A vehicle (e.g. a plasmid) used to transfer the genetic material such as DNA sequences from the donor organism to the target cell of the recipient organism

Schematic diagram of a generic viral vector

A viral vector is a virus which has been modified in a laboratory environment for purpose of introducing genetic material into a cell.

To form a viral vector, remove the genes in the virus that cause disease.

Then replace those genes with genes encoding the desired effect (for instance, insulin production in the case of diabetics)..

This procedure must be done in such a way that the genes which allow the virus to insert its genome into its host's genome are left intact

Converting a virus into a vector

A packaging (helper) construct, containing viral genes derived from the parental virus that encode structural proteins and proteins that are required for vector genome replication, is introduced into a packaging cell line along with a construct that contains the vector genome

The helper DNA can be delivered as a plasmid or helper virus, or it can be stably integrated into the chromatin of the packaging cell.

Pathogenicity functions and the sequences that are required for encapsidation are eliminated from the helper construct so that it cannot be packaged into a viral particle.

The vector genome contains the transgenic expression cassette and is flanked by inverted terminal repeats and cis acting sequences that are required for genome encapsidation.

Some vector genomes retain viral genes that are relatively inactive, as a result of the elimination of viral early genes that are required for their transcription.

Viral structural proteins and proteins that are required for replication of the vector DNA are expressed from the packaging construct and the replicated vector genomes are packaged into virus particles

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Origin of replication

Promoter

Cloning site Protein

purification tags

Reporter genes:

Targeting sequence

Antibiotic resistance

Epitope

Genetic markers

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Origin of replication

Promoter

Cloning site

for replication and maintenance of vector in host cell.

•to drive transcription of vector's transgene

•Also to drive transcription of other genes in vector such as the antibiotic resistance gene

•allow for the insertion of foreign DNA into the vector through ligation

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Protein purification tags

Some expression vectors include proteins or peptide sequences that allows for easier purification of expressed protein.

Reporter genes:

Targeting sequence

that allow for identification of plasmid that contains inserted DNA sequence.

that directs the expressed protein to a specific organelle in the cell or specific location

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Antibiotic resistance

Epitope

allow for survival of cells that have taken up the vector in growth media containing antibiotics through antibiotic selection.

allows for antibody identification of cells expressing the target protein.

Genetic markers

allow for confirmation that the vector has integrated with the host genomic DNA.

Vectors perform their functions in two ways mostly:

Transcription:Expression:

Prokaryotes expression vector:

Promoter Ribosome Binding Site (RBS)Translation initiation site

•there are two types of expression vector:Prokaryotes expression vectorEukaryotes expression vector

Viruses are highly evolved natural vectors for the transfer of foreign genetic information into cells.

But to improve safety, they need to be replication defective.

So the viruses can be used as vehicles to carry 'good' genes into a human cell.

Eukaryotes expression vectors:

These require sequences that encode for: Polyadenylation tail Minimal UTR length Kozak sequence

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Key properties for a viral vector

Safety

Low toxicity

Stability

Cell type specificity

Identification

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Safety

Viral vector should be modified in such a way as to minimize the risk of handling them.

Usually involves the deletion of a part of the viral genome critical for viral replication

Low toxicityviral vector should have a minimal effect on the physiology of the cell it infects.

Stability

Some viruses are genetically unstable & can rapidly rearrange their genomes.

This is detrimental to predictability and reproducibility of work conducted using a viral vector and is avoided in their design.

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Cell type specificity

Most viral vectors are engineered to infect as wide a range of cell types as possible.

viral receptor can be modified to target the virus to a specific kind of cell. Viruses modified in this manner are said to be pseudo typed.

Identification

Viral vectors are often given certain genes that help identify which cells took up the viral genes. These genes are called Markers

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What are main types of viral vectors?

DNA viral vectors

Adenovirus

Adeno-Associated virus (AAV)

Herpes virus

RNA viral vectors

Lentivirus

Retrovirus

Retroviral Vectors• Retroviral vectors are commonly used and known to integrate into

the genome of the infected cell in a stable and permanent fashion.

• Reverse transcriptase in the virus allows integration into the hostgenome.

• There are two types

of retroviral vectors:

replication-competent and

replication defective.

• Usually replication-defective vectors are preferred in practice asthey allow for several rounds of replication due to their codingregions.

Lentiviral Vectors• Lentiviruses are a type of retrovirus that are able to integrate

into non-dividing cells and do not require mitotic cell division in order to function.

• Instead, the genome enters the cell DNA via reverse transcription and is incorporated in a random position of the cell genome.

Adenoviral Vectors

• Adenoviral vectors have a wide rangeof action and are able to delivernucleic acids to both dividing andnon-dividing cells.

• Adenoviruses are often responsiblefor respiratory, gastrointestinal andeye infection that affect humans.

• As a result, research is currentlybeing conducted to investigate theuse of adenoviral vectors inapplications of gene therapy andvaccination.

Adeno-associated viral vectors

• Similarly to adenoviral vectors, adeno-associated viral (AAV) vectors can deliver genetic material to dividing and non-dividing cells.

• It is a small virus that is known to affect humans with a very mild immune response.

• As a result AAV vectors have beneficial properties for gene therapy that are effective with limited negative effects. However, the utility of this type of vector is significantly limited by its restricted capacity of DNA.

