Recombinant viral vectors are genetic engineering tools commonly used for gene transfer purpose with high transfection efficiency and site specific gene insertion.

1. VIRAL VECTORS FOR GENE
TRANSFER
Seminar
on
ANKUR SHARMA

2. 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

3. 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.

4. 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

5. • 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.

7. 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

8. 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.

9. 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.

10. • 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

11. 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.

12. 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.

14. 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.

17. 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.

19. • 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).

20. Recombinant Vaccinia
virus Expression Vector:

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

22. 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.

23. pBamC
(oriP, bacterial origin of replication, ampR gene, neo)
pHEBO
( contain hyg instead of neo)
+ EBNA-1
p201

24. • EBV replicons have been used to express epidermal growth
factor receptor, TNF receptor and Na+K+ ATPase.
• Used for construction of episomal cDNA libraries.

25. 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

26. 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.

27. Early generations of adenoviral vectors
(replication defective)

28. Gutless Adenoviral vector

29. 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

30. 30
Retrovirus
Enveloped 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 translational
machinery.
Reverse transcriptase to generate DNA

31. 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-Matrix
CA- Capsid
NC- Nucleocapsid
PRO- Protease
RT- Reverse transcriptase
IN- Integrase
SU- surface envelope protein
TM- transmembrane envelope protein.
PBS- primer binding site
PPT- polypurine tract
R - repeat sequence
U3 - promoter/enhancer
U5 - reverse transcription/
integration.

32. Retroviral Structural genes
Gene Proteins Function
gag = group specific antigen (internal structural proteins)
matrix (MA), binds envelope, organization
capsid (CA), protects genome and enzymes
nucleocapsid (NC) chaperones RNA, buds
pol = polymerase enzymes
reverse transcriptase + RNA to DNA
RNAase H (RT) degrades template RNA
protease (PR) maturation of precursors
integrase (IN) provirus integration
env = envelope proteins
surface glycoprotein (SU) receptor binding
transmembrane protein (TM) virus-cell fusion

33. 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.

35. • 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