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Gene Expression Regulation and Vectors
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6. Gene Expression
Kushal Saha
Gene expression is the combined process of the transcription of a gene into mRNA, the processing of that
mRNA, and its translation into protein (for protein-encoding genes).
Genes encode proteins and proteins dictate cell function. Therefore, the thousands of genes expressed in a
particular cell determine what that cell can do. Moreover, each step in the flow of information from DNA to
RNA to protein provides the cell with a potential control point for self-regulating its functions by adjusting
the amount and type of proteins it manufactures.
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene
product. These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA
genes, the product is a functional RNA.
Levels of regulation of gene expression:
Several steps in the gene expression process may be modulated, including the
1. Transcription, 3. Translation,
2. RNA splicing 4. Post-translational modification of a protein.
7. An overview of the flow of information
from DNA to protein in a eukaryote:
First, both coding and noncoding regions of
DNA are transcribed into mRNA. Some regions
are removed (introns) during initial mRNA
processing. The remaining exons are then
spliced together, and the spliced mRNA
molecule (red) is prepared for export out of the
nucleus through addition of an endcap
(sphere) and a polyA tail. Once in the
cytoplasm, the mRNA can be used to construct
a protein.
Process of alteration of gene expresses been studied has been studied in in detail & involves modulation of
gene transcription. Transcription control can result in tissue specific gene expression and influenced by
hormones, heavy metals.
Purpose of regulation of gene expression:
Regulated expression of genes is required for
1)Adaptation-Cells of multicellular organisms respond to varying conditions. Such cells exposed to hormones
and growth factors change substantially in –
shape,
growth rate, and
other characteristics
2) Tissue specific differentiation and development-
The genetic information present in each somatic cell of a metazoan organism is practically identical.
Cells from muscle and nerve tissue show strikingly different morphologies and other properties, yet
they contain exactly the same DNA.
These diverse properties are the result of differences in gene expression.
Expression of the genetic information is regulated during ontogeny and differentiation of the
organism and its cellular components.
8. In simple terms, regulation of gene expression is of two types-
1.Positive regulation: When the expression of genetic is quantitatively increased by the presence of specific
regulatory element is known as positive regulation. Element modulating positive regulation is known as
activator or positive regulator.
2.Negative regulation: When the expression of genetic information diminished by the presence of specific
regulatory element. The element or molecule mediating the negative regulation is said to be repressor.
Biological systems exhibits 3 types of temporal responses:
Type A response: Increased extent of gene expression is continued in presence of inducing signal. This is
commonly observed in prokaryotes in response to intracellular conc. of nutrient.
TYPE B response: increased amount of gene expression is transient even in presence of regulatory signal.
This is seen in commonly during development of organism.
9. Type c response: Increased gene expression that persists even after termination of signal. It is seen in development
of tissue or organ.
Differences between gene expression in prokaryotes and eukaryotes:
Gene regulation is significantly more complex in eukaryotes than in prokaryotes for a number of reasons:
1) The genome being regulated is significantly larger:
The E. coli genome consists of a single, circular chromosome containing 4.6 Mb.
This genome encodes approximately 2000 proteins.
In comparison, the genome within a human cell
Contains 23 pairs of chromosomes ranging in size from 50 to 250 Mb.
Approximately 40,000 genes are present within the 3000 Mb of human DNA.
It would be very difficult for a DNA-binding protein to recognize a unique site in this vast array of DNA
sequences.
More-elaborate mechanisms are required to achieve specificity.
2) Different cell types:
Different cell types are present in most eukaryotes.
Liver and pancreatic cells, for example, differ dramatically in the genes that are highly expressed.
Different mechanisms are involved in the regulation of such genes.
10. 3) Absence of operons:
The eukaryotic genes are not generally organized into operons as are there in prokaryotes
Instead, genes that encode proteins for steps within a given pathway are often spread widely across
the genome.
