Gene Transfer Technologies

T.Manoj Kumar

Presentation on

What is gene therapy ????

• Gene therapy is an experimental technique that uses genes to

treat or prevent disease.

• In the future, this technique may allow doctors to treat a

disorder by inserting a gene into a patient’s cells instead of

using drugs or surgery.

Researchers are testing several approaches to gene

therapy, including:

• Replacing a mutated gene that causes disease with a healthy

copy of the gene.

• Inactivating or “knocking out,” a mutated gene that is

functioning improperly.

• Introducing a new gene into the body to help fight a disease.

• Although gene therapy is a promising treatment option for a

number of diseases, the technique remains risky and is still

under study to make sure that it will be safe and effective.

• Gene therapy is currently only being tested for the treatment

of diseases that have no other cures.

Gene transfer ????

• It is defined simply as a technique to efficiently and stably

introduce foreign genes into the genome of target cells.

• The insertion of unrelated, therapeutic genetic information in

the form of DNA into target cells.

Introduction

• There are different reasons to do gene transfer. Perhaps

foremost among these reasons is the treatment of diseases

using gene transfer to supply patients with therapeutic genes.

• There are different ways to transfer genes. Some of these

methods involve the use of a vector such as a virus so it can

take the gene along with it when it enters the cell.

• It provides a novel approach for the investigation and

potential treatment of a variety of disease.

History

• During the 1970’s Rogers made it became possible to introduce

exogenous DNA constructs into higher eukaryotic cells in vitro.

• Mammalian transgenesis was first achieved in the early 1980’s. The

model used in this study was mice.

• In 1990’s, first approved gene therapy case in The United States took

place on 14th September 1990, at the national institute of health, under

the direction of professor William French Anderson.

• In 2012, Glybera became the first gene therapy treatment to be approved

for clinical use in either Europe or The United States after its

endorsement by the European commission.

• Gene transfer may help treat type 1 diabetes (which is due to

failure of the pancreas to produce enough insulin).

• Among the key factors that decide whether the gene

for insulin is turned on or off is the gene PDX-1.

• Using a vector virus the PDX-1 gene has been transferred (into

mice) where the gene is expressed by pancreatic cells which

now produce insulin.

• Gene transfer of PDX-1 may reprogram tissues other than the

pancreas to make insulin and control the abnormally high blood

sugar levels in diabetes.

Gene Transfer Techniques

Based on the vectors used the gene transfer techniques can be

divided as,

• Non-viral methods.

• Viral methods.

Non-Viral Delivery Systems

• Non-viral vectors using mechanical or chemical approaches

can efficiently transfect cells in vitro.

• Mechanical methods involve direct injection or the use of

“gene gun technology” to introduce the plasmid DNA.

Limitations:

Low levels of gene expression.

Inability to use for systemic administration due to the presence

of serum nucleases.

• Electroporation using electrical mediated disruption of cell

membranes to effect transfection is used mainly for in vitro

applications.

• The success of non-viral delivery will be greatly dependent

on the ability to design systems that can transfect cells with

high efficiency, increased stability in presence of serum

proteins and reduced toxicity to cells both in vitro and in

vivo.

• One advantage of this system is they have no constraints on

size of the gene that can be delivered.

Generally there are two approaches for DNA transfer

1. Natural methods of DNA transfer.

2. Artificial methods of DNA transfer.

ELECTROPORATION:

• It is an efficient process to transfer DNA into cells.

• Microscopic pores are induced in biological membrane by the

application of electric field. These pores are known as

electropores which allow the molecules, ions and water to

pass from one side of the membrane to another.

• Electroporation has been reported to enhance the level of

gene expression and significantly improve immune responses

elicited to DNA vaccines in both large and small animals.

General applications of electroporation:

• Introduction of exogeneous DNA into animal cell lines, plant

protoplast, yeast protoplast and bacterial protoplast.

• Electroporation can be used to increase efficiency of

transformation or transfection of bacterial cells.

• Wheat, rice, maize, tobacco have been stably transformed with

frequency upto 1% by this method.

• Electroporation of early embryo may result in the

production of transgenic animals.

• Hepatocytes, epidermal cells, haematopoietic stem cells,

fibroblast, mouse T and B lymphocytes can be transformed

by this technique.

• Naked DNA may be used for gene therapy by applying

electroporation device on animal cells.

Advantages of electroporation

1. Method is fast.

2. Less costly.

3. Applied for a number of cell types.

4. Simultaneously a large number of cell can be treated.

5. High percentage of stable transformants can be produced.

MICROINJECTION

• Microinjection where the DNA is directly injected into plant

protoplasts or cells (specifically into the nucleus or cytoplasm)

using fine tipped (0.5 - 1.0 micrometer diameter) glass needle

or micropipette.

