ctors in Gene Therapy

WHY VECTORS
Are the vehicles
used to carry the
beneficial genetic
material to the
mutated cells
A completely
reliable, safe and
efficient vector is
not yet available
A perfect vector
would target
specific cell types,
insert their genetic
information into a
safe site in the
genome, and be
regulated by
normal
physiological
signals 3D Animation of Systemic BacterialDeliveryof Gene Therapy in Cancer - YouTube.FLV
 Commonly used Vectors
The two major strategies of gene delivery into mammalian cells
(viral mediated and non viral mediated)
These methods differ in accuracy, efficiency, and stability of gene
expression
Are the vehicles used to carry the
beneficial genetic material to the Vectors under
mutated cells study as gene
A completely reliable, safe and delivery vehicles
Aefficient
perfectvector
vectoriswould
not yet available
target
specific cell types, insert their
genetic information into a
safe site in the
genome, and
Virotherapy Liposomes Naked DNA
be regulated by
normal physiological
signals

Adeno-
Bacteria,
Retroviruses Adenoviruses Associated
Macropha
Viruses
Summary of Existing Vectors (1)
 Viral Vector
VIRUSES
Viruses have naturally evolved an efficient mechanism to deliver their nucleic acids into
eukaryotic cells
Cancer researchers have envied this selectivity for years: if they could only target
cancer therapies to tumor cells and avoid damaging normal ones, they might be able to
eliminate many side effects of cancer treatment.
Modified viruses are currently the most popular vehicles for gene delivery

WHY VIRUSES?
Viruses through the time of evolution have evolved to infect the cells with great
specificity
Viruses tend to be very efficient at transfecting their own DNA into the host cell genome
This allows them to produce new viral particles at the period of synthesis of the cell

WHAT VIRUSES TO USE?

how well they transfer the genes to cells
which cells they can recognize and infect
and whether they alter the cells DNA permanently or temporarily
 Summary of Success with Different Viral Vector

AAV (Adenovirus associated virus)

Vectors
Good expression for hemophilia B
Safe

Retroviral vectors
Good expression for hemophilia A and B
Safe

Adenoviral vectors
Great expression for hemophilia A or B
VIDEO
Toxic (caused one death in a human patient
 Retroviral infection cycle
The retrovirus deposits its
RNA into the cell as the Are single-stranded RNA viruses with
virus uncoats
Viral ssDNA is then reverse
a small genome.
transcribed into dsDNA They are relatively easy to construct, infect
using viral reverse
transcriptase replicating target cells efficiently, and
The linear DNA is
transported into the nucleus,
integrate securely in the host genome.
where it integrates randomly
into the hosts genome

However, the random nature of viral integration into the host

genome results in insertional mutagenesis of each infected cell
Ideal Vector Life Cycle
 Viral vector strategy
Replication & virulence genes can be substituted with therapeutic genes
 Retroviral vectors
Purposes
Designed to enter cell
and deposit genes
Specially constructed to
prevent the generation of
replication competent
retroviruses (RCR)

Advantages
long-term expression
low toxicity
high capacity
low antivector
immunity

Disadvantages
Lack of cell
specificity
Random splicing into
host DNA
 Basic Recombinant Construction
High specificity
Efficient for transfer and combining genetic materials in host cells

GENETHEURAPHY1.exe

GENETHEURAPHY2.exe

Verma and Weitzman (2005)

 VECTORS IN GENE THERAPY: Lentiviruses
Many viral vector systems have been developed for gene therapy applications, but
most of thesevectors are non-integrating, which limits their usefulness for
transgenesis.
The best characterized integrating viral vectors originate from the Retroviridae
family, because of their intrinsic ability to integrate into genomic DNA. However,
oncoretroviral vectors have shown limited success because of the requirement of
cell replication for integration.
Lentiviral vectors (LV) have overcome these limitations showing the ability to
transduce nondividing cells and provide a more efficient transgene expression if
compared with oncoretroviral vectors.
Higher efficiency of LV vectors is likely due to the stability of transcripts derived
from the lentiviral provirus because of the strong 3 poly(A) signal that causes an
increased cytoplasmic accumulation of the RNA of the transgene [24].
Naldini et al. were the first to demostrate that a LV vector derived from human
mmunodeficiency virus type 1 (HIV-1) could provide efficient in vivo delivery, stable
integration and long-term expression of transgenes into non-mitotic cells such as
neurons [25,26].

