Table 1: Properties of different viral vector systems

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Table 1 Properties of different viral vector
systems
AdV AAV HSV Rv Lv
Genetic material dsDNA ssDNA dsDNA RNA RNA
Insert size 8-30kb lt5kb 40-150kb 8kb 8kb
Tropism broad broad Strong for neurones Dividing cells only broad
Inflammatory potential high low high low low
Integration into host genome no No/yes no yes yes
Main limitations Inflammatory response, toxicity Small packaging capacity Inflammation, transient expression Only dividing, possible oncogenesis Possible oncogenesis in some applications
Main advantages Efficient transduction Non-inflammatory/ non pathogenic Large packing capacity, strong neuronal tropism Persistent gene transfer Persistent gene tran
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Adenovirus
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Figure 1. The most common viral morphologies.
Left to Right. A naked icosahedral virus (e.g.
poliovirus), an enveloped icosahedral virus (e.g.
herpes virus), a naked helical virus (e.g.
tobacco mosaic virus) and an enveloped helical
virus (e.g. influenza virus). Individual
capsomeres are arranged to form a capsid which
encloses the nucleic acid (DNA or RNA) of the
virus. Many animal viruses also contain an
envelope, which is partly derived from the host
cell membtrane but which always contains unique
viral proteins drawn here as "spikes".
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5
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polygon
Helical ???
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Figure 2. Comparative size and shape of various
groups of viruses representing diversity of form
and host range. A. Smallpox virus  B. Orf virus 
C. Rhabdovirus D. Paramyxovirus  E. Bacteriophage
T2  F. Flexuous-tailed bacteriophage  G. Herpes
virus  H. Adenovirus  I. Influenza virus  J.
Filamentous flexuous virus K. Tobacco mosaic
virus  L.  Polyoma/papilloma virus  M. Alflafa
mosaic virus  N. poliovirus  O. Bacteriophage
phiX174. Viruses have fundamentally three
morphologies 1. polygonal, the most common
polygon being the icosahedron (E, F, G,  H, L,
N)  2. helical, wherein the capsomeres assemble
as a helix enclosing the nucleic acid) (D, I, J,
K, M B is controversial) 3.  complex, wherein
the proteins are laid down in patches or layers
(A).  Some animal viruses have envelopes which
enclose their nucleocapsid (D, G, I). The
envelopes are embedded with viral proteins that
secre their entry and exit in cells. Only
bacteriophages have tails which  are used for
adsorption and penetration of their host cell.
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adsorption
 penetration
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Table 1. Viral vectors used for gene delivery
Vector
Packaging capacity
Host range
Clinical trials

Broad, infects both non-dividing and dividing
cells
AAV
Low ,4 kb

Adenovirus
Medium ,7.5 kb
Broad low transduction of neurons

Broad, neuron and glial cell-pecific strains
Medium ,7.5 kb
Alphaviruses
-
Herpes simplex virus
High .30 kb
Broad, neurons, stem cells, muscle cells
-
Lentivirus
Medium 8 kb
Broad, dividing and non-dividing cells

Retrovirus
Medium 8 kb
Restricted, dividing cells only t
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Adenovirus

• High transfer efficiency in a wiled variety of
  cell type and tissue
• Infect both dividing and nondividing cell
• Very high titer
• Large scale production available,
• Larger insert,
• Well studied,
• Short-lived(1-3 weeks),
• Immune response

