Genetic Technology Applied to Medicine — Cambridge AS & A Level Biology 9700 Revision Notes & Topical Questions

Section 1 of 17Recombinant human proteins

Recombinant proteins

• Recombinant proteins proteins made from recombinant DNA in microorganisms or animal cells in culture.
  • (bacteria, yeast)

They are used for research and treatment, e.g. diabetes, cancer, infectious diseases and haemophilia.

Most are made in eukaryotic cells, which carry out post-translational modification using the Golgi apparatus and enzymes.

Advantages:

• Cost-effective in large volumes with a reliable supply.
• Engineered to be identical to human proteins, or modified beneficially.
• Avoids moral, ethical or religious objections to cow or pig proteins.

Section 2 of 17Making recombinant human insulin

1

Modify bacterial plasmids to include the human insulin gene.

2

Cut plasmids open with restriction endonucleases and splice the gene in with DNA ligase.

3

Insert the recombinant plasmids into Escherichia coli by transformation.

4

Identify the transgenic bacteria with markers, isolate and purify them.

5

Grow them in fermenters under optimal conditions; they multiply by binary fission and express insulin, which is extracted and purified.

Section 3 of 17Other recombinant proteins

Other recombinant proteins

Insulin

• Insulin in 1982 the first recombinant human protein approved for diabetes;
  • identical to human insulin and can be modified to act faster or slower.

Factor VIII

• Factor VIII blood-clotting protein made by genetically modified hamster kidney and ovary cells in fermenters,
• giving less risk of transmitting infections such as HIV.

Adenosine deaminase (ADA)

• Adenosine deaminase (ADA) made by modified cabbage looper moth larvae (virus vector) to treat ADA deficiency, a cause of SCID, until gene therapy is possible.

Section 4 of 17Genetic screening

Genetic screening

• Genetic screening testing an embryo, foetus or adult to analyse the DNA and detect particular alleles.

DNA samples come from tissue samples, IVF embryos, chorionic villus sampling or amniocentesis.

• Chorionic villus sampling takes placental tissue at 10-13 weeks;
  • amniocentesis takes amniotic fluid at 15-16 weeks.

Genetic counsellors explain results and discuss chances of an affected child, termination, treatments, finances, siblings and ethics.

Examples:

• BRCA1 and BRCA2 faulty alleles raise breast and ovarian cancer risk;
  • preventive measures or earlier screening are possible.
• Huntington's disease is autosomal dominant with no cure;
  • screening aids planning and reproductive decisions (50% risk).
• Cystic fibrosis is autosomal recessive (faulty CFTR); screening shows whether couples are carriers.

Benefits: informed reproductive decisions and participation in research and clinical trials.

Section 5 of 17Gene therapy

Gene therapy

• Gene therapy altering a person's genetic material to treat or cure disease by replacing, inactivating or inserting a gene.

Most therapies are still in clinical trials because delivery systems and correct expression are hard to achieve.

Vectors

• Vectors viruses are most common; liposomes and naked DNA are non-viral options.
  • (retroviruses, lentiviruses, adeno-associated viruses)

Somatic gene therapy

• Somatic gene therapy targets body cells, is not inherited and its effects are often short-lived.
• Ex vivo — cells are removed, given the gene by a virus and returned by injection.
• In vivo — the gene is delivered to cells inside the body.

Germline therapy would be permanent and heritable; it is illegal in humans.

Section 6 of 17Gene therapy examples

Gene therapy examples

SCID

• SCID ex vivo therapy puts a normal ADA allele into T-lymphocytes, which are replaced over time, so repeat treatment is needed every 3 to 5 months.
• Early retroviral vectors inserted randomly and caused leukaemia, so lentiviruses and adeno-associated viruses are now used.

Leber congenital amaurosis

• Leber congenital amaurosis in vivo therapy injects adeno-associated viruses carrying a normal allele into the retina, improving eyesight.

Cystic fibrosis

• Cystic fibrosis normal CFTR delivered by liposomes or viruses to airway epithelium;
  • the effect is short-lived because the cells are replaced.

Section 7 of 17Social and ethical issues of screening

Preventive action

• Preventive action e.g. elective mastectomy when BRCA mutations are found.

Pre-implantation genetic diagnosis (PGD) selects embryos without faulty alleles, raising fears of designer babies.

Risk of miscarriage from sampling, and the decision whether to terminate a pregnancy.

Whether to know the risk of an incurable disease, and at what age to screen children.

Stigmatisation, discrimination by insurers or employers and confidentiality of data.

Section 8 of 17Social and ethical issues of gene therapy

1

Side effects that can be fatal, e.g. leukaemia in SCID trials.

2

Whether germline therapy should be allowed, since it is passed on and could be a cure or cause long-term effects.

3

Commercial viability for rare diseases, e.g. Glybera was withdrawn for too few patients.

4

High expense of repeated treatments limits access.

5

Who decides which genes may be altered, and enhancement or gene doping, e.g. erythropoietin.

