Choose 2 of the following to answer in as much detail as possible. Please be sure to number your answers.  

1. Discuss and describe in detail the different categories of viral vectors. Discuss when and how they are used in gene therapy and the advantages and/or disadvantages of each.
2. Discuss the importance of drug interactions due to cytochrome P450 isozymes and the implications for gene therapy. Give specific examples!
3. Discuss in detail genome editing. Includes the engineered nucleases used and their use in NHEJ or HDR. Also include how each is currently used in gene/cell therapies.

 

1) Categories of viral vectors

• lentiviral vectors
• retroviral vectors
• adenoviral vectors
• herpes simplex vectors
• chimeric vectors

2)

• production of cholesterol, steroids, prostacyclins.
• detoxification of foreign chemicals
• metabolism of drugs
• contain heme pigment

3)

Genome editing is a type of genetic engineering. The engineered nucleases used are;

• zinc finger nucleases(ZFNs)
• transcription activator like effector nucleases (TALENs)
• Enineered meganucleases

Step-by-step explanation

1) The categories of viral vectors are;

1. Lentiviral vectors - types of retrovirus that are able to integrate into non-dividing cells and do not require mitotic cell division for their functioning. They are used in gene therapy when, the genome enters the cell DNA via reverse transcription and is incorporated in a random position. The advantage of this in gene therapy is that it demonstrates low negative consequences. The disadvantage of this is that it leads to cellular function disturbance which can lead to cancer.
2. Retroviral vectors - they integrate the genome of the infected cell in stable and permanent fashion. Reverse transcriptase in the virus allows integration into the host genome. Examples are replication-defective and replication competent. The practice allows several rounds of replication through coding. It is disadvantage in the way that some cells such as neurons are resistant to the effect of retroviral vectors.
3. Adenoviral vectors - they can deliver range of actions and able to deliver nucleic acids to both diving and non-dividing cells. They have a beneficial properties to gene therapy that are effective with limited negative effects. However, the utility of this type of vector is significantly limited by its restricted capacity of DNA.
4. Herpes simplex vectors - has ability to deliver large scale quantities of exogenous DNA. The primary concerns with the use of herpes simplex virus to deliver genetic material are cytotoxicity and the maintenance of transgene expression.
5. Chimeric vectors - combines the properties of several types to manipulate the advantages and disadvantages of the vectors. Use at present but are promising solution for the future solution for the future when their properties have been established further.

2) The importance of drug interractions due to cytochrome P450

1. They are essential enzymes for the production of cholesterol, steroids, prostacyclins and thromboxane in the body.
2. The are necessary for the detoxification of foreign chemicals that may cause toxic to the body.
3. They are also involved in metabolism of drugs in the human body.
4. They contain heme pigment that absorbs light at wavelength of 450nm when exposed to carbon monoxide.

The implication of gene therapy are;

1. the new gene might be inserted in the wrong location in the DNA which might cause harmful mutations to the DNA or even cancer.
2. Affects the development of a fetus in unexpected ways.

3)

Genome editing is a type of genetic engineering which DNA is inserted, deleted, modified or replaced in genome of a living organism. Genome editing with engineered nucleases i.e all the three major classes of these enzymes;

1. zinc finger nucleases(ZFNs)
2. transcription activator like effector nucleases (TALENs)
3. Enineered meganucleases

Common methods such editing used in engineered nucleases creates site-specific double-strand breaks at the desired locations in the genome. The induced double-strand breaks are repaired through non-homologous end joining resulting in target mutations.

• ZFNs are generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Can be used to alter genomes of higher organisms.
• TALENs have been used to introduce specific human somatic and pluripotent stem cells using double-stranded donor templates.
• Engineered meganucleases are used to replace, eliminate or modify sequences in a highly targeted way.