Friday, November 22nd Lecture Notes

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

Humans have used selective breeding to alter the genotypes and phenotypes of domesticated animal species for over 10,000 years

In the 1980s, scientists began to develop techniques for introducing specific cloned genes into the chromosomal DNA of both animals and plants

• Such genetically modified organisms (GMOs) became widely used in scientific research

  • But GMOs are also developed for use in agriculture and industry

    • Genetically improved crops and livestock

    • Production and sale of pharmaceutical drugs synthesized from cloned genes

GMOs

• Agriculture

  • AquaAdvantage salmon

  • Golden rice

  • Rainbow papaya

• Gene therapy

  • Severe combined immunodeficiency

  • Hereditary blindness

  • Cancer

  • Epidermolysis Bullosa

In the 1990s, the bicotechnology company AquaBountry gained FDA approval to generate a breed of genetically modified Atlantia salmon

• Salmon primarily grow during summer months (regardless of water temperature) because that is the only time that their pituitary glands produce growth hormones

  • It takes 3 years for an Atlantic salmon hatchling to grow to its full adult size

  • To circumvent this limitation, AquaBountry isolated a cDNA encoding salmon growth hormone, and cloned it into a plasmid with a constitutive promoter obtained from another fish, the ocean pout

• The pout lives in cold north Atlantic waters, and produces an antifreeze protein to keep its blood from freezing

  • The antifreeze gene has a strong, constitutive promoter transcribed year around

  • The fusion gene was injected into fertilized salmon eggs, and some embryos inserted the transgene into their chromosomal DNA

• The experiment was a success; continuous expression of growth hormone allowed the genetically modified salmon to reach adult size in half the time

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)

• After nearly 20 years of study, the FDA concluded that the AquaAdvantage salmon is safe for human consumption, equivalent to wild type salmon in nutrition, and approved its sale in 2015

• Eggs are pressure-treated to induce triploid, to make the progeny sterile

  • This prevents escape and intermating with wild population

• Sale of this salmon is widespread in Canada and in 2019 farming began in the US

Genetically Modified Livestock and Crops

• Why are some people concerned?

  • Environmental impact

    • The FDA restricts how and where GMOs are raised

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

  • Gene transfer to other strains/species

    • Transgenes could be transferred to other strains of the same species by mating or to other species by means of viral or transposon vectors

    • Example: genes that provide pesticide resistance in crops would be a major nuisance if introduced into weed species

  • Unpredictable 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, phytonutrients, 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

Some GMOs provide novel solutions to hunger and nutrition-related diseases in underdeveloped countries

• WHO estimated 250,000-500,000 vitamin-A-deficient children become blind every year, half of them dying within 12 months of losing their sight

Golden Rice

• Vitamin A deficiencies are a particular problem in rice-based cultures

• Wild-type rice contains neither vitamin A nor its precursor B-carotene

• Golden rice and its successor Golden rice 2 were genetically modified to synthesize and deposit B-carotene within the edible part of the rice grain

• A 100 g serving of Golden rice 2 contains 80% of the daily vitamin A requirement for an 8 year old child

  • Why does rice not produce vitamin A?

    • Most of the rice is endosperm, a starchy tissue that would serve as a food source for the rice embryo if the seed were to germinate

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

• To generate Golden rice, scientists:

  • 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

Critical Point: 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

Golden Rice

• Not sold for profit, and its seed is provided free of charge to subsistence farmers in developing countries by the International Rice Research Institute

• Opponents of GMOs have tried to block these efforts, including the obstruction of fields where Golden rice is being grown

• To date, Golden rice has been approved for growth and sale in the Philippines and Bangladesh

Rainbow Papaya

• Saved the Hawaiian papaya industry from being wiped out by the ringspot virus

• Virus

  • Lethal to papaya plants

  • Spread quickly by aphids

  • Cannot be controlled by culling diseased trees

• Resistance induced via a transgenic construct

  • Coat protein of the ringspot virus expressed in the papaya plant

  • Confers resistance to virulent ringspot virus

  • The rainbow papaya is an F1 hybrid of a transgenic papaya (Sunset) and non-transgenic papaya (Kapoho)

  • Presence of virus coat protein not harmful to humans

    • Broken down in stomach

    • Extensive studies showing no allergic reactions

    • Most non-Rainbow papaya has viral content (harvested from infected trees)

The FDA’s interpretation of genetically modified is that foreign DNA has been introduced into the organism’s genome

• This can be nothing more than DNA sequence from a plasmid used in cloning the gene

• But 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

Gene Therapy

• The term gene therapy refers to the genetic modification of humans or human cells in order to ameliorate disease conditions

