Gene Therapy Manik

Genes, which are carried on chromosomes, are the basic
physical and functional units of heredity. Genes are specific
sequences of bases that encode instructions on how to make
proteins. Although genes get a lot of attention, it’s the proteins
that perform most life functions and even make up the majority of
cellular structures. When genes are altered so that the encoded
proteins are unable to carry out their normal functions, genetic
disorders can result.
Gene therapy is a technique for correcting defective genes
responsible for disease development. There are several
approaches for correcting the faulty genes.
Gene Therapy

Defective Gene – Genetic Disorder
Gene is said to be defective if there is change in the inherited
gene. It is clear that any heritable change in a gene is brought
about by mutation. Mutation can be defined as any change in the
base sequence of the DNA. The consequences of mutation is-
# premature termination in the growth of the peptide chain,
# synthesis of non-functional protein.
In either events, the absence of the normal protein can lead to a
variety of clinical manifestations depending on the structural or
enzymatic role that normally plays in the cell. Such conditions
range from mild disorders that require no treatment (e.g., color
blindness) to life threatening disease (e.g., hemophilia, cystic
fibrosis).

Over 45,000 human diseases have been identified related
directly to the genetic disorders.
Treatment of Genetic Disorders-Gene therapy versus
conventional therapy
Limitations of the conventional therapy
Therapy based on the replacement of the missing or defective
protein is available for only a few of these disorders. Example:
Factor VIII for hemophilia, adenosine deaminase for SCIS,
transfusion for sickle cell disease.

Limitations of the conventional therapy
Conventional therapies are only partially effective in ameliorating the
manifestations of the disease and are accompanied by significant
complications.
For most genetic disease, providing the missing protein in a therapeutic
fashion is not feasible due to the complex and fragile nature of the
protein and the need to deliver the protein to a specific subcellular
localization (i.e., cell surface expression, lysosomal localization,
etc.).
Transplantation of the major affected organ has been done in some
instances (e.g., bone marrow transplantation for sickle cell
disease, liver transplantation for hyperlipidemia), but this has also
severe limitations of organ availability and adverse consequences
arising from the immune suppression required to prevent rejection
of an allogenetic tissue.

Providing a normal copy of the defective gene to the affected tissues
would circumvent the problem of delivering complex proteins, as the
protein could be synthesized within the cells using the normal cellular
pathways.
The limited number of tissues are affected by most inherited disorders,
this greatly simplifies the requirements for effective gene therapy,
since a functional copy of the gene need to be provided only to those
tissues that actually require it. So gene therapy is generally applied to
correct defect in only part of the body and thus targeting of the
therapeutic gene to a specialized area is important in gene therapy.
If the gene transfer can be targeted to the major affected organs, thus
side effects arising from ectopic gene expression in nontargeted cells
might be avoided.
Problems overcome by gene therapy

As with other pharmaceutical agents, cell-specific targeting has the
advantage of decreasing the effective volume of distribution and
the amount of gene transfer agent needed.
 The majority of gene therapy trials underway are for the treatment of
acquired disorders such as AIDS, cancer, CVS and inherited disorder
arising from single gene defects, such as AD, LSD, etc.
 In case of inherited disorder, the defective gene that cause the
disorder is the subject of intervention, and-
 In the case of acquired diseases, either a defective gene that
contributes to the disorder or a gene that mediates an unrelated
biochemical process may be the basis for intervention. Example:
Treatment of HIV infection potentially could rely on the interruption of
viral processes that contribute to the pathogenesis of AIDS,
antisense mRNA, a dominant negtaive mutant protein.
Where is gene therapy applied?Where is gene therapy applied?

The ability to transfect genes into cells and to cause their expression
is the basis of gene therapy. So gene medicines are generally
based on gene expression system that contains a therapeutic
gene and a delivery gene. The success of gene therapy is largely
dependent on the development of a vector or vehicle that can
selectively and efficiently deliver a gene to target cell with minimal
toxicity.
Two major methods have been described for gene transfer:
- Viral-mediated gene transfer : various viruses are used as
carrier, e.g., retrovirus, adenovirus, adenoassociated virus, etc.
- Non-viral-mediated gene transfer : various types of synthetic
vectors are used as carrier, e.g., cationic lipid, cationic polymer,
etc.
Basis of Gene Therapy

Candidate Disease for Gene Therapy
Diseases wherein gene therapy has been focused upon include-
Genetic Disease Defect Target cells
1. Severe Combined Adenosine deaminase Bone marrow
immunodeficiency(SCID/ADA) cell or T-cell
2. Hemophilia- A Factor VIII deficiency Liver, fibroblast
- B Factor IX deficiency or bone marrow
3. Cystic fibrosis Loss of CFTR gene Airways in lung
4. Familial hypercholesterimia Def. LDL receptor Liver
5. Hemoglobinopathy
Thalessimia structural defect bone marrow
Sickel cell anemia of alpha/beta- globin gene

Disease-Acquired Defect Target cells
6. Neurologic diseases
Alzheimer’s disease APP gene Neuron/gilal
Parkinson’s disease alpha-Syn gene Neuron
7. Cardiovascular disease
Atheroscleorosis HDL Vascular endothelial cell
8. Infectious disease
AIDS T-cell
Hepatitis B Liver

Approaches for gene therapy
Different approaches have been tried for effective gene therapy. They
are:
1. Gene modification- a) Replacement therapy
b) Corrective gene therapy
2. Gene transfer- a) Physical (microinjection, electroporation, etc.)
b) Chemical (Liposome, polymer, etc.)
c) Biological (Viral and non-viral vector
3. Gene transfer in specific cell lines-
a) Somatic gene therapy
b) Germline gene therapy
4. Eugenic approach (gene insertion)

A) Replacement Therapy
In this therapy, a defective gene is inserted somewhere in the genome so
that its product could replace that of a defective gene. This approach
may be suitable for recessive disorders which are marked by
deficiency of an enzyme or others proteins. It may not be suitable for
dominant disorders which are associated with the production of an
abnormal gene product.
B) Corrective Gene Therapy
In this therapy it requires replacement of a mutant gene or a part of it with
a normal. This can be achieved by using recombinant DNA
technology. Another form of corrective therapy involves the
suppression of a particular mutation by a transfer RNA that is
introduced in a cell.
1. Gene modification

2. Gene Transfer:
Gene transfer can be brought about by- A) Physical, B)
Chemical, and C) Biological means.
A) Physical: Several physical approaches have been developed
to enhance the efficiency of gene transfer via naked DNA.
These physical approaches allow DNA directly to penetrate
cell membrane. For example: Electroporation, gene gun, etc.
B) Chemical: Synthetic chemicals have been developed for gene
transfer efficiently. Example: Cationic lipid, cationic polymer,
etc.
C) Biological: Viruses have been developed as a logical tools for
gene transfer, because they have evolved mechanisms to
enter the cell. Example: Adenovirus, retrovirus, etc.

3. Gene Transfer in specific cell lines:
Somatic Gene Therapy
Somatic gene therapy involves the insertion of genes into
specific somatic cells. They can function during the life time of
an individual and hence correct a genetic disease. However, it
presents a number of practical problems.
Germ Line Therapy
Germ line therapy involves injection or insertion of gene into
germ cells, into fertilized eggs. Here, the inserted gene would
be passed onto future generations too.
4. Eugenic Approach (gene insertion)
It is brought about by inserting genes to alter or improve complex
traits of a person. For example: intelligence. However, it is far
beyond the current technologial feasibility.

Essential Prerequisites for Gene Therapy
Before the patient is being subjected to gene therapy, several
essential prerequisites are to be fulfilled. They are:
1. It must be possible to isolate the appropriate gene and to
define its major regulatory regions.
2. Identification and harvesting of appropriate target cells,
3. Development of safe and efficient vectors with which the new
gene could be introduced.
4. Clear evidence of experimental data on the adequate
functioning of the inserted gene, life span of the recipient cell
and that no untoward effect exists, should be ensured.
5. Last but not the least, the patient or their family must be fully
counselled.

An ideal delivery system would be one-
- Which could accommodate a broad size range of inserted DNA,
- Which could be targeted to specific types of cells,
- Which would not permit replication of the DNA,
- Which could provide long-term gene expression,
- Which could be easily produced,
- Which will be non-toxic and non-immunogenic
Still such a delivery system does not exist.
There are two major delivery systems for gene transfer:
- Viral-mediated gene delivery: various viruses are used as
carrier, e.g., retrovirus, adenovirus, adenoassociated virus, etc.
- Non-viral-mediated gene delivery : various types of synthetic
vectors are used as carrier, e.g., cationic lipid, cationic polymer, etc.
Qualities of an ideal delivery system

Viral Mediated Gene Delivery
Why virus is used as a tool for gene delivery-
1. Viruses have evolved mechanisms to enter into the cell.
2. They can replicate inside the cell,
3. They use the cellular machinery to express their genes.
The natural life cycle of mammalian viruses has made them a vehicle for
the transfer of therapeutic gene.
But for viral vectors to be useful, several viral functions must be altered-
1. At a minimum, the virus must be rendered replication-incompetent to
prevent uncontrolled spread of the transgene,
2. Some elements of its own genome must be removed to allow for
insertion of the transgene.
3. Beyond this, additional modifications are dependent on the specific
virus to avoid its toxicity.

