Gene expression systems Non viral methods for gene transfer Gene therapy Gene transfer

1. GENE EXPRESSION SYSTEMS
(NON VIRAL GENE TRANSFER)
Presented by
Simran
M.Pharmacy 2nd sem
GGSCOP, YNR

2. INTRODUCTION
 GENE EXPRESSION
 It is the process by which a gene’s DNA sequence is converted into the
structures and functions of a cell.
 Non-protein coding genes are not translated into protein.
 Gene expression refers to a complex series of processes in which the
information encoded in a gene is used to produce a functional product such
as a protein that dictates cell function.
 Genetic information, chemically determined by DNA structure is transferred
to daughter cells by DNA replication and expressed by Transcription
followed by Translation.

3. WHAT IS GENE THERAPY?
 Gene therapy is an Experimental technique that uses genes to treat or prevent
disease.
 Basically gene therapy is an intracellular delivery of genomic materials
(transgene) into specific cells to generate a therapeutic effect by correcting
an existing abnormality or providing the cells with a new function.

4. GENE TRANSFER TECHNIQUES
 Based on the vectors used the gene transfer techniques can be divided as
1. Viral methods
2. Non Viral methods

5. NON-
VIRAL
Chemical
Methods
Oligonucleotides
Liposomes
Cationic liposomes
Lipoplexes
Polymerosomes
Polyplexes
Dendrimers
Physical
Methods
•Naked DNA
•Gene gun
•Electroporation
•Sonoporation
•Magnetofection
•Hydrodynamic

6. PHYSICAL METHODS
 Physical methods applied for in vitro and in vivo gene delivery are based on
making transient penetration in cell membrane by mechanical, electrical,
ultrasonic, hydrodynamic, or laser-based energy so that DNA entrance
into the targeted cells is facilitated.

8. NAKED DNA
 Simplest method of non-viral transfection
 Expression rate is very low as compared to other methods.
 Naked DNA alone is able to transfer a gene (2–19 kb) into skin,
thymus, cardiac muscle, and especially skeletal muscle and liver
cells when directly injected.
 Only possible for certain tissues. Requires large amount of DNA.
 Different methods like Electroporation,Sonoporation and the use
of a “gene gun”, can be used for direct injection of Naked DNA.
 Although naked DNA injection is a safe and simple method, its
efficiency for gene delivery is low so it is only proper for some
applications, such as DNA vaccination.

9. DNA PARTICLE BOMBARDANT BY
GENE GUN
 DNA particle bombardant by gene gun is an ideal alternative
technique to injection of naked DNA.
 Gold or tungsten spherical particles (1–3 µm diameter) are coated
with plasmid DNA and then accelerated to high speed by pressurized
gas to penetrate into target tissue cells.
 It is a modification of a technique called “biolistic,” originally
developed for plant transgenesis, but now used for in vitro and in
vivo gene delivery into mammalian cells too,such as skin, mucosa,
or surgically exposed tissue and for DNA-based immunization or
vaccination.

11. ELECTROPORTION
 It is a method that uses short pulses of high voltage to carry DNA across the cell
membrane and this shock causes temporary formation of pores in the cell
membrane, allowing DNA molecules to pass through.
 Causes a high rate of cell death following electroporation has limited its use,
including clinical applications
 A newer method of electroporation, termed electron-avalanche transfection, has
been used in gene therapy experiments. By using a high-voltage plasma discharge,
DNA was efficiently delivered following very short (microsecond) pulses.
Compared to electroporation, the technique resulted in greatly increased efficiency
and less cellular damage.
 Electroporation has been used in vivo for many types of tissues, such as skin,
muscle, lung, HGPRT gene delivery, and tumor treatment.

13. SONOPORATION
 Sonoporation uses ultrasonic frequencies to deliver DNA into cells.
 The process of acoustic cavitation is thought to disrupt the cell membrane and
allow DNA to move into cells.
 Ultrasound can make some nanomeric pores in membrane to facilitate
intracellular delivery of DNA particles into cells of internal organs or tumors.
 The ultrasound wave’s cavitate the micro bubble within the microcirculation
of target tissue, that result in deposition of targeted transfection of therapeutic
gene.

14.  Micro bubbles are composed of gas filled core [air/nitrogen/inert
gas) with high molecular weight such as per fluorocarbon or
sulfur hexafluoride .The outer shell consists of biocompatible
compounds like lipids, proteins or synthetic biopolymers
 Generally used in brain, cornea, kidney, peritoneal cavity, and
muscle and heart tissues.

