Gene expression systems

GENE EXPRESSION
SYSTEMS
Presented by:
Miss. Mrinal R. Gite
M. PHARM. 1st YEAR
(Department of Pharmaceutics)
Guided by:
Dr. P. N. Kendre
HOD, Department of Pharmaceutics
Rajarshi Shahu College of Pharmacy, Buldana 443001
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CONTENTS
➢ Introduction
➢ Vectors used for gene transfer
I. Viral vectors
II. Non-viral vectors
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INTRODUCTION
➢ Gene therapy-experimental technique-uses genes to treat or
prevent disease
➢ Process in which information is encoded in a gene is used to
produce functional product such as proteins
➢ Involves different steps through which DNA is converted
into RNA.
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➢ Key phases in gene expression:
1. Transcription- conversion of DNA to RNA
2. Post transcriptional modifications and splicing
3. RNA transport
4. Translation/ protein synthesis
5. Protein binding
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➢ Expression system is made up of gene which is encoded by
DNA and machinery needed to make mRNA from the DNA
and translate that into protein
➢ There are 2 major categories of gene therapy
1. Somatic gene therapy
2. Germline gene therapy
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Cell type
Germline SomaticVector
type
Non-
viral
Viral
PHYSICAL
•Balistic DNA
•Needle
•Electroporation
•Sonoporation
•Magnetofection
CHEMICAL
•Cationic lipids
•Cationic polymers
•Polymer- lipid system
• Retroviral
• Adeno-association
vectors
• Adenoviral vectors
• Helper dependent
adenoviral
• Herpes simplex virus
• Pox virus
• Lentivirus
• Hybrid adenoviral
vector
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1. Germline gene therapy
➢ Genetic abnormalities can be corrected by direct
manipulation of germline cells – with no targeting
2. Somatic gene therapy
➢ Insertion of genes into diploid cells of an individual where
the genetic material is not passed on to its progeny
➢ It affects only targeted cells in the patient and is not passed
on future generations
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Three types
i. Ex-vivo delivery
ii. In situ delivery
iii. In vitro delivery
Vectors used for gene delivery
❖ Viral vectors
❖ Non-viral vectors
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A. Viral vectors
1. Retroviral
2. Adeno-association vectors (AAV)
3. Adenoviral vectors
4. Helper dependent adenoviral
5. Herpes simplex virus
6. Pox virus
7. Lentivirus
8. Hybrid adenoviral vector
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1. Retroviral vector
➢ Commonly employed vectors, derived from moloney murine
leukaemia virus (MMLV)
➢ Typical feature of retroviruses and retroviral vector is their
ability to integrate into host DNA
➢ Viral RNA is reversibly transcribed and integrated in the
form of provirus
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➢ They co-operate with enzymes of host cell and they use it
for their own replication and long term expression of viral
proteins
➢ Useful for Ex-vivo delivery of somatic cells
➢ Retroviral vectors also have been applied for familial
hyperlipidaemia gene therapy and tumour vaccination
➢ Type of RNA virus
➢ Contain enzyme called reverse transcriptase.
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Step 1
Retrovirus attaches to cell membrane of
the host and outer envelope of
retrovirus is lipid bilayer and
It is fused together & as it fused
together retrovirus is taken into the host
cell
Capsid is dissolved &begins to
breakdown and releases the enzyme of
retrovirus and RNA nucleic acid of
retrovirus
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Step 2- reverse transcription- DNA used
to create RNA molecule
Reverse transcriptase bind to viral RNA
and perform reverse transcription
Viro RNA will be used to build a molecule
of viro DNA and eventually the enzyme
reverse transcriptase is degraded and
broken down but more importantly the
retroviruses created viro DNA .
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Step 3- viral DNA fused with host
DNA(provirus created)
Viral DNA enters into the nucleus along
with enzyme Integrase
Integrase- cut upon the DNA of host cell
and it is called Integrase because it helps to
integrate the viro DNA into host cell DNA
This combination of host DNA and viro
DNA is called provirus.
