Gene Therapy MANIK

Taught By
Department of Pharmacy
University of Rajshahi
Rajshahi-6205, Bangladesh

Gene Therapy: GS SIR
Printing Management By: Md. Imran Nur Manik ; Ph.G.(R.U.) Page | 1
Gene Therapy
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.
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 event, 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
1. 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 deaminizes for SCIS, transfusion for
sickle cell disease.
2. Conventional therapies are only partially effective in ameliorating the manifestations of the disease
and are accompanied by significant complications.
3. 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.).
4. 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.
Problems overcome by gene therapy
A. 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.
B. The limited number of tissues is 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.
Md.
Imran
Nur
Manik

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.
C. 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.
D. 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.
Where is gene therapy applied?
1. 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.
2. In case of inherited disorder, the defective gene that cause the disorder is the subject of intervention
and
3. 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.
Basis of Gene Therapy
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:
1. Viral-mediated gene transfer: various viruses are used as carrier, e.g., retrovirus, adenovirus,
adenoassociated virus, etc.
2. Non-viral-mediated gene transfer: various types of synthetic vectors are used as carrier,. e.g.,
cationic lipid, cationic polymer, etc.
Candidate Disease for Gene Therapy
Diseases wherein gene therapy has been focused upon include-
Genetic Disease Genetic Disease Target cells
1. Severe Combined immunodeficiency(SCID/ADA) Adenosine deaminase Bone marrow cell
or T-cell
2. Hemophilia- A
- B
Factor VIII deficiency
Factor IX deficiency
Liver, fibroblast
or bone marrow
3. Cystic fibrosis Loss of CFTR gene Airways in lung
4. Familial hypercholesterimia Def. LDL receptor Liver
5. Hemoglobinopathy
Thalessimia
Sickel cell anemia
structural defect
of alpha/beta- globin
gene
bone marrow
Md.
Imran
Nur
Manik

Defective Gene – Genetic Disorder:
Disease-Acquired Defect Target cells
6. Neurologic diseases: Alzheimer’s disease
Parkinson’s disease
APP gene alpha-Syn
gene
Neuron/gilal
Neuron
7. Cardiovascular disease; Atheroscleorosis HDL Vascular endothelial cell
8. Infectious disease:
AIDS ; Hepatitis B
T-cell
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)
Gene modification
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.
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.
Md.
Imran
Nur
Manik

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.
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 4ethnological 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 counseled.
Qualities of an ideal delivery system
An ideal delivery system would be one-
1. Which could accommodate a broad size range of inserted DNA?
2. Which could be targeted to specific types of cells?
3. Which would not permit replication of the DNA?
4. Which could provide long-term gene expression?
5. Which could be easily produced?
6. 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:
1. Viral-mediated gene delivery: various viruses are used as carrier,
e.g., retrovirus, adenovirus, adenoassociated virus, etc.
2. Non-viral-mediated gene delivery : various types of synthetic vectors are used as carrier,
e.g. cationic lipid, cationic polymer, etc.
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.
Md.
Imran
Nur
Manik

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.
Retrovirus
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,
6. It could carry up to 8Kb of coding information,
7. Above all, it establishes one way, nonreplicative infection of target cells,
8. It lacks 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.
Replication Cycle of Retroviruses
(Source: http://sgugeneticsgroup12.pbworks.com/w/page/226078/FrontPage)
Md.
Imran
Nur
Manik

Genome Structure of Retroviruses
Retrovirus
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; y, 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.
(Source: http://enni82.hubpages.com/hub/Overview-of-Retrovirus-Life-Cycle# )
Genome Structure of Retroviruses
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.
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.
pol: 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.
Md.
Imran
Nur
Manik

env: 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 leukemia 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.
►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
Md.
Imran
Nur
Manik

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 or 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.
Md.
Imran
Nur
Manik

Clinical Administration of Retrovirus
The clinical administration of retroviruses has been accomplished by:
● - ►Ex vivo transduction of patient’s 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 targeted.
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. 1. 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. (Source:http://vir.sgmjournals.org/content/81/11/2573.full)
Basic Characteristics of Adenovirus
►1. Adenovirus is a DNA virus having 60-90 nm in diameter. It has double stranded DNA genome
and do not possess a lipid envelope
►2. 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.
Md.
Imran
Nur
Manik

►3. Adenoviral genome encodes approximately 15 proteins.
►4. 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.
►5. 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
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.
2. At 200 nucleotides of the 5Ꞌ 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.
Md.
Imran
Nur
Manik

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.
Design and Construction of Replication defective 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 subgroup C 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 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 5ꞌ 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.
Md.
Imran
Nur
Manik

