Vectors Flashcards
what is the genetic map importance
fundamental resource in the future of biomedical research into disease
how many base pairs does the human chromosome house
3 billion base pairs of DNA that contains only 30,000 - 40,000 protein-coding genes (5% of the genome)
what part of genetic disease is good for gene therapy
Most of the genetic diseases are defined by a mutation in one gene
what makes some diseases harder to cure
some diseases e.g. diabetes and asthma, more then one gene so understanding disease, is more difficult and hence so to a cure
what is sickle-cell anemia
inherited, chronic blood disease in which of red blood cells become crescent shaped and function abnormally
why do patients become anemic due to sickle-cell
Patients become anemic because of decreased ability of red blood cells to provide adequate oxygen supplies to body tissues, usually die between the ages of 20-40
what is the ratio of sickle cell anemia of african origin
1:500
what causes sickle cell-anemia
Autosomal recessive mutation in HBB (human beta-globin) gene, chromosome 11
normal haemoglobin is called A
what is the defect in sickle-cell
Haemoglobin molecule-S caused by a single base change results in a E –> V causes sickled red blood cells
what is A/S sickle cell
heterozygotes, have sickle cell trait are carriers, (have mild anemia, intermediate inheritance)
what is S/S sickle cell
S/S are homozygotes, have disease
what is the treatment for sickle cell
no cure for sickle cell, since the gene has been mapped, a prenatal diagnosis is available using RFLP analysis
what does RFLP analysis allow
This allows parents to determine if fetus will be A/A, A/S or S/S
whether to keep or abort
what is A/A sickle cell
normal adult haemoglobin
why is sickle cell still present
Sickle cell people are resistant to malaria so provides an advantage
what is gene therapy
transfer of selected genes into a host with the hope of curing a disease state
what is traditional pharmaceutical development
Pharamceuticals that will interact with gene products
what is gene therapy
if gene is corrected
what is cystic fibrosis treated with
antibiotics
what does antibiotic treatment of cystic fibrosis do
clear lungs - traditional
what is the gene therapy of cystic fibrosis
genetic treatment correct DNAse gene
what is SCID
Severe combined Immunodificiency Disease
what has caused SCID
number of mutations have been attributed to causing SCID
what causes SCID
Genetic mutations occurs on Autosomes 19 and 20 and also on X Chromosome (where boys would be effected, since girls have 2 copies of the X chromosome, second of which is likely to be normal)
what are SCID children called
bubble babies
why are SCID children called bubble babies
they must live in a sterile environment as they can not cope with infections
where are viral vectors used
Used in both ex vivo or in vivo therapies
how do viral vectors propagate
Normally are replication defective, need to propagate in special cell lines
what do vectors include
Retroviral
adenoviral
Adeno-associated
Herpes virus
what is ex vivo gene transfer
transfer of genetic material to cells isolated from host
following transfer cells are implanted back
indirect gene transfer
what is the advantage of ex vivo gene transfer
can take out cancer cells
what is in-vivo gene transfer
Transfer of genetic material to cells located within host
aka direct gene transfer
what are the advantages of retroviral vectors
- Easy to work with, plasmids are easy to engineer
- Packaging lines available to simplify generation of viral particles
- Gives high number of transfected cells
what are the disadvantages of retroviral vectors
- Limited gene capacity (6KB)
- Integrate into human genome and therefore are potentially oncogenic (cancer) due to insertional mutagenisis
what happens in ex-vivo gene therapy
- Bone marrow cells removed
- Bone marrow enriched to isolate parent cells or immune system
- Cells infected with virus containing gene therapy
- Cells take up gene, normal version of which is missing in boys
- Cells returned to baby boy
- After 2 weeks immune system develops top protect baby from infection
ADA expressing cells are transferred back
what is one of the mutations in SCID
in gene Adenosine deaminase
what is ADA
adenosine deaminase
what effect does a loss of function mutation in ADA have
causes a build up of adenosine which causes death of B and T cells lymphocytes which are crucial to our ability to fight infections