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Step 4-
Viral RNA
created- each time
the host cell
transcribes its
DNA
Normal
transcription of
provirus is done
and it forms a
copy of viro RNA
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After this capsid of virus is created by ribosomes
Viro RNA attaches to ribosomes and they read viro RNA and
create viro protein –such as reverse transcriptase and also
create integrase as process continues
In cytoplasm of cell they posses ribosomes.
Step 5 –Viral DNA is translated into viral proteins
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Step 6- viral proteins self
assemble into immature
viruses
Step 7- viruses released
through cell membrane
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2. Adenoviral vectors
➢ These are non enveloped DNA viruses, linear genome and
double stranded DNA molecule
➢ Adenoviral vectors have been isolated from large number of
different species and more than 100 different serotypes have
been reported
➢ Adenoviruses type 2 and type 5 can be utilized for
transferring both dividing and non dividing cells and have
low specificity.
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Entry of adenoviruses into the host cell
involves two sets of interactions
between the virus and the host cell.
Entry into the host cell is initiated by the
knob domain of the fiber protein
binding to the cell receptor.
The two currently established receptors are:
CD46 for the group B human adenovirus
serotypes and the coxsackievirus
adenovirus receptor (CAR) for all other
serotypes.
secondary interaction, where a
specialized motif in the penton base
protein interacts with an integrin
molecule.
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It is the co-receptor interaction that
stimulates internalization of the adenovirus.
This co-receptor molecule is αv integrin
Attachment to αv integrin stimulates cell
signaling and thus induces actin
polymerization resulting in entry of the virion
into the host cell within an endosome
Then endosome acidifies, which alters
virus topology by causing capsid
components to disassociate
These changes as well as the toxic nature
of the pentons results in the release of the
virion into the cytoplasm.
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With the help of cellular microtubules
the virus is transported to the nuclear
pore complex whereby the
adenovirus particle disassembles.
Viral DNA is subsequently released
which can enter the nucleus via the
nuclear pore.
After this the DNA associates with
histone molecules.
Thus viral gene expression can occur
and new virus particles can be
generated.
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3. Lentiviruses
➢ Subclass of retroviruses
➢ Recently used as gene delivery vectors due to their ability
to naturally integrate with non-dividing cells
➢ They can deliver 8 kb of sequence because they have strong
tropism for neural stem cells extensively used as ex-vivo
gene transfer in CNS with no significant immune responses
and no unwanted side effects .
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➢ Lentiviruses vectors have the advantages of high
efficiency infection of dividing and non –dividing cells,
long-term stable expression of a transgene, low
immunogenicity and the ability to accommodate larger
transgenes.
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4. Adeno-Association Vector (AAV)
➢ Adeno associated vectors (AAV) are like adenoviral
vectors in their features but because of having some
deficiency in their replication and pathogenicity they are
safer than adenoviral vector
➢ In human, AAVs are no associated with any disease
➢ They have ability to integrate into a specific site on
chromosome 19 with no noticible effects cause long-term
expression in vivo.
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➢ Major advantage is complicated process of vector
production and limited transgene capacity of the particle.
➢ AAVs have been used in treatment of some diseases like
CF (Cystic fibrosis), haemophilia B and AAT(alpha-1-
antitrypsine) Deficiency.
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B. Non-viral vectors
❖ Physical
1. Ballistic DNA
2. Needle
3. Electroporation
4. Sonoporation
5. Magnetofection
❖ Chemical
i. Inorganic particles
ii. Synthetic/ biodegradable.
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❖ Physical
1. Biolistic DNA
➢ Particle bombardment, micro projectile gene transfer/gene
gun are used for ballistic DNA
➢ Method is first used as gene transfer technique to plant
➢ Based on principle-delivering DNA coated heavy metal
particles by crossing target tissue at certain speed
➢ Sufficient speed is achieved by high voltage electronic
discharge, spark discharge/helium pressure discharge.
