Current Trends In Gene Delivery

Presented By: Vidya Dhonde.

M.Pharm (IP) Sem-II

C U SHAH COLLEGE OF PHARMACY.

Contents

•Gene therapy•History•Gene delivery•Types of gene delivery•Vectors •Conclusion•References

Gene therapy•Gene therapy is an experimental technique that uses genes to

treat or prevent disease.

•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.

1960concepts of Gene Therapy was introduced

1970: Friedmann and Roblin first attempt to perform gene therapy

1990: 1.The first approved gene therapy case at the National Institute of Health, U.K. It was performed on a four year old girl named Ashanti DaSilva. 2.Sickle cell disease is successfully treated in mice

1992: Doctor Claudio Bordignon working at the Vita-Salute San Raffaele University, Italy performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary diseases.

1999: Death of Jesse Gelsinger in a gene-therapy experiment resulted in a significant setback to gene therapy research in the United States

History of gene therapy

2006: Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients.

2007- 2011: Research is still ongoing and the number of diseases that has been treated successfully by gene therapy increases.• Retinal disease

• Colour blindness

2013European officials have approved the Western world's first gene therapy drug from a small Dutch biotech company

Types of

gene therap

y

Somatic gene

therapy

Germ line

gene therapy

Somatic gene therapy • The therapeutic genes are

transferred into the somatic cells, or body, of a patient.

• Affects only the targeted cells in the patient, and is not passed to future generations.

• Appropriate and acceptable for many disorders, including cystic fibrosis, muscular dystrophy, cancer, and certain infectious diseases

Germ line gene therapy • Sperm or eggs, are modified by

the introduction of functional genes, which are integrated into their genomes.

• This would allow the therapy to be heritable and passed on to later generations.

• Highly effective in counteracting genetic disease and hereditary disorders.

IN VIVO

EX VIVO

Gene delivery

• In gene delivery, a normal gene is inserted into the genome to replace an abnormal, disease causing gene.

• The challenge for gene delivery is ‘how to get the new or replace gene into the patient’s target cells’.

• So a carrier molecule called vector must be used for the above purpose.

Characteristics of gene delivery vectors:

• The ideal gene delivery vector should be very specific, capable of efficiently delivering one or more genes of the size needed for clinical application.

• Once the vector is inserted into the patient, it should not induce an allergic reaction or inflammation.

• It should be safe for the patient.

• Vector should be able to target specific cell types

• Finally a vector should be able to express the gene for as long as is required, generally the life of the patient

How It Works???

•A vector delivers the therapeutic gene into a patient’s target cell.

•The target cells become infected with the viral vector.

•The vector’s genetic material is inserted into the target cell.

•Functional proteins are created from the therapeutic gene causing the

cell to return to a normal state.

Types of gene delivery

Viral gene delivery Non- viral gene delivery

Physical method• Electroporation• Gene gun • Ultrasound • Hydrodynamic

therapy

Chemical method• Polymer• Liposomes• Lipoplexes• polyplexes

Viral gene delivery systems consist of viruses: • Adenoviruses• Retroviruses• Adeno associated

viruses• Herpes simplex viruses

Viral gene delivery: Viruses

• Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner.

• Scientists have tried to take advantage of this capability and manipulate the viral genome and replace them with working human gene.

• This altered virus can then be used to transfer genes into cells with great efficiency.

• Some of the viruses insert their genes into the host genome, but do not actually enter the cell.

• Others penetrate the cell membrane hidden as protein molecule and enter the cell.

MOST COMMON VIRAL VECTORS

• Retro virus

• Adeno viruses

Most common viral vectors• Adeno-associated virusesSs DNA viruses that can insert their genetic material at a specific site on chromosome 19. It is now being used in preliminary studies to treat hereditary blood disease, hemophilia, muscle and eye disease.

• Herpes simplex virusesDs DNA viruses.It is a human neurotropic virus, which is mostly used for gene transfer in nervous system. It has a large genome compared to other viruses.

Retroviruses• Create double stranded DNA copies from RNA genome.

• These copies of its genome can be integrated into the chromosome of host cell by another enzyme carried the virus called integrase.