what are viral vectors grown with
viral vectors have been grown which contain ADA
where are viral vectors that contain ADA used
transferred to totipotent stem cells (bone marrow cells)
cells were the transferred to patient and ADA expressing cells corrects defect
what happens in retrovirus life cycle
Virus comes in, inserts ssRNA in which is converted to DNA integrated into genome = provirus
As a provirus it can express its genes
Normally in retrovirus late phase it transcribes, translates is assembled to be moved out of cell
how are the early and late phases differentiated
provirus stays in cell in early phase and passes out in late phase
what would happen in gene therapy retrovirus cycle
In gene therapy would not want it to leave and make a virus
what happens in early retrovirus life cycle
Produce DNA copy of RNA genome by using RT carried within virion
transport of vDNA to nucleus
integrating vDNA into host genome
what happens in late retrovirus life cycle
viral mRNA transcription and progeny viral RNA from vDNA, driven by promoter in 5’ Long Terminal Repeat
Translation in cytoplasm of Viral proteins
Formation and packaging of viral capsid with two RNA strands and RT molecules
Budding of enveloped virons to exterior of cell
what is gag region
gag region encodes capsid proteins
what is pol region
pol encodes reverse transcriptase and integrase proteins
what is the env region
env encodes envelope and proteins needed for receptor recognition and envelope anchoring
what is psi
packaging signal
what is the long terminal region important for
LTR is important in initiating viral DNA synthesis and integration
what happens when virus infected into human cells
Once infected into human cells virus will integrate but can’t make virus as doesn’t contain genes for virus (gag, pol and env)
what happens in MMLV ADA expressing virus
package vector RNA (encoding ADA) into viruses which then can then be used to deliver genes to human cells
what is used to make a ‘hollowed out’ virus genome
MMLV vector
what does MMLV vector genome contain
packaging signals psi Ψ+ and LTRs from Virus
what selectable marker gene does MMLV vector contain
neomysin
what is the transgene in MMLV vector
adenosine deaminase
why can MMLV vector get out of the cell
Does not encode viral genes gag, pol and env
what is a selectable marker
Often to select transgene, one must co-express a selectable marker, allowing only cells expressing transgene to be expressed
what is a transgene
selected gene tested in a gene transfer experiment
what does a viral vector contain
Psi packaging signal
Long terminal repeats
Gene x – e.g. ADA
Promoter e.g. SV40 Important for transcription enhancement and drives neomycin resistance
what happens in MMLV therapy for SCID
- MMLV vector expressing ADA is constructed
- Vector transfected onto packaging cell line, producing a Producer cell line
- Virus produced from Producer cell line that expresses ADA
- virus used to infect stem cells, isolated from bone marrow
- cells, expressing ADA are then transferred to SCID patient
what does the packaging line encode
gag
pol
env
what does the packaging line synthesise
viral proteins but since lines contain no encapsidation signal (ΔΨ) in either gene only empty capsids are produced
what is the producer cell line in MMLV
producing recombinant virus which contains ADA and neomycin resistance
how is a retroviral vector RNA made
packaging cell line, want to add: ADA, long terminal repeats and neomycin
Genes encapsulated into a virus by psi
vector transfected onto packaging cell producing specific producer cell line
virus is produced encodes ADA
what happens after transfection of packaging cell line with ADA retroviral vector DNA
DNA integrates into chromosome
what is transcribed and put into viral capsids
Full length RNA from the retrovirus vector that carries the encapsidation signal psi (Ψ+)
can viral particles replicate after made
can only replicate on packaging cell lines as don’t have gag, pol and env genes to make a virus
what is the ADA expressing virus used for
infect stem cells isolated from bone marrow cells
once virus in stem cell can ADA and neo still be expressed
Once in stem cell cant make anymore virus can just express these genes
ADA expressing virus can integrate and express (ADA and neo)
can not make a virus can only do so in packaging cell line, stem cells do not express gag, pol and env