• This method of gene transfer is used to introduce DNA into

large cells, normally performed under a specialized optical

microscope setup called a micromanipulator.

• The process is frequently used as a vector in genetic

engineering and transgenetics to insert genetic material into

a single cell.

• Computerized control of holding pipette, needle, microscope

stage and video technology has improved the efficiency of

this technique.

Advantages of microinjection:

• Frequency of stable integration of DNA is far better as

compare to other methods.

• Method is effective in transforming primary cells as well as

cells in established cultures.

• The DNA injected in this process is subjected to less extensive

modifications.

• Mere precise integration of recombinant gene in limited copy

number can be obtained.

Limitations of microinjection:

1. Costly.

2. Skilled personal required.

3. More useful for animal cells.

4. Embryonic cells preferred for manipulation.

5. Knowledge of mating timing, oocyte recovery is essential.

6. Method is useful for protoplasts and not for the walled

cells.

Applications of microinjection

• Process is applicable for plant cell as well as animal cell but

more common for animal cells.

• Technique is ideally useful for producing transgenic animal

quickly.

• Procedure is important for gene transfer to embryonic cells.

• Applied to inject DNA into plant nuclei.

• Method has been successfully used with cells and protoplast

of tobacco, alfalfa etc.

• Microinjection is potentially a useful method for

simultaneous introduction of multiple bioactive compounds

such as antibodies, peptides, RNAs, plasmids, diffusion

markers, elicitors, Ca2+ as well as nucleus and artificial

micro or Nano particles containing those chemicals into the

same target single-cells.

MACRO INJECTION

• Macroinjection is the method tried for artificial DNA

transfer to cereals plants that show inability to regenerate

and develop into whole plants from cultured cells.

• Needles used for injecting DNA are with the diameter

greater than cell diameter.

• DNA injected with conventional syringe into region of plant

which will develop into floral tillers.

• Around 0.3 ml of DNA solution is injected at a point above

tiller node until several drops of solution came out from top

of young inflorescence.

• Timing of injection is important and should be fourteen

days before meiosis.

• This method was found to be successful with rye plants.

• It is also being attempted for other cereals plants.

Advantages and limitations of macroinjection

• This technique does not require protoplast.

• Instrument will be simple and cheap.

• Methods may prove useful for gene transfer into cereals which

do not regenerate from cultured cell easily.

• Technically simple.

Limitations

1. Less specific.

2. Less efficient.

3. Frequency of transformation is very low.

BIOLISTICS OR MICROPROJECTILES FOR DNA TRANSFER

• Biolistics or particle bombardment is a physical method that

uses accelerated micro projectiles to deliver DNA or other

molecules into intact tissues and cells.

• Biolistics transformation is relatively new and novel method

amongst the physical methods for artificial transfer of

exogenous DNA.

• This method avoids the need of protoplast and is better in

efficiency. This technique can be used for any plant cells, root

section, embryos, seeds and pollen.

• The gene gun is a device that literally fires DNA into target

cells.

• The DNA to be transformed into the cells is coated onto

microscopic beads made of either gold or tungsten. Beads are

carefully coated with DNA.

• The coated beads are then attached to the end of the plastic

bullet and loaded into the firing chamber of the gene gun.

• An explosive force fires the bullet down the barrel of the gun

towards the target cells that lie just beyond the end of the

barrel.

• When the bullet reaches the end of the barrel it is caught and

stopped, but the DNA coated beads continue on toward the

target cells.

• Some of the beads pass through the cell wall into the

cytoplasm of the target cells.

• Here the bead and the DNA dissociate and the cells become

transformed.

• Once inside the target cells, the DNA is solubilised and may

be expressed.

original 22-caliber biolistic gun

• DNA is bound to the microprojectiles, which impact the

tissue or immobilized cells at high speeds

General applications of biolistics

• Biolistics technique has been used successfully to transform

soyabean, cotton, spruce, sugarcane, papaya, sunflower, rice,

maize, wheat, tobacco etc.

• Genomes of subcellular organelles have been accessible to

genetic manipulation by biolistic method.

• Method can be applied to filamentous fungi and yeast

(mitochondria).

• The particle gun has also been used with pollen, early stage

embryoids, meristems and somatic embryos.

Advantages and limitations of biolistics

1. Requirement of protoplast can be avoided.

2. Walled intact cells can be penetrated.

3. Manipulation of genome of subcellular organelles can be

achieved.

