Viral vectors: Viruses as vehicles for gene transfer: · Decouple virus from pathology. Virus -> vector. Viruses often pathogenic to the host. · Replication-defective viral vectors- prevent self- replication in the host - normally pathogenic. Complementing transactor producer cell systems. Safety mechanism. Decouple viral propagation from the therapeutic gene. Need to propagate virus to produce it, but don't want it to do that when it is used. · Tissue specificity: viral vector tropism - which cell virus infects, promoters- genetic elements to target gene delivery. Want to target specific tissues/cells e.g. photoreceptors. · Episomal vs integrated transgene delivery: dif means by which virus can deliver its genes to the host cell. · Ex-vivo vs in-vivo strategies for gene therapy: direct or take cells out and treat then put back. VIRUSES AS VEHICLES FOR GENE TRANSFER 1 VIRAL PROPAGATION PATHOLOGY, DISEASE I VIRAL PROPAGATION THERAPEUTIC GENE TENS OF MILLIONS . OF RECOMBINANT VIRUSES LIVER MUSCLE BRAIN BLOOD Factors required for successful gene therapy: Factors Required for Successful Gene Therapy 1. Uptake of vector by appropriate cells 2. Vector entry into nucleus Receptor Endosome MHCII Nuclear Matrix O Golgi Nucleus CTL Cytoplasm 3. Evade Host Defense · Uptake of vector by appropriate cells: if in vivo. Genes can get to the cell. If virus is injected systemically, needs to over come the host immune system. · Vector entry into nucleus: needs to bind to the cell to be internalised -> endosome -> select what enters and exists. Foreign material is trafficked to a lysosome and destroyed - need to evade this to get out of the endosome. Need to be able to process the DNA into a form that can be used by a cell -> nucleus -> processing in the nucleus must occur. · Evade host defence. Native mechanisms of entry: · Adenovirus S: broad. Binds to the CAR receptor- broadly exp across many cells in the body. General tropism to the liver: filters the blood, good access to the whole systemic., tendency for any virus injected to end up in the liver, many virus infect liver cells. Capsid - interacts w receptors, aspects can be changed in the same way as the envelope.
· AAV2: broad. Bind to heparin sulfate proteoglycan (HSPG) - broadly exp across many cells in the body. Capsid - aspects can be changed in the same way as the envelope. · Retrovirus (lentivirus): HIV1 v specific to CDffi+ cells. Mechanism of entry: depends on cell exp CDffi on cell surface. Specific. WT HIV not good for any condition that doesn't involve CDffi. Envelope virus · Tropism: which cells can the virus infect - Natural: useful, broad range of dif types of viruses can be used - Engineered: if there is no virus/tropism that is appropriate to the cell type wanted, can engineer it. Change aspects of the virus to make it more appropriate to the target. Use HIV1 by changing envelope glycoproteins they don't have wt HIV1 glycoproteins on the envelope. This process is called sudotyping,