Lecture 18 Thursday November 15 2007

Thursday November 15, 2007

gene therapy

  • somatic cell gene therapy
  • single gene defect diseases
  • gene identity, sequance and function must be known in order for gene therapy to work
  • you have to understand the biology of the disease really well
  • you have to be certain that adding back copy of the gene will fix the problem
  • often adding back causes a host of unwanted side effects
  • delivery of the new gene to affected cells is also a challenge

successful gene therapy

  • you have to target the gene to the right cell
  • the new gener has to activate in the DNA
  • integration inserts the genome into
  • ensure there are no harmful side effects

Vectors

  • a vector is a carrier
  • a carrier of the the replacement gene
  • the vector also has to be harmless
  • easy to make large quantities
  • targeted to certain cells or tissues
  • heritable change

2 main ways gene therapy is done

  • EX vivo or Invivo

Exvivo

  • target cells manipulated outside the body then returned to patient
  • taking cells out of the body
  • growing the cells in a dish with a virus containing the vector dna treated with Gene Therapy

In vivo

  • target gene delivered to target cell in the patient
  • injected into the body

types of vectors

  • VIral vectors
  • they make the virus as harmless as possible
  • non viral vectors
  • DNA vectors

viral vector advantages

  • viruses enter cells efficiently
  • they can be engineered to target specific cell types
  • can be modified so the virus is inhibited from replicating or destroying cells
  • viruses can stably integration
  • often they remain as episomes - which is in the nucleus and replicated during mitosis but does not last forever

viruses

  • immune system recognizes the virus and tries to destroy it
  • integration can cause mutation - viruses may be able to accumulate mutations and become harmful (HIIV accumulates rapid mutations)
  • limited insert size - some genes are quite large
  • some viruses are small
  • viruses have preferential places in DNA that they like to bind to - often thesei niches are near activly replicating genes

non viral

advantages

no immune response
no size limit

diasdvantages

  • do not integrate into the dna
  • not very efficient at getting into the cell

retroviruses

  • used in more thn 30% of current clinical trials
  • its denome is RNA (reverse transcription)

reverse transcription

  • it is an exception to the central dogma
  • DNA - RNA - PROTEIN
  • reverse transcription PROTEIN - RNA - DNA

*virus waits until the cell divides to infect the nucleus

adenovirus

  • used in about 30% of current clinical trials
  • causes the common cold
  • double stranded DNA genome
  • infects dividing and non-dividing cells
  • no integration
  • very immunogenic - causes a strong immune response (which is dangerous and can lead to death

Adeno-associated virus

  • single stranded DNA genome
  • does not cause any known diseas in humans
  • can infect both dividing and non dividing cells
  • this virus is ineffective and can not activate without a helper virus
  • Integrates into specific site on chromosome 19 (95% of time)
  • often this virus is genetically engineered not to integrate

herpes simplex virus

  • single stranded DNA genome
  • infects neurones

liposomes

  • artificial lipid sphere with aqueous core
  • plasmid DNA (double stranded circular DNA)
  • infects any cell type
  • no immune response but not very efficient

Manipulating DNA in the Lab

plasmids are found in bacteria
start off with circular DNA

  • DNA fragment to be cloned

recombinant DNA - dna that has been manipulated

restriction enzymes

  • restriction enzymes cut DNA at specific sequences (staggered or blunt cuts, palendromes)
  • if these enzymes cut DNA and plasmid then they can be pasted back very easy
  • often then annealing can occur (aatt = ttaa)

cDNA

  • complementary
  • reverse tran
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