4) Chromatin structure:
The DNA in eukaryotic cells is extensively folded and packed into the protein-DNA complex call
chromatin.
Histones are an important part of this complex since they both form the structures known as
nucleosomes and also contribute significantly into gene regulatory mechanisms.
5) Uncoupled transcription and translation processes:
In prokaryotes, transcription and translation are coupled processes, the primary transcript is
immediately translated.
The transcription and translation are uncoupled in eukaryotes, eliminating some potential gene
regulatory mechanisms.
The primary transcript in eukaryotes undergoes modifications to become a mature functional m RNA.
Vectors for gene therapy:
Vectors for gene therapy can be classified into two types:
1. Viral vectors
2. Non-viral
Viral vectors:
Viral gene delivery systems consist of viruses that are modified to be replication-deficient which were made
unable to replicate and can deliver the genes to the cells to provide expression. Viral systems have advantages
such as constant expression and expression of therapeutic genes. Adenoviruses, retroviruses, lentiviruses,
etc. are used for viral gene delivery. There are some limitations that restrict the use of these systems, which
includes the use of viruses in production, immunogenicity, toxicity and lack of optimization in large-scale
production.
11. Viruses have appropriate mechanisms for transfer of genetic material to the target cell; it involves the use of
viral vectors that provide high transduction effectiveness and advanced level of gene expression. The optimal
design of a viral vector depends on the types of virus to be used.
I. Retroviral Vectors:
First viruses used as vectors in this gene therapy experiments were retroviruses. These belong to a class of
viruses (RNA as genetic material) that creates double stranded DNA copies with the enzyme reverse
transcriptase. The first and the most commonly used retrovirus is Molony murine leukemia virus (MuLV). It is
cultured in vitro but is inactive in in-vivo experiments.
The advantages of retroviral vectors are determined by their stability integration into the host genome,
generation of viral for efficient gene transfer, infectivity of the recombinant viral particles.
II. Adenoviral Vectors:
Adenoviruses (Ad) were first discovered in 1953 by isolation from human adenoid tissue cultures. Adenovirus
has a 36 kb, double stranded DNA genome packaged in a 100 nm icosahedral capsid. Wild-type adenovirus
infects cells in the upper respiratory tract and results in mild cold. Adenovirus infects dividing and nondividing
cells, which is important for in-vivo gene delivery. They are commonly used as gene vectors. Adenoviruses
replicate and produce virions, which contain the nucleus of the infected cell. Adenoviral particles do not
contain lipid or membrane and are therefore stable in solvents such as ether or ethanol. Adenoviruses are
one of the largest and most complex viruses, whose Ad structure was analyzed with cryo-electron microscopy
and X-ray diffractometry.
Crystal structures of single Ad protein contain fiber knob, shaft, domains, penton base, hexon, and cysteine
protease. Ad capsid consists of 252 sub-units called capsomeres, which contain 240 hexonproteins and 12 of
the penton base.
III. Baculovirus:
Baculoviruses are a diverse group of DNA viruses which is capable of infecting more than 500 insect species.
Among them Autographa californica baculovirus which is multiple nucleopolyhedrovirus (AcMNPV) contains
a circular double-stranded DNA genome of ≈134 kb and is the most widely used. Budded AcMNPV is highly
infectious to cultured insect cells, thus recombinant baculoviruses are engineered for carrying exogenous
genes to infect insect cells for the production of numerous recombinant proteins.
Baculovirus neither replicates nor is toxic inside the transduced mammalian cells.
12. Baculovirus DNA degrades in the cells over time and there is no evidence of baculoviral DNA integration into
host chromosomes unless selective pressure is applied.
IV. Lentiviral systems:
Lentivirals are viral systems without small retrovirus-like viral proteins and has no capacity for replication.
They provide gene delivery to non-dividing cells. This application is used in targeting in post-mitotic and highly
differentiated cells. The most important advantage of lentiviruses is their ability for gene transfer to non-
dividing cells.