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➢ Gas pressure, particle size, dose frequency are the critical
parameters in determining the efficiency of gene transfers
➢ Gold, tungsten and silver are used as metal particles-
typically 1μm diameter
➢ The major advantage of gene gun is precise delivery of
DNA doses.
➢ Gene gun based gene transfer has been extensively tested
for intramuscular, intradermal, and intratumor genetic
immunisation.
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➢ It was demonstrated that this approach is able to produce
more immune response with lower dose comparing to
needle injection in large animal models and in clinical
human trials.
➢ It is most commonly used in gene therapy research in
ovarian cancer.
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2.Needle
➢ Genetic material of interest is administered through a
needle carrying syringe into tissue/systemic injection
from a vessel.
➢ Without any carrier it is the simplest and safest method
of gene transfer.
➢ Attractive candidate tissues are muscle, skin, liver,
cardiac muscle and solid tumours.
➢ However, the efficiency is low due to rapid degradation
by nucleases in serum and cleared by mononuclear
phagocyte system.
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3.Electroporation
➢ Other terms used for electroporation are gene electro
injection, gene therapy, electro gene transfer
➢ Electric filed applied greater than the membrane capacitance
which will cause charges of opposite polarity to line up on
the either side of cell membrane thus forming a potential
difference at a specific point on the cell surface
➢ As a result membrane breakdown form a pore and allows
the molecule to pass.
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➢ Pore formation occurs in approximately 10 nano seconds.
➢ The pore of the membrane can be reversible based on the
field strength and pulse duration.
➢ If it is reversible cells remain viable, otherwise cell death
results.
➢ Irreversible electroporation is used in cancer treatment to
destroy cancer cells.
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➢ Permeability of membrane to the gene transfer is controlled
by the amplitude and duration of pulse
➢ Generally cancer cells required low field strength
(<700vlcm) with long pulse where as muscle cells need
short pulse with high field strength.
➢ Electroporation has emerged as a reliable physical method
for delivering plasmid DNA.
➢ The therapy can be delivered by intradermal,
intramuscularly or as intratumoural.
11-05-2020 34

❖ Chemical
A. Inorganic Particles
They are generally nanoparticles that can be engineered by
varying size, shape and porosity in order to escape from reticulo
endothelial system or to protect an entrapped molecule from
degradation.
Carbon nanotubes, fullerenes, supra molecular system are most
studied in this category.
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1.Calcium Phosphate
➢ Calcium Phosphate particles were the first ones to used in
this system
➢ It is biocompatible and biodegradable
➢ Calcium plays a vital role in endocytosis and has the
advantage of being readily absorbed and it pose high
binding affinity
➢ Calcium Phosphate nano crystals grow with time reducing
its capacity to store
➢ Later overcome by adding magnesium.
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2. Silica
➢ Major component of materials like sand and glass.
➢ The most commonly used silica as gene delivery agent is
obtained by functionalizing nanoparticles with amino
silicones due to its low toxicity.
➢ It decreased delivery efficiency in the presence of serum
containing media due to the interaction between serum
proteins is a major limiting factor.
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B. Synthetic/Natural Biodegradable
1.Lipid Nano Emulsion
➢ It is a dispersion of one immiscible liquid in another stabilized
by emulsifying agent
➢ They are particles of around 200nm comprises of oil, water
and surfactant
➢ It is considered to be superior to liposomes in scaling up and
stability thus making it more serum resistant
➢ They are less toxic than liposomes.
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2.Solid Lipid Nanoparticles
➢ This particles are made from lipids are solid at both room
and body temperature.
➢ It has advantages of both cationic lipids and lipid nano
emulsion.
➢ It is shown that cationic solid lipid nanoparticle can
effectively protect.
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Polymer Based Vectors
a.Polyethylenimine(PEI)
➢ PET is considered as a gold standard for in vivo and in-
vitro gene transfer
➢ Cationic polymers have high density amine groups
which exert protein sponge effect that ultimately stops
the addification of endosomal pH.
➢ This leads to the influx of chloride within the
compartment and increases the osmotic pressure, leading
to the swelling and rapture of endosomal membrane.
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