Limitations

1. Integration is random.

2. Requirement of equipments.

LIPOSOME MEDIATED GENE TRANSFER

• Liposomes are spheres of lipids which can be used to

transport molecules into the cells.

• These are artificial vesicles that can act as delivery agents

for exogenous materials including transgenes.

• They are considered as sphere of lipid bilayers surrounding

the molecule to be transported and promote transport after

fusing with the cell membrane.

• Cationic lipids are those having a positive charge are used for

the transfer of nucleic acid.

• These liposomes are able to interact with the negatively

charged cell membrane more readily than uncharged

liposomes, with the fusion between cationic liposome and the

cell surface resulting in the delivery of the DNA directly

across the plasma membrane.

• Cationic liposomes can be produced from a number of cationic

lipids, e.g. DOTAP and DOTMA.

• These are commercially available lipids that are sold as an in

vitro-transfecting agent, as lipofectin.

• Liposomes for use as gene transfer vehicles are prepared by

adding an appropriate mix of bilayer constituents to an

aqueous solution of DNA molecules.

• The liposomes are then ready to be added to target cells.

• Germline transgenesis is possible with liposome mediated

gene transfer and ES cells have been successfully transfected

by liposomes also.

Advantages of liposome mediated DNA transfer

1. Simplicity.

2. Long term stability.

3. Low toxicity.

4. Protection of nucleic acid from degradation.

CALCIUM PHOSPHATE MEDIATED DNA TRANSFER

• The process of transfection involves the admixture of isolated DNA

(10-100ug) with solution of calcium chloride and potassium

phosphate under condition which allow the precipitate of calcium

phosphate to be formed.

• Cells are then incubated with precipitated DNA either in solution or

in tissue culture dish. A fraction of cells will take up the calcium

phosphate DNA precipitate by endocytosis.

• Transfection efficiencies using calcium phosphate can be quite low,

in the range of 1-2 %. It can be increased if very high purity DNA

is used and the precipitate allowed to form slowly.

Limitations of calcium phosphate mediated DNA transfer

• Frequency is very low.

• Integrated genes undergo substantial modification.

• Many cells do not like having the solid precipitate adhering

to them and the surface of their culture vessel.

• Due to above limitations transfection applied to somatic

gene therapy is limited.

DNA TRANSFER BY DAE-DEXTRAN METHOD

• DNA can be transferred with the help of DAE Dextran also.

DAE-Dextran may be used in the transfection medium in which

DNA is present.

• This is polycationic, high molecular weight substance and

convenient for transient assays in cos cells.

• It does not appear to be efficient for the production of stable

transfectants.

• If DEAE-Dextran treatment is coupled with Dimethyl

Sulphoxide (DMSO) shock, then upto 80% transformed cell can

express the transferred gene.

• It is known that serum inhibits this transfection so cells are

washed nicely to make it serum free.

• Stable expression is very difficult to obtain by this method.

• Treatment with chloroquinine increases transient expression

of DNA.

• The advantage of this method is that, it is cheap, simple and

can be used for transient cells which cannot survive even

short exposure of calcium phosphate.

POLYETHYLENE GLYCOL MEDIATED TRANSFECTION

• This method is utilized for protoplast only. Polyethylene glycol

stimulates endocytosis and therefore DNA uptake occurs.

• Protoplasts are kept in the solution containing PEG.

• Calcium chloride is added and sucrose and glucose acts as

osmotic buffering agent.

• After exposure of the protoplast to exogenous DNA in presence

of PEG and other chemicals, PEG is allowed to get removed.

Intact surviving protoplasts are then cultured to form cells with

walls and colonies in turn.

• After several passages in selectable medium frequency of

transformation is calculated. PEG based vehicles were less

toxic and more resistant to nonspecific protein adsorption

making them an attractive alternative for non-viral gene

delivery.

Viral Delivery Systems

• Viruses are naturally evolved vehicles that efficiently

transfer their genes into host cells.

• This ability has made them attractive as tools for gene

delivery purposes.

• Viral vectors that have been extensively studied and

genetically manipulated for safety concerns in laboratory

research and for in vivo gene transfer protocols include

retroviruses, adenoviruses, herpes simplex viruses,

lentiviruses, adeno associated viruses and Sindbis viruses.

• Each of the viral vectors has their own individual

advantages, problems, and specific applications.

• Choice of viral vectors is dependent on gene transfer

efficiency, capacity to carry foreign genes, toxicity, stability,

immune responses towards viral antigens and potential viral

recombination.

