Gene Therapy

• The first GMO was made

  1973
• DNA manipulation


  • Most has been done using short fragments in test tubes and bacterial systems
  • DNA can be transferred to human / animal cells to add something new to their genome
  • Insertion is random → unexpected consequences
  • Could be inserted into fertilized ovum to make an animal
• Transgenic animal


  First targeted transgenic animal (mouse) made in 1987
• Polymerase chain reaction (PCR) and chemical synthesis of DNA


  • Made things faster
  • Still a long way off making mammals
• RNAi


  Allowed genes to be turned down, but has to be constantly present
• Achieving a desired genetic change - even in cells in tissue culture remained practically impossible

  
• Gene therapy


  Could only add new DNA
• Limitations of previous DNA cutting methods


  • Most cutting and modification done in vitro not in cells
  • Enzymes used to cut had defined recognition sites, rarely cut exactly where you want, and cut at many other places

  
• Targetted nucleases


  Zinc finger nucleases and TALENs: A new protein required for each site to be cut, proprietary technology, expensive, efficiency limited

  
• CRISPR/Cas9


  Open source, cheap, highly efficient
• Two ways to edit DNA with CRISPR/Cas9

  1. Non-homologous end joining (NHEJ)

  2. Homology-directed repair (HDR)

  
• There are genetic constructs that make Cas9 and have a site to add DNA for any gRNA you like

  
• You can buy Cas9 protein and any gRNA you like, mix them in a tube, and 'transfect' them into a cell
• Off-target effects


  • Considered the main limitation, cutting at undesired locations, partly addressed by combining two Cas9 'nickases'
• Gene therapy


  Can now include gene correction

  
• Targeting the CRISPR/Cas9 is still a major hurdle for in vivo gene therapy

  
• There are CRISPR clinical trials on the US NIH database
• Gene drives


  Genes that are inherited at greater than Mendelian rates
• Normal (Mendelian) inheritance


  There are two copies of the genome, one copy of each gene comes from each parent, the gene will be in 1/4 of next generation
• Gene drives


  A gene drive can cause a gene to be duplicated
• CRISPR has to do with gene drives

  
• Applications of gene drives

  • Inhibiting / controlling insect vectors of disease
  • Pest animal control
  • Controlling pests of agriculture
• Any release of gene drives requires enormous care and consideration, and public scrutiny
• Strategic issues


  • How does Australia ensure we are active participants and not bystanders?
  • How will we decide when genome engineering is considered safe for human medicine?
  • How will we balance potential health benefits against environmental concern (e.g. gene drives)?
  • How will we ensure that policy and the public are informed by science?
  • How will we respond to international regulatory moves (e.g. licencing or moratoria)