Transgenics 3

Question 1

What is β-carotene = 4

Answer 1

1 *precursor of vitamin A

2 *found in yellow vegetables & greens

3 *vitamin A deficiency 400 million people

4 *skin disorders, blindness

Question 2

β-carotene pathway
transformed into rice

Answer 2

*4 enzymes: 2 from daffodil,
2 from a bacterium

Daily requirement of vitamin
A in 300 g cooked rice

Question 3

Golden Rice: follow up projects address
micronutrient malnutrition

Answer 3

1 * Iron deficiency (rice contains phytate, which binds iron - absorption in
gut inhibited)

2 * Rice also transformed with genes encoding:
- fungal enzyme that breaks down phytate
- iron storage protein (ferritin) from French bean
- metallothionen-like protein from basmati rice to facilitate
absorption in gut

3 * 7 introduced genes - 6 from 4 unrelated species, 1 from another
rice strain

4 * Others: Zinc, protein, vitamin E

Question 4

Herbicide Resistant Crop Plants
1. weed destroy
2. Glyphospahte
3. EPSP synthase

Answer 4

1. Weeds destroy ~10% of crops worldwide.
   >100 different herbicides used:
   * >10 billion dollars annually
   * environmental damage & human health
2. Glyphosate
   * non-toxic to humans
   * broken down by soil bacteria
   * effective at low concentrations
3. EPSP synthase – target of glyphosphate
   * amino acid synthesis in bacteria & plants
   * inhibited by the herbicide glyphosate

**Crop plants transformed with gene encoding
glyphosate-resistant EPSP* synthase

*EPSP = 5-enolpyruvylshikimate-3-phosphate

Question 5

Edible Vaccines and Plants?

Answer 5

Edible parts of a plant that has been genetically modified to produce specific
components of a particular pathogen

Question 6

Edible Vaccines - Subunit vaccines?

Answer 6

Subunit vaccines:
* fragment of a pathogen – eg surface proteins from virus or bacterium

• act as antigens & stimulate immune system to make antibodies against the
  pathogen

Question 7

Problem of Edible Vaccines vs SOLUTION?

Answer 7

PROBLEM: need refrigeration and sterile conditions

SOLUTION: transform plant with genes encoding surface proteins

Question 8

Edible Vaccines - TOBACCO?

Answer 8

• Tobacco plant transformed with hepatitis B subunit - subunit detected in leaf tissue with commercial anti-hepB antibody & colour reaction
• Moved to edible plants, eg potato, lettuce

Question 9

Edible Vaccines - Edible vaccines in clinical trials

Answer 9

1 *Cholera: leading cause of death in infants & children in developing countries

*E. coli enterotoxin - similar in structure to cholera enterotoxin
*inserted into potato - immunity conferred when raw potatoes eaten

2.* Rabies - transgenic spinach

Question 10

Edible Vaccines -* more research needed: QUESTIONS =4

Answer 10

1 - necessary antigen conc?

2. • possible overstimulation of immune system after long
     term consumption

3 - how to overcome oral
tolerance acquisition

5 - convenient, but costs are
complex

Question 11

New interest of edible vaccines?

Answer 11

New interest – ebola (antibodies that recognize Ebola virus surface
glycoproteins isolated from individuals who survived Ebola infections)

Question 12

Edible vaccines: how to make?

Answer 12

1. Gene from a human pathogen is inserted into bacterium that infects plants
2. Bacteria infect potato leaf segments
3. leaf segments sprout into whole plants carrying gene from human pathogen
4. eating raw potato triggers immune response to pathogen

Question 13

WHAT IS Gene therapy - Treatment of human
diseases

WHAT IS ITS GOAL?

Answer 13

1 * “Any procedure intended to treat or alleviate disease by genetically modifying the cells of a patient” Strachan and Read

2. The general goal of gene therapy is to attack the genetic basis of disease at its source: to “ cure ” or correct an abnormal condition caused by a mutant allele by
   introducing wild-type allele into cells

Question 14

A boy is born with a disease
that makes his immune
system ineffective.