  • 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

  • Thus, in contrast to many non-human GMOs, currently approved gene therapy procedures are restricted to somatic tissues, and do not produce heritable changes in the genome

The most widely used form of gene therapy is to introduce a functional wild-type copy of a gene into a patient whose gene carries a loss-function mutation

The first successful human gene therapy was performed in 1990

• 4-year-old Ashanti DeSilva suffered from Severe Combined Immunodeficiency (SCID), a condition in which the immune system fails due to a lack of functioning lymphocytes

• SCID patients are at constant risk of fatal infection by viruses and bacteria that are a mere annoyance to the populace at large

Several genetic abnormalities can lead to SCID

• Ashanti was homozygous for loss-of-function alleles of a gene that encodes the enzyme adenine deaminase (ADA)

• While most cells in the human body are affected by an ADA deficiency, without this enzyme the T lymphocytes of the immune system are severely disabled and have a severely shortened life span

Prior to gene therapy, the inly successful treatment for ADA-SCID was bone marrow transplantation

• Bone marrow contains hematopoetic stem cells capable of generating all blood cell types, including lymphocytes

• But bone marrow transplants are risky

  • Immune reactions between host and transplant are common, and can be fatal

To treat Ashanti, physicians

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

• Genetically modified these 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

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

To integrate the wild-type ADA gene into the chromosomal DNA of Ashanti’s T cells, doctors used a genetically engineered retrovirus as the vector

• To ensure that the virus itself did not serve as a disease agent several potentially deleterious genes were deleted from it

To be safe and effective, this sort of gene replacement therapy must address four important considerations:

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

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

    • In Ashanti’s case gene therapy was not a cure, but it alleviated her disease to such a degree that she is now fine with only oral medications

    • Dozens of subsequent patients have been effectively cured by using hematopoietic stem cells, not T cells

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

  • Blood cells are ideal for gene therapy

  • They can be removed from the body, genetically modified in vitro, and returned to the bloodstream

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

  • For Ashanti, the strong viral promoters drove expression in the modified cells

• Will transgene insertion or expression have deleterious side effects?

  • In Ashanti’s case, there were no significant side effects

  • However, there have been a few cases in which patients became ill or died from gene therapy procedures

• In 2000, a group of patients were treated in for a different type of SCID caused by a genetic deficiency for the yc subunit of a cytokine receptor

  • 3 of 20 patients in this study developed leukemia, and 1 died of complications

• Insertion of viral vectors into the host genome is essentially random, and has a potential pitfall: insertional mutagenesis

In at least 2 of these patients, the retrovirus inserted in or near the gene LMO2, which controls blood cell development

• Due to this insertion site, a strong enhancer sequence in the viral DNA apparently caused excessive transcription of LMO2

• Over-expression of LMO2 led to unregulated overgrowth of the transgenic lymphocytes—resulting in leukemia

The high rate of complications observed in this one clinical study is not typical of most human gene therapy

• Subsequent work has shown that the specific retrovirus used in this study has an unusually high probability of inserting into gene regulatory regions, possibly accounting for the observed effects

Further Developments in Gene Therapy

• Unlinke blood, most tissues cannot be removed, genetically modified, and reintroduced into the body

• Thus, to treat genetic diseases in other tissues it is often necessary to inject the patient with the transgene in a viral vector that can infect cells throughout the body

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)

• Patients were homozygous deficient for the enzyme RPE65A, which is required od to synthesize visual pigments in this retina

• A viral vector carrying the wild-type RPE65A gene was injected into the eye, infecting and genetically modifying the patients’ cells

• The RPE65A transgene was under the control of its own wild-type promoter, and was therefore only expressed in the photoreceptor cells where this protein is normally made

Critical Point

• 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.

In addition to replacing deficient genes, gene therapy has been successfully used to modify the T lymphocytes of cancer patients so that they will destroy cancer cells more effectively

• As with othe blood cell therapies, the T lympohocytes are modified in vitro, then reintroduced into the bloodstream

• But in this case, the genetic modification can be personalized to specifically target that patient’s malignancy

Epidermolysis Bullosa

• Skin blistering disease

• Mutations in Laminin B3 (component of extracellular matrix)

• Cell layers do not adhere

• Successful trials published in 2006, work halted due to government red tape

Strategy

• 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

Other recent advances in gene therapy

• Sickle cell anemia

• Angelman syndrome

  • Use CRISPR technology

Mini Study Guide

• Most genetically modified organisms are used in laboratory research

• Name 3 examples of genetically modified crops/livestock that benefit the food supplly/human health and how theose mmodifications were made

• Name three medical conditions that can be corrected with gen e therapy and how they are corrected

• Why did one gene therapy trial go awry?

• Know why choice of vector and promoter are critical to gene therapy