Viruses as Therapeutics Flashcards
Distinguish between somatic and germline gene therapy.
How is exogenous genetic material introduced to a cell?
Both involve transfer of genetic material. If the transfer affects cells that will be passed onto subsequent generations, it is germline. Otherwise, it is somatic.
Viruses are typical, but there are alternatives (chemical shock, liposomes, physical injection/gene guns)
Describe how gene therapy was used to treat a patient with X-linked SCID.
A defective gene for the common gamma chain (common to some interleukins) was replaced in CD34+ cells.
Explain how retroviral vectors are designed and harvested.
A group of cells is infected gag-pol-env genes (by retroviral infection?). A vector plasmid containing the exogenous genes of interest (bound by LTRs) is transfected into this cell line, where it hopefully integrates with the viral genome and is picked up by new virions.
What advantages and disadvantages are conferred by using retroviruses as the gene vector?
Delivery is efficient and easy, and tropism can be specified.
Insertion is haphazard and may interrupt other genes, and the inserted genes may not always be expressed.
Distinguish lentivirus vectors from retrovirus vectors.
Lentiviruses are retroviruses. They are able to infect nondividing cells.
How are adenovirus vectors different from either retro or lentiviruses in their mechanism of gene delivery?
How are these vectors produced?
Integration never occurs; the virus stays in the cell and expresses the desired genes separately (can specify early/late).
Transfection of 293 cell lines produces vector particles. Note that insertion of genes into the adenoviral genome results in deletion of others (to make room).
What are some problems associated with using adenoviruses as a gene vector?
What are “conditionally replicative adenoviruses”?
The expression is short-term since there is no integration. The virus is immunogenic and will elicit a response, limiting the level of infection (better with E3 deletions?). The genome is limited.
These are adenoviruses that are generally specific for tumor cells, and will only replicate under conditions such as those in a cell without cell checkpoints or tumor suppressors.
To what family does vaccinia belong?
How is its mechanism of gene delivery distinct from either retro or adenoviruses?
It is a poxvirus.
It is the only virus mentioned that is replication-competent. It replicates in the cytoplasm and can carry a much larger exogenous gene load.
How are vaccinia vectors produced?
How is hepatitis B being considered for treatment using vaccinia virus?
Name some other poxviruses.
Exogenous genes are introduced either on a plasmid, or are inserted/recombined in its genome (eg at the TK gene).
Hepatitis B has an antigen that can be expressed by cells infected with vaccinia viruses carrying the gene for it. This would promote long-term immunity if introduced into a host cell.
Canarypox, modified vaccinia ankara (MVA). These do not multiply in human cells.
What illnesses could be treated using engineered viruses, asides from gene deficiencies like SCID or infections like hepatitis B?
How do CRAd and reovirus target tumor cells?
Many cancers. Certain viruses are already known to be oncolytic, and we could feasibly engineer viruses to have tumor tropisms.
CRAd exhibits early gene deletions which make it incapable of lytic replication except in wildly proliferating cells (eg via hTERT promoter). They may also have specific adhesional tropisms (eg RGD binds tumor integrins).
Reoviruses are more selective for cells with elevated Ras activity, and are inhibited by PKR (in normal cells).
