Week 9 Flashcards
advantages of viral vector systems (3)
high efficiency and expression
virus coating DNA.
can be broad or selective
Adenoassociated virus are used for? Why?
Therapy in humans. It does not integrate.
What virus are used in research?
Retrovirus and lentivirus
disadvantages of viral vector systems?
Activation strategies complex due to high security requirements Therapeutic concerns (can insert themselves into oncogene, can have allergic reaction...)
Two types of genes in virus?
Early: involved in replication. Promote entry of host into S-phase.
Late: structural. Capsid proteins and envelope proteins.
Replication-competent addition what is it? what is a disadvantage?
Add gene of interest.
Limited by packaging size of virus
Replication-competent replacement what is it? what is a disadvantage?
Replaces virus gene w gene of interest.
It is helper-independent.
Helper-dependent vectors
Cannot replicate on its own and needs function supplied in trans.
In vitro cloning
Cut with nucleases and put in transgene w ligase
In vivo cloning
Insertion through homologous recombination
How to make Simian Virus SV40 safe?
Take out T-antigen complex, which is what signals for unwinding of DNA. Needs COS cell line to replicate now.
Adenovirus Genome changes to make it safe.
Packaging signal (psi) needed for the virus to package. Can make virus w no psi, or w psi and missing late genes to prevent unwanted gene replication.
Adenovirus genome advantages (3)
Broad host range among vertebrates
Dividing and non-dividing cells can be infected
Low pathogenicity
Adenovirus genome disadvantages
Previous exposure can lead to reduced efficacy
Inflammation
Leaky expression
Lytic cycle basics?
Phage DNA into bacterium, host cell transcribes and translates, phage particles assemble, lyse host cell and is released.
Lysogenic cycle basics
Phage accidentally acquires fragments of host DNA and transduction occurs upon infection of new host (integration of fragments into host)
Adeno-associated viruses (AAV) advantages
Different tropisms allows for programming of genome anyway wanted.
Change of capsid gene structure allows to target different organs.
What are 4 challenges for gene therapy?
Maximise impact but minimise risks
Strategies that work on animal models may not work in humans
Research is time consuming and difficult (many steps)
How is ex gene therapy usually made?
Isolate haematopoietic stem cells and then put then back in the organism. Has limited cell divisions.
Gammaretroviral vector characteristics
Replicate in host cells via reverse transcription
BUT can integrate into proto-oncogenes
CAR-T cell therapy
take blood, target cancer, culture, put receptor cell that can select against cancer and then put it back. Can bind to cancer and kill it.
Advantages of CAR-T cell therapy
ex vivo, which is ideal, and can be used with CRISPR CAS9, better than virus.
