Week 8

Question 1

Transfection

Answer 1

• 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

Question 2

How to initiate viral replication

Answer 2

1. Viral particles (infection)
2. • viral RNA (transfection)
3. cDNA synthesis and cloning + reverse transcription –> DNA –> plasmid (transfection) –> new progeny virion
4. • 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

Question 3

When using viral vectors, you need to….

Answer 3

• Remove capacity of virus to replicate and release new infectious particles
• Made defective by deletion of one or more genes required for replication

Question 4

Viral vector design requirements

Answer 4

• Appropriate promoter e.g. CMV
• Terminate RNA e.g. polyA signal
• Maintenance of genome size within packaging limit of virus - delete non essential genes

1. Remove viral sequences responsible for pathogenicity and replication
2. Separate viral sequences required for replication and production of virus particles–> packaging construct
3. Flank transgene by essential cis-acting sequences and packaging signals
4. Provide viral proteins required for packaging and replication in packaging cell

• ​Need RNA packaging signal = retrovirus containing foreign gene

Question 5

Comparison of different viral vectors

Answer 5

Question 6

Retro and lentivirus vectors

Answer 6

• 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

Question 7

Reverse transcription of RNA to DNA

Answer 7

1. 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
2. RNaseh removes hybridised RNA
3. cDNA hybridisees to R sequences at 3’ end of genome
4. Extension of DNA strand
5. Most of hybrid RNA removed
6. 3’ end of second DNA strand synthesised
7. Balance of RNA genome and tRNA primer removed
8. PBS sequences hybridise
9. both DNA strands completed
10. LTR repeats - made up of u3, R, U5
11. Integrates into cellular DNA integrated form called provirus

Question 8

Issues with retroviral gene transfer vectors

Answer 8

• 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

Question 9

Vector plasmid vs integrated vector after RT

Answer 9

• 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

Question 10

Applications of retroviral/lentiviral vectors

Answer 10

• Gene correction therapy: no vector-induced immune response or cytotoxicity
• Cancer therapy: transient high-level expression
• Vaccines

Question 11

DNA virus expression vectors

Answer 11

• 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

Question 12

Adenovirus vectors

Answer 12

• 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

Question 13

Jesse Gelsinger

Answer 13

• 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)

Question 14

Safer adenoviral vectors (gutless)

Answer 14

• Remove structural genes by using cre-lox remobination system

Question 15

Adenovirus expression vectors

Answer 15

• 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

Question 16

ZFNs

Answer 16

• Tethered to fok1 - when dimerised, cleaves ccr5 part of DNA
• Make CCR5 defective –> HIV can’t enter

Question 17

CRISPR- Cas9

Answer 17

• Guide RNA directs annealing, recruits binding of Cas9 proteins which direct editing

Question 18

adeno advantages vs. disadvantages

Answer 18

Advantages:

• High titres
• Dividing and non-dividng cells
• Wide tissue tropism
• Can modify penton spikes to tune tropism

Disadvantages:

• Transient expression
• Highly immunogenic
• High titers of virus can be toxic
• Suitable for cancer immunotherapy

Question 19

AAV vectors

Answer 19

• Parvovirus
• Have TR sequences at either end, rep and cap protein
• Vectors only need to retain TR, can include RNA pol II specific promoter, cDNA, polyA
  *

Question 20

AAV vector: advantage vs disadvantages

Answer 20

Advantages:

• Integration and persistent expression
• No insertional mutagenesis
• Infect dividing and non-dividing
• Safe

Disadvantages:

• Small size
• Low virus titer, low gene expression
• 1 trial recipient death

Question 21

Herpes vectors

Answer 21

• Low risk
• Big genome - 40kb of foreign DNA
• Neurotropic

Question 22

DNA recombination to prepare herpesvirus

Answer 22

• Homologous DNA recombination produces mutant strains of Herpesvirus for production of packaging cell lines
• Vectors produced by recombination with highly deleted defective virus or using large plasmids with an HSV Ori S and packaging sequence

Question 23

RNA replicons

Answer 23

• Self-replicating Rnas
• replication in cytoplasm
• High level expression of heterologous genes
• Can be delivered as: VLP, naked RNA, naked DNA
• e.g. +ssRNA vectors - togavirus, flavivirus

Question 24

Kunjin vector

Answer 24

• West nile flavivirus
• High level of expression, noncytopathic, cytoplasmic replication (no integration)
• No recombination
• Vaccines for HIV, ebola, cancer
• Limitations: packaging constrains, pre-existing immunity