Retroviruses are composed of an RNA genome that is packaged in an
envelope derived from cell membrane and viral proteins. Retroviruses
were first described for gene transfer applications in 1981 and first
utilized in clinical trials in 1989.
Retroviruses have had the greatest clinical use so far and offer the
potential for long-term gene expression from a stably integrated
transgene. Characteristically, it offers a number of advantages in
effective gene delivery to the cell:
1. It provides for official entry of genetic materials into a wide variety of
cells,
2. It is well understood with simple molecular biology,
3. It integrates into host genome,
4. It has a potential control over the range of cells to be infected,
5. It is capable of gene expression,
Retrovirus

6. It could carry upto 8Kb of coding information,
7. Above all, it establishes one way, nonreplicative infection of target
cells,
8. It lack irrelevant and potentially immunogenic proteins.
Drawbacks in use of retrovirus:
1. Retrovirus is limited to dividing cells,
2. Large scale production is technically possible, although purification
and concentration potentially are problematic due to the instability
of the virus.
Based on genomic structure, retroviruses are of two types:
1. Simple retrovirus, eg, most oncoviruses.
2. Complex retrovirus, e.g., lentivirus, spumavirus
Retrovirus

Replication Cycle of Retroviruses

Genome Structure of Retroviruses
LTRLTR
ATTATT
U3 R U5U3 R U5

Figure 1. Proviral genome structure of the Murine Leukemia Virus
(MLV), a simple retrovirus. Indicated are the 5’ and 3’ long
terminal repeat (LTR; open boxes) regions comprising U3, R and
U5, as well as open reading frames (filled boxes) for gag, pol and
envelope (env) proteins. Processed protein subunits are indicated
in bold. att, attachment site; cap, 5’RNA capping site; pA,
polyadenylation site; PBS, primer binding site; SD, splice donor;
, packaging signal; SA, splice acceptor; PPT, polypurine tract;
MA, matrix; CA, capsid; NC, nucleocapsid; PR, protease; RT,
reverse transcriptase; IN, integrase; SU, surface; TM, trans-
membrane; E, enhancer; P, promoter.
Retrovirus

Genome Structure of Retroviruses
LTRLTR
U3 R U5U3 R U5
Essential characteristics of a simple retrovirus:
1. The viral DNA contains large redundant sequences at the two ends
of the genome designated long terminal repeats (LTRs). LTRs can
be further divided into U3 (unique 3), R (repeat), and U5 (unique 5)
regions.
U3: The viral promoters and transcriptional enhancers are located in the
U3 region; R: The R region is essential for reverse transcription and
replication of all retroviruses. In addition, the R regions of some viruses
also contain elements important for gene expression. U5: The U5 region
contains sequences that facilitate the initiation of reverse transcription.
2. Immediately downstream of the 5’ LTR is a primer binding site
(PBS) that has sequence complementarity to a portion of a cellular
tRNA. Different tRNAs are used by different viruses as primers for
the initiation of reverse transcription.

Essential characteristics of a simple retrovirus:
3. The packaging signal (C) or encapsidation signal (E) are sequences
that interact with the viral proteins to accomplish specific packaging of
the viral RNA.
4. The coding regions of all retroviruses contain at least three genes. Gag:
The gag gene near the 5’ end of the viral genome codes for Gag
polyproteins that make up the viral capsid. The pol gene encodes reverse
transcriptase and integrase. Reverse transcriptase copies the viral RNA to
generate the viral DNA, whereas integrase integrates the viral DNA into the
host chromosome to form a provirus. The env gene codes for the envelope
polyprotein, which is cleaved into the transmembrane domain and the surface
domain (SU). The sequences that encode the viral protease (Pro) are always
located between gag and pol and are most often expressed as either a part of
the Gag polyprotein or as a part of the Gag-Pol polyprotein.
5. The region between env and the 3’ LTR contains a purine-rich region
known as polypurine tract (PPT) that is important for reverse
transcription.
6. Short sequences at the two ends of the LTR are important for
integration and are referred to as attachment sites (att). The att
interact with integrase and are necessary for efficient integration of the
viral DNA.

DEVELOPMENT OF RETROVIRAL VECTOR
The first retroviral vector systems were derived from murine leukaemia
virus (MLV).
There were (and are still) a number of reasons for choosing MLV as the
basis for such gene delivery systems, including (i) the biology of this
retrovirus is particularly well understood, (ii) the MLV genome was
among the earliest retroviral genomes molecularly cloned and (iii)
these viruses are able to infect cells efficiently.
Retroviral vector systems consist of two components: (i) a vector construct
that carries the gene to be delivered and provides the genome for the
recombinant virus, and (ii) a cell line that provides the viral proteins
required to produce the recombinant virus, known as packaging cells
Construction of vector
-Retroviral vectors are constructed from the proviral form of the virus. The
gaga, pol, and env genes are removed by the selectable marker to
make room for the gene(s) of therapeutic interest and to eliminate the
replicative functions of the virus. Upto 8 Kilobases of heterologous
DNA can be incorporated into the retroviral vector.

Construction of vector
-Along with the gene of therapeutic interest, gene encoding antibiotic
resistance often are included in the recombibant as a means of
selecting the virus-harbouring cultured cells. For example,
aminoglysoside-3’-phosphotransferase gene for kanamycin,
hygromycin B transferase gene for hygromycin. The antibiotic
resistance gene confers resistance to antibiotics.
- Sequences containing promoter and enhancer functions may also be
included with the transgene to facilitate the efficient expression,
and in some circumstances, to provide for tissue-specific
expression after administration in vivo. Alternatively, the promoter
and enhancer functions contained in the LTR of the virus may be
used for this purpose.
Marker gene Therapeutic gene with separate internal
promoter to drive high level expression
LTR NeoR CMV ADA LTR

Packaging Cell Lines or Helper Cell
To produce recombinant retroviral vector virions, the vector construct
carrying the gene(s) to be delivered is introduced by physical gene
transfer methods (such as transfection, electroporation etc.) into a
retroviral packaging cell line or helper cell. Helper cells are engineered
culture cells expressing viral proteins needed to propagate retroviral
vectors; this is generally achieved by transfecting plasmids expressing
viral proteins into culture cells. Most helper cell lines are derived from
cell clones to ensure uniformity in supporting retroviral vector
replication. These helper or packaging cells produce the viral
structural (Gag and Env) proteins and enzymes (pol-encoded RT, IN),
but are not able to package the viralRNA encoding these proteins
since the region required for encapsidation has been deleted. Instead
the proteins recognise and associate with genomic length RNA from
the introduced vector construct, which carries an intact region, to
form recombinant virus particles. The recombinant virus particles
carrying the retroviral vector genome bud out of the packaging cell
line into the cell culture medium.
The virus-containing medium is either directly filtered to remove cells and
cellular debris and then used to infect the target cell, or virus is
purified and concentrated before infecting target cells.

After the virus has bound to the receptor on the cell surface, the viral capsid
is delivered into the cell and the viral RNA is reverse transcribed into a
DNA form which integrates into the host cell DNA. The integrated viral
DNA (provirus) functions essentially as any other cellular gene and
directs the synthesis of the products of the delivered gene(s).
Major Problem with Retroviral Vector
The major problem with two-component retroviral vector systems arises as
a result of the naturally occurring phenomenon of homologous
recombination. If the vector provirus and the provirus providing the
structural proteins in the packaging cells recombine, there is a
possibility that replication- competent retrovirus will arise. Such virus
is essentially a wild-type retrovirus and no longer carries the delivered
gene(s). Replication-competent virus rapidly infects many cells and
may eventually cause insertional mutagenesis. Consequently,
considerable effort has been devoted to the design of superior
packaging systems that drastically reduce the possibility of
recombination occurring, as well as to produce improved, safer
vectors that cannot replicate even if recombination occurs.

IMPROVEMENTS TO PACKAGING CELLS
1. Improvements to packaging cells have involved removing as much of
the retroviral information as possible to reduce the possibility of
homologous recombination occurring. The retroviral promoter and
termination sequences can be replaced by heterologous promoters
and termination sequences. This has the additional advantage of
allowing the use of promoters that are more strongly active than the
retroviral promoter, thereby giving rise to higher levels of viral protein
production.
2. The coding information for the viral proteins cannot be removed by
necessity, but these proteins can be made from separate constructs
so that additional recombination events are required to recreate a
complete replication-competent retrovirus. This has been achieved
by expressing the Gag and Pol proteins from one construct and the
Env proteins from a second construct.
3. In addition to the improvements to packaging cells, safer retroviral vector
constructs also have been produced that carry an artificially inserted
stop codon in the Gag reading frame. This ensures that even if
replication-competent virus is generated, it will not be able to express
its Gag and Pol proteins and thus virus assembly and release will be
inhibited.

CLINICAL ADMINISTRATION OF RETROVIRUS
The clinical administration of retroviruses has been accomplished by:
- Ex vivo transduction of patients cell,
- Direct injection of virus into tissue,
- Administration of retroviral producer cells.
1. Ex vivo gene transfer: The ex vivo approach has been most widely
employed in human clinical trials. Although cumbersome in that it
requires the isolation and maintenance in tissue culture of the
patients cell. It has the advantage that the extent of gene transfer
can be quantified readily and a specific population of cells can be
targetted.
2. In vivo gene transfer: Retroviruses are being tested as potential agents
to treat brain tumors which, in many circumstances, are relatively
inaccessible. Although the direct stereotactic injection of
recombinant retrovirus into the target tissue is possible, the
efficiency of gene transfer is very low.

CLINICAL ADMINISTRATION OF RETROVIRUS
The clinical administration of retroviruses has been accomplished by:
- Ex vivo transduction of patients cell,
- Direct injection of virus into tissue,
1. Ex vivo gene transfer: The ex vivo approach has been most widely
employed in human clinical trials. Although cumbersome in that it
requires the isolation and maintenance in tissue culture of the
patients cell. It has the advantage that the extent of gene transfer
can be quantified readily and a specific population of cells can be
targetted.
2. In vivo gene transfer: Retroviruses are being tested as potential agents
to treat brain tumors which, in many circumstances, are relatively
inaccessible. Although the direct stereotactic injection of
recombinant retrovirus into the target tissue is possible, the
efficiency of gene transfer is very low.