Herpes Simplex Virus Vectors

• This type of viral vector has the ability to deliver large-scale quantities of exogenous DNA.

• The primary concerns with the use of herpes simplex virus to deliver genetic material are cytotoxicity and the maintenance of transgene expression.

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In Gene therapy

In vaccines production:

Main applications of viral vectors

Applications;Cancer- transfer suppressor geneImmunology-Transduce haemotopoietic stemcell(HSC)Stem cell biology -Transfer Human stemcell

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In Gene therapy:

Gene therapy is a technique for correcting defective genes responsible for disease development.

There are following delivery systemfor gene therapy:• Physical methods• Non-viral vectors• Viral vectors

Virus is usually “crippled” to disable its ability to cause disease and viral methods have proved to be the most efficient to date

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In Gene therapy:

If the pathogenicity of a specific virus, such as adenovirus, can beeliminated while the efficiency of gene transfer and expression is retained,the gene may be well suited for gene therapy.

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In vaccines production:

Viruses expressing pathogen proteins are currently being developedas vaccines against these pathogens, based on the same rationale asDNA vaccines.

T-lymphocytes recognize cells infected with intracellular parasitesbased on the foreign proteins produced within the cell.

A viral vaccine induces expression of pathogen proteins within hostcells. Since viral vaccines contain only a small fraction of pathogengenes, they are much safer and sporadic infection by the pathogen isimpossible.

Adenoviruses are being actively developed as vaccines.

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This vaccine approach involves:

inserting influenza virus genes into a different carrier virus, or vector, that is used as a vaccine.

DNA or RNA-encoding influenza proteins, such as hemagglutinin, are engineered into a vector that infects humans but does not cause disease.

With a microbial vector vaccine, the vector itself, including the influenza genetic material, is injected directly into a person.

The harmless vector virus can then express the proteins necessary to prompt an immune response.

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Retrovirus Structure:

Outer coat: Inner core:

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Retroviral mediated gene transfer:

Transfection of packaging cell line

Virus Collection

Transduction in target cells

The production of replication defective virus is accomplished when packaging cells are transfected with plasmids that express all of the viral proteins necessary to generate infectious particles,

as well as the nucleic acid sequence of interest that will be packaged within them for delivery.

retroviral vectors produce replication defective, or self-inactivating, particles.

This allows for delivery of the desired sequence, without continued viral replication in the target cells.

1.Transfection of packaging cell line

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The packaging cells produce infectious particles, whose genome onlyencodes sequences from the transfer plasmid, which can be used totransduce the target cells.

The highest concentration of virus is typically produced between 48-72hours.

2-Virus Collection

To collect the virus, removethe supernatant from thepackaging cell line.

The purified virus containingthe desired gene can bestored at -80 °C until needed.

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3-Transduction in target cells

Viral Envelope glycoprotein interacts with the cellular receptor and enters the actively dividing cells.

The viral dsDNA of retroviruses is not capable of passing through the nuclear pore complex and requires breakdown of the nuclear membrane during mitosis for integration of nucleic acid.

The viral genome enters the nucleus and desired gene sequence is integrated into the host genome through the activity of restriction enzymes and Integrase while the other portion of viral genome is self inactivated and replication defective.

Some transfer vectors also contain a marker such as a fluorescent reporter or drug selection marker for the selection of transducedcells.

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Principle of retrovirus vector gene transfer

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The structural genes gag, poland env are deleted from the virus vector which instead carries the therapeutic gene and the psi packaging signal.

Therefore, the vector requires complementation of the deleted structural genes for formation of recombinant infectious virus particles, which is mediated by the helper cell.

These cells harbour the ‘wild-type’retrovirus lacking the psi packaging signal.

The retroviral vector is transduced into the helper cell where recombinant virus particles are produced.

These particles can be used to infect the target cells via specific receptors to start reverse transcription for random integration of the proviral DNA into the host genome where the vector starts expression of the therapeutic gene.

Adenovirus vectors are based on serotypes 2 and 5.

Therapeutic genes are placed into the deleted E1 region of the viral genome, driven by internal promoters.

The function of E1 for production of viral particles is provided by the complementing cell line expressing E1.

Structure of adenoviral vectors and principle of adenovirus production.

This cell line produces viralstocksat high titres for infection of desired cells and tissues.

After infection of target cells, viral particles enter the cytoplasmicendosome and deliver the viral DNA harbouring the therapeutic gene.

Gene expression is performed from the epichromosomal viral DNA.

The wild-type AAV consists of the viral genes rep and cap coding for the different rep (Rep78, Rep68, Rep52, Rep42) and cap (VP1, VP2, VP3) proteins, the AAV promoters (p5, p19, p40), the polyadenylation site (pA) and the inverted terminal repeats (ITR).

In rAAV vectors, the viral rep and cap genes are replaced by a transgene cassette carrying the promoter, the transgene and the pA-site.

Structure of adeno-associated virus (AAV) vectors

Comparison between bacterial & viral mediated gene transfer:

Viral mediated

DNA insert length is upto30kbp

More transfectioneffeciency

Long term persistence and stable gene transfer

Bacterial mediated

DNA insert length is upto 10 kbp

comparitively less transfection effeciency

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