Download - Viral Vectors in Gene Therapy 97-2003
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VIRAL VECTORS IN GENE THERAPY
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Gene therapy
Aim of gene therapy: To correct a genetic defect bytransferring of a functional normal copy of the gene intocells
Applies to: Genetic disorder (deficiency)
Cancer Genetic predisposition
Mutation in oncogene or tumor suppressor gene
Autoimmunity diseases: rheumatoid arthritis
Delivery of counteracting gene Viral infections
Delivery of DNA molecules that will produce RNAor proteins against the virus
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Use virus particles to
carry nucleic acids into
cells. Compared to naked
DNA, virus particles
provide a relatively
efficient means of
delivering nucleic acids
into cells, for expression
as recombinant genes
(example = adenovirus)
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Virus are obligatory intracellular parasites
Very efficient at transferring viral DNA into host cells
Target specific cells: depending on the viral
attachment proteins (capsid or glycoproteins) Gene replacement: non-essential genes of virus are
deleted and exogenous genes are inserted.
Viral Vectors: Adenovirus
Retrovirus Lentivirus
Adeno-associated virus (AAV)
Herpes simplex virus (HSV)
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Replication-competent virus (unsafe) Replication-defective virus (rep-)
Doesnt encode viral structural proteins
Cant replicate beyond the first cycle of
infection Elements needed to generate rep- Viral Vector
Transfer Vector: plasmid (promoter,gene of interest, ori, packaging signal)
Packaging vector (large plasmids or celllines): provide the viral structural proteins forpackaging of transfer vector
Helper virus : provides additional
sequences necessary for virus formation.
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Viral particles must infect cells & transport
recombinant viral DNA into nucleus, but
must not be able to produce & release newinfectious particles that could transfer
foreign gene(s) to other cells/tissues or from
patient to another human
Viruses are made defective by deletion ofone or more genes required for replication
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Retroviral vectors
Moloney murine leukemia virus (MuLV) Generation of replication defective retroviral vector
Transfer plasmid vector:-
Gene of interest
Long terminal repeats (LTR) promoter, polyA,
integration, replication and reverse transcription
Primer binding site (PBS) (origin of replication)
RNA packaging signal
Polypurine tract(important for reverse transcription)
Packaging vector:
Cell line stably transfected with plasmid constructs
containing Gag, Pol and Env
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Retroviral vectors- Limitations
A critical limitation of retroviral vectors is their inability to infectnondividing cells, such as those that make up muscle, brain, lungand liver tissue.
The cells from the target tissue are removed, grown in vitro andinfected with the recombinant vector, the target cells are producing
the foreign protein are then transplanted back into the animal (exvivo gene therapy).
Problems with expression being shut off, prolonged expression isdifficult to attain.
Expression is reduced by inflammatory interferons acting on viralLTRs, as the retroviral DNA integrates, viral LTR promoters areinactivated.
Possibility of random integration of vector DNA into the hostchromosome.
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Belong to the retrovirus family but can infect both dividingand non-dividing cells.
They are more complicated than retroviruses, containing anadditional six proteins, tat, rev, vpr, vpu, nef and vif.
Human immunodeficiency virus (HIV) has been disabled anddeveloped as a vector for in vivo gene delivery.
Low cellular immune response, thus good possibility for invivo gene delivery with sustained expression over six months.
No potent antibody response.
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Double-stranded DNA viruses,usually cause benign respiratory disease.
Can infect dividing and non-dividing cells,
can be produced at high titers.
Replication-deficient adenovirus vectors
can be generated by replacing the E1 or
E3 gene, which is essential for replication.
Cells infected with recombinant adenovirus can express the
therapeutic gene, but because essential genes for replication are
deleted, the vector cant replicate.
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Advantages Disadvantages
High titers
Both dividing and
non-dividing cells
Wide tissue tropism
Can modify viral
pentons to alter
tissue tropism
Transient expression (notgood for genetic diseases)
Highly immunogenic High titers of virus can be
toxic
Gene transcription with E1-negative virus is leaky:many genes expressed at low
level CD8 CTL responses to these
processed adenoviralpeptides leads to eliminationof infected cells expressingforeign genes
Newer, gutted adenoviralgenomes lacking majority ofgenes are better, but requirespecial packaging cell lines
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