• There is a wide variety of vectors used to deliver DNA or

oligo nucleotides into mammalian cells, either in vitro or in

vivo.

• The most common vector systems are based on retroviruses,

adeno -associated virus (AAV), adenovirus, herpes simplex

virus (HSV), cationic liposomes, and receptor-mediated

polylysine-DNA complexes.

• Other viral vectors that are currently under development are

based on lenti viruses, human cytomegalovirus (CMV),

Epstein-Barr virus (EBV), poxviruses, negative-strand RNA

viruses (influenza virus), alpha viruses etc.

The three commonly used viral gene transfer systems are

1. Retrovirus (RV).

2. Adenovirus (AV).

3. Adeno Associated Virus (AAV).

Retro Virus Vectors

• Commonly employed vectors.

• Derived from Murine Leukemia Virus (MuLV).

• Not associated with pathology in humans.

• Virus genome has two single copy RNA molecules,

complexed with viral core proteins, surrounded by lipid

envelope.

Recombinant Retrovirus

Properties:

• Infect wide variety of cells.

• Proviral copy – stable integration into the host cell –

lifelong correction.

• Viral replication sequences – cis and Trans acting

elements – generation of replication defective

recombinant retrovirus.

Contains two building blocks

• Retroviral Vector (Transfer Gene).

• Retrovirus Packing Cell (Replication Defective Virus

production).

• The packing signal and other cis acting sequences are

removed.

• Transfected into the packing cells.

• Grown in culture medium, released into the medium.

• Harvested and added to the cells to be genetically corrected.

Applications

• Ex-vivo gene therapy.

• Treatment of T-lymphocyte deficiency (ADA), Tumor

Infiltrating Lymphocytes (TIL), Bone marrow cells (ADA

deficiency, Gauchers disease), hepatocytes (LDL receptor

deficiency), and melanoma.

• In-vivo gene transfer using retroviral vectors for suicide

genes - used in Brain Tumor.

Adeno Virus Vectors

• These are non-enveloped DNA viruses, linear genome and

double stranded DNA molecule of about 36kb.

• 49 distinct subtypes (serotypes).

• Genome Regions: Distinguished into,

• Early (E), and Late (L) – transcription of regions – prior to

or after onset of DNA replication.

• The extremity – consists of a short sequence, The Inverted

Terminal Repeat (ITR) - viral replication and for

encapsidation.

• The Early (E1) genes – Trans activation of other E genes (E2 &

E4) – shut down host cell protein synthesis – starts replication

of adenovirus DNA.

• Late genes L2 – L5 – activated – code for structural proteins of

virus.

• New virion particles are released by host cell lysis.

• These virus enters the Nucleus and remains extra

chromosomally and will not integrate into the host cell

chromosomes.

Recombinant Adenovirus

Features

• Biology of Adenovirus – characterized.

• Not associated with severe human pathology.

• Efficient in introducing its DNA into host cells.

• Can infect a variety of cells & have a broad host range.

• Replication of defective recombinant Adenoviruses, lacking

E1 region can be propagated in-vitro in human cells

harboring E1 sequence in the genome.

Applications

• In-vivo gene therapy – transduce non-dividing and

terminally differentiated cells.

• Transfect cells in-vivo in the intact organ.

• Gene therapy of Cystic Fibrosis.

• Gene therapy of muscle in liver (Blood clotting disorders)

and therapy of diseases of CNS.

Adeno Associated Virus Vectors (AAV)

• Members of Parvo virus family.

• Lack envelope.

• Heat stable and resistant to various chemicals.

• Single stranded DNA molecule.

• Depend on virus – cannot replicate on its own – another virus

is necessary for replicate, uses Adeno/Herpes virus for

effective replication.

• Establishes latent infection – integrates its genome into the

host cell DNA.

Applications

• Used in hematopoietic stem cells for treatment of -

Thalassemia and sickel cell anaemia.

• - Thalassemic erythrocyte contains insufficient - globin

chain whereas, mutant - globin chains are produced in

sickel cell.

References:

• Daan J. A. Crommelin and Robert D. Sindelar, “

Pharmaceutical Biotechnology”, 1st Edition, 1997, Harwood

Academic Publishers, page no-167 – 181.

• James D. Watson, Michael Gilman, Jan Witkowski, Mark

Zoller, “Recombinant DNA”, 2nd Edition, Scientific American

books, 1998, Page no-567 –579.

• http://encarta.msn.com/media_461561269/Gene_Therapy.html

• ASIAN J. EXP. BIOL. SCI. VOl 1 (1) 2010:208-218.