They contain accessory genes which regulate viral gene expression, control combination of infectious
particles and modulate viral replication in infected cells.
For example, HIV-1 is one of the most widely used lentiviral vectors, and contains six accessory genes.
Non-viral gene delivery systems:
It develops gene expression to specific cells for treatment of human diseases or for transfer of genetic
material to inhibit the production of a target protein. The nonviral gene delivery system uses synthetic or
natural compounds or physical forces for delivering a piece of DNA into a cell. The materials used are less
toxic and immunogenic than the viral gene. Nonviral vectors can overcome the cell membrane barrier and
intracytoplasmic compartmentalization of the administered genetic material. These are efficient for
increasing membrane penetration in vitro. It uses a variety of lipid formulations for delivering DNA to host
cells. Nonviral systems comprise all the physical and chemical systems except viral systems and also include
either chemical methods (such as cationic liposomes and polymers) or physical methods (such as gene gun,
electroporation, particle bombardment, ultrasound utilization).
Non-viral methods for transfection:
There are three categories of non-viral systems are available:
• Inorganic particles
• Synthetic or natural biodegradable particles
• Physical methods
13. Physical methods for gene delivery:
This methoduses a physical force to overcome the membrane barrier of the cells and facilitate intracellular
gene transfer. These make transient penetration in cell membrane by mechanical, electrical, ultrasonic,
hydrodynamic, or laser-based energy for DNA entry into the targeted cells.
These methods include the following:
Needle and Jet injection:
Needle injections are localized injection of naked DNA which were first demonstrated intramuscularly in 1990
and then in several other tissues, including liver, skin, and brain. DNA uptake is mostly localized in the area
where needle track is applied. Physical damage induced by needle insertion is responsible for the uptake of
DNA. Different agents such as transferrin, water-immiscible solvents, nonionic polymers, surfactants, or
nuclease inhibitors have been tested to enhance the overall gene expression by this procedure.
The jet injection was first come in 1947 as a needle-free drug delivery method. Jet injection is done through
a high-speed, ultrafine stream of DNA solution driven by a pressurized gas, usually CO2. The injection
generates pores on membranes of target cells and allows intracellular gene transfer.
The jet injection-based gene transfer is ideal for DNA-based vaccine development and for topical
immunization purpose. This method has been used to directly transfect skin cancer cells to facilitate
conventional chemotherapy.
Hydroporation:
Hydroporation also called hydrodynamic gene delivery method. It is used for gene delivery to hepatocytes in
rodents. Hydrodynamic gene delivery is based on the principle of characteristics and structure of capillaries
and the fluids passing through blood veins. The hydrodynamic method employs the high pressure as a driving
force for gene transfer.
Hydrodynamic gene delivery uses the hydrodynamic pressure created by the injection of the large volume of
DNA solution with blood pressure inside veins and then the permeability of the capillary endothelium
increases and poreform in the plasma membrane of parenchyma cells.
14. Gene Gun:
Delivery with gene gun method is also called as ballistic DNA delivery or DNA-coated particle bombardment.
It was first used for gene transfer to plants in 1987. This method depends on the impact of heavy metal
particles on target tissues and delivery of coated DNA on particles in passing. The particles are accelerated to
sufficient velocity by highly pressurized inert gas, usually helium. Macroparticles made of gold, tungsten or
silver have been used for gene delivery through gene gun.
Gene gun-based gene transfer has been extensively tested for intramuscular, intradermal and intratumor
genetic immunization.
This method has been used in vaccination against the influenza virus and in gene therapy for treatment of
ovarian cancer.
Electroporation:
Electroporation includes controlled electric application to increase cell permeability. Electroporation was first
developed in 1960s, with studies on the degradation of cell membrane with electric induction.