• Integrase inserts the gene anywhere because it has no specific site.

• Integrase may cause insertional mutagenesis.

• Gene therapy trial using retroviral vector to treat X-linked SCID represent the most successful application till date.

Adenoviruses • Adenoviruses are one of the largest and most complex viruses, whose

Ad structure was analyzed with cryo-electron microscopy and X-ray diffractometry.

• Ad2 and Ad5 are the most widely studied adenoviruses.

• Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans.

•Advantage: Beneficial in in-vivo condition.

•Disadvantage: Strong immune and inflammatory response at high dose.

Adenoviruses:

Adeno-associated virus:• Small, single stranded DNA that insert genetic material at a specific

point on chromosome 19.

• From parvovirus family- causes no known disease and doesn't activate patient immune response.

• It is now being used in preliminary studies to treat hereditary blood disease hemophilia, muscle and eye disease.

• Hemophilia treatments, for example, a gene-carrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding.

• Disadvantage: It can produce unintended genetic damage because the virus inserts its genes directly into host cell’s DNA.

Herpes Simplex Viruses•Double stranded DNA viruses that infect neurons eg: Herpes simplex virus type 1

• It is a human neurotropic virus, which is mostly used for gene transfer in nervous system.

• It has a large genome compared to other viruses, which enable scientist to insert more than one therapeutic gene into a single virus, declining the way for treatment of disorders caused by more than one gene defect.

•HSV makes an ideal vector as it can infect a wide range of tissues including muscle, liver, pancreas, and nerve and lung cells.

•Antibodies to HSV-1 are common in humans, however complications due to herpes infections are somewhat rare.

NON-VIRAL GENE DELIVERY

It involve direct introduction of therapeutic DNA.

Advantages:

• Ease of fabrication

• cell / tissue targeting

• Low immune response

• Less toxic

• Immunogenic

• Ease of production

Disadvantage:The biggest disadvantage of non-viral vectors in clinical use is low transfection efficiency.

•Electroporation includes controlled electric application to increase cell permeability.

•Electroporation introduces foreign genes into the cell by electric pulses.

• In this method, pores are formed on the membrane surface to enable the DNA to enter the cell.

•However, high-frequency electrical fields applied for a long duration can cause local tissue damage and inflammation

•The most promising application of electroporation is to enhance plasmid-mediated gene expression in skeletal muscle, which might be useful for inherited muscular dystrophies.

Electroporation

Advantages:

•Reliable and more efficient method.

Disadvantages:

•Difficult to use in internal organ.

•High temp due to high voltage application can cause irreversible tissue damage.

Ex Vivo Electroporation

Gene gun method:•Delivery with gene gun method is also termed ballistic DNA delivery or DNA-coated particle bombardment.

•This method is based on the principle of delivery of DNA coated heavy metal particles by crossing them from target tissue at a certain speed.

• The particles achieve sufficient speed due to a pressurized inert gas.

•Generally, gold, tungsten or silver microparticles were used as the gene carrier .

•Gas pressure, particle size and dose frequency are critical factors in determining the degree of tissue damage and penetration effectiveness of the application .

•Gene-gun-based gene transfer is a widely tested method for intramuscular, intradermal and intratumoral genetic immunization .

The advantages of gene gun delivery systems :

• It does not use toxic chemicals or complex biological systems.

• Delivery is achieved without the need for a receptor.

• DNA fragments of various sizes, including large ones, are transported.

• It has high repeatability.

Ultrasound • Ultrasound has many clinical advantages as a gene delivery system, due its

easy and reliable procedure.

• In recent years, it was found that microbubbles applied by ultrasound increased gene expression. (perfluoropropane-loaded albumin microbubbles )

• The microbubbles were modified with plasmid DNA before the injection and then ultrasound was applied.

• The transfection efficiency of this system is based on frequency, time of ultrasound treatment, the plasmid DNA mount used, etc.

• In conclusion, size and local concentration of DNA plays an important role in the effectiveness of transfection.

Hydrodynamic therapy

• It is based on the principle of understanding the characteristics and structure of capillaries, an understanding of the dynamic characteristics of the fluids passing through blood veins.