CLINICAL APPLICATION OF RETROVIRUS
Gene therapy approaches involving retroviral vectors can be used to treat
several different types of human diseases. Retroviral vectors are
being tested in many clinical trials. A few examples of gene therapy
clinical trials involving retroviral vectors are briefly described below:
1. The first clinical trials of human gene therapy using retroviral vector was
designed to correct a genetic disorder known as adenosine
deaminase (ADA) deficiency. The patients lack adenosine
deaminase which results in SCID. The lymphocytes of the patients
were harvested and transduced or transfected ex-vivo with a
retrovirus containing a functional ADA gene and then the
lymphocytes were reinfused into the host.
2. Hepatic genetic deficiencies have been experimentally treated with
genes introduced via retroviral vectors. A case of
hypercholesterolemia in rabbits have been treated ex vivo and
reimplantation of hepatocytes with the LDL-receptor gene.

Structure of Adenovirus
Fig. Structure of adenovirus. The locations of the capsid and cement
components are reasonably well defined. In contrast, the disposition of
the core components and the virus DNA is largely conjectural.

BASIC CHARACTERISTICS OF ADENOVIRUS
Adenovirus is a DNA virus having 60-90 nm in diameter. It has double
stranded DNA genome and do not possess a lipid envelope
Unlike retrovirus, adenoviruses have a characteristic morphology, with an
icosahedral capsid consisting of three major proteins, hexon (II),
penton base (III) and a knobbed fibre (IV), along with a number of
other minor proteins, VI, VIII, IX, IIIa and IVa2. The virus genome is
a linear, double-stranded DNA with a terminal protein (TP) attached
covalently to the 5´ termini, which have inverted terminal repeats
(ITRs). The virus DNA is intimately associated with the highly basic
protein VII and a small peptide termed mu. Another protein, V, is
packaged with this DNA–protein complex and appears to provide a
structural link to the capsid via protein VI. The virus also contains a
virus-encoded protease (Pr), which is necessary for processing of
some of the structural proteins to produce mature infectious virus.
Adenoviral genome encodes approximately 15 proteins.

Adenovirus infects a wide variety of human tissues such as respiratory
epithelium, vascular endothelium, cardiac and skeletal muscle,
peripheral and central nervous system, hepatocytes, the exocrine
pancreas and many tumor types.
Adenovirus deliver their genomes to the nucleus and can replicate with
high efficiency, they are prime candidates for the expression and
delivery of therapeutic genes.
ADENOVIRUS-MEDIATED INFECTION OF TARGET CELLS
Infection takes place when the fiber protein of capsid binds a cell surface
receptor.
Subsequently, peptide sequences in the penton base portion of the capsid
engage integrin receptor domains (alpha3, beta3; alpha3, beta5) on
the cell surface. This leads to virus internalization via endosomal
pathway where the viral particles begin to disassemble.
The virus escapes the endosome prior to its fusion with lysosomal
compartment and thus avoid digestion.
The viral DNA is able to enter the target cell nucleus and begin
transcription of viral mRNA.

Genomic Organization of Human Adenoviruses
U3 R U5U3 R U5
FIGURE 1. Simplified transcription map of the Ad5 genome.
Essential characteristics of adenoviruses:
1. Most Ad vectors are based on the well-characterized human Ad serotypes 2
or 5. The genome consists of a 36 kb linear, double-stranded DNA
molecule. Each end of the genome has an inverted terminal repeat (ITR)
of 100-140 bp to which the terminal protein is covalently linked. ITRs are
required for viral DNA replication.
L1 L2 L3 L4 L5
LTR LTR
Y

Essential characteristics of adenoviruses
2. At 200 nucleotides of the 5’ extreme is located packaging signal (Y),
sequence that directs the packaging of the viral genome through its
interaction with various viral and cellular proteins. Gene are encoded
on both strands of the DNA in a series of overlapping transcription
units.
3. The viral genome is classified based on the timing of their expression
(Fig. 1). Early genes (E1, E2, E3, and E4) are expressed before the
onset of DNA replication. Proteins encoded by the early genes
function to activate other Ad genes, replicate the viral DNA, interfere
with immune recognition of infected cells, and modify the host-cell
environment to make it more conducive to viral replication. The late
genes (L1-L5) are expressed after DNA replication and primarily
encode proteins involved in capsid production and packaging of the
Ad genome.
E1 = E1A genes activate the cascade of all other viral genes because only this
gene needs the presence of cellular factors for its transcription. Furthermore,
E1A proteins inhibit cell replication, which contributes to viral genome
replication more efficiently. The region E1B codies for proteins that inhibit
apoptosis and prepare the intranuclear environment for adenoviral replication
(E1B 55K and E1B 19K).

E2= The E2 region encodes proteins required for viral DNA replication,
including a single stranded DNA binding protein (E2a) and both the
viral DNA polymerase and the 55 kDa terminal protein (E2b).
E3= The E3 region encodes proteins that prevent the cellular immune
response and thus, the adenovirus gets the time required to complete
the infection cycle.
E4= the E4 region encodes 7 open reading frames (ORFs) with clearly
different functions. These functions include participation in the viral
genome replication, splicing, mRNA transport, inhibition of cellular
protein synthesis, regulation of apoptosis and cell lysis.
Expression of the early genes leads to DNA replication, approximately
eight hours after infection, and subsequent activation of the late genes
under the transcriptional control of the major late promoter, production
of virus progeny, and finally, death of the host cell and virus release.
Essential characteristics of adenoviruses

DESIGN AND CONSTRUCTION OF REPLICATIONDEFECTIVE
HUMAN ADENOVIRAL VECTORS
Replication-deficient adenoviral vectors, similar to other viral vectors, are
composed of the virion structure surrounding a modified viral genome.
To date, most vector particles are based on the wild-type capsid
structure which, in addition to protecting the viral DNA, provides the
means to bind and enter (transduce) target cells. However, the viral
genome has been modified substantially. These changes are
designed to disable growth of the virus in target cells, by deleting viral
functions critical to the regulation of DNA replication and viral gene
expression, while maintaining the ability to grow in available packaging
or helper cells. Deletion of such sequences provides space within the
viral genome for insertion of exogenous DNA that encodes and
enables appropriate expression of the gene of interest (transgene).
The subgroupC adenoviruses, serotypes 2 and 5 (Ad2 and Ad5), are
among the best studied adenoviruses, and the viruses used most
commonly as gene transfer vectors. The vast majority of adenoviral
vectors for gene therapy are E1 replacement vectors, where the

DESIGN AND CONSTRUCTION OF ADENOVIRAL VECTOR
transgene is inserted in place of the E1 region. This E1 region deletion
includes the entire E1a gene and approximately 60% of the E1b gene.
The vectors retain the immediate 5 end of the viral genome, including
the left inverted terminal repeat (ITR) and encapsidation signal (Y),
sequences required for packaging, and the overlapping E1 enhancer
region, in addition to the remainder of the viral genome (Figure 5.1).
As the E1 gene products lead to sequential activation of the major
transcription units, deletion of this region greatly reduces early and
late gene expression and renders the virus severely replication
impaired.
To provide more space within the adenoviral vectors for insertion of the
transgene, the E3 region, not required for viral replication or growth, is
also frequently deleted. Occasionally, the transgene is inserted into
this E3 region deletion. Adenoviral vectors lacking only E1 and E3
regions are referred to as first generation, or Av1, vectors.
Adenoviruses can effectively package DNA up to 105% of the
genome size, allowing the accommodation of up to 8 kb of exogenous
DNA in E1/E3 deleted Av1 vectors.

The transgene transcriptional unit consists of the elements required to
enable appropriate expression of the transgene such as the promoter,
the gene of interest, and a polyadenylation signal, and, in most
instances, is designed to maximize the expression of the exogenous
gene. A large variety of promoters have been utilized for transgene
expression, the choice of which depends on the application and the
target tissue.
Strong, constitutively expressed viral promoters such as the adenovirus
major later promoter, the Rous sarcoma virus promoter, the
cytomegalovirus (CMV) promoter and a hybrid CMV enhancer/-actin
promoter have been incorporated into recombinant adenoviral
vectors. More recently, the use of cellular, tissue-specific promoters
such as the liver-specific albumin promoter, lung-specific cystic
fibrosis transmembrane conductance regulator promoter, the cardiac
muscle-specific myosin light chain-2 promoter, and the
hepatomaspecific -fetoprotein promoter has been described. Finally,
regulatable promoters responsive to hormonal or pharmacological
agents have been incorporated into adenoviral vectors. The inclusion
of tissue-specific and/or regulatable promoters to the transgene
expression
IMPROVEMENT OF TRANSGENE EXPRESSION

cassette avoids the unknown consequences of overexpression of genes in
tissues other than the targeted organ, and may, therefore, increase
the safety of such vectors. An additional approach shown to increase
the potency of the transgene in adenoviral vectors is the introduction
of genomic elements into the expression cassette. For example, the
addition of an intron to the human factor VIII (FVIII) cDNA boosted in
vivo expression approximately 10-fold, and the inclusion of the
human factor IX (FIX) truncated first intron and 5 and 3 untranslated
regions to the human FIX cDNA functioned synergistically to increase
human FIX plasma levels in transduced mice approximately 2000-
fold. Finally, a variety of signals have been used to direct
polyadenylation such as the simian virus 40 polyadenylation signal.
IMPROVEMENT OF TRANSGENE EXPRESSION

