, in four numbered stages, how human insulin is produced today.…

Summary

Gene therapy introduces or corrects genes in a patient's cells; mRNA vaccines temporarily induce the synthesis of an antigen to activate immunity; they do not modify the genome and are not gene therapy.
Recombinant proteins (such as human insulin) are produced by introducing the gene of interest into bacteria or yeast, which make it on a large scale.
GM crops (such as Bt corn) incorporate genes from another species to resist pests or herbicides, with advantages in productivity and also criticisms that are worth knowing about.
Bioremediation uses microorganisms to break down pollutants, from water treatment plants to hydrocarbon spills.
DNA fingerprinting compares STRs in DNA to identify people with great reliability; the editing of human embryos remains the subject of an intense ethical debate.

Let's practice! 2 medium exercises in this section

★★☆ Bt corn incorporates a gene from the bacterium Bacillus thuringiensis that produces an…★★☆ The diagram summarizes, in four numbered stages, how human insulin is produced today.…

Let's practice! 3 hard exercises in this section

★★★ During the development of an mRNA vaccine against an enveloped pathogenic virus, the…★★★ In a forensic investigation, a minute bloodstain is found on a victim's clothing…