Castellani and Conese (2010) Lentiviral

Vectors and Cystic Fibrosis Gene
 VECTORS IN GENE THERAPY: Lentiviruses

Although various lentiviruses from different species (human, simian, equine, ovine,
bovine, feline, caprine) have been used to generate gene transfer vectors (reviewed
in [27]) only HIV-1 and feline immunodeficiency virus (FIV) have been considered in
the context of airway epithelium and cystic fibrosis gene therapy.
Lentiviruses, such as HIV-1, have a diploid genome with two copies of singlestranded
plus RNA and a nucleocapsid constituted by the proteins derived from the gag gene,
viral enzymes such as a protease derived from the pro gene, Rnase H, reverse
transcriptase and integrase derived from the pol gene.
The virion is enveloped by the host cell membrane containing the viral protein
derived from the env gene. After virus entry into the cell, reverse transcription
produces viral double strand DNA which enter in the nucleus and integrate randomly
and permanently in the host genome, resulting in a provirus state.
In the provirus, the lentiviral genome is flanked on both sides by the long terminal
repeats (LTR), which contain the U5, R and the U3 regions. The 5-LTR can act like an
RNA pol II promoter. The 3-LTR acts to terminate transcription and promote
polyadenylation. The LTR also has recognition sequences necessary for integration
into the genome. Tat is an accessory viral protein which activates the 5 LTR
promoter, whereas Rev controls the amount of RNA splicing as well as the RNA
export into the cytoplasm.
At the end of cycle infection, viral genomes are packaged, enveloped and released
from the cells. Two important cis-acting DNA elements, the central polypurine tract
sequence (cPPT) and the postregulatory element (PRE), derived from woodchuck
hepatitis B virus, are also included in the transfer vector to enhance the transduction
efficiency and transcript stability. In particular, cPPT has been and
Castellani shown
Coneseto (2010)
facilitate
Lentiviral
nuclear translocation of preintegration complexes, andVectors and Cystic Fibrosis Gene
PRE acts at the post-
 Lentivirus (FIVV-HIV)
Minimal HIV vector plasmid

