Week 8 Flashcards
1
Q
Transfection
A
- Viral infection initiated by naked nucleic acid
- Has come to mean transfer of any nucleic acid into eukaryotic cells
- Uses molecular conjugates e.g. CaPO4 precipitate, Cationic liposomes
- By viral vectors = transduction
2
Q
How to initiate viral replication
A
- Viral particles (infection)
- viral RNA (transfection)
- cDNA synthesis and cloning + reverse transcription –> DNA –> plasmid (transfection) –> new progeny virion
- sense strand of RNA, lacking VPG protein (transfection)
- Can use cDNA cloned into plasmid with suitable RNA pol II
- RNA synthesised enzymatically using bacteriophage polymerase e.g. T7 RNA pol, SP6 RNA pol
3
Q
When using viral vectors, you need to….
A
- Remove capacity of virus to replicate and release new infectious particles
- Made defective by deletion of one or more genes required for replication
4
Q
Viral vector design requirements
A
- Appropriate promoter e.g. CMV
- Terminate RNA e.g. polyA signal
- Maintenance of genome size within packaging limit of virus - delete non essential genes
- Remove viral sequences responsible for pathogenicity and replication
- Separate viral sequences required for replication and production of virus particles–> packaging construct
- Flank transgene by essential cis-acting sequences and packaging signals
- Provide viral proteins required for packaging and replication in packaging cell
- Need RNA packaging signal = retrovirus containing foreign gene
5
Q
Comparison of different viral vectors
A
6
Q
Retro and lentivirus vectors
A
- Inserts integrated (permanently transduce cells)
- Lentivirus has a lot more inessential genes - get rid of many of genes
- VPR - assists in infecting non-dividing cells
- psi signal = packaging signal
- Rev retained in lentivirus
7
Q
Reverse transcription of RNA to DNA
A
- tRNA primer extended to form DNA copy of genomic 5’ end, tRNA primer binds to PBS region (primer binding signal), repeated sequence at either end (R), make a copy of R and U5
- RNaseh removes hybridised RNA
- cDNA hybridisees to R sequences at 3’ end of genome
- Extension of DNA strand
- Most of hybrid RNA removed
- 3’ end of second DNA strand synthesised
- Balance of RNA genome and tRNA primer removed
- PBS sequences hybridise
- both DNA strands completed
- LTR repeats - made up of u3, R, U5
- Integrates into cellular DNA integrated form called provirus
8
Q
Issues with retroviral gene transfer vectors
A
- Regeneration of replication competent retrovirus
- Inactivate LTR as a promoter, express structural proteins for packaging from separate plasmids
- Packaging cells must not express endogenous retoviruses
- Use alternative internal promoter (self-LTR inactivating vector, doesn’t rely on LTR) or remove Tat from HIV
- Insertional mutagenesis - carcinogenic
- Limited envelope tropism - add pseudotype vector particles, VSV-G protein (pantropic), can bind to sialic acid –> capable of infecting any cell
- Heteologous gene expression wanes with time
9
Q
Vector plasmid vs integrated vector after RT
A
- Vector plasmid: remove LTR to serve as a promoter
- don’t need all of R sequence or all of UR but DO need ends of U5 (att) which interacts with integrase and retain packaging site
- U3 has other attachment site for integrase, delte TATAA box and enhancer sequences that form promoter
10
Q
Applications of retroviral/lentiviral vectors
A
- Gene correction therapy: no vector-induced immune response or cytotoxicity
- Cancer therapy: transient high-level expression
- Vaccines
11
Q
DNA virus expression vectors
A
- Most have very big genomes - too big to clone into plasmid
- Make shuttle vector e.g. in e.coli
- Contransfect of recombinant plasmid with viral DNA or transfection of recombinant plasmid DNA into virus infected cells to facilitate homologous recombination
12
Q
Adenovirus vectors
A
- Replication defective form of human adenovirus type 5
- Retain essential elements (inverted terminal repeats)
- Non-essential : E3
- Supplied by packaging construct/cell line : E1A, E1B
13
Q
Jesse Gelsinger
A
- Liver disease: OTC deficiency
- treated with adenoviral vector high dose (e1 and e4 genes deleted), carried normal copy of OTC gene
- Death: toxicity of high titer, high immunogenicity of adenoviral vector (immune response)
14
Q
Safer adenoviral vectors (gutless)
A
- Remove structural genes by using cre-lox remobination system
15
Q
Adenovirus expression vectors
A
- Efficient transduction, highly immunogenic, high titres
- Gene therapy, vaccines, cancer therapy
- LImitations: pre-existing immunity, strong T cell response (short term expression), srong humoral response against viral capsid prevents re immunization
