Lecture 21 - Viral Vectors Flashcards
Difference between transfection and transduction
Transfection is inserting naked DNA into a cell.
Transduction is inserting DNA into a cell using a viral vector.
How to initiate viral replication from a nucleic acid
1)
2)
3)
1) Infect cells with virus
2) Reverse transcribe RNA genome into DNA, place into a plasmid. Transfect plasmid into cells
3) Enzymatically synthesise RNA from viral genome inserted into a plasmid using bacteriophage polymerase. Transfect cell with RNA.
Things that viral vectors can be engineered for 1) 2) 3) 4)
1) Protein expression
2) Functional RNA expression
3) Gene therapy
4) Vaccines
Necessary measure for biosafety of viral vectors
Vectors must be replication-deficient
Ways to make viral vectors replication-deficient
Deletion of one or more genes necessary for replication.
Can separate replication and packaging of genome.
What is needed to make a viral genome incorporate into a virion?
Packaging sequence (psi)
Type of cells that vectors should ideally be able to infect
Terminally-differentiated as well as dividing cells
Basic viral vector design requirements 1) 2) 3) 4)
1) Include appropriate promotor elements (CMV immediate-early promotor common)
2) Include foreign gene of interest and a poly-adenylation sequence
3) Genome size must be within packaging limit of virus
4) Virus mustn’t be able to replicate
Necessary steps involved in creating a viral vector 1) 2) 3) 4)
1) Remove viral sequences for pathogenicity, replication
2) Separate viral sequences required for replication and for production of viral particles.
3) Flank transgene (transgene cassette) with essential cis-acting sequences and packaging signals (EG: psi)
4) Provide viral proteins required for packaging that have been deleted from the viral genome in either the cell line, or in a helper plasmid.
Viral vector that produces the highest titres
Adenovirus (10^11 virions)
Viral vectors with a good ability to manipulate tropism
1)
2)
3)
1) Adenovirus
2) Retrovirus
3) Lentivirus
Viral vectors with a poor ability to manipulate tropism
1)
2)
3)
1) Adeno-associated viruses
2) Herpesviruses
3) RNA replicons (not viruses)
Viral vector with a high immunogenicity
Adenovirus
Viral vector that can’t infect non-dividing cells
Retrovirus
Size of possible retrovirus insert
1-7kb
Size of adenovirus possible insert
2-38kb
Size of lentivirus possible insert
7-18kb
Size of herpesvirus possible insert
30kb
Size of AAV possible insert
4.5kb
Design and manufacture of integrating viral vectors 1) 2) 3) 4) 5)
1) Use lentiviruses or retroviruses
2) Essential to retain LTRs, poly-purine primer near U3
3) Remove LTR promoter in U3.
4) Replace gag, pol and env with heterologous gene (gag, pol, env supplied by packaging cell line)
5) Remove vif, vpu, vpr, nef, tat (vpr is necessary for infection of non-dividing cells)
Vectors that can integrate into non-dividing cells
Lentivectors
Problems with retroviral gene vectors 1) 2) 3) 4) 5)
1) Regeneration of replication-competent retrovirus
2) Viral transcription elements are a biosafety concern, as they might insert into endogenous retroviruses in genome and reactivate them
3) Insertional mutagenesis can result in cancers
4) Limited envelope tropism
5) Heterologous tropism wanes with time
Solutions to the danger of regeneration of replication retrovirus from a retroviral vector
1)
2)
3)
1) Inactivate LTR promoter
2) Express structural proteins for packaging in different vectors (gag, env, pol)
3) Packaging cells used to amplify virus must not express endogenous retroviruses
Solutions to the safety concerns about retroviral transcription elements in retroviral vectors
1)
2)
1) Use an alternative internal promoter or remove LTR promoter
2) Remove tat from HIV vectors
Solution to limited envelope tissue tropism of retroviral vectors
Pseudotype vector particles with pantropic env proteins, such as VSV-G
Pantropic env proteins
Env proteins that can bind to a wide variety of receptors on most cell types
Ways to modify retroviral LTR so that Rt and integration are preserved, but LTR replication is prevented 1) 2) 3) 4) 5) 6) 7)
1) Replace U3 with CMV immediate-early promoter
2) R must be retained, for RT jumps
3) Psi packaging site must be retained
4) A heterologous promoter can be used to increase transcription of heterologous gene
5) U3 enhancer sequences must be deleted
6) Delete TATAA, use a non-viral poly-adenylation signal
7) Must retain U3, U5 attachment site for integration
Requirement of self-inactivation of LTR in retroviral vectors
Heterologous gene also include a non-viral gene promoter
Best way to express functional small RNAs
1)
2)
1) Using a functional RNA pol III promoter
2) By placing the small RNA into an intron of RNA pol II transcripts that require processing
Advantages of retro- and lentiviral vectors in gene therapy
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2)
1) Little immunogenicity
2) Integrates into cell genomes
Diseases that could be treated with lentiviral or retroviral vectors
1)
2)
1) Chronic granulomatous disease (NADPH oxidase mutation)
2) X-linked SCID
DNA viruses used for vectors 1) 2) 3) 4)
1) Adenovirus (Ad5)
2) Adeno-associated virus (AAV)
3) Poxviruses (modified vaccinia Ankara, fowlpox, canarypox)
4) Herpesvirus (HSV-1)
How does design of DNA vectors differ from RNA vectors?
