1. Human Health and Disease
Lecture 4

2. "We used to think that our fate was in our stars, but now we know that, in large measure, our fate is in our genes, "quotes James Watson.

3. Genes, DNA, Alleles
Our genetic program is made up of thousand of genes, stretches of DNA, that generally code for different proteins that do particular jobs in the cells in our body.
The process of decoding a gene into a protein occurs in two basic stages:
‘Transcription', where specific mRNA molecules are generated from the DNA stretch concerned; and
‘Translation', where the specific mRNA molecules are used as a template to make the specific protein.
For any particular gene there are usually two copies (sometimes the term 'allele' is used instead of copy), one from the mother and a second passed on from the father. For most genes, only one normal copy is required for normal function.

4. Genetic Disorders
When genes are altered so that encoded proteins are unable to carry out their normal functions, genetic disorders can result.
Silent mutations are mutations in DNA that do not significantly alter the phenotype of the organism in which they occur. Silent mutations can occur in non-coding regions (outside of genes or within introns), or they may occur within exons.
In genetics, a missense mutation is a point mutationin which a single nucleotide change results in a codon that codes for a different amino acid. It is a type of nonsynonymous substitution.
In genetics, a nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and usually nonfunctional protein product. It differs from a missense mutation, which is a point mutation where a single nucleotide is changed to cause substitution of a different amino acid. Some genetic disorders, such as thalassemia and DMD, result from nonsense mutations.

5. Types of Mutations
Substitution: is a mutation that exchanges one base for another (i.e., a change in a single "chemical letter" such as switching an A to a G).
Insertions: are mutations in which extra base pairs are inserted into a new place in the DNA.
Deletions: are mutations in which a section of DNA is lost, or deleted.
Frameshift: Since protein-coding DNA is divided into codons three bases long, insertions and deletions can alter a gene so that its message is no longer correctly parsed. These changes are called frameshifts.

6. Classification of Genetic Disorders
Level 1: Single Gene Disorders
Disorders which result when a mutation causes the protein product of a single gene to be altered, differentiated, or missing.
Level 2: Chromosome Abnormalities
Entire or whole chromosomes, or large segments of the chromosomes, are missing, duplicated, or altered.
Level 3: Multifactorial Disorders
Multifactorial disorders are those which result from mutations in multiple genes. They’re complex, often coupled with environmental causes.

7. Gene Therapy Definition: 1.‘The use of genes as medicines’
2. The introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders
GT ‘will be’ used to treat genetic disorders, viral infections and mutations in cancer.

8. How it is done?
Replacing a mutated gene that causes disease with a healthy copy of the gene.
Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
Introducing a new gene into the body to help fight a disease.

9. Basic concept

10. What are the ways to deliver the genes?
Ex-vivo Gene Therapy - In ex-vivo gene therapy, cells are first cultured or synthesized outside of an organism (for gene therapy), and then inserted into the organism to provide the treatment. Ex- vivo gene therapy is more common than in-vivo therapy. Ex-vivo therapy significantly reduces many risks involved with gene therapy. At the same time, however, ex-vivo gene therapy holds some limitations - indirectly introducing the desired-containing cells into an organism may trigger immune responses. The cells also may not function as desired, malfunction, or not entirely work at all.
In-vivo Gene Therapy - In in-vivo gene therapy, the gene is directly delivered to the organism, such as through a vector or other means. In-vivo gene therapy is less commonly used than ex- vivo gene therapy. In-vivo gene therapy holds more risks than its ex-vivo counterpart, such as a possible immune reaction from the organism. Vectors used in in-vivo gene therapy include viruses, bacterial plasmids, nanoparticles, and more.

11. Types of gene therapy
There are two different types of gene therapy depending on which types of cells are treated:
Somatic gene therapy: transfer of a section of DNA to any cell of the body that doesn’t produce sperm or eggs. Effects of gene therapy will not be passed onto the patient’s children.
Germline gene therapy: transfer of a section of DNA to cells that produce eggs or sperm. Effects of gene therapy will be passed onto the patient’s children and subsequent generations.

12. Somatic gene therapy

13. Somatic Gene Therapy
Advantages - Somatic gene therapy is relatively effective as a procedure. Many developments, such as possible treatments and cures, have been developed using gene therapy. Also, somatic gene therapy is significantly less controversial than germ line gene therapies.
Disadvantages - If a treatment or cure is successful (or any other trait modification using somatic gene therapy, for that matter), it will not be passed on to the patient/organism's offspring. In addition, the methodology of somatic gene therapy, such as the use of viral vectors, is difficult.

