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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