Intro to gene therapy NOTE

  • plasmid=transfection

    Form of transportation of gene to a patient

  • Infection=transduction

    Form of transportation of gene to a patient

  • Example of genetic issues gene therapy helps with:

    • Cystic Fibrosis

    • “bubble boy disease”

    • Haemophilia

  • What is gene therapy?

    A treatment of a disease by introduction of genetic material into cells of an affected patient to modify specific gene expression. All present gene therapy is based on gene addition

    • What changes this?

      CRSPR-9 gene editing

  • What genetic material is used?

    DNA RNA, OLIGONUCLEOTIDES

  • How is instructed DNA manufactured?

    Like a drug with strict requirements.

  • What is germ line therapy?

    It is specific oocyte pronucleus injection or perivitelline infection. It is not currently allowed. Many ethical debates.

  • what is somatic gene therapy?

    Only aiming at somatic cells this is allowed.

  • How does injection reach the oocyte injection?

    There is perivitelline space between the oocyte plasma membrane and the zona pellucida, gene can be injected there and they reach the oocyte.

    • Diagram

  • What is pronuclear injection?

    It can be used to make transgenic animals, which is a direct injection of DNA into the nucleus

    • What equipment is used?

      A micropipette and an inverted microscope with 200x magnification.

  • What is nuclear transplantation?

    Also known as the dolly technique, it allow for correct homologous recombination but has risks of technology unacceptable.

    • Why is the risk unacceptable?

      Clones have higher health issues at younger ages alongside possible misuse due to ethical concerns.

  • How are embryonic stem cell manipulated?

    Cells are removed from blastocyst to enable homologous recombination. The implantation of a manipulated ES cell back to the blastocyst can produce chimeric offspring.

  • What is somatic gene therapy?

    It is a non-targeted delivery that has potential to affect all cells of an organ or organism.

    • What are the issues?

      it may reduce efficiency of transfer. and cause harmful effect of ectopic expression.

  • What are targets of gene therapy?

    • Cancer

      It can kill off cancer cells via suicide genes or immune stimulation. It can also provide correction by inactivation of oncogenic sequences.

    • Infectious disease

      It can kill of infections using immunisation i.e. DNA-vaccine, or it can kill a gene product using antisense.

  • What are the ideal vectors for gene therapy?

    It should enable safe and permanent transfer with efficient, physiologically regulated gene expression. It must also be able to target affected cells only by a single application.

    • What is the theoretical best strategies to avoid random integration?

      homologous recombination, autonomously replicating episomal element.

  • What are the advantages and disadvantages of non-viral vectors?

    • Advantage

      • Unlimited DNA- packaging capacity

      • Low toxicity

      • ow immunogenicity

      • Non-infectious

      • Easy chemical production

      • Easily modifiable, highly versatile

      • targetable

    • Disadvantages

      • Long process of optimisation to reach high efficiency

      • Low efficiency

      • Transient persistence

  • What are the advantages and disadvantages of adenovirus vectors?

    • Advantage

      • Suitable for in vivo use especially in lungs

      • Very high virus titres

      • Biology well understood

    • Disadvantages

      • No integration into host genome

      • Virus-proteins can cause dose dependent inflammatory reactions

      • Complicated vector design and limited insert capacity.

  • What are the advantages and disadvantages of adeno-associated vectors?

    • Advantages

      • small, not complicated genome

      • Preferential integration of wt into human chromosome

      • Long term gene expression

      • Non-pathogenic

      • Humans are natural hosts

      • High titre

    • Disadvantages

      • Limited capacity for foreign genes

      • Biology not well understood

      • Mechanism in recombinant unclear

      • Requires adenovirus as helper for replication

  • What are the advantages and disadvantages of retroviral vectors?

    • Advantages

      • Small and simple genome

      • Good insert capacity for foreign DNA

      • Stable co-linear integration into host genome

      • High efficiency of transfer

      • No toxic effect on host cells

    • Disadvantages

      • Infects only dividing cells lentiviral group

      • Random integration may interfere with cellular genes

      • May recombine to replication competent virus

      • Often only transient expression

      • Relatively low virus titre

  • Induced pluripeurtant stem cells

    Reversing a skin cell to a STEM cell