Improvement of Transgene Expression
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 cassette avoids the unknown consequences of over expression 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.
Propagation and Purification of Adenoviral Vector
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 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.
Md.
Imran
Nur
Manik

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, 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 non-dividing cells.
Md.
Imran
Nur
Manik

♦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
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.
Md.
Imran
Nur
Manik

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:
1. 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.
2. These viruses can insert genetic material at a specific site on chromosome 19 with near 100%
certainty.
3. 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.
4. This type of virus is being used, however, because it is non-pathogenic (most people carry this
harmless virus).
5. 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.
Md.
Imran
Nur
Manik

Genomic Organization of AAV Vectors
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.
Application Of Adenoassociated Viral Vectors
1. 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.
2. 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.
3. 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.
Limitations of Adenoassociated Viral Vectors
1. 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.
Md.
Imran
Nur
Manik

2. 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.
[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.]
Herpes Simplex 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, 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.
Application of Herpes Simplex Viral Vector
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.
Md.
Imran
Nur
Manik

Alpha Virus 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 takes place in the cytoplasm of the
host cells.
Application of Alphavirus Viral Vector
1. 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.
2. Alphavirus vectors carrying therapeutic or toxic genes used for intratumoral injections have
demonstrated efficient tumor regression.
3. 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.
Limitation 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 extends 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.
Md.
Imran
Nur
Manik

Non- Viral Vectors
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.
Advantages
The advantages of nonviral carriers over their viral counterparts are:
(1) They are easy to prepare and to scale-up;
(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.
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 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.
Md.
Imran
Nur
Manik

☼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.
Application of Naked DNA
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 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.
Gene Transfer by Physical Methods
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.
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.
Md.
Imran
Nur
Manik

Above: Helios Gene Gun schematic (Above left and right images courtesy of BioRad)
(Source: http://www.bio.davidson.edu/courses/molbio/molstudents/spring2003/mcdonald/gene_gun.html)
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
1. The level of reporter gene expression obtained was 2 to 3 orders of magnitude higher than that with
plasmid DNA alone.
2. DNA as large as 100 kb has been effectively delivered into muscle cells.
Electroporation Process
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 Electroporation 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.
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.
Md.
Imran
Nur
Manik

Electroporator with square wave and exponential decay waveforms for in vitro, in vivo, adherent cell and 96 well
electroporation applications. Manufactured by BTX Harvard Apparatus, Holliston MA USA.
Cuvettes for electroporation. These are plastic with aluminium electrodes and a blue lid. They hold a maximum of 400 μl.
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 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 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.
Ultrasound-Facilitated Gene Transfer
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.
Md.
Imran
Nur
Manik

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 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.
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
Cationic Lipid and Polymer
1. 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).
Md.
Imran
Nur
Manik

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 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,
(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-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.
Md.
Imran
Nur
Manik

►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 Polymer
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 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.
Md.
Imran
Nur
Manik

The transfection efficiency mediated by cationic polymers possessing 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.
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.
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.
Md.
Imran
Nur
Manik

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.
{RNase H-dependent antisense mechanism. Single-stranded oligonucleotides are transported across the plasma membrane, by
either poorly characterized natural processes or by the use of facilitators such as cationic lipids (step 1). Once in the cytoplasm, single-
stranded oligonucleotides rapidly accumulate in the cell nucleus (steps 2 and 3), where they bind to their targeted RNA (step 4). Once
bound to the RNA, RNase H recognizes the oligonucleotide/RNA duplex as a substrate, cleaving the RNA strand and releasing the
antisense oligonucleotide (step 5). Although the cleavage of the RNA by RNase H is shown to occur in the nucleus, RNase H is also present
in the cytosol, allowing for cleavage to occur in that cellular compartment as well}
(Source: http://www.nature.com/onc/journal/v22/n56/fig_tab/1207231f1.html)
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.
Md.
Imran
Nur
Manik

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.
Backbone of Oligonucleotide
Md.
Imran
Nur
Manik

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 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.
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.
Vitrvene Injection
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.
Md.
Imran
Nur
Manik

Binding of fomivirisen to the target mRNA, results in inhibition of IE2 protein synthesis, subsequently
inhibiting virus replication.
siRNA (Small Interfering RNA)
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:
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).
Md.
Imran
Nur
Manik

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.
www.pharmacydocs.blogspot.com
Md.
Imran
Nur
Manik

Gene Therapy MANIK

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Gene Therapy MANIK

Similar to Gene Therapy MANIK (20)

More from Imran Nur Manik

More from Imran Nur Manik (20)

Recently uploaded

Recently uploaded (20)

Gene Therapy MANIK