All genes coding for enzymatic or structural HIV proteins have been
removed.
(1) consisting of the CMV/HIV LTR hybrid promoter followed by the
packaging signal ( ), the rev-binding element RRE for cytoplasmic
export of the RNA, the transgene expression cassette consisting of internal
promoter(s) and transgene(s), and the 3' self-inactivating (SIN) LTR.
Together with the HIV vector plasmid (1), the HIV packaging plasmid (2),
Lentivirus (FIV-HIV)
Single-stranded
RNA (9.2 kb) virus
of the retrovirus
family
Change from wild
type: (HIV
genes)
Does not require
replicating cells for
gene transfer
Packaging cells:
HIV (gag+, pol+,
tat+, rev+),
MoMLV env+, or
VSV-G env+
Virus propagation:
Transient
transfection
Transgene
expression : stable
 Adenoviruses
Are double Electron Micrograph
Adenovirus of Purified
stranded DNA Structure
genome that Adenoviruses Particles
cause
respiratory,
intestinal, and
eye infections
in humans Courtesy of Dr. Pierre
Boulanger, Montpellier
The inserted School of Medicine, France
DNA is not
incorporate
into genome Adenovirus
Not replicated Attachment
though
Has to be
reinserted
when more
cells divide
Ex. Common Adenovirus
cold Internalization
 Adenoviruses
Unlike retroviruses, adenoviruses
do not integrate their DNA into
the genes of cells they infect
The genes carried into a cell
usually work only for a while and
then break down
Scientists have investigated
adenoviruses extensively in
gene therapy approaches to
treat cancer.
The viruses are armed with
genes that make cancer cells
more susceptible than normal
cells to chemotherapy, and
therefore would reduce the
amount of chemotherapy
needed, and the number of
healthy cells killed.
They can easily be rendered
replication-defective by ADENOVIRUSES have been genetically altered
deletion of critical regulatory to kill tumor cells but to spare healthy neighbors
 Advantages of using a recombinant Adenovirus
1. Broad host range
Can infect a broad range of mammalian cells
Allows for the expression of recombinant proteins in most mammalian cell lines and tissues
Have been used extensively to express human as well as non-human proteins
2. Infection and expression of genes in replicative and non-replicative cells
Transfection cannot be done in non-replicative cells
Best system to study gene expression in primary non-replicative cells
Allows for a direct comparison of results obtained with transformed cell lines and primary cells
3. Replicates efficiently to high titers
Production of 1010 to 1011 VP/mL
Can be concentrated up to 1013 VP/mL
4. Viral can accommodate up to 7.5 kb of foreign DNA
Ad can normally encapsidate a viral DNA molecule slightly bigger than the normal DNA
(105%)
To provide additional cloning space, the E1 and E3 early regions of Ad have been deleted
5. Homologous system for human genes
Human virus as a vector and human cells as a host
Proper folding and exact post-translational modifications of human proteins
Most human proteins are expressed at high levels and are fully functional.
6. Propagation in suspension cultures
293 cells can be adapted to grow in suspension
Allow production scale-up: > 20L
7. Simultaneous expression of multiple genes
Insertion of two genes in a double expression cassette
 Adenoviral vectors

do not insert into

genome
temporary
lack of specificity
strong immune
response

http://en.wik
ipedia.org/wi
ki/Gene_thera
 Adenoviral Vectors
Double stranded
DNA (36 kb) virus
of the adenovirus
family

Change from wild

type: (E1. E3)

Large cloning
capacity

Packaging cells:
(E1A+, E1B+)

Virus
propagation:
Infection

Transgene
expression :
Transient
 Virotherapy is a new strategy to
treat cancer by selectively
infecting and killing tumor cells
The viruses used in
virotherapy can either kill
tumor cells by bursting them
open or delivering genes that
make the
cells more
susceptible
to
Two chemotherapies
main
strategies
are

being
explored.

VIROTHERAPY
 Virotherapy with Transductional Targeting
Scientists are
attempting to
engineer viruses
to selectively
infect and
destroy only
cells that have
turned cancerous
They are
attaching adapter
molecules onto
the viral outer
coat protein or
directly
modifying these
proteins to try
and prevent
them from
entering normal
cells
 Virotherapy with Transcriptional Targeting
This approach
involves placing
a tumor-specific
promoter next to
one of the
adenoviruss
essential genes
The promoter
acts as an on
switch that
permits the gene
to function only
in cancer cells
The virus can
enter normal
cells, but they
cannot
reproduce and
kill them
 Adeno-associated Viruses
A unique animal virus that requires coinfection with an unrelated helper virus, such as an
Adenovirus, for infection into a cell
Without a helper virus they insert into a host genome and remain as a provirus
Transcription occurs with the help from the second virus
Therefore this virus is less likely to produce a immune response in the host
They do not require the target cell to be actively replicating in order to infect the cell
And have been able to infect all human cell cultures tested so far.

Many of the characteristics of AAV that make it a desirable gene therapy vector can be discussed
in terms of its life cycle. When AAV infects a cell it has two options:
1.In the presence of a helper virus, such as adenovirus or herpes virus, it can replicate and
produce progeny virions.
2.In the absence of a helper virus, AAV integrates site specifically into the host genome and
become latent until such time as superinfection with a helper virus allows it to replicate.
Two major characteristics beneficial to gene therapy vectors are realized:
1.Primarily, it is a defective virus. Without a helper virus, AAV cannot propagate and has been
not been implicated in any diseases. It is therefore considered a harmless virus.

