VIRAL VECTORS FOR GENE TRANSFER

Seminar on

ANKUR SHARMA

Why use viral vectors?

• Virus are obligate intracellular parasites

• Very efficient at transferring viral DNA into host cells

• Specific target cells: depending on the viral attachment proteins (capsid or glycoproteins)

• Gene replacement: non-essential genes of virus are deleted and exogenous genes are inserted

• Gene therapy

Why mammalian cells?

• Production of recombinant proteins with authentic post

translational modifications (-s-s- bond formation,

glycosylation)

• Used for commercial production of antibodies, hormones,

cytokines, growth factors and vaccines.

SV 40 based Vectors

• First Animal virus based vector developed

• Small virus with icosahedral capsid and circular dsDNA genome.

• Genome size – 5243bp

• Cause lytic infection in permissive host cells resulting in the cell lysis and the release of thousands of progeny virions.

• Origin of replication is 75 bases long and marked by unique BglI

• Replication occurs bidirectionally and no termination signal is required

• The viral genome has two transcription units- early and late regions which face in opposite directions.

• Due to alternative splicing multiple mRNA are produced from both transcripts.

• Early region produces regulatory proteins while the late region produces components of viral capsid.

Lytic infection cycle of SV 40

• Three steps:- A) Early mRNA synthesis• Two partially overlapping genes code for : T-antigen ( large T) :- 90kDa Role in initiation of replication and control of its own

transcription. t-antigen (small t) :- 18kDa Required for interaction of virus with non permissive cells B) Viral DNA replication

C) Late mRNA synthesis• The primary transcript is processed to yield 3 mRNA 16S mRNA,

19S mRNA and 18S mRNA encoding capsid proteins VP1, VP2 and VP 3 respectively.

• Viral DNA is packaged and intact viral particles are released as a result of cell lysis.

SV 40 Vectors• There are two fundamental systems for SV 40 virus based vector

construction :

A) Viral genome as Vector • Used for transduction of foreign gene in permissive cells.

• Either early or late region could be replaced with foreign DNA and a co-introduced helper virus provides the necessary trans function for the required gene product.

COS cell line (derivative of African green monkey cell line CV-1 contain T antigen encoding sequence of the SV 40 genome.

• 3.7 to 5.3 kb (70-101%) length can be packaged into viral particles so limited to small genes only.

• Adv. - Introduction of recombinant DNA to susceptible cells is more

efficient high copy no. 105 genome/cell allows transient expression of cloned

genes and harvesting of large amount of recombinant protein. late SV 40 genes are efficiently expressed in infected cells

SVGT5• Derived due to deletion of late region.• Express both 16S and 19S mRNA.• HindIII site at position 1493 and BamHI at 2533 were used for

vector construction.• SV 40 genomic DNA containing 6 restriction sites for HindIII

was partially digested with HindIII• 6 fragments were obtained and were separated by gel

electrophoresis• These molecules were digested with BamHI and EcoRI.• Resulting SVGT5 vector (4203bp in length) was obtained.

SVGT7 • Contain same BamHI site as SVGT5 but different HindIII site

at position 1046.• Lacks 3’ accepter site for 16S mRNA at position 1463 so

doesn’t express this mRNA.

B) Plasmid based Vectors• These are shuttle vectors containing origin of replication from both

SV40 genome and E.coli plasmid vectors like pBR322.

• Replicate within the permissive host cells but due to large size are unable to be packaged in viral particles.

• Plasmid vectors pSV1GT5 and pSV1GT7 contain fragments from SVGT5 and 7 and the expression is regulated by late SV40 promoter.

Vaccinia Virus Vector

• Vaccinia virus contain large dsDNA as genetic material and replicate within cytoplasm of host cells.

• Genome size:- 187kb

• Contain enzymes for its replication and transcription so recombinant genome introduced by transfection are non infectious.

• Recombinant virus are generated in vivo by homologous recombination using a targeting plasmid transfected into virus infected cells.

• Direct ligation vectors can be used for transfection of cells containing a helper virus providing replication and transcription enzymes in trans.

• For selection of recombinants, transgene may be inserted in TK gene and its analog 5-bromodeoxyuridine can be used for negative selection.

• Selectable markers like neo or screenable markers like lacZ may also be co-introduced for recombinant identification.

• Not suitable for expression of genes containing introns.• For highest expression endogenous late promoters such as P 11

are used (~1ug/106 cells).