Answer 14

1. Recessive genetic disorder
   called severe combined
   immunodeficieny disorder
   (SCID)
2. Caused by mutation in the
   gene coding for the blood enzyme adenosine deaminase (ADA) (can be Xlinked also - IL-2 deficiency).
3. Loss of protein = loss of precursor cells for various cells of the immune
   system.
4. No ability to fight infections - live in completely sterile environment
5. No conventional therapy available - bone marrow transplant would not work - graft vs host against precursor cell

Question 15

Techniques have been developed - different kind of
transplantation

Answer 15

1. A normal gene is “transplanted” into cells of
   the body, thereby permitting their function and survival

To understand this approach consider:
- An example, how gene therapy corrected a growth-hormone deficiency in mice.

• Mice with recessive mutation little (lit) are dwarves because they lack a protein (growth-hormone-releasing hormone receptor, or GHRHR)
• Necessary to induce the pituitary to secrete mouse growth hormone into the circulatory system.

Question 16

Construct 5kb linear DNA fragment with coding sequence for rat growth
hormone fused to regulatory sequences & mouse metallothionein gene.

Inject 5000 copies of transgene into homozygous lit/lit eggs

Leads to expression of adjacent genes in the presence of heavy metals

Answer 16

Construct 5kb linear DNA fragment with coding sequence for rat growth
hormone fused to regulatory sequences & mouse metallothionein gene.

Inject 5000 copies of transgene into homozygous lit/lit eggs

Leads to expression of adjacent genes in the presence of heavy metals

Eggs were implanted into uteri of surrogate mother mice, baby mice born and raised.

Approx 1% babies turned out to be transgenic, increased size when heavy metals were administered during development

• A representative transgenic mouse was then crossed with a homozygous lit/lit female.
• Mice two to three times weight of their lit/lit relatives produced in subsequent generations. Larger mice heterozygote
• Rat growth hormone transgene dominant allele…..

Question 17

The transgenic mice:
Mice are siblings but mouse on left was derived from egg transformed by
injection with “
new
” gene. (44g vs untreated sibling 29g)

Answer 17

The transgenic mice:
Mice are siblings but mouse on left was derived from egg transformed by
injection with “
new
” gene. (44g vs untreated sibling 29g)

Question 18

Mice transgenic example raises important points about gene therapy.

What is it and explain

Answer 18

The genetic defect occurs in GHRHR, the gene encoding a
regulator of mouse growth-hormone production

However, the gene therapy did not attempt to correct the original defect

Rather, gene therapy bypasses the need for GHRHR by producing growth hormone by another route

Two basic types of gene therapy can be applied to humans
– germline and somatic

Question 19

Understanding GERMLINE THERAPY

Answer 19

More ambitious - introduce transgene into germ line as well as into the somatic cells.

Would achieve a cure for the person treated but his or her
children would also carry the therapeutic transgene

“Cure” of mouse lit recessive defect example of germ-line
therapy

Question 20

What does GERMLINE GENE THERAPY DO?

Answer 20

produces permanent,
transmissible modification. Edit embryo, then IVF.

Question 21

Germline gene therapy produces permanent,
transmissible modification. Edit embryo, then IVF…

ARGUMENTS AGAINST…5

Answer 21

1 o Unnecessary –
preimplantation genetic
diagnosis can screen for
embryos with genetic defect

2 o Risky – unpredictable and permanent

3 o Equitable access

4 o Designer babies/regulation

5 o Embryo can’t consent for
genetic material to be
modified

Question 22

Germline gene therapy produces permanent,
transmissible modification. Edit embryo, then IVF

Arguments for…3

Answer 22

1 o For people homozygous for a dominant disorder (eg
Huntingtons), no healthy
embryos. Some couples
don’t produce enough
embryos to screen.

