Lecture 35: Gene Therapy and Genetically Modified Organisms

True or False: With the advent of modern molecular biology, it became feasible to introduce genes into an organism’s genome with the intention of manipulating its phenotype.

true

What has been used to alter the genotypes and phenotypes of domesticated animals for over 10,000 years?

selective breeding

What is the use of GMOs in agriculture?

genetically improved crops and livestock

What is the use of GMOs in industry?

production and sale of pharmaceutical drugs synthesized from cloned genes

What are three agriculture GMO examples?

• AquaAdvantage salmon

• Golden rice

• Rainbow papaya

What are the four gene therapy GMO examples?

• severe combined immunodeficiency

• hereditary blindness

• cancer

• epidermolysis

True or False: In the 1990s, the biotechnology company AquaBountry gained FDA approval to generate a breed of genetically modified Atlantia salmon.

true

Why do salmon only primarily grow during summer months?

summer is the only time that their pituitary glands produce growth hormones

How long does it take for an Atlantic salmon hatchling to grow to its full adult size?

3 years

How did AquaBounty generate larger salmon faster?

AquaBounty isolated a cDNA encoding salmon growth hormone, and cloned it into a plasmid with a constitutive promoter

Where did the constitutive promoter come from?

the ocean pout

Where does the ocean pout live?

in cold north Atlantic water

What does the ocean pout produce to keep its blood from freezing?

an antifreeze protein

What is so important about the antifreeze gene?

it has a strong, constitutive promoter that’s transcribes all year round

True or False: The fusion gene was injected into fertilized salmon eggs, and some embryos inserted the transgene into their chromosomal DNA.

true

Was the AquaBounty experiment a success?

yes, the salmon were modified to continuously express growth hormone

True or False: The development and marketing of genetically modified plants and animals is under governmental regulation, and in the US is overseen by the Food and Drug Administration (FDA).

true

Did the FDA conclude that the AquaAdvantage salmon is safe for human consumption?

yes, salmon is safe for human consumption and is equivalent to wild type salmon

What measure does AquaAdvantage take to ensure that modified salmon don’t breed with wild-type salmon?

the modified salmon eggs are pressure-treated to induce triploid—making the progeny sterile

What are some environmental impacts that people are concerned about?

• how and where GMOs are raised

• individuals might escape into the wild and potentially harm natural ecosystems

Why are people concerned about gene transfer to other strains/species?

transgenes can be transferred to other strains of the same species by mating or to other species by means of viral or transposon vectors

Why are some people concerned about the unpreditable health risks?

• Science is still learning new things about human nutrition, and GMO foods could differ from the original strain in their relative content of hormones, growth factors, nutrients, etc

• The nutritional content and dietary effects of GMOs approved for sale have been extensively tested for safety and similarity to unmodified organisms

• Some consumers fear that there could be consequences

What is an example of how GMOs have helped in underdeveloped countries?

the production of golden rice to feed vitamin-A-deficient children

What is a problem in rice-based cultures?

vitamin A deficiencies

True or False: Wild-type rice contains neither vitamin A nor its precursor B-carotene.

true

Golden rice and its successor Golden rice 2 were genetically modified to synthesize and deposit what within the edible part of the rice grain?

B-carotene

A 100 g serving of Golden rice 2 contains what percentage of the daily vitamin A requirement for an 8 year old child?

80%

What is most of the rice?

endosperm

What is endosperm?

a starchy tissue that would serve as a food source the rice embryo if the seed were to germinate

Why does rice not produce vitamin A?

in wild-type rice, the endosperm does not express two enzymes that are essential for the biosynthesis of B-carotene

What did scientists do to generate golden rice?

• Obtained cloned genes for these two biosynthesis enzymes (1 from daffodil and 1 from bacterium)

• Fused each of these genes with a promoter sequence known to drive transcription in the endosperm

• Introduced these fused constructs into the rice genome as transgenes

Note the critical point of GMOs:

Once an experimenter has identified a promoter that yields the appropriate expression pattern, any cloned gene product can be expressed precisely when and where it is needed

Where has Golden rice been approved for growth and sale?

the Philippines and Bagladesh

What virus was affecting the Hawaiian papaya?

the ringspot virus

What papaya was developed to save the Hawaiian papaya?

the rainbow papaya

What are some characteristics of the ring spot virus?

• Lethal to papaya plants

• Spread quickly by aphids

• Cannot be controlled by cutting diseased trees

How did scientists introduce resistance in the papaya?

the coat protein of the ringspot virus was expressed in the papaya plant—conferring resistance to the virulent ringspot virus

What is the rainbow papaya an F1 hybrid of?

a transgenic papaya (Sunset) and non-transgenic papaya (Kapoho)

Is the virus coat protein harmful to humans?

no, the virus coat protein is broken down in the stomach and does not show any allergic reactions

What is the FDA’s interpretation of genetically modified?

foreign DNA that has been introduced into the organism’s genome

True or False: The FDA has decided it has no jurisdiction to regulate crops or live stocks whose genomes have been modified by gene editing techniques that do not introduce foreign DNA, and dozens of gene-edited food plants are currently being developed for sale.

true

What does the term gene therapy mean?

the genetic modification of humans or human cells in order to ameliorate disease conditions

True or False: Due to ethical concerns, most developed countries have passed laws that directly or indirectly prevent genetic modification of the human germ line or of developing embryos; currently approved gene therapy procedures are restricted to somatic tissues, and do not produce heritable changes in the genome.

true

What is the most widely used form of gene therapy?

to introduce a functional wild-type copy of a gene into a patient whose gene carries a loss-function mutation

Who was the first successful human gene therapy patient?