Section 9 of 17What gene therapy is

Gene therapy

• Gene therapy altering a person's genetic material to treat or cure disease.

Possibilities: replace a faulty gene, inactivate a faulty gene or insert a new gene.

Experimental treatments are being tried for SCID, Leber congenital amaurosis (a rare blindness), β-thalassaemia and haemophilia B.

Most are still in clinical trials because delivery systems that transfer normal alleles and ensure correct expression are hard to find.

Delivery vectors

• Delivery vectors modified viruses, liposomes or plasmids.

Section 10 of 17Somatic and germline therapy

Somatic and germline therapy

Somatic gene therapy has two types:

• Ex vivo — cells are removed, the normal allele is added with a vector in the laboratory and the corrected cells are returned.
• In vivo — the vector carrying the allele is introduced directly into the patient's tissues.

Somatic therapy is not permanent because treated cells die and are replaced, so it may need repeating.

Germline therapy changes are permanent and inherited, which raises serious ethical concerns and is not permitted in many countries.

Section 11 of 17Example: SCID

1

SCID can be caused by inability to make adenosine deaminase (ADA), an enzyme key to the immune system.

2

Without it, children can die from common infections and are kept in plastic bubbles.

3

A functional ADA allele is inserted into bone marrow stem cells ex vivo and the cells returned.

4

This allows production of T lymphocytes and a working immune system.

Section 12 of 17Genetic screening

Genetic screening is used to analyse the DNA of people with a family history of a genetic disease and find whether they are at risk or carriers.

It is also used to test embryos before implantation, for prenatal diagnosis and for newborn screening.

Benefits

• Benefits early diagnosis, preventative treatment and informed family planning.

Issues

• Issues psychological stress, privacy, discrimination by insurers and employers, false results and the ethics of selecting embryos.

Section 13 of 17Why genetic screening is used

1

Screening can show whether someone carries an allele for an inherited disorder or has a higher disease risk.

2

This supports prevention, monitoring and informed reproductive decisions.

3

Pre-implantation genetic testing examines IVF embryos before implantation so an embryo without a disease-causing allele may be selected.

4

Genetic counselling helps families understand what a result does and does not mean.

Section 14 of 17Screening decisions and privacy

1

A person may want to know a risk to plan ahead, but learning of an untreatable condition can cause distress.

2

Screening children raises questions of future choice and consent.

3

Genetic information is sensitive.

• Misuse can cause stigma or discrimination.
• Confidentiality and control of access matter.

4

Prenatal sampling carries procedural risks that must be weighed.

Section 15 of 17Questions about gene therapy

Somatic therapy changes cells in the treated person; germline changes could pass to future generations.

Benefits must be weighed against side effects and uncertain long-term outcomes.

Cost and repeat treatment can limit access, especially for rare diseases or short-lived treated cells.

Other questions

• Other questions which traits should be altered, whether enhancement is acceptable and how to ensure fair access.

Section 16 of 17Key terms

Term	Meaning
Recombinant protein	Protein made from recombinant DNA in host cells.
Genetic screening	Testing DNA for particular alleles.
Somatic gene therapy	Gene therapy in body cells that is not inherited.
Germline gene therapy	Alteration of gametes or embryos, passed to future generations.
Ex vivo	Treatment of cells outside the body before returning them.
In vivo	Treatment carried out inside the body.
PGD	Pre-implantation genetic diagnosis of embryos made by IVF.
SCID	Severe combined immunodeficiency, from lack of adenosine deaminase.

Term	Meaning
Gene therapy	Treating disease by altering genetic material.
Ex vivo	Gene added to cells outside the body.
In vivo	Gene delivered to cells inside the body.
Germline therapy	Alteration of gametes or embryos, inherited.
SCID	Severe combined immunodeficiency.
Adenosine deaminase	Enzyme needed for immune system function.

Term	Meaning
Pre-implantation genetic testing	Screening of IVF embryos before implantation.
Genetic counselling	Explaining results and options to families.
Somatic therapy	Treatment of body cells, not inherited.
Germline change	Change passed to future generations.
Enhancement	Altering traits beyond treating disease.

Section 17 of 17Exam technique

1

Saying gene therapy is inherited — somatic therapy is not; only germline changes are.

2

Saying germline therapy is legal in humans — it is illegal.

3

Saying genetic screening cures disease — it only gives information.

4

Saying bacteria are best for all recombinant proteins — eukaryotic cells give post-translational modification.

5

Forgetting that early retroviral vectors inserted randomly and caused leukaemia.

1

Saying somatic gene therapy is inherited — it affects only treated cells.

2

Saying SCID is caused by too much ADA — it is caused by lack of ADA.

3

Saying ex vivo means inside the body — it means outside the body.

4

Listing screening benefits without ethical issues.

1

Saying screening gives certain answers — a risk result does not mean disease will definitely occur.

2

Ignoring privacy and discrimination when discussing screening.

3

Forgetting that sampling carries a risk of miscarriage.

4

Treating enhancement as the same as treating disease.