The propagation of Av1 adenoviral vectors, rendered almost
completely replication defective by the deletion of the E1
region, requires the generation of cell lines to complement the
E1 functions in trans. Several human cell lines that
constitutively express the E1 proteins have been established.
To date, the most widely used cell line, 293, consists of
human embryonic kidney cells transformed with sheared Ad5
DNA that express the left 11% of the Ad5 genome. While 293
cells allow replication of Av1 vectors to high titers, this cell line
is not ideal for large-scale vector production. Recombination
between homologous E1 region sequences encoded in the
vectors with those inserted in the 293 cell genome has the
potential to generate replication-competent adenoviruses
(RCA). Furthermore, the presence of RCA in preparations of
adenoviral vectors was shown to induce significant tissue
damage in vivo. The generation of RCA may be prevented by
elimination of sequence homology between the vector DNA
PRPAGATION AND PURIFICATION OF ADENOVIRAL VECTOR

and the adenovirus sequences in the genome of the
complementing cells.
Unlike retroviral vectors, the stability of the adenovirus virion allows
extensive purification and concentration without significant
loss of activity. Procedures for adenoviral vector purification
involve harvest and disruption of infected cells using multiple
freeze and thaw cycles, or sonication, and removal of the cell
debris by centrifugation. Vector is purified and concentrated in
one to three CsCl centrifugation steps, followed by dialysis or
chromatography. However, for large-scale manufacturing,
chromatographic methods which avoid CsCl centrifugation are
desirable. Vector concentration is determined
spectrophotometrically, to evaluate particle number, and
biologically, to measure infectivity by gene transfer or plaque
assay. Concentrated vector preparations containing 1011
plaque forming units per milliliter can be obtained routinely.
PRPAGATION AND PURIFICATION OF ADENOVIRAL VECTOR

LIMITATIONS AND IMPROVEMENTS OF ADENOVIRAL
VECTORS
Although recombinant adenoviral vectors have become increasingly
popular gene delivery vehicles, there are two major limitations that
could hamper their eventual use in human gene therapy.
First, the adenoviral vectors usually mediate a short-term gene
expression;
Second, adenoviral vectors tend to elicit strong immune and
inflammatory responses in vivo. A single large dose of adenovirus can
efficiently provoke production of neutralizing antibodies directed to the
viral particle, which in turn would preclude or reduce the efficiency of
repeated systemic administration.
Several approaches have been explored to circumvent the immunogenicity
of adenoviral vectors by changing the vector designs. For instance, a
new generation of adenoviral vectors has been constructed with
deletion of E1, E2 and E4 genes in order to avoid expression of
immunogenic viral proteins in host cells.

Alternatively, constitutive expression of E3 gp19K protein in E1-deleted
vector has provided encouraging results with more stable transgene
expression in the liver and lung of animal models. The function of
gp19K is to inhibit the transport of major histocompatibility complex
class I molecules to cell surface, leading to the impairment of
antigen-presenting cells’ function and reduced clearance of
adenoviral infected cells by CTL immune responses.
The recently developed gutless adenoviral vectors, which have most or all
adenoviral genes deleted, have shown significantly reduced
immunogenicity and prolonged expression of homologous
transgenes in mice.
Many approaches are being developed to control host immune responses
at the time of infection. One of them is to transiently block cell
adhesion and co-stimulatory molecules, such as CD40 ligand, in
order to prevent both cytotoxic response and production of virus-
specific neutralizing antibodies. Immunomodulating cyto-kines, such
as IL-10 and IL-12, were also used to disrupt the balanced Th
activation towards either Th1 (cytotixic) or Th2 (humoral) subset,
thereby reducing antibody production and cellular immune response,
LIMITATIONS AND IMPROVEMENTS OF ADENOVIRAL VECTORS

respectively. Because TNF-alpha has been shown to play a key role in
adenovirus-induced immune response, inhibition of this pathway may
offer a particularly promising prospect in overcoming host cell immune
responses.
Manipulation of virus capsid components by genetic engineering may
provide another alternative to circumvent pre-existing humoral
response to the commonly used adenovirus serotype 5. In fact, vector
capsids displaying chimeric Ad5/Ad12 hexon monomers were shown
to overcome neutralizing antibodies in C57BL/7 mice primed with Ad5.
Interestingly, such chimeric capsid may also change the binding
affinity to host cells. For instance, chimeric capsid Ad5/Ad7 exhibited
an enhanced binding affinity for human lung epithelial cells but
significantly diminished efficiency for liver-directed gene transfer.
IMPROVEMENT OF EXPRESSION LEVEL

Long-term expression of transgenes is desirable for replacement gene
therapy. Several strategies have been developed to address the
drawback of adenovirus-mediated transient gene expression. For
example, a chimeric adenoviral-retroviral vector has been constructed
in order to maintain transgenes within actively dividing cells. This
chimeric virus was shown to infect cells and produce recombinant
retroviruses that can infect surrounding cells and integrate into host
chromosome. Similarly, a hybrid adenoviral/ adeno-associated virus
(AAV) was engineered and shown to integrate the transgene at a
specific locus of human chromosome 19. The major difference
between the two types of chimeric viruses is that AdV/AAV vector may
also maintain efficient and lasting transgene expression in nondividing
cells.
The transgene transcriptional unit consists of the elements required to enable
appropriate expression of the transgene such as the promoter, the gene of
interest, and a polyadenylation signal, and, in most instances, is designed
to maximize the expression of the exogenous gene. A large variety of
promoters have been utilized for transgene expression, the choice of
which depends on the application and the target tissue.

While adenoviral vectors can efficiently transfer genes into a broad
spectrum of cell types, this wide tropism also represents an apparent
drawback when gene delivery to a specific tissue is needed.
Moreover, the transgene expression mediated by adenoviral vectors
may require fine-tuned regulation for some, if not all, therapeutic
applications. Currently, expression of most transgenes is driven by
ubiquitous promoters of viral origin, such as the immediate-early
promoter from human cytomegalovirus (CMV) and the Rous sarcoma
virus long terminal repeat (LTR). Although these promoters provide
high levels of transgene expression, it is not always desirable and
specific enough for a wide variety of therapeutic applications. In this
respect, endogenous promoters have been used to restrict transfer
gene expression to particular cell types at a physiologically relevant
level. For instance, tissue-specific transgene expression was achieved
when a transactivator was coupled with a tissue-specific promoters
such as the liver-specific albumin promoter, lung-specific cystic
fibrosis transmembrane conductance regulator promoter, the cardiac
muscle-specific myosin light chain-2 promoter, and the
hepatomaspecific -fetoprotein promoter has been described.

An additional approach shown to increase the potency of the transgene in
adenoviral vectors is the introduction of genomic elements into the
expression cassette. For example, the addition of an intron to the
human factor VIII (FVIII) cDNA boosted in vivo expression
approximately 10-fold, and the inclusion of the human factor IX (FIX)
truncated first intron and 5 and 3 untranslated regions to the human
FIX cDNA functioned synergistically to increase human FIX plasma
levels in transduced mice approximately 2000-fold. Finally, a variety of
signals have been used to direct polyadenylation such as the simian
virus 40 polyadenylation signal.
FORMATION OF REPLICATION-COMPETENT ADENOVIRUS
Recombination between homologous E1 region sequences encoded in the
vectors with those inserted in the helper cell, 293 cell genome has the
potential to generate replication-competent adenoviruses (RCA).
Furthermore, the presence of RCA in preparations of adenoviral
vectors was shown to induce significant tissue damage in vivo. The
generation of RCA may be prevented by elimination of sequence
homology between the vector DNA and helper cell DNA.

APPLICATION OF ADENOVIRAL VECTOR
One of the major advantages of adenoviral vectors is that, for a wide
variety of cell types, they provide more efficient gene transfer
compared with other gene delivery approaches. This is especially
true for in vivo gene transfer. Recombinant adenoviruses can transfer
genes into both proliferating and quiescent cells. One limitation of
adenovirus-mediated gene expression is that it is transient, ranging
from two weeks to a few months, largely because recombinant
adenoviruses are replication-deficient and do not integrate into the
host genome. Thus, adenovirus vectors may not be suitable for long-
term correction of chronic disorders but should be adequate for
therapeutic strategies that require high and transient gene
expression.
Use of Adenoviral Vectors for Cancer Gene Therapy
In the majority of cancer gene therapy trials, adenoviral vectors have been
administered in vivo, and have been used to transfer drug-sensitive
genes (such as the herpes virus thymidine kinase), immuno-
modulators (such as IL-2, IL-12, FasL), melanoma tumor antigens
(such as MART-1), or tumor suppressor genes (such as p53). To
date, many tumor

types have been treated with adenoviral based gene transfer. These
include melanoma, prostate cancer, mesothelioma, pancreatic
cancer, lung cancer, neuroblastoma, glioblastoma, etc
Use of Adenoviral Vectors for Non-cancer Gene Therapy
Monogenic Diseases
Adenoviral vectors have also played an important role in gene transfer
studies directed to several monogenic diseases. For example, cystic
fibrosis (transmembrane conductance regulator). Adenoviral vectors
expressing CFTR have been used in phase I clinical studies on the
biosafety and efficacy of gene transfer.
Genetic and Metabolic Liver Diseases
Because adenoviral vectors have been shown to mediate efficient gene
transfer to hepatocytes, adenoviral directed gene delivery has been
pursued as potential therapies for a wide variety of genetic and
metabolic liver disorders, such as lysosomal storage diseases,
glycogen storage diseases, phenylketonuria, and Tay-Sachs disease.

Neurodegenerative Diseases
Recently, the efficacy of adenoviral vectors has been demonstrated in
several models of neurodegenerative diseases including Parkinson’s
disease (PD) and motor neuron diseases. In rat PD models,
adenoviral vectors expressing either tyrosine hydroxylase,
superoxide dismutase or glial-derived neurotrophic factor improved
the survival and functional efficacy of dopaminergic cells.
Cardiovascular Diseases and Tissue Regeneration
At least in animal models, adenoviral vectors have also been shown to
effectively transducer therapeutic genes for several cardiovascular
diseases, such as atherosclerosis, cerebral ischemia, familial
hypercholesterolemia, hypertension, and cardiac arrhythmias.

ADENOASSOCIATED VIRUS
Basic Characteristics of Adenoassocited Virus:
Adeno-associated viruses, from the parvovirus family, are small
nonenveloped viruses (20-25 nm) with a genome of single
stranded DNA (ssDNA), which is approximately 4.7 kb in size.
These viruses can insert genetic material at a specific site on
chromosome 19 with near 100% certainty.
There are a few disadvantages to using AAV, including the small
amount of DNA it can carry (low capacity) and the difficulty in
producing it.
This type of virus is being used, however, because it is non-
pathogenic (most people carry this harmless virus).
In contrast to adenoviruses, most people treated with AAV will not
build an immune response to remove the virus and the cells
that have been successfully treated with it.

