Genetherapy

Question 1

Define
Transformation
Transfection
Transduction

Answer 1

Transformation - naked DNA into Bacteria
Transfection - naked DNA into cells
Transduction - DNA in viral vector into Cell

Question 2

Give an Example of non-viral gene therapy

Answer 2

DNA or RNA in lipid layers.
Ex: siRNA against miss folded Proteins (disease - ATTR, drug patisiran)

Question 3

How does the gene transduction of Adeno viruses work?

Answer 3

Virus binds to extracellular receptor –> internalization of the complex –> Virus escapes form the endosome –> Trafficked to the nucleus –> docking at the nuclear pore complex –> insertion of vDNA (viral DNA)

Question 4

Name the main Advantages and Disadvantages of Adenoviral (AV) gene transduction. Compare to Adeno Associated Viral (AAV) gene transduction

Answer 4

AV:
- High efficient transduction and high levels of gene expression
- Easy production
- Rarely integrate
But - needs special receptor not on present on cells
- Neutralizing immune responds
- Non persistent (since not integrating)
AAV:
- non pathogenic = lower Immunogenicity
- Integrates at even lower frequency
But - small packing capacity

Question 5

What is the main difference between AV and AAV compared to Retroviruses?

Answer 5

AV, AAV non integrating, Retrovirus integrating

Question 6

Outline the main features and disadvantages of Lentiviruses, what is the difference between Retroviruses and Lentiviruses?

Answer 6

Lentivrus (e.g. HIV) = subsection of Retrovirus, that are also able to integrate into non dividing cells
- Integrates
- potential risk of insertional mutations but higher probability to integrate into active gene and not in the enhancer (ADA-SCID lentivirus > other Retovirus)

Question 7

Explain how splitting the genome of a Virus can make it safer when used as a gene therapy?

Answer 7

env, gag pol on differend plasmides than the trangene between the LTR –> inject all three plasmides into one cell –> viral vectors are generated but only carry the transgene to the next cells that they infect.

Question 8

What is CRISPR;Cas9? And how can it be used in gene therapy (three examples)?

Answer 8

Bacaterial immune respons, can induce sequence specific ds brakes. tracrRNA provides the specific sequence and Cas9 is the protein complex, that engulfes the tracrRNA and induces the cut in the DNA.
Can be used to induce a transgene (homology directed repair HDR) or silence a gene (non homologous end joining NHEJ).
Can also be altered, so that the complex dose not induce ds breaks but only associates sequence speciffic –> can be used to modulate the effector region of a gene or to induce epegenetic modifications

Question 9

What is an CAR and what are its advantages? What are disadvantages of the treatment?

Answer 9

Chimereic antibody receptor (CAR) is an artificial receptor designed from an Ab to recognize an Ag of e.g. a cancer cell, that induces a cytotoxic response against the target cell carrying the Ag. The Ag can be MHC independent and tumor specific.
Disadvantage –> Autogenic = expensive and takes a long time to produce

Question 10

What is Multi antigen recognition?

Answer 10

Combines the recognition of multiple Ag, resulting in higher efficacy and memory cell creation.
SynNotch recognizes Ag1 –> releases transcription factor for the expression of CAR against Ag2 –> induction of cytotoxic response