GENE THERAPY 1

GENE THERAPYPRESENTED BY

SUBODH S SATHEESH

MPHARM

PHARMACEUTICS

ECPS

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Experimental technique for correcting defective genes that are responsible for disease development

The most common form of gene therapy involves inserting a normal gene to replace an abnormal gene

Other approaches used: 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

INTRODUCTION

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Each human being carries normal as well as some defective genes

In case of cystic fibrosis the mutant gene causes the development of cysts and fibrous tissue in the patient’s pancreas

If the defective gene, however, is dominant, the disease is expressed in any people that carry the defective gene

Diseases such as heart disease do have a genetic component, but are largely dependent on diet and lifestyle.

Genetic defects

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Severe combined immuno-deficiencies (SCID) Hemophilia Parkinson's disease Cancer HIV Brain tomour Lung cancer Prostate cancer asthma

Researches are done on

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TYPES OF GENE

THERAPY

GERM LINE GENE

THERAPY

SOMATIC GENE

THERAPY

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It involves the introduction of corrective genes to reproductive cells or zygotes.

It offers possibility for true care for several diseases. Result in permanent changes. Potential for offering a permanent therapeutic effect for

all who inherit the target gene. Possibility of eliminating some diseases from a particular family.

Germ line therapy

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

Short-lived because the cells of most tissues ultimately die and are replaced by new cells.

Transporting the gene to the target cells or tissue is also problematic.

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

Stem cell therapy

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Gene augmentation therapy Targeted killing of specific cells Targeted inhibition of gene expression Targeted gene mutation

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Gene therapy strategies

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Types of somatic gene therapy

Ex vivo

cells are modified outside the body and

then transplanted back in again

called ex vivo because the cells are treated

outside the body

In vivo

genes are changed in cells when the cells are

still in the body

called in vivo because the gene is transferred

to cells inside the patient’s body

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Genes are transferred to the cells grown in culture, transformed cells are selected, multiplied and then introduced into the patient.

The use of autologous cells avoids immune system rejection of the introduced cells.

The cells are sourced initially from the patient to be treated and grown in culture before being reintroduced into the same individual.

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EXVIVO GENE THERAPY

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It involves the transfer of cloned genes directly into the tissues of the patient.

Its done in case of tissues whose individual cells cannot be cultured in vitro in sufficient numbers (like brain cells) and/or where re-implantation of the cultured cells in the patient is not efficient.

Liposomes and certain viral vectors are employed for this purpose because of lack of any other mode of selection.

In case of viral vectors such type of cultured cells were often used which have been infected with the recombinant retrovirus in vitro to produce modified viral vectors regularly.

The efficiency of gene transfer and expression determines the success of this approach.

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INVIVO GENE THERAPY

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Plasmids or viruses used to move recombinant DNA from one cell to other

Adenoviruses retroviruses and liposomes are commonly used Ability to transfer and integrate genes into new cells Classified to viral Non viral

Vectors of gene therapy

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Retroviruses Adenoviruses Liposomes Herpes simplex virus

Viral vectors

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First used ones Retroviruses are diploid, single-stranded, circular-enveloped

RNA viruses of the family Retroviridae, with a genome of 7–11 kb, and a diameter of approximately 80–120 nm

Retroviruses cause diseases such as AIDS, leukemia, and cancer

Retroviruses are viruses that integrate with host genome to produce viral proteins (gag, pol, env) that are extracted during gene delivery.

Commonly used retroviruses are the Moloney murine leukemia virus species, which have the capacity to deliver exogenous genetic material up to approximately 9 kb

Retroviruses

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Viruses with double stranded DNA They can infect a broader variety of cells than

retroviruses It can cause respiratory, intestinal and eye infection adenoviruses Ad2 and Ad5 are The most widely studied

adenoviruses Penton and fiber proteins of virus capsid interact with

the coxsackievirus-adenovirus receptor cell surface protein to provide cell binding

Adenoviruses

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Lentivirals are viral systems without small, retrovirus-like viral proteins and no capacity for replication

The most important advantage of lentiviruses compared with other retroviruses is their ability for gene transfer to non-dividing cells

Genome of lentiviruses have a more complicated structure; they contain accessory genes which regulate viral gene expression.

HIV-1 is one of the most widely used lentiviral vectors, and contains six accessory genes (tat, rev, vif, vpr, nef, vpu).

Lentiviral vectors do not require degradation of the nuclear membrane for integration

lentivirus

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Nuerotropic virus Gene transfer to nervous system Can infect a wide range of tissues Complications are rare

Herpes simplex virus

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Many studies that used viral vectors reported unsatisfactory results, due to the immunologic and oncogenic adverse effects of these vectors.