Ashanti DeSilva

What did Ashanti DeSilva suffer from?

severe combined immunodeficiency (SCID)

What is SCID?

a condition in which the immune system fails due to a lack of functioning lympohocytes

What is Ashanti’s genetic abnormality that lead to SCID?

Ashanti was homozygous for loss-of-function alleles for adenine deaminase (ADA)

Does ADA affect all cells in the human body?

no, they only affect the immune system

What happens to the immune system if a person has an ADA deficiency?

without ADA, T lymphocytes are severely disabled and have a severely shortened life span

Prior to gene therapy, what was the only successful treatment for ADA-SCID?

bone marrow transplantation

In regard to ADA-SCID, what does bone marrow contain?

hematopoietic stem cells that are capable of generating all blood cell types

What is the problem with bone marrow transplants?

immune reactions between host and transplant are common and can be fatal

How did physicians treat Ashanti?

• withdrew samples of her blood and isolated her ADA-deficient T lymphocytes

• genetically modified T cells by integrating a cloned copy of the wild-type ADA gene into their genome

• reintroduced the genetically modified T cells into her bloodstream

True or False: Following nine injections of transgenic lymphocytes over a period of 2 years, Ashanti’s blood ADA level rose from zero to the normal range.

true

How did doctors integrate the wild-type ADA gene into the chromosomal DNA of Ashanti’s T cells?

a genetically engineered retrovirus was used as a vector

True or False: How did doctors ensure that the retrovirus wouldn’t serve as a disease agent?

potentially deleterious genes were deleted from it

What four considerations must gene replacement therapy address for safety reasons?

• Can the disease condition be improved by simply adding a missing gene product?

• Can the transgene be effectively delivered to and integrated into the DNA of the cells or tissues where it is needed?

• How will expression of the transgene be regulated in the recipient cells?

• Will transgene insertion or expression have deleterious side effects?

Can the disease condition be improved by simply adding a missing gene product?

For Ashanti and other ADA-SCID patients, introducing a wild-type ADA gene has proven sufficient to restore immune system function

True or False: Dozens of subsequent patients have been effectively cured by using hematopoietic stem cells, not T cells.

true

What other genetic abnormality can SCID be caused by?

a genetic deficiency for the yc subunit of a cytokine receptor

True or False: Insertion of viral vectors into the host genome is essentially random and can cause insertional mutagenesis.

true

What happened to 2 out of 4 of the yc-SCID patients?

the retrovirus was inserted in or near the LMO2 gene

What does the LMO2 gene control?

blood cell development

What did insertion of the retrovirus in or near the LMO2 gene cause?

the strong enhancer sequence in the viral DNA caused excessive transcription of LMO2

What does over-expression of LMO2 cause?

unregulated overgrowth of the transgenic lymphocytes—resulting in leukemia

True or False: Research has shown that the specific retrovirus used in the yc-SCID clinical trial has an unusually high probability of inserting into gene regulatory regions, possibly accounting for the observed effects.

true

How are genetic diseases in tissues beside blood treated?

patients are injected with a transgene in a viral vector that will, in turn, infect cells throughout the entire body

True or False: In 2008, Bainbridge reported success using gene therapy to partially rescue vision in patients suffering from a type of hereditary blindness (Leber’s Congenital Amaurosis).

true

What are LCA patients homozygous deficient for?

the enzyme RPE65A

What is RPE65A required for?

to synthesize visual pigments in the retina

True or False: A viral vector carrying the wild-type RPE65A gene was injected into the eye, infecting and genetically modifying the patients’ cells.

true

What was the RPE65A transgene under the control of?

its own wild-type promoter

Why is it important that RPE65A was under the control of its own wild-type promoter?

RPE65A was only expressed in photoreceptor cells where the protein was normally made

Note the critical point of gene therapy:

As with the development of GMOs, once a clinical researcher has identified a promoter that yields expression in the appropriate tissues, that promoter can in theory be used to target gene therapy to only the cells that need it.

True or False: Gene therapy has been successfully used to modify the T lymphocytes of cancer patients so that they will destroy cancer cells more effectively.

true

What is epidermolysis bullosa?

a skin blistering disease caused by mutations of Laminin B3

What do mutations in Laminin B3 cause?

causes the cell layers to not adhere together

What is the strategy for treating epidermolysis bullosa?

• Harvest healthiest bit of skin can find on patient

• Introduce functional copy of LAMB3 into patient’s own skills cells; some will be stem cells

• Grown skin from these cells in culture

• Graft stem cell-derived skin to severely blistered area

• Corrected stem cells will take over the replenishment of the skin

What are some other recent advances in gene therapy?

• sickle cell anemia

• angelman syndrome

• CRISPR technology