2 o Potentially ‘eradicate
human disease’

3 o We can do it, therefore we
should offer it

Question 23

Somatic therapy ..transgene …transgenic clones

Answer 23

Attempts to correct disease phenotype by treating some somatic cells in affected person

At present not possible to render entire body transgenic, so method
addresses diseases caused by genes expressed predominantly in one
tissue

Question 24

Somatic gene therapy on diseases… THE METHOD

Answer 24

1. In such diseases, likely that not all the cells of that tissue need to become
   transgenic; a portion of cells carrying transgene will relieve symptoms
2. Method: removing some cells from patient with defective genotype,
   making cells transgenic by introducing copies of cloned wild-type gene.
3. Transgenic cells reintroduced into patient’s body, provide normal gene
   function

Question 25

What may SOMATIC CELL GENE THERAPY INVOLVE?

Answer 25

1 *Gene supplementation (augmentation)

2 *Gene replacement

3 *Targeted inhibition of gene expression

4 *Targeted killing of specific cells

Question 26

Somatic cell gene therapy may involve..Problems of safety and efficacy;

Answer 26

Problems of safety and efficacy;delivering the
right to the appropriate cells in such a way that
its expression pattern will correct the defect
without other unwanted effects.

Question 27

Delivery Systems
Retroviral System: Method 3 and problems 2

Answer 27

1 *Virus containing transgene of interest spliced into its genome, replacing
most of the viral genes, therefore virus unable to form progeny viruses ….”disarmed”

2 *Natural cycle of retrovirus maintained so integrates at some location into the host cell’s chromosomes, carrying the transgene along with it

3 *Cells are removed from person (BM, stem cells), the retroviral vector containing transgene added, transgenic cells reintroduced

***Problems:

1 * Integrating virus inserts in some unknown resident gene, inactivate it (insertional mutagenesis).

2 * Retrovirus infects only proliferating cells (eg blood cells), cannot be used to treat many disorders that effect tissues in which cells
rarely divide

Question 28

Delivery Systems
Adenoviral System: Method 3 and Advanatge 1

Answer 28

1 * Normally infects respiratory epithelia, injecting its genome into the
epithelial cells lining surface of lung

2 * Viral genome does not integrate, persists extra-chromosomally in
cells

3 * Eliminates problem of vector inactivating resident gene

• Advantage - attacks non dividing cells, most tissues susceptible

Question 29

Case study - SCID gene therapy

Answer 29

• First condition to be treated with gene therapy 1992:
  hematopoietic stem cells were transduced with a corrective
  transgene using a retrovirus
• Successful, but T cell leukemia in 3/20 patients (Insertional
  mutagenesis – DNA integrated into sensitive spot in genome, eg tumor suppressor gene -> tumour )
• Mamcarz et al, NEJM April 2019
• Functioning copy of IL2RG -> bone marrow cells. Different
  vector - lentiviral vector (modeled on HIV). Insulator sequence
  at the end of IL2RG to prevent it from turning on nearby genes
  (still untargeted)
• novel step - pretreatment with low dose chemotherapy. Kills off
  immune precursors - make space around bone marrow for gene-edited cells to get in and take hold

Question 30

Gene therapy - Conclusion

success, accepted/not, promise? Concerns?

Answer 30

Germline generally not accepted

• Many approaches for somatic GT being investigated (>500 trials underway – severe, single gene disorders)
• Some show great promise, but there are many details to
  be worked out with regard to methods of delivery and
  vectors. CRISPR/Cas9 – new promise (targeted)
• Concerns include:
• Insertional mutagenesis – oncogenic (SCID). CRISPR –
  more precise genome changes
• Immune reaction to viral proteins (has caused deaths)
  -Problems with stable integration – multiple treatments
• Multigene disorders tricky
• Some somatic therapies may modify germline too
• Cost