DNA virus with a genome consisting of a
linear double-stranded DNA molecule of
approximately 36,000 nt
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Structure The icosahedral capsid (70 to 100 nm)
is made up of 252 capsomeres 240 hexons forming
the faces and 12 pentons at the vertices. Each
penton bears a slender fiber. The double-stranded
linear DNA is associated with two major core
proteins and carries a 55-kDa protein covalently
attached to its 5' end.
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Trafficking of Adenovirus from Membrane To Nucleus
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Adenoviruses
Clinical Manifestations Adenoviruses cause acute
respiratory disease (usually), pneumonia
(occasionally), acute follicular conjunctivitis,
epidemic keratoconjunctivitis, cystitis, and
gastroenteritis (occasionally). In infants,
pharyngitis and pharyngeal-conjunctival fever are
common
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Host Defenses Most adolescents and adults have
circulating neutralizing antibodies immunity is
widespread. Cytotoxic T lymphocytes destroy
adenovirus-infected cells
Diagnosis Adenovirus infection is suggested
clinically by fever, upper respiratory tract
infections, and conjunctivitis the diagnosis is
confirmed by a rise in antibody titers and by
virus isolation
Vector in Gene Therapy Adenoviral genomes have
been developed into vectors in experimental
therapy since adenoviruses readily infect human
and other mammalian cells. Vector genomes carry
deletions in the E1 and E3 regions the gaps in
the genome are used to take up foreign genes,
e.g., the gene for the cystic fibrosis
transmembrane conductance regulator (CFTR).
Deletions in E1 minimize the potential of these
vector genomes to elicit an infection cycle in
human cells. The first clinical applications in
patients suffering from the genetic disease
cystic fibrosis have been reported but problems
with adenovirus toxicity remain.
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Most of the adenovirus genes (Fig. 67-3) are
transcribed by the host DNA-dependent RNA
polymerase II in a complex transcriptional
program Genes in the E1A region of the adenovirus
genome are the first to be transcribed. One
protein product of this gene region is a
transactivator that is essential for the
activation of all other viral genes. This
immediate-early viral function can also activate
or inactivate certain cellular genes. The jointly
controlled E2A and E2B regions code for proteins
that are essential for viral DNA replication.
Among the E3-encoded functions, one is a 25,000
(19,000)-molecular-weight glycoprotein
responsible for the interaction with cell
membrane-associated proteins (major
histocompatibility complex). The E3
region-encoded functions may be unnecessary for
viral replication in cell culture, but essential
for the interaction with the intact defense
system of an organism and for the modulation of
host functions. The late viral L1 region can also
be transcribed early in the infection cycle,
probably to a limited extent. Genes encoded in
the L1 region of Ad5 DNA are essential for virion
assembly. All the late viral functions are under
the control of the major late promoter (MLP)
components, which are located at about 17, 20 and
27 map units on the viral genome. The gene
encoding the fiber structural protein can also be
controlled by the x, y, and z leaders (Fig. 67-3)
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synthesis of early proteins This refers to the
transcription and translation of a section of the
phage DNA to make a set of proteins that are
needed to replicate the phage DNA. Among
the early proteins produced are a repair enzyme
to repair the hole in the bacterial cell wall, a
DNAase enzyme that degrades the host DNA into
precursors of phage DNA, and a virus specific DNA
polymerase that will copy and replicate phage DNA

