viral vectors in gene therapy 97-2003

7/31/2019 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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viral vectors in gene therapy 97-2003

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