ANIMAL VIRAL BASED VECTORS

By Prema . B 14PGZ002

Introduction• Viral vectors- They are more efficient gene delivery

vehicles.• Other than non viral and physical method of gene

transferring system, it gives efficient transfection.• Main disadvantage is that insert size limitation ,

immunogenicity and manufacture.

Why we use viral vectors• Virus are obligate intracellular parasites.• Very efficient at transferring DNA into host cells• It has specific target cells.• Non-essential genes of virus are deleted and genes of

interests are inserted.

• However transfection using viral vector is still a choice as construction of this vector is a laborious process.

Conts…..• The first animal vector was devised from the primate

papova virus, simian virus 40 (SV40) and it was used for cloning in 1979.

• But after that many vectors have been developed from many other virus.

• E.g., papilloma virus, adenoviruses, the Epstein-Barr herpes virus, vaccinia viruses, and baculo viruses.

Types of viral vectors• Adeno viral vector• Sv40 vector• Adeno associated vector• Retro viral vector – lenti viral vector• Baculo virus vector etc.,

1.Adenoviral vector• Adenovirus :

• Non enveloped DNA virus.• Genome : linear, ds DNA of 36 kbp in size .∼

Vector • Doesn’t integrate into host genome.• Replicates as an episomal element in the nucleus.

Conts..• Has six early transcription units – essential for viral replication.• Major late transcript – encodes for capsid components.

AV is widely used as gene transfer and expression vector...?? 1.Stability

2.High capacity for foreign DNA3.Wide host range

4.Ability to produce high titre stock • Suitable for transient expression in dividing cells – they do not integrate into

the genome.• EXCEPTION – Prolonged expression in post mitotic cells (e.g., neuron).

Genome organizationImmediate early – E1AEarly – E1B, E2A , E2B, E3 & E4Late – L1 to L5The 3 main goal of these proteins are ; to alter the expression of host proteins that are necessary for DNA synthesis ,to activate other virus genes , to avoid premature death of infected cell by host immune defenses ,to activate other virus genes ( such virus encoding DNA polymerase).

IR- helps in linearizing the strandPsi site – packaging signal site – helps to package DNA in a perfectly designed structure.

Generation of adeno virus expression system

Construction of recombinant adeno virus

First generation- deletion in E1 encoding productsSecond generation – also deletion in E2 / E4Third generation – called as gutless vector containing inverted terminal repeats and packaging signal (without E1 ,E2 & E4)..

Significance of last generation ad vector

2. Adeno - associated virus• Its a simple, non-pathogenic, single stranded non

enveloped DNA virus (4.5 kb in size).• Infect non dividing cells like hematopoietic progenitor cells

and post mitotic cells of neurons.• Stably integrate into host genome at a specific site in the

human chromosome 19.

Genome organization• It contains two genes (cap and rep), sandwiched between

inverted terminal repeats that define the beginning and the end of the virus and contain the packaging sequence.

• The cap gene - encodes viral capsid proteins • The rep gene - involved in viral replication and integration• ITR- primase independent synthesis of second strand

(helps in converting ss to ds DNA )

also required for integration into host cell and rescue from it

Conts…

It contains 3 promoters – each producing Rep / Cap transcript of different lengths

Rep 78/68- genome replication(transcripts using the P5 promoter)

Rep 52/40- genome packaging(using the P19 promoter.)

Vp – capsid proteins.(three viral capsid proteins (VP1, VP2, and VP3) using the P40 promoter)

To produce an recombinant AAV vector, the rep and cap genes are replaced with a transgene.

Production of the recombinant vector requires that rep and cap are provided in trans along with the helper virus gene products

AAV vector

3.Retroviral vector• Single stranded.• Has two copies of the genome, which resemble eukaryotic mRNAs.• The viral genome is reverse transcribed by reverse transcriptase into

a DNA double-strand copy inside the host cells. Features of RV vectoro Contains gene for replication, expression and packaging (ψ

sequences). o Gene of interest may inserted in the nonessential coding region or it

may replace some essential gene (gag). o genomes are used as vectors, generally as shuttle vectors.

RV vector

Lentiviral vector• Belong to the retrovirus family but can infect both dividing

and non-dividing cells.• It act as a vector for in vivo gene delivery.• Long term gene expression.

• They are more complicated than retroviruses, containing an 9 virulence proteins.

• Out of it 6 genes (tat, rev, vpr, vpu, nef and vif.) will be removing without altering gene transfer ability.

Making of lentiviral vector• Lentiviral vector is produced by co transfecting 3 tpes of

plasmid in HEK 293T cells.• Includes envelope plasmid , packaging plasmid, transfer

plasmid.• In the last generation the rev & tat genes are not included.

Generation of LV vector• 1st- composed of all HIV genes except the envelope

protein.• 2nd- deletion of 5 to 9 genes (leaving only gag/pol or

tat/rev – responsible for structural and enzymatic components , transcription and post transcriptional genes respectively.

• 3rd- safest vector – contains only gag , pol , rev and utilizes 5 prime LTR’s in the absence of tat.

Further modification• To make the virus not to revert back to its original form .• Make it unable to reconstitute its promoter.• In higher generation vector the transfer vector contains

WPRE to increase the level of transcripts.• These modifications are made to make it inactivate.• Involves removal of U3 from 3’LTR.

Applications of viral vector• The applications of cloning vectors in recombinant DNA

technology is immerse. • Cloning DNA fragments.• Discovering disease genes.• It is used for making Vaccines.• It is used in making transgenic animals.

Conclusion• With a aid of more advanced models and broader base

immunological knowledge , correction of genetic disease currently untreatable may someday be achieved through the use of viral vectors.

Book references• P.Ramadass., ‘Animal Biotechnology’., 2008 MJP publishers chennai.• Richard J Reece., Analysis of genes and genomes.,John

Wiley & Sons Ltd (2006).

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