 synthesis of late proteins. Each of the several
replicated copies of the phage DNA can now be
used for transcription and translation of a
second set of proteins called the late proteins.
The late proteins are mainly structural proteins
that make up the capsomeres and the various
components of the tail assembly. Lysozyme is also
a late protein that will be packaged in the tail
of the phage and be used to escape from the host
cell during the last step during the replication
process.
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Regulation of apoptosis in adenovirus-infected
cells. Adenovirus infection and E1 expression
produces the three oncoproteins of the virus,
E1A, E1B 55K and E1B 19K.1, 2 Yellow symbols
indicate proapoptotic, blue symbols indicate
antiapoptotic function, and faded symbols
indicate failed signaling events. E1A induces a
cellular DNA damage response that results in p53
accumulation, which can induce apoptosis through
transcriptional upregulation of the BH3-only
proteins PUMA and NOXA.68 The E1B 55K protein
binds p53 inhibiting its apoptotic and also
growth arrest functions.1, 2 Induction of a DNA
damage response by E1A also triggers the
proteasome-mediated degradation of the BAK
inhibitor MCL-1.55 E1B 19K binds BAK and inhibits
apoptosis. In the absence of BAK, BAX can
functionally substitute for BAK and the E1B 19K
protein can also bind and inhibit activated
BAX.44 In death receptor-mediated apoptosis where
both BAX and BAK are activated, E1B 19K binds and
inhibits both BAX and BAK.41, 42, 43 Inhibition
of this BAX and BAK pathway for apoptosis extends
the survival of infected cells to the end of the
replication cycle thereby facilitating virus
production
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How to construct Adenovirus?
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Homologous Recombination from a Molecular
Perspective
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Recombinant Adenovirus construction
100/0
(01.3)
pBRX
?
Ad5 (9.25-16)
ITR
p
pA
COX-1
?
pAd-pgk-cDNA
pJM17
pBR322
pBR322
293 cells (0 12 of Ad5) Homologous
recombination
ITR
ITR
p
pA
?
COX-1
rAd-cDNA E1 and E3 (partial) deleted
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Restoration of Deficient Membrane Proteins in the
Cardiomyopathic Hamster by In Vivo Cardiac Gene
Transfer
BackgroundOne of the most important problems in
developing in vivo cardiac gene transfer has been
low transfection efficiency. A novel in vivo
technique was developed, tested in normal
hamsters, and the feasibility of restoring a
deficient structural protein (-sarcoglycan) in
the cardiomyopathic (CM) hamster evaluated
Methods and ResultsAdenoviral (AdV) vectors
encoding either the lacZ gene or -sarcoglycan
gene were constructed. Hypothermia was achieved
in hamsters by external body cooling to a rectal
temperature of 18 to 25C. Through a small
thoracotomy, the ascending aorta and the main
pulmonary artery were occluded with snares, and
cardioplegic solution containing histamine was
injected into the aortic root viral constructs
were delivered 3 to 5 minutes later followed by
release of the occluders and rewarming. Four days
later, homogeneous -galactosidase expression was
detected throughout the ventricles of the normal
hearts
(Circulation. 2002105502-508.)
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Expression of -Galactosidase in Normal Hamsters
(Circulation. 2002105502-508.)
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Modified Adenovirus Gene Therapy
Promoter specific
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Evolutionary conservation of the kidney- specific
cadherin (Ksp-cadherin) promoter.
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An in vivo method for adenovirus-mediated
transduction of thick ascending limbs
(A) Confocal fluorescence micrographs showing
specific green fluorescent protein (GFP)
expression in medullary thick ascending limbs
(THAL) from a rat kidney transduced with the
Na/K/2 Cl (NKCC2) cotransporter promoter
(Ad-NKCC2GFP) (scale bar 25 m). (B and C)
Fluorescent and transmittance micrographs showing
specific GFP expression in a cortical THAL from a
kidney transduced with Ad-NKCC2GFP (N 4). Green
, GFP red, Tamm-Horsfall (scale bar 25 m).
Kidney International, Vol. 63 (2003), pp.
11411149
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Genetic Modification of the Fiber knob
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Adapter-based Targeting
The Ad infection pathway. Shown are the basic
steps of initial high-affinity binding of the
virion fiber to its primary cellular receptor,
CAR (left). Alternatively, a generalized adapter
molecule ablates native CAR-based tropism and
targets Ad to an alternate cellular receptor
molecule (right). The dual specificity of the
adapter molecule for both the Ad and the
alternative receptor provides novel,
CAR-independent cell binding.
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(a). Adenoviral particles can be re-targeted to
TAAs, using bifunctional adapter molecules.
Examples of such adapter molecules are chemical
conjugates of a Fab fragment derived from an
antiknob monoclonal antibody with a whole
antibody directed against the target antigen (I),
or naturally occurring ligands such as folate or
basic fibroblast growth factor (II). The other
class of adapter molecules consists of
recombinant bispecific fusion proteins, examples
of which include so-called diabodies, comprised
of two single-chain antibodies (scFv), with one
scFv recognizing the fiber knob and the other the
TAA (III). Another example is the trimeric
sCAR-fibritin-scFv, which uses the soluble
ectodomain of the native Ad receptor CAR to bind
to the Ad, thus ablating CAR recognition (IV).
(b) Adenoviral particles can be re-targeted to
TAAs using genetic targeting strategies in which
the targeting ligands are incorporated into
capsid proteins. The Ad5 knob protein can, for
instance, be replaced with that of other
serotypes (V), or it can be altered by
incorporation of targeting ligands, such as
RGD-containing peptide sequences or six histidine
residues, at the C-terminus of the protein (VI).
As an alternative to the C-terminus, peptide
sequences can also be incorporated in the HI-loop
of the Ad5 knob (VII). The entire fiber and knob
domain can be replaced by an artificial fiber,
for instance, consisting of the bacteriophage T4
fibritin trimerization domain and a targeting
motif (VIII). Finally, multiple genetic
modifications can be combined in a single
particle, forming a so-called 'complex mosaic
Ad'. An example is a particle containing Ad3 knob
instead of Ad5 knob, in which a six-histidine
targeting motif is incorporated at the C-terminus
(IX).
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Treatment Responsive Promoter
Schematic of the tet-regulatable system. In the
presence of doxycycline (a tetracycline analog),
the system is switched on, allowing for
expression of the oncogene H-RASV12G. Without
doxycycline, the oncogene expression is turned
off
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primary cutaneous melanoma was measured at
intervals and the longest dimension was plotted
against time in days. Time 0 represents initial
induction with doxycycline. Red brackets indicate
periods during which the animal was ON
doxycycline induction. Blue lines indicate period
without doxycycline administration. Insets
Representative photographs of the tumor taken on
respective time points (ON and 12 days OFF) and
the corresponding histopathologic sections (HE,
200x) during the first cycle of doxycycline
treatment. Note in the ON dox biopsy, tumor cells
completely replaced all normal architecture of
the skin. Compare to same site biopsied 12 days
later, with intact epidermis and hair follicle
(asterix) and residual foci of melanoma cells
(arrows).
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Regression of melanoma is accompanied by
activation of cell suicide program, apoptosis.
Dying cells stained brown (TUNEL staining, 400x)
in these photomicrographs
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A lentiviral microRNA-based system for
single-copy polymerase II-regulated RNA
interference in mammalian cells
Introduction