Figure 1. Genome organisation of Adeno-associated viruses.
The AAV genome is built of single-stranded deoxyribonucleic acid
(ssDNA), either positive- or negative-sensed, which is about
4.7 kilobase long. The genome comprises inverted terminal
repeats (ITRs) at both ends of the DNA strand, and two open
reading frames (ORFs): rep and cap (see figure 1). The
former is composed of four overlapping genes encoding Rep
proteins required for the AAV life cycle, and the latter
contains overlapping nucleotide sequences of capsid
proteins: VP1, VP2 and VP3, which interact together to form
a capsid of an icosahedral symmetry.
GENOMIC ORGANIZATION OF AAV VECTORS

The Inverted Terminal Repeat (ITR) sequences comprise 145
bases each. They were named so because of their symmetry,
which was shown to be required for efficient multiplication of
the AAV genome. Another property of these sequences is their
ability to form a hairpin, which contributes to so-called self-
priming that allows primase-independent synthesis of the
second DNA strand. The ITRs were also shown to be required
for both integration of the AAV DNA into the host cell genome
and rescue from it, as well as for efficient encapsidation of the
AAV DNA combined with generation of a fully-assembled,
deoxyribonuclease-resistant AAV particles.
With regard to gene therapy, ITRs seem to be the only sequences
required in cis next to the therapeutic gene: structural (cap)
and packaging (rep) genes can be delivered in trans. With this
assumption many methods were established for efficient
production of recombinant AAV (rAAV) vectors containing a
reporter or therapeutic gene.
LIMITATIONS AND IMPROVEMENTS OF ADENOVIRAL VECTORSGENOMIC ORGANIZATION OF AAV VECTORS

Several trials with AAV are on-going or in preparation, mainly
trying to treat muscle and eye diseases; the two tissues where
the virus seems particularly useful.
However, clinical trials have also been initiated where AAV vectors
are used to deliver genes to the brain. This is possible
because AAV viruses can infect non-dividing (quiescent) cells,
such as neurons in which their genomes are expressed for a
long time.
In recent human trials, CD8+ immune cells have recognized the
AAV infected cells as compromised and killed these cells
accordingly. This action appears to be triggered by part of the
capsid or outer coat of the type 2 virus.
APPLICATION OF ADENOASSOCIATED VIRAL VECTORS

One of the major limitations for the use of AAV as a gene delivery vehicle is
the relatively small packaging capacity. The unique ability of AAV
vectors to become joined into concatamers by head-to-tail
recombination of the ITRs has been exploited as a means to increase
the coding capacity. In this approach, either the gene itself or the
different elements of the transgene expression cassette are split over
two AAV vectors that are administered simultaneously. Transgene
expression is obtained only after recombination between the two viral
genomes, but the efficiency is often reduced as compared to single
vector transduction.
The AAV vectors do not contain any viral coding regions, and therefore,
there is no toxicity associated with gene expression. However, a
single injection of AAV vector elicits a strong humoral immune
response against the viral capsid, which will interfere with re-
administration of the vector. Furthermore, natural infections have
resulted in a high prevalence of circulating neutralizing antibodies
against AAV in the majority of the population, which may inhibit
transduction.
LIMITATIONS OF ADENOASSOCIATED VIRAL VECTORS

Herpes simplex viruses (HSV) belong to the subfamily of
Alphaherpesvirinae. Herpes viruses consists of a relatively large linear
DNA genome of double-stranded DNA 150 kb in length, encased
within an icosahedral protein cage called the capsid, which is wrapped
in a lipid bilayer called the envelope. The envelope is joined to the
capsid by means of a tegument. This complete particle is known as
the virion.
The genome of Herpes viruses encodes some 100-200 genes. These
genes encode a variety of proteins involved in forming the capsid,
tegument and envelope of the virus, as well as controlling the
replication and infectivity of the virus.
Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are two species of the
herpes virus family, which cause infections in humans. An infection by
a herpes simplex virus is marked by watery blisters in the skin or
mucous membranes of the mouth, lips or genitals. The genomes of
HSV-1 and HSV-2 are complex, and contain two unique regions called
the long unique region (UL) and the short unique region (US). Of the
74 known ORFs, UL contains 56 viral genes,
HERPES SIMPLEX VIRAL VECTORS

HERPES SIMPLEX VIRAL VECTOR
whereas US contains only 12. Transcription of HSV genes is catalyzed by
RNA polymerase II of the infected host. Immediate early genes,
which encode proteins that regulate the expression of early and late
viral genes, are the first to be expressed following infection. Early
gene expression follows, to allow the synthesis of enzymes involved
in DNA replication and the production of certain envelope
glycoproteins. Expression of late genes occurs last, this group of
genes predominantly encode proteins that form the virion particle.
Herpes viruses are currently used as gene transfer vectors due to their
specific advantages over other viral vectors. Among the unique
features of HSV derived vectors are the very high transgenic capacity
of the virus particle allowing to carry long sequences of foreign DNA,
the genetic complexity of the virus genome, allowing to generate
many different types of attenuated vectors possessing oncolytic
activity, and the ability of HSV vectors to invade and establish lifelong
non-toxic latent infections in neurons from sensory ganglia from
where transgenes can be strongly and long-term expressed.
APPLICATION OF HERPES SIMPLEX VIRAL VECTOR

Alphaviruses, like Sindbis Virus and Semliki Forest Virus, belong to the
Togaviridae family of viruses. There are 27 alphaviruses, able to infect
various vertebrates such as humans, rodents, birds, and larger mammals
such as horses as well as invertebrates. Alphaviruses particles are
enveloped have a 70 nm diameter, tend to be spherical and have a 40 nm
isometric nucleocapsid.
The genome of alphaviruses consists of a single stranded positive sense RNA.
The total genome length ranges between 11 and 12 kb, and has a 5’ cap,
and 3’ poly-A tail. There are two open reading frames (ORF’s) in the
genome, non-structural and structural. The first is non structural and
encodes proteins for transcription and replication of viral RNA, and the
second encodes four structural proteins: Capsid protein C, Envelope
glycoprotein E1, Envelope glycoprotein E2, and Envelope glycoprotein
E3. The expression of these proteins and replication of the viral genome
all
ALPHA VIRUS VIRAL VECTOR

takes place in the cytoplasm of the host cells.
Alphaviruses are of interest to gene therapy researchers, in particular the
Ross River virus, Sindbis virus, Semliki Forest virus, and Venezuelan
Equine Encephalitis virus have all been used to develop viral vectors
for gene delivery. Application of replication-deficient vectors leads to
short-term expression, which makes these vectors highly attractive
for cancer gene therapy. Alphavirus vectors carrying therapeutic or
toxic genes used for intratumoral injections have demonstrated
efficient tumor regression.
Of particular interest are the chimeric viruses that may be formed with
alphaviral envelopes and retroviral capsids. Such chimeras are
termed pseudotyped viruses. Alphaviral envelope pseudotypes of
retroviruses or lentiviruses are able to integrate the genes that they
carry into the expansive range of potential host cells that are
recognized and infected by the alphaviral envelope proteins E2 and
E1. The stable integration of viral genes is mediated by the retroviral
interiors of these vectors.
APPLICATION OF ALPHAVIRUS VIRAL VECTOR

There are limitations to the use of alphaviruses in the field of gene
therapy due to their lack of targeting, however, through the
introduction of variable antibody domains in a non-conserved
loop in the structure of E2, specific populations of cells have been
targeted. Furthermore, the use of whole alphaviruses for gene
therapy is of limited efficacy both because several internal
alphaviral proteins are involved in the induction of apoptosis upon
infection and also because the alphaviral capsid mediates only
the transient introduction of mRNA into host cells. Neither of
these limitations extend to alphaviral envelope pseudotypes of
retroviruses or lentiviruses.
However, the expression of Sindbis virus envelopes may lead to
apoptosis, and their introduction into host cells upon infection by
Sindbis virus envelope pseudotyped retroviruses may also lead to
cell death. The toxicity of Sindbis viral envelopes may be the
cause of the very low production titers realized from packaging
cells constructed to produce Sindbis pseudotypes.
LIMITATION OF ALPHAVIRUS VIRAL VECTOR

An alternative to the use of viral vectors for gene delivery is to
deliver genetic material in the form of bacterial plasmid DNA.
In the simplest form, naked plasmid DNA can be injected into
skeletal muscle leading to transfection of muscle fibers close
to the site of delivery. Though the transfection efficiency by
nonviral vectors is relatively lower than that by viral vectors,
synthetic nonviral vectors are designed to overcome many of
the problems associated with viral vectors, such as risk of
generating the infectious form or inducing tumorigenic
mutations, risk of immune reaction, limitation to the size of
genes incorporated, and difficulty for the production to scale
up.
The advantages of nonviral carriers over their viral counterparts
are:
 (1) they are easy to prepare and to scale-up;
NON- VIRAL VECTORS

 (2) they are generally safer in vivo;
 (3) they do not elicit a specific immune response and can
therefore be administered repeatedly;
 (4) nonviral vectors allow for the delivery of large DNA
fragments and are also particularly suitable to deliver
oligonucleotides to mammalian cells, which is an excellent
feature for the application of antisense strategies to
downregulate the expression of certain genes; and
 (5) they are better for delivering cytokine genes because they
are less immunogenic than viral vectors.
NONVIRAL VECTORS