2.Secondly, AAV can integrate into the host chromosome via its terminal repeats. This feature
predicts stability of the virus within a cell and propagation of the virus along with the host cell
chromosome. While the site specificity of integration requires the AAV rep protein, maintenance
of AAV within the host chromosome does not require any protein expression allowing complete
deletion of all AAV open reading frames in recombinant vectors.
 VECTORS IN GENE THERAPY: ADENO-ASSOCIATED VIRUS (1)

AAV
Lytic
1960
Nonpathogenic

Adenovirus

Latent
Hoggan,M.D.,etal.Continuouscarriageofadenovirusassociated
virusgenomeincellcultureintheabsenceofhelperadenovirus.in
ProceedingsoftheFourthLepetiteColloquium.1972.Cocoyac,Mexico. 10,000/cell
R.JudeSamulski,Ph.D.
 Adeno-associated viral (AAV) Vectors

Nature Reviews Genetics

1; 91-99 (2000);

Integrate into
genome but
small in size
 Adeno-associated virus (AAV) Vectors
Small single-
stranded DNA
(4.5 kb) virus of
the parvovirus
family
Change from
wild type:
(rep, cap)
Does not require
replicating cells
for gene transfer
Packaging cells:
HIV (rep+,
cap+)
Virus
propagation:
Transient
transfection
Transgene
expression :
stable
Macrophage as Gene
Therapy Vehicle
 Macrophage as Gene Therapy Vehicle, a Novel System of Delivery
Act as defensive agent in body
Eat foreign cell like bacteri
Release cytokines to call another macrophage and resulted in defensive (immune)
response
Macrophage activated
rapidly in the development
of some serious disease like
Cancer, Atherosclerosis, and
so on.
Thus research to make
macrophage act as vehicle
to transfer theurapetic gene
using virus as vector like
adenovirus.
Adenovirus a popular
method of gene transfer to
primary macrophages due to
their ability to infect
nondividing cell with high
efiiciency and reasonable
longevity of transgene
expression
 Adenovirus & Macrophage
Adenovirus can infect human macrophage with efficiency of 80%
By stimulating integrin expression (adenovirus receptor), efficiency increased over
90%
 Limitation of using Adenoviral as Gene Vector in Macrophage
Can incorporate only 7.5 Kb the amount of foreign DNA that can be inserted.
adenoviral low specificity to infect macrophage, instead the viral infect a broad range
of cell types.
Macrophage adoptive transfer relatively risk-free procedure unfortunately only
minimal therapeutic benefit (Andreesen et al 1998).
Attention to focus enhancing the ability of adoptively transferred macrophages to
kill tumor cells or ameliorate other disease states
Macrophage migrate into diseased tissues carrying therapeutic DNA solve the
problem

In Conclutons:
Gene Therapy using adenoviral as vector has many benefit, mainly because of its
nature as virus that can integrate its DNA into host DNA easily
Using adenoviral as vector increase efficiency to transfer gene therapy into target cell
Using macrophage become novel system to deliver gene therapy to target cell in some
disease
Using macrophage with adenoviral as vector has some limitation due to their small
vector size and low specificity to infect macrophage cell
In the end, combination of macrophage and adenoviral vector may open new path to
cure some serious disease with gene therapy
Microbe & Macrophage
 VECTORS IN GENE THERAPY: Lentiviruses
The development of lentiviral vector system, in order to achieve a gene transfer
agent, is based on modifications of a native lentivirus genome.
Presently, the lentivirus-based gene delivery system isconstituted by four
components: 1) the packaging elements like structural proteins and enzymes involved
in the formation of the viral particles, derived from the gag-pol genes; 2) a
posttranscriptional regulator for gag and pol expression as well as nuclear RNA
export encoded by the rev gene; 3) the vector carrying the transgene to be
transferred to the target cells; 4) an heterologous glycoprotein i.e., vesicular
stomatitis virus glycoprotein G (VSV-G) which pseudotypes the vector in order to
increase its tropism.
All structural proteins are HIV-1-derived, except the envelope because of the
restricted host range of the HIV-1 env glycoprotein. Association of VSV-G with viral
coresderived from lentiviruses, results in an high titer production and in a broad
tropism of the vectors.
Finally, deletion of the coding sequences for viral gene products renders the LV gene
transfer vectors replication incompetent and makes 68 kb available for the insertion
of desired genes, in this case a large transcriptional units such as an epithelial-
specific promoter expressing the CFTR gene [25].
Lentivirus vectors only recently made their debut in human clinical trials for
hematological disorders, such as -thalassemia and sickle cell disease [32].
In the first reported clinical trial with LV vectors, an HIV-1 derived vector expressing
an antisense gene against the HIV-1 envelope gene (termed VRX496) was transferred
to autologous CD4 T cells. A single intravenous infusion of these gene-modified
autologous T cells was well tolerated in all five patients enrolled in the clinical trial;
Castellani and Conese (2010) Lentiviral
self-limiting mobilization of the vector and improvement of immune
Vectors and Cysticfunction was
Fibrosis Gene
 VECTORS IN GENE THERAPY: Lentiviruses
LV vectors are endowed with interesting features which make them promising agents for
application to gene therapy of CF lung disease. However, they present some drawbacks which
need to
be addressed before they can enter into the clinic. Although the issue of attachment and entry
mechanisms has not been studied in detail, it seems that, at least in in vitro cultures of human
polarized
airway epithelia and small conventional animal models, the apical surface of the airway
epithelium
poses a strenous barrier to efficient transduction from this side, due to the lack of apical
receptors.
Moreover, while other viral vectors have been considered in light of the extracellular barriers,
such as
the thick and dehydrathed mucus layer covering the airway epithelium, comprehensive studies on
this
aspect are still lacking. The alternative to the use of tight junction disrupting agents and
membrane
detergents, which could possibly enhance the leakage of bacterial products and inflammatory
mediators into the submucosa and further damage the CF lung, is the pseudotyping of LV
envelope
with heterologous proteins from other viruses. This approach should be evaluated in terms of the
safety
issue, when pathogen viruses are used [57]. Another approach is to use physical methods in order
to
enhance the residence time of viral vectors onto the airway epithelium, avoiding their wash out
by the
mucociliary clearance. For example, magnetofection is a nucleic acid delivery technique to cells
supported and site-specifically guided by the attractive forces of magnetic fields acting on viral
vectors
which are associated with magnetic nanoparticles. This field isCastellani
actively and Conese (2010)
investigated Lentiviral
by us and
Vectors and Cystic Fibrosis Gene
 VECTORS IN GENE THERAPY: ADENO-ASSOCIATED VIRUS (1)
Many of the characteristics of AAV that make it a desirable gene therapy vector can be
discussed in terms of its life cycle. When AAV infects a cell it has two options:
1.In the presence of a helper virus, such as adenovirus or herpes virus, it can replicate
and produce progeny virions.

2.In the absence of a helper virus, AAV integrates site specifically into the host genome
and become latent until such time as superinfection with a helper virus allows it to
replicate.

Two major characteristics beneficial to gene therapy vectors are realized:

1.Primarily, it is a defective virus. Without a helper virus, AAV cannot propagate and
has been not been implicated in any diseases. It is therefore considered a harmless
virus.

2.Secondly, AAV can integrate into the host chromosome via its terminal repeats. This
feature predicts stability of the virus within a cell and propagation of the virus along
with the host cell chromosome. While the site specificity of integration requires the
AAV rep protein, maintenance of AAV within the host chromosome does not require
any protein expression allowing complete deletion of all AAV open reading frames in
recombinant vectors.