• RNA interference (RNAi)

A gene silencing mechanism which suppresses
specifically the homologous genes by dsRNA
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RNA Interference
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Endogenous micro(mi)RNA pathway
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Inhibition of renal cancer cell growth in vitro
and in vivo with oncolytic adenovirus armed short
hairpin RNA targeting Ki-67 encoding mRNA
Cancer Gene Therapy (2009) 16, 2032
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Table 1. Viral vectors used for gene delivery
Vector
Packaging capacity
Host range
Clinical trials

Broad, infects both non-dividing and dividing
cells
AAV
Low ,4 kb

Adenovirus
Medium ,7.5 kb
Broad low transduction of neurons

Broad, neuron and glial cell-pecific strains
Medium ,7.5 kb
Alphaviruses
-
Herpes simplex virus
High .30 kb
Broad, neurons, stem cells, muscle cells
-
Lentivirus
Medium 8 kb
Broad, dividing and non-dividing cells

Retrovirus
Medium 8 kb
Restricted, dividing cells only t
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• Adeno-Associated Virus(AAV)
• Integrating genome, Stable transgene expression
• Infect both dividing and nondividing
• Poor Immunogenicity
• Smaller insert,
• Integration is random

small (20 to 25 nm in diameter),
nonenveloped, single-stranded (ss) DNA virus
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AAV is a single stranded DNA virus whose genome
is flanked by two inverted terminal repeats
(ITRs). The genome contains two ORFs, that direct
the synthesis of 6 major RNA species, due to the
presence of 3 different transcription promoters
and alternative splicing signals (Fig2). The rep
ORF encodes four regulatory proteins that are
involved in AAV DNA replication, gene expression
and packaging. The cap gene encodes  three
structural proteins, VP1, VP2 and VP3, that
constitute the capsid.
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Integration
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The Expression of Adenovirus in Lung
Human Gene Ther 1997 899-109
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Robust Adenoviral and Adeno-Associated Viral Gene
Transfer to the In Vivo Murine Heart
(Circulation. 20031082790-2797.)
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Adeno-associated viral vector-mediated hypoxia
response element-regulated gene expression in
mouse ischemic heart model
PNAS July 9, 2002 vol. 99 no. 14
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Adeno-associated virus serotype 8 Efficiently
Delivers Genes to Muscle and Heart
Nature biotechnology 23 p321
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Time course of GFP gene expression and the
presence of vector DNA in liver and heart after
I,p injection of AAV8
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Retrovirus
Integration, Gene expression for extended time
period Infect dividing cell
lack of sufficient methods for production of
high-titer virus stocks
safety concerns related to their origin from HIV
Why use lentivirus? Lentiviruses integrate into
the cellular genome allowing inheritance of the
introduced gene of interest. Lentiviruses infect
dividing and non-dividing cells, and so may be a
good choice for stem cells that are dividing
slowly. Lentiviruses have previously been shown
to infect haematopoietic and neural stem cells
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Retrovirus Replication
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Lentivirus construction
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Functional Expression of Mouse Relaxin and Mouse
Relaxin-3 in the Lung from an Ebola Virus
Glycoprotein- Pseudotyped Lentivirus via Tracheal
Delivery
relaxin is a known modulator of connective tissue
and the extracellular matrix by virtue of its
ability to regulate matrix metalloproteinases
(MMPs). Relaxin knockout mice exhibit age-related
pulmonary fibrosis,
Endocrinology 147(8)37973808
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Thanks for Listening
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PCR
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Infection of Human Fetal Cardiac Myocytes by a
Human Immunodeficiency Virus-1Derived Vector
(Circ Res. 199883738-742.)
Freshly isolated human fetal cardiac myocytes
(HFCMs)
HIV-1 infection using wild-type HIV-1
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