GENE TRANSFER WITH NAKED DNA
The simplest approach to nonviral delivery systems is direct gene
transfer with naked plasmid DNA. Simple injection of plasmid
DNA directly into a tissue without additional help from either a
chemical agent or a physical force is able to transfect cells.
Local injection of plasmid DNA into the muscle, liver, or skin,
or airway instillation into the lungs, leads to low-level gene
expression. Specific or nonspecific receptors on the cell
surface that bind and internalize DNA have been implicated as
a mechanism.
Plasmid Design
The design and engineering of plasmids to obtain maximum transfection
has been extensively researched. In addition to the transgene of
interest, plasmid DNA molecules typically contain several regulatory
signals such as promoter and enhancer sequences that play an
important role in regulating gene expression. In viral delivery vectors,

such signals can be endogenously present or artificially engineered in the
virus genome. In addition, splicing and polyadenylation sites are
present in the transgene construct that help in the correct processing
of the mRNA generated after transcription. Some vectors also have
introns that may increase premRNA processing and nuclear
transport.
Promoter sequences play a vital role in initiating gene transcription.
Promoter sequences offer recognition sites for the RNA polymerase
to initiate the transcription process. Higher efficiency can be
obtained by engineering the plasmid with strong tissue- or tumor-
specific promoters. Commonly used promoter sequences are
derived from viral origins such as cytomegalovirus (CMV) and roux
sarcoma virus, or are obtained from human origins such as alpha
actin promoter. However, sometimes promoters can lose their
activity upon immune stimulation. Promoter sequences may also
play an important role in determining the immune response of the
cell to the gene product. For example, it was demonstrated that
human muscle creatine kinase promoter has no immunostimulatory
PLASMID DESIGN

effect in mice, as opposed to the commonly used CMV promoter
sequence, during the expression of a gene vaccine encoding the
hepatitis B surface antigen.
Enhancers are regions in the plasmid DNA that enhance the production of
the gene of interest by as much as several hundred times. Enhancers
can be tissue specific and can be present on the plasmid locus either
upstream or downstream from the promoter region. Transcription
efficiency can be substantially improved by the choice of suitable
enhancers. For example, promoters and enhancers derived from
immunoglobulin genes have been used to increase gene transduction
in hematopoietic cells and to improve specificity of viral vectors useful
in the treatment of hematological malignancies. Muscle creatine
kinase enhancer has been useful for enhanced targeted expression of
transgenes for gene therapy to correct for muscular dystrophy.
LIMITATIONS AND IMPROVEMENTS OF ADENOVIRAL VECTORSPLASMID DESIGN

Gene transfer with naked DNA is attractive to many researchers
because of its simplicity and lack of toxicity. Practically, airway
gene delivery and intramuscular injection of naked DNA for the
treatment of acute diseases and DNA-based immunization,
respectively, are 2 areas that are likely to benefit from naked
DNA-mediated gene transfer, provided that further
improvements are made in delivery efficiency and duration of
transgene expression.
LIMITATIONS OF NAKED DNA AND HOW TO OVERCOME
A broad application of naked DNA–mediated gene transfer to gene
therapy may not be conceivable because DNA, being large in
size and highly hydrophilic, is efficiently kept out of the cells in
a whole animal by several physical barriers. These include the
blood endothelium, the interstitial matrices, the mucus lining
and specialized ciliate/tight junction of epithelial cells, and the
plasma membrane of all cells. In addition, DNA degradation by
APPLICATION OF NAKED DNA

intra- and extracellular nuclease activities further reduces the
chance that DNA entering nuclei will be intact and functional.
The current strategy for improving naked DNA–based gene transfer
is to include in DNA solution substances capable of enhancing
the efficiency of DNA internalization by target cells. For
example, transferrin has been shown to enhance transfection
in vitro. The addition of water-immiscible solvents, non-ionic
polymers, or surfactants, or the use of hypotonic solution, has
also been shown to elevate gene transfer across cell
membranes. Also, several nuclease inhibitors have been
shown to enhance naked DNA–mediated gene transfer in
cultured cells, muscle, and lungs.
LIMITATIONS OF NAKED DNA

Physical approaches have been explored for gene transfer into cells in vitro
and in vivo. Physical approaches induce transient injuries or defects on
cell membranes, so that DNA can enter the cells by diffusion. Gene
delivery employing mechanical (particle bombardment or gene gun),
electric (electroporation), ultrasonic, hydrodynamic (hydrodynamic gene
transfer), or laser-based energy has been explored in recent years.
GENE TRANSFER BY PHYSICAL METHODS
Transfer by Gene Gun
Particle bombardment through a gene gun is an ideal
method for gene transfer to skin, mucosa, or
surgically exposed tissues within a confined area.
DNA is deposited on the surface of gold particles,
which are then accelerated by pressurized gas and
expelled onto cells or a tissue. The momentum allows
the gold particles to penetrate a few millimeters deep
into a tissue and release DNA into cells on the path.
This method has been successfully used to deliver
genes into liver, skin, pancreas, spleen and tumours.
This method may be used for DNA-based
immunization.

GENE TRANSFER BY ELECTROPORATION
It is one of the several physical methods used for gene transfer. In
this process, DNA is transferred into cells in suspension by
applying pulses of high voltage electricity that created pores in
cell membrane or increase the permeability of protoplast
membrane thereby facilitating the entry of DNA molecules ino
the cells.
Electroporation is a versatile method that has been extensively
tested in many types of tissues in vivo, among which skin and
muscles are the most extensively investigated, although the
system should work in any tissues into which a pair of
electrodes can be inserted.
ADVANTAGES OF ELECTROPORATION
The level of reporter gene expression obtained was 2 to 3 orders of
magnitude higher than that with plasmid DNA alone.
DNA as large as 100 kb has been effectively delivered into muscle
cells.

Long-term expression over 1 year after a single electroporation
treatment was seen.
Gene transfer by electroporation showed less variation in effi ciency
across species than did direct DNA injection.
LIMITATIONS OF ELECTRPORATION PROCESS
Several major drawbacks exist for in vivo application of electroporation.
First, it has a limited effective range of ~1 cm between the
electrodes, which makes it difficult to transfect cells in a large area
of tissues.
Second, a surgical procedure is required to place the electrodes deep
into the internal organs.
Third, high voltage applied to tissues can result in irreversible tissue
damage as a result of thermal heating. Ca 2+ influx due to
disruption of cell membranes may induce tissue damage because
of Ca 2+ -mediated protease activation.
ELECTROPORATION PROCESS

The possibility that the high voltage applied to cells could affect the
stability of genomic DNA is an additional safety concern.
However, some of these concerns may be resolvable by
optimizing the design of electrodes, their spatial arrangement,
the field strength, and the duration and frequency of electric
pulses.
ELECTRONIC PULSE DELIVERY (EPD) TECHNOLOGY
EPD is a sophisticated method which subjects the target cells to a
precisely controlled pulses of electrical field in a computer
controlled molecular transfer system. The EPD system
consists of –a controller, and –a reaction chamber.
-The controller, driven by an EPD computer accurately controls the
output of electronic pulse, which in turn are controlled by
different parameters.
- The reaction chamber, where the DNA transfer takes place holds
LIMITATION OF ELECTROPORATION

the target cells and therapeutic gene as a mixture. The target cells
suitable for EPD delivery include haematopoietic stem cells,
hepatocyte, fibroblasts, and myoblasts.
ADVANTAGES OF EPD TECHNOLOGY
1. Using this technology, large molecules can be transferred into
target cells (e.g., DNA-15kb).
2. EPD inserts only the therapeutic genes into the target cells
without the involvement of any other molecule.
3. It has no toxic effect on the target cells and is completely free
from other potential hazards.
4. EPD technology has remarkable transfer efficiency (80-90%).
5. It can be operated in a batch or continuous process.
6. The time taken by this method for transfer is very short (from
few seconds to a minute).
EPD TECHNOLOGY

EPD technology has been confused with electroporation
procedure. In electroporation, the target cells are damaged
after which the molecules transfer takes place. Furthermore,
electroporation often applies a significant electric current and
need no contact by electrode to the mixture of genes and the
target cells.
EPD TECHNOLOGY

Ultrasound can facilitate gene transfer at cellular and tissue levels.
A 10- to 20-fold enhancement of reporter gene expression
over that of naked DNA has been achieved.
The transfection efficiency of this system is determined by several
factors, including the frequency, the output strength of the
ultrasound applied, the duration of ultrasound treatment, and
the amount of plasmid DNA used. The efficiency can be
enhanced by the use of contrast agents or conditions that
make membranes more fluidic. The contrast agents are air-
filled microbubbles that rapidly expand and shrink under
ultrasound irritation, generating local shock waves that
transiently permeate the nearby cell membranes.
Unlike electroporation, which moves DNA along the electric field,
ultrasound creates membrane pores and facilitates
intracellular gene transfer through passive diffusion of DNA
across the membrane pores. Consequently, the size and local
ULTRASOUND-FACILITATED GENE TRANSFER

concentration of plasmid DNA play an important role in determining
the transfection efficiency. Efforts to reduce DNA size for gene
transfer by methods of standard molecular biology or through
proper formulation could result in further improvement.
Interestingly, significant enhancement has been reported in
cell culture and in vivo when complexes of DNA and cationic
lipids have been used.
Since ultrasound can penetrate soft tissue and be applied to a
specific area, it could become an ideal method for noninvasive
gene transfer into cells of the internal organs.
So far, the major problem for ultrasound-facilitated gene delivery is
low gene delivery efficiency.
ULTRASOUND-FACILITATED GENE TRANSFER

GENE DELIVERY BY CHEMICAL METHODS
As has been discussed, gene expression can be achieved by direct
intratissue injection of naked plasmid DNA, gene transfer via
other routes of administration such as intrathecal and
intravenous injection will generally require the use of a delivery
vector or vehicle.
Various types of synthetic vectors have been developed for gene
transfer. Theses include:
1. Cationic lipids; 2. Cationic synthetic polymers;
3. Peptides that act in a nonspecific manner;
4. Peptide and carbohydrate based targeting ligand
Among these, cationic lipid and polymers have been studied most
extensively. In this system, DNA is formulated into condensed
particles of cationic lipid or polymer. The DNA containing particles are
subsequently taken up by cells via endocytosis in the form of
intracellular vesicles, from which a small fraction of the DNA is

released into the nucleus, where transgene expression takes place.
CATIONIC LIPID AND POLYMER