It is overcome by NVGDS non-viral vectors have many advantages, such as easy of

fabrication, cell/tissue targeting, and low immune response biggest disadvantage of non-viral vectors in clinical use is

low transduction efficiency. the biggest difficulty in gene therapy is the development of

physical methods to ensure gene transfer to target cells of the gene delivery vectors and delivered gene.

Non viral methods

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Physical methods Chemical methods

Nonviral methods

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Encapsulation Lipoplexes Dendrimers polyplexes

Chemical methods

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Gene guns Electroporation Ultrasound polymers

Physical methods

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Delivery with gene gun method is also termed ballistic DNA delivery or DNA-coated particle bombardment, and was first used for gene transfer to plants in 1987.

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

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

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

Gene gun

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Ultrasound has many clinical advantages as a gene delivery system, due its easy and reliable procedure

Microbubbles or ultrasound contrast agents decrease cavitation threshold with ultrasound energy.

Mostly perfluoropropane-loaded albumin microbubbles were used.

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

Ultrasound

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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.

If the molecule is smaller than the pore size , it can be transferred to the cell cytosol through diffusion

loaded molecules and ions can be transported from the membrane via electrophoretic and electro-osmotic means via the effect of electric regions

Electroporation

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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.

Biodegradable polymers are non-water soluble, and undergo chemical or physical change in biologic environment.

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

polymers

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CHEMICAL METHODS

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An alternative to electrostatic condensation of DNA is encapsulation of DNA with a biodegradable polymer.

Polymers that have an ester linkage in their structures (like polyesters) are hydrolytically degraded to short oligomeric and monomeric compounds, which are more easily discharged from the body.

The degradation mechanism and DNA release can be controlled by changing the physicochemical characteristics and composition of the polymer.

DNA is protected from enzymatic degradation by encapsulation.

Encapsulation

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 Deactivate the genes involved in the disease process  Uses antisense specific to the target gene to disrupt the

transcription of the faulty gene Double stranded oligodeoxynucleotides as a decoy for

the transcription factors that are required to activate the transcription of the target gene

 The oligonucleotide is designed to anneal with complementarity to the target gene with the exception of a central base, the target base, which serves as the template base for repair.

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Oligonucleotides

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Highly branched macromolecule with spherical shape Cationic dentrimers associate with nucleic acid the lack of ability to transfect some cell types, the lack of

robust active targeting capabilities, incompatibility with animal models, and toxicity of cationic lipids are absent in dendrimers

Dendrimers offer robust covalent construction and extreme control over molecule structure, and therefore size

Producing dendrimers has been aslow and expensive process

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Dendrimers

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Anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors

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

 Helper lipids (usually electroneutral lipids, such as DOPE) were added to form lipoplexes, much higher transfection efficiency was observed

The most common use of lipoplexes has been in gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes

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lipoplexes

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Complexes of polymers with DNA are called polyplexes.  polyplexes cannot directly release their DNA load into the

cytoplasm.  low toxicity, high loading capacity, and ease of fabrication,

polycationic nanocarriers. co-transfection with endosome-lytic agents such as

inactivated adenovirus must occur. Use of polymers and copolymers help in the ease of

controlling the size, shape, surface chemistry of these polymeric nano-carriers gives them an edge in targeting capability and enhanced permeability.

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POLYPLEXES

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To decide what is normal and what is disability To decide whether somatic gene therapy is more or less ethical

than germ line therapy Will the therapy only benefit the wealthy due to its high cost? Could the widespread use of gene therapy make the society

less accepting of people who are different? Should people be allowed to use gene therapy to enhance

basic human traits such as height, intelligence, or athletic ability?

Ethical issues

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Give a chance of a normal life to baby born with genetic disease.

Give hope of healthy life to cancer patient. For certain disease that do not have any cure except

gene therapy, it could save many lives.

Advantages

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The genetic testing, screening and research in finding the availability of certain gene is very controversy.

May increase rate of abortion if prenatal test regarding baby with genetic disease is done.

The cost is very high and the patient might need an insurance to cover the treatment.

Cosmetic industry may monopolized this gene therapy if it is used in enhancing beauty and in vanishing the aging effect, rather than used for treatment of a disease.

Problems with viral vectors

Disadvantages

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Remington The science and practice of pharmacy volume 1

Nejm Gene therapy and novel drug delivery page 1-36 Intechopem Gene therapy and viral and nonviral

vectors 387-402 Japi.org Human gene therapy 1-17 Anderson Germ line therapy spring 2013 1-25 Nptel.ac.in gene therapy page 1-24

REFERENCE

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