Gene Therapy Drug Delivery Flashcards
What is meant by nucleic acid therapeutics
RNA can be used for either:
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gene expression or
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Antisense oglionucleotides
- knock down gene expression by stopping RNA translation
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Antisense oglionucleotides
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silencing
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Small interferfering RNA (siRNA)
- small synthetic double stranded RNA (dsRNA) molecules for RNA interference.
- silence genes
- small synthetic double stranded RNA (dsRNA) molecules for RNA interference.
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Small interferfering RNA (siRNA)
Explain the mechanism of action of RNA interference.
- Degradation of dsRNA into siRNA by a Dicer (RNase III-like activity)
- The siRNA generated are subsequently unwound and assembled into the RNA-induced effector complex (RISC) which acts on the specific target mRNA to be cleaved & degraded (translational repression).
What was a fundamental problem of using DNA as a theapeutic agent for gene therapy?
- The DNA must be delivered to the nucleus.
- But the nucleus possesses a nuclear membrane that has the nuclear pore complex which controls entry of macromolecules into the nucleus.
- Viruses can by-pass this membrane
- Hence, viral gene-therapy
Describe THREE potential therapeutic outcomes of gene therapy.
- Delivery of DNA as a therapeutic agent
- Replacement of a missing protein
- ’cure’ of diseases caused by single gene defects
- i.e. Cystic fibrosis
- ’cure’ of diseases caused by single gene defects
- Expression of a protein for other therapeutic purposes
- modulation of the immune system
- cancer, autoimmune diseases
- over-expression of protective proteins
- neurodegenerative diseases
- modulation of the immune system
What is an issue using attenuated viruses for gene therapy?
- Safety issue
- Although attenuated viruses are replication incompetent, there is still that possibility of having a competent replicating virus.
What is an advantage and disadvantage of using RNA for gene therapy?
- Advantage:
- Delivery of RNA to the cytoplasm avoids a major barrier (nuclear membrane)
- The RNA will directly be translated to the protein. Therefore, don’t have to deliver DNA to the nucleus.
- Delivery of RNA to the cytoplasm avoids a major barrier (nuclear membrane)
- Disadvantage
- There is a challenge in delivering RNA to the cytoplasm of the target cells
“Gene therapy has only a transient effect.” True or False. Discuss.
- False.
- Permanent treatment can be provided by integration of the DNA into the target cell genome
- Here, it corrects single gene defects (such as cystic fibrosis)
What are some possible consequences of integrating DNA into the target cell genome?
- A promoter gene for cancer can be switched on if a retrovirus is placed upstream of the promoter gene.
- Possilbe to introduce oncogensis
Why is transient gene therapy preferable?
- Transient treatment is more like drug therapy, where the coding sequence is not integrated, but expressed as episomal.
- This allows us to assess side effects and adjust dose
Transient therapy is the preferable treatment to correct single gene defects. True or False.
False. Transient therapy is preferable for all therapies apart from correction of single gene defects because it’s not possible to do this, and can only be done via permanent gene therapy.
List and briefly describe FOUR varieties of vectors used for gene therapy.
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Retrovirus
- integrating vectors
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Adenovirus
- non-integrating
- non-dividing cells
- immunogenic
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Adeno-associated virus (AAV)
- integrating AND non-integrating
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Non-viral
- non-integrating
- plasmid delivery
Explain FOUR safety problems using attenuated viruses.
- Finite chance of replication-competent viral particles being harvested during production
- Finite chance of recombination happening in the patient
- Viral particles have immunogenic proteins which prevents multiple doses being used
- Insertion of viral gene promoter upstream of host cell oncogenes could promote cancer
What is meant by ‘packaging cell line’ for the production of retroviruses?
- Packaging cells allow the production of retroviral vectors in the absence of replication-competent virus (deleted viral genes).
- To produce a retrovirus, the retroviral vector is transfected into a packaging cell line.
- Viral assembly occurs.
- Infection of target cells
Briefly describe TWO formulations for non-viral gene delivery.
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Lipoplexes
- Cationic lipid/DNA complexes
- Micelle/Lipid structure enclosing the DNA
- Transfect a cell via lipofection where it diffuses across the membrane, then endocytosis of the lipoplex occurs and the DNA is released into the cytoplasm.
- The cationic lipids also protect against degradation of the DNA by the cell.
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Polyplexes
- Cationic polymer/DNA complexes
- Weakly basic polymers that promote endosomal escape
- Once Polyplexes are diffused across the membrane, they are enclosed in an endosome
- Here, the “Proton sponge” effect occurs:
- A drop in pH of the endosome (caused by a proton pump) will cause an expansion of the polymer which disrupts the endosomal membrane, thus creating pores to allow the DNA to escape
Lipoplexes and Polyplexes can be used to transfect cells in vitro. True of False.
- True
- Both these complexes only have poor activity in vivo because they only be used in dividing cells performed in vitro.
Compare the advantages and disadvantages between Plasmid/Lipid complexes and Adenovirus.