Recombinant Vaccinia virus Expression Vector:

Construction of an infectious vaccinia virus expressing the influenza virus HA gene

Epstein-Barr virus

• It is a herpesvirus with a large ds DNA genome (approximately 170kb)

• Predominantay infects primates and canine cells.• Naturally lymphotropic, infecting human B lymphocytes and

cause infectious mononucleosis.• In cultured lymphocytes it is established as an episomal

replicon with ~1000 copies per cell.• Latent origin oriP and gene encoding trans acting regulator

Epstein Barre nuclear antigen 1 (EBNA1) are required for episomal maintenance.

pBamC (oriP, bacterial origin of replication, ampR gene, neo)

pHEBO( contain hyg instead of neo)

+ EBNA-1p201

• EBV replicons have been used to express epidermal growth factor receptor, TNF receptor and Na+K+ ATPase.

• Used for construction of episomal cDNA libraries.

Adenovirus structure• Nonenveloped particle , 36 kbp• Contains linear double stranded DNA• Does not integrate into the host genome• Replicates as an episomal element in the nucleus

•Causes a benign respiratory infections in human•Serotypes 2 and 5 are commonly used as vectors

Gene Structure and Organization

• Early transcripts are represented by E1–E4 regions• Late transcripts are represented by L1–L5 regions

• MLP: major late promoter;

• C: packaging signal.

Early generations of adenoviral vectors

(replication defective)

Gutless Adenoviral vector

Characteristics of adenoviral vector

• Advantages – High titers

– Efficient transduction of Both dividing and non-dividing cells

– Wide tissue tropism

– No insertional mutagenesis; remains epichromosomal

• Disadvantages– Transient expression ( not good for genetic diseases)– Highly immunogenic– High titers of virus can be toxic– More suitable for cancer immunotherapy

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RetrovirusEnveloped virus with lipid bilayer and viral spike glycoproteins.

Genome: Two copies of single stranded positive-stranded RNA (8-10kb).

Have outer matrix protein and inner core capsid containing viral genome.

Viral genes are integrated into host genome.

Progeny virus produced using host cell transcriptional and translationalmachinery.

Reverse transcriptase to generate DNA

AAAA 3’

CA

R U5 U3 R5’m7GpppG gag pol env

y ( Packaging Signal)

PBS

PPT

MA CA NC

PRO RT IN

SU TM

MA-MatrixCA- CapsidNC- NucleocapsidPRO- ProteaseRT- Reverse transcriptaseIN- Integrase

SU- surface envelope proteinTM- transmembrane envelope protein.

PBS- primer binding sitePPT- polypurine tractR - repeat sequenceU3 - promoter/enhancerU5 - reverse transcription/ integration.

Retroviral Structural genesGeneProteins Functiongag = group specific antigen (internal structural proteins)

matrix (MA), binds envelope, organizationcapsid (CA), protects genome and enzymesnucleocapsid (NC) chaperones RNA, buds

pol = polymerase enzymesreverse transcriptase + RNA to DNARNAase H (RT) degrades template RNAprotease (PR) maturation of precursorsintegrase (IN) provirus integration

env = envelope proteinssurface glycoprotein (SU) receptor binding transmembrane protein (TM) virus-cell fusion

Retroviral vectors• Retroviral vectors are based on Moloney murine leukemia virus (Mo-MLV) which is capable of infecting both mouse and human cells.

• The viral genes, gag, pol and env, are replaced with the transgene of interest and expressed on plasmids in the packaging cell line.

• Because the non-essential genes lack the packaging sequence, they are not included in the virion particle

• Transcription could be under the control of LTRs or enhancer promoter elements might be engineered in with the transgene.

•The culture medium in which these packaging cells have been grown is then applied to the target cells, resulting in transfer of the transgene. 

• A critical limitation of retroviral vectors is their inability to infect nondividing cells, such as those that make up muscle, brain, lung and liver tissue. 

• The cells from the target tissue are removed, grown in vitro and infected with the recombinant vector, the target cells are producing the foreign protein are then transplanted back into the animal (ex vivo gene therapy).

 • Expression is reduced by inflammatory interferons acting on viral LTRs, as the retroviral DNA integrates, viral LTR promoters are inactivated.

• Possibility of random integration of vector DNA into the host chromosome.

Limitations