It is by generating pores on a cell membrane through electric pulses.The efficiency is determined by the
intensity of the pulses, frequency and duration.Electroporation is temporary destabilization of the cell
membrane targeted tissue by insertion of a pair of electrodes into it so that DNA molecules in the surrounding
media of the destabilized membrane would be able to penetrate into cytoplasm and nucleoplasm of thecell.
Ultrasound:
Ultrasound has many clinical advantages as a gene delivery system, due its easy and reliable procedure.
Microbubbles applied by ultrasound increased gene expression. Microbubbles or ultrasound contrast agents
decrease cavitation threshold with ultrasound energy.
The microbubbles were modified with plasmid DNA before the injection and then ultrasound was applied.
Magnetofection:
Magnetofection is a simple and efficient transfection method that has the advantages of the nonviral
biochemical and physical transfection systems.In this method the magnetic fields are used to concentrate
particles containing nucleic acid into the target cells.
15. DNA is complexed with magnetic nanoparticles which is made of iron oxide and coated with cationic lipids or
polymers through electrostatic interaction. The magnetic particles are then concentrated on the target cells
by the influence of an external magnetic field.
Synthetic or natural biodegradable particles:
Synthetic or natural biocompatible particles may be composed by cationic polymers, cationic lipids or cationic
peptides, and also the combination of these components. The potential advantages of biodegradable carriers
are their reduced toxicity and avoidance of accumulation of the polymer in the cells.
Polymer-based non-viral vectors:
Polymers are long-chained structures composed of small spliced molecules called monomers. Polymers that
are composed of a repeated monomer are called homopolymers, while those composed of two monomers
are called copolymers. Natural and synthetic polymers are used in drug delivery systems. Biodegradable and
non-biodegradable polymers are used according to the type of controlled release mechanism. Biodegradable
polymers are non-water soluble, and undergo chemical or physical change in biologic environments.
Polyamides, dextran, and chitosan are examples of biodegradable polymers.
Non-biodegradable polymers are not degraded in biological environments.
Hydrophilic polymers are hydrogels, which are non-water soluble and swell in water, while hydrophobic
polymers are non-water soluble and do not swell.
Examples of hydrophilic hydrogel polymers include polyvinylalcohol, polyvinylacetate, poly-ethyleneglycol,
polyacrylic acid, polyhydroxy-ethyl methacrylate, and polymethacrylic acid.
Examples of hydrophobic polymers include silicones, and polyethylene vinyl acetate.
For successful delivery, polymers should package DNA in small sizes. So the extracellular and intracellular
stability of DNA is increased; cellular uptake by endocytosisis enabled; and, by transporting it to the nucleus,
the active form of DNA can be released within the nucleus.
Liposomes:
Liposomes are colloidal drug delivery systems. They are biologic membrane-like sacs in sphere form, formed
by one or more lipid layers, and include an aqueous phase. The phospholipid phase consists of principle
components like aqueous phase and cholesterol. Liposomes are classified according to the number of layers
16. they contain. The advantages of liposomes include effectiveness at small doses, extended dosing interval,
and ideal transport for active substances with a short half-life. Cellular uptake mechanism of active
substances in liposomes can be categorized as endocytosis, combination by melting, and adsorption.
Liposomes are used for application of carcinogenic, antifungal, ant parasitic, antiviral, and anti-inflammatory
drugs, hormones, DNAs, and cosmetics. Liposomes are divided into three categories according to their
charges:
Cationic, anionic, or neutral. Compared to viral vectors, liposome delivery systems are non-pathogenic and
nonimmunogenic, with ease of preparation.
Cationic lipids are included in 6 subcategories:
(1) Monovalent cationic lipids
(2) Polyvalent cationic lipids
(3) Guanidine containing
(4) Cholesterol derivative compounds
17. (5) Cationic polymers: Poly (ethylenimine) (PEI), Poly-l-lysine) (PLL)
(6) Lipid-polymer hybrid
Mechanism of gene delivery by cationic particles:
The mechanism of gene delivery by cationic systems includes 4 steps:
(1)Nonspecific interaction between cationic particles and cell surface
(2)Endocytosis into endocytosis vesicles (endosomes)
(3)Compaction and release of the DNA particle from endosomes
(4)Translocation of the DNA particle to nucleus by membrane receptors and transgenic expression of it.