DNA vectors are too large to place into a plasmid
Need to make into a ‘shuttle vector’.
Shuttle vector
A vector for DNA viruses.
Clone DNA virus genome into an E coli shuttle vector within viral flanking sequences, under control of a promoter
Most common adenovirus vector used
Replication-deficient adenovirus serotype 5
Proportion of adults with neutralising antibodies to Ad5
~40%
Cloning capacity of Ad5 vectors
~8kb
Ad5 vector design and manufacture 1) 2) 3) 4)
1) ITRs essential and are retained in vector
2) Heterologous genes are cloned in the place of E1, E3 and E4 genes
3) Ad viral particles are produced by transfection into a complementary cell line that expresses E1 and E4 genes (HEK 293 cells)
4) Ad viral particles purified form cells using a caesium chloride gradient
New, safer type of Ad vectors
‘Gutless’ adenoviral vectors (safer, can have bigger inserts)
Formation of ‘gutless’ Ad vectors
Use cre-recombinase to remove expression of adenoviral structural proteins
Main features of adenoviral vectors
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2)
3)
1) Efficient transduction of mammalian cells
2) Highly-immunogenic in animals
3) Grow to high titres (10^13) in cell suspensions
Applications of adenoviral vectors
Gene therapy (limited use), cancer therapy, vaccines
Limitations of adenoviral vectors
1)
2)
3)
1) Pre-existing immunity reduces effectiveness
2) Strong T-cell response to vector proteins produced in transduced cells leads to clearance of these cells
3) Strong humoral immunity to viral capsid prevents re-immunisation
Possible anti-HIV adenoviral vector application
Adenovirus used to deliver zinc-finger endonucleases and TALEN proteins that cleaves CCR5 gene
Advantages of adenoviral vectors 1) 2) 3) 4)
1) High titres
2) Infect replicating and non-replicating cells
3) Wide tissue tropism
4) Can modify penton spikes to target tissue tropism
AAV vector features 1) 2) 3) 4) 5)
1) Parvovirus (ssDNA, 4.5kb genome)
2) Can infect non-replicating cells
3) Replace viral rep and cap genes with heterologous gene. Express rep and cap (essential proteins) in 293 cell line, with assistance of adenoviral helper proteins.
4) Insert length restricted to 4.7kb
5) Might integrate into host genome as a concatemer
Structure of an AAV vector 1) 2) 3) 4)
1) ITRs retained
2) Replace rep, cap genes with heterologous gene.
3) CMV immediate-early gene promoter, poly adenylation sequence
4) Need to deliver AAV vector with adenoviral helper plasmids (for packaging) and AAV packaging plasmids (that express rep and cap). Alternatively, rep and cap can be expressed by 293 cell line.
Advantages of AAV vectors 1) 2) 3) 4)
1) Integration, persistent expression
2) No insertional mutagenesis
3) Infects dividing, non-dividing cells
4) Safe
Disadvantages of AAV vectors
1)
2)
1) ~4.7kb insert limit
2) Low titre of virus and gene expression
Herpesvirus vectors
1)
2)
3)
1) ~40-50kb foreign DNA can be incorporated
2) Can target nervous system, as is a neurotropic virus
3) Hard to make recombinantly
Production of herpesvirus vectors
1)
2)
Either:
1) Recombination with highly-deleted defective virus
2) Using large plasmids with and HSV OriS and packaging sequence
RNA replicons 11) 2) 3) 4)
1) Self-replicating RNAs
2) Derived from viral RNA genomes
3) Replicate in the cytoplasm
4) Capable of high levels of heterologous-gene expression
Ways to deliver RNA replicons
1)
2)
3)
1) Virus-like particles
2) Naked RNA
3) Naked DNA
Promising (+)RNA virus as an RNA replicon vector
Kunjin (a West Nile virus, flaviviridae)
How can replicons be generated?
1)
2)
3)
1) Replace structural genes of RNA viruses with heterologous genes. This leaves only replicative machinery.
2) Deliver to cells as a plasmid under promoter control. Cell line must express structural genes.
3) RNA replicon transcribed, replicated in the cytoplasm, translated into VLPs.
Kunjin RNA replicon features 1) 2) 3) 4)
1) High levels of expression
2) non-cytopathic
3) Cytoplasmic replication
4) No recombination
Kunjin RNA replicon applications
1)
2)
Vaccines
Cancer therapy
Limitations of Kunjin RNA replicons
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2)
1) Packaging constraints - VLPs have small cloning capacity
2) Pre-existing immunity to West Nile virus exists in America, Africa