• Hydrodynamic gene delivery uses the hydrodynamic pressure created by the injection of the large volume of DNA solution with blood pressure inside veins.

• Thus the permeability of the capillary endothelium increases and pores form in the plasma membrane encircling parenchyma cells.

• DNA or other related molecules can reach the cell from these pores.

•Membrane pores are later closed, and these molecules are retained within the cell.

• In HGT, the principle reason for targeting parenchyma cells is that capillary endothelium and parenchyma cells are closely related, and when the endothelial barrier is ruptured, this allows DNA to easily reach parenchyma cells.

• In addition, the capillary’s thin wall structure has a stretchable and easily fragmented structure.

•The effectiveness of hydrodynamic procedure depends on capillary structure, the structure of the cells encircling capillaries, and the hydrodynamic force applied.

Chemical methods:Polymers:

• For successful delivery, polymers should package DNA in small sizes.

• Thus, extracellular and intracellular stability of DNA is increased.

• Cellular uptake by endocytosis enabled.

• And, by transporting it to the nucleus, the active form of DNA can be released within the nucleus.

Liposomes:• These are lipid bilayers surrounding an aqueous vesicle.

• Can be used to introduce foreign DNA into a target cell.

• Liposomes are used for application of carcinogenic, antifungal, antiparasitic, antiviral, and anti-inflammatory drugs, hormones, DNAs, and cosmetics.

• Advantages:

• Safer when compared to Viral vectors.

• Can carry large DNA molecules.

• effectiveness at small doses.

• Disadvantages:

• Inefficient transfer.

• Transient expression.

Lipoplexes•Complexes formed by self assembly of DNA with liposomes are generally known as lipoplexes.

Lipoplexes: = Complexes of Liposome +DNA

Cationic lipid + Neutral Lipid

• Chemical stable• Biodegradable • Eg. Amino acids Aromatic rings

•Stabilizes the Complexes•Reduces toxicity •Eg. Cholesterol

v. Polyplexes

•Polyelectrolyte complexes formed by self assembly of DNA with cationic polymers are referred to as polyplexes.

Polyplexes: Complexes of Cationic polymers + DNA

Consist of polymer , DNA binding proteins with ligands attached to them

Eg. Poly-L-lysine polyethelineimine

Eg. Histone.synthetic polypeptide.

Polyplexes Mediated Transfection Mechanism:

Summary of approved and published current clinical gene therapy protocols

Conclusion For certain disease that do not have any cure except gene therapy, it

could save many lives.

The cost is very high.

In Cosmetic industry may used in enhancing beauty and in vanishing the aging effect.

European officials have approved the Western world's first gene therapy drug from a small Dutch biotech company, in a milestone for the novel medical technology that fixes faulty genes.

Although some cellular therapy products have been approved, CBER has not yet approved any human gene therapy product for sale.

References Gene Therapy for Cancer Treatment: Past , Present and Future Deanna Cross,

PhD and James K. Burmester, PhD Clinical Medicine & Research Volume 4, Number 3 pg.218-227 .

Gene Delivery Systems: Recent Progress in Viral and Non-Viral Therapy, Erdal Cevher, Ali Demir Sezer and Emre sefik caglar chapter 16, pg.437-456.

Review article: gene therapy, recent developments and future prospects in gastrointestinal oncology , Y. Touchefeu K. J. Harrington, J. P. Galmiche & G. Vassaux ,2010 ,Blackwell Publishing Ltd, pg.953-968.

Human Gene Therapy : A Brief Overview of the Genetic Revolution Sanjukta Misra JAPI february 2013 ,VOL. 61 , pg.40-53.

Nonviral gene delivery: techniques and implications for molecular medicine Alan L. Parker, Christopher Newman, Simon Briggs, Leonard Seymour and Paul J. Sheridan Vol. 5; 3 September 2003, pg.1-15.

Review Article Recent Trends of Polymer Mediated Liposomal Gene Delivery System Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 934605, pg. 15 .

www.genetherapynet.com

http://www.fda.gov/BiologicsBloodVaccines/CellularGeneTherapyProducts/default.htm