Cationic liposomes are the more important current nonviral polycationic systems, which compact negatively,
charged nucleic acids lead to the formation of nanomeric complexes. Cationic liposomes have unique
characteristics, such as capability to incorporate hydrophilic and hydrophobic drugs, low toxicity, no
activation of immune system, and targeted delivery of bioactive compounds to the site of action.
Cationic liposomes are being used in gene delivery into lung, skeletal muscles, spleen, kidney, liver, testis,
heart, and skin cells. PEI is considered one of the most effective polymer-based transfection agents. PEI was
first used in gene transfer in 1995. It exists in either branched or linear structures. PEI has a high density of
amine groups of which majority are nonprotonated at the physiological pH.
Transfection efficiency and toxicity of PEI depends on its molecular weight, configuration, and the charge
ratio of polymer to DNA used. PLL is among the first synthetic polymers being used for constructing target-
specific gene carrier.
Antisense Technologies:
Antisense technologies are techniques thatform a very powerful weapon for studying gene function and for
discovering new specific treatments of diseases in humans, animals, and plants. In Antisense technology,
synthetically produced molecules seek out and bind to messenger RNA (mRNA), blocks the final step of
protein production. mRNA is the nucleic acid molecule that carries genetic information from the DNA to the
other cellular machinery involved in the protein production. By binding to mRNA, the antisense drugs
interrupt and inhibit the production of specific disease-related proteins. ―Sense‖ refers to the original
18. sequence of the DNA or RNA molecule. ―Antisense‖ refers to the complementary sequence of the DNA or
RNA molecules.
Antisense Drug Discovery:
Inserting Antisense into cells:
1 • Therapeutic goal
2 • Identification of target gene sequence
3 • Design antisense inhibitors
4 • Synthesis of antisense inhibitors
5 • Cell culture screening
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• Lead inhibitor of target gene
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• Sufficient quantities of drug
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• Animal efficiency and dose response, time course study
• Toxicology in animal phase I, II, III trials
19. Endocytosis: One of the simplest methods to get nucleotide in the cell, it relies on the cells natural
process of receptor mediated endocytosis. The drawbacks to this method are the long amount of time
for any accumulation to occur, the unreliable result, and the inefficiency.
Micro-Infection: As the name implies, the antisense molecule would be injected into the cell. The yield
of this method is very high, but because of the precision needed to inject a very small cell with smaller
molecules only about 100 cells can be injected per day.
Liposome–Encapsulation: This is the most effective method, but also a very expensive one. Liposome
encapsulation can be achieved by using products such as lipofect ACE to create a cationic
phospholipids bilayer that will surround the nucleotide sequence. The resulting liposome can merge
with the cell membrane allowing the antisense to enter the cell.
Electroporation: The conventional method of adding a nucleotide sequence to a cell can also be used.
The antisense molecule should transverse the cell membrane offer a shock is applied to the cells.
Antisense technology suppresses the gene for the protein that makes tomatoes – Poil Flavor saur tomatoes
were transgenic tomatoes constructed to have artificial DNA that coded for aRNA that was complementary
to the RNA that coded for the protein that caused spoiling. Antisense technology is used in fundamental
research where the introduction of antisense oligonucleotides can help determine the role of a specific gene
in a specific physiological process.
Antisense technology blocks the production of disease technology and production of disease causing
proteins.
This is done by creating a synthetic ―antisense‖ or complementary nucleotide sequence of DNA or RNA that
interacts and binds to the ―sense‖ or original mRNA sequence.
This ―mRNA‖- antisense complex‖ can no longer be translated and the disease causing protein cannot be
produced.
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