L30 - New and Emerging Drug Technologies

• When can nucleic acid based treatments be used?

  1 - DNA mutation causes a defective protein

  
  

  2 - not enough protein produced (sub-physiological levels) eg insulin

  
  

  3 - too high levels of protein that exacerbates disease
• what are oligonucleotides


  short nucleic acid sequences
• Why use gene therapy?


  - what do mAbs/therapeutic proteins do in the body?- what happens when gene therapy is used?- mAbs (proteins) bind to either protein or receptor, to prevent natural protein binding and elciting an effect

  - OR proteins are soluble receptors

  
  

  - Use gene therapy so body produces its OWN therapeutic proteins

  
  

  - deliver a GENE that codes for the protein rather than the protein itself

  
• Why is it better to use gene therapy than to deliver protein directly?

  - overcomes difficulties of producing/purifying recombinant proteins as pt's own cells do it!

  
  

  - transient, so need repeated injections of gene/vector

  
• what is siRNA?

  
  

  what does it do?

  Small interfering RNA.

  
  

  short sequence of double-stranded RNA that block expression of a gene with the same sequence as the RNA at translation stage

  
• How does siRNA interfere with gene expression?

  1)DICERcomplex chops up double stranded RNA in cells intosiRNA

  2) siRNA unwinds tosingle stranded RNA(complementary to mRNA)

  3) shortsiRNAbinds tomRNA

  4) InducesRNA Induced Silencing Complex(RISC) to cleave mRNA

  5) double stranded RNA and mRNA derived from it are both degraded, so can't produce proteins

  
• What is DICER?


  an enzyme that cleaves double stranded RNA into siRNA

  
• What is RISC?


  RNA induced silencing complex
• What can we do therapeutically to interfere with gene expression?

  - design siRNAs complementary to mRNAs of receptors

  
  

  - siRNAs bind to their complementary mRNA of the receptor, and a RISC complex forms that cleaves mRNA

  
  

  - hence protein production prevented
• What is miRNA?

  
  

  what's it derived from?

  
  

  what do they do?

  micro RNA

  
  

  - similar to siRNA: short nucleotide coded for by genes in genome

  
  

  - derived from specific genes that code miRNAs, not proteins!

  
  

  - regulate gene expression by repressing/destabilising target mRNA

  
• If miRNAs are expressed poorly in cancer tissues, how can we overcome?

  deliver miRNAs to cancer cells to overcome

  
• If miRNAs are overexpressed in cancer tissues, how can we overcome?


  deliver anti-microRNAs

  
• What are anti-microRNAs?


  complementary strands that bind to miRNAs so they can't bind to the receptor

  
• Describe how the mRNA covid vaccines works

  - mRNA delivered to cell endoplasmic reticulum, and is translated into spike proteins from virus.

  
  

  - spike protein released from cell and enters circulation

  
  

  - protein recognised as foriegn and immune response occurs

  
• What are cell therapies?

  
  

  Eg?

  taking a cell sample from pt, engineering cells in lab (eg so they code for therapeutic proteins) and implanting new cells in pt

  
  

  
  

  Eg - hematopoietic stem cells in bone marrow transferred from donor to recipient

  
• What are CAR T cells?

  Chimeric antigen receptors T-cells

  
  

  used to treat blood cancers

  
• How are CAR T cells used in cell therapy?

  - pt T cells harvested and genetically engineered with CAR (chimeric antigen receptor)

  
  

  - CAR T cells grown in lab, inserted into T cells and given back to pt

  
  

  - receptors can recognise antigens/cancer cells and kill them

  
• What are mesenchymal stem cells?

  
  

  What do they do?

  stem cells found throughout body that are attracted to cytokines secreted by tumours

  
  

  - at tumours, mesenchymal stem cells release a ligand which kills cancer cells
• How can we engineer mesenchymal stem cells?

  
  

  What's an issue with this?

  
  

  What's a benefit of mesenchymal stem cells?

