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Gene therapy and viral vector
Lecture 3
Viral Vectors
Retrovirus
 Adenovirus
 Adeno-associated virus
 Herpes virus
 Pox virus
 Human foamy virus (HFV)
 Lentivirus
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Adenovirus
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Medium-sized (90-100nm)
Icosahedral viruses
Nonenveloped (without an outer lipid bilayer)
Double stranded DNA
Derived from Human Adenoids (tonsils) in 1953
They have a broad range of vertebrate hosts; in humans, 100
distinct adenoviral serotypes have been found to cause a wide
range of illnesses, from mild respiratory infections in young
children (known as the common cold) to life-threatening multiorgan disease in people with a weakened immune system.
Serotype or serovar are distinct variations within a species of bacteria
or viruses or among immune cells of different individuals.
Different types/serotypes are associated with different conditions:
respiratory disease (mainly species HAdV-B and C)
conjunctivitis (HAdV-B and D)
gastroenteritis (HAdV-F types 40, 41, HAdV-G type 52)
obesity or adipogenesis (HAdV-A type 31, HAdV-C type 5, HAdV-D types 9, 36,
37)
Structure of Adenovirus
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The 38 genes in the Human Adenovirus E genome are organized in
17 transcription units, each containing 1-8 coding sequences.
Alternative splicing during processing of the pre-mRNAs produced
by each transcription unit enable multiple different mRNAs to be
produced from one transcription unit.
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The E1A, E1B, E2A, E2B, E3, and E4 transcription units are
successively transcribed early in the viral reproductive cycle. The
proteins coded for by genes within these transcription units are
mostly involved in regulation of viral transcription, in replication of
viral DNA, and in suppression of the host response to infection.
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The L1-L5 transcription units are transcribed later in the viral
reproductive cycle, and code mostly for proteins that make up
components of the viral capsid or are involved in assembly of the
capsid. The L1-L5 transcription units are all regulated by the same
promoter region and share the same transcription start site. As a
result, transcription of all five late transcription units begins at the
same point in the viral reproductive cycle.
The functions of many adenovirus proteins are known
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Structural proteins include capsid proteins II (hexon), III (penton
base), IIIa, IV (fiber), VI,VIII, and IX; and core proteins V,VII, X, and
the terminal protein TP.
Encapsidation proteins IVa2, 52K, and L1, and hexon assembly
protein 100K are involved in assembly of viral capsids.
The L3 protease cleaves viral precursor proteins pTP, pVI, pVII,
pVIII, and IIIa to produce the mature viral proteins.
Control protein E1A activates transcription of a number of viral
genes as well as genes of the host cell.
Control protein E1B 19K suppresses apoptosis by mimicking the
action of cellular protein Bcl-2.
Control protein E1B 55K binds to and inactivates the
transcriptional regulator p53, thus blocking transcription of
genes normally activated by p53 and contributing to the
suppression of apoptosis.
The functions of many adenovirus
proteins are known
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The three proteins coded for by the E2A and E2B transcription units are
all involved in replication of viral DNA. Adenovirus DNA replication
begins at each end of the viral DNA, using the TP protein (rather than
RNA) as a primer, so the viral DNA polymerase replicates every base of
the genome.
Membrane protein E3 RID-alpha and membrane protein E3 RID-beta
performs a variety of molecular functions that contribute to inhibiting
apoptosis.
CR1 beta membrane glycoprotein modulates the host immune response.
Membrane glycoprotein E3 gp19K inihibits the insertion of class I MHC
proteins in the host-cell membrane, thereby preventing T-cell
lymphocytes from recognizing that the host cell has been infected by a
virus.
Control protein E3 14.7K protects the virus from host antiviral
responses.
The control proteins of the E4 transcription unit are involved in
regulating transcription of viral DNA.
How Adenovirus enters the host cell?
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Entry of adenoviruses into the host cell involves two sets of
interactions between the virus and the host cell.
Entry into the host cell is initiated by the knob domain of the
fiber protein binding to the cell receptor. The two currently
established receptors are: CD46 for the group B human
adenovirus serotypes and the coxsackievirus adenovirus
receptor (CAR) for all other serotypes.
This is followed by a secondary interaction with
an integrin molecule. It is the co-receptor interaction that
stimulates entry of the adenovirus.
Binding to integrin results in endocytosis of the virus particle
via clathrin-coated pits.
Attachment to integrin stimulates cell signaling and thus
induces actin polymerization resulting in entry of the virion into
the host cell within an endosome.
Entry in the nucleus
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Once the virus has successfully gained entry into the
host cell, the endosome acidifies, which alters virus
topology.
These changes, as well as the toxic nature of the
pentons, destroy the endosome, resulting in the
movement of the virion into the cytoplasm.
With the help of cellular microtubules the virus is
transported to the nuclear pore complex, whereby the
adenovirus particle disassembles.
Viral DNA is subsequently released, which can enter
the nucleus via the nuclear pore.
After this the DNA associateswith histone molecules.
Thus, viral gene expression can occur and new virus
particles can be generated.
Benefits
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Their basic biology has been studied extensively,
The viral genome can accommodate large
heterologous transgene insertions,
They readily infect quiescent and dividing cells,
They can be amplified to high titers and they have
previously been shown to be relatively safe for
use in humans.
The family Adenoviridae consists of five genera,
including genus Mastadenovirus and genus
Aviadenovirus, which infect mammals and birds
respectively.
The adenovirus vector most commonly used for
clinical trials and experimental gene therapy
applications is species C adenovirus, HAdV-C5.
Drawbacks
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Adenovirus delivered genes can be lost due
to genetic instability therefore repeated
doses are necessary to maintain the
expression of transgene.
They would not integrate into the host
genome, their gene expression is too short
term.
Immunologic responses against adenoviruses
have made their clinical application limited to
a few tissues, such as liver, lung (especially
for CF(Cystic Fibrosis) treatment), or
localized cancer gene therapy.
Drawbacks
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Although the risk of serious disease
following natural adenovirus infection is
rare and the viral genome would not
integrate into the host genome, gene
therapy by adenoviral vectors has caused
serious bad side effects and even death of
some patients.
Lecture prepared from
http://www.ncbi.nlm.nih.gov/pmc/articles/
PMC3507026/#!po=5.42169
 http://www.genetherapynet.com/viralvector/adenoviruses.html
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