14. Germ line gene therapy

15. Germline gene therapy
Advantages - Germ line gene therapy is done before the organism has grown or developed; therefore, the cure is inherited by future generations of that organism. Also, germ line gene therapy allows for a desired gene to become fully incorporated into the organism before activation; therefore, one of the factors influencing unwanted immune responses (to the gene) is removed.
Disadvantages - Germ line gene therapy is very controversial. In addition, due to this controversial nature and other limiting factors, germ line gene therapy is not fully pursued for development. Germ line gene therapy also holds numerous risks, such as a margin for possible error during the gene 'transplant‘. Banned in countries throughout the world

16. History of Gene Therapy
Gene therapy was first proposed nearly forty years ago as a method of manipulating cells at the molecular level in order to cure rare genetic diseases like cystic fibrosis, phenylketonuria and cancers.
Following an explosion in cancer, virology and bacterial genetics research in the late 1960s and with the biochemical characterisation of human genetic diseases, it was proposed that synthetic and viral vectors could be used to deliver corrected versions of genes into cells. It was proposed that this gene transfer would induce a corrected phenotype on the cell.
The transfer of large genetic sequences into cells presents a fundamental problem because DNA is negatively charged and will not readily cross the plasma membranes. Therefore in addition to identifying the genetic basis of a disease and cloning a corrective copy of the sequence, much effort has gone into the production of vector systems for transporting genes into cells and expressing the genes in-vivo.

17. Types of Gene Therapy

18. Replication deficient viruses were proposed as ideal gene transfer systems because they have the ability to infect cells, partially evade immune responses and express their genome in the host cell. A range of viral vectors including retroviruses, lentiviruses, adenoviruses and adeno associated viruses have been used in gene therapy trials.
In addition non-viral gene transfer systems using cationic liposome/DNA complexes have been developed which allow the transfection of cells with potentially much larger pieces of DNA and none of the immune response problems associated with viral gene therapy.

19. Gene Therapy Techniques
1. Gene augmentation therapy
This is used to treat diseases caused by a mutation that stops a gene from producing a functioning protein.
This therapy adds DNA containing a functional version of the lost gene back into the cell.
The new gene produces a functioning product at sufficient levels to replace the protein that was originally missing.
This is only successful if the effects of the disease are reversible or have not resulted in lasting damage to the body.
For example, this can be used to treat loss of function disorders such as cystic fibrosis by introducing a functional copy of the gene to correct the disease.
Cell with non-functioning gene
Cell with functioning gene

20. 2. Gene inhibition therapy
Suitable for the treatment of infectious diseases, cancer and inherited disease caused by inappropriate gene activity.
The aim is to introduce a gene whose product either:
inhibits the expression of another gene
interferes with the activity of the product of another gene.
The basis of this therapy is to eliminate the activity of a gene that encourages the growth of disease-related cells.
For example, cancer is sometimes the result of the over- activation of an oncogene (gene which stimulates cell growth). So, by eliminating the activity of that oncogene through gene inhibition therapy, it is possible to prevent further cell growth and stop the cancer in its tracks.

21. 3. Killing of specific cells
Suitable for diseases such as cancer that can be treated by destroying certain groups of cells.
The aim is to insert DNA into a diseased cell that causes that cell to die.
This can be achieved in one of two ways:
the inserted DNA contains a “suicide” gene that produces a highly toxic product which kills the diseased cell
the inserted DNA causes expression of a protein that marks the cells so that the diseased cells are attacked by the body’s natural immune system.
It is essential with this method that the inserted DNA is targeted appropriately to avoid the death of cells that are functioning normally.

23. How is DNA transfer done?
A section of DNA/gene containing instructions for making a useful protein is packaged within a vector, usually a virus?, bacterium? or plasmid? Or nanoparticles.
The vector acts as a vehicle to carry the new DNA into the cells of a patient with a genetic disease.
Once inside the cells of the patient, the DNA/gene is expressed by the cell’s normal machinery leading to production of the therapeutic protein and treatment of the patient’s disease.

25. Challenges of Gene Therapy
Delivering the gene to the right place and switching it on:it is crucial that the new gene reaches the right cell
delivering a gene into the wrong cell would be inefficient and could also cause health problems for the patient
even once the right cell has been targeted the gene has to be turned on
cells sometimes obstruct this process by shutting down genes that are showing unusual activity.

26. Avoiding the immune response:
The role of the immune system is to fight off intruders.
Sometimes new genes introduced by gene therapy are considered potentially-harmful intruders.
This can spark an immune response in the patient, that could be harmful to them.
Scientists therefore have the challenge of finding a way to deliver genes without the immune system ‘noticing’.
This is usually by using vectors that are less likely to trigger an immune response.

27. Making sure the new gene doesn’t disrupt the function of other genes:
Ideally, a new gene introduced by gene therapy will integrate itself into the genome of the patient and continue working for the rest of their lives.
There is a risk that the new gene will insert itself into the path of another gene, disrupting its activity. 
This could have damaging effects, for example, if it interferes with an important gene involved in regulating cell division, it could result in cancer.

28. The cost of gene therapy:
Many genetic disorders that can be targeted with gene therapy are extremely rare.
Gene therapy therefore often requires an individual, case-by-case approach. This may be effective, but may also be very expensive.

29. Lecture prepared from
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