  - engineer to express specific genes that can be used to kill tumour cells

  
  

  issue = scaling up production

  
  

  benefit = "off the shelf" therapy; not pt specific
• What is gene editing?

  
  

  Why is it desired?

  = permanently editing the genome to cure disease state

  desired bc most cell therapies are transient

  viagene disruption(stops gene expression),gene correction(inserting desired base sequence in place of mutated one) orgene insertion

  
• Gene Editing Eg - what is CRISPR/Cas9?

  what's it used for?bacterial defense mechanism which can be used to edit DNA

  
  

  - used to inactivate, correct and insert genes in human cells

  
  

  
  

  - we can develop target sequences as we know the human genome

  
• Eg: How can we use gene editing to cure HIV?

  - HIV enters T helper cells

  
  

  - Th cells must coexpress CD4 with CCR5

  
  

  - HIV needs CD4 and CCR5 proteins to be present to enter cell

  
  

  - use gene editing to stop CCR5 being expressed in the cell of HIV+ pt

  
• Eg how can cell therapy be used to treat blindness due to corneal burning?

  - deliver limbal stem cells on fibrin to repair damage

  
  

  - fibrin placed over damaged cornea

  
  

  - over time, limbal stem cells can repair damage

  
• Tissue engineering


  - what does it do?- how can we do this?

  
  

  Eg trachea transplant, Claudia Castillo- aims to replace diseases/damaged living tissue with living tissue designed specifically for that pt

  1.Isolate cells from pt

  2.Expand no. of cells

  3.Grow on 3D scaffold with growth factors and stimuli

  4.Implant in pt

  
• What are benefits to controlled drug delivery?

  - improved compliance: don't have to remember to take it multiple times a day

  
• MR drugs are great for oral delivery, but not for long term release. How can we overcome this?

  Polymeric systems: entrap drug in polymer!

  
  

  gives sustained therapeutic [plasma] by:

  
  

  - polymer degrading over time to release drug

  OR

  - drug slowly diffuses out of polymer
• How do microchips work in controlled drug delivery?

  - microchips have wells with resovoirs which contain the drug

  
  

  - delivery orifice at top sealed by membrane

  
  

  - can be programmed/deprogrammed to release the drug

  
• What does targeted delivery aim to do?

  - concentrate drug at target tissue (site of action)

  
  

  - minimise loss of drug before it reaches target

  
  

  - minimise elimination

  
  

  - aid intracellular delivery

  
  

  
  

  = ensuring as much drug gets to the target as possible

  
• How can we specifically target drugs?

  Antibody-drug conjugates!

  
  

  - mAbs are bound to small drug chemo/radiotherapy

  
  

  - mAbs bind to specific targets, so can be used to deliver highly toxic drugs to target

  
  

  - ADC taken up into cell, then linker between antibody and drug is cleaved

  
• What's an example of an antibody-drug conjugate (ADC) used in breast cancer?

  Kadcyla = trastuzumab/herceptin + cytotoxic drug

  
• What are oncolytic viruses?

  
  

  Eg?

  viruses which destroy tumour cells

  
  

  Eg Imlygic infects cells deficient in p53 tumour suppressor genes and kills them

  
• What's the EPR effect?

  passive drug targeting strategy:Enhanced Permeability and Retention

  - blood vessels in tumours are leaky due to fenestrations (gaps between endothelial cells)

  - macromolecules/nanoparticles can easily enter, but are retained bc vessels are poorly drained

  - particles can accumulate in tumours: as tumours get large, they're poorly vascularised at core so gaps between endothelial cells close

  
• How can bacteria be used to fight cancer?

  hypoxic areas of tumours are resistant to chemo/radiotherapy

  
  

  SO inject anaerobic bacteria into hypoxic regions, then use antibiotics to remove when finished

  
  

  - bacteria tag tumours with antigens that can be recognised by CAR T cells

  
  

  - tag recognised by proCAR T cells, which bind to tumour and destroy them