15. MAGNETOFECTION
 Magnetofection is a simple and efficient transfection method.
 In this method the magnetic fields are used to concentrate particles containing
nucleic acid into the target cells.
 In this way, the magnetic force allows a very rapid concentration of the entire
applied vector dose onto cells, so that 100% of the cells get in contact with a
significant vector dose.
 Magnetofection has been adapted to all types of nucleic acids (DNA, siRNA,
dsRNA, mRNA), nonviral transfection systems (transfection reagents) and
viruses.

16. HYDRODYNAMIC DELIVERY
 Hydrodynamic is a simple and highly efficient method for direct intracellular delivery of any
water-soluble compounds and particles into internal organs.
 It is also called as Hydroporation.
 The technique uses hydrodynamic pressure to penetrate the cell membrane.
 Hydrodynamic pressure is created by injecting large volume DNA solution in a fraction of
time.
 This creates increased permeability of capillary endothelium and forms pores in plasma
membrane.
 The therapeutic gene of interest can reach the cell through these pores.
 This method has been successful for gene delivery into rodent liver and expression of
hemophilia factors cytokines, erythropoietin, and hepatic growth factors, in mouse and rat.

17. CHEMICAL NONVIRAL DELIVERY
SYSTEMS
 Chemical systems are more common than physical methods and generally are
nanomeric complexes, which include compaction of negatively charged nucleic
acid by polycationic nanomeric particles, belonging to cationic
liposome/micelle or cationic polymers.

18. OLIGONUCLEOTIDES
 To inactivate genes involved in disease process.
 Different approaches are used for Oligonucleotides based Gene Therapy.
1. Using antisense specific to the target gene which disrupts the transcription
of faulty genes.
2. By Using siRNA small molecules of RNA) to signal the cell to cleave
specific sequences in the mRNA transcript of the faulty genes which
results in disruption of translation.
 Eg. Fomivirsen, Mipomersen

19. LIPOSOMES
 It is an artificially prepared spherical vesicle made up of lipid bilayer.
 DNA is encapsulated within liposome.
 Can carry any size of DNA fragment.
 Liposome easily enters the cell membrane and delivers the genes to the
target cell.
 It does not cause an immune response.
 This method is less efficient.

20. CATIONIC LIPOSOMES
 Cationic liposomes are more important current non viral polycationic systems,
which compact negatively charged nucleic acids lead to the formation of
nanomeric complexes.
 Cationic liposomes have unique characteristics: Capability incorporate
hydrophilic and hydrophobic drugs, Low toxicity and no activation of immune
system.
 Targeted delivery of active 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.

22. LIPOPLEXES
 Cationic Lipids due to their positive charge, were first used
to condense negatively charged DNA molecules so as to
facilitate the encapsulation of DNA into liposomes.
 Cationic lipids significantly enhanced the stability of
lipoplexes.
 Cationic liposomes interact with the cell membrane, and was
widely believed as the major route by which cells uptake
lipoplexes.
 The most common use of lipoplexes has been in gene transfer
into cancer cells.

23. POLYMEROSOMES
 Polymerosomes are synthetic versions of liposomes (vesicles with a lipid
bilayer), made of amphiphillic block copolymers.
 They can encapsulate either hydrophilic or hydrophobic contents and can be
used to deliver DNA, proteins, or drugs to cells.
 Advantages of polymersomes over liposomes include
 greater stability,
 mechanical strength,
 blood circulation time, and
 storage capacity.

24. POLYPLEXES
 Complexes of polymers with DNA are called polyplexes.
 Most polyplexes consist of cationic polymers and their fabrication
is based on self-assembly by ionic interactions.
 Difference between polyplexes and lipoplexes is that polyplexes
cannot directly release their DNA load into the cytoplasm.
 As a result, co-transfection with endosome-lytic agents such as
inactivated adenovirus was required to facilitate nanoparticle
escape from the endocytic vesicle made during particle uptake.
 However, Polyethyleneimine and chitosan show proton sponge
effect, in disruption of endosomes and there is no need of
transfection by adenoviruses.

25. DENDRIMERS
 A dendrimer is a highly branched macromolecules
with a spherical shape.
 It is possible to construct a cationic dendrimer, i.e.
one with a positive surface charge
 DNA or RNA with opposite charge binds with
cationic dendrimer.
 On reaching its destination the dendrimer nucleic
acid complex is then taken into the cell via
endocytosis.

27. Thank You…