LIMITATIONS AND IMPROVEMENTS OF ADENOVIRAL VECTORS1. CATIONIC LIPID
In 1987, Felgner et al. first reported that a double chain monovalent
quaternary ammonium lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-
trimethylammonium chloride, effectively binds and delivers DNA to
cultured cells, hundreds of new cationic lipids have been developed,
differ by the number of charges in their hydrophilic head group and by the
detailed structure of their hydrophobic moiety.
Cationic lipid, as the name indicates consists mainly of a positively
charged lipid, thus it has been used to reduce the net negative surface
charge on DNA plasmid based gene expression system, in order to
reduce charge-charge repulsion at the surface of biological membranes.
Such lipids form a stable complexes with the gene expression system.
Although some cationic lipids alone exhibit good transfection activity, they
are often formulated with a noncharged phospholipid or cholesterol as a
helper lipid to form liposomes. For example, LIPOFECTIN, a novel and
highly efficient DNA transfection system consisted of the positively

charged quarternery amino lipid, DOTMA in a 1:1 weight mixture
with dioleyl-phosphatidyl choline (DOPE). As DOPE can fuse
with endosomal membrane, it is generally included to effect the
endosomal release of a gene expression system. Spontaneous
mixing between cationic lipids and cellular lipids in the
membrane of the endocytic vesicles is crucial to the
endosome-releasing process. Spontaneous lipid mixing in
endosomes becomes more profound when a non-bilayer-
forming lipid such as dioleoylphosphatidylethanolamine
(DOPE) is used as the helper lipid, rather than a bilayer-
forming lipid, dioleoylphosphatidylcholine. Inclusion of DOPE is
believed to increase membrane fluidity and facilitate lipid
exchange and membrane fusion between lipoplexes and the
endosomal membrane. A high local concentration of DOPE,
which has a strong tendency to form an inverse hexagonal
phase, may lead to a nonbilayer lipid structure and cause
membrane perturbation and endosome destruction. However,
some
CATIONIC LIPID

multivalent lipids have intrinsic transfection activity, and a helper
lipid does not have a major impact on overall transfection
activity, indicating that multivalent cationic lipids work on a
different mechanism.
Upon mixing with cationic liposomes, plasmid DNA is condensed
into small quasi-stable particles called lipoplexes. DNA in
lipoplexes is well protected from nuclease degradation.
Lipoplexes are able to trigger cellular uptake and facilitate the
release of DNA from the intracellular vesicles before reaching
destructive lysosomal compartments.
The transfection efficiency of lipoplexes is affected by:
1) the chemical structure of the cationic lipid,
(2) the charge ratio between the cationic lipid and the DNA,
(3) the structure and proportion of the helper lipid in the complexes,
(4) the size and structure of the liposomes,
CATIONIC LIPID

(5) the total amount of the lipoplexes applied, and
(6) the cell type.
The first 4 factors determine the structure, charge property, and transfection
activity of the lipoplexes. The remaining 2 define the overall toxicity to the
treated cells, and the susceptibility of the cells to a particular lipid-based
transfection reagent.
Figure 6 Model of the intracellular delivery of plasmid mediated by cationic lipid
in plasmid DNA-cationic lipid complexes (lipoplexes). Following binding
(step 1) and endocytosis (step 2) into a target cell, the lipoplexes are
transferred to endosomal compartments (step 3). The membrane of the
lipoplex then fuses with the endosomal membrane due to the tendency of
positively charged and negatively charged membranes to adhere and
fuse, leading to lipid mixing between lipoplex and endosomal lipids.
Anionic lipids from the endosomal membrane then displace cationic lipids
from the plasmid DNA, leading to release of plasmid and further
formation of non-bilayer structures (step 4).
CATIONIC LIPID

CATIONIC LIPID-UNDESIRABLE EFFECT
Cationic lipids as oligonucleotide carriers have several
disadvantages. The main disadvantages of cationic lipids are
their toxicity and markedly decreased activity in the presence
of serum.
Once administered in vivo, lipoplexes tend to interact with
negatively charged blood components and form large
aggregates that could be absorbed onto the surface of
circulating red blood cells, trapped in a thick mucus layer, or
embolized in microvasculatures, preventing them from
reaching the intended target cells in the distal location. Newer
cationic lipid formulations are available that exhibit decreased
toxicity.e.g. The inclusion of a helper lipid (DOPE or
cholesterol)in new formulation reduces the effective charge
ratio required to deliver oligonucleotides into cells and permits
delivery in the presence of high serum concentration.

Toxicity related to gene transfer by lipoplexes has been observed. Acute
inflammation reactions have been reported in animals treated with airway
instillation or intravenous injection of lipoplexes. Symptoms include acute
pulmonary hypotension, induction of inflammatory cytokines, tissue
infiltration of neutrophils in lungs, decrease in white cell counts, and in
some cases tissue injury in liver and spleen. In humans, various degrees
of adverse inflammatory reactions, including flulike symptoms with fever
and airway inflammation, were noted among subjects who received
aerosolized GL67 liposomes alone or lipoplexes. These early clinical data
suggest that these lipoplex formulations are inadequate for use in
humans.
APPLICATION OF CATIONIC LIPID BASED GENE TRANSFER
Despite these undesirable characteristics, lipoplexes have been used for in vivo
gene delivery to the lungs by intravenous and airway administration.
Transgene expression was clearly detectable but in most cases was
insufficient for a meaningful therapeutic outcome. For airway gene
delivery to the lungs, animal studies using lipoplexes prepared from 3 b -
[N-(N′,N′-dimethylaminoethane) carbamoyl]cholesterol (DC-Chol) and
CATIONIC LIPID-UNDESIRABLE EFFECT

DOPE have shown that this procedure was mild to the host and
partially effective in correcting genetic defects in a cystic
fibrosis transmembrane regulator mutant model.
Polymeric gene carriers have been studied because of some
advantanges over the lipid systems:
 (1)Relatively small size and narrow distribution of complex.
 (2) High stability against nucleases; and
 (3) Easy control of physical factors (e.g. hydrophilicity and
charge) by co-polymerization
Like cationic lipid, positively charged polymer form complex by
interacting with negatively charged DNA. The complex is
called polyplex, which results from the electrostatic attraction
between the cationic charge on the polymer and the
CATIONIC POLYMER

negatively charged DNA. These polyplexes effect efficient gene delivery
into a variety of cell types. A cationic polymer/DNA complex may
contain one or more plasmid DNA. The formation of cationic
polymers/DNA complexes protects DNA from both extracellular and
intracellular degradation during the trasfection. For in vitro gene
delivery, cell membrane is the first barrier. The complexes are taken
up by cells usually through endocytosis, and the route of uptake
determines the subsequent DNA release, trafficking and lifetime in
the cells. Endocytosis is a multi-step process including binding,
internalization, forming of endosomes, fusion with lysosomes and
lysis. Since DNA and associated complexes are easily degraded in
endosomes and lysosomes due to the extremely low pH and
enzymes, they must rapidly escape from endosomes. Chloroquine is
a weak base. The chloroquine added into the polymer/DNA
complexes or cell culture medium raises the pH value of endosomes
(normal pH of endosome is 5.5) after uptake by cells, facilitates
endosome escaping and improves the trasfection efficiency. The
transfection efficiency mediated by cationic polymers possessing
CATIONIC POLYMER

high buffer capacity at pH 5 7, such as polyethylenimine (PEI) and
polyamidoamine dendrimer, is high due to their ability to
capture the protons entering the endosomes during their
acidification leading to swelling and destabilization of
endosomes. After escaping from endosomes, DNA or
polymer/DNA complexes move through cytoplasm to the
nucleus by diffusion[8]. Cell nuclear membrane is another
potent tight barrier for foreign DNA, which isolates the
nucleoplasm from cytoplasm in order to control important cell
activities such as DNA replication and transcription. The
nuclear pores on the nuclear membrane have huge and
complex structures. Molecules smaller than 300 kg/mol can
pass through the nuclear pores by passive diffusion, whereas
the larger molecules are generally transported by the active
signal-mediated process. DNA or polymer/DNA complexes
enter the nucleus probably through the nucleus pores.
CATIONIC POLYMER

Over the past two decades, various
natural and synthetic cationic
polymers were applied in gene
delivery[11] including
polyethylenimine (PEI),
polyamidoamine dendrimer,
chitosan, gelatin, cationic peptides,
cationic polyesters (PAGA), cationic
polyphosphoesters (PPE), poly(vinyl
pyridine), poly[(2-
dimethylamino)ethyl methacrylate],
etc. Among the polymer vectors
currently used, PEI is the most
successful one and has become the
gold standard of polymer gene
delivery vector.
CATIONIC POLYMER

OLIGONUCLEOTIDES
Oligonucleotides are short single-stranded segments of DNA. In
general, they are 13–25 nucleotides in length and hybridize to
a unique sequence in the total pool of targets present in cells.
Due to their antigene and antisense application they are used
either as therapeutic agents or as tools to study gene function.
Although it is not a complicated matter to synthesize
phosphodiester oligonucleotides, their use is limited as they
are rapidly degraded by the intracellular endonucleases and
exonucleases, usually via 3’-5’ activity. In addition, the
degradation products of phosphodiester oligonucleotides,
dNMP mononucleotides, may be cytotoxic and also exert
antiproliferative effects. Upon cellular internalization it can
selectively inhibit the expression of a single protein.

OLIGONUCLEOTIDES
Mechanism of action
Oligonucleotides are in theory designed to specifically modulate the transfer of
the genetic information to protein, but the mechanisms by which an
oligonucleotide can induce a biological effect are subtle and complex. However,
they produce the biological effects through two mechanisms, antigene and
antisense mechanisms.
For antigene applications, oligonucleotides must enter the cell nucleus, form a
triplex with the double-stranded genomic DNA, and inhibit the translation as well
as the transcription processes of the protein.
On the basis of antisense mechanism of action, two classes of antisense
oligonucleotide can be discerned: (a) the RNase H-dependent oligonucleotides,
which induce the degradation of mRNA; and (b) the steric-blocker
oligonucleotides, which physically prevent or inhibit the progression of splicing or
the translational machinery.
The majority of the antisense drugs investigated in the clinic function via an
RNase H-dependent mechanism. RNase H is a ubiquitous enzyme that
hydrolyzes the RNA strand of an RNA/DNA duplex. Oligonucleotide-assisted
RNase H-dependent reduction of targeted RNA expression can be quite
efficient, reaching 80–95% down-regulation of protein and mRNA expression.

OLIGONUCLEOTIDE
An antisense drug inhibits production of a disease-causing protein based on
protein’s mRNA and gene sequence
Antisense Strand
Sense-Antisense Duplex
RNase H
Cell Membrane
Cytoplasm
Nucleus
DNA
mRNA

LIMITATIONS AND IMPROVEMENTS OF ADENOVIRAL VECTORSOLIGONUCLEOTIDES
Furthermore, in contrast to the steric-blocker oligonucleotides, RNase H-
dependent oligonucleotides can inhibit protein expression when targeted to
virtually any region of the mRNA. Whereas most steric-blocker oligonucleotides
are efficient to bind to key sequences in the target which may inhibit the target’s
ability to interact with the cellular machinery required for protein synthesis. For
example, oligonucleotide interaction with the initiation codon or with the splicing
sequences can cause transcriptional arrest by this route.
Design of Oligonucleotides
Design is critical for the clinical efficacy of oligonucleotides and should include
consideration of length, chemistry, conformation, and ability to hybridize with the
target mRNA.
Length.
The suggested optimal length for oligonucleotides with efficient antisense activity
ranges from 12 to 28 bases. There is a consensus that very short sequences are
likely to be nonspecific and sequences longer than 25 bases would experience
difficulty in cellular uptake. As the length of the molecule increases, hydrogen
bonding between the base pairs and stacking interactions increases, leading to

An increased overall affinity for the target. However, longer
oligonucleotides are inherently difficult to import into cells because of
their size and tend to self-hybridize, thereby affecting their uptake.
Even in cell culture experiments, the optimum length suggested for
oligonucleotides varies considerably.
Backbone Modifications
Oligonucleotides having the endogenous phosphodiester backbone are
susceptible to degradation by nucleases and hence have limited use
for antisense applications. Various chemical modifications to the
backbone have been used to improve oligonucleotide stability. The
most common modifications include the introduction of
phosphorothioate and methyl phosphonate linkages in the backbone.
Phosphorothioate analogs are chosen for their stability against
nucleases and the methylphosphonate backbone for its relative
hydrophobicity and ease of diffusion across membranes.
Phosphorothioate backbone oligonucleotides can have significantly
increased biological half-life compared to their corresponding
unmodified phosphodiester oligonucleotides.
DESIGN OF OLIGONUCLEOTIDE

Hybridization Capacity
Antisense oligonucleotides are designed primarily for their ability to
hybridize with the mRNA of interest. The mRNA has a complex
secondary and tertiary spatial structure that restricts the accessibility
of certain segments of the molecule to hybridize with the
oligonucleotide. Consequently, although it is theoretically possible to
design antisense constructs to virtually any part of the mRNA, not all
oligonucleotides are efficient in inhibiting protein synthesis. Complex
algorithms predicting mRNA structure, “gene-walking,” RNase H
mapping, and combinatorial screening are some of the strategies that
are being explored to predict hybridization-accessible sites on mRNA
molecules.
Secondary Conformation
The presence of some specific sequences in the oligonucleotide can allow it
to have a preferred conformation. For example, it was demonstrated
that the presence of continuous guanines at the 3'-end of an
oligonucleotide made it resistant to degradation because of the ability
of these guanines to assemble into hyperstructures in vivo. These
DESIGN OF OLIGONUCLEOTIDE

hyperstructures were resistant to nuclease activity and were efficiently taken
up by cells. The secondary structure of oligonucleotides can be affected
by divalent cations such as Ca2+ and Mg2+ that typically occur in the
cellular environment, thereby possibly affecting their activity or cellular
uptake.
Clinical Applications of Oligonucleotides
For therapeutic purposes, oligonucleotides can be used to selectively block the
expression of proteins that are implicated in diseases. With successful
antisense inhibition of proteins in animal models, the first antisense drug,
fomivirsen sodium (Vitravene, Isis Pharmaceuticals, Carlsbad, CA ) was
approved for the treatment of cytomegalovirus retinitis in AIDS patients in
1998. Fomivirsen sodium is formulated as an intravitreal aqueous
injection in sodium bicarbonate buffer at pH 8.7.
Antisense oligonucleotides such as MG98 and ISIS 5132, designed to
inhibit the biosynthesis of DNA methyltransferase and c-raf kinase,
respectively, are in human clinical trials for cancer. ISIS 2302, targeting
ICAM-1, is being investigated for the treatment of ulcerative colitis.
OLIGONUCLEOTIDES

OLIGONUCLEOTIDE
Two other drug candidates, Affinitak and Alicaforsen (Isis
Pharmaceuticals), are in phase 3 clinical trials for non-small cell lung
cancer and Crohn’s disease, respectively. Genasense (oblimersen
sodium), developed by Aventis (Bridgewater, NJ) and Genta
(Berkeley Heights, NJ), is being investigated in advanced phase 2
trials in combination with other chemotherapy regimens for a range of
cancers including malignant melanoma, chronic lymphocytic
leukemia, and multiple myeloma.
VITRAVENE TM INJECTION
(Fomivirisen sodium intravitreal injection)
Vitravene is a sterile, aqueous, preservative-free, bicarbonate buffered
solution for intravitreal injection. It is represented by the following
structural formula-
5’-GCG TTT GCT CTT CTT CTT GCG-3’
So fomivirisen sodium is a phosphorotioate oligonucleoltie, twenty one
nucleotides in length.

Indications
Vitravene is indicated for the local treatment of cytomegalovirus (CMV) retinitis
in patients with AIDS, who are intolerant of or have a contraindication to
other treatment for CMV retnitis or who were insufficiently responsive to
previous treatments for CMV retinitis.
Mechanism of action
Vitravene inhibits human cytomegalovirus (HCMV) replication through an
antisense mechanism. The nucleotide sequence of fomivirisen is
complementary to a sequence in mRNA transcripts of the major
immediate early region 2 (IE2) of HCMV. This region of mRNA encdes
several proteins responsible for regulation of viral gene expression that
are essential for production of infectious CMV. Binding of fomivirisen to
the target mRNA, results in inhibition of IE2 protein synthesis,
subsequently inhibiting virus replication.
VITRVENE INJECTION

A number of technologies have been used in an attempt to mediate the
down-regulation of gene expression. For example, anti-sense
oligonucleotides and ribozymes have been used for more than a
decade to target specific RNAs for degradation. Although these
methods worked satisfactorily in some simple experimental models,
they have generally not delivered effective gene silencing in complex
mammalian systems. However, in the past several years,
extraordinary developments in RNA interference (RNAi)-based
methodologies have seen small interfering RNAs (siRNAs) become
the primary means by which most researchers attempt to target
specific genes for silencing. RNAi was first discovered in
Caenorhabditis elegans, when it was noted that introducing a double-
stranded RNA (dsRNA) that was homologous to a specific gene
resulted in the post-transcriptional silencing of that gene. The
obvious therapeutic potential of RNAi resulted in rapid elucidation of
its mechanism of action and it is now known that gene silencing is
mediated via the following basic mechanisms:
siRNA (Small Interfering rna)

In essence, RNAi is initiated by long stretches of dsRNA that undergo
processing by an enzyme referred to as Dicer. Dicer cuts the long
stretches of dsRNA into duplexes with 19 paired nucleotides and two
nucleotide overhangs at both 3-ends. These duplexes are called
siRNA (short interfering RNA). The double-stranded siRNA then
associates with RISC (RNA-induced silencing complex), a fairly large
(approx. 160 kDa) protein complex comprising Argonaute proteins as
the catalytic core of this complex. Within RISC, the siRNA is
unwound and the sense strand removed for degradation by cellular
nucleases. The antisense strand of the siRNA directs RISC to the
target mRNA sequence, where it anneals complementarily by
Watson–Crick base pairing. Finally, the target mRNA is degraded by
RISC endonuclease activity.
Although early investigations found that the introduction of long dsRNA had
the potential to mediate the down-regulation of any gene, it was also
found that in mammalian cells an anti-viral interferon response (IR)
that resulted in the cessation of all protein synthesis was also elicited.
Synthetic siRNAs (21–23 nucleotides in length) were shown not to
elicit this IR and have hence been used in studies of

mammalian gene function.
This mechanism is
fundamentally different
from PTGS (post-
transcriptional gene
silencing) by ASOs (Table
1). Binding of ASOs to
their target mRNA
prevents protein
translation either by steric
hindrance of the
ribosomal machinery or
induction of mRNA
degradation by Rnase H
(ribonuclease H).

In brief,
1. Long dsRNA precursors derived from endogenous genes or
artificially introduced plasmids are cleaved by Dicer yielding
siRNA.
Alternatively, synthetic siRNA can be transfected into cells.
2. siRNA is incorporated into RISC, followed by unwinding of the
double-stranded molecule by the helicase activity of RISC.
3. The sense strand of siRNA is removed and the antisense strand
recruits targeted mRNA, which is cleaved by RISC and
subsequently degraded by cellular nucleases.

Gene Therapy Manik

Empfohlen

Empfohlen

Weitere ähnliche Inhalte

Was ist angesagt?

Was ist angesagt? (20)

Ähnlich wie Gene Therapy Manik

Ähnlich wie Gene Therapy Manik (20)

Mehr von Imran Nur Manik

Mehr von Imran Nur Manik (20)

Kürzlich hochgeladen

Kürzlich hochgeladen (20)

Gene Therapy Manik