Adenoviruses

Adenoviruses

1. Intro

   • dsDNA viruses
   • ~35kb
   • Enveloped
   • Targets many cell types

2. Genome

   • Early Genes
     • E1, E2, E3 and E4 clusters
       • E1A and E1B crucial for activation of transcription of all other genes
   • Late Genes

3. Life Cycle

   • Binds cells via CAR receptor
     • Enters cells by r.m.e
       • Escapes endosome through lysis of endosomal membrane
         • Virion translocates to the nucleus via microtubules
           • Genome released into nucleus, where it remains as a non-integrating episome (no insertional mutagenesis)

4. Production of Vectors

   • Human pathogen - must be disabled to use as a vector
   • Deleting early genes required for replication and replacing with therapeutic transgene
   • Uses packaging cell lines where viral proteins expressed on plasmids to assemble viral particle, whilst deleting them from the genome to make replication incomptent
   • 1st Gen
     • E1A and B genes deleted (transcriptional activators) --> replication incompetent
     • Allowed incorporation of up to 6kb of DNA
     • Failed in clinical trials, E1-like cellular proteins were able to activate expression of other adenoviral genes, which produced a large immune response
   • 2nd Gen
     • Deleted E1, E2 and E3 genes --> Replication incompetent
     • Allowed packaging of 18kb DNA
     • Only transient transgene expression possible
     • Still produced an immune response which prevented repeat administration
   • 3rd Gen 'Gutless' vectors
     • Removes as much viral dna as possible, leaving only the packaging sequence and ITR
     • Packaging of up to 36kb, stuffer DNA must be added to make this up
     • Uses packaging cell line which expresses Cre recombinase - deletes any material between lox P sites
       • A helper adenovirus containing lox p sites is transfected into cell line - all its DNA is removed including its packaging sequence
         • Therefore genome doesnt get incorporated into vector. However, transgene engineered to have packaging sequence and is packaged into new virus particle
           • Cre recombinase only 99.9% effective, 0.01 contamination of live adenovirus --> In large titres, this is a lot --> Big immune response
   • Manipulating tropism
     • CAR receptor not expressed well in cancer cells --> Minimal infectivity
       • Can use bispecific conjugates --> Cover glycoproteins on the surface with PEG to prevent CAR binding, then add a ligand to target other receptors such as FGF2, which is upregulated in many cancers
   • Pseudotyping
     • Many serotypes of adenoviruses with different tropisms, e.g. May use glycoproteins on F40 and F41, which are specific for GI tract, on an Ad5 vector to influence infectivity of specific cell types

5. Problems: Jesse Gelsinger

   • Trial run by scientists at PennState
     • James wilson and colleagues now banned from conducting trials on humans
   • Aimed at patients with ornithine transcarbamylase (OTC) deficiency --> 2nd enzyme in usea cycle.
     • Deficiency leads to accumulation of ammonia, a neurotoxin
       • Babies normal at birth as mother has functional OTC, onset features 24-72 hours after feeding commences
         • Total lack of OTC leads to death shortly after birth
           • Partial OTC deficiency may usually be controlled by drugs and dietary intake
             • Wanted a short, sharp burst of OTC expression to give time for doctors to formulate a drug plan
   • Phase 1 trial - 18 patients with partial OTC loss were given a 2nd gen vector in increasing doses
     • Jesse received 6x 10^13 particles straight into liver --> Died 2 days later from multiple organ failure
       • Due to massive immune response - First death in a gene therapy clinical trial
   • FDA inquiry found 4 major errors during the trial:
     • 1. Jesse was not eligible. Liver function was abnormal after a viral infection. Trial changed criteria without FDA approval so he could be included
       • 2. A number of primates had died via this approach in pre clinical trials - patients were not informed of this
         • 3. Other patients had experienced grade III toxicity at lower doses than given to Jesse - trial should have stopped here
           • 4. There was pressure on the team from investors

6. In Cancer

   • Want to use a vector whose replication is blocked in normal cells, but allowed in cancer cells
   • Onyx-015
     • 50% of tumours mutated in p53
       • E1B 55K inhibits p53
         • E1B 55K deletion allows normal p53 to inhibit virus replication, but mutant p53 (in cancer cells) cannot.
     • Targeted at inoperable head and neck tumours to wash out after surgery
     • Early clinical trials show virus has little effect in first 10 days, but after this, once virus is replicating, there is a dramatic decrease in tumour size
       • Carries on for 3-4 weeks before virus cleared by immune system
         • Treatment may be enhanced by in combination with chemotherapy to depress the immune system
           • Very promising
     • Thought that it may have another mechanism as virus has been shown to replicate in p53+ cells too
       • E1B 55K has ub. ligase activity and targets a specific cellular protein to enhance viral mRNA export, which cancer cells are already upregulated in
         • Combination of this and p53 which gives activity
   • Genedicine - Spearheaded by China, reintroduces WT p53 - startling results in clinical trials that haven't been seen in clinic thus far
   • Advexin - Reintroduces WT p53 for germline mutations (Li Fraumeni). Used in clinic in USA, direct injection into tumours seems very effective

7. As gene therapy vectors

   • Cons
     • Immune response (cellular and humoural) - adenoviruses cause colds- everyone will have antibodies
     • Lack of persistance of the episome - not replicated with genome, may only be used for short, sharp gene expression
   • Pro's
     • Transduce a wide range of cells (CAR present on many cells)
     • No insertional mutagenesis
     • Infects both dividing and non-dividing cells
     • 3rd generation vectors can contain large amount of DNA